Acetic acid-and furfural-based adaptive evolution of Saccharomyces cerevisiae strains for improving stress tolerance and lignocellulosic ethanol production

Acetic acid and furfural are known as prevalent inhibitors deriving from pretreatment during lignocellulosic ethanol production.They negatively impact cell growth,glucose uptake and ethanol biosynthesis of Saccharomyces cerevisiae strains.Development of industrial S.cerevisiae strains with high tolerance towards these inhibitors is thus critical for efficient lignocellulosic ethanol production.In this study,the acetic acid or furfural tolerance of different S.cerevisiae strains could be significantly enhanced after adaptive evolution via serial cultivation for 40 generations under stress conditions.The acetic acid-based adaptive strain SPSC01-TA9 produced 30.5 g·L^(-1)ethanol with a yield of 0.46 g·g^(-1)in the presence of 9 g·L^(-1)acetic acid,while the acetic acid/furfural-based adaptive strain SPSC01-TAF94 produced more ethanol of 36.2 g·L^(-1)with increased yield up to 0.49 g·g^(-1)in the presence of both 9 g·L^(-1)acetic acid and 4 g·L^(-1)furfural.Significant improvements were also observed during non-detoxified corn stover hydrolysate culture by SPSC01-TAF94,which achieved ethanol production and yield of 29.1 g·L^(-1)and 0.49 g·g^(-1),respectively,the growth and fermentation efficiency of acetic acid/furfural-based adaptive strain in hydrolysate was 95%higher than those of wildtype strains,indicating the acetic acid-and furfural-based adaptive evolution strategy could be an effective approach for improving lignocellulosic ethanol production.The adapted strains developed in this study with enhanced tolerance against acetic acid and furfural could be potentially contribute to economically feasible and sustainable lignocellulosic biorefinery.

Postbiotics from Saccharomyces cerevisiae fermentation stabilize microbiota in rumen liquid digesta during grain-based subacute ruminal acidosis(SARA) in lactating dairy cows

Background Subacute ruminal acidosis(SARA)is a common metabolic disorder of high yielding dairy cows,and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation.This study evaluated the impact of two postbiotics from Saccharomyces cerevisiae fermentation products(SCFP)on rumen liquid associated microbiota of lactating dairy cows subjected to repeated grain-based SARA challenges.A total of 32 rumen cannulated cows were randomly assigned to 4 treatments from 4 weeks before until 12 weeks after parturition.Treatment groups included a Control diet or diets supplemented with postbiotics(SCFPa,14 g/d Original XPC;SCFPb-1X,19 g/d Nutri Tek;SCFPb-2X,38 g/d Nutri Tek,Diamond V,Cedar Rapids,IA,USA).Grain-based SARA challenges were conducted during week 5(SARA1)and week 8(SARA2)after parturition by replacing 20%DM of the base total mixed ration(TMR)with pellets containing 50%ground barley and 50%ground wheat.Total DNA from rumen liquid samples was subjected to V3–V416S r RNA gene amplicon sequencing.Characteristics of rumen microbiota were compared among treatments and SARA stages.Results Both SARA challenges reduced the diversity and richness of rumen liquid microbiota,altered the overall composition(β-diversity),and its predicted functionality including carbohydrates and amino acids metabolic pathways.The SARA challenges also reduced the number of significant associations among different taxa,number of hub taxa and their composition in the microbial co-occurrence networks.Supplementation with SCFP postbiotics,in particular SCFPb-2X,enhanced the robustness of the rumen microbiota.The SCFP supplemented cows had less fluctuation in relative abundances of community members when exposed to SARA challenges.The SCFP supplementation promoted the populations of lactate utilizing and fibrolytic bacteria,including members of Ruminococcaceae and Lachnospiraceae,and also increased the numbers of hub taxa during non-SARA and SARA stages.Supplementation with SCFPb-2X prevented the fluctuations in the abundances of hub taxa that were positively correlated with the acetate concentration,andα-andβ-diversity metrics in rumen liquid digesta.Conclusions Induction of SARA challenges reduced microbiota richness and diversity and caused fluctuations in major bacterial phyla in rumen liquid microbiota in lactating dairy cows.Supplementation of SCFP postbiotics could attenuate adverse effects of SARA on rumen liquid microbiota.

