Effect Of Increasing Fermentation Culture PH On Aerobic Respiration In Lactococcus Lactis

Lactococcus lactis is the best characterized member of the lactic acid bacteria(LAB)and is widely used in the dairy fermented products processing,and has also become famous as the first genetically modified organism to be used alive for the treatment of human diseases.L.lactis is generally considered to be aerotolerant anaerobes,but when the hemin is provided,it is able to undergo respiratory growth.Respiration confers L.lactis cells new characteristics such as increased biomass,and resistance to environmental stress such as oxidative stress,acid stress and low temperature stress,which are beneficial to improve the application value of L.lactis.Previously we showed that the leaky expression of the transglutaminase gene in L.lactis subsp.cremoris NZ9000 resulted in ammonia formation and increase of intracellular cytoplasmic pH(pHin).This led to significantly higher biomass production under aerobic conditions.This phenomenon resembled to the reported aerobic respiration of L.lactis,and we assumed that the increased pHin might help to induce L.lactis aerobic respiration.In this dissertation,we took the recombinant strain L.lactis NZ9000(pFL010)and the control strain NZ9000(pNZ8148)as research objects,and assessed the role of the increased fermentation liquid pH on aerobic respiration of L.lactis.The main results were as followed:1.To establish an optimized system to analyze L.lactis NZ9000(pFL010)proteomics,the protein extraction method,the pH of ampholyte,SDS-PAGE gel concentration,and the focusing conditions were optimized.Our results showed that ideal electrophotogram can be gotten when the electrophoresis conditions were as followed: broking cell walls with ultrasonic vibration,the pH of ampholyte ranged from 4 to 7,SDS-PAGE gel concentration 12.5%,and the focusing conditions8000 Vh.2.Comparative proteomic analysis showed that the levels of 59 proteins in strain NZ9000(pFL010)cells grown in aerated cultures were significantly altered compared with that in static cultures.Notably,5 of the differentially expressed proteins fell into the cell respiration process,including succinate dehydrogenase iron-sulfur subunit(SDHB),thioredxoin reductase,flavodoxin FldA,fumarate hydratase,and NADH dehydrogenase I subunit nuoB.3.SDH activity in L.lactis NZ9000(pFL010)cells and the control strain NZ9000(pNZ8148)cells cultured under different conditions were determined,and we deduced that higher pHin might be conducive to the sdhB gene expression and enzyme activity in L.lactis NZ9000.4.In order to validate the higher pH of fermentation liquid can improve cells' respiration,the growth and metabolic characteristics,including the oxygen consumption,glucose utilization,culture pH,and the OD600 of fermentation liquid,ofL.lactis NZ9000(pNZ8148)grown under different conditions were assessed.Results showed that L.lactis NZ9000(pNZ8148)grown aerobically with pH control at6.5±0.1 displayed similar characteristics to aerated hemin-grown cells,and it's energy production efficiency was higher than the fermentative cells,which were grown with a constant culture pH of 6.5±0.1 but without aeration.5.Results showed the exponential-phase(2 h and 4 h)of L.lactis NZ9000(pNZ8148)cells grown aerobically with pH control at 6.5±0.1 possess hemin,whereas no hemin activity was measured in the merely aerobic cells.Measurement of the ATP level in the exponential-phase(2h and 4h)cells showed that the hemin concentration was best correlated with the energy level,that is,the intracellular ATP level of the former cells was 6.24-fold and 4.97-fold that of the latter,respectively.6.Our results showed that the growth of L.lactis NZ9000(pNZ8148)cells grown aerobically with pH control at 6.5±0.1 and cells grown merely aerobically were significantly reduced in the presence of 10 mM DNP,however,the growth superiority of the former cells over the latter cells was significant,as the maximal biomass achieved by the former cells grown in the present of DNP was 58.2% of that in the absence of DNP,whereas the corresponding data for the latter cells was only 32.6%.7.Results showed that catalase was present in L.lactis NZ9000 strains.But the catalase is insensitive to environmental pH and is inactive in the absence of hemin,suggesting the catalase in L.lactis NZ9000 strains belongs to hemin-dependent catalase.8.The survivability of L.lactis NZ9000(pNZ8148)cells grown aerobically with pH control at 6.5±0.1 and cells grown merely aerobically were compared.Results showed that the fomer displayed improved resistance against the low temperature stress compared with the latter cells,as the survival rate of the former cells at the 5th day,10 th day and 15 th day was 1.09-fold,1.11-fold and 1.15-fold that of the latter cells,respectively.When exposed to acid stress(pH 4.0,2 h),the survival rate of the former cells increased 29.6% than the latter.