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What Is Fermentation?In its strictest sense fermentation is the energy-yielding anaerobic metabolic breakdown of a nutrient molecule, such as glucose, without net oxidation. Fermentation yields lactate, acetic acid, ethanol, or some other simple product. Fermentation is also used much more broadly to refer to the bulk growth of microorganisms on some medium. No distinction is made between aerobic and anaerobic metabolism when the word is used in this sense. This process is often used to produce or preserve food. Fermentation typically refers to the fermentation of sugar to alcohol using yeast, but other fermentation processes include the making of yogurt. The science of fermentation is known as zymology. Fermentation usually implies that the action of the microoganisms is desirable. French chemist Louis Pasteur was the first zymologist, when in 1857 he connected yeast to fermentation. Pasteur originally defined fermentation as respiration without air. Pasteur performed careful research and concluded, "...I am of the opinion that alcoholic fermentation never occurs without simultaneous organization, development and multiplication of cells... ...if asked, in what consists the chemical act whereby the sugar is decomposed... ...I am completely ignorant of it...." The German Eduard Buchner, winner of the 1907 Nobel Prize in chemistry, later determined that fermentation was actually caused by a yeast secretion that he termed zymase. The research efforts undertaken by the Danish Carlsberg scientists greatly accelerated the gain of knowledge about yeast and brewing. The Carlsberg scientists are generally acknowledged with kick-starting the entire field of molecular biology. With few exceptions, all dietary carbohydrates and proteins can serve as substrates for microbial fermentation. Nonetheless, the crucial advantage of being a herbivore is the ability to efficiently extract energy from cellulose and other components of plant cell walls. Cellulose fibers account for 40-50% of the total dry weight of stems, leaves and roots. These fibers are embedded in a matrix of hemicelluloses and phenolic polymers (lignin-carbohydrate complexes) that are covalently crosslinked. Cellulose itself is a linear polymer of glucose molecules linked to one another by beta[1-4] glycosidic bonds and herein lies the problem for the vertebrate digestive system. As far as is known, no enzyme able to hydrolyze beta[1-4] glycosidic bonds has evolved in vertebrates. However, a variety of such beta-glucanases are synthesized by microbes. Thus, the diverse population of bacteria and protozoa in the rumen or hindgut produce all the enzymes necessary to digest cellulose and hemicellulose. The glucose released in this process is then taken up and metabolized by the microbes, and the waste products of microbial metabolism are passed on to the host animal. Sugars derived from digestion of soluble carbohydrates such as starch are processed similarly. Fermentation occurs under anaerobic conditions. As a consequence, sugars are metabolized predominantly to volatile fatty acids (VFAs). Additional major products include lactic acid, carbon dioxide and methane. The principle VFAs are acetic, proprionic and butyric acids, which collectively provide for the majority of a herbivore's energy needs. The ratio of these VFA's vary with diet, although the majority product is always acetate. On a diet high in fiber, the molar ratio of acetic to proprionic to butyric acids is roughly 70:20:10. As described above, proteins are also important substrates for fermentation. In caudal fermenters, much of the dietary protein is digested and absorbed prior to the large gut, but in ruminants, all dietary protein enters the rumen. The bulk of this protein is digested by microbial proteases and peptidases. The resulting peptides and amino acids are taken up by microbes and used in several ways, including microbial protein synthesis. However, a large quantity of amino acids ingested by fermentative microbes are deaminated and enter some of the same pathways used for carbohydrate metabolism. The net result is that much of dietary protein is metabolized to VFAs. Clearly, from the standpoint of the host animal, VFAs are the important product of fermentation. These small lipids are used for many purposes, but the paramount importance of VFAs to herbivores is that they are absorbed and serve as the animal's major fuel for energy production, serving much the same function that glucose does in you. Additional information on the way in which VFAs are put to use is presented in subsequent sections. Glycolysis (via the traditional Embden-Meyerhoff pathway) is the enzymatic breakdown of glucose into pyruvate. This process requires ADP and NAD+ as co-factors , and produces ATP and NADH inside the cell. A cell can always find a way to hydrolyze ATP back into ADP when needed (no oxidation/reduction is involved in ATP hydrolysis), but NAD+ is a more difficult problem, as its formation involves oxidation of NADH. Under aerobic conditions, the electrons from NADH can ultimately be passed to oxygen, but under anaerobic conditions, another electron acceptor is needed. During the primary fermentation (H), the fermentable sugars, mainly maltose and glucose are converted to ethanol and carbon dioxide. This action is performed by the brewing yeast, which during the brewing process also produces many of the characteristic aroma compounds found in beer. At the end of the primary fermentation, the yeast cells flocculate and sediment at the bottom of the fermenter and can be cropped and used for a new fermentation. Not all yeast cells sediment; some will remain in suspension, and these cells are responsible for maturation of the beer. During this process the off-flavour, diacetyl is degraded to below the taste threshold. The fermentation characteristics of brewer's yeast are strain-dependent and are genetically inherited. Much of the genetics of Saccharomyces yeasts has been elucidated, and the knowledge gained, forms the basis for breeding of brewing yeast. Thus, new types of beer with altered aromas can be produced with yeast strains selected through breeding. Malolactic fermentation (MLF, or malo) is an important winemaking process conducted on most red grape wines and some white grape wines. It is also used with some fruit wines. The following article gives information concerning the conditions necessary for MLF, its affects, prevention, progress, suitable wine type candidates for it and yeast compatibility. Malolactic fermentation bacteria MLF is conducted by lactic acid bacteria (LAB), of the genera Lactobacillus, Pediococcus, and Leuconostoc. Not all LAB are desirable for MLF. Leuconostoc oenos is the most beneficial, and probably the most frequently occurring species of LAB in wine. Species associated with wine spoilage are generally members of Lactobacillus and Pediococcus genera. The Lactobacillus genus, for example, can cause acescence (excessive acetic acid) by metabolising sugar or tartaric acid [Radler and Yannissis, 1972]. Many LAB metabolise pentoses, tartaric acid and glycerol. The term "malolactic fermentation bacteria" (MLB) is commonly used to refer to those LAB strains which are more desirable for MLF. They are more resistant to low pHs such as those in wine (and in which other LAB find it more difficult to live) and they prefer to metabolise malic acid over sugars and citric acid (and they do not metabolise tartaric acid or glycerol). In brewing, fermentation is the conversion of sugar into carbon dioxide gas (CO2) and ethyl alcohol. This process is carried out by enzymes within a yeast cell. It is in fact a complex series of conversions that bring about the conversion of sugar to CO2 and alcohol. Yeast is a member of the plant family and in brewing we use the sugar fungi form of yeast. These cell gain energy from the break down of the sugar. The by-product, CO2, bubbles through the liquid and dissipates into the air. The alcohol remains in the liquid which is great for us but not for the yeast, as the yeast dies when the alcohol exceeds its tolerance level. Brewer's yeast tolerate up to about 5% alcohol. Beyond this alcohol level the yeast cannot continue fermentation. Wine yeast on the other hand tolerates up to about 12% alcohol. The level of alcohol tolerance by yeast varies from 5% to about 21% depending on yeast strain. The fermentation process has other limits such as temperature. Greater than 27C kills the yeast less and than 15C results in yeast activity which is too slow. The amount of sugar in the solution can be too much and this can prevent fermentation. Some recipes suggest adding the sugar in parts throughout fermentation rather that all at the beginning. This is especially true if the brew is aimed at producing a high level of alcohol. Some yeast strains have evolved to handle higher sugar levels. Yeast such as Tokay and Sauterne. J Ind Microbiol Biotechnol . 2005 Jan 15; {Epub ahead of print}Reducing by-product formation in L: -lactic acid fermentation by Rhizopus oryzae; Wang X et al.; During L: -lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l(-1) to 0.6 g l(-1) KH(2)PO(4) reduced the maximal concentration of L: -lactic acid and fumaric acid from 85 g l(-1) to 71 g l(-1) and from 1.36 g l(-1) to 0.18 g l(-1), respectively; and it decreased the fermentation time from 72 h to 52 h . Phosphate at 0.40 g l(-1) KH(2)PO(4) was suitable for both minimizing fumaric acid accumulation and benefiting L: -lactic acid production. J Dairy Sci, 2005 Feb, 88(2), 757 - 65 Influence of extruded soybeans with or without bicarbonate on milk performance and Fatty Acid composition of goat milk; Schmidely P et al.; The effects of extruded soybeans (ESB) included at 0, 10, or 20% of dry matter (DM) of the diet in combination with sodium bicarbonate (0 vs . 1% bicarbonate added to DM) on rumen fermentation characteristics, production parameters, and fatty acid (FA) profiles of milk fat were examined in 30 midlactation goats and 6 rumen-cannulated goats fed high-concentrate diets (30:70 forage-to-concentrate ratio) ad libitum in a 3 x 2 factorial design . Diets were fed as total mixed rations . The trial lasted 13 wk with the final 9 wk as the test period . Milk yield and composition were recorded each week throughout the trial . Individual samples of milk were taken in wk 4, 7, 10, 11, and 13 to determine FA profile of milk fat . Dry matter intake and intake of net energy for lactation were not affected by dietary treatments . Feeding ESB did not modify ruminal pH or volatile fatty acids concentration in the rumen fluid, but it increased the molar proportion of propionate . Feeding ESB increased fat-corrected milk, milk fat content, and fat yield compared with the control diets . There was no change in milk protein content when ESB were fed . Feeding ESB increased the proportions of oleic, linoleic, and linolenic acids in milk fat at the expense of most of the saturated FA . It also increased the n-6 to n-3 FA ratio of milk . The largest changes in milk yield and milk composition were generally obtained with ESB included at 20% of DM . The addition of sodium bicarbonate tended to increase ruminal pH, VFA concentrations in the rumen fluid, and the molar proportions of acetate . The addition of sodium bicarbonate increased milk fat content and fat yield, with no change in milk FA composition . It is concluded that during midlactation, the inclusion of ESB to 20% of DM prevented low milk fat content for goats fed high-concentrate diets, with no decrease in milk protein content . The addition of sodium bicarbonate may enhance the effects of ESB on milk fat content and fat yield. J Dairy Sci, 2005 Feb, 88(2), 698 - 710 Effect of different dietary geometric mean particle length and particle size distribution of oat silage on feeding behavior and productive performance of dairy cattle; Leonardi C et al.; Twenty lactating Holstein cows (5 primiparous and 15 multiparous) were used in a 5 x 5 Latin Square design, with 5 treatments and 3 periods of 21 d each . Diets contained 25% corn silage, 25% oat silage, and 50% concentrate (dry matter basis) . The 5 treatments tested in the experiment were long oat silage (LOS), medium oat silage (MOS), fine from long oat silage (FLOS), fine from medium oat silage (FMOS), and half LOS plus half FLOS (LFLOS) . The geometric mean particle length (GMPL) of the diets was 6.68, 5.19, 4.46, 4.35, and 5.39 mm for LOS, MOS, FLOS, FMOS, and LFLOS, respectively . The LFLOS was designed to provide dietary GMPL similar to MOS, but with a more bimodal particle size distribution (PSD) . Linear and quadratic effects of GMPL were tested, based on the mean GMPL of the feed actually consumed (cGMPL) . Contrasts were used to test for the effect of different PSD (MOS vs . LFLOS) and to test for differences between FMOS and FLOS, which would indicate unequal fermentations in the MOS and LOS silos . No differences were detected between FMOS and FLOS in most of the variables measured . Increasing cGMPL linearly decreased dry matter intake, milk production, and milk protein percentage and yield without affecting milk fat percentage, milk fat yield, ruminal pH, and ruminal volatile fatty acid concentration . Although cows fed diets with increasing cGMPL spent more time eating and chewing per day and per kilogram of dry matter