Saccharomyces cerevisiae prevents postoperative recurrence of Crohn's disease modeled by ileocecal resection in HLA-B27 transgenic rats

BACKGROUND Postoperative recurrence(POR)after ileocecal resection(ICR)affects most Crohn's disease patients within 3-5 years after surgery.Adherent-invasive Escherichia coli(AIEC)typified by the LF82 strain are pathobionts that are frequently detected in POR of Crohn's disease and have a potential role in the early stages of the disease pathogenesis.Saccharomyces cerevisiae CNCM I-3856 is a probiotic yeast reported to inhibit AIEC adhesion to intestinal epithelial cells and to favor their elimination from the gut.AIM To evaluate the efficacy of CNCM I-3856 in preventing POR induced by LF82 in an HLA-B27 transgenic(TgB27)rat model.METHODS Sixty-four rats[strain F344,38 TgB27,26 control non-Tg(nTg)]underwent an ICR at the 12th wk(W12)of life and were sacrificed at the 18th wk(W18)of life.TgB27 rats were challenged daily with oral administration of LF82(109 colony forming units(CFUs)/day(d),n=8),PBS(n=5),CNCM I-3856(109 CFUs/d,n=7)or a combination of LF82 and CNCM I-3856(n=18).nTg rats receiving LF82(n=5),PBS(n=5),CNCM I-3856(n=7)or CNCM I-3856 and LF82(n=9)under the same conditions were used as controls.POR was analyzed using macroscopic(from 0 to 4)and histologic(from 0 to 6)scores.Luminal LF82 quantifications were performed weekly for each animal.Adherent LF82 and inflammatory/regulatory cytokines were quantified in biopsies at W12 and W18.Data are expressed as the median with the interquartile range.RESULTS nTg animals did not develop POR.A total of 7/8(87%)of the TgB27 rats receiving LF82 alone had POR(macroscopic score≥2),which was significantly prevented by CNCM I-3856 administration[6/18(33%)TgB27 rats,P=0.01].Macroscopic lesions were located 2 cm above the anastomosis in the TgB27 rats receiving LF82 alone and consisted of ulcerations with a score of 3.5(2-4).Seven out of 18 TgB27 rats(39%)receiving CNCM I-3856 and LF82 had no macroscopic lesions.Compared to untreated TgB27 animals receiving LF82 alone,coadministration of CNCM I-3856 and LF82 significantly reduced the macroscopic[3.5(2-4)vs 1(0-3),P=0.002]and histological lesions by more than 50%[4.5(3.3-5.8)vs 2(1.3-3),P=0.003].The levels of adherent LF82 were correlated with anastomotic macroscopic scores in TgB27 rats(r=0.49,P=0.006),with a higher risk of POR in animals having high levels of luminal LF82(71.4%vs 25%,P=0.02).Administration of CNCM I-3856 significantly reduced the levels of luminal and adherent LF82,increased the production of interleukin(IL)-10 and decreased the production of IL-23 and IL-17 in TgB27 rats.CONCLUSION In a reliable model of POR induced by LF82 in TgB27 rats,CNCM I-3856 prevents macroscopic POR by decreasing LF82 infection and gut inflammation.

Reducing Yield of Fusel Alcohols by Saccharomyces cerevisiae of Compound Mutagenesis Through UV-MPMS Method

Excessive fusel alcohol contents will cause the beer to produce off-flavors and cause dizziness and headaches.Reducing the contents of fusel alcohols in beer is very important to people's health.The excessive fusel alcohol contents in beer is a common problem in the industry.How to control the contents of fusel alcohols in a reasonable range is of great significance for improving beer quality.After one round of ultraviolet(UV)and one round of multifunctional plasma mutagenesis system(MPMS)mutagenesis,the yeast strains with lower fusel oil yield and more stablility could be screened.According to the relationship between the fusel alcohol Harris metabolic pathway of brewer's yeast and lactic acid metabolism,excellent strains were obtained by triple screening with lactic acid medium,calcium carbonate medium and 2,3,5-triphenyl tetrazolium chloride upper medium.The content of fusel alcohol in the finished beer fermentation test of screened strain Z43 was 52.1±0.142 mg•L^(-1),which was 43%lower than that of the starting strain,and other fermentation properties remained unchanged.After eight passages,it was verified that the strain was stable and heritable.These results showed that strain Z43 presented promising characteristics for use in the production of beer with a potentially low contents of fusel alcohols.

Overexpression of a novel gene , Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.

