Screening Of Gossypol-degrading Strain , Mechanism Of Gossypol Degradation And Its Application In Fermentation Of Cottonseed Meal

Cottonseed meal is an important protein material compared with soybean meal, but the use of cottonseed meal in feed industry has been limited by the gossypol and some other anti-nutritional factors. Gossypol is a toxic polyphenolic binaphthyl dialdehyde, it can depress the growth and fertility of animal. There were several methods to remove the gossypol in cottonseed meal, including solvent extraction of free gossypol, chemical treatment with calcium hydroxide or ferrous sulfate. These methods play an important role in detoxification of cottonseed meal but many drawbacks still exist. Microbial fermentation as a new detoxification method get more and more attention, because the way is not only get the safe criteria, but also it can highly enhanced the untilization of nutrient substance. In this study, four strains with high efficiency of gossypol detoxification were screened out, the process parameters of fermentation detoxification were optimized, the factors of affecting gossypol detoxification were researched, and the mechanism of fermentation detoxification was also investigated in molecular level. The main results are as follows:1. The screen of strains for gossypol degradation. The PDA medium containing different concentration of acetic gossypol and MRS medium with acetic gossypol were used for preliminary screening and rescreening. Cottonseed meal was then applied for practically microbial fermentation. By this means, four strains with high efficiency of gossypol detoxification were screened out from the samples of soil, rotten cottonseed meal and probiotics, then were identified as Enterococcus faecalis, Arthrobacter protophormiae, Lactobacillus casei and Candida parapsilosis by morphology and molecular biology methods. C. parapsilosis KDN0118 was then used as the main production strain.2. The composition of Culture medium and fermentation conditions of C. parapsilosis KDN0118 seed under liquid fermentation were optimized by single-factor experiment and statistical method. In the first step, the optimal of carbon source, the nitrogen source and the range of initial pH, temperature, rotating speed, volumes of culture medium, and fermentation time were determined by single-factor experiment. As key factors of microbial growth, yeast extract, K₂HPO₄, MgSO₄ and fermentation time were then determined by Plackett-Burman design experiment. Finally, Box-Behnken design and SAS software were applied in the establishment of quadratic polynomial regression model between microbial growth and the four key factors. The optimized fermentation conditions were: yeast extract 44.3 g/L, K2HPO₄ 7.3 g/L, MgSO₄ 0.96 g/L, after culturing of 31.1 h, the microbial growth reached the maximum of 6.55×109cfu/mL.3. C. parapsilosis KDN0118 was then cultured for the biodegradation of free gossypol under solidstate fermentation. Single-factor experiment and statistical methods were used in the optimization of culture medium and fermentation conditions. Carbon source and its concentration, water content, initial pH, inoculum size, temperature, stirring times and fermentation time were optimized separately. Plackett-Burman design experiment was then applied in the screening of key factors that affect the degradation of gossypol. As a result, Na₂CO₃, FeSO₄, MgSO₄, water content and fermentation time infulenced fermentation detoxification significantly. Finally, CCD design and SAS software were applied in the establishment of quadratic polynomial regression model between gossypol degradation and these key factors. Optimized culture medium and fermentation conditions obtained from model calculation were as follows: Na2CO₃ 0.56 g/L, FeSO₄ 72.7 mg/L, MgSO₄ 0.56 g/L, moisture content 58.1%, after culturing of 31.6 h, the gossypol degradation reached the maximum of 60.94%.4. Based on parameters of solidstate fermentation, mechanism of degradation of free gossypol in microorganism was investigated. LC-MS/MS was used in the monitoring of gossypol metabolites at intervals during fermentation. MassLynx V4.1 software analysis revealed the appearance of new products during the biodegradation process. The previously research of microbe-induced gossypol degradation was very limited. The elemental composition tool and massfragment software were used for the first time to identify the biodegradation products, as a result 18 metabolites were identified, and two degradation pathways of gossypol were proposed.5. Mixed fermentation technology was researched by means of the combination of functional bacteria and degrading bacteria. Four strains capable of degrading gossypol and six functional strains that screened out were fermented in various combinations. The optimum combination of strains for fermentation was determined by the consideration of detoxification and nutritional value of cottonseed meal. The role of each strain in fermentation was also evaluated. Finally, stability trial of the mixed fermentation process was tested. The results showed that both the iteration and the stability were very excellent.