Both viable Bifidobacterium longum subsp. infantis B8762 and heat-killed cells alleviate the intestinal inflammation of DSS-induced IBD rats.

In view of the safety concerns of probiotics, more and more attention is paid to the beneficial effects of dead probiotics cells. Herein, we investigated and compared the alleviation effects of viable Bifidobacterium longum subsp. infantis B8762 (B. infantis B8762) and its heat-killed cells on dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) rats. Four groups of rats (n = 12 per group) were included: normal control, DSS-induced colitis rats without bacterial administration (DSS), DSS-induced colitis rats with viable B. infantis B8762 administration (VB8762), and DSS-induced colitis rats with dead B. infantis B8762 administration (DB8762). Our results showed that both VB8762 and DB8762 administration exerted significant protective effects on DSS-induced IBD rats, as evidenced by a reduction in mortality, disease activity index score, body weight loss, as well as decreased histology score, which were companied by a significant decrease in serum pro-inflammatory factors compared with DSS group, and a stronger effect on modulating the fecal microbiota alpha-diversity and beta-diversity compared with DSS group. Additionally, the fecal metabolome results showed that both VB8762 and DB8762 interventions indeed altered the fecal metabolome profile and related metabolic pathways of DSS-induced IBD rats. Therefore, given the alleviation effects on colitis, the DB8762 can be confirmed to be a postbiotic. Overall, our findings suggested that VB8762 and DB8762 had similar ability to alleviate IBD although with some differences. Due to the minimal safety concern of postbiotics, we propose that the postbiotic DB8762 could be a promising alternative to probiotics to be applied in the prevention and treatment of IBDs.IMPORTANCEInflammatory bowel disease (IBD) has emerged as a global disease because of the worldwide spread of western diets and lifestyles during industrialization. Up to now, many probiotic strains are used as a modulator of gut microbiota or an enhancer of gut barrier to alleviate or cure IBD. However, there are still many issues of using probiotics, which were needed to be concerned about, for instance, safety issues in certain groups like neonates and vulnerable populations, and the functional differences between viable and dead microorganisms. Therefore, it is of interest to investigate the beneficial effects of dead probiotics cells. The present study proved that both viable Bifidobacterium longum subsp. infantis B8762 and heat-killed cells could alleviate dextran sodium sulfate-induced colitis in rats. The findings help to support that some heat-killed probiotics cells can also exert relevant biological functions and can be used as a postbiotic.

The inhibition mechanism of PostbioYDFF-3 on quality deterioration of refrigerated grass carp fillets from the perspective of endogenous enzyme and microorganisms changes.

The protective mode of PostbioYDFF-3 (referred to as postbiotics) on the quality stability of refrigerated fillets was explored from the aspects of endogenous enzyme activity and the abundance of spoilage microorganisms. Compared to the control group, the samples soaked in postbiotics showed significant reductions in TVC, TVB-N and TBARS values by 39.6%, 58.6% and 25.5% on day 5, respectively. In addition, the color changes, biogenic amine accumulation and texture softening of the fish fillets soaked in postbiotics were effectively suppressed. Furthermore, the activity of endogenous enzyme activities was detected. The calpain activities were significantly inhibited (p < 0.05) after soaking in postbiotics, which declined by 23%. Meanwhile, high throughput sequencing analysis further indicated that the growth of spoilage microorganism such as Acinetobacter and Pseudomonas were suppressed. Overall, the PostbioYDFF-3 was suitable for preserving fish meat.

Postbiotic Administration Ameliorates Colitis and Inflammation in Rats Possibly through Gut Microbiota Modulation.

Postbiotics are preparations of inanimate microorganisms and/or their components that are beneficial to host health. Compared with probiotics, the postbiotic dose required for exerting obvious protective effects is unknown. Thus, we conducted a dose-dependent postbiotic intervention study in dextran sulfate sodium (DSS)-induced colitis rats. The trial included five rat groups, including: control without DSS/postbiotic treatment, group C; 7-day DSS treatment, group D; 14-day low, medium, and high probiotic doses (0.1, 0.2, 0.4 g/kg; groups L, M, H, respectively) after DSS induction. We found that postbiotic intervention effectively mitigated the symptoms and inflammation in colitis rats, evidenced by the improved spleen index, less severe colon tissue damage, and changes in serum cytokine levels (decreases in tumor necrosis factor-α and interleukin-1ß; increase in interleukin-10) in postbiotic groups compared with group D. Moreover, the therapeutic effect was dose-dependent. Fecal metabolomics analysis revealed that the postbiotic recipients had more anti-inflammatory metabolites, namely, salicyloyl phytophingosine, podophylloxin, securinine, baicalein, and diosmetin. Fecal metagenomics analysis revealed that the postbiotic recipients had more beneficial microbes and less pro-inflammatory bacteria. This study confirmed that postbiotics are effective in alleviating colitis in a dose-dependent manner. Our findings are of interest to food scientists, clinicians, and the health food industry.

