Effect Of Lactose On The Growth And Metabolism Of Lactobacillus Helveticus And Its High Cell Density Culture

Lactobacillus helveticus belongs to the group of lactic acid bacteria (LAB) and possesses high proteolysis activity. It can grow well in skim milk medium. To achieve high cell density culture, medium is very important. It adds yeast extract or vitamins into medium to increase biomass. Alkali neutralization is usually used in fermentation industry to remove the effect of acid on the growth of LAB. In this paper, the cell density increased by adjusting the concentration of lactose to8.0%. This indicated that skim milk contained enough nitrogen sources but lack carbon source. The effect of carbon source on the growth and metabolism of strain CICC22171was observed. Transcriptome sequencing, proteomics, fluorescent quatititive PCR were conducted in this study. Meantime, viable count and the activity of key enzyme were also monitored during fermentation. Thus, the aim of this study was:(1) to reveal mechanism of how the carbon affect the growth of L. helveticus;(2) to optimize medium and conditions of fermentation process to improve biomass production. The main results were concluded as follows:L. helveticus CICC22171can grow well in skim milk medium and there was a significant increase (P<0.05) of cell density compared with compared with whey medium and casein medium. The consumption NaOH was about141.2mL and the viable count could reach to8.32×108cfu/mL. The optimum nitrogen source was skim milk. Similar result also obtained from SDS-PAGE. Further research indicated that the viable count increased significantly (P<0.01) when the concentration of lactose increased from4.5%to8.0%. The final viable count was1.14×109cfu/mL. This demonstrated that carbon source was relatively insufficient and affected the growth of strain CICC22171.In this section, the activities of β-Galactosidase (P-GAL), hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were detected during fermentation. The activities of these key enzyme were significant increased when the strain grown in skim milk medium with high lactose (8.0%). Meantime, fluorescent quatititive PCR was performed to monitor the expression of these genes during fermentation. It indicated that the expression of genes (PK and LDH) increased16.015-fold and8.444-fold compared with their initial values. This demonstrated that the cell with high level of energy charge which could promote cell proliferation.Two-dimensional electrophoresis and tandem mass spectrometry (2D-Maldi-TOF-TOF-MS) was employed to detect full-cell solubility protein. Finally,11differentially expressed protein spots were identified Maldi-TOF-TOF-MS, which are involved in glucose metabolism, amino acid biosynthesis, nucleotide biosynthesis and materials transport.The differentially expressed genes between L. helveticus CICC22171cultured in high lactose culture condition (H) and low lactose culture condition (L), were analyzed based on RNA-Seq data.1979genes were differentially expressed (p-value<0.05) with1250up-regulated and729down-regulated. With the threshold of Iog2Ratio>1, there were307up-regulated genes and167down-regulated genes predicted to be the differentially expressed genes. After gene annotation and classification, there were1621genes annotated on GO (gene ontology),241genes annotated on KEGG (Kyoto Encyclopedia of Genes and Genomes). The up-regulated genes involved in fatty acid biosynthesis, amino acid biosynthesis, transmembrane transport, energy production and conversion and inorganic ion transport. The down-regulated genes involved in DNA hypermethylation, DNA restriction to modify and glyconeogenesis. This demonstrated that the cell cultured in high lactose medium showed high level in gene expression and metabolism.In this experiment, high cell concentration and substrate consumption could achieve by batch fermentation. The viable count could reach to3.98x109cfu/mL which increased significantly (p<0.01) compared with batch and continuous processes. In addition, the strain CICC22171was sensitive to oxygen. So it must control the ventilation and select low speed of agitator.