intake, there was no effect of cGMPL on rumen pH . Feeding medium oat silage increased milk fat percentage and yield compared with feeding a mixture of long and fine oat silage. J Dairy Sci, 2005 Feb, 88(2), 690 - 7 Ruminal fermentation and amino Acid flow in holstein steers fed whole cottonseed with elevated concentrations of free Fatty acids in the oil; Sullivan HM et al.; The influence of feeding whole cottonseed (WCS) containing elevated concentrations of free fatty acids (FFA) in the oil on ruminal fermentation and amino acid (AA) flow to the abomasum was evaluated in a 4 x 4 Latin square trial . Four ruminally and abomasally cannulated Holstein steers were fed diets containing 12.5% of dry matter as WCS with concentrations of 8.0, 11.3, 14.7, or 18.0% FFA in the oil . Intake, ruminal digestibility, and flow to the abomasum of dry matter, organic matter, and acid detergent fiber were not affected by FFA level of WCS . Intake of neutral detergent fiber and total kilograms of neutral detergent fiber digested in the rumen were similar for all treatments . Ruminal neutral detergent fiber digestibility was lower for 8 and 14.7% FFA, resulting in a cubic effect on flow to the abomasum . Ruminal pH, molar proportions of isobutyrate, and total branched-chain volatile fatty acids (VFA) decreased linearly, whereas molar proportions of acetate and acetate:propionate ratio increased linearly as FFA in WCS increased . Total VFA were lower, and molar proportions of propionate were higher, for 8 and 14.7% FFA, resulting in a cubic effect . Intake of N, total N flow, and nonmicrobial N flow to the abomasum were similar among treatments . Flow of microbial N was lower for the 11.3% FFA treatment, resulting in a quadratic response . Only nonsignificant differences were observed in AA flow to the abomasum . Results of this trial indicate that WCS with FFA up to 18% may result in small changes in rumen fermentation. J Biotechnol, 2005 Mar 2, 116(1), 79 - 89 Epub 2004 Dec 28. Separation and purification of benzylpenicillin produced by fermentation using coupled ultrafiltration and nanofiltration technologies; Tessier L et al.; The purpose of this study was to evaluate the capacity of using coupled ultrafiltration-nanofiltration technologies for separation and purification of benzylpenicillin (BP) . More specifically, we verified the efficiency of three ultrafiltration (UF) membranes (cut-off of 5000, 30,000 and 100,000 Da) to remove impurities that cause stable emulsion during the chemical extraction of the antibiotic . We also tested the effectiveness of a nanofiltration (NF) membrane (cut-off of 300 Da) to concentrate the benzylpenicillin recovered from permeates and to decrease the osmotic pressure by reducing the ionic charge of the broth . Results have shown that high recovery (89.0-91.0%) can be obtained in permeate generated by the 30,000 and 100,000 UF membranes, but a slight emulsion will be formed during phase separation . With the 5000 UF membrane, lower recovery is obtained (81.0%) but no emulsion is produced, leading to a high solvent extraction yield (94.6%) . The nanofiltration of 30,000 and 100,000 UF permeates leads to very high recovery (98.0%), but stable emulsions are formed, reducing the chemical extraction yield (80.0-82.6%) . For the nanofiltration of 5000 UF permeate, excellent recovery of the antibiotic is noted (97.4%) leading to high extraction yield (92.4%) with no emulsion formed . Diafiltration step should be applied during UF procedure in order to increase the antibiotic recovery in the generated permeates. Appl Microbiol Biotechnol . 2005 Jan 14; {Epub ahead of print} Monoxenic production of the entomopathogenic nematode Steinernema carpocapsae using culture media containing agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp) . Effects of the contents of nitrogen, carbohydrates and fat on infective juvenile production; Islas-Lopez MA et al.; The production of infective juvenile stages (IJ) of the entomopathogenic nematode Steinernema carpocapsae in the presence of its symbiotic bacterium Xenorhabdus nematophilus was carried out in orbitally agitated bottles . Four complex culture media (M1-M4) were used, containing from 8% to 28% (by vol.) agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp) as the main carbohydrate source . After 20 days of fermentation, a maximum viable IJ concentration of 249,000 IJ/ml and an initial nematode population multiplication factor of x620 were achieved when medium M4 was used (aguamiel concentration in this medium was 28% by vol.) . M4 medium contained (w/v): 0.3% total nitrogen, 3.2% total carbohydrates and 3.0% total fat . According to the results obtained, total carbohydrates concentration appeared to be of great importance in obtaining high IJ concentrations. Comp Biochem Physiol B Biochem Mol Biol, 2005 Feb, 140(2), 259 - 69 Gut carbohydrases from the New Zealand marine herbivorous fishes Kyphosus sydneyanus (Kyphosidae), Aplodactylus arctidens (Aplodactylidae) and Odax pullus (Labridae); Skea GL et al.; Carbohydrase activities were examined in Odax pullus (Labridae), Kyphosus sydneyanus (Kyphosidae) and Aplodactylus arctidens (Aplodactylidae) collected from subtidal reefs in northeastern New Zealand . Enzyme extracts were prepared using two methods from gut wall, gut fluid and microbial pellet samples taken serially along the gut, and assayed against the substrates starch, laminarin, carrageenan, alginate and agarose . In all three fish species, starch degradation activity was substantially higher than for any other substrate tested . Activities of 500, 1294 and 3326 units g tissue(-1) were measured in anterior gut wall extracts of O . pullus, K . sydneyanus and A . arctidens, respectively . Starch degrading activity in gut fluid declined from 37, 313 and 284 units ml(-1) in anterior gut sections of O . pullus, K . sydneyanus and A . arctidens, respectively, to less than 50 units ml(-1) in terminal gut section of each species . Activity against structural polysaccharides was much lower than against starch and was detected mainly in posterior gut sections . The two methods of sample preparation differed little in enzyme activities; however, method of sample preparation did affect isoform patterns as displayed by zymogram analysis . Results suggest that these fish species fall on a continuum from maximizing throughput and digesting easily hydrolysed substrates in the foregut in A . arctidens to relying more heavily on microbial fermentation in the hindgut in K . sydneyanus. J Biol Chem . 2005 Jan 12; {Epub ahead of print} New functions for parts of the krebs cycle in procyclic trypanosoma brucei, a cycle not operating as a cycle; van Weelden SW et al.; It was investigated whether substrate availability influences the type of energy metabolism in procyclic T . brucei . We showed that absence of glycolytic substrates (glucose and glycerol) did not induce a shift from a fermentative metabolism to complete oxidation of substrates . We also showed that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic T . brucei procyclic cells . Furthermore, absence of glucose did not result in increased degradation of amino acids . Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance . We argue why it is likely that in procyclic cells, phosphoglycerate kinase (PGK) is not only located in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle . We demonstrated that procyclic T . brucei uses parts of the Krebs cycle for other purposes than for complete degradation of mitochondrial substrates . We suggest that citrate synthase, plus pyruvate dehydrogenase and malate dehydrogenase, are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we showed to occur in proliferating procyclic cells . The part of the Krebs cycle consisting of a-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate . We also showed that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate to malate, which can then be used in gluconeogenesis. Oral Health Prev Dent, 2004, 2 Suppl 1, 259 - 64 The caries balance: the basis for caries management by risk assessment; Featherstone JD; Dental caries progression or reversal depends upon the balance between demineralization and remineralization . The 'Caries Balance' is determined by the relative weight of the sums of pathological factors and protective factors . Minimally invasive dentistry aims at the least possible removal of enamel or dentin, including reducing pathological factors and enhancing remineralization to avoid any removal of hard tissues . A structured caries risk assessment should be carried out based upon the concept of the caries balance . Following the risk assessment a treatment plan is devised which leads to the control of dental caries for the patient . The balance between pathological and preventive factors can be swung in the direction of caries intervention and prevention by the active role of the dentist and his/her auxiliary staff . Much is now understood about the mechanism of dental caries . We have known for a long time that demineralization of enamel, dentin or cementum is caused by organic acids that are generated by so-called acidogenic bacteria in the plaque when these bacteria feed upon fermentable carbohydrates (Silverstone, 1973; Featherstone, 2000; Loesche, 1986) . The natural repair process is remineralization, which occurs when the pH rises again and calcium and phosphate from saliva together with fluoride enter the subsurface region of the lesion and form a new veneer on the existing crystal remnants in the lesion (Ten Cate and Featherstone, 1991) . This veneer is less soluble than the original mineral and resists further acid attacks . The key to improved dental health for all is now for the dental profession to embrace this knowledge and put it into practice in the real world, to inhibit caries formation and progression, and to enhance the natural repair process. J Assoc Physicians India, 2004 Jun, 52, 500 - 1 Symmetrical peripheral gangrene due to viral gastroenteritis; Kashyap R et al.; A case of multifactorial symmetrical peripheral gangrene due to viral gastroenteritis, shock, dopamine infusion, exposure to low temperature and nonlactose fermenters septicaemia is presented for its rarity and devastating consequences. Int J Cancer . 2005 Jan 11; {Epub ahead of print} Bile acid deoxycholate induces differential subcellular localisation of the PKC isoenzymes beta(1), epsilon and delta in colonic epithelial cells in a sodium butyrate insensitive manner; Looby E et al.; Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC) . Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer . Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC) . We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB . HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta . DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr . To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used . Stimulation with 300 muM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min . Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered . Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane . Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells . Supplementary material for this article can be found on the International Journal of Cancer website at (c) 2004 Wiley-Liss, Inc. Can J Microbiol, 2004 Oct, 50(10), 821 - 826 Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter; Tchorbanov AI et al.; Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope–Lingood meat extract based medium . The World Health Organization advises the use of the semisynthetic one, as it has important advantages . Data on the composition of casein-based media and their ability to support high toxin production are not freely available . Important factors affecting toxin production during C . diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+ . We established that the optimal pH for toxin production is 7.2 . The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05–0.41 microg/mL . Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope–Lingood was produced. J Anim Sci, 2005 Feb, 83(2), 344 - 9 Effects of propylene glycol on carcass traits and its related gene expression in Korean native steers; Kim YK et al.; The effects of propylene glycol (PEG) on performance, ruminal fermentation, blood glucose and insulin, carcass traits, and abundance of IGF-1 mRNA in LM and leptin mRNA in adipose tissue were examined in 20 Korean native steers, with 10 each in control and PEG-fed groups, respectively . Propylene glycol mixed with concentrate diet was provided daily at a rate of 2.5 mL/kg BW(0.75) . Experimental animals were fed a concentrate diet to 1.8% of BW twice daily plus rice straw ad libitum during the 4-mo period before marketing . Daily DMI and ADG did not differ between control and PEG-fed steers . Steers receiving PEG displayed an increase (P = 0.044) in propionate concentration, whereas acetate concentration decreased (P = 0.032) . Although blood glucose was not affected, serum insulin was increased (P = 0.047) by PEG feeding . Propylene glycol did not affect carcass weight, 13th-rib fat depth, marbling score, or lipid content of LM . The backfat of PEG-fed steers did not differ in leptin mRNA from control steers, whereas increased leptin mRNA was found in i.m . fat with PEG feeding . There was no treatment effect on the level of IGF-1 mRNA in the LM of the tested steers . These results indicate that the amount of PEG fed to steers was not sufficient to improve marbling score through enhanced ruminal propionate and insulin . The role of increased i.m . leptin mRNA level in PEG-fed steers remains to be further elucidated. Acta Neurol Scand, 2005 Feb, 111(2), 75 - 83 Influences of brain tumor-associated pH changes and hypoxia on epileptogenesis; Schaller B; Schaller B . Influences of brain tumor-associated pH changes and hypoxia on epileptogenesis . Acta Neurol Scand 2004 DOI: 10.1111/j.1600-0404.2004.00355.x . (c) Blackwell Munksgaard 2004.Objective -The pathophysiological changes related to epileptic activity in peri- and intra-tumoral tissue are complex and have been only partly understood until now; possible mechanisms involve different structural, biochemical and histological tumor-related alterations . Methods -Medical databases were searched for evidence on influence of brain tumor-associated pH changes and hypoxia on epileptogenesis . Results -During the perioperative period, tumor-related hypoxia and acidity related to tumor neovascularization by vascular endothelial growth factor (VEGF) in combination with angiopoietins 1/2 (ang 1/2), may be a major factor contributing to outcomes involving epileptic activity after surgical tumor removal . Because anaerobic fermentation produces far less ATP than oxidative phosphorlyation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell . In mammalian cells, this metabolic switch is regulated by the transcription factor hypoxia-inducible factor-1 . Conclusions -From the molecular point of view, therapeutic implications for the perioperative period may have relevance for the future. Biotechnol Bioeng . 2005 Jan 10; {Epub ahead of print} Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source; Bhargava S et al.; For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations . In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation . The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity . To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae . The total cycle time for substrate pulsing was varied over a wide range (30-2,700 s), with substrate added only during the first 30% of each cycle . As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added . Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time . This led to reduced broth viscosity and increased oxygen uptake rate . However, high values of cycle time (i.e., 900-2,700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein . In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors . (c) 2005 Wiley Periodicals, Inc. Planta Med, 2004 Dec, 70(12), 1103 - 1114 Clifford MN. This paper seeks to catalyse a reappraisal of the nature, fate and biological significance in humans of phenols, polyphenols and tannins (PPT) consumed in normal diets, and in particular questions the primacy of PPT radical-scavenging mechanisms for the supposed health benefits of diets rich in fruits and vegetables . PPT are classified by structure and function . Arguments are presented to show that cinnamates and derived polyphenols make significantly larger contributions to the total PPT intake than the flavonols and flavones upon which the vast majority of attention has been focussed previously . Daily intakes of total PPT may range from less than 100 mg to in excess of 2 g, and the critical importance of coffee and black tea as the major dietary sources is shown . Only some 5 % of the dietary PPT is absorbed in the duodenum, and of this only some 5 %, mainly flavanols, reaches the plasma unchanged, the balance being mammalian conjugates . Over 95 % of the intake passes to the colon and is fermented by the gut microflora . A fraction of the resulting microbial metabolites is absorbed and appears in the plasma primarily as mammalian conjugates . Even following high intakes of PPT, the plasma metabolites collectively make a very small (less than 5 %) and transient contribution to the total concentration of redox active substances in plasma . This explains the failure of most studies that sought to detect an increase in plasma antioxidant power after consuming a PPT-rich meal or supplement . The powerfully antioxidant PPT aglycones, much used in in vitro studies, do not reach the plasma . The redox potential of those unchanged PPT and PPT metabolites that reach the plasma enables them to scavenge damaging radicals, but the endogenous plasma antioxidants, especially ascorbate, are required for disposal of the resultant phenoxyl radicals . Black tea and coffee, the major sources of PPT, are poor sources of ascorbate . It is suggested that if diets rich in fruits and vegetables are health-promoting, and if these effects are due to PPT, then alternatives to radical-scavenging mechanisms must be sought . Evidence is presented to show that some mammalian metabolites of PPT may indeed be able to protect the vascular endothelium and that diets rich in PPT may in humans at normal dietary levels have the ability to protect against Type II diabetes and the metabolic syndrome through effects on glucose absorption and associated hormones . Such effects are recommended for further investigation. Protein Expr Purif, 2005 Feb, 39(2), 237 - 46 Optimization of BLyS production and purification from Escherichia coli; Laird MW et al.; B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines . When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation . Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials . In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism . Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system . This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435mg/L of properly folded, trimeric, biologically active rsBLyS . This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production . Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin. Q J Nucl Med Mol Imaging, 2004 Dec, 48(4), 258 - 66 Design and manufacture of monoclonal antibodies for radioimmunotherapy; Hale G et al.; antibodies is fundamental to their use for radioimmunotherapy . Besides the right selection of antibody specificity and affinity, recombinant antibodies can be designed to simplify manufacture and minimise unwanted side effects . Although many innovative new technologies have been developed in recent years, antibodies are still most commonly produced from mammalian cells and purified by column chromatography . Purification methods have to be designed and validated to remove potential contaminants, especially retroviruses, which in principle might be present in mammalian cell lines . Adherence to relevant ''Good Manufacturing Practices'' is mandatory in the production of any medicinal product and there are numerous guidelines regarding the manufacture of antibodies . This article outlines some methods used for fermentation, purification and quality control of antibodies intended for radiolabelling. Appl Biochem Biotechnol, 2005 Jan, 120(1), 37 - 50 Enhancing profitability of dry mill ethanol plants: process modeling and economics of conversion of degermed defibered corn to ethanol; Rajagopalan S et al.; An Aspen Plustrade mark modeling platform was developed to evaluate the performance of the conversion process of degermed defibered corn (DDC) to ethanol in 15- and 40-million gallons per year (MGPY) dry mill ethanol plants . Upstream corn milling equipment in conventional dry mill ethanol plants was replaced with germ and fiber separation equipment . DDC with higher starch content was fed to the existing saccharification and fermentation units, resulting in higher ethanol productivity than with regular corn . The results of the DDC models were compared with those of conventional dry mill ethanol process models . A simple financial analysis that included capital and operating costs, revenues, earnings, and return on investment was created to evaluate each model comparatively . Case studies were performed on 15- and 40-MGPY base case models with two DDC process designs and DDC with a mechanical oil extraction process. Appl Environ Microbiol, 2005 Jan, 71(1), 255 - 260 Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions; Cheraiti N et al.; Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market . The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated . We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions . The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S . cerevisiae and Saccharomyces uvarum . We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture . The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture . Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures . The S . cerevisiae-S . uvarum hybrid strain produced large amounts of acetaldehyde for which the S . cerevisiae strain acted as a receiving strain in the mixed culture . Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others . Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S . cerevisiae. J Biotechnol, 2005 Feb 23, 115(4), 425 - 434 Epub 2004 Nov 11. Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol; Panagiotou G et al.; The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose . However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product . In this study, the growth characteristics of F . oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations . Under aerobic conditions the maximum specific growth rate was found to be 0.043h(-1), and the highest ethanol yield (1.66mol/mol) was found under anaerobic conditions . During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions . The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden-Meyerhof-Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions . The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation . On the other hand, the presence of high levels of gamma-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions. Biotechnol Bioeng . 2005 Jan 5; {Epub ahead of print} Maintaining rapid growth in moderate-density Escherichia coli fermentations; Zawada J et al.; A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented . High-density fermentations are a common and successful means of producing biological products . However, acetate accumulation can be a substantial problem in these procedures . To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates . If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity . It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate . We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins . The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture . Amino acids and vitamins were supplied in the medium to increase the growth rate . We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation . Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids . Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate . Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control . Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations . (c)2004 Wiley Periodicals, Inc. Appl Microbiol Biotechnol . 2005 Jan 6; {Epub ahead of print} Strategies for efficient production of heterologous proteins in Escherichia coli; Jana S et al.; In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically . Production of these proteins has a remarkable demand in the market . Escherichia coli offers a means for the rapid and economical production of recombinant proteins . These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone . These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost. J Clin Microbiol, 2005 Jan, 43(1), 452 - 4 Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates; Bielaszewska M et al.; A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 microg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of </=4 microg/ml, did not grow on Te-containing media, and lacked ter genes . Optimal detection of SF EHEC O157:NM strains requires Te-independent strategies. FEBS J, 2005 Jan, 272(1), 269 - 81 Structural basis for stereo-specific catalysis in NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans; Martins BM et al.; NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2-oxoglutarate to (R)-2-hydroxyglutarate and belongs to the d-2-hydroxyacid NAD(+)-dependent dehydrogenase (d-2-hydroxyacid dehydrogenase) protein family . Its crystal structure was determined by phase combination to 1.98 A resolution . Structure-function relationships obtained by the comparison of HGDH with other members of the d-2-hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride transfer reaction . A model for substrate recognition and turnover is discussed . The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2-oxoglutarate . The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates) . Arg235 interacts with the substrate's alpha-carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the gamma-carboxylate group can dock at an arginine cluster . The proton-relay system built up by Glu264 and His297 permits His297 to act as acid-base catalyst and the 4Re-hydrogen from NADH is transferred as hydride to the carbonyl group Si-face leading to the formation of the correct enantiomer (R)-2-hydroxyglutarate. World J Gastroenterol, 2005 Jan 14, 11(2), 242 - 8 Screening of stimulatory effects of dietary risk factors on mouse intestinal cell kinetics; Shivshankar P et al.; AIM: Although epidemiological and experimental studies validate influence