【摘要】：為研制出高活性飼用益生菌制劑替代仔豬飼料中添加的抗生素。本研究從健壯仔豬糞便中篩選出具有較強(qiáng)產(chǎn)酸能力的乳酸菌,并對菌株生長特性,耐受動物消化道逆環(huán)境能力、抑制病原菌、粘附小腸上皮細(xì)胞等益生特性及菌劑制備工藝進(jìn)行研究。主要研究成果如下:(1)豬源乳酸菌的篩選及鑒定。通過添加溴甲酚紫指示劑的MRS進(jìn)行初次篩選,得到26株疑似目標(biāo)菌株;經(jīng)過耐酸耐膽鹽復(fù)篩,得到7株對低pH和高濃度膽鹽具有一定耐受能力的菌株。通過生理生化、分子生物等手段將其中耐酸耐膽鹽性能最好的兩株菌分別鑒定為植物乳桿菌NCU156和戊糖片球菌NCU301。(2)NCU156生長特性。NCU156能夠產(chǎn)生大量的乳酸使培養(yǎng)基pH降低至3.6左右。0-2 h是生長延滯期,2-12 h是對數(shù)期,12-32 h是穩(wěn)定期,32 h以后為衰亡期。(3)NCU156動物胃腸道環(huán)境耐受性。NCU156在pH2.5的MRS中4 h存活率為87.31%。在3 g/L的膽鹽濃度下4 h存活率仍有41.88%;在模擬胃液中處理4 h菌數(shù)稍有增加(P0.05),接入模擬腸液后10 h活菌數(shù)在108 CFU/mL以上,說明NCU156能夠順利達(dá)到動物腸道。(4)NCU156粘附性能。NCU156粘附能力在一定范圍內(nèi)呈菌濃度和Ca2+濃度依賴性,在作用2 h粘附達(dá)到平衡,在穩(wěn)定期中期(20 h)粘附最穩(wěn)定;對病原菌粘附的抑制作用中,排斥性的粘附抑制作用效果最強(qiáng),其次是競爭性的粘附抑制,最后是置換性的粘附抑制。(5)NCU156抑菌性能。NCU156對常見7株致病菌的抑菌性研究表明,對銅綠假單胞菌抑制效果最好,抑菌圈的直徑達(dá)23 mm以上,菌體沒有抑菌性;發(fā)酵液抑菌性不受高溫、過氧化性酶、蛋白酶等處理方式的影響,故其抑菌性可能主要來源于所產(chǎn)生的乳酸;NCU156藥敏結(jié)果顯示其對四環(huán)素類、青霉素類抗生素敏感,對頭孢類、糖肽類、喹諾酮類抗生素均耐藥。(6)NCU156菌劑制備工藝優(yōu)化。以活菌數(shù)為指標(biāo)通過單因素實(shí)驗(yàn)和BOX-Behnken響應(yīng)面實(shí)驗(yàn)設(shè)計(jì)優(yōu)化菌劑制備的工藝,最佳發(fā)酵條件為:接種量4.45%、料液比1:0.61、發(fā)酵溫度30.6℃、發(fā)酵時間46.40 h。優(yōu)化后的烘干條件為熱風(fēng)干燥50℃、4 h。
[Abstract]:In order to develop a high-activity probiotic agent for feeding to replace the antibiotics added in the feed of piglets. In this study, lactic acid bacteria with strong acid-producing ability were screened from the feces of healthy piglets, and the growth characteristics of the strains, tolerance to the adverse environment of the digestive tract of animals, and inhibition of pathogenic bacteria were studied. The probiotics of adhesion to intestinal epithelial cells and the preparation of bacteria were studied. The main results are as follows: (1) screening and identification of porcine lactic acid bacteria. Through the initial screening of MRS with bromocresol violet indicator, 26 suspected target strains were obtained, and 7 strains with certain tolerance to low pH and high concentration bile salt were obtained by repeated screening with acid tolerance and bile salt tolerance. Through physiology and biochemistry, The two strains with the best acid-tolerant bile salt tolerance were identified as Lactobacillus plantarum NCU156 and NCU301. (2) NCU156 by molecular biology. NCU156 can produce a large amount of lactic acid to reduce the pH to 3. The growth delay period was about 0.6 h and 0.2 h, respectively. 2-12 h was logarithmic phase, 12-32 h was stable phase, and 32 h later was decaying stage. (3) NCU156 animal gastrointestinal environment tolerance. The survival rate of NCU156 in MRS of pH2.5 was 87.31 h. The survival rate of 4 h was still 41.88 at the concentration of 3 g / L of bile salt. The number of bacteria in simulated gastric juice increased slightly after 4 h treatment (P0.05), and the number of live bacteria was more than 108 CFU/mL after 10 hours of inoculation. The results showed that NCU156 could reach the animal intestinal tract smoothly. (4) the adhesion ability of NCU156 was in a certain range of concentration and Ca2 concentration dependent, the adhesion of NCU156 reached equilibrium at 2 h, and the most stable in the middle of stable phase (20 h). In the inhibition of the adhesion of pathogenic bacteria, the repellent adhesion inhibition was the strongest, followed by the competitive adhesion inhibition. The results showed that NCU156 had the best inhibitory effect on Pseudomonas aeruginosa, the diameter of inhibition circle was over 23 mm, and the bacteriostasis of NCU156 was not. The bacteriostasis of fermentation broth is not affected by high temperature, superoxide enzyme, protease and so on, so its bacteriostasis may mainly come from the lactic acid produced. The results of NCU156 showed that it was sensitive to tetracyclines penicillin antibiotics and resistant to cephalosporins glycopeptides and quinolones. (6) the preparation process of NCU156 bacteria was optimized. The optimum fermentation conditions were as follows: inoculation amount 4.45, ratio of material to liquid 1: 0.61and fermentation temperature 30.6 鈩，

本文編號：2360317