| Objective: In this study,a 4 factor 4 level orthogonal design was used,and the content of free gossypol(FG)in cottonseed meal after gossypol degradation enzyme digestion was used as an indicator,optimize the parameters of gossypol-degrading enzymatic hydrolysis of cottonseed meal,and compared with that by solid-state fermentation of Candida tropicalis ZD-3,at the same time,the effects of gossypol degrading enzyme and probiotics on the biological detoxification of cottonseed meal were compared,to evaluate the effect of gossypol degrading enzymes on enzymatic detoxification of cottonseed meal,so as to provide a theoretical basis for the efficient utilization of cottonseed meal biological enzyme detoxification.Method: This paper consists of three experiments.Experiment 1: 4 factor 4 level orthogonal design is used,the enzymolysis parameters include enzymolysis temperature(25 ?,30 ?,35 ?,40 ?),enzymolysis time(1.5 h,2 h,2.5 h,3 h),and the amount of enzyme added.(1%,3%,5%,7%),substrate moisture(35%,40%,45%,50%),and 16 experimental treatments were designed,by measuring the free gossypol content of cottonseed meal of different treatment combinations,orthogonal test analysis of variance and multiple comparisons of LSD were used to obtain the parameters of the optimal hydrolysis conditions for the enzymolysis temperature,the enzymolysis time,the addition amount,and the substrate moisture.Experiment 2: according to the parameters of the optimal enzymolysis conditions in Experiment 1,high-quality cottonseed meal was prepared by gossypol degrading enzyme and Candida tropicalis ZD-3,which was divided into cooked meal group and raw meal group,each group was divided into Ctrl group(control group),GE group(gossypol degrading enzyme group)and CT group(Candida tropicalis ZD-3 group),the content of free gossypol,neutral washing fiber(DNF),crude ash(Ash),crude protein(CP),acid soluble protein(TCA-N)in cottonseed meal and cottonseed meal protein molecular weight distribution were measured.Experiment 3: cottonseed meal was prepared with gossypol degrading enzyme,gossypol degrading enzyme and Candida tropicalis ZD-3,gossypol degrading enzyme and mixed bacteria(Candida tropicalis ZD-3,Bacillus subtilis and Lactobacillus acidophilus).Each group was divided into cooked meal group and raw meal group.Each group was divided into Ctrl group(control group),GE group(gossypol degrading enzyme group),CT group(Candida tropicalis ZD-3 group),CBL groups(mixed bacteria group),GC1 group(gossypol degrading enzyme + Candidatropicalis ZD-3 group,detoxification conditions are the same as GE groups),GC2groups(gossypol degradation enzyme + Candida tropicalis ZD-3 group,detoxification conditions are the same as CT groups),GCBL1 groups(gossypol degradation enzyme + mixed bacteria group,detoxification conditions are the same as GE groups),GCBL2 groups(gossypol degradation enzyme + mixed bacteria group,detoxification conditions are the same as CBL groups),and the detection indexes are the same as experiment 2.To compare the effect of gossypol degrading enzyme and probiotics on the detoxification of cottonseed meal,and to evaluate the detoxification effect of gossypol degrading enzyme on cottonseed meal.Results: Experiment 1: Through orthogonal design,the optimal conditions for detoxification of cooked cottonseed meal by gossypol degrading enzymes were temperature 40 ?,time 2 h,addition amount 5%,and substrate moisture 35%.The verification test results showed that,The free gossypol content was reduced from341.02 mg / kg to 25.58 mg / kg,and the detoxification rate of cottonseed meal reached 92.49%;the optimal conditions for the detoxification of raw cottonseed meal by gossypol degrading enzyme were temperature 35 ?,time 1.5 h.addition amount3% and substrate moisture 35%.The verification test results showed that the free gossypol content was reduced from 341.02 mg / kg to 37.01 mg / kg,and the detoxification rate of cottonseed meal reached 89.15%.Experiment 2: Compared with the control group,the content of free gossypol in the cooked meal group and the raw meal group was extremely significant reduce(p<0.01),and the difference between the treatment groups is very significant(P < 0.01);Compared with the control group,the NDF and CP of each treatment group were significant(p <0.05),and no significant difference between treatment groups(p >0.05);Compared with the control group,the Ash content of the treatment group was reduced,and the TCA-N content was increased,but the differences were not significant(p> 0.05);The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis showed that the degradation of macromolecular proteins in cottonseed meal into small molecular peptides was not obvious after treatment.Experiment 3: Compared with the control group,the content of free gossypol in the cooked meal group and the raw meal group was extremely significant reduce(p<0.01),the difference between the compound detoxification group and the gossypol degrading enzyme group was very significant(p <0.01);Compared with the control group,the NDF and CP of each treatment group were significant(p <0.05),and no significant difference between treatment groups(p > 0.05);Compared with the controlgroup,the Ash content of the treatment group was reduced,and the TCA-N content was increased,but the differences were not significant(p> 0.05);SDS-PAGE analysis showed that only after the fermentation of mixed bacteria group,the degradation of large molecular protein into small molecular peptide was obvious.Conclusion: The optimal conditions for detoxification of cooked cottonseed meal by gossypol degrading enzymes were temperature 40 ?,time 2 h,addition amount 5%,and substrate moisture 35%;the optimal conditions for the detoxification of raw cottonseed meal by gossypol degrading enzyme were temperature 35 ?,time1.5 h.addition amount 3% and substrate moisture 35%.Gossypol degradation enzyme can be a good substitute for Candida tropicalis ZD-3 for detoxification of cottonseed meal,However,the effect of mixed treatment of gossypol degrading enzyme and probiotics on detoxification of cottonseed meal was lower than that of using gossypol degradation enzyme alone. |
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