Studies On Selection, Physiological Characteristics And Application Of An Aeortolerant Bifidobacterium

This thesis presents my research work on selection of an aerotolerant bifidobacterium and study on its physiological characteristics and application. Firstly, an oxygen-resistant bifidobacterium has been selected, and polyphasic taxonomy approaches are employed to identify the selected strain such as phenotypic analysis, 16S rRNA and 16S to 23S internally transcribed spacer sequence analyses. On the basis of the oxygen-resistant bifidobacterium, physiological functions have been studied. And response surface analysis method is used to optimize the culture conditions and the medium for the selected strain. Freeze-drying protective agents have been optimized, and then the freeze-dried powder is applied in the yogurt. In addition, the mechanism of oxygen tolerance of bifidobacterium has been preliminary investigated. The main research results are listed as follows:A rapid screening method based on RBGR value is used to screen oxygen tolerant bifidobacterium from nine strains. To choose the most oxygen-resistant bifidobacterial strain, the oxygen concentration is increased in the further scre ening.The selected strain also grows well in liquid culture when the oxygen i ntake. Using morphological, physiological and biochemical,16S rRNA and 16S-23S ITS sequence analysis methods, the selected strain is identified as Bifidob-acterium.animalis.subsp.lactis. It was named as:Bifidobacterium.animalis.subsp. lactis Qq08, for short as Bl Qq08.The physiological features of Bl Qq08, including acid and bile-resistant properties, in vitro adhesion and cholesterol-lowering ability, antibacterial properties have been studied. The results compared with the commercial strain, Bl Qq08 shows better resistance to acid and bile resistant properties. The adhesion number to Caco-2 cell surface is 8.57±3.74 CFU/cell. The rate of degradation of cholesterol in vitro is 53.8% which is one of the highest result compared with current report in China as known. The fermentation broth of Bl Qq08 can inhibit of pathogenic bacteria including Escherichia coli, Staphylococcus aureus and Salmonella with different degrees. These excellent features, making Bl Qq08 has the potential to be used as probiotics.The mechanism of bifidobacteria resistance to oxygen has been studied. The results showes that the oxygen tolerance of bifidobacteria is relevant to its sensitivity to hydrogen peroxide, the accumulation of hydrogen peroxide is the main factor in inhibition of the growth of bfidobacteria. The activity of SOD has no direct relevance with the oxygen tolerance ability of bifidobacteria, and the existence of NADH-oxidase and NADH-peroxidase can reduce the accumulation of hydrogen peroxide.The cultivation conditions and medium compositions have been optimized with response surface method (RSM). The experimental results show that the optimum culture medium for incubation Bl Qq08 was composed of:0.55%(W/V)whey powder,1.11%(W/V) poly peptone,0.2%(W/V)K2HPO4, (W/V)MgCl2.6H2O0.1%, 0.002%(W/V)CaC12,0.001%(W/V)FeCl3,0.1%(W/V) NaHCO3 and 0.1%(W/V) FOS at pH value of 7.2. After incubation 24h in the optimum fermentation medium, the populations of the viable organisms can reach 3.45×109CFU/mL which is 5.39 times of the results when fermented in the primary medium.The cryoprotectants for freeze drying process of Bl Qq08 are optimized. The optimum freeze-drying protective agents for Bl Qq08 are:15% skim milk,8% lactose, 1.5% ascorbic acid (Vc),3% glycerol. In the triplicate lyophilization tests, the average survival rate of Bl Qq08 reached 91.23% which was higher than the current report as known. The resultant lyophlized powder has been stored in non-strict anaerobic respectively at 4℃and room temperature for 1 month, there is no significant decrease in bacterial number, the results show that Bl Qq08 freeze-dried powder has good storage stability.The resultant lyophlized powder of Bl Qq.08 is added to the yogurt at the end of fermentation and the effect of Bl Qq08 on yogurt quality has been analyzed. The results show that Bl Qq08 had no significant effect on the chemical and physical properties of yogurt, including pH, acidity, viscosity and soluble solids. After storage in 4℃for 1 month, the viable cells of Bl Qq08 maintained 109CFU/mL, the results show that bacteria can be added to yogurt as probiotics.