Fermentations

Plant Physiol, 1994 Nov, 106(3), 1015 - 1022
Differential Transcript Levels of Genes Associated with Glycolysis and Alcohol Fermentation in Rice Plants (Oryza sativa L.) under Submergence Stress; Umeda M et al.; Expression of genes encoding enzymes involved in specialized metabolic pathways is assumed to be regulated coordinately to maintain homeostasis in plant cells . We analyzed transcript levels of rice (Oryza sativa L.) genes associated with glycolysis and alcohol fermentation under submergence stress . When each transcript was quantified at several times, two types (I and II) of mRNA accumulation were observed in response to submergence stress . Transcripts of type I genes reached a maximum after 24 h of submergence and were reduced by transfer to aerobic conditions or by partial exposure of shoot tips to air . In a submergence-tolerant rice cultivar, transcript amounts of several type I genes, such as glucose phosphate isomerase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, and enolase, increased significantly compared to an intolerant cultivar after 24 h of submergence . This suggests that the mRNA accumulation of type I genes increases in response to anaerobic stress . mRNA accumulation of type II genes, such as aldolase and pyruvate kinase, reached a maximum after 10 h of submergence . Following transfer to aerobic conditions, their transcript levels were not so rapidly decreased as were type I genes . These results suggest that the mRNA levels of genes engaged in glycolysis and alcohol fermentation may be regulated differentially under submergence stress.

Plant Physiol, 1994 Jun, 105(2), 651 - 657
Improved Cytoplasmic pH Regulation, Increased Lactate Efflux, and Reduced Cytoplasmic Lactate Levels Are Biochemical Traits Expressed in Root Tips of Whole Maize Seedlings Acclimated to a Low-Oxygen Environment; Xia JH et al.; We tested the hypothesis (J.-H . Xia and P.H . Saglio {1992} Plant Physiol 100: 40-46) that the enhanced ability of maize (Zea mays) root tips to survive anoxia, elicited by a 4-h exposure to 3% O2 ("acclimation"), is due to less cytoplasmic acidosis early in anoxia . Cytoplasmic pH and fermentation reactions were monitored in excised and intact (attached) maize root tips by simultaneous in vivo 13C- and 31P-NMR spectroscopy . We demonstrate that both excised and intact acclimated root tips have significantly higher cytoplasmic pH values under anoxia . This reduction in cytoplasmic acidosis is greater in intact root tips . Remarkably, cytoplasmic pH does not change when root tips are transferred from 3% O2 to anoxia . The earlier observation of considerable lactate efflux and lowered intracellular lactate in excised, acclimated root tips (ibid.) was extended to intact seedlings . The predominant fermentation end product retained in the cells of acclimated root tips is alanine . We discuss the relationship between cytoplasmic pH and levels of intracellular lactate and alanine in sugar-replete roots, and the role of cytoplasmic pH in determining survival under anoxia.

Plant Physiol, 1994 May, 105(1), 61 - 67
Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L; Johnson JR et al.; Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25{deg}C . Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions . Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h . During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2 . Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips . During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips . These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.

Plant Physiol, 1993 Apr, 101(4), 1163 - 1168
Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism); Good AG et al.; The onset of anaerobiosis in barley root tissue (Hordeum vulgare L . cv Himalaya) results in the following metabolic responses . There are rapid increases in the levels of pyruvate, lactate, and ethanol . Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue . Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate . The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine . Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions . Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis . Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue . The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.

Plant Physiol, 1993 Feb, 101(2), 553 - 560
Evidence for a Large and Sustained Glycolytic Flux to Lactate in Anoxic Roots of Some Members of the Halophytic Genus Limonium; Rivoal J et al.; Soil salinity and anaerobiosis often occur together . This led us to investigate the fermentative metabolism in roots of species from the halophytic genus Limonium (Plumbaginaceae) . Root segments from hypoxically induced plants were incubated for 8 h under strict anoxia in the presence of {U-14C}glucose . In three species (Limonium latifolium, L . nashii, and L . humile), the pattern of 14C-labeled end products was typical of higher plants, with a 14C flux to ethanol higher than that to lactate . However, in four species (L . ramosissimum, L . gougetianum, L perezii, and L . sinuatum), the rate of lactate fermentation was exceptionally high, and in the latter two species the 14C flux to lactate exceeded that to ethanol . These two species secreted most of the lactate produced into the medium . Calculations indicated that the cytoplasm would have been lethally acidified had this secretion not occurred . The effects of factors that might control lactate fermentation or secretion (O2 partial pressure, pH, salt concentration) were studied in two contrasting species: L . sinuatum and L . latifolium . In both species, the lactate:ethanol ratio was higher under hypoxia (0.1-3 kPa O2 partial pressure) than under strict anoxia . In L . sinuatum, this ratio was slightly increased by increasing the pH of the medium from 5.5 to 7.5, but salinity treatment had no effect . The potential contribution of lactate fermentation to the overall carbon and energy metabolism of halophytes is discussed.

Plant Physiol, 1993 Feb, 101(2), 407 - 414
Hypoxic and Anoxic Induction of Alcohol Dehydrogenase in Roots and Shoots of Seedlings of Zea mays (Adh Transcripts and Enzyme Activity); Andrews DL et al.; Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferentially under low O2 conditions . We examined levels of Adh1 transcripts and of ADH activity in 5-mm root tips, root axes (the remainder of the seminal root), and shoots of maize (Zea mays L . cv TX 5855) seedlings . Seedlings with roots averaging about 60-mm long were transferred from fully aerobic conditions (solutions sparged with 40% {v/v} O2) to anaerobic (O2-free) conditions, or to an intermediate O2 concentration . There was no prior acclimation to low O2 . In root tips, anoxia induced Adh1 transcripts and enzyme activity at 6 h, but this was followed by a rapid decline so that at 12 to 18 h neither were detectable and the root tips were dead . In contrast, higher levels of Adh1 transcripts and enzyme activity were maintained for at least 48 h in root axes and shoots . When induction at 6 h was measured over a wide range of O2 concentrations, a peak in ADH activity occurred in all tissues at 4% (v/v) O2 . Maximum levels of transcripts, however, were in the range of 0 to 4% O2, depending on the tissue . The time course of hypoxic induction (at 4% O2) in root tips showed a peak in transcript levels at 6 h, whereas ADH activity continued to rise throughout the 24-h experiment . These results show that in root tips, ADH induction by anoxia was small and transient relative to induction by hypoxia.

Steroids, 2002 Sep, 67(10), 869 - 72
Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282; Faramarzi MA et al.; The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated . This potential had not been previously examined . The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one . Each microbial metabolite was purified and characterized using spectroscopic methods.

Eur J Biochem, 2002 Sep, 269(18), 4495 - 504
Directed evolution of a glutaryl acylase into an adipyl acylase; Sio CF et al.; Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA) . Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process . To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative . Such an adipyl acylase can be generated from known glutaryl acylases . The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis . For selection the mutants were grown on an adipyl substrate . The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency . The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase . In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy . Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate . The strategy herein described may be generally applicable to all beta-lactam acylases.

Pak J Sci, 1968 Jan-Mar, 20(1 and 2), 64 - 7
Contraceptives and other steroid drugs: their production from steroidal sapogenins; Fazli FR; PIP: Sterols, steroidal sapogenins, steroidal alkaloids and alkaloidal amines derived from plant sources provide the starting materials for steroid production . Sarmentogenin (III) a cardiac glycoside, was first used, but the source was limited . Hecogenin (IV), a saponin (Agave sislana), was manufactured to cortisone by the process of Spensley et al . Introduction of an oxygen atom at carbon 11 by microbiological means gave a new series of starting compounds, among them, diosgenin (V) which converts to progesterone, to 11 hydroxyprogesterone by fermentation, cortisone, hydrocortisone and delta compounds . Reviews on the development, physiological and biochemical aspects of oral contraceptives were mentioned . A steroid with activity equivalent to progesterone was made by Ehrenstein in a 12-step synthesis from a cardiac aglycone strophanthidin . Estradiol converted to 19-nor testosterone by Birch and Mukherji provided a breakthrough in production of 19-nor steroids and led to production of 19-nor progesterone (VI) with a higher activity than progesterone . 19-nor-17alpha-ethynyltestosterone (VIII), its acetate derivative, and a related compound (I) account for 80% of consumption of oral contraceptives in the United States . Reviews of nor-steroids by Colton and Kilmstra, and of the chemical developments leading to currently used steroid contraceptives by Djerassi are mentioned .

Plant Physiol, 1995 Nov, 109(3), 1069 - 1076
Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions; Guglielminetti L et al.; An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability . The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound . Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of {alpha}-amylase (EC 3.2.1.1), {beta}-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and {alpha}-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely . We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia . Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo . This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ({alpha}-amylase, {beta}-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, {alpha}-glucosidase) . These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.

Plant Physiol, 1995 Oct, 109(2), 659 - 665
Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls; Darley CP et al.; Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated . Following chilling at 4{deg}C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures . Vacuolar ATPase levels did not change significantly throughout the chilling regime . However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ . Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein . Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.

Plant Physiol, 1995 Oct, 109(2), 353 - 361
Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation); Vanlerberghe GC et al.; Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity . After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer . High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain {alpha}-keto acids, particularly pyruvate . Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate . Our evidence suggests that NADPH may be specifically required for AOX reduction . All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well . Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms . Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

Biol Rev Camb Philos Soc, 2002 Aug, 77(3), 443 - 53
Carbohydrates: a limit on bacterial diversity within the colon; Rabiu BA et al.; The human large intestine is recognised as a physiologically important organ responsible for the conservation of water and salts . Through its resident bacteria, it is also capable of complex, enzyme catalysed, hydrolytic-digestive functions that have a high biological impact on the host . These microorganisms metabolise dietary components, principally complex carbohydrates that are not hydrolysed or absorbed in the upper gastrointestinal tract, and in this way, sequester energy for the host, through fermentation . This process involves a series of anaerobic, energy-yielding, catabolic reactions which complete digestive processes in the gut, resulting in end products that in turn influence the distribution of microbial species present as well as having some systemic effects . Some of the bacteria are thought to possess important health-promoting activities, especially with respect to their influence on mucosal and systemic immune responses to disease . These bioactivities can be modulated by substrates that support and influence microbial development, growth and survival . For these reasons, it is necessary to review dietary factors that may delimit bacterial diversity, to be able to predict responses and sensitivities to various environmental pressures and manipulations that occur in this area of human microbiology.

Appl Microbiol Biotechnol, 2002 Sep, 59(6), 721 - 6 Epub 2002 Jul 19.
Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol; Stevenson DM et al.; The direct fermentation of cellulosic biomass to ethanol has long been a desired goal . To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium . One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1) . This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol . Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium . The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose . Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed . Strain A10 was also found to utilize (aerobically) a wide range of carbon sources . In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants . The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.

Plant Physiol, 2002 Sep, 130(1), 256 - 64
Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance; Gong D et al.; The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway . SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3 . In this study, we examined some of the biochemical properties of the SOS2 in vitro . To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli . Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain . These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation . The three enzymes had similar peptide substrate specificity and catalytic efficiency . Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium . The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation . Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop . Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.

Plant Physiol, 1997 Mar, 113(3), 925 - 932
Dynamics of Acetaldehyde Production during Anoxia and Post-Anoxia in Red Bell Pepper Studied by Photoacoustic Techniques; Zuckermann H et al.; Acetaldehyde (AA), ethanol, and CO2 production in red bell pepper (Capsicum annum L.) fruit has been measured in a continuous flow system as the fruit was switched between 20% O2 and anaerobic conditions . Minimum gas phase concentrations of 0.5 nL L-1, 10 nL L-1, and 1 mL L-1, respectively, can be detected employing a laser-based photoacoustic technique . This technique allows monitoring of low production rates and transient features in real time . At the start of anaerobic treatment respiration decreases by 60% within 0.5 h, whereas AA and ethanol production is delayed by 1 to 3 h . This suggests a direct slow-down of the tricarboxylic acid cycle and a delayed onset of alcoholic fermentation . Reexposure of the fruit to oxygen results in a 2- to 10-fold upsurge in AA production . A short anoxic period leads to a sharp transient peak lasting about 40 min, whereas after numerous and longer anoxic periods, post-anoxic AA production stays high for several hours . High sensitivity of the fruit tissue to oxygen is further evidenced by a sharp decrease in post-anoxic AA production upon an early return to anaerobic conditions . Ethanol oxidation by the "peroxidatic" action of catalase is proposed to account for the immediate post-anoxic AA upsurge.

Plant Physiol, 1997 Feb, 113(2), 657 - 661
Molecular Genetic Evidence of the Ability of Alternative Oxidase to Support Respiratory Carbon Metabolism; Vanlerberghe GC et al.; With the cytochrome pathway inhibited, AOX was able to support considerable growth of cultured tobacco (Nicotiana tabacum cv Petit Havana SR1) cells but the efficiency of carbon utilization decreased dramatically . Antisense cells with decreased AOX protein did not grow, whereas sense cells with elevated AOX protein had higher growth and respiration rates than the wild type . In antisense cells a large accumulation of pyruvate resulted in aerobic ethanolic fermentation.

Int J Food Microbiol, 2002 Sep 15, 78(1-2), 119 - 31
Commercial bacterial starter cultures for fermented foods of the future; Hansen EB; Starter cultures for fermented foods are today developed mainly by design rather than by screening . The design principles are based on knowledge of bacterial metabolism and physiology as well as on the interaction with the food product . In the genomics era, we will obtain a wealth of data making design on a rational basis even simpler . The design tools available are food grade tools for genetic, metabolic and protein engineering and an increased use of laboratory automation and high throughput screening methods . The large body of new data will influence the future patterns of regulation . It is currently difficult to predict in what direction the future regulatory requirements will influence innovation in the food industry . It can either become a promoting force for the practical use of biotechnology to make better and safer products, or it can be limiting the use of starter cultures to a few strains with official approval . Successful cultures based on modern technology is expected to be launched in the areas of: probiotics, bioprotection, general improvement of yield and performance for the existing culture market and probably the introduction of cultures for fermenting other food products . A scientific basis for dramatic innovations that could transform the culture industry is currently being established.

J Nutr, 2002 Sep, 132(9), 2638 - 43
A poorly fermented gel from psyllium seed husk increases excreta moisture and bile acid excretion in rats; Marlett JA et al.; Psyllium seed husk (PSH) increases stool output and lowers blood cholesterol levels in humans . PSH and three fractions isolated from it were meal-fed to colectomized rats and fermented in vitro to test the hypothesis that viscous, gel-forming fraction B was responsible for these physiological actions . Control rats were fed 50 g/kg cellulose . The concentration of each PSH fraction in the test meals was equivalent to its concentration in PSH . Yields of the fractions were: A, 171; B, 575; and C, 129 g/kg of PSH . The wet weight and moisture content of ileal excreta (IE) from rats fed test meals containing PSH or fraction B were greater than those measured in excreta from rats fed meals containing cellulose or the other two PSH fractions . Total bile acids in IE did not differ between rats fed PSH or fraction B and were greater in these groups than in the other groups . Fraction A was not fermented during 3 d of incubation; fraction B was poorly fermented, with approximately 30% of the constituent sugars disappearing; and fraction C was rapidly and nearly completely fermented . These results indicate that the gel-forming fraction we isolated from PSH is the physiologically active component of the husks.

Trends Biotechnol, 2002 Oct, 20(10), 426 - 32
Meeting the consumer challenge through genetically customized wine-yeast strains; Pretorius IS et al.; Wine producers are facing intensifying competition brought about by a widening gap between wine production and wine consumption, a shift of consumer preferences away from basic commodity wine to top quality wine, and economic globalization . Consequently, they are calling for a total revolution in the 'magical' world of wine . The process of transforming the wine industry from a production- to a market-orientated industry results in an increasing dependence on, amongst others, biotechnological innovation . Market-orientated wine-yeast strains are currently being developed for the cost-competitive production of wine with minimized resource inputs, improved quality and low environmental impact . The emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine.

Anal Bioanal Chem, 2002 Sep, 374(1), 167 - 72 Epub 2002 Aug 10.
Determination of yeast assimilable nitrogen content in wine fermentations by sequential injection analysis with spectrophotometric detetection; Muik B et al.; An automated method for measuring the primary amino acid concentration in wine fermentations by sequential injection analysis with spectrophotometric detection was developed . Isoindole-derivatives from the primary amino acid were formed by reaction with o-phthaldialdehyde and N-acetyl- L-cysteine and measured at 334 nm with respect to a baseline point at 700 nm to compensate the observed Schlieren effect . As the reaction kinetic was strongly matrix dependent the analytical readout at the final reaction equilibrium has been evaluated . Therefore four parallel reaction coils were included in the flow system to be capable of processing four samples simultaneously . Using isoleucine as the representative primary amino acid in wine fermentations a linear calibration curve from 2 to 10 mM isoleucine, corresponding to 28 to 140 mg nitrogen/L (N/L) was obtained . The coefficient of variation of the method was 1.5% at a throughput of 12 samples per hour . The developed method was successfully used to monitor two wine fermentations during alcoholic fermentation . The results were in agreement with an external reference method based on high performance liquid chromatography . A mean-t-test showed no significant differences between the two methods at a confidence level of 95%.

J Dairy Sci, 2002 Aug, 85(8), 1976 - 87
Whole linted cottonseed as a forage substitute fed with ground or steam-flaked corn: digestibility and performance; Harvatine DI et al.; Six ruminally and duodenally cannulated Holstein cows were used in a 6 x 6 Latin square design . The objective was to evaluate any potential interactions in site of nutrient digestion when neutral detergent fiber (NDF) from cottonseed was incrementally substituted for forage NDF (FNDF) from alfalfa silage and when starch availability was varied by feeding ground (G) or steam-flaked (SF) corn . Iso-NDF diets were forage control with G corn (21% FNDF), 5% whole cottonseed (WCS) with G or SF corn (18% FNDF), 10% WCS with G or SF corn (15% FNDF), and 15% WCS with G corn (12% FNDF) . Ruminal or total tract digestibilities of organic matter (OM) or nonstructural carbohydrate (NSC) were unaffected, but efficiency of microbial protein synthesis decreased as WCS increased . Ruminal NDF digestibility was not affected despite a linear decrease in pH, but postruminal NDF digestibility decreased with increasing WCS . Ruminal digestibilities of OM and NSC were higher for SF than G corn but did not affect efficiency of microbial N synthesis . Dry matter intake increased quadratically with increasing level of WCS but decreased when SF replaced G corn . Milk yield did not differ across treatments . Milk fat percentage was affected quadratically and milk protein increased linearly with increasing WCS . Milk fat percentage decreased but milk protein was not affected when SF replaced G corn . Lack of an interaction between corn source and level of WCS substitution suggests that WCS was equally effective in maintaining ruminal fermentation and digestibility in diets varying in ruminal starch availability.

J Dairy Sci, 2002 Aug, 85(8), 1969 - 75
The effect of delayed ensiling and application of a propionic acid-based additive on the fermentation of barley silage; Mills JA et al.; Prolonged exposure to air can adversely affect the silage fermentation process . To investigate a possible method to overcome this problem, we determined if a buffered propionic acid-based additive, applied to chopped, whole-plant barley exposed to air before ensiling, would affect the subsequent fermentation . Wilted forage was chopped and treated with nothing, or with 0.1% (wt/wt wet forage) of a buffered propionic acid-based additive and ensiled immediately in quadruplicate 20-L laboratory silos . Portions of the chopped forage, untreated and treated, were left in loose piles in a barn for 24 h before ensiling . Another portion of the untreated silage exposed to air for 24 h was also treated with 0.1% of the additive just before ensiling . Prolonged exposure to air before ensiling increased the numbers of yeasts on forages by more than 1,000-fold . The concentrations of water-soluble carbohydrates decreased by more than 50%; the ammonia-N concentrations increased 40%, and pH increased by more than 1 unit as a result of exposure to air . These changes were less in forage that was treated with the additive at chopping . After 60 d, silages of forages that were exposed to air before ensiling had a higher pH, higher concentrations of ammonia-N and butyric acid, and lower concentrations of lactic and acetic acids than silages of forage that had been ensiled immediately after harvest . In situ DM digestibility was lowest in untreated silages that had been exposed to air before ensiling . In contrast, treatment with the additive, applied before or after exposure to air, prevented the reduction in in vitro digestion.

J Dairy Sci, 2002 Aug, 85(8), 1947 - 57
Effects of forage particle size and grain fermentability in midlactation cows . II . Ruminal pH and chewing activity; Krause KM et al.; Our study investigated the effects of, and interactions between, level of dietary ruminally fermentable carbohydrate (RFC) and forage particle size on rumen pH and chewing activity for dairy cows fed one level of dietary NDF . Also, correlations between intake, production, chewing, and ruminal pH parameters were investigated . Eight cows (61 days in milk) were assigned to four treatments in a double 4 x 4 Latin square . Treatments were arranged in a 2 x 2 factorial design; finely chopped alfalfa silage (FS) and coarse alfalfa silage (CS) were combined with concentrates based on either dry, cracked-shelled corn (DC; low RFC) or ground, high-moisture corn (HMC; high RFC) . Diets were fed ad libitum as a total mixed rations with a concentrate:forage ratio of 60:40 . Diets averaged 18.7% crude protein, 24.0% neutral detergent fiber, 18.3% , acid detergent fiber and 27.4% starch on a DM basis . Mean particle size of the four diets were 6.3, 2.8, 6.0, and 3.0 mm for DCCS, DCFS, HMCCS, and HMCFS, respectively . Decreasing forage particle size decreased ruminal pH from 6.02 to 5.81, and increasing level of RFC decreased pH from 5.99 to 5.85 . Minimum daily ruminal pH decreased from 5.66 to 5.47 when level of RFC was increased, and decreased from 5.65 to 5.48 when forage particle size decreased . Time below pH 5.8 per day increased from 7.4 h to 10.8 h when level of RFC increased, and increased from 6.4 h to 11.8 h when forage particle size was decreased . Area below 5.8 showed the same relationship with RFC and forage particle size . Also, forage particle size affected the postprandial pH pattern . Cows spent more time eating when fed CS compared with FS (274 vs . 237 min/d), and time spent eating decreased when level of RFC was increased (271 vs . 241 min/d) . Decreasing forage particle size decreased time spent ruminating (485 vs . 320 min/d), rumination periods (15.3 vs . 11.7), and duration of rumination periods (29 vs . 26 min) . Increasing level of RFC increased time spent ruminating per kg NDF intake (68.5 vs . 79.5 min/kg) . Milk fat percentage was correlated to mean ruminal pH (r = 0.41), time spent below pH 5.8 (r = -0.55), and area below 5.8 (r = -0.57), but not to intake or chewing variables . DMI of particles retained on a screen equivalent in size to the top screen of the Penn State particle separator was the intake parameter explaining most of the variation in mean ruminal pH (r = 0.27) and was correlated to time spent ruminating (r = 0.61) and chewing (r = 0.61).

Microbiology, 2002 Sep, 148(Pt 9), 2783 - 8
Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization; Hamacher T et al.; For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important . As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed . After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost . Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Gal2 are important xylose-transporting proteins . Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose . Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions . The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated.

Wien Klin Wochenschr, 2002 May 15, 114(8-9), 289 - 300
Short-chain fatty acids: bacterial mediators of a balanced host-microbial relationship in the human gut; Saemann MD et al.; The luminal compartment of the gastrointestinal tract is colonized by a large and highly complex microflora providing not only nutritional advances, but also representing a potential immunological challenge for the host . Under physiological conditions, the immune cells of the colonic mucosa do not defeat the microflora . In contrast, in cases of inflammatory bowel disease (IBD), the intestinal microflora appears to be the target of immune reactivity as demonstrated in various genetic studies and animal models of mucosal inflammation . The mechanisms responsible for the maintenance of this immunological unresponsiveness in the mucosal compartment are still largely enigmatic though recent studies indicate that luminal components might control this peculiar state . The bacterial fermentation product n-butyrate has been identified as such as critical molecule . Apart from its essential nutritional function for colonocytes, an anti-inflammatory activity of this short-chain fatty acid (SCFA) has been recognized in vitro and in vivo . Regarding its molecular mode of action, an interference with transcription factors critical for the production of pro-inflammatory cytokines has been found . This overview discusses the physiological functions of this bacterial metabolite and its emerging role as a potent regulator of mucosal homeostasis . Special emphasis is laid on potential therapeutic implications of SCFA in the treatment of several forms of colitis.

Environ Technol, 2002 Aug, 23(8), 863 - 75
Fermentation of municipal primary sludge: effect of SRT and solids concentration on volatile fatty acid production; Bouzas A et al.; Laboratory bench-scale experiments were conducted to investigate the performance of primary sludge fermentation for volatile fatty acids production . Primary sludges from two major wastewater treatment plants located in Valencia (Pinedo and Carraixet) were used . Experiments were performed at solids retention times between 4 and 10 days, and total volatile solids concentrations between 0.6% and 2.8% . Operation at two temperatures (20 degrees C and 30 degrees C) was also checked . Results indicated the importance of feed sludge characteristics on volatile fatty acids yields, being approximately double for the Carraixet wastewater treatment plant sludge than for the Pinedo plant . In both cases, higher volatile fatty acids yields were observed at higher total volatile solids concentrations . Solids retention times above 6 days scarcely improve volatile fatty acids yields, while experiments conducted at 4 days of solids retention times show an important decrease in volatile fatty acids yields . On raising temperature an increase in volatile fatty acids yields was observed, mainly due to an improvement in the hydrolysis of particulate organic matter.

Electrophoresis, 2002 Jul, 23(14), 2216 - 22
Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins; Nandakumar MP et al.; Filamentous fungal fermentations are used to produce billions of dollars of biochemical and pharmaceutical products annually, yet are plagued by a number of poorly understood problems that would benefit from proteomic analysis . Unfortunately, few publications are available which describe extraction of filamentous fungal proteins for two-dimensional electrophoresis . The goal here was to develop protocols for extraction of fungal proteins, from both wild-type and a recombinant strain of the industrially important filamentous fungi Aspergillus oryzae, to be used for both one- and two-dimensional electrophoresis (1-DE and 2-DE) . Because fungal cell walls are exceptionally resistant to fragmentation, four lysis protocols were tested: (i) boiling in strong alkali solution, (ii) boiling in Sodium dodecyl surfate (SDS), (iii) chemical lysis in Y-PER(R) reagent, and (iv) mechanical lysis via rapid agitation with glass beads in a Mini-BeadBeater(R) . For both 1-DE and 2-DE, rapid agitation with glass beads was found to be the most efficient extraction method, yielding both mini- and large-format gels with little streaking or spot tailing, and proteins comprising a broad range of molecular weights and pI values.

Biotechnol Bioeng, 2002 Sep 20, 79(6), 694 - 700
Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors; Persson P et al.; This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE) . The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used . Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield . This knowledge has given rise to the need to identify and remove the inhibiting compounds . Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates . Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates . Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract . The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae . Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives .

Biotechnol Bioeng, 2002 Sep 20, 79(6), 674 - 81
Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments; Visser D et al.; In this article we present a novel device, the BioScope, which allows elucidation of in vivo kinetics of microbial metabolism via perturbation experiments . The perturbations are carried out according to the continuous-flow method . The BioScope consists of oxygen permeable silicon tubing, connected to the fermentor, through which the broth flows at constant velocity . The tubing has a special geometry (serpentine channel) to ensure plug flow . After leaving the fermentor, the broth is mixed with a small flow of perturbing agent . This represents the start of the perturbation . The broth is sampled at different locations along the tubing, corresponding to different incubation times . The maximal incubation time is 69 s; the minimally possible time interval between the samples is 3-4 s . Compared to conventional approaches, in which the perturbation is carried out in the fermentor, the BioScope offers a number of advantages . (1) A large number of different perturbation experiments can be carried out on the same day, because the physiological state of the fermentor is not perturbed . (2) In vivo kinetics during fed-batch experiments and in large-scale reactors can be investigated . (3) All metabolites of interest can be measured using samples obtained in a single experiment, because the volume of the samples is unlimited . (4) The amount of perturbing agent spent is minimal, because only a small volume of broth is perturbed . (5) The system is completely automated . Several system properties, including plug-flow characteristics, mixing, oxygen and carbon dioxide transfer rates, the quenching time, and the reproducibility have been explored, with satisfactory results . Responses of several glycolytic intermediates in Saccharomyces cerevisiae to a glucose pulse, measured using a conventional approach are compared to results obtained with the BioScope . The agreement between the results demonstrates that the BioScope is indeed a promising device for studying in vivo kinetics .

Biotechnol Bioeng, 2002 Sep 20, 79(6), 664 - 73
Mass transfer effects on the reaction rate for heterogeneously distributed immobilized yeast cells; Gutenwik J et al.; Here we examine the efficiency of different immobilized cell gradients applied to immobilized Saccharomyces cerevisiae fermenting glucose to ethanol . We developed a simulation model to fully study the competing effects of mass transfer hindrance and kinetics . It is based on a diffusion-reaction model and can be used to analyze the different cell concentration profiles inside an immobilized gel bead, in terms of effectiveness factors, productivity, and mass flux . The internal diffusion coefficient, which varies with the local cell concentration, as well as the external mass transfer, is taken into account when describing the efficiency . Although the diffusion hindrance is greater at higher cell concentrations, high cell concentration is still advantageous in the present case because the increase in reaction rate outweighs the diffusion hindrance . Thus, high cell concentrations contribute to increased productivity . The influence of the cell concentration gradient on the efficiency of the beads is negligible . Within the range of cell profiles studied it has been established that the location of the cells within the bead is of lesser importance . However, a steep cell gradient increases the importance of the external mass transfer .

Biotechnol Bioeng, 2002 Sep 20, 79(6), 653 - 63
Water and glucose gradients in the substrate measured with NMR imaging during solid-state fermentation with Aspergillus oryzae; Nagel FJ et al.; Gradients inside substrate particles cannot be prevented in solid-state fermentation . These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies . We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images . We found that moisture gradients in the solid substrate remain small when evaporation is minimized . This is corroborated by predictions of a diffusion model . In contrast, strong glucose gradients developed . Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels . Integration of the glucose profile gave an average concentration close to the measured average content . On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity . The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment .

Biotechnol Bioeng, 2002 Sep 20, 79(6), 628 - 40
Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale; Junker B et al.; Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures . Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags . Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature . For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum . Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L . In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium . It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium . Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages . Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes .

Biotechnol Bioeng, 2002 Sep 30, 79(7), 804 - 15
Pattern analysis techniques to process fermentation curves: application to discrimination of enological alcoholic fermentations; Roger JM et al.; In fermentation processes, kinetic curves are generally aimed at control purposes . However, these curves could also contain information about inherent features of the product (such as origin, quality, etc.) . This article presents several pattern analysis techniques used to classify fermentation curves . An application to alcoholic fermentation is presented as an illustration: it aims at retrieving the origin of a must from its fermentation curve . The fermentation kinetics of five vineyard musts, harvested over 9 years on the same parcels, were recorded . From these curves two sets of variables were generated: The first (p(1)) gathers all the kinetic curve points . The second (p(2)) contains a restrained number of variables, generated by the expert knowledge of the enologist . The set p(2) was processed by two very different techniques: a linear one (factorial discriminant analysis) and a nonlinear one (artificial neural networks) . The set p(1) was processed by a new chemometric technique, the discriminant partial least-squares regression . For all the sets and the techniques used the selection of variables was studied . The interest in the latter is largely demonstrated both by theoretical and practical discussions . The discrimination results (up to 94% of good predictions) enhance the interest of the on-line measurements and their use in such pattern analysis tools .

