为了提高乳酸乳球菌发酵生物量,该文首先筛选了最优碳氮源,分析了乳酸乳球菌偏好利用的氮源特征,定向制备了酪蛋白肽,进一步分析酪蛋白肽的添加对菌株增殖的影响;然后通过测定菌株生长速率被抑制时的碳氮消耗比和耐渗透压能力确定了初始发酵培养基的碳氮源添加比例和添加量。结果表明,葡萄糖是乳酸乳球菌的最佳碳源,酪蛋白肽和酵母浸粉FM 803以1:2的质量比复合为最佳氮源,进一步优化微量元素的添加量和恒pH分批培养策略,得到最优培养基和培养工艺。乳酸乳球菌CCFM 1093的最优培养基:葡萄糖137 g/L、复合氮源53 g/L、MgSO4·7H2O 0.043 g/L、MnSO4·H2O 0.03 g/L、吐温-80 1 mL/L;乳酸乳球菌CCFM 1032最优培养基:葡萄糖106 g/L、复合氮源51 g/L、MgSO4·7H2O 0.043 g/L、MnSO4·H2O 0.03 g/L、吐温-80 1 g/L,活菌数分别可达到(1.01±0.06)×1012 CFU/mL和(9.78±0.38)×1011 CFU/mL,是MRS培养基静置培养的12.6倍和12倍。该研究结果将会显著提高乳酸乳球菌的工业生产效率。
Lactococcus lactis is widely used in food, medicine and other fields, which is also one of the common starters in the dairy industry. The strains must reach a certain number of viable bacteria in order to exert the corresponding effect in vivo, which requires high culture density by optimizing the culture medium and culture process. Most of the traditional culture models are constant pH batch culture, but this model has two major problems: low substrate utilization and osmolarity inhibition. This leads to the difficulty of breaking the 1011 CFU/mL level of viable bacteria in the fermentation broth. The amount of substrate in the medium is a direct determinant of the final biomass, but when the concentration of substrate in the initial medium is too high, the osmotic pressure inhibition may result in slow or even stopped growth of the strain. Finding a balance between the two factors has become a key issue. Therefore, two strains of Lactococcus lactis were selected to study the effects of substrate type, substrate amount and culture process to improve the fermentation biomass. In this study we firstly screened the carbon and nitrogen sources in the medium and found that glucose is the most suitable carbon source for strain proliferation. Monosaccharides are more easily absorbed and utilized. The characteristics of nitrogen source were then studied to find that L. lactis prefers to utilize. The results showed that L. lactis preferred to use Angel® yeast extract FM803, which is rich in peptides with small molecules. In order to prepare the nitrogen source rich in small molecular peptides, casein was chosen as a substrate and trypsin and papain complex were chosen as enzymes for enzymatic digestion. Further analysis of the effect of casein peptide addition on strain proliferation were studied. When casein peptide and yeast extract FM803 were compounded in the ratio of 1:2, the highest biomass was achieved. The above strategy resulted in the viable counts of (6.80±0.46)×1011 and (6.31±0.29)×1011 CFU/mL of the two strains, respectively, which was 8.48 and 7.74 times higher compared to that cultured in MRS medium. In addition, the requirements of the trace elements manganese and magnesium for L. lactis were analyzed. Trace elements can be involved in cellular metabolism as cofactors, structural stabilization of metalloproteins, and binding to proteins as signal transduction factors and regulation of gene expression. Therefore, maintaining the relative stability of intracellular metal ions is essential for the survival of bacteria. The results showed that both trace elements were required for the growth of the strain. The experiments described above yielded optimal substrates, and the effect of carbon to nitrogen ratio on the proliferation of the strain must be elucidated. The carbon to nitrogen ratio directly affects the growth and metabolism of microorganisms. Too high or low carbon to nitrogen ratio not only caused waste of substrate, but also inhibited strain growth. The consumption ratios of carbon and nitrogen sources were determined to be 2.56 and 2.07 when the growth rate of the strain was inhibited. This ratio was adopted as the ratio of carbon and nitrogen sources added to the initial medium. The total amount of carbon and nitrogen added to the medium was determined by measuring the tolerance of the strain to osmotic pressure. The growth rate of L. lactis CCFM 1093 and L. lactis CCFM 1032 were inhibited at an osmotic pressure of 1 500 and 1 200 mOsm/kg, respectively. The medium prepared according to this principle allowed the substrate to be used as fully as possible for proliferation without waste. Finally, the optimal culture conditions for the two strains were obtained by optimizing the constant pH batch culture strategy. Optimal medium for L. lactis CCFM 1093 was as follows: glucose 137 g/L, complex nitrogen source 53 g/L, MgSO4·7H2O 0.043 g/L, MnSO4·H2O 0.03 g/L, tween-80 1 mL/L. Optimal medium for L. lactis CCFM 1032 was as follows: glucose 106 g/L, complex nitrogen source 51 g/L, MgSO4·7H2O 0.043 g/L, MnSO4·H2O 0.03 g/L, tween-80 1 mL/L. The viable bacteria count could reach (1.01±0.06)×1012 and (9.78±0.38)×1011 CFU/mL, respectively. Compared with MRS static culture, the number of viable counts increased by 12.6 and 12 times, respectively. This study reveals the consumption pattern of substrate in the process of strain culture, which provides a theoretical basis for high-density culture and is believed to have great potential in the industrial expansion of strain culture.
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