飼用益生菌的篩選及其菌劑制備技術(shù)研究
發(fā)布時(shí)間：2018-10-15 19:23
【摘要】：羅伊氏乳桿菌作為一種重要的動(dòng)物微生態(tài)制劑中的益生菌,其菌體本身及代謝產(chǎn)物能夠抑制仔豬腸道過路菌和病原菌的粘附及生長(zhǎng),因此能夠提高仔豬的免疫力和降低仔豬死亡率,從而大大的改善了養(yǎng)殖行業(yè)的質(zhì)量。本課題是從畜禽糞便中有針對(duì)性、目的性地篩選出產(chǎn)酸菌株,且對(duì)其進(jìn)行體外性能評(píng)價(jià)。主要研究如下:(1)飼用益生菌的篩選。經(jīng)過初篩及耐酸耐膽鹽復(fù)篩,從仔豬糞便中篩選出三株產(chǎn)酸菌株,通過菌體特征、糖發(fā)酵等生理生化實(shí)驗(yàn)以及分子生物學(xué)鑒定,最后確定其為羅伊氏乳桿菌,且其中耐受性最強(qiáng)的為NCU801。(2)NCU801生長(zhǎng)曲線繪制。羅伊氏乳桿菌NCU801屬于異型發(fā)酵,能夠利用葡萄糖代謝產(chǎn)生乳酸、乙酸,同時(shí)還能夠產(chǎn)生乙醇、二氧化碳等,0-4 h、4-16 h、16-24 h及24 h后分別為延滯期、對(duì)數(shù)期、穩(wěn)定期和衰亡期。(3)NCU801的動(dòng)物胃腸道耐受性能研究。L.reuteri NCU801在pH 2.5動(dòng)物胃液時(shí),菌株D值大于60 min;通過模擬動(dòng)物腸液耐受性實(shí)驗(yàn),菌體在腸液作用4 h后,存活率仍有38.6%。在膽鹽濃度為0.3%的改良MRS培養(yǎng)基中作用24 h后,活菌數(shù)下降一個(gè)多數(shù)量級(jí),但活菌數(shù)仍有9.7×105 CFU/m L�？梢�,L.reuteri NCU801具有較強(qiáng)抵抗動(dòng)物胃腸道逆環(huán)境的能力。(4)NCU801的抑菌性能研究。通過對(duì)腸道中常見的9種致病菌的抑菌實(shí)驗(yàn),發(fā)現(xiàn)羅伊氏乳桿菌NCU801的菌體、發(fā)酵液和無菌上清液均具有抑菌性,且發(fā)酵液和發(fā)酵液的無菌上清液的抑菌效果較菌體更強(qiáng),其中發(fā)酵液和無菌上清液對(duì)大腸埃希氏菌抑制作用最強(qiáng),抑菌圈直徑達(dá)18 mm以上,由此可以判斷出羅伊氏乳桿菌NCU801的抑菌作用主要依賴于本身的代謝產(chǎn)物。(5)NCU801腸道粘附性能研究。NCU801對(duì)Caco-2細(xì)胞的粘附,其粘附指數(shù)同菌體濃度、菌體發(fā)酵時(shí)間、孵化時(shí)間及pH值都有對(duì)應(yīng)的關(guān)系;而NCU801對(duì)Caco-2細(xì)胞粘附指數(shù)最大值達(dá)(5.08±0.15)CFU/cell。NCU801活菌、死菌均能在置換、競(jìng)爭(zhēng)和排斥性的抑制病原菌對(duì)Caco-2細(xì)胞粘附;其中抑制粘附作用最大的為活菌懸液對(duì)大腸桿菌排斥粘附抑制,抑制粘附率達(dá)86.8±4.5(%)。(6)NCU801高密度培養(yǎng)條件研究。通過單因素試驗(yàn),確定了L.reuteri NCU801最佳碳源、氮源、緩沖鹽、無機(jī)鹽離子分別為葡萄糖、牛肉膏和酵母膏、NaAc、Mg2+;而正交試驗(yàn)優(yōu)化的培養(yǎng)基配方為:牛肉膏1.5%,酵母膏1.5%,葡萄糖5%,MgSO4 0.06%,MnSO4 0.01%,Na Cl 0.001%,K2HPO4 0.2%,檸檬三銨0.2%,CH3COONa 0.3%。菌液濃度達(dá)1.3×109 CFU/mL。
[Abstract]:Lactobacillus Roy is one of the most important probiotics in animal microecology. Its bacteria and metabolites can inhibit the adhesion and growth of intestinal tract bacteria and pathogens in piglets. Therefore, it can improve the immunity of piglets and reduce the mortality of piglets, thus greatly improving the quality of the breeding industry. The purpose of this study was to screen acid-producing strains from animal feces and evaluate their performance in vitro. The main results are as follows: (1) screening of probiotics for feeding. Three strains of acid-producing strains were screened from piglets' feces after primary screening and repeated screening of acid-tolerant bile salt. The strains were identified as Lactobacillus Roy by physiological and biochemical experiments, such as sucrose fermentation and molecular biological identification. Among them, NCU801. (2) NCU801 