Effects of different forms of yeast Saccharomyces cerevisiae on growth performance,intestinal development,and systemic immunity in early-weaned piglets

The present study was conducted to determine effects of different forms of yeast （Saccharomyces cerevisiae, strain Y200007） on the growth performance, intestinal development, and systemic immunity in early-weaned piglets. A total of 96 piglets （14-d old, initial average body weight of 4.5 kg） were assigned to 4 dietary treatments： （1） basa diet without yeast （Control）; （2） basal diet supplemented with 3.00 g/kg live yeast （LY）; （3） basal diet supplemented with 2.66 g/kg heat-killed whole yeast （HKY）; and （4） basal diet supplemented with 3.00 g/kg superfine yeast powders （SFY）. Diets and water were provided ad libitum to the piglets during 3-week experiment. Growth performance of piglets was measured weekly. Samples of blood and small intestine were collected at days 7 and 21 of experiment. Dietary supplementation with LY and SFY improved G：F of piglets at days ]-21 of the experiment （P 〈 0.05） compared to Control group. Serum concentrations of growth hormone （GH）, triiodothyronine （T3）, tetraiodothyronine （T4）, and insulin growth factor 1 （iGF-1） in piglets at day 21 of the experiment were higher when fed diets supplemented with LY and SFY than those in Control group （P 〈 0.05）. Compared to Control group, contents of serum urea nitrogen of piglets were reduced by the 3 yeast-supplemented diets （P 〈 0.05）. Diets supplemented with LY increased villus height and villus-to-crypt ratio in duodenum and jejunum of piglets （P 〈 0.05） compared to other two groups at day 7 of the experiment. Feeding diets supplemented with LY and SFY increased （P 〈 0.05） serum concentrations of IgA, IL-2, and IL-6 levels in piglets compared to Control. The CD4＋/CD8＋ ratio and proliferation of T-lymphocytes in piglets fed diets supplemented with LY were increased compared to that of Control group at day 7 of the experiment （P 〈 0.05）. In conclusion, dietary supplementation with both LY and SFY enhanced feed conversion, small intestinal development, and systemic immunity in early-weaned piglets, with better improvement in feed conversion by dietary supplementation with LY, while dietary supplementation with SFY was more effective in increasing systemic immune functions in early-weaned piglets.

Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Feeding a Saccharomyces cerevisiae fermentation product improves udder health and immune response to a Streptococcus uberis mastitis challenge in mid-lactation dairy cows

Background:We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product(NTK)in response to a mastitis challenge.Eighteen mid-lactation multiparous Holstein cows(n=9/group)were fed the control diet(CON)or CON supplemented with 19 g/d NTK for 45 d(phase 1,P1)and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis(phase 2,P2).After 36-h,mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2(9 d post challenge).Cows were then followed until day 75(phase 3,P3).Milk yield(MY)and dry matter intake(DMI)were recorded daily.Milk samples for somatic cell score were collected,and rectal and udder temperature,heart and respiration rate were recorded during the challenge period(P2)together with blood samples for metabolite and immune function analyses.Data were analyzed by phase using the PROC MIXED procedure in SAS.Biopsies were used for transcriptomic analysis via RNA-sequencing,followed by pathway analysis.Results:DMI and MY were not affected by diet in P1,but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON.NTK reduced rectal temperature,somatic cell score,and temperature of the infected quarter during the challenge.Transcriptome data supported these findings,as NTK supplementation upregulated mammary genes related to immune cell antibacterial function(e.g.,CATHL4,NOS2),epithelial tissue protection(e.g.IL17C),and anti-inflammatory activity(e.g.,ATF3,BAG3,IER3,G-CSF,GRO1,ZFAND2A).Pathway analysis indicated upregulation of tumor necrosis factorα,heat shock protein response,and p21 related pathways in the response to mastitis in NTK cows.Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows.Conclusions:Overall,results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.