Antibacterial and antibiofilm activities of protocatechualdehyde and its synergy with ampicillin against methicillin-resistant Staphylococcus aureus.

Protocatechualdehyde (PA) is a phenolic acid present in many plants and has many biological activities. Herein, the antagonistic effects and the action mechanism of PA against methicillin-resistant Staphylococcus aureus (MRSA) were studied. The results showed that PA had both significant antibacterial and anti-biofilm activities against MRSA. Additionally, PA had synergy with ampicillin against MRSA. It was elucidated that PA was prominent in destroying cell membranes, increasing cell membrane permeability and intracellular ROS production, thus leading to bacterial cell damage. Transcriptome analysis showed that PA disrupts many physiological pathways, including increasing cell membrane permeability, inhibiting biofilm formation, decreasing resistance to antimicrobial agents, and impairing DNA replication. Finally, the antimicrobial preservation test showed that PA could inhibit the growth of MRSA and prevent the corruption of beef. In summary, PA is an effective natural antibacterial substance and has a good application potential in food preservation, even in tackling antibiotic resistance problems.

Antibacterial and Antibiofilm Activities of Chlorogenic Acid Against Yersinia enterocolitica.

Nowadays, developing new and natural compounds with antibacterial activities from plants has become a promising approach to solve antibiotic resistance of pathogenic bacteria. Chlorogenic acid (CA), as a kind of phenolic acid existing in many plants, has been found to process multifunctional activities including antibacterial activity. Herein, the antibacterial and antibiofilm activities of CA against Yersinia enterocolitica (Y. enterocolitica) were tested for the first time, and its mechanism of action was investigated. It was demonstrated that CA could exert outstanding antibacterial activity against Y. enterocolitica. Biofilm susceptibility assays further indicated that CA could inhibit biofilm formation and decrease the established biofilm biomass of Y. enterocolitica. It was deduced that through binding to Y. enterocolitica, CA destroyed the cell membrane, increased the membrane permeability, and led to bacterial cell damage. In addition, the transcriptomic analysis revealed that CA could disorder many physiological pathways, mainly including the ones of antagonizing biofilms and increasing cell membrane permeability. Finally, the spiked assay showed that the growth of Y. enterocolitica in milk was significantly inhibited by CA. Taken together, CA, as an effective bactericidal effector with application potential, exerts antagonistic activity against Y. enterocolitica by mainly intervening biofilm formation and membrane permeability-related physiological pathways.

Mesopic fermentation contributes more to the formation of important flavor compounds and increased growth of Lactobacillus casei Zhang than does high temperature during milk fermentation and storage.

Probiotic fermented milk is more and more popular due to their positive health associated properties. However, fermentation temperature and other process conditions may affect the growth and metabolism of probiotic strains, thereby affecting quality of the final products. In this study, the growth behaviors and metabolomic profiles of yogurts induced by Lactobacillus casei Zhang at fermentation termination (FT) and d 10 of storage (S10d) under different fermentation temperatures at 37°C (low) and 42°C (high) were analyzed and compared using liquid chromatography-mass spectrometry (MS)- and gas chromatography-MS-based metabolomics approaches. At 37°C, the growth of L. casei Zhang at FT and S10d was significantly increased, and the potential relationship between riboflavin, starch, and sucrose metabolism and growth of L. casei Zhang may be mutually promoting. Fermentation temperature (37°C and 42°C) affected volatile and nonvolatile metabolomic profiles and pathways. The levels of acetaldehyde, 2,3-butanedione, acetoin, butyric acid, decanoic acid, hexanoic acid, and octanoic acid were significantly higher at 37°C than at 42°C at FT and S10d. This indicates that the low temperature (37°C) most likely contributes more to the formation of important flavor compounds during the fermentation process and production of short-chain fatty acids during storage.

