Fermentations

Appl Biochem Biotechnol, 2004 Feb, 112(2), 111 - 22
Production of antitumoral retamycin during fed-batch fermentations of Streptomyces olindensis; Pamboukian CR et al.; Fed-batch runs were performed in order to correlate the production of retamycin, an anthracycline antibiotic produced by Streptomyces olindensis in submerged cultures, with the specific growth rate . Maximum retamycin production was achieved with an exponential feed rate, controlling the specific growth rate at a low value (0.03 h-1, about 10% of the maximum specific growth rate) . Control of the specific growth rate at higher values (0.10 and 0.17 h-1) caused a decrease in antibiotic production . Morphology, assessed by image analysis, was shown to be highly relevant in this process . Cell growth mainly in the form of clumps (90% clumps and 10% free filaments) led to better results than growth as clumps (75%) and free filaments (25%).

Clin Appl Thromb Hemost, 2004 Jan, 10(1), 27 - 37
Molecular and pharmacologic profile of tinzaparin and a comparable low-molecular-weight bacterial sulfaminoheparosan; Maddineni J et al.; Low-molecular-weight heparins (LMWH) represent depolymerized porcine mucosal heparin derivatives, which are commonly used for the management of thrombotic disorders . Because of their widespread usage, the supplies of the raw material namely unfractionated heparin are nearly exhausted . Porcine mucosal tissue is almost exclusively used for the preparation of these agents . Thus, there is a timely need for the production of heparin like drugs from other sources . Fermentation techniques have been used to produce carbohydrates such as dextran and innulin for therapeutic purposes . Bacterial cell wall polysaccharide mimics the linear hexose units, which constitute heparin . Utilizing Escherichia coli cell membranes produced by fermentation technology, chemical sulfation and enzymatic epimerization, sulfaminoheparosan type of polymer mimicking the structure of heparin has been produced . These semi-synthetic sulfaminoheparosans exhibit biologic actions comparable to that observed with heparin . The sulfaminoheparosan core can also be degraded to obtain low-molecular-weight (LMW) derivatives mimicking LMWHs . Using this technique, a novel LMW sulfaminoheparosan derivative (Q93C/239) was produced by Inalco, Milan, Italy . To compare this heparin analogue, a LMWH, namely tinzaparin, was used to determine the relative anticoagulant, antiprotease, and molecular profile . Additional studies were carried out to determine the susceptibility of this agent to heparinase-I . These comparative studies exhibited both antiprotease and anticoagulant properties similar to those of tinzaparin . However LMW sulfaminoheparosan resisted heparinase-I digestion at low heparinase-I concentrations . These studies demonstrate that the sulfaminoheparosan derived LMW components exhibit similar molecular and anticoagulant profile as tinzaparin and warrant additional preclinical and clinical development to determine their potential usefulness as antithrombotic agents.

J Gene Med, 2004 Feb, 6 Suppl 1, S54 - 66
Plasmid DNA purification; Stadler J et al.; The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA . However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA . Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing . The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA.The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material . The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA . Master and working cell banks need to be characterised and established . Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms . Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised . To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform . A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i) . different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii) . the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii) . the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins).We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy .

J Gene Med, 2004 Feb, 6 Suppl 1, S45 - 53
Animal-free production of ccc-supercoiled plasmids for research and clinical applications; Schleef M et al.; The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application . Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances.Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP) . Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g . gene gun or jet injection) . The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms .

Infect Immun, 2004 Mar, 72(3), 1657 - 65
Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6; Okusawa T et al.; The lipopeptide FSL-1 {S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF} synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8 . FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did . The level of the activity of FSL-1 was higher than that of MALP-2 . This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter . To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity . Both portions did not reveal the activity . A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity . Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid . These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.

J Air Waste Manag Assoc, 2004 Feb, 54(2), 242 - 9
Performance of an innovative two-stage process converting food waste to hydrogen and methane; Han SK et al.; This study was conducted to evaluate the performance of an innovative two-stage process, BIOCELL, that was developed to produce hydrogen (H2) and methane (CH4) from food waste on the basis of phase separation, reactor rotation mode, and sequential batch technique . The BIOCELL process consisted of four leaching-bed reactors for H2 recovery and post-treatment and a UASB reactor for CH4 recovery . The leaching-bed reactors were operated in a rotation mode with a 2-day interval between degradation stages . The sequential batch technique was useful to optimize environmental conditions during H2 fermentation . The BIOCELL process demonstrated that, at the high volatile solids (VS) loading rate of 11.9 kg/m3 x day, it could remove 72.5% of VS and convert VS(removed) to H2 (28.2%) and CH4 (69.9%) on a chemical oxygen demand (COD) basis in 8 days . H2 gas production rate was 3.63 m3/m3 x day, while CH4 gas production rate was 1.75 m3/m3 x day . The yield values of H2 and CH4 were 0.31 and 0.21 m3/kg VS(added), respectively . Moreover, the output from the post-treatment could be used as a soil amendment . The BIOCELL process proved to be stable, reliable, and effective in resource recovery as well as waste stabilization.

Environ Technol, 2003 Dec, 24(12), 1471 - 8
Kinetics of glucose fermentation by a mixed culture in the presence of linoleic, oleic, and stearic acid; Lalman JA et al.; The effects of long chain fatty acids (LCFAs) on glucose degradation were examined at 21 degrees C . A competitive inhibition model was used to determine the kinetics of glucose degradation . Half velocity constants (Ks) were a function of LCFA concentration only at 100, 300 and 500 mg l(-1) . The inhibitor constants (KI) for individual and mixed LCFAs were statistically the same . Glucose degradation rates for cultures receiving saturated (stearic acid (SA)) and monounsaturated (oleic acid (OA)) LCFAs were statistically the same but statistically different when compared to cultures fed with a polyunsaturated LCFA (linoleic acid (LA)) . Individual and mixed LCFAs inhibited glucose degradation at threshold levels of 300 and 500 mg l(-1), respectively.

J Econ Entomol, 2003 Dec, 96(6), 1967 - 73
Knockdown and mortality of adults of eight species of stored-product beetles exposed to four surfaces treated with spinosad; Toews MD et al.; Contact toxicity of a commercial bacterial fermentation insecticide, spinosad, to adults of eight stored-product beetles was evaluated on four different surfaces . Aqueous spinosad suspension was sprayed with an airbrush to 30.5-cm2 surfaces of concrete, galvanized steel, unwaxed floor tile, or waxed floor tile to obtain deposits of 0.05 or 0.1 mg (AI)/cm2 . Control surfaces were sprayed with distilled water . Approximately 24 h after distilled water or spinosad application, 30 adult beetles were confined, by species, to each untreated and spinosad-treated surface . Insects on surfaces were exposed for 24 h to assess knockdown at 25 +/- 1 degree C and 50 +/- 10% RH, and then were held on food for an additional 24 h to assess mortality . Knockdown and mortality of each insect species on all four surfaces were significantly greater on spinosad-treated surfaces than on distilled water-treated surfaces . Knockdown and mortality of all species on all surfaces was similar at the two spinosad deposit levels . Except for Tribolium spp., mortality of all other species exposed to spinosad was 99-100% . Tribolium spp . were highly susceptible to spinosad on concrete (98-100% mortality); however, on unwaxed floor tile, steel, and waxed floor tile recovery on food after knockdown resulted in only 72-92% mortality . Our results suggest that spinosad has excellent contact activity against adults of stored-product insects, especially on concrete, and has potential for use as a general surface, spot, or crack/crevice spray to control insects in empty bins, warehouses, food-processing facilities, and retail stores.

Rev Argent Microbiol, 2003 Oct-Dec, 35(4), 219 - 23
{Degradation of pine needles by Stereum hirsutum}; Mouso N et al.; Pine-needle degradation by Stereum hirsutum was studied under conditions of solid state fermentation with the aim of accelerating its decomposition, avoiding the accumulation in situ and in view of the possible utilization of the residual organic matter . Three experimental systems were tested: pine needles alone and with the addition of either a nitrogen source or barley grain . Determinations were made at 14 and 28 days of incubation . All treatments showed substrate degradation . The addition of a nitrogen source raised enzymatic activities measured but not the degree of degradation . Grain addition resulted in higher biomass, enzyme activities, sugar accumulation and degradation of the substrate . Fungal biomass estimated as N-acetyl glucosamine allowed calculation of the actual degradation of the substrate, that reached 19% at 28 d of culture without additions and 44% at 14 d in pine-needles with grain.

Folia Microbiol (Praha), 2003, 48(5), 633 - 8
Kinetics of soluble glucan production by Claviceps viridis; Flieger M et al.; Among 18 tested strains of Claviceps spp., 7 produced significant amounts of exocellular polysaccharide (EPS) . The maximum production of EPS was found in fermentation broth of Claviceps viridis . The kinetics of growth, substrate consumption, and EPS production in the batch, aerobic, submerged culture of this fungus were investigated in detail . The experimental data were processed by a simple mathematical model describing mass balance of growth, substrate consumption, formation of intermediates, and production of EPS . The parameters of the model were estimated from data obtained in cultivation performed in flasks and two laboratory fermentors of different size . Physiological similarity was obtained during process scale-up in volumetric ratio 1:100 . The sugar consumption efficiency (52%) and observed EPS productivity (1.9 kg/m3 per d) were comparable with literature data.

J Nutr Sci Vitaminol (Tokyo), 2003 Dec, 49(6), 428 - 33
Bitter melon malt vinegar increases daily energy turnover in rats; Ichikawa M et al.; Vinegar is generally believed to be good for health . A mash consisting of 35% ethanolic extract from bitter melon malt vinegar-water (8:50:42) was subjected to further acetate fermentation and the resulting vinegar was converted to dried vinegar powder by spray drying after adsorption on dextrin, which was mixed with a commercial rat chow (CRF-1) in the ratio of 1:19 so as to prepare an experimental diet . Male 12-wk old rats of LETO and OLETF strains were fed this experimental diet in parallel with CRF-1 (control) and examined for respiratory quotient (RQ) and blood or plasma parameters associated with diabetes mellitus . Administration of the experimental diet increased daily food intake as well as daily energy expenditure in both strains . RQ significantly lessened in the vinegar diet-fed group of LETO strain, which was reflected not only in the increased energy consumption from fat but also in the decreased energy consumption from carbohydrate, while no significant difference was observed between both dietary groups of OLETF strain in this respect . The profiles of diurnal energy expenditure in both dietary groups of LETO strain exerted two peaks before lights-on and lights-off . Nevertheless, there was a clear difference between both dietary groups of OLETF strain: interestingly the reproduction of the two peaks became conspicuous in the vinegar diet-fed group despite the lack of such peaks in the control . As a consequence of blood or plasma inspection, it turned out that there was no change in HbA1c but a significant increase in plasma cholesterol in the vinegar diet-fed OLETF rats . From these results, a long-term administration of bitter melon malt vinegar can be expected to suppress a lowering of energy turnover inherent with aging and thereby improve anorexia rather than to bring about a preventive effect against the manifestation of NIDDM.

J Nutr Sci Vitaminol (Tokyo), 2003 Dec, 49(6), 422 - 7
Evidence suggesting that difructose anhydride III is an indigestible and low fermentable sugar during the early stages after ingestion in humans; Tamura A et al.; We investigated the influences of difructose anhydride III (DFAIII), a novel commercially available disaccharide, on sugar metabolism, breath hydrogen and serum acetate in the early stages after ingestion to determine whether DFAIII is an indigestible sugar and to what degree it is fermentable in humans . This study was designed as a randomized controlled single-blind crossover test with 9 healthy subjects, who drink a 200 mL water solution containing 10 g of DFAIII, lactulose or sucrose following overnight fasting . Blood samples (for analysis of glucose, fructose, insulin, triacylglycerol, free fatty acids, and acetate) were collected at 0, 0.5, 1, 2, 4, 8 h after the ingestion and breath samples (for analysis of hydrogen and methane gases) were collected at 1 h intervals until 8 h after the ingestion . We also interviewed each subject hourly about the incidence and severity of specific abdominal complaints and other symptoms . The results revealed that ingestion of 10 g of DFAIII did not change the serum levels of glucose, fructose, and insulin, similarly to the case with lactulose, and no increase in breath hydrogen excretion was comparable to the case with sucrose . The incidence of specific abdominal symptoms tended to be lower after DFAIII ingestion than after lactulose ingestion . It thus turned out that DFAIII was indigestible and low fermentable in the early stages after ingestion.

J Nutr Sci Vitaminol (Tokyo), 2003 Dec, 49(6), 414 - 21
Stimulation of butyrate production in the large intestine of weaning piglets by dietary fructooligosaccharides and its influence on the histological variables of the large intestinal mucosa; Tsukahara T et al.; Fructooligosaccharides (FOS) reach the large intestine and are fermented into short-chain fatty acids (SCFA), lactate, and carbon dioxide . As the major energy source for the epithelial cells of the large intestine, n-butyrate stimulates the proliferation of cells as well as mineral and water absorption from the lumen . We examined the effect of dietary FOS supplementation on luminal SCFA production and its influence on the morphometrical variables of mucosa of the large intestine in commercially available pigs . Six weaning piglets were used . After 7 d of adaptation, three pigs were given a test diet containing FOS (10%) ad libitum for 10 d . The other three remained on the basal diet and were used as controls . At the end of the experiment, their large intestines were removed, and the cecum, gyri centripetales, gyri centrifugales, and rectum were separated . The contents of each portion were collected and measured for SCFA concentration, pH, and moisture . A micrometer was used to measure the crypt depth . The numbers of epithelial and mitotic cells in the crypt columns were also counted . The concentration of SCFA was significantly higher in piglets fed FOS than in the controls . The concentration of n-butyrate was markedly stimulated by FOS . The number of epithelial . mitotic, and mucin-containing cells was higher in piglets fed FOS than in the controls . Accordingly, the crypt depth was larger in the FOS-fed piglets . The luminal n-butyrate concentration showed a significantly positive correlation with the crypt depth and the number of epithelial, mitotic, and mucin-containing cells.

Public Health Nutr, 2004 Feb, 7(1A), 201 - 26
Diet, nutrition and the prevention of dental diseases; Moynihan P et al.; Oral health is related to diet in many ways, for example, nutritional influences on craniofacial development, oral cancer and oral infectious diseases . Dental diseases impact considerably on self-esteem and quality of life and are expensive to treat . The objective of this paper is to review the evidence for an association between nutrition, diet and dental diseases and to present dietary recommendations for their prevention . Nutrition affects the teeth during development and malnutrition may exacerbate periodontal and oral infectious diseases . However, the most significant effect of nutrition on teeth is the local action of diet in the mouth on the development of dental caries and enamel erosion . Dental erosion is increasing and is associated with dietary acids, a major source of which is soft drinks . Despite improved trends in levels of dental caries in developed countries, dental caries remains prevalent and is increasing in some developing countries undergoing nutrition transition . There is convincing evidence, collectively from human intervention studies, epidemiological studies, animal studies and experimental studies, for an association between the amount and frequency of free sugars intake and dental caries . Although other fermentable carbohydrates may not be totally blameless, epidemiological studies show that consumption of starchy staple foods and fresh fruit are associated with low levels of dental caries . Fluoride reduces caries risk but has not eliminated dental caries and many countries do not have adequate exposure to fluoride.It is important that countries with a low intake of free sugars do not increase intake, as the available evidence shows that when free sugars consumption is <15-20 kg/yr ( approximately 6-10% energy intake), dental caries is low . For countries with high consumption levels it is recommended that national health authorities and decision-makers formulate country-specific and community-specific goals for reducing the amount of free sugars aiming towards the recommended maximum of no more than 10% of energy intake . In addition, the frequency of consumption of foods containing free sugars should be limited to a maximum of 4 times per day . It is the responsibility of national authorities to ensure implementation of feasible fluoride programmes for their country.

Biodegradation, 2004 Feb, 15(1), 9 - 18
Degradation of the radioactive and non-labelled branched 4(3',5'-dimethyl 3'-heptyl)-phenol nonylphenol isomer by sphingomonas TTNP3; Corvini PF et al.; The degradation of the 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) nonylphenol isomer in cultures of Sphingomonas TTNP3 supplemented with the technical mixture of nonylphenol was first assessed . Then the radioactive and non-labelled form of these diastereomers were both synthesised . The radioactive isomers were synthesised using {ring-U-14C}-labelled phenol and 3,5-dimethyl-3-heptanol by Friedel and Crafts alkylation . The time-course of degradation was performed with and without 14C-p353NP; balancing of radioactivity was calculated from different soluble fractions (organic, aqueous), bacterial biomass, and 14CO2 evolved as mineralization product . The noticeable portion of 14C bound to biomass showed that at least the aromatic ring of 14C-p353NP was degraded and served as energy source and probably as carbon source for bacterial growth . In addition, the appearance of 3,5-dimethyl-3-heptanol, the nonanol corresponding with the side-chain of p353NP, was demonstrated in the bacterial media, and its concentration determined during the course of fermentation . Besides the parent 14C-p353NP, no other radioactive compounds, i.e . metabolites of 14C-p353NP were detected in the media.

J Agric Food Chem, 2004 Feb 25, 52(4), 980 - 6
Novel fibrinolytic enzyme in fermented shrimp paste, a traditional asian fermented seasoning; Wong AH et al.; A novel fibrinolytic enzyme was purified from fermented shrimp paste, a popular seasoning used in Asian countries . The enzyme is a monomer with an apparent molecular weight of 18 kDa, and it is composed primarily of beta-sheet and random coils . The N-terminal amino acid sequence was determined to be DPYEEPGPCENLQVA . It is a neutral protease with an optimal activity from pH 3 to 7 . No inhibition was observed with PMSF, Pepstatin A, E64, and 1,10-phenanthroline, but the enzyme was slightly inhibited by EDTA and Cu(2+) . It was relatively specific to fibrin or fibrinogen as a protein substrate, yet it hydrolyzed none of the plasma proteins in the studies . In vitro, the enzyme was resistant to pepsin and trypsin digestion . It also had an anticoagulant activity measured with activated partial thrombin time and prothrombin time tests . The novel fibrinolytic enzyme derived from traditional Asian foods is useful for thrombolytic therapy . In addition, this enzyme has a significant potential for food fortification and nutraceutical applications, such that its use could effectively prevent cardiovascular diseases.

J Agric Food Chem, 2004 Feb 25, 52(4), 891 - 7
Effect of antioxidant protection of must on volatile compounds and aroma shelf life of Falanghina (Vitis vinifera L.) wine; Moio L et al.; Two vinification methods involving different degrees of antioxidant protection of Falanghina must during prefermentative steps, and referred as HAMP (high antioxidant must protection) and LAMP (low antioxidant must protection), were compared in terms of fermentation performances of four different yeast strains, composition of the volatile fraction of wines at the end of alcoholic fermentation, and shelf life of wines during storage . The use of HAMP technology resulted in wines with lower volatile acidity and higher concentrations of medium-chain fatty acid ethyl esters, acetates, and volatile fatty acids . For two of the four strains a lower concentration of isoamyl alcohol was also observed . HAMP wines also revealed increased shelf life because of the higher concentration of odor active esters at the end of storage and better preservation of varietal aromas.

Biotechnol Lett, 2003 Dec, 25(24), 2103 - 5
Scale-up of erythritol production by an osmophilic mutant of Candida magnoliae; Kohl ES et al.; Erythritol production by an osmophilic mutant of Candida magnoliae was performed in fermentations of up 50 l to develop an optimized commercial process . By simultaneous feeding glucose and yeast extract, erythritol productivity of 1.2 g l(-1) h(-1) was reached giving 200 g erythritol l(-1) with a yield of 0.43 g g(-1).

Yeast, 2004 Feb, 21(3), 201 - 10
Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae; Paiva S et al.; To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed . The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters . Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity . Disruption of ADY2 in S . cerevisiae abolished the active transport of acetate . Microarray analyses of ady2Delta strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport . Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate .

J Microbiol Methods, 2004 Mar, 56(3), 297 - 314
PCR-DGGE fingerprinting: novel strategies for detection of microbes in food; Ercolini D; Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting was recently introduced into food microbiology . This paper describes the technique and reports on the state-of-the-art application of this technique to food and food-related ecosystems . Applications of PCR-DGGE in several fields of food microbiology are reviewed: the identification of microorganisms isolated from food, the evaluation of microbial diversity during food fermentation, and microbiological and commercial food quality assessment . Potentials and limitations of this culture-independent approach in food microbiology are indicated and future perspectives are discussed.

Biotechnol Bioeng, 2004 Mar 20, 85(6), 638 - 46
Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis; Zelic B et al.; Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation . This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium . Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested . Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities . The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach . To separate pyruvate from fermentation broth a fully integrated continuous process was developed . In this process electrodialysis was used as a separation unit . Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved .

J Biol Chem, 2004 Apr 2, 279(14), 13293 - 6 Epub 2004 Feb 13.
Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids; Miyauchi S et al.; SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known . Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids . The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes . Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions . Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes . Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner . These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm . The substrate-induced currents decreased as the carbon chain length of the substrates increased . The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process . Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule . These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.

Mini Rev Med Chem, 2004 Feb, 4(2), 179 - 88
Biomedical applications of chemically and microbiologically synthesized poly(glutamic acid) and poly(lysine); Shih IL et al.; This review article deals with the synthesis, physiochemical properties, and potential biomedical applications of two homo-poly amino acids . Poly-alpha-glutamic acid (alpha-PGA) and poly-alpha-lysine (alpha-PL) were synthesized by chemical synthesis . poly-gamma-glutamic acid (gamma-PGA) and poly-epsilon-lysine (epsilon-PL) were naturally occurring bio-materials that were produced by microbial fermentation . Poly(glutamic acid) (PGA) and poly(lysine) (PL) are water soluble, biodegradable, edible and nontoxic toward humans and the environment . As a result, they are suitable for various applications and have recently attracted considerable interest of the chemical industry . The distinguished features of PGA and PL also make them promising candidates for biomedical applications . The applications of PGA and PL in the areas of biomedical materials, drug delivery carriers and biological adhesives have been studied extensively and will be discussed in this review.

Br Poult Sci, 2003 Dec, 44(5), 710 - 8
Effects of microbial phytase, produced by solid-state fermentation, on the performance and nutrient utilisation of broilers fed maize- and wheat-based diets; Wu YB et al.; 1 . The influence of a microbial phytase on the performance, toe ash contents and nutrient utilisation of male broilers fed diets based on maize and wheat was investigated . The experiment was conducted as 2 x 2 x 2 factorial arrangement of treatments . Within the factorial, two diet types (maize-soy or wheat-soy) containing two levels of non-phytate phosphorus (3.0 or 4.5 g/kg) were evaluated and each level of non-phytate phosphorus was supplemented with 0 or 500 PU phytase/kg diet . Each of the 8 dietary treatments were fed to 6 pens of 8 birds from d 1 to 21 post-hatching . 2 . Main effects of diet type and phytase were observed for all parameters . Main effect of non-phytate phosphorus was significant only for feed/gain and toe ash contents . Phytase addition improved weight gains irrespective of diet type or non-phytate phosphorus level, but the magnitude of improvement in the phosphorus-deficient wheat-soy diet was greater, resulting in a diet type x non-phytate phosphorus interaction . Responses in toe ash contents were noted only in phosphorus-deficient diets, as indicated by a non-phytate phosphorus x phytase interaction . 3 . Phytase addition improved apparent metabolisable energy values of wheat-based diets, but had little effect on the apparent metabolisable energy of maize-based diets as shown by a diet type x phytase interaction . The apparent metabolisable energy was not influenced by dietary non-phytate P . 4 . Phytase improved ileal nitrogen digestibility in both diet types, but the responses to added phytase tended to be higher in wheat-based diets, as shown by a diet type x phytase interaction . 5 . Increasing the dietary non-phytate phosphorus level reduced phosphorus digestibility and increased excreta phosphorus content . Addition of phytase improved phosphorus digestibility, but the increments were higher in low phosphorus diets resulting in a non-phytate phosphorus x phytase interaction . Phytase addition tended to lower the excreta phosphorus content, but the effects were greater in birds fed low phosphorus diets, as shown by a non-phytate phosphorus x phytase interaction.

Wei Sheng Yan Jiu, 2003 Nov, 32(6), 602 - 5
{Study on the production of citrinin by Monascus strains used in food industry}; Li F et al.; In order to screen strains with less or nearly no production of citrinin, thirty-five Monascus strains used in food industry were selected to investigate the effect of cultivation condition and the medium composition on citrinin production . The results from the study indicated that all strains produced citrinin on the rice with the levels ranging from 0.28 to 2458.80 mg/kg (201.60 mg/kg for the average and 61.99 mg/kg for the median, respectively), while 30 strains (85.71%) yielded this toxin on the submerged culture with the concentration between 0.09 and 55.65 mg/kg (11.99 mg/kg for the average and 3.51 mg/kg for the median, respectively) . Therefore, citrinin production in rice in this study was higher than that in the liquid . In addition, the red pigment production in rice was 3-509 (average 93) times higher than that in the liquid . One strain with the highest color value (1134 U/g) but lower citrinin production in rice was obtained . These results suggested that it is necessary to make the safety evaluation of microorganisms for the production of foods and food ingredients, to investigate the ability of citrinin production by Monascus strains preserved by either the food manufacturer or the national culture collection units and, to survey the citrinin contamination in Monascus products countrywide . It is urgent for China to establish the tolerance limit of citrinin in foods fermented by Monascus species.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 175 - 86 Epub 2004 Feb 13.
Biotechnological advantages of laboratory-scale solid-state fermentation with fungi; Holker U et al.; Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented . This is due to the lack of proper standardisation that would allow objective comparison with other processes . Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported . On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects . However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O2, substrate and moisture gradients . Hence, most published reviews also focus on progress towards industrial engineering . The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology . This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF . In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.

Nutrition, 2004 Feb, 20(2), 187 - 91
Fasting breath hydrogen concentration in short bowel syndrome patients with colon incontinuity before and after antibiotic therapy; Justino SR et al.; OBJECTIVE: Nutrition success in short bowel syndrome (SBS) depends on the intake nutrients and the intestinal absorption capacity . An evaluation of energy expenditure and oxidation of substrate can be obtained with indirect calorimetry by measuring O(2) and CO(2) in the respiration . Elevated colonic fermentation can occur in SBS, producing H(2) and CO(2), which can also be eliminated through respiration and as a consequence affect the results from indirect calorimetry . The objective of this study was to determine the fasting breath H(2) concentration and alterations before and after antibiotic therapy in patients with severe SBS with colon in continuity . METHODS: The study was conducted in two phases . In phase 1, the fasting breath H(2) concentrations were measured in 10 patients with severe SBS with colon incontinuity and a control group of 10 healthy volunteers . In phase 2, the fasting breath H(2) concentrations were re-evaluated after treatment for 7 d with antibiotics in six patients with high rates of H(2) . The analyses were performed with a gas chromatograph (microanalyzer DP; Quintron Instruments, Milwaukee, WI, USA), with results of breath hydrogen and methane concentration expressed in parts per million (ppm) . RESULTS: In phase 1, the levels of fasting breath H(2) were higher in the patients with severe SBS with colon incontinuity than in the healthy controls (32.00 +/- 17.77 versus 5.30 +/- 3.31 ppm; P < 0.001), with 7 of 10 patients presenting levels of H(2) above the normal rate (12 ppm) . The presence of an ileocecal valve did not modify the results significantly . In phase 2, all six patients treated with antibiotics presented normalization in the levels of fasting breath H(2) (from 43.50 +/- 6.90 ppm to 1.33 +/- 1.03 ppm; P < 0.001) and concomitant improvement in the gastrointestinal symptoms . CONCLUSIONS: In relation to the healthy controls, patients with SBS with colon incontinuity presented higher levels of fasting breath H(2) . Antibiotic therapy normalized the levels of fasting breath H(2) and improved the gastrointestinal symptoms . We suggest that the breath H(2) test may be performed routinely in patients with SBS to diagnose elevated intestinal fermentation, prevent errors in the interpretation of the indirect calorimetry, and treat eventual associated gastrointestinal symptoms.

Med Hypotheses, 2004, 62(2), 291 - 3
Too much short chain fatty acids cause neonatal necrotizing enterocolitis; Lin J; Nenatal necrotizing enterocolitis (NEC) is a disease mainly affects premature infants . It is well known that prematurity, enteral formula feeding, and bacterial colonization are three major risk factors for NEC . Acetic acid, propionic acid and butyric acid are short chain fatty acids (SCFAs), which are produced mainly in the colon by bacterial fermentation of undigested carbohydrates . Although luminal production of modest quantities of SCFAs is essential for normal colonic mucosal function, excessive production/accumulation of SCFAs may arise in premature infants due to increased luminal carbohydrates malabsorption and poor gastrointestinal motility, and may have deleterious effects on mucosal integrity . Therefore, it is proposed that too much luminal short chain fatty acids cause neonatal NEC.

Lett Appl Microbiol, 2004, 38(3), 239 - 44
Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR; Torriani S et al.; AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S . bayanus and their hybrids . METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used . A single amplicon of 1710 bp or 329 bp was obtained with species S . cerevisiae and S . bayanus, respectively, while the presence of both bands was observed in S . pastorianus because of its hybrid nature . Both amplification products were also obtained after amplification from DNA of several laboratory S . cerevisiae x S . bayanus hybrid strains . CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S . cerevisiae, S . bayanus and their hybrids . SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

Arch Biochem Biophys, 2004 Mar 1, 423(1), 170 - 81
HOCl-mediated cell death and metabolic dysfunction in the yeast Saccharomyces cerevisiae; King DA et al.; The nature of oxidative damage to Saccharomyces cerevisiae caused by levels of HOCl that inhibit cell replication was explored with the intent of identifying the loci of lethal lesions . Functions of cytosolic enzymes and organelles that are highly sensitive to inactivation by HOCl, including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the mitochondrion, were only marginally affected by exposure of the yeast to levels of HOCl that completely inhibited colony formation . Loss of function in membrane-localized proteins, including the hexose transporters and PMA1 H(+)-ATPase, which is the primary proton pump located within the S . cerevisiae plasma membrane, was also marginal and K(+) leak rates to the extracellular medium increased only slowly with exposure to increasing amounts of HOCl, indicating that the plasma membrane retained its intrinsic impermeability to ions and metabolites . Adenylate phosphorylation levels in fermenting yeast declined in parallel with viability; however, yeast grown on respiratory substrates maintained near-normal phosphorylation levels at HOCl doses several-fold greater than that required for killing . This overall pattern of cellular response to HOCl differs markedly from that previously reported for bacteria, which appear to be killed by inhibition of plasma membrane proteins involved in energy transduction . The absence of significant loss of function in critical oxidant-sensitive cellular components and retention of ATP-synthesizing capabilities in respiring yeast cells exposed to lethal levels of HOCl suggests that toxicity in this case may arise by programmed cell death.

Food Nutr Bull, 2003 Dec, 24(4), 360 - 7
Dietary assessment of refugees living in camps: a case study of Mae La Camp, Thailand; Banjong O et al.; This study presents data on consumption patterns, methods of food procurement, and adequacy of dietary intake among Burmese refugee camp households living along Thailand's border with Burma . Households established for one or more years and with children under 15 years of age were sampled . A questionnaire was used to determine economic, food-consumption, and dietary intake patterns; foods consumed were weighed and measured using a 24-hour recall for the household unit; and nutritional status was determined by a Microtoise tape and digital standing scales . In total, 182 households containing 1,159 people were surveyed . The average household energy and protein intakes were 96.6% and 111.4%, respectively, of the recommended daily allowance (RDA) for healthy Thais . Twelve percent of protein was derived from animal sources . Carbohydrate, protein, and fat accounted for 84%, 9%, and 7% of total energy, respectively . The intake of vitamins A, B1, B2, and C and of calcium ranged from 24.2% to 53.1% of the RDA . Iron intake was 85.3% of the RDA, derived mainly from rice, fermented fish, mung beans, green leafy vegetables, and eggs . Ration foods supplied 60.5% to 98.18% of all nutrients consumed in the households, with the exception of vitamins A and C . Among children under five years of age, 33.7% were underweight, 36.4% were studied, and 8.7% were wasted . Although the refugees were able to procure some nonration foods by foraging, planting trees and vegetables, raising animals, and purchasing and exchanging ration foods for other items, the quantity and quality were not sufficient to compensate for the nutrients that were low or lacking in the ration . The overwhelming majority of dietary nutrients were provided by ration foods, and although the ration and the overall diet may be adequate for short-term subsistence, they do not suffice for long-term survival and optimal growth, especially for younger children.