Saccharomyces cerevisiae CNCM I-3856 in irritable bowel syndrome: An individual subject meta-analysis

AIMTo confirm previous conclusions on Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 for irritable bowel syndrome (IBS) management.METHODSAn individual patient data meta-analysis was performed on two randomized clinical trials studying the effect of S. cerevisiae CNCM I-3856 supplementation on gastrointestinal (GI) symptoms in IBS subjects. A total of 579 IBS subjects were included. Outcomes were the daily Likert scale scores of abdominal pain/discomfort and bloating [area under the curve (AUC) and weekly means], responder status, and bowel movements (stool frequency and consistency). Statistical analyses were conducted in Intent to Treat (ITT) population, IBS-C subjects and IBS-C subjects with an abdominal pain/discomfort score higher than or equal to 2 at baseline (“IBS-C ≥ 2 subpopulation”).RESULTSS. cerevisiae CNCM I-3856 significantly improved abdominal pain/discomfort and bloating during the second month of supplementation [AUC (W5-W8)] with improvement up to the minimal clinically relevant threshold of 10%: a 12.3% reduction of abdominal pain/discomfort in the ITT population compared to the Placebo group (P = 0.0134) has been observed. In the IBS-C ≥ 2 subpopulation, there were a 13.1% reduction of abdominal pain/discomfort and a 14.9% reduction of bloating compared to the Placebo group (P = 0.0194 and P = 0.0145, respectively). GI symptoms significantly decreased during supplementation but no statistical differences were reported between groups at the end of the supplementation period. Responder status was defined as a subject who experienced a decrease of 1 arbitrary unit (a.u.) or 50% of the abdominal discomfort score from baseline for at least 2 wk out of the last 4 wk of the study. A significant difference between groups was reported in the ITT population, when considering the first definition: subjects in the Active group had 1.510 higher odds to be a responder (reduction of 1 a.u. of abdominal pain/discomfort) compared with subjects in the Placebo group (P = 0.0240). At the end of supplementation period, stool consistency in the Active group of the ITT population was significantly improved and classified as “normal” compared to Placebo (respectively 3.13 ± 1.197 a.u. vs 2.58 ± 1.020 a.u., P = 0.0003). Similar results were seen in the IBS-C ≥ 2 subpopulation (Active group: 3.14 ± 1.219 a.u. vs Placebo group: 2.59 ± 1.017 a.u., P = 0.0009).CONCLUSIONThis meta-analysis supports previous data linking S. cerevisiae I-3856 and improvement of GI symptoms, in IBS overall population and in the IBS-C and IBS-C ≥ 2 subpopulations.

Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae

Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.

Computational Analysis of Signal Peptide-Dependent Secreted Proteins in Saccharomyces cerevisiae

Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.

Effect of yeast Saccharomyces cerevisiae supplementation on serum antioxidant capacity, mucosal sIgA secretions and gut microbial populations in weaned piglets

This study was conducted to determine the effect of different forms of yeasts Saccharomyces cerevisiae supplementation on serum antioxidant capacity, mucosal secretory immunoglobulin A（s Ig A） secretions and gut microbial populations in weaned piglets. A total of 96 piglets weaned at 14 d of age were randomly allotted to 4 dietary treatments：（1） basal diet without yeast（Control）;（2） basal diet supplemented with 3.00 g kg–1 live yeast（LY）;（3） basal diet supplemented with 2.66 g kg–1 heat-killed whole yeast（HKY）; and（4） basal diet supplemented with 3.00 g kg–1 superfine yeast powders（SFY）. Each treatment had 4 replicates（pens）, with 6 piglets per replicate. The experiment lasted for 3 wk. At d 7 and 21 of the experiment, the samples of serum, mucosa and mesenteric lymph node（MLN） from jejunum, and digesta from the ileum and cecum were collected for determinations. Compared with the Control, dietary SFY supplementation increased serum superoxide dismutase（SOD） activity and lysozyme levels at d 7, and jejunum mucosal s Ig A secretions at d 21 of the experiment（P〈0.05）. Dietary LY supplementation increased serum SOD activity and jejunum mucosal s Ig A secretions, but decreased serum malondialdehyde（MDA） concentration at d 7 and 21（P〈0.05）. Piglets fed diets supplemented with LY and SFY had lower p H values and decreased numbers of Escherichia coli in the ileum and cecum contents at d 21 compared with the Control（P〈0.05）. Moreover, the ratio of Lactobacilli to E. coli in the ileum and cecum contents was increased by dietary LY and SFY supplementations（P〈0.05）. Collectively, different forms of yeasts, especially LY and SFY, may modulate body antioxidant capacity and enhance the intestinal immunity by regulation of secretions of mucosal s Ig A and reduction of pathogenic bacteria colonization, thus improving intestinal health of weaned piglets.