Comparative effects of the single and binary probiotics of Lacticaseibacillus casei Zhang and Bifidobacterium lactis V9 on the growth and metabolomic profiles in yogurts.

In the present study, comparative effects of the single and binary probiotics of Lacticaseibacillus casei Zhang (L. casei Zhang) and Bifidobacterium lactis V9 (B. lactis V9) on the growth and metabolomic profiles during milk fermentation and storage has been analyzed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The growth of B. lactis V9 can be significantly increased (p < 0.001) by co-cultivation with L. casei Zhang at the end of fermentation and storage 10 days, and valine, leucine and isoleucine biosynthesis may be a major contributor to the growth promotion of B. lactis V9. However, the growth of L. casei Zhang was not affected by co-cultivation with B. lactis V9. There were notable distinctions in volatile and non-volatile metabolomic profiles and pathways between the single and binary probiotics cultures; binary probiotics L. casei Zhang and B. lactis V9 significantly affected the volatile, non-volatile metabolic profiles compared to the single probiotics. The levels of acetic acid, hexanoic acid, butanoic acid and pentanoic acid were significantly higher (p < 0.05) in binary probiotics cultures compared to the single probiotic cultures at the storage 10 days, which indicates that binary probiotics had additive effects on the production of short-chain fatty acids during storage. This work provides a detailed insight into metabolomic profiles and growth that differ between the single and binary probiotics cultures, and it can be helpful to develop probiotic yogurt with high probiotic viability and distinct metabolomic profiles.

The Bacterial Diversity of Spontaneously Fermented Dairy Products Collected in Northeast Asia.

Spontaneously fermented dairy products have a long history, and present diverse microorganisms and unique flavors. To provide insight into the bacterial diversity, 80 different types of spontaneously fermented dairy product samples' sequence data that were downloaded from MG-RAST and NCBI and 8 koumiss and 4 shubat were sequenced by the PacBio SMRT sequencing platform. All samples including butter, sour cream, cottage cheese, yogurt, koumiss, shubat, and cheese, were collected from various regions in Russia, Kazakhstan, Mongolia and Inner Mongolia (China). The results revealed that Firmicutes and Proteobacteria were the most dominant phyla (>99%), and 11 species were identified with a relative abundance exceeding 1%. Furthermore, Streptococcus salivarius, Lactobacillus helveticus, Lactobacillus delbrueckii, Enterobacter xiangfangensis, and Acinetobacter baumannii were the primary bacterial species in the fermented dairy product samples. Principal coordinates analysis showed that koumiss and shubat stood out from the other samples. Moreover, permutational ANOVA tests revealed that the types of fermented dairy products and geographical origin significantly affected microbial diversity. However, different processing techniques did not affect microbial diversity. In addition, results of hierarchical clustering and canonical analysis of the principal coordinates were consistent. In conclusion, geographical origin and types of fermented dairy products determined the bacterial diversity in spontaneously fermented dairy product samples.

Different growth behaviors and metabolomic profiles in yogurts induced by multistrain probiotics of Lactobacillus casei Zhang and Bifidobacterium lactis V9 under different fermentation temperatures.

The growth behaviors and metabolomic profiles in yogurts induced by multistrain probiotics of Lactobacillus casei Zhang (LCZ) and Bifidobacterium lactis V9 (V9) at the fermentation termination and 10 d of storage at 4°C under different fermentation temperatures (37°C and 42°C) were compared using metabolomics based on liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The growths of LCZ and V9 were affected by fermentation temperatures; the viable cell density of LCZ was higher at 37°C than that at 42°C; however, V9 was higher at 42°C. Multistrain probiotics had higher contribution to the changes in volatile and nonvolatile metabolomic profiles at 42°C than those at 37°C. At fermentation termination, there were 2 common enriched pathways increased by multistrain probiotics at 37°C and 42°C, which were biosynthesis of peptides and amino- and nucleotide-sugar metabolism. At 10 d of storage, 4 common increased enriched pathways were alanine, aspartate and glutamate metabolism; tyrosine metabolism; valine, leucine, and isoleucine degradation; and valine, leucine, and isoleucine biosynthesis. This work provided a detailed insight into different effects of different multistrain probiotics of LCZ and V9 fermentation temperatures on the growth behaviors and volatile and nonvolatile metabolomic profiles of yogurts.

Comparison of the composition and function of the gut microbiome in herdsmen from two pasture regions, Hongyuan and Xilingol.

