Fermentation Condition And Functional Study Of Exopolysaccharides From Bacillus Amyloliquefaciens

Bacillus amyloliquefaciens is a GRAS(generally recognized as safe)strain,with the ability to inhibit bacteria and fungi.At present,researches are focused on antibacterial and antifungi compounds such as baciamin,peptide lipid antibiotics,macrolactin,produced by B.amyloliquefaciens.However,little attention is paid to the exopolysaccharides(EPS)from B.amyloliquefaciens,though microbial polysaccharides have good physical and chemical properties as well as biological activity.In this paper,the fermentation condition,structural characterization,chemical modification and the related functions in vitro of the EPS from B.amyloliquefaciens PB6 were investigated.The main research contents were as follows:1.The fermentation of EPS from B.amyloliquefaciens:The fermentation conditions of EPS were optimized using single factor experiments and response surface methodology(RSM)design.As a result,the optimal culture medium ingredients were sucrose 400 g/L,yeast extract 2.5 g/L and sodium chloride 10 g/L.The optimum cultivation conditions of initial pH value,inoculation amount,fermentation temperature and rotate speed were 7.0,4%,30℃,150 r/min,respectively.The maximum yield of EPS was 104.37 g/L after fermentation for 26 h under these conditions.2.The purification of EPS from B.amyloliquefaciens:The crude EPS extract from the fermentation broth of B.amyloliquefaciens PB6 was purified by ethanol precipitation,sevag deproteinization,dialyzation and gel permeation chromatography on Superdex G 200.The structure and functions of the fraction with the higher yield(named the P)were further studied.Gel permeation chromatography analysis indicated that P was a homogeneous polysaccharide with a relative molecular weight of 6.02 kDa.The content of sugar and protein in P was determined as 99%and 0.25%,and uronic acid was not detected.3.The structural characterization of the polysaccharide P:The results from complete hydrolysis and HPLC analysis showed that P was mainly composed of fructose and a small amount of glucose.FT-IR spectra showed that fructose units existed in P as fructofuranose,NMR spectra showed that P was a typical levan type polysaccharide,consisted of(2→6)-linked β-D-Fruf residues with(1,2→6)-linked P-D-fructose branch.By Methylation reaction and GC-MS analysis the branch degree of P was found as 3%and there was a little glucose at the end of its main chain.4.The chemical modification and the corresponding bioactivities of P:four chemically modified polysaccharides from P were prepared as acetylated polysaccharide(YP),sulfated polysaccharide(SP),sulfonylated polysaccharide(HP)and phosphorylated polysaccharide(PP).Their antioxidant,antibacterial and antitumor activities were evaluated by various methods in vitro.The result showed that chemical modification could significantly improve the bioactivities of the exopolysaccharide P,and different derivatives showed different behaviors.After chemical modification,the scavenging ability on ABTS free radical,DPPH radical and the iron reducing ability all increased,the antioxidant capacity was in the order of SP>HP>YP>PP>P.Meanwhile,PP showed an obvious inhibitory effect to Staphylococcus aureus,and a better inhibitory effect than SP to Bacillus subtilis,but only SP had an inhibitory effect to Salmonella.In addition,the antitumor activity in vitro on Hela was in the order of YP>HP>SP>P>PP.