Study On Breeding And Application Of High Bacteriocin Producing Of Lactobacillus Acidophilus NX2-6

Lactic acid bacteria is widely recognized as safe(GRAS)food grade level microorganisms,it has the capability of producing diacetyl,organic acids,bacteriocins and class bacteriocins and other antibacterial substances,which can kill food-borne pathogens and spoilage bacteria.Adding antimicrobial substances produced by the Lactic acid bacteria into food for preservation,which can improve food safety effectively,and it has potential to become a natural food preservative.The yield of bacteriocins produced by lactic acid bacteria are generally low,which can not satisfy the needs for industrial production.Therefore,increasing the yield of bacteriocins is particularly important.Genome shuffling is a new microbial breeding technique which is developing rapidly.The application of genome shuffling technology to lactic acid bacteria in biological breeding plays an important role for increasing bacteriocin production.On this study,Lactobacillus acidophilus NX2-6 as the original strain,was choosen to enhance the ability to produce bacteriocins by using a variety of mutation ways.1.High-yield mutagenesis strain breeding of Lactobacillus acidophilus NX2-6 was completed by using physical and chemical mutagens,including 60Co? rays,DES mutagenesis and protoplast mutagenesis of UV mutagenesis.According to death rate and the positive mutation rate,conditions of these three methods were optimized as follows:optimal dose of 60Co? ray mutagenesis was 2.0 kGy;the optimum concentration of DES mutagenesis was 0.7%and the best time was 10min;the optimum time of protoplast UV radiation mutagenesis was 20 s.Three high-yield strains ?56,D47 and UV156 were screened out due to high antibacterial potency which were increased by 2.56 times,2.04 times and 2.96 times,respectively,compared to the original strain.2.The best condition for protoplast preparation,regeneration and inativation of Lactobacillus acidophilus NX2-6 were determined.The optimum conditions for protoplast preparation were determined as follows:collecting the bacteria culture after 6h;using high-salt buffer as washing hypertonic;the best sucrose concentration of regeneration medium was 0.2mol/L;the optimum lysozyme concentration was 30 mg/mL;the optimum reaction temperature was 37?;the best time of hydrolysis was 40 min.The optimum conditions for inactivated protoplasts were determined as follows:heat-inactivation at 58?for 30 min;UV inactivation for 110 s.3.Genome shuffling was applied to high-yield strains ?56,D47 and UV156 for strain optimization.The optimum conditions for PEG 6000 induced protoplast fusion were 50%PEG 6000,pH 9.0 and induced for 10 min.By using the optimized condition,F50 were screened out as its high inhibitory potency(30912.15 U/mL)which increased by 5.6 times compared to the original strain(5495.41 U/mL)and the strain was still stable after 10 generations.4.The antibacterial substance produced by Lactobacillus acidophilus NX2-6 was applied on preservation of apple juice.The results showed that bacterial growth in apple juice was prevented in 30 days at 28?,0.1%bacteriocins and 45?,0.2%bacteriocins.The sugar acid ratio,pH value,clarity and fruity flavour of apple juice were maintained.