耐氧性双歧杆菌的筛选及其生理特性与应用研究

发布时间：2018-01-16 07:20

  本文关键词：耐氧性双歧杆菌的筛选及其生理特性与应用研究　出处：《浙江大学》2010年博士论文　论文类型：学位论文

  更多相关文章： 双歧杆菌 耐氧 生理特性 响应面 酸奶

【摘要】：本文从筛选一株耐氧的双歧杆菌出发,并利用形态学、生理生化和分子生物学手段对该菌进行鉴定。在此基础上,对耐氧双歧杆菌的各项生理功能进行了研究,并利用响应面的分析方法对该菌最适的培养条件和培养基进行了优化,对该菌的冷冻干燥保护剂进行了优化设计,并将冻干后的菌粉在酸奶中的应用特性进行了研究,另外,对双歧杆菌对氧胁迫的抵抗机理进行了初探。论文的主要研究结果如下： 利用快速筛选耐氧性双歧杆菌的方法对9株双歧杆菌的耐氧性进行考察,通过增加氧气浓度的方式进行复筛,从中选出一株最耐氧的双歧杆菌,该菌能够在抵抗液体培养时氧气的摄入,获得较高的生长量。利用形态学分析、生理生化特性和16S rRNA及16S-23S ITS序列分析相结合的方法,鉴定得出该耐氧双歧杆菌为bifidobacterium.animalis.subsp.lactis命名为：Bifidobacterium.animalis.subsp.lactis Qq08,简称Bl Qq08. 对Bl Qq08的几项生理功能特性包括耐酸、耐胆汁特性,体外粘附能力及清除胆固醇能力,对常见致病菌的抑制特性进行了研究。结果表明,Bl Qq08与商业菌株相比,表现出更优良的耐酸与耐胆汁特性。能够粘附于Caco-2细胞表面,粘附数为8.57±3.74个/细胞。体外对胆固醇的降解率为53.8%,处于国内先进水平。Bl Qq08的发酵液对常见致病菌大肠杆菌、金黄色葡萄球菌、沙门氏菌具有不同程度的抑制作用。这些优良的特性,使得Bl Qq08具备了成为潜在益生菌的先决条件。 对双歧杆菌抵抗氧胁迫的机理进行了初探。表明双歧杆菌对氧气耐受性的程度不同,是由于其对过氧化氢的敏感程度不同所致,过氧化氢的累积是导致双歧杆菌生长受抑制最主要的因素,SOD的活性与双歧杆菌对氧气的耐受性无直接相关性,而NADH-oxidase和NADH-peroxidase的存在与双歧杆菌对氧气的耐受性有相关性。 利用响应面实验设计的科学方法对Bl Qq08的培养条件进行了系统优化,最终确定培养Bl Qq08的最佳培养基为：0.55%(W/V)乳清粉,1.11%(W/V)聚蛋白胨,0.2%(W/V) K2HPO4,0.1%(W/V) MgCl2.6H2O,0.002%(W/V) CaCl2, 0.001%(W/V) FeCl3,0.1%(W/V) NaHCO3,0.1%(W/V)低聚果糖,pH值7.2。在该条件下培养24h后,活菌数为：3.45×109CFU/mL,较优化前提高了5.39倍。 对Bl Qq08的冷冻干燥保护剂进行了优化设计,适合Bl Qq08的冷冻干燥保护剂配方为：15%脱脂乳,8%乳糖,1.5%抗坏血酸(Vc),3%甘油。冷冻干燥存活率高达91.23%,高于目前文献报道的水平。冻干后的菌粉在非严格厌氧常温及4℃分别储存1个月,菌数无明显降低,B1 Qq08冻干粉具有良好的储存稳定性。 将B1 Qq08添加至酸奶中,研究B1 Qq08的加入在保质期内对酸奶品质的影响。结果表明B1 Qq08对酸奶的理化特性包括pH值、酸度、粘度及可溶性固形物无明显影响,且在储存过程中能够保持109CFU/mL的活菌数,该菌可以作为益生菌添加至酸奶制品中。
[Abstract]:Based on the screening of a bifidobacterium strain with oxygen tolerance and the identification of the strain by morphological, physiological, biochemical and molecular biological methods, the physiological functions of Bifidobacterium were studied. The optimum culture conditions and culture medium were optimized by the method of response surface analysis, and the freeze-drying protectant was optimized. In addition, the mechanism of bifidobacterium resistance to oxygen stress was studied. The main results were as follows: The oxygen tolerance of 9 bifidobacterium strains was investigated by rapid screening of bifidobacterium, and one of the most oxygen-tolerant bifidobacterium was selected by re-screening by increasing oxygen concentration. The bacteria could gain high growth rate by using morphological analysis when it could resist the oxygen intake in liquid culture. Methods of combining physiological and biochemical characteristics with 16s rRNA and 16S-23S ITS sequence analysis. It was identified that the oxygen-tolerant bifidobacterium was named as bifidobacterium.animalis.subsp.lactis:. Bifidobacterium.animalis.subsp.lactis Qq08. Short for Bl Qq08. Several physiological functional characteristics of Bl Qq08 including acid resistance, bile resistance, in vitro adhesion and cholesterol scavenging ability were studied. The inhibitory properties of common pathogenic bacteria were studied. Compared with commercial strains, Bl Qq08 exhibited better acid and bile resistance and could adhere to the surface of Caco-2 cells. The adhesion number was 8.57 卤3.74 / cell and the degradation rate of cholesterol in vitro was 53.8%. The fermentation broth with advanced level of .Bl Qq08 in China was used to treat common pathogenic bacteria E. coli. Staphylococcus aureus and Salmonella have different degrees of inhibition. These excellent properties make Bl Qq08 a prerequisite for potential probiotics. The mechanism of bifidobacterium resistance to oxygen stress was studied. The results showed that the different degree of oxygen tolerance of Bifidobacterium was due to the different sensitivity of bifidobacterium to hydrogen peroxide. The accumulation of hydrogen peroxide is the most important factor to inhibit the growth of Bifidobacterium. There is no direct correlation between SOD activity and oxygen tolerance of Bifidobacterium. The presence of NADH-oxidase and NADH-peroxidase was associated with oxygen tolerance of Bifidobacterium. The culture conditions of Bl Qq08 were systematically optimized by using the scientific method designed by response surface experiment. Finally, the best culture medium for Bl Qq08 was: 1. 11% W / V) W / V) 0.2W / V) K2HPO4. The best medium for the culture of Bl Qq08 was:: 0.55% W / V) WR / V) 1.11% W / V) peptone 0.2% W / V) K2HPO4. MgCl 2.6H 2O 0.002: W / V) CaCl _ 2, 0.001) W / V) FeCl3. The pH value of fructose oligosaccharides was 7.2. After 24 hours of cultivation, the number of live bacteria was 3.45 脳 10 9 CFU / mL. It was 5.39 times higher than that before optimization. The optimized design of Bl Qq08 freeze-drying protectant was carried out. The formula of freeze drying protectant suitable for Bl Qq08 was: 1 15% skimmed emulsion and 8% lactose. The survival rate of 1.5% ascorbic acid was 3% glycerol. The survival rate of freeze-drying was up to 91.23%. The freeze-dried bacteria powder was stored at non-strict anaerobic room temperature and 4 鈩，

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