粗糙脉孢菌固态发酵稻草产饲料蛋白的研究

发布时间：2018-03-10 01:26

本文选题：粗糙脉孢菌　切入点：稻草　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：我国稻草等农业废弃物数量巨大且利用率低,采用微生物固态发酵技术可将其转化为高营养的动物饲料,以实现资源的可持续利用,也为饲料行业的发展提供了一个新的思路。在众多木质纤维素降解真菌中,粗糙脉孢菌(Neurospora crassa)具有较强的降解和利用木质纤维素的能力,因而可用于产乙醇等能源物质,此外也被广泛应用于遗传或分子等生物学方面的研究,但是通过优化Neurospora crassa发酵条件以获取大量饲料蛋白的研究却极少。Neurospora crassa可产三种纤维素酶、多种半纤维素酶和一些木质素酶,培养简单且生长迅速,并具备强大的蛋白质表达和分泌能力,此外其安全性也已得到了证实,因此可被用作固态发酵的菌种。本论文以稻草为原料,利用Neurospora crassa 14-8作为固态发酵的关键菌种,并结合其它真菌协同发酵降解稻草,通过优化发酵条件和工艺以提高发酵产物中真蛋白的含量。首先研究了单独接种Neurospora crassa 14-8时不同发酵条件对真蛋白产量的影响,先后利用单因素试验以及正交试验优化了温度、初始pH、发酵时间、接种量、水料比、淀粉添加量、氮源种类及其添加量。通过对真蛋白产量的测定和分析发现,在固体发酵培养基中添加0.2%尿素和1%可溶性淀粉,调整培养基初始pH为6,料水比保持1:2,并接种10%Neurospora crassa 14-8于32℃进行120 h的发酵可获得较高的真蛋白产量。采用混菌发酵系统即Neurospora crassa 14-8和产朊假丝酵母(Candida utilis)协同发酵降解稻草,研究了混合发酵时两种菌的接种比例、Candida utilis接种时间和发酵时间这三种因素对真蛋白产量的影响,确定了最优的发酵条件。为了进一步提高真蛋白的产量,采用响应面法对固态发酵基础培养基中的不同阳离子和Tween 80添加量进行了优化。通过Plackett-Burman试验从七种不同阳离子中筛选出Mn2+、Cu2+和Zn2+三个显著性因素,这三种阳离子显著影响真蛋白的产量。依据Plackett-Burman试验的结果,先后经最陡爬坡试验和Box-Behnken试验确定了三种阳离子的最优浓度为:Mn2+0.06 g/L,Zn2+0.15 g/L,Cu2+0.2 g/L。在该优化条件下进行试验,测得的真蛋白产量高达9.48%,这与未经发酵的稻草真蛋白含量(3.98%)相比,增长率为147.24%。结果表明,阳离子的种类和浓度经优化后添加到发酵培养基中可促进酶的水解,提高了稻草的降解速率,并可增加微生物蛋白质的积累。选取Neurospora crassa 14-8、Candida utilis、米曲霉(Aspergillus oryzae)和黄孢原毛平革菌(Phanerochaete chrysosporium)四个菌种进行不同组合发酵,发现Neurospora crassa 14-8、Candida utilis和Phanerochaete chrysosporium混合发酵后真蛋白产量最高。通过研究不同诱导剂对Phanerochaete chrysosporium产木质素酶的影响,最终确定以5 mM Mn2+和50 mM藜芦醇作为木质素过氧化物酶(LiP)的诱导剂,以提高酶促水解的效率。与此同时,对多菌种混合发酵的条件进行了优化,当Neurospora crassa 14-8晚于Phanerochaete chrysosporium 1天再接种,总接种量为10%,并且Neurospora crassa 14-8与Phanerochaete chrysosporium的接种比例为1:2时可获得最大的真蛋白产量。实验探讨了不同Ca(OH)_2预处理浓度和脱毒工艺对稻草降解和发酵产真蛋白的影响。结果表明以5 g/L Ca(OH)_2进行预处理可有效分解木质素,提高稻草的降解率。经碱预处理后水洗并添加2%的漆酶进行发酵,可使发酵产物的木质素含量明显减少,真蛋白的产量有所提高,由此说明发酵前通过水洗并加入漆酶的方法可解除部分预处理水解液中的抑制物。本研究主要利用Neurospora crassa 14-8固态发酵稻草产饲料蛋白,提供了一种经济、环保的方式,并且在大规模饲料蛋白转化中具有潜在应用价值。
[Abstract]:China's rice straw and other agricultural waste huge quantity and low utilization rate, by microbial solid fermentation technology can be transformed into animal feed with high nutrition, to achieve the sustainable use of resources, but also provides a new way for the development of feed industry. Among the many lignocellulolytic fungus, Neurospora crassa (Neurospora crassa) has strong ability of degradation and utilization of lignocellulose, and thus can be used for ethanol production such as energy material, research also has been widely used in genetic or molecular biological aspects of the study, but through the optimization of fermentation conditions for Neurospora crassa to get a lot of protein feed little.Neurospora crassa can produce three kinds of Cellulase, hemicellulase variety and some ligninolytic enzyme, simple cultivation and rapid growth, and have strong ability of protein expression and secretion, in addition to its safety has also been Confirmed, therefore can be used as solid fermentation bacteria. This paper using straw as raw material, using Neurospora crassa 14-8 as the key strains solid state fermentation, and in combination with other fungal synergistic fermentation straw degradation, by optimizing the fermentation conditions and the process to improve the content of protein fermentation products. First studied the effects of different crassa Neurospora single inoculation fermentation conditions of true protein yield 14-8, successively by single factor experiment and orthogonal test to optimize the temperature, initial pH, fermentation time, inoculation amount, ratio of water to material, adding amount of starch, nitrogen source and its adding