Researches Of Isolation, Catalyst Kinetics, Application And High Cell Density Fermentation Of Lactobacillus Salivarius XH4B With α-galactosidase Activity

In this study, we took Chinese traditional foods as material to isolate lactic acid bacteria (LAB) with a-galactosidase activity. Both catalyst activity and LAB strain properties were tested and a correlationship between catalyst activity and biomass were established. After culture medium optimization as well as biofilm inducement, LAB high cell density fermentation was attained which contributed to the stable, high a-galactosidase activity whole cell Lactobacillus salivarius XH4B probiotic.8LAB strains with higher a-galactosidase activity were obtained through incubation by semi-selective culture medium PY-SE, staining by X-a-Gal and catalyst activity measurement by pNPG. After16S rDNA identification,3out of8strains were named as:Lactobacillus salivarius XA1R (JX125455), L. salivarius XH4B (JX125456), Pediococcus acidilactici XS1B (JQ927329). According to the growth viability and a-galactosidase activity of the strains, as well as the Chinese edible fungi regulation in foods, we eventually chose the strain of L. salivarius XH4B for the following researches.The best carbohydrate for a-galactosidase activity inducement was stachyose, followed by raffinose, a-lactose. As to extraction, the lysozyme method could attain48.2%activity, while the methods of freeze-thawing, supersonic extraction and protease only attained25.5-36.1%activity compared with whole-cell a-galactosidase activity. The purifying ratios of hyper-filtration and Sephadex G200column chromatography were149.80%and391.91%respectively compared with crude enzyme extracted by supersonic. The best catalyst activity conditions were:citric buffer pH value5.5, ion strength0.15mol/L and temperature52℃. The Km values of the a-galactosidase were0.817to pNPG and6.672to raffinose. Only K+and Na+activated the catalyst activity positively.When SE (Soybean meal extract) added into the culture medium, there was floating biofilm instead of attached biofilm formation which observed by crystal violet staining as well as electronic scanning microscope. The biofilm formation enhanced the LAB cells resistance to circumstantial stresses, and changed the α-galactosidase accumulation trends. There was a positive correlation between L. salivarius XH4B biomass and a-galactosidase activity with an index of0.936, which promised the high cell density fermentation for higher a-galactosidase activity.The best hydrogen for biomass yield was yeast extract (4.38g/L), and carbohydrates were lactose (5.01g/L), glucose (4.91g/L) and sucrose (4.89g/L). Sodium acetate, sodium citrate and disodium hydrogen phosphate could buffer the culture media pH value and did not inhibit the L. salivariu.s XH4B growth. The culture media of30～50%PY-SE balanced nitro-and carbon-hydrates and adequate protein coagulation to induce floating biofilm which was the best ones for high cell density fermentation. With1-2-3fed-batch method when fermented iluid was discarded, the biomass could reach17.89g/L. When the fermented iluid remained in batches, two fed-batches (1-2-3and3-2-1) were not different significantly, however, the1-2-3fed-batch could thoroughly dilute the former metabolic waste and the biomass was higher than3-2-1.With the additive of α-galactosidase or freeze dried L. salivasrius XH4B could hydrate stachyose and raffinose90.63%and69.89%respectively, which significantly higher than the starter only.This study isolated L. salivarius XH4B with high α-galactosidase activity and broadened the method to LAB high cell density fermentation with SE inducement to floating biofilm. This would benefit the fermentation industry in yoghurt or probiotic manufacturing.