Improve Ethanol Yield Through Minimizing Glycerol Yield in Ethanol Fermentation of Saccharomyces cerevisiae

In ethanol fermentation of Saccharomyces cerevisiae （S. cerevisiae）, glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synth.etase, respectively,were overexpressed using two-step gene replacment in fpsl△gpd2△ mutant.The fermentation properties of ZAL69（fpsl△：：LEU2 gpd2△：：URA3） and ZAL808 （fps1△：：LEU2 gpd2△：：URA3 PPGK1-GLT1 PPGK1-GLN1） under microaerobic conditions were investigated and compared with those of wild type（DC124）. Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by. 13.17% and 6.66 %, respectively, whereas glycerol yield decreased by 37.4 % and 41.7 %. Meanwhile, acetic acia yield and pyruvic acid yield aecreasea aramatlcally comparea to wild type. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield, but simultaneous overexpression of GLT1 and GLN1 infps1△gpd2△ mutant did not have a higher ethanol yield thanfps1△gpd2△ mutant.

Seroreactivity against Saccharomyces cerevisiae in patients with Crohn's disease and celiac disease

AIM:To explore whether there was anti-Saccharomyces cerevisiae antibodies (ASCA) positivity in our patients with biopsy-confirmed celiac disease. METHODS:A cohort of patients with inflammatory bowel diseases (42 patients with Crohn's disease and 10 patients with ulcerative colitis) and gluten sensitive enteropathy (16 patients) from Debrecen,Hungary were enrolled in the study. The diagnosis was made using the formally accepted criteria. Perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA), antiendomysium antibodies (EMA),antigliadin antibodies (AGA) and anti human tissue transglutaminase antibodies (tTGA) were investigated. RESULTS:The results showed that ASCA positivity occurred not only in Crohn's disease but also in Celiac disease and in these cases both the IgG and IgA type antibodies were proved. CONCLUSION:It is conceivable that ASCA positivity correlates with the (auto-) immune inflammation of small intestines and it is a specific marker of Crohn's disease.

Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage

Background： A possible option to meet the increased demand of forage for dairy industry is to use the agricultural byproducts, such as corn stover. However, nutritional value of crop residues is low and we have been seeking technologies to improve the value. A feeding trial was performed to evaluate the effects of four levels of Saccharomyces cerevisiae fermentation product（SCFP; Original XP; Diamond V） on lactation performance and rumen fermentation in mid-lactation Holstein dairy cows fed a diet containing low-quality forage. Eighty dairy cows were randomly assigned into one of four treatments： basal diet supplemented with 0, 60, 120, or 180 g/d of SCFP per head mixed with 180, 120, 60, or 0 g of corn meal, respectively. The experiment lasted for 10 wks, with the first 2 weeks for adaptation.Results： Dry matter intake was found to be similar（P 〉 0.05） among the treatments. There was an increasing trend in milk production（linear, P ≤ 0.10） with the increasing level of SCFP supplementation, with no effects on contents of milk components（P 〉 0.05）. Supplementation of SCFP linearly increased（P 〈 0.05） the N conversion, without affecting rumen pH and ammonia-N（P 〉 0.05）. Increasing level of SCFP linearly increased（P 〈 0.05） concentrations of ruminal total volatile fatty acids, acetate, propionate, and butyrate, with no difference in molar proportion of individual acids（P 〉 0.05）. The population of fungi and certain cel ulolytic bacteria（Ruminococcus albus, R. flavefaciens and Fibrobacter succinogenes）increased linearly（P 〈 0.05） but those of lactate-utilizing（Selenomonas ruminantium and Megasphaera elsdeni） and lactate-producing bacteria（Streptococcus bovis） decreased linearly（P ≤ 0.01） with increasing level of SCFP. The urinary purine derivatives increased linearly（P 〈 0.05） in response to SCFP supplementation, indicating that SCFP supplementation may benefit for microbial protein synthesis in the rumen.Conclusions： The SCFP supplementation was effective in maintaining milk persistency of mid-lactation cows receiving diets containing low-quality forage. The beneficial effect of SCFP could be attributed to improved rumen function; 1）microbial population shift toward greater rumen fermentation efficiency indicated by higher rumen fungi and cel ulolytic bacteria and lower lactate producing bacteria, and 2） rumen microbial fermentation toward greater supply of energy and protein indicated by greater ruminal VFA concentration and increased N conversion. Effects of SCFP were dose-depended and greater effects being observed with higher levels of supplementation and the effect was more noticeable during the high THI environment.