Nutr Cancer, 2003, 47(1), 24 - 33
Soyasaponins: the relationship between chemical structure and colon anticarcinogenic activity; Gurfinkel DM et al.; Soyasaponins are bioactive compounds found in many legumes . Although crude soyasaponins have been shown to have anti-colon carcinogenic activity, there have been no structure-activity studies . In this study, therefore, purified soyasaponins and soyasapogenins were tested for their ability to suppress the growth of HT-29 colon cancer cells, as determined by the WST-1 assay, over a concentration range of 0-50 ppm . Soyasaponin I and III, soyasapogenol B monoglucuronide, soyasapogenol B, soyasaponin A1, soyasaponin A2, and soyasapogenol A were evaluated . Also tested were mixtures comprising acetylated group A soyasaponins, deacetylated group A soyasaponins, and group B soyasaponins . The most potent compounds were the aglycones soyasapogenol A and B, which showed almost complete suppression of cell growth . The glycosidic soyasaponins by comparison were largely inactive . Soyasaponin A(1), A(2), and I, group B and deacetylated and acetylated group A fractions had no effect on cell growth . Soyasaponin III and soyasapogenol B monoglucuronide were marginally bioactive . These results suggested that the bioactivity of soyasaponins increased with increased lipophilicity . Results from in vitro fermentation suggested that colonic microflora readily hydrolyzed the soyasaponins to aglycones . These observations suggest that the soyasaponins may be an important dietary chemopreventive agent against colon cancer, after alteration by microflora.

Nutr Cancer, 2003, 47(1), 1 - 12
Soy consumption and colorectal cancer; Spector D et al.; We explored the postulated association between soy foods and colorectal cancer incidence by analyzing 13 epidemiological studies: 3 ecological, 1 cohort, and 9 case control . Seven case-control studies evaluated the association between soy intake and colon or colorectal cancer (2,008 cases) . Point estimates generally suggest an inverse association between higher soy consumption and colon cancer onset, although nearly all of the confidence intervals overlap 1.0 . Two of the nine case-control studies focused on adenomas as the outcome (675 total cases), and results for these studies also showed inverse associations . Of the six case-control studies that evaluated the association between soy consumption and rectal cancer (732 cases), the point estimates generally suggest an inverse association with unfermented soy consumption and rectal cancer onset but not fermented soy products . These studies have many limitations, particularly with regard to dietary measurement issues, such as incomplete assessment of soy intake, inadequate quantification, and inappropriate time period for cancer prevention as well as inadequate adjustment for confounders . Most of these issues would contribute to underestimations of any association . In spite of the methodological issues, the available evidence is compelling enough to warrant further study utilizing stronger methodology.

Yakugaku Zasshi, 2004 Jan, 124(1), 31 - 6
{Chemical profiles of methylpyrazines contained in commercially available natto}; Kanuma M et al.; Structures and amounts of methylpyrazines contained in commercial natto, a fermented soybean food in Japan, were determined using HPLC equipped with an acid-resistant reversed phase column, Capcell Pak C18 ACR (Shiseido) . Mobile phase solvent mixtures consisted of acidic phosphate buffer solution (pH 2.0) containing 2% acetonitrile gave satisfactory results with baseline separation of the authentic specimens, such as naked pyrazine, monomethylpyrazine, 2,3-, 2,5-, and 2,6-dimethylpyrazine, trimethylpyrazine, and tetramethylpyrazine . We used the mobile-phase solvent with a flow rate of 1 ml/min at 15.0 degrees C . Before HPLC, commercial natto samples were treated with water to prepare diluted suspensions of surface mucous materials . The suspensions were treated on Sep-Pac C18 Cartridges (Waters) with phosphate buffer solutions containing 2-7% acetonitrile . The extracts were then injected into the analytical column to obtain chromatograms that were used to determine the structures and amounts of methylpyrazines . The results showed that a commercialiy packed natto contains a considerable amount of 2,5-dimethylpyrazine instead of the tetrametyl- and trimethylpyrazines in the traditional products . This may be a result of recent efforts of natto makers whose interests have been focused on new methods for preparing odorless products.

J Basic Microbiol, 2004, 44(1), 49 - 58
Extracellular chitinase production by Trichoderma harzianum in submerged fermentation; Sandhya C et al.; Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using submerged fermentation was studied . Colloidal chitin (1.5% w/v) was used as sole carbon source . Maximum chitinase production (14.7 U/ml) was obtained when fermentation was carried out at 30 degrees C for 96 h using 72 h old mycelium in a medium containing colloidal chitin 1.5% (w/v) as carbon source and 0.42 (% w/v) peptone as nitrogen source (pH 5.5) . Supplementation of additional carbon sources (0.75% w/v) showed no further enhancement in chitinase production while supplementation of nitrogen sources (0.42% w/v) such as peptone and tryptone in the fermentation medium showed a marked increase in production . The process parameters that controlled chitinase production by the fungus were studied and presented here.

J Basic Microbiol, 2004, 44(1), 42 - 8
Biosynthesis of tannase and gallic acid from tannin rich substrates by Rhizopus oryzae and Aspergillus foetidus; Mukherjee G et al.; Modified solid-state fermentation (MSSF) of tannin-rich substrates for production of tannase and gallic acid was carried out using two fungal cultures, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557) . The tannin rich substrates included powdered fruits of Terminalia chebula and Caesalpinia digyna pod cover powder . The different environmental parameters for the maximum production of tannase and gallic acid were optimized through media engineering . The highest yield of tannase and gallic acid was obtained after 60 h in case of Rhizopus oryzae and after 72 h by Aspergillus foetidus with 3 ml of induced inoculum . The optimum initial pH of the fermentation was found to be 4.5 in case of Rhizopus oryzae and 5.0 for Aspergillus foetidus . MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity . Collectively, the data reveal the potential of the modified solid-state fermentation process for the production of tannase and gallic acid from tannin-rich substrates with R . oryzae and A . foetidus.

Angew Chem Int Ed Engl, 2004 Feb 6, 43(7), 788 - 824
Industrial methods for the production of optically active intermediates; Breuer M et al.; Enantiomerically pure amino acids, amino alcohols, amines, alcohols, and epoxides play an increasingly important role as intermediates in the pharmaceutical industry and agrochemistry, where both a high degree of purity and large quantities of the compounds are required . The chemical industry has primarily relied upon established chemical methods for the synthesis of these intermediates, but is now turning more and more to enzymatic and biotechnological fermentation processes . For the industrial implementation of many transformations alternative methods are available . The advantages of the individual methods will be discussed herein and exemplified by syntheses of relevant compounds.

Appl Environ Microbiol, 2004 Feb, 70(2), 1238 - 41
Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli; Kim P et al.; Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK) . In E . coli K-12, PEPCK overexpression had no effect on succinate fermentation . In contrast, in the phosphoenolpyruvate carboxylase mutant E . coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.

Appl Environ Microbiol, 2004 Feb, 70(2), 1207 - 12
Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme; Fujita Y et al.; A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae . When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin . Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope . A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h . The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield . This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.

Bioresour Technol, 2004 May, 92(3), 285 - 90
Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor packed with loofah; Yang Y et al.; A fixed-bed reactor with acclimated methanogens immobilized on a loofah support was studied on a laboratory scale to evaluate the system producing methane from the mixture of CO(2) and H(2) gas, with the production of vitamin B(12) as a by-product . Fermentation using CO(2)/H(2) acclimated methanogens was conducted in a jar fermentor with hydraulic retention times (HRTs) of three and six days . The performance of the reactor was mainly dependent on the HRT . With an HRT of three days, the methane production rate and the vitamin B(12) concentration in the culture broth were 6.18 l/l-reactor/h and 2.88 mg/l-culture liquid; these values were 11.96 l/l-reactor/h and 37.54 mg/l-culture liquid for an HRT of six days . A higher total cell mass of methanogens retained 42.5 g dry cell/l-culture liquid was achieved in the HRT of six days . The loofah carrier immobilized almost 95% of the methanogens, which led to a more effective bio-reaction . It was also observed that the fermentation system had a better ability to buffer pH, especially for an HRT of six days.

Bioresour Technol, 2004 May, 92(3), 251 - 60
Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae; Najafpour G et al.; Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process . The fermentation set-up was comprised of a column packed with beads of immobilized cells . The immobilization of S . cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase . The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate . The production of ethanol was steady after 24 h of operation . The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h . In addition, batch fermentation was carried out with 50 g/l glucose concentration . Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared . In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time . Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time . A yield of 38% was obtained with 150 g/l glucose . The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h . The cell growth rate was based on the Monod rate equation . The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively . The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively . Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively . The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh . Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold . The performance of the two reactors was compared and a respective rate model was proposed . The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol . The achieved results in ICR with high substrate concentration are promising for scale up operation . The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.

J Dairy Sci, 2004 Jan, 87(1), 112 - 21
Methane production by mixed ruminal cultures incubated in dual-flow fermentors; Eun JS et al.; This study evaluated the effects of dilution rate and forage-to-concentrate ratio on gas production by rumen microbes . Continuous cultures were used to monitor methane production at three liquid dilution rates (3.2, 6.3, or 12.5%/h) and three forage-to-concentrate ratios (70:30, 50:50, or 30:70) . Filtered ruminal contents were allowed 6 d of adaptation to diets followed by 7 d of data collection . Forage consisted of pelleted alfalfa and the concentrate mix included ground corn, soybean meal, and a mineral and vitamin premix . The experiment was replicated in a split-plot design . Total volatile fatty acid production averaged 58.0 mmol/d and was not affected by treatment . Molar proportion of acetate increased with increasing forage-to-concentrate ratio . Molar proportion of propionate tended to decrease at dilution rate of 12.5%/h and increased with the medium and low forage-to-concentrate ratio . Culture pH tended to be greater at a dilution rate of 12.5%/h . Methane production that was calculated from stoichiometric equations was not affected by treatments . However, methane production based on methane concentration in fermentor headspace resulted in an interaction effect of treatments . Stoichiometric equations underestimated methane output at higher dilution rates and with high forage diets . Total diet fermentability was lowest at dilution rate of 3.2%/h . Increasing dilution rates increased microbial yield; increasing the proportion of concentrate improved microbial efficiency . Dilution rate and forage-to-concentrate ratio altered the partition of substrate by microbes . Methane production based on actual concentrations differed from values estimated using stoichiometry of end-product appearance.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 393 - 6
Static magnetic fields enhancement of Saccharomyces cerevisae ethanolic fermentation; da Motta MA et al.; Magnetic effects induced in ethanolic fermentation by Saccharomyces cerevisiae strain DAUFPE-1012 were studied during a 24 h exposure to 220 mT steady magnetic fields (SMF) at 23 +/- 1 degrees C, produced by NdFeB rod magnets . The magnets were attached diametrically opposed (N to S) to a cylindrical tube reactor . The biomass growth in the reactor culture media (yeast extract + glucose 2%) during 24 h was monitored by measurements of optical density, which was correlated to cell dry weight . Ethanol concentration and glucose level were measured every 2 h . The pH of the culture media was maintained between 4 and 5 . As a result, biomass (g/L) increased 2.5-fold and ethanol concentration 3.4-fold in magnetized cultures (n = 8) as compared with SMF nonexposed cultures (n = 8) . Glucose consumption was higher in magnetized cultures, which correlated to the ethanol yield.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 269 - 76
Application of vortex flow adsorption technology to intein-mediated recovery of recombinant human alpha1-antitrypsin; Ma J et al.; Vortex flow is a secondary flow pattern that appears above a critical rotation rate in the annular gap between an inner rotating solid cylinder and an outer stationary cylindrical shell . By suspending adsorbent resin in the vortices, a novel unit operation, vortex flow adsorption (VFA), is created . In VFA, the rotation of the inner cylinder facilitates the fluidization of the adsorbent resin . Similar to expanded bed processes, VFA has high fluid voidage so that it can be used to recover biochemical products directly from fermentation broths or cell homogenates without removing cells or cell debris first . In this study, recombinant human alpha1-antitrypsin (alpha1-AT) was expressed in Escherichia coli as a fusion with a modified intein containing a chitin-binding domain . Therefore, the fusion protein can be recovered by chitin resin affinity adsorption . The intein can be induced to undergo in vitro peptide bond cleavage to specifically release alpha1-AT from the bound fusion protein . The capture efficiency of the fusion protein, 26.2%, was obtained in the VFA process . In addition, the specific activity of alpha1-AT was dramatically improved from 0.3 to 205.2 EIC/(mg total protein) after adsorption and cleavage . Therefore, vortex flow adsorption is an integrative technology to combine the primary clarification, concentration, and purification steps in conventional downstream processing into a single unit operation to efficiently recover and purify biochemical products.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 134 - 9
Optimal experimental condition for hemicellulosic hydrolyzate treatment with activated charcoal for xylitol production; Mussatto SI et al.; Rice straw was hydrolyzed into a mixture of sugars using diluted H(2)SO(4) . During hydrolysis, a variety of inhibitors was also produced, including acetic acid, furfural, hydroxymethylfurfural, and lignin degradation products (several aromatic and phenolic compounds) . To reduce the toxic compounds concentration in the hydrolyzate and to improve the xylitol yield and volumetric productivity, rice straw hemicellulosic hydrolyzate was treated with activated charcoal under different pH values, stirring rates, contact times, and temperatures, employing a 2(4) full-factorial design . Fermentative assays were conducted with treated hydrolyzates containing 90 g/L xylose . The results indicated that temperature, pH, and stirring rate strongly influenced the hydrolyzate treatment, temperature and pH interfering with all of the responses analyzed (removal of color and lignin degradation products, xylitol yield factor, and volumetric productivity) . The combination of pH 2.0, 150 rpm, 45 degrees C, and 60 min was considered an optimal condition, providing significant removal rates of color (48.9%) and lignin degradation products (25.8%), as well as a xylitol production of 66 g/L, a volumetric productivity of 0.57 g/L.h, and a yield factor of 0.72 g/g.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 122 - 7
Precursor-directed biosynthesis of novel triketide lactones; Regentin R et al.; Precursor-directed biosynthesis was used to produce different triketide lactones (R-TKLs) in a fermentation process . Plasmids expressing engineered versions of the first subunit of 6-deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains . The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1 degrees ) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE) . Different synthetic precursors were examined for their effect on R-TKL production . An overproducing strain of S . coelicolor expressing the M2+TE protein was found to be best for production of R-TKLs . Racemic precursors were as effective as enantiomerically pure precursors in the fermentation process . The R group on the precursor significantly affected titer (propyl >> chloromethyl > vinyl) . The R-TKLs were unstable in fermentation broth at pH 6-8 . A two-phase fermentation with a pH shift was implemented to stabilize the products . The fermentation pH initially was controlled at optimal values for cell growth (pH 6.5) and then shifted to 5.5 during production . This doubled peak titers and stabilized the product . Finally, the concentration of synthetic precursor in the fermentation was optimized to improve production . A maximum titer of 500 mg/L 5-chloromethyl-TKL was obtained using 3.5 g/L precursor.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 57 - 64
Model-based analysis and optimization of an ISPR approach using reactive extraction for pilot-scale L-phenylalanine production; Takors R; Based on experimental data from fermentation runs, as well as from L-phenylalanine (l-Phe) separation studies, a simple model is presented that describes the total ISPR approach for on-line L-Phe separation . While fermentation process modeling via a macrokinetic model revealed an L-Phe inhibition constant of 20 +/- 1.35 g/L using recombinant E . coli cells, the reactive-extraction process modeling identified the L-Phe cation diffusion in the aqueous donor film and the transport of the lowly soluble carrier/L-Phe complex in the aqueous acceptor film as the most dominant transfer steps . The corresponding mass transfer coefficients were estimated as k(PheD) = 128 x 10(-7) cm/s (extraction) and k(CPheA) = 178 x 10(-5) cm/s (back-extraction) . Simulation studies were performed for the total ISPR approach, which gave hints for strategies of further process optimization.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 38 - 43
Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues; Desai RP et al.; A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies . A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection . Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed . These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor . The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production . Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process . Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity . The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium . The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor . The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process . By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

J Antibiot (Tokyo), 2003 Nov, 56(11), 909 - 16
R176502, a new bafilolide metabolite with potent antiproliferative activity from a novel Micromonospora species; Laakso JA et al.; During the course of a screening program intended to identify new antiproliferative agents, a new bafilolide metabolite was discovered . R176502 (1) was isolated from the liquid fermentation cultures of a novel Micromonospora species found in African river bottom sediment . It was purified from ethyl acetate extracts using a series of countercurrent chromatographic steps . The structure was determined using 1- and 2-D NMR experiments . Three previously described bafilomycins (bafilomycins A1 (2), B1 (3), and B2 (4)) were also isolated (from other microbial strains) . R176502 exhibited potency for inhibition of tumor cell proliferation in the nM range of concentrations.

J Antibiot (Tokyo), 2003 Nov, 56(11), 899 - 904
Oximidine III, a new antitumor antibiotic against transformed cells from Pseudomonas sp . I . Taxonomy, fermentation, isolation, physico-chemical properties and biological activity; Hayakawa Y et al.; Our screening for antitumor antibiotics against transformed cells resulted in the isolation of a new active metabolite, oximidine III, from Pseudomonas sp . QN05727 . This substance selectively inhibited the growth of rat 3Y1 fibroblasts transformed with various oncogenes . In ras- or src-transformed cells, oximidine III arrested the cell cycle at G1 phase and increased the expression of p21WAF1.

J Dairy Sci, 2004 Feb, 87(2), 399 - 405
Effect of supplemental L-lysine-HCL and corn source on rumen fermentation and amino acid flow to the small intestine; Bernard JK et al.; Four lactating Jersey cows fitted with ruminal and duodenal cannulae were used in a 4 x 4 Latin square design trial to determine the effect of supplemental lysine in diets containing dry ground (GC) or steam-flaked (SFC, 360 g/L) corn on ruminal fermentation and amino acid (AA) flow to the duodenum . Supplemental L-lysine-HCL provided 10 g/d of additional Lys to the total mixed rations . There were no interactions between supplemental Lys and corn source . Supplemental Lys increased Lys intake, but did not alter nutrient intake and digestibility or N flow to the duodenum . Intake of dry matter (DM), organic matter (OM), and neutral detergent fiber (NDF) and ruminal digestibility of starch tended to be higher, whereas ruminal digestibility of DM, OM, acid detergent fiber, and NDF was lower for diets supplemented with SFC compared with GC . Whole-tract digestibility was similar for both corn supplements . Ruminal pH and molar proportions of volatile fatty acids were not affected by supplemental Lys or corn source; however, ruminal NH(3) concentrations were lowest when SFC was fed . Intake of N tended to be higher and the flow of total N and individual AA to the duodenum was higher for diets supplemented with SFC . There was a trend for increased flow of microbial N for diets supplemented with SFC . Supplemental L-lysine-HCL did not alter ruminal fermentation, flow of amino acid to the small intestine, or nutrient digestibility, but feeding SFC reduced ruminal fiber digestion and increased microbial protein synthesis and flow of amino acid to the duodenum.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 611 - 7 Epub 2004 Feb 03.
Polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha cells: a computer simulation; Jurasek L et al.; Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA . It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer . A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on published experimental data . The results are applicable to R . eutropha cells or other microorganisms and transgenic plants, where polyhydroxybutyrate production is made possible by heterologous expression systems . The simulation starts at the outset of the PHA accumulation phase when the cells are small and contain no PHA granules . In the presence of abundant glucose, the cell responds to phosphorus limitation by producing 3-hydroxybutyryl-CoA which undergoes polymerization on the few PHA synthase molecules present in the cytoplasm . The amphiphilic PHA synthase-PHA complex attracts additional PHA synthase molecules and granules begin to grow from these initiation sites . Phosphorus limitation and the appearance of PHA in the cytoplasm also stimulate production of phasin molecules that attach themselves to the growing granules . As the granules grow bigger, they begin to touch each other and move to optimize their packing . The phasin coat prevents the granules from coalescing . The size of the cell increases and its prolate ellipsoid shape becomes closer to spherical . The accumulation process stops either when the supply of glucose is exhausted or when the granules become tightly packed within the cell, so that access to their surface is limited . All important variables, such as cell dimensions, granule size, counts of granule-associated molecules, PHA yield, degree of polymerization of the PHA molecules, etc., are recorded in real time during the simulation . Examples of virtual experiments with the cell and their results are shown.

Plant J, 2004 Feb, 37(4), 539 - 53
Transcript profiles and deduced changes of metabolic pathways in maternal and filial tissues of developing barley grains; Sreenivasulu N et al.; Different aspects of barley grain development have been studied in detail, but a more global analysis of gene expression patterns is still missing . We have employed macro arrays, containing 1184 unique sequences from 1421 barley cDNA fragments, to study gene expression profiles in maternal and filial tissues of developing barley caryopses from fertilization to early storage phase . Principle component analysis (PCA) defined distinct expression networks in the pre-storage (0, 2, and 4 days after flowering (DAF)) and early storage phase (10 and 12 DAF) . During an intermediate phase (6 and 8 DAF), PCA visualizes a dramatic re-programming of the transcriptional machinery . In maternal tissues, a large set of protein-mobilizing enzyme mRNAs, together with upregulated lipid-mobilizing enzyme and downregulated reactive oxygen species (ROS)-scavenging enzyme genes, suggests mobilization of stored compounds and programmed cell death (PCD) . In the filial tissue fraction, a set of genes highly expressed during the pre-storage phase is involved in growth processes, including cell wall biosynthesis . The data suggest that the necessary UDP-glucose is provided both by sucrose synthase (isoform 3) and an invertase-driven pathway . Further, major developmental changes in pathways producing energy are predicted . A bell-shaped expression profile with a peak during the intermediate phase is characteristic for genes associated with photosynthesis and ATP production . The photosynthesis-determined increase of ATP concentration could be a prerequisite for the initiation of grain filling, dominated by starch and storage protein synthesis . Storage product accumulation is accompanied by high transcriptional activity of genes involved in glycolysis and fermentation, as well as in the citric acid cycle.

Biotechnol Bioeng, 2004 Mar 5, 85(5), 524 - 38
Restructuring upstream bioprocessing: technological and economical aspects for production of a generic microbial feedstock from wheat; Koutinas AA et al.; Restructuring and optimization of the conventional fermentation industry for fuel and chemical production is necessary to replace petrochemical production routes . Guided by this concept, a novel biorefinery process has been developed as an alternative to conventional upstream processing routes, leading to the production of a generic fermentation feedstock from wheat . The robustness of Aspergillus awamori as enzyme producer is exploited in a continuous fungal fermentation on whole wheat flour . Vital gluten is extracted as an added-value byproduct by the conventional Martin process from a fraction of the overall wheat used . Enzymatic hydrolysis of gluten-free flour by the enzyme complex produced by A . awamori during fermentation produces a liquid stream rich in glucose (320 g/L) . Autolysis of fungal cells produces a micronutrient-rich solution similar to yeast extract (1.6 g/L nitrogen, 0.5 g/L phosphorus) . The case-specific combination of these two liquid streams can provide a nutrient-complete fermentation medium for a spectrum of microbial bioconversions for the production of such chemicals as organic acids, amino acids, bioethanol, glycerol, solvents, and microbial biodegradable plastics . Preliminary economic analysis has shown that the operating cost required to produce the feedstock is dependent on the plant capacity, cereal market price, presence and market value of added-value byproducts, labor costs, and mode of processing (batch or continuous) . Integration of this process in an existing fermentation plant could lead to the production of a generic feedstock at an operating cost lower than the market price of glucose syrup (90% to 99% glucose) in the EU, provided that the plant capacity exceeds 410 m(3)/day . Further process improvements are also suggested .

Biotechnol Bioeng, 2004 Mar 5, 85(5), 463 - 74
High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain; Chen C et al.; During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed . In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E . coli strains carrying mutations in genes encoding periplasmic proteases . Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment . In order to eliminate the possible effects of mutations in other genes, we constructed E . coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations . Extensive light chain proteolysis persisted in degP strains . However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains . These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E . coli, and that DegP (HtrA) makes a minor contribution to this degradation as well . The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations .

Curr Genet, 2004 Apr, 45(4), 187 - 96 Epub 2004 Feb 04.
Genetic analysis of apomictic wine yeasts; Castrejon F et al.; The Saccharomyces cerevisiae wine yeast IFI256 was selected because of its high fermentative capacity and tolerance to ethanol . Sporulation of the IFI256 strain produced two-spore asci unable to conjugate, but able to sporulate again and the spores produced two-spore asci in all cases . That process was studied for at least five generations . The electrophoretic karyotype showed a pattern of 21 chromosomal bands, which was identical both in the parental and in all the descendants analyzed, from the first to the fifth generation . The DNA content of the parental and the descendants was of 1.7 n, which indicates that the capacity for sporulation shown by all descendants was due to apomixis rather than homothallism of the strain . Different concentrations of glucose and acetate and the addition of zinc salts to the presporulation and sporulation media increased the frequency of four-spore asci by up to 9% . However, the tetrads formed were in fact two dyads that resulted from induced endomitosis . Crosses of IFI256 with laboratory strains produced hybrids giving four-spore asci after sporulation, thus indicating the mutation to be recessive . Transformation of IFI256 with plasmids carrying either SPO12 or SPO13 functional genes and crosses with strains carrying functional or mutated SPO12 and/or SPO13 genes indicated that IFI256 carries several mutations, one of which was located to the SPO12 gene . Parasexual cycles and chromosome loss induced after crossing IFI256 with cir0 strains indicated that apomictic mutations were exclusively located at chromosome VIII . The high frequency of wine strains which are apomictic suggests apomixis to be an advantageous phenotype which allows the formation of stress-resistant asci but prevents the loss of favored chromosomal rearrangements.

J Agric Food Chem, 2004 Feb 11, 52(3), 602 - 8
Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae; Coghe S et al.; The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process . Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt . To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt . Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process . During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling . Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype . In beer made with barley malt, ferulic acid was mainly released during the brewing process . Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers . As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.

J Agric Food Chem, 2004 Feb 11, 52(3), 415 - 20
On-line multisensor monitoring of yogurt and filmjölk fermentations on production scale; Navratil M et al.; Near-infrared (NIR) spectrometry and electronic nose (EN) data were used for on-line monitoring of yogurt and filmjolk (a Swedish yogurt-like sour milk) fermentations under industrial conditions . The NIR and EN signals were selected by evaluation of principal component analysis loading vectors and further analyzed by studying the variability of the selected principal components . First principal components for the NIR and the EN signals were used for on-line generation of a process trajectory plot visualizing the actual state of fermentation . The NIR signals were also used to set up empirical partial least-squares (PLS) models for prediction of the cultures' pH and titratable acidity (expressed as Thorner degrees, degrees T) . By using five or six PLS factors the models yielded acceptable predictions that could be further improved by increasing the number of reliable and precise calibration data . The presented results demonstrate that the fusion of the NIR and EN signals has a potential for rapid on-line monitoring and assessment of process quality of yogurt fermentation.

J Anim Sci, 2004 Jan, 82(1), 307 - 18
Interactions between supplement energy source and tall fescue hay maturity on forage utilization by beef steers; Fieser BG et al.; This experiment was conducted to determine the effects of tall fescue hay maturity on intake, digestion, and ruminal fermentation responses to different supplemental energy sources fed to beef steers . Twelve ruminally cannulated, crossbred steers (initial BW = 228 +/- 21 kg) were used in a split-plot experiment with a 3 x 4 factorial treatment arrangement . Steers were assigned randomly to three supplement treatments: 1) no supplement, 2) pelleted soybean hulls, or 3) coarse cracked corn . The second treatment factor was fescue hay maturity: 1) vegetative (VEG), 2) boot-stage (BOOT), 3) heading-stage (HEAD), and 4) mature (MAT) . Supplements were fed once daily at 0.67% of BW (OM basis) and tall fescue hay was offered once daily at 150% of average intake . Supplement type x forage maturity interactions were not detected (P > or = 0.25) for forage, total, or digestible OM intake, which generally decreased (P < 0.01) with advancing forage maturity . Supplementation decreased (P < 0.01) forage and increased (P < 0.01) total OM intake . Supplement type had no effect (P = 0.56) on substitution ratio (unit change in forage intake per unit of supplement intake) . Digestible OM intake was increased (P < 0.01) by supplementation and was greater (P = 0.05) with soybean hulls than with corn . Supplement type x forage maturity interactions (P < or = 0.10) were observed for OM and NDF digestibilities and N retention . Increases in digestibility with soybean hulls relative to corn were greater and supplementation elicited greater increases in N retention with more mature forages . Compared with soybean hulls, corn supplementation resulted in greater (P < 0.01) negative associative effects on OM digestibility . Supplementation did not affect (P > or = 0.10) ruminal pH, total VFA concentrations, or acetate:propionate ratio . Corn supplementation decreased (P < or = 0.07) ruminal NH3-N concentrations compared with control and soybean hulls; however, decreases in ruminal NH3-N concentrations were not consistent with the presence of negative associative effects . Thus, mechanisms not involving ruminal pH or NH3-N concentration seem responsible for negative associative effects observed with corn supplementation . Within the range of forage quality in this study, increases in digestible OM intake from starch- or fiber-based supplements were independent of forage maturity . When fed at similar levels of OM, soybean hull supplementation provided an average of 6% greater digestible OM intake than corn supplementation.

J Anim Sci, 2004 Jan, 82(1), 170 - 8
Fermentation of eastern gamagrass (Tripsacum dactyloides {L.} L.) by mixed cultures of ruminal microorganisms with or without supplemental corn; Eun JS et al.; Five dual-flow fermentors (700 mL) were used to determine the effects of eastern gamagrass (Tripsacum dactyloides {L.} L.) diets on microbial metabolism by mixed rumen cultures . Fermentors were incubated with filtered ruminal contents and allowed to adapt for 4 d to diets followed by 3 d of sample collection . Five dietary treatments were tested: 1) gamagrass hay (GH) + no corn (GHNC), 2) gama grass silage (GS) + no corn (GSNC), 3) GS + low corn (GSLC), 4) GS + medium corn (GSMC); and 5) GS + high corn (GSHC) . The experiment was conducted as a randomized complete block design with five treatments and three replications . Total VFA concentrations were not affected by diets . Corn addition linearly decreased (P < 0.001) molar proportion of acetate . In contrast, molar proportion of propionate was reduced in GSLC (cubic effect, P < 0.001) but remained similar across other diets . Corn supplementation linearly increased molar proportion of butyrate (P < 0.001) . The acetate + butyrate-to-propionate ratio was highest in cultures offered GSLC (cubic effect, P < 0.001) but similar across other diets . Feeding GSNC resulted in a higher ruminal pH compared with GHNC (P < 0.03) . Increasing the level of corn supplementation in GS linearly decreased culture pH (P < 0.001) . All diets resulted in similar methane production, with the exception of GSMC, which lowered methane output (quadratic effect, P < 0.004) . Total substrate fermented to VFA and gas tended to be greater with GHNC than with GSNC (P < 0.06) and linearly increased with the addition of corn (P < 0.004) . Neutral detergent fiber digestibility was similar between GH and GS and was not affected by supplemental corn . Microbial N flow increased in cultures offered GSHC (quadratic effect, P < 0.02) . Corn supplementation at the medium and high level linearly decreased C 18:0 (P < 0.02) and increased trans-C18:1 (P < 0.004) . Including corn at the high level with GS did not have a detrimental effect on fermentation in dual-flow fermentors.

Space Med Med Eng (Beijing), 2003 Oct, 16(5), 374 - 6
{Effect of space flight on yield of Monascus purpureus}; Yin H et al.; Objective: To select high Lovastatin-producing microbial breed by space flight . Method: Monascus purpureus species was carried into space by the recoverable spaceship, "Shenzhou 3" . After flight, the strain was rejuvenized, segregated and selected . The content of Lovastatin produced in the solid fermentation was examined . Result: Mutants with high productivity of Lovastatin were obtained . A series of tests showed that the acquired character of the mutants was stable . Conclusion: Space flight is an effective method for the selection of fine strains.