of genetic, environmental and dietary factors in the causation of various types of cancers including colon, results from all these sources are inconclusive . Hypothesizing that high fat diet and obesity are among the major predisposing factors in the incidence of colon cancer, we evaluated the role of diet constituted with food material derived from a tropical plant, Tamarindus indica Linn (TI) . METHODS: A two part randomized double-blind study was conducted employing inbred Swiss albino mice from a single generation for the whole investigation . One day-old neonates (n = 12) were subcutaneously administered with monosodium glutamate (MSG) to induce obesity (OB) . At weaning these animals were maintained on modified AIN-76 diet supplemented with 10% TI and 10% fat bolus (w/w, TIFB) for 8 wk . Subsequently, in the second part of study, four groups of animals belonging to the same generation, age and gender (n = 12 per group), were maintained on: AIN-76 control diet (CD); AIN-76 mixed with 10% TI extract (TI); and, mixed with 10% TI and 10% FB (TIFB) for 8 wk, to determine intestinal crypt cell proliferation, functionally-specific enzyme activities, fermentation profile, and energy preferences . RESULTS: We observed a significant increase in the crypt cell production rate in distal colonic segment of experimental animals when compared with the controls . This segment also contained significantly low butyrate levels compared to control and TIFB groups . All the experimental groups showed a gross decrease in the enzyme activities viz., succinate dehydrogenase, acid-galactosidase and dipeptidyl amino peptidase IV demonstrating pathological stress caused by the test regimens, and an altered metabolic flux in the cellular environment . CONCLUSION: We have demonstrated a cumulative response to the three dietary factors, one of which (TI) is reported, herein, for the first time to modulate kinetics of large intestinal mucosa, contributing to total risk posed by these test agents. J Agric Food Chem, 2005 Jan 12, 53(1), 170 - 5 Evaluation of citrinin occurrence and cytotoxicity in monascus fermentation products; Liu BH et al.; Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability . However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health . In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products . CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts . When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity . Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels . In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay . These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line. J Agric Food Chem, 2005 Jan 12, 53(1), 98 - 102 Moderate decrease of pH by sourdough fermentation is sufficient to reduce phytate content of whole wheat flour through endogenous phytase activity; Leenhardt F et al.; Whole wheat bread is an important source of minerals but also contains considerable amounts of phytic acid, which is known to impair their absorption . An in vitro trial was performed to assess the effect of a moderate drop of the dough pH (around 5.5) by way of sourdough fermentation or by exogenous organic acid addition on phytate hydrolysis . It was shown that a slight acidification of the dough (pH 5.5) with either sourdough or lactic acid addition allowed a significant phytate breakdown (70% of the initial flour content compared to 40% without any leavening agent or acidification) . This result highlights the predominance of wheat phytase activity over sourdough microflora phytase activity during moderate sourdough fermentation and shows that a slight drop of the pH (pH value around 5.5) is sufficient to reduce significantly the phytate content of a wholemeal flour . Mg "bioaccessibility"of whole wheat dough was improved by direct solubilization of the cation and by phytate hydrolysis. Appl Microbiol Biotechnol, 2005 Feb, 66(5), 465 - 474 Epub 2004 Nov 17. Archaea in protozoa and metazoa; Lange M et al.; The presence of Archaea is currently being explored in various environments, including extreme geographic positions and eukaryotic habitats . Methanogens are the dominating archaeal organisms found in most animals, from unicellular protozoa to humans . Many methanogens can contribute to the removal of hydrogen, thereby improving the efficiency of fermentation or the reductive capacity of energy-yielding reactions . They may also be involved in tissue damage in periodontal patients . Recent molecular studies demonstrated the presence of Archaea other than methanogens in some animals-but so far, not in humans . The roles of these microorganisms have not yet been established . In the present review, we present the state of the art regarding the archaeal microflora in animals. Biofactors, 2004, 22(1-4), 279 - 87 Food technology: Challenge for health promotion; Kwon TW et al.; The food technology has brought countless benefits to today's food supply . Despite its many positive contributions, it has also brought unintended negative consequences . It is the time to mobilize the food technology to help the food supply more secure, safer and healthier, and here three possible approaches are foreseeable: First, we should continue to improve the conventional technologies . Many wholesome foods have been prepared and preserved using natural materials simply by fermentation . Second, we have to enhance the minimal processing as much as applicable . Third, new ingredients, intelligent packaging and functional foods should be explored to improve food supply and health . Today, consumer interest in the functional foods has been increased tremendously, and the future of food lies in the functional foods . However, the situations in the developing world are different from this . As food resource is limited in this region, food technology has to be emphasized to increase food supply . To help solve such complex problems, not only new technologies, but also conventional technologies have to be mobilized . Simultaneously, even higher technical capabilities have to be built up by applying new findings from the related disciplines to allow the food technology to play its vital role. Biofactors, 2004, 22(1-4), 93 - 7 Induction of apoptosis in human leukemia cells by naturally fermented sugar cane vinegar (kibizu) of Amami Ohshima Island; Mimura A et al.; Naturally fermented vinegar such as Kibizu (sugar cane vinegar in Amami Ohshima, Japan), Kurozu (black rice vinegar in Kagoshima, Japan), Kouzu (black rice vinegar in China) and red wine vinegar in Italy had potent radical-scavenging activity analyzed by DPPH method . For the elucidation of food factor for cancer prevention contained in naturally fermented vinegar, the induction of apoptosis in human leukemia cell HL-60 was investigated with sugar cane vinegar Kibizu . Fraction eluted by 40% methanol from Amberlite XAD 2 chromatography of sugar cane vinegar showed potent radical scavenging activity . The fraction also showed the activity repressing growth of typical human leukemia cells such as HL-60, THP-1, Molt-4, U-937, Jurkat, Raji and K-562 . On the other hand, the fraction did not have any growth inhibition activity against human fetal lung cell TIG-1 . The most potent radical-scavenging activity and the growth repression activity of the leukemia cell were observed in the same chromatographic fraction of methanol 40% . From cell sorting FACS analyses, electron microscopic observations and cytochemical staining of chromatin and nuclear segments in human leukemia cell HL-60 treated with the active fraction, it was concluded that apoptosis was induced in the leukemia cell by the fraction of sugar cane vinegar and resulted in the repression of growth of the human leukemia cells . Chromatographic fraction of sugar cane juice eluted by 20% methanol showed potent activities of radical-scavenging and growth repression of HL-60 . These results led us the consideration that active components in sugar cane juice could be converted to more lipophilic compounds with activity to induce apoptosis in HL-60 by microbial fermentation with yeast and acetic acid bacteria. Biofactors, 2004, 22(1-4), 67 - 9 A fermented grain food mixture, AOBtrade mark, inhibits liver metastasis in the metastasis model of rat colon cancer; Minamiyama Y et al.; Natural antioxidants have been shown to be rich sources of microchemicals with the potential to prevent human cancers . We examined whether dietary supplement of a fermented grain food mixture (AOB), which has been shown to have a strong antioxidative effect, may protect against colon cancer metastasis . At day 5 of AOB (6.5%) supplementation in a basal diet, the rat colon cancer cells (RCN-H4) were injected beneath the capsule of the spleen and one min later rats were splenectomized . All rats without AOB supplementation had multiple liver metastases . However, the number and the size of liver tumors were reduced by about 80% in AOB group . Combined use of cisplatin and AOB enhanced an anti-metastatic effect . The inhibition of liver metastasis by AOB was caused by the regulation of cell cycle . This product may be used as an adjuvant in the therapy of malignant neoplasia. Biofactors, 2004, 22(1-4), 5 - 19 Studies on action of menaquinone-7 in regulation of bone metabolism and its preventive role of osteoporosis; Tsukamoto Y; The effect of menaquinone-7 (MK-7) on bone components and bone resorbing factors induced-bone resorption using the femoral-diaphyseal and - metaphyseal tissues obtained from elderly female rats in vitro were examined . Calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) in the diaphyseal and metaphyseal tissues in elderly females rats were significantly decreased as compared with that of young rats, indicating that aging causes a deterioration of bone formation . The presence of MK-7 (10(-6)-10(-5) M) caused a significant prevention of reduction of biochemical components . On the other hand, the bone-resorbing factor, parathyroid hormone (1-34) (PTH; 10(-7) M) and prostaglandin E(2) (PGE(2); 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues . This decreases was completely inhibited in the presence of MK-7 (10(-7)-10(-5) M) . In addition, MK-7 (10(-7)-10(-5)} M) completely prevented the PTH (10(-7) M) or PGE(2) (10(-5) M) induced increases in medium glucose consumption and lactic acid production by bone tissues, Furthermore, the effect of the prolonged intake of dietary MK-7 on bone loss in ovariectomized rats was investigated . As a result, it was found that the intake of experimental diets containing the fermented soybean (natto) with supplemental MK-7 caused significant elevations of MK-7 and gamma-carboxylated osteocalcin concentration, a bio marker of bone formation, in the serum of both ovariectomized rats and normal subjects, suggesting that MK-7 may play an important role in the prevention of age-related bone loss. Biofactors, 2004, 21(1-4), 89 - 94 Novel physiological function of fructooligosaccharides; Tokunaga T; Two key properties of short chain fructooligosaccharides (sc-FOS) which lead to physiological functions are indigestibility in the small intestine and fermentability in the colon . Sc-FOS is converted into short chain fatty acids (SCFAs) by intestinal bacteria in the colon and absorbed . Through the metabolic pathway, sc-FOS improves gastrointestinal (GI) condition such as relief from constipation, formation of preferable intestinal microflora and intestinal immunomodulation those are known as prebiotics' function . Besides improvement of GI condition, dietary sc-FOS influences on calcium and magnesium absorption in the colon . A major mineral absorption site is the small intestine, but the colon also works as a Ca and Mg absorption site with an aid of SCFAs made from sc-FOS . Furthermore dietary sc-FOS influences on bioavailability of soy-isoflavones . Plasma and urinal concentration of Genistein and Daidzein, aglycones of Daidzin and Genistin, are higher in the rat fed with sc-FOS than the control rat . An additive effect of dietary isoflavone and sc-FOS was observed on the bone mineral density in OVX mice and moreover sc-FOS increased ceacal beta-glycosidase activity and equol production . These results suggest that FOS increase the bioavailability of isoflavones. Biofactors, 2004, 21(1-4), 179 - 84 Suppressive activity of a fermented grain food product on dextran sulfate sodium-induced experimental colitis in mice; Kwon KH et al.; Oxidative stress has been shown to play pivotal roles in the onset of inflammatory bowel disease . We evaluated the effects of a dietary anti-oxidant, Antioxidant Biofactor (AOB(R)), a processed grain food, on dextran sulfate sodium (DSS)-induced colitis in mice . Female ICR mice were fed a diet containing 0.1% or 1% AOB for 2 weeks, during which they were given 5% DSS in drinking water for the latter 1 week to induce colitis . A diet containing 1% AOB, but not that with 0.1% AOB, attenuated DSS-induced body weight loss and colon shortening (each, P < 0.05), and dramatically improved colitis histologic scores . In addition, DSS-induced increases in colonic mucosal IL-1beta, but not TNF-alpha, protein levels were significantly abrogated in 1% AOB-fed mice (P < 0.05) . Further, 1% dietary AOB abolished the expression of IL-1beta mRNA levels in colonic mucosa (P < 0.01) . Our results suggest that AOB is effective for the prevention of DSS-induced colitis in mice. Bioresour Technol, 2005 May, 96(8), 949 - 53 Microbial transformation of tannin-rich substrate to gallic acid through co-culture method; Banerjee R et al.; Modified solid-state fermentation (MSSF) of tannin-rich substrate yielding tannase and gallic acid was carried out using a co-culture of the filamentous fungi, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557) . Powdered fruits of Terminalia chebula and powdered pod cover of Caesalpinia digyna was used in the process and the different process parameters for maximum production of tannase and gallic acid by co-culture method were optimized through media engineering . MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity . The optimal pH and incubation period was 5.0 and 48h respectively . Through the co-culture technique the maximum yield of tannase and gallic acid was found to be 41.3U/ml and 94.8% respectively. Natl Toxicol Program Tech Rep Ser, 2004 Aug, (510), 1 - 346 Toxicology and carcinogensis . Studies of urethane, ethanol, and urethane/ethanol (urethane, CAS No . 