| Lactic acid bacteria exopolysaccharides?EPS?is a polysaccharide polymer composed of monosaccharides and derivatives secreted by lactic acid bacteria into extracellular environment during growth.Lactic acid bacteria exopolysaccharides have received increased attention and widely used in food and medicine industry,because of their good physicochemical properties and biological activity,At present,some studies have shown that polysaccharides from different sources have a good effect on promoting plant growth and improving plant resistance to stress.lactic acid bacteria as a safe food-grade microorganism,Hence,the development and application of EPS in agriculture has broad application prospect and research value.In this study,we studied fermentation,characterization and induction of stress resistance in rice by screening high-yield exopolysaccharide from lactic acid bacteria.The main results are as follows:?1?LPC-1 was screened for the highest EPS production by combining“ropy”strand colony formed on the screening plate and the polysaccharide production,which was identified as Lactobacillus plantarum by morphological,physiological,biochemical and16S rDNA tests.Effects of various culture conditions on EPS production were investigated firstly by single factor method.For EPS production with,the prefernable culture conditions were 30?and pH 6.5 for 24 h with sucrose 30 g·L-1,soybean peptone 10 g·L-1,as the carbon,nitrogen of MRS meadium,respectively.The fermentation conditions of EPS produced by L.plantarum LPC-1 were optimized.There main factors were screened by Plackett-Burman design from fourteen nutritional and cultural factors affecting L.plantarum LPC-1 EPS yield.The PB experimental results showed that temperature,pH and concentration of diammonium citrate,were significant factors for the production of EPS.The screened significant factors were then optimized by response surfacemethodology?RSM?.The optimal conditions were confirmed by RSM as follows:temperature 34?,pH 6.7 and diammonium citrate 3 g·L-1.Under these optimal conditions,the L.plantarum LPC-1 EPS yield reached 2064.69 mg·L-1.It is in good agreement with the predicted value and increased by 48.64%compared with the pre-optimization.?2?To remove proteins,two methods were tested:Sevage method and trichloroaceticacid?TCA?method.The results indicated that TCA method is better.TCA finalconcentration 4%was selected and the deproteinization rate was 80.62%.The optimalconditions of ethanol precipitation of EPS produced by L.plantarum LPC-1 were investigated by single-factor experiments and response surface methodology.A mathematical model was then developed to show the significant effect of each factor andtheir interactions on the yield of EPS.The optimal parameters for ethanol precipitation of L.plantarum LPC-1 EPS were confirmed as follows:ethanol concentration 96%,precipitation time 12 h and precipitation time 6?.Under the optimal condition,the model-predicted and experimental values of EPS yield were 2147.25 mg·L-1.?3?The deproteinized and dialysed EPS was further purified by DEAE-Cellulose-52and Sephadex G-100 to afford two polysaccharide fractions?EPS-1 and EPS-2?.The structural characterizations of EPS-1 and EPS-2 were investigated by various methods.SEM image of the EPS-1 showed smooth and folded,and there were a lot of big holes.SEM image of the EPS-2 showed smooth and clastic and there were a few smaller holes.The results of UV spectrum scanning show that EPS-1 has a small amount of protein and nucleic acid,and EPS-2 were absence of protein and nucleic acid.The FT-IR spectrum of EPS-1 and EPS-2 were have the structual of pyranose and?-mannose bond.In addition,EPS-1 has?glucosidic bond.The monosaccharide composition of EPS were determined by GC,the EPS-1 and EPS-2 were mainly composed of mannose and glucose,with different molar ratio of 2.40:15.01 and 3.02:11.63.The results showed that both EPS-1 and EPS-2 had certain antioxidant activity,especially the scavenging ability of DPPH radical.When the concentration of EPS-1 was more than 8 mg·L-1,the scavenging ability of EPS-1could be higher than that of VC.?4?The effects of seed-soaking treatment with different concentrations of lactic acid bacterial exopolysaccharides on the seed germination and seedling growth of rice were investigated.The experimental results indicated that the seed-soaking treatment with lactic acid bacterial exopolysaccharides had no significant effects on the germination rate and germination vigor of rice seeds,but it could significantly increase the germinal root length,and also had significant effects on the soluble protein content and amylase activity in seeds,To investigate the effects of exopolysaccharides?EPS?from lactic acid bacteria on biological traits of rice seedlings and physicochemical properties of rice rhizosphere soil.It was shown that application of EPS could promote the growth of rice seedlings and improve rhizosphere soil environment,enhance soil structure,improve soil aggregate stability,mitigate soil acidification,increase soil enzyme activities,and improve soil nutrients.Correlation analysis and principal component analysis showed that the mechanism of rice growth promotion by exopolysaccharides was different from that by glucose.The exopolysaccharides of lactic acid bacteria could be used as a new plant growth regulator.?5?Different concentrations of EPS of lactic acid bacteria was applied to rice seed in their germination stage.The results indicated that the EPS can significantly increase seed germination potential,germination rate and germination index,root growth were also increased significantly.As the the EPS concentration increased,the trend of rice seed germination increased firstly and then decreased under different stress.The content of 200mg·L-1 EPS significantly alleviated the damage caused by acid and salt stress.Specifically,germination potential,germination rate and germination index were increased by 178.2%,38.7%and 114.4%under acid stress,respectively.Under salt(7 mg·L-1 NaCl)stress,compared with without EPS,germination potential,germination rate and germination index were also increased by 152.9%,73.8%and 103.0%,respectively.Their difference reached at a significant level?P<0.05?.Further analysis showed that the process of soaking in EPS would reduce malondiadehyde?MDA?content and enhance the activity of antioxidant enzymes in varying degree such as catalase?CAT?activity,peroxidase?POD?activity,superoxide dismutase?SOD?activity of rice shoots,which could effectively alleviate the toxic effects of stress resistance.At the same time,EPS could also promote the growth of rice seedlings under stress.This study provides theoretical and practice foundation for improving crops stress resistance by microbe-derived.?6?In order to investigate effect of EPS of lactic acid bacteria on the growth of rice seedlings and soil environment in rhizosphere under copper stress,different concentrations of EPS of lactic acid bacteria was applied to the soil under copper.The results indicated that the growth indexes and soil environment of rice seedlings under copper stress were significantly improved by exogenous addition of extracellular polysaccharides of lactic acid bacteria.At the seedling age of 10d,the content of 2 g·Kg-1 EPS significantly alleviated the damage caused by copper stress Specifically,plant height,leaf length,leaf width,root length,root number and fresh weight increased 34.28%,44.66%,22.22%,41.81%,21.43%,46.15%,respectively.At the seedling age 20d,under high concentration of copper stress,compared with without EPS,the content of chlorophyll a,chlorophyll b,carotenoid and total chlorophyll were increased by 61.01%,45.11%,211.72%,58.06%.At the same time,the content of soluble sugar and protein and the activity of antioxidant enzyme were significantly increased by without EPS under copper stress.On the other hand,the physicochemical properties and soil enzyme activity of rhizosphere soil were also improved to a certain extent.The results of real-time fluorescence quantitative PCR showed that lactic acid bacteria EPS could promote the expression of Sulphate transporter,CatA,OsHMA4,Cytochrome P450 gene under low concentration of copper stress. |
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