Spore-producing Breeding Of Lactobacillus Reuteri Of Avian Origin

Protoplast fusion technology is an important method in microbial breeding and a whole genome engineering method to rapidly improve cell phenotype.It has been widely used in many fields.The purpose of this study was to conduct fusion breeding of spore producing lactic acid bacteria for Lactobacillus reuteri of avian origin through protoplast fusion technology,to improve its environmental tolerance and transgastric capacity,and enhance its probiotic function.In this way,the spore-producing ability of Bacillus licheniformis was introduced into the Lactobacillus reuteri of avian origin,and the digestive enzyme capacity was endowed with high yield.So as to provide a feasible method for spore-producing lactic acid bacteria breeding,and a high-quality strain for the production of microecologics.In this study,a Lactobacillus reuteri of avian origin,named LRC,was isolated from chicken cecum contents.With LRC and Bacillus licheniformis as the parent strain preparation of protoplast,PEG-6000 to promote protoplast fusion,40 suspected fusants was obtained on regeneration medium.Strains of FRL1-FRL40,first by morphological observation,spore staining,produce acid and identification ofβ-galactose glucoside enzyme test,and then according to the gyr B gene of Lactobacillus reuteri and Bacillus licheniformis’ s 16S-ITS interval sequence design specific primers P1/P2 and P3/P4,establish a double PCR identification method of fusion.The identification results showed that all the 40 suspected fusants produced spores,and 34 of them were positive for acid production.The double PCR results showed that all the 34 fusants produced two specific electrophoretic bands,and but only 6 of them were positive for the identification of β-galactose glucoside enzyme test.The digestive enzyme production ability of 6 strains of fusants positive for β-galactosidase test was analyzed: the ability of produce amylase,Bacillus licheniformis(27.11 U/m L)> fusants(16.32U/m L-19.05 U/m L)> LRC(1.76 U/m L);the ability of produce extracellular neutral protease,Bacillus licheniformis(9.46 U/m L)> fusants(3.92-5.78 U/m L)> LRC(2.34 U/m L);the capacity of produce extracellular alkaline protease,Bacillus licheniformis(37.16 U/m L)> fusants(25.14-31.25U/m L)> LRC(1.27 U/m L).Two fusants FRL22 and FRL32 with good genetic stability were obtained by genetic stability test.After artificial gastric juice simulation for 2 h,the survival rate of each strain was LRC(27.22%)> FRL22(18.74%)> FRL32(17.47%)> Bacillus licheniformis(11.72%),and the spore survival rate,Bacillus licheniformis(47.46%)> FRL3(44.75%)> FRL22(84%).The results of feed fermentation with single strain inoculation showed that the number of viable bacteria of LRC decreased continuously during fermentation.The viable number of Bacillus licheniformis,FRL22 and FRL32 increased continuously and reached the peak in the first seven days of fermentation then decreased,but the number of spores increased gradually.The number of viable bacteria at 14 d of fermentation was FRL32(5.82×108 CFU/g)>Bacillus licheniformis(4.43×108 CFU/g)>FRL22(3.64×108 CFU/g).In conclusion,although the Lactobacillus reuteri of avian origin isolated in this study has a strong ability to pass through the stomach,it is not suitable for feed fermentation.After fusion with Bacillus licheniformis,its ability to pass through the stomach is further improved,and it is suitable for the fermentation of raw feed,and it has a higher ability to produce digestive enzymes.Two strains of spore-producing Lactobacillus reuteri FRL22 and FRL32 were obtained by genetic stability test,which laid a foundation for the preparation of microecologics in the future.