| Xenorhabdus nematophilus,which produce a variety of metabolites in the process of interaction with nematodes andinsects,is an important biological resources.Xenorhabdus nematophilus are carried by the infestative nematode into the insect host,and grow in insect haemolymph carriing higher free amino acids,carbohydrates and inorganic salts,which forces the growth and the production of antimicrobial substances of Xenorhabdus nematophilus into a higher osmotic pressure environment.The production of metabolites of Xenorhabdus nematophilus is closely related to the external environmental conditions(temperature,pH,etc).Osmotic pressure as an important environmental factor,affect the growth of microbial cells and product synthesis,but the effect of osmotic pressure on Xenorhabdus nematophilu YL001 is rarely reported.This study,Xenorhabdus nematophila YL001 strain isolated and identified early with high antimicrobial activity was used to investigate the effects of osmotic pressure on growth,antimicrobial activity and active substances Nematophin and Xcns.And the effect of osmotic pressure on the fermentation characteristics of YL001 was further elucidated by adding externally compatible solutes,providing a theoretical basis for utilization of the strain.The main results and conclusions of the thesis are as follows:1.The growth,antimicrobial activity and metabolites of YL001 cells were measured at 0,5,10,20,30 and 40 g/L of NaCl,respectively.Results showed that NaCl concentration at 0 g/L was beneficial to the growth,antimicrobial activity and the production of active metabolites Nematophin and Xcns of YL001.Biomass of YL001 was highest at 0 g/L of NaCl,which was 1.01,1.13,1.50,1.85 and 2.98 times higher than that of 5,10,20,30 and 40 g/L of NaCl respectively.Antimicrobial activity of YL001 the fermentation supernatant and the methanol extract declined with the increase of NaCl.The diameter of the inhibition zone of YL001 fermentation supernatant and methanol extract against Bacillus subtilis were 21.83 and 21.17 mm,respectively,which were significantly higher than those of other treatments;The inhibitory rate of the fermentation broth on M.grisea,B.cinerea,Valsa mali Miyabe &Yamada,P.piricola,T.ucumeris were more than 85% at 0 g/L of NaCl,which were 0.26,1.74,0.12,2.02,1.06 times higher than that of 40 g/L;The protective effect of fermentation supernatant and its methanol extract of YL001 on B.cinerea were 68.98% and 63.26% at 0 g/L,respectively,76.06% and 318.94% higher than that of NaCl at 40 g/L,and therapeutic effects were 43.37% and 44.18%,respectively,which were 671.71% and 197.31% higher than NaCl 40 g/L;The content of Nematophin was 13.84 mg/L,which was 1.37,1.63,2.59,5.47,8.82 times than that of 5,10,20,30,40 g/L,respectively.The relative content of XCN1 at 0 g/L of NaCl,was 1.26,1.98,3.84,7.69,28.55 times than that of 5,10,20,30 and 40 g/L respectively.2.The compatibility solute L-proline could affect the growth,antimicrobial activity and the production of active metabolites Nematophin and Xcns of YL001.Compatible solute Lproline at 50 mM in the TSB medium treated with NaCl stress(0,5,10,20,30,40 g/L),could increase YL001 cell growth by 71.74%,83.33%,234.48%,219.23% and 93.75%,respectively;Antibacterial activity of YL001 with L-proline to Bacillus subtilis increased by 18.08%,19.97%,24.20%,22.22%,34.93% and 50.12%,respectively;L-proline can promote the production of Nematophin when NaCl concentration in culture is less than 10 g/L.When NaCl concentration is 0,5,10 g/L,the accumulation of Nematophin at the present of L-proline increased by 85.06%,38.40%,27.72%;The relative contents of XCN1 at the present of Lproline decreased by 44.09%,35.30% and 3.69%,respectively.3.The growth,antimicrobial activity and metabolites of YL001 cells were measured at 0,125,250,500,750,1000 mmol/L of sorbitol,respectively.Results showed that the growth of YL001 increased first and then decreased with the increase of sorbitol concentration,higest at 500 mmol/L of sorbitol,which was 21.43% and 134.48% higher than that of 0,1000 mmol/L,respectively;The antibacterial activity against B.subtilis was also the highest,which was 54.46% and 14.16% higher than that of 0,1000 mmol/L;The accumulation of Nematophin decreased with the increase of sorbitol concentration,as the content of Nematophin was 13.68 mg/L at 0 mmol/L of sorbitol,but the relative content of XCN1 at 250 mmol/L of sorbitol increased by 54.12%,48.87% than that of 0,1000mmol/L.4.Addition of L-proline could promote the growth of YL001 as the concentration of sorbitol was less than 500 mmol/L,and the growth of YL001 increased by 92.85%,66.13%,47.69%,13.24%,respectively,when the concentration of sorbitol was 0,125,250 and 500 mmol/L;L-proline could increase the antibacterial activity of YL001 as the concentration of sorbitol was less than 250 mmol/L,which increased by 23.57%,34.82% at 0,125 mmol/L of sorbitol;The production of Nematophin also increased by 32.16%?12.58%?9.54% at 0,125,250 mmol/L of sorbitol with the present of L-proline;But the relative content of XCN1 all decreased by 8.06%?3.97%?1.34% at 0,250,1000 mmol/L of sorbitol with the present of L-proline.5.The fermentation kinetics of YL001 under different osmotic pressure conditions was analyzed by 5L fermentor,results showed that the dry weight and the average specific growth rate were 18.08 g/L and 0.027 h-1 when the concentration of NaCl was 0 g/L,respectively,which was 2.32 and 2.52 times higher than that of 30 g/L,respectively;At the same time,the content of Nematophin and the average ratio of Nematophin were 15.17 mg/L and 0.030 h-1,respectively,which were 4.34 and 2.00 times higher than 30 g/L,respectively;Antibacterial activity was 330.0 U/mL at 0 g/L of NaCl,which was 30.28%,45.57% and 519.14% higher than that of 10,20 and 30 g/L;The relative content of XCN1 at 0 g/L of NaCl was 0.07,1.37 and 50.28 times higher than that of 10,20 and 30 g/L,respectively. |
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