| Cholesterol, also known as cholesterin, is a derivative of multi-cyclopentane hydrogen phenanthrene. It is widely found in animal tissue cells as an indispensable substance..However, excessive intake of the cholesterol will increase its presence in serum over the normal level, which will easily result in various cardiovascular diseases that are threatening to human health., including atherosclerosis, coronary heart disease, stroke, hypertension, etc.Thus, how to reduce serum cholesterol is essential to the treatment of related diseases.Lactobacillus is a kind of probiotics, widely existing in the human bodies and animals. It can promote the growth of animals, regulate the normal flora of the gastrointestinal tract, maintain the micro-biological balance, inhibit the growth of spoilage bacteria in the gut, and strengthen the immunity of the organism. Thus it has a number of important physiological functions..Some studies have also indicated that some lactic acid bacteria could degrade cholesterol,which offers a new direction from which to reduce the level of cholesterol in serum.This paper studies the separation and purification as well as the identification of lactic acid bacteria which can reduce cholesterol. Besides, it mentions their application in fermented kimchi, the optimization of the medium and the preparation of probiotics. It lays the foundation for the development and use of the cholesterol-removal lactic acid bacteria.This thesis includes the following four aspects.1. The screening and identification of cholesterol-removal lactic acid bacteriaFirst, cholesterol-removal lactic acid bacteria has to be screened and identified. The experiment adopts 99 screened bacterial strains. They are negative in the catalase test and positive in the gram-stain test; they have obvious transparent calcium circle. 8 of them are found more than 30% cholesterol degradation rate. I4 has the highest degradation rate of 37.3± 1.50(%, n = 3). Through physiological and biochemical tests and the 16 S rRNA sequence analysis, the I4 strain is identified as Lactobacillus I4 strains(Lactobacillus plantarum).Meanwhile, this strain has underwent acid-proof and cholate-proof tests. The experiment shows that Lactobacillus I4 has a great acid-resisting capability and a certain cholate-resisting capability.2. 2.The response surface optimization of cholesterol-removal Lactobacillus I4 enrichment mediumResponse surface methodology is adopted to optimize cholesterol-removal Lactobacillus I4 enrichment medium. The main factors affecting bacterial concentration are screened out by Plackett-Burman. They are sucrose, yeast extract and sodium acetate. After the response surface optimization, the optimum concentration of the main three factors are 2.05%, 1.54%,and 0.31%, respectively. The density of OD620 in the optimized enrichment medium can reach a point of 2.458. Compared with MRS medium, the density has increased by 23.7%, and the colony units has improved 3.91 times.3. The preparation of I4 cholesterol-removal Lactobacillus bacteria and the observation on its stabilityThe related conditions of I4 cholesterol-removal Lactobacillus bacteria and the observation on its stability have been studied.Through the selection of the preparation conditions of bacteria, the best formula which provides the most viable bacteria(1.15 × 1012CFU/g) is prepared. It includes gelatin, which functions as the protection,and bacteria mash, with the proportion of 1:5(w: v). Meanwhile,the mixture has to be pre-cooled for 60 min under 40 ℃ and the volume is 3 ml. The test of the cholesterol-removal ability of the formula indicates a degrading rate of 36.9 ± 1.80(%, n= 3), which shows that lyophilized Lactobacillus I4 is hardly affected in terms of cholesterol-lowering ability. Through the observation of bacteria stability, it is concluded that the number of living bacterium is 0.978x1011cfu/g, 2.223 x1011cfu/g, and 1.125 x 1011cfu/g,after different cultivations of normal temperature, 3 ℃, and- 18 ℃, each for 90 days. The figures indicate that this kind of bacteria has a good stability.4. The application of I4 cholesterol-removal Lactobacillus probiotics in the fermentation of kimchiThe application of I4 cholesterol-removal Lactobacillus probiotics in the fermentation of kimchi is also studied. During fermentation of kimchi, the variations of pH values, total acid amount, amino nitrogen amount and nitrite amount as well as the sensory changes of different amount of the added bacteria are tested. Meanwhile, the amount of all bacteria, lactic acid bacteria and E. coli in the fermentation liquid is measured after 7 days’ fermentation. The results indicate that with the help of the I4, the period of fermentation is greatly shortened and the Kimchi is safer and more stable. In the fermented liquid, the amount of all bacteria and lactic acid bacteria is 2.72×106CFU/mL and 2.33×106CFU/mL, which means a majority of microorganism in the liquid is lactic acid bacteria. Zero E-coli indicates no morbigenous microorganism, which conforms to the related requirements of GB2714-2003 ", the Pickles Health Standards" on the amount of microorganism. |
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