Eur J Clin Nutr, 2004 Feb, 58(2), 350 - 5
Bioavailability of selenium from bovine milk as assessed in subjects with ileostomy; Chen J et al.; OBJECTIVE: To assess the absorption of dietary selenium in humans, especially of milk selenium . DESIGN:: 1-day meal studies in subjects with ileostomy . SETTING: Hospital outpatient clinics . SUBJECTS: Three subjects in the pilot study and nine subjects in the main study (eight men/ four women) . INTERVENTION: Different beverages, 1 l/day, were given in addition to basal diets (soft drink, 1 week; low-fat milk, 3 weeks; fermented low-fat milk, 3 weeks and soft drink, 1 week) . Ileostomy effluents were collected during the last 2 days in each of the four periods . RESULTS: On days when the subjects were given 1 l of low-fat milk, the estimated fractional absorption of total dietary selenium was 65.5 (2.3)% (mean (s.d.), n=18), which was similar to the value when fermented low-fat milk was given (64.1 (3.2)%) . However, both the calculated amount of milk selenium absorbed (10.9 (2.4) vs 9.4 (1.7) microg selenium) and its fractional absorption (73.3 (16.1) vs 64.1 (11.2)%, n=18) were significantly higher for milk than for fermented milk . CONCLUSIONS: Selenium from milk and other sources is well absorbed in subjects with ileostomy . The real absorption may be even higher than the values shown.

J Gen Appl Microbiol, 2003 Dec, 49(6), 321 - 8
Role of some fermentation parameters on cyclosporin A production by a new isolate of Aspergillus terreus; Sallam LA et al.; A local isolate of Aspergillus terreus was selected among different microorganisms as a new cyclosporin A (Cy A) producing culture . The formation of Cy A was investigated under different fermentation conditions (including selection of the cultivation medium, fermentation time course, inoculum nature, medium volume, agitation rate, pH value) . Relatively high Cy A productivities were maintained when the fermentation process was carried out using a medium composed of (g/L): glucose, 50; bactopeptone, 10; KH(2)PO(4), 5; KCl, 2.5; pH 5.3, inoculated with 2% standard inoculum of 48 h age, shaken at 200 rpm for 10 days.

Anal Bioanal Chem, 2004 Mar, 378(5), 1369 - 75 Epub 2004 Jan 28.
Solvent extraction of amino acids into a room temperature ionic liquid with dicyclohexano-18-crown-6; Smirnova SV et al.; Amino acids Trp, Gly, Ala, Leu are extracted efficiently from aqueous solution at pH 1.5-4.0 (Lys and Arg at pH 1.5-5.5) into the room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF(6)) with dicyclohexano-18-crown-6 (CE) . The most hydrophilic amino acids such as Gly are extracted as efficiently as the less hydrophilic (92-96%) . The influence of pH, amino acid and crown ether concentration, volume ratio of aqueous and organic phases, and presence of some cations on amino acid recovery were studied . The ratio of amino acid to crown ether in the extracted species is 1:1 for cationic Trp, Leu, Ala, and Gly and to 1:2 for dicationic Arg and Lys . This ionic liquid extraction system was used successfully for the recovery of amino acids from pharmaceutical samples and fermentation broth, and was followed by fluorimetric determination.

J Nutr, 2004 Feb, 134(2), 479 - 82
Microbial degradation products influence colon cancer risk: the butyrate controversy; Lupton JR; All dietary fiber, by definition, escapes digestion in the small intestine and thus arrives relatively intact in the large intestine . Its fate in the large intestine depends upon the type of fiber and the colonic microflora . Highly fermentable fibers result in short chain fatty acids including butyrate, which is thought by some to be protective against colon cancer . However, not all studies support a chemopreventive effect for butyrate and the lack of agreement (particularly between in vivo and in vitro studies) on butyrate and colon cancer has been termed the "butyrate paradox." There are a number of reasons for this discrepant effect including differences between the in vitro and in vivo environments, the timing of butyrate administration, the amount of butyrate administered, the source of butyrate (usually dietary fiber) as a potential confounder, and an interaction with dietary fat . Collectively, the studies suggest that the chemopreventive benefits of butyrate depend in part on amount, time of exposure with respect to the tumorigenic process, and the type of fat in the diet.

Lett Appl Microbiol, 2004, 38(2), 118 - 24
Rapid detection of Oenococcus oeni in wine by real-time quantitative PCR; Pinzani P et al.; AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation . METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O . oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples . Conventional CFU countings were also performed . The PCR assay confirmed to be specific for O . oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively) . CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O . oeni in wine without the necessity for sample plating . SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O . oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.

Yeast, 2004 Jan 15, 21(1), 75 - 86
The high general stress resistance of the Saccharomyces cerevisiae fil1 adenylate cyclase mutant (Cyr1Lys1682) is only partially dependent on trehalose, Hsp104 and overexpression of Msn2/4-regulated genes; Versele M et al.; The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in general stress resistance . Previously we identified a mutant which is deficient in fermentation-induced loss of stress resistance (fil1), as a partially inactivating mutant in adenylate cyclase . We have now investigated possible causes of its high stress resistance . Deletion of the TPS1 gene, encoding the first enzyme in the biosynthesis of trehalose, or the heat shock protein gene HSP104 only resulted in a minor effect on heat stress resistance compared with deletion of these genes in a wild-type background . A strain with a deletion of both genes still showed a higher stress resistance in the fil1 background compared to the corresponding wild-type background . Deletion of the transcription factor genes MSN2 and MSN4, which are required for the expression of STRE-regulated genes, resulted in a dramatic drop in heat resistance in the wild-type background but had much less effect in the fil1 mutant . The fil1 msn2Deltamsn4Delta strain remained more heat-resistant than a wild-type strain . A strain in which all four genes, TPS1, HSP104, MSN2 and MSN4, are deleted was very sensitive to heat stress and also to oxidative and salt stress . Presence of the fil1 mutation in such a strain, however, still clearly enhanced heat, oxidative and salt stress resistance . These results indicate that, in addition to trehalose, Hsp104 and the Msn2/4-controlled genes, other factors exist in S . cerevisiae that can, significantly and independently of the known factors, enhance general stress resistance . The mutants described in this work provide a tool to identify these novel components .

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 25 - 32 Epub 2004 Jan 27.
Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations; Luparia V et al.; Although yeasts are known to be able to incorporate a wide variety of exogenous sterols under strict anaerobiosis, no data are available on the assimilation of grapevine phytosterols under enological conditions and the eventual impact on fermentation kinetics . We used therefore a mixture of pure phytosterols, in a proportion representative of the different grape skins phytosterols, to supplement a synthetic fermentation medium simulating a grape must . Under anaerobiosis, normal biomass formation was achieved with 5 mg phytosterols l(-1) . Similar results were obtained in comparison with the observed maximal fermentation rates . These results clearly indicated that grape phytosterols may efficiently act as a substitute for ergosterol in the yeast membrane for promoting yeast growth and initial fermentative activity . Analysis of total yeast sterols indicated that phytosterols are accumulated without further modification, mainly in their esterified form . However, all the fermentations performed with synthetic media supplemented with phytosterols led to stuck fermentations, linked to a correlative strong decrease in cell viability during the stationary phase . Therefore, grape phytosterols are easily incorporated by yeast cells under enological conditions for promoting initial growth and fermentative activity, but rapidly perturb the yeast membrane properties by being the predominant sterols.

Int J Vitam Nutr Res, 2003 Nov, 73(6), 403 - 9
Citrus pectin and oligofructose improve folate status and lower serum total homocysteine in rats; Thoma C et al.; Low folate status leads to increased total homocysteine (tHcy) concentration, and this has been associated with an increased risk of several diseases . Many colonic bacteria are capable of synthesizing folate, and certain dietary fibers may enhance this effect . We assessed the ability of non-fermentable (cellulose) and fermentable (citrus pectin and oligofructose) fibers to improve folate status and lower tHcy in rats . Weanling Sprague-Dawley rats were fed a folate-deficient diet with 5% cellulose for four weeks . Rats were then randomly assigned to one of five folate-adequate (400 micrograms/kg diet) test diets for 24 days . Diets were as follows: Basal; Basal + Sulfa Drug (succinylsulfathiazole); Cellulose; Citrus Pectin; and Oligofructose . High-fiber diets were formulated by diluting the basal diet such that the final diets contained 10% of the added fiber . Twenty-one days later, 3H-p-aminobenzoic acid was injected into the cecum, and rats were terminated three days later . Rats receiving the Citrus Pectin diet had significantly higher plasma (p = 0.011), erythrocyte (p = 0.035), and colonic tissue folate concentrations (p = 0.013) and lower tHcy (p = 0.003) than rats given the Cellulose diet . Rats receiving the Oligofructose had significantly higher plasma folate (p < 0.001) and lower tHcy (p = 0.032) concentrations than rats receiving the Cellulose diet . 3H-folate was detected in the livers of all rats except those receiving Sulfa Drug . Our study indicates that Citrus Pectin and Oligofructose, but not Cellulose, can significantly increase indices of folate status in rats and lower tHcy . It also confirms the ability of the large bowel to absorb folate.

J Biotechnol, 2004 Feb 19, 108(1), 31 - 9
Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis; Ross A et al.; A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of {13C} and {15N} into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies . Isotope labelling of the culture was performed throughout the complete process, starting from preculture . Sufficient biomass is first generated in a batch phase . Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process . Thereby a quantitative utilisation of the most expensive component {13C}-glucose is achieved, while the approximate amount of the {15N}-ammonium chloride to be incorporated is calculated from the scheduled biomass . The usefulness of the strategy is demonstrated with production of uniformly {13C/15N}-labelled tryparedoxin of Crithidia fasciculata . Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.

J Dairy Sci, 2003 Dec, 86(12), 4020 - 32
Nitrogen supplementation of corn silages . 2 . Assessing rumen function using fatty acid profiles of bovine milk; Cabrita AR et al.; The effects of N supplementation strategies on milk fatty acid profiles of dairy cows and their use as a noninvasive technique to diagnose rumen function, and to guide protein feeding decisions on-farm were evaluated in three experiments . Each experiment was designed according to three 3 x 3 Latin squares with 9 Holstein cows receiving total mixed rations based on corn silage . Experiment 1 was designed to study effects of diets with different ratios of effective rumen-degradable protein (ERDP; g) to fermentable metabolizable energy (FME; j) providing, respectively, a large deficiency, a slight deficiency, and a slight excess in relation to the target level of 11 g of ERDP/MJ FME for lactating cows . Experiment 2 evaluated effects of different proportions of quickly and slowly rumen-degradable protein achieved by replacing soybean meal with urea in the concentrates (0, 0.5, and 1% urea for U0, U5, and U10, respectively) . Experiment 3 investigated effects of synchronizing the availability of FME and ERDP in rumen by offering the protein-rich concentrate once or twice per day before the meal (corn silage, ryegrass hay, and energy-rich concentrate), or included in the total mixed ration . Milk fatty acid profiles were significantly affected by dietary N and carbohydrate supply . Principal component factor analysis provided a reasonable description of the data, clearly discriminating between fatty acids that are synthesized by different metabolic pathways . Several sources/pathways were distinguished: de novo synthesis in the mammary gland (short- and medium-chain fatty acids), delta9-desaturase activity (monoenoic fatty acids), direct absorption from the blood stream (long-chain fatty acids), and de novo synthesis by the rumen microbial populations (odd-chain fatty acids) . Discriminant canonical analysis showed that milk odd-chain fatty acids had a higher ability to discriminate between diets than even-chain fatty acids . The anteiso C15:0 increased in line with increasing sugar supply, and C17:0 appears to be a marker of protein deficiency . Additionally, iso C17:0 and anteiso C17:0 were associated with the NDF and CP contents of diets . The results suggests that milk odd-chain fatty acids have the potential to be used as a noninvasive technique to assess rumen function in terms of microbial populations, substrates and interactions.

Appl Microbiol Biotechnol, 2004 Jun, 64(6), 823 - 8 Epub 2004 Jan 22.
Over-expression system for secretory phospholipase D by Streptomyces lividans; Ogino C et al.; The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector-pUC702-for Escherichia coli and S . lividans . The transformant was found to secrete a large amount of PLD (about 2.0x10(4) U/l, 42 mg/l) when cultured in a jar fermentor . Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5x10(4) U/l, 118 mg/l) after about 60 h . In contrast to the original producer, Stv . cinnamoneum, which secretes only a small amount of PLD (about 1.1x10(3) U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.

Angiogenesis, 2003, 6(2), 121 - 8
Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo; Toi M et al.; We previously showed pomegranate seed oil and fermented juice polyphenols to retard oxidation and prostaglandin synthesis, to inhibit breast cancer cell proliferation and invasion, and to promote breast cancer cell apoptosis . Here we evaluated the anti-angiogenic potential of these materials in several ways . We checked a possible effect on angiogenic regulation by measuring vascular endothelial growth factor (VEGF), interleukin-4 (IL-4) and migration inhibitory factor (MIF) in the conditioned media of estrogen sensitive (MCF-7) or estrogen resistant (MDA-MB-231) human breast cancer cells, or immortalized normal human breast epithelial cells (MCF-10A), grown in the presence or absence of pomegranate seed oil (SESCO) or fermented juice polyphenols (W) . VEGF was strongly downregulated in MCF-10A and MCF-7, and MIF upregulated in MDA-MB-231, overall showing significant potential for downregulation of angiogenesis by pomegranate fractions . An anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (HUVEC) and in myometrial and amniotic fluid fibroblasts, and inhibition of HUVEC tubule formation demonstrated in an in vitro model employing glass carrier beads . Finally, we showed a significant decrease in new blood vessel formation using the chicken chorioallantoic membrane (CAM) model in vivo . 'In sum, these varied studies employing different models in different laboratories overall demonstrate for the first time an anti-angiogenic potential of pomegranate fractions, suggesting further in vivo and clinical investigations (for updates: info@rimonest.com).

Pediatr Res, 2004 May, 55(5), 847 - 54 Epub 2004 Jan 22.
Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells; Mally P et al.; Circulating short-chain fatty acids (SCFAs) are primarily derived from bacterial fermentation of carbohydrates in the colon where they function as physiologic modulators of epithelial cell maturation . Butyrate has been shown to induce tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, and enkephalin neuropeptide gene transcription, suggesting a role in perinatal sympathoadrenal stress-adaptation . We sought to determine whether there were SCFA structural requirements for this effect . Nine biologically relevant SCFAs and butyrate derivatives were tested in an in vitro model (PC12, rat pheochromocytoma cells) for their ability to regulate neurotransmitter-related gene expression . Our results revealed that among all the studied SCFAs, only propionate and butyrate increased tyrosine hydroxylase and proenkephalin mRNA levels . The functional activity was selective to the carbon atom chain length and associated with the presence of an ethyl moiety in the carbon atom backbone chain . Modifications or absence of this domain affected the gene induction response, suggesting a receptor-mediated mechanism(s) . Moreover, propionate, butyrate, and the drug 4-phenylbutyrate were each shown to regulate transmitter genes via at least three independent mechanisms: histone hyperacetylation, cAMP signaling, or peroxisome proliferator-activated receptor gamma-mediated pathways . Thus, the biologic impact of SCFAs on catecholaminergic and opioid systems depend on the activation of SCFA-specific, dose-specific, and gene-specific molecular mechanisms . We speculate that 1) circulating levels of SCFAs may influence sympathoadrenal transmitter biosynthesis and hence whole animal stress-adaptive responsiveness after birth, and 2) the adverse effects of antibiotics on delayed acquisition of postnatal gut flora may affect this apparent evolutionary advantage of gut colonization.

J Biotechnol, 2004 Feb 5, 107(3), 245 - 53
Lack of interaction between AFLR and AFLJ contributes to nonaflatoxigenicity of Aspergillus sojae; Chang PK; Aspergillus sojae, which is believed to be a domesticated strain of Aspergillus parasiticus, contains all of the aflatoxin biosynthetic genes but is unable to produce aflatoxins and is generally recognized as safe (GRAS) for producing fermented foods . In A . parasiticus both aflR, the aflatoxin pathway-specific regulatory gene, and aflJ, a co-activator gene, are necessary for transcription of genes encoding the aflatoxin biosynthetic enzymes . A . sojae aflR differs from A . parasiticus aflR in that it encodes extra His and Ala, and has a pretermination defect that causes truncation of the carboxyl terminus of the predicted protein . A . sojae aflJ differs from A . parasiticus aflJ in that it encodes a predicted protein with Ser39 replaced by Ala and Ser283 replaced by Pro . Steady-state levels of aflatoxin biosynthetic gene transcripts of aflR, aflJ, pksA, nor1, ver1 and omtA in A . sojae as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were much lower than those of A . parasiticus . Yeast two-hybrid assays showed that the truncated A . sojae AFLR did not interact with AFLJ of A . sojae and A . parasiticus but that an A . sojae AFLR reverted to the putative ancestral form interacted normally with AFLJ of A . sojae and A . parasiticus . Deletion analysis showed that both amino- and carboxy-terminal regions of the A . sojae AFLJ were important for the R-J interaction . The truncated A . sojae AFLR thus not only was impaired in its ability to activate transcription of aflatoxin biosynthetic genes, but also was unable to interact with AFLJ, in A . parasiticus both of which are required for normal expression of the aflatoxin biosynthetic genes . Consequently, the lack of aflatoxin-producing ability of A . sojae resulted primarily from two defects in the regulatory mechanism responsible for gene transcription.

Arch Microbiol, 2004 Mar, 181(3), 231 - 6 Epub 2004 Jan 21.
In Saccharomyces cerevisiae, the effect of H2O2 on ATP, but not on glyceraldehyde-3-phosphate dehydrogenase, depends on the glucose concentration; Osorio H et al.; As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively . Depending on the glucose concentration in the medium, the effect of the addition of H2O2 on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed . In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged . The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H2O2 . Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose.

Appl Biochem Biotechnol, 2004 Jan, 112(1), 37 - 54
Production of beta-carotene from beet molasses by Blakeslea trispora in stirred-tank and bubble column reactors: development of a mathematical modeling; Goksungur Y et al.; The effect of aeration rate and agitation speed on beta-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentor and optimization of the production of the pigment in a bubble column reactor were investigated . In addition, a central composite design was employed to determine the maximum beta-carotene concentration at optimum values for the process variables (aeration rate, sugar concentration, linoleic acid, kerosene) . By image analysis of the morphology of the fungus, a quantitative characterization of the hyphae and zygospores formed was obtained . The hyphae were differentiated to intact hyphae, vacuolated hyphae, evacuated cells and degenerated hyphae . An increased proportion of zygospores was correlated to high beta-carotene production . In the stirred-tank fermentor, the highest concentration of the carotenoid pigment (92.0 mg/L) was obtained at an aeration rate of 1.5 vvm and agitation speed of 60 rpm . In the bubble column reactor, the aeration rate and concentration of sugars, linoleic acid, kerosene, and antioxidant significantly affected the production of beta-carotene . In all cases, the fit of the model was found to be good . Aeration rate, sugar concentration, linoleic acid, and kerosene had a strong positive linear effect on beta-carotene concentration . Moreover, the concentration of the pigment was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions . Maximum beta-carotene concentration (360.2 mg/L) was obtained in culture grown in molasses solution containing 5% (w/v) sugar supplemented with linoleic acid (37.59 g/L), kerosene (39.11 g/L), and antioxidant (1.0 g/L).

FEMS Yeast Res, 2004 Jan, 4(4-5), 511 - 9
Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum; Echavarri-Erasun C et al.; A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth . Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X . dendrorhous . Four X . dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX . Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant . The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU . These E . nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414 . Systematic rDNA homology alignments were performed to identify fungi related to E . nigrum . Stimulation of carotenogenesis by E . nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X . dendrorhous and Phaffia rhodozyma.

J Agric Food Chem, 2004 Jan 28, 52(2), 380 - 4
Effect of fermentation on Sweetpotato (Ipomoea batatas) toxicity in mice; Thibodeau MS et al.; Unfortunate bovine fatalities occurring after ingestion of mold-damaged sweetpotatoes preclude the use of the culled tubers in livestock feed . In cattle, mold-damaged sweetpotatoes induce an acute respiratory distress syndrome resulting in asphyxiation . Because of this potential toxicity and the general abundance of culled sweetpotatoes, the detoxification efficacy of ensiling was explored since it is an easy and economically viable technique often applied to preserve livestock feed . Sweetpotato slices with or without mold damage were stored either frozen (to represent unfermented samples) or fermented for 6 weeks at room temperature . Following fermentation, organic extracts were generated for administration to mice . Thirty hours following administration of the extracts, mice were evaluated for gross and microscopic lesions affecting the lungs, liver, and kidneys . Fermentation of 6 weeks duration was observed to inadequately eliminate the lung, liver, and kidney toxicity caused by mold-damaged sweetpotatoes . In fact, fermentation exacerbated the hepatotoxicity of mold-damaged sweetpotatoes . This is also the first demonstration that sweetpotato regions lacking visible mold damage can induce lung and kidney injury, which, however, is preventable by fermentation.

J Agric Food Chem, 2004 Jan 28, 52(2), 350 - 4
Influence of baking conditions and precursor supplementation on the amounts of the antioxidant pronyl-L-lysine in bakery products; Lindenmeier M et al.; The influence of baking conditions and dough supplements on the amounts of the antioxidant and Phase II-Enzyme modulating, protein-bound 2,4-dihydroxy-2,5-dimethyl-1-(5-acetamino-5-methoxycarbonyl-pentyl)-3-oxo-2H-pyrrol (pronyl-L-lysine) in bakery products was investigated in quantitative studies . These studies revealed high amounts of the antioxidant in bread crust, only low amounts in the crumb, and the absence of this compound in untreated flour . The amounts of pronyl-L-lysine were found to be strongly influenced by the intensity of the thermal treatment . For example, increasing the baking time from 70 to 210 min or increasing the baking temperature from 220 to 260 degrees C led to a 5- or 3-fold increase in the concentrations of this antioxidant in the crust, respectively . In addition, modifications in the recipe showed to have a major impact on pronyl-L-lysine formation . For example, substituting 5% of the flour with the lysine-rich protein casein or with 10% of glucose increased the amounts of the antioxidant by more than 200% . Quantitative analyses of commercial bread samples collected from German bakeries revealed the highest amount of 43 mg/kg for a full grain bread, followed by a rye/wheat bread, both of which have been sourdough fermented . A mixed-grain bread as well as pale wheat bread, both prepared without sourdough fermentation, contained significantly lower amounts of pronyl-L-lysine, and German pretzels, which are treated with a dilute sodium hydroxide solution prior to baking, contained only trace amounts of pronyl-L-lysine (e.g., less than 5 mg/kg were detectable in pretzels) . Systematic studies revealed that the decrease of the pH value induced by microbial acid formation during sourdough fermentation is the clue for producing high amounts of pronyl-L-lysine in baking products . These data clearly demonstrate for the first time that the amounts of the antioxidant and chemopreventive compound pronyl-L-lysine in bakery products is strongly dependent on the manufacturing conditions as well as the recipe.

Phytochemistry, 2004 Jan, 65(2), 233 - 41
Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry; van Elswijk DA et al.; During recent years, phytoestrogens have been receiving an increasing amount of interest, as several lines of evidence suggest a possible role in preventing a range of diseases, including the hormonally dependent cancers . In this context, various parts of the pomegranate fruit (Punica granatum; Punicaceae), e.g . seed oil, juice, fermented juice and peel extract, have been shown to exert suppressive effects on human breast cancer cells in vitro . On-line biochemical detection coupled to mass spectrometry (LC-BCD-MS) was applied to rapidly profile the estrogenic activity in the pomegranate peel extract . The crude mixture was separated by HPLC, after which the presence of biologically active compounds, known or unknown, was detected by means of an on-line beta-estrogen receptor (ER) bioassay . Chemical information, such as molecular weight and MS/MS fingerprint, was obtained in real time by directing part of the HPLC effluent towards a mass spectrometer . Using this approach in total three estrogenic compounds, i.e . luteolin, quercetin and kaempferol, were detected and identified by comparing the obtained molecular weights and negative ion APCI MS/MS spectra with the data of an estrogenic compound library . Although well known in literature and widely distributed in nature, the presence of these phytoestrogenic compounds in pomegranate peel extract was not reported previously . Compared to traditional screening approaches of complex mixtures, often characterized by a repeating cycle of HPLC fractionation and biological screening, LC-BCD-MS was shown to profoundly accelerate the time required for compound description and identification.

Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2641 - 3
Suppression by Hydrangeae Dulcis Folium of D-galactosamine-induced liver injury in vitro and in vivo; Nakagiri R et al.; Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER . var . thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days . The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder . Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.

Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2533 - 40
Variable interactions between sucrose non-fermented 1-related protein kinases and regulatory proteins in higher plants; Nozawa A et al.; WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins . We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4 . Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A . thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them . Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL) . This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2 . In contrast, WPK4 interacted with none of the CBLs . In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay . These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A . thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.

Toxicol Appl Pharmacol, 2004 Jan 1, 194(1), 41 - 8
Plasmid DNA damage caused by stibine and trimethylstibine; Andrewes P et al.; Antimony is classified as "possibly carcinogenic to humans" and there is also sufficient evidence for antimony carcinogenicity in experimental animals . Stibine is a volatile inorganic antimony compound to which humans can be exposed in occupational settings (e.g., lead-acid battery charging) . Because it is highly toxic, stibine is considered a significant health risk; however, its genotoxicity has received little attention . For the work reported here, stibine was generated by sodium borohydride reduction of potassium antimony tartrate . Trimethylstibine is a volatile organometallic antimony compound found commonly in landfill and sewage fermentation gases at concentrations ranging between 0.1 and 100 microg/m3 . Trimethylstibine is generally considered to pose little environmental or health risk . In the work reported here, trimethylstibine was generated by reduction of trimethylantimony dichloride using either sodium borohydride or the thiol compounds, dithioerythritol (DTE), L-cysteine, and glutathione . Here we report the evaluation of the in vitro genotoxicities of five antimony compounds-potassium antimony tartrate, stibine, potassium hexahydroxyantimonate, trimethylantimony dichloride, and trimethylstibine-using a plasmid DNA-nicking assay . Of these five antimony compounds, only stibine and trimethylstibine were genotoxic (significant nicking to pBR 322 plasmid DNA) . We found stibine and trimethylstibine to be about equipotent with trimethylarsine using this plasmid DNA-nicking assay . Reaction of trimethylantimony dichloride with either glutathione or L-cysteine to produce DNA-damaging trimethylstibine was observed with a trimethylantimony dichloride concentration as low as 50 microM and L-cysteine or glutathione concentrations as low as 500 and 200 microM, respectively, for a 24 h incubation.

AAPS PharmSciTech . 2000 Jul 23;1(3):E22.
The potential of organic-based amylose-ethylcellulose film coatings as oral colon-specific drug delivery systems; Siew LF et al.; Amylose-ethylcellulose film coatings obtained from organic-based solvents were investigated as potential vehicles for colonic drug delivery . Amylose, in the form of an amylose-butan-1-ol dispersion, and ethylcellulose, dissolved in either ethyl lactate, ethanol, or propanol and plasticized with dibutyl sebacate, were mixed in various proportions and applied using a fluidized bed coater to achieve a range of film thicknesses on 5-aminosalicylic acid pellets . Drug release from the coated pellets was assessed under gastric and small intestinal conditions in the presence and absence of pepsin and pancreatin using dissolution methodology, and also within a simulated colonic environment involving fermentation testing with human feces in the form of a slurry . Under upper gastrointestinal tract conditions, the rate and extent of drug release were found to be related to the thickness of the coating and the ratio of amylose to ethylcellulose within the film . Modeling of the drug release data revealed that the ratio was more important than coat thickness in controlling drug release, irrespective of the solvent used for coating . Coatings with a thick film and/or low amylose content were relatively impermeable and able to delay drug release under conditions mimicking the upper gastrointestinal tract . Furthermore, drug release was unaffected by the presence of pepsin and pancreatin and by long-term storage . Under simulated colonic conditions, drug release was more pronounced from coating formulations containing higher proportions of amylose . Colon-specificity can therefore be achieved using such systems by judicious choice of the appropriate ratio of amylose to ethylcellulose and coat thickness.

J Ind Microbiol Biotechnol, 2004 Jan, 31(1), 5 - 10 Epub 2004 Jan 16.
Carnocin KZ213 produced by Carnobacterium piscicola 213 is adsorbed onto cells during growth . Its biosynthesis is regulated by temperature, pH and medium composition; Khouiti Z et al.; Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213 . The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate) . The optimal pH for growth is between 6 and 7 . The maximum bacteriocin productivity in the supernatant occurs at pH 7 . Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH . No bacteriocin production is observed below pH 6.5 . Temperature has a dramatic effect on carnocin KZ213 production . Growth is optimal at 25 degrees C and 30 degrees C, although no bacteriocin production is detected at 30 degrees C . Also, bacteriocin production is observed at 25 degrees C in MRS*, but not in complex APT broth, where growth is optimal . The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis . Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition . Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth . Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.

J Biol Chem, 2004 Mar 26, 279(13), 12414 - 20 Epub 2004 Jan 13.
Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol . Role of acetyl CoA synthetase in anaerobic ATP synthesis; Takasaki K et al.; Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions . The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H . (2002) J . Biol . Chem . 277, 1892-1896) . We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants . The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation . We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs) . This is similar to the mechanism suggested in F . oxysporum except A . nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F . oxysporum . The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes . We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity . Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate . Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation . These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.

Exp Cell Res, 2004 Jan 1, 292(1), 29 - 39
The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells; Jones SA et al.; Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease . Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease . In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis . TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation . Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation . Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death . Reduction in cell death was associated with maintenance of viable cell number . Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers . TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk . In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance . We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.

Biotechnol Lett, 2003 Nov, 25(22), 1953 - 6
Calculation of fermentation parameters from the results of a batch test taking account of the volume of biomass in the fermenting medium; Borzani W; The values of fermentation parameters calculated from the measured concentrations of substrates and/or products may be significantly affected by the volume of biomass in the fermenting medium . Corrections proposed in this paper should be evaluated and, depending on their magnitude, considered in order to obtain more representative results.

Biotechnol Lett, 2003 Nov, 25(22), 1875 - 80
Isolation and characterisation of resistant-to-fermentation carbohydrate polymers from cultures of Rhizopus nigricans grown on agrofood waste materials; Hellin P et al.; Rhizopus nigricans was cultivated in a liquid medium using lemon, mandarin, orange, pear and melon peel or artichoke bracts as the carbon source . In all cultures, a carbohydrate polymer fraction remained resistant to fermentation . These fractions were isolated in gram amounts and characterised . The molecular weight distribution of the fractions and its sugar composition resembles those of the hairy-regions of the pectins . In the fractions, four main carbohydrates were found: 4-7 mol% Rha, 42-59 mol% Ara, 7-14 mol% Gal, 17-33 mol% GalA.

Biotechnol Lett, 2003 Dec, 25(23), 1989 - 92
Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli; Khalilzadeh R et al.; Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter . Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process . Cell density of recombinant E . coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates . In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process . The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.

Biotechnol Lett, 2003 Dec, 25(23), 1983 - 7
Rhizopus arrhizus--a producer for simultaneous saccharification and fermentation of starch waste materials to L(+)-lactic acid; Jin B et al.; Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents . Lactic acid at 19.5-44.3 g l(-1) with a yield of 0.85-0.96 g g(-1) was produced in 40 h using 20-60 g starch l(-1) . Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.

J Food Prot, 2004 Jan, 67(1), 117 - 23
Biogenic amine formation and "zapatera" spoilage of fermented green olives: effect of storage temperature and debittering process; Garcia PG et al.; The effects of temperature and the debittering process on amine formation and other chemical changes related to "zapatera" spoilage of fermented green table olives during storage, without any chemical correction, were studied . Unwashed olive brines were more concentrated in all analyzed compounds, except NaCl . No changes in formic, acetic, and succinic acids or in ethanol, hydroxytyrosol, or tyrosol were observed in the olive brines during storage . The concentration of putrescine in the brine at the beginning of storage and end of fermentation was about 38 mg/liter, and it did not change during storage . This amine only seems to be produced during the active fermentation phase . The effects of temperature and the type of debittering process and time and its interactions (except the time x temperature x debittering process on pH) had significant effects on the production of cadaverine and tyramine, as well as on changes of pH and lactic and propionic acids . Storage at 15 degrees C produced a complete stabilization of the fermented olives . However, storage of washed olives at 20 and 28 degrees C produced a gradual decrease of lactic acid content, an increase in pH, production of propionic acid, and formation of cadaverine and tyramine, the effect becoming greater as the temperature rose . It appears that formation of cadaverine and tyramine only occurs during storage and might be related to zapatera spoilage . Changes were always significantly lower in unwashed olives, which leads to a practical stabilization of the product.

