【作者】 周生飞；

【导师】 汝应俊；

【作者基本信息】 甘肃农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 棉籽粕具有较高的蛋白含量,是重要的植物源蛋白饲料原料,但其中含有棉酚及其他一些抗营养因子,这些抗营养因子对动物具有一定的毒害作用,从而极大地限制了其在畜牧生产中的饲用价值和使用量。为了提高棉籽粕的营养价值,早期研究提出了硫酸亚铁处理、氢氧化钙处理和有机溶剂浸提等一些脱毒方法,对棉籽粕的脱毒起到了重要作用,但却也存在着较多弊端。微生物发酵法是一种新型的脱毒方法,它不仅可以很好地达到脱毒的效果,同时还可以提高棉籽粕的营养价值,具有广阔的发展前景,近年来受到越来越多的重视。本课题通过高效降解棉酚菌种的选育,发酵工艺参数的优化,对微生物发酵法脱毒效果及其影响因素进行了考察分析,并采用分子生物学方法对微生物发酵脱毒的机理进行了研究。主要研究成果如下:1、棉酚降解菌的筛选。通过采样、初筛和复筛等系统方法对棉酚降解菌株进行了筛选,得到四株棉酚降解率较高的菌株,然后采用形态学观察和分子生物学方法进行了菌种鉴定,最终确定四株菌分别为粪肠球菌（Enterococcus faecalis ）,原玻璃蝇节杆菌（ Arthrobacter protophormiae ）,干酪乳杆菌（Lactobacillus casei）,近平滑假丝酵母菌（Candida parapsilosis）,并选用近平滑假丝酵母菌（C. parapsilosis KDN0118）作为主要生产菌株。2、利用单因素法和统计学方法对C. parapsilosis KDN0118种子液体发酵的培养基成分和发酵条件进行了优化。首先利用单因素法确定了最佳碳源、最佳氮源以及初始pH、温度、转速、装液量、发酵时间的最适范围,然后采用Plackett-Burman（PB）实验设计筛选出对发酵菌量具有显著影响的关键因素酵母膏、K₂HPO₄、MgSO₄和发酵时间,最后通过Box-Behnken实验设计和SAS软件回归分析建立发酵菌量关于四个关键因素的二次多项式回归模型,并通过响应面分析法和典型性分析得出培养基最佳成分和最优发酵条件:酵母膏44.3 g/L、K₂HPO₄ 7.3 g/L、MgSO₄ 0.96 g/L,发酵时间为31.1 h,预测最大发酵菌量Ymax为6.55×109 cfu/mL。3、利用单因素法和统计学方法对C. parapsilosis KDN0118固态发酵的培养基成分和发酵条件进行了优化。首先对碳源及其浓度、水分含量、初始pH、接种量、温度、翻曲次数和发酵时间进行了优化,然后利用Plackett-Burman（PB）实验设计筛选对棉酚降解率具有显著影响的关键因素Na2CO₃、FeSO₄、MgSO₄、水分含量和发酵时间,最后通过CCD实验设计和SAS软件回归分析建立棉酚降解率与关键因素之间的的二次多项式回归模型,并通过模型求解确定培养基最佳成分和最优发酵条件:Na₂CO₃ 0.56 g/L,FeSO₄ 72.7 mg/L,MgSO₄ 0.56 g/L,水分含量58.1%和发酵时间31.6 h,预测最大棉酚降解率Ymax为60.94%。4、以固态发酵工艺参数为基础,对微生物发酵代谢棉酚的降解机理进行了研究。运用LC-MS/MS对不同时间发酵液中的棉酚降解产物进行检测,根据MassLynx V4.1软件分析获得18种棉酚降解产物,用“元素组成”工具进行分析,推导出相应分子结构,并用Massfragment软件进行碎片分析和验证匹配,提出了C. parapsilosis KDN0118降解棉酚的代谢途径,包括与赖氨酸的结合途径及苯环的开环降解途径。5、进行棉酚降解菌与功能菌相结合的混菌发酵工艺进行了研究。通过四株棉酚降解菌和六株功能菌的组合实验,考察了各菌株之间的相互关系,确定了棉酚降解菌与功能菌的最佳组合,并综合评价了混合菌发酵的效果以及各菌株在发酵中的作用,并通过实验验证了该混合菌组合发酵的稳定性。更多还原

【Abstract】 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.更多还原