| Lactococcus lactis has already acquired the status of generally recognized as safe(GRAS), and has been considered as probiotic bacteria which was beneficial tohumans.In the practical application, it will face a lot of unsuitable living environments,such as low temperature, high osmotic pressure, bad human intestinal microenvironment,etc. People keep in trying to improve the resistance of L. lactis in the stress environmentand to expand its application field.In previous research, it had been found that L. lactis NZ9000(pFL010) mtg geneleaky expression can make the cell wall thicker. Base on that, in this paper, the differenceof the recombinant strain and the control strain’s stress resistance in unsuitableenvironment, including the acid, bile salt, low temperature and osmotic pressure wascompared. Then, to find the inner relation between the thickened cell wall and theresistance ability to multiple stresses of the recombinant strain, the two strains weretreated with cystamine, which is the competitive inhibitor of MTG and might decreasethe extent of the cell wall thickening. The results were as follows:With the increase of dissolved oxygen concentration, the biomasses (OD600) of therecombinant strain significantly rose. The highest biomass in15ml medium underagitation condition was3.5times larger than that of40ml medium under static condition,4.5times larger than that of the control strain under agitation condition, and thelogarithmic phase of the recombinant strain in higher dissolved oxygen concentrationenvironment increased to16h.The fermentation liquid pH of the recombinant strain was close to neutral at the endof fermentation. The highest biomass in GM17medium at pH5.0was88.8percent ofthat under normal conditions. For the control strain, the highest biomass in GM17medium at pH5.0was48percent of that under normal conditions. The acid stressresistance of the recombinant strain at stationary phase and logarithmic phase wasbasically the same. But for the control strain, the acid resistance of cells at stationaryphase was stronger than that at logarithmic phase. When confronted by pH3.0for10min,the survival rates of the two strains were largely different, i.e., the recombinant strain was2296.8times higher than that of the control strain. When the stress time was prolonged,the survival rates of the both strains suddenly dropped off. Cultivation without containing chloramphenicol and induced by pH5.0could enhance the acid resistance of the twostrains.When the temperature was under4℃, the recombinant strain could slowly growth,but the viable cell number of the control strain had always been a downward trend andwas almost zero finally, which indicated that the cell vitality of the recombinant strainwas stronger than the control strain at4℃. At-20℃the survival rates of the recombinantstrain and the control strain both declined. The difference between the two strains wassmall.At30℃, the growth of the recombinant strain cultivated in the medium containing5%bile salts was not affected. It was worth noting that the biomass obtained at37℃washigher than that at30℃. When exposed to0.5%bile salt for2h, the survival rate of therecombinant bacteria was48.46%±4.02%. However, the control strain was sensitive to bilesalt, whose bile salt tolerance level was lower than0.01%.When exposed to0.03%bile saltfor2h, the control strain failed to grow. When decreasing the bile salt concentration to0.02%, the survival rate of the control strain was57.33%±4.62%.For the recombinant strain and the control strain, the growth performance ofstationary phase cells under high osmotic conditions was similar to that of logarithmicphase cells. The growth performance of the recombinant strain cultivated in GM17medium containing1%NaCl was better than the control group (no NaCl). In the presenceof4%NaCl, The highest biomass (OD600) of the recombinant strains was77.35percentof that under non-stress conditions. While no growth of the control strain was observedunder the same osmotic stress conditions.The qualitative test of the bile salt hydrolase activity indicated that the strong bilesalt resistance of the recombinant strain was not due to the existence of bile salt hydrolase.Exposure of the recombinant strain pretreated with40mmol/L cystamine to0.5%bilesalt, the survival rates deceased from48.46%±4.02to14.2%±1.76%; the viable cellnumbers stressed by4%NaCl deceased by13.55%; after removing cell wall withlysozyme, during the gram stain process, the time of restraining cells to red was ahead of1h. For the control strain, almost no change was observed under the same conditions.These results demonstrated that the resistance ability to bile salt and high concentrationof NaCl of the recombinant strain might be related to the thickened cell wall. |
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