【摘要】：對(duì)豬樣品及東部地區(qū)苜蓿樣品中的乳酸菌進(jìn)行系統(tǒng)的分類鑒定及理化性能研究;篩選具有較強(qiáng)抑菌活性、耐受人工胃腸液環(huán)境并快速產(chǎn)酸的優(yōu)良乳酸菌應(yīng)用于保育豬生產(chǎn)中,評(píng)價(jià)其對(duì)保育豬生長(zhǎng)過(guò)程中排泄物及腸道微生物多樣性,以及對(duì)保育豬生長(zhǎng)性能的影響。為抗生素低減型生豬生產(chǎn)找到了新的途徑,為乳酸菌添加劑的制作提供優(yōu)良的菌種。根據(jù)乳酸菌的生理生化特征、16S r RNA基因序列分析,可以從豬樣品中一共分離到52株乳酸菌,這些菌株經(jīng)過(guò)鑒定,提示一共包括了Lactobacillus reuteri,Lactobacillus acidophilus,Lactobacillus amylovorus,Lactobacillus johnsonii,Lactobacillus fermentum,Lactobacillus,Bifidobacterium,Lactobacillus salivarius,Weissella,Morganella morganii,Streptococcus alactolyticus,Enterococcus faecalis,Lactococcus lactis和Enterococcus在內(nèi)的14種乳酸菌菌種。通過(guò)雙層牛津杯平板法從這些菌株中篩選到2株對(duì)大腸桿菌、沙門(mén)氏菌及李斯特氏菌均有較強(qiáng)抑菌活性的乳酸菌P83和P84。對(duì)其抑菌活性物質(zhì)做初步分析的時(shí)候可以發(fā)現(xiàn),兩株菌株的發(fā)酵上清液在排除酸和過(guò)氧化氫的作用后依然保持較強(qiáng)的抑菌活性;以上的結(jié)果表明P83和P84的發(fā)酵上清液中除了酸和過(guò)氧化氫外,還可能存在著細(xì)菌素等抑菌活性物質(zhì)。從我國(guó)東部河北,上海,遼寧,江蘇、山東和北京6個(gè)地區(qū)共收集到苜蓿樣品125份,并且從其中分離出242株的乳酸菌,采用雙層牛津杯法評(píng)定了這242株乳酸菌的抑菌活性,并且從中篩選出來(lái)了62株抑菌活性比較強(qiáng)的優(yōu)良的乳酸菌;對(duì)這62株乳酸菌進(jìn)行16S r RNA基因序列分析發(fā)現(xiàn),分離到的桿菌較少共13株,其中包括魏斯氏菌屬5株,共4個(gè)種:Weissella,Weissella mino,Weissella confusa和Weissella viridescens;乳酸桿菌屬一共8株,一共有3個(gè)種:Lactobacillus curvatus,Lactobacillus sakei和Lactobacillus plantarum。分離到的球菌數(shù)量比較多,一共有49株球菌,其中包括了腸球菌屬9株,一共有4個(gè)種:Enterococcus faecalis,Enterococcus mundtii,Enterococcus和Enterococcus hirae;明串珠菌屬一共17株,一共有3個(gè)種:Leuconostoc,Leuconostoc citreum和Leuconostoc mesenteroides;戊糖片球菌屬一共9株,共1個(gè)種:Pediococcus pentosaceus;乳酸乳球菌屬一共14株,共3個(gè)種:Lactococcus lactis,Lactobacillus sakei和Lactococcus garvieae;通過(guò)生理生化實(shí)驗(yàn)以及人工模擬胃腸液實(shí)驗(yàn)對(duì)這些乳酸菌的一些生長(zhǎng)的特性和對(duì)胃腸液耐受的能力進(jìn)行進(jìn)一步研究,其中菌株ZZU A190在p H3.0~10.0,鹽濃度0~6.5%,人工腸液和人工胃液等條件下都表現(xiàn)出很強(qiáng)的耐受能力。通過(guò)生理生化實(shí)驗(yàn)、抑菌活性檢測(cè)實(shí)驗(yàn)以及人工胃腸液耐受實(shí)驗(yàn),對(duì)從豬樣品和苜蓿樣品中分離到的及實(shí)驗(yàn)室保存的乳酸菌進(jìn)行篩選,篩選到抑菌活性較強(qiáng)和對(duì)人工胃腸液有較好耐受性能的菌株ZZU A190和ZZU A203作為制備乳酸菌添加劑的菌種。將它們作為添加劑進(jìn)行保育豬飼喂研究的結(jié)果顯示,抗生素或抗生素與乳酸菌發(fā)酵飼料聯(lián)合使用對(duì)保育豬糞便中乳酸菌的數(shù)量無(wú)顯著影響。單獨(dú)使用乳酸菌發(fā)酵飼料不僅能夠顯著增加保育豬糞便中乳酸菌的含量,而且使得糞便中大腸桿菌的數(shù)量顯著減少。這可能與抗生素的長(zhǎng)期使用導(dǎo)致產(chǎn)生了致病菌的抗性菌株,致使腸道微生物菌群失調(diào)有關(guān)。與抗生素陰性對(duì)照相比,日糧中只添加乳酸菌發(fā)酵飼料或抗生素與乳酸菌發(fā)酵飼料聯(lián)合添加均可以顯著提高保育豬的日增重;與抗生素陽(yáng)性對(duì)照相比,僅單獨(dú)添加乳酸菌發(fā)酵飼料能夠顯著提高保育豬的日增重,降低腹瀉率與死亡率。
[Abstract]:This * is a systematic identification and identification of Lactobacillus in pig samples and alfalfa samples in the eastern region, and screening of excellent lactic acid bacteria with strong antibacterial activity, tolerance to the artificial gastrointestinal fluid environment and rapid acid production. The * * is used to evaluate the diversity of feces and intestinal microflora in the growing process of conservation pigs. And the effect on the growth performance of the * * pigs. It has found a new way for the production of low production of antibiotics, and provided excellent strains for the production of lactic acid bacteria additives. According to the physiological and biochemical characteristics of lactic acid bacteria, 16S * r RNA gene sequence analysis, 52 strains of lactic acid bacteria can be isolated from pig