Proliferation Of Lactic Acid Bacteria Supplemented With Protein Hydrolysates And High-Cell-Density Culture System In Milk

Lactic acid bacteria (LAB) lack various biosynthetic pathways so that they require nutrients, especially amino acids and vitamins. The growth of LAB in milk should be limited strictly because of less proteolytic activities of LAB and low concentration of essential amino acids in milk.Based on nutritional metabolism mechanism of LAB, proliferation of LAB by adjunct WPH and SPH was-used to explore the internal relation between biological characteristic of LAB involving in metabolism, growth, propagation and physiology and high-cell-density growth in milk.The high-cell-density culture system and rapid growth of LAB by adjunct protein hydrolysates in reconstituted skim milk can improve research level of direct-vat-set (DVS) yogurt starter culture and promote development of the starter culture industry in China.The main results can be concluded as follows:Whey protein hydrolysate (WPH) and soy protein hydrolysate (SPH) were obtained hydrolyzing whey protein concentrates and soy protein concentrates by alkaline proteiase, the degree of hydrolysis (DH) were 16.30% and 20.15%, respectively. The DH of whey protein hydrolyzed by pepsin was 12.51%, while the optimum craft parameters for this was pH 2.2,60℃, substrate concentration 2%, and ratio of enzyme to substrate 7%.The special isolating constituents of WPH and SPH were obtained by ultra filtration, they were a complex mixture of 2 to 8 peptides and free amino acids analyzing by high performance liquid chromatography (HPLC) and mass spectrometry (MS), essential amino acids was high content and ratio of vary amino acid was proper to growth of LAB. Furthermore, various peptides with molecular weight of 200 to 1500 Dalton was obtained via isolating WPH by SephadexG-25 and G-15 as second and third steps components. Growth of LAB was accelerated by SPH and WPH significantly, and this effect on Streptococcus thermophilus was superior to that on Lactobacillus delbrueckii subsp. bulgaricus. The effect of adding protein hydrolytes alone on Growth of LAB was less than yeast extracts, furthermore, the coaction of them can make the cell amount enhance 1.0-1.5 log cycle.The result showed that the proliferation effect of on Lactobacillus acidophilus B was advanced much more than whey protein and whey protein acidic hydrolytes, while the mainly composition in WPH was peptides with molecular weight less than 5000 Dalton, and 2 to 8 amino acids residuals with molecular weight of 200 to 1500 Dalton played the most important role.The cell count of S. thermophilus can reach to 1010 cfu per ml on growing medium consisting of 12% skim milk,1.2%SPH,0.6% CaCO3, and 0.5% yeast extracts. While the equal level for L. bulgaricus can be achieved on the medium constituting of 12% skim milk,0.5% yeast extracts,0.8% SPH, and 0.6%CaCO3. It is indicated that the medium of 12% skim milk,0.8%yeast extracts,0.9% CaCO3 and 0.9%SPH displayed significant proliferation effect on the co-growth of both S. thermophilus and L. delbrueckii, and the cell count reached to 1011 cfu per ml.The growth kinetics of LAB will be analyzed by chemically defined media (CDM).It indicated that 8 essential amino acids minimal medium can just meet basic requirements for growth of LAB, which kept a low level.The maximum growth rates (μmax) of S. thermophilus and L. bulgaricus on CDM with 20 amino acids reached to 0.10 h-1 and 0.12 h-1, respectively.The cell density of LAB will increase five times as 20 amino acids were replaced by SPHIV, meanwhile, the maximum growth rates of L. bulgaricus and S. thermophilus reached to 0.215 h-1 and 0.262 h-1 respectively, and it is two times over which obtained by 20 amino acids. Thus SPH can be applied as a kind of additional nitrogen resource ingredients to enhance growth rate of LAB.The result of ferment test can be summarized as follows:firstly, acid production of LAB on nonfat milk supplemented with yaest extracts and multiplication complex were 2 and 3.12 times than that of control. Then the cell count of LAB reached to 1010 cfu/ml on skim milk supplemented with multiplication complex, which increased 1.0-1.5 log cycle than control. Thirdly, the parameter of accumulate additional alkali volume and interval additional alkali volume showed that the growth curve of LAB on skim milk ascend slowly, and three waves trend as supplemented with yeast extract, and went up dramatically with addition of multiplication complex. The addition of yeast extract alone can not support the continual growth of LAB, however, the addition of yeast extract and protein hydrolytes together can accelerate it and made it to keep in a high growth level. Finally, much more biomass of LAB on milk with multiplication complex would be obtained and the protein in milk would be utilized sufficiently.