| Streptococcus thermophilus is known as a starter culture widely used in the production of dairy products. At present, Streptococcus thermophilius is mainly applied in the form of direct investment of dry powder in dairy industry. One of the key technologies of direct vat set starter preparationis the high-density cultivation of lactic acid bacteria. During this process, Streptococcus thermophilus mainly goes through two aspects of stress factors---The gradually decrease of intracellular p H and increase of extracellular sodium lactate concentrations. These two stress factors directly affect the growth and biomass acquisition of Streptococcus thermophilus. This thesis mainly investigated the change of intracellular physiological state, amino acid consumption and demand patterns of Streptococcus thermophilus cultivated in chemical synthetic medium, which could lay a foundation for subsequent group stud y, and for the design of high density culture medium of Streptococcus thermophilus.This study targeted the growth dynamics of Streptococcus thermophilus TF96 cultivated in chemical synthetic medium. With the application of modern analytical technologies, the change of intracellular p H and extracellular sodium lactate concentration at fermentation key points were studied; Using kit method and high performance liquid chromatography(HPLC), the change rules of of intracellular ATP concentration, H+-ATPase activity, NADH, NADPH and NH4+ concentration were evaluated; Amino acid analysis technique was used to study the amino acid consumption and demand pattern of Streptococcus thermophilus during cultivation. The study content illuminates the Streptococcus thermophilus intracellular physiological state in the process of cultivation and consumption patterns of amino acids and how the demand patterns, with the reduce of intracellular p H and extracellular sodium lactate concentrations increased cases of.In the complete chemically defined medium, with the extracellular p H invariably, the cellular p H of Streptococcus thermophilus TF96 decreased from 6.43 in the early of logarithmic phase to 5.92 in the stable phase; while the cellular concentration of sodium lactate increased from 89.45×10-4mol/L in the early of logarithmic phase to 199.77×10-4mol/L in the stable phase. Under different growth pressure, the physiological state were different. With the concentration of H+ increased, both the cellular ATP and the activity of H+-ATPase increased. By the time of 2.3 h, both the content of ATP and the activity of H+-ATPase reached a peak of 12.08μmol/gprot and 0.67μmol/mgprot/hour. While from 4.7 to 9 h, the cellular ATP and the activity of H+-ATPase increased from 2.20μmol/gprot to 6.65μmol/gprot and 0.13μmol/mgprot/hour to 0.40μmol/mgprot/hour, respectively. however, the content of NADH and NADPH were decreased with the the concentration of H+ increasing. At 2.3 h, the content of NADH and NADPH were low. From 4.7 to 9 h, the content of NADH and NADPH were decreased from 5.93nmol/107 cell to 3.95nmol/107 cell and 2.07nmol/107 cell to 1.07nmol/107 cell, respectively. While the concentration of NH4+ was increased from 16.74 mg/L to 36.78 mg/L. The results indicate, to adjust to the decreasing p H value and increasing sodium lactate, Streptococcus thermophilus TF96 showed different physiological status to maintain the growth.Study on Streptococcus thermophilus TF96 amino acid consumption patterns changing with the growth rate showed that in the whole growth process, consumption of Asx(aspartic acid and asparaginate), Glx(glutamic acid and glutamine), serine and arginine were the greatest. As the Asx(aspartic acid and asparaginate), Glx(glutamic acid and glutamine) and serine were 17.15%, 18.73% and 13.37%, and the consumption of arginine were 98% of arginine supplementation amount of medium; Secondly, threonine, valine, leucine, tyrosine, phenylalanine and lysine amino acid consumption accounted for 3%~10% of the total, while the remaining amino acids(Glycine, alanine, methionine, histidine and proline) consumption was low. In average growth rate at μ1=0.12h-1, cells were in in the phase of adjustment, so all amino acid consumption were significantly higher. While in the average growth rate at μ2=0.36h-1, μ3=0.52h-1, μ4=0.08h-1, and amino acid consumption increased with the increase of the μ. The results showed that Streptococcus thermophilus TF96, in order to adapt to the continuous reduction of intracellular p H and the gradual increase in the concentration of sodium lactate growth, adjusted the amount of amino acid consumption for better growth.Compared the amino acid demand pattern and growth rate of Streptococcus thermophilus TF96, the amino acid demand changed significantly with the increasing μ. Additionally, the most demanded amino acids were Asx(aspartic acid and asparaginate), Glx(glutamic acid and glutamine) and Lys with percentage of 14.41%, 15.64% and 10.37% of the total demands, respectively; besides, the demands of threonine、serine、glycine、alanine、methionine、isoleucine and leucine were4%~8% totally; the rest, valine、tyrosine、phenylalanine、histidine and arginine were in a low level. From μ3=0.52h-1 to μ4=0.08h-1, the demands of amino acids were significantly increased. Results show, under double pressure, Streptococcus thermophilus TF96 regulated the cell state by synthesis of emergency protein.During the cultivation of Streptococcus thermophilus TF96, the consumption of amino acids were 37.31mmol/g. In the whole consumption, the growth demand was 13.24mmol/g, which was far less than the total consumption. The results show not all the amino acids were used for the cell growth, but the waste of nitrogen. And the nitrogen waste may be due to the excessive consumption of Asx(aspartic acid and asparaginate), Glx(glutamic acid and glutamine), serine and arginine. This research illustrated the regularity of variability of the intracellular physiol ogical state and the consumption and demand of amino acid with the intracellular p H value reducing and the extracellular sodium lactate concentration increasing, in the cultivation of Streptococcus thermophilus TF96. |
PDF Full Text Request