amount. By measuring and true protein yield analysis found that adding 0.2% and 1% urea soluble culture starch based in solid fermentation, the adjustment of the initial pH of the medium was 6, the ratio of material to water 1:2, and inoculation of 10%Neurospora crassa 14-8 at 32 DEG C for 120 h fermentation can get higher Really. The protein yield of mixed fermentation system is 14-8 Neurospora crassa and Candida utilis (Candida utilis) Co fermentation of straw degradation, inoculation ratio of two kinds of bacteria mixed fermentation was studied. The effects of these three factors Candida utilis inoculation time and fermentation time on true protein yield, the optimum fermentation conditions sure. In order to further improve the true protein yield, using the response surface method based on solid state fermentation medium with different cations and the addition of Tween 80 was optimized. Through Plackett-Burman test from seven Mn2+ screened different cations, Cu2+ and Zn2+ three significant factors, these three kinds of cationic protein really significant effect on the basis of production. The results of the Plackett-Burman trial, has the steepest ascent test and Box-Behnken test to determine the optimal concentration of three kinds of cations: Mn2+0.06 g/L, Zn2+0.15 g/L, Cu2+0.2 g/ L. test was carried out under the optimal conditions, the measured true protein yield as high as 9.48%, and this without straw true protein content of fermentation (3.98%) compared with the growth rate of 147.24%.. The results show that the type and concentration of cations after optimization is added to the fermentation medium can promote enzyme hydrolysis, improve the degradation the rate of straw, and can increase the microbial protein accumulation. Select Neurospora crassa 14-8, Candida utilis, Aspergillus oryzae (Aspergillus oryzae) and Phanerochaete Chrysosporium (Phanerochaete Chrysosporium) four strains of different combinations of fermentation, Neurospora crassa 14-8, Candida utilis and Phanerochaete Chrysosporium mixed fermentation of true protein yield was the highest. Through the study of different revulsants effect on Phanerochaete Chrysosporium production of ligninolytic enzyme, determined by 5 mM Mn2+ and 50 mM of resveratrol as lignin peroxidase (Li P) inducing agent, to improve the efficiency of enzymatic hydrolysis. At the same time, the fermentation conditions were optimized, when the Neurospora crassa 14-8 Phanerochaete Chrysosporium 1 days later than before vaccination, the total inoculation rate was 10%, and the ratio of Neurospora crassa 14-8 and Phanerochaete inoculated Chrysosporium for maximum protein yield of 1:2. Experimental study of different Ca (OH) _2 concentration pretreatment and detoxification process of rice straw degradation and fermentation of true protein. The results show that the effect of 5 g/L Ca (OH) _2 pretreatment can effectively improve the quality of wood decomposition, the straw degradation rate. After pretreatment by alkali washing and add 2% the laccase fermentation, the fermentation product of lignin content was significantly reduced, true protein yield was increased, indicating that the method of pre fermentation by washing and adding laccase can release part pretreatment in Hydrolysate This study mainly used Neurospora crassa 14-8 solid-state fermentation to produce feed protein from rice straw, providing an economic and environmental friendly way, and has potential application in large-scale feed protein transformation.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816

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