Biotechnol Bioeng, 2002 Sep 30, 79(7), 713 - 23
Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER; Smith JD et al.; Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging . Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae . To determine whether a eukaryotic host, S . cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus . We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity . We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture . All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck . Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck . Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association . Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields .

Biotechnol Bioeng, 2002 Oct 20, 80(2), 139 - 43
Recovery and separation of cell lysate proteins using hydrogels guided by aqueous two-phase extraction principles; Putka CS et al.; The addition of poly(ethylene glycol) and salts to clarified cell lysates of Thiosphaera pantotropha increases sorption of microbial proteins into dextran hydrogels, consistent with the thermodynamics of aqueous two-phase extraction . Addition of 12 wt% PEG-10,000 to the lysate increased total sorption of protein by the dextran gel from 5.2 mg/g dextran to 37 mg/g; addition of either 0.1 M potassium iodide or tetrabutylammonium fluoride along with PEG to the lysate increased protein sorption to more than 63 mg/g, a 12-fold increase . SDS-PAGE demonstrated that the type of salt added controls which proteins are absorbed by the gel . Previously demonstrated only with model solutions, these results suggest another approach to recovery and separation strategies for proteins produced by fermentation .

Microbiol Mol Biol Rev, 2002 Sep, 66(3), 506 - 77, table of contents
Microbial cellulose utilization: fundamentals and biotechnology; Lynd LR et al.; Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature . The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures . Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics . A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations . Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step . Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance . A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Biochem Biophys Res Commun, 2002 Sep 6, 296(5), 1148 - 51
The antioxidant cocktail effective microorganism X (EM-X) inhibits oxidant-induced interleukin-8 release and the peroxidation of phospholipids in vitro; Deiana M et al.; The antioxidant beverage EM-X is derived from the ferment of unpolished rice, papaya, and sea-weeds with effective microorganisms . Oxidative stress enhances the expression of proinflammatory genes, causing the release of the chemokine interleukin-8 (IL-8), which mediates a multitude of inflammatory events . Human alveolar epithelial cells (A549) were treated with H(2)O(2) (100 microM) or TNF-alpha (10ng/ml) alone or with the addition of EM-X (100 microl/ml), incubated for 20h, and the release of IL-8, measured using ELISA . EM-X inhibited the release of IL-8 at the transcriptional level in A549 cells . EM-X also decreased the iron/ascorbate dependent peroxidation of ox-brain phospholipids in a concentration dependent manner . A TEAC value of 0.10+/-0.05mM was obtained for EM-X, indicating antioxidant potential . We suggest that the anti-inflammatory and antioxidant properties of EM-X are dependent on the flavonoid contents of the beverage.

J Agric Food Chem, 2002 Sep 11, 50(19), 5378 - 85
Quantitation of odor-active compounds in rye flour and rye sourdough using stable isotope dilution assays; Kirchhoff E et al.; Application of the aroma extract dilution analysis on a flavor distillate prepared from freshly ground rye flour (type 1150) revealed 1-octen-3-one (mushroom-like), methional (cooked potato), and (E)-2-nonenal (fatty, green) with the highest flavor dilution (FD) factors among the 26 odor-active volatiles identified . Quantitative measurements performed by stable isotope dilution assays and a comparison to the odor thresholds of selected odorants in starch suggested methional, (E)-2-nonenal, and hexanal as contributors to the flour aroma, because their concentrations exceeded their odor thresholds by factors >100 . Application of the same approach on a rye sourdough prepared from the same batch of flour revealed 3-methylbutanal, vanillin, 3-methylbutanoic acid, methional, (E,E)-2,4-decadienal, 2,3-butanedione, and acetic acid as important odorants; their concentrations exceeded their odor thresholds in water and starch by factors >100 . A comparison of the concentrations of 20 odorants in rye flour and the sourdough made therefrom indicated that flour, besides the fermentation process, is an important source of aroma compounds in dough . However, 3-methylbutanol, acetic acid, and 2,3-butanedione were much increased during fermentation, whereas (E,E)-2,4-decadienal and 2-methylbutanal were decreased . Similar results were obtained for five different flours and sourdoughs, respectively, although the amounts of some odorants in the flour and the sourdough differed significantly within batches.

J Agric Food Chem, 2002 Sep 11, 50(19), 5318 - 25
Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol; Persson P et al.; Treatment with alkali, particularly overliming, has been widely used as a method for the detoxification of lignocellulose hydrolysates prior to ethanolic fermentation . However, the mechanisms behind the detoxification effect and the influence of the choice of cation have not been well understood . In this study, a dilute acid hydrolysate of spruce and an inhibitor cocktail consisting of six known inhibitors were used to investigate different alkali detoxification methods . The various treatments included the addition of calcium hydroxide, sodium hydroxide, potassium hydroxide, and ammonia to pH 10.0 and subsequent adjustment of the pH to 5.5 with either sulfuric or hydrochloric acid as well as treatment with the corresponding amounts of calcium, sodium, and potassium as sulfate or chloride salts at pH 5.5 . An RP-HPLC method was developed for the separation of 18 different inhibitors in the hydrolysate, including furaldehydes and phenolics . Detection and quantification were carried out by means of UV, DAD, and ESI-MS in negative mode . Treatment of the spruce hydrolysate with alkali resulted in up to approximately 40% decrease in the concentration of furaldehydes . The effects on the aromatic compounds were complex . Furthermore, SFE was performed on the precipitate formed during alkali treatment to evaluate the inhibitor content of the precipitate, and the following RP-HPLC analysis implied that potential inhibitors were removed mainly through conversion rather than through filtration of precipitate . Parallel experiments in which sulfuric acid or hydrochloric acid was used for acidification to pH 5.5 after alkali treatment indicated that the choice of anion did not affect the removal of inhibitors . Detoxification with calcium hydroxide and ammonia resulted in better fermentability using Saccharomyces cerevisiae than detoxification with sodium hydroxide . The results from the experiments with the inhibitor cocktail indicated that the positive effects of alkali treatment are difficult to explain by removal of the inhibitors only and that possible stimulatory effects on the fermenting organism warrant further attention.

J Dairy Sci, 2002 Jul, 85(7), 1767 - 76
Efficacy of carbohydrate sources for milk production by cows fed diets based on alfalfa silage; Broderick GA et al.; The effectiveness of three carbohydrate sources, high-moisture ear corn (HMEC), cracked shelled corn (CSC), and a 50:50 mixture of HMEC plus dried citrus pulp (DCP), fed with or without supplemental rumen-undegraded protein as expeller soybean meal (ESBM), was assessed in 48 multiparous dairy cows . All diets contained (dry mater {DM} basis) 50% alfalfa silage, 10% ryegrass silage, 28% NDF, and one of six concentrates: A) 38% HMEC; B) 38% CSC; C) 19% DCP plus 19% HMEC; D) 27% HMEC plus 12% ESBM; E) 27% CSC plus 12% ESBM; or F) 13% DCP, 13% HMEC, and 12% ESBM . Diets A, B, and C averaged 19% crude protein, of which 53% was nonprotein nitrogen (NPN), and diets D, E, and F averaged 22% crude protein, of which 40% was NPN . Cows were fed a high-energy covariate diet for 2 wk, blocked into eight groups of six, based on covariate protein yield, then randomly assigned to diets that were fed for 12 wk . Feeding ESBM increased DM intake, yields of milk, fat-corrected milk, fat, protein, SNF, and milk and blood urea concentration and decreased weight loss . There were no production differences between HMEC and CSC . However, DM intake, yields of milk, fat-corrected milk, fat, protein, lactose, SNF, and milk SNF content all were lower on the diets containing DCP versus HMEC and CSC . A 6 x 6 Latin square trial conducted at the same time with six ruminally cannulated cows showed similar effects of diet on DM intake and milk production . Ruminal ammonia was elevated by ESBM but not ruminal total amino acids and branched-chain volatile fatty acids . Ruminal propionate was highest on HMEC diets and lowest on DCP diets; acetate, butyrate and acetate-to-propionate ratio were lowest on HMEC diets and highest on DCP diets . These results indicated that, compared to HMEC and CSC, feeding the pectin-rich carbohydrate source DCP altered ruminal fermentation but depressed intake and milk production in lactating cows.

Water Sci Technol, 2002, 45(12), 167 - 74
Application of membrane-coupled anaerobic volatile fatty acids fermentor for dissolved organics recovery from coagulated raw sludge; Kim JO et al.; To investigate the treatment performance of membrane-coupled anaerobic volatile fatty acids fermentor system, the effects of operational parameters for volatile fatty acids production were evaluated through experiments and a mathematical model . The volatile fatty acids recovery ratio was largely affected by the change of hydraulic retention time, reaching its maximum value at 12 hrs . Over the range of hydraulic retention time 8 to 96 hrs, the volatile fatty acids recovery ratio decreased with the increase of hydraulic retention time above 12 hrs, while the ratio of mineralization and gasification increased . Hydraulic retention time and membrane filtration ratio should be maintained less than 1 day and above 0.9, respectively, to attain over 40% of organic materials recovery ratio at 10 days of solids retention time . When the hydrolysis rate constant was 0.01 hr-1, the organic loading rate should be maintained at above 1.0 (kgC/m3/day) to attain over 45% of volatile fatty acids recovery ratio . Based on experimental and simulated results, membrane-coupled anaerobic volatile fatty acids fermentor system was thought to be effective for dissolved organics recovery from coagulated sewage sludge.

Appl Environ Microbiol, 2002 Sep, 68(9), 4534 - 8
Fumarate-mediated inhibition of erythrose reductase, a key enzyme for erythritol production by Torula corallina; Lee JK et al.; Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism . The increased production of erythritol by Torula corallina with trace elements such as Cu(2+) has been thoroughly reported, but the mechanism by which Cu(2+) increases the production of erythritol has not been studied . This study demonstrated that supplemental Cu(2+) enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses . Erythrose reductase, a key enzyme that converts erythrose to erythritol in T . corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography . In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration . In contrast, the enzyme activity remained almost constant regardless of Cu(2+) concentration . This suggests that supplemental Cu(2+) reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol . This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T . corallina.

Appl Environ Microbiol, 2002 Sep, 68(9), 4448 - 56
Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae; Cronwright GR et al.; Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries . To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S . cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed . Kinetic parameters were collected from published values . Maximal enzyme activities and intracellular effector concentrations were determined experimentally . The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model . Values calculated by the model agreed well with those measured in independent experiments . The model also mimics the changes in the rate of glycerol synthesis at different phases of growth . Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway . Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50) . Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08) . The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.

Ann Allergy Asthma Immunol, 2002 Aug, 89(2), 197 - 202
Glucoamylase: another fungal enzyme associated with baker's asthma; Quirce S et al.; BACKGROUND: Aspergillus-derived enzymes are widely used as dough additives in the baking industry . These enzymes may give rise to immunoglobulin (Ig)E-mediated sensitization and occupational asthma . Glucoamylase (or amyloglucosidase) is an important industrial enzyme obtained from Aspergillus niger and used to provide fermentable sugars for yeast to improve loaf volume and texture . OBJECTIVE: The aim of our study was to investigate the potential allergenic role of glucoamylase in baker's asthma . METHODS: We report four subjects with work-related allergic respiratory symptoms who were exposed to glucoamylase and other starch-cleaving enzymes used as baking additives . The causative role of glucoamylase in work-related asthma was investigated by immunologic tests and specific inhalation challenges (SIC) . Glucoamylase allergenic components were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting . RESULTS: Skin prick tests to glucoamylase (10 mg/mL) gave a positive response in all patients . Further, a positive skin prick test to alpha-amylase was obtained in the four patients and to hemicellulase in two of them . SIC to glucoamylase elicited isolated early asthmatic responses in the three patients tested, and SIC to alpha-amylase elicited early asthmatic responses in two patients and a dual asthmatic response in another patient . Immunoblotting with glucoamylase showed several IgE-binding bands with molecular masses between 33 and 96 kD . IgE-inhibition assays showed scarce to moderate allergenic cross-reactivity between glucoamylase and alpha-amylase . CONCLUSIONS: These bakers had developed IgE-mediated occupational asthma to glucoamylase and alpha-amylase . Fungal glucoamylase is widely used as a baking additive and this enzyme may give rise to allergic respiratory reactions among exposed workers.

Life Sci Space Res, 1969, 7, 102 - 9
Intestinal hydrogen and methane of men fed space diet; Calloway DH et al.; Intestinal bacteria form two gases, hydrogen (H2) and methane (CH4), that could constitute a fire hazard in a closed chamber . So H2 and CH4 pass from the anus but these gases are also transported by the blood to the lungs and removed to the atmosphere . Several factors affect gas formation: 1) amount and kind of fermentable substrate; 2) abundance, types, and location of microflora; and 3) psychic and somatic conditions that affect the gut . We evaluated the first factor by studying men fed different diets and have also recorded influences of uncontrollable factors . One group of 6 men ate Gemini-type diet (S) and another received a bland formula (F), for 42 days . Breath and rectal gases were analyzed during the first and final weeks . Flatus gases varied widely within dietary groups but much more gas was generated with diet S than with F . In the first 12-hour collection, subjects fed S passed 3 to 209 ml (ATAP) of rectal H2 (avg 52) and 24 to 156 ml (avg 69) from the lungs (assuming normal pulmonary ventilation) . With F, these values were 0 to 3 ml (avg 1) and 6 to 36 ml (avg 20) . Subjects were calmer during the second test . Gas production was lower with S than initially; F values were unchanged . Methane differed idiosyncratically, presumably due to differences in flora . Computed from 12-hour values, maximum potential daily H2 and CH4 are per man: for S, 730 ml and 382 ml; for F, 80 and 222 ml . Volumes would be larger at reduced spacecraft and suit pressures.

J Antibiot (Tokyo), 2002 Jun, 55(6), 565 - 70
Arylomycins A and B, new biaryl-bridged lipopeptide antibiotics produced by Streptomyces sp . Tü 6075 . I . Taxonomy, fermentation, isolation and biological activities; Schimana J et al.; New lipopeptide antibiotics, colourless arylomycins A series and yellow arylomycins B series were detected in the culture filtrate and mycelium extracts of Streptomyces sp . Tu 6075 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening . Arylomycins are a family of lipohexapeptide antibiotics, which represent the first examples of biaryl-bridged lipopeptides . They show antibiotic activities against Gram-positive bacteria.

J Infect Dis, 2002 Aug 15, 186(4), 493 - 500 Epub 2002 Aug 02.
Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study; Gerber A et al.; Hemolytic-uremic syndrome (HUS) is mainly associated with foodborne infections by Shiga toxin-producing Escherichia coli (STEC) . From January 1997 through December 2000, 394 children with HUS were evaluated in a prospective multicenter surveillance study in Germany and Austria (incidences, 0.7/100,000 and 0.4/100,000 children <15 years old, respectively) . Blood leukocytosis was associated with increased detection of STEC in stool cultures (P<.01) and a more severe disease course . Risk of death was associated with cerebral involvement (P<.01) . Most strikingly, non-O157:H7 STEC were detected in 43% of stool cultures of patients with HUS: O26 was detected in 15%, sorbitol-fermenting O157:H(-) in 10%, O145 in 9%, O103 in 3%, and O111 in 43% . Patients with O157:H7 serotypes required dialysis for a longer time and had bloody diarrhea detected more frequently, compared with patients with non-O157:H7 serotypes (P<.05) . This large study in children with HUS underlines the rising importance of non-O157:H7 serotypes, and, despite increased public awareness, the number of patients remained unchanged.

J Nat Prod, 2002 Aug, 65(8), 1091 - 5
Durhamycin A, a potent inhibitor of HIV Tat transactivation; Jayasuriya H et al.; Tat is a small HIV protein essential for both viral replication and the progression of HIV disease . In our efforts to discover Tat inhibitors from natural product screening of microbial fermentation extracts, we discovered durhamycin A (1) as a potent inhibitor (IC(50) = 4.8 nM) of Tat transactivation . Detailed NMR and MS/MS studies were utilized to elucidate the structure of 1 as a new member of the aureolic acid family of antibiotics . It consists of tetrasaccharide and disaccharide moieties attached to the aglycone, which is hitherto unknown in the aureolic acid family . Three other novel analogues, durhamycin B (2), compound (3), and the aglycone (4), were also discovered or chemically prepared that were less potent than durhamycin A.

Curr Microbiol, 2002 Oct, 45(4), 281 - 6
Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH; Mnatsakanyan N et al.; Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4 . These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H(2) and CO(2) during fermentative growth . FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH . In this study, regulation of activity of these pathways by formate has been investigated . In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H(2) became mostly Hyd-3 dependent . These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4.

J Agric Food Chem, 2002 Aug 28, 50(18), 5236 - 40
Characterization of resistant starch type III from banana (Musa acuminata); Lehmann U et al.; Banana starch (Musa acuminata var . Nandigobe) was evaluated for its use in generating resistant starch (RS) type III . Structural, physicochemical, and biological properties of these products were analyzed . The investigated process includes debranching of the native starch and retrogradation under different storage temperatures and starch concentrations . After enzymatic debranching, a high amount of low-molecular-weight polymers with a degree of polymerization between 10 and 35 glucose units beside a higher molecular weight fraction were found . The resulting products comprised RS contents of about 50% . After heat-moisture treatment, the RS yield increased up to 84% . Peak temperatures of about 145 degrees C found in DSC measurements pointed to a high thermal stability of the RS products . In vitro fermentations of the RS products, carried out with intestinal microflora of healthy humans, resulted in a molar ratio of acetate:propionate:butyrate of about 49:17:34 . The established method allowed the production of a high-quality RS with prebiotic properties for health preventing applications.

Water Sci Technol, 2002, 45(10), 163 - 8
Influence of pulsation on start-up of UASB reactors; Franco A et al.; The aim of this work is to study the influence of pulsation on the start-up of lab-scale UASB reactors . Pulsation was produced by an Elastic Membrane Pulsator (EMP) . The application of this device in previous works improved the performance of continuous fixed-bed fermentors and reduced the formation of preferential pathways, the retention of gas metabolites within the bed and the resistance to mass transfer . These characteristics seem to be suitable for feeding UASB reactors . In this work, the influence of pulsation frequency was studied in two pulsed UASB reactors operated in parallel with a non-pulsed one . One of them (P1) operated at high frequencies (periods of 50 and 200 s between each pulsation) and the other (P2) at low frequencies (periods of 3600 and 900 s between each pulsation) . An important improvement of the removal efficiency for pulsed reactors with respect to the non-pulsed one was obtained . The structure of the biomass was observed at the end of the process by scanning electron microscopy . In general, granulation of biomass was improved when operating in pulsing form.

Biochemistry, 2002 Aug 27, 41(34), 10603 - 7
In vitro evolution of amphioxus insulin-like peptide to mammalian insulin; Guo ZY et al.; By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP . After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: {A2Ile}ILP, {B12Val, B16Tyr}ILP, {B25Phe}ILP, {A2Ile, B12Val, B16Tyr, B25Phe}ILP (four-mutated ILP), {A2Ile, B12Val, B16Tyr, B24Phe, B25Phe}ILP (five-mutated ILP), and {A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr}ILP (six-mutated ILP) . Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP . The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency . The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable . By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity . These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory.

Yeast, 2002 Jun 15, 19(8), 713 - 26
The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation; Luyten K et al.; We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions . A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions . No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain . The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation . However, these two carriers have different functions . The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone . The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation . The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation . These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation . This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle.

Infect Immun, 2002 Sep, 70(9), 4925 - 35
Identification and functional mapping of the Mycoplasma fermentans P29 adhesin; Leigh SA et al.; Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes . Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved . We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M . fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not . Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop . A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity . Together these results suggest that the direct interaction of M . fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein . Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype . Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species . This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Protein Expr Purif, 2002 Aug, 25(3), 379 - 88
cDNA cloning, high-level expression, purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck; Liu W et al.; As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing . By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver . Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature . Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS . After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained . Duck SOD is a homodimer with 153 residues for each subunit . The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart . Five charge isomers were observed on isoelectricfocusing (IEF) . The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C . These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.

J Food Prot, 2002 Aug, 65(8), 1297 - 303
Microbial quality of water supply to an urban community in Trinidad; Agard L et al.; A microbiological study was conducted to determine the quality of the water supply to an urban community in San Fernando proper in south Trinidad using total coliforms and thermotolerant coliforms as indicators of water pollution . The membrane filter technique was used to detect total coliforms and thermotolerant coliforms on endo agar and MFc agar, respectively . The residual chlorine levels in water from the reservoir, from standpipes along the distribution line, and from households were determined with a commercial test kit . Of a total of 104 drinking water samples obtained from households, 84 (80.8%), 56 (53.8%), and 70 (67.3%) tested positive for total coliforms, thermotolerant coliforms, and Escherichia coli, respectively . The difference was statistically significant (P < 0.05, chi2) . Of the 81 water samples collected from the Water and Sewerage Authority (WASA) main supply to households, 38 (46.9%), 13 (16.0%), and 27 (33.3%) were contaminated by total coliforms, thermotolerant coliforms, and E . coli, respectively, and the difference was statistically significant (P < 0.05, chi2) . Eight (20.5%) of 39 water samples from standpipes along the distribution line tested positive for total coliforms, compared with 4 (10.3%) samples testing positive for thermotolerant coliforms . All five samples of treated water obtained from the reservoir tested negative for coliforms . There was a significant difference (P = 0.004) in the mean residual chlorine levels in water from the reservoir, water from standpipes, and water from households . Similarly, as the level of residual chlorine decreased, there was a statistically significant (P = 0.004) increase in the prevalence of total coliforms in water from 0.0% (treated reservoir water) to 15.2% (standpipe) to 53.5% (household mains) to 80.0% (household drinking water) . There was also a statistically significant difference (P < 0.001, chi2) in the prevalence of total coliforms in drinking water and in water from the WASA main supply to households . Of the 105 E . coli strains tested, 7 (6.7%), 16 (15.2%), and 22 (21.0%) were mucoid, hemolytic, and non-sorbitol fermenters, respectively . It was concluded that the high degree of contamination of drinking water in households poses a health hazard to consumers.

Lett Appl Microbiol, 2002, 35(3), 195 - 202
Production of red pigment by submerged culture of Paecilomyces sinclairii; Cho YJ et al.; AIMS: From a survey of submerged culture of edible mushrooms, a high pigment-producing fungus Paecilomyces sinclairii was selected and its optimal culture conditions investigated . METHODS AND RESULTS: The optimal culture conditions for pigment production were as follows: inoculum age, 3 d; temperature, 25 degrees C; initial pH, 6.0; carbon source, 1.5% (w/v) soluble starch; nitrogen source, 1.5% (w/v) meat peptone . Although addition of 10 mmol l(-1) CaCl2 to the culture medium slightly increased pigment production, most of the bio-elements examined had no notable or detrimental effect on pigment production . CONCLUSIONS: Under the optimal conditions obtained in the flask culture tested, a ninefold increase in pigment production (4.4 g l(-1)) was achieved using a 5(-l) batch fermenter . Paecilomyces sinclairii secreted water-soluble red pigment into the culture medium . The pigment colour was strongly dependent on the pH of the solution: red at pH 3-4, violet at pH 5-9 and pink at pH 10-12 . SIGNIFICANCE AND IMPACT OF THE STUDY: The high concentration of pigment (4.4 g l(-1)) produced by P . sinclairii demonstrates the possibility of commercial production of pigment by this strain, considering its relatively high production yield and light stability.

Biochemistry, 2002 Aug 20, 41(33), 10462 - 71
Enzymes involved in fatty acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and FabD and their acyl carrier protein specificity; Florova G et al.; Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens . Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH . In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis . We have shown that sgFabH activity is present in S . glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo . Isotope incorporation studies with {CD3}acetate and {13CD3}acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C . These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process . Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S . glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E . coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S . glaucescens PKS ACP) . In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM) . This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes . Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols . The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes . In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.

J Environ Manage, 2002 May, 65(1), 25 - 38
Wastewater management in a cane molasses distillery involving bioresource recovery; Nandy T et al.; Waste management involving bioresource recovery in a cane molasses-based distillery engaged in the manufacture of rectified spirit (alcohol) is described . The spentwash generated from the distillation of fermenter wash is highly acidic (pH 4.0-4.3) with high rates of biochemical and chemical oxygen demand (BOD: 52-58, COD: 92-100 kg/m3) and suspended solids (2.0-2.5 kg/m3) . Biogas is recovered from high strength raw spentwash through the full-scale application of a biomethanation system as pretreatment option, comprising anaerobic fixed film reactors . This, combined with subsequent concentration through multiple effect evaporators (MEE), and utilization of concentrated effluent for biocomposting of pressmud (another by-product of the industry) for production of biomanure contributes to the elimination of effluent discharges.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 501 - 8 Epub 2002 Jun 22.
Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae; Yoshimoto H et al.; Branched-chain alcohols, such as isoamyl alcohol and isobutanol, and isoamyl acetate are important flavor components of yeast-fermented alcoholic beverages . Analysis of a null mutant of the BAT2 gene encoding cytosolic branched-chain amino acid aminotransferase, and a transformant with multi-copy plasmids containing the BAT2 gene showed that the BAT2 gene product plays an important role in the production of branched-chain alcohols and isoamyl acetate . Fermentation tests using the bat2 null mutant transformed with multi-copy plasmids carrying the ATF1 gene, which encodes alcohol acetyltransferase, indicated that modified expression of BAT2 and ATF1 genes could significantly alter the proportion of branched-chain alcohols and isoamyl acetate synthesized . Furthermore, fermentation tests using different ratios of nitrogen source and RNA blot analyses demonstrated that transcription of L-leucine biosynthetic ( LEU) and BAT genes is co-regulated by nitrogen source, that production of isoamyl alcohol depends on this transcription, and that ATF transcription increased with increased concentrations of nitrogen source . Our data suggest that changes in isoamyl alcohol production by nitrogen source are due to transcriptional co-regulation of LEU and BAT genes, and that production of isoamyl acetate is dependent on isoamyl alcohol production and ATF transcription.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 443 - 8 Epub 2002 Jun 27.
The influence of bark on the fermentation of Douglas-fir whitewood pre-hydrolysates; Robinson J et al.; Douglas-fir ( Pseudotsuga menziesii) whitewood was supplemented with increasing concentrations of bark (0-30%) and was pretreated using SO(2)-catalysed steam explosion . The presence of bark in the feedstock resulted in the decreased recovery of total sugars, furfural and 5-hydroxymethylfurfural in the resultant pre-hydrolysate . No detrimental impact on monomer sugar recovery was observed . The concentration of lipophilic extractives present in the pre-hydrolysate increased with increasing bark loading, to a maximum of 0.43 g x l(-1) . The water-soluble pre-hydrolysates were fermented by Saccharomyces cerevisiae to determine the impact of bark on sugar consumption and ethanol production . Despite the inclusion of bark, fermentation of all pre-hydrolysates resulted in the complete consumption of hexose sugars within 48 h . Ethanol yields were greater than 0.43 g x g(-1) for all pre-hydrolysates regardless of bark content, indicating that, up to a content of 30%, bark had a negligible impact on the fermentation of the pre-hydrolysates to ethanol.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 436 - 42 Epub 2002 Jul 03.
Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae; Zaldivar J et al.; Heterologous genes for xylose utilization were introduced into an industrial Saccharomyces cerevisiae, strain A, with the aim of producing fuel ethanol from lignocellulosic feedstocks . Two transformants, A4 and A6, were evaluated by comparing the performance in 4-l anaerobic batch cultivations to both the parent strain and a laboratory xylose-utilizing strain: S . cerevisiae TMB 3001 . During growth in a minimal medium containing a mixture of glucose and xylose (50 g/l each), glucose was preferentially consumed . During the first growth phase on glucose, the specific growth rates were 0.26, 0.32, 0.27 and 0.30 h(-1) for strains TMB 3001, A (parental strain), A4, and A6, respectively . The specific ethanol productivities were 0.04, 0.13, 0.04 and 0.03 g/g.per hour, for TMB 3001, A, A4 and A6, respectively . The specific xylose consumption rates were 0.06, 0.21 and 0.14 g/g.per hour, respectively for strains TMB 3001, A4 and A6 . Xylose consumption resulted mainly in the formation of xylitol, with biomass and ethanol being minor products . The metabolite profile of intermediates in the pentose phosphate pathway and key glycolytic intermediates were determined during growth on glucose and xylose, respectively . The metabolite pattern differed depending on whether glucose or xylose was utilized . The levels of intracellular metabolites were higher in the industrial strains than in the laboratory strain during growth on xylose.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 409 - 18 Epub 2002 Jul 03.
Microbial alkaline pectinases and their industrial applications: a review; Hoondal GS et al.; The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes . Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations . The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment . It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential . The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology.

Cytotherapy, 2001, 3(3), 233 - 42
Manufacture and quality control of CAMPATH-1 antibodies for clinical trials; Phillips J et al.; BACKGROUND: CAMPATH-1 Abs have been used for T-cell depletion in stem-cell transplantation since the early 1980s . During that time there has been substantial progress in manufacturing techniques and quality control procedures . This article summarizes the methods used to produce the Abs for clinical use and describes results of quality control tests on representative batches . METHODS: Rat hybridoma and recombinant CHO cells were cultured in hollow-fiber fermentors . Antibodies were purified from the culture supernatant by fractionation with ammonium sulphate, or by column chromatography . Additional steps were added to assure the removal of DNA and viruses . A range of analytical methods was used to characterize the antibodies . Samples were stored frozen at -70 degrees C and re-analyzed many years later to assess the long-term stability . RESULTS: Hollow-fiber fermentors provided a simple and reliable means for antibody production, with yields between 3-10 mg/h and a convenient concentration for further processing (0.6-2.0 mg/mL) . All of the CAMPATH-1 Abs (rat IgM, rat IgG2b and human IgG1) could be purified by affinity chromatography on Protein A, but the low pH required for elution caused unacceptable aggregation of the IgM . CAMPATH-1H contained approx . 20% dimeric IgG, which could be removed by size exclusion chromatography . Antibodies were stable for at least 6 years at -70 degrees C, but there was unacceptable aggregation of CAMPATH-1M in one batch stored for 9 years . DISCUSSION: Pilot-scale production of MAbs for clinical studies is feasible in a small academic center, but regulatory requirements now demand that great attention is paid to all aspects of manufacturing and quality assurance . Although the underlying principles of cell culture and protein chemistry remain the same, the level of documentation, validation and quality control has increased greatly over the last 20 years.

Curr Pharm Des, 2002, 8(19), 1707 - 12
Epothilones: a novel class of non-taxane microtubule-stabilizing agents; Altaha R et al.; The epothilones are a novel class of non-taxane microtubule-stabilizing agents obtained from the fermentation of the cellulose degrading myxobacteria, Sorangium cellulosum . Preclinical studies have shown that the epothilones are more potent than the taxanes and active in some taxane-resistant models . Similar to paclitaxel and other taxanes, the epothilones block cells in mitosis, resulting in cell death . The chief components of the fermentation process are epothilones A and B, with epothilones C and D found in smaller amounts . Trace amounts of other epothilones have also been detected . Pre-clinical studies have shown that epothilone B is the most active form, exhibiting significantly higher antitumor activity than paclitaxel and docetaxel . Several phase I and phase II clinical trials are ongoing with epothilone B and BMS 247550, an epothilone B analog . Preliminary reports indicate these agents are active against human cancers in heavily pre-treated patients . The epothilones appear to be well tolerated, with a side effect profile that is similar to that reported with the taxanes . This article will review some basic aspects of epothilone chemistry and biology, and pre-clinical and preliminary clinical experience with epothilone B and its analog, BMS 247550.