Correlation between Saccharomyces cerevisiae DNA in intestinal mucosal samples and anti-Saccharomyces cerevisiae antibodies in serum of patients with IBD

AIM： To investigate the correlation between ASCA and presence of mucosal S. cerevisiae DNA in a population of CD, ulcerative colitis （UC） patients and controls. METHODS： S. cerevisiae-specific primers and a fluorescent probe were designed for a 5＇ exonuclease real time PCR （TaqManTM） assay, which is a homogenous system using a fluorescent-labelled probe for the detection of PCR product in real time. We analyzed the relation of the PCR results with the ASCA findings in a group of 76 inflammatory bowel disease （IBD） patients （31 CD, 45 UC） and 22 healthy controls （HC）. RESULTS： ASCA （IgA or IgG） were positive in 19 （61%） patients with CD, 12 （27%） with UC and none of the HC. PCR amplification was inhibited and excluded from the final results in 10 （22%） UC patients, 7 （22%） CD patients, and 6 （30%） HC. In only 15 of the mucosal samples, S. cerevisiae DNA was detected by real time PCR, including 7 （29%） in CD, 7 （19%） in UC, 1 （6%） in HC. In 4 CD and in 4 UC patients, ASCA and mucosal S. cerevisiae were positive. Hucosal S. cerevisiae was present in combination with negative ASCA IgA and IgG in 3 UC, and 3 CD patients. CONCLUSION： We conclude that since the presence of S. cerevisiae in colonic mucosal biopsy specimens is very rare, ASCA is unlikely to be explained by continuous exposure to S. cerevisiae in the mucosa. Therefore, ASCA formation must occur earlier in life and levels remain relatively stable thereafter in immunological susceptible persons.

Construction of Saccharomyces cerevisiae Strain Stably Expressing a Fusion Protein Containing Ten Tandem Recombinant Human Glucagon-like Peptide-1 Analogues

The recombinant Saccharomyces cerevisiae strain stably expressing recombinant human glucagon-like peptide-l（rhGLP-1） analogue, as a potential oral drug delivery system for diabetes type II treatment, was successfully constructed by the homologous recombination between chromosomal DNA and yeast and integrating vector pNK-GLP containing yeast ribosomal DNA fragments. The amount of rhGLP-I analogue fusion protein in transformant SG2 reached ca. 0.84 mg per gram of packed cells when SG2 was grown for 24 h in the YPD medium with a inoculum and medium ratio of 1：1. Oral administration of 5 g lyophilized SG2/kg to hyperglycemic rats decreased serum glucose from （24.8±1.40） to （21.2±1.36） mmol/L.

The effect of Saccharomyces cerevisiae on digestion and mortality in the volcano rabbit(Romerolagus diazi)

An experiment was conducted to evaluate whether supplementation with a probiotic could enhance digestion and reduce mortality in the volcano rabbit in captivity. Two enclosures at Chapultepec Zoo, Mexico（114 individuals） were used in a crossover design（two periods of 60 days） with the following treatments： control group and supplementation with Saccharomyces cerevisiae（2×108 CFU/exhibit/day）. Supplementation with the probiotic negatively affected（P〈0.01） the digestibility of dry matter, organic matter, neutral detergent fiber（NDF） and energy. Mortality increased（P〈0.04） following supplementation with the probiotic（4.26% vs. 8.89%）, primarily in the juvenile rabbits. The results indicate that yeast supplementation in the volcano rabbit negatively affects digestion and mortality in captivity.

Asymmetric Synthesis of (-)-1-Trimethylsilyl-ethanol with Immobilized Saccharomyces Cerevisiae Cells in Water/Organic Solvent Diphasic System

Asymmetric synthesis of (-)-1-trimethylsilyl-ethanol with immobilized Saccharomyces cerevisiae cells in water/organic solvent biphasic system was studied. The effects of shake speed, hydrophobicity of organic solvent, volume ratio of water phase to organic phase, pH value of aqueous phase and reaction temperature on the initial reaction rate, maximum yield and enantiomeric excess (ee) of the product were systematically explored. All the above-mentioned factors had significant influence on the reaction. n-Hexane was found to be the best organic solvent for the reaction. The optimum shake speed, volume ratio of water phase to organic phase, pH value and reaction temperature were 150 r.min-1, 1/2, 8 and 30 ℃ respectively, under which the maximum yield and enantiomeric excess of the product were as high as 96.8% and 95.7%, which are 15% and 16% higher than those of the corresponding reaction performed in aqueous phase. To our best knowledge, this is the most satisfactory result obtained.