The Research Of Nutrient Requirements, High Cell Density Cultivation And Multiplication Mechanism Of S. Thermophilus SP1.1

The growth of Lactic acid bacteria(LAB) is inhibited by the consumption ofnutrients and the accumulation of metabolites. High cell density cultivation(HCDC) canreduce the amount of leavening agents, shorten the production cycle and reduceproduction costs, which is the the necessary conditions of lactic acid bacteria orprobiotics commercial production. Discussing limitations of nutrients during the growthof LAB, and revealing the significance of strains cell division mechanism for achievingHCDC of LAB is more and more important.This study mainly described the optimization of chemical synthetic mediumlaboratory when culturing isolated S. thermophilus sp1.1, and researched41kinds ofingredients that affected cell density by single factor experiment, and19of which had agreater impact on cell density. Using Plackett-Burman to screen5most influentialfactors, which were isoleucine, leucine, cysteine, magnesium and potassium dihydrogenphosphate. Steepest ascent drew that the respectively optimal dosage of isoleucine,leucine, cysteine, magnesium sulfate and potassium dihydrogenphosphate were0.20g/L,1.12g/L,0.34g/L,0.92g/L and2.20g/L, and cell density could improve2.7%. Thenresponse surface experiments were based on the optimal dosage center, showing thatisoleucine and magnesium, cysteine and magnesium interacted significantly.5kinds ofimportant components'final optimal concentration were determined:0.22g/Lisoleucine,1.16g/L leucine,0.36g/L cysteine,0.48g/L magnesium sulfate,2.25g/Lpotassium dihydrogen phosphate, and the cell density increased by1.2%.41kinds of ingredients were inspected by single-omission growth experiments onthe basis of optimization of chemical synthesis of medium, and the result showed thatglucose, histidine, methionine, calcium pantothenate, niacin, riboflavin and potassiumwere the growth essential nutrients of S. thermophilus sp1.1. Then the couple-omissiongrowth experiments was used to research the interaction between components, andshowed that bacteria could not grow without glutamate or glutamine concurrently.Serine, tryptophan and tyrosine significantly inhibited growth of S. thermophilus sp1.1.The41kinds of ingredients streamlined into38kinds (serine, tryptophan and tyrosineomission), the cell density increased by15.1%.The study discussed the effects of variable temperature and variable pH on celldensity and fermentation activity at the end of lag time and logarithmic time. Resultsshowed that the impact on fermentation activity was greater than cell density whenchanging temperature and pH. The strategy that initial incubation temperature is39?,then varying temperature to36?at the end of the lag time, last changing to39?at the end of log phase was the most optimum staged cultivation temperature. And thestrategy that initial incubation pH was6.0, then varying pH to5.5at the end of the lagtime until to stationary phase was the most optimum staged cultivation pH. Cell densityincreased by8.7%, fermentation activity increased by23.9%for staged cultivation.The study of the relationship between penicillin-binding proteins(PBP) and cellgrowth shows that fluorescent penicillin can mark the penicillin-binding protein of S.thermophilus sp1.1specially, and identifies12kinds of penicillin-binding protein byfluorescence imaging, named PBP1to PBP10, and their molecular weight are139kD,118kD,94kD,78kD,67kD,62kD,58kD,56kD,52kD and43kD respectively. Theviability of quantitative analysis of penicillin-binding protein is verified by method offluorescence-labeled penicillin, and the conclusion shows that protein concentration andthe fluorescence intensity has a good correlation coefficient. with the growth of cells,the type of penicillin binding protein and content may change. The content ofpenicillin-binding protein changed at different growth time. Cell divided fast inlogarithmic phase, the biosynthesis of PBP1and PBP2did not increase significantly(P>0.05), indicating that it did not participate in the fission process of cell. And thephenomenon of synthesis of cell increased in stationary phase may be concerned withcell elongation. The content of PBP3and PBP4in logarithmic phase and stationaryphase increased significantly(P<0.05), and had great effect on cell proliferation andelongation. PBP6synthesis increased with rapid proliferation of cell, while stayed stablein stationary phase, and involved in cell division and elongation. The change trend ofPBP5, PBP7, PBP8, PBP9, PBP10, PBP11and PBP12was approximately the same.The content of logarithmic phase increased significantly(P<0.05) and the plateaureduced significantly which indicated the function was mainly related to bacteria splitprocess, an extension of the bacteria was not involved.