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