Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography

A method previously used for the analysis of organic acids in silage has been applied to the detection and quantification of acidic fermentation products (C₁ to C₆ volatile fatty acids, lactic and succinic acid) of rumen bacteria and anaerobic fungi grown in pure culture. The acids were converted to tertiary butyldimethylsilyl derivatives prior to separation on a 30 m DB1 capillary gas chromatographic column. The quantitative recoveries of formic and succinic acids were found to be comparable to the recoveries of other acids reported in the original study. The quantitative recovery of lactic acid was found to be dependent on storage of the samples at ambient temperature for at least 24 h following derivatization. The simultaneous determination of a wide range of volatile and non-volatile acidic products is an important feature of this method.     

Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media

A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C-1-C-7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 mumol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of toxonomic significance.     

Coupled lactic acid fermentation and adsorption

Polyvinylpyridine (PVP) and activated carbon were evaluated for coupled lactic acid fermentation and adsorption, to prevent the product concentration from reaching inhibitory levels. The lactic acid production doubled as a result of periodical circulation of the fermentation broth through a PVP adsorption column. The adsorbent was then regenerated and the adsorbed lactate harvested, by passing 0.1 N NaOH through the column. However, each adsorption-regeneration cycle caused about 14% loss of the adsorption capacity, thus limiting the practical use of this rather expensive adsorbent. Activated carbon was found much more effective than PVP in lactic acid and lactate adsorption. The cells of Lactobacillus delbrueckii subsp. delbrueckii (LDD) also had strong tendency to adsorb on the carbon. A study was therefore conducted using an activated carbon column for simultaneous cell immobilization and lactate adsorption, in a semi-batch process with periodical medium replacement. The process produced lactate steadily at about 1.3 g l(-1)h(-1) when the replacement medium contained at least 2 g l(-1) of yeast extract. The production, however, stopped after switching to a medium without yeast extract. Active lactic acid production by LDD appeared to require yeast extract above a certain critical level (<2 g l(-1)).     

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.     

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.     

Lactic acid fermentation by cells of Lactobacillus rhamnosus immobilized in polyacrylamide gel

Summary The process of lactic acid fermentation of lactose to lactic acid by Lactobacillus rhamnosus ATCC 7469 has been studied. The following processes have been explored: growth kinetics, as well as lactose utilization, production of lactic acid and further degradation of lactic acid. The immobilization experiments were conducted with microbial cells entrapped in polyacrylamide gels. Gels with different ratios of the monomer (acrylamide) and the cross-linking agent (N,N′-methylene-bis-acrylamide) have been tested. These were used in a repeat-batch process. The current processes inside and outside the gel particles were subjects of examination. The evolution of the activity of immobilized cells with repeated use showed that the particles served mainly as a donor of cells for the free culture. In all experiments a very high degree of conversion, 85–90% was observed. After several runs however, the particles were exhausted for microbial cells. A kinetic model of the process of lactic acid production was developed. This model allowed the evaluation of the effect of microbial growth and diffusion limitations inside the gel particles on the process rate and the separate contribution of the free and immobilized cells to the overall fermentation process upon multiple use.     

Impact of lactic acid on cell proliferation and free radical‐induced cell death in monolayer cultures of neural precursor cells

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic‐acid‐based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices. Biotechnol. Bioeng. 2009;103: 1214–1223. © 2009 Wiley Periodicals, Inc.     

Soybean milk residue ensiled with peanut hulls: fermentation acids, cell wall composition, and silage utilization by mixed ruminal microorganisms

Preservation of soybean milk residue (SMR) by ensiling with peanut hulls (PEH) and subsequent utilization of silage by mixed ruminal microorganisms were investigated. Treatments were combinations of SMR with PEH at the following ratios: 100:0, 78:22, 71:29, and 60:40 (fresh weight basis). After eight weeks of ensiling, silage lactic acid, crude protein, ether extract, and non-fiber carbohydrates were highest when SMR was ensiled alone and reduced as amounts of SMR decreased. Similar trends were observed for silage in vitro dry matter digestibility, and gas and volatile fatty acid production by ruminal microorganisms. Conversely, silage pH, dry matter, neutral detergent fiber, acid detergent fiber, cellulose, and lignin increased accordingly. The ensiling treatment appeared to alter silage cell wall composition. In particular, silage treated with PEH at the low level (78:22) resulted in reduced fiber contents and lignification. The silage (SMR:PEH=78:22) had enhanced efficiency of both silage fermentation and in vitro ruminal fermentation pattern.     

狭叶红景天引种驯化后抗氧化能力、主要活性物质含量及挥发性组分的变化

以甘肃省甘南藏族自治州碌曲县栽培和野生狭叶红景天为材料,分别对根茎(主根和侧根)的抗氧化能力、可溶性糖、总黄酮、酚类和红景天苷含量,以及挥发性组分进行研究.结果表明: 狭叶红景天栽培根茎的自由基抑制率(I)、铁离子还原/氧化能力(FRAP),以及可溶性糖、总黄酮、酚类和红景天苷含量均显著大于野生根茎;栽培和野生根茎中均含有21种挥发性组分,相对含量较高的为苯乙酮、2-糠醛、棕榈酸等;与野生根茎相比,栽培根茎有15种组分的相对含量增加,主根中相对变化量较大的为3-羟基月桂酸、2,4-二叔丁基苯酚、亚麻酰氯等,侧根为2-乙酰呋喃、对羟苯基乙醇、丁子香酚等.栽培和野生侧根的I、FRAP值,以及总黄酮、酚类和红景天苷含量均显著大于主根.与主根相比,侧根有12种挥发性组分相对含量增加,变化量较大的为3-羟基月桂酸、10,13-十八碳二炔酸甲酯、对羟苯基乙醇等.栽培驯化后狭叶红景天根茎的抗氧化能力、主要活性物质含量及挥发性组分相对含量高于野生狭叶红景天,且侧根的品质更佳.     

