高效降解棉酚的酵母菌的筛选及其发酵工艺研究
发布时间：2018-11-17 21:00
【摘要】：棉粕中有毒物质棉酚的存在极大抑制了棉粕在畜禽饲料业中的应用,通过微生物固态发酵法可以对棉粕中的棉酚起到降解作用。本研究从自然界中筛选得到数株对棉酚具有强降解能力的饲用酵母菌株。通过对不同菌株的棉酚降解能力进行定量测定后,选择最优菌株用于棉粕固态发酵;并对其发酵工艺和影响因素进行了深入研究。以果皮作为主要筛选分离的样本,从中筛选出酵母菌共33株,通过26S rDNA D1/D2区域序列分析对其进行了分类鉴定,鉴定结果为:其中热带假丝酵母(Candida tropicalis)9株,马克斯克鲁维酵母(Kluyveromyces marxianus)2株,库德毕赤酵母(Pichia kudriavzevii)6株,杰丁毕赤酵母(Pichia jadinii)1株,季也蒙毕赤酵母(Meyerozyma guilliermondii)2株,Candida rugosa 2株,Candida mesorugosa 2株,Candida orthopsilosis 2株,未测序的7株。将筛选到的这33株酵母菌和实验室现存的酵母菌共87株,接种到以醋酸棉酚为唯一碳源的固体培养基上,观察并记录它们的生长情况,其中东方伊萨酵母(Issatchenkia orientalis)和库德毕赤酵母(Pichia kudriavzevii)生长情况良好,产朊假丝酵母(Candida utilis)长势稍弱。从每种酵母菌中,各选取1-2株接种以醋酸棉酚为唯一碳源的液体培养基中,测定总菌数,进一步判断其对醋酸棉酚的利用情况。最终挑选出对醋酸棉酚利用情况良好的库德毕赤酵母(Pichia kudriavzevii)SD2A和产朊假丝酵母(C.utilis)SH87作为固体棉粕发酵的主要菌株。对棉粕进行生料固态发酵,通过比较不同发酵条件下SD2A和SH87的棉酚降解率,确定菌株SH87的最适发酵条件为:时间36h、发酵物水分42%、温度30℃;菌株SD2A在接种量为10%,pH 6.0、水分42%、温度37℃、时间36h时棉酚降解率最高(83.42%)。枯草芽孢杆菌和酵母菌混菌发酵结果表明,酵母菌接种比例的增加有助于提高棉酚降解率,表明了酵母菌对棉酚的降解作用。选择菌株SH87制备固体菌剂,并对其制备条件进行优化。以活菌数为指标,确定其最佳条件为:固态发酵培养基在以盒装的发酵方式发酵2 d,固态发酵培养基含水量45%,发酵完的物料在45℃下烘干至含水量13%左右;此时最大活菌数可达6.13×108个·g-1。以SD2A为出发菌株,经过紫外诱变与筛选,得到突变菌株SD2A-UV65;其棉酚降解率高达93.95%,比出发菌株提高了10.71%。菌株SH87对生料固态发酵棉粕的放大实验发酵44h后,粗蛋白含量增加了7.25%,棉酚的降解率为87.02%。对菌株SD2A发酵液中蛋白质进行提取纯化后,将提取物和醋酸棉酚一起水浴,水浴后醋酸棉酚含量有极大降低;此结果表明酵母菌胞外酶在棉酚降解过程中起到了重要作用。用葡萄糖替代液体发酵培养基中作为唯一碳源的醋酸棉酚,提取后的物质对醋酸棉酚也有降解作用;猜测此胞外酶不是诱导型的。
[Abstract]:The presence of gossypol in cottonseed meal greatly inhibited the application of cottonseed meal in livestock and poultry feed industry. Gossypol in cottonseed meal could be degraded by microbial solid fermentation. In this study, several forage yeast strains with strong degradation of gossypol were obtained from nature. After quantitative determination of gossypol degradation ability of different strains, the optimal strain was selected for solid fermentation of cottonseed meal, and the fermentation process and influencing factors were studied. A total of 33 strains of yeast were isolated from the pericarp and identified by 26s rDNA D1/D2 sequence analysis. The results were as follows: 9 strains of Candida tropicalis were identified. There were 2 (Kluyveromyces marxianus) strains, 6 (Pichia kudriavzevii) strains, 1 (Pichia jadinii) strain of Pichia pastoris, 2, Candida rugosa 2 strains, Candida rugosa 2, Candida mesorugosa 2 strains and 7 unsequenced, Candida mesorugosa strains. A total of 87 of the 33 yeasts screened and the existing yeasts in the laboratory were inoculated on solid medium with gossypol acetate as the sole carbon source, and their growth was observed and recorded. (Issatchenkia orientalis) and (Pichia kudriavzevii) of Pichia pastoris grew well, and (Candida utilis) of Candida prion was slightly weak. From each yeast, 1-2 strains were inoculated in liquid medium with gossypol acetate as the sole carbon source, the total number of bacteria was determined, and the utilization of gossypol acetate was further judged. Finally, Pichia pastoris (Pichia kudriavzevii) SD2A and Candida prion (C.utilis) SH87, which had good utilization of gossypol acetate, were selected as the main strains for solid cottonseed meal fermentation. Cotton meal was fermented by solid state fermentation of raw meal. By comparing the degradation rate of gossypol between SD2A and SH87 under different fermentation conditions, the optimum fermentation conditions of strain SH87 were determined as follows: time 36 h, moisture 42 鈩，
本文编号：2339023
[Abstract]:The presence of gossypol in cottonseed meal greatly inhibited the application of cottonseed meal in livestock and poultry feed industry. Gossypol in cottonseed meal could be degraded by microbial solid fermentation. In this study, several forage yeast strains with strong degradation of gossypol were obtained from nature. After quantitative determination of gossypol degradation ability of different strains, the optimal strain was selected for solid fermentation of cottonseed meal, and the fermentation process and influencing factors were studied. A total of 33 strains of yeast were isolated from the pericarp and identified by 26s rDNA D1/D2 sequence analysis. The results were as follows: 9 strains of Candida tropicalis were identified. There were 2 (Kluyveromyces marxianus) strains, 6 (Pichia kudriavzevii) strains, 1 (Pichia jadinii) strain of Pichia pastoris, 2, Candida rugosa 2 strains, Candida rugosa 2, Candida mesorugosa 2 strains and 7 unsequenced, Candida mesorugosa strains. A total of 87 of the 33 yeasts screened and the existing yeasts in the laboratory were inoculated on solid medium with gossypol acetate as the sole carbon source, and their growth was observed and recorded. (Issatchenkia orientalis) and (Pichia kudriavzevii) of Pichia pastoris grew well, and (Candida utilis) of Candida prion was slightly weak. From each yeast, 1-2 strains were inoculated in liquid medium with gossypol acetate as the sole carbon source, the total number of bacteria was determined, and the utilization of gossypol acetate was further judged. Finally, Pichia pastoris (Pichia kudriavzevii) SD2A and Candida prion (C.utilis) SH87, which had good utilization of gossypol acetate, were selected as the main strains for solid cottonseed meal fermentation. Cotton meal was fermented by solid state fermentation of raw meal. By comparing the degradation rate of gossypol between SD2A and SH87 under different fermentation conditions, the optimum fermentation conditions of strain SH87 were determined as follows: time 36 h, moisture 42 鈩，
本文编号：2339023
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