51-79-6; ethanol, CAS No . 64-17-5) in B6C3F1 mice (drinking water studies); The Energetic Conversion Competence of Escherichia coli during Aerobic Respiration Studied by 31P NMR Using a Circulating Fermentation System; Fermentation & Biotechnology Laboratories and Institute of Life Sciences, Ajinomoto Co . Inc., Kawasaki-ku, Kawasaki, 210-8681To determine the actual potential of the energetic conversion efficiency of Escherichia coli during aerobic respiration, apparent P/O ratios (P/O(app)) under either limited or standard glucose-feeding conditions were estimated . The previously reported circulating fermentation system (CFS) was used, and (31)P NMR saturation-transfer (ST) techniques were employed . By coupling with on-line NMR observations, CFS allowed us to evaluate cellular energetics directly, with both the dissolved oxygen tension and glucose feeding precisely controlled to prevent the effect of substrate-level phosphorylation based on aerobic or anaerobic acidogenesis in E . coli cells . Phosphate consumption rates under standard and limited glucose-conditions were estimated as 4.62 +/- 0.46 and 1.99 +/- 0.11 micromol/s g of dry cell weight (DCW), respectively . Using simultaneously assessed O(2) consumption rates, the P/O(app) values under these two conditions were estimated as 1.4 +/- 0.3 and 1.5 +/- 0.1, respectively . To correlate the obtained P/O(app) values with the potential efficiency of respiratory enzymes, we determined the activities of two NADH dehydrogenases (NDH 1 and 2) and two ubiquinol oxidases (bo- and bd-type) during the periods when ST was performed . NDH-1 activities in standard or limited glucose cultures were maintained at 57% or 58% of the total NADH oxidizing activity . The percentages of bo-type oxidase activity in relation to the total ubiqinol oxidizing activity under the standard and limited glucose conditions were 32% and 36%, respectively . These percentages of enzymatic activities represent the respiratory competence of E . coli cells, suggesting that, during the NMR observatory period, the enzymatic activity was not at a maximum, which could also explain the estimated P/O(app) values . If this is the case, enhancing the expression of the bo-type oxidase or disrupting of the bd-type oxidase gene could be effective approach to increasing both the P/O ratio and cellular yields. Anal Chem, 2005 Jan 1, 77(1), 290 - 303 Closed-loop, multiobjective optimization of analytical instrumentation: gas chromatography/time-of-flight mass spectrometry of the metabolomes of human serum and of yeast fermentations; O'Hagan S et al.; The number of instrumental parameters controlling modern analytical apparatus can be substantial, and varying them systematically to optimize a particular chromatographic separation, for example, is out of the question because of the astronomical number of combinations that are possible (i.e., the "search space" is very large) . However, heuristic methods, such as those based on evolutionary computing, can be used to explore such search spaces efficiently . We here describe the implementation of an entirely automated (closed-loop) strategy for doing this and apply it to the optimization of gas chromatographic separations of the metabolomes of human serum and of yeast fermentation broths . Without human intervention, the Robot Chromatographer system (i) initializes the settings on the instrument, (ii) controls the analytical run, (iii) extracts the variables defining the analytical performance (specifically the number of peaks, signal/noise ratio, and run time), (iv) chooses (via the PESA-II multiobjective genetic algorithm), and (v) programs the next series of instrumental settings, the whole continuing in an iterative cycle until suitable sets of optimal conditions have been established . Genetic programming was used to remove noise peaks and to establish the basis for the improvements observed . The system showed that the number of peaks observable depended enormously on the conditions used and served to increase them by as much as 3-fold (e.g., to over 950 in human serum) while in many cases maintaining or reducing the run time and preserving excellent signal/noise ratios . The evolutionary closed-loop machine learning strategy we describe is generic to any type of analytical optimization. Huan Jing Ke Xue, 2004 Sep, 25(5), 65 - 9 {Anaerobic fermentation biohydrogen production from molasses, starch and milk wastewaters}; Liu M et al.; Research of anaerobic fermentation biohydrogen production from molasses, starch and milk wastewater in CSTR was conducted . The theoretic possibility of biohydrogen production from three kinds of organic materials and the hydrogen producing stability was also discussed . The results indicated that molasses and starch wastewaters are preferable feeds for anaerobic biohydrogen fermentation . The hydrocarbon is the optimal feed in the main three kinds of organic materials at present time . The sugars which dissolving better are preferable materials for biohydogen production than starch at present, but the starch is better than sugars for biohydrogen production in the future if the conditions could been controlled good . The niches are variety along with the different of feeds . The optimal pH values of stability operational controlling is 4.5 +/- 0.3 for sugars wastewater and 4.0 +/- 0.2 for starch wastewater . The ORP value must lower than -220 mV and the optimal value is about -300 mV . The milk wastewater is not the suitable biohydrogen production material in CST reactor. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 257 - 64 The nagA gene of Penicillium chrysogenum encoding beta-N-acetylglucosaminidase; Diez B et al.; We purified the beta-N-acetylglucosaminidase from the filamentous fungus Penicillium chrysogenum and its N-terminal sequence was determined, showing the presence of a mixture of two proteins (P1 and P2) . A genomic DNA fragment was cloned by using degenerated oligonucleotides from the Nt sequences . The nucleotide sequence showed the presence of an ORF (nagA gene) lacking introns, with a length of 1791 bp, and coding for a protein of 66.5 kDa showing similarity to acetylglucosaminidases . The NagA deduced protein includes P1 and P2 as incomplete forms of the mature protein, and contains putative features for protein maturation: an 18-amino acid signal peptide, a KEX2 processing site, and four glycosylation motifs . The sequence just after the signal peptide corresponds to P2 and that after the KEX2 site to P1 . The nagA transcript has a size of about 2.1 kb and is present until the end of the fermentation process for penicillin production . NagA is one of the most largely represented proteins in P . chrysogenum, increasing along the fermentation process . The suitability of the nagA promoter (PnagA) for gene expression in fungi was demonstrated by expressing the bleomycin resistance gene (ble(R)) from Streptoalloteichus hindustanus in P . chrysogenum. Anal Biochem, 2005 Jan 15, 336(2), 164 - 71 Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly (13)C-labeled cell extracts as internal standards; Wu L et al.; A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) (13)C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis . This mixture of fully U (13)C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on fully U (13)C-labeled substrates . The obtained labeled cell extract contained, in principle, the whole yeast metabolome, allowing the quantification of any intracellular metabolite of interest in S . cerevisiae . We have applied the labeled cell extract as IS in the analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates in S . cerevisiae sampled in both steady-state and transient conditions following a glucose pulse . The use of labeled IS effectively reduced errors due to variations occurring in the analysis and sample processing . As a result, the linearity of calibration lines and the precision of measurements were significantly improved . Coextraction of the labeled cell extract with the samples also eliminates the need to perform elaborate recovery checks for each metabolite to be analyzed . In conclusion, the method presented leads to less workload, more robustness, and a higher precision in metabolome analysis. Life Sci, 2005 Jan 21, 76(10), 1167 - 76 Profound difference of metabolic pharmacokinetics between pure glycyrrhizin and glycyrrhizin in licorice decoction; Hou YC et al.; To investigate the difference of metabolic pharmacokinetics between pure glycyrrhizin (GZ) and GZ in licorice decoction, six New Zealand White rabbits were orally given pure GZ and licorice decoction containing equivalent content of GZ in a randomized crossover design . HPLC methods were used for the quantitation of GZ and glycyrrhetic acid (GA) in serum . The results indicated that the areas under curves (AUCs) of GZ and GA after administration of licorice decoction were significantly higher than those after pure GZ . This result was contradictory with that obtained in rats . To explore the mechanism of the pharmacokinetic difference, feces of rabbits, rats, pigs and humans were used to investigate the presystemic metabolism of pure GZ and GZ in licorice decoction . The results indicated that pure GZ was hydrolyzed to GA more rapidly and to a greater extent than that in licorice decoction by various feces . In addition, when pure GZ was fermented, the metabolic profiles of GA and 3-dehydroGA in rabbit feces were quite different from other feces . In conclusion, the bioavailabilities of GZ and GA are significantly better from licorice than from pure GZ in rabbits but the presystemic metabolism of pure GZ in rabbit is rather different from that in rat, pig and human. J Nat Prod, 2004 Dec, 67(12), 2086 - 9 A phthalide with in vitro growth inhibitory activity from an oidiodendron strain; Mullady EL et al.; A screening campaign was implemented utilizing capillary electrophoresis as a primary assay to discover binders to the cancer target Akt1 from a crude natural extract library . Fungal extracts with binding activities were characterized for biochemical inhibition of Akt1 to phosphorylate the downstream substrate protein Bad . One of the crude extracts with bioactivity selected for isolation and structure elucidation from fermentation of the fungal culture Oidiodendron sp . F01895 yielded a new trihydroxy phthalide (1) . The structure of 1 was determined by a combination of 1D and 2D NMR spectroscopic data along with high-resolution mass spectrometric data . Compound 1 displays inhibition of Akt1 biochemical activity in vitro and confers growth inhibition on some cancer-derived cell lines in culture. J Environ Sci Health B, 2004, 39(5-6), 889 - 902 Reduction of greenhouse gases from anaerobic piggery wastewater treatment by bromochloromethane in Taiwan; Su JJ et al.; This work establishes methods of reducing the amount of methane produced from the anaerobic treatment of piggery wastewater by either reducing the storage time before solid/liquid separation or inhibiting the activity of methanogens in anaerobic wastewater treatment system . Experimental results showed these two methods can be adopted effectively to reduce methane production resulting from anaerobic piggery wastewater treatment . First, the wastewater must be processed using solid/liquid separation immediately after washing pig houses . This process can reduce by 62% the biogas production and indirectly decrease the methane production from the anaerobic wastewater treatment reactor . Second, adding 10 mg L(-1) bromochloromethane (BCM) daily into the anaerobic wastewater treatment reactor can significantly reduce the amount of biogas and methane produced during the anaerobic fermentation process . Furthermore, biogas production can be completely inhibited after 4 days . Adding BCM (< or =10 mg L(-1)) to wastewater only slightly affected the efficiency of the anaerobic wastewater treatment process . Results in this study can provide the basis for further research on reduction of the amount of methane produced from anaerobic wastewater treatments. Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2477 - 83 Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji; Yoshimoto M et al.; Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses . Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content . Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato . Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production . Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation . The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar . These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions . Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid. Med Hypotheses, 2005, 64(3), 658 - 60 The change of the staple diet of black South Africans from sorghum to maize (corn) is the cause of the epidemic of squamous carcinoma of the oesophagus; Isaacson C; The change of the staple diet of Black South Africans from sorghum to maize (corn) is the cause of the epidemic of squamous carcinoma of the oesophagus . For many years sorghum was the staple diet of Black South Africans . From approximately the early part of the twentieth century, maize gradually replaced sorghum . Squamous carcinoma of the oesophagus was infrequent in the first half of the twentieth century, rising slowly to current epidemic proportions . Fusarium fungi grow freely on maize, producing fumonisins, which reduce nitrates to nitrites and synthesise cancer-producing nitrosamines . Nitrosamines are the presumed carcinogens . Fusarium fungi do not grow well on sorghum, the production of fumonisin from sorghum being two orders of magnitude lower than maize . The higher incidence of oesophageal cancer in Black males is ascribed to their greater consumption of traditional beer, which is produced by fermenting maize . Patients with oesophageal cancer consume more beer than controls . Countries in Africa, in which the staple food is sorghum, have a low incidence of squamous carcinoma of the oesophagus . Crops from various parts of the country should be examined for Fusarium fungi and nitrosamines . The nitrosamine content of traditional beer should be assessed . If nitrosamines are detected, their carcinogenic potential should be studied experimentally . Should these tests prove positive, it would be vital to break the Fusarium-nitrosamine-cancer chain. Med Hypotheses, 2005, 64(3), 599 - 601 Do the mitochondria of malaria parasites behave like the phoenix after return in the mosquito? regeneration of degenerated mitochondria is required for successful Plasmodium infection; Bongaerts G; Mitochondria are energy generators in eukaryotic organisms like man and the pathogenic malaria parasites, the Plasmodium spp . From the moment a mosquito-mediated malaria infection occurs in man the parasite multiplies profusely, but eventually the oxygen supply becomes the limiting factor in this process . Consequently, the parasite will increasingly generate energy (and lactic acid) from sugar fermentation . Simultaneously, the cristate structure of Plasmodium mitochondria degenerates and becomes acristate . The degenerated acristate mitochondria of mammalian Plasmodium parasites seem to be able to revitalise by transforming to cristate mitochondria inside the oxygen-rich mosquito, like the rebirth of the old phoenix . In this way the infectivity of the parasite is revitalised. Nature, 2004 Dec 23, 432(7020), 1058 - 61 Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1; Schawalder SB et al.; Regulation of ribosome biogenesis is central to the control of cell growth . In rapidly growing yeast cells, ribosomal protein (RP) genes account for approximately one-half of all polymerase II transcription-initiation events, yet these genes are markedly and coordinately downregulated in response to a number of environmental stress conditions, or during the transition from fermentation to respiration . Although several conserved signalling pathways (TOR, RAS/protein kinase A and protein kinase C) impinge upon RP gene transcription, little is known about how initiation at these genes is controlled . Rap1 (refs 6, 7) and more recently Fhl1 (ref . 8) were shown to bind upstream of many RP genes . Here we show that the essential protein Ifh1 binds to and activates many RP gene promoters under optimal growth conditions in Saccharomyces cerevisiae . Ifh1 is recruited to RP gene promoters through the forkhead-associated domain of Fhl1 . Ifh1 binding decreases when RP genes are downregulated either by TOR inhibition or nutrient depletion, and is restored after release from starvation or upon regulated induction of IFH1 expression . These findings indicate a central role for Ifh1 and Fhl1 in RP gene regulation. Zhongguo Zhong Yao Za Zhi, 2003 Nov, 28(11), 1038 - 40 {Studies on chemical constituents of the mycelia from fermented culture of Flammulina velutipes}; Kang J et al.; OBJECTIVE: To study the chemical constituents from the mycelia of Flammulina velutipes . METHOD: The compounds were isolated with silica gel column chromatography and their structures were elucidated on the basis of spectral analysis (IR, EL-MS, FAB-MS, 1H-NMR, 13NMR) . RESULT: Seven compounds were identified as cyclo-(R-pro-R-leu) (1), cyclo-(R-isoleu-R-leu) (2), phenylalanine (3), alanine (4), leucine (5), guanosine (6), adenosine (7), CONCLUSION: The compounds 1-6 were isolated from the mycelia of Flammulina velutipes for the first time. Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print} In vitro gas production as a surrogate measure of the fermentability of cellulosic biomass to ethanol; Weimer PJ et al.; Current methods for measuring ethanol yields from lignocellulosic biomass are relatively slow and are not well geared for analyzing large numbers of samples generated by feedstock management and breeding research . The objective of this study was to determine if an in vitro ruminal fermentation assay used in forage quality research was predictive of results obtained using a conventional biomass-to-ethanol conversion assay . In the conventional assay, herbaceous biomass samples were converted to ethanol by Saccharomyces cerevisiae cultures in the presence of cellulase enzymes . Cultures were grown in sealed serum bottles and gas production monitored by measuring increasing head space pressure . Gas accumulation as calculated from the pressure measurements was highly correlated (r(2)>0.9) with ethanol production measured by gas chromatography at 24 h or 7 days . The same feedstocks were also analyzed by in vitro ruminal digestion, as also measured by gas accumulation . Good correlations (r(2) approximately 0.63-0.82) were observed between ethanol production during simultaneous saccharification and fermentation and gas accumulation in parallel in vitro ruminal fermentations . Because the in vitro ruminal fermentation assay can be performed without sterilization of the medium and does not require aseptic conditions, this assay may be useful for biomass feedstock agronomic and breeding research. J Vet Sci, 2004 Dec, 5(4), 325 - 30 Transactivation of peroxisome proliferator-activated receptor alpha by green tea extracts; Lee K; Tea is a popular beverage . Recently, green tea was reported to increase the number of peroxisomes in rats . In this study, to find out whether the green tea-induced proliferation of peroxisomes is mediated by PPARalpha , a transient transfection assay was carried out to investigate the interactions of tea extracts (green tea, black tea,oolong tea and doongule tea) and tea components (epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin and gallic acid), with mouse cloned PPARalpha . Green tea and black tea extracts, and epigallocatechin gallate, a major component of fresh green tea leaves, increased the activation of PPAalpha 1.5-2 times compared with the control . It is suggested that the green tea induced-peroxisomal proliferation may be mediated through the transactivation of PPARalpha and that epigallocatechin gallate may be an effective component of green tea leaves . This would account for the increase in the number of peroxisomes and the activity of peroxisomal enzymes previously reported . However, black tea, a fully fermented product, had a stronger effect than oolong tea extract . These results also suggest, that in addition to epigallocatechin gallate, green tea leaves may possess some active chemicals newly produced as a result of the fermentation process, which act on PPARalpha like other peroxisome proliferators. Lett Appl Microbiol, 2005, 40(1), 37 - 43 Avoidance of oxidative-stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies? Wiseman A. Abstract a . wiseman . 2004.Aims: Bioprocess oxidative stress caused by many reactive oxygen species (ROS) can lead to largely irreversible perturbation of yeast bioprocesses . These include the production of proteins derived from recombinant DNA yeast technology (aerobically grown Saccharomyces cerevisiae) . These proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t-PA), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors VIII and XIII . In addition, there may be a demand for severe acute respiratory syndrome-coronavirus antigens, hepatitis A, B and C viral-selected antigens, HIV retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens . Prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing Cu/Zn cytosolic, Mn mitochondrial and Fe bacterial) glutathione peroxidases (and other Se-containing proteins and enzymes such as the thioredoxins), catalases (Fe-containing), cytochrome c peroxidases (Fe-containing), ceruloplasmins (Cu-containing), metallothionines (these cysteine thiol-rich proteins bind ions of cadmium and mercury) and tyrosinases(Cu-containing) . Methods and Results: ROS are generated inadvertently by single metal valency couples such as FeII/FeIII and by FeIII/FeV present in 2700 (including 57 human) isoforms in cytochromes P450 mixed-function oxidases (EC 1.14.14.1; O(2) : mono-oxygenase NADPH/NADH requiring) . In addition, mixed-metal couples such as valency unmatched forms in CuI/FeII and FeIII/MnIV can recycle electrons . Moreover, proteins/protein chaperone couples can recycle electrons, often where futile-recycling systems have been instigated . Furthermore, oxidized membrane phospholipids (R) can form ROOH (lipid hydroperoxides) and ROH (lipid alkoxides) that can generate ROS through Fenton chemistry (iron-catalysed) chain reactions . Utilization of chain-breaking antioxidants such as vitamin E (alpha-tocopherol) in the lipid phase and vitamin C (ascorbate) in the aqueous phase can terminate these ROS-producing reactions . Conclusions: The main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ROS of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes) . The main impact of the study is that the utilization of genetically modified (GM) yeast produced by recombinant DNA technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ROS, which is avoidable by deployment of appropriate antioxidants such as vitamins E, C and D (and antioxidant proteins and enzymes often of microbial origin via recombinant DNA technology). Fermentation - process by which the living cell is able to obtain energy through the breakdown of glucose and other simple sugar molecules without requiring oxygen. Fermentation is achieved by somewhat different chemical sequences in different species of organisms. Two closely related paths of fermentation predominate for glucose. When muscle tissue receives sufficient oxygen supply, it fully metabolizes its fuel glucose to water and carbon dioxide. However, at times of strenuous activity, muscle tissue uses oxygen faster than the blood can supply it. During this anaerobic condition, the six-carbon glucose molecule is only partly broken down to two molecules of the three-carbon sugar called lactic acid. This process, called lactic acid fermentation, also occurs in many microorganisms and in the cells of most higher animals. In alcoholic fermentation, such as occurs in brewer’s yeast and some bacteria, the production of lactic acid is bypassed, and the glucose molecule is degraded to two molecules of the two-carbon alcohol, ethanol, and to two molecules of carbon dioxide. Many of the enzymes of lactic acid and alcoholic fermentation are identical to the enzymes that bring about the metabolic conversion known as glycolysis. Alcoholic fermentation is a process that was known to antiquity. Before 2000 B.C. the Egyptians apparently knew that crushed fruits stored in a warm place would produce a substance with a pleasant intoxicating power. By 1500 B.C. the production of beer from germinating cereals (malt) and the preparation of wines from crushed grapes were established arts in most of the Middle East. Aristotle believed that grape juice was an infantile form of wine and that fermentation was, therefore, the maturation of the grape extract. Interest in the process of fermentation has continued through the ages, and much of modern biochemistry, especially enzyme studies, has emerged directly from early studies on the fermentation process. One of the earliest laboratories established for the study of biological chemistry was that founded in Copenhagen in 1875 and financed by the brewing family of Jacob Christian Jacobsen. Fermentation is of different types and takes place under anaerobic conditions mostly in saprophytic microorganisms like certain bacteria and fungi. However, it may also take place in higher organisms under certain conditions. The two most common types of fermentation are (1) alcoholic fermentation and (2) lactic acid fermentation. (1) Alcoholic fermentation : the type of fermentation in which ethyl alcohol is the main end product .This is very common