J Clin Microbiol, 2004 Jan, 42(1), 354 - 8
Nissui glucose fermentative gram-negative rod identification system EB-20 gives a unique profile for typical non-sorbitol-fermenting Escherichia coli O157:H7; Kodaka H et al.; The 98 non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 strains identified on a Nissui glucose fermentative gram-negative rod identification system (EB-20) gave a unique biochemical profile number that was not detected in 85 pathogenic and 13 nonpathogenic E . coli strains . Thus, EB-20 is useful for the identification of NSF E . coli O157:H7 and provides a simple, cost-effective, and reliable tool for clinical laboratories.

J Ind Microbiol Biotechnol, 2003 Dec, 30(12), 699 - 704 Epub 2004 Jan 09.
Malolactic bioconversion using a Oenococcus oeni strain for cider production: effect of yeast extract supplementation; Herrero M et al.; Yeast extract addition to reconstituted apple juice had a positive impact on the development of the malolactic starter culture used to ensure malolactic fermentation in cider, using active but non-proliferating cells . In this work, the reuse of fermentation lees from cider is proposed as an alternative to the use of commercial yeast extract products . Malolactic enzymatic assays, both in whole cells and cell-free extracts, were carried out to determine the best time to harvest cells for use as an inoculum in cider . Cells harvested at the late exponential phase, the physiological stage of growth corresponding to the maximum values of specific malolactic activity, achieved a good rate of malic acid degradation in controlled cider fermentation . Under the laboratory conditions used, malic acid degradation rates in the fermentation media turned out to be near 2.0 and 2.5 times lower, compared with the rates obtained in whole-cell enzymatic assays, as useful data applicable to industrial cider production.

J Ind Microbiol Biotechnol, 2003 Dec, 30(12), 721 - 31 Epub 2004 Jan 09.
Isolation, structure, and HIV-1-integrase inhibitory activity of structurally diverse fungal metabolites; Singh SB et al.; HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies . In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described . These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity . The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid . These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M . The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.

Appl Environ Microbiol, 2004 Jan, 70(1), 159 - 66
Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast; van Maris AJ et al.; The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism . However, Pdc(-) S . cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose . We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects . An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed . Transcriptome analysis did not reveal the mechanisms behind the C(2) independence . Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S . cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source . Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose . In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate . By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).

Cell Microbiol, 2004 Feb, 6(2), 187 - 99
Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation; Into T et al.; Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll-like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-kappaB, but also initiate apoptotic responses . The aim of this study was to clarify the apoptotic machineries . Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid . The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs . Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-kappa B activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively . Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins . In addition, both dominant-negative forms also downregulated the activation of both NF-kappa B and caspase-8 in the cells . Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK . These findings suggest that mycoplasmal lipoproteins can trigger TLR2- and TLR6-mediated sequential bifurcate responses: NF-kappa B activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

J Agric Food Chem, 2003 Jul 30, 51(16), 4788 - 98
Multielement composition of wines and their precursors including provenance soil and their potentialities as fingerprints of wine origin; Almeida CM et al.; The influence of the provenance soil and vinification process on the wine multielemental composition was investigated . For this purpose, two different vineyards from the Douro wine district, Portugal, were selected . Monovarietal grapes from a 10 year old vineyard were used to produce a red table wine, in a very modern winery . Polyvarietal grapes from a 60-70 year old vineyard were used to produce a red fortified wine, similar to Port, through a traditional vinification process . The multielement compositions (Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Hf, Li, Mn, Mo, Nb, Ni, Pb, Rb, Sb, Sc, Sr, Ti, Th, Tl, U, V, W, Y, Zn, Zr, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) of soil, grape juices (prepared in the laboratory), and samples collected in the different steps of each winemaking process were measured . Inductively coupled plasma mass spectrometry was used, after suitable pretreatment of the samples (by UV irradiation for liquid samples and high-pressure microwave digestion for soil) . Both vinification processes influenced the multielement composition of the wines . Most of the elements presented similar or even lower concentrations in the wine as compared to that observed in the respective grape juice, probably as a result of precipitation or coprecipitation with suspended particles during fermentation and/or wine aging . Evidence of effective contamination during grape pressing, fermentation, and/or fining of wines (depending on the element) was observed for Cd, Cr, Cu, Fe, Ni, Pb, V, and Zn in the fortified wine and Al, Cr, Fe, Ni, Pb, and V in the table wine . Nevertheless, significant correlations were obtained between the multielement composition of the wine and the respective grape juice (R = 0.997 and 0.979 for the fortified and table wines, respectively, n = 31, P < 0.01), as well as between that in the wine (median of the two studied wines) and the provenance soil (R = 0.994, n = 19, P < 0.01), for the set of elements determined in common in the different types of samples . These results are promising concerning the usefulness of the elemental patterns of both soil and wine as fingerprints of the origin of the studied wines . Nevertheless, more wines from the same and other wine districts must be studied in order to consolidate this conclusion . The multielement compositions of the studied wines were compared with those of wines of different characteristics and origins, as well as with the respective legal threshold limit values, when available . Relatively low metal levels, below their threshold limit values, were found in all cases.

Biotechnol Bioeng, 2004 Jan 5, 85(1), 103 - 13
Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing; Tin Lee C et al.; In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium . We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs . The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs . Cross-flow microfiltration using a 0.2 microm ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate . The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase digestion of nucleic acids following chemical extraction . Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer . The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification . Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration . In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations .

Biotechnol Bioeng, 2004 Jan 20, 85(2), 155 - 65
Effect of fermentation broth and biosurfactants on mass transfer during liquid-liquid extraction; Pursell MR et al.; Mass transfer rates in liquid-liquid extraction processes can be seriously affected by the presence of surface-active contaminants . This is especially true of applications of a biotechnological origin, where the microorganism used in the process may produce the surface-active contaminants . An investigation into the effects of soluble and insoluble fermentation broth components on mass transfer using chloramphenicol extraction into octanol as the model system was conducted . Soluble components produced during fermentation were found to adsorb to the interface, where they reduced the overall mass transfer coefficient by up to 70% . After fractionation it was found that components in the weight range from 10-30 kDa had the greatest effect on mass transfer . Protein and phospholipid compounds of similar size were found to reduce the overall mass transfer coefficient to a similar extent to the broth components at concentrations around 0.001mg/l . The biomass produced during the fermentation also reduced mass transfer substantially, and it is likely that this was due to physical blockage of the interface .

Med Dosw Mikrobiol, 2003, 55(3), 219 - 24
{Characteristic of saccharose fermentation by pathogenic Escherichia coli strains--phenotypic and genotypic elements}; Skwark M et al.; The purpose of the study was to characterize fermentation of sucrose by Escherichia coli strains and to answer why some of these strains doesn't utilize this disaccharide . Investigations included 16 E . coli strains . Only 5 of these strains utilized sucrose . Genotypic analysis demonstrated the presence of cscB gene (encoding the sucrase permease which catalyzes transport of sucrose through the plasma membrane of the cell) in 5 strains of E . coli and cscA gene (encoding an enzyme sucrase that catalyzes the utilization of sucrose) in 6 strains of E . coli . These 5 of E . coli strains which possessed a chromosomally encoded sucrose metabolic pathway utilized sucrose with a different time . 3 of them destroyed this disaccharide after 24 h and 2 of them destroyed it after 48 h . Ten of E . coli strains hadn't cscA gene and 11 of them had not cscB genes . The lack of these genes can be the prove that it is not possible for 11 of E . coli strains to synthesize sucrose permease and for 10 of them to synthesize sucrase and it may be the reason of not utilize disaccharide sucrose by these bacteria.

Microbiology, 2004 Jan, 150(Pt 1), 109 - 15
Aspirin commits yeast cells to apoptosis depending on carbon source; Balzan R et al.; The effect of aspirin on the growth of a wild-type Saccharomyces cerevisiae strain (EG103), containing both copper,zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), a strain deficient in MnSOD (EG110) and a strain deficient in CuZnSOD (EG118) was measured in media containing different carbon sources . Aspirin inhibited the fermentative growth of all three strains in glucose medium . It inhibited the non-fermentative growth of the MnSOD-deficient strain very drastically in ethanol medium and had no effect on this strain in glycerol or acetate medium . The non-fermentative growth of the other two strains was not affected by aspirin . The growth inhibition of strain EG110 was associated with early necrosis in glucose medium and late apoptosis in ethanol medium . The apoptosis was preceded by a pronounced loss of cell viability . The growth inhibitory effect of aspirin was not reversed by the antioxidants N-acetylcysteine and vitamin E . Furthermore, aspirin itself appeared to act as an antioxidant until the onset of overt apoptosis, when a moderate increase in the intracellular oxidation level occurred . This suggested that reactive oxygen species probably do not play a primary role in the apoptosis of cells exposed to aspirin.

J Biol Chem, 2004 Apr 16, 279(16), 16677 - 86 Epub 2003 Dec 29.
Transcription factors and nuclear receptors interact with the SWI/SNF complex through the BAF60c subunit; Debril MB et al.; Transcriptional activity relies on coregulators that modify chromatin structure or serve as bridging factors between transcription factors and the basal transcription machinery . We identified a new coregulator of peroxisome proliferator-activated receptor gamma, BRG1/Brm-associated factor of 60 kDa, subunit c2 (BAF60c2), in a yeast two-hybrid screen of a human adipose tissue cDNA library . BAF60c2 represents a new isoform of BAF60c, a component of the SWI/SNF (mating type switching/sucrose non-fermenting) chromatin remodeling complex . This new isoform as well as the previously identified protein, renamed BAF60c1, is localized primarily in the cell nucleus and is expressed in a wide variety of tissues . Both BAF60c isoforms bind to several nuclear receptors and transcription factors of various families . BAF60c proteins interact in a ligand-independent manner with peroxisome proliferator-activated receptor gamma and enhance its transcriptional activity . Both isoforms are enriched in the central nervous system and also modulate the transcriptional activity of retinoic acid-related orphan receptor alpha1 . In conclusion, BAF60c represents a new coregulator that constitutes an important anchoring point by which the SWI/SNF complex is recruited to nuclear receptors and other transcription factors.

Int J Food Microbiol, 2004 Jan 15, 90(2), 237 - 48
High efficiency transformation of Penicillium nalgiovense with integrative and autonomously replicating plasmids; Fierro F et al.; Penicillium nalgiovense is a filamentous fungus that is acquiring increasing biotechnological importance in the food industry due to its widespread use as starter culture for cured and fermented meat products . Strains of P . nalgiovense can be improved by genetic modification to remove the production of penicillin and other potentially hazardous secondary metabolites, to improve its capacity to control the growth of undesirable fungi and bacteria on the meat product, and other factors that contribute to the ripening of the product in order to get safer and better quality foods . Genetic manipulation of P . nalgiovense has been limited by the lack of molecular genetics tools that were available for this fungus, particularly for "self-cloning" avoiding the use of exogenous DNAs . In this article we describe a series of vectors, selectable markers and transformation methods that can be used for efficient transformation of P . nalgiovense, gene cloning and expression . A uridine auxotrophic P . nalgiovense mutant with an inactive pyrG gene has been isolated . The P . nalgiovense wild-type pyrG gene was cloned and sequenced, and vectors carrying the gene were shown to complement the pyrG mutant . Autonomously replicating plasmids carrying the AMA1 region from Aspergillus nidulans transformed P . nalgiovense very efficiently; these plasmids were shown to be maintained as stable extrachromosomal elements in P . nalgiovense and could be rescued in Escherichia coli . The mitotic stability of self-replicative AMA1 plasmids in P . nalgiovense was higher than that reported for Penicillium chrysogenum.

Phytochemistry, 2004 Jan, 65(1), 31 - 41
Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites; Willits MG et al.; A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa . Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions . Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro . Additionally, using mixed cultures of E . coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed . This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.

Biofactors, 2003, 18(1-4), 237 - 44
Caenorhabditis elegans ubiquinone biosynthesis genes; Rodriguez-Aguilera JC et al.; Ubiquinone (coenzyme Q, Q) is an essential lipid electron carrier in the mitochondria respiratory chain, and also functions as antioxidant and participates as a cofactor of mitochondrial uncoupling proteins . Caernorhabditis elegans synthesize Q9, but both dietary Q8 intake and endogenous Q9 biosynthesis determine Q balance . Thus, it is of current interest to know the regulatory mechanisms of Q9 biosynthesis in this nematode . Here we review results that leaded to identification of genes involved in Q9 biosynthesis in this nematode using the RNA interference technology . C . elegans coq genes were silenced and depletion of Q content was observed, indicating that the genes related here participate in Q9 biosynthesis . Silenced populations showed an extension of adult life span, probably by the decrease of endogenous oxidative stress produced in mitochondria . We also report the heterologous complementation of C . elegans coq-5 and coq-7 genes in their homologue yeast coq null mutants, leading to restore its ability to growth in non-fermentable sugars . These complemented yeast strains accumulated Q6 but also the intermediate demethoxy-Q6 . These findings support the conservative functional homology of these genes.

J Nat Prod, 2003 Dec, 66(12), 1527 - 30
Isolation and structure elucidation of Sch 642305, a novel bacterial DNA primase inhibitor produced by Penicillium verrucosum; Chu M et al.; A novel primase inhibitor, Sch 642305 (1), was isolated from the fermentation broth of the fungal culture Penicillium verrucosum . The structure of 1 was elucidated on the basis of MS and NMR spectroscopic data as a new and unusual bicyclic 10-membered macrolide . The absolute configuration of the asymmetric centers was determined by X-ray crystallographic analysis of the p-bromobenzoate derivative (3) . Compound 1 exhibited inhibitory activity against bacterial DNA primase enzyme with an EC(50) of 70 microM.

Bioresour Technol, 2004 Apr, 92(2), 163 - 71
Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor; Helle SS et al.; Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol . SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used . The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids . Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation . With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains . For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed . By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.

Proc Nutr Soc, 2003 Aug, 62(3), 703 - 10
The medical management of intestinal failure: methods to reduce the severity; Nightingale JM; A new definition of intestinal failure is of reduced intestinal absorption so that macronutrient and/or water and electrolyte supplements are needed to maintain health or growth . Severe intestinal failure is when parenteral nutrition and/or fluid are needed and mild intestinal failure is when oral supplements or dietary modification suffice . Treatment aims to reduce the severity of intestinal failure . In the peri-operative period avoiding the administration of excessive amounts of intravenous saline (9 g NaCl/l) may prevent a prolonged ileus . Patients with intermittent bowel obstruction may be managed with a liquid or low-residue diet . Patients with a distal bowel enterocutaneous fistula may be managed with an enteral feed absorbed by the proximal small bowel while no oral intake may be needed for a proximal bowel enterocutaneous fistula . Patients undergoing high-dose chemotherapy can usually tolerate jejunal feeding . Rotating antibiotic courses may reduce small bowel bacterial overgrowth in patients with chronic intestinal pseudoobstruction . Restricting oral hypotonic fluids, sipping a glucose-saline solution (Na concentration of 90-120 mmol/l) and taking anti-diarrhoeal or anti-secretory drugs, reduces the high output from a jejunostomy . This treatment allows most patients with a jejunostomy and > 1 m functioning jejunum remaining to manage without parenteral support . Patients with a short bowel and a colon should consume a diet high in polysaccharides, as these compounds are fermented in the colon, and low in oxalate, as 25% of the oxalate will develop as calcium oxalate renal stones . Growth factors normally produced by the colon (e.g . glucagon-like peptide-2) to induce structural jejunal adaptation have been given in high doses to patients with a jejunostomy and do marginally increase the daily energy absorption.

Nutr Cancer, 2003, 46(2), 202 - 11
Sodium butyrate inhibits cell growth and stimulates p21WAF1/CIP1 protein in human colonic adenocarcinoma cells independently of p53 status; Kobayashi H et al.; Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines . The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells . Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type) . NaB significantly inhibited cell growth in all four cell lines . NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase . A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 . In all cell lines, p21 mRNA levels were immediately elevated after NaB exposure, and p21 protein levels were increased within 6 h . NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence . NaB did not influence p21 mRNA stability . Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21 mRNA by NaB was not accompanied by relaxation of the chromatin in the region of the p21 gene.

J Agric Food Chem, 2003 Dec 31, 51(27), 7930 - 5
Multiple forms of xylose reductase in Candida intermedia: comparison of their functional properties using quantitative structure-activity relationships, steady-state kinetic analysis, and pH studies; Nidetzky B et al.; The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR) . To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme . dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM) . msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM) . A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR . This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms . Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes . A lower pK of 7.4 is seen for dsXR-NADPH . Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.

J Biotechnol, 2004 Jan 8, 107(1), 41 - 54
Polyhydroxybutyrate synthesis in transgenic flax; Wrobel M et al.; Flax (Linum usitatissimum L.) is an annual plant species widely cultivated in temperate climates for bast fibres and linseed oil . Apart from traditional textile use, the fibres are fast becoming an integral part of new composite materials utilized in automobile and constructive industry . Especially attractive for environmental safety demands are biodegradable and renewable biocomposities based on polyhydroxybutyrate (PHB) polymer as a matrix and reinforced with the flax fibres . Manufacturing of PHB by bacteria fermentation is however substantially more expansive as compared to technologies producing conventional plastics . We report for the first time generation of transgenic plants which produce both components of flax/PHB composites, i.e . the fibres and the thermoplastic matrix in the same plant organ of a crop . The flax (cv . Nike) plants were transformed using constructs bearing either single cDNA, encoding the beta-ketothiolase enzyme (C plants), or all three of the genes necessary for poly-beta-hydroxybutyrate (PHB) synthesis (M plants) . Both constructs contained a plastidial targeting sequence . The amount of PHB produced by the transgenic plants was up to over 70-fold higher than in wild-type plants, when analysed using the gas chromatography/mass spectrometry (GC-MS method) . The PHB accumulation in plastids caused change both in their shape and size . The use of a stem-specific promoter for transgene expression protected the transgenic plant from growth retardation and also provided higher PHB synthesis than in the case of constructs governed by the 35S CaMV constitutive promoter . None toxic effects that could lead to stunted growth or the loss of fertility were observed, when 14-3-3 promoter was used as the stem-specific . Significant modifications in stem mechanical properties were accompanied to the PHB accumulation in growing cell of fibres in the transgenic plants . The Young's modulus E, the average measure of stem tissues resistance to tensile loads increased up to twice in M plants as compared to a single gene transformed ones . However, a wide range of E values, from 24.1 to 54.4 MPa, was observed in dependence of tested strain . Potential commercial significance of the genetic manipulation approach enabling synthesis of thermoplastic in crops cultivated for fibres is discussed.

Am J Gastroenterol, 2003 Dec, 98(12), 2700 - 4
Breath testing to evaluate lactose intolerance in irritable bowel syndrome correlates with lactulose testing and may not reflect true lactose malabsorption; Pimentel M et al.; OBJECTIVES: An increased prevalence of lactose intolerance is seen in irritable bowel syndrome (IBS) . Recently, we demonstrated a high prevalence of abnormal lactulose breath test results in IBS suggesting bacterial overgrowth . Because symptoms of lactose intolerance result from bacterial fermentation, the purpose of this study was to determine whether an abnormal lactose breath test is reflective of malabsorption or early presentation to bacteria . METHODS: Subjects with diarrhea-predominant IBS were enrolled . On day 1, subjects underwent a lactulose breath test after an overnight fast . Within 1 wk, subjects returned after fasting for a lactose breath test with simultaneous blood glucose measurements every 15 min to complete a lactose tolerance test (LTT) . Symptoms were evaluated 3 h after lactose administration . RESULTS: Twenty subjects completed the study . One subject inadvertently received dextrose through the intravenous and was excluded . Of the remaining 19 subjects, three (16%) had an abnormal LTT suggesting malabsorption . In all, 10 subjects (53%) had an abnormal lactose breath test, 14 (74%) had an abnormal lactulose breath test, and 11 (58%) had symptoms after lactose administration . The agreement with symptoms was moderate (kappa = 0.47) and fair (kappa = 0.24) when compared to the lactose breath test and LTT, respectively . There was a fair correlation between lactose breath test and LTT (kappa = 0.29) . However, lactose breath test hydrogen levels >166 ppm were universally predictive of abnormal LTT . Finally, a significant correlation was seen between the hydrogen production on lactose and lactulose breath test (r = 0.56, p = 0.01) . CONCLUSIONS: Lactose breath testing in IBS subjects does not seem to reflect malabsorption; it may be an indicator of abnormal lactulose breath test, suggesting bacterial overgrowth.

J Am Geriatr Soc, 2004 Jan, 52(1), 3 - 12
Nutritional formula enhanced immune function and reduced days of symptoms of upper respiratory tract infection in seniors; Langkamp-Henken B et al.; OBJECTIVES: To assess whether an experimental nutritional formula, given as a supplement, would reduce days of symptoms of upper respiratory tract infection (URTI) and affect antibody and lymphocyte proliferative responses to influenza vaccine . DESIGN: A prospective, randomized, double-blind, controlled trial was conducted between October 1999 and April 2000 . SETTING: Assisted- and independent-living facilities in North Central Florida . PARTICIPANTS: Sixty-six individuals, aged 65 and older . INTERVENTION: Subjects received 8 oz/d of an experimental formula containing antioxidants, zinc, selenium, fermentable oligosaccharides, and structured triacylglycerol or an isoenergetic, isonitrogenous control formula for 183 days . MEASUREMENTS: Subjects recorded daily symptoms of URTI . Antibody titers and lymphocyte proliferation to three influenza vaccine components were measured on Days 57 and 183 . RESULTS: Eighteen subjects in the control group and 16 subjects in the experimental group consumed an average of 7 ounces of formula daily and completed the 183-day study . Median days of symptoms of URTI were 3 (range 0-69, total days=156) and 0 (range 0-49, total days=78) for the control and experimental groups, respectively (P=.049) . On Day 57, seven of 17 (41%) subjects in the control group and 13 of 15 (87%) subjects in the experimental group achieved a fourfold or greater increase in serum antibody titer to A/Beijing (P=.012) . Lymphocyte proliferation to influenza vaccine components was greater in the experimental (median=1,365 cpm, range=0-14,955 cpm) than the control group (median=136 cpm, range=0-4,270 cpm) (P=.013) . CONCLUSION: Subjects consuming an experimental nutritional formula experienced enhanced immune function and fewer days of URTI symptoms.

Lett Appl Microbiol, 2004, 38(1), 50 - 5
Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii; Gutierrez-Sanchez G et al.; AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture . METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used . Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation . An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation . Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced . Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions . CONCLUSIONS: Caffeine degradation as well as biomass production were characterized . SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation . Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.

Lett Appl Microbiol, 2004, 38(1), 32 - 7
Streptomyces sp . 173, an insecticidal micro-organism from marine; Xiong L et al.; AIMS: To find new insecticidal antibiotics from marine micro-organisms . METHODS AND RESULTS: Strains isolated from seawater and sea sediments from Beidiahe and Dagang of the east coast of China were screened for their insecticidal qualities . The screening was carried out using bioassay of brine shrimp and the insect pest Helicoverpa armigera . The fermentation, preliminary extraction and isolation of Streptomyces sp.173 were carried out . CONCLUSIONS: In total 331 isolates were examined through bioassay of brine shrimp and 40 isolates (12.08%) showed potential insecticidal activities . Of the 40 isolates, one isolate, designated Streptomyces sp.173, was found to have strong insecticidal activity against both brine shrimp and H . armigera, similar to that of avermectin B1 . SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated Streptomyces sp.173 has great insecticidal potency . This work indicated that marine micro-organisms could be an important source of insecticidal antibiotics and the improved anti-brine shrimp bioassay is suitable for primary screening.

Yi Chuan Xue Bao, 2003 Aug, 30(8), 723 - 9
{Cloning and identification of the priming glycosyltransferase gene involved in exopolysaccharide 139A biosynthesis in Streptomyces}; Wang LY et al.; Recently in our laboratory, Streptomyces sp . 139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity . The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway, i.e . the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer . Degenerate primers-based PCR approach was adopted to isolate the putative priming glycosyltransferase gene in Streptomyces sp . 139 . According to the genes encoding the priming glycosyltransferases that have been identified in several microorganisms, a multiple alignment of the amino acid sequences of these genes was used to identify regions conserved between all genes . To clone the priming glycosyltransferase gene in Streptomyces sp . 139, degenerate primers were designed from these conserved regions taking into account information on Streptomyces codon usage to amplify an internal DNA fragment of this gene . A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces sp . 139 total genomic DNA . Sequence analysis showed that it is part of a putative priming glycosyltransferase gene and contains the predicted conserved domain B . To isolate the complete priming glycosyltransferase gene, a Streptomyces sp . 139 genomic library was constructed in the E . coli--Streptomyces shuttle vector pOJ446 . Using the 0.3 kb PCR product of priming glycosyltransferase gene as a probe, 17 positive colonies were isolated by colony hybridization . A 4.0 kb BamHI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed the complete priming glycosyltransferase gene . The priming glycosyltransferase gene ste5 (GenBank under accession number AY131229) most likely begins with GTG, preceded by a probable ribosome binding site (RBS), GGGGA . It encodes a 492-amino-acid protein with molecular weight of 54 kDa and isoelectric point of 10.6 . The G + C content of ste5 is 73%, close to the average of G + C content (74%) for Streptomyces . Moreover, the preference usage of G or C as third base of codons are found in the ste5, which is in accordance with the Streptomyces codon usage . A BlastP search showed that the C-terminal region of Ste5 shows highly homology with a number of priming glycosyltransferases from many different organisms . Ste5 contains two putative catalytic residues, Glu and Asp (residues 423 and 474) with a spacing of approximately 50 amino acids that conserved in various beta-glycosyltransferases . Moreover, the C-terminal one third of Ste5 contains three domains, A, B and C that is reported to be common to glycosyltransferases . By hydrophilicity plot prediction, the N-terminal two thirds of Ste5 exhibits 5 putative transmembrane domains . To investigate the involvement of the identified polysaccharide gene cluster in EPS 139A biosynthesis, the gene ste5 encoding priming glycosyltransferase was insertionally disrupted by a single-crossover homologous recombination event . A 0.85 kb internal fragment of ste5 was cloned into vector pKC1139 to yield pLY5015 that was transduced into Streptomyces sp . 139 . Correct integration in Streptomyces LY1001 ste5- mutant strain was confirmed by Southern hybridization . After fermentation, no EPS 139A could be detected in the cultures of ste5- mutant strain Streptomyces LY1001 . Therefore, the gene ste5 identified in this work is involved in the synthesis of the Streptomyces sp . 139 EPS.

Ukr Biokhim Zh, 2003 Sep-Oct, 75(5), 77 - 84
{Mechanisms of acetylcholine-dependent production of H2O2 and NO2- by stromal cells of endometrium}; Danylovych IuV; The metabolism of NO(NO2-) and H2O2(O2-) by stroma cells of pig endometrium is NAD(P)H and glutathione-dependent process . The efficiency of biosynthesis and utilization of these metabolites appreciably depends on the state of SH-groups of the conforming ferment systems . And the reversible oxidation of SH-groups (maybe by the reaction products) results in the drop of biosynthesis rate . The NO and H2O2 metabolism is also defined by the state of oxidative metabolism of arachidonic acid (depending on salicylate), and also intensity of a course of redox-processes on plasmalemma (is regulated by cytochrome c) . The NO2- biosynthesis by stroma cells is strongly inhibited by the agents, which super produce H2O2(O2-) (salicylate and cytochrome c) . The NO(NO2-) and H2O2(O2-) metabolism at stimulation by acetylcholine is of cyclic character, and the infringement of any link during biosynthesis or utilization of these compositions results in losses of cyclicity . In contrast to this the formation of nitrosoglutathione with time achieves the saturation, which reflects its buffer and depositing with respect to NO function and permits to consider formation of the latter as one of mechanisms of effective utilization of NO(NO2-)--by the stroma cells.

Ukr Biokhim Zh, 2003 Sep-Oct, 75(5), 69 - 76
{Role of ATP-sensitive potassium channel activators in liver mitochondrial function in rats with different resistance to hypoxia}; Tkachenko HM et al.; Effects of ATP-sensitive potassium (KATP) channels opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) in rats with different resistance to hypoxia on indices of ADP-stimulation of mitochondrial respiration by Chance, calcium capacity and processes of lipid peroxidation in liver has been investigated . We used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate . Additional analyses contain the next inhibitors: mitochondrial fermentative complex I-10 mkM rotenone, succinate dehydrogenase 2 mM malonic acid . It was shown that effects of pinacidil induced the increasing of oxidative phosporylation efficacy and ATP synthesis together with lowering of calcium capacity in rats with low resistance to hypoxia . Effects of pinacidil were leveled by glibenclamide . These changes are connected with the increasing of respiratory rate, calcium overload and intensification of lipid peroxidation processes . A conclusion was made about protective effect of pinacidil on mitochondrial functioning by economization of oxygen-dependent processes, adaptive potentialities of organisms with low resistance to hypoxia being increased.

Ukr Biokhim Zh, 2003 Sep-Oct, 75(5), 17 - 27
{Interrelation between thrombin structure and its stability}; Kolodzeiskaia MV et al.; Data concerning peculiarities of fermentative nature and structure of thrombin in water-salt solution have been generalized; regularities of stabilizing effect made on thrombin by various polyols and other substances have been analyzed . It has been shown that formation of thrombin optimum macrostructure is one of the methods of its stabilization . Presence of different dissolving additives changes this enzymes hydration and this affects its stability and activity . There exist some systems to stabilize thrombin solutions . The systems consist of various salts, low-molecular and high-molecular polyols, surfactants, protein chain, composition buffer, etc . It has been shown that optimal concentrations of polyols, buffer salts and surfactants, as well as protein interaction increase considerably thrombin stability, preserving secondary structure even under its low concentration in the solution.

Protein Expr Purif, 2004 Jan, 33(1), 123 - 33
Expression and purification of the recombinant diphtheria fusion toxin DT388IL3 for phase I clinical trials; Urieto JO et al.; A genetically engineered fusion toxin targeted to acute myeloid leukemia (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human interleukin-3 . The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BLR (DE3) Escherichia coli . A single transformed colony was grown in Superbroth with ampicillin; bacteria were centrifuged at an OD(650) of 1.3; master cell bank aliquots of bacteria in 30% glycerol/Superbroth were frozen and stored at -80 degrees C . Master cell bank bacteria were diluted 1500-fold into Superbroth and recombinant protein was induced with 1 mM IPTG at an OD(650) of 0.6 . After two additional hours of fermentation, inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride and dithioerythritol . Recombinant protein was refolded by diluted 100-fold in cold buffer with arginine and oxidized glutathione . After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymyxin B affinity chromatography . The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C . Seventy-five 3-L bacterial culture preparations were made and pooled for the AT-1 batch (568 mL) and twenty-four 3-L bacterial culture preparations were made and pooled for the AT-2 batch (169 mL) . The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS-PAGE, limulus amebocyte lysate endotoxin assay, human AML TF/H-ras cell cytotoxicity assay, sterility, tandem mass spectroscopy, IL3 receptor binding affinity, ADP ribosylation activity, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, peptide mapping, stability, HPLC TSK3000, N-terminal sequencing, E . coli DNA contamination, C57BL/6 mouse toxicity, cynomolgus monkey toxicity, and immunohistochemistry . Yields were 25.7+/-5.6 mg/L bacterial culture of denatured fusion toxin . After refolding and chromatography, final yields were 20+/-11% or 5 mg/L . Vialed product was sterile . Batches were in 0.25 M sodium chloride/5 mM Tris, pH 8, and had protein concentrations of 1.8-1.9 mg/mL . Purity by SDS-PAGE was 99+/-1% . Aggregates by HPLC were <1 % . Potency revealed a 48 h IC(50) of 6-8 pM on TF/H-ras cells . Endotoxin levels were 1 eu/mg . The remaining chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule . The LD(10) in mice was 250 microg/kg/day every other day for six doses . The MTD in monkeys was 60 microg/kg/day every other day for six doses . Drug did not react with tested frozen human tissue sections by immunohistochemistry . There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 and -20 degrees C . Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 24 h and at 37 degrees C in human serum for 24 h . The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development.