Mycopathologia, 2002, 154(3), 127 - 38
Effect of liquid culture media on morphology, growth, propagule production, and pathogenic activity of the Hyphomycete, Metarhizium flavoviride; Fargues J et al.; Two isolates of Metarhizium spp . were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M . flavoviride (or M . anisopliae var . acridum) strain IMI 330189, and Mf324 = M . flavoviride strain ARSEF324) . Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects . Shake-flask experiments were carried out at 28 degress C in the dark . Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity . Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules . Submerged propagule yields were higher with Mf189 than with Mf324 in all seven media . While high concentrations of propagules (1.4 to 2.4 x 10(8) propagules ml(-1) for MF189 and 1.4 to 8.3 x 10(7) propagules ml(-1) for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins-Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media) . Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules . In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides . In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia . Infection potential of submerged propagules was assayed on Schistocerca gregaria . Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10(7) propagules ml(-1) . All seven media produced submerged propagules that were highly infectious for S . gregaria larvae . Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins-Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control.

Eur J Gastroenterol Hepatol, 2002 Jul, 14(7), 753 - 6
Evaluation of hydrogen excretion after lactulose administration as a screening test for causes of irritable bowel syndrome; Sen S et al.; OBJECTIVE : To determine whether it is possible to separate cases of irritable bowel syndrome associated with excess total hydrogen production (as a surrogate of colonic fermentation; these patients may be offered an exclusion diet as treatment) from other causes of irritable bowel syndrome by determining the amount of hydrogen excreted on patients' breath after oral administration of lactulose . DESIGN : Comparison of 24-hour hydrogen excretion and breath hydrogen following lactulose in untreated patients fulfilling the Rome criteria for irritable bowel syndrome, normal controls and irritable bowel syndrome patients who had previously failed to improve on an exclusion diet . METHODS : Colonic fermentation was measured by indirect calorimetry over 24 h . Immediately after calorimetry, the patients who were fasting received 20 g lactulose; end-expiratory breath samples were then collected every 30 min for 3 h . Hydrogen concentrations were determined by an electro-chemical cell . RESULTS : The total 24-hour excretion of hydrogen was significantly greater in the irritable bowel syndrome group (median 333.7 ml/24 h, interquartile range 234.7-445.67) compared to the normal volunteers (median 203.1 ml/24 h, interquartile range 131.4-256; P = 0.002) or the failed-diet group (median 204.5 ml/24 h, interquartile range 111.35-289.13; P = 0.015) . No difference was detected in breath excretion of hydrogen following lactulose in any group . CONCLUSION : Total hydrogen production over 24 h is increased in some patients with irritable bowel syndrome who may respond to exclusion diets . However, this sub-group of patients cannot be identified by measuring breath-hydrogen excretion after lactulose.

Anticancer Res, 2002 May-Jun, 22(3), 1569 - 74
Diverse biological activities of fermented pine seed shell extract; Mihara S et al.; Intraperitoneal administration of fermented pine seed shell extract (PSSE) (up to 2 g/kg) induced no apparent acute toxicity to mice . Pretreatment of mice with PSSE protected them from the lethality of Escherichia coli infection . PSSE showed a very weak cytotoxic activity against both normal and tumor cells and no anti-HIV activity, but stimulated the mouse macrophage-like Raw 264.7 cells to produce nitric oxide (NO) and citrulline . ESR spectroscopy showed that PSSE produced no detectable radicals, but effectively scavenged O2- (generated by the hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by the Fenton reaction) and NO (generated by NOC-7) . Comparison of PSSE with other natural products, such as polyphenols and vitamins, further confirmed the close association between radical intensity and radical scavenging activity, suggesting the bimodal action of natural products . Although the biological activities of PSSE were relatively lower than those of other natural products, the present study suggests the possible medicinal efficacy of PSSE.

J Agric Food Chem, 2002 Aug 14, 50(17), 4895 - 9
Content of biogenic amines in a Chardonnay wine obtained through spontaneous and inoculated fermentations; Torrea D et al.; This paper describes the content of biogenic amines in wines obtained from a Chardonnay must inoculated with different strains of Saccharomyces cerevisiae and in a wine fermented with the indigenous yeasts (control wine) . The concentrations of nonvolatile amines and phenethylamine in the wines from the inoculated must were superior to those of the control wine . This was probably due to the fact that consumption of the precursor amino acids of these amines, during fermentation, was also greater in the inoculated samples than in the control sample . Furthermore, from the results obtained it may be said that, at least to some extent, the nonvolatile amines were formed by yeasts during fermentation . The concentrations of dimethylamine, ethylamine, and pyrrolidine (volatile amines) were different for the different wines, although they did not reach concentrations sufficiently high to have any effect on the aroma.

FEMS Microbiol Rev, 2002 Aug, 26(3), 285 - 309
Enzymology and bioenergetics of respiratory nitrite ammonification; Simon J; Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions . In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite . The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase . The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products . This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification . High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA . Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes . The NrfH protein of W . succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA . Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters . In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products . The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif . The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue . The attachment of the lysine-ligated heme group requires specialized proteins in W . succinogenes and Escherichia coli that are encoded by accessory nrf genes . The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.

J Altern Complement Med, 2002 Jun, 8(3), 315 - 23
A fermentation product of Cordyceps sinensis increases whole-body insulin sensitivity in rats; Balon TW et al.; OBJECTIVE: CordyMax trade mark Cs-4 (Cs-4) is a standardized mycelial fermentation product of Cordyceps sinensis, a fungus that has been used for various pharmacologic, metabolic, and ergogenic purposes . The goal of this investigation was to determine the effects of oral Cs-4 administration on whole-body insulin sensitivity, skeletal muscle glucose transport, and endurance performance . DESIGN: We studied different indices of carbohydrate metabolism in rats that received Cs-4 orally at a dose of 2 g/kg of body weight daily for 30 days . RESULTS: C-peptide response observed during the oral glucose tolerance test (OGTT) after 10 days of treatment was significantly decreased in the Cs-4-treated group (Cs-4, 52,802 +/- 4,124 vs . control, 70,696 +/- 6309 pM x 120 min; p < 0.05) . The integrated insulin area under the curve (53.3 +/- 4.9 ng/mL x 120 minutes) and the glucose-insulin index (6.6 +/- 0.6 units) obtained from the OGTT were significantly decreased (p < 0.01) in the Cs-4-treated group compared to their vehicle-treated counterparts (82.1 +/- 8.1 ng/mL x 120 minutes; 9.9 +/- 0.7 units) after 20 days of treatment . Neither integrated glucose area under the curve observed during either OGTT, basal- or insulin-stimulated 2-deoxyglucose transport nor skeletal muscle GLUT-4 concentrations were affected by Cs-4 treatment . In addition, swim time to exhaustion did not differ between groups in this animal model . CONCLUSION: We conclude that CordyMax Cs-4 may have potential beneficial effects by maintaining whole-body glucose disposal with a less pronounced increase in insulin secretion after a carbohydrate challenge, however, its effects on endurance performance remain questionable.

J Altern Complement Med, 2002 Jun, 8(3), 309 - 14
CordyMax Cs-4 improves glucose metabolism and increases insulin sensitivity in normal rats; Zhao CS et al.; OBJECTIVE: To evaluate effects of CordyMax trade mark Cs-4, a mycelial fermentation product of Cordyceps sinensis, on improving glucose metabolism and insulin sensitivity . DESIGN: An in vivo pharmacology study . Subjects and Study Interventions: Adult Wistar rats, male and female, were given CordyMax 250 or 500 mg/kg per day or placebo for 17 days by gavage . OUTCOME MEASUREMENTS: Fasting blood glucose, fasting plasma insulin, glucose-insulin index, and oral glucose tolerance . RESULTS: Rats fed Cs-4 at either 250 or 500 mg/kg showed significantly reduced fasting blood glucose after the 17-day treatment, by 27% and 24% from baselines respectively (both p < 0.001) . Examination of fasting plasma insulin demonstrated a 37% decrease in the high dose treatment groups (p = 0.012) . Glucose-insulin index, an index of insulin sensitivity, increased by 10% and 17% in both 250 and 500 mg/kg groups (p = 0.008 and p = 0.0001, respectively) . Oral glucose tolerance tests showed significantly improved glucose tolerance at 0.5, 1.0, and 2.0 hours after oral administration of a bolus of glucose (the area under the glucose curve: p = 0.05-0.006), but no change at 5 hours . CONCLUSION: CordyMax Cs-4 is effective in lowering basal blood glucose and plasma insulin, improving glucose metabolism by enhancing insulin sensitivity, and improving oral glucose tolerance.

J Ind Microbiol Biotechnol, 2002 Aug, 29(2), 93 - 8
Extracellular production of L-ascorbic acid by Chlorella protothecoides, Prototheca species, and mutants of P . moriformis during aerobic culturing at low pH; Running JA et al.; Nine strains of Chlorella protothecoides and 43 strains representing the five species of Prototheca were screened in flask culture for their ability to synthesize L-ascorbic acid (AA) . Ascorbic acid was detected in all strains, ranging from 4.8 to 0.38 mg AA g x (-1) of dry cells . Organisms selected for further study grew well and maintained their AA productivity above a pH of 3.5 . They can produce AA using a variety of carbon and nitrogen sources . Aerobic fermentation of selected strains resulted in extracellular accumulation of AA up to 76 mg x l(-1) . By classical mutagenesis and selection methods, we created mutants of Prototheca moriformis ATCC 75669 that produced greater quantities of AA than the wild-type strain (78.4 vs 21.9 mg AA g x (-1) of cells) . A process based on extracellular production could greatly reduce the cost of AA manufacture by eliminating the need for extraction of the AA from the cells.

Biochim Biophys Acta, 2002 Jul 1, 1554(3), 159 - 69
cAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism; Dejean L et al.; The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast . By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways . Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content . In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP . (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins . This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield . (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield . It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.

Ultrasonics, 2002 May, 40(1-8), 19 - 23
Application of low intensity ultrasonics to cheese manufacturing processes; Benedito J et al.; Ultrasound has been used to non-destructively assess the quality of many foods such as meat, fish, vegetables and dairy products . This paper addresses the applications of low intensity ultrasonics in the cheese manufacturing processes and highlights the areas where ultrasonics could be successfully implemented in the future . The decrease of ultrasonic attenuation during the renneting process can be used to determine the optimum cut time for cheese making . The ultrasonic velocity increases during maturation for those types of cheese that become harder during this manufacturing stage, thus being an indicator of the maturity degree . Moreover, ultrasonic measurements could be linked to sensory parameters . From the ultrasonic velocity measurements at two different temperatures, it is possible to assess cheese composition, thus allowing an improvement in the quality and uniformity of cheese commercialization . In addition, in pulse-echo mode it is possible to detect cracked pieces due to abnormal fermentations and also to assess the distance of the crack from the surface.

Oncogene, 2002 Aug 12, 21(35), 5358 - 60
DNA methylation and cancer; Jones PA; There is tremendous ferment in the field of epigenetics as the relationships between chromatin structure and DNA methylation patterns become clearer . Central to this activity is the realization that the 'histone code', which involves the post-translational modification of histones and which has important ramifications for chromatin structure, may be linked to the DNA cytosine methylation pattern . New discoveries have suggested that histone lysine 9 methylation is implicated in the spread of heterochromatin in Drosophila and other organisms . Very recently it has been found that histone lysine 9 methylation is also necessary for some DNA methylation in Neurospora and plants . There is therefore the possibility that these two processes are closely linked, suggesting ways in which DNA methylation patterns may be established during normal development . Understanding these processes is fundamental to understanding what goes awry during the process of aging and carcinogenesis where DNA methylation patterns become substantially altered and contribute to the malignant phenotype.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 815 - 20
Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption; Dainiak MB et al.; Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths . However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads . To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA) . The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions . PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent . Thus, the shielding layer of PAA was adsorbed only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study . PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which otherwise bound strongly to the native adsorbent at neutral conditions . Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells . The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 745 - 51
Process design for enzymatic adipyl-7-ADCA hydrolysis; Schroen CG et al.; Adipyl-7-ADCA is a new source for 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the substrates for antibiotics synthesis . In this paper, a novel process for enzymatic 7-ADCA production is presented . The process consists of a reactor, a crystallization step, a membrane separation step, and various recycle loops . The reactor can either be operated batch-wise or continuously; with both types of processing high yields can be obtained . For batch reactors chemical degradation of 7-ADCA can be neglected . For continuous reactors, chemical stability of 7-ADCA is a factor to be taken into account . However, it was shown that the reaction conditions and reactor configuration could be chosen in such a way that also for continuous operation chemical degradation is not important . Downstream processing consisted of crystallization of 7-ADCA at low pH, followed by a nanofiltration step with which, at low pH, adipic acid could be separated from adipyl-7-ADCA and 7-ADCA . The separation mechanism of the nanofilter is based on size exclusion combined with charge effects . Application of this filtration step opens possibilities for recycling components to various stages of the process . Adipic acid can be recycled to the fermentation stage of the process while both adipyl-7-ADCA and 7-ADCA can be returned to the hydrolysis reactor . In this way, losses of substrates and product can be minimized.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 734 - 8
Efficacy of a hot washing process for pretreated yellow poplar to enhance bioethanol production; Nagle NJ et al.; Cost reductions for pretreatment and bioconversion processes are key objectives necessary to the successful deployment of a bioethanol industry . These unit operations have long been recognized for their impact on the production cost of ethanol . One strategy to achieve this objective is to improve the pretreatment process to produce a pretreated substrate resulting in reduced bioconversion time, lower cellulase enzyme usage, and/or higher ethanol yields . Previous research produced a highly digestible pretreated yellow poplar substrate using a multistage, continuously flowing, very dilute sulfuric acid (0.07% (w/v)) pretreatment . This process reduced the time required for the bioconversion of pretreated yellow poplar sawdust to ethanol . This resulted in a substantially improved yield of ethanol from cellulose . However, the liquid volume requirements, steam demand, and complexity of the flow-through reactor configuration were determined to be serious barriers to commercialization of that process . A reconfigured process to achieve similar performance has been developed using a single-stage batch pretreatment followed by a separation of solids and liquids and washing of the solids at a temperatures between 130 and 150 degrees C . Separation and washing at the elevated temperature is believed to prevent a large fraction of the solubilized lignin and xylan from reprecipitating and/or reassociating with the pretreated solids . This washing of the solids at elevated temperature resulted in both higher recovered yields of soluble xylose sugars and a more digestible pretreated substrate for enzymatic hydrolysis . Key operating variables and process performance indicators included acid concentration, temperature, wash volume, wash temperature, soluble xylose recovery, and performance of the washed, pretreated solids in bioconversion via simultaneous saccharification and fermentation (SSF) . Initial results indicated over a 50% increase in ethanol yield at 72 h for the hot washed material as compared to the control (no washing, no separation) and a 43% reduction of in the bioconversion time required for a high ethanol yield from cellulose

Biotechnol Prog, 2002 Jul-Aug, 18(4), 723 - 7
High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture; Shi XM et al.; The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea . In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source . As a result, a maximum cell dry weight concentration of 48 g/L was achieved . This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions . In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture . Such a modification resulted in an enhanced lutein production without significantly lowering biomass production . The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture . The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose . This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein . The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L) . The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment . The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.

Ukr Biokhim Zh, 2002 Mar-Apr, 74(2), 10 - 20
{Thermobiosensors: features of design and function and prospects for their use}; Bevza OV et al.; Thermobiosensors are biosensors, which utilize thermal sensors (thermistors, thermocouple) for measuring changes of heat arising during ferment, immune or metabolic reactions . Thermosensors apply to an estimation of a condition of ferment systems, interactions of immune components, kinetics of a biocatalysis, and also for application in biochemical diagnostics, at definition of quality of products, monitoring of an environment and control of biotechnological processes . In the review the data, concerning of the major principle of development and creation mini- and microthermobiosensors, are given on the basis of modern achievement in the field of technology of micromanufacturing . The various types mini-, micro- and multithermobiosensors, and also their application in separate areas of the biochemical and clinical analysis and monitoring of an environment are surveyed . The development and modern achievement in creation of separate variants thermosensors is in detail described on the basis of thermistors and thermocouples . The place thermosensors in general development of biosensor controls is allocated and the attention to prospect of their further improvement and application is given.

Bioorg Med Chem, 2002 Oct, 10(10), 3129 - 34
Biological effects of G1 phase arrest compound, sesquicillin, in human breast cancer cell lines; Jeong HW et al.; Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells . During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7 . Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53 . Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.

J Am Soc Mass Spectrom, 2002 Jul, 13(7), 862 - 74
Structural elucidation studies on 14- and 16-membered macrolide aglycones by accurate-mass electrospray sequential mass spectrometry; Roddis M et al.; Collision induced dissociation sequential mass spectrometry was used to investigate the fragmentation of the heptaketide macrolide aglycones, 6-deoxyerythronolide B (6-dEB), erythronolide B (EB), and acetate-starter EB (Ac-EB) . The fragmentations of two previously reported octaketide analogs produced by "stuttering" of the erythromycin polyketide synthase, stuttered-6-dEB and acetate-starter stuttered-6-dEB were also studied . The accuracy with which the mass of each fragment was measured allowed it to be attributed to an unambiguous formula . Most of the experiments were repeated using samples dissolved in deuterated solvents . These data were then used to deduce plausible fragmentation pathways of the five compounds which were shown to have a high degree of similarity . Preliminary fragmentation analysis of a novel octaketide analog was performed and the structure was predicted as stuttered EB . Subsequent scale-up of the bacterial fermentations, followed by isolation and characterization by nuclear magnetic resonance spectroscopy confirmed this prediction . Further fragmentation experiments were then performed on this compound, which provided further evidence of the similarity of the fragmentation schemes . These results demonstrate the utility of collision induced dissociation sequential mass spectrometry analysis in the preliminary screening of bacterial fermentations for new polyketides . These studies were performed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1341 - 7
Geovibrio thiophilus sp . nov., a novel sulfur-reducing bacterium belonging to the phylum Deferribacteres; Janssen PH et al.; Strain AAFu3T (= DSM 11263T = ATCC BAA-311T), a Gram-negative, non-sporulating bacterium, was isolated from a methanogenic mixed culture growing with acetone, in which acetate was the major intermediate . The cells of strain AAFu3T were slender spirilla, usually of less than one turn, and were motile by means of a single polar flagellum . The cells contained c-type cytochromes and the G+C content of the genomic DNA was 50.2 mol% . Sulfur, nitrate, fumarate, DMSO and oxygen (microaerophilically) were used as electron acceptors, but sulfate, sulfite, thiosulfate and ferric iron were not . Sulfide, hydrogen, formate and acetate acted as electron donors for respiratory growth, while fumarate, maleate and L-malate supported fermentative growth . Neither fermentative nor respiratory growth was supported by carbohydrates, fatty acids more than two carbons long, alcohols or amino acids . The strain was a mesophile . Comparative sequence analysis of the 165 rRNA gene and comparison of phenotypic characteristics showed that strain AAFu3T is closely related to Geovibrio ferrireducens, within the phylum Deferribacteres . Strain AAFu3T was designated as the type strain of a new species, for which the name Geovibrio thiophilus is proposed.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1285 - 9
Fulvimonas soli gen . nov., sp . nov., a gamma-proteobacterium isolated from soil after enrichment on acetylated starch plastic; Mergaert J et al.; Six deep-yellow-pigmented strains were isolated from soil after enrichment on plasticized acetylated starch granules as a source of carbon . They showed very similar and unique fatty acid profiles, consisting almost exclusively of branched fatty acids . The strains consisted of small, motile rods, were oxidase- and catalase-positive, did not ferment sugars and were able to depolymerize starch and suspended acetylated starch in overlayer plates . The 16S rDNA sequence of a representative strain, strain LMG 19981T, showed 96.7% sequence similarity to that of Rhodanobacter lindaniclasticus, 96.0% to that of Frateuria aurantia and less than 92% to sequences of other members of the gamma-Proteobacteria . Repetitive extragenic palindromic DNA PCR fingerprinting revealed two groups, representatives of which showed 93% DNA-DNA reassociation with each other and less than 10% with Frateuria aurantia LMG 1558T . On the basis of phenotypic characteristics and the G+C content of the DNA, the strains could be differentiated from Frateuria aurantia and Rhodanobacter lindaniclasticus . The name Fulvimonas soli gen . nov., sp . nov . is proposed, with the type strain LMG 19981T (= DSM 14263T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1257 - 61
Weissella koreensis sp . nov., isolated from kimchi; Lee JS et al.; A taxonomic study was carried out on two strains that came from kimchi, a traditional Korean fermented-vegetable food . The DNA G+C content of these strains was 37 mol % . Both strains contained Lys-Ala-Ser in the cell walls . On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rDNA sequence comparisons and DNA-DNA reassociation, it is proposed that these strains represent a novel species of the genus Weissella, Weissella koreensis sp . nov . The type strain is strain S-5623T (= KCTC 3621T KCCM 41516T = JCM 11263T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1239 - 46
Final classification of Bisgaard taxon 9 as Actinobacillus arthritidis sp . nov . and recognition of a novel genomospecies for equine strains of Actinobacillus lignieresii; Christensen H et al.; Phenotypic characterization of bacteria from diseased and healthy horses identified 18 isolates as Bisgaard taxon 9 and 11 isolates as Actinobacillus lignieresii . All strains of taxon 9 were alpha-galactosidase- and raffinose-positive and showed variable fermentation of (+)L-arabinose and (-)D-sorbitol . Strains of A . lignieresii were negative for these characteristics, with the exception of raffinose . Two strains from the (-)D-sorbitol-negative group of taxon 9 showed a 16S rRNA similarity of 99-6%, while 99.5% similarity was found between two strains of the (-)D-sorbitol-positive group . DNA-DNA hybridization between the two strains representing the (-)D-sorbitol-negative group showed 98% binding, and their closest relationship was to a strain of A . lignieresii (64%) . The two strains of the (-)D-sorbitol-positive group showed 83% binding and were related to the (-)D-sorbitol-negative group at a 76% DNA binding level . Actinobacillus arthritidis sp . nov . is proposed for 12 strains of the (-)D-sorbitol-positive group . Actinobacillus genomospecies 2 is suggested for the six strains of the (-)D-sorbitol-negative group . Phenotypically, strains of A . arthritidis and Actinobacillus genomospecies 2 differ in (-)D-sorbitol fermentation and can be separated from Actinobacillus equuli by being trehalose-negative, while a positive reaction for alpha-galactosidase separates the taxa from A . lignieresii . The type strain of A . arthritidis, CCUG 24862T, was isolated from a joint of a horse . Three equine isolates of A . lignieresii that could not be separated from the type strain by means of phenotypic characteristics showed 98.6-100% 16S rRNA similarity, but only 96.4-96.7% similarity to the type strain . DNA-DNA hybridization between two strains of this group showed 92% binding but only 70% binding to the type strain of A . lignieresii . Consequently, these equine isolates of A . lignieresii represent a new genomospecies of Actinobacillus, suggested as genomospecies 1 because phenotypic characteristics are not presently available to separate it from the type strain of A . lignieresii.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 239 - 41
{Study of alginate/chitosan microcapsules for immobilization of Escherichia coli DH5 alpha}; Fu YL et al.; Objective proteins synthesized from genetically recombined Escherichia coli strain (E . coli) have been successfully produced by microbe fermentation, but complicated separation and purification steps always make against the maintenance of activities as well as increase the cost . Aiming at simplifying the process, an idea of administrating directly the microencapsulated genetically recombined E . coli is proposed . In this paper, study on culture of E . coli DH5 alpha immobilized in alginate/chitosan (ACA) microcapsule is presented . It was found that E . coli DH5 alpha grew well in the microcapsule with stable growth period longer than that of suspension culture, and cell aggregation phenomenon was observed . In vivo experiments showed that ACA microcapsules with E . coli DH5 alpha stayed over 48 h in mouse intestine, and the morphology of microcapsules was kept intact . These preliminary results have demonstrated that administration of microencapsulated E . coli DH5 alpha is safe, which laied the foundation for microencapsulated genetically recombined E . coli as carriers of gene engineering drugs.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 221 - 4
{Study of immobilization of Aspergillus oryzae mycelium aided by artificial neural network}; Wang Y et al.; Aspergillus oryzae mycelium pellets with abundant aminoacylase were cultured by liquid fermentation . Mycelium pellets were immobilized by crosslinking method with reagent of gelatin and formaldehyde . Basing on the orthogonal design table L16 (4(5)), artificial neural network(ANN) model with back-propagation(BP) of error structured 4-10-15-1 was used to optimize the immobilization conditions . And the optimal conditions were obtained . Then the activity of the immobilized cells prepared under the optimal conditions was assayed . Compared with the free mycelium activity of 1800 u, the residual activity rate was 83%, which was in good accordance with that predicted by ANN . It illustrated that artificial neural network can be used to find optimal conditions for cell immobilization.

Bioresour Technol, 2002 Oct, 85(1), 31 - 3
Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up; Sharma SK et al.; Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification . Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol . Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.

J Am Diet Assoc, 2002 Jul, 102(7), 993 - 1000
Position of the American Dietetic Association: health implications of dietary fiber; Marlett JA et al.; Dietary fiber consists of the structural and storage polysaccharides and lignin in plants that are not digested in the human stomach and small intestine . A wealth of information supports the American Dietetic Association position that the public should consume adequate amounts of dietary fiber from a variety of plant foods . Recommended intakes, 20-35 g/day for healthy adults and age plus 5 g/day for children, are not being met, because intakes of good sources of dietary fiber, fruits, vegetables, whole and high-fiber grain products, and legumes are low . Consumption of dietary fibers that are viscous lowers blood cholesterol levels and helps to normalize blood glucose and insulin levels, making these kinds of fibers part of the dietary plans to treat cardiovascular disease and type 2 diabetes . Fibers that are incompletely or slowly fermented by microflora in the large intestine promote normal laxation and are integral components of diet plans to treat constipation and prevent the development of diverticulosis and diverticulitis . A diet adequate in fiber-containing foods is also usually rich in micronutrients and nonnutritive ingredients that have additional health benefits . It is unclear why several recently published clinical trials with dietary fiber intervention failed to show a reduction in colon polyps . Nonetheless, a fiber-rich diet is associated with a lower risk of colon cancer . A fiber-rich meal is processed more slowly, which promotes earlier satiety, and is frequently less calorically dense and lower in fat and added sugars . All of these characteristics are features of a dietary pattern to treat and prevent obesity . Appropriate kinds and amounts of dietary fiber for the critically ill and the very old have not been clearly delineated; both may need nonfood sources of fiber . Many factors confound observations of gastrointestinal function in the critically ill, and the kinds of fiber that would promote normal small and large intestinal function are usually not in a form suitable for the critically ill . Maintenance of body weight in the inactive older adult is accomplished in part by decreasing food intake . Even with a fiber-rich diet, a supplement may be needed to bring fiber intakes into a range adequate to prevent constipation . By increasing variety in the daily food pattern, the dietetics professional can help most healthy children and adults achieve adequate dietary fiber intakes.

Huan Jing Ke Xue, 2002 May, 23(3), 53 - 8
{The solid-state fermentation with double dynamic of gas phase for celluloses}; Xu F et al.; To utilize the advantages of solid-state fermentation for cellulose production, the solid-state fermentation with double dynamic of gas phase was proposed . The studies showed as follows: under the optimum operating conditions, the air pulsation range, the air pulsation periods and the air internal circulation, the fermentation temperatures were controlled quite well and the maximum temperature difference was 0.12 degree C/cm at a bed height of 9.0 cm; the water activity in the fermentation with steam-exploded straw as substrate was very suitable for the fungal growth; and with the observation of microbial growth by ESEM . It was discovered that air-pulsating fermentation was more advantageous to cellulose production and microorganism growth than static fermentation . The period of air-pulsating fermentation (60 h) was almost shorter 1/3 and its filter paper activity of enzymes (20.36 IU/mg) was larger 1 time than in the static fermentation with the fermentation period 84 h and the enzyme activity (10.82 IU/mg) . Thus, this double dynamic of gas laid strong basis for cellulose production on a large scale.

Br J Nutr, 2002 Aug, 88(2), 159 - 69
Evaluation of large-intestinal parameters associated with dietary treatments designed to reduce the occurrence of swine dysentery; Durmic Z et al.; Diets containing soluble NSP (sNSP) and resistant starch (RS) increase hindgut fermentation in pigs, which in turn increases the incidence of swine dysentery (SD) after infection with the intestinal spirochaete Brachyspira hyodysenteriae . In the present study pigs were fed diets based on either wheat or sorghum, fed either raw or treated by extrusion, and/or with the addition of dietary enzymes to reduce RS and/or sNSP content . The aim was to determine the effects of these treatments on pig performance, large intestinal fermentation and expression of SD . Weaned pigs (n 132) were fed experimental diets for 4 weeks, when half the pigs in each treatment group were euthanased and samples collected to assess the influence of the diet on hindgut fermentation . The remaining pigs then were infected with B . hyodysenteriae, and monitored for development of SD . In general, compared with pigs fed raw wheat, fermentation in all parts of the large intestine was reduced either by feeding raw sorghum-based diets, or by feeding diets that were extruded . The addition of enzymes that degrade RS or sNSP reduced fermentation only in the distal parts of the large intestine . The incidence of SD was lower in pigs fed sorghum-based diets, and some of the extruded diets, but none of the dietary treatments offered full protection against SD . Multiple regression analysis of the results from all three experiments showed that colonisation by spirochaetes was highly related to dietary sNSP concentrations, whilst development of SD was similarly influenced by RS content of the diet.

Eur J Clin Nutr, 2002 Aug, 56 Suppl 3, S57 - 9
Probiotics as a help in children suffering from malnutrition and diarrhoea; Solis B et al.; Malnutrition is associated with a significant impairment of cell-mediated immunity, phagocyte function, immunoglobulin A concentrations and cytokine production . This situation-which is unfortunately very frequent in infants and children from developing countries-leads to a high risk of infection, and often to episodes of diarrhoea, hence aggravating the nutritional status . Fermented milks are known to exert a beneficial influence on the host's health . They act by modulating gut microflora, regulating any alteration of gut mucosa and stimulating immune response . A number of studies have shown that probiotics shorten the duration of diarrhoea and prevent recurrence of other episodes . Furthermore, probiotics can prevent diarrhoea from infection in infants with malnutrition . In addition, it has been shown that cytokines could be used as biological markers of both impaired immune system and the immune stimulation of probiotics . The aim of this review is to update the effect of fermented milks in situations of malnutrition and diarrhoea as a consequence of infection as well as the role of cytokines produced by fermented milks in these deficient conditions.

J Nat Prod, 2002 Jul, 65(7), 1039 - 40
Ergot alkaloids produced by submerged cultures of Claviceps zizaniae; Kantorova M et al.; Two ergopeptine alkaloids, alpha-ergocryptine (1) and its C(8) epimer alpha-ergocryptinine, have been isolated from the mycelium and fermentation broth of submerged cultures of Claviceps zizaniae CCM 8240 . The structure of 1 was determined by HPLC/positive ion APCI MS and NMR analysis . Alkaloid concentrations of 10 microg/mL in 14-day-old fermentation broth and 1 mg/g of dry mycelium mass were found . These results are of considerable biotechnological interest since these were the only detectable alkaloids produced . Toxicity of naturally occurring sclerotia of C . zizaniae cannot be excluded.

