本文選題：粗糙脈孢菌　切入點：稻草　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：我國稻草等農業(yè)廢棄物數(shù)量巨大且利用率低,采用微生物固態(tài)發(fā)酵技術可將其轉化為高營養(yǎng)的動物飼料,以實現(xiàn)資源的可持續(xù)利用,也為飼料行業(yè)的發(fā)展提供了一個新的思路。在眾多木質纖維素降解真菌中,粗糙脈孢菌(Neurospora crassa)具有較強的降解和利用木質纖維素的能力,因而可用于產乙醇等能源物質,此外也被廣泛應用于遺傳或分子等生物學方面的研究,但是通過優(yōu)化Neurospora crassa發(fā)酵條件以獲取大量飼料蛋白的研究卻極少。Neurospora crassa可產三種纖維素酶、多種半纖維素酶和一些木質素酶,培養(yǎng)簡單且生長迅速,并具備強大的蛋白質表達和分泌能力,此外其安全性也已得到了證實,因此可被用作固態(tài)發(fā)酵的菌種。本論文以稻草為原料,利用Neurospora crassa 14-8作為固態(tài)發(fā)酵的關鍵菌種,并結合其它真菌協(xié)同發(fā)酵降解稻草,通過優(yōu)化發(fā)酵條件和工藝以提高發(fā)酵產物中真蛋白的含量。首先研究了單獨接種Neurospora crassa 14-8時不同發(fā)酵條件對真蛋白產量的影響,先后利用單因素試驗以及正交試驗優(yōu)化了溫度、初始pH、發(fā)酵時間、接種量、水料比、淀粉添加量、氮源種類及其添加量。通過對真蛋白產量的測定和分析發(fā)現(xiàn),在固體發(fā)酵培養(yǎng)基中添加0.2%尿素和1%可溶性淀粉,調整培養(yǎng)基初始pH為6,料水比保持1:2,并接種10%Neurospora crassa 14-8于32℃進行120 h的發(fā)酵可獲得較高的真蛋白產量。采用混菌發(fā)酵系統(tǒng)即Neurospora crassa 14-8和產朊假絲酵母(Candida utilis)協(xié)同發(fā)酵降解稻草,研究了混合發(fā)酵時兩種菌的接種比例、Candida utilis接種時間和發(fā)酵時間這三種因素對真蛋白產量的影響,確定了最優(yōu)的發(fā)酵條件。為了進一步提高真蛋白的產量,采用響應面法對固態(tài)發(fā)酵基礎培養(yǎng)基中的不同陽離子和Tween 80添加量進行了優(yōu)化。通過Plackett-Burman試驗從七種不同陽離子中篩選出Mn2+、Cu2+和Zn2+三個顯著性因素,這三種陽離子顯著影響真蛋白的產量。依據(jù)Plackett-Burman試驗的結果,先后經(jīng)最陡爬坡試驗和Box-Behnken試驗確定了三種陽離子的最優(yōu)濃度為:Mn2+0.06 g/L,Zn2+0.15 g/L,Cu2+0.2 g/L。在該優(yōu)化條件下進行試驗,測得的真蛋白產量高達9.48%,這與未經(jīng)發(fā)酵的稻草真蛋白含量(3.98%)相比,增長率為147.24%。結果表明,陽離子的種類和濃度經(jīng)優(yōu)化后添加到發(fā)酵培養(yǎng)基中可促進酶的水解,提高了稻草的降解速率,并可增加微生物蛋白質的積累。選取Neurospora crassa 14-8、Candida utilis、米曲霉(Aspergillus oryzae)和黃孢原毛平革菌(Phanerochaete chrysosporium)四個菌種進行不同組合發(fā)酵,發(fā)現(xiàn)Neurospora crassa 14-8、Candida utilis和Phanerochaete chrysosporium混合發(fā)酵后真蛋白產量最高。通過研究不同誘導劑對Phanerochaete chrysosporium產木質素酶的影響,最終確定以5 mM Mn2+和50 mM藜蘆醇作為木質素過氧化物酶(LiP)的誘導劑,以提高酶促水解的效率。與此同時,對多菌種混合發(fā)酵的條件進行了優(yōu)化,當Neurospora crassa 14-8晚于Phanerochaete chrysosporium 1天再接種,總接種量為10%,并且Neurospora crassa 14-8與Phanerochaete chrysosporium的接種比例為1:2時可獲得最大的真蛋白產量。實驗探討了不同Ca(OH)_2預處理濃度和脫毒工藝對稻草降解和發(fā)酵產真蛋白的影響。結果表明以5 g/L Ca(OH)_2進行預處理可有效分解木質素,提高稻草的降解率。經(jīng)堿預處理后水洗并添加2%的漆酶進行發(fā)酵,可使發(fā)酵產物的木質素含量明顯減少,真蛋白的產量有所提高,由此說明發(fā)酵前通過水洗并加入漆酶的方法可解除部分預處理水解液中的抑制物。本研究主要利用Neurospora crassa 14-8固態(tài)發(fā)酵稻草產飼料蛋白,提供了一種經(jīng)濟、環(huán)保的方式,并且在大規(guī)模飼料蛋白轉化中具有潛在應用價值。
[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.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S816

【參考文獻】

相關期刊論文 前1條

1 Changyou Shi;Jun He;Jie Yu;Bing Yu;Zhiqing Huang;Xiangbing Mao;Ping Zheng;Daiwen Chen;;Solid state fermentation of rapeseed cake with Aspergillus niger for degrading glucosinolates and upgrading nutritional value[J];Journal of Animal Science and Biotechnology;2015年03期

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本文編號：1591167