Protein Expr Purif, 2004 Jan, 33(1), 11 - 8
An improved purification procedure for soluble processing alpha-glucosidase I from Saccharomyces cerevisiae overexpressing CWH41; Faridmoayer A et al.; Processing alpha-glucosidase I, which is encoded by CWH41, regulates one of the key steps in asparagine-linked glycoprotein biosynthesis by cleaving the terminal alpha-1,2-linked glucose from Glc(3)Man(9)GlcNAc(2), the common oligosaccharide precursor . This cleavage is essential for further processing of the oligosaccharide to the complex, hybrid, and high mannose type carbohydrate structures found in eukaryotes . A method is described for the purification of the soluble form of the alpha-glucosidase I, from recombinant Saccharomyces cerevisiae overexpressing CWH41 . A homogeneous enzyme preparation was obtained in higher yield than previously reported . Cultivation of recombinant S . cerevisiae in a fermenter increased the biomass 1.7 times per liter and enzyme production 2 times per liter compared to cultivation in shake flasks . Ammonium sulfate precipitation with three chromatography steps, including chromatography on an N-(5'-carboxypentyl)-1-deoxynojirimycin column, resulted in highly purified enzyme with no detectable contamination by other alpha- and beta-aryl-glycosidases . The purification procedure reproducibly yielded 40 microg of pure enzyme per gram wet biomass . Enzyme that was purified using an alternative procedure contained minor impurities and was hydrolyzed by an endogenous proteolytic activity to peptides that retained full catalytic activity . Controlled trypsin hydrolysis of the highly purified enzyme released polypeptide(s) containing the alpha-glucosidase I catalytic domain, with no loss of catalytic activity . This suggests that the catalytic domain of yeast alpha-glucosidase I is resistant to trypsin hydrolysis and remains fully functional after cleavage.

J Bacteriol, 2004 Jan, 186(1), 192 - 9
pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12; Yohannes E et al.; During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH . Additional modes of pH regulation were revealed under anaerobiosis . E . coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels . A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures . The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA) . High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity . Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration . This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate . On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd) . The acid induction of NikA (nickel transporter) is of interest because E . coli requires nickel for anaerobic fermentation . The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions . Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

J Appl Microbiol, 2004, 96(1), 84 - 95
Screening and typing of Patagonian wine yeasts for glycosidase activities; Rodriguez ME et al.; AIMS: The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines . METHODS AND RESULTS: The study was carried out in oenological autochthonous yeasts from Comahue region (Argentinean North Patagonia) . A set of screenable yeast phenotypic characteristics indicative of their potential usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-Saccharomyces populations . Twelve isolates showing six different glycosidase phenotypes were selected and they were characterized at species and strain levels using molecular methods . A close correlation between molecular and phenotypic characteristics was observed . Five strains belonging to Candida guilliermondii, C . pulcherrima and Kloeckera apiculata with highest constitutive beta-glucosidase activity levels without anthocyanase activity were discriminated . Some of them also showed constitutive beta-xylosidase and inductive alpha-rhamnosidase activities . CONCLUSIONS: The extension of the selection of oenological yeast to non-Saccharomyces species provided strains possessing novel and interesting oenological characteristics which could have significant implications in the production of more aromatic young red wine . SIGNIFICANCE AND IMPACT OF THE STUDY: As these non-Saccharomyces are indigenous to wine, they can be used in mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine nuance that is typical of this specific area.

J Appl Microbiol, 2004, 96(1), 76 - 83
Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts; Ganga MA et al.; AIMS: The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile . METHODS AND RESULTS: Microvinifications were carried out with grape musts of two areas . In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts . The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique . In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation . Furthermore, a study of the production of extracellular enzymes was done . The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase . CONCLUSION: The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area . Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.

Biotechnol Lett, 2003 Nov, 25(21), 1819 - 26
A novel, repeated fed-batch, ethanol production system with extremely long term stability achieved by fully recycling fermented supernatants; Lu X et al.; Using Saccharomyces cerevisiae, a novel, repeated fed-batch ethanol production system from corn flour by fully recycling fermented supernatants is demonstrated . With recovery of ethanol by evaporation coupled with consecutive removal of the insoluble and soluble inhibitory substances accumulated, either completely or partially by filtration, the concentrations of the soluble inhibitors in the system could be maintained at their equilibria . As a result, a sustained high concentration of ethanol (up to 15% v/v) and significant pollution control performance were obtained.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 636 - 43 Epub 2003 Dec 16.
Effects of CO2 on the formation of flavour volatiles during fermentation with immobilised brewer's yeast; Shen HY et al.; Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems . However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages . One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO2, which has inhibitory effects on yeast growth and metabolism . This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO2 from the liquid medium, which results in a low level of dissolved CO2 during fermentation . Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system . By sparging a CO2 flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10-30% during the active fermentation stage, while the fermentation rate was unaffected . The uptake of branched-chain amino acids was reduced by 8-22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively . The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO2 during fermentation with immobilised yeast.

Biochem J, 2004 Apr 1, 379(Pt 1), 47 - 55
NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing; Brondijk TH et al.; Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ) . In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity . We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis . The residual rate of nitrate reduction (approx . 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation . Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices . Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm . An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway . A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC . As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm . An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex . These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA.

J Zhejiang Univ Sci, 2004 Feb, 5(2), 173 - 9
A pair of two-component regulatory genes ecrA1/A2 in S . coelicolor; Li YQ et al.; Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor . DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway . ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover . Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain . However, the change of the actinorhodin (Act) production level was not significant compared with wild type . Thus, these experiment results confirmed that the two-component system ecrA1/A2 was positive regulatory element for red gene cluster.

J Biol Chem, 2004 Mar 5, 279(10), 9432 - 9 Epub 2003 Dec 12.
Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase; Oyedotun KS et al.; Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain . The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits . We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation . The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties . Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site . We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562) . Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment . This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes . Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.

J Food Prot, 2003 Dec, 66(12), 2267 - 75
A model study of Escherichia coli O157:H7 survival in fermented dry sausages--influence of inoculum preparation, inoculation procedure, and selected process parameters; Naim F et al.; The influence of inoculum preparation, inoculation level, and inoculation procedure on Escherichia coli O157:H7 inactivation during the manufacture of fermented sausage was evaluated in a model study . Prior growth in glucose-enriched tryptone soya broth, which provided exposure to mildly acidic conditions (pH 4.8), had no effect on the later survival of E . coli O157: H7 strains 5-1 and ATCC 43894 under extremely acidic conditions (pH 2), but the same strains became sensitive to acidity after 7 days of incubation on the surface of refrigerated beef (as per the normal contamination route from slaughter to further processing) . In subsequent sausage production trials, the extent of destruction observed for E . coli O157:H7 strains F-90, 5-1, and ATCC 43894 inoculated directly into the meat batter was unchanged when the inoculation level was decreased from 7.3 to 4.7 log CFU/g, but the level of inactivation was ca . 1 log higher when the surfaces of beef cuts, rather than the batter, were inoculated 7 days prior to processing . Regardless of processing conditions (fermentation to a pH of < or = 5.0 at 24 or 37 degrees C, drying at 14 degrees C to a water activity {a(w)} value of 0.91 or 0.79), strains F-90, 5-1, and ATCC 43894 showed similar survival capacities during the manufacture of sausage . A approximately 2-log reduction in pathogen numbers was generally obtained after samples were dried to an a(w) of 0.91, irrespective of fermentation temperature . The addition of a 5-day predrying holding stage at the fermentation temperature significantly (P < 0.05) increased pathogen inactivation when fermentation was carried out at 37 degrees C (but not when it was carried out at 24 degrees C) . However, significant pathogen reductions (4 to 5 log CFU/g) were achieved only for extensively dried products (a(w) = 0.79).

J Dairy Sci, 2003 Nov, 86(11), 3675 - 84
Effects of corn silage processing and amino acid supplementation on the performance of lactating dairy cows; Ouellet DR et al.; This experiment was conducted to determine the effect of crop processing and amino acid supplementation on dairy cow performance . Corn silage processed (PCS) or unprocessed (UCS) was used as the main forage (45% of dry matter, DM) in a total mixed ration (TMR) . Each TMR was either supplemented (AA) or not (AAO) with ruminally protected amino acids (lysine, 3 g/d and methionine, 14 g/d) . Thirty-two (551 kg) Holstein cows were randomly assigned to four treatments: PCS-AA, PCS-AA0, UCS-AA, and UCS-AA0 in a 2 x 2 factorial structure . Between wk 7 and 17 of lactation, cows were fed ad libitum TMR comprising 45% of corn silage plus 1 kg of grass hay once a day . The UCS presented better fermentation characteristics than PCS . Dry matter intake (DMI) of the TMR was not affected by treatment and averaged 22.7 kg/d . Energy-corrected milk (ECM) production was 9% higher with UCS than with PCS (33.1 vs . 30.1 kg/d) . Milk efficiency was therefore 6% higher with UCS than with PCS (1.43 vs . 1.35 kg ECM/kg of DMI) . The concentration of major milk constituents (fat, protein, lactose, urea) was not affected by treatments . Apparent digestibility of DM, organic matter, N, starch, acid detergent fiber, and neutral detergent fiber were similar among treatments . The effective ruminal degradability of DM, starch, and protein, however, was greater with PCS than with UCS . Amino acid supplementation had no effect on milk production nor on milk constituents, whether it was used with processed corn silage or with unprocessed corn silage . These data indicate that feeding UCS resulted in a greater milk production compared with PCS . The numerically higher DMI, a potentially greater intestinal digestion of starch or the better conservation of UCS could have contributed to the greater milk production.

J Dairy Sci, 2003 Nov, 86(11), 3661 - 6
Ruminal fermentation and bacterial protein synthesis of whole cottonseed coated with combinations of gelatinized corn starch and urea; Bernard JK et al.; Four ruminally and duodenally cannulated Jersey cows were used in a 4 x 5 incomplete Latin square study to determine the effects of including urea in the gelatinized corn starch coating applied to whole cottonseed (WCS) on ruminal fermentation, fiber digestion, and bacterial protein synthesis . Treatments included uncoated WCS (control) and four coated WCS treatments . The coatings provided two concentrations each of gelatinized corn starch (2.5 {2S} or 5% {5S}) and feed grade urea (0.25 {2U} or 0.5% {5U}) . Treated WCS comprised 15% of the ration dry matter that was fed as a total mixed ration once daily . Ruminal pH and molar proportions of isobutyrate was higher and NH3-N concentrations lower for control compared with coated WCS . Molar proportions of propionate tended to be higher and valerate was lower with 2S compared with 5S . Molar proportions of acetate tended to be lower, whereas butyrate was higher for 5U than 2U . Nutrient intake was lower for WCS coated with 5S5U compared with 2S5U . Ruminal NDF digestibility of NDF tended to be higher with 5U compared with 2U, but no differences were observed in ruminal or total tract apparent digestibility of nutrients . No differences were observed in the flow of total N or bacterial N to the duodenum, but the flow of nonbacterial N tended to be higher for WCS coated with 5U . Coating WCS appears to slightly alter ruminal metabolism while providing similar amounts of N flowing to the duodenum without altering fiber digestion.

J Dairy Sci, 2003 Nov, 86(11), 3562 - 70
Pelleted beet pulp substituted for high-moisture corn: 3 . Effects on ruminal fermentation, pH, and microbial protein efficiency in lactating dairy cows; Voelker JA et al.; The effects of increasing concentrations of dried, pelleted beet pulp substituted for high-moisture corn on ruminal fermentation, pH, and microbial efficiency were evaluated using eight ruminally and duodenally cannulated multiparous Holstein cows in a duplicated 4 x 4 Latin square design with 21-d periods . Cows were 79 +/- 17 (mean +/- SD) DIM at the beginning of the experiment . Experimental diets with 40% forage (corn silage and alfalfa silage) and 60% concentrate contained 0, 6.1, 12.1, or 24.3% beet pulp substituted for high-moisture corn on a DM basis . Diet concentrations of NDF and starch were 24.3 and 34.6% (0% beet pulp), 26.2 and 30.5% (6% beet pulp), 28.0, and 26.5% (12% beet pulp), and 31.6 and 18.4% (24% beet pulp), respectively . Substituting beet pulp for corn did not affect daily mean or minimum ruminal pH but tended to reduce pH range . Ruminal acetate:propionate responded in a positive exponential relationship to added beet pulp . Rate of valerate absorption from the rumen was not affected by treatment . Substituting beet pulp for corn up to 24% of diet DM did not affect efficiency of ruminal microbial protein production, expressed as microbial N flow to the duodenum as a percentage of OM truly digested in the rumen . Microbial efficiency was not correlated to mean pH or daily minimum pH . While microbial efficiency was not directly related to concentration of beet pulp fed, it was positively correlated with passage rate of particulate matter, as represented by starch and indigestible NDF, probably due to reduced turnover of microbial protein in the rumen.

Chemotherapy, 2003 Dec, 49(6), 298 - 302
Comparative study of the sensitivity of lymphoblastoid and transformed monocytic cell lines to the cytotoxic effects of Viscum album extracts of different origin; Duong Van Huyen JP et al.; Viscum album (VA) preparations consist of aqueous extracts of V . album, the European mistletoe . VA extracts contain mistletoe lectins, which are members of the ribosome-inactivating protein type II family . VA preparations have cytotoxic and immunomodulatory properties . Cytotoxicity induced by VA extracts may differ greatly according to the origin of the preparation (host tree, fermented extract) and the cell type . This work was performed to assess the cytotoxicity of various VA preparations, i.e . VA Qu FrF, Qu Spez, M Spez and VA P, in lymphoblastoid and monocytic cell lines . VA Qu FrF, Qu Spez and M Spez induced dose-dependent cell death and inhibition of cell proliferation in lymphoblastoid T cell lines and in transformed monocytic lines . In contrast, the majority of B cell lines tested were resistant to cytotoxicity induced by VA extracts . While VA Qu FrF, Qu Spez and M Spez were potent inducers of cell death, extracts of VA P, derived from mistletoe plants growing on pine trees, failed to induce any cell death in any of the cell lines examined .

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 613 - 25 Epub 2003 Dec 11.
Biotechnology and molecular biology of the alpha-glucosidase inhibitor acarbose; Wehmeier UF et al.; The alpha-glucosidase inhibitor acarbose, O-{4,6-dideoxy-4{1 s-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl}-amino-alpha-D-glucopyranosyl}-(1-->4)- O-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose, is produced in large-scale fermentation by the use of strains derived from Actinoplanes sp . SE50 . It has been used since 1990 in many countries in the therapy of diabetes type II, in order to enable patients to better control blood sugar contents while living with starch-containing diets . Thus, it is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market . Cultures of Actinoplanes sp . also produce various other acarbose-like components, of which component C is hard to separate during downstream processing, which is one of the most modern work-up processes developed to date . The physiology, genetics and enzymology of acarbose biosynthesis and metabolism in the producer have been studied to some extent, leading to the proposal of a new pathway and metabolic cycle, the "carbophore" . These data could give clues for further biotechnological developments, such as the suppression of side-products, enzymological or biocombinatorial production of new metabolites and the engineering of production rates via genetic regulation in future.

Br J Nutr, 2003 Nov, 90(5), 895 - 906
Steroids in the intestinal tract of rats are affected by dietary-fibre-rich barley-based diets; Dongowski G et al.; The aim of the present study was to investigate the influence of dietary-fibre (DF)-rich barley-based diets on bile acids (BA) and neutral sterols (NS) in the intestinal tract of rats . For this purpose, young male Wistar rats (n 50; ten per group) weighing about 67 g were fed either a barley-free diet (control group) or diets containing 500 g barley meal extrudates/kg or a barley meal-Novelose mixture (groups A-D) for 6 weeks . These barley products contained 7-24 g resistant starch/100 g and 7-12 g (1 --> 3),(1 --> 4)-beta-glucan/100 g . More steroids were transported towards the lower parts of the intestinal tract when higher concentrations of macromolecular DF were present in the diets (P < 0.001) . Tauroconjugated and primary BA dominated in the contents of the small intestine . Intense enzymic conversion of BA occurred in the caecum and colon . The fermentation of DF affected indirectly the amount of formed secondary BA . The main BA present in the caecal contents were muricholic acids, hyodeoxycholic acid and cholic acid . The BA spectrum in the colonic contents was different from that in the caecum . A higher concentration of NS appeared in the intestinal contents of the groups fed the barley-based diets than in the controls (P < 0.005) . The microbial conversion of cholesterol to coprostanol, cholestanone and coprostanone was influenced by the amount and composition of the DF in the gut . DF in the diet may affect the concentration and spectrum of steroids in the intestinal tract . The results are relevant for the discussion of mechanisms behind the cholesterol-lowering effects of DF.

Appl Biochem Biotechnol, 2003 Dec, 111(3), 129 - 38
Fermentation process optimization of recombinant Saccharomyces cerevisiae for the production of human interferon-alpha2a; Chu J et al.; The effects of different culture conditions on the expression level of human interferon-alpha2a (IFN-alpha2a) by using recombinant yeast were investigated in a 2.6-L jar fermentor . Appropriate supplement of glucose and the maintenance of residual glucose at a low level resulted in the reduction of ethanol formation and enhancement of the bioactivity of IFN-alpha2a to 4.9 x 106 from 3.1 x 10(6) IU/mL . When adenine was added evenly for 10-20 h of fermentation into the basal culture medium at a speed of 2 microg/mL of medium/h, OD600 was greatly increased to 24, and the protein increased to 276 mg/L . The content of ethanol generated was also reduced tremendously during the process, and as a result, 1.3 x 10(7) IU/mL of biologic activity was achieved . In the expression phase, pH had an important impact on expression level, which should be controlled at 5.5.

Biotechnol Adv, 2004 Jan, 22(3), 261 - 79
Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance; Salehizadeh H et al.; Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms . PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers . To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes . The use of mixed cultures and cheap substrates can reduce the production cost of PHA . Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence . The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used . This work reviews the development of PHA research . Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.

Yeast, 2003 Dec, 20(16), 1369 - 85
Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation; Rossignol T et al.; The transcriptome of a wine yeast was monitored throughout an alcoholic fermentation under conditions mimicking an enological environment . Major changes in gene expression occurred during fermentation, affecting more than 2000 genes, as the yeast adapted to changing nutritional, environmental and physiological conditions . The genes of many pathways are regulated in a highly coordinated manner, and genes involved in the key metabolic pathways of fermentation are strongly expressed . We showed that, during fermentation of a synthetic medium mimicking a natural must in which growth arrest was caused by nitrogen exhaustion, entry into the stationary phase triggered major transcriptional reprogramming . Many TOR target genes involved in nitrogen utilization or other functions are induced at this stage, suggesting that this signalling pathway plays a critical role in changes in gene expression in response to nitrogen depletion . Entry into stationary phase is a key physiological event and is followed by a general stress response . The superimposition of multiple stresses, including starvation and ethanol stress, gives rise to a unique stress response, involving hundreds of genes encoding proteins involved in various cellular processes, many of unknown function .

Can J Microbiol, 2003 Oct, 49(10), 650 - 4
Attempts to inhibit ruminal methanogenesis by blocking pyruvate oxidative decarboxylation; Ungerfeld EM et al.; The inhibition of pyruvate oxidative decarboxylation as a means of decreasing ruminal methanogenesis in vitro was studied . In the first experiment, the addition of adenosine and adenine (with and without ribose) to ruminal batch cultures did not decrease methanogenesis . In the second experiment, the addition of oxythiamin decreased methanogenesis by 23% . In the third experiment, three pyruvate derivatives did not inhibit methanogenesis, although hydroxypyruvate improved organic matter fermentation from 57.8% to 64.2% . The additives did not seem to inhibit pyruvate oxidative decarboxylation.

Microbiology, 2003 Dec, 149(Pt 12), 3575 - 86
The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects; Gordon DM et al.; Escherichia coli was isolated from more than 2300 non-domesticated vertebrate hosts living in Australia . E . coli was most prevalent in mammals, less prevalent in birds and uncommon in fish, frogs and reptiles . Mammals were unlikely to harbour E . coli if they lived in regions with a desert climate and less likely to have E . coli if they lived in the tropics than if they lived in semi-arid or temperate regions . In mammals, the likelihood of isolating E . coli from an individual depended on the diet of the host and E . coli was less prevalent in carnivores than in herbivores or omnivores . In both birds and mammals, the probability of isolating E . coli increased with the body mass of the host . Hosts living in close proximity to human habitation were more likely to harbour E . coli than hosts living away from people . The relative abundance of E . coli groups A, B1, B2 and D strains in mammals depended on climate, host diet and body mass . Group A strains were uncommon, but were isolated from both ectothermic and endothermic vertebrates . Group B1 strains could also be isolated from any vertebrate group, but were predominant in ectothermic vertebrates, birds and carnivorous mammals . Group B2 strains were unlikely to be isolated from ectotherms and were most abundant in omnivorous and herbivorous mammals . Group D strains were rare in ectotherms and uncommon in endotherms, but were equally abundant in birds and mammals . The results of this study suggest that, at the species level, the ecological niche of E . coli is mammals with hindgut modifications to enable microbial fermentation, or in the absence of a modified hindgut, E . coli can only establish a population in 'large-bodied' hosts . The non-random distribution of E . coli genotypes among the different host groups indicates that strains of the four E . coli groups may differ in their ecological niches and life-history characteristics.

Appl Microbiol Biotechnol, 2003 Dec, 63(3), 267 - 73 Epub 2003 Jul 09.
Optimization of recombinant aminolevulinate synthase production in Escherichia coli using factorial design; Xie L et al.; The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111 . The effects of initial succinate, glucose and isopropyl-beta-d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied . One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors . Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG . ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655 . Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).

Appl Environ Microbiol, 2003 Dec, 69(12), 7535 - 40
Application of genome-wide expression analysis to identify molecular markers useful in monitoring industrial fermentations; Higgins VJ et al.; Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae identified the YOR387c and YGL258w homologues as highly inducible in zinc-depleted conditions . Induction was specific for zinc deficiency and was dependent on Zap1p . The results indicate that these sequences may be valuable molecular markers for detecting zinc deficiency in industrial fermentations.

J Pept Sci, 2003 Nov-Dec, 9(11-12), 701 - 13
A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum; Reiber K et al.; Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound . The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin . Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet . By improving the culture conditions it was possible to grow S . ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times . The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation . Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs) . Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib) . substantiating their involvement in Ampullosporin biosynthesis . Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster . Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2079 - 83
Candida zemplinina sp . nov., an osmotolerant and psychrotolerant yeast that ferments sweet botrytized wines; Sipiczki M; Four yeast strains isolated from fermenting botrytized grape musts in the Tokaj wine region of Hungary are shown to represent a new osmotolerant and psychrotolerant species . The new species, Candida zemplinina (type strain 10-372(T)=CBS 9494(T)=NCAIM Y016667(T)), is closely related to Candida stellata, a yeast common on overripe grapes and in sweet fermenting wines . The sequence of the D1/D2 domain of the C . zemplinina 10-372(T) 26S rDNA shows 8.1 % sequence difference when compared to its counterpart in C . stellata CBS 157(T) . In the conserved 5.8S gene of the ITS1-5.8S-ITS2 region the difference is 8 % . The D1/D2 domain differs only at two nucleotides from the homologous sequence of a yeast strain isolated from botrytized grapes in California, suggesting that C . zemplinina is a wine yeast that occurs in geographically distant localities.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1991 - 8
Propionicimonas paludicola gen . nov., sp . nov., a novel facultatively anaerobic, Gram-positive, propionate-producing bacterium isolated from plant residue in irrigated rice-field soil; Akasaka H et al.; Two propionate-producing strains (Wd(T) and Wf) that were isolated anaerobically from plant residue of irrigated rice-field soil in Japan were characterized phenotypically and phylogenetically . The growth rate of strain Wd(T) was very slow in basal medium, but both growth and propionate production were stimulated significantly by the addition of cyanocobalamin . Strain Wf grew well in basal medium and produced substantial amounts of fermentation products, including propionate . Other phenotypic and phylogenetic characteristics of the two isolates were almost identical . Both were facultatively anaerobic, but much better growth was observed under anaerobic conditions . Cells were Gram-positive, non-motile, non-spore-forming and pleomorphic rods with irregular V- or crescent-shaped cell arrangements . Fermentation products from glucose in the presence of excess cyanocobalamin were acetate, lactate, a small amount of succinate and CO(2), in addition to propionate . Both oxidase and catalase activities were negative . The strains possessed meso-diaminopimelic acid in their peptidoglycan and their major cellular fatty acids were C(13 : 0), anteiso-C(15 : 0) and C(15 : 0) . The isolates had high genomic DNA G+C contents (68.7 and 67.4 mol%, respectively) . Menaquinones MK-9(H(4)) and MK-10(H(4)) were the predominant respiratory quinones . Phylogenetic analysis based on 16S rDNA sequences placed both strains in the Actinobacteria, with Micropruina glycogenica as their closest relative (sequence similarity values of 95.8 and 95.7 %, respectively) . Microlunatus phosphovorus and Friedmanniella antarctica were also related closely to the isolates . As their morphological, physiological and chemotaxonomic characteristics were distinctly different from those of any related species, Propionicimonas paludicola gen . nov., sp . nov . is proposed to accommodate these strains . The type strain of the novel species is Wd(T) (=JCM 11933(T)=DSM 15597(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1973 - 7
Rheinheimera pacifica sp . nov., a novel halotolerant bacterium isolated from deep sea water of the Pacific; Romanenko LA et al.; An aerobic, Gram-negative, non-fermentative, rod-shaped, motile, non-pigmented bacterium, KMM 1406(T), was isolated from a sample of Pacific deep sea water and investigated for phenotypic characteristics, chemotaxonomic features and phylogenetic relationships . The deep-sea isolate exhibited growth in 0-8 % (w/v) NaCl and at 4-37 degrees C, hydrolytic activity on gelatin, Tween 80 and starch and lack of D-glucose utilization . The major fatty acids were C(16 : 0), C(16 : 1)omega9c, C(17 : 1)omega8c and C(18 : 1)omega7c . The DNA G+C content was 49.6 mol% . 16S rRNA gene sequence analysis revealed that strain KMM 1406(T) was related closely to Rheinheimera baltica DSM 14885(T) within the gamma-Proteobacteria, with 96.8 % sequence similarity . On the basis of phenotypic and molecular data, a novel species, Rheinheimera pacifica sp . nov., is proposed . The type strain is KMM 1406(T) (=IAM 15043(T)=JCM 12090(T)=NRIC 0539(T)=CCUG 46544(T)).

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1837 - 41
Evaluation of ion exchange resins for removal of inhibitory compounds from corn stover hydrolyzate for xylitol fermentation; Maciel de Mancilha I et al.; The use of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar constituents is well-known and effective . However, a major problem associated with this pretreatment is the poor fermentability of the produced hydrolyzate as a result of the presence of the microorganism's inhibitory compounds . In the present work, seven ion-exchange resins were tested in order to detoxify corn stover hydrolyzate . Regarding xylose recovery, it was observed that more than 92% recovery was feasible . Furfural removal varied from 53.% to 99.%, and hydroxymethylfurfural (HMF) removal was effective between 37% and 100% . Acetic acid was totally removed by Purolite A 103 S resin . Corn stover hydrolyzate (CSH) treated with Purolite A 103 S, and Finex CS 14 GC resins, was tested as substrate for xylitol production using a yeast, Candida mogii . Product yields, Yp/s, of 0.41 and 0.37 g/g and cellular yields, Yx/s, of 0.24 and 0.13 g/g, respectively, were obtained using the two types of resin-treated hydrolyzates.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1786 - 91
Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity; Sanz R et al.; Important oenological properties of wine depend on the winemaking yeast used in the fermentation process . There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed . Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use . Laboratory-scale fermentations were performed using two different S . cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation . The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye . In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells . Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L . During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed . Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF . These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1683 - 8
An optimized method for Aspergillus niger spore production on natural carrier substrates; Bapat PM et al.; Aspergillus niger spores have wide ranging applications in the fermentation industry as well as in wastewater treatment . We present an optimized method for production of A . niger spores on natural substrates such as rice, split pea, and millet . The specific productivity (number of spores per gram of dry substrate) was 31-fold greater and volumetric productivity was 750-fold greater compared to agar slopes . The important process variables were incubation temperature, moisture content, and inoculum quantity . We find that the optimal condition for total spore count is different from the viable spore count for millet . The optimum lies in a narrow region defined by the process parameters . Of the three substrates tested split pea gave the highest specific spore productivity of 3.1 x 10(10) spores per gram of dry substrate . This is the first report of systematic study on the effect of process parameters on spore viability . The method of A . niger spore production on natural substrate appears advantageous as compared to the currently practiced method in terms of scale-up, cost, and ease of operation.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1677 - 82
Reduced formation of byproduct component C in acarbose fermentation by Actinoplanes sp . CKD485-16; Choi BT et al.; Acarbose fermentation was conducted by cultivation of Actinoplanes sp . CKD485-16 . Approximately 2,300 mg/L of acarbose was produced at the end of cultivation along with 600 mg/L of the acarbose byproduct component C . Maltose, a known moiety of acarbose, should be maintained at high concentration levels in culture broths for efficient acarbose production . The acarbose yield increased with an increasing osmolality of the culture medium, with a maximum value of 3,200 mg/L obtained at 500 mOsm/kg . Component C was also produced in proportion to the osmolality . Conversion of acarbose to component C was accomplished with resting whole cells . Inhibitors of the conversion of acarbose to component C were sought since component C is probably derived from acarbose . Valienamine was found to be a potent inhibitor, resulting in a more than 90% reduction in component C formation at a 10 microM concentration . Effects were similar in a 1,500-L pilot fermentor with acarbose and component C yields of 3,490 and 43 mg/L at 500 mOsm/kg, respectively.

Environ Sci Technol, 2003 Nov 15, 37(22), 5186 - 90
The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production; Oh SE et al.; Hydrogen gas can be recovered from the microbial fermentation of organic substrates at high concentrations when interspecies hydrogen transfer to methanogens is prevented . Two techniques that have been used to limit methanogenesis in mixed cultures are heat treatment, to remove nonsporeforming methanogens from an inoculum, and low pH during culture growth . We found that high hydrogen gas concentrations (57-72%) were produced in all tests and that heat treatment (HT) of the inoculum (pH 6.2 or 7.5) produced greater hydrogen yields than low pH (6.2) conditions with a nonheat-treated inoculum (NHT) . Conversion efficiencies of glucose to hydrogen (based on a theoretical yield of 4 mol-H2/mol-glucose) were as follows: 24.2% (HT, pH = 6.2), 18.5% (HT, pH = 7.5), 14.9% (NHT, pH = 6.2), and 12.1% (NHT, pH = 7.5) . The main products of glucose (3 g-COD/L) utilization (> or = 99%) in batch tests were acetate (3.4-24.1%), butyrate (6.4-29.4%), propionate (0.3-12.8%), ethanol (15.4-28.8%), and hydrogen (4.0-8.1%), with lesser amounts of acetone, propanol, and butanol (COD basis) . Hydrogen gas phase concentrations in all batch cultures reached a maximum of 57-72% after 30 h but thereafter rapidly declined to nondetectable levels within 80 h . Separate experiments showed substantial hydrogen losses could occur via acetogenesis and that heat treatment did not prevent acetogenesis . Heat treatment consistently eliminated the production of measurable concentrations of methane . The disappearance of ethanol produced during hydrogen production was likely due to acetic acid production as thermodynamic calculations show that this reaction is spontaneous once hydrogen is depleted . Overall, these results show that low pH was, without heat treatment, sufficient to control hydrogen losses to methanogens in mixed batch cultures and suggest that methods will need to be found to limit acetogenesis in order to increase hydrogen gas yields by batch cultures.

FEMS Yeast Res, 2003 Dec, 4(3), 339 - 47
Application of the reuseable, KanMX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences; Walker ME et al.; The characterisation of wine yeasts and the complex metabolic processes influencing wine fermentation and the quality of wine might best be achieved by exploiting the standard classical and recombinant genetic techniques which have been successfully used with laboratory strains . However, application of these techniques to industrial strains has been restricted because such strains are typically prototrophic and often polyploid . To overcome this problem, we have identified commercial wine strains with good mating and sporulation properties from which heterothallic derivatives were constructed by disruption of the HO gene . Consequently, these haploids are amenable to genetic analysis, whilst retaining desirable wine-making properties . The approach used was an adaptation of a previously published gene disruption procedure for laboratory yeast and is based on the acquisition of geneticin resistance from a removable KanMX marker . The present work is the first report of the application of a construct of this type to the disruption of the HO gene in wine yeasts that are in common commercial use . Most of the 4.9-kb disruption construct was successfully removed from the genome of the haploid derivative strains by loop-out of the KanMX marker through meiotic recombination . Sequencing of the HO region confirmed the reduction of foreign sequences to a 582-bp fragment comprised largely of a single direct repeat at the target gene . The removal of the active foreign gene (conferring antibiotic resistance) allows the application of other constructs based on the KanMX module without the need to resort to other selectable marker systems . Laboratory-scale fermentation trials typically showed minimal differences between the HO disruptants and the parental wine strains in terms of fermentation kinetics and formation of key metabolites.