in yeast (unicellular fungus) and also seen in some bacteria. Yeast cells release enzymes called zymase complex which bring about the fermentation. The reactions are similar to anaerobic respiration. (2) Lactic acid fermentation : The type of fermentation in which lactic acid is the end product. It is carried out by some bacteria (e.g. lactic acid bacteria), and also by animals (muscle glycolysis in animals, under oxygen deficiency, results in the formation of lactic acid this is whay we experience in muscle cramps, or "Charley horse"). Lactic acid bacteria can ferment milk sugar lactose (C12H22O11) to lactic acid. The process is extracellular. This brings about curdling of milk. Commercial application of fermentation : There are various kinds of fermentation carried on by different microorganisms. Many of these result in highly useful end products. Such useful microbial activity is used on large industrial scale to obtain the useful end products for the benefit of mankind. Some of the industrial products of the microbial fermentation activities are (a) antibiotics (b) vitamins (c) industrial alcohol (d) bakery products (e) some dairy products (f) tanning of leather (g) curing of tea and coffee (g) lactic acid (h) butric acid, (i) acetic acid etc. The ancient Greeks understood that important chemical changes took place during this type of fermentation. Their name for this change was "alchemy." Like the fermentation of dairy products, preservation of vegetables and fruits by the process of lacto-fermentation has numerous advantages beyond those of simple preservation. The proliferation of lactobacilli in fermented vegetables enhances their digestibility and increases vitamin levels. These beneficial organisms produce numerous helpful enzymes as well as antibiotic and anticarcinogenic substances. Their main by-product, lactic acid, not only keeps vegetables and fruits in a state of perfect preservation but also promotes the growth of healthy flora throughout the intestine. Other alchemical by-products include hydrogen peroxide and small amounts of benzoic acid. A partial list of lacto-fermented vegetables from around the world is sufficient to prove the universality of this practice. In Europe the principle lacto-fermented food is sauerkraut. Described in Roman texts, it was prized for both for its delicious taste as well as its medicinal properties. Cucumbers, beets and turnips are also traditional foods for lacto-fermentation. Less well known are ancient recipes for pickled herbs, sorrel leaves and grape leaves. In Russia and Poland one finds pickled green tomatoes, peppers and lettuces. Lacto-fermented foods form part of Asian cuisines as well. The peoples of Japan, China and Korea make pickled preparations of cabbage, turnip, eggplant, cucumber, onion, squash and carrot. Korean kimchi, for example, is a lacto-fermented condiment of cabbage with other vegetables and seasonings that is eaten on a daily basis and no Japanese meal is complete without a portion of pickled vegetable. American tradition includes many types of relishes—corn relish, cucumber relish, watermelon rind—all of which were no doubt originally lacto-fermented products. The pickling of fruit is less well known but, nevertheless, found in many traditional cultures. The Japanese prize pickled umeboshi plums, and the peoples of India traditionally fermented fruit with spices to make chutneys. Lacto-fermented condiments are easy to make. Fruits and vegetables are first washed and cut up, mixed with salt and herbs or spices and then pounded briefly to release juices. They are then pressed into an air tight container. Salt inhibits putrefying bacteria for several days until enough lactic acid is produced to preserve the vegetables for many months. The amount of salt can be reduced or even eliminated if whey is added to the pickling solution. Rich in lactic acid and lactic-acid-producing bacteria, whey acts as an inoculant, reducing the time needed for sufficient lactic acid to be produced to ensure preservation. Use of whey will result in consistently successful pickling; it is essential for pickling fruits. During the first few days of fermentation, the vegetables are kept at room temperature; afterwards, they must be placed in a cool, dark place for long-term preservation. It is important to use the best quality organic vegetables, sea salt and filtered or pure water for lacto-fermentation. Lactobacilli need plenty of nutrients to do their work; and, if the vegetables are deficient, the process of fermentation will not proceed. Likewise if your salt or water contains impurities, the quality of the final product will be jeopardized. Lacto-fermentation is an artisanal craft that does not lend itself to industrialization. Results are not always predictable. For this reason, when the pickling process became industrialized, many changes were made that rendered the final product more uniform and more saleable but not necessarily more nutritious. Chief among these was the use of vinegar for the brine, resulting in a product that is more acidic and not necessarily beneficial when eaten in large quantities; and of subjecting the final product to pasteurization, thereby effectively killing all the lactic-acid-producing bacteria and robbing consumers of their beneficial effect on the digestion. The recipes presented in Nourishing Traditions are designed to be made in small quantities in your own kitchen. They require no special equipment apart from a collection of wide-mouth, quart-sized mason jars and a wooden pounder or a meat hammer. (For special sauerkraut crocks that enable you to make large quantities, see Sources.) We recommend adding a small amount of whey to each jar of vegetables to ensure consistently satisfactory results. Concentrated whey and dried whey can be purchased at health food stores, but the whey you make yourself (pages 86-87) is far superior because it still contains its valuable enzyme content. Whey proteins are very fragile and easily denatured by the drying process. About one inch of space should be left between the top of your vegetables with their liquid and the top of the jar, as the vegetables and their juices expand slightly during fermentation. Be sure to close the jars very tightly. Lacto-fermentation is an anaerobic process and the presence of oxygen, once fermentation has begun, will ruin the final product. We have tried to keep the recipes in Nourishing Traditions as simple as possible without undue stress on ideal temperatures or precise durations. In general, a room temperature of about 72 degrees will be sufficient to ensure a lactic-acid fermentation in about two to four days. More time will be needed if your kitchen is colder and less if it is very warm. After two to four days at room temperature, the jars should be placed in a dark, cool spot, ideally one with a temperature of about 40 degrees. In days gone by, crocks of lacto-fermented vegetables were stored in root cellars or caves. A wine cellar or small refrigerator kept on a "warm" setting is ideal; failing that, the top shelf of your refrigerator will do. Lacto-fermented fruit chutneys need about two days at room temperature and should always be stored in a refrigerator. Lacto-fermented vegetables increase in flavor with time—according to the experts, sauerkraut needs at least six months to fully mature. But they also can be eaten immediately after the initial fermentation at room temperature. Lacto-fermented vegetable condiments will keep for many months in cold storage but lacto-fermented fruits and preserves should be eaten within two months of preparation. Throughout human history, bacteria and fungi have been intimately involved with both the success and failure of daily life. Microorganisms cause disease, but they also play a wider role in sustaining life. We know that certain bacteria play a major part in recycling chemical elements as well as compounds. For example, bacteria help return the materials of dead organisms back to the earth, so those materials can be used by living organisms. What's more, without bacterial action, living things would not be able to use certain compounds found in soil, water, and even the atmosphere. f, e. Modified versions of these microorganisms are now essential in modern day pharmaceutical production. Fermentation is one of the oldest ways humans have used microbes. In his book From Caveman to Chemist, Hugh W. Salzberg retells some of the ancient history of fermentation. Since grains, fruits, juices, milk, and other organic liquids ferment naturally, some speculate that fermented drinks and dairy products must have been available not long after early people developed agriculture. The Bible refers to vineyards, wine, and the effects of too much alcohol consumption. Hannah, the mother of the prophet Samuel, was mistakenly admonished by the high priests to “put away thy wine from thee.” Ashurbanipal (668-626 BC), the last great Assyrian emperor, was something of an expert on wines. He compiled a list of what he considered the best wines. In ancient Egypt tax inspectors assessed the quality of the wines. Even poor Egyptians were sometimes buried with jars of beer. In both ancient Egypt and ancient Mesopotamia wines were put into jars at the place of origin in the presence of government officials and sealed with an official seal. In Mesopotamia, brewing was a home industry. The brewers were often women, who carried out the business of selling beer as well. These women-brewers had a relatively privileged position in society during the period of about 4000-2600 BC. The code of law handed down by the Babylonian lawgiver King Hammurabi (reigned 1792-50 BC) included regulations in terms of the sale price of beer and its minimum alcohol content. The brewer-women were also cautioned about allowing political conspiracies to be formulated on their tavern premises. Fermentation is carried out by both bacteria (prokaryotes) and fungi (eukaryotes) during their metabolism. Both groups of organisms also figure in the world of human disease, as they are both sources of antibiotics, as we'll see throughout this module. Fermentation results in a number of byproducts that have many different uses. As mentioned, the most well-known product of fermentation is ethyl alcohol. This substance is both a beverage as well as a starter molecule for synthesizing other compounds. Since fermentation is carried out in the absence of oxygen, we call it an anaerobic process. It is a method by which organisms such as yeast obtain their energy by converting sugars into other chemical compounds, particularly carbon dioxide and water. Interestingly, our bodies also use this same anaerobic fermentation to obtain energy from sugars when oxygen is in low supply in our blood, such as during vigorous exercise. The products of this process are lactic acid and water rather than the carbon dioxide and water that human metabolism normally produces. In this day and age, pharmaceutical companies utilize the fermentation carried out by microorganisms to produce antibiotics, hormones,and specialized proteins such as antibodies and insulin. This wide range of products is possible because the bacterium or fungus involved in fermentation has been genetically changed to produce a specific substance. You have the opportunity to explore fermentation as a chemical process through a series of activities linked below. The chemistry, although occurring in a living organism, can be examined outside a yeast or bacterial cell if you are able to extract certain enzymes (known as catalysts in the world of chemistry) from the cells. The fact that enzymes could carry out fermentation outside of a living organism was first shown by Eduard Buchner, a contradiction to the idea of Louis Pasteur that only living things could carry on the fermentation process. Buchner added sugar to juices extracted from yeast cells, even cells that had been killed by heating. Fermentation would begin within an hour. Buchner theorized that the yeast juice contained just one enzyme for converting sugar to carbon dioxide and water. Today we know that yeast cells contain more than a dozen enzymes involved in fermentation. You don't need to understand the science of fermentation to make good spirits and liqueurs in the home unless you want to experiment with the fermentation system, i.e., fermenting larger volumes or higher alcohol levels. But know how is the base for improvements. If you read this whole document, you will know even more then most home brew shop owners (except those who also have read it)! Seeing fermentation from the yeast's perspective helps in understanding the science. Yeast is a living organism very similar to the individual cells in our own bodies. It is easy to think of dried yeast as "just another ingredient" like nutrients or sugar. Nothing could be further from the truth. Yeast's sole aim in life is to reproduce. It does this by " budding" to produce a daughter cell identical to the parent. Given a plentiful supply of oxygen, sugar, minerals, enzymes and amino acids, it will reproduce itself every 30 minutes and one thus ends up with a bucket full of yeast! Take away the oxygen and you get much less growth and a bucket full of alcohol. As far as the yeast is concerned, sugar (a sugar molecule) is a source of energy the yeast cell imports (eats). Glucose has 6 carbon atoms joined together