Study On Isolation,Identification And Degradation Mechanism Of Gossypol-degrading Strain

With the arrival of twenty-first century,people began to pay attention to the collocation of nutrition,milk,meat and other food demand gradually increased,the development of animal husbandry has played a stimulating effect on the upgrade,at the same time also promoted the development of feed industry,so the demand for a large increase in feed raw materials.China is vast territory and abundant resources,cottonseed meal is very rich in resources,its output ranked first in the world.The study found that the protein content of cottonseed meal is relatively high,the crude protein content can reach more than 40%,which is a high feed value of development and application,but because the presence of gossypol,which greatly limited the application of cottonseed meal,and caused the waste of resources.While,due to the cottonseed meal has higher protein content and the price is lower,so to find a feasible method of green will make full use of cottonseed meal,to a great extent,will play a great role in solving the serious shortage of protein resources in china.Compared to the traditional cottonseed meal detoxification method,microbial fermentation is a kind of new green detoxification methods,its detoxification can also improve the nutritional value of cottonseed meal,increase palatability and feed intake.Therefore,the research direction is screened for degrading gossypol strains by microbial fermentation method for detoxification of cottonseed meal,and to explore the optimal conditions of microbial fermentation of cottonseed meal process parameters,using molecular biology method of polyacrylamide gel(SDS-PAGE),and combined with LC-MS/MS on microbial fermentation cottonseed meal detoxification mechanism was studied,the main results are as follows:1.48 strains of gossypol-biodegrading strains,using acetic acid gossypol as the sole carbon source,were screened in soil samples from different cotton fields surrounding a cottonseed oil factory in Xin xiang,China.Six strains with a high ability to degrade gossypol were obtained by secondary screening,and morphological observation and molecular identification of 18 S rDNA was performed.The results confirmed that strain 1 was Aspergillus niger,strain 2 was Aspergillus flavus,strain 3 was Penicillium purpureum,strain 4 was Aspergillus terreus,strain 5 was Penicillium sp.,and strain 6 was Saccharomyces cerevisiae.These strains were used as the objects to be studied in the following experiments.2.Gossypol degradation was compared using the phloroglucinol method,and Aspergillus terreus was displayed the highest activity,with a pure gossypol degradation rate of 33.6%.In order to further explore the potential of Aspergillus terreus in the practical application of degradation of gossypol,In this paper,the cottonseed meal was solidified by Aspergillus terreus,and different test factors such as fermentation time,inoculation amount,ratio of material to water,carbon source and nitrogen source five factors on the impact of cotton meal detoxification,in order to control the principle of a single variable for single factor detoxification conditions.The results showed that the medium was 10% maltose as the carbon source,3% peptone was nitrogen source,the ratio of material to water was 1: 0(g / ml)and the inoculation amount was 6%.The medium was incubated at 30? 72 h,Aspergillus terreus had the best detoxification efficiency to cottonseed meal,and the virus-free rate was 88.3%.3.Under optimum conditions preliminary studies fermentation of cottonseed meal,cottonseed meal fermented with the control group,the fermentation time was 24 h-72 h of sampling samples were polyacrylamide gels(SDS-PAGE).SDS-PAGE showed that there were six bands with significant difference before and after fermentation.After fermentation significant change before and after the bands were excised and further processed by LC-MS/MS for its detection and analysis were detected 37 kinds of proteins.Through the in-depth study on the mechanism of degradation of gossypol,the paper found that 11 kinds of interest proteins may be related to degradation of gossypol,These proteins/ enzymes were actin,tubulin,ATP a/? subunit,triosephosphate isomerase,phosphoglycerate kinase,malate dehydrogenase,glutamate dehydrogenase,inorganic pyrophosphatase,xylanase,?-1,4-endo-xylanase.Gossypol degradation requires more energy,most of which proteins are associated with energy metabolism.Among them,xylanase and ?-1,4-endo-xylanase can indirectly degrade lignin,and the molecular structure of lignin monomer contains a variety of active functional groups,among which there are phenolic hydroxyl groups,gossypol belongs to phenolic hydroxyl compounds,suggesting that xylanase and ?-1,4-endo-xylanase may play an important role in the degradation of gossypol.The present study initially explains the degradation mechanism of Aspergillus terrus fermented cottonseed metabolized gossypol,which provides a theoretical basis for the further study of microbial detoxification gossypol and the related application of cottonseed meal.