Lancet, 2003 Nov 29, 362(9398), 1808 - 10
Food-aid cereals to reduce neurolathyrism related to grass-pea preparations during famine; Getahun H et al.; Neurolathyrism is a spastic paraparesis that can be caused by excessive consumption of the drought-resistant grass pea (Lathyrus sativus) . Devastating neurolathyrism epidemics have occurred during major famine crises in various parts of the world . We investigated in a case-control study the effects of food aid on risk of paralysis . Risk increased with consumption of boiled grass pea (adjusted odds ratio 2.78, 95% CI 1.09-7.13 with cereals; 5.22, 2.01-13.55 without cereal) and raw unripe green grass pea (1.96, 1.16-3.31; p=0.011), but not with the fermented pancake, unleavened bread, and gravy preparations . In a correlational study there was an inverse relation between the number of new cases and the amount of food-aid cereals distributed per person . During famine, cereals and nutritional information should reach people before they have grass pea as the only food.

Haematologia (Budap), 1996, 27(2), 55 - 84
Deoxycoformycin (pentostatin): clinical pharmacology, role in the chemotherapy of cancer, and use in other diseases; Spiers AS; Pentostatin (2'-deoxycoformycin, dCF) is a purine nucleoside analog and a product of the fermentation of Streptomyces antibioticus . It is a tight-binding inhibitor of adenosine deaminase (ADA), an enzyme essential in the cellular metabolism of purines . Children with congenital absence of ADA suffer from atrophy of lymphoid tissues and severe combined immune deficiency (SCID) syndrome . It was hypothesized that pentostatin would be lymphocytotoxic and this proved to be true; this finding prompted its investigation in lymphoid neoplasms . It was anticipated that pentostatin would be most active in neoplasms with high intracellular concentrations of ADA, e.g . acute lymphocytic leukemia (ALL), particularly of the T-cell variety . Although pentostatin proved to be active in ALL, large doses were required and major toxic effects outweighed therapeutic benefits . By contrast, pentostatin proved to be exceptionally active in hairy cell leukemia (HCL), a B-cell neoplasm with low intracellular concentrations of ADA . Pentostatin has since been shown to possess activity in chronic lymphocytic leukemia, prolymphocytic leukemia, cutaneous T-cell lymphomas, adult T-cell lymphoma-leukemia, and low grade non-Hodgkin's lymphomas . It potentiates the activity of vidarabine against viruses and against the cells of acute myeloid leukemia . Pentostatin is inactive in melanoma and renal carcinoma, but has not been adequately evaluated in other solid tumors . The toxic effects of pentostatin include renal failure, central nervous system (CNS) depression, immunosuppresion, keratoconjunctivitis, and opportunistic infections . In the absence of pre-existing bone marrow compromise, pentostatin produces only mild myelosuppression . Aside from its use as an antineoplastic agent, pentostatin has potential applications as an immunosuppressive drug, as an antiviral agent, as an antimalarial compound, and in the protection of cells of the CNS from damage induced by ischemia and anoxia . Clinical studies with pentostatin are ongoing, and its roles in the management of neoplastic and non-neoplastic diseases have yet to be fully defined.

Chem Pharm Bull (Tokyo), 2003 Dec, 51(12), 1458 - 9
Microbial transformation of terreusinone, an ultraviolet-A (UV-A) protecting dipyrroloquinone, by Streptomyces sp; Li X et al.; Biotransformation study was conducted on the marine dipyrroloquinone, terreusinone (1) isolated from the marine-derived fungus Aspergillus terreus . Preparative-scale fermentation of terreusinone with Streptomyces sp . has resulted in the isolation of a new oxidized metabolite, terreusinol (2) . The structure was elucidated as 2-{(1R)-1-hydroxyisobutyl}-6-{(1R)-1,2-dihydroxyisobutyl}-1H,5H-pyrrolo{2,3-b}indole-4,8-dione (2) on the basis of physicochemical evidence . Terreusinol (2) showed an ultraviolet-A (UV-A) (320-390 nm) protecting activity with ED(50) values of 150 microM, which is more active than oxybenzone (ED(50), 350 microM) currently being used as sunscreen.

Appl Biochem Biotechnol, 2003 Nov, 111(2), 113 - 28
Activity and survival of spray-dried Beijerinckia sp . microencapsulated in different carbohydrates; Boza Y et al.; This study examined the possibility of preserving Beijerinckia cultures by encapsulation using a spray drier, for use in biotechnological processes in the production of biopolymers . An adequate choice of the wall (coating) material is one of the factors that will determine the degree of cell survival and the maintenance of fermentative activity in the encapsulated inoculum . Malt dextrin, dehydrated glucose syrups, modified starch, and acacia (gum arabic) were used as wall materials . The results showed that spray-dried Beijerinckia encapsulated in malt dextrin, stored for 2 mo, and inoculated into sterile must after rehydration presented the greatest stability with respect to fermentative activity, although the glucose-encapsulated cells showed the highest percentage of viability during spray drying and during the storage period.

Bioresour Technol, 2004 Mar, 92(1), 97 - 101
Enhancement of citric acid production with ram horn hydrolysate by Aspergillus niger; Kurbanoglu EB; The potential use of ram horn hydrolysate (RHH) as a supplement for improvement of citric acid production by Aspergillus niger NRRL 330 was studied . For this purpose, first RHH was produced . Ram horns were hydrolyzed by treating with acid (6 N-H2SO4) and the RHH was obtained . With the addition of RHH to the fermentation medium with a final concentration of 4% (optimal concentration), citric acid value reached a maximum value (94 g/l), which is 52% higher than that of the control experiment . The addition of 4% (v/v) RHH enhanced citric acid accumulation, reduced residual sugar concentration and stimulated mycelial growth . Adding 4% RHH had no adverse effects on A . niger . As a result, RHH was found to be suitable as a valuable supplement for citric acid production in the submerged fermentation.

Bioresour Technol, 2004 Mar, 92(1), 41 - 8
Production of Botrytis cinerea for potential introduction into a vineyard; Akau HL et al.; Botrytis cinerea was produced in solid-phase fermentation, liquid fermentation and on potato dextrose agar . Stored products were evaluated for grape colonization in grape bioassays and in field trials, and for B . cinerea density using colony forming unit analyses and a nucleic-acid-based method . B . cinerea colony forming unit density was significantly correlated to the probability of successful grape colonization in grape bioassays (p-value=0.0002) . Solid fermentation products could be stored longer than liquid fermentation and potato dextrose agar products . There was little difference in the rate of grape colonization in laboratory bioassays among solid-phase fermentation, liquid fermentation and plate culture products . Although the initial B . cinerea colonization rate of field grapes was slightly greater on vines treated with solid-phase fermentation and plate culture products compared to vines treated with product from liquid fermentation, there was no significant difference in final colonization between vines treated with solid-phase fermentation, liquid fermentation and plate culture products and untreated vines.

Bioresour Technol, 2004 Mar, 92(1), 15 - 9
Ethanol recovery from corn fiber hydrolysate fermentations by pervaporation; O'Brien DJ et al.; Corn fiber, a byproduct of corn wet milling, is an attractive feedstock for biomass ethanol production . Corn fiber was hydrolyzed by dilute sulfuric acid and neutralized by one of two methods: conventional lime treatment or neutralization by strongly basic anion exchange . The anion exchange neutralized (AEN) hydrolysate contained substantially lower levels of the inhibiting compounds furfural, 5-hydroxymethylfurfural, and acetic acid compared to the lime neutralized hydrolysate . In batch fermentations the ethanol yields and final ethanol concentration of the two hydrolysates were similar at 0.32-0.43 g/g and 29-44 g/l, respectively . Sugar consumption in the AEN fermentations was superior . Coupling of a membrane pervaporation unit to a fed-batch fermentation of AEN hydrolysate maintained the ethanol concentration below 25 g/l with complete sugar utilization for approximately 5 days . A concentrated ethanol stream of 17 wt.% ethanol was produced by the pervaporation unit.

Metab Eng, 2003 Oct, 5(4), 277 - 83
Metabolic engineering for microbial production of shikimic acid; Kramer M et al.; Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections . An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms . Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid . An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate . Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.

Metab Eng, 2003 Oct, 5(4), 264 - 76
The effects of alternate optimal solutions in constraint-based genome-scale metabolic models; Mahadevan R et al.; Genome-scale constraint-based models of several organisms have now been constructed and are being used for model driven research . A key issue that may arise in the use of such models is the existence of alternate optimal solutions wherein the same maximal objective (e.g., growth rate) can be achieved through different flux distributions . Herein, we investigate the effects that alternate optimal solutions may have on the predicted range of flux values calculated using currently practiced linear (LP) and quadratic programming (QP) methods . An efficient LP-based strategy is described to calculate the range of flux variability that can be present in order to achieve optimal as well as suboptimal objective states . Sample results are provided for growth predictions of E . coli using glucose, acetate, and lactate as carbon substrates . These results demonstrate the extent of flux variability to be highly dependent on environmental conditions and network composition . In addition we examined the impact of alternate optima for growth under gene knockout conditions as calculated using QP-based methods . It was observed that calculations using QP-based methods can show significant variation in growth rate if the flux variability among alternate optima is high . The underlying biological significance and general source of such flux variability is further investigated through the identification of redundancies in the network (equivalent reaction sets) that lead to alternate solutions . Collectively, these results illustrate the variability inherent in metabolic flux distributions and the possible implications of this heterogeneity for constraint-based modeling approaches . These methods also provide an efficient and robust method to calculate the range of flux distributions that can be derived from quantitative fermentation data.

Biochemistry (Mosc), 2003 Nov, 68(11), 1159 - 70
Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli; Bagramyan K et al.; Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen . Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms . One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable . Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt) . Its formation also depends on the external pH and the presence of formate . Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force . It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.

J Agric Food Chem, 2003 Dec 3, 51(25), 7472 - 4
Unfermented rooibos tea: quantitative characterization of flavonoids by HPLC-UV and determination of the total antioxidant activity; Bramati L et al.; Unfermented rooibos originates from the leaves and the stems of the indigenous South African plant, Aspalathus linearis, and it has been reported to have a higher content of flavonoids compared to that of fermented rooibos . The HPLC/UV method developed in our laboratory for the analysis of the fermented rooibos was applied to the quantitative characterization of the major flavonoids present in the unfermented rooibos . Main compounds determined were aspalathin (49.92 +/- 0.80 mg/g), isoorientin (3.57 +/- 0.18 mg/g), orientin (2.336 +/- 0 . 049 mg/g), and rutin (1.69 +/- 0.14 mg/g), followed in order by isovitexin, vitexin, isoquercitrin and hyperoside, quercetin, luteolin and chrysoeryol . The identity of detected flavonoids was confirmed by comparing their retention times and UV spectra with those of corresponding standards . The total antioxidant activity (TAA) of the tea infusions was measured by the ABTS*+ radical cation decolorization assay . The TAA of unfermented rooibos (0.8 Trolox meq/g) resulted 2-fold higher than that of the fermented rooibos . When compared with different water infusions of Camellia sinensis (green and black tea), this TAA value was about 50% lower.

J Agric Food Chem, 2003 Dec 3, 51(25), 7385 - 90
Approaches to spirit aroma: contribution of some aromatic compounds to the primary aroma in samples of orujo spirits; Dieguez SC et al.; Terpenes and C(13) norisoprenoids are among the most important aromatic compounds found in a volatile and nonvolatile form in grapes . Aromatic typicity of a spirit could be attributed to these compounds despite the very important presence of volatile compounds produced during alcoholic fermentation . In this study, following a solid phase extraction stage, the determination of the varietal aromatic compounds by gas chromatography was performed on 15 samples of Galician orujo spirits . The results show that significant differences (p < 0.05) exist in the concentrations of varietal aromatic compounds in Galician spirits obtained from different varieties of grapes . alpha-Ionona is the varietal aromatic compound that is most likely to contribute to the aroma of all of the spirits studied . The spirits from Catalan Roxo are the most aromatic, with floral and fruity nuances, while the spirits from Godello were the less aromatic group as far as the varietal compounds studied are concerned . Spirits from Mencia and Treixadura show a similar profile, but the former has a more intensive aroma due to beta-pinene, citronellol, and alpha-ionone . Albarino spirits stand out because of their profile that is marked by the contributions of eugenol and linalool.

J Ind Microbiol Biotechnol, 2003 Dec, 30(12), 682 - 90 Epub 2003 Nov 29.
Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology; Trupkin S et al.; A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation . Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared . The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels . The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes . Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process . The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization . The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R . This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes . It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.

Plant Physiol, 2003 Dec, 133(4), 2048 - 60 Epub 2003 Nov 26.
Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape; Vigeolas H et al.; The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds . Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds . (a) . By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds . At ambient external oxygen (21% {v/v}) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds . A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds . (b) . The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen . (c) . To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques . The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations . In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels . The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% {v/v}) external oxygen concentrations . (d) . When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v) . (e) . Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites . (f) . Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase . (g) . The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state . (h) . The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis . The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.

J Exp Bot, 2004 Jan, 55(394), 145 - 6 Epub 2003 Nov 28.
The rice pyruvate decarboxylase 3 gene, which lacks introns, is transcribed in mature pollen; Li Y et al.; The rice pyruvate decarboxylase 3 gene (PDC3), which has no introns, was previously postulated to be a pseudogene because no PDC3 mRNA had been detected, even under anaerobic conditions . However, in this study, it was found that rice PDC3 transcripts accumulated in panicles after heading . Within anthers obtained from the panicles, PDC3 was shown to be transcribed in mature pollen by in situ hybridization . These results suggest that the rice PDC3 is a functional gene . Its product may play a role in aerobic alcoholic fermentation in mature pollen.

J Exp Bot, 2004 Jan, 55(394), 35 - 42 Epub 2003 Nov 28.
Highly conserved protein kinases involved in the regulation of carbon and amino acid metabolism; Halford NG et al.; It has been clear for over a decade and a half that ancient signalling pathways controlling fundamental cellular processes are highly conserved throughout the eukaryotes . Two plant protein kinases, sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK1) and general control non-derepressible 2 (GCN2)-related protein kinase are reviewed here . These protein kinases show an extraordinary level of conservation with their fungal and animal homologues given the span of time since they diverged from them . However, close examination of the signalling pathways in which they operate also reveals intriguing differences in activation and function.

J Agric Food Chem, 2003 Dec 3, 51(25), 7495 - 503
Capillary electrophoretic determination of theanine, caffeine, and catechins in fresh tea leaves and oolong tea and their effects on rat neurosphere adhesion and migration; Chen CN et al.; Theanine, caffeine, and catechins in fresh tea leaves and oolong tea were determined by using capillary electrophoresis (CE) . CE separated these tea polyphenols from three other tea ingredients, namely, caffeine, theophylline, and theanine, within 8 min . The young leaves (apical bud and the two youngest leaves) were found to be richer in caffeine, (-)-epigallocatechin gallate (EGCg), and (-)-epicatechin gallate (ECg) than old leaves (from 5th to 7th leaves) . On the other hand, the old leaves (from 8th to 10th leaves) contained higher levels of theanine, (-)-epigallocatechin (EGC), and (-)-epicatechin (EC) . Results from a comparison of fresh young tea and oolong tea compositions indicated oolong tea contained more theanine and catechins than fresh young tea . Furthermore, it was found that the levels of theanine, EGC, and EGCg in young leaves rose markedly with the withering process . Caffeine did not markedly change . However, fully or partially fermented teas (oolong tea or pauchong tea) have a common initial step in the withering process . Fresh tea leaves or oolong tea extract (0.1%, w/v) markedly inhibited neurosphere adhesion, cell migration, and neurite outgrowth in rat neurospheres . Theanine (348 micrograms/mL) and caffeine at high concentration (50 micrograms/mL) did not inhibit neurosphere adhesion or migration activities, but EGCg at 20 micrograms/mL effectively inhibited neurosphere adhesion for 24 h . These results indicated that EGCg might affect neural stem cell survival or differentiation.

J Agric Food Chem, 2003 Dec 3, 51(25), 7402 - 9
Pyruvic acid and acetaldehyde production by different strains of Saccharomyces cerevisiae: relationship with Vitisin A and B formation in red wines; Morata A et al.; The production of pyruvate and acetaldehyde by 10 strains of Saccharomyces cerevisiae was monitored during the fermentation of Vitis vinifera L . variety Tempranillo grape must to determine how these compounds might influence the formation of the pyroanthocyanins vitisin A and B (malvidin-3-O-glucoside-pyruvate acid and malvidin-3-O-glucoside-4 vinyl, respectively) . Pyruvate and acetaldehyde production patterns were determined for each strain . Pyruvate production reached a maximum on day four of fermentation, while acetaldehyde production was at its peak in the final stages . The correlation between pyruvate production and vitisin A formation was especially strong (R (2) = 0.80) on day 4, when the greatest quantity of pyruvate was found in the medium . The correlation between acetaldehyde production and the formation of vitisin B was strongest (R (2) = 0.81) at the end of fermentation when the acetaldehyde content of the medium was at its highest . Identification and quantification experiments were performed by HPLC-DAD . The identification of the vitisins was confirmed by LC/ESI-MS.

Water Sci Technol, 2003, 48(6), 241 - 7
Evaluation of the start-up of an integrated municipal solid waste and leachate treatment system; Libanio PA et al.; A pilot-scale experiment was set up in the laboratory with the purpose of investigating the most relevant aspects of the anaerobic digestion of municipal solid wastes (MSW), which are necessary for the preparation of future feasibility studies and cost-benefit analyses . The experiment consisted of a comparative analysis among three different lines of operation, each one consisting of three anaerobic MSW reactors (with unit volume of 700 L): conventional landfill (line 1), raw leachate recycling (line 2), and integrated treatment, with seeded leachate recycling (line 3) . So, a UASB reactor was installed in the integrated treatment line with the purpose of removing the organic load of the leachate of the MSW reactors and utilising the biological sludge produced for inoculation of the waste digestion . The endogenous inoculation promoted in line 3, by means of recirculation of the leachate and return of the exceeding biological sludge produced in the UASB reactor, has favoured the initial fermentation stage and also the acceleration of the methanogenic phase.

Nippon Yakurigaku Zasshi, 2003 Dec, 122(6), 527 - 38
Pharmacological effects of ivermectin, an antiparasitic agent for intestinal strongyloidiasis: its mode of action and clinical efficacy; Ikeda T; Ivermectin is an oral semi-synthetic lactone anthelmintic agent derived from avermectins isolated from fermentation products of Streptomyces avermitilis . Ivermectin showed a concentration-dependent inhibitory effect on motility of a free-living nematode, Caenorhabditis elegans (C . elegans) . There exist specific binding sites having a high affinity for ivermectin in the membrane fraction of C . elegans, and a strong positive correlation was detected between the affinity for these binding sites and the suppressive effect on motility of C . elegans in several ivermectin-related substances . These results suggested that the binding to these binding sites is important for the nematocidal activity of ivermectin . In oocytes of Xenopus laevis injected with the Poly (A)(+) RNA of C . elegans, expression of a chloride channel, which is irreversibly activated by ivermectin, was recognized . The pharmacological properties of this channel suggest that the ivermectin-sensitive channel is a glutamate-activated chloride channel . As to the glutamate-activated chloride channel, two subtypes (GluCl-alpha and GluCl-beta) were cloned, suggesting these subtypes constitute the glutamate-activated chloride channel . These findings suggest that ivermectin binds to glutamate-activated chloride channels existing in nerve or muscle cells of nematode with a specific and high affinity, causing hyperpolarization of nerve or muscle cells by increasing permeability of chloride ion through the cell membrane, and as a result, the parasites are paralyzed to death . In experimental infections in sheep and cattle, ivermectin exhibited potent dose-dependent anthelmintic effects on Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Oesphagostomum, and Dictyocaulus . Anthelmintic effects were reported also in dogs, horses, and humans infected with Strongyloides . In the clinical Phase III trial in Japan, 50 patients infected with Strongyloides stercoralis were administered approx . 200 microg/kg of ivermectin to be given orally twice at an interval of 2 weeks . As a result, the Strongyloides stercoralis-eradicating rate was 98.0% (49/50).

FEMS Microbiol Rev, 2003 Dec, 27(5), 663 - 93
Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics; Krause DO et al.; The degradation of plant cell walls by ruminants is of major economic importance in the developed as well as developing world . Rumen fermentation is unique in that efficient plant cell wall degradation relies on the cooperation between microorganisms that produce fibrolytic enzymes and the host animal that provides an anaerobic fermentation chamber . Increasing the efficiency with which the rumen microbiota degrades fiber has been the subject of extensive research for at least the last 100 years . Fiber digestion in the rumen is not optimal, as is supported by the fact that fiber recovered from feces is fermentable . This view is confirmed by the knowledge that mechanical and chemical pretreatments improve fiber degradation, as well as more recent research, which has demonstrated increased fiber digestion by rumen microorganisms when plant lignin composition is modified by genetic manipulation . Rumen microbiologists have sought to improve fiber digestion by genetic and ecological manipulation of rumen fermentation . This has been difficult and a number of constraints have limited progress, including: (a) a lack of reliable transformation systems for major fibrolytic rumen bacteria, (b) a poor understanding of ecological factors that govern persistence of fibrolytic bacteria and fungi in the rumen, (c) a poor understanding of which glycolyl hydrolases need to be manipulated, and (d) a lack of knowledge of the functional genomic framework within which fiber degradation operates . In this review the major fibrolytic organisms are briefly discussed . A more extensive discussion of the enzymes involved in fiber degradation is included . We also discuss the use of plant genetic manipulation, application of free-living lignolytic fungi and the use of exogenous enzymes . Lastly, we will discuss how newer technologies such as genomic and metagenomic approaches can be used to improve our knowledge of the functional genomic framework of plant cell wall degradation in the rumen.

J Appl Microbiol, 2003, 95(5), 1087 - 95
Controlling grape must fermentation in early winemaking phases: the role of electrochemical treatment; Lustrato G et al.; AIMS: To contribute to an understanding of the phenomena related to the effect of low electric current (LEC) in grape must fermentation during laboratory and pilot plant scale winemaking, with selected co-culture yeasts (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermodii strain 465) . METHODS AND RESULTS: LEC (10, 30, 50 and 100 mA) was applied to fresh grape must as an alternative method to the usual addition of SO2 . Parameters such as polarity, treatment duration (24-96 h) and type of inoculum yeast were varied one at a time . LEC decreased the survival time and increased the death rate of H . guilliermondii strain 465 in co-cultures, whereas it did not affect the growth and survival of S . cerevisiae strain 40 . A final comparison was made of the main physico-chemical parameters on wine obtained after the different tests . CONCLUSIONS: The results have demonstrated that the low voltage treatment using a pair of graphite electrodes had a positive effect on grape juice fermentation (yeast microflora) during the early stages of winemaking, even with the potential of being an alternative method to the usual addition of SO2 . SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing new winemaking technologies for an innovative yeast fermentation control process for 'biological wine'.

J Antibiot (Tokyo), 2003 Sep, 56(9), 747 - 54
Jenamidines A to C: unusual alkaloids from Streptomyces sp . with specific antiproliferative properties obtained by chemical screening; Hu JF et al.; Three new naturally occurring bicyclic alkaloids, jenamidines A (1), B (2) and C (3), were discovered and isolated from the culture broth of Streptomyces sp . (strain HKI0297) via the chemical screening approach . Fermentation, isolation, structure and biological activities of these three new secondary metabolites are reported . The jenamidines have an unusual octahydro-pyrido{1,2-a}pyrimidine skeleton . Jenamidine A (1) shows antiproliferative effects against the chronic myeloid leukaemic cell line K-562 . In addition, the new tricyclic sesquiterpenoid, africantriol (4) was isolated from the same strain.

Food Chem Toxicol, 2004 Jan, 42(1), 93 - 105
An assessment of the genotoxicity and human health risk of topical use of kojic acid {5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one}; Nohynek GJ et al.; Kojic acid (KA), a natural substance produced by fungi or bacteria, such as Aspergillus, Penicillium or Acetobacter spp, is contained in traditional Japanese fermented foods and is used as a dermatological skin-lightening agent . High concentrations of KA (>or=1000 microg/plate) were mutagenic in S . typhimurium strains TA 98, TA 100, TA 1535, TA102 and E . coli WP2uvrA, but not in TA 1537 . An Ames test following the "treat and plate" protocol was negative . A chromosome aberration test in V79 cells following a robust protocol showed only a marginal increase in chromosome aberrations at cytotoxic concentrations after prolonged (>or=18 h) exposure . No genotoxic activity was observed for hprt mutations either in mouse lymphoma or V79 cells, or in in vitro micronucleus tests in human keratinocytes or hepatocytes . All in vivo genotoxicity studies on KA doses were negative, including mouse bone marrow micronucleus tests after single or multiple doses, an in vivo/in vitro unscheduled DNA synthesis (UDS) test, or a study in the liver of the transgenic Muta(TM) Mouse . On the basis of pharmacokinetic studies in rats and in vitro absorption studies in human skin, the systemic exposure of KA in man following its topical application is estimated to be in the range of 0.03-0.06 mg/kg/day . Comparing these values with the NOAEL in oral subchronic animal studies (250 mg/kg/day), the calculated margin of safety would be 4200- to 8900-fold . Comparing human exposure with the doses that were negative for micronuclei, UDS and gene mutations in vivo, the margins of safety are 16000 to 26000-fold . In conclusion, the topical use of KA as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.

J Ind Microbiol Biotechnol, 2003 Nov, 30(11), 669 - 76 Epub 2003 Nov 19.
Improvement of monacolin K, gamma-aminobutyric acid and citrinin production ratio as a function of environmental conditions of Monascus purpureus NTU 601; Wang JJ et al.; Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement . Its metabolic products monacolin K and gamma-aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent . Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public . In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied . When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg . In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design . When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30 degrees C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.

Bioprocess Biosyst Eng, 2003 Dec, 26(2), 103 - 7 Epub 2003 Nov 18.
Batch xylitol production by Candida guilliermondii FTI 20037 from sugarcane bagasse hemicellulosic hydrolyzate at controlled pH values; Rodrigues RC et al.; Batch fermentation of sugarcane bagasse hemicellulosic hydrolyzate by the yeast Candida guilliermondii FTI 20037 was performed using controlled pH values (3.5, 5.5, 7.5) . The maximum values of xylitol volumetric productivity ( Q(p)=0.76 g/l h) and xylose volumetric consumption ( Q(s)=1.19 g/l h) were attained at pH 5.5 . At pH 3.5 and 7.5 the Q(p) value decreased by 66 and 72%, respectively . Independently of the pH value, Y(x/s) decreased with the increase in Y(p/s) suggesting that the xylitol bioconversion improves when the cellular growth is limited . At the highest pH value (7.5), the maximum specific xylitol production value was the lowest ( q(pmax)=0.085 g/l h.), indicating that the xylose metabolism of the yeast was diverted from xylitol formation to cell growth.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 237 - 42 Epub 2003 Nov 18.
Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity; Skory CD; Rhizopus oryzae is capable of producing high levels of lactic acid by the fermentation of glucose . Yields typically vary over 60-80%, with the remaining glucose diverted primarily into ethanol fermentation . The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate . Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment, were transformed into R . oryzae . This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in a high-copy number . Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection . Isolates transformed with ldhA containing plasmid were compared with both the wild-type parent strain and the auxotrophic recipient strain containing vector only . All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls . Integration of plasmid pLdhA48XI increased the stability of the strain, but did not seem to offer any benefit for increasing lactic acid production . Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid . Plasmid pLdhA71X, containing a large ldhA fragment (6.1 kb), routinely yielded higher levels of lactic acid than the smaller region (3.3 kb) used to construct plasmid pLdhA48XI . The greatest levels of ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII . However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared with the control.

Biosens Bioelectron, 2003 Dec 30, 19(5), 423 - 31
Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase; Inaba Y et al.; A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA) . The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala . The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction . The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala . It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1) . A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM . The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala . The D-Ala content in some fish sauces was also determined using the proposed sensor system . The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method . From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).

Arch Tierernahr, 2003 Oct, 57(5), 347 - 57
Effects of stage of maturity on ensiling characteristics and ruminal nutrient degradability of oat silage; Mustafa AF et al.; A study was conducted to determine the effects of stage of maturity on ensiling characteristics and ruminal nutrient degradability of oat silage . Oat was field grown and forage was harvested at the boot or soft dough stage and ensiled in mini-silos for 0, 2, 4, 8, 16 and 45 days . Two lactating Holstein cows fitted with ruminal fistulas were used determine ruminal nutrient degradability . Regardless of the stage of maturity, ensiled forages went through a rapid fermentation with a sharp decline in pH during the first 2 days of ensiling . Extensive proteolysis took place between 0 and 2 days as indicated by a reduction in true protein and neutral detergent insoluble protein (NDICP) and an increase in non-protein nitrogen (NPN) . Chemical analysis of the 45 days silage showed that stage of maturity had no effect on neutral detergent fibre (NDF) and acid detergent fibre (ADF) of oat silage . However, oat harvested at the boot stage contained more crude protein (CP) and less starch than that harvested at the soft dough stage . Distribution of protein fractions showed that oat harvested at the boot stage contained lower NPN, NDICP and acid detergent insoluble protein than oat harvested at the soft dough stage . Results of the in situ incubation experiment indicated that oat harvested at the soft dough stage had lower ruminal dry matter (60.6 vs . 66.4%) . CP (81.3 vs . 88.7%) and NDF (35.4 vs . 42.2%) degradabilities than oat harvested at the boot stage . It was concluded that chemical composition and ruminal nutrient degradability of oat silage are significantly influenced by stage of maturity.

Dev Biol (Basel), 2003, 113, 101 - 4; discussion 115-6
Re-processing strategies for biologicals API manufacturing processes; Opitz U; During the past 10 years, the production of biotechnological products has reached large scale starting from fermentor volumes of several thousand litres . Due to this large scale production, the value of a single batch has significantly increased . Sometimes even in routine production, technical or human failures occur . Without a re-processing procedure in place, such a failed batch has to be discarded leading to a waste of raw materials, time, energy and to a waste of money . This can be avoided by a well-defined re-processing strategy . On the other hand re-processing always raises concerns regarding product quality and stability . Therefore, re-processing should be adequately validated to exclude a negative impact on the product . An example for such a validation study is discussed.

Dev Biol (Basel), 2003, 113, 37 - 44; discussion 111-2
Validation of fermentation processes; Lubiniecki AS et al.; The ability to prepare consistent biopharmaceutical products depends extensively on possession of banked and characterized cell substrates and on development of production processes which can be validated . While the attributes that define cell characterization have been extensively detailed by ICH and the regulatory agencies in the past decade, little has been specified regarding process validation for biological processes . The extent to which validation concepts can be applied to biological processes varies depending on the nature of the process, the nature of the product, and the level of knowledge regarding the relationship between process parameters and product quality . Expectations concerning the rigour of the validation programme should be adjusted accordingly . There is no single approach that is appropriate for all processes and products . At a minimum, there should be an attempt to define which process parameters are critical, and to focus the attention of validation efforts on these parameters.

Yeast, 2003 Nov, 20(15), 1263 - 72
The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains; Jeppsson M et al.; Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255 . In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation . We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level . G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain . Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose) . Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization . Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors . Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001 .

Yeast, 2003 Nov, 20(15), 1243 - 53
Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway; Remize F et al.; Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2 . The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively . We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation . We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20% . In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain . The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions . However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly . The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant . These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast . The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation .

Medicina (Kaunas), 2003, 39 Suppl 2, 25 - 9
{Technology and analysis of "Askoeziuofito" tablets}; Bernatoniene J et al.; The objective of this work is to produce chewable tablets out of Echinacea purpurea liquid extract (1:1) and ascorbic acid: to create the technology, to select methods of analysis and to examine stability . The paper describes the technology of tablets: a method of condensation is chosen; the influence of additional substances over tableting is established; pressing characteristics of tableting mixtures are examined . The quality of tablets is evaluated in terms of appearance and technological rates: average tablet mass, hardness against pressure, hardness against wearing, time of disintegration, and speed of ascorbic acid secretion . The identity of ascorbic acid, ferments and hydroxycinamon acid was established . Quantities of ascorbic and chicory acids were defined . The tablets produced were named "Askoeziuofito" tablets . The name consists of abbreviated terms of ascorbic acid, Echinacea plant and phytochemical preparation.

