本文選題：羽毛粉 + 蛋白酶　； 參考：《河南農(nóng)業(yè)大學》2015年博士論文

【摘要】：隨著飼料蛋白資源的日益短缺和價格飛速上漲,低質蛋白資源的開發(fā)成為研究的熱點。羽毛粉蛋白質含量豐富,但其結構穩(wěn)定,不溶于水,不能被一般的蛋白酶水解,難以被動物消化吸收。本文通過篩選得到對羽毛粉降解效果較好的米曲霉和枯草芽孢桿菌菌株,對兩種菌株的中性蛋白酶和堿性蛋白酶基因進行DNA改組和克隆,將改組后的蛋白酶基因成功轉入畢赤酵母X33中,并對重組酵母蛋白酶活力和酶學性質進行分析。最后利用重組畢赤酵母發(fā)酵羽毛粉并將其應用到肉雞生產(chǎn)中,對發(fā)酵羽毛粉替代魚粉對肉雞的飼養(yǎng)效果進行研究,為緩解蛋白資源短缺和提高羽毛粉的飼用價值提供了新的技術途徑。主要結果如下:(1)從五種微生物菌株中初篩出產(chǎn)蛋白酶活力較高的米曲霉和枯草芽孢桿菌菌株。通過測定蛋白酶活力和羽毛粉中可溶性蛋白含量,對米曲霉和枯草芽孢桿菌對羽毛粉的降解效果進行研究。結果顯示,兩種微生物菌株均可對羽毛粉起到降解作用。發(fā)酵72 h后,米曲霉產(chǎn)蛋白酶活力達到3256.61 U/g(P0.05),枯草芽孢桿菌產(chǎn)蛋白酶活力達到690.03 U/g(P0.05)。米曲霉發(fā)酵羽毛粉48 h時可溶性蛋白含量,由最初的2.21 mg/g提高到362.07 mg/g(P0.05)。(2)使用RT-PCR技術,成功克隆得到米曲霉中性和堿性蛋白酶基因、枯草芽孢桿菌中性和堿性蛋白酶基因,四種蛋白酶基因與NCBI公布的蛋白酶基因同源性均大于99.5%。(3)對克隆得到的四種蛋白酶基因進行DNA改組,將改組蛋白酶基因與pMD19-T質粒連接并轉入大腸桿菌中,通過酪蛋白平板篩選,得到三個來自米曲霉的DNA改組中性蛋白酶基因NPGA-S1、NPGA-S2、NPGA-S3和一個來自枯草芽孢桿菌的DNA改組中性蛋白酶基因NPGB-S1。(4)將四種DNA改組蛋白酶基因和兩種中性蛋白酶基因電擊轉入畢赤酵母X33中,通過重組酵母蛋白酶活力測定,篩選出NPGA-S2和NPGA-S3兩種改組效果較好的蛋白酶基因。(5)將NPGA-S2和NPGA-S3與四種初始蛋白酶基因一起進行第二輪DNA改組,初篩出一個來自米曲霉的中性蛋白酶DNA改組基因NPGA-S4,將其電擊轉化至畢赤酵母X33中,通過對三種含米曲霉中性蛋白酶改組基因的重組畢赤酵母產(chǎn)蛋白酶酶活力的測定,最終選出NPGA-S2作為最好的DNA改組蛋白酶基因。與初始蛋白酶基因相比,DNA改組蛋白酶基因在1022 bp處的核苷酸T突變?yōu)镃,相應的氨基酸由半胱氨酸突變?yōu)榫彼�。DNA改組蛋白酶的酶活力比初始蛋白酶活力提高36.4%(P0.05),pH和熱穩(wěn)定性也得到了明顯提高,在pH6.5-8.0和30-70℃之間保持穩(wěn)定(P0.05)。(6)通過雞強飼試驗對發(fā)酵羽毛粉蛋白質、鈣和磷代謝率進行測定,米曲霉和含蛋白酶基因重組酵母發(fā)酵羽毛粉的蛋白質和鈣磷表觀代謝率顯著高于普通羽毛粉和不含蛋白酶基因重組酵母發(fā)酵羽毛粉(P0.05)。與普通羽毛粉相比,發(fā)酵羽毛粉(含玉米)中各種氨基酸含量均有所提高,含改組蛋白酶基因的重組酵母發(fā)酵羽毛粉賴氨酸含量增長接近1倍。(7)用米曲霉和含蛋白酶基因重組酵母發(fā)酵羽毛粉替代魚粉添加到肉雞日糧中,通過測定黃羽肉雞生長性能、屠宰性能、肉品質、腸道微生物、血液生化指標和經(jīng)濟效益分析,研究發(fā)酵羽毛粉替代魚粉對肉雞生產(chǎn)的可行性。飼養(yǎng)試驗分為前期(1-28d)和后期(29-56d)兩個階段。前期和后期分別選擇250只黃羽肉雞,分為五個處理,每個處理5個重復,每個重復10只雞。其中A組為含魚粉的基礎日糧(對照組),B組用等量米曲霉發(fā)酵羽毛粉替代基礎日糧中的魚粉,C組用等量不含蛋白酶基因的pGAPZαA重組畢赤酵母發(fā)酵羽毛粉替代基礎日糧中魚粉(負對照組),D組用等量含米曲霉蛋白酶基因的pGAPZαA重組畢赤酵母發(fā)酵羽毛粉替代基礎日糧中魚粉,E組用等量含米曲霉DNA改組蛋白酶基因(NPGA-S2)的pGAPZαA重組畢赤酵母發(fā)酵羽毛粉替代基礎日糧中魚粉。結果表明,飼養(yǎng)試驗前期和后期,除負對照組外,添加米曲霉和含蛋白酶基因發(fā)酵羽毛粉各組和對照組在日增重、日采食量、飼料轉化率和營養(yǎng)物質代謝率方面差異不顯著(P0.05)。含DNA改組蛋白酶基因重組酵母發(fā)酵羽毛粉組屠宰率、全凈膛率和肉品質雖然高于其他各組,但各組之間差異不顯著(P0.05)。米曲霉和含蛋白酶基因重組酵母發(fā)酵羽毛粉組腸道蛋白酶活力顯著高于對照組和負對照組(P0.05)。各組在腸道微生物數(shù)量、腸道微生物菌群分布和微生物多樣性方面基本一致,其中含DNA改組蛋白酶基因重組酵母發(fā)酵羽毛粉組對腸道微生物生長和穩(wěn)定有促進作用。除對照組外,各組血清中總蛋白、球蛋白和白蛋白差異不顯著(P0.05),添加發(fā)酵羽毛粉對肉雞血清酶活和免疫系統(tǒng)無明顯影響。另外,經(jīng)濟效益分析也證明,使用發(fā)酵羽毛粉替代魚粉飼喂肉雞是可行的,為羽毛粉等非常規(guī)蛋白質飼料資源的開發(fā)和利用奠定基礎。
[Abstract]:With the increasing shortage of feed protein resources and the rapid rise in price, the development of low quality protein resources has become a hot spot of research. The protein content of feather powder is rich, but its structure is stable and insoluble in water. It can not be hydrolyzed by the ordinary protease, and it is difficult to be digested and absorbed by animals. This paper has been screened to get better degradation effect of feather powder. The strains of Bacillus subtilis and Bacillus subtilis were reformed and cloned for the neutral protease and alkaline protease genes of two strains. The protease genes were successfully transformed into Pichia pastoris X33, and the recombinant yeast protease activity and the properties of the protease were analyzed. Finally, the featherpowder was fermented by the recombinant Pichia pastoris and applied to the DNA. In the production of broilers, the feeding effect of fermented