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Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.

Animal Feed

Pilot-scale semisolid fermentation of straw.

Semisolid fermentation of ryegrass straw to increase its animal feed value was successfully performed on a pilot scale. The pilot plant, which could handle 100 kg of straw per batch, was designed so that all major operations could take place in one vessel. The straw was hydrolyzed at 121 degrees C for 30 min with 0.5 N H2SO4 (7:3 liquid:solid), treated with ammonia to raise the pH to 5.0, inoculated with Candida utilis, and fermented in a semisolid state (70% moisture). During fermentation the straw was held stationary with air blown up through it. Batch fermentation times were 12 to 29 h. Semisolid fermentation did not require agitation and supported abundant growth at 20 to 40 degrees C even at near zero oxygen tensions. Fermentation increased the protein content, crude fat content, and in vitro rumen digestibility of the straw.

Animal Feed

Consumption of traditional Sardinian fermented milk promotes changes in the rat gut microbiota composition and functions.

BACKGROUND: Fermented milk products are part of the staple diet for many Mediterranean populations. Most of these traditional foods are enriched with lactobacilli and other lactic acid bacteria, as well as with metabolites resulting from lactose fermentation. Currently, there is very little scientific knowledge on how dietary supplementation with fermented milk affects the composition of the gut microbiota and its metabolic activities. RESULTS: We integrated 16 S rRNA gene-based taxonomic profiling with metaproteomics-based functional analysis to investigate gut microbiota changes in rats exposed to an 8-week dietary supplementation with casu axedu, a traditional fermented milk produced within rural communities in Sardinia (Italy). Several microbial taxa showed a significantly increased abundance at the end of the dietary treatment, including Phascolarctobacterium, Prevotella, Blautia glucerasea, and Lactococcus lactis, while Bacteroides dorei and Helicobacter rodentium were decreased compared to the control rats. Metaproteomic analysis highlighted a striking reshaping of the Prevotella proteome in agreement with its blooming in casu axedu-fed animals, suggesting an increase of the glycolytic activity through the Embden-Meyerhof-Parnas pathway over the Entner-Doudoroff pathway. Moreover, an increased production of enzymes involved in succinate biosynthesis was observed, which in turn significantly boosted the abundance of Phascolarctobacterium and its production of propionate. Fermented milk consumption also promoted microbial synthesis of branched chain essential amino acids L-valine and L-leucine. Finally, metaproteomic data indicated a reduction of bacterial virulence factors and host inflammatory markers, suggesting that the consumption of casu axedu can have beneficial effects on the gut mucosa health. CONCLUSIONS: Our integrated multi-omics approach reveals that dietary supplementation with the traditional Sardinian fermented milk, casu axedu, induces significant shifts in the rat gut microbiota composition and function, characterized by the enrichment of beneficial taxa and metabolic pathways associated with improved gut health and reduced inflammation.

Animals

Absorption of colostral proteins by newborn calves fed unfermented, fermented, or buffered colostrum.

Unfermented, frozen colostrum from the first three postpartum milkings of 10 cows was thawed, pooled, and treated to produce three diets: 1) unfermented, 2) fermented (7 days at 25 to 27 C), and 3) fermented (as in 2) with pH adjusted to match that of unfermented colostrum. Eighteen newborn, unsuckled Holstein calves were assigned randomly to one of the three diets. Colostrum diets were thawed and fed at 0, 8, 16, 24, and 36 h. Blood was sampled at 0, 4, 8, 16, 24, and 48 h. Minimal breakdown of colostral gamma-globulin and immunoglobulin G (IgG) occurred during fermentation. Protein breakdown during fermentation was associated primarily with the casein fraction. Concentrations of gamma-globulin in serum of calves receiving unfermented colostrum were higher than those of calves fed fermented colostrum at all sampling times beyond 0 h. Concentrations of gamma-globulin in serum of calves fed buffered colostrum were intermediate. Concentrations of IgG followed a similar trend. Health problems were not encountered, indicating potential for passive immunization of newborn calves via fermented, buffered colostrum in emergency situations.

Absorption

[Effect of carbon composition of the fermented medium on the synthesis of volatile acids by the yeast Saccharomyces carlsbergensis 776].