J Vet Sci, 2001 Apr, 2(1), 15 - 24
Immunostimulatory effects of anionic alkali mineral complex solution Barodon in porcine lymphocytes; Yoo BW et al.; The anionic alkali mineral complex solution, Barodon (Barodon-S.F . Corp., Korea), was evaluated for its effectiveness as a nonspecific immunostimulator in pigs . The effects of Barodon were determined by analysis of feed efficiency, growth rate, and phenotype of leukocyte subpopulations using monoclonal antibodies specific to porcine leukocyte differentiation antigens and flow cytometry (FC) . The study was focused to investigate the change in proportion of the CD4+CD8+ double positive T lymphocyte subpopulation (dpp) which exists uniquely in pigs . In addition, the mitogen-stimulated lymphoproliferative response, tissue distribution in lymphoid organs and the adjuvant effect of Barodon on hog cholera vaccine efficiency were determined . The study has revealed the average daily gain rates and feed conversion rates were significantly (p<0.05) improved in either group of pigs fed with 0.05% Barodon-spray feed (Tx-1) or pigs fed with 3% Barodon-fermented feed (Tx-2) in comparison with group of pigs fed with feed containing no Barodon (control) . The proportion of cells expressing CD4+ antigen in Barodon-treated group increased from 3 weeks posttreatment and was significantly higher (p<0.05) than that of control at 8 weeks posttreatment . Particularly, the significantly higher proportion was maintained from 8 weeks through 13 weeks posttreatment in Tx-1 group (p<0.05) . The proportion of cells expressing CD8+ antigen was significantly higher at 3 weeks posttreatment in Tx-2 (p<0.01) . Proportion of MHC class II-expressing cells was significantly higher in Tx-1 and Tx-2 group at 11 weeks and 8 weeks posttreatment (p<0.05), respectively . In addition, the proportion of Non T/Non B (N) cells was also significantly higher in Tx-2 at 3 weeks posttreatment (p<0.01) and maintained to 13 weeks posttreatment (p<0.1) . Between Barodon-treated groups, the proportion of MHC class II-expressing cells was observed to be larger in Tx-2 than Tx-1 from 3 weeks to 8 weeks posttreatment (p<0.05) . However, there were no significant difference in the proportions of CD2+ cells, B cells, monocytes and granulocytes between Barodon-treated and control group during the experiment . Dual-color FC analysis, study has revealed an increased proportion of dpp present in lymphocytes obtained from peripheral blood (PB) and mesenteric lymph node (MLN) of Barodon-treated group at 8 and 11 weeks posttreatment . The proportion of dpp in PB was 27.5% and 32.1% in Tx-1 and Tx-2, respectively, but only 2.2% in control group at 8 weeks posttreatment . In MLN, the proportion was 45.1% and 52.1% in Tx-1 and Tx-2, respectively, otherwise 16.5% in control group at 8 weeks posttreatment . The mitogen-stimulated activity was significantly higher in Tx-1 than in the control group at 11 weeks posttreatment when cells were stimulated with Con A and PHA, respectively (p<0.01) . Also, Con A-, PHA and PWM-stimulated activity was significantly higher in Tx-2 than in the control group at the same time (p<0.05) . The tissue distribution of CD4+, CD8+ and CD4+CD8+ dpp in MLN and spleen was significantly larger in Tx-1 and Tx-2 than in the control group (p<0.01) . Also, a larger proportion of dpp was observed in Tx-2 than Tx-1 in spleen between Barodon-treated groups (p<0.01) . In conclusion, the study has demonstrated that Barodon had an immunostimulatory effect on pigs through proliferation and activation of porcine immune cells, specially CD4+CD8+ dpp lymphocytes.

FEMS Yeast Res, 2003 Nov, 4(2), 175 - 84
Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene; Heo JH et al.; The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha . A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed . The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP) . Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol . The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth . A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol . The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H . polymorpha.

Int J Mol Med, 2003 Dec, 12(6), 923 - 8
Relationship between peroxyl radical scavenging capability measured by the chemiluminescence method and an aminocarbonyl reaction product in soy sauce; Ando M et al.; Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis . Because of this a radical scavenger as an antioxidant has been sought in food materials . Soy sauce is a traditional fermented seasoning of East Asian countries and is available throughout the world . The relationship between the peroxyl radical scavenging capability using the luminol chemiluminescence method and melanoidin, the main product from aminocarbonylation, i.e., Maillard reaction, from soy sauce was examined . In this study, we report that soy sauce has a very high antioxidative capacity and from the comparisons of the IC50 values of 26 soy sauces in the case of the optical density of the soy sauce's color being standardized, it was found that not only melanoidin is an antioxidative product, but also other products have strong antioxidative properties.

J Ethnopharmacol, 2003 Dec, 89(2-3), 251 - 60
Screening of entomopathogenic Deuteromycetes for activities on targets involved in degenerative diseases of the central nervous system; Schmidt K et al.; A selection of 32 fungal strains, belonging to 8 genera of entomopathogenic Deuteromycetes collected in various provinces of China, were screened for activities on targets involved in degenerative diseases of the central nervous system . The strains were grown under various fermentation conditions, and a total of 256 different extracts were obtained . The bioassays included functional screens for NMDA antagonistic activity in stably transfected fibroblasts, for neuritogenic activities in PC-12 cells, and tests for MAO inhibitory and radical scavenging properties . Several extracts with promising activities were identified . Some Paecilomyces extracts induced pronounced axonal-like outgrowths in PC-12 cells . In Paecilomyces militaris RCEF 0095, the neuritogenic activity could be linked to yellow pigments . Three Beauveria and Paecilomyces strains showed radical scavenging properties, which could be localized in the extract by a bioautographic assay on TLC . An extract obtained from the mycelium of Paecilomyces tenuipes RCEF 0275 showed moderate MAO inhibitory activity, whereas extracts of Sporothrix chondracris RCEF 0187 antagonized NMDA receptor mediated cell toxicity.

EMBO J, 2003 Nov 17, 22(22), 5975 - 82
Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine; Marobbio CM et al.; The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified . Here the identification of the mitochondrial carrier for S-adenosylmethionine (SAM) Sam5p is described . The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties . In confirmation of its identity, (i) the Sam5p-GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM-requiring Bio2p) on fermentable carbon sources and a petite phenotype on non-fermentable substrates; and (iii) both phenotypes of the knock-out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.

Arch Pharm Res, 2003 Oct, 26(10), 805 - 8
Microbial metabolism of the environmental estrogen bisphenol A; Yim SH et al.; Preliminary microbial metabolism studies of bisphenol A (BPA) (1) on twenty six microorganisms have shown that Aspergillus fumigatus is capable of metabolizing BPA . Scale-up fermentation of 1 with A . fumigatus gave a metabolite (2) and its structure was established as bisphenol A-O-beta-D-glucopyranoside (BPAG) based on spectroscopic analyses.

Nahrung, 2003 Oct, 47(5), 339 - 44
Improvement of the digestibility of the proteins of the red alga Palmaria palmata by physical processes and fermentation; Marrion O et al.; Palmaria palmata (dulse) is an edible red alga constituting a potential protein source in human diet . However, previous studies showed that the digestibility of dulse proteins is bad because of the cell-wall encapsulating cytoplasmic proteins and the presence of fibers . The water-soluble xylan, present in high proportions in dulse, could be involved to explain the weak digestibility of proteins . To limit the influence of fibers and to improve the nutritional quality of these proteins, we have treated dulse by physical processes or by fermentation by moulds . After a 30 min predigestion by pepsin followed by a 6 h digestion into a cell dialysis containing porcine pancreatin, the corrected in vitro digestibility of crude dulse was very low (about 1.5% after correction by digestibility blank) . The in vitro protein digestibility was estimated to 58% of that of casein for dulse samples obtained after washing in demineralized water and grinding in liquid nitrogen . The in vitro protein digestibility of fermented samples was 45%-65% of that of casein . After physical treatment, the digestibility improvement was related to the elimination of soluble molecules such as xylan and mineral salts . The improvement observed after fermentations seemed due to the degradation of insoluble fibers.

Arch Microbiol, 2003 Dec, 180(6), 465 - 70 Epub 2003 Nov 08.
Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni; Richter H et al.; The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO(2)) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor . In this study, {U-(13)C}glucose, {U-(13)C}fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses . In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively . Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive ( K(i)=180 microM) and uncompetitive ( K'(i)=350 microM) inhibition by 6-phosphogluconate . Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively ( K(i)=1.3 microM) with a low contribution of uncompetitive inhibition ( K'(i)=13 microM) . The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 microM) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 microM) . We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.

J Nutr, 2003 Nov, 133(11), 3523 - 8
Acetylated, propionylated or butyrylated starches raise large bowel short-chain fatty acids preferentially when fed to rats; Annison G et al.; Maize starch was acylated with acetic, propionic or butyric anhydride to produce the corresponding acylated starch . In the first experiment, butyrylated starch at a degree of substitution (DS) of 0.25 (i.e., 1 acyl unit per 4 glucosyl units) was fed to rats for 3 d . Cecal and distal colonic SCFA concentrations were 170 and 78% higher, respectively, in rats fed the butyrylated starch . However, the greatest increase was in butyrate with corresponding increases of 460 and 212% . Subsequently, acetylated, propionylated or butyrylated starches with DS of approximately 0.18 were prepared on a larger scale . Body weight gain did not differ between rats fed these acylated starches or a control starch for 14 d . Large bowel pH was significantly lower and digesta mass significantly higher throughout the large bowel in rats fed the acylated starches . Cecal + distal colonic starch averaged 12 mg in rats fed the control starch and 103, 134 and 135 (pooled SEM = 6) mg in rats fed acetylated, propionylated or butyrylated starch, respectively . Large bowel SCFA concentrations and pools were significantly higher in rats fed the three acylated starches and were disproportionately greater in the SCFA that had been esterified to the starch . In the cecum, acetate, propionate and butyrate pools were 280, 690 and 1060% higher, respectively, in rats fed the corresponding acylated starch than in those fed the control diet . In the distal colon, the corresponding increases were 320, 940 and 1370% . These data indicate that acylated starches are resistant starch (RS) and raise large bowel SCFA, apparently through bacterial release of the esterified fatty acid and fermentation of the residual starch.

Bioresour Technol, 2004 Feb, 91(3), 273 - 81
Wet oxidation pretreatment for the increase in anaerobic biodegradability of newspaper waste; Fox M et al.; Wet oxidation was investigated for its process performance on methane fermentation of newspaper waste . The mechanisms of solubilization of newspaper waste were investigated using the following criteria: destruction of total COD (TCOD), production of soluble COD (SCOD), production of volatile fatty acids, production of soluble carbohydrates, production of soluble lignin derivatives (SLD), production of furan (F) and destruction of lignin and cellulose . Wet oxidation was carried out at 170, 190, and 210 degrees C, with a retention time of 1 h . The highest removal efficiencies of TCOD and cellulose were achieved at 210 degrees C, approximately 40% and 69% were destroyed, respectively . On the other hand, highest lignin removal efficiency was achieved at 190 degrees C in which approximately 65% was removed . Batch methane fermentation tests were performed in 2-l glass bottles filled with the wet oxidized newspaper samples . Methane fermentation of newspaper pretreated at 190 degrees C gave the highest CH(4) conversion efficiency (59% of the initial TCOD was recovered as CH(4) gas) . Anaerobic cellulose removals varied from 74% to 88%.

Bioresour Technol, 2004 Feb, 91(3), 259 - 62
High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue; Liming X et al.; Cellulase production using corn cob residue from xylose manufacture as substrate was carried out by Trichoderma reesei ZU-02 . It was found that on the same cellulose basis, the cellulase activity and yield produced on corn cob residue were comparable with that on purified cellulose . Under batch process, the optimum concentration of substrate was 40 g/l and the optimum C/N ratio was 8.0 . In 500 ml flasks, cellulase activity reached 5.25 IU/ml (213.4 IU/g cellulose) after seven days' cultivation . In a 30 m(3) stirred fermenter for large scale production, cellulase and cellobiase activity were 5.48 IU/ml (222.8 IU/g cellulase) and 0.25 IU/ml (10.2 IU/g cellulose), respectively, after four days' submerged fermentation . The produced cellulase could effectively hydrolyze the corn cob residue, and the yield of enzymatic hydrolysis reached 90.4% on 10% corn cob residue (w/v) when the cellulase dosage was 20 IU/g substrate.

J Org Chem, 2003 Nov 14, 68(23), 8902 - 5
Total synthesis of the depsipeptide FR-901375; Chen Y et al.; The first total synthesis of FR-901375, a novel bicyclic depsipeptide isolated from the fermentation broth of Pseudomonas chloroaphis No . 2522, has been achieved . The synthetic approach involves 13 reaction steps and is achieved in 12% overall yield . The key points in the successful synthetic strategy are a concise asymmetric synthesis of the key building block (3R,4E)-3-hydroxy-7-mercapto-4-heptenoic acid, a mild Mitsunobu macrolactonization step, and an I(2)-mediated deprotection with concomitant disulfide-bridge formation.

Appl Environ Microbiol, 2003 Nov, 69(11), 6650 - 8
Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae); Lemke T et al.; The soil macrofauna plays an important role in the carbon and nitrogen cycle of terrestrial ecosystems . In order to gain more insight into the role of the intestinal microbiota in transformation and mineralization of organic matter during gut passage, we characterized the physicochemical conditions, microbial activities, and community structure in the gut of our model organism, the humus-feeding larva of the cetoniid beetle Pachnoda ephippiata . Microsensor measurements revealed an extreme alkalinity in the midgut, with highest values (pH > 10) between the second and third crown of midgut ceca . Both midgut and hindgut were largely anoxic, but despite the high pH, the redox potential of the midgut content was surprisingly high even in the largest instar . However, reducing conditions prevailed in the hindgut paunch of all instars (E(h) approximately -100 mV) . Both gut compartments possessed a pronounced gut microbiota, with highest numbers in the hindgut, and microbial fermentation products were present in high concentrations . The stimulation of hindgut methanogenesis by exogenous electron donors, such as H(2), formate, and methanol, together with considerable concentrations of formate in midgut and hemolymph, suggests that midgut fermentations are coupled to methanogenesis in the hindgut by an intercompartmental transfer of reducing equivalents via the hemolymph . The results of a cultivation-based enumeration of the major metabolic groups in midgut and hindgut, which yielded high titers of lactogenic, propionigenic, and acetogenic bacteria, are in good agreement not only with the accumulation of microbial fermentation products in the respective compartments but also with the results of a cultivation-independent characterization of the bacterial communities reported in the companion paper (M . Egert, B . Wagner, T . Lemke, A . Brune, and M . W . Friedrich, Appl . Environ . Microbiol . 69:6659-6668, 2003).

Appl Environ Microbiol, 2003 Nov, 69(11), 6569 - 76
Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli; Overhage J et al.; The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp . strain HR199, respectively . Resting cells of the corresponding recombinant strain E . coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1) . This biotransformation was scaled up to a 30-liter fermentation volume . The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture . The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol . In a two-step biotransformation, E . coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E . coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J . Overhage, H . Priefert, and A . Steinbuchel, Appl . Environ . Microbiol . 65:4837-4847, 1999) . This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1) . The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp . strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E . coli XL1-Blue.

J Anim Sci, 2003 Nov, 81(11), 2675 - 85
Influence of steam-peeled potato-processing waste inclusion level in beef finishing diets: effects on digestion, feedlot performance, and meat quality; Radunz AE et al.; Inclusion of potato-processing waste (PW) from the frozen potato products industry in high-grain beef cattle finishing diets was evaluated in two studies . In a randomized complete block design, 125 crossbred yearling heifers (365 +/- 0.3 kg initial BW; five pens per treatment; five heifers per pen) were used to evaluate PW level on feedlot performance and meat quality . Heifers were fed for 85 (two blocks) or 104 d (three blocks) . In a digestion study, four ruminally, duodenally, and ileally cannulated Holstein steers (474.7 +/- 26.6 kg initial BW) were used in a 4 x 4 Latin square design to evaluate effects of PW level on ruminal fermentation, site of digestion, and microbial protein synthesis . The control diet for both studies contained 80% corn, 10% alfalfa hay, 5% concentrated separator by-product (CSB), and 5% supplement (DM basis) . Potato waste replaced corn and separator by-product (DM basis) in the diet at 0, 10, 20, 30, and 40% in the feedlot study, and at 0, 13, 27, and 40% in the digestion study . In the feedlot study, DMI decreased (linear; P = 0.007) with increasing inclusion of PW . Increasing PW decreased ADG and feed efficiency from 0 to 30% and then increased at 40% (quadratic; P < 0.01) . Calculated dietary NEg concentrations did not differ among treatments (P = 0.18) . Hot carcass weight decreased as PW increased from 0 to 30% and then increased at 40% PW (cubic; P < 0.01) . Fat thickness and longissimus muscle area decreased with increasing PW (linear; P < 0.05) . Level of PW did not affect marbling or liver scores (P > 0.30) . No difference (P > 0.20) was observed for Warner-Bratzler shear force at 0, 10, 20, and 30% PW levels; however, 40% PW resulted in lower (P = 0.05) shear force values . Taste panel scores for juiciness and flavor intensity did not differ with increasing PW (P > 0.30) . Steaks from cattle fed 0% were scored less tender than 10 and 40% PW (cubic; P < 0.05) . In the digestion study, DMI decreased (quadratic; P < 0.01) with increasing PW . Ruminal pH and total VFA concentration increased (linear; P < 0.05) and true N disappearance from the stomach complex and apparent total-tract N disappearance decreased with increasing level of PW (linear; P < 0.01) . Starch intake and ruminal disappearance decreased with increasing level of PW (quadratic; P < 0.05) . Inclusion of PW decreased feedlot performance, with little effect on carcass characteristics or meat quality . Optimal inclusion of PW in finishing diets may depend on the cost of transportation and other dietary ingredients.

Poult Sci, 2003 Oct, 82(10), 1608 - 15
In vitro fermentation characteristics of two mushroom species, an herb, and their polysaccharide fractions, using chicken cecal contents as inoculum; Guo FC et al.; In vitro fermentabilities of two mushrooms (Lentinus edodes--LenS; Tremella fuciformis--TreS), an herb (Astragalus membranaceus--AstS), and their polysaccharide fractions (LenE, TreE, and AstE) were investigated using microflora from chicken ceca . Polysaccharides were extracted using the hot water method . The mushrooms had lower polysaccharide yields (8 to 10%) than the herb (31%) . Fermentation kinetics were determined using the in vitro cumulative gas production technique . End-products, such as gas, volatile fatty acids (VFA), and ammonia, were also determined . The gas profiles of intact materials were similar for AstS and LenS . The TreS had a diphasic digestion pattern . The extracts had similar profiles to the intact materials though gas production rates were faster . Intact materials tended to produce less VFA than the extracts though LenS and AstE had the highest total VFA production overall . Intact materials contained more protein than the extracts, and therefore resulted in more branched-chain fatty acids and ammonia . Fermentation kinetics and end-point products demonstrated differences in availability of substrates between the mushrooms and herb . These medicinal mushroom and herb materials, particularly their polysaccharide extracts, show promise in altering microbial activities and composition in chicken ceca . In vivo experiments are necessary for confirmation of this hypothesis.

Poult Sci, 2003 Oct, 82(10), 1596 - 601
Impact of galactose, lactose, and Grobiotic-B70 on growth performance and energy utilization when fed to broiler chicks; Douglas MW et al.; Three chick assays were conducted to determine the effect of increasing dietary galactose (GAL), lactose (LAC), and Grobiotic-B70, a LAC fermentation product, on growth performance, toxicity, and energy utilization when fed to commercial broiler chicks . One-day-old male commercial broiler chicks were randomly assigned to treatments in each assay . In all assays, a 22% CP corn-soybean meal-dextrose basal diet containing a growth-promoting antibiotic (Bacitracin methylene disalicylate) was fed . In assay 1, GAL was added at 2, 4, 6, 10, or 15% . In assay 2, Grobiotic-B70 was added at 5% . In assay 3, GAL, LAC, and Grobiotic-B70 were each added at 2, 4, and 6% . All additions were made in place of dextrose, and diets were fed from 0 to 20 or 21 d of age . In assay 1, the 15% GAL treatment resulted in high mortality (27%) by d 3 and was terminated . The 10% GAL treatment also resulted in increased mortality, most of which occurred during the 7-to-14-d period . Inclusion of 2 and 4% GAL resulted in an improvement (P < 0.05) in growth compared to the basal diet . Inclusion of 2, 4, 6, and 10% GAL resulted in a significant linear decrease in MEn (r2 = 0.85) . In chick assay 2, 5% Grobiotic-B70 increased growth during the first 2 wk . In chick assay 3, 6% Grobiotic increased weight gain (P < 0.05) from 0 to 14 d, and addition of 2 or 4% GAL, 2 or 4% LAC, and 4 or 6% Grobiotic-B70 increased weight gain (P < 0.08) from 0 to 21 d . Our results indicate that levels of 10 to 15% GAL are toxic . In contrast, low levels of GAL and Grobiotic-B70, and possibly LAC, may increase growth of commercial broiler chicks.

J Biol Chem, 2004 Feb 6, 279(6), 3956 - 79 Epub 2003 Nov 03.
The yeast mitochondrial proteome, a study of fermentative and respiratory growth; Ohlmeier S et al.; Saccharomyces cerevisiae is able to switch from fermentation to respiration (diauxic shift) with major changes in metabolic activity . This phenomenon has been previously studied on the transcriptional level . Here we present a parallel analysis of the yeast mitochondrial proteome and the corresponding transcriptional activity in cells grown on glucose (fermentation) and glycerol (respiration) . A two-dimensional reference gel for this organelle proteome was established (available at which contains about 800 intense spots . From 459 spots 253 individual proteins were identified, among them low abundant and hydrophobic proteins, and 37 proteins previously deemed hypothetical, with partially unknown cellular localization . After the diauxic shift, mitochondrial levels of only 18 proteins were changed (17 increased, with 1 decreased), among them proteins involved in the tricarboxylic acid cycle (Sdh1p, Sdh2p, and Sdh4p) and the respiratory chain (Cox4p, Cyb2p, and Qcr7p), proteins contributing to other respiratory pathways (Ach1p, Adh2p, Ald4p, Cat2p, Icl2p, and Pdh1p), and two proteins with unknown function (Om45p and Ybr230p) . Apart from an overall increase in mitochondrial protein mass, the mitochondrial proteome remains remarkably constant, even in a major metabolic adaptation . This seemingly disagrees with results of the DNA microarray analyses, where a rather heterogenous up- or down-regulation of genes encoding mitochondrial proteins implies large changes in the proteome . We propose that the discrepancy between proteome and transcriptional regulation, apart from different translation efficiency, indicates a changed turnover rate of proteins in different physiological conditions.

Biotechnol Bioeng, 2003 Dec 20, 84(6), 723 - 31
Genetically engineered binding proteins as biosensors for fermentation and cell culture; Ge X et al.; The signal-transduction properties and the potential applications of two engineered binding proteins from E . coli were extensively studied . Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP) . Both proteins respond to their ligands in the micromolar range . The proteins can be stored at 4 degrees C for at least 5 months . Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges . The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours . Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes . The potential applications of GBP were studied in yeast fermentation and E . coli fermentations in three different scales: 150 mL, 5 mL, and 100 microL . The results were compared with an YSI 2700 Chemistry Analyzer . Although the latter could not give reliable results for the E . coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation . Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 microL) and the results were also compared with those obtained with YSI . Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 microL) prevented it from being used in the 100-microL cell culture . Because of their small sample volume requirements (less than 1 microL) and high sensitivity, the assays described here might find wide applications in high-throughput bioprocessing .

Biotechnol Bioeng, 2003 Dec 20, 84(6), 639 - 46
Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera; Eksteen JM et al.; Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source . Eight constructs containing the L . kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S . fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared . The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators . The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators . These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b) . Southern-blot analysis confirmed the stable integration of the different gene constructs into the S . cerevisiae genome and plate assays revealed amylolytic activity . The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media . This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation . The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity . It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S . cerevisiae . This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S . cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries .

Biotechnol Bioeng, 2003 Dec 20, 84(6), 619 - 26
Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora; Hussy I et al.; For the transition to the hydrogen economy, hydrogen must be produced sustainably, e.g., by the fermentation of agricultural material . Continuous fermentative production of hydrogen from an insoluble substrate in nonsterile conditions is yet to be reported . In this study hydrogen production using mixed microflora from heat-treated digested sewage sludge in nonsterile conditions from a particulate co-product of the wheat flour industry (7.5 g L(-1) total hexose) at 18- and 12-hour hydraulic retention times, pH 4.5 and 5.2, 30 degrees C and 35 degrees C was examined . In continuous operation, hydrogen yields of approximately 1.3 moles hydrogen/mole hexose consumed were obtained, but decreased if acetate or propionate levels rose, indicating metabolism shifted towards hydrogen consumption by homoacetogenesis or propionate producers . These shifts occurred both at pH 4.5 and 5.2 . Sparging the reactor with nitrogen to reduce hydrogen in the off-gas from 50% to 7% gave stable operation with a hydrogen yield of 1.9 moles hydrogen /mole hexose consumed over an 18-day period .

Acta Microbiol Pol, 2003, 52(2), 149 - 58
Phenotypic and molecular characteristics of typical and atypical Escherichia coli O157, clinical and food isolates; Sadowska B et al.; Enrichment, colony isolation and confirmation are three general phases of a standard diagnostic method . E . coli O 157 (the main member of EHEC group) differs metabolically from other strains of E . coli in a number of ways . Most isolates are slow- or non-fermenters of sorbitol and lack the enzyme beta-glucuronidase (GUD) . But, a variety of atypical strains of E . coli O157 (sorbitol-fermenting variants, nonmotile and GUD-positive) have been reported . The discovery of these atypical pathogenic strains brings into question the validity of testing for the pathogen only by biotyping . Using classical cultivation and immunomagnetic separation, we have isolated from food a few atypical E . coli O157 (sorbitol-fermenting strains, GUD positive, nonmotile O157 strain which does not agglutinate with O157 latex and does not produce Shiga toxin) . On the other hand, non-O157 VTEC (O26 serotype) producing Shiga toxin was isolated from meat . Molecular markers of E . coli O157 and virulence-associated factors of strains with aberrant biochemical properties were studied by PCR . This method helped us in the final identification of isolates . Since it was suggested that the production of verotoxins (VT) is accompanied by the production of enterohemolysin (Ehly) such correlation has also been evaluated in respect to the collection of VTEC of human, animal and food origin.

J Dairy Sci, 2003 Oct, 86(10), 3343 - 53
The effect of corn silage particle size on eating behavior, chewing activities, and rumen fermentation in lactating dairy cows; Kononoff PJ et al.; The objective of this experiment was to evaluate effects of reducing corn silage particle size on eating behavior, chewing activity, and rumen fermentation in lactating dairy cows . Four cannulated, multiparous cows averaging 110 +/- 4 d in milk and weighing 675 +/- 70 kg were randomly assigned to a 4 x 4 Latin square . During each of four 14-d periods, animals were offered one of four diets that were chemically similar but varied in corn silage particle size: short (SH), mostly short (MSH), mostly long (MLG), and long (LG), with a geometric mean particle length of 7.4, 7.8, 8.3, and 8.8 mm, respectively . Reducing particle size increased dry matter intake (DMI) linearly (28.0, 26.8, 26.8, and 25.7 kg/d for SH, MSH, MLG, and LG respectively) . At 8, 16, and 24 h postfeeding, the neutral detergent fiber (NDF) concentration of feed remaining in the bunk decreased linearly with reduced particle size . Time spent eating or ruminating was not different across treatments; however, total chewing activity (TC; sum of time spent eating and ruminating) exhibited a quadratic response with highest chewing activities observed for diets with shortest and longest particle size . Eating or ruminating time per kilogram of DMI was not affected by corn silage particle size, but TC per kilogram of DMI decreased linearly with decreasing particle size . In comparison, when expressed as minutes per unit of NDF intake (NDFI), ruminating, and TC were linearly reduced as particle size decreased . Rumen pH was not affected by corn silage particle size even though total concentration of volatile fatty acids increased linearly from 89.1 mM/L to 93.6 mM/L as diet particle size decreased . A quadratic effect was observed in molar proportion of acetate and propionate with the highest concentration observed in animals consuming diets of intermediate particle size . Results of this experiment suggest that reducing corn silage particle size may increase DMI, positively affect rumen fermentation, and reduce sorting behavior . Because both chewing activity and sorting tendencies increased when proportion of TMR particles > 19.0 mm increased, results suggest that particle size measurement as estimated by the PSPS is useful in understanding some factors that affect feeding behavior.

J Dairy Sci, 2003 Oct, 86(10), 3337 - 42
In situ evaluation of hen mortality meal as a protein supplement for dairy cows; Kim WK et al.; A study was conducted to evaluate the nutritional composition and in situ degradation of hen mortality meals . There were four treatments: control autoclaved hen meal (C-HM), enzyme-treated, fermented, autoclaved hen meal (E-HM), NaOH-treated, fermented, autoclaved hen meal (NaOH-HM), and soybean meal (SBM) . For the E-HM or NaOH-HM, hen mortality was treated with a feather digesting enzyme or NaOH to improve digestibility of feathers on the carcass . After the enzyme or NaOH treatment, treated hen mortality was preserved by a fermentation procedure . The crude protein levels of the C-HM and SBM were higher than the E-HM and NaOH-HM, and the concentration of fat in the C-HM was higher than the other treatments . Levels of Lys, Thr, Arg, Ile, Leu, Val, and Phe for the C-HM and SBM were higher than in the E-HM and NaOH-HM . The Met, Cys, and Gly levels in the C-HM were higher than the soybean meal . In situ ruminal degradation data showed that the C-HM had lower dry matter and crude protein degradation than the other treatments, whereas the E-HM or NaOH-HM was more susceptible to ruminal degradation . These results indicate that the C-HM has higher levels of crude protein, amino acids, and resistance to ruminal degradation, whereas the E-HM or NaOH-HM was more digestible to ruminal microorganisms.

J Dairy Sci, 2003 Oct, 86(10), 3330 - 6
Effect of sarsaponin on ruminal fermentation with particular reference to methane production in vitro; Lila ZA et al.; This experiment was designed to investigate the effects of different concentrations (0, 1.2, 1.8, 2.4, and 3.2 g/L) of sarsaponin on ruminal microbial methane production using the substrates soluble potato starch, cornstarch, or hay plus concentrate (1.5:1) . Ruminal fluid was collected from a dairy cow, mixed with phosphate buffer (1:2) and incubated (30 ml) anaerobically at 38 degrees C for 6 and 24 h with or without sarsaponin . Excluding the lower level of sarsaponin, pH of the medium was slightly decreased . Ammonia-N concentration and numbers of protozoa were decreased in a dose-dependent manner . Total volatile fatty acids and total gas production were increased . Molar proportion of acetate was decreased and propionate was increased with a corresponding decrease in acetate:propionate ratio . Hydrogen production was decreased . As the concentration of sarsaponin increased from 1.2 to 3.2 g/L, fermentation of soluble potato starch, cornstarch, or hay plus concentrate decreased methane production from 20 to 60% (6 h) and 17 to 50% (24 h), 21 to 58% (6 h) and 18 to 52% (24 h), and 23 to 53% (6 h) and 15 to 44% (24 h), respectively . Excluding the lower dose concentration (1.2 g/L) of sarsaponin, in vitro disappearance of dry matter of hay plus concentrate was decreased after 24 h . In conclusion, these results show that sarsaponin stimulated the mixed ruminal microorganism fermentation as well as to inhibit methane production in vitro.