growth curve was the most tolerant. Lactobacillus Roy NCU801 is a kind of abnormal fermentation, which can produce lactic acid, acetic acid, ethanol, carbon dioxide and so on by glucose metabolism. After 0-4 h, 4-16 h, 16-24 h and 24 h, the delay period is logarithmic phase, respectively. (3) the animal gastrointestinal tolerance of NCU801. When pH 2.5 animal gastric juice, the strain D value was greater than 60 min;. The bacteria survived 38.6% after the intestinal fluid was exposed for 4 h. After 24 h exposure to the modified MRS medium with 0.3% bile salt concentration, the number of viable bacteria decreased by one order of magnitude, but the number of live bacteria was still 9.7 脳 10 ~ 5 CFU/m 路L ~ (-1). It can be seen that L.reuteri NCU801 has a strong ability to resist the adverse environment of gastrointestinal tract. (4) the bacteriostasis of NCU801. The bacteriostatic experiments of 9 common pathogenic bacteria in intestinal tract showed that the bacteria of Lactobacillus Roy NCU801, fermentation broth and aseptic supernatant were all bacteriostasis, and the bacteriostasis effect of aseptic supernatant of fermentation broth and fermentation broth was stronger than that of bacteria. Among them, fermentation broth and aseptic supernatant had the strongest inhibitory effect on Escherichia coli, and the diameter of inhibition circle was more than 18 mm. It can be concluded that the bacteriostatic effect of Lactobacillus Roy NCU801 mainly depends on its metabolites. (5) the adhesion of NCU801 to Caco-2 cells is studied. The adhesion index of NCU801 to Caco-2 cells is the same as the cell concentration and fermentation time. The maximum adhesion index of NCU801 to Caco-2 cells was (5.08 鹵0.15) CFU/cell.NCU801, and the dead bacteria could inhibit the adhesion of pathogenic bacteria to Caco-2 cells by replacement, competition and rejection. Among them, the most effective adhesion inhibition was the rejection of adhesion by live bacteria suspensions to Escherichia coli, and the inhibition rate of adhesion was 86.8 鹵4.5 (%). (6) NCU801 high density culture conditions. The optimum carbon source, nitrogen source, buffer salt and inorganic salt ion of L.reuteri NCU801 were determined as glucose, beef paste and yeast extract, NaAc,Mg2 respectively. The optimum medium formula of orthogonal experiment was: beef paste 1.5, yeast extract 1.5, glucose 5 and MgSO4 0.06, MnSO4 0.0110, Na Cl 0.001, K2HPO4 0.2cm, lemon triammonium 0.2, CH3COONa 0.3. The concentration of bacteria solution was 1.3 脳 10 ~ 9 CFU/mL..
