【摘要】：棉粕中有毒物質(zhì)棉酚的存在極大抑制了棉粕在畜禽飼料業(yè)中的應(yīng)用,通過微生物固態(tài)發(fā)酵法可以對棉粕中的棉酚起到降解作用。本研究從自然界中篩選得到數(shù)株對棉酚具有強降解能力的飼用酵母菌株。通過對不同菌株的棉酚降解能力進行定量測定后,選擇最優(yōu)菌株用于棉粕固態(tài)發(fā)酵;并對其發(fā)酵工藝和影響因素進行了深入研究。以果皮作為主要篩選分離的樣本,從中篩選出酵母菌共33株,通過26S rDNA D1/D2區(qū)域序列分析對其進行了分類鑒定,鑒定結(jié)果為:其中熱帶假絲酵母(Candida tropicalis)9株,馬克斯克魯維酵母(Kluyveromyces marxianus)2株,庫德畢赤酵母(Pichia kudriavzevii)6株,杰丁畢赤酵母(Pichia jadinii)1株,季也蒙畢赤酵母(Meyerozyma guilliermondii)2株,Candida rugosa 2株,Candida mesorugosa 2株,Candida orthopsilosis 2株,未測序的7株。將篩選到的這33株酵母菌和實驗室現(xiàn)存的酵母菌共87株,接種到以醋酸棉酚為唯一碳源的固體培養(yǎng)基上,觀察并記錄它們的生長情況,其中東方伊薩酵母(Issatchenkia orientalis)和庫德畢赤酵母(Pichia kudriavzevii)生長情況良好,產(chǎn)朊假絲酵母(Candida utilis)長勢稍弱。從每種酵母菌中,各選取1-2株接種以醋酸棉酚為唯一碳源的液體培養(yǎng)基中,測定總菌數(shù),進一步判斷其對醋酸棉酚的利用情況。最終挑選出對醋酸棉酚利用情況良好的庫德畢赤酵母(Pichia kudriavzevii)SD2A和產(chǎn)朊假絲酵母(C.utilis)SH87作為固體棉粕發(fā)酵的主要菌株。對棉粕進行生料固態(tài)發(fā)酵,通過比較不同發(fā)酵條件下SD2A和SH87的棉酚降解率,確定菌株SH87的最適發(fā)酵條件為:時間36h、發(fā)酵物水分42%、溫度30℃;菌株SD2A在接種量為10%,pH 6.0、水分42%、溫度37℃、時間36h時棉酚降解率最高(83.42%)。枯草芽孢桿菌和酵母菌混菌發(fā)酵結(jié)果表明,酵母菌接種比例的增加有助于提高棉酚降解率,表明了酵母菌對棉酚的降解作用。選擇菌株SH87制備固體菌劑,并對其制備條件進行優(yōu)化。以活菌數(shù)為指標,確定其最佳條件為:固態(tài)發(fā)酵培養(yǎng)基在以盒裝的發(fā)酵方式發(fā)酵2 d,固態(tài)發(fā)酵培養(yǎng)基含水量45%,發(fā)酵完的物料在45℃下烘干至含水量13%左右;此時最大活菌數(shù)可達6.13×108個·g-1。以SD2A為出發(fā)菌株,經(jīng)過紫外誘變與篩選,得到突變菌株SD2A-UV65;其棉酚降解率高達93.95%,比出發(fā)菌株提高了10.71%。菌株SH87對生料固態(tài)發(fā)酵棉粕的放大實驗發(fā)酵44h后,粗蛋白含量增加了7.25%,棉酚的降解率為87.02%。對菌株SD2A發(fā)酵液中蛋白質(zhì)進行提取純化后,將提取物和醋酸棉酚一起水浴,水浴后醋酸棉酚含量有極大降低;此結(jié)果表明酵母菌胞外酶在棉酚降解過程中起到了重要作用。用葡萄糖替代液體發(fā)酵培養(yǎng)基中作為唯一碳源的醋酸棉酚,提取后的物質(zhì)對醋酸棉酚也有降解作用;猜測此胞外酶不是誘導型的。
[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，

本文編號：2339023