【作者】 庄国宏；

【导师】 秦爱建； 江国托；

【作者基本信息】 扬州大学 ， 兽医， 2007， 硕士

【摘要】 本研究从人和动物肠道分离、筛选出一株能产生具有广谱抗菌活性的嗜酸乳杆菌,对其抗菌活性物质(细菌素)从生物学特性、分离提取、基因诱变和发酵工艺等方面进行了初步研究,为食品的天然生物防腐和动物肠道细菌性疾病防治产品的开发及应用提供参考依据。1.从健康人群粪便、动物肠道及新鲜粪便中分离获得一株产生抗菌活性物质的乳酸杆菌,通过形态学及生理、生化鉴定确认其为嗜酸乳杆菌。对该菌株产生的抗菌物质所进行的生物学特性的初步研究结果表明:分离菌株Sr-1产生的抗菌物质主要成分是蛋白质类物质,对大肠杆菌(E.coli )、沙门氏菌(S.pullorum)、金黄色葡萄球菌(S.aureus)和温和气单胞菌(A.sobria)等革兰氏阴性菌和革兰氏阳性菌都具有抗菌作用,初步判断该抗菌物质属细菌素类。2.采用硫酸铵沉淀方法,将发酵液中的细菌素进行沉淀、溶解,通过生物胶Bio-Gel?P-6分析并对各峰进行收集、制备,采用琼脂扩散方法对收集各峰进行抑菌试验,发现在收集的蛋白峰中有两个抗菌蛋白峰,这表明嗜酸乳杆菌Sr-1产生的细菌素是由两种不同分子量的蛋白质组成的。通过Tricine-SDS-PAGE检测,只能检测到一条分子量相对较大的细菌素,分子量在8,100Da左右。对分离出的细菌素性质的研究表明,细菌素在pH值大于5.0以上的环境下,具有较好的热稳定性和酸、碱稳定性,在温度90℃处理30min对活性没有影响,121℃处理30min活性仅损失11.1%。经过蛋白酶处理后细菌素的抗菌活性有不同程度的损失。并对细菌素的抗菌效果进行了测定,表明其抗菌作用显著,具有广谱的抗菌效果。3.以分离获得产细菌素的一株嗜酸乳杆菌(Sr-1)为出发菌株,经紫外线照射(UV)和亚硝基胍(NTG)多轮复合诱变,遴选出一株高产类细菌素的目的突变株(SUN-211),根据对SUN-211发酵的代谢产物进行检测,结果显示,发酵液产生的细菌素效价较未诱变前提高了47.12%。经过多次传代,该性状且具有良好的遗传稳定性。4.培养基的组成和培养条件对嗜酸乳杆菌Sr-1的生长及细菌素的产生有很大的影响。对产细菌素的嗜酸乳杆菌Sr-1发酵培养基和培养条件的优化研究结果表明:以MRS为基础的发酵培养基对细菌的生长和细菌素的产生分别提高了53.6%和35.3 %,筛选发酵培养基配方的主要成分为:大豆蛋白胨2.5 %、酵母粉0.4 %、葡萄糖2 %和维生素0.3 %,培养基的最适初始pH值为6.5;产细菌素最适温度为32℃;发酵时间为24h;大分子蛋白对细菌素的产生具有明显的诱导作用。5.对嗜酸乳杆菌Sr-1产生的细菌素粗提物在食品防腐和动物肠道疾病治疗的应用效果的考察结果表明,细菌素在消毒鲜牛奶的保藏中具有明显的防腐效果,添加细菌素试验组在37℃保存24h、48h后与对照组相比细菌总数相差4-5个数量级。细菌素对经济动物和猪等胃肠道疾病具有良好的防治效果,相对于抗生素而言,细菌素的治疗恢复期缩短了1-2天,愈后生长良好。更多还原

【Abstract】 One local strain of Lactobacillus acidophilus (L. acidophilus) with broadspectrum antimicrobial activity was seperated and screened out from intestine of human and animals in the present studies. The antimicrobial substance (namely bacteriocin) secreted by the bacterium was studied in the aspects of biological character, extraction, mutagenesis and fermentation. Results lay foundation for developing and applying of food biological antisepsis and medicament for animal intestinal bacterial diseases.1. One local strain of Lactobacillus with antimicrobial activity seperated from feces of human and animals was confirmed as L. acidophilus by the tests on its morphological, physiological and biochemical character. Results of the biological character of the secreted antimicrobial substance showed that the antimicrobial substance secreted by the separated stain Sr-1 is protein. The antimicrobial substance was confirmed as bacteriocin for its broadspectrum antimicrobial property to both gram-positive and gram-negative bacteria such as E. coli, S.pullorum, S. aureus and A. sobria.2. Bacteriocin was precipitated by ammonium sulfate from fermented broth and further separated by Bio-Gel?P-6 chromatography, then the antimicrobial activity of all the collected fractions was tested by gel diffusion test, results showed that bacteriocin secreted by L. acidophilus Sr-1 contain two types of protein since two of them has antimicrobial property. Only the one with higher molecular weight can be showed on Tricine-SDS-PAGE gel, and its molecular weight is 8100Da or so. The purified bacteriocin has fine stability against heat alkale when pH is above 5.0. The antimicrobial activity stay still after being heated at 90℃for 30min, lost only 11.1% after being heated at 121℃for 20min, and lost to a certain extent after being digested by proteinase. The purified bacteriocin has perfect and broadspectrum antimicrobial effect.3. The segregated Lactobacillus acidophilus (Sr-1) with the capability of producing the bacteriocin as original strain was developed into a mutant stock (SUN-211) with higher production of bacteriocin by selected strictly, after repeated induction and mutation with the combination of UV administer radiation and NTG,the detection of SUN-211 metabolic product demonstrated bacteriocin valency induced by the broth increased 47.12%, compared with that absent of induction and mutation. The gained character was with excellent genetic stability, which was indicted after the strain passages.4. The composition of medium and condition of cultivate took great influence on the growth of Lactobacillus acidophilus Sr-1 and the product of bacteriocin were tested. The studies on optimization of media composition and cultivate condition for the fermentation of Lactobacillus acidophilus Sr-1 indicated the fermentation medium based on MRS could increase 53.6% and 35.3% of the growth of bacteria and the production of bacteriocin, respectively. The major composition of this fermentation medium included: soy peptone2.5%, yeast powder 0.4%, glucose 2% and vitamin 0.3%; the optical incipient pH of medium was 6.5; the optical temperature for induction of bacteriocin was 32℃; the best fermentation stage was 24h; and the giant molecule protein played the important role on the induction of bacteriocin.5. The examination of effect on bacteriocin crude extract secreted by Lactobacillus acidophilus Sr-1 to food antisepticise and cure of animal intestinal tract diseases demonstrated that bacteriocin induced the conspicuous antiseptic effect on the conservation of sterilized fresh milk: the number of bacteria in groups with bacteriocin differed in 4 to 5 order of magnitude from that in control groups under 37℃after 24, 48h, respectively, indicating bacteriocin good at antisepticising. Bacteriocin also showed the excellent prevention and cure effect on gastrointestinal tract diseases of economic animal and pig or other animals. Comparing with antibiotic, bacteriocin shorted 1-2 days of convalescence, showed the better growth after bacteriocin treatment.更多还原