There is a close relationship between the gut microbiome and health in humans including regulation of immunity and energy metabolism. This study investigated differences in the gut microbiome of herdsmen from two regions: Hongyuan pasture in Sichuan and Xilingol pasture in Inner Mongolia. We found significant differences in the gut microbiome between the two groups. The main discriminatory species between the two groups were Bifidobacterium longum, Bifidobacterium breve, Phascolarctobacterium succinatutens, Prevotella stercorea, Prevotella copri, Eubacterium biforme, and Fusobacterium prausnitzii. The abundances of Bifidobacterium longum and Bifidobacterium breve were significantly lower in the gut microbiomes of Hongyuan herdsmen than in the gut microbiomes of Xilingol herdsmen. Functional metagenomic analysis showed that more genes were enriched in glycoside hydrolase and transposase in the gut microbiome of Hongyuan herdsmen compared with Xilingol herdsmen, suggesting a higher energy demand in the gut microbiome of Hongyuan herdsmen. Significantly more genes associated with glycolysis, starch degradation, and sucrose degradation were also found in the gut microbiome of Hong yuan herdsmen compared with Xilingol herdsmen. These results indicate that herdsmen from different pastoral regions had distinct gut microbiome composition and functions.

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation.

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry-based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.

Comparison of the effects of single probiotic strains Lactobacillus casei Zhang and Bifidobacterium animalis ssp. lactis Probio-M8 and their combination on volatile and nonvolatile metabolomic profiles of yogurt.

There has been a growing interest in cofermentation of starter cultures with probiotics in milk. In this study, we analyzed the effects of adding the single probiotics Lactobacillus casei Zhang (Zhang) or Bifidobacterium animalis ssp. lactis Probio-M8 (M8) or a combination of Zhang and M8 to starter cultures on volatile and nonvolatile metabolomic profiles after 14 d of storage at 4°C and compared using a liquid chromatography-tandem mass spectrometry (LC-MS) and GC-MS-based metabolomics approach. Principal component analysis, heatmap plots, and Spearman correlation results showed that Zhang alone had a greater effect on volatile and nonvolatile metabolomic profiles than M8 alone. The combination of Zhang and M8 had additive effects on the production of metabolites. For volatile metabolites, the levels of acetaldehyde, diacetyl, acetoin, and acetic acid were higher for the combination of Zhang and M8 compared with either single probiotic culture. Significantly increased nonvolatile components induced by adding Zhang were identified were enriched in the galactose, amino- and nucleotide sugar, fructose and mannose, purine, phenylalanine metabolism, and arginine biosynthesis pathways. The metabolism and biosynthesis of starch, sucrose, tyrosine, galactose metabolism, and aminoacyl-tRNA biosynthesis were significantly upregulated by adding the combination of Zhang and M8. This work provides a detailed insight into different effects of Zhang and M8 used alone or in combination on the volatile and nonvolatile metabolomic profiles of yogurts.

Meta-Analysis: Randomized Trials of Lactobacillus plantarum on Immune Regulation Over the Last Decades.

Lactobacillus (L.) plantarum strains, belong to lactic acid bacteria group, are considered indispensable probiotics. Here, we performed meta-analysis to evaluate the regulatory effects of L. plantarum on the immunity during clinical trials. This meta-analysis was conducted by searching across four most common literature databases, namely, Cochrane Central Register of Controlled Trials, Web of Science, Embase, and PubMed. Clinical trial articles that met the inclusion and exclusion criteria were analyzed by Review Manager (version 5.3). p-value < 0.05 of the total effect was considered statistically significant. Finally, total of 677 references were retrieved, among which six references and 18 randomized controlled trials were included in the meta-analysis. The mean differences observed at 95% confidence interval: interleukin (IL)-4, -0.48 pg/mL (-0.79 to -0.17; p < 0.05); IL-10, 9.88 pg/mL (6.52 to 13.2; p < 0.05); tumor necrosis factor (TNF)-α, -2.34 pg/mL (-3.5 to -1.19; p < 0.05); interferon (IFN)-γ, -0.99 pg/mL (-1.56 to -0.41; p < 0.05). Therefore, meta-analysis results suggested that L. plantarum could promote host immunity by regulating pro-inflammatory and anti-inflammatory cytokines.

Analysis of the response of the cell membrane of Saccharomyces cerevisiae during the detoxification of common lignocellulosic inhibitors.