feather powder instead of fish meal on broilers was studied. A new technical approach was provided to alleviate the shortage of protein resources and improve the feeding value of feather powder. The main results are as follows: (1) from the first screening of five strains of microorganism, Aspergillus oryzae and Bacillus subtilis producing high activity of protease have been screened. Determination of protease activity and soluble protein content in feather powder, the degradation effect of Aspergillus oryzae and Bacillus subtilis on feather powder was studied. The results showed that two microbial strains could degrade feather powder. After 72 h fermentation, the protease activity of Aspergillus oryzae reached 3256.61 U/g (P0.05), Bacillus subtilis producing protease The activity reached 690.03 U/g (P0.05). The soluble protein content of Aspergillus oryzae Fermented feathers 48 h increased from 2.21 mg/g to 362.07 mg/g (P0.05). (2) RT-PCR technology was used to clone the neutral and alkaline protease genes of Aspergillus oryzae, Bacillus subtilis neutral and alkaline protease genes, and four protease genes published with NCBI. The homology of the protease gene is greater than 99.5%. (3) to reorganize the four protease genes obtained from the cloned gene. The protease gene is reorganized by DNA. The protease gene is connected to the pMD19-T plasmid and transferred into the Escherichia coli. By screening the casein plate, three DNA reformed neutral egg white enzyme genes from Aspergillus oryzae, NPGA-S1, NPGA-S2, NPGA-S3, and one from the blight are obtained. The DNA modified neutral protease gene NPGB-S1. (4) of Bacillus buds (4) converted four DNA protease genes and two neutral protease genes into Pichia pastoris X33. Through recombinant yeast protease activity determination, two protease genes of NPGA-S2 and NPGA-S3 were screened out. (5) NPGA-S2 and NPGA-S3 and four species were first introduced. The initial protease gene was reorganized in second rounds of DNA. A neutral protease DNA restructure gene NPGA-S4 from Aspergillus oryzae was screened, and its electric shock was converted to Pichia pastoris X33. The activity of protease production of the recombinant Pichia pastoris containing three species of Aspergillus oryzae neutral protease was determined, and NPGA-S2 was selected as the best. DNA reformed protease gene. Compared with the initial protease gene, the nucleotide T of the DNA reformed protease gene at 1022 BP mutation was C. The corresponding amino acids increased by 36.4% (P0.05), and pH and thermal stability were significantly increased from the cysteine to the arginine.DNA reformed protease, and the pH and thermal stability were also significantly increased in pH6.5-8.0. Stability (P0.05) was maintained between 30-70 and 6. (6) the protein, calcium and phosphorus metabolism of fermented feather powder was measured by the strong feeding test of chicken. The apparent metabolic rate of protein and calcium phosphorus of Aspergillus oryzae and protease gene recombinant yeast feathers was significantly higher than that of ordinary feather powder and recombinant yeast fermented feathery powder without protease gene (P0.05). Compared with ordinary feather powder, all