摘要
随着我国发酵乳制品工业的迅猛发展,积极研究并大力开发高效浓缩型酸奶发酵剂,对于推动我国乳酸菌发酵剂产业化进程,促进我国发酵乳制品工业的发展,具有重要的意义。菌体的富集浓缩是发酵剂制备的关键技术之一,目前工业生产中常用的是离心分离的方法,这种方法对菌体损伤大,回收率低,而且与菌体一起沉淀下来的丙酮酸盐和二乙酰等羰酰基化合物,会与细胞内的氨基反应,从而加速细胞死亡。本研究采用膜分离代替传统的离心分离来富集浓缩菌体,探讨该法是否可以弥补离心分离的不足。
研究内容:(1)通过对乳品科学教育部重点实验室保藏的3株保加利亚乳杆菌和3株嗜热链球菌进行生理生化鉴定。(2)通过观察灭菌后优化培养基的状态和pH的测定,来确定乳清粉优化培养基灭菌前应调至的pH。(3)以KLDS1.8501和KLDS3.8501两株菌为研究对象,通过最佳反应条件的确定,建立光吸收值与活菌数的线性回归方程,探讨MTT比色分析法用于快速细菌计数的可行性。(4)通过试验比较不同孔径的膜渗透通量的大小及其衰减幅度,来确定使用陶瓷膜的类型。(5)通过观察过滤过程中膜渗透通量和死亡率的变化,确定微滤膜的最佳工作条件。(6)选择五种化学清洗剂:HNO_3,NaOH,NaClO,Na-EDTA,SDS,分别进行单步化学清洗和两步化学清洗,确定最佳膜清洗方案。(7)比较膜分离与离心分离两种方法的分离存活率、回收率,以及分离后的发酵活力。
实验结果:(1)通过生理生化实验,鉴定了菌株KLDS1.9201,KLDS1.9205,KLDS1.8501为保加利亚乳杆菌;菌株KLDS3.8501,KLDS3.0503,KLDS3.9207为嗜热链球菌。(2)确定了保加利亚乳杆菌和嗜热链球菌的乳清粉优化培养基灭菌前pH应分别调至6.6和6.9。(3)若想用MTT法精确反映活菌数,一定要把KLDS1.8501的活菌数调到5.5×10~6～2.18×10~7CFU/mL之间,把KLDS3.8501的活菌数调到6.4×10~6～5.12×10~7CFU/mL之间。在最佳反应条件下,KLDS1.8501菌株的线性回归方程是y=1.222x-0.06;KLDS3.8501菌株的线性回归方程是y=5.759x+0.034。但在实际应用中,MTT比色分析法与平板菌落计数两种方法检测的活菌数差异显著。(4)确定采用0.2μm的无机陶瓷膜对嗜热链球菌发酵液进行分离和浓缩。(5)膜过滤嗜热链球菌的最佳工作条件为:操作压力0.15MPa,操作温度45℃。在这种操作条件下,对嗜热链球菌发酵液进行分离和浓缩,菌体存活率可达到90%。(6)最佳清洗方案为:先用1%NaC1O清洗15min,再用1.5%SDS清洗15min,通量恢复率可达到99%。(7)与离心分离相比,膜分离可以显著提高分离的存活率、回收率和发酵活力。
结论:MTT比色分析法只可以粗略的表示乳酸菌活菌数的变化趋势,但不能准确定量检测保加利亚乳杆菌和嗜热链球菌的活菌数量。膜分离技术可以用于乳酸菌菌体的富集浓缩,与离心分离法相比,膜分离具有菌体分离存活率高和分离后发酵活力高的优点。
With the rapidly development of fermented dairy product industry, It's very important to research and develop high activity concentrated yoghourt starter culture for promoting the industrial process of lactic acid bacteria starter and development of fermented dairy industry. cells enriching concentration is one of the critical technology of high-activity concentrated freeze-drying yoghourt starter. At present centrifugal separation is commonly used in the industrial production, but this method has large damage to the cells and low recovery rate, and pyruvate and carbonylacyl group like diacetyl which are precipitated with cells can react with amino group in cells, resulting in the acceleration of cell death. In this paper, new membrane separation technology instead of traditionally centrifugal separation is utilized to concentrate cells, so as to determine whether this method could improve the shortage of centrifugal separation.
The topics of the research include: I. six strains of Lactabacillus delbrueckii ssp. Bulgaricus and Streptococcus thermopHilus, three for each, are identified by pHysiological biochemistry test such as carbohydrate fermentation, gelatin liquefaction, hydrogen sulfide reduction, azotate reduction, amylohydrolysis, salt-tolerated, and acid-tolerated test. II. The pH of optimizing whey powder mediums before sterilization are determined through observing the state of the optimizing mediums after sterilizing and adjusting their’s pH. III. KLDS1.8501 and KLDS3.8501 were used in this study. Through confirming of the optimum reaction conditions, equation of linear regression was constructed between light absorption value and viable count, with the feasibility of applying MTT method to bacteria-counting to be discussed. IV. By comparing the flux intensity and attenuation of membranes which have different apertures, the type of ceramic membrane is determined. V. The best manipulation parameters of microfiltration membrane are studied by measuring the flux intensity and the change of death rate. VI. Through one-step and two-step chemical clean respectively, five chemical abluents, HNO_3,NaOH,NaClO,Na-EDTA,and SDS are choosed to determine the best program of membrane cleaning.Ⅶ. Compare survival, recovery and fermented activities of membrane Separation with the ones of centrifugal separation.
The results of the experiments are: I. Strains KLDS1.9201,KLDS1.9205, KLDS1.8501 are identified as Lactabacillus delbrueckii ssp. Bulgaricus; KLDS3.8501,KLDS3.0503,KLDS3.9207 as Streptococcus thermopHilus by pHysiological biochemistry test. II. The PH of the optimizing mediums of Lactabacillus delbrueckii ssp. Bulgaricus and Streptococcus thermopHilus should be respectively adjusted to 6.6 and 6.9 before sterilization. III. As to reflect the viable count exactly by MTT method, the count range of KLDS1.8501 and KLDS3.8501 must be adjusted to 5.5×106~2.18×10~7CFU/mL, 6.4×10~6~5.12×10~7CFU/mL, respectively. Equation of linear regressions of them were y=1.222x-0.06, y=5.759x+0.034, respectively, under the optimum reaction conditions. However, the difference between MTT colorimetric analysis antigenic and plate count method was significant in the actual application. IV. The 0.2μm(dimension) inorganic ceramic membrane is choosed to use for separating and concentrating fermentation broth of St. thermopHilus. V. The best working parameters of membrane when St.thermopHilus are separated and concentrated are determined as follows, the pressure is 0.15MPa, temperature 45℃, And in this operational condition the viability of cells could be 90%. VI. The best method of cleaning is to use 1% NaC1O×15min firstly and then by 1.5%SDS×15min. After cleaning, the water filtration ratio is 99%.Ⅶ. Compared with centrifugal separation, membrane Separation can increase significantly the survival, recovery and fermented activities.
MTT colorimetry can express rudely changing trend of lactic acid bacteria, but it can not detect accurately the survival of Lactobacillus bulgaricus and Streptococcus thermopHilus.We concluded that membrane separation technology can be used for cells enriching concentration of LAB, and that comparing with the centrifugal separation, it has the advantages of higher separating viability and stronger fermentation ability of cells.
引文
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