Gaining an in-depth understanding of the response of Saccharomyces cerevisiae to the different inhibitors generated during the pretreatment of lignocellulosic material is driving the development of new strains with higher inhibitor tolerances. The objective of this study is to assess, using flow cytometry, how three common inhibitors (vanillin, furfural, and acetic acid) affect the membrane potential, the membrane permeability and the concentration of reactive oxygen species (ROS) during the different fermentations. The membrane potential decreased during the detoxification phase and reflected on the different mechanisms of the toxicity of the inhibitors. While vanillin and furfural caused a metabolic inhibition and a gradual depolarization, acetic acid toxicity was related to fast acidification of the cytosol, causing an immediate depolarization. In the absence of acetic acid, ethanol increased membrane permeability, indicating a possible acquired tolerance to ethanol due to an adaptive response to acetic acid. The intracellular ROS concentration also increased in the presence of the inhibitors, indicating oxidative stress. Measuring these features with flow cytometry allows a real-time assessment of the stress of a cell culture, which can be used in the development of new yeast strains and to design new propagation strategies to pre-adapt the cell cultures to the inhibitors.

Characterization and association of bacterial communities and nonvolatile components in spontaneously fermented cow milk at different geographical distances.

In the ecosystem of spontaneously fermented cow milk, the characteristics and relationship of bacterial communities and nonvolatile components at different scales of geographical distances (provincial, county, and village levels) are unclear. Here, 25 sampling sites from Xin Jiang and Tibet, 2 provinces of China, were selected based on the distribution of spontaneously fermented cow milk and used for metagenomic and metabolomic analysis. At the provincial geographical distance, the same predominant species, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, were detected in Xin Jiang and Tibet. Further, the richness of the bacterial composition of samples from Tibet was higher than those from Xin Jiang; specifically, at the species level, 28 species were identified in Tibet samples but only 7 species in Xin Jiang samples. At the provincial geographical level, we detected significant differences in bacterial structure, shown in principal coordinate analysis plots, and significant differences (Simpson index) in bacterial diversity were also detected. However, at the county and village levels, no significant differences were detected in bacterial communities and diversity, but a difference in bacterial compositions was detectable. This indicates that bacterial communities and diversity of spontaneously fermented milk dissimilarity significantly increased with geographic distance. For the nonvolatile component profiles, the partial least squares discriminant analysis plot (R2Y > 0.5 and Q2 > 0.5 for the goodness-of-fit and predictive ability parameter, respectively) showed that samples from different geographical distances (provincial, county, and village) were all separated, which indicated that all the discriminations in nonvolatile components profiles were from different geographical distances. Investigating relationships between lactic acid bacteria and discriminatory nonvolatile components at the county level showed that 9 species were positively correlated with 16 discriminatory nonvolatile components, all species with low abundance rather than the predominant species L. delbrueckii ssp. bulgaricus and Strep. thermophilus, which indicates the importance of the selection of autochthonous nonpredominant bacteria.

Metabolic footprint analysis of volatile metabolites to discriminate between different key time points in the fermentation and storage of starter cultures and probiotic Lactobacillus casei Zhang milk.

Interest has been growing in the co-fermentation of starter cultures with probiotic bacteria in milk. However, the representative metabolites and metabolic changes at different key time points during milk fermentation and storage in starter cultures and probiotic bacteria are still unclear. In this study, we used gas chromatography/mass spectrometry-based metabolomics to identify volatile metabolites and discriminate between 6 different time points [fermentation initiation (FI), fermentation curd (FC), fermentation termination (FT), storage 1 d (S1d), storage 7 d (S7d), and storage 14 d (S14d)] during the fermentation and storage of starter cultures and Lactobacillus casei Zhang milk. Of the 52 volatile metabolites identified, 15 contributed to discrimination of the 6 time points. Then, using the profile from the different time points, we analyzed pairwise comparisons (FI vs. FC; FC vs. FT; FT vs. S1d; S1d vs. S7d; S7d vs. S14d); these time-lapse comparisons showed metabolic progressions from one fermentation stage to the next. We found representative and exclusive metabolites at specific fermentation and storage time points. The greatest difference in metabolites occurred between FC and FT, and the metabolic profiles between S7d and S14d were most similar. Interestingly, decanoic acid, octanoic acid, and hexanoic acid reached their highest level at storage 14 d, indicating that the post-fermentation storage of fermented milk with L. casei Zhang may add more probiotic functions. This work provides detailed insight into the time-specific profiles of volatile metabolites and their dynamic changes; these data may be used for understanding and eventually predicting metabolic changes in milk fermentation and storage, where probiotic strains may be used.

