| The extracellular polysaccharides（EPSs）produced by microbial fermentation,as another branch of polysaccharides of animals,plants and algae have incomparable advantages compared with other sources,such as controlled growth conditions during fermentation,simple extraction method of crude EPSs in fermentation broth,and short-time production of polysaccharides,and so on.With the continuous development of microbial resources,more and more microbial EPSs with rich application prospects have been found.The excellent properties of microbial EPSs can enhance the value and application range of many products,but the available microbial EPSs in the market are limited.Therefore,novel microbial EPSs with strong application potential are important to promote their further development.In contrast to common environmental microorganisms,extreme microorganisms may express certain silent genes under the influence of the environment,which may activate novel metabolic pathways to produce new EPSs.Given the potential to produce novel applied EPSs,extremophiles were screened in high osmotic pressure environments（low water activity bacon and water-rich marine）.On this basis,the EPSs were fermented in high salt mediums,and then their isolation,purification,preliminary structure and application potential were investigated.In addition,this paper also took advantage of the growth of extreme microorganisms to preliminarily verify the feasibility of their open fermentation for EPSs production.The main contents and results were as follows:（1）Two high EPSs-producing strains LR-1 and HS-5 were obtained from Hunan bacon and Jiaozhou Bay seawater of Qingdao by high-salt screening medium and phenol-sulfuric acid method.Subsequently,the phenotypic characteristics,physiological and biochemical characteristics,and molecular biology confirmed that LR-1 and HS-5 were gram-positive and long rod-shaped bacteria.Both of them could tolerate 160 g/L Na Cl,and the optimal concentration of LR-1 and HS-5 was 0 g/L and 40 g/L Na Cl,respectively.Therefore,LR-1 and HS-5 were defined as extreme salt-tolerant and moderate halophilic strains.According to 16S r RNA identification,LR-1 and HS-5 were named Bacillus Subtilis LR-1（B.Subtilis LR-1）and Bacillus sp.HS-5,respectively.（2）The crude EPS of B.subtilis LR-1,an extreme salt-tolerant strain from bacon,was obtained in fermentation broth containing 20 g/L Na Cl,and then the isolation,purification and alcohol precipitation optimization of crude EPS were studied.First,the crude EPS（BL）was extracted by centrifugation,Sevage method,ethanol precipitation,dialysis,anhydrous ethanol and acetone washing.After that,the optimal ethanol precipitation process（4°C precipitation temperature,80 m L/d L ethanol concentration,and12 h precipitation time for maximum yield of 53.43 g/L）of BL was determined based on single factor and response surface methodology.Finally,the main purified component BL-P1 was obtained by column purification.（3）The feasibility of open fermentation of Bacillus sp.HS-5,a moderate halophilic bacterium from Jiaozhou Bay seawater,was studied by coating plate method and microbial community analysis.The open fermentation conditions of HS-5 were determined by fermentation medium adding different Na Cl concentrations.The final conditions were as follows:50 g/L glucose,10 g/L yeast extract,20 g/L peptone,90 g/L Na Cl,and 10%inoculation amount.After that,the crude EPS in the open fermentation broth was extracted and purified by the procedure described in（2）,and two major purified components,BH-1and BH-2,were obtained.（4）The structures of BL-P1,BH-1 and BH-2 were studied by HPLC,HPGPC,FT-IR and NMR.The monosaccharide composition of BL-P1 was mainly glucose and mannose,and the ratio was Glc:Man=7:1.It was a multi-branched heteropolysaccharide with an average molecular weight（Mw）of 41.11 k Da.The residues that made up the EPS chain mainly included:1,4,6-α-D-Glcp,1,4-β-D-Glcp,1,2-α-D-Manp,1,3-β-D-Glcp and T-α-D-Glcp at different locations.BL-P1 contained→4,6)-α-D-Glcp-（1→4）-β-D-Glcp-（1→backbone.The monosaccharide composition of BH-1 was the same as that of BL-P1（Glc:Man=7:1）and Mw was 41.6 k Da.BH-1 was mainly composed of T-α-D-Glcp,T-α-D-Manp-（1-OH）,1,6-α-D-Glcp and 1,3-β-D-Glcp.Its long-chain was→6）-α-D-Glcp-（1→3）-β-D-Glcp-（1→6）-α-D-Glcp-（1→3）-β-D-Glcp-（1→.BH-2 was a heteropolysaccharide mainly composed of mannose,rhamnose,glucuronic acid and glucose（Man:Rha:Glc A:Glc=1:1:2:1）,with Mw of 22.6 k Da.The structure of BH-2 was more complex than BH-1.It was mainly composed of 1,2-α-D-Manp,1,2,6-α-D-Manp,1,2-α-L-Rhap,T-β-D-Glcp A,1,4-β-D-Glcp,1,3-β-D-Glcp and 1,6-β-D-Glc.There could be→2,6）-α-D-Manp-（1→2,6）-α-D-Manp-（1→main chain.（5）The potential applications of BL-P1,BH-1 and BH-2 in antioxidant,rheological,flocculating orα-amylase inhibitory activities were studied in vitro.BL-P1 had advantages in antioxidant and rheological properties.It showed strong scavenging ability on DPPH（77.65%）and hydroxyl radical（93.23%）.The good rheological properties were as follows:1）presenting rheological stability during the cycle of heating and gradually cooling as well as under high salt conditions（Na Cl and Ca Cl2）;2)synergistic thickening among BL-P1and gellan gum solution at a ratio of 1:1（v/v）.BH-1 and BH-2 were water-soluble and water-holding polysaccharides,which were highly soluble in water.They had no significant flocculation activity,but both hadα-amylase inhibitory activity（32.98±2.85%,37.05±3.98%）.（6）The morphological characteristics of BH-1 and BH-2 in open fermentation were studied by thermogravimetric analysis,scanning electron microscopy and X-ray analysis.BH-1 was a kind of partially crystalline EPS with a rough surface and holes of different sizes and flat edges.BH-2 was an amorphous or semi-crystalline structure with smooth film morphology.BH-1 had better heat resistance than BH-2 at temperatures ranging from 30℃to 700℃,with weight losses of 25%and 53%,respectively.In conclusion,two strains of tolerant（halophilic）B.subtilis LR-1 and Bacillus sp.HS-5 were screened from the extreme salt environment of high osmotic pressure given the potential of producing new EPSs by extreme microorganisms.On this basis,the EPSs were obtained in high-salt mediums.After that,the extraction and purification process,optimization of the extraction procss,its structure and application potential of EPSs were introduced in detail,which can further enrich the information on EPSs of tolerant（halophilic）strains.Moreover,the strong antioxidant capacity and gelation properties of BL-P1 made it the potential for application as an antioxidant and additive in products.BH-1 and BH-2 had applications ability inα-amylase inhibitory and had application potential as hypoglycemic products.In addition,the feasibility of open fermentation using Bacillus sp.HS-5 was verified,which laid the foundation for open fermentation of EPSs. |
PDF Full Text Request