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.     

Comparison of isotopic fractionation in lactic acid and ethanol fermentations

Pure D(-) and L(+) enantiomers of lactic acid were prepared by fermentation reactions with specific bacteria. In addition, naturally deuterated ethanol was prepared and converted into diastereoisomers using mandelic acid. Various sugars and nutrients were fermented into lactic acid in water having different deuterium contents and ethanol samples were obtained from yeast fermentation of sugars from different botanical origins. The methine and methylene groups in lactic acid and ethanol respectively show similar deuterium contents which are related to that found in the fermentation water. However, the methyl groups of both molecules are significantly different whatever the botanical origin of the carbon source in the fermentation medium.     

Kinetics and mass transfer for lactic acid recovered with anion exchange method in fermentation solution

An anion exchange method for lactic acid recovered from lactic acid-glucose solution in an ion-exchange membrane-based extractive fermentation system was examined. The exchange isotherms of anion exchange resins for lactic acid recovered were measured batchwise, and the exchange-desorption kinetics of lactic acid passing through the exchange column was investigated. The determined typical breakthrough and elution curves were measured and simulated by conventional mode. The mass transfer coefficients were identified by numberical method. The effects of the velocity of the fluid on the dynamics were studied. Aqueous NaOH solution was found to be the best solvent for elution. An experiment on anioun exchange from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the ion exchange system from a borth. The ion-exchange mass-transfer coefficient and efficiency from the fermentation broth is found to be lower when compared with aqueous solutions of pure lactic acid. The results show that the separation method with anion exchange resins may be used in the production of lactic acid by fermentation.(c) 1995 John Wiley & Sons, Inc.     

The ratio of L-form to D-form of lactic acid as a criteria for the identification of lactic acid bacteria

Stereoisomers of lactic acid produced by lactic acid bacteria were determined by HPLC by using an enantiomeric resolution column. In general, the ratios of L-form to D-form (the type of stereoisomer) obtained were compared with those in references. Values of the type of stereoisomer of lactic acid were discussed from the viewpoint of identification of lactic acid bacteria.     

Effect of wilting and the additives,straw, molasses and urea on the fermentation pattern of maize silage

The process of ensiling was studied in fresh maize (15% dry matter (DM)), wilted maize (18 and 24% DM) and maize mixed with 5–20% of wheat straw (18, 25 and 29% DM). Silages with 24% DM were preserved better than those with lower dry matter content. There was a significant change, with time, in pH, titrable acidity, volatile fatty acids, lactic acid, number of lactic acid bacteria, volatile nitrogen and soluble sugars in all the treatments. There was a significant decline in volatile fatty acids (P<0.05) and ammonia (P<0.01) production, and a significant increase in soluble sugar (P<0.01) in silages made after wilting. A significant decline in titrable acidity (P<0.01), volatile fatty acid production (p<0.05) and ammonia nitrogen (P<0.01), and a significant increase in pH (P<0.01) were found in silages of maize mixed with wheat straw. The overall rate of fermentation decreased during the first few days of fermentation in wilted and wheat straw silages, but the final products had characteristics of a good silage. In the second experiment the effect of urea and molasses was studied on wheat straw plus maize (15:85) silage with an initial DM content of 31–34%. Three levels of molasses (0, 3 and 6% of fresh weight) and two levels of urea (0 and 0.5% of fresh weight) were studied. Urea treatment with 3% molasses was found to be the best on the basis of silage characteristics.     

A 44-kDa of protein identical to the N-terminal amino acid sequence of MCT1 in human circulation

A family of specific carrier protein designated as monocarboxylate transporter (MCT) has been known to transport the lactate and other moncarboxylates in mammalian cells. We hypothesized the presence of serum protein in human circulation that may works as a lactate carrier and that biochemical structure would possesses common structure with MCT on the plasma membrane.Immunoblot analysis with an anti-MCT1 polyclonal antibody suggested the presence of a 44-kDa protein in human circulation and N-terminal amino acid sequencing exhibited a stretch of 14 amino acids which is completely identical to MCT1. The unbound fractions from the GST-MCT1 fusion protein-immobilized glutathione sepharose 4B column demonstrated that lactic acid concentration began to increase with one fraction delay compared to Sepharose 4B and GST-immobilized column. When lactic acid was washed away with PBS, lactic acid concentrations in the effuluent constantly decreased in both Sepharose 4B and GST-immobilized column. However, GST-MCT1-immobilized column showed specific convex curve from fraction approximately 3 mM of lactate and demonstrated wash out delay compared to Sepharose 4B and GST-immobilized column.These observations demonstrated biochemical and immunological similarities between a 44-kDa protein purified from human serum and MCT1 present on the plasma membrane. The studies on MCT1-fusion protein suggested possible functional properties of a 44-kDa protein as a lactate buffer by holding and unhand a lactate according to the lactate concentration in human blood. The experiments described herein have suggested the existence of lactate carrier in human circulation which is free from plasma membrane.     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.