Fermented milk containing Lactobacillus casei Zhang and Bifidobacterium animalis ssp. lactis V9 alleviated constipation symptoms through regulation of intestinal microbiota, inflammation, and metabolic pathways.

Studies suggest that probiotics and fermented milk can improve defecation in constipated patients. However, the mechanism of fermented milk containing probiotics on constipation remains poorly understood. Volunteers with chronic constipation symptoms were recruited and given 200 g/d of fermented milk containing Lactobacillus casei Zhang and Bifidobacterium animalis ssp. lactis V9 (PFM) for 4 wk. Clinical symptoms, cytokines, metagenomics, and metabolomics were evaluated in constipated participants before and after PFM intervention. After PFM intervention, we observed significant improvement of constipation symptoms. In the serum samples, the anti-inflammatory cytokine IL-10 increased and the proinflammatory cytokine C-reactive protein and lipopolysaccharides decreased. Metagenomics results showed that the increase of B. animalis was correlated with an increase in defecation frequency. Fatty acid biosynthesis and bile acid biosynthesis in stool samples as well as carnitine shuttle, vitamin E metabolism, and ascorbate and aldarate metabolism were identified as significantly altered metabolic pathways. Acylcarnitine, located on the carnitine shuttle pathway, had a significantly positive correlation with defecation frequency. It was speculated that PFM may contribute to alleviating constipation symptoms through 3 potential mechanisms: fine-tuning gastrointestinal microbiota, fighting inflammation, and regulating metabolic pathways.

Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotolerans.

The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.

Metabolic footprint analysis of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations.

INTRODUCTION: There has been a growing interest towards creating defined mixed starter cultures for alcoholic fermentations. Previously, metabolite differences between single and mixed cultures have been explored at the endpoint of fermentations rather than during fermentations. OBJECTIVES: To create metabolic footprints of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations. METHODS: 1H NMR- and GC-MS-based metabolomics was used to identify metabolites that discriminate single and mixed cultures of Lachancea thermotolerans (LT) and Saccharomyces cerevisiae (SC) during alcoholic fermentations. RESULTS: Twenty-two metabolites were found when comparing single LT and mixed cultures, including both non-volatiles (carbohydrate, amino acid and acids) and volatiles (higher alcohols, esters, ketones and aldehydes). Fifteen of these compounds were discriminatory only at the death phase initiation (T1) and fifteen were discriminatory only at the death phase termination (T2) of LT in mixed cultures. Eight metabolites were discriminatory at both T1 and T2. These results indicate that specific metabolic changes may be descriptive of different LT growth behaviors. Fifteen discriminatory metabolites were found when comparing single SC and mixed cultures. These metabolites were all volatiles, and twelve metabolites were discriminatory only at T2, indicating that LT-induced changes in volatiles occur during the death phase of LT in mixed cultures and not during their initial growth stage. CONCLUSIONS: This work provides a detailed insight into yeast metabolites that differ between single and mixed cultures, and these data may be used for understanding and eventually predicting yeast metabolic changes in wine fermentations.

Potential of Glycosidase from Non-Saccharomyces Isolates for Enhancement of Wine Aroma.

The aim of this work was to rapidly screen indigenous yeasts with high levels of ß-glucosidase activity and assess the potential of glycosidase extracts for aroma enhancement in winemaking. A semiquantitative colorimetric assay was applied using 96-well plates to screen yeasts from 3 different regions of China. Isolates with high ß-glucosidase activity were confirmed by the commonly used pNP assay. Among 493 non-Saccharomyces isolates belonging to 8 generas, 3 isolates were selected for their high levels of ß-glucosidase activity and were identified as Hanseniaspora uvarum, Pichia membranifaciens, and Rhodotorula mucilaginosa by sequence analysis of the 26S rDNA D1/D2 domain. ß-Glucosidase in the glycosidase extract from H. uvarum strain showed the highest activity in winemaking conditions among the selected isolates. For aroma enhancement in winemaking, the glycosidase extract from H. uvarum strain exhibited catalytic specificity for aromatic glycosides of C13 -norisoprenoids and some terpenes, enhancing fresh floral, sweet, berry, and nutty aroma characteristics in wine.