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S816.3
[Abstract]:Lactobacillus Roy is one of the most important probiotics in animal microecology. Its bacteria and metabolites can inhibit the adhesion and growth of intestinal tract bacteria and pathogens in piglets. Therefore, it can improve the immunity of piglets and reduce the mortality of piglets, thus greatly improving the quality of the breeding industry. The purpose of this study was to screen acid-producing strains from animal feces and evaluate their performance in vitro. The main results are as follows: (1) screening of probiotics for feeding. Three strains of acid-producing strains were screened from piglets' feces after primary screening and repeated screening of acid-tolerant bile salt. The strains were identified as Lactobacillus Roy by physiological and biochemical experiments, such as sucrose fermentation and molecular biological identification. Among them, NCU801. (2) NCU801 growth curve was the most tolerant. Lactobacillus Roy NCU801 is a kind of abnormal fermentation, which can produce lactic acid, acetic acid, ethanol, carbon dioxide and so on by glucose metabolism. After 0-4 h, 4-16 h, 16-24 h and 24 h, the delay period is logarithmic phase, respectively. (3) the animal gastrointestinal tolerance of NCU801. When pH 2.5 animal gastric juice, the strain D value was greater than 60 min;. The bacteria survived 38.6% after the intestinal fluid was exposed for 4 h. After 24 h exposure to the modified MRS medium with 0.3% bile salt concentration, the number of viable bacteria decreased by one order of magnitude, but the number of live bacteria was still 9.7 脳 10 ~ 5 CFU/m 路L ~ (-1). It can be seen that L.reuteri NCU801 has a strong ability to resist the adverse environment of gastrointestinal tract. (4) the bacteriostasis of NCU801. The bacteriostatic experiments of 9 common pathogenic bacteria in intestinal tract showed that the bacteria of Lactobacillus Roy NCU801, fermentation broth and aseptic supernatant were all bacteriostasis, and the bacteriostasis effect of aseptic supernatant of fermentation broth and fermentation broth was stronger than that of bacteria. Among them, fermentation broth and aseptic supernatant had the strongest inhibitory effect on Escherichia coli, and the diameter of inhibition circle was more than 18 mm. It can be concluded that the bacteriostatic effect of Lactobacillus Roy NCU801 mainly depends on its metabolites. (5) the adhesion of NCU801 to Caco-2 cells is studied. The adhesion index of NCU801 to Caco-2 cells is the same as the cell concentration and fermentation time. The maximum adhesion index of NCU801 to Caco-2 cells was (5.08 鹵0.15) CFU/cell.NCU801, and the dead bacteria could inhibit the adhesion of pathogenic bacteria to Caco-2 cells by replacement, competition and rejection. Among them, the most effective adhesion inhibition was the rejection of adhesion by live bacteria suspensions to Escherichia coli, and the inhibition rate of adhesion was 86.8 鹵4.5 (%). (6) NCU801 high density culture conditions. The optimum carbon source, nitrogen source, buffer salt and inorganic salt ion of L.reuteri NCU801 were determined as glucose, beef paste and yeast extract, NaAc,Mg2 respectively. The optimum medium formula of orthogonal experiment was: beef paste 1.5, yeast extract 1.5, glucose 5 and MgSO4 0.06, MnSO4 0.0110, Na Cl 0.001, K2HPO4 0.2cm, lemon triammonium 0.2, CH3COONa 0.3. The concentration of bacteria solution was 1.3 脳 10 ~ 9 CFU/mL..
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S816.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 王玲;蒲萬霞;扎西英派;孟曉琴;郭福存;李金善;鄧海平;;“斷奶安”防治仔豬腹瀉的作用機(jī)理及應(yīng)用研究[J];安徽農(nóng)業(yè)科學(xué);2009年04期
2 杜丹,王斌,陳丙波 ,魏泓;兩株乳酸桿菌益生菌特性的研究[J];氨基酸和生物資源;2005年01期
3 唐芳索;;微生態(tài)制劑在犬腹瀉病治療中的應(yīng)用[J];今日畜牧獸醫(yī);2008年02期
4 倪學(xué)勤;Joshua Gong;曾東;Hai Yu;Weido Si;周小秋;;豬源乳酸桿菌對(duì)鼠傷寒沙門氏菌DT104在豬腸道上皮細(xì)胞IPEC-J2上粘附的影響[J];微生物學(xué)通報(bào);2008年07期
5 王紅艷,賈繼輝,邢詠梅,陳春燕,周亞濱,唐偉,程軼U，
本文編號(hào)：2273580
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2273580.html
最近更新
教材專著