Nutr Hosp, 2002, 17 Suppl 2, 7 - 10
{The colon as an organ: habitat of bacterial flora}; Guarner F; The intestinal flora constitutes a complex ecosystem comprising several hundred species of micro-organism, most of them in the genus bacteria . This ecosystem includes some micro-organism considered to be pathogenes for their capacity to invade the host, but they also contain numerous species capable of promoting beneficial effects on health . Bacterial flora begins to be acquired inmmediately after birth . By the age of two years, the flora etablished is practically definitive . There are transiet modifications derived from the use of antibiotics or in connection with changes in diet, but these are normally reversible so that every individual tends to have a relatively stable flora . The composition of bacterial flora varies from one individual to another but it is metabolic functions show less diversity . The flora in the human colon is like an organ with intense metabolic activity due to the action of bacterial enzymes on the substrates present in the intestine . Many authors have considered it more relevant to identify the enzymatic activity of the bacterial flora than the variety of species contained . The main function of the flora are: 1) fermentation of dietary waste and endogenous mucins; 2) energy recovery through the generation of short-chain fatty acids; 3) protection against colonization and invasion by pathogens (barrier effect), and 4) development, stimulation and modulation of the immune system . Special attention has been paid in recent years to the recognition of certain bacterial species that can have such salutary effects on the host . Probiotics are living microorganism that are consumed in order to obtain a beneficial effect regardless of their intrinsic nutritional value . Considerable experimental evidence has been gathered to suggest that probiotics are useful in many health-related spheres . There is good documentation to the effect that the consumption of yoghurt reduces the signs and symptoms of intestinal lactase deficiency . Some probiotics have been shown to be useful in the prevention and treatment of acute diarrhoea due to rotavirus in children and adolescents . The consumption of certain probiotics has also been seen to reinforce certain functions in the human immune system.

Nutr Hosp, 2002, 17 Suppl 2, 11 - 6
{Colonic metabolism of fiber}; Garcia Peris P et al.; Over the last few years, the colon has come to be considered more and more as a central digestive organ . This is where bacterial fermentation takes place to eliminate the substrates, mostly carbohydrates, that have avoided digestion by human enzymes within the small intestine . Of these, fibre plays a prominent role . As a result of this colonic fermentation, short-chain fatty acids are created and these have an important function at the level of the colon (reduction in pH, trophic effect, energy production, etc.) and at the systemic level (lipid metabolism, glycaemic metabolism, etc.) No less important are the effects of this colonic metabolism of fibre on proliferation of bacteria, thus giving fibre probiotic effects.

Tsitol Genet, 2002 Mar-Apr, 36(2), 53 - 9
{Genetic polymorphism of blood and milk enzymes and proteins and their association with economically valuable traits of black-white cattle of the western region of the Ukraine}; Sirats'kyi IZ et al.; The structure of genetic polymorphism of the blood and milk proteins and blood enzymes was studied . It was shown that in the Western region of Ukraine Black-and-White breed cattle had seven phenotypes of transferrin, one phenotype of hemoglobin, three phenotypes of amylase, five phenotypes of alkaline phosphatase, ceruloplasmine, beta-lactoglobuline, and alpha si-caseine, and three phenotypes of beta-caseine . The effects of homo- and heterozygous levels on the heifer growth, milk production and fertility of cows were found . Complex analysis of polymorphism of blood and milk ferments and proteins gives possibility to find a closer association of genetic tests with growth, milk production, and fertility of animals of Black-and-White breed.

J Antibiot (Tokyo), 2002 May, 55(5), 472 - 9
Cyclipostins: novel hormone-sensitive lipase inhibitors from Streptomyces sp . DSM 13381 . I . Taxonomic studies of the producer microorganism and fermentation results; Wink J et al.; The cyclipostins are a group of hormone-sensitive lipase inhibitors produced by a Streptomyces species . Having verticillate spore chains this strain exhibits significant differences to the known species of the former genus Streptoverticillium . Taxonomic studies and fermentation results are presented.

J Antibiot (Tokyo), 2002 May, 55(5), 467 - 71
Novel alpha-glucosidase inhibitors, CKD-711 and CKD-711a produced by Streptomyces sp . CK-4416 . III . Physico-chemical properties and structure elucidation; Chang HB et al.; We have isolated two novel a-glucosidase inhibitors, O-{4-deoxy-4-(2,3-epoxy-3-hydroxymethyl-4,5,6-trihydroxycyclohexane-1-yl-amino)-alpha-D-glucopyranosyl}-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (named CKD-711) and its hexameric analog CKD-711a, from the fermentation broth of Streptomyces sp . CK-4416 . HRFAB-MS and NMR analyses reveal that molecular formulae of CKD-711 and CKD-711a are C25H43NO20 and C37H63NO30, respectively with the latter containing two more glucose moieties than the former . Detailed chemical structures of both compounds have been characterized by high-resolution two-dimensional NMR methods.

J Antibiot (Tokyo), 2002 May, 55(5), 457 - 61
Novel alpha-glucosidase inhibitors, CKD-711 and CKD-711a produced by Streptomyces sp . CK-4416 . I . Taxonomy, fermentation and isolation; Kim JG et al.; New alpha-glucosidase inhibitors, CKD-711 and CKD-711a were produced from the fermentation broth of Streptomyces sp . CK-4416 which was isolated from a forest soil of Jeju Island, South Korea . CKD-711 and CKD-711a were purified by Dowex 50W-2X and Sephadex G-10 column chromatography . In in vitro studies, CKD-711 showed a potent inhibitory activity against a-glucosidase from mammalian, but less inhibition against a-amylase from microorganism and mammalian . CKD-711a showed a lower inhibitory activity than CKD-711.

Anal Biochem, 2002 Aug 1, 307(1), 138 - 46
Application of 2H NMR to the study of natural site-specific hydrogen isotope transfer among substrate, medium, and glycerol in glucose fermentation with yeast; Pionnier S et al.; 2H NMR is a very useful tool in isotope tracing studies . This technique was applied to a quantitative study of a site-specific deuterium affiliation among the substrate, the medium, and a product (glycerol), in glucose fermentation with yeast . The quality of the results depends on the quantitative 2H NMR analysis of glycerol . After comparing several potential analysis probe molecules, the derivative of glycerol, 2,2-dimethyl-1,3-dioxolane-4-methanol, was chosen as the most advantageous . Using this probe in a set of isotope-labeling experiments, we describe how a complete quantitative site-specific hydrogen isotope transfer model, which connects the site-specific isotopic ratios of the substrate, the medium, and the products, can be established . This model can provide information on complex hydrogen transfer mechanisms during biochemical reactions and can be useful for the prediction of site-specific hydrogen isotopic ratios at natural abundance of the products, based on that of the substrate or reactants and the medium.

J Agric Food Chem, 2002 Jul 31, 50(16), 4713 - 7
Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination; Romero R et al.; Pattern recognition techniques, such as principal component analysis, cluster analysis, and linear discriminant analysis, have been applied to samples of red, white, and rose wines to determine whether some biogenic amines could be considered as chemical descriptors . Eight amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) were determined by RP-HPLC, after derivatization with dabsyl chloride . However, only putrescine, cadaverine, histamine, tyramine, spermidine, and spermine were found in the wines analyzed . From the association between variables obtained by principal component analysis and clustering and from the relationship found by linear discriminant analysis, it can be deduced that the amines generated during malolactic fermentation (putrescine, histamine, and tyramine) could be used as chemical descriptors to characterize table wine samples.

Bioresour Technol, 2002 Aug, 84(1), 97 - 100
Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium; Adham NZ; Natural oils with high unsaturated fatty acids content when added at concentrations of 2% and 4% (v/v) to beet molasses (BM) medium caused a considerable increase in citric acid yield from Aspergillus niger . The fermentation capacities were also examined for production of citric acid using BM-oil media under different fermentation conditions . Maximum citric acid yield was achieved in surface culture in the presence of 4% olive oil after 12 days incubation.

Fungal Genet Biol, 2002 Aug, 36(3), 224 - 33
Inactivation of a cytochrome P-450 is a determinant of trichothecene diversity in Fusarium species; Brown DW et al.; Species of the genus Fusarium produce a great diversity of agriculturally important trichothecene toxins that differ from each other in their pattern of oxygenation and esterification . T-2 toxin, produced by Fusarium sporotrichioides, and nivalenol (NIV), produced by some strains of F . graminearum, contain an oxygen at the C-4 position . Deoxynivalenol (DON), produced by other strains of F . graminearum, lacks a C-4 oxygen . NIV and DON are identical except for this difference, whereas T-2 differs from these trichothecenes at three other carbon positions . Sequence and Northern analyses of the F . sporotrichioides genomic region upstream of the previously described core trichothecene gene cluster have extended the cluster by two genes: TRI13 and TRI14 . TRI13 shares significant similarity with the cytochrome P-450 class of enzymes, but TRI14 does not share similarity with any previously characterized proteins . Gene disruption and fermentation studies in F . sporotrichioides indicate that TRI13 is required for the addition of the C-4 oxygen of T-2 toxin, but that TRI14 is not required for trichothecene biosynthesis . PCR and sequence analyses indicate that the TRI13 homolog is functional in NIV-producing strains of F . graminearum but nonfunctional in DON-producing strains of the fungus . These genetic observations are consistent with chemical observations that biosynthesis of T-2 toxin and NIV requires a C-4 hydroxylase while biosynthesis of DON does not.

Z Naturforsch {C}, 2002 May-Jun, 57(5-6), 452 - 8
Extracellular polysaccharides of Penicillium vermiculatum; Kogan G et al.; Extracellular Polysaccharide, Isolation, Characterization Extracellular polysaccharide mixture that forms a viscous mucilate in the fermentation medium during industrial cultivation of Penicillium vermiculatum was isolated by ethanol precipitation and its structural characteristics were investigated by a combination of physicochemical methods . The mixture contained two structurally different polysaccharides similar to those previously described for some fungal species . This is the first report of the fully structurally characterized extracellular polysaccharides of Penicillium vermiculatum.

Eur J Cancer Prev, 2002 Jun, 11(3), 271 - 81
Butyrate and aspirin in combination have an enhanced effect on apoptosis in human colorectal cancer cells; Menzel T et al.; Laboratory and epidemiological studies suggest that butyrate, a metabolic product of microbial fermentation of dietary fibre, and aspirin, a non-steroidal antiphlogistic drug, both reduce the risk of developing colon cancer . Notably, few data exist on potential interactions of these two substances . In this study, the effects of a butyrate-aspirin combination on human colon cancer cells were compared with treatment with aspirin or butyrate alone . Both substances decreased proliferation and induced differentiation and apoptosis . Butyrate reduced mutant p53 expression, whereas aspirin did not affect p53 expression . Butyrate-induced apoptosis correlated with an increase in Bak expression and a decrease in the expression of Bcl-XL . Aspirin had no effect on the investigated apoptosis-controlling factors . The antiproliferative and pro-apoptotic effects of the butyrate-aspirin combination were markedly enhanced . The combination resulted in a stronger decrease in the expression of PCNA and cdk2 . Our data suggest that the anticarcinogenic effect of aspirin might effectively be augmented by combination with the short-chain fatty acid butyrate.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 59 - 65
Enhanced production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli; Gao HJ et al.; Wild-type bacteria including Escherichia coli normally do not produce extracellular D-(-)-3-hydroxybutyric acid (3HB) . To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), phosphor-transbutyrylase (ptb) and butyrate kinase (buk) in E . coli strain DH5alpha . E . coli DH5alpha containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phbA and phbB genes produced small amounts of 3HB, ranging from 75 to 400 mg l(-1), while E . coli DH5alpha harboring p68CMPTK containing genes of phbA, phbB, ptb and buk increased the 3HB concentration to 1.4 g l(-1) in shake flasks supplemented with LB broth and 20 g l(-1) glucose . 3HB production was further improved to over 2 g l(-1) in shake flasks when E . coli DH5alpha hosted two plasmids simultaneously that separately contained phbA and phbB in one plasmid while ptb and buk in the other . A batch fermentation run in a 5-l fermenter produced approximately 5 g l(-1) 3HB after 24 h . A fed-batch process increased 3HB production to 12 g l(-1) after 48 h of fermentation.

Adv Biochem Eng Biotechnol, 2002, 76, 233 - 55
Continuous annular chromatography; Wolfgang J et al.; In recent years the demand for process scale chromatography systems in the industrial downstream process has been increasing steadily . Chromatography seems to be the method of choice when biological active compounds must be recovered from a mixture containing dozens of side products and contaminants as it is for example the case when processing fermentation broths . Since chromatography can solve almost any separation problem under mild operating conditions, a continuous chromatography system represents an extremely attractive and powerful option for such large-scale applications . The increasing number of biotechnological products forces system suppliers of the downstream processing side to develop new and improved high throughput purification technologies . Continuous Annular Chromatography (CAC) has been shown to be the only continuous chromatography technique to fulfill the high demands raised by modern biotechnological productions . In recent years Prior Separation Technology has transferred the principle of Continuous annular chromatography from the research laboratories to the fully developed industrial downstream process scale . The technology is now called Preparative Continuous Annular Chromatography--P-CAC . It can be placed at any stage in the downstream line starting at the very early stages where capturing and concentration of the desired product is required down to the polishing steps, which assure a sufficient final purity of the end product.

J Vet Med A Physiol Pathol Clin Med, 2002 Jun, 49(5), 251 - 5
Effect of vitamin E on ruminal fermentation in vitro; Naziroglu M et al.; The effects of vitamin E on pH value, total protozoa counts, volatile fatty acid (VFA), ammonia nitrogen and lactate levels were examined using an in vitro ruminal incubation system . The ruminal fluid (100 ml) of the first and second group was supplemented with 0.4 mg or 0.8 mg of vitamin E, respectively . Samples were taken immediately before and following 3, 6, 12 and 24 h of incubation at 39 degrees C and analysed for the total protozoa counts, the pH and the levels of ammonia nitrogen, lactate and VFA . Levels of propionate at 24 h and ammonia nitrogen at 12 and 24 h were significantly higher in the second group than in the control . In contrast, the levels of butyrate at 6, 12 and 24 h and lactate at 6, 12 and 24 h were lower in the second group than in the control . Propionate at 24 h, acetate levels at 6, 12 and 24 hand ammonia nitrogen levels at 6, 12 and 24 h and total rumen protozoa counts at 6, 12 and 24 h were significantly higher in the second group as compared with control . In contrary, butyrate levels at 6, 12 and 24 h, lactate levels at 6, 12 and 24 h were lower in second group than in control . There was no statistically significant difference among the groups in the pH values . In conclusion, the addition of vitamin E to in vitro ruminal fluid was found to increase the concentrations of acetate and propionate, total counts of protozoa, levels of ammonia nitrogen, but to decrease the butyrate and lactate levels of the ruminal aliquots in in vitro ruminal fermentation.

J Mol Microbiol Biotechnol, 2002 Jul, 4(4), 453 - 61
The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea; Deppenmeier U et al.; The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines) . Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g . in rice paddies . The 4,096,345 base pairs circular chromosome of M . mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997) . 3,371 open reading frames (ORFs) were identified . Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain . 544 of these ORFs reach significant similarity values only in the bacterial domain . They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response . Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes . These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.

Yeast, 2002 Aug, 19(11), 923 - 32
Dissection of the promoter of the HAP4 gene in S . cerevisiae unveils a complex regulatory framework of transcriptional regulation; Brons JF et al.; In S . cerevisiae, the heteromeric Hap2/3/4/5 complex is necessary for induced transcription of a large number of genes involved in oxidative metabolism on non-fermentable carbon sources . The Hap4p subunit is the activator subunit and at the same time also the regulatory part of the complex, since it is the only one whose level is regulated by carbon source itself . HAP4 promoter analysis shows a 265 bp activating region at position -1006/-741 bp upstream of the ATG start codon . Specific and differential protein-binding to a 30 nt CSRE-like sequence within this region was observed with extracts from repressing and inducing carbon sources . Carbon source-dependent activation mediated by the 265 bp fragment, as well as protein binding to the 30 nt CSRE-like region, is dependent on the presence of CAT8 function, unveiling a complex framework by which the expression of the HAP4 gene is coordinated .

J Physiol, 2002 Jul 15, 542(Pt 2), 493 - 500
Loads, capacities and safety factors of maltase and the glucose transporter SGLT1 in mouse intestinal brush border; Lam MM et al.; Safety factors are defined as ratios of biological capacities to prevailing natural loads . We measured the safety factor of the mouse intestinal brush-border hydrolase maltase in series with the glucose transporter SGLT1, for comparison with previous studies of sucrase and lactase . Dietary maltose loads increased 4-fold from virgin to lactating mice . As in previous studies of intestinal adaptive regulation, that increase in load without change in dietary composition resulted in an increase in maltase and SGLT1 capacities mediated non-specifically by an increase in intestinal mass, without change in maltase or SGLT1 activities per milligram of tissue . Maltase and SGLT1 capacities increased only sublinearly with load during lactation, such that safety factors decreased with load: from 6.5 to 2.4 for maltase, and from 1.1 to 0.5 for SGLT1 . The apparently high safety factor for maltase may be related to the multiple natural substrates hydrolysed by the multiple sites of maltase activity . The apparently low safety factor for SGLT1 is made possible by the contribution of hindgut fermentation to carbohydrate digestion . SGLT1 activity is paradoxically higher for mice consuming sucrose than for mice consuming maltose, despite maltose hydrolysis yielding double the glucose load yielded by sucrose hydrolysis, and despite glucose constituting the load upon SGLT1.

Chem Commun (Camb), 2002 Feb 7, (3), 204 - 5
The biosynthesis of bisvertinolone: evidence for oxosorbicillinol as a direct precursor; Abe N et al.; Biosynthetic incorporation of labeled sodium acetate into oxosorbicillinol in Trichoderma sp . USF-2690 suggests that oxosorbicillinol is derived from six acetate units, and subsequent bioconversion of the labeled oxosorbicillinol to bisvertinolone in the fermentation of the strain suggests that bisvertinolone is biosynthesized from oxosorbicillinol and sorbicillinol in a Michael-type reaction.

Bioresour Technol, 2002 Sep, 84(3), 299 - 301
Studies on desorption of individual textile dyes and a synthetic dye effluent from dye-adsorbed agricultural residues using solvents; Robinson T et al.; Two solvents, A and B (A: methanol, chloroform, water in the ratio 1:1:1; B: 50% methanol), were used to extract textile dyes adsorbed onto substrates for the purpose of future analyses of the amount of dyes degraded through solid state fermentation (SSF) using white rot fungi . Barley husk, apple pommace and corncob were separately soaked in five different dye solutions and a synthetic textile effluent . A maximum value of 93% desorption of Cibacron Red from corncob was achieved using solvent A . Barley husk was the only substrate from which the synthetic textile effluent could be desorbed, with 82% being recovered using solvent A.

Carcinogenesis, 2002 Jul, 23(7), 1131 - 7
The colonic response to genotoxic carcinogens in the rat: regulation by dietary fibre; Hu Y et al.; The apoptotic response to DNA damage appears to be an innate biological mechanism for protection against tumourigenesis . It is possible that agents that protect against colorectal cancer act by enhancing the apoptotic deletion of cells suffering DNA damage, with consequent removal of those with tumourigenic mutations . We examined the acute apoptotic response to genotoxic carcinogens ("AARGC") in colonic epithelium and the possibility that dietary fibres of different fermentability might regulate AARGC . To fully define the time-course and nature of AARGC in response to the carcinogen azoxymethane (AOM), a single injection of AOM (10 mg/kg) was given to rats and apoptosis monitored in the colon by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling staining over a 72 h period . Having defined the site and time of maximum response, two groups of eight rats were fed diets containing 10% wheat bran fibre (WB; fermentable) or 10% methylcellulose (MC; poorly fermentable) for 4 weeks . Colonic AARGC was compared by light microscopy; lumenal short chain fatty acids (SCFAs) and pH were measured as indicators of the fermentative environment . AOM-induced AARGC was maximal at 8 h and greater in distal compared with proximal colon . Apoptotic cells were situated predominantly in the lower half of the crypt, with the median at position 9 indicating involvement of daughter as well as stem cells . There was no "second wave" of apoptosis within 72 h as follows irradiation in small intestine . Distal colonic AARGC in rats fed WB was twice that in rats fed MC (P < 0.01) . Compared with MC, WB significantly lowered lumenal pH and increased all SCFAs including butyrate, while proliferation did not differ between the fibres . Certainly, dietary fibres can regulate AARGC and further studies are warranted to determine if this biological effect is the way in which dietary factors regulate tumourigenesis . Lumenal generation of butyrate may enhance AARGC as butyrate is proapoptotic in vitro.

Br J Nutr, 2002 Jul, 88(1), 73 - 80
Influence of the pattern of peptide supply on microbial activity in the rumen simulating fermenter (RUSITEC); Russi JP et al.; The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation . The energy source was fibre prepared from sugar-beet pulp . N was added as NH3 continuously infused (AC) or peptides (Bacto(R) Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS) . Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment . Treatments had no effect on DM degradation . Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment . Microbial N flow tended to be higher with the peptide additions (P<0.061), with no significant difference between the two peptides treatments . The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0.05) and the proportion of LAM derived from NH3 lower (P<0.05) . However, LAM only accounted for 20-30 % total microbial population . Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply . This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

J Cell Physiol, 2002 Jul, 192(1), 81 - 92
Cystathionine beta-synthase is coordinately regulated with proliferation through a redox-sensitive mechanism in cultured human cells and Saccharomyces cerevisiae; Maclean KN et al.; Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration . Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells . In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth-arrested cultures or those growing non-fermentatively on ethanol or glycerol had approximately 3-fold less activity . CBS activity assays and Western blotting indicated that growth-specific regulation of CBS is evolutionarily conserved in a range of human cell lines . CBS activity was found to be maximal during proliferation and was reduced two- to five-fold when cells became quiescent at confluence . In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum-starvation, nutrient depletion, or the induction of differentiation . Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation . The intracellular level of the CBS regulator compound, S-adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation-specific regulation of human CBS . Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo .

Biotechnol Bioeng, 2002 Jul 20, 79(2), 165 - 9
Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate; Carvalho W et al.; Candida guilliermondii cells, immobilized in Ca-alginate beads, were used for batch xylitol production from concentrated sugarcane bagasse hydrolyzate . Maximum xylitol concentration (20.6 g/L), volumetric productivity (0.43 g/L . h), and yield (0.47 g/g) obtained after 48 h of fermentation were higher than similar immobilized-cell systems but lower than free-cell cultivation systems . Substrates, products, and biomass concentrations were used in material balances to study the ways in which the different carbon sources were utilized by the yeast cells under microaerobic conditions . The fraction of xylose consumed to produce xylitol reached a maximum value (0.70) after glucose and oxygen depletion while alternative metabolic routes were favored by sub-optimal conditions .

Biotechnol Bioeng, 2002 Aug 5, 79(3), 284 - 94
Three-dimensional simulation of grain mixing in three different rotating drum designs for solid-state fermentation; Schutyser MA et al.; A previously published two-dimensional discrete particle simulation model for radial mixing behavior of various slowly rotating drums for solid-state fermentation (SSF) has been extended to a three-dimensional model that also predicts axial mixing . Radial and axial mixing characteristics were predicted for three different drum designs: (1) without baffles; (2) with straight baffles; and (3) with curved baffles . The axial mixing behavior was studied experimentally with video- and image-analysis techniques . In the drum without baffles and with curved baffles the predicted mixing behavior matched the observed behavior adequately . The predicted axial mixing behavior in the drum with straight baffles was predicted less accurately, and it appeared to be strongly dependent on particle rotation, which was in contrast to the other drum designs . In the drum with curved baffles complete mixing in the radial and axial direction was achieved much faster than in the other designs; that is, it was already achieved after three to four rotations . This drum design may therefore be very well suited to SSF . It is concluded that discrete particle simulations provide valuable detailed knowledge about particle transport processes, and this may help to understand and optimize related heat and mass transfer processes in SSF .

Biotechnol Bioeng, 2002 Aug 20, 79(4), 450 - 6
Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker; Woodgate J et al.; The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E . coli DH5alpha fermentations is addressed . A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described . This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid . It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously . Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated . Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein . The resulting complex was adsorbed directly onto glutathione-Sepharose . Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins .

Biotechnol Bioeng, 2002 Aug 20, 79(4), 429 - 37
Differential rates of gene expression monitored by green fluorescent protein; Lu C et al.; The use of green fluorescent protein (GFP) as a reporter gene has made a broad impact in several areas, especially in studies of protein trafficking, localization, and expression analysis . GFP's many advantages are that it is small, autocatalytic, and does not require fixation, cell disruption, or the addition of cofactors or substrates . Two characteristics of GFP, extreme stability and chromophore cyclization lag time, pose a hindrance to the application of GFP as a real-time gene expression reporter in bioprocess applications . In this report, we present analytical methods that overcome these problems and enable the temporal visualization of discrete gene regulatory events . The approach we present measures the rate of change in GFP fluorescence, which in turn reflects the rate of gene expression . We conducted fermentation and microplate experiments using a protein synthesis inhibitor to illustrate the feasibility of this system . Additional experiments using the classic gene regulation of the araBAD operon show the utility of GFP as a near real-time indicator of gene regulation . With repetitive induction and repression of the arabinose promoter, the differential rate of GFP fluorescence emission shows corresponding cyclical changes during the culture .

Biotechnol Bioeng, 2002 Aug 20, 79(4), 398 - 407
The response of GS-NS0 myeloma cells to single and multiple pH perturbations; Osman JJ et al.; Animal cells are cultured in several types of vessels at laboratory and industrial scale the most common being the stirred tank and the air-lift . Economically, it is preferable to culture animal cells at the largest possible scale but the perceived sensitivity of animal cells to hydrodynamic shear has, until now, limited the aeration and agitation rates used . This has been reported to cause inhomogeneities in operational parameters such as dissolved oxygen concentration, temperature and pH . pH is of special interest during the latter stages of many animal cell fermentation because alkali additions, used for pH control, can cause large local pH perturbations of varying size and duration . The effect of single and multiple pH perturbations on the cell growth of a widely used GS-NS0 mouse myeloma cell line grown in batch culture was investigated . The effect of perturbation amplitude and duration was investigated using a single stirred tank reactor (STR) . In the single STR system cells were subjected to one pH 8.0 or 9.0 perturbation ranging in duration from 0-90 minutes . No measurable decrease in viable cell number was seen for pH 8.0 perturbations of any duration whereas pH 9.0 perturbations lasting for 10 minutes caused a 15% decrease in viable cell number . The proportion of viable cells decreased with increasing perturbation time and a 90-minute exposure killed all of the cells . The effect of multiple pH perturbations on GS-NS0 cells was investigated using two connected STR's . More specifically the number of perturbations and the perturbation frequency were investigated . Cells were subjected to between 0 and 100 perturbations at pH 8.0; the time between each perturbation (frequency) was 6 minutes and each perturbation lasted for 200 seconds . Viable cell number decreased with increasing perturbation number, with 100 perturbations causing death of 27.5% of cells . Cells were also exposed to 10 perturbations at pH 9.0, each of 200 second duration at frequencies of either 6, 18 or 60 minutes . Approximately 8 times more cells were killed with perturbations at a 6-minute frequency (28.3% cell death) than at a 60-minute frequency (3.4% cell death) .

Biotechnol Bioeng, 2002 Jun 5, 78(5), 539 - 44
Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system; Rahardjo YS et al.; Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation . This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF . In cultures of A . oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus . This is due to the fact that A . oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid . It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A . oryzae than was previously reported for R . oligosporus and C . minitans . It also means that the overall oxygen uptake rate for A . oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R . oligosporus and C . minitans . This would imply that cooling problems become more pronounced . Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF .

Biotechnol Bioeng, 2002 Jun 5, 78(5), 527 - 38
Monitoring of complex industrial bioprocesses for metabolite concentrations using modern spectroscopies and machine learning: application to gibberellic acid production; McGovern AC et al.; Two rapid vibrational spectroscopic approaches (diffuse reflectance-absorbance Fourier transform infrared {FT-IR} and dispersive Raman spectroscopy), and one mass spectrometric method based on in vacuo Curie-point pyrolysis (PyMS), were investigated in this study . A diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi producing the natural product gibberellic acid, were analyzed directly without a priori chromatographic separation . Partial least squares regression (PLSR) and artificial neural networks (ANNs) were applied to all of the information-rich spectra obtained by each of the methods to obtain quantitative information on the gibberellic acid titer . These estimates were of good precision, and the typical root-mean-square error for predictions of concentrations in an independent test set was <10% over a very wide titer range from 0 to 4925 ppm . However, although PLSR and ANNs are very powerful techniques they are often described as "black box" methods because the information they use to construct the calibration model is largely inaccessible . Therefore, a variety of novel evolutionary computation-based methods, including genetic algorithms and genetic programming, were used to produce models that allowed the determination of those input variables that contributed most to the models formed, and to observe that these models were predominantly based on the concentration of gibberellic acid itself . This is the first time that these three modern analytical spectroscopies, in combination with advanced chemometric data analysis, have been compared for their ability to analyze a real commercial bioprocess . The results demonstrate unequivocally that all methods provide very rapid and accurate estimates of the progress of industrial fermentations, and indicate that, of the three methods studied, Raman spectroscopy is the ideal bioprocess monitoring method because it can be adapted for on-line analysis .

Trends Mol Med, 2002 Jul, 8(7), 324 - 9
Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine; Mason HS et al.; The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems . Genes encoding bacterial and viral antigens are faithfully expressed in edible tissues to form immunogenic proteins . Studies in animals and humans have shown that ingestion of transgenic plants containing vaccine proteins causes production of antigen-specific antibodies in serum and mucosal secretions . In general, the technology is limited by low expression levels for nuclear-integrated transgenes, but recent progress in plant organelle transformation shows promise for enhanced expression . The stability and immunogenicity of orally delivered antigens vary greatly, which necessitates further study on protein engineering to enhance mucosal delivery . These issues are discussed with regard to the further development of plant-based vaccine technology.

Acta Vet Hung, 2002, 50(2), 217 - 29
Effect of different fat sources on in vitro degradation of nutrients and certain blood parameters in sheep; Febel H et al.; This study was designed to determine the effects of calcium salt of palm oil fatty acids (CS), hydroxyethylsoyamide (HESA), butylsoyamide (BSA) and soybean oil (SO) on degradation of crude protein and fibre in vitro, and on the blood plasma lipid parameters in vivo . Five mature wethers (body weight 75 kg) were fed five diets in a 5 x 5 Latin square experiment . The control diet consisted of 50% meadow hay and 50% concentrate with no added fat . The control diet was supplemented with CS, HESA, BSA, or SO . Fat was added at 3.5% of dietary dry matter (DM) . The final ether extract content of the ration was near 6% . Each period lasted 20 days . Fat supplements, except HESA, consistently decreased the in vitro DM disappearance of soybean meal as compared to control . In contrast to the effect of other treatments, crude protein degradation was greatest in the test tubes with inocula obtained from sheep fed diet with HESA . Fat supplements equally inhibited the DM and fibre breakdown of alfalfa pellet . CS and HESA seemed to be less detrimental to in vitro fermentation of neutral detergent fibre (NDF) than BSA and SO . All fat supplements increased blood plasma triglyceride, cholesterol and total lipid content . Plasma concentration of cholesterol and total lipid was highest with SO . The inclusion of CS in the diet increased 16:0, while all fat supplements increased plasma 18:0 and decreased 16:1 and 18:1 fatty acid content . Plasma 18:2n-6 was not changed by feeding CS and SO . However, compared to the control diet, 18:2n-6 increased with 12 and 41% in plasma fatty acids when sheep were fed HESA and BSA, respectively . The results showed that plasma concentration of linoleic acid was enhanced more when the amide was synthesised from butylamine than when from ethanolamine.