by chemical bonds. It breaks these bonds one by one, each time liberating energy, which is then used for growth. Without oxygen, it can only break one bond and so liberates only a little energy (also little growth). What's left is thrown out of the cell as a waste product: ethanol. So, if you want to make alcohol, keep the oxygen out! To grow, yeast also needs amino acids, enzymes and minerals as well as the energy it extracts from sugar. l, a. These are needed to build new proteins (by creating bonds between amino acids) and carry out the many enzymatic reactions within the cell. A good Turbo sachet will contain all of these essential growth ingredients collectively we call these "yeast nutrients". If you have ever tried to ferment pure sugar with just yeast, you will know that you get very little alcohol, this is because yeast needs these other nutrients as well as sugar. So yeast is a living organism which uses sugar to make energy for growth. If there is no oxygen around yeast cannot extract all the energy from sugar and throws out ethanol as a waste product. To function, yeast also needs amino acids, enzymes and minerals, which collectively we call nutrients. As well as throwing out ethanol as a waste product, yeast throws out another 1300 other compounds, which we can call "volatiles". These volatiles fall into chemical categories Silage Fermentation. Once oxygen is depleted in the forage mass, fermentation begins. Fermentation is the lowering of the pH in the forage to a point where no organism (mold or bacteria) can function. The pH is lowered by lactic acid which is a byproduct of lactobacillus bacteria. The lactobacillus bacteria are on the forage when it is mowed and multiply rapidly until the forage is fermented. Lactobacillus bacteria consume forage carbohydrates for their energy source and excrete lactic acid. The lactobacillus bacteria continue to produce lactic acid and lower the forage pH until they can not function anymore. At this pH level, the forage is fermented and can exist unchanged for many years as long it is not exposed to oxygen. f, h. Clostridia bacteria are also on the forage when it is mowed and are put in the silo with the forage. Clostridia bacteria consume forage carbohydrates, forage proteins, and lactic acid as their energy source and excrete butyric acid. Butyric acid is associated with rotten or putrefied silage. Situations that might benefit clostridia growth are insufficient forage carbohydrate levels (rain while forage is wilting, extended respiration period due to poor packing, seepage due to excessive forage moisture) to complete the fermentation process and/or low lactobacillus bacteria levels. The debates over open versus closed fermentation will no doubt continue as long as there are interested brewers to debate. I intend to present some of my feelings, opinions, and experiences with using open fermenters, and point out some of the inherent pros/cons of using this technique. I want to emphasize one thing about this issue: the choice of fermenters is not going to be *the* deciding factor in your finished product, many other factors will play a more important part in the character of your beer. Namely, malt choices, mashing programs, and above all, yeast strain/viability/cleanliness will be the dominant influences on the finished beer. Having said this, there are instances where breweries who changed from open fermenters to closed unitanks have noted distinct changes in the perceived quality of the beers, when judged by experienced taste panels. [1] Open fermentation is a concept that most homebrewers think is a sure route to infected beer, or as something to be employed in some dark cellar in an old European brewery. I say nonsense! Think for a minute about some of the best world class beers and then think of how many are made using open fermenters: Sierra Nevada, Anchor, numerous English, Belgian and yes, even German brewers use them. It is a common sight in Bavaria to see a brewer mucking around in the thick krausen on top of the open fermenter, collecting samples, skimming yeast, generally doing things that homebrewers are told to avoid. Eric Warner has noted in his excellent book on Wheat beers that open fermenters are the preferred method of German weizen production [2], and that when open fermenters are used the yeast can be repitched for many more generations than when a closed fermenter is used. So whats an open fermenter? At the simplest, it is a vessel with an open top. Depending on the size of the fermenter, they are often covered by some form of lid. The bigger versions are truly open, large shallow vessels, some are lined with stainless steel or an enamel like coating that is usually used over a concrete/block foundation. Often the fermenters are just large stainless steel cylinders. Most, but not all, have some form of attemperater device, to combat the temperature rise during ferments. This can be in the form of exterior jacketing, or metal piping that is immersed in the wort, cold water or glycol is pumped inside the pipes, cooling the ferment. Probably the most classic open fermenters are the Yorkshire Squares used at the Samual Smiths brewery in Tadcaster, England. These are made of flat slate walls, sealed together, with a collecting lid where the excess krausen is contained. OK, so your thinking open fermentation only works in big breweries since they are filtering the air, and keeping the whole room under positive pressure, and nobody is allowed in. Yes, and no. Sure, lots of breweries go to the extreme of maintaining a separate room with filtered air. Lots more don't do anything. Certainly, the breweries in England that I visited never went to the extreme of filtered air, nor did the breweries in Bavaria and Belgium. Belgian methods of brewing may seem strange , but the dominant flavor profiles found in Belgium beers are a result of the choice of a yeast strain(s) that throws high levels of esters and phenolics, and rarely a result of some infection in the fermenter (even though this is the way to produce lambics, the word infection is a misnomer in this context). Certainly, the Bavarian brewmasters would recoil in horror if any foreign bacteria or wild yeast were to be found in the open fermenter, and in practice, they are not a problem. I did not always use open fermenters, the first hundred or so of my beers were made with a "closed carboy" system. I put closed in quotes since the carboy can be fitted with a blowoff tube, resulting in a kind of hybrid closed/open fermenter. Since fall '92, I have been using a open fermenter exclusively, and I am a devoted fan of the concept. My fermenter is a stainless steel cylinder, of roughly equal height to width, with a heavy lid. If you brew with a 10 or 15 gallon stainless steel kettle, this can double as your fermenter, once you remove the hot break. Some brewers employ modified 1/2 BBl Sankey kegs, and these too make excellent open fermenters. I have also read of brewers modifying Golden Gate kegs and using these as fermenters. The least desirable, but easiest to start with, is the plain plastic bucket. The reason I say least desirable is that cleaning plastic is more difficult than stainless, and the inevitable scratches in the plastic walls can be harder to sanitize. Even so, I know of an award winning homebrewer who ferments in food grade plastic trash cans, and another 2 BBl brewpub who ferments in large High Density Poly- Ethelyne (HDPE) containers. I have found that as you increase the brew length (volume of beer produced), it is easier to fabricate some sort of fermenter that can hold the entire batch. In this way, you will be limiting the number of vessels to sanitize and clean up. It is far cheaper and easier to fabricate or modify a container to be an open ferementer than to make a closed one, particularly as the volume increases. An important consideration when sizing the fermenter is to account for a large amount of krausen that can develop during the ferment. Head space of 30% is optimum, but less can be used, with the result being some possible loss of product (which also occurs when using the blowoff carboy method). Of course, there are some limitations to using open fermenters. I believe they are no more prone to infections than using carboys, but there is an increased chance for infection if one has numerous fruit flies or other animals around the fermenter, provided the lid is off. Probably the biggest limitation is that of time, I do not advise leaving the beer in the fermenter for more than 2 weeks. Of course, any ferment should be racked by the second week, so maybe this isn't such a limitation after all. The reason time is more important in open fermenters is not so much the proximity of the still beer to dead yeast, but of the danger of oxidation reactions occurring as the beer sits. In a closed system, this will not be a problem, but as long as the beer is moved in a timely manner, the CO2 produced during open fermentation will protect the beer. Another important factor to consider is the overall cleanliness of the fermentation area. It need not be sterile, but a reasonable degree of cleanliness is in order, in particular for fermentation inside of a refrigerator. Many brewers use a temperature control device to moderate the ferment temperature inside of a refrigerator. If you use an open fermenter inside of a refrigerator, be sure to clean all obvious sources of contamination and general dirt. Some may even want to sponge down the interior of the refrigerator with a mild sanitizer such as chlorine/water. At the very least, all spilled trub, yeast and wort should be thoroughly cleaned up. Household pets should also be prevented from crawling into the fermenting beer, they may like the results too much! My fermenter is located in the basement, a few feet off the ground, away from large drafts and any foreign debris sources. Heres a summary of how I use my open fermenter. Since I use a stainless fermenter, I don't want to use a chlorine based sanitizer, due to problems with corrosion. So, I prepare a solution of Iodophor, at 12.5 ppm (1 oz in 10 gallons), of a few gallons. Using rubber gloves, I sponge the sanitizer over the sides of the fermenter. I let it run out the drain, then back over the sides of the fermenter. I also run Iodophor through my wort chiller into the fermenter, followed by a hot water rinse. Once the hot water is drained, the vessel is ready for cast out wort. I fill the fermenter from the wort chiller, oxygenate and add thick yeast slurry. As in any fermentation, there is no substitute for pitching enough viable clean yeast. d, c, l, e, d, j. The key to success with an open fermenter (or closed) is a sanitized vessel, and an adequate amount of pitching yeast. Remember to use significantly more yeast if the original gravity of the wort is higher than 1.060. If one is using enough yeast, visible fermentation is evident within 12 hours (ale wort, fermented between 60-70 F). As soon as the fermenter is full and the yeast is pitched, place the lid on. Once the fermentation is generating a thick head of krausen, I have found it helpful to leave the lid partially cracked, allowing an airspace for the large amounts of CO2 to vent. With the ferment in high krausen, the classic dense rocky heads will form. At this stage, trub will be scrubbed from the ferment, and rise to the surface, along with other solid matter that was carried over into the fermenter. This scum can be skimmed off with a sanitized spoon (I leave a long handled stainless steel spoon in some Iodophor and just rinse it off when needed). The ability to skim the trub and yeast that rise to the top of the fermenter is one of the main advantages of open fermentation. Don't overdo it, but about once a day or every other day, depending on the rate of ferment, skim the top. Many ale yeasts tend to flocculate at the top of the ferment as the ferment diminishes. This yeast is excellent to skim and store in a sanitized container, in a cold fridge (as close to 32F as possible). When choosing yeast to save, be sure to wait a few days into the ferment so that the trub is scrubbed away and the harvested yeast is clean. As the ferment dies down, keep the lid over the vessel. Another great plus of open fermenting is the ease of dry hopping. What I do is let the main fermentation subside and when the yeast clumps to the surface, skim as much off as possible, then add the loose whole hops (I find that whole hops give better aroma and are easier to use with an open fermenter). Allow at least 3 days time for the dry hopping to take affect. I would avoid leaving the beer in the primary for longer than 2 weeks, and aim for 10 days when dry hopping, and a mere 5 days otherwise. These are optimum figures for ale ferments, and are often not realistic in homebrewing, the primary cause being inadequate oxygenation of the cast out wort, and/or insufficient yeast cell densities/viabilities in the pitching yeast. To rack off of the hops, use a sanitized copper/brass or stainless "choreboy" scouring pad, held over the racking cane with a rubber band. Alternatively, the hops can be removed with a sanitized strainer, provided a minimum of air is introduced to the still beer. Fermentation is a two-stepped process that begins with the Putrefaction of the hermaphroditic "child" from the Conjunction resulting in its death an |