Screening Of Dominant Cellulose-degradation Strains And Condition Optimization Of Mixed Strains Fermentation Of Pleurotus Eryngii Spent Mushroom Substrate

In this study,the dominant cellulose-degradation strains were screened from the Pleurotus eryngii spent mushroom substrate（PESMS）and mixed fermentation agent with Pediococcus acidilactic and Saccharomyces cerevisiae was used to ferment PESMS.Response surface methodology（RSM）was used to optimize the fermentation conditions of PESMS and improve the degradation of cellulose.The feeding safety and nutritional value of PESMS before and after mixed fermentation were explored.Exp.1 Screening of dominant cellulose-degradation strainsThe dominant cellulose-degradation strains were screened by CMC screening medium,combined with carboxymethyl cellulase activity（CMCA）and filter paper enzyme activity（FPA）.Bacterial 16S rDNA fungal 18S rDNA homologous sequence alignment was used to determine the strain species,and its enzyme production conditions were determined.（1）Three dominant cellulose-degradation strains were screened,Bacillus subtilis,Aspergillus niger and Trichoderma reesei.Among them,P12 and L6 can be used as feed additives in the feed according to the Catalogue of Feed Additives Varieties（2013）published by the Ministry of Agriculture of the People’s Republic of China.（2）The optimum enzyme production time,temperature and pH of P12 were 48 h,37℃and 6.5-7 respectively,while that of L6 were 48 h,30℃and 6.5-7 respectively.Exp.2 Response surface methodology to optimize the mixed strains fermentation conditions of PESMSIn this experiment,the dominant cellulose-degradation strains（P12 and L6）were mixed with Pediococcus acidilactic and Saccharomyces cerevisiae as compound microorganism agents.The fermentation conditions of PESMS were optimized by response surface methodology,and design the Plackett-Burman experiment,the steepest climbing experiment and the Box-Behnken experiment optimize the fermentation conditions,to improve the rate of the degradation of cellulose.The optimum fermentation conditions were 1.94:1 for the volume ratio of Bacillus subtilis to Aspergillus Niger,4.7%for the inoculation of the dominant strains of cellulose decomposition,10.4%for the addition of bran,46.1%for the water content,and the fermentation time was 8 days.Under the optimum fermentation conditions,the measured NDF degradation rate was 21.08%,which was not significantly different from the model predicted results（21.86%）（P>0.05）,and the degradation rate of NDF was increased by15.33%.Exp.3 Analysis of feeding quality and safety of mixed microorganism fermentation of PESMS.In this experiment,the mycotoxin content,heavy metal content,conventional nutrients,volatile fatty acids and amino acid components in the natural fermentation group and the compound fermentation group of SMS were determined,and the feeding quality and safety of the mixed microorganism fermentation of PESMS were investigated.The results showed that:（1）Fermentation treatment significantly reduced the content of neutral detergent fiber and acid detergent fiber in PESMS（P<0.05）,and the experimental group was significantly lower than the control group（P<0.05）;the fermentation treatment significantly increased the content of crude protein and crude fat（P<0.05）,and the experimental group was significantly higher than the control group（P>0.05）;the dry matter recovery rate of the experimental group was significantly higher than that of the control group（P<0.05）.（2）The content of lactic acid in the experimental group was significantly higher than that in the control group（P<0.05）,and the contents of pH,acetic acid,propionic acid,butyric acid,total volatile fatty acid and ammonia nitrogen in the experimental group were significantly lower than those in the control group（P<0.05）.（3）Fermentation treatment decreased the contents of vomiting toxin and zearalenone in PESMS（P>0.05）,and there was no significant difference between the experimental group and the control group（P>0.05）.However,the content of aflatoxin B₁ in the experimental group was significantly lower than that in the control group（P<0.05）.After fermentation,the content of heavy metals in the control group and the experimental group was significantly higher than that of the raw material group（P<0.05）.Before and after fermentation,the contents of mycotoxin and heavy metals in feed of raw material group,experimental group and control group all met the National Feed Hygiene Standard（GB 13078-2017）.（4）Sixteen amino acids were detected in the three groups of PESMS before and after fermentation,but no tyrosine was detected.The contents of essential amino acids and total amino acids in experimental group were significantly higher than those in raw materials and control group（P<0.05）.（5）The mixed fermentation group had strong bread flavor and sour flavor,soft texture and light brown color,which was better than the natural fermentation group.