【摘要】：為了得到高抗逆性、高生物活性的益生菌菌株,研究其生理特性,以期進(jìn)一步地在生產(chǎn)上應(yīng)用,本研究從健康豬的新鮮糞便及腸道內(nèi)容物、傳統(tǒng)食物等來源分離篩選目標(biāo)益生菌,分離得到兩株抗逆性強(qiáng)、具有潛在益生性能的菌株ZZS16-3和菌株LYP06-2,兩者都具有較強(qiáng)的抗逆性,在人工胃液、人工腸液、不同濃度的膽鹽環(huán)境中存活,并保持較高的存活率。細(xì)菌的菌落形態(tài)觀察、染色鏡檢、生化試驗(yàn)以及16S rDNA序列分析及同源性比較,綜合確定菌株ZZS16-3為枯草芽孢桿菌,菌株LYP06-2為植物乳桿菌�？莶菅挎邨U菌ZZS16-3能夠產(chǎn)生多種酶類,如蛋白酶、脂肪酶、淀粉酶、纖維素酶和木聚糖酶等,并且能夠抑制金黃色葡萄球菌,菌株LYP06-2具有產(chǎn)酸的特性,能夠抑制大腸桿菌,具有潛在的益生性能。從毒理學(xué)角度評(píng)價(jià)枯草芽孢桿菌ZZS16-3和植物乳桿菌LYP06-2這兩種菌的安全性,進(jìn)行急性毒性試驗(yàn),發(fā)現(xiàn)試驗(yàn)昆明小鼠未出現(xiàn)中毒癥狀,無小鼠死亡,剖檢未見異常,兩者的急性毒性試驗(yàn)最大耐受劑量MTD15000 mg/Kg,按照GB15193.3-2003中急性毒性(LD50)劑量分級(jí)表,屬于無毒級(jí)別。30天大鼠實(shí)驗(yàn)結(jié)果表明,與對(duì)照組比較,復(fù)合菌液對(duì)大鼠生長(zhǎng)發(fā)育、代謝無不良影響,臟器指數(shù)、血常規(guī)、血液生化及病理學(xué)檢查均未見異常。通過單因素分析,確定最佳碳源為酵母浸粉,最佳氮源為明膠,最佳無機(jī)鹽為CaCl2,通過正交實(shí)驗(yàn)分析得到最佳培養(yǎng)基配方為：0.5%酵母浸粉、1.5%明膠、1.5%CaCl2;通過發(fā)酵條件分析確定發(fā)酵初始pH為7.2,發(fā)酵溫度37℃,轉(zhuǎn)速為150 r/min。按照1%的接種量接種到裝有6L發(fā)酵培養(yǎng)基的發(fā)酵罐中,初始pH為7.2,培養(yǎng)溫度37℃,轉(zhuǎn)速150 r/min,罐壓0.05 Mpa,通風(fēng)比：1：0.8,培養(yǎng)18 h,得到芽孢生成率90%以上,活菌數(shù)為3.1×109 CFU/mL的枯草芽孢桿菌發(fā)酵液。飼料中添加含有枯草芽孢桿菌ZZS16-3和植物乳桿菌LYP06-2的復(fù)合微生態(tài)制劑在一定程度上提高了仔豬的生長(zhǎng)性能,減少仔豬腹瀉,降低料重比。
[Abstract]:In order to obtain probiotic strains with high resistance to stress and high biological activity, the physiological characteristics of probiotics were studied in order to be further applied in production. Two strains of probiotics, ZZS16-3 and LYP06-2, which have potential probiotics, were isolated and screened from traditional food sources. High survival rate was maintained in different concentrations of bile salt. The colony morphology, staining microscopic examination, biochemical test, 16s rDNA sequence analysis and homology comparison showed that ZZS16-3 was Bacillus subtilis and LYP06-2 was Lactobacillus plantarum. Bacillus subtilis ZZS16-3 can produce a variety of enzymes, such as protease, lipase, amylase, cellulase and xylanase, and can inhibit Staphylococcus aureus. Potential probiotics. The safety of Bacillus subtilis (ZZS16-3) and Lactobacillus plantarum (LYP06-2) were evaluated from the perspective of toxicology. The acute toxicity test was carried out in Kunming mice. The maximum tolerated dose (MTD15000 mg/Kg,) of both acute toxicity tests was according to the acute toxicity (LD50) dose classification table in GB15193.3-2003. The results showed that the compound bacteria solution had no adverse effects on the growth and development and metabolism of the rats compared with the control group. Organ index, blood routine, blood biochemical and pathological examination were not abnormal. Through single factor analysis, the optimum carbon source is yeast powder, and the best nitrogen source is gelatin. The optimum inorganic salt was CaCl2,. The best medium was obtained by orthogonal experiment. The optimum medium was: 0.5% yeast extract 1.5% gelatin, 1.5Cl2, the initial pH was 7.2, the fermentation temperature was 37 鈩，

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