Development Of High GSH Yeast Culture And Its Effects On Growth,Rumen Fermentation Function And Mechanism Of Heat Stress Beef Cattle

Glutathione（GSH）is an active tripeptide formed by the condensation of glycine,cysteine and glutamate,which has antioxidant and detoxifying effects.Yeast cultures（YC）,on the other hand,are a product containing a variety of active nutrients with important effects such as alleviating stress,improving production performance and improving rumen fermentation function in ruminants.In this study,we aimed to obtain a high GSH-producing yeast strain,optimize the fermentation conditions to produce yeast cultures,and study the effects of adding this high GSH-producing yeast culture to the diet on the performance,nutrient digestibility,rumen fermentation characteristics and serum indexes of beef cattle under heat stress,and investigate its mechanism of action through rumen microbial macrogenomic and metabolomic techniques,so as to provide theoretical basis and support for the production application of high GSH yeast cultures on beef cattle.The study was divided into four parts.The study was divided into four parts and the results of the experiments are as follows:Experiment 1 Screening of GSH-producing yeast strains and optimization of fermentation conditionsIn this experiment,a brewer’s yeast strain was screened for GSH production,and a high GSH yeast culture was obtained after optimization of culture conditions.Firstly,a high GSH-producing Saccharomyces cerevisiae strain KF-Y-A was screened from five yeast strains based on GSH content and cell dry weight,and then the liquid fermentation medium was optimized by single-factor optimization of nitrogen source,carbon source and cysteine addition level,and the liquid fermentation conditions（fermentation time,fermentation temperature and inoculation level）were optimized by response surface method using GSH content as the response value to obtain the optimal liquid medium（4 g/L ammonium sulfate,40 g/L molasses,0.02%cysteine）and culture conditions（fermentation time of 42 h,fermentation temperature of 30°C,and inoculum level of 8%）,under which the GSH content reached 196.8 mg/L.Finally,the changes of nutrients during solid-state fermentation were studied,and with the progress of fermentation,the crude protein and acid soluble protein contents in the substrate were As the fermentation progressed,the crude protein and acid soluble protein contents in the substrate increased and the reducing sugar level gradually decreased,reaching a maximum value of 4.27 billion yeast/g at 40 h and a maximum GSH content of 123.5 mg/kg at 48 h.This indicated that the solid-state fermentation reached its optimum state at the 40-48 h stage.Experiment 2 Effect of different levels of high GSH yeast cultures on rumen in vitro fermentation parametersIn this experiment,the effect of cultures produced by the high GSH yeast strains obtained in Experiment 1 on rumen fermentation characteristics was investigated by in vitro fermentation method in order to preliminarily derive the appropriate amount of high GSH yeast cultures to be used as additives in beef cattle.Ruminal fluid was harvested from Jinjiang cattle and the fermentation substrate had a 4:6 concentrate to crude ratio.Five groups were set up in the experiment,namely,control,0.125%YC,0.25%YC,0.5%YC,1.0%YC and 1.5%YC groups,with five replicates in each group.Ruminal fluid p H,ammonia nitrogen（NH₃-N）,microbial protein（MCP）and volatile fatty acids（VFA）were measured.The results showed that the addition of yeast culture significantly increased the contents of MCP,acetic acid,propionic acid,butyric acid and total volatile fatty acids in rumen fluid compared with the control group（P<0.05）,increased the B/C ratio and improved the rumen fermentation function.Under the conditions of this test,the 0.5%,1.0%and 1.5%addition levels performed well.Considering the actual cost of use,two levels of 0.5%and 1.0%were selected for the follow-up test.Trial 3 Effect of high GSH yeast culture on productive performance,serum index and rumen fermentation function in heat-stressed beef cattleSummer heat stress in beef cattle has always been an important issue,which seriously affects beef cattle health and breeding efficiency.Therefore,how to improve the production performance of beef cattle under summer heat stress has been a hot topic of research in the industry.This experiment aimed to investigate the effects of high GSH yeast cultures on the production performance,nutrient digestibility,serum index and rumen fermentation function of beef cattle under heat stress.Twenty-four6-month-old Jinjiang bulls weighing 110±5.7 kg were randomly divided into three groups,i.e.,control group,low-dose supplementation group（0.5%supplementation）and high-dose supplementation group（1.0%supplementation）,with 8 animals in each group and 4 replicates of 2 animals in each group.The experiment was conducted in summer and the temperature and humidity values of the barn were monitored daily during the pre-testing period of 7 days and the regular test period of 62 days.The results showed that:（1）the addition of yeast culture increased the daily weight gain of beef cattle under heat stress and significantly reduced the feed to meat ratio compared with the control group（P<0.05）;（2）the digestibility of NDF and CP increased with the increase of yeast culture addition level at the end of 62 d of the trial.Among them,the 1.0%addition group significantly increased the feed protein digestibility（P<0.05）.（3）At the end,total antioxidant capacity（T-AOC）was elevated with increasing levels of yeast culture addition（P<0.05）,and serum thyroxine T4 content was significantly higher than that of the control group（P<0.05）.（4）Addition of yeast cultures significantly increased the content of acetic acid,propionic acid and total volatile fatty acids in rumen fluid（P<0.05）,but had no significant effect on rumen p H,NH₃-N and MCP content.The results suggest that high GSH yeast cultures improved rumen fermentation function and antioxidant function of the organism in heat-stressed beef cattle.Experiment 4:Effect of yeast culture on rumen microbiota and metabolomics of heat-stressed beef cattleThe results of experiment 3 showed that the addition of yeast culture to the diet could improve the growth performance and rumen fermentation function of beef cattle.To elucidate the mechanism of the effect of high GSH yeast cultures on rumen fermentation function.In this experiment,we analyzed the effects of high GSH yeast culture on rumen microflora structure and metabolites of heat-stressed beef cattle using rumen fluid samples collected from the control and 1%high GSH yeast culture addition groups in experiment 3,using macrogenomics and metabolomics techniques.The results showed that（1）effects on microbiota and diversity:addition of high GSH yeast cultures significantly increased the abundance of s＿bacterium＿F082,s＿bacterium＿P201 and s＿bacterium＿F083 at the species level.（2）Effect on functional genes:The addition of high GSH yeast cultures to the diet at the KEGG level significantly increased the functional gene enrichment levels of fructose and mannose metabolism in carbohydrate metabolism,and the trend increased the functional gene enrichment levels of butyric acid and nitrogen metabolism,with no effect on other metabolic pathways.（3）Effect on the metabolism of rumen microorganisms:The synthesis of nicotinic acid metabolism of rumen microorganisms was significantly enhanced by the addition of high GSH yeast cultures,and the metabolism ofγ-tocotrienols,tripeptides and other active substances was elevated.It showed that the high GSH yeast culture may exert its function mainly through vitamin and amino acid metabolism.In conclusion,a high GSH Saccharomyces cerevisiae strain KF-Y-A was obtained from this test,which reached 196.8 mg/L of GSH under the test culture conditions.its optimal liquid culture conditions were:42 h,30°C,8%inoculation.This high GSH yeast culture product significantly improved the daily weight gain of heat-stressed beef cattle,reduced growth performance indexes such as feed-to-weight ratio,improved the antioxidant capacity of the organism and enhanced rumen fermentation function.The high GSH yeast culture had no significant effect on the composition of rumen microbial abundance in beef cattle;numerous differential metabolites and metabolic pathways were detected by rumen metabolomics and were mainly enriched in vitamins,amino acids and other metabolisms.