J Basic Microbiol, 2002, 42(3), 201 - 6
Influence of pH on the xylose reductase activity of Candida guilliermondii during fed-batch xylitol bioproduction; Godoy De Andrade Rodrigues DC et al.; The influence of pH on the xylose reductase (XR) activity was studied . The fed-batch fermentation using exponential feeding rate was employed to produce xylitol . The feeding started when the xylose concentration in the fermenter reached 50 g/l and the pH was adjusted to 2.5, 4.0 or 6.0 . The best results for XR activity (0.567 U/mg(protein)) and xylitol volumetric productivity (1.06 g/l.h) were achieved with pH 6.0.

J Basic Microbiol, 2002, 42(3), 162 - 71
Alcohol production from starch by mixed cultures of Aspergillus awamori and immobilized Saccharomyces cerevisiae at different agitation speeds; Farid MA et al.; The influence of different agitation speeds on alcoholic fermentation by free and immobilized cells of Saccharomyces cerevisiae in a co-culture with free cells of Aspergillus awamori was investigated . Starch hydrolysis and glucose, glucoamylase and alpha-amylase accumulation in the fermentation medium was affected by agitation speed . Maximum amounts of the enzymes were obtained at 150-200 rpm after 72 h where, maximum growth and glucose accumulations were noticed at 200-300 rpm . Alcohol production was stimulated by low agitation speed (50 rpm) and decreased at higher speeds . The results also showed that the amount of produced alcohol was affected by the time of yeast inoculation . When the inoculation of yeast was carried out after the growth of fungi for 72 h, the amounts of produced alcohol increased by 84, 75, 89 and 68% at 50, 100, 150 and 200 rpm, respectively than that produced when the two organisms were inoculated together at the beginning of the fermentation process . A batch culture of the two organisms produced about 2% (v/v) alcohol from 12% (w/v) corn starch in 72 h at 50 rpm . On the other hand, the immobilized yeast and suspended A . awamori produced more alcohol reaching 3.7% (v/v) at 200 rpm under the same cultivation conditions . The fermentation process was less affected by alginate and cell concentrations of the immobilized yeast . Repeated batch fermentation by co-culture of A . awamori and immobilized yeast cells were successfully used for 12 times without a significant loss in alcohol production.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 252 - 8 Epub 2002 May 01.
Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii; Komeda T et al.; The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated . Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded . To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii . A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C . boidinii and placed downstream of the C . boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium . The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter . Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction . When the recombinant C . boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.

Equine Vet J, 2002 May, 34(3), 302 - 5
Dietary soyabean oil depresses the apparent digestibility of fibre in trotters when substituted for an iso-energetic amount of corn starch or glucose; Jansen WL et al.; The aim of the present study was to establish whether the inhibitory effect of fat feeding on fibre digestion has been underestimated due to the substitution of fat for corn starch . A high fat intake has been shown to lower total intestinal tract apparent digestibility of crude fibre in horses but, since fat was substituted for nonstructural carbohydrates, including starch, the specific effect of fat could not be ascertained . The possibility could not be excluded that starch also inhibits fibre digestibility, so that the fat effect observed earlier would have been underestimated . In this study, the intakes of iso-energetic amounts of soyabean oil, corn starch or glucose were compared as to fibre digestibility . Unlike starch, glucose is fully absorbed by the small intestine and, therefore, is not expected to influence fibre fermentation in the caecum and colon . Six trotters were fed rations high in soyabean oil (158 g/kg dry matter), corn starch (337 g/kg dry matter) or glucose (263 g/kg dry matter) according to a 3 x 3 Latin square design . Apparent crude fibre digestibility was similar for the rations with corn starch (mean +/- s.d., 70.7 +/- 3.06% of intake, n = 6) or glucose (71.0 +/- 1.90%), but was significantly depressed by fat feeding (56.5 +/- 7.65%) . Similar observations were made for apparent digestibilities of neutral and acid detergent fibre and of cellulose . It was concluded that the addition of fat to the feed ration of horses has a specific inhibitory effect on fibre utilisation and, therefore, reduces the amount of energy provided by dietary fibre.

Water Res, 2002 May, 36(9), 2337 - 41
Operating conditions for the determination of the biochemical acidogenic potential of wastewater; Ruel SM et al.; The aim of this work was to study the test conditions for the determination of the biochemical acidogenic potential (BAP) of wastewater, which should be useful to predict the performance of enhanced biological phosphorus removal (EBPR) . Proposed operating conditions for a simple and reproducible BAP test in 250 ml serum bottles (equipped with black butyl stoppers and magnetic bars) are: use of either frozen or fresh water, no inoculum addition, fermentation carried out in the dark during 15 days, addition of 1 mM bromo-ethane sulfonate (BES) and 2 mM barium chloride (BaCl2), stirring speed strong enough to maintain vortex conditions, no pH control and controlled temperature of 20 degrees C.

J Gastroenterol, 2002, 37(6), 442 - 8
Efficacy of lactulose plus 13C-acetate breath test in the diagnosis of gastrointestinal motility disorders; Urita Y et al.; BACKGROUND: We designed a new method of measuring gastric emptying and orocecal transit time (OCTT) at the same time to assess the influence of gastric emptying upon OCTT . METHODS: Twenty-five dyspeptic patients (6 men, 19 women) with a mean age of 64.8 years (range, 25-80 years) were studied . The patients received a liquid test meal, containing 100 mg of 13C-acetate and 12g of lactulose, while they were in the sitting position after an overnight fast . Breath samples were collected at 10-min intervals of 120 min and both 13CO2 and hydrogen (H2) levels were measured . Subsequently, H2 concentrations were measured at 30-min intervals, for a total of 240 min . RESULTS: The results of gastric emptying were expressed as the time of peak 13CO2 excretion . OCTT was defined as the period between the ingestion of lactulose and a H2 peak rise of 5 ppm above the baseline value . The onset of H2 enrichment in the breath began at 90-110 min, whereas 13CO2 levels increased from the beginning, with peak enrichment values being reached after 60-80 min . OCTT was related to 13CO2 peak time . In 5 of the 25 patients, H2 breath enrichment in the 10-min sample was more than 5 ppm over the baseline value . All these 5 patients had double or triple peaks in serial breath H2 concentrations . CONCLUSIONS: The combination of the lactulose hydrogen breath test (LHBT) with the 13C-acetate breath test, which requires only breath samples, provides us with much information on the gastrointestinal tract; gastric emptying, OCTT, bacterial overgrowth in the small intestine, colonic fermentation, and oropharyngeal flora . The 13C-acetate breath test can be useful as an adjuvant test when LHBT is performed for measuring OCTT.

Nahrung, 2002 Jun, 46(3), 204 - 8
Content of strontium, lithium and calcium in selected milk products and in some marine smoked fish; Nabrzyski M et al.; The strontium, lithium and calcium contents have been determined in 87 samples of five kinds of milk-fermented products, as well as in 93 samples of ten kinds of marine smoked fish . The samples were purchased on the local market . The representative samples were dry ashed in quartz crucibles and the ash was treated with suitable amounts of conc . HCl and a few drops of conc . HNO3 . The obtained sample solution was then used for the determination of Sr, Li and Ca by the flame atomic absorption spectrometry (AAS) method . Ca and Li were determined using the air-acetylene flame and Sr with nitrous oxide-acetylene flame, according to the manufacturer's recommendations . The contents of Sr in the fermented milk products ranged from 0.21 to 0.79 (mean 0.44 +/- 0.07) and in the marine smoked fish from 0.02 to 4.63 (mean 1.16 +/- 0.24) mg/kg of the edible form of both products . Li contents in the milk products ranged from 0.01 to 0.50 (mean 0.07 +/- 0.04) and in the smoked fish from 0.00 to 0.58 (mean 0.11 +/- 0.08) mg/kg . The calcium was highest in the milk products and ranged from 1,010 to 2,020.0 (mean 1,377 +/- 143) mg/kg . In the smoked fish calcium varied strongly and ranged from 40 to 1,052 (mean 303 +/- 53) mg/kg of the edible form . The calculated average ratio of strontium to calcium (mg Sr/1 g Ca) in the milk beverages and yogurts amounted 0.32 and in the smoked fish was 12 times as high and amounted 3.84 . The main purpose of this work was to present Ca, Sr and Li together . This is justified because Sr and to some extent also Li are able to modulate Ca metabolism and vice versa.

Nahrung, 2002 Jun, 46(3), 158 - 66
Structure, physicochemical properties and in vitro fermentation of enzymatically degraded cell wall materials from apples; Forster S et al.; Cell wall materials (CWM) prepared from apple parenchyma tissue by treatment with commercial enzymes for maceration, mash fermentation and liquefaction were characterised with regard to their composition and structure as well as their physicochemical and physiological properties . Increasing enzymatic degradation of the CWM resulted in growing loss of the pectin matrix, decreasing porosity as well as increasing particle aggregation . Due to these structural alterations the water binding, the viscoelastic properties of the CWM-water-suspensions and the in vitro fermentation, forming short chain fatty acids, were reduced . The investigations showed that interrelations exist between enzymatic treatment and changes of (i) structure and state of matrices (evaluated by means of thermal analysis), (ii) physicochemical properties and (iii) physiological properties . So the application of liquefying enzymes can lead to a complete removal of the pectin matrix, causing an essentially improved thermal stability of the CWM preparation, but strongly reduced water binding and reduced structure-forming properties into the CWM-water-suspensions . The formation of short-chain fatty acids during in vitro fermentation of the CWM preparations by fresh human faeces flora depended on the portion and the state of the pectin matrix and the cellulose network, respectively.

Microbes Infect, 2002 Jul, 4(9), 963 - 8
Choline-containing lipids in mycoplasmas; Rottem S; Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic cells, and to induce cytokine secretion by cells of the immune system . These findings suggest that choline-containing lipids are important mediators of tissue pathology in the infectious process caused by M . fermentans.

J Agric Food Chem, 2002 Jul 17, 50(15), 4303 - 9
"Untypical aging off-flavor" in wine: synthesis of potential degradation compounds of indole-3-acetic acid and kynurenine and their evaluation as precursors of 2-aminoacetophenone; Hoenicke K et al.; Kynurenine (1) and indole-3-acetic acid (2) are considered as potential precursors of 2-aminoacetophenone (3), which is regarded to be the aroma impact compound causing an "untypical aging off-flavor" (UTA) in Vitis vinifera wines . The mechanism of the formation of 3 was studied using model fermentation and model sulfuration media spiked with 1 or 2 as potential precursors . Possible degradation products such as kynurenamine (4) and kynurenic acid (5), or skatole (6), 2-oxoskatole (7), 2-formamidoacetophenone (8), 2-oxindole-3-acetic acid (9), and 3-(2-formylaminophenyl)-3-oxopropionic acid (10) were evaluated by HPLC-UV of the fermentation and sulfuration media and comparison with synthesized 7, 8, 9, and 10 . The synthesis of the possible precursor 4-(2-aminophenyl)-2,4-dioxobutanoic acid (11), a proposed metabolite of 1 failed because a spontaneous cyclization yields 5 and N-oxo-kynurenic acid (12), but not 11 . It could be shown that the formation of 3 is triggered by an oxidative degradation of 2 after sulfuration with potassium bisulfite via the intermediates 10 and 8 . However, no formation of 3 occurred during sulfuration of a model wine spiked with 1 or during fermentation of a model must spiked with 1 or 2.

J Agric Food Chem, 2002 Jul 17, 50(15), 4221 - 5
Chemical and biochemical transformations during the industrial process of sherry vinegar aging; Palacios V et al.; The work described here concerns a study of the chemical and biochemical transformations in sherry vinegar during the different aging stages . The main factors that contribute to the nature and special characteristics of sherry vinegar are the raw sherry wine, the traditional process of acetic acid fermentation in butts (the solera system), and the physicochemical activity during the aging process in the solera system . A number of chemical and biochemical changes that occur during sherry vinegar aging are similar to those that take place in sherry wine during its biological activity process (where the wine types obtained are fino and manzanilla) or physicochemical activity process (to give oloroso wines) . Significant increase in acetic acid levels was observed during the biological activity phase . In addition, the concentrations of tartaric, gluconic, succinic, and citric acids increased during the aging, as did levels of amino acids and acetoin . A color change was also produced during this stage . Glycerol was not consumed by acetic acid bacteria, and levels of higher alcohols decreased because of the synthesis of acetates . On the other hand, in the physicochemical phase the microbiological activity was lower . Concentrations of some cations increased because of evaporation of water through the wood . A color change was also produced in this stage . Concentrations of different amino acids decreased because of reaction with carbonyl compounds . A precipitation of potassium with tartaric acid was also observed.

Biomol Eng, 2002 Jun, 19(1), 5 - 15
Metabolomics: quantification of intracellular metabolite dynamics; Buchholz A et al.; The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism . A process by which this information can be derived from dynamic fermentation experiments is presented . By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state . With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus . Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods . Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated . The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.

Mol Biotechnol, 2002 Jul, 21(3), 279 - 92
Identification of yeasts present in sour fermented foods and fodders; Middelhoven WJ; This paper deals with rapid methods for identification of 50 yeast species frequently isolated from foods and fodders that underwent a lactic acid fermentation . However, many yeast species present in olive brine, alpechin, and other olive products were not treated . The methods required for identification include light microscopy, physiological growth tests (ID32C system of BioMerieux), assimilation of nitrate and of ethylamine as sole nitrogen sources, vitamin requirement, and maximum growth temperature . An identification key to treated yeast species is provided . In another table characteristics of all yeast species treated are listed.

Lett Appl Microbiol, 2002, 35(2), 117 - 20
The effect of nitrogen and carbon sources on growth of the biocontrol agent Pantoea agglomerans strain CPA-2; Costa E et al.; AIM: The effect of several nitrogen and carbon sources on the growth of Pantoea agglomerans (strain CPA-2) was studied for the first time . METHODS AND RESULTS: Synthetic nitrogen and carbon sources were tested to obtain a suitable medium . Synthetic yeast extract provided maximum growth and disaccharides such as sucrose, lactose and trehalose improved this growth significantly from 3.2 x 10(9) to 5.5 x 10(9) cfu ml-1 . CONCLUSION: Pantoea agglomerans can be produced in a combination of nitrogen sources such as yeast extract with carbohydrates such as sucrose in shake flask and a laboratory fermenter (5 l) . SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest good production of this biocontrol agent on a laboratory scale and the potential of scaling up the process.

Lett Appl Microbiol, 2002, 35(2), 98 - 101
Protective agents used to reverse the metabolic changes induced in wine yeasts by concomitant osmotic and thermal stress; Caridi A; AIMS: The reversion of the metabolic changes induced in wine yeasts by stressors . METHODS: Six strains of Saccharomyces were inoculated in grape must containing over 400 g l-1 of sugar and incubated at 35 degrees C, both with and without the addition of 100 mg l-1 of catechin, inositol or SO2 . RESULTS: Significant correlations between addition of the stress-protectants and change in the metabolic behaviour of the wine yeasts were observed . Depending on strain and protectant, and expressing data as a percentage of increase or decrease compared to the control, fermentation vigour after 3 d increased by up to 10%, titratable acidity of the wines increased by up to 7%, ethanol content increased by up to 20%, unitary acetic acid production decreased by up to 35%, and unitary glycerol production decreased by up to 20% . IMPACT OF STUDY: By using protective agents it is possible to minimize the abnormal fermentation performance that wine yeasts exhibit under thermal and osmotic stress.

Ann Bot (Lond), 2002 May, 89(5), 551 - 8
Parameters affecting the early seedling development of four neotropical trees under oxygen deprivation stress; Kolb RM et al.; Some of the parameters that determine flooding resistance-and consequently habitat zonation-were investigated in four neotropical trees (Schizolobium parahyba, Sebastiania commersoniana, Erythrina speciosa and Sesbania virgata) . The constitutive parameters of seeds (size, nature and amount of reserves) only partly influenced resistance to flooding, mainly through a high carbohydrate : size ratio . Parameters describing metabolic efficiency under stress conditions were more important . Among them, fermentation capacity and levels of ATP and of total adenylates played a key role . The highest resistance to anoxia was associated with increased availability of free sugars, elevated alcohol dehydrogenase activity and corresponding mRNA levels, more efficient removal of ethanol and lactate, and higher adenylate levels . Finally, as a lethal consequence of energy shortage, free fatty acids were released on a massive scale in the flooding-sensitive species Schizolobium parahyba, whereas lipid hydrolysis did not occur in the most resistant species Sesbania virgata.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 526 - 31
Expression of gene encoding GL-7ACA acylase in Escherichia coli; Wang ED et al.; Glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp.130 catalyzes hydrolysis of glutaryl 7-amino cephalosporanic acid to produce 7-amino cephalosporanic acid (7-ACA) . 7-ACA is the starting material for the industrial production of most cephalosparonic derivatives . Six plasmids for expression of GL-7ACA acylase were constructed and these recombin ant plasmids presented different expression characteristics in Escherichia coli . The acylase gene from plasmid pKKCA1 was inserted into plasmid pMFT7-5 and the resulting plasmid pMFT7CA1 has higher expression in E.coli . The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7CA1 was near 5 u/g, so the over produced enzyme was easily purified by a single-step anion exchange column chromatography . The enzyme could be purified by immobilized ion affinity chromatography after fused by 6xHis in the N-terminal of its alpha-subunit . Because plasmid pSMLCA1 brings tc(R) and p15A origin, it is special useful plasmid in fermentation . Two secretory expression plasmids, pSUCA1S and pETCA1pelB, could secrete the acylase to periplasmic space of bacteria . The whole cells containing the secretory expression plasmid may be used for production of 7-ACA directly.

J Org Chem, 2002 Jul 12, 67(14), 5001 - 4
Novel sesquiterpenoids from the fermentation of Xylaria persicaria are selective ligands for the NPY Y5 receptor; Smith CJ et al.; Neuropeptide Y (NPY) is a polypeptide found in the peripheral and central nervous system and is involved in the regulation of feeding . Antagonists of NPY receptor activation could therefore have potential for development as antiobesity drugs . Fermentation of an isolate of Xylaria persicaria yielded two novel eremophilane sesquiterpenoids xylarenals A (1) and B (2) . These compounds are selective for the NPY Y5 receptor but have only modest affinity . The isolation, structure elucidation, and biological activities of these compounds are described.

J Nutr, 2002 Jul, 132(7), 2082 - 6
Resveratrol enhances the differentiation induced by butyrate in caco-2 colon cancer cells; Wolter F et al.; Butyrate, a short-chain fatty acid produced in the colon by microbial fermentation of fiber, inhibits growth of colonic carcinoma cells while inducing differentiation . Resveratrol, a plant polyphenol found in red wine and peanuts, has been shown to exert chemopreventive properties on colon cancer cells . The aim of this study was to determine whether resveratrol modulates the effects of butyrate on Caco-2, a colonic adenocarcinoma cell line . The growth inhibitory effect of resveratrol (50 micromol/L) was more powerful than that of butyrate (2 mmol/L) . Butyrate did not intensify the inhibition of proliferation exerted by resveratrol . Although the polyphenol enhanced the differentiation-inducing effect of butyrate, it did not elevate alkaline phosphatase activity or E-cadherin protein expression, markers of epithelial differentiation, when applied alone . Butyrate-induced transforming growth factor-beta1 secretion was inhibited by resveratrol . Treatment with the combination of resveratrol and butyrate attenuated levels of p27(Kip1), whereas resveratrol enhanced butyrate's effect on the induction of p21(Waf1/Cip1) expression . These data demonstrate a possible combined chemopreventive effect of two substances naturally occurring in the colonic lumen after ingestion of fibers and resveratrol-containing food.

Bioresour Technol, 2002 Jul, 83(3), 229 - 33
Microbial production of extra-cellular phytase using polystyrene as inert solid support; Gautam P et al.; Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support . Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A . ficuum and 4.52 U/gds for R . oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1.3 x 10(7) spores/5 g substrate, for 72 h for A . ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1 x 10(6) spores/5 g substrate for 96 h for R . oligosporus . Results indicated scope for production of phytase using polystyrene as inert support.

Folia Microbiol (Praha), 2002, 47(3), 283 - 6
Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium; Verma P et al.; Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production by Phanerochaete chrysosporum under solid-state fermentation conditions . Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL . The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated . Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.

Trop Anim Health Prod, 2002 May, 34(3), 231 - 9
Assessment of molasses-urea blocks for goat and sheep production in the Sultanate of Oman: intake and growth studies; Forsberg NE et al.; The goal of this study was to develop a molasses-urea block (MUB) for purposes of supplementing trace minerals to domestic ruminant livestock in Oman . To accomplish this, the utility of molasses and date syrup as fermentable energy sources, of straw, date flakes and wheat bran as fibre sources, and of cement and lime as binders were evaluated . The proportion of cement needed for adequate hardening of the block was also studied . Molasses- and date syrup-based blocks hardened equally well . However, the higher cost of date syrup precluded its use . Wheat straw yielded a low-density block that hardened slowly . Date fibre retained moisture and hardened extremely slowly . Wheat bran-based blocks hardened quickly and yielded dense blocks . Hence, wheat bran was judged to be the superior source of fibre . Lime did not effectively bind the blocks . A cement content of 15% allowed hardening of the blocks within 2-3 weeks . A level of 10% cement in the block reduced the hardening rate by about 50% . Sheep and goats consumed both the straw- and wheat bran-based blocks but at different rates . Consumption of the straw-based block by sheep ranged from 50 to 179 g/head per day, whereas the denser wheat bran-based block was consumed at a rate of 8-20 g/head per day . Consumption of the straw-based block by goats was low (8 g/head per day) compared to that of wheat bran-based blocks (16-24 g/head per day) . On the basis of the intake of the bran-based block by sheep, a block was designed that would provide approximately 50% of an animal's trace mineral requirements per day . This block consisted of 45% molasses, 10% urea, 5% trace minerals, 2.5% NaCl, 22.5% wheat bran and 15% cement . Sheep consuming this block gained more weight than sheep fed a conventional mineral block or sheep receiving no mineral supplementation . MUBs are inexpensive (9.5 US cents/kg) . We conclude that MUBs have utility for providing trace elements in ruminant diets.

Trop Anim Health Prod, 2002 May, 34(3), 215 - 30
Ruminal fermentation kinetics in ewes offered a maize stover basal diet supplemented with cowpea hay, groundnut hay, cotton seed meal or maize meal; Chakeredza S et al.; The effect on ruminal digestive kinetics of supplementing maize stover (MS) with cowpea hay (CW), groundnut hay (GN), cotton seed meal (CSM) or maize meal (MM) was evaluated in a 5 x 5 Latin square using five Merino ewes with ruminal cannulae . CW and GN were offered at 30% w/w to MS, while CSM and MM were given at 15 g/kg0.75 per day . There were significant time (p < 0.001) and time x diet (p < 0.05) effects on pH but the effect of diet alone was not significant (p > 0.05) . There was a significant (p < 0.05) increase in the concentration of ammonia with time on the supplemented diets . There were no significant (p > 0.05) differences in the concentrations of total volatile fatty acids (VFAs) . Butyrate increased with supplementation (p < 0.05) . Supplementation increased the concentrations of branched-chain and total VFAs with time (p < 0.05) . The non-glucogenic ratio was not significantly (p > 0.05) lower on supplemented diets owing to the higher butyrate concentration . The degradation curve constants were not significantly different (p > 0.05), the average effective dry matter degradation being 27.80% +/- 0.618% at 0.05 h-1 flow rate . The chemical and physical characteristics of the cell wall of MS may have presented few sites for microbial colonization, so limiting degradation . These results showed that small quantities of forage supplements can improve the ruminal environment in animals subsisting on low-quality forages and form a basis for comparison with bought-in protein and energy sources.

Food Nutr Bull, 2002 Jun, 23(2), 190 - 5
Effect of amylase-rich flour (ARF) treatment on the viscosity of fermented complementary foods; Uvere PO et al.; Grains of cowpea and maize and slices of fresh cassava, cocoyam, plantain, and yam were steep-fermented in water, while flours from the same plant materials were fermented by backslopping for 24 to 30 hours . The pH and apparent viscosity of the gruels from the resulting flours were determined . Loss in weight due to fermentation was higher in fresh tubers than in dry grains . The pH of the flours decreased during fermentation . Measurements showed that the apparent viscosity only of gruels from flours produced by backslopping decreased after 24 hours . The apparent viscosity of gruels from steep-fermented flours was higher than that of the unfermented flours and those produced by backslopping . The apparent viscosity reduction of gruels from steep-fermented flours using amylase-rich flour (ARF) from five-day white sorghum malt was better with the prepared gruels than when applied to the flour-in-tap-water suspension before it was used to prepare gruels . Viscosity reduction using ARF was also better with the fermented gruels than with gruels from unfermented flours . The implications of these results for the formulation of complementary flour blends for infant feeding are discussed.

Nutr Cancer, 2001, 41(1-2), 156 - 64
Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal; Ebert MN et al.; Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells . The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment . As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells . We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein . The genotoxic impact of HNE (100-200 microM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis . Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24-72 h of incubation with 4 mM butyrate . Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate . Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway . Most effects were greater in HT29 parent cells than in clone cells . In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity . Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.

Nutrition, 2002 Jul-Aug, 18(7-8), 636 - 42
Ingestion of water-soluble soybean fiber improves gastrectomy-induced calcium malabsorption and osteopenia in rats; Shiga K et al.; OBJECTIVE: Total gastrectomy produces calcium malabsorption and osteopenia . We examined the effects of feeding water-soluble soybean fiber (WSSF), a highly fermentable dietary fiber with low viscosity, on calcium absorption and bone characteristics in totally gastrectomized rats . METHODS: Male Sprague-Dawley rats were assigned to two groups: sham operation and gastrectomy . After the postoperative recovery period, rats of each group were fed diets with or without WSSF (50 g/kg diet) for 4 wk . RESULTS: Net calcium absorption and femoral dry weight and calcium content and maximum breaking force were lower in the gastrectomy groups than in the sham groups . In the gastrectomy groups, calcium absorption and these femoral parameters were higher in rats fed the WSSF diet than in rats fed the WSSF-free diet . Feeding WSSF increased the concentrations of short-chain fatty acids and soluble calcium and decreased the pH in cecal contents . Net calcium absorption in gastrectomized rats correlated positively with the concentration of total short-chain fatty acids (the sum of acetic, propionic, and butyric acids) in cecal contents (r = 0.545, P < 0.05) and negatively with cecal pH (r = -0.628, P < 0.05) . CONCLUSIONS: These findings indicate that ingestion of WSSF partly prevents the diminished calcium absorption after total gastrectomy in rats, resulting in the improvement of postgastrectomy osteopenia . The increase of calcium absorption may be associated with cecal fermentation of WSSF.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(6), 1087 - 97
Effect of agitation and aeration on bioconversion of domestic wastewater sludge in a batch fermenter; Alam MZ et al.; Effects of agitation and aeration rate on microbial treatment of domestic wastewater sludge were investigated in a batch fermenter using mixed culture of Penicillium corylophilum and Aspergillus niger . It was found that liquid state bioconversion (LSB) of wastewater sludge was highly influenced by the effects of agitation and aeration . The maximum production of sludge cake and reduction of organic substances in treated sludge were recorded at 150-200 rpm of agitation speed and 0.5 vvm of aeration rate after 72 h of treatment . No effective results were observed at higher rate of agitation (300 rpm) and aeration (1.5 vvm) as compared to optimum values . The results showed that the minimum level of air saturation (pO2) was adequate to maintain the bioconversion process.

Int J Food Sci Nutr, 2002 Jul, 53(4), 343 - 9
Loss of residual cyanogens in a cassava food during short-term storage; Onabolu AO et al.; Residual cyanogens in gari, the most popular cassava food in West Africa, is implicated in the causation of tropical ataxic neuropathy . Gari is eaten by soaking its granules in cold water or by adding boiling water to make a food called eba . This study was conducted to determine whether loss of the residual cyanogens in gari during short-term storage and when gari is made into eba will reduce dietary cyanide load in consumers . Fifteen samples of gari, nine roasted from cassava mash fermented for at least 4 days (type A) and six roasted from cassava mash fermented for only 1 day (type B), were stored for 4 weeks . Free cyanide, linamarin, and cyanohydrin in gari and in eba made from the gari were determined at weekly intervals for 4 weeks . Free cyanide was absent in all samples of gari . Mean cyanohydrin dropped from 8.4 mg HCN Eq/kg dry weight to 4.6 mg HCN Eq/kg dry weight in type A gari, while it dropped from 3.0 mg HCN Eq/kg dry weight to 1.3 mg HCN Eq/kg dry weight in type B gari . Mean linamarin dropped from 6.6 mg HCN Eq/kg dry weight to 2.8 mg HCN Eq/kg dry weight in type A gari, while it dropped from 1.7 mg HCN Eq/kg dry weight to 0.4 mg HCN Eq/kg dry weight in type B gari . Loss of linamarin and cyanohydrin was significant at P < 0.001 for type A gari and at P < 0.002 for type B gari when the weekly levels were compared with initial values . When gari was made into eba, 36% of cyanohydrin and 47% of linamarin were lost from type A gari, while 38% of cyanohydrin and 5% of linamarin were lost from type B gari . Loss of linamarin and cyanohydrin when gari was made into eba was significant for both types of gari at P < 0.001 . This study shows that the loss of cyanohydrin and linamarin in gari during short-term storage and when gari is made to eba will reduce dietary cyanide load in consumers.

Appl Environ Microbiol, 2002 Jul, 68(7), 3467 - 77
Genotypic and phenotypic diversity within species of purple nonsulfur bacteria isolated from aquatic sediments; Oda Y et al.; To assess the extent of genotypic and phenotypic diversity within species of purple nonsulfur bacteria found in aquatic sediments, a total of 128 strains were directly isolated from agar plates that had been inoculated with sediment samples from Haren and De Biesbosch in The Netherlands . All isolates were initially characterized by BOX-PCR genomic DNA fingerprinting, and 60 distinct genotypes were identified . Analyses of 16S rRNA gene sequences of representatives of each genotype showed that five and eight different phylotypes of purple nonsulfur bacteria were obtained from the Haren and De Biesbosch sites, respectively . At the Haren site, 80.5% of the clones were Rhodopseudomonas palustris, whereas Rhodoferax fermentans and Rhodopseudomonas palustris were numerically dominant at the De Biesbosch site and constituted 45.9 and 34.4% of the isolates obtained, respectively . BOX-PCR genomic fingerprints showed that there was a high level of genotypic diversity within each of these species . The genomic fingerprints of Rhodopseudomonas palustris isolates were significantly different for isolates from the two sampling sites, suggesting that certain strains may be endemic to each sampling site . Not all Rhodopseudomonas palustris isolates could degrade benzoate, a feature that has previously been thought to be characteristic of the species . There were differences in the BOX-PCR genomic fingerprints and restriction fragment length polymorphisms of benzoate-coenzyme A ligase genes and form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes between benzoate-degrading and non-benzoate-degrading genotypes . The ability to distinguish these two Rhodopseudomonas palustris groups based on multiple genetic differences may reflect an incipient speciation event resulting from adaptive evolution to local environmental conditions.