kinds of amino acids in fermented feather powder (corn) were improved, and the content of lysine content of the recombinant yeast fermented feathery powder containing the reorganized protease gene increased nearly 1 times. (7) using Aspergillus oryzae and protease gene recombinant yeast fermented feathery feathers added to the broiler diet by measuring Huang Yurou. The growth performance, slaughter performance, meat quality, intestinal microorganism, blood biochemical indexes and economic benefits were analyzed. The feasibility of fermented feather meal instead of fish meal on broiler production was studied. The feeding experiment was divided into two stages (1-28d) and later stage (29-56d). In the early and late stages, 250 yellow feathered broilers were divided into five treatments, and 5 were treated with each treatment. Repeat, 10 chickens were repeated each, of which group A was a basal diet containing fish meal (control group), and the B group used Aspergillus oryzae Fermented feathers to replace fish meal in basal diet, and group C with pGAPZ alpha A recombinant Pichia pastoris fermented feathery powder with equal amount of non protease gene instead of fish meal in basal diet (negative control group), and D group with Aspergillus oryzae in the same amount. The gene pGAPZ alpha A recombinant Pichia pastoris fermented feathers to replace the fish meal in the basal diet, and the E group used the pGAPZ alpha A recombinant Pichia pastoris feathers from the same amount of Aspergillus oryzae DNA to replace the fish meal in the basal diet with the same amount of Aspergillus oryzae DNA (NPGA-S2). The results showed that the early and later period of the feeding test, except for the negative control group, added Aspergillus oryzae and protease. There was no significant difference in daily weight gain, daily feed intake, feed conversion rate and nutrient metabolism rate (P0.05) in each group and control group (P0.05), but the total net rate and meat quality were higher than those of other groups, but the difference was not significant (P0.05), Aspergillus oryzae. The protease activity of the enteric fermented feather meal group containing the protease gene was significantly higher than that of the control group and the negative control group (P0.05). The number of intestinal microbes, the distribution of intestinal microflora and the microbial diversity in each group were basically the same, including the DNA reorganized protease gene regrouping yeast fermented feather powder group to the intestinal microorganism growth. In addition to the control group, there was no significant difference in total protein, globulin and albumin in the serum (P0.05), but the addition of fermented feather powder had no obvious effect on the serum enzyme activity and immune system of broilers. In addition, the analysis of economic benefits also proved that it was feasible to feed the chicken with fermented feather powder instead of fish meal, and the feather powder was very important. Laying the foundation for the development and utilization of protein feed resources.
【學位授予單位】：河南農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S831

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