| This paper was aimed to study the degradation of cottonseed hull lignocellulose by pleurotus ostreatus,the effect of cottonseed hull fermented by pleurotus ostreatus on the digestion and metabolism of sheep nutrients and fecal microbial diversity,and the degradation of free gossypol of cottonseed hull by pleurotus ostreatus and its mechanism,providing reference for the microbial treatment technology of cottonseed hull detoxification and the development and application of roughage.The main research results are as follows.Experiment 1:Screening of high-efficient lignocellulose-degrading strains and optimization of liquid medium compositionThe Pleurotus ostreatus strain with higher lignocellulose degradation rate was obtained by preliminary screening through plate color producing reaction,decolorization reaction and re-screening through liquid enzyme production trial.The results of optimizing medium composition showed that the strain had the best biomass and enzyme-producing on the 4th day of culture with the conditions of 10%(v/v)inoculation,10 g/L glucose as carbon source,2.0 g/L peptone as nitrogen source,1.0 g/L VB1 as growth factor,1.5 g/L KH2PO4 and MgSO4 as inorganic salt.Experiment 2:Effect of Pleurotus ostreatus fermentation on lignocellulose and free gossypol content in cottonseed hullsPleurotus ostreatus strain was inoculated into cottonseed hull for solid fermentation.The results showed that when the inoculation amount was set to 10%(v/m),the mycelia grew full of bags at 21 days after inoculation.Cottonseed hull fermented by Pleurotus ostreatus strain for 21 days significantly decreased the contents of cellulose(P<0.05),hemicellulose(P<0.01),lignin(P<0.01),and free gossypol(P<0.01),and significantly increased the crude protein content(P<0.01).Experiment 3:Transcriptionomic and metabonomic analysis of degradation of free gossypol by Pleurotus ostreatusFree gossypol(400 mg/L)was added to culture medium for the liquid culture,The transcriptome and metabolites of free Gossypol-degrading fungus Pleurotus ostreatus after 72 hours of culture were analyzed.The results showed that 13781 Uni genes were annotated in GO analysis,and cluster analysis were conducted from three aspects,such as the biological processes,molecular functions,and cell components.The percentage of them was 80.85%,87.18%,and 49.54%,respectively.KEGG analysis showed that 5499 Unigenes were annotated,12 pathways involved in material metabolism,9 in organic system,4 in genetic information processing,4 in cellular process,and 2 in environmental information processing.Metabolism accounted for the largest proportion of pathways with significant differences in gene expression.Differential expression analysis showed that 1862 genes were up-regulated and 3314 genes were down-regulated in the experimental group compared with the control group.Laccase produced by Pleurotus ostreatus may be involved in the degradation of free gossypol under this experimental conditions.Metabolite analysis showed that free gossypol might inhibit intracellular glucose metabolism in Pleurotus ostreatus.Experiment 4:Effects of enlarged fermentation of Pleurotus ostreatus on lignocellulose in cottonseed hulls,digestion and metabolism of sheep and fecal microorganismsTen Suffolk rams with average body weight 60.09±4.60 kg,age 2±0.5 years old,and healthy were selected and divided into two groups(control group and experimental group),five in each group.On the basis of feeding concentrate supplements according to 1.2%body weight,the control group was fed with cottonseed hulls treated by 1%CaO according to 2%(air-dried weight)body weight per day,while the experimental group was fed with cottonseed hulls fermented by Pleurotus ostreatus after treated by 1%CaO according to 2%(air-dried weight)body weight per day;the pre-experimental period is 14 days,and the experimental period is 7 days.The results showed that the apparent digestibility of dry matter(DM),organic matter(OM),crude protein(CP),calcium(Ca),and total energy(TE)in the experimental group was significantly lower than that in the control group(P<0.01),and decreased by 17.13%,25.10%,16.22%,30.63%,10.32%,respectively.The apparent digestibility of neutral and acid detergent fibers was significantly lower than that of control group(P<0.05),but there was no difference between hemicellulose and phosphorus compared with control group(P>0.05).The calcium intake in the experimental group was higher than in the control group(P<0.01);Nitrogen and calcium contents in the feces of the experimental group were significantly higher than those of the control group(P<0.01),and increased by 37.21%,8.05%,respectively.The deposition rate of calcium in the experimental group was significantly higher than that in the control group(P<0.05),increased by 36.96%;there was no significant difference in nitrogen and phosphorus deposition rate between the experimental group and the control group(P>0.05).The dominant bacteria in the feces of sheep fed with 2%BW cottonseed hulls were Firmicutes,Proteobacteria,Bacteroidetes,Spirochaetes,and Verrucomicrobia.The dominant bacteria in the feces of sheep fed with 2%·BW fermented cottonseed hulls were Firmicutes,Proteobacteria,Bacteroidetes,Spirochaetes,Verrucomicrobia,and Actinobacteria.The dominant fungi in the feces of sheep fed with 2%·BW cottonseed hulls were Ascomycetes,Basidiomycetes,Zygomycota,conjugates,and Neocallimastigomycota.The dominant fungi in the feces of sheep fed with 2%·BW fermented cottonseed hulls were Ascomycetes,Basidiomycetes,and Zygomycota.In summary,Pleurotus ostreatus can produce the enzymes related to lignocellulose decomposition,can significantly reduce the content of cellulose,hemicellulose,lignin and free gossypol in cottonseed hulls,and significantly increase the crude protein content.Feeding fermented cottonseed hulls can increase the feed intake and calcium deposition rate of sheep;After induced by free gossypol,13 781 Unigenes were annotated by GO analysis,1862 genes were up-regulated and 3314 genes were down-regulated.Laccase produced by Pleurotus ostreatus may be involved in the degradation of free gossypol under this trial conditions.Free gossypol may be inhibit intracellular glucose metabolism in Pleurotus ostreatus. |
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