Appl Environ Microbiol, 2002 Jul, 68(7), 3287 - 92
Process and metabolic strategies for improved production of Escherichia coli-derived 6-deoxyerythronolide B; Pfeifer B et al.; Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated . In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described . The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E . coli were studied in shake flask cultures . The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E . coli . The fermentation strategy employed consistently produced approximately 100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter . The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E . coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter) . In addition to reinforcing the potential for using E . coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.

Br J Nutr, 2002 May, 87 Suppl 2, S255 - 9
Inulin and oligofructose modulate lipid metabolism in animals: review of biochemical events and future prospects; Delzenne NM et al.; Inulin and oligofructose, besides their effect on the gastro-intestinal tract, are also able to exert 'systemic' effect, namely by modifying the hepatic metabolism of lipids in several animal models . Feeding male Wistar rats on a carbohydrate-rich diet containing 10 % inulin or oligofructose significantly lowers serum triacylglycerol (TAG) and phospholipid concentrations . A lower hepatic lipogenesis, through a coordinate reduction of the activity and mRNA of lipogenic enzymes is a key event in the reduction of very low-density lipoprotein-TAG secretion by oligofructose . Oligofructose is also able to counteract triglyceride metabolism disorder occurring through dietary manipulation in animals, and sometimes independently on lipogenesis modulation: oligofructose reduces post-prandial triglyceridemia by 50 % and avoids the increase in serum free cholesterol level occurring in rats fed a Western-type high fat diet . Oligofructose protects rats against liver TAG accumulation (steatosis) induced by fructose, or occurring in obese Zucker fa/fa rats . The protective effect of dietary inulin and oligofructose on steatosis in animals, would be interesting, if confirmed in humans, since steatosis is one of the most frequent liver disorders, occurring together with the plurimetabolic syndrome, in overweight people . The panel of putative mediators of the systemic effects of inulin and oligofructose consists in either modifications in glucose/insulin homeostasis, the end-products of their colonic fermentation (i.e . propionate) reaching the liver by the portal vein, incretins and/or the availability of other nutrients . The identification of the key mediators of the systemic effects of inulin and oligofructose is the key to identify target function(s) (or dysfunction(s)), and finally individuals who would take an advantage of increasing their dietary intake.

Br J Nutr, 2002 May, 87 Suppl 2, S159 - 62
Inulin and oligofructose in the dietary fibre concept; Cherbut C; Dietary fibre consists of remnants of edible plant cell polysaccharides and associated substances resistant to hydrolysis by human alimentary enzymes, which may benefit health through a wide range of physiological effects . Inulin and oligofructose are storage carbohydrates found in a number of vegetables, fruits and whole grains . They resist digestion and absorption in the stomach and small intestine of humans, as shown by their almost full recovery at the end of the ileum of healthy or ileostomised volunteers . Inulin and oligofructose thus enter into the large intestine where they are available to fermentation, as demonstrated by increased breath hydrogen . Fermentation of both substrates is complete and no residue is found in human stools . Inulin and oligofructose improve laxation . Their bulking capacity comprised between 1.2 and 2.1 g of stool per g of ingested substrate, results mainly from increases in microbial biomass in the colon . As water content of bacterial cells is high, stools are softer and easier to expulse . Stool frequency is thus increased, particularly in slightly constipated individuals . In addition, likely due to their fermentation properties, inulin and oligofructose also affect the intestinal epithelium (trophicity, mucin expression, etc.), that may strengthen mucosal protection and reduce the risk of gastrointestinal diseases . In summary, inulin and oligofructose are plant carbohydrates, resistant to digestion in the human small intestine and fermented by colonic bacteria . They exert several intestinal physiological effects contributing to maintenance of health . Therefore, inulin and oligofructose fit well within the current concept of dietary fibre.

Environ Technol, 2002 Apr, 23(4), 429 - 35
Reduced ammonia emissions from slurry after self-acidification with organic supplements; Clemens J et al.; Ammonia volatilisation from field applied slurries causes environmental hazards and loss of fertilizer value . Acidification of slurry, usually with inorganic or organic acids has previously been used to reduce NH3 emissions . In this study, we present an alternative technique for the acidification of slurry, namely the use of fermentation by endogenous microbes to form organic acids from readily degradable organic compounds . In laboratory experiments, the addition of different sugars (sucrose in dosages of 0.003, 0.01, 0.03, 0.1 and 0.3 mol l(-1), glucose in dosages of 0.05 and 0.1 mol l(-1)) and organic residues (sugar beet residues in dosages of 33 and 330 g fresh weight l(-1), biowaste at 50 g fresh weight l(-1)) to cattle slurry resulted in a considerable decrease in pH, with a minimum pH of 4.7 . A subsequent pH increase indicated that the organic acids were probably further degraded with a resultant loss of acidity in the slurry . In a field study, the NH3 emissions from untreated and acidified (pH = 6) slurries were compared after field application (20 m3 ha(-1)) . During the first 20 hours, the acidified slurry showed NH3 emissions of less than 5% of the applied ammonia compared to a 26% loss from the untreated slurry . The total emissions of NH4+-N were 32% for acidified and 54% for untreated slurry . Easily degradable organic amendments therefore have the potential to effectively reduce NH3 emissions from slurries and may be an alternative for the use of acids.

J Dairy Sci, 2002 May, 85(5), 1176 - 82
Chewing activity, saliva production, and ruminal pH of primiparous and multiparous lactating dairy cows; Maekawa M et al.; Four multiparous (MP) and four primiparous (PP) ruminally cannulated lactating Holstein cows were used in a double 4 x 4 Latin square design to study the chewing behavior, saliva production, and ruminal pH of cows in the first or subsequent lactation . Cows were fed one of four diets; three total mixed rations containing 40, 50, or 60% silage (DM basis), and a separate ingredient diet containing 50% concentrate . Dry matter intake was higher for MP cows than for PP cows (19.2 vs . 17.1 kg/d) but not as a percentage of body weight (2.97 +/- 0.06%) . Multiparous cows spent more time eating than PP cows (260 vs . 213 min/d, respectively), even after adjustment for dry matter intake (13.8 vs . 12.4 min/kg DM) . Multiparous cows also spent more time ruminating per day than PP cows (560 vs . 508 min/d, respectively) . Eating salivation rate was not affected by parity, but resting salivation rate was higher for MP cows than for PP . Although MP cows spent more time chewing than PP cows, total daily saliva production was only numerically higher for MP cows because the increase in saliva produced during chewing was accompanied by a decrease in saliva produced during resting . Furthermore, pH profiles tended to be lower for MP cows than for PP cows . Multiparous cows may have a greater risk of incurring acidosis than PP cows because increased salivary secretion associated with increased chewing may not sufficiently compensate the increment of fermentation acids produced in the rumen due to high feed intake.

J Agric Food Chem, 2002 Jul 3, 50(14), 4126 - 30
Improvement of flour quality through carbohydrases treatment during wheat tempering; Haros M et al.; Wheat flour is obtained by the milling process, which includes several steps such as cleaning, tempering, and milling . In the tempering the moisture content of wheat grains is increased to 15.5% by adding an adequate amount of water . The addition of different enzymes (cellulase, xylanase, and beta-glucanase) to the tempering solution has been tested in order to modify the quality of the resulting flour . Rheological and fermentative properties were measured by the farinograph, amylograph, and rheofermentometer . The data show that the technological parameters of the resulting flours were greatly modified by the addition of enzymes to the tempering solution . The quality of the fresh bread obtained from the carbohydrase-treated wheat was improved with regard to specific bread volume, bread shape, and crumb firmness . This method is revealed as an excellent tool to ensure a good distribution of the enzymes in the resulting flour, to control dosage during milling, and to obtain flour of specific characteristics according to their final use.

J Agric Food Chem, 2002 Jul 3, 50(14), 3971 - 6
Ellagic acid and ellagitannins affect on sedimentation in muscadine juice and wine; Lee JH et al.; A mechanism for the formation of water-insoluble sediments in wines and juices made from red and white muscadine grapes (Vitis rotundifolia) was investigated as a function of processing methodology and storage . Sediments are considered quality defects in muscadine grape products, and their presence may influence consumer acceptability and expansion of retail markets . Processing regimes included both hot (70 degrees C) and cold (25 degrees C) press techniques for wine or juice production, and fermentations in contact with grape skins for 3, 5, and 7 days . Relationships between free ellagic acid (FE), total ellagitannins (ET), and total ellagic acid (TE) concentrations were evaluated initially in each product and in sediments that formed during storage for 50 and 120 days at 20 degrees C . Processing techniques influenced initial concentrations of these compounds and the extent of sediment formation . Following storage, juices generally had higher concentrations of FE in sediments compared to wines, but sedimentation was independent of initial FE or TE concentrations . Decreases in ET were observed for hot-pressed juice and skin-fermented wines after storage indicating their hydrolysis during storage and possible contribution to FE in sediments . However, quantitative analysis of the collected sediments revealed that no more than 12% FE by weight was actually present in the sediments, with the remainder consisting of either unidentified compounds or conjugated forms of ellagic acid . This work elucidated a potential mechanism for the presence of FE in muscadine wine and juice sediments through ellagitannin hydrolysis and suggests that sedimentation from mechanisms other than ellagic acid precipitation may also contribute to wine and juice quality.

Mol Cell Biochem, 2002 Apr, 233(1-2), 153 - 8
Cholestin inhibits cholesterol synthesis and secretion in hepatic cells (HepG2); Man RY et al.; Hyperlipidemia is a well-known risk factor for atherosclerosis and statins are widely used to treat patients with elevated levels of lipids in their plasma . Notwithstanding the proven benefits of statin drugs on both primary and secondary prevention of heart disease, the high cost of statin treatment, in addition to possible side effects such as liver function abnormalities, may limit their widespread use . We conducted a study on a natural product as an alternative to statin treatment . Cholestin, a dietary supplement, is prepared from rice fermented with red yeast (Monascus purpureus), which has been shown to significantly decrease total cholesterol levels in hyperlipidemic subjects . Our objective was to determine the cellular effect of Cholestin on cholesterol synthesis in human hepatic cells (HepG2) and the mechanism by which it caused a change in lipid metabolism . Cholestin had a direct inhibitory effect on HMG-CoA reductase activity (78-69% of control) . Cholesterol levels in HepG2 cells treated with Cholestin (25-100 microg/mL) were significantly reduced in a dose-dependent manner (81-45% of control, respectively) . This reduction was associated with decreased synthesis and secretion of both unesterified cholesterol (54-31 and 33-14% of control, respectively) and cholesteryl ester (18-6 and 37-19% of control, respectively) . These results indicate that one of the anti-hyperlipidemic actions of Cholestin is a consequence of an inhibitory effect on cholesterol biosynthesis in hepatic cells and provide the first documentation of a biomolecular action of red yeast rice.

J Biol Chem, 2002 Sep 6, 277(36), 32505 - 9 Epub 2002 Jun 24.
Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene; Xiang L et al.; The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of l-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants . We now report its characterization for the first time in a bacterium . The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A . The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin . Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.

J Bacteriol, 2002 Jul, 184(14), 3909 - 16
The glycolytic flux in Escherichia coli is controlled by the demand for ATP; Koebmann BJ et al.; The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism . We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism . Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular {ATP}/{ADP} ratio . This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells . By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux . The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP . These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E . coli . We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited . Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.

Dermatol Surg, 2002 Jun, 28(6), 491 - 4
Safety data of injectable nonanimal stabilized hyaluronic acid gel for soft tissue augmentation; Friedman PM et al.; BACKGROUND: Nonanimal hyaluronic acid gel was recently developed for soft tissue augmentation and volume expansion and has been shown to offer several advantages in comparison to other augmentation materials . There are rare reports of adverse events believed to be secondary to trace amounts of proteins in the hyaluronic acid raw material . OBJECTIVE: To determine the safety profile of nonanimal stabilized hyaluronic acid gel (Restylane, Perlane, Restylane Fine Lines, Q-Med AB, Uppsala, Sweden) for soft tissue augmentation using a retrospective review of all adverse events data from Europe, Canada, Australia, South American, and Asia from 1999 and 2000 . RESULTS: Data from an estimated 144,000 patients treated in 1999 indicated the major reaction to injectable hyaluronic acid was localized hypersensitivity reactions, occurring in approximately 1 of every 1400 patients treated . In 1999 there was an adverse event reported for 1 of every 650 patients (0.15%) treated . These were temporary events that included redness, swelling, localized granulomatous reactions, bacterial infection, as well as acneiform and cystic lesions . For 2000 there was an estimated 262,000 patients treated with hyaluronic acid gel . The total number of adverse events was 144, corresponding to one adverse event for every 1800 patients (0.06%) treated . The major adverse event was again hypersensitivity, occurring in 1 of every 5000 patients treated . CONCLUSION: According to the reported worldwide adverse events data, hypersensitivity to nonanimal hyaluronic acid gel is the major adverse event and is most likely secondary to impurities of bacterial fermentation . According to data from 2000, the incidence of hypersensitivity appears to be declining after the introduction of a more purified hyaluronic acid raw material.

Lett Appl Microbiol, 2002, 35(1), 47 - 51
Biological activities of Saccharomyces cerevisiae and fermented rice bran as feed additives; Koh JH et al.; AIMS: The objective of the present study was to examine for the various biological activities for feed additives of Saccharomyces cerevisiae and fermented rice bran . METHODS: The feed additives (100 g of each: YE, brewery yeast; PM, mixture of YE and SF; SF, fermented rice bran) were decocted with 2 l water at 100 degrees C to a half volume . RESULTS: The hot-water extract of PM was found to increase the stimulation of the macrophage activation (2.0-fold) and the intestinal immune modulating activity (1.8-fold), compared with those of control . When the hot-water extracts of PM, SF and YE were fed to the rats for 8 d (1 g kg-1 d-1), PM and SF showed the more effective anti-stress effect on spleen, thyroid and thymus gland . SIGNIFICANCE AND IMPACT OF THE STUDY: The hot-water extract of PM can have a significant impact on strengthening the living body's immune system and proliferating anti-stress activities with relation to enhance immunity.

J Ind Microbiol Biotechnol, 2002 Jul, 29(1), 28 - 33
Influence of Saccharomyces cerevisiae strains on fermentation and flavor compounds of white wines made from cv . Emir grown in Central Anatolia, Turkey; Nurgel C et al.; The effect of inoculation with selected Saccharomyces cerevisiae strains was studied on fermentation and flavor compounds of wines made from Vitis vinifera L . cv . Emir grown in Central Anatolia, Turkey . Flavor compounds were analysed and identified by GC-FID and GC-MS, respectively . The total concentrations of flavor compounds did not increase with the addition of indigenous and commercial wine yeasts, but differences were noted in individual volatile compounds . Cluster and factor analyses of flavor compounds also showed that wines produced were different depending on the wine strain used . Wines were completely fermented to less than 1.4 g/l residual sugar . Yeasts other than S . cerevisiae survived longer than previously reported . Inoculation with selected strains increased the ethanol level.

J Anim Sci, 2002 Jun, 80(6), 1683 - 9
Sodium chlorate supplementation reduces E . coli O157:H7 populations in cattle; Callaway TR et al.; Cattle are a natural reservoir of the food-borne pathogen Escherichia coli O157:H7 . Therefore, strategies that reduce E . coli O157:H7 prior to slaughter will reduce human exposures to this virulent pathogen . When bacteria that can anaerobically respire on nitrate (e.g., E . coli) are exposed to chlorate, they die because the intracellular enzyme nitrate reductase converts nitrate to nitrite, but also co-metabolically reduces chlorate to cytotoxic chlorite . Because chlorate is bactericidal only against nitrate reductase-positive bacteria, it has been suggested that chlorate supplementation be used as a strategy to reduce E . coli O157:H7 populations in cattle prior to harvest . Cattle (n = 8) were fed a feedlot-style high-grain diet experimentally infected with three strains of E . coli O157:H7 . Cattle were given access to drinking water supplemented with 2.5 mM KNO3 and 100 mM NaCl (controls; n = 4) or 2.5 mM KNO3 and 100 mM NaClO3 (chlorate-treated; n = 4) . Sodium chlorate treatment for 24 h reduced the population of all E . coli O157:H7 strains approximately two logs (10(4) to 10(2)) in the rumen and three logs (10(6) to 10(3)) in the feces . Chlorate treatment reduced total coliforms and generic E . coli from 106 to 10(4) in the rumen and by two logs throughout the rest of the gastrointestinal tract (ileum, cecum, colon, and rectum) . Chlorate treatment reduced E . coli O157:H7 counts throughout the intestinal tract but did not alter total culturable anaerobic bacterial counts or the ruminal fermentation pattern . Therefore, it appears that chlorate supplementation is a viable potential strategy to reduce E . coli O157:H7 populations in cattle prior to harvest.

J Anim Sci, 2002 Jun, 80(6), 1538 - 44
Developmental changes in ketogenic enzyme gene expression during sheep rumen development; Lane MA et al.; Ketogenesis is the conversion of acetyl-CoA to the ketone bodies acetoacetate and beta-hydroxybutyrate (BHBA) . In hepatic ketogenesis, which occurs during fasting in both nonruminant and ruminant animals, the source of acetyl-CoA is the mitochondrial oxidation of predominantly long-chain fatty acids . In the mature, fed ruminant animal, the ruminal epithelium is also capable of producing ketone bodies . In this case, the source of acetyl-CoA is the mitochondrial oxidation of butyrate produced by the microbial fermentation of feed . The purposes of this study were to determine ontogenic and dietary effects on ketogenic enzyme gene expression in developing lamb ruminal epithelium . Twenty-seven conventionally reared lambs and twenty-seven milk-fed lambs were slaughtered between 1 and 84 d of age . Six additional milk-fed lambs were weaned (the fed group) or maintained on milk replacer with a volatile fatty acid gavage (the VFA group) until 84 d of age . At slaughter, total RNA was extracted from samples of ruminal epithelium . The expression of the genes encoding acetoacetyl-CoA thiolase, the first enzyme in the ketogenic pathway, and 3-hydroxy-3-methylglutaryl-CoA synthase, the rate-limiting enzyme in the ketogenic pathway in nonruminant liver, were examined . Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase mRNA concentrations increased with age independent of diet . 3-Hydroxy-3-methylglutaryl-CoA synthase mRNA levels in ruminal epithelium obtained from milk-fed lambs were low before 42 d of age, but a marked increase occurred by 42 d of age . At 84 d of age, there were no differences in acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase expression due to diet . The pattern of the expression of these genes, in particular, 3-hydroxy-3-methylglutaryl-CoA synthase, parallels the rate of production of BHBA by rumen epithelial cells isolated from the same lambs, which increased to conventionally reared adult levels at 42 d of age and did not differ with diet . In conclusion, development of the ketogenic capacity of the ruminal epithelium occurs as the animal ages, regardless of dietary treatment . Thus, the expression of the genes encoding the ketogenic enzymes are not affected by the presence of VFA in the ruminal lumen.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1998, 16(2), 113 - 6
{Histological changes of Cysticercus cellulosae under the action of proteinase of Omphalia lapidescens in vitro}; Zhao G et al.; AIM: For the purpose of finding an anthelmintic with high efficacy, low toxicity and low cost, especially from the Chinese traditional medicines . METHODS: Forty Cysticercus cellulosae were separately incubated with proteinase from artificially fermented Omphalia lapidescens for 2 h, 4 h and 8 h, and then examined for histological changes by light microscopy and compared with those of proteinase from natural dry Omphalia lapidescens after similar treatment . RESULTS: Cysticercus cellulosae were morphologically and structurally impaired after exposure to the action of proteinase of Omphalia lapidescens, and the degree of impairment was proportionally paralleled to the duration of treatment . CONCLUSION: The anti-cysticercus effect of proteinase of Omphalia lapidescens and the homogeneity of proteinases from artificially fermented and natural Omphalia lapidescens were confirmed.

Zhongguo Zhong Yao Za Zhi, 1999 Jan, 24(1), 12 - 4, 62
{RP-HPLC determination of adenosine in fermented Cordyceps}; Li X et al.; OBJECTIVE: A method for determination of adenosine in four fermented Cordyceps products by RP-HPLC is described . METHOD: C18 column was used with methanol-0.06 mol/L potassium phosphate monobasic-tetrahydrofuran (10:150:1.5) as mobile phase . The flow rate was 1 ml/min and detected at 260 nm . RESULTS: The average recovery was 98.7% and relative standard deviation was 1.10% (n = 5) . CONCLUSION: This method is simple, rapid and sensitive . The paper introduces the feasibility of supersonic extraction method taking the place of reflux method.

Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1226 - 8 Epub 2002 Jun 20.
Purification, crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, the fourth enzyme in the dTDP-L-epivancosamine biosynthetic pathway; Merkel AB et al.; The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria . The compounds used clinically are very complex organic molecules and are made by fermentation . The biosynthesis of these is complex and fascinating . Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues . In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism . The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6-trideoxy-3-C-methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here . The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin . The crystals of EvaD diffract to 1.5 A and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1 A with space group P2(1)2(1)2 . Data to this resolution were collected at the European Synchrotron Radiation Facility.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 155 - 9
Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni; Carrete R et al.; Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions . This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity . Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively . It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.

Microb Cell Fact . 2002 May 13;1(1):2.
Stress responses and replication of plasmids in bacterial cells; Wegrzyn G et al.; Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology . For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering . Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids . However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist . Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins . Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly . Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood . There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication . In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons . Nevertheless, recent results of studies on other plasmids are also discussed shortly.

Environ Sci Technol, 2002 Jun 1, 36(11), 2530 - 5
Biological hydrogen production measured in batch anaerobic respirometers; Logan BE et al.; The biological production of hydrogen from the fermentation of different substrates was examined in batch tests using heat-shocked mixed cultures with two techniques: an intermittent pressure release method (Owen method) and a continuous gas release method using a bubble measurement device (respirometric method) . Under otherwise identical conditions, the respirometric method resulted in the production of 43% more hydrogen gas from glucose than the Owen method . The lower conversion of glucose to hydrogen using the Owen protocol may have been produced by repression of hydrogenase activity from high partial pressures in the gastight bottles, but this could not be proven using a thermodynamic/rate inhibition analysis . In the respirometric method, total pressure in the headspace never exceeded ambient pressure, and hydrogen typically composed as much as 62% of the headspace gas . High conversion efficiencies were consistently obtained with heat-shocked soils taken at different times and those stored for up to a month . Hydrogen gas composition was consistently in the range of 60-64% for glucose-grown cultures during logarithmic growth but declined in stationary cultures . Overall, hydrogen conversion efficiencies for glucose cultures were 23% based on the assumption of a maximum of 4 mol of hydrogen/ mol of glucose . Hydrogen conversion efficiencies were similar for sucrose (23%) and somewhat lower for molasses (15%) but were much lower for lactate (0.50%) and cellulose (0.075%).

J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 74 - 80
Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation; Jordan GL et al.; Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli . These proteases have been linked to stresses, such as the stringent and heat-shock responses . This study reports the dramatic up-regulation of protease activity in an industrial recombinant E . coli fermentation upon induction . The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction . AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes . AXOKINE was expressed in both the soluble and inclusion body fractions in E . coli . Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity . Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions . Protease inhibitors were used to characterize the proteases . The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive . The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor . The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent . A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.

J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 65 - 9
Containment in industrial biotechnology within wastewater treatment plants; Noordover JA et al.; Both physical and biological containment are considered to be essential parts in the risk analysis of industrial Good Industrial Large-Scale Practice (GILSP) processes using genetically modified organisms (GMOs) . Biological containment of industrial microorganisms has become a more important issue since the introduction of recombinant DNA techniques . In the event of an accidental discharge in the production plant, a large amount of organisms could be released into the wastewater treatment (WWT) system . This WWT system should therefore be considered as a part of the containment . This study demonstrates both a hydrodynamic and a microbiological model for the containment aspects of industrial WWT plants . The models are verified by measurements using industrial hosts of GILSP GMOs at full scale . Both models describe the full-scale equipment accurately . The results are supplemented with microcosm studies on survival of GMOs in defined niches . It is shown that WWT plants can be considered as useful additional parts of the containment of microorganisms, in case of an accidental discharge . The effect of drainage of an enormous amount of microorganisms (several tons) through the WWT plant into the environment is shown to be comparable to the direct drainage of a small-scale fermenter . Microcosm experiments correlate well with the survival rates in the WWT and therefore can be of use to predict the behaviour of GMOs in this environment.

Ann Chim, 2002 Apr, 92(4), 457 - 67
Size characterization of barley starch granules by gravitational field-flow fractionation: a rapid, low-cost method to assess the brewing capability of different strains; Reschiglian P et al.; Cereal starch occurs as two types of micrometer-sized granules, large and small . Large starch granules are more susceptible to enzymatic hydrolysis . When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred . Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time . In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented . For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF . As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Protein Expr Purif, 2002 Jun, 25(1), 97 - 104
Large-scale expression and purification of high-molecular-weight glutenin subunits; Dowd C et al.; The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough . The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours . Consequently, it is difficult to purify these proteins separately in appreciable amounts . Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins . However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized . Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained . In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter . Our results include optimization of expression conditions to increase yield and stability of proteins . Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression . We also developed a large-scale purification procedure . The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour . Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour . These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality .

Curr Microbiol, 2002 Aug, 45(2), 88 - 93
Microbiological transformation of L-tyrosine to 3,4-dihydroxyphenyl L-alanine (L-dopa) by a mutant strain of Aspergillus oryzae UV-7; Ikram-Ul-Haq et al.; The present study deals with the microbiological transformation of L-tyrosine to 3,4-dihydroxyphenyl L-alanine by a mutant strain of Aspergillus oryzae UV-7 . Sixteen different mutant strains of Aspergillus oryzae (GCB-6) were isolated through UV-irradiation . These mutant strains were screened for the production of mold mycelia by submerged fermentation in 250-ml Erlenmeyer flasks . Of all the mutant strains examined, UV-7 gave maximum production of L-dopa (1.28 mg/ml) . The reaction was carried out using mold mycelium as a source of enzyme tyrosinase in shake flasks . The maximum production of L-dopa was obtained when glucose (25 mg/ml) was used as the carbon source and NH(4)Cl (3 mg/ml) was used as the nitrogen source . The optimum pH for mycelium development was 5.0; L-dopa production was maximum at pH 3.5 of the reaction mixture . The reaction by mold mycelium (75 mg/ml) was carried out under acidic conditions . Optimum temp, time, and L-tyrosine concentration were 55 degrees C, 60 min, and 3.0 mg/ml, respectively.

J Microbiol Methods, 2002 Sep, 51(1), 73 - 82
In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture; Noguchi Y et al.; To optimize an appropriate microbial culture in a fermentor, precise control of the medium's dissolved oxygen tension (DOT) is crucial . In particular, to study the effect of DOT on cellular metabolic status by using in vivo nuclear magnetic resonance (NMR) measurements, the set-up of the experiment must be optimized to maintain DOT in the culture . In the conventional method, DOT is monitored by a sensor inside a fermentor and is controlled by changing the agitation rate . Here, we report a novel and accurate system that minimizes time lag by an automated aeration flow control device, allowing an NMR spectrometer to monitor representative metabolites in real-time . To fulfill these two objects, the fermentor was composed of a fermentation vessel and two outer tubes, through which the medium was circulated by rotary pumps . One tube monitored DOT in via a sensor, and at the same time the other tube monitored metabolites via an NMR spectrometer.In this study, we used this system to analyze the responses of Escherichia coli cells under various oxygen conditions . The results validated the use of this system in the study of microbial metabolism.

Free Radic Res, 2002 Apr, 36(4), 485 - 90
The regulation of COQ5 gene expression by energy source; Hagerman RA et al.; The degree of severity of cardiomyopathy is inversely correlated with tissue levels of coenzyme Q (Q), suggesting that Q synthesis may impact the progression of the disease . It has been suggested that Q functions as an endogenously synthesized anti-oxidant, in addition to regenerating the potent anti-oxidants, vitamins E and C . However, very little is known about the mechanisms that regulate Q synthesis . Using the simple eukaryote Saccharomyces cerevisiae as a model, experiments have been designed to investigate the regulation of Q synthesis at the genetic level . To investigate the regulation of COQ5 gene expression by energy source, mRNA content was evaluated in yeast cells treated with dextrose, glycerol or oleic acid . After 1.5 h, more COQ5 mRNA is produced by oleic acid treated cells than by glycerol treated . Experiments performed using COQ5 promoter deletion/reporter constructs demonstrate a specific response to oleic acid . Additional promoter deletion analysis demonstrates that a non-fermentable carbon source element is also present, responding to both glycerol and oleic acid . The specific oleic acid response appears to be regulated by the Rtg family of transcription factors . This family of proteins is required for oleic acid-induced expression of genes of beta-oxidation and peroxisomal proliferation, and plays an important role in co-ordinating mitochondrial/peroxisomal/nuclear communication in response to oleic acid, as well as defects in cellular respiration.

Arch Tierernahr, 2001, 55(2), 103 - 20
Effects of rumen fluid collection site on microbial population structure during in vitro fermentation of the different substrates quantified by 16S rRNA hybridisation; Muetzel S et al.; Rumen fluid samples from a cow were withdrawn manually from the feed mat (solid phase) or the liquid phase below this mat and incubated in vitro with wheat straw, sorghum hay and a concentrate mixture . From the inoculum and several samples collected during in vitro incubation RNA was extracted to assess microbial population size and structure . RNA content recovered from the solid phase rumen fluid was significantly higher than from the liquid phase . The composition of the microbial population in the solid phase material was characterised by a high proportion of Ruminococci . Neither the proportion of other cell wall degrading organisms (Fibrobacter and Chytridiomycetes) nor the Eukarya and Archaea populations differed between the two sampling sites . Gas production was higher when substrates were incubated with solid phase than with liquid phase rumen fluid regardless of sampling time . However, the higher level of gas production was not accompanied by a corresponding increase in true digestibility . The RNA probes showed that during in vitro incubation with liquid phase rumen fluid, the eukaryotic population was inactive no matter which substrate was used and the activity of methanogens (Archaea) was lower than with solid phase rumen fluid . The population pattern of the cell wall degrading organisms was influenced mainly by the substrate fermented, and to a smaller extent by the inoculum used for in vitro fermentation.

J Biotechnol, 2002 Aug 7, 97(2), 133 - 45
Optimizing high strength acetic acid bioprocess by cognitive methods in an unsteady state cultivation; Arnold S et al.; Methods of adapting micro-organisms to an inhibiting factor in an active industrial bioprocess were examined with an acetic acid fermentation as model . With the aim of automatic control, a fuzzy-logic system was developed on the basis of the collected knowledge of skilled vinegar brewers . In a first step, this fuzzy system was to assess the actual adaptation degree of the bacteria on the basis of data from robust and reasonably priced sensors . From this information an appropriate setpoint value for the inhibiting factor 'final acid concentration' was derived for each batch cycle . As a result a further acid tolerance was found after several batch cycles . This adaptation effect should be used to increase the product concentration to more than 20 g per 100 ml acetic acid with a high productivity . The stepwise adapted culture was productive over the aimed acetic acid concentration, a 10% improvement of the product formation rate could be found compared with the status before conditioning . High product concentration and increased productivity finally result in shorter cycle times, less transport and storage volumes, an improved utilization of energy and material resources, and, last but not least, they are an essential steps towards the fulfillment of economical and ecological demands.

