本文關(guān)鍵詞：耐氧性雙歧桿菌的篩選及其生理特性與應(yīng)用研究　出處：《浙江大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 雙歧桿菌 耐氧 生理特性 響應(yīng)面 酸奶

【摘要】：本文從篩選一株耐氧的雙歧桿菌出發(fā),并利用形態(tài)學(xué)、生理生化和分子生物學(xué)手段對該菌進(jìn)行鑒定。在此基礎(chǔ)上,對耐氧雙歧桿菌的各項(xiàng)生理功能進(jìn)行了研究,并利用響應(yīng)面的分析方法對該菌最適的培養(yǎng)條件和培養(yǎng)基進(jìn)行了優(yōu)化,對該菌的冷凍干燥保護(hù)劑進(jìn)行了優(yōu)化設(shè)計(jì),并將凍干后的菌粉在酸奶中的應(yīng)用特性進(jìn)行了研究,另外,對雙歧桿菌對氧脅迫的抵抗機(jī)理進(jìn)行了初探。論文的主要研究結(jié)果如下： 利用快速篩選耐氧性雙歧桿菌的方法對9株雙歧桿菌的耐氧性進(jìn)行考察,通過增加氧氣濃度的方式進(jìn)行復(fù)篩,從中選出一株最耐氧的雙歧桿菌,該菌能夠在抵抗液體培養(yǎng)時(shí)氧氣的攝入,獲得較高的生長量。利用形態(tài)學(xué)分析、生理生化特性和16S rRNA及16S-23S ITS序列分析相結(jié)合的方法,鑒定得出該耐氧雙歧桿菌為bifidobacterium.animalis.subsp.lactis命名為：Bifidobacterium.animalis.subsp.lactis Qq08,簡稱Bl Qq08. 對Bl Qq08的幾項(xiàng)生理功能特性包括耐酸、耐膽汁特性,體外粘附能力及清除膽固醇能力,對常見致病菌的抑制特性進(jìn)行了研究。結(jié)果表明,Bl Qq08與商業(yè)菌株相比,表現(xiàn)出更優(yōu)良的耐酸與耐膽汁特性。能夠粘附于Caco-2細(xì)胞表面,粘附數(shù)為8.57±3.74個(gè)/細(xì)胞。體外對膽固醇的降解率為53.8%,處于國內(nèi)先進(jìn)水平。Bl Qq08的發(fā)酵液對常見致病菌大腸桿菌、金黃色葡萄球菌、沙門氏菌具有不同程度的抑制作用。這些優(yōu)良的特性,使得Bl Qq08具備了成為潛在益生菌的先決條件。 對雙歧桿菌抵抗氧脅迫的機(jī)理進(jìn)行了初探。表明雙歧桿菌對氧氣耐受性的程度不同,是由于其對過氧化氫的敏感程度不同所致,過氧化氫的累積是導(dǎo)致雙歧桿菌生長受抑制最主要的因素,SOD的活性與雙歧桿菌對氧氣的耐受性無直接相關(guān)性,而NADH-oxidase和NADH-peroxidase的存在與雙歧桿菌對氧氣的耐受性有相關(guān)性。 利用響應(yīng)面實(shí)驗(yàn)設(shè)計(jì)的科學(xué)方法對Bl Qq08的培養(yǎng)條件進(jìn)行了系統(tǒng)優(yōu)化,最終確定培養(yǎng)Bl Qq08的最佳培養(yǎng)基為：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。在該條件下培養(yǎng)24h后,活菌數(shù)為：3.45×109CFU/mL,較優(yōu)化前提高了5.39倍。 對Bl Qq08的冷凍干燥保護(hù)劑進(jìn)行了優(yōu)化設(shè)計(jì),適合Bl Qq08的冷凍干燥保護(hù)劑配方為：15%脫脂乳,8%乳糖,1.5%抗壞血酸(Vc),3%甘油。冷凍干燥存活率高達(dá)91.23%,高于目前文獻(xiàn)報(bào)道的水平。凍干后的菌粉在非嚴(yán)格厭氧常溫及4℃分別儲(chǔ)存1個(gè)月,菌數(shù)無明顯降低,B1 Qq08凍干粉具有良好的儲(chǔ)存穩(wěn)定性。 將B1 Qq08添加至酸奶中,研究B1 Qq08的加入在保質(zhì)期內(nèi)對酸奶品質(zhì)的影響。結(jié)果表明B1 Qq08對酸奶的理化特性包括pH值、酸度、粘度及可溶性固形物無明顯影響,且在儲(chǔ)存過程中能夠保持109CFU/mL的活菌數(shù),該菌可以作為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 鹵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，

本文編號(hào)：1432093