The effect of glucose, maltose and sucrose on the synthesis of volatile oils during fermentation of model carbohydrate solutions (6, 8 and 11%) by the yeast Saccharomyces carlsbergensis 776 was studied. The composition and concentration of carbohydrates affected the build-up of volatile fatty acids during fermentation. The accumulation of biomass and volatile fraction of fatty acids reached maximum on the medium containing 11% glucose. There was a certain correlation between the biomass synthesis and accumulation of volatile fatty acids, i.e. with an increase in the biomass the content of volatile fatty acids in the medium increased. During fermentation of disaccharide solutions cell multiplication diminished and the fermentation process accelerated. Ethanol, residual sugar and acidity of fermented solutions increased with the initial concentration of carbohydrates. The pH value of the fermented must remained essentially unaltered independent of the sugar amount used.

Culture Media

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.

Adenosine Triphosphate

Transcriptomic shift in ethanol and amino acid metabolic genes regulated by Med15 during alcoholic fermentation.

Organisms that thrive in extreme environments provide natural experiments in evolution, revealing the genetic regulators that orchestrate complex phenotypic change. Wine yeast are specialized strains that are adapted to survive in the wine making environment while producing high concentrations of ethanol. In addition to large genomic changes that differentiate wine yeast from yeast used in other industries, single nucleotide and polyglutamine tract polymorphisms in the transcriptional regulator Med15 are associated with the fermentation efficiency and stress response phenotypes of wine yeast. In this study we investigated the transcriptional differences during wine fermentation in transgenic lab strain yeast having integrated wine yeast MED15 alleles. Compared to the unmodified lab strain (LAB or MED15 LAB ), the same strain in which the MED15 locus was replaced with a MED15 allele from yeast isolated from palm wine, the fermented sap of palm (oil, date, coconut) trees, (WY23, or MED15 WY23 ) exhibited enhanced expression of glycolytic, fermentation, and amino acid biosynthesis genes. Our experimental data confirms the importance of arginine biosynthetic genes during the fermentation process and suggests that the improvement in fermentation efficiency in strains with MED15 alleles from some wine yeast strains may be related to the role of Med15 in expression of the genes of the arginine biosynthetic pathway. The global benefit conferred by polymorphisms in a single transcriptional regulator, makes Med15 a prime target for engineering of strains devoted to various types of alcohol production.

Journal Article

Metagenomic Insights into Microbial Assembly and Key Metabolic Genes Driving Flavor Formation in Spontaneously Fermented Zhejiang Rosy Vinegar.

The spontaneous fermentation of Zhejiang rosy vinegar (ZRV) is driven by environmental microbiota, but the processes underlying its flavor formation remain poorly understood. Using metagenomic sequencing, we investigated microbial community assembly, environmental drivers, and metabolic networks during industrial-scale ZRV fermentation. Acetic acid dominated the final organic acids. Community assembly shifted toward deterministic selection with rising acidity, with a slight rebound of stochastic processes in the late stage (R2 values of 0.442 and 0.346 for bacteria and fungi, respectively). Mantel tests confirmed that environmental factors significantly regulated microbial assembly. Co-occurrence networks grew more complex, with positive interactions accounting for 85.24% (bacteria) and 90.10% (fungi) in the late stage. Key genes (ldh, gapA, pgk) from Acetobacter pasteurianus and Lactobacillus acetotolerans dominated late-stage fermentation, while genes (adhP, SDH) from Aspergillus oryzae and Saccharomyces cerevisiae supported early- and mid-stage fermentation. These findings elucidate microbiota-driven metabolic pathways in ZRV, supporting the fermentation window optimization and industrial vinegar quality standardization.

Acetic Acid

Genomic signatures of dairy adaptation in Saccharomyces cerevisiae from traditional Yaghnob goat-cheese fermentation.

The growing interest in studying Saccharomyces cerevisiae strains from previously unexplored niches is greatly expanding our understanding of this yeast's ecology and evolution. While strains involved in alcoholic fermentation are the most studied, S. cerevisiae has also been isolated from milk fermentations and their products, suggesting a potential evolutionary specialization for dairy environments. These fermentations are characterized by the predominant presence of lactose, a carbon source that S. cerevisiae cannot metabolize directly but can exploit through the enzymatic activity of co-occurring microorganisms that convert lactose into fermentable substrates, such as glucose and galactose. In this study, we analyzed S. cerevisiae strains isolated from an unexplored and remote niche: traditional goat fermented milk produced by the Yaghnob people, an ethnically and geographically partly isolated population living in the Upper Zarafshan area of the Republic of Tajikistan. Comparative analyses with published S. cerevisiae genomes positioned the Yaghnob strains at the base of the phylogenetic dairy clade. These strains revealed distinctive coding sequences and strain-specific single-nucleotide variants present in all Yaghnob strains but absent from the other 1,053 strains analyzed. Further investigation of variants in key genes involved in galactose metabolism provided insights into the genomic and protein-level evolution of Yaghnob strains, uncovering unique genomic signatures of adaptation to the dairy environment.