samples. Including Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus, Bifidobacterium, Lactobacillus salivarius, Weissella, Morganella morganii, Streptococcus alactolyticus, Enterococcalis, Lactobacillus lactius and Enterococcus Two strains of lactic acid bacteria P83 and P84 with strong antibacterial activity against Escherichia coli, Salmonella and Listeria were screened from these strains by double-layer Oxford cup plate method. The results showed that there might be bacteriostatic substances such as bacteriocins in the supernatant of P83 and P84 besides acid and hydrogen peroxide. 125 alfalfa samples were collected from 6 areas of Hebei, Shanghai, Liaoning, Jiangsu, Shandong and Beijing in eastern China and were separated from them. A total of 242 strains of lactic acid bacteria were isolated and evaluated by double-layer Oxford cup method, and 62 strains of lactic acid bacteria with strong antibacterial activity were screened out. Weissella, Weissella mino, Weissella confusa and Weissella viridescens; Lactobacillus, Lactobacillus curvatus, Lactobacillus sakei and Lactobacillus plantarum. A total of 49 strains of cocci were isolated, including 9 strains of Enterococcus, and 4 species: Enterococcus FA FA fa. Eccalis, Enterococcus mundtii, Enterococcus and Enterococcus hirae; Leuconostoc, Leuconostoc citreum and Leuconostoc mesenteroides; Pediococcus pentosaceus; Lactococcus lactis, Lactococcus lactis, Lactococci, Lactococcus lactis, Lactobacillus enteroides; Pediococcus pentosaceus Lus sakei and Lactococcus garvieae. Some growth characteristics of these lactic acid bacteria and their ability to tolerate gastrointestinal juice were further studied by physiological and biochemical experiments and artificial gastrointestinal juice simulation experiments. Strain ZZU A190 showed strong tolerance under the conditions of pH 3.0-10.0, salt concentration 0-6.5%, artificial intestinal juice and artificial gastrointestinal juice. Through physiological and biochemical experiments, bacteriostatic activity test and artificial gastrointestinal juice tolerance test, * lactic acid bacteria isolated from pig and alfalfa samples and lab preserved were screened. Strain ZZU A190 and ZZU A203, which had strong antibacterial activity and good tolerance to artificial gastrointestinal fluid, were screened for Lactobacillus preparation. The results of the study showed that antibiotics * or antibiotics combined with Lactobacillus fermented feed had no significant effect on the number of lactic acid bacteria * in the pig feces. Lactobacillus fermented feed alone could significantly increase the content of lactic acid bacteria in pig feces, and * This may be related to the long-term use of antibiotics resulting in the production of pathogenic bacteria resistant strains, resulting in the imbalance of intestinal microbial flora. * the daily gain of raising pigs is higher than that of the positive antibiotics. Only adding lactic acid bacteria fermented feed alone can significantly increase the daily gain * and reduce the diarrhoea rate and mortality of the pigs.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828

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