| Numerous studies have shown that some lactic acid bacteria possess many important biological functions such as reducing cholesterol,anti-oxidation,enhancing immunity and anti-tumor,which makes these lactic acid bacteria have the potential as probiotics in human's gastrointestinal tract.It is widely acknowledged that probiotics must be present at an adequate level at mucosal sites to achieve beneficial effects.To guarantee cell viability during passage through the gastrointestinal tract,it is necessary to increase LAB tolerance to physiological concentrations of bile salts.In addition,lactic acid bacteria will encounter many other environmental stresses,such as osmotic stress,low-temperature stress and antibiotic stress,which seriously reduce cell activity,therefore,how to increase lactic acid bacteria's ability to endure environmental stresses has become an important research focus.Our previous studies have shown that the recombinant Lactococcus lactis NZ9000(pFL010),which heterologously produced transglutaminase,exhibited a thickened cell wall relative to the control strain 8148.Limited studies have demonstrated that cell wall thickness may be responsible for the resistant phenotype of some microbes.Thus,we set out to compare the bile salt resistance difference between the combinant strain FL010 and the control strain 8148,and explore the correlation between this difference,the thickened cell wall and transglutaminase.The influences of exogenous transglutaminase on the tolerance of various lactic acid bacteria strains to environmental stresses,including bile salt stress,erythromycin stress,NaCl stress and low temperature stress,were evaluated.The main results were as follows:(1)Plate count method and spectrophotometer turbidometry can both be used to evaluate the resistance to bile salt.The former can be used to analyse quantitatively,but error prone;the latter is convenient and possesses good data-parallelism.Strain FL010 can tolerate 20% of bile salt(the minimum tolerance was 38.35%),whereas strain 8148 can only tolerate 0.04% bile salt(the minimum tolerance was 10.77%),which indicated that there was great differences in bile salt resistance between the two strains.(2)Addition of cystamine into the culture medium significantly inhibited the growth of strain FL010,as the tolerance rate of cells treated with 12 mM cystamine reduced by about 50% compared with that of the control(without cystamine treatment),and tolerance rate decreased with the increaseing of cystamine concentration.Nevertheless,cystamine had no effect on the growth of strain 8148.Similarly,the bile salt tolerance rate of strain FL010 obviously declined after the treatment of cystamine,but the bile salt tolerance rate of strain 8148 almost kept unchanged before and after the cystamine treatment.The synchronous culture of strain FL010 showed that the bile salt tolerance of young cells was 52.79%,significantly lower than the 74.07% tolerance of aged cells,indicating that the bile salt resistance of strain FL010 had something to do with the expression of mtg gene.Strain 8148 cells treated with transglutaminase displayed obvious improved resistance against bile salts compared with the control(without transglutaminase treatment).(3)Tansmission electron microscope(TEM)showed that the cell wall of strain 8148 appeared to be much more thick compared to the control,when it was cultivated with a final concentration of 9 U/mL transglutaminase,and 18 mM cystaminse existed in the medium of strain FL010 exert an influence on its cell wall as the cell wall was obviously thinned.(4)Analysis of the animo acid composition of the peptidoglycan revealed that in strain 8148 cells there was 6.02 ± 0.42 ?g glutamine and 51.6 ± 3.05 ?g lysine per mg of peotidoglycan,respectively,and Lactobacillus plantarum YL contained 13.36 ?g glutamine and 19.05 ?g lysine per mg cell wall peptidoglycan.Lactococcus lactis ssp.lactis NFL contained 64.62 ?g lysine per mg peptidoglycan,but no glutamine was detected in its peptidoglycan.Addition of transglutaminase into the medium could improve bile salt tolerance of Lactobacillus plantarum.Nevertheless,Variations of transglutaminase level did not influence persistence of L.lactis ssp.lactis NFL in bile salts.Results indicated that transglutaminase can also increase bile salt resistance of lactic acid bacteria whose cell wall peptidoglycan contains glutamine and lysin.(5)Results revealed that strain 8148 cells cultivated supplemented with transglutaminase showed higher viability after 0.5 mg/mL erythromycin treatment compared to the control cells,as survival of the former was 1.9-fold and 9.6-fold that of the latter when exposed to 500 ?g/mL erythromycin for 1 h and 2 h,respectively.(6)The sensitivity of strain FL010,strain 8148 and Lactobacillus plantarum YL were different when exposed to different concentration of Na Cl.Cystamine could significantly reduce the maximum biomass of strain FL010 cultivated with NaCl,but had little effect on strain 8148 under the same conditions.Cystamine could increase the tolerance rate of strain 8148 and Lactobacillus plantarum YL when exposed to NaCl stress.(7)Strain 8148 and Lactobacillus plantarum YL had higher tolerance rate under 4? than under-20?.And,their low temperature stress tolerance rate could be increased to some extent when treated with transglutaminase.In conclusion,addition of transglutaminase into culture medium can increase environmental stress tolerance of lactic acid bacteria with the presence of glutamine and lysine in the peptidoglycan of the cell wall. |
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