Saccharomyces cerevisiae

Exploring the mechanism of aroma production in fermented cherry juice by L. brevis LD1.0600 using flavomics and whole genome analysis.

This study focused on L.brevis LD1.0600 with excellent fermentation traits: it analyzed genome-wide key regulatory genes for micro-metabolites, combined with fermented cherry juice flavor metabolomics data, and used machine learning to explore correlations between gene regulation, metabolite production, and flavor formation. The SVM model screened and verified fermented cherry juice VOCs; through OAV and flavor wheel analysis, LD1.0600 emerged as the top-performing strain, with a sweet, fruity dominant aroma. Key aroma-active components (OAV > 100) included 2-methoxy-4-vinylphenol, benzaldehyde, 2-methyl-butanoic acid and hexanoic acid, and 2-methoxy-4-vinylphenol and hexanoic acid elevated by LD1.0600-regulated genes (Chrom1-001884, Chrom1-000925, fabF and Chrom1-000199). At the same time, through research, a "strain screening-SVM screening of DVCs-OAV screening of key aroma components-whole genome sequencing of flavor regulatory genes" system was established. This system can not only be applied to the screen fermentation strains, but also can be extended to the application of other fermentation products.

Fermentation

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2&#x3b5;fattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2&#x3b5;fattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550&#x2030; 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200&#x2030;. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2&#x3b5;fattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2&#x3b5;fattyacid/water variations (<30&#x2030;) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Saccharomyces cerevisiae

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.

Amino Acids

Fungal growth and acid production during fermentation and refermentation of organic acid treated corn silages.

Chopped corn (35% dry matter) treated with either propionic, formic, 60% propionic plus 40% formic, or 80% propionic plus 20% acetic acids at 0, .5, 1, and 2%, was placed in polyethylene bags inside metal drums, and evacuated. During fermentation silages were sampled and temperatures determined. On day 40 of fermentation silages were placed in open containers at 25 C and were sampled during refermentation. All samples were analyzed for volatile and lactic acids, pH, and number and type of fungi. Lactate fermentation was totally inhibited at 2% addition of all acids, but formic acid was more effective than propionic acid at .5 and 1%. Acerate production was equally depressed by both propionic and formic additions. Heating, growth of fungi, and days until spoilage were delayed by all acid additions during refermentation, with propionic more effectivethan formic. The large increases in lactate and acetate of treated silages during refermentation reflect a protection of soluble sugars during fermentation and subsequent use by microbes after exposure to air. Silages treated with more than 1%propionic (as propionic or a mixture) did not increase in fungal colonies during refermentation. At initiation of fermentation yeasts werehigher than other fungi but decreases by day 40. During refermentation, yeasts again grew. Geotrichum comprised 35% on day 40 of refermentation but was lower at other times. Aspergillus proliferated during refermentation and few Penicillium were detected.

Acetates

Protein, methionine, lysine and a fermentation residue as variables in diets of young turkeys.

A study was conducted to determine the effects of adding methionine, lysine and a fermentation residue to practical-type diets containing 24, 27, and 30% protein for young turkeys. A 2 X 2 X 2 X 3 factorial design of variables was used in each of two seven-week experiments. A total of 864 poults were divided into groups such that the 24 diets were each fed to 9 males and 9 females in each experiment. Seven-week body weights were increased 11.4% from 0.1% added DL-methionine and decreased 13.7% from 1.5% added fermentation residue, L-Lysine added at 0.157% failed to increase significantly body weights. Increasing the protein from 24 to 27 and 30% increased seven-week body weights 16.7 and 28.9%, respectively. As dietary protein increased, the increases in body weight from added methionine became smaller, i.e., 16.9, 14.4 and 4.8% in diets containing 24, 27, and 30% protein, respectively. Plots of body weight on (1) amount of methionine and on (2) amount of total sulfur amino acids in the diet show a closer relationship with the latter. The sulfur amino acid requirement of poults to seven weeks of age appears to be at least 1.03%, the highest quantity used in this study. The fermentation residue, a product not currently marketed, remains after the isolation of spectinomycin from controlled fermentation of Streptomyces flavopersicus and contains 14% dry matter. The cause of the depressed growth from the added fermentation residue has been discussed.

