H~+-ATPase Activity Of Lactobacillus Delbrueckii Subsp. Lactis Able To Postpone Postacidification Of Yogurt

Yogurt can still product lactic acid during storage, which usually leads to postacidification. Flavor and sensory quality of yogurt will be affected by postacidification. Persistent acid production by Lactobacillus delbrueckii subsp.lactis during refrigerated storage is a major cause of postacidification. Therefore, the aim of this study was to research the relation between physiology activity of L. delbrueckii subsp. lactis and postacidification to improve the flavor and sensory quality of yogurt during storage.1. Three mutant strains of L. delbrueckii subsp. lactis (L5) with reduced H⁺-ATPase activities were isolated with MRS (neomycin sulfate, 400μg/mL). The H⁺-ATPase activities of mutant strains 5M6, 5M1 and 5B1 were reduced to 51.3%, 27.7% and 34.3% that of the parental strains. The four strains were cultured for 24h at 37℃in MRS. Results of neomycin sensitivity, biomiss and medium acidification showed that: the mutant strains 5M6, 5M1 and 5B1 were proved to be genetic stability.2. Compared to parental strain L5, physiology specialities of 5M6, 5M1 and 5B1 were performed:â‘ 5M6, 5M1 and 5B1 were more slenderer than parental strain whever cultured in MRS or skim milk.â‘¡Theβ-galactosidase activities of 5M6, 5M1 and 5B1 were obviously reduced to 3.42%,2.56%,2.12% that of parental strain.â‘¢Cultured in MRS for 24h at 37℃, the ATP level of parental strain L5 was obviously lower than the mutant strains 5M6, 5M1 and 5B1. The ATP level of L5 was 0.63×10^-18mol/cell; the ATP levels of the mutant strains were 1.18×10^-18mol/cell,1.60×10^-18mol/cell and 1.38×10^-18mol/cell.â‘£Cultured for 24h in MRS of pH4.2, 5.2 and 6.2 at 37℃, the biomass reductions of mutant strains 5M1, 5M6 and 5B1 were more than parental strain L5. The biomass of 5M1, 5M6 and 5B1 reduced by 83.9%, 73.6% and 69.6% and the biomass of L5 reduced by 52.0%.⑤Cultured for 24h in MRS with NaCl 1%, 3% and 5% at 37℃, compared to the cultures in MRS without NaCl, the biomass reductions of mutant strains 5M1, 5M6 and 5B1 were more than parental strain L5. The biomass of 5M1, 5M6 and 5B1 reduced by 94.1%, 88.5% and 79.7% and the biomass of L5 reduced by 50.5%.â‘¥Cultured for 48h in skim milk at 37℃, the acidity of L5 was 2.02% and the pH of midium was 3.28; the acidities of 5M6, 5M1 and 5B1 were 0.48%, 0.76% and 0.50%, and the pH of midium were 5.40, 5.22 and 4.52.⑦Metabolism of amino acid were determined by HPLC. Cultured for 12h in MRS at 37℃, the conversion rate about Glu to GABA of parental strain was 13.9%, which is slightly lower than 5M1,5M6 and 5B1. There were also differentencees berween 5M1,5M6 and 5B1. The conversion rates of 5M6, 5M1 and 5B1 were 16.7%, 15.6% and 15.4%. In addition, the four strains all had a higher conversion rate after they were cultered for 18h. The conversion rate of L5 was 15.1%, which was still lower than other three strains; the conversion rates of 5M6, 5M1 and 5B1 were 17.3%, 16.2% and 17.0% respectively.3. Effects of temperature, pH, time of reaction, substrate(ATP), metal ions, culture time and DCCD on H⁺-ATPase activity were studied.â‘ The molecular weight of H⁺-ATPase is 126.0KD; The maximum activity of H⁺-ATPase occurred within 5 min at 42℃and pH 7.5 when 3mmol/L of ATP was addied.â‘¡The activity of H⁺-ATPase was influenced by metal ions. H⁺-ATPase activity was increased by K⁺, Na⁺and Mg²⁺. The H⁺-ATPase activities of 5M1, 5M6, 5B1 and L5 were increased by Mg²⁺by 107.0%, 45.9%, 163% and 16.7%. H⁺-ATPase activity was reduced by Ba²⁺, Mn²⁺, Al²⁺ and Li⁺. The H⁺-ATPase activities of 5M1, 5M6, 5B1 and L5 were reduced by Al³⁺ by 76.0%, 81.7%, 60.4% and 93.4%.â‘¢It was also found out that H⁺-ATPase activity was partially inhibited by DCCD. H⁺-ATPase activities of L5,5M6,5M1 and 5B1 were obviously reduced by 87.6%, 63.9%, 32.8% and 72.1% with DCCD(60mmol/L) added to the reaction mixture.â‘£The four strains all had higher activities of H⁺-ATPase when cultured for 8h and 24h in MRS at 37℃. When cultured for 8h, the H⁺-ATPase activities of 5M1, 5M6, 5B1 and L5 were 0.25U, 0.40U, 0.24U and 4.55U. When cultured for 24h, the H⁺-ATPase activities of 5M1, 5M6, 5B1 and L5 were 0.18U, 0.30U, 0.19U and 3.80U.4.Each of the four strains above and S.thermophillus were inoculated with a percentage of 2% to skim milk and cultured at 42℃until curdling.Then the yogurt were stored at 4℃for 30d.â‘ There were no significant differences about curdling time and curdling pH between parental strain and mutant strains. After a storage of 5d at 4℃, the acidity of yogurt fermented by L5 was 1.22% and pH was 3.87; the acidities of yogurt fermented by 5M1, 5M6 and 5B1 were 0.76%, 0.76% and 0.88% and pH were 4.26, 4.24 and 4.22. After a storage of 10d at 4℃, the acidity of yogurt fermented by L5 was 1.35% and pH was 3.75; the acidities of yogurt fermented by 5M1, 5M6 and 5B1 were1.12%, 1.17% and 1.13% and pH were 4.15,4.05 and 4.08. Obviously, yogurt fermeted by mutant strains can keep lower acidity whin shelf life of yogurt, which postponed the postacidification of yogurt for 5 days at least.â‘¡Sensory evaluations of the four strains indicated that: yogurt fermeted by 5M6, 5M1 and 5B1 had a good flavor. Results of SDS-PAGE showed that: there were no significant differences in protein composition of yogurt fermented by mutant strains and parental strain.â‘¢Inoculated Lactobacillus acidophilus NCFM to the yogurt above, The viable counts of NCFM about yogurt fermented by mutant strains 5M1, 5M6, 5B1 and parental strain were 1.5×10⁸cfu/mL, 1.3×10⁸cfu/mL, 1.5×10⁸cfu/mL and 9.0×10⁷cfu/mL.Obviously, there was a increase in viable counts(>10⁸) of NCFM about yogurt fermented by mutant strains. So, mutant strains with reduced H⁺-ATPase activity were very useful to improve the survival of probiotics.