J Appl Microbiol, 2002, 93(1), 163 - 8
Phenotypic degeneration occurs during sector formation in Metarhizium anisopliae; Ryan MJ et al.; AIMS: The formation of sectors was observed during subculturing of an isolate of the entomopathogenic fungus Metarhizium anisopliae, a fungus used for biological control of insect pests . The aim of the investigation was to establish whether sector formation was accompanied by changes in physiological characters . METHODS AND RESULTS: Four degenerative morphological states, with reduced sporulation capacity, were characterized . Subcultures were taken from each sector and four new culture lines established . The new lines were further subcultured every 21 d . A physiological assessment of each line was undertaken after 42 d using TLC of secondary metabolites and fluorogenic enzyme tests . Full sporulation capacity was not regained on subculture, although some cultures recovered partially . Changes in secondary metabolite profiles and the loss in detection of activity of specific enzymes were observed . CONCLUSIONS: Sector formation was frequently accompanied by changes in the ability to produce secondary metabolites and enzymes . SIGNIFICANCE AND IMPACT OF THE STUDY: The results illustrate the importance of maintaining the stability of important cultures during routine subculture . The consequences could have significant implications if degenerate cultures are used as inocula for liquid fermentation cultures or industrial scale production.

Artif Cells Blood Substit Immobil Biotechnol, 2002 May, 30(3), 199 - 218
Properties of hydrogel materials used for entrapment of microbial cells in production of fermented beverages; Navratil M et al.; Approaches using immobilized biological materials are very promising for application in different branches of the food industry, especially in the production of fermented beverages . Materials tested by our team for the process of entrapment belong to the family of charged polysaccharides able to form beaded hydrogels by ionotropic gelation (e.g . alginate, pectate, kappa-carrageenan) and synthetic polymers (e.g . polyvinyl alcohol) forming bead- and lens-shaped hydrogels by thermal sol/gel transition . Concentration of a gel, conditions and instrumentation of gelation process, bead and size distribution, porosity, diffusion properties, mechanical, storage and operational stability, and many other parameters were followed and optimized . Our work has been oriented especially to practical applications of immobilized cells . Brewing yeast cells were successfully immobilized by entrapment materials and used in a process of batch and continual production of beer, including primary and secondary fermentation of wort . Other applications include continual production of ethanol by fermentation of different saccharide substrates (molasses, glucose syrup, wheat hydrolysate), mead and non-alcoholic beverages production.

Oncol Rep, 2002 Jul-Aug, 9(4), 817 - 22
Preventive effect of fermented brown rice and rice bran against colon carcinogenesis in male F344 rats; Katyama M et al.; Epidemiological and preclinical studies demonstrate that nutrition plays an important role in the etiology of cancer . It has been reported that rice components, especially rice germ plays a key role in prevention of cancer . The experiments described here examined the potential anticancer properties of brown rice fermented by Aspergillus Oryzae (FBRA) in male F344 rats using inhibition of the formation of azoxymethene (AOM) induced aberrant crypt foci (ACF) and tumors in the colon as the measure of preventive efficacy . The agent was administered at 2.5 and 5% levels in the diet during the initiation phase (during and until 1 week after carcinogen treatment) and/or post-initiation phase (beginning 1 week after carcinogen treatment) of carcinogenesis . In the ACF and tumor studies, rats were sacrificed 5 or 40 weeks after the initiation of AOM treatment (15 mg/kg body weight, once weekly for 3 weeks), respectively . Colonic ACF and tumors were evaluated histopathologically . Administration of 2.5 and 5% FBRA in the diet continuously during initiation and post-initiation period significantly inhibited the ACF formation in rats treated with AOM, compared with rats treated with AOM alone (99+/-24.1 and 79+/-18.4 vs . 139.5+/-27.7, respectively) . In addition, administration of 5% FBRA in the diet during the post-initiation phase significantly suppressed the incidence (44 vs.18%) and multiplicity (0.93+/-0.96 vs . 0.18+/-0.40) of colon adenocarcinomas as compared to those given the control diet . In addition, 5% FBRA in the diet during post-initiation phase caused significant inhibition of cell proliferation in the colonic mucosa as compared to the group fed the control diet (81% reduction, p<0.05) . These observations demonstrated for the first time that FBRA inhibits colon tumor development in rats, and suggest that it is a promising dietary supplement for prevention of human colon cancer.

Science, 2002 Jun 14, 296(5575), 2026 - 8
RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance; Baxter-Burrell A et al.; Transient soil flooding limits cellular oxygen to roots and reduces crop yield . Plant response to oxygen deprivation involves increased expression of the alcohol dehydrogenase gene (ADH) and ethanolic fermentation . Disruption of the Arabidopsis gene that encodes Rop (RHO-like small G protein of plants) guanosine triphosphatase (GTPase) activating protein 4 (ROPGAP4), a Rop deactivator, elevates ADH expression in response to oxygen deprivation but decreases tolerance to stress . Rop-dependent production of hydrogen peroxide via a diphenylene iodonium chloride-sensitive calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is necessary for induction of both ADH and RopGAP4 expression . Tolerance to oxygen deprivation requires Rop activation and RopGAP4-dependent negative feedback regulation . This Rop signal transduction rheostat balances the ability to increase ethanolic fermentation with survival.

Infect Immun, 2002 Jul, 70(7), 3785 - 92
In vivo effects of a synthetic 2-kilodalton macrophage-activating lipopeptide of Mycoplasma fermentans after pulmonary application; Luhrmann A et al.; Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals . Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes . The present study deals with the question of which mycoplasmal components cause this host reaction . The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection . To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2 . Application of 2.5 microg induced a pronounced leukocyte accumulation in the bronchoalveolar space . At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3 . Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation . The leukocyte count of the lung interstitium was increased on day 3 after treatment . After 10 days all investigated cell populations returned to control levels . Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates . MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2 . Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction . The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.

Trends Biotechnol, 2002 Jul, 20(7), 299 - 305
Biotechnological approaches for L-ascorbic acid production; Hancock RD et al.; Over the past decade there has been increasing pressure to develop alternatives to the Reichstein process, a largely chemical synthesis by which the vast majority of world vitamin C (L-ascorbic acid, L-AA) is produced . The pressures include increasing environmental concerns and legislation, and the need to increase process efficiency and reduce capital costs . The development of efficient fermentation processes in the past ten years has also represented a catalyst for change . Here, we describe the development of biotechnological alternatives for the synthesis of Reichstein intermediates by industrial microorganisms . The recent elucidation of the plant biosynthetic pathway represents new opportunities not only for the direct synthesis of L-AA by fermentation but also for the production of human crop plants and animal fodder with enhanced nutritional value . We discuss the potential for these developments in the light of recent findings concerning L-AA biosynthesis in plants.

Microbiol Immunol, 2002, 46(4), 265 - 76
Mycoplasmal lipoproteins induce toll-like receptor 2- and caspases-mediated cell death in lymphocytes and monocytes; Into T et al.; Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT-4 and Raji, and in one monocytic cell line, THP-1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL-60 . These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA . The lipoproteins activated caspase-3 in both MOLT-4 and HL-60 cells, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase . The cytotoxicity to MOLT-4 and HL-60 cells was inhibited by various caspase inhibitors, Ac-DMQD-CHO, Ac-IETD-CHO, and Z-VAD-FMK . The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll-like receptor 2 . Thus this study demonstrated that mycoplasmal lipoproteins induce caspases-dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2-mediated signaling.

J Antibiot (Tokyo), 2002 Apr, 55(4), 391 - 5
Radamycin, a novel thiopeptide produced by streptomyces sp . RSP9 . II . Physico-chemical properties and structure determination; Castro Rodriguez J et al.; The new cyclic peptide antibiotic, radamycin (1) and the known thiopeptide methylsulfomycin I (2) have been isolated from the fermentation broth of a Streptomyces sp . RSP9 . The structure of radamycin was elucidated by NMR, LC-MS and FAB-MS and was established as a thiopeptide with oxazole and thiazole moieties, and several unusual amino acids.

J Antibiot (Tokyo), 2002 Apr, 55(4), 377 - 82
Neosordarin and hydroxysordarin, two new antifungal agents from Sordaria araneosa; Davoli P et al.; Two novel antifungal agents belonging to the sordarin family have been isolated from fermentations of Sordaria araneosa by bioassay-guided purification and their structures elucidated by NMR techniques . Neosordarin (1) is closely related to the recently discovered hypoxysordarin (2), with only small differences on the aliphatic side chain acylating the hydroxyl in the 3'-position of the sordarose moiety . Hydroxysordarin (3) closely resembles sordarin (4), the only slight difference being the replacement of sordarose with altrose as the sugar unit.

J Antibiot (Tokyo), 2002 Apr, 55(4), 355 - 62
Indocarbazostatin and indocarbazostatin B, novel inhibitors of NGF-induced neuronal differentiation in PC12 cells . I . Screening, taxonomy, fermentation and biological activities; Matsuura N et al.; During the course of our screening for modulators of signal transduction of mammalian cells, we discovered two novel indolocarbazole antibiotics, indocarbazostatin and indocabazostatin B, from a culture broth of a Streptomyces sp . as inhibitors of NGF-induced neuronal differentiation in rat pheochromocytoma PC12 cells . Indocarbazostatin and indocarbazostatin B inhibited NGF-induced neurite outgrowth from PC12 cells at 6 nM and 24 nM, respectively, whereas K-252a inhibited at 200 nM under our assay conditions.

J Agric Food Chem, 2002 Jun 19, 50(13), 3840 - 8
Investigation of volatiles evolution during the alcoholic fermentation of grape must using free and immobilized cells with the help of solid phase microextraction (SPME) headspace sampling; Mallouchos A et al.; A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on delignified cellulosic material (DCM) . Repeated batch fermentations were conducted using these biocatalysts and free cells, separately, at temperatures of 20, 15, and 10 degrees C . Solid phase microextraction (SPME) was used in monitoring the formation of volatile alcohols, acetate esters, and ethyl esters of fatty acids . The kinetics of volatile production were similar for free and immobilized cells . In all cases immobilized cells showed a better rate of volatile production, which was directly connected to sugar consumption . The main difference observed was in propanol production, which increased with temperature decrease for the immobilized cells, whereas it remained constant for the free ones . In the case of immobilized cells significant amounts of esters were also produced . It is well-known that esters contribute to the fruity aroma of wine . It was also established that SPME is a very sensitive, accurate, and reliable technique and can be used without any reservation in the characterization of volatile constituents of wine.

J Agric Food Chem, 2002 Jun 19, 50(13), 3822 - 7
Analysis of grape Vitis vinifera L . DNA in must mixtures and experimental mixed wines using microsatellite markers; Siret R et al.; Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality . Microsatellite markers have already been applied to the authentication of grape juices (Faria, M . A.; Magalhaes, R.; Ferreira, M . A.; Meredith, C . P.; Ferreira Monteiro, F . J . Agric . Food Chem . 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J . M.; Merle, M . H.; Cabanis, J . C.; This, P . J . Agric Food Chem . 2000, 48, 5035-5040) . In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures . The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected . Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations . DNA was extracted along the fermentation process and analyzed using five microsatellite loci . The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation . The applications of these results to commercial purposes are discussed.

J Agric Food Chem, 2002 Jun 19, 50(13), 3818 - 21
Investigation of the beta-damascenone level in fresh and aged commercial beers; Chevance F et al.; This study investigated the increase of beta-damascenone content during aging in a variety of commercial Belgian beers . Quantities detected in fresh beers were generally low (from 6 ng/g to 25 ng/g) . After 5 days at 40 degrees C, the level increased (to as much as 210 ng/g) in most of the beers studied, according to the type of beer . Further experiments showed that wort initially contains large quantities of beta-damascenone (450 ng/g), but that degradation of the compound during fermentation accounts for the low concentrations observed in fresh beers . Production during beer aging can be partially explained by acid hydrolysis of glycosides.

J Agric Food Chem, 2002 Jun 19, 50(13), 3647 - 53
Automatic flow system with voltammetric detection for diacetyl monitoring during brewing process; Rodrigues PG et al.; Diacetyl can be determined by adsorptive stripping voltammetry after derivatization with o-phenylenediamine . The method may be applicable to the determination of diacetyl in different foods, being a good alternative to other analytical methods . In this work an on-line automated analytical system for diacetyl determination in beer is described . A hanging mercury drop electrode voltammetric flow detector was used, and the analyte was determined without the traditional deoxygenation procedure . The method was successfully applied to the determination of diacetyl during beer fermentation and in the final product . The automation strategy used was based on a flow network similar to those used in multicommutated flow systems, with a pervaporation unit used for diacetyl separation . The developed system was tested in real conditions in the monitoring of brewing processes . The results obtained were similar to those obtained with the usual GC-ECD methodology in the 5-600 ppb range . The analytical rate of the developed method is about 12 determinations/h.

J Chromatogr A, 2002 Apr 12, 953(1-2), 1 - 6
Headspace solid-phase microextraction analysis of 3-alkyl-2-methoxypyrazines in wines; Sala C et al.; A procedure to determine 3-alkyl-2-methoxypyrazines in wines is described . It is based on the headspace solid-phase microextraction (HS-SPME) technique after a clean-up of the sample by distillation (previously acidified to pH 0.5) to remove ethanol and other volatile compounds that can interfere in the SPME . Determination is performed by means of capillary gas chromatography using a nitrogen-phosphorus detector . The method allows quantification of 3-isobutyl-2-methoxypyrazine, 3-sec-butyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine at their natural concentration levels and below their sensory thresholds in Cabernet Sauvignon and Merlot wines . The method was successfully applied to experimental red wines and the evolution of their pyrazine contents during the winemaking process was monitored . Pyrazine content increased during the first maceration day but did not change significantly during alcoholic and malolactic fermentation . Final contents in wines were 12-27 ng/l of 3-isobutyl-2-methoxypyrazine and 5-10 ng/l of 3-sec-butyl-2-methoxypyrazine.

Bioresour Technol, 2002 May, 83(1), 1 - 11
Hydrolysis of lignocellulosic materials for ethanol production: a review; Sun Y et al.; Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil . There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol . The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process . Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials . Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose . Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis . Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.

Waste Manag Res, 2002 Apr, 20(2), 110 - 8
Enhanced acidogenic fermentation of food waste in a continuous-flow reactor; Han SK et al.; This study was performed to improve acidogenic fermentation of food waste in a continuous-flow reactor . The fermentation of food waste is affected by the fermentation constraints such as the biodegradability of substrate, the degrading capability of microorganisms and the environmental conditions . The key factors were, therefore, examined to control the fermentation constraints, such as the effect of seed inoculation and the effect of adjusting dilution rate . Acidogenic fermentation of food waste employing rumen microorganisms resulted in the enhanced efficiency (71.2%) as compared with that (59.8%) employing mesophilic acidogens . In addition, the fermentation efficiency increased from 71.2 to 82.0% by adjusting dilution rate from 3.0 to 1.0 d(-1) depending on the state of the fermentation . The main component of the acidified product was shifted from butyric to acetic acid . This meant that the increase of the fermentation efficiency was mainly caused by the enhanced degradation of vegetables and meats . The control of the fermentation constraints was, therefore, very effective in improving the fermentation efficiency of food waste.

Folia Microbiol (Praha), 2002, 47(2), 171 - 7
Effect of plant oils and organic acids on rumen fermentation in vitro; Jalc D et al.; We determined the effect of plant oils (rapeseed, sunflower, linseed) and organic acids (aspartic and malic) on the fermentation of diet consisting of hay, barley and sugar beet molasses . Rumen fluid was collected from two sheep (Slovak Merino) fed with the same diet twice daily . Mixed rumen microorganisms were incubated in fermentation fluid, which contained rumen fluid and Mc Dougall's buffer . All supplemented diets significantly increased pH, molar proportion of propionate, and numerically decreased methane production . Lactate production was also decreased significantly (except with malate) . Incorporation of plant oils into aspartate- and malate-treated incubations negated the decrease of butyrate, lactate and the increase of pH and ammonia with malate treatment, as well as in vitro dry matter digestibility and pH with aspartate treatment . The effect of combined additives on methane production and molar proportion of propionate was lower compared with additives supplemented separately . Combination of additives had no additive effect on rumen fermentation . All additives decreased total protozoan counts in rumen fluid.

Control Clin Trials, 2002 Jun, 23(3), 333 - 53
The antibiotic treatment trial of Gulf War Veterans' Illnesses: issues, design, screening, and baseline characteristics; Collins JF et al.; Many veterans who were deployed to the Persian Gulf during the 1990-1991 Gulf War developed multiple unexplained symptoms such as pain, fatigue, and neurocognitive problems . This constellation of symptoms has been termed Gulf War Veterans' Illnesses (GWVI) . Although there is no proven explanation for the cause of GWVI, one fairly widespread explanation is systemic Mycoplasma fermentans infection . The Antibiotic Treatment Trial of GWVI is a randomized placebo-controlled trial to determine whether a 1-year course of doxycycline treatment in deployed Gulf War veterans with GWVI and testing as Mycoplasma species positive will improve their overall functional status as measured by the Physical Component Summary of the SF-36V questionnaire . The study of a multisymptom illness such as GWVI is complicated by the nonspecific nature of the illness, the unknown etiology, and the lack of a widely accepted outcome measure . The presumption of mycoplasma infection raises concerns regarding the methodology for determination of mycoplasma infection, the choice of treatment, and the duration of treatment . However, such a presumption allows the formulation of a clear testable hypothesis that can be tested with treatments with known rates of adverse events and known activity against Mycoplasma species . This paper describes the major issues faced by the investigators during planning, the study design, the patient screening results, and the baseline characteristics of the study patients . There were 2712 patients screened for study entry at 26 Department of Veterans Affairs and two Department of Defense medical centers . Of these, 491 met all study entry criteria and were randomized to either 1 year of doxycycline (200 mg/day) or 1 year of placebo . All patients were seen monthly during treatment and at 6 months after the end of treatment . Study patients had a mean age of 41 years and were mostly male (86%), white (64%), married (68%), and employed full-time (71%).

BMC Musculoskelet Disord . 2002 Jun 3;3(1):15 . Epub 2002 Jun 03.
Experimental arthritis induced by a clinical Mycoplasma fermentans isolate; Rivera A et al.; BACKGROUND: Mycoplasma fermentans has been associated with rheumatoid arthritis . Recently, it was detected in the joints and blood of patients with rheumatoid arthritis, but it is not clear yet how the bacteria enter the body and reach the joints . The purpose of this study was to determine the ability of M . fermentans to induce experimental arthritis in rabbits following inoculation of the bacteria in the trachea and knee joints . METHODS: P-140 and PG-18 strains were each injected in the knee joints of 14 rabbits in order to evaluate and compare their arthritogenicity . P-140 was also injected in the trachea of 14 rabbits in order to test the ability of the bacteria to reach the joints and induce arthritis . RESULTS: M . fermentans produced an acute arthritis in rabbits . Joint swelling appeared first in rabbits injected with P-140, which caused a more severe arthritis than PG-18 . Both strains were able to migrate to the uninoculated knee joints and they were detected viable in the joints all along the duration of the experiment . Changes in the synovial tissue were more severe by the end of the experiment and characterized by the infiltration of neutrophils and substitution of adipose tissue by connective tissue . Rabbits intracheally injected with P-140 showed induced arthritis and the bacteria could be isolated from lungs, blood, heart, kidney, spleen, brain and joints . CONCLUSION: M . fermentans induced arthritis regardless of the inoculation route . These findings may help explain why mycoplasmas are commonly isolated from the joints of rheumatic patients.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 713 - 8
Mitsuokella jalaludinii sp . nov., from the rumens of cattle in Malaysia; Lan GQ et al.; Five strains of phytase-producing, gram-negative, non-spore-forming, non-motile, small, stout, rod-shaped, strictly anaerobic, fermentative bacteria were isolated from the rumens of cattle in Malaysia . All five strains had morphological, physiological and biochemical features in common . Although these strains had many physiological and biochemical characteristics that were identical to those of the Mitsuokella multacida type strain (ATCC 27723T), they could be distinguished from this species by means of the following characteristics: a smaller cell size (1.2-2.4 microm long and 0.6-0.8 microm wide); a lower final pH value (3.8-4.0) in peptone/yeast extract/glucose broth; inhibition by 0.001% brilliant green; insensitivity to kanamycin (100 microg ml(-1)) and penicillin (10 microg ml(-1)); a higher optimum growth temperature (approx . 42 degrees C); the ability to grow at 45 and 47 degrees C; the ability to ferment glycerol, sorbitol and amidon; and the inability to ferment mannitol, rhamnose, D-tagatose and melezitose . The G+C content of the type strain (M 9T) of these five strains was 56.9 mol% . Analysis of the 16S rRNA gene sequence of type strain M 9T indicated that the strain falls within the genus Mitsuokella . The sequence similarity between type strain M 9T and Mitsuokella multacida was 98.7% . The DNA-DNA relatedness between type strain M 9T and Mitsuokella multacida type strain DSM 20544T (= ATCC 27723T) was 63.8%, indicating that, in spite of a high level of similarity for the 16S rRNA gene sequence, type strain M 9T is independent of Mitsuokella multacida at the species level . On the basis of these results, a new species, Mitsuokella jalaludinii sp . nov., is proposed for these strains . The type strain is M 9T (= DSM 13811T = ATCC BAA-307T).

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 1023 - 32
Bannoa hahajimensis gen . nov., sp . nov., and three related anamorphs, Sporobolomyces bischofiae sp . nov., Sporobolomyces ogasawarensis sp . nov . and sporobolomyces syzygii sp . nov., yeasts isolated from plants in Japan; Hamamoto M et al.; Fourteen ballistoconidium-forming yeast strains were isolated from leaves of plants collected in the Ogasawara Islands, which are isolated islands in the Pacific Ocean, about 1,000 km south of the Japanese mainland, in the subtropical zone . The 14 isolates were characterized by the absence of xylose in whole-cell hydrolysates, the presence of Q-10(H2) as the major ubiquinone isoprenologue, G+C contents of 47.6-52.0 mol%, the inability to ferment sugars or to assimilate nitrate and positive Diazonium blue B and urease reactions . They formed a phylogenetically coherent cluster within the Erythrobasidium lineage in the Urediniomycetes of the Basidiomycota based on 18S rDNA sequences . Analyses of the nucleotide sequences of internal transcribed spacer regions and DNA complementarity showed that four genospecies were recognized among the 14 isolates . A mating reaction was observed in one of the four genospecies, which produced one-celled basidia on dikaryotic hyphae with clamp connections . On the basis of the morphological, physiological, chemotaxonomic and phylogenetic analyses, a new teleomorphic genus, Bannoa, is proposed, in which one novel species is described, Bannoa hahajimensis gen . nov., sp . nov . (type strain OK-248(T) = JCM 10336T = CBS 9039(T)) . The other three anamorphic genospecies are described as Sporobolomyces bischofiae sp . nov . (type strain OK-257T = JCM 10338T =CBS 9041T), Sporobolomyces ogasawarensis sp . nov . (type strain OK-14T = JCM 10326T = CBS 9038T) and Sporobolomyces syzygii sp . nov . (type strain OK-227T = JCM 10337T = CBS 9040T.

J Biotechnol, 2002 Jul 17, 97(1), 59 - 68
Reactive extraction of lactic acid using alamine 336 in MIBK: equilibria and kinetics; Wasewar KL et al.; Lactic acid is an important commercial product and extracting it out of aqueous solution is a growing requirement in fermentation based industries and recovery from waste streams . The design of an amine extraction process requires (i) equilibrium and (ii) kinetic data for the acid-amine (solvent) system used . Equilibria for lactic acid extraction by alamine 336 in methyl-iso-butyl-ketone (MIBK) as a diluent have been determined . The extent to which the organic phase (amine +MIBK) may be loaded with lactic acid is expressed as a loading ratio, z={HL}(o)/{B}(i,o) . Calculations based on the stoichiometry of the reactive extraction and the equilibria involved indicated that more lactic acid is transferred to the organic phase than would be expected from the (1:1) stoichiometry of the reaction . The extraction equilibrium was interpreted as a result of consecutive formation of two acid-amine species with stoichiometries of 1:1 and 2:1 . Equilibrium complexation constant for (1:1) and (2:1) has been estimated . Kinetics of extraction of lactic acid by alamine 336 in MIBK has also been determined . In a first study of its kind, the theory of extraction accompanied by a chemical reaction has been used to obtain the kinetics of extraction of lactic acid by alamine 336 in MIBK . The reaction between lactic acid and alamine 336 in MIBK in a stirred cell falls in Regime 3, extraction accompanied by a fast chemical reaction occurring in the diffusion film . The reaction has been found to be zero order in alamine 336 and first order in lactic acid with a rate constant of 1.38 s(-1) . These data will be useful in the design of extraction processes.

Curr Top Med Chem, 2002 Jul, 2(7), 675 - 99
Evaluation and development of spinosyns to control ectoparasites on cattle and sheep; Kirst HA et al.; The spinosyns are a novel family of fermentation-derived natural products that exhibit potent insecticidal activities . Spinosad, a naturally-occurring mixture of spinosyn A and spinosyn D, has successfully established its utility for crop protective applications in the agrochemical field . Potential applications of this unique chemical family of macrolides also have been investigated in the field of animal health . Applications for the control of blowfly strike and lice on sheep have now been commercially developed and registered in Australia and potential applications for the control of ectoparasites on cattle are being studied.

Biotechnol Prog, 2002 May-Jun, 18(3), 675 - 8
Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations; Gibson LH et al.; Of five strains of Aureobasidium pullulans studied, NRRL Y-2311-1 yielded the highest titer (26.2 g/L) of pullulan and formed the lowest amount of melanin-like pigment . Sucrose was superior to glucose as the carbon and energy source on the basis of yield and titer of pullulan produced . Pullulan titer was higher (26.2 vs 5.1 g/L), biomass concentration was lower (6.9 vs 12.7 g/L), and DO was lower (0 vs 60% of saturation) when the fermenter was agitated by a marine propeller compared to Rushton impellers . Pullulan produced by strain NRRL Y-2311-1 ranged in weight-average molar mass (M(w)) from 486 KDa and number-average molar mass (M(n)) from 220 Da on day 1 of growth to 390 KDa and 690 Da on day 6; M(w) declined by about 35% from day 1 to day 3, the day of maximum pullulan titer . For the other strains, the ranges of molar mass on the day of maximum pullulan titer were 338-614 KDa (M(w)) and 100-6820 Da (M(n)).

Biotechnol Prog, 2002 May-Jun, 18(3), 666 - 7
Control and fault diagnosis in bottom fermentation systems using parity space approach; Riverol C et al.; The main objective of this note is to describe a real-time fault diagnosis and control for a cylindroconical fermenter to laboratory scale as an alternative to the classic systems . Development of a good controller for a fed-batch reactor is not enough without a fault diagnosis system . We are working to expand this idea . The failure detection system is based on the parity space approach joined with a fuzzy controller with more robustness and of superior performance in MIMO systems compared to conventional strategies . A 2-week period is reported.

Biotechnol Prog, 2002 May-Jun, 18(3), 514 - 23
Extractive bioconversion of 2-phenylethanol from L-phenylalanine by Saccharomyces cerevisiae; Stark D et al.; The bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA) by the yeast Saccharomyces cerevisiae is limited by the toxicity of the product . PEA extraction by a separate organic phase in the fermenter is the ideal in situ product recovery (ISPR) technique to enhance productivity . Oleic acid was chosen as organic phase for two-phase fed-batch cultures, although it interfered to some extent with yeast viability . There was a synergistic inhibitory impact toward S . cerevisiae in the presence of PEA, and therefore a maximal PEA concentration in the aqueous phase of only 2.1 g/L was achieved, compared to 3.8 g/L for a normal fed-batch culture . However, the overall PEA concentration in the fermenter was increased to 12.6 g/L, because the PEA concentration in the oleic phase attained a value of 24 g/L . Thus, an average volumetric PEA production rate of 0.26 g L(-1) h(-1) and a maximal volumetric PEA production rate of 0.47 g L(-1) h(-1) were achieved in the two-phase fed-batch culture . As ethanol inhibition had to be avoided, the production rates were limited by the intrinsic oxidative capacity of S . cerevisiae . In addition, the high viscosity of the two-phase system lowered the k(l)a, and therefore also the productivity . Thus, if a specific ISPR technique is planned, it consequently has to be remembered that the productivity of this bioconversion process is also quickly limited by the k(l)a of the fermenter at high cell densities.

Biotechnol Prog, 2002 May-Jun, 18(3), 437 - 44
Effects of increased impeller power in a production-scale Aspergillus oryzae fermentation; Li ZJ et al.; The goal in this study was to determine how increased impeller power affects enzyme expression in large-scale (80 m(3)), fed-batch Aspergillus oryzae fermentations . An approximate 50% increase in average impeller power was achieved by increasing impeller diameter approximately 10%, while operating at slightly reduced speed . Measured decreases in terminal (95%) mixing time show increased power improved bulk mixing . However, batches operated at increased power had lower recombinant enzyme productivity . Biomass assays and image analysis tests showed no significant difference between "high power" and control batches, suggesting that slower growth, altered morphology, or increased hyphal fragmentation were not the cause of reduced productivity . Off-line tests on the shear-thinning, highly viscous broth show oxygen limitation occurred after transport through the air-liquid interface and imply the limitation may involve bulk mixing . Specifically, oxygen transfer may be limited to a small zone surrounding each impeller . When this is the case, oxygen mass transfer will be determined by both impeller shear and fluid circulation, which have been characterized with the energy dissipation/circulation function (EDCF) . EDCF values during control fermentations were approximately constant at 25 kW m (-3) s(-1), while EDCF values during "high power" batches fell linearly from 40 to 15 kW m (-3) s(-1) . The point at which "high power" EDCF values drop below those in control fermentations corresponds almost exactly with the point at which product titer stops increasing . Thus, our findings suggest oxygen mass transfer was less efficient during the latter half of "high power" fermentations because of reductions in impeller speed and subsequent decreases in EDCF values . This observation has clear implications during the scale-up of viscous fungal fermentations, implying that not only is the level of impeller power important, but also relevant is how this power is applied.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 296 - 302
Construction of a Novel Engineering Strain E.coli G830,which is Adoptable to High-cell-density Fermentation; Yang YG et al.; A novel engineered strain G830 adoptable to high-cell-density fermentation by integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin gene) into thr operon in the chromosome of PA1 blocking Pta-Ack metabolic pathway through the homologous recombination between the homologous fragments of integrated plasmid and that of chromosome . The engineered strain G830 was characterized by phenotype observation, PCR, thr mutant, acetate acid detection, Western blotting and VHb activity assays . In high dentity fermentation, the cellular respiration, energy metabolism, highest bacterial density and dry bacteria weight of the G830 strain were markedly better than control strains PA1 and BL21 . The expression of recombinant prolyl endopeptidase (PEP) in G830 and PA1 under the above condition was high and stable . Their growth situation and fermentation parameters were similar with their parental strains without plasmids, and resided plasmids maintained stably in those strains . It revealed that the integral VHb and acetate metabolism pathway (Pta-Ack) block improved the growth of host strain under low-dissolved-oxygen conditions, enhanced the recombinant proteins production and reduced the accumulation of acetate harmful to bacterial growth . In conclusion, the novel engineering strain G830 was adoptable to high cell density fermentation.

Biophys Chem, 2002 Jun 19, 97(2-3), 213 - 31
Coherent and robust modulation of a metabolic network by cytoskeletal organization and dynamics; Aon MA et al.; In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence . The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway . Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively . The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network . Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e . a high degree of polymerization relieves the negative control . The stability of the model's steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP . The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.