Animal Feed

Microbial diversity, functional activities, and safety risks in fermented tea: a comprehensive review.

Microbial fermented teas are gaining global popularity due to their unique sensory profiles and health benefits. The quality and safety of these products are governed by complex microbial ecosystems that orchestrate the biotransformation of tea leaf components. This review addresses a critical paradox in the field: the same microbial activities that generate desirable bioactive metabolites, such as theabrownins and organic acids, also create ecological niches for mycotoxigenic fungi, posing significant health risks from contaminants like ochratoxin A, citrinin, and aflatoxins. While extensive research has cataloged the microbial diversity in these systems, a comprehensive framework linking processing environments to microbial community assembly, functional outcomes, and quantifiable safety risks remains elusive. This review systematically bridges this gap by synthesizing current knowledge on the microbial consortia-dominated by Aspergillus, Penicillium, Bacillus, and Lactiplantibacillus species-that drive tea fermentation. We critically analyze their functional roles in enhancing flavor, bioactivity, and potential probiotic activity while simultaneously evaluating the mechanisms of mycotoxin production and accumulation. By integrating microbial ecology, biochemistry, and food safety, we propose a forward-looking perspective focused on transitioning the industry from traditional, spontaneous fermentation to modern, controlled biotechnological processes. This approach, centered on the use of defined starter cultures, predictive modeling, and active biocontrol strategies, provides a roadmap for ensuring the consistent quality and safety of fermented tea products, ultimately unlocking their full potential as high-quality functional foods.

Tea

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization.

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an in&#xa0;vivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with &#x3b1;-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Animals

Viral community in Aspergillus spp. isolated from commercially available fermented dried bonito.

Katsuobushi is a traditional processed seafood product used in Japanese-style cooking, and when it is produced through fermentation by fungi, it is called karebushi. The fungi involved in katsuobushi fermentation are collectively referred to as katsuobushi molds. We previously discovered seven novel viruses from katsuobushi molds and determined their genome sequences. However, our previous explorations used only nine fungal strains available from culture collections, leaving the diversity of viruses infecting fungi involved in katsuobushi fermentation unclear. Therefore, in this study, we aimed to isolate fungi from commercially available karebushi and clarify the prevalence of viruses in the isolates. Karebushi produced by three manufacturers was obtained, and 30 fungal strains (including Aspergillus spp.) were isolated from each. Double-stranded RNA (dsRNA) fractions were prepared from the mycelia of the isolated strains. Electrophoresis suggested that a relatively high proportion of the isolates harbored dsRNA elements consistent with RNA virus infection (30-70% per manufacturer; 59% overall). Furthermore, dsRNA sequencing identified four novel viruses in isolates of Aspergillus chevalieri and Aspergillus montevidensis: a beny-like virus, a gammapartitivirus, a narnavirus, and a victorivirus, in addition to two previously reported viruses. Notably, this represents the first report of a beny-like virus in Aspergillus spp. This study provides insights into the diversity of viruses infecting fungi involved in katsuobushi fermentation.

Aspergillus

Transcriptomic shift in ethanol and amino acid metabolic genes regulated by Med15 during alcoholic fermentation.

Organisms that thrive in extreme environments provide natural experiments in evolution, revealing the genetic regulators that orchestrate complex phenotypic change. Wine yeast (WY) are specialized strains that are adapted to survive in the wine making environment while producing high concentrations of ethanol. In addition to large genomic changes that differentiate WY from yeast used in other industries, SNP and polyglutamine tract polymorphism in the transcriptional regulator Med15 are associated with the fermentation efficiency and stress response phenotypes of WY. In this study, we investigated the transcriptional differences during wine fermentation in transgenic lab strain yeast having integrated WY MED15 alleles. Compared to the unmodified lab strain (MED15 LAB), the same strain in which the MED15 locus was replaced with a MED15 allele from yeast isolated from palm wine, the fermented sap of palm (oil, date, coconut) trees (MED15 WY23), exhibited enhanced expression of amino acid biosynthesis genes as well as stress resistance and metabolic adaptation genes. Our experimental data confirm the role of arginine in efficient fermentation and suggest that certain MED15 alleles alter the expression patterns of arginine pathway genes in some cases improving carbon flux under nitrogen stress. The global benefits conferred by natural polymorphisms in a single transcriptional regulator highlight Med15 as a target for engineering of strains devoted to various types of alcohol production.

Ethanol