Screening And High-cell Density Culture Of Lactic Acid Bacteria With β-Galactosidase Activity

Lactic acid bacteria are a kind of microbiology which are no-pathogen-inducedand gram-positive, and they can metabolize carbohydrates to secrete plenty of lacticacid. Lactic acid bacteria are generally regarded as safe microbiology, so they haveexcellent properties used for producing foods. Moreover, Lactic acid bacteria possesshigher β-galactosidase activity, which can relieve symptom of lactose intolerantprevailing in human crowd. As a result, lactic acid bacteria gradually become a focusin β-galactosidase research. High cell density culture can enhance lactic acid bacteriato multiply generally by generally in a indefinitely volume container, and it issignificant of enlarging the application scale of lactic acid bacteria production inmicrobiology industry. This research was provided by Modern AgriculturalTechnology System Special Funds(serial number: CRAS-37).In this research, Lactobacillus bulgaricus and Streptococcus thermophilus areused as the start objects of my study, aiming at detecting their β-galactosidase activity,resistance to severe circumstance such as low pH value and high concentrationcholate.After screening the ideal strains, their probiotic properties were detected.hydrophobic property, and screening the ideal bacteria strain. By adjusting andoptimizing the ingredients of MRS broth, it can stimulate the growth of L. bulgaricuseffectively; and through adding functional oligosaccharides to M17broth, S.thermophilus realize their multiplication, too.There are mainly several results as follow through my experiments and analyses.(1) β-galactosidase activities are significantly different among diversity lacticacid bacteria. What is moer, most lactic acid bacteria are sensitive to pH2.0, whileresistant to pH3.0. Even though, L. bulgaricus can grow lightly in pH3.0liquidmedium. As shown in bile salt resistance trials, tolerance to bile of lactic acid bacteriadecreases gradually accompanying with the cholate concentration become higher andhigher. Two excellent lactic acid bacteria, L bulgaricus L2and S. thermophilus S2,were screened by comparing their β-galactosidase activity and resistance to severeenvironment such as low pH value and high cholate conditions. The β-galactosidaseactivities of L2and S2are925U and138U, respectively. At the same time, thehydrophobic properties and autoaggregation abilities of L2and S2in vitro showedthat they can adhere to small intestine of human, meaning that they are potential to be used for food productions.(2) The morphologies of L2and S2are possessing typical characteristics of lacticacid bacteria. All of them are surface smooth and present small pots which are lightwhite. All of them can metabolize lactose effectively, and L2and S2can denature67.53%and73.94%of lactose in skimmed milk in42℃in6h. This is relate to theirβ-galactosidase activity. The combination tests for a better ferment show that, theoptimized proportion of inoculation of L2and S2is2:1(v/v). Under such proportion,they can curd skimmed milk the in4h, endowing the yoghurt with beautiful flavourcharacteristics and better storability. Titration acid of the yoghurt produced by thecombination conserved in4℃do not change notably, showing that the yoghurt do notexist the phenomenon which the yoghurt will become sourer and sourer during theshelf life.(3) The growth curve of L2was obtained by determining its colony-forming unitper milliliter every2h, finding that the optimal time of gathering thallus was10-12h.The effects of different carbon source, nitrogen source and mineral salt on the growthof L2were observed, and the optical carbon source, nitrogen source and mineral saltswere lactose, beef extract, K2HPO4and sodium citrate, respectively. The results oforthogonal test showed that the modified MRS can maintain a rapid growth velocityof L2. L2can grow up to38.87×10~8cfu/mL in modified-MRS after12h under37℃.The viability rate of L2, which was cultured in modified-MRS for12h, was56.40%after freeze-drying, leaving colony-forming unit up to219.22×10~8cfu/mL.(4) The growth curve of S2was obtained by determining its colony-forming unitand the optimal colleting time for thallus was shown to be8-10h. The optical growthsituations in M17were pH6.5and42℃, with the inoculation size of3%. The effectsof different functional oligosaccharides on the growth of S2were detected, and theirstimulation functions ranges from large to small showed as follows:FOS>melitose>lactulose>inulin. The orthogonal test showed that S2can grow up to147×10~8cfu/mL in M17where2%of FOS was added as probiotic after9h. Comparedwith M17, its colony-forming unit increased a log value. The viability rate of S2,which was cultured in modified-M17for9h, was much more24.12%than in M17after freeze-drying, leaving colony-forming unit up to996.10×10~8cfu/mL.