Extraction And Component Analysis Of The Anti-oxidative "Protective Coat" Produced By The Oxygen-tolerant Mutant Strain With Isoflavone Reducing Activity

Strain Lactobacillus sp.Niu-O16,which is capable of converting isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein under anaerobic conditions,is an obligate anaerobic Gram-positive bacterium isolated from bovine rumen gastric juice.After a long-term oxygen-tolerant domestication process,we obtained an oxygen-tolerant mutant strain,which we named Aeroto-Niu-O16.The oxygen-tolerant mutant strain Aeroto-Niu-O16 forms a “protective coat” when being incubated aerobically.The“protective coat” is mainly composed of exopolysaccharide.In this study,the method of extracting exopolysaccharide from the Aeroto-Niu-O16 protective membrane of the mutant strain was discussed,and the species and proportion of monosaccharide of the protective membrane polysaccharide were analyzed,which lay the foundation for further study about the formation mechanism of the protective membrane outside the mutant strain.The proper extraction method of exopolysaccharide was determined by orthogonal test.The contents of total sugar,protein and ketone sugar were determined by phenol sulfuric acid method,coomassie brilliant blue method and resorcinol method respectively.The monosaccharide components were analyzed by PMP precolumn derivatization high performance liquid chromatography,and the scavenging capacity of DPPH,superoxide anion and hydroxyl radical in vitro were tested.The results are as follows:(1)The addition of cellulase could effectively extract the protective membrane components of exopolysaccharide.The proper extraction temperature was 50?C,and when the enzyme concentration was 10 mg/mL and the enzyme dosage was 5~20 ?L,the extracted total sugar concentration increased with the increase of enzyme dosage.After the enzyme dosage exceeded 20 ?L,the extracted exopolysaccharide concentration did not increase with the increasing of enzyme dosage;when the extraction time was extended from 0.5 h to 1.5 h,the total sugar concentration increased significantly,but the total sugar concentration did not change significantly when the extraction was longer.Through the orthogonal test,the order of the three factors was: extraction temperature > enzyme dosage > extraction time;the optimum extraction conditions were as follows: extraction temperature 50?C,enzyme dosage 20 ?L,extraction time 2 h,and the concentration ofexopolysaccharide extracted under the proper condition was 44.8 ?g/mL.(2)The component of the extracted exopolysaccharide samples was analysised.By the coomassie brilliant blue method,it was found that there were 7.6% protein components in the exopolysaccharide sample,and the resorcinol rest of sample was positive,indicating that the component contained ketone sugar and the proportion was 9.4%.The standard curves of the inregral area of high performance liquid chromatography correspending to the concentration of monosaccharide-PMP derivatives were drawn.Raffinose,which was composed of two monosaccharides,was hydrolyzed by trifluoroacetic acid and analysised by PMP precolumn of column.The components and proportion of raffinose were measured by high performance liquid chromatography.The species of monosaccharide components and the ratio of the three sugars were found to be in full agreement with the actual structure of the three sugar.The results showed that acid hydrolysis,PMP precolumn derivatization and high performance liquid chromatography were feasible.The exopolysaccharide samples were hydrolyzed by acid and analysised by high performance liquid chromatography.The HPLC analyses showed that glucose and galactose were the two main components of the exopolysaccharide extracted from the “protective coat” of strain Aeroto-Niu-O16.The ratio of the two monosaccharides was glucose: galactose =1.02:1.(3)The antioxidant activity of the extracted exopolysaccharides was analyzed.The results showed that the extracted exopolysaccharide was able to scavenge DPPH free radical,hydroxyl radical and superoxide anion radical.When the concentration of the exopolysaccharide samples was 3 mg/mL,the scavenging rate of DPPH free radical and hydroxyl radical reached the maximum and were 62.8% and 43.9% respectively,and when the concentration of the exopolysaccharide samples was 4 mg/mL,the scavenging rate of superoxide anion reached the maximum and was 32.6%.