| Under salt stress, lactic acid bacteria needs to transport compatible solutes from the environment to relieve the salt stress, but the different types of lactic acid bacteria transport different types of compatible solutes. The present study focused on the compatible solutes which salt tolerant strains can transport. However, the study on the compatible solutes which salt sensitive strains(such as L. bulgaricus) will transport and the effects of compatible solutes on cell membrane is less. Therefore, this paper will focus on the study of the compatible solutes which salt sensitive strains will transport under salt stress and the effect of the compatible solutes on the cell membrane.This paper use L. bulgaricus 3 as the experimental object, determining the sub lethal concentration of salt is 0.2 M. Based on this, using the amino acid analyzer to analyse the kinds and contents of amino acids transfered to the vivo of the bacteria,getting that L. bulgaricus 3 by transfering methionine, lysine lysine, histidine and proline to alleviate the salt stress; by adding the exogenous compatible solutes determining that L. bulgaricus 3 can transport ectoine and L-carnitine under salt stress.By response surface analysis method to determine the optimal compatible solutes combination which can protect L. bulgaricus 3 under salt stress: ectoine is 5.30 m M,methionine is 6.74 m M and histidine is 6.48 m M. And compared to the contrast sample,there is a significant increase in the number of live bacteria(P<0.05) after adding compatible solutes. At this time the cell density than salt increased by 100 times,increasing bacteria effect is significant(P<0.05).By amino acid analyzer and HPLC analysis the compatible solutes that transported during different growth period of L.bulgaricus 3 under salt stress showing taht during the growth lag phase L. bulgaricus 3mainly transported histidine; during the early logarithmic growth L. bulgaricus 3transports histidine, methionine and ectoine as the compatible solutes. The content of ectoine is significantly(P<0.05) higher than the amount of histidine, methionine.During the mid and stage logarithmic growth, and the stable stage, L. bulgaricus 3 transports ectoine and methionine as the compatible solutes. The content of ectoine is significantly(P<0.05) higher than methionine.By scanning electron microscopy(SEM) to observe on cell morphology, showing that the bacteria diameters were decreased significantly(P<0.05) after adding compatible solutes than under salt stress and were not significant(P>0.05) with non-salt-stress conditions cell diameters, indicating that the compatible solutes can protect the cell by changing the morphology of cell under salt stress and after adding the compatible solutes the morphology of cell trends to the morphology of the cell in non-stress condition. By confocal laser scanning microscope(CLSM) to observe the proportion of living bacteria, showing that after the addition of compatible solutes live bacteria ratio increases significantly(P<0.05) compare to that under salt stress and is not significant(P>0.05) compare to the live bacteria ratio under non-stress condition,indicating that after adding compatible solutes the live bacteria ratio will be increased significantly(P<0.05), and it also proves that the salt stress compatible solutes really does the protect to the cell. By flow cytometry to observe the cell membrane permeability showing that after the addition of compatible solute the integrity of cell membrane increased significantly(P<0.05) than the integrity of cell membrane under salt stress and and is not significant(P>0.05) compare to the integrity of cell membrane under non-stress condition of difference, indicating that after adding the compatible solutes under salt stress, the cell membrane permeability is adjusted to the normal self regulation range and providing protective effect to the cell.By zeta potential analyzer to measure the zeta potential of the cell membrane, showing that after adding the compatible solutes the zeta potential value of the cell membrane are less than that under salt stress and the zeta potential value decreases significantly(P<0.05) and is not significant(P>0.05) with the zeta potential value under non-stress condition, showing that after the addition of compatible solutes cell membrane stability is improved. It is also proved that the protective effect of the compatible solutes on cell under salt stress.Using fluorescence spectrophotometer to measure the cell membrane fluidity,showing that the cell fluorescence polarization degree(P) and cell membrane micro viscosity(η) become significantly(P<0.05) compare that under salt stress and are not significant(P>0.05) compare to that under non-stress condition, indicating that after the addition of compatible solute, the membrane fluidity increases significantly(P<0.05)and trends to the cell membrane fluidity under non-stress condition. |
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