Trends Plant Sci, 2002 Jun, 7(6), 246 - 50
Hydrogenases in green algae: do they save the algae's life and solve our energy problems?
Happe T, Hemschemeier A, Winkler M, Kaminski A.
Green algae are the only known eukaryotes with both oxygenic photosynthesis and a hydrogen metabolism . Recent physiological and genetic discoveries indicate a close connection between these metabolic pathways . The anaerobically inducible hydA genes of algae encode a special type of highly active {Fe}-hydrogenase . Electrons from reducing equivalents generated during fermentation enter the photosynthetic electron transport chain via the plastoquinone pool . They are transferred to the hydrogenase by photosystem I and ferredoxin . Thus, the {Fe}-hydrogenase is an electron 'valve' that enables the algae to survive under anaerobic conditions . During sulfur deprivation, illuminated algal cultures evolve large quantities of hydrogen gas, and this promises to be an alternative future energy source.

Early Sci Med, 2002, 7(1), 31 - 64
Fermentation, phlogiston and matter theory: chemistry and natural philosophy in Georg Ernst Stahl's Zymotechnia Fundamentalis; Chang KM; This paper examines Georg Ernst Stahl's first book, the Zymotechnia Fundamentalis, in the context of contemporary natural philosophy and the author's career . I argue that the Zymotechnia was a mechanical theory of fermentation written consciously against the influential "fermentational program" of Joan Baptista van Helmont and especially Thomas Willis, Stahl's theory of fermentation introduced his first conception of phlogiston, which was in part a corpuscular transformation of the Paracelsian sulphur principle . Meanwhile some assumptions underlying this theory, such as the composition of matter, the absolute passivity of matter and the "passions" of sulphur, reveal the combined scholastic and mechanistic character of Stahl's natural philosophy . In the conclusion I show that Stahl's theory of fermentation undermined the old fermentational program and paved the way for his dualist vitalism.

J Dairy Res, 2002 Feb, 69(1), 163 - 71
Evaluation of volatile compounds in different types of ghee using direct injection with gas chromatography-mass spectrometry; Wadodkar UR et al.; Desi ghee (DG) was prepared from fermented cream followed by heat clarification (desi method) in the laboratory and butter oil (BO) was prepared from fresh butter by melting and centrifugation . Fresh samples of three brands of industrial ghee (IG-1, IG-2, IG-3) were collected from the local market . Volatile compounds of desi and industrial ghee and butter oil were isolated and concentrated using direct injection and cryofocussing techniques; separation and identification was by Gas Chromatography Mass Spectrometry (GCMS) . A maximum of 36 compounds were detected in desi ghee whereas compounds detected in three industrial ghee samples varied from 22-29 . The lowest number of compounds (16) was detected in butter oil . Of the identified compounds, maltol, 5-hydroxymethyl furfuraldehyde, dihydrodihydroxypyranone, 1,3-butanediol and 1-octanol were identified only in desi ghee volatiles . The concentration of acetic acid was found to be remarkably higher in desi ghee volatiles than in industrial ghee . Also the levels of identified fatty acids, methyl ketones, aldehydes, lactones and alcohols were high in desi ghee volatiles compared with industrial ghee and butter oil . In total, 62 compounds were detected, which included 6 aldehydes, 12 ketones, 8 each of fatty acids, alcohols and lactones, 4 each of esters and hydrocarbons or other compounds, and 12 compounds remained unidentified.

J Dairy Res, 2002 Feb, 69(1), 103 - 11
Effect of long-term intake of milk products on blood pressure in hypertensive rats; Sipola M et al.; The effect of long-term intake of two fermented milk products on the development of hypertension was compared in young spontaneously hypertensive rats (SHR) . The products contained tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP), which have been shown to possess angiotensin converting enzyme (ACE) inhibitory activity . Six-week-old SHR were divided into four groups to receive orally ad libitum water, skim milk or two fermented milk poducts (fermented milk A or fermented milk B; the latter is commercially available in Japan with trade name Calpis) for 14 weeks . The calculated intake of IPP was 0.4 mg/d and 0.2 mg/d in the groups receiving fermented milk A and B, respectively, whereas the corresponding amounts for VPP were 0.6 mg/d and 0.3 mg/d . Systolic blood pressure (SBP) was monitored weekly by tail-cuff method . The development of hypertension was significantly attenuated in both groups receiving fermented milk products, whereas skim milk did not affect blood pressure . The effect was detectable after 6 weeks of treatment . At the end of the experiment, the lowest blood pressure level was found in the group receiving fermented milk A: the SBP was 21 mm Hg lower than in the group receiving water and 10 mm Hg lower than in the group receiving fermented milk B . This difference could be explained by larger intake of ACE inhibitory tripeptides in the group receiving fermented milk A as compared with fermented milk B.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(4), 439 - 49
Volatile fatty acids as electron donors for the reductive dechlorination of chloroethenes; Lu XX et al.; Uses of a mixture of six volatile fatty acids (VFAs) including acetic, propionic, butyric, isobutyric, valeric and isovaleric acids as electron donors for the reductive dechlorination of chloroethenes have been investigated by both microcosm and column studies . The fates of tetrachloroethene (PCE), cis-dichloroethene (cis-DCE) and vinyl chloride (VC) in the presence of VFAs and in the absence of VFAs were respectively documented . The results showed that VFAs stimulated complete reductive dechlorination of chloroethenes, either as direct substrates for the dechlorinating bacteria or via H2 formed during VFAs-degradation . There were sequential utilizations of different VFAs by fermenting bacteria . In the microcosm, propionic acid was the first to be used, followed by acetic, butyric, isobutyric, valeric, and isovaleric acids, and their mean first-order degradation rates obtained were 0.128, 0.048, 0.016, 0.027, 0.025 and 0.003 day(-1), respectively . In the column, propionic acid was the first to be used, followed by butyric and valeric acids, and their calculated first-order degradation rates were 0.833, 0.403 and 0.260 day(-1), respectively.

J Environ Sci (China), 2002 Apr, 14(2), 204 - 9
A novel technology for quick acclimation of an anaerobic microbial consortia used for biodegrading teraphthalic acid (TA); Li XM et al.; The seed sludge originated from a methane fermentation reactor was enriched and acclimated with TA as sole carbon source under nitrate respiration mode first for 6 week, and then can be turned to methane fermentation conditions . After 6 weeks processing, the specific rate of TA degradation under nitrate respiration mode reached 23.8 mgTA/(L.gVSS), more than two times of that under methane fermentation mode 11.4 mg TA/(L.gVSS) . The TA reductive cleavers' population density increased about 6 times and 20 times after 30 days and 90 days acclimation . After a total of 90 days for the enrichment and acclimation, the fermentative bacteria which originally existed in the seed sludge nearly disappeared, and instead of them, the TA reductive and cleaving bacteria group was formed in the new consortia, which was confirmed by the MPN counts and roll tube counts . Compared with the control experiment, the acclimation period can be shortened by about 50%.

FEMS Microbiol Lett, 2002 May 7, 210(2), 221 - 5
Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions; Gallmetzer M et al.; Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics . Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria . However, succinate excretion is also widespread among fungi . In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions . The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)) . A strong increase of succinate excretion was observed if the respiratory chain was inhibited . It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle . Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive . A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.

Biochemistry, 2002 Jun 11, 41(23), 7199 - 208
Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus; Meng L et al.; The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli . The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth . The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis . This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes . When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected . The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(II) . Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I . Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide . The substrates MSSHRWDW and MP-p-NA showed the smallest K(m) values while the substrates MGMM and MP-p-NA provided the highest turnover . The catalytic efficiency (k(cat)/K(m)) of PfMetAP-II for MP-p-NA at 30 degrees C was 799 500 and 340 930 M(-1) s(-1) for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively . Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 50 +/- 15 and 20 +/- 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively . Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 +/- 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site . The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry . PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be approximately 85 degrees C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer . The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM . Both specific activity and K(m) values increased with increasing temperature . An Arrhenius plot was constructed from the k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature . Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively) . The thermodynamic parameters calculated at 25 degrees C are as follows: DeltaG++ = 46.23 kJ/mol, DeltaH++ = 10.79 kJ/mol, and DeltaS++ = -119.72 J.mol(-1).K(-1) for Co(II)-loaded PfMetAP; DeltaG++ = 46.44 kJ/mol, DeltaH++ = 16.94 kJ/mol, and DeltaS++ = -99.67 J.mol(-1).K(-1) for Fe(II)-loaded PfMetAP . Interestingly, at higher temperatures (> 50 degrees C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degrees C) than Co(II)-loaded PfMetAP-II.

Food Addit Contam, 2002 Jun, 19(6), 542 - 6
Determination of total arsenic, inorganic and organic arsenic species in wine; Herce-Pagliai C et al.; Forty-five wine samples from the south of Spain of different alcoholic strength were analysed for total arsenic and its inorganic {As(III), As(V)} and organic (monomethylarsonic acid {MMAA}, dimethylarsinic acid {DMAA}) species . The As levels of the wine samples ranged from 2.1 to 14.6 microg l(-1) . The possible effect of the alcoholic fermentation process on the levels of the total arsenic and arsenical species was studied . The average total arsenic levels for the different samples were very similar, without significant differences between all types of wines . In table wines and sherry, the percentages of total inorganic arsenic were 18.6 and 15.6%, with DMAA or MMAA being the predominant species, respectively . In most samples, DMAA was the most abundant species, but the total inorganic aresenic fraction was considerable, representing 25.4% of the total concentration of the element . The estimated daily intakes of total arsenic and total inorganic arsenic for average Spanish consumers were 0.78 and 0.15 microg/person day(-1), respectively . The results suggest that the consumption of these types of wines makes no significant contribution to the total and inorganic arsenic intake for normal drinkers . However, wine consumption contributes a higher arsenic intake than through consumption of beers and sherry brandies.

Int J Food Microbiol, 2002 May 25, 75(3), 213 - 29
Impact of small scale fermentation technology on food safety in developing countries; Motarjemi Y; Fermentation is one of the oldest technologies used for food preservation . Over the centuries, it has evolved and been refined and diversified . Today, a variety of food products is derived from this technology in households, small-scale food industries as well as in large enterprises . Furthermore, fermentation is an affordable food preservation technology and of economic importance to developing countries . In the report of an FAO/WHO Workshop (FAO/WHO, 1996), fermentation was reviewed and the nutritional and safety aspects of fermentation technologies and their products were assessed . Fermentation enhances the nutritional quality of foods and contributes to food safety particularly under conditions where refrigeration or other foods processing facilities are not available . Hazard Analysis and Critical Control Point (HACCP) studies of some fermented products have demonstrated that depending on the process and the hygienic conditions observed during preparation, some fermented foods, e.g . togwa prepared in Tanzania, may pose a safety risk . Fermented foods must therefore be studied following HACCP principles and small-scale food industries and households must be advised on the critical control points of fermentation processes and the control measures to be applied at these points . This paper reviews the risks and benefits of fermentation and demonstrates the application of the HACCP system to some fermented foods in developing countries.

Int J Food Microbiol, 2002 May 25, 75(3), 197 - 212
Appropriate starter culture technologies for small-scale fermentation in developing countries; Holzapfel WH; Modern food biotechnology has moved a long way since ancient times of empirical food fermentations . Preservation and safeguarding of food are, however, still major objectives of fermentation . In addition, other aspects, such as wholesomeness, acceptability and overall quality, have become increasingly important and valued features to consumers even in developing countries where old traditions and cultural particularities in food fermentations are generally well maintained . Due to limitations in infrastructure and existing low technologies, rural areas in most developing countries have not been able to keep abreast of global developments toward industrialisation . At the same time, fermented foods play a major role in the diet of numerous regions in Africa and Asia . In many traditional approaches, the advantages of some form of inoculation of a new batch, e.g . by back-slopping or the repeated use of the same container (e.g . a calabash) is appreciated and generally practised . Still, the benefits of small-scale starter culture application as a means of improved hygiene, safety and quality control, in support of HACCP approaches, are not yet realised in small-scale fermentation operations . Approaches and considerations for the selection of pure cultures for small-scale, low-tech applications may differ in some respects from the large-scale industrial approaches practised since 100 years . Selection criteria should take account of the substrate, technical properties of the strain, food safety requirements and quality expectations . Lack of experience in the application of starter cultures in small-scale operations and under rural conditions presents a major obstacle but also an exciting challenge to food microbiologist and technologist . Culture preservation, maintenance and distribution demand special logistic and economic considerations . Quality, safety and acceptability of traditional fermented foods may be significantly improved through the use of starter cultures selected on the basis of multifunctional considerations, also taking into account the probiotic concept and possibilities offered for improved health benefits.

Int J Food Microbiol, 2002 May 25, 75(3), 181 - 7
Basic requirements for the transfer of fermentation technologies to developing countries; Rolle R et al.; Traditional small-scale fermentation technologies offer considerable potential for stimulating development in the food industry of developing countries in light of their low cost, scalability, minimal energy and infrastructural requirements and the wide consumer acceptance of fermented products in these countries . Efficient transfer and adaptation of these technologies is, however, often limited by inadequate basic scientific knowledge of the processes involved and the lack of appropriate biological inoculants and process controls for these technologies . Basic infrastructures, such as suitably equipped laboratories with consistent working conditions, a constant supply of good quality water and reliable power supplies, are critical elements of a minimal technology base for transfer and adaptation of these technologies . Building the institutional capacity in developing countries to facilitate research and development geared toward a better understanding of the technologies applied in small-scale traditional fermentations is essential, as is the encouragement of governments to formulate supportive national policies, which promote small-scale agro-industrial development . Socioeconomic considerations play a critical role in the successful and sustainable transfer and adoption of technologies and their products in developing countries.

Ukr Biokhim Zh, 2001 Jul-Aug, 73(4), 73 - 8
{Features of lipid peroxidation in brain and liver tissues from aging rats under stress}; Kul'chitskii OK et al.; The lipid peroxidation (LPO) level between in the adult and old rats brain and liver was determined as to be essentially undiffering . Stress activated the LPO independence the age of animals and tissues investigated . The concentration changes of LPO products testify to it . In the adult rats under the stress capability of tissues to induction in vitro ferment and ascorbat-depending LPO, in comparison with the control, decreases, at old--does not change in the brain and considerably grows in the liver . Stress is accompanied by an oppression of Na, K-ATP-ase PM activity of hepatocytes, more expressed in the old animals.

Ukr Biokhim Zh, 2001 Jul-Aug, 73(4), 100 - 3
{Activity of glutathione reductase and glutathione-S-transferase in rabbit brain tissue in the long-term post-traumatic period}; Cherchenko AP et al.; The glutathione-reductase and glutathione-S-transferase activities in the cortex and brain stem tissues of rabbits with post-traumatic epileptic reality (1 year after the light brain injury) was defined . An increase of glutathione-reductase activity in the cortex microsomal and stem mitochondrial fractions, and increase of glutathione-S-transferase activity in cortex and stem mitochondrial fractions was obtained . The conclusion is made that the activation of the anti-oxidant glutathione fermentative system is a long-term metabolic CNS adaptation in the case while mitochondrial oxidation and oxidative phosphorilation are disturbed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 629 - 633
Expression, Purification, Characterization of Amphioxus Insulin-like Peptide and Preparation of Polyclonal Antibody to It; Shen L et al.; To elucidate the origination and evolution as well as structure-function relationship of insulin and IGF-1, the gene of amphioxus insulin-like peptide (ILP), a common ancestor of insulin and IGF-1, was chemically synthesized and cloned into the expression vector pVT102-U . In the recombinant ILP, the C-terminus of B-domain and the N-terminus of A-domain of amphioxus ILP deduced from cDNA were linked together by a tripeptide, Ala-Ala-Lys, and its B28Arg residue was also replaced by Lys to facilitate its conversion to a double-chain form by Lys-C cleavage . The expression vector was transformed into yeast cells and the recombinant ILP was expressed efficiently . The purified single-chain and double-chain ILP was obtained by fermentation, purification and enzymatic cleavage . Molecular weight measurement and amino acid composition analysis showed that the primary structure of ILP was correct . Circular dichroism analysis showed that the secondary and tertiary structure of double-chain ILP was similar to that of insulin, while double-chain ILP had no measurable insulin activity in insulin receptor binding assay . Additionally, New Zealand Rabbits were immunized with single-chain ILP and high titre polyclonal antibody was obtained . This work was important for further investigating the molecular evolution of insulin and IGF-1 and the distribution and existence of ILP in amphioxus.

J Agric Food Chem, 2002 Jun 5, 50(12), 3597 - 601
Antioxidative functions of natto, a kind of fermented soybeans: effect on LDL oxidation and lipid metabolism in cholesterol-fed rats; Iwai K et al.; Natto water-soluble fractions, low-molecular-weight viscous substance, and soybean water extract, which had an inhibitory effect on the oxidation of low-density lipoproteins (LDL) in vitro, were fed to rats for 3 weeks . These fractions had no influence on the growth of rats, which were fed a basal diet containing 1% cholesterol, but lowered plasma triglyceride and total cholesterol . Inhibition of copper-oxidation of plasma and LDL ex vivo, and a reduction in lipid peroxidation in liver and aorta in vivo, were also observed . The antioxidant enzymes were not induced in rats fed natto fraction diets . These results demonstrate that ingestion of the natto fractions led to inhibition of LDL oxidation, and that the fractions perform direct antioxidant action in the body . It is suggested that natto fractions might help to prevent arteriosclerosis, as they appear to reduce lipid peroxidation and improve lipid metabolism.

J Agric Food Chem, 2002 Jun 5, 50(12), 3592 - 6
Inhibitory effect of natto, a kind of fermented soybeans, on LDL oxidation in vitro; Iwai K et al.; The oxygen radical scavenging activity of natto (fermented soybeans) and its inhibitory effect on the oxidation of rat plasma low-density lipoprotein (LDL) in vitro were investigated to evaluate the usefulness of the antioxidant properties of natto, which has been shown to have antioxidant activity . Natto was separated into three water-soluble fractions: high-molecular-weight viscous substance (HMWVS; Mw > 100 000), low-molecular-weight viscous substance (LMWVS; Mw < 100 000), and soybean water extract (SWE) . LMWVS had the strongest radical scavenging activity for hydroxyl and superoxide anion radicals, as assessed by electron spin resonance . The increase of conjugated dienes in LDL oxidized by copper and an azo pigment was depressed by the addition of LMWVS and SWE . These results demonstrate that natto fractions have inhibitory effects on LDL oxidation as a result of their radical scavenging activity.

J Agric Food Chem, 2002 Jun 5, 50(12), 3527 - 32
Chemical composition and flavor of ecuadorian cocoa liquor; Luna F et al.; The contribution of the chemical composition to the flavor of cocoa liquor from an Ecuadorian selfed population of clone EET 95 was investigated . Polyphenols, purine alkaloids, organic acids, and sugars were quantified, and the key sensory characteristics of cocoa were scored by a trained panel . Despite the short bean fermentation (2 days) commonly used for Arriba cocoa, acetic acid content was closely correlated to liquor pH, demonstrating its essential role in cocoa liquor acidification . Polyphenols were positively correlated to astringency, bitterness, and the green note and negatively correlated to the fruity character . Alkaloid and polyphenol levels fluctuated significantly within the selfed progeny and tended to be lower than those of the heterozygous clone EET 95 (inbreeding effect) . These results support the idea that polyphenols might be essential to the overall perception of cocoa liquor characteristics and indicate that the composition and the sensory quality of cocoa liquor are the result of both a genotypic contribution and the conditions of fermentation and roasting.

J Agric Food Chem, 2002 Jun 5, 50(12), 3412 - 8
Method development and fate determination of pesticide-treated hops and their subsequent usage in the production of beer; Hengel MJ et al.; The fate of residues of seven agrochemicals (chlorfenapyr, quinoxyfen, tebuconazole, fenarimol, pyridaben, and E- and Z-dimethomorph) from the treatment on hops to the brewing of beer was studied . First, a multi-residue analytical method was developed for the determination of pesticide residues in spent hops, trub, wort, and beer . Each matrix was validated over at least two levels of fortification, for all seven compounds, in the ranges 0.05-5.0, 0.001-1.0, 0.001-0.05, and 0.0005-1.0 ppm for spent hops, trub, wort, and beer, respectively . Recoveries ranged from 73 to 136% . Second, the matrixes prepared from hops, which were treated under commercial practices with each compound, were analyzed using the method developed . The use of treated hops resulted in the carryover of 0.001 ppm of tebuconazole, 0.008 Z-dimethomorph, and 0.005 ppm of E-dimethomorph into the wort . The bulk of the remaining residues of all seven compounds was found on the spent hops . Following fermentation, all compounds were found in levels less than 0.0005 ppm in beer, except Z- (0.006 ppm) and E-dimethomorph (0.004 ppm) . Third, when all seven pesticides were spiked prior to the pitching of yeast into clean wort, most of the nonpolar compounds (chlorfenapyr, quinoxyfen, and pyridaben) partitioned into the organic material (trub) which settled to the bottom, while the more polar compounds (fenarimol, tebuconazole, and E- and Z-dimethomorph) were generally distributed evenly between the beer and the trub.

J Ind Microbiol Biotechnol, 2002 Jun, 28(6), 325 - 32
Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions; Vemuri GN et al.; We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase . AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node . Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase . Activities of six key enzymes were also determined for these aerobic fermentations . From these results, six transition times based on physiological states were selected for studying dual-phase fermentations . The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition . Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h.

Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 15 - 22
Cellobiose quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium is produced by intracellular proteolysis of cellobiose dehydrogenase; Raices M et al.; The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products . After 24 h of induction, CDH was detected in the culture supernatant and a protein was recognized by a specific anti-CDH polyclonal antibody in the sonicated biomass . Northern blot experiments performed with several fungal RNA samples showed, after 24 h of induction, only one single species of an mRNA transcript corresponding in size to the cdh gene (2.5 kb) The relative amount of this transcript decreased as a function of time . Southern blot experiments done with genomic DNA and database search in the recently available genome information also ruled out the presence in this strain of a separate cbq gene distinct from the cdh gene . Taken together, these results demonstrated that CBQ originates from the cdh gene . Furthermore, it is not produced by differential splicing but by a posttranslational, predominantly intracellular, proteolytic cleavage.

Curr Microbiol, 2002 Jul, 45(1), 1 - 4
Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae; Oda Y et al.; Thirty-eight strains of the fungus Rhizopus oryzae were grown on potato pulp, an agricultural by-product of the starch industry . Either lactic acid or fumaric acid and ethanol were formed, and the ratio differed among the strains tested . The highest amount of L(+)-lactic acid (10 mg/g fresh matter) was observed in the pulp fermented for six days by Rhizopus oryzae IFO 4707 . The IFO 4707 strain rapidly reduced the hardness and pH of potato pulp within one day followed by the gradual synthesis of lactic acid . A composition analysis showed that the enzymes secreted from the fungal cells hydrolyzed starch efficiently with partial degradation of the cell wall . Rhizopus oryzae may be used as an inoculant for ensiling potato pulp and other agricultural by-products containing starch.

J Bacteriol, 2002 Jun, 184(12), 3348 - 59
Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase; Hao T et al.; Omega4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter . DNA upstream of the insertion site was cloned, and the promoter was localized . The promoter resembles vegetative promoters in sequence, and sigma(A) RNA polymerase, the major form of RNA polymerase in growing M . xanthus, initiated transcription from this promoter in vitro . Two complete open reading frames were identified downstream of the promoter and before the Omega4514 insertion . The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators . ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation . Tn5 lac Omega4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase . An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Omega4514 in orf3 caused a defect . Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases . ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant . Also, multiple upstream DNA elements are necessary for full developmental expression . These results suggest that transcriptional activation also regulates the operon . Omega4514 is the first example of a developmentally regulated M . xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.

Lett Appl Microbiol, 2002, 34(6), 389 - 93
Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairii; Kim SW et al.; AIMS: The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii . METHODS AND RESULTS: The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30 degrees C and 6.0, respectively . Sucrose (60 g l(-1)) and corn steep powder (10 g l(-1)) were the most suitable carbon and nitrogen source for exopolysaccharide production . CONCLUSIONS: Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7.4 g l(-1), which was approximately three times higher than that in basal medium . The maximum specific growth rates (micro max) and yield coefficient (Y(P/S)) in the optimal culture medium was 0.16 h(-1) and 0.19, respectively . SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms.

J Nat Prod, 2002 May, 65(5), 786 - 8
New tricyclic sesquiterpenes from the fermentation broth of Stereum hirsutum; Yun BS et al.; In search for novel metabolites from the culture broth of the mushroom Stereum hirsutum, three new tricyclic sesquiterpenes named hirsutenols A (1), B (2), and C (3) have been isolated . Their structures were assigned on the basis of various spectroscopic studies.

J Nat Prod, 2002 May, 65(5), 742 - 4
14'-Hydroxymytoxin B and 16-hydroxyroridin E, two new cytotoxic trichothecenes from Myrothecium roridum; Alvi KA et al.; Two new trichothecenes, 14'-hydroxymytoxin B (1) and 16-hydroxyroridin E (3), were isolated from a fermentation extract of Myrothecium roridum . The structures of 1 and 3 were determined by spectral data interpretation . Both compounds showed potent cytotoxic activity against primary soft-tissue sarcoma cells.

J Nutr Sci Vitaminol (Tokyo), 2002 Feb, 48(1), 1 - 5
Resistant protein; its existence and function beneficial to health; Kato N et al.; The remnants of proteins themselves or complexes with protein remaining no longer indigestible in the intestine are referred to as resistant proteins, which exert physiological functions similar to dietary fibers and are also better for health . In recent years, noticeable functions attributable to resistant proteins have become gradually apparent with regard to several proteinous items . Recent investigations have revealed that the relevant ingredients are either condensed in isolates or concentrates of vegetable proteins or causally brought about as a consequence of denaturation and/or entanglement in the process of preparation . Some protein components inherently insusceptible to mammalian digestive enzymes also belong to the group of resistant proteins in case of edibility irrespective of their sources . Among the medicinal benefits of several resistant proteins hitherto pointed out by animal experiments, there were preventive effects against hypercholesterolemia, constipation, corpulence, tumorigenesis (colon, liver, mammary gland), gallstone formation or poisoning, and wholesome improvements in enteric fermentation of short-chain fatty acids.

Chronobiol Int, 2002 Mar, 19(2), 361 - 75
Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae; Uno T et al.; Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae . To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved . Neither the protein levels of major Hsps nor the expression of the beta-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation . The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism . This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance . These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps . The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level . The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.

Mikrobiologiia, 2002 Mar-Apr, 71(2), 200 - 4
{Growth-coupled lipid synthesis in Mortierella alpina LPM 301, a producer of arachidonic acid}; Eroshin VK et al.; Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of intense lipid synthesis in the period of active mycelium growth . Under batch cultivation of this strain in glucose-containing media with potassium nitrate or urea, the bulk of lipids (28-35% of dry biomass) was produced at the end of the exponential growth phase and remained almost unaltered in the stationary phase . The ARA content of lipids comprised 42-50% at the beginning of the stationary phase and increased continuously after glucose depletion in the medium due to the turnover of intracellular fatty acids; by the end of fermentation (189-210 h), the amount of ARA reached 46-60% of the total fatty acids (16-19% of dry mycelium) . Plausible regulatory mechanisms of the growth-coupled lipid synthesis in microorganisms are discussed.

Microb Ecol, 2001 Oct, 42(3), 238 - 247
Sulfate Reduction at a Lignite Seam: Microbial Abundance and Activity; Detmers J et al.; In a combined isotope geochemical and microbiological investigation, a setting of multiple aquifers was characterized . Biologically mediated redox processes were observed in the aquifers situated in marine sands of Tertiary age and overlying Quaternary gravel deposits . Intercalated lignite seams define the aquitards, which separate the aquifers . Bacterial oxidation of organic matter is evident from dissolved inorganic carbon characterized by average carbon isotope values between ?18.4 per thousand and ?15.7 per thousand (PDB) . Strongly positive sulfur isotope values of up to +50 per thousand (CTD) for residual sulfate indicate sulfate reduction under closed system conditions with respect to sulfate availability . Both, hydrochemical and isotope data are thus consistent with the recent activity of sulfate-reducing bacteria (SRB) . Microbiological investigations revealed the presence of an anaerobic food chain in the aquifers . Most-probable-number (MPN) determinations for SRB and fermenting microorganisms reached highest values at the interface between aquifer and lignite seam (1.5 x 103 cells/g sediment dry mass) . Five strains of SRB were isolated from highest MPN dilutions . Spore-forming bacteria appeared to dominate the SRB population . Sulfate reduction rates were determined by the 35S-radiotracer method . A detailed assessment indicates an increase in the reduction rate in proximity to the lignite seam, with a maximum turnover of 8.4 mM sulfate/a, suggesting that lignite-drived compounds represent the substrate for sulfate reduction.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1921 - 7
Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase; Vessillier S et al.; The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system . The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant . The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66% . Analysis of the kinetic parameters revealed efficient activities (k(cat)/K(m) ratios of >10(6) M(-1) s(-1)) toward most penam and carbapenem compounds, with the exception of the 6-alpha-methoxypenam derivative temocillin and of biapenem, which were poorer substrates . Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds . BlaB was also able to hydrolyze serine-beta-lactamase inhibitors, including beta-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

J Dairy Sci, 2002 Apr, 85(4), 909 - 18
Effect of casein infusion in the rumen, duodenum or both sites on factors affecting forage intake and performance of dairy cows fed red clover-grass silage; Khalili H et al.; Four lactating dairy cows were used in a balanced 4 x 4 Latin square design to examine the effects of casein infusion in the rumen, duodenum, or both on the intake of red clover-grass silage, chewing behavior, diet digestion, microbial protein synthesis, rumen fermentation, digestion, and passage kinetics, milk production, and milk composition . Duodenal infusion increased eating time, silage intake, rumen neutral detergent fiber pool, yields of milk, protein, and lactose, and concentration of milk protein, and tended to decrease that of milk fat . Ruminal infusion tended to decrease eating time, and increased significantly microbial protein synthesis, rumen ammonia-N concentration, molar proportions of isovalerate and valerate, digestion rate of digestible neutral detergent fiber, passage rate of indigestible neutral detergent fiber, and milk urea content, and decreased rumen neutral detergent fiber pool . Silage intake, and yields of milk, protein, and lactose were highest when casein was simultaneously infused in both sites.

J Dairy Sci, 2002 Apr, 85(4), 879 - 88
Effects of sucrose supplementation on dry matter intake, milk yield, and blood metabolites of periparturient Holstein dairy cows; Ordway RS et al.; Thirty-four multiparous Holstein dairy cows (780 +/- 17.2 kg body weight; 3.4 +/- 0.08 body condition score) were used in a completely randomized design to evaluate the effects of fermentable carbohydrate source on dry matter (DM) intake, milk production, and blood metabolites of transition cows . Treatments were initiated 30 d before expected calving date . After calving, all cows went onto a similar lactating cow diet . Dry matter intake was measured daily, and milk production and composition were measured weekly for 56 d after calving The control diet consisted of 11.5% ground corn, whereas the treatment diet consisted of sucrose replacing 2.7% of ground corn on a DM basis . Prepartum plasma glucose concentrations were higher (66.3 vs . 69.3 +/- 1.13 mg/dl) for cows fed the diet containing sucrose . Glucose concentrations were not different postpartum . Prepartum and postpartum nonesterified fatty acids, blood urea N, and insulin did not differ between treatments . Substitution of sucrose as a readily available carbohydrate source for ground corn did not affect prepartum or postpartum DM intake . Milk yield, 3.5% fat-corrected milk yield, and milk components did not differ between treatments . Results from this research demonstrated that partially replacing ground corn with sucrose did not enhance prepartum or postpartum intake or performance.

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