Reasearch On The Effect Of D-amino Acid On Acid Resistance And The Nisin Yield Of Lactococcus Lactis F44

Nisin has the characteristics of stability,safety and antibacterial,and is recognized as a safe and non-toxic food additive.Lactococcus lactis F44 is a production strain of nisin.Lactococcus lactis produces a large amount of lactic acid during the production of nisin fermentation,which continuously reduces the p H of its fermentation liquid.This acidic condition is not conducive to the growth of the strain,which in turn reduces the production of nisin.The preliminary study showed that the acid resistance of Lactococcus lactis cell wall peptidoglycan could be improved and nisin yield could be increased.The cell wall is the first barrier for cells to resist external pressure,and Damino acids are closely related to the biosynthesis of peptidoglycan.The effects of 19 D-amino acids on Lactococcus lactis F44 were analyzed by acid resistance and nisin yield.The composition of cell wall peptidoglycan was analyzed by vancomycin fluorescence marker and liquid-factor-mass spectrometry.The strains expressing mur F and ddl related genes for cell wall synthesis were constructed by molecular biology.The correlation between D-amino acid and Lactococcus lactis cell wall synthesis was investigated.The results of acid resistance test and nisin valence analysis show that the survival rate and nisin yield of D-amino acids added to the medium have increased.And the possibility of D-amino acid conversion to L-amino acid participation in metabolism was ruled out by nisin valence detection.Experimental data on Nisin high-yield bacteria G-02 further indicate that the addition of D-amino acids can indeed increase nisin production.The results of contrast analysis of the fluorescence intensity of the strain with DMet or D-Phe in the medium were found to be weak.It is shown that D-Met or D-Phe may bind to the fourth or fifth position of the cell wall peptidoglycan.The results of liquid-mass spectrometry(LC-MS)test showed that the components of diglycotetrapeptide and diglycopentapeptide extracted by cell wall peptidoglycan contained D-Met or D-Phe,which verified the hypothesis of fluorescence markers.The results of over-expression of mur F and ddl genes show that the overexpression of MurF and Ddl proteins increases the probability that D-Met and D-Phe participate in cell wall synthesis.Moreover,the recombinant strains expressing MurF and Ddl showed good acid resistance and high nisin yield in the medium containing D-Met and D-Phe.Among them,the survival rate of the patient condition strain was increased threefold,and the nisin yield reached 4384.17 IU/m L,which was 64.57 % higher than that of the original strain.The expression of mur F gene in high-yield bacteria G-02 and the addition of D-Met and D-Phe mixed amino acids can increase the nisin yield to about 12,000 IU/m L,which is the highest yield reported in the current literature.The results of this study show that the excessive expression of genes related to the addition of D-amino acids and cell wall synthesis can increase the acid resistance and nisin yield of the strains,and provide new ideas for the construction of high-yielding nisin strains.