Study On The Preparation Of Antioxidant Substances With Millet Bran By Bacillus Natto Solid State Fermentation Method

As a by-product of millet processing,millet bran is rich in nutrients,while China is a large grain production country,the annual yield of millet is about 4.5 million tons the annual output of millet bran is up to 0.4 million tons.Millet bran is mainly used for feed and fertilizer,the utilization rate is low,resulting in a waste of resources,so the in-depth studied of millet bran to explore its potential is of great significance.In this paper,we used defatted millet bran as raw material,using Bacillus natto to degrade the high molecular compounds in millet bran by solid state fermentation,which could enhance the antioxidant capacity of antiradical agents seprated from millet bran.The main contents of the research involve the optimization of solid state fermentation condition,comparison of antioxidant activities of extracts from millet bran before and after fermentation,the synergetic effect between the main antioxidant of fermented millet bran,purification and antioxidant capacity of the main antioxidant were studied.The main results of the studied were found,the single factor test was based on the medium of defatted millet bran 5₀0 g,NH2CONH2 0.10 g,K2HPO4 · 3 H2O 0.02 g and distilled water 6.00 mL.The best fermentation conditions were inoculum size 8%,average particle size 0.22 mm?60-80 mesh?,pH 7.0,temperature 34 ?,time 24 h.Under these conditions of single factor experiment,T-AOC?Total antioxidant capacity?was used as the evaluation index to carry out Plackett-Burman test.It was found that the effect of each factor was as follows:fermentation temperature>fermentation time>average particle size>initial pH>inoculum amount.Due to the limitation in the screening process,the particle size can not be accurately controlled,so the fermentation temperature,fermentation time and initial pH are chosen as the factors of Box-Behnken design for response surface methodology.The optimal fermentation conditions were pH 6.7,time 26 h,temperature 35 ? inoculum size 8%,average particle size 0.22 mm.The actual value of T-AOC was?344.51 ±8.02 U/g?,which was about 2.18 times higher than the unfermented millet bran?108.12 ± 3.10 U/g?.Then the scavenging rate of DPPH?1,1-diphenyl-2-picrylhydrazyl?free radical was 83.11 ±0.94%.Antioxidant activity?T-AOC,DPPH free radical scavenging ability and reducing ability?of fermented and unfermented millet bran extracts were determined and compared.The results showed that the T-AOC,DPPH free radical scavenging ability and reducing ability of millet bran extracted from fermented millet bran were significantly higher than that of unfermented millet bran.The interaction among polysaccharides,polypeptides and polyphenolics were determined by compounding method,results showed that experimental treatments that had been studied have significant effect on all determined parameters.It was showed that these treatments had additive effect on T-AOC however there was a remarkable antagonistic effect on DPPH free radical scavenging ability and an obvious synergistic effect was found on reducing ability under the experimental concentration range The polysaccharides,polypeptides and polyphenolics were compared with fermented millet bran extract.The results showed that the main antioxidant substance was polypeptides.Finally,by comparing the extraction effects of petroleum ether,hexane,butanol,ethyl acetate and chloroform,the extraction rate of butanol,ethyl acetate and chloroform were higher,the ethyl acetate extracts had the highest antioxidant capacity among the three solvents whereas T-AOC was 7.8910.33 U/mL?1 mg/mL of polypeptides?and DPPH free radical scavenging ability rate was 38.03±0.43%?0.1 mg/mL of polypeptides?,so the best extraction solvent was pure ethyl acetate.After the solid state fermentation,the millet bran was purified by water extraction,ammonium sulfate precipitation,ethyl acetate extraction and semi preparation high performance liquid chromatography?Waters 600?.The purity analysis was conducted by high performance liquid chromatography?Ultimate3000?.A kind of antioxidant peptide was isolated with the extraction rate of 0.15 mg/g millet bran,the T-AOC was 8.26±0.24 U/mL?1 mg/mL of polypeptides?and DPPH radical scavenging rate was 40.84±1.61%?0.1 mg/mL of polypeptides?.LC-MS/MS was used to determine the molecular weight of 358.22 Da and the amino acid sequence was Gly-Pro-Trp.By comparing the antioxidant capacity of antioxidant peptide from millet bran and GSH?Glutathione?,results showed that both the antioxidant peptides and GSH had a good dose dependant antioxidant capacity effect with concentration ranges?0-1.0mg/mL of polypeptides?.With the increase of polypeptides concentration,the ABTS?Agreement of basic telecommunications services?free radical scavenging ability and FRAP?Ferric reducing antioxidant power?also increases.The GSH had better scavenging ability of ABTS free radical,but the antioxidant peptide from millet bran had higher FRAP capacity.