Study on the Breeding of High Efficient Detoxification Strains and Suggestion Parameters of Solid Fermentation on Cottonseed Meal
|School||Nanjing Agricultural College|
|Course||Animal Nutrition and Feed Science|
|Keywords||Strains Mutation FG degradation rate ITS sequence Cottonseed meal Fermentation parameters of detoxification|
The experiment was conducted to investigate the breeding of high efficient detoxification strains of cottonseed meal. Meanwhile, DNA of mutant in the genus level was identitied and compared. The optimal fermentation parameters were determined.Some referenced information of this work was provided for the new good detoxification strains development and efficient and safe utilization of cottonseed meal. The study included three parts and the results were presented as follows:In the first part, the experiment was designed to study the breeding of high efficient detoxification strains of cottonseed meal.The aim was to improve and screen strains with high free gossypol (FG) degradation rate. With Candida oleophila, Kloeckera lindneri, Rhodothece glutinis as the starting strains, the mutant with high free gossypol (FG) degradation rate were bred after the starting strains were treated by ultraviolet(UV) diethylsulfate(DES) and UV+DES mutation. And the optimal conditions for mutation were confirmed. The results showed that six available mutant strains were screened through primary screening and secondary screening (GJ-Z-25, GJ-D-25, GJ-Z-25-D-25, GJ-Z-20-D-20, HJ-D-20, HJ-Z-20-D-20). The FG degradation rate of mutant strains were 90.69%,91.40%,89.64%,93.78%,90.78% and 89.56% respectively, increased 50.00%, 51.20%,48.29%,55.14%,26.33% and 24.63% compared with the starting strains. The composite mutation of Candida oleophila was more effective than alone mutation. But alone DES mutagenesis of Rhodothece glutinis was more effective than the compound mutation. The experiment obtained highly concentrations gossypol tolerated strains, enhanced the degradation rate of gossypol significantly and increased nutritional value of cottonseed meal.In the second part, another experiment was conducted to identity and compare DNA of mutant in the genus level.And used a pair of primers,named ITS1 and ITS4 amplify the 5.8S rRNA gene and ITS2 regions,determined the sequence,Then submit the sequence to the NCBI BLAST database to compare so as to assay those mutant strains to the different level.The results showed that GJ-D-25, GJ-Z-25, GJ-Z-25-D-25 was the same species through BLAST analysis.As Query coverage and Max ident were both 100%, mutant sequence was most approach to Uncultured fungus ITS1,5.8S rRNA gene and ITS2 region, clone f4.As Query coverage was 99%,Max ident was 100%, mutant sequence was most similar to Candida railenensis 18S rRNA gene (partial), ITS1,5.8S rRNA gene. Through BLAST 2 SEQUENCE analysis, HJ-D-20 sequence was only 94% similar to starting strain. After BLAST SEQUENCE analysis, HJ-D-20 was most similar to Rhodosporidium paludigenum strain.In the third part, another experiment was conducted to determine the suggestion fermentation parameters. Considering the detoxification and CP content as index, the effect of different carbon source, nitrogen source, inoculation amount, fermentation temperature and fermentation time on the fermentation were studied. The suggestion fermentation parameters of GJ-D-25 were determined that the substrate included 70% cottonseed meal, 20% corn flour and 10% bran,1% ammonium chloride, the proportion of substrate and water was 1:0.8, inoculation amount was 5% and the pH was in nature.