J Dairy Sci, 2003 Oct, 86(10), 3260 - 70
Effects of casein and glucose on responses of cows fed diets based on restrictively fermented grass silage; Vanhatalo A et al.; This study was conducted to investigate whether well fed dairy cows given restrictively fermented grass silage diet will respond to incremental glucose and amino acid supply at early stage of lactation . Four rumen-cannulated Finnish Ayrshire cows were used in a 4 x 4 Latin square experiment with 14-d periods . The cows were fed good quality restrictively fermented grass silage ensiled with a formic acid additive for ad libitum intake . A concentrate mixture consisting of barley (85%) and solvent extracted rapeseed meal (11.4%) was given at a rate of 9 kg/d . The four treatments were continuous abomasal infusions of water (control), casein 300 g/d, glucose 300 g/d, and casein 300 g/d + glucose 300 g/d . The infusions had only minor effects on feed intake, diet digestibility, or rumen fermentation pattern . Both casein and glucose infusions increased milk, protein and lactose yields the effects being partly additive on the combined infusion . Infused casein increased milk protein and urea as well as plasma urea concentrations . Both casein and glucose tended to increase plasma glucose concentration . Casein increased arterial plasma concentrations of essential amino acids (EAA), branched-chain AA (BCAA), and total AA (TAA) . Both casein and glucose, although glucose usually less than casein, increased arteriovenous differences of EAA, nonessential AA, BCAA, and TAA . Extraction efficiencies of AA were higher for glucose than for casein . Mammary plasma flow was at highest on the control diet, but reduced owing to infused nutrients, the reduction being less with combined rather than separate infusions of casein and glucose . Based on the partly additive increases in milk production parameters and changes in plasma metabolites, it is suggested that glucose alone increased milk protein yield by sparing AA from hepatic utilization, while casein increased both supply of AA and glucose . It was concluded that cows at early stage of lactation fed diets comprising of restrictively fermented grass silage and a cereal-based concentrate suffer from both limited AA and glucose supplies.

J Dairy Sci, 2003 Oct, 86(10), 3249 - 59
Effects of various glucogenic sources on production and metabolic responses of dairy cows fed grass silage-based diets; Vanhatalo A et al.; Four rumen cannulated Finnish Ayrshire cows in midlactation were used in an experiment designed as a 4 x 5 incomplete Latin square with 2-wk periods to compare effects of glucogenic substrates on grass silage-based diets . The five treatments were continuous infusions of 1) water (control), 2) casein 300 g/d, 3) glucose 300 g/d, 4) propionic acid 247 g/d, and 5) barley starch 270 g/d . Substrates were infused either into the rumen (propionic acid) or into the abomasum (other substrates) . As a basal diet, cows were fed a formic acid treated grass silage ad libitum (digestible organic matter 690 g/kg dry matter {DM}, crude protein {CP} 131 g/kg DM) and a barley-rapeseed concentrate (CP 141g/kg DM) at a rate of 7 kg/d . Production responses to glucogenic substrates other than casein were negligible, suggesting that glucose supply of the cows did not primarily limit milk production . However, with casein cows produced significantly more milk, milk protein, and lactose than with other glucogenic substrates . Casein increased urea and essential amino acid (EAA), and decreased nonessential AA (NEAA) in arterial plasma compared with other substrates, suggesting that casein provided precursors both in terms of NEAA for gluconeogenesis and EAA for milk protein synthesis . This puts forward that providing the AA needs of the mammary gland for milk protein synthesis are met, glucose supply may become the next limiting factor for milk protein synthesis in cows fed diets based on restrictively fermented grass silage . The limited supply of AA from the basal diet, and possibly the low production levels of cows partly invalidated the hypothesis of monitoring differing glucogenic substrates for grass silage-based diets.

Prikl Biokhim Mikrobiol, 2003 Sep-Oct, 39(5), 501 - 8
{Physiological and biochemical characteristics of immobilized champagne yeasts and their participation in sparkling processes.}; Martynenko NN et al.; Methods for immobilizing champagne yeasts, physiological and biochemical characteristics of the immobilized cells, and problems of their utilization in the production of quality champagne wines are reviewed . Studies aimed at the development of efficient biocatalysts for champaignizing wines using bottle fermentation (method champenoise) and tank processing (bulk, or Charmat process), based on the use of immobilized yeast cells, are described . Data on the industrial use of such biocatalysts in countries manufacturing champagne wines are presented . Problems and prospects of further research in this field are discussed.

Bioresour Technol, 2004 Jan, 91(2), 179 - 88
A bioethanol process development unit: initial operating experiences and results with a corn fiber feedstock; Schell DJ et al.; Interest in bioethanol production from lignocellulosic feedstocks for use as an alternative fuel is increasing, but near-term commercialization will require a low cost feedstock . One such feedstock, corn fiber, was tested in the US Department of Energy (DOE)/National Renewable Energy Laboratory (NREL) bioethanol pilot plant for the purpose of testing integrated equipment operation and generating performance data . During initial runs in 1995, the plant was operated for two runs lasting 10 and 15 days each and utilized unit operations for feedstock handling, pretreatment by dilute sulfuric-acid hydrolysis, yeast inoculum production, and simultaneous saccharification and fermentation using a commercially available cellulase enzyme . Although significant operational problems were encountered, as would be expected with the startup of any new plant, operating experience was gained and preliminary data were generated on corn fiber pretreatment and subsequent fermentation of the pretreated material . Bacterial contamination was a significant problem during these fermentations.

Bioresour Technol, 2004 Jan, 91(2), 153 - 6
Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass; Kang SW et al.; To investigate the production of cellulases and hemicellulases from Aspergillus niger KK2, solid state fermentation (SSF) was performed by using different ratios of rice straw and wheat bran . When A . niger KK2 was grown on rice straw alone as a solid support in SSF, the maximum FPase activity was 19.5 IU g(-1) in 4 days . Also, CMCase (129 IU g(-1)), beta-glucosidase (100 IU g(-1)), xylanase (5070 IU g(-1)) and beta-xylosidase (193 IU g(-1)) activities were concurrently obtained after 5-6 days of fermentation . The higher enzyme activities produced by A . niger KK2 is a significant advantage from the viewpoint of practical saccharification reaction . Cellulases and hemicellulases produced by A . niger KK2 might be applied to pulp and paper industry, feed industry and chemical industry.

Bioresour Technol, 2004 Jan, 91(2), 111 - 5
Citrus waste recovery: a new environmentally friendly procedure to obtain animal feed; Tripodo MM et al.; Citrus juice centrifugation pulp is the semi-solid product obtained from the industrial centrifugation of juices, to obtain a clear juice . This waste causes many economic and environmental problems because of its fermentability . In this paper we describe a method which makes it possible to obtain animal feed from citrus juice centrifugation pulp . To this end, alkaline and/or enzymatic treatments were carried out on the centrifugation pulp . These treatments facilitate pressing and so help to produce a material which, using suitable methods, may be dried . Enzyme treatment proved to be the most efficient of the methods under investigation designed to favour the pressing of the pulp . The product obtained with this method showed excellent digestibility in vitro and its protein content, although not especially high, compared favourably with that of many other agroindustrial waste products currently used as components of animal feed.

Rev Argent Microbiol, 2003 Jul-Sep, 35(3), 128 - 32
{Rennet production by Rhizomucor miehei NRRL 3169}; Mariani DD et al.; The production of rennet was studied, using different strains of the fungus Rhizomucor miehei . The selection and preservation of strains, type of growth, media design and operation conditions were evaluated . The experiments were carried out in Erlenmeyer flasks in rotary shaker at 250 rpm and 2.5 eccentricity, and in mechanically stirred fermentors of the New Brunswick type, at 30 degrees C . In the studies concerning strain selection, the best strain was Rhizomucor miehei NRRL 3169 . The major titles of enzyme were obtained in batch process at 168 h, with 884 SU/ml, whereas in mechanically stirred fermentors the best value was 1160 SU/ml . These values were far more superior to former ones published by various experts.

Mar Biotechnol (NY), 2004 Jan-Feb, 6(1), 95 - 103 Epub 2003 Nov 05.
Exocellular cyclic dipeptides from a Ruegeria strain associated with cell cultures of Suberites domuncula; Mitova M et al.; From cell cultures of Suberites domuncula was isolated a bacterial strain, SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an alpha-Proteobacterium of the genus Ruegeria . The occurrence of the strain in sponge cell culture could be explained by its resistance to the antibiotics used in the isolation of sponge cell cultures or by the preservation of SDC-1 by host sponge cells . The fatty acid composition of SDC-1 is characterized by branched C-12 methyl fatty acids . Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1 . Cyclodipeptides are one of the families of cell-cell signaling compounds and may have some role to play in sponge-bacteria interactions.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2124 - 31
3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway; Adachi O et al.; 3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time . In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation . Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase . Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation . 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells . Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other . Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.

Bioresour Technol, 2004 Jan, 91(1), 105 - 9
Reusing soy residue for the solid-state fermentation of Ganoderma lucidum; Hsieh C et al.; Lingzhi has been a popular oriental medicine used to treat various human diseases . Soy residue from the waste of tofu manufacturing was used to culture Ganoderma lucidum in solid-state fermentation . The solid-state fermentation was conducted in three types of containers: test tube, 500-ml flask, and sterilize-able polypropylene plastic bag . The highest rate of mycelial growth of 6 mm/day was observed in the medium of carbon/nitrogen (C/N) ratio of 80 using test tubes . However, a growth rate of 7.5 mm/day was found at the C/N ratio of 70-80 in the 500-ml flasks . In the tests using plastic bags, the fruiting bodies were fully developed only for the C/N ratios of 70 and 80 . The components of fruiting bodies obtained from different media were also analyzed and compared . The contents of ash, polysaccharides, and crude protein of fruiting bodies were found higher in the media of C/N ratio of 80.

Bioresour Technol, 2004 Jan, 91(1), 41 - 51
Mixed acid fermentation of paper fines and industrial biosludge; Domke SB et al.; This paper uses countercurrent fermentation to anaerobically convert paper fines and industrial biosludge to carboxylate salts using a mixed culture of acid-forming microorganisms . Using the MixAlco process, the carboxylate salts can be thermally converted to ketones and hydrogenated into mixed alcohol fuels . Continuum particle distribution modeling (CPDM) correlated batch fermentation data to countercurrent fermentation data, allowing the prediction of product concentrations and conversions over a wide range of solid loading rates and liquid residence times . For 80% paper/20% biosludge, the predicted product concentrations agreed with the data within 7.7% . The predicted conversion agreed with the actual conversion within 27.8% . By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.2% . For 40% paper/60% biosludge, the predicted product concentrations agreed with the data within 9.6% . The predicted conversion agreed with the actual conversion within 28.3% . By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.4% . For both the 80/20 and 40/60 cases, CPDM predicts that 90% conversion is possible with a 20 g/l product concentration, 300 g/l substrate concentration, 16 day liquid residence time, and 2.5 g/(ld) solids loading rate . Before proceeding to an industrial plant, these predictions must be verified in a pilot plant.

Bioresour Technol, 2004 Jan, 91(1), 31 - 9
Hydrolysis of animal manure lignocellulosics for reducing sugar production; Wen Z et al.; Converting animal manure into value-added products provides a potential alternative for treatment and disposal of such materials . Lignocellulosics are a major component of animal manure and represent an undeveloped bioresource . In this work, a process was developed for hydrolyzing manure lignocellulosics into fermentable sugars . When raw dairy manure was pre-treated with 3% sulfuric acid at 110 degrees C for 1 h, hemicellulose was completely degraded into mainly arabinose, galactose and xylose . The pretreated materials were then treated with cellulolytic enzymes, Celluclast-1.5L and Novozyme-188, to hydrolyze the cellulose . The optimal enzyme loadings were identified as 13 FPU cellulase/g substrate and 5 IU beta-glucosidase/g substrate . The optimal temperature and pH were determined to be 46 degrees C and 4.8, respectively . A substrate concentration of 50 g/l favored both glucose concentration (in hydrolysate) and glucose yield (based on per 100 g manure) . It was also found that a reduced particle size of 590-mum resulted in a high glucose yield with further decreases in particle size not increasing the yield . For each particle size investigated, the addition of 2% tween-80 resulted in at least 20% improvement in glucose yield . The optimized hydrolysis process achieved a glucose yield of 11.32 g/100 g manure, which corresponded to about 40% cellulose conversion.

J Med Food, 2003 Fall, 6(3), 151 - 6
Kimchi and an active component, beta-sitosterol, reduce oncogenic H-Ras(v12)-induced DNA synthesis; Park KY et al.; The Korean fermented vegetable food, kimchi, has been demonstrated to have anticancer functional properties . This study examined the effect of kimchi samples, methanol extracts of commercially grown baechu cabbage kimchi (CK) and organically grown baechu cabbage kimchi (OK), as well as the dichloromethane fraction (DCM fr.) from CK, and the active compound (AC), which has been identified as largely beta-sitosterol, from DCM fr., on the Ras-dependent signaling pathway . CK, OK, and DCM fr . exhibited a greater inhibition against the proliferation of Rat2 fibroblasts transformed with Ras(v12) (HO6) than parental Rat2 fibroblasts . In addition, OK and DCM fr . showed a higher inhibitory effect than CK . Furthermore, we employed the single-cell microinjection technique, combined with 3-bromo-5'-deoxyuridine incorporation, to examine the effects of kimchi samples on DNA synthesis induced by microinjected oncogenic Ras(v12) . When the DCM fr . and AC were used to treat Rat1 fibroblasts overexpressing human insulin receptors (HIRc-B) and microinjected with oncogenic H-Ras(v12), the DNA synthesis of injected cells was decreased, suggesting that kimchi might block the signaling pathway of oncogenic Ras(v12), thus preventing the proliferation of transformed cells . This study provides additional evidence that kimchi and its active components, including beta-sitosterol, have potential in both the prevention and treatment of cancer, and presents convincing evidence that the anticancer effects may be a result of an inhibition of Ras oncogene signaling.

Biotechnol Lett, 2003 Oct, 25(19), 1677 - 83
Morphological quantification of pellets in Streptomyces hygroscopicus var . geldanus fermentation broths using a flatbed scanner; O'Cleirigh C et al.; An image analysis technique has been developed to allow high throughput morphological characterisation of microbial fermentation broths containing spherical pellets greater than 100 microm in diameter . Images of stained Streptomyces hygroscopicus var . geldanus culture samples at three different inoculum levels were captured using a flatbed scanner, at a resolution of 21 microm per pixel (1200 dots per inch) and subsequently analysed leading to the generation of a morphological profile of each sample . The time taken for image capture and analysis of a prepared sample, containing approx . 2000 particles, was 3 min 6 s.

Biochem J, 2004 Feb 1, 377(Pt 3), 693 - 700
Structure and function of human Vps20 and Snf7 proteins; Peck JW et al.; Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function . In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans . Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant . In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles . Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting . To explore possible protein-protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function . Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 {ALG-2 (apoptosis-linked gene 2) interacting protein 1}, a mammalian Bro1p {BCK1 (bypass of C kinase)-like resistance to osmotic shock}-containing protein involved in cellular vacuolization and apoptosis . Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2 . Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1 . Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures . Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.

J Agric Food Chem, 2003 Nov 5, 51(23), 6906 - 10
Prevention of patulin toxicity on rumen microbial fermentation by SH-containing reducing agents; Morgavi DP et al.; Patulin, a toxic fungal metabolite, negatively affects rumen fermentation . This mycotoxin has also been associated with intoxication cases in cattle . This study investigates the use of SH-containing reducing compounds to prevent patulin's negative effects on the rumen microbial ecosystem . The effect of 50 microg/mL patulin on the fermentation of alfalfa hay was measured in batch cultures with and without reducing agents . Sulfhydryl-containing cysteine and glutathione prevented the negative effects of the toxin on dry matter degradation, gas, and volatile fatty acid production (P < 0.01) . However, non-sulfhydryl-containing ascorbic and ferulic acids did not protect against patulin's toxicity (P > 0.01) . Patulin was unstable in buffered rumen fluid as the concentration decreased by half after 4 h of incubation . In the presence of sulfhydryl groups, the toxin disappeared rapidly and was not detected after 1 h of incubation . The utilization of sulfhydryl-containing compounds such as cysteine to avert patulin toxicity could have practical implications in ruminant nutrition.

J Agric Food Chem, 2003 Nov 5, 51(23), 6782 - 90
Evolution of chemical and sensory properties during aging of top-fermented beer; Vanderhaegen B et al.; The aging and consequent changes in flavor molecules of a top-fermented beer were studied . Different aging conditions were imposed on freshly bottled beer . After 6 months of aging, the concentration changes were recorded for acetate esters, ethyl esters, carbonyls, Maillard compounds, dioxolanes, and furanic ethers . For some flavor compounds, the changes with time of storage were monitored at different temperatures, either with CO(2) or with air in the headspace of the bottles . For some molecules a relationship was determined between concentration changes and sensory evaluation results . A decrease in volatile esters was responsible for a reduced fruity flavor during aging . On the contrary, various carbonyl compounds, some ethyl esters, Maillard compounds, dioxolanes, and furanic ethers showed a marked increase, due to oxidative and nonoxidative reactions . A very high increase was found for furfural, 2-furanmethanol, and especially the furanic ether, 2-furfuryl ethyl ether (FEE) . For FEE a flavor threshold in beer of 6 mug/L was determined . In the aged top-fermented beer, FEE concentrations multiple times the flavor threshold were observed . This was associated with the appearance of a typical solvent-like flavor . As the FEE concentration increased with time at an almost constant rate, with or without air in the headspace, FEE (and probably other furanic ethers) is proposed as a good candidate to evaluate the thermal stress imposed on beer.

J Agric Food Chem, 2003 Nov 5, 51(23), 6761 - 6
GC-ITMS determination and degradation of captan during winemaking; Angioni A et al.; Captan and its metabolite tetrahydrophthalimide (THPI) were determined in grapes, must, and wine by GC-ITMS . Pesticides were extracted with acetone/petroleum ether (50:50 v/v) . Because of the high selectivity of the ITMS detector, no interferent was found and cleanup was not necessary . Recoveries from fortified grapes, must, and wines ranged between 90 and 113% with a maximum coefficient of variation of 11% . Limits of quantitation were 0.01 mg/kg for both compounds . In model systems, captan and its metabolites, THPI, cis-4-cyclohexene-1,2-dicarboxylic acid, and 1,2,3,6-tetrahydrophthalamic acid, were determined by HPLC . The degradation of captan during winemaking was studied . Captan degraded in must, giving 100% THPI, and at the end of fermentation, only THPI was found in wine . The degradation of captan to THPI was due to the acidity in must and wine . This metabolite was present at low levels on grapes, and, unlike captan, it had no negative effect on the fermentative process . Model systems showed that the mechanism of disappearance of captan in grapes was due to photodegradation and codistillation.

Z Naturforsch {C}, 2003 Sep-Oct, 58(9-10), 708 - 12
Influence of carbon, nitrogen and phosphorous sources on glucoamylase production by Aspergillus awamori in solid state fermentation; Bertolin TE et al.; It was the objective of the present study to increase the production of glucoamylase by Aspergillus awamori through solid state fermentation, using wheat bran as the main carbon source and (NH4)2SO4, urea, KH2PO4, glucose, maltose and starch as additional nitrogen, phosphorus, and carbon sources . The production of glucoamylase is strongly influenced by N and C sources . A 100% increase was observed when the (NH4)2SO4 was replaced by urea, with C/N = 4.8, using maltose as the additional carbon source . C/P ratios in a range of 5.1 to 28.7 did not induce glucoamylase production under the studied conditions.

Extremophiles . 2003 Oct 24; {Epub ahead of print}
Production of C(35) isoprenoids depends on H(2) availability during cultivation of the hyperthermophile Methanococcus jannaschii; Manquin BP et al.; A series of five progressively saturated C(35) isoprenoids has been identified in cell-free extracts of the deep-sea methanogen Methanococcus jannaschii . Production and relative abundance of the isoprenoids were dependent on culture conditions; significant production occurred in a 16-l fermentor (12-l working volume) and a 2.5-l fermentor (2-l working volume) but could not be duplicated in serum bottles . Several factors were investigated and shown not to account for the different production levels, including medium composition, pH, and temperature . However, the interphase mass transfer rate was shown to significantly affect the production of C(35) isoprenoids in a fermentor . The structures of the novel isoprenoids were confirmed by hydrogenation reactions and mass spectra of the isoprenoids . Indirect evidence based on genomics and mass spectrometry data implicates head-to-head condensation of farnesyl pyrophosphate (C(15)) with geranylgeranyl pyrophosphate (C(20)) as the mechanism for C(35 )synthesis.

Bioresour Technol, 2003 Dec, 90(3), 265 - 73
De-icers derived from corn steep water; Yang BY et al.; Corn steep water (CSW) and other byproducts derived from fermentations and sugar productions are presently forming the base of compositions for de-icing and anti-icing materials . Since the de-icing and anti-icing values are in part a colligative property, increase in the molar concentration of ionic species has been frequently necessary to decrease further the freezing point of this byproducts stream . In the present study this has been achieved by the generation of biodegradable organic acid salts in situ, without the use of chloride or other inorganic salts, by the alkaline degradation of reducing sugars added to corn steep water, which alone is not an efficient de-icer . Reducing sugars, such as glucose, react with alkali metal hydroxides to produce principally hydroxy carboxylic acids that react with the alkali metal hydroxide to form a mixture of organic acid salts . The ionic strength of the resulting solution is increased since each sugar molecule produces nearly two acid molecules upon degradation . The ionic strength necessary to achieve the desired freezing point depression is determined by the amount and concentration of the alkali metal hydroxide used, with the necessary counter anions being derived from the degradation of the reducing sugar . The amount of the sugar used is that required to result in a near to neutral final solution . The well-known anti-corrosive property of CSW is used in the de-icer preparations, either by conducting the alkaline degradation of the sugar in this medium, or by using water for the degradation of the sugar followed by dilution of the resulting solution with CSW to adjust the viscosity of the final solution to meet the requirements for spraying . The monovalent metal hydroxides are more efficient in producing de-icer solutions than the divalent metal hydroxides.

J Nat Prod, 2003 Oct, 66(10), 1373 - 7
Komodoquinone A, a novel neuritogenic anthracycline, from marine Streptomyces sp . KS3; Itoh T et al.; A novel anthracycline, komodoquinone A (1), and its aglycone, komodoquinone B (2), were isolated from the solid-state fermentation of the marine Streptomyces sp . KS3, which was isolated from marine sediment . The absolute stereostructures of 1 and 2, except for the sugar portion, were elucidated on the basis of chemical and physicochemical evidence . Komodoquinone A (1) is a unique anthracycline, in which a new amino sugar is connected to the D-ring of the anthracyclinone skeleton, and was found to induce neuronal cell differentiation in the neuroblastoma cell line, Neuro 2A.

J Nat Prod, 2003 Oct, 66(10), 1313 - 7
Isolation and characterization of new epothilone analogues from recombinant Myxococcus xanthus fermentations; Starks CM et al.; Nine new epothilone analogues (6-8, 10, 11a, 11b, 12, 13, and 15) were isolated from fermentations of Myxococcus xanthus strains engineered with modified polyketide synthase genes . The epothilone structures were elucidated primarily through interpretation of 1D and 2D NMR data . 4-Desmethyl-10,11-didehydroepothilone D (6) displayed activity against several tumor cell lines, including a multi-drug-resistant cell line.

Biotechnol Bioeng, 2003 Nov 20, 84(4), 494 - 503
Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers; Kumar A et al.; Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions . The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems . In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML . The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol% . When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E . coli cells . Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer . His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively . The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer . The SDS-PAGE pattern showed significant purification of the antibody fragments .

Biotechnol Bioeng, 2003 Nov 20, 84(4), 474 - 84
Further studies related to the scale-up of high cell density Escherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when using aqueous ammonia to control pH; Onyeaka H et al.; In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model . For the first time, the effect on high cell density Escherichia coli fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously . To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level . It was demonstrated that for E . coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case . However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained . The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7 .

Biotechnol Bioeng, 2003 Nov 20, 84(4), 459 - 66
Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat; Chao YP et al.; To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat . The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity . By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock . With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein . In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C . By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process . Consequently, this resulted in Pck production equivalent to 15% of total cell protein . The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale . Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins .

J Food Prot, 2003 Oct, 66(10), 1857 - 63
Infectivity of Trichinella nativa in traditional northern (country) foods prepared with meat from experimentally infected seals; Forbes LB et al.; The infectivity of Trichinella nativa larvae in three traditional northern (country) foods was assessed . Foods were prepared with meat from seals experimentally infected with Trichinella nativa and evaluated over a 317-day period during which this food was fed directly to cats while mice were orally inoculated with larvae recovered following the digestion of the food in a solution containing 1% pepsin and 1% HCl at 37 degrees C . Foods examined were igunaq (meat and blubber placed in a seal skin bag and allowed to ferment), nikku (air-dried meat), and sausage (meat, fillers, salt, and spices) . Sausage was examined both in a raw state and after partial cooking . Infective T . nativa larvae survived in igunaq, nikku, raw frozen sausage, and poorly cooked sausage for at least 5 months under controlled laboratory conditions . Core temperatures of partially cooked sausage never exceeded 50 degrees C . Caution should be exercised in using these data to establish guidelines for the consumption of raw products, since the survival of infective larvae could be unpredictably extended under field conditions . These data indicate significant food safety risks associated with igunaq, nikku, and sausage prepared with Trichinella-infected meat and provide information for use in risk management and in directing future research.

Biotechnol Lett, 2003 Sep, 25(18), 1485 - 90
Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe); Prakoso B et al.; Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp . The amount of RUR soybean was quantified using an ELISA plate test . The RUR signal decreased during fermentation . In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal . The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R . oryzae, Mucor rouxii and Aspergillus awamori.

Biodegradation, 2003 Oct, 14(5), 331 - 46
Predicting biogeochemical calcium precipitation in landfill leachate collection systems; VanGulck JF et al.; Clogging of leachate collection systems within municipal solid waste landfills can result in greater potential for contaminants to breach the landfill barrier system . The primary cause of clogging is calcium carbonate (CaCO3(s)) precipitation from leachate and its accumulation within the pore space of the drainage medium . CaCO3(s) precipitation is caused by the anaerobic fermentation of volatile fatty acids (VFAs), which adds carbonate to and raises the pH of the leachate . An important relationship in modeling clogging in leachate collections systems is a yield coefficient that relates microbial fermentation of VFAs to precipitation of calcium carbonate . This paper develops a new, mechanistically based yield coefficient, called the carbonic acid yield coefficient (Y(H)), which relates the carbonic acid (H2CO3) produced from microbial fermentation of acetate, propionate, and butyrate to calcium precipitation . The empirical values of Y(H) were computed from the changes in acetate, propionate, butyrate, and calcium concentrations in leachate as it permeated through gravel-size material . The theoretical and empirical results show that the primary driver of CaCO3(s) precipitation is acetate fermentation . Additionally, other non-calcium cations (e.g., iron and magnesium) precipitated with carbonate (CO3(2-)) when present in the leachate . A common yield between total cations bound to CO3(2-) and H2CO3 produced, called the calcium carbonate yield coefficient (Yc), can reconcile the empirical yield coefficient for synthetic and actual leachates.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 127 - 31
Production, survival and efficacy of Coniothyrium minitans conidia produced in shaken liquid culture; Cheng J et al.; Coniothyrium minitans is a fungal biocontrol agent of the plant pathogen Sclerotinia sclerotiorum . Growth and sporulation of 21 strains of C . minitans were examined on potato dextrose agar (PDA) and compared with that in potato dextrose broth (PDB) in shaken culture after 12 days at 20 degrees C, to identify strains with potential for inoculum production in liquid culture . Four strains that produced high numbers of pycnidia in PDA also formed pycnidia on mycelial strands in PDB and 10(7) conidia ml(-1) broth were produced . The other strains formed pellets during shaking, resulting in production of less than 10(5) conidia ml(-1) . Conidia from shaken PDB culture had the same ability to infect and rot sclerotia of S . sclerotiorum as conidia produced routinely on PDA, and survived well in dry kaolin dust for 6 months at temperatures less than 8 degrees C with less than 1 log(10) colony forming units mg(-1) loss . These results suggest that it might be possible to identify useful strains of C . minitans for future commercial conidial production in liquid fermentation systems based on morphological characteristics on agar.

Pathophysiology, 2003 Sep, 9(4), 221 - 227
Fermented grain products, production, properties and benefits to health; Minamiyama Y et al.; Fermented foods such as Japanese traditional food "miso (fermented soy bean paste)" have been shown to be rich source of micronutrients with the potential to prevent various human diseases . We have introduced effects of a new dietary supplement of fermented grain foods mixture containing extracts from wheat germ, soybeans, rice bran, tear grass, sesame, wheat, citrus lemon, green tea, green leaf extract and malted rice under the trade name of antioxidant biofactor (AOB) . Chemical analysis of AOB shows the presence of various phenolic compounds (catechins, rutin, genistin, daidzin, etc.) . AOB has strong antioxidant properties and additional biological effects, which might be of importance in context with the prevention of degenerative diseases . This paper focuses on the effect of supplementing AOB in various animal models and humans.

Planta, 2004 Jan, 218(3), 350 - 9 Epub 2003 Oct 15.
Cyanobacterial H(2) production -- a comparative analysis; Schutz K et al.; Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production . These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H(2) evolution . The uptake hydrogenase was identified in all N(2)-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme . In N(2)-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia . Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation . It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions . With regard to large-scale hydrogen evolution by N(2)-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase . On the other hand, fermentative H(2) production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 340 - 5 Epub 2003 Oct 16.
Evaluation of different organic phases for water-in-oil xanthan fermentation; Kuttuva SG et al.; Water-in-oil (W/O) fermentation technology has the potential for overcoming the problems related with high broth viscosity in xanthan fermentations . By dispersing the aqueous broth in a continuous organic phase, the broth-thickening mechanisms are confined within the aqueous droplets without significantly increasing the overall viscosity . In this study, xanthan fermentations were made with perfluorocarbon (PFC) or vegetable oil as the organic phase . The results were compared with those obtained previously using n-hexadecane as the organic phase, to evaluate the effects of various properties . PFC provided easy phase separation at the end of fermentation but required higher power input for agitation, a direct result of its high density . The aqueous droplets formed were large (400-450 microm), limiting the cell concentration employable due to the occurrence of oxygen starvation in the inner core . One main advantage of using vegetable oil was its low cost . In addition, vegetable oil provided much finer droplets (<120 microm) and produced high xanthan concentrations (>100 g l(-1)) . However, complete phase separation for product recovery was difficult to achieve . Fermentations in both organic phases were terminated by the occurrence of phase inversion to highly viscous O/W dispersions at aqueous-phase volume fractions of 0.53-0.56 . The initial fraction was 0.3 but changed due to base addition for pH adjustment and nutrient addition for prolonged production.

J Antibiot (Tokyo), 2003 Aug, 56(8), 694 - 9
The novel gluconeogenesis inhibitors FR225659 and FR225656 from Helicomyces sp . No . 19353 . III . Structure determination; Zenkoh T et al.; During the course of screening for novel gluconeogenesis inhibitors, FR225659 and its related compounds were isolated from a fermentation broth of Helicomyces sp . No . 19353 . Spectroscopic analysis concluded that FR225659 is an N-acyl tripeptide consisting of a novel acyl, a 3-chloro-4-hydroxyarginine, a 3-hydroxy-3-methylproline and a dehydrovaline . Degradation study allowed assignment of the absolute configuration of the 3-hydroxy-3-methylproline to be (2S,3R) . FR225656 was shown to possess a dehydroisoleucine instead of the dehydrovaline of FR225659.

J Antibiot (Tokyo), 2003 Aug, 56(8), 682 - 8
The novel gluconeogenesis inhibitors FR225659 and related compounds that originate from Helicomyces sp . No . 19353 . I . Taxonomy, fermentation, isolation and physico-chemical properties; Ohtsu Y et al.; FR225659 and four related compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids . They were isolated from the culture broth of Helicomyces sp . No . 19353 and can be purified by absorptive resin and reverse-phase column chromatography . They are potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents.

Eur Cell Mater, 2001 Nov 19, 2, 30 - 5
Interaction between Saccharomyces cerevisiae and chrysotile; Cassiola F et al.; The interaction between Saccharomyces cerevisiae and chrysotile fibers was studied by scanning electron microscopy . The yeast cells adhere preferentially to the fibrils . In the extreme case, all the adhered fibrils were broken, resulting in a complete coverage of the surface . The chrysotile covered cells showed less buds, but retained metabolic capacities, and were fully active in fermentation experiments after one year . The interaction degree was depending on contact time and adhesion medium . The longer the contact period, the stronger the interaction between the cells and the fibers . Cells adhered in water show poor entrapment after short contact time, but were highly entrapped after longer periods and did not show any agglomerates . Cells adhered in the presence of nutrients showed a lower entrapment and a higher degree of cellular growth.

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