Enzymatic Modification And Activity Of Grifola Frondosa On Rice Bran Polysaccharides

Rice bran polysaccharides(RBPSs)are the major effective constituents of rice bran(RB),which have good biological activity.However,high molecular weights,low solubility and unexposed active sites make these polysaccharides difficult to absorb and are the mainfactors that limit the use of these polysaccharides in functional foods.In this study,the intracellular enzymes from Grifola frondosawere used to modify RBPSs.The changes of monosaccharide compositions,molecular weights,NK cell activity and antioxidant activity in vitro before andafter modification were investigated.1.The components of G.frondosa liquid medium were optimized by singel factor test.The biomass of G.frondosa and the content of soluble proteins in mycelium were used as indicators.The results showed that the optimal carbon source was glucose,nitrogen source was peptone,inorganic salt was magnesium sulfate and p H value was 6.0.On the basis of these,orthogonal test was carried out,and the optimal medium for liquid fermentation were determined as follows:glucose 4%,peptone 1.5%,magnesium sulfate 0.1%,p H=6.0.The cultivation conditions were optimized as temperature as 28?,the number of cultivation days as 8d and the rotating speed as160 r/min respectively.Mycelia were collected under this condition.The intracellular enzymes ware extracted by 85% ammonium sulfate precipitation method.The intracellular protein concentration was 65.6 mg/g.The activity of cellulase was 133.50±1.68 U/g,the activity of pectinase was60.46±0.20 U/g,the activity of amylase was 44.74±0.54 U/g and the activity of xylanase was 9.72±0.15 U/g.2.In this paper,the extraction of RBPSs by enzyme combined with ultrasound wasdetermined.The optimalratio of RB to water is 1:15.The mixture was heated to 60? and incubated with ?-amylase(70 U/m L)at p H 6 for 2 h to remove the starch.Then,the temperature was reduced to 50?,and the mixture was incubated with glucoamylase(200 U/m L)at p H 5 for1 h to remove the dextrin.Proteins were removed by treating the mixture with pepsin(20 U/m L)at 37? at p H 4 for 1 h.All the reactions were terminated by heating at 100? for 10 min.The digested samples were extracted by ultrasound(225 W)at 70? for 80 min.The extracts were centrifuged at 4,000 r/min for 10 min,and the supernatant as A liquidwas collected.The precipitate was added to water at a ratio of 1:15;the p H was adjusted to 7;and the extract was treated with ultrasound(225 W)at 70? for 80 min.Then,the extract was centrifuged at 4,000r/min for 10 min,and the supernatant as B liquidwas collected.Liquid A and B were mixed.After precipitation with ethanol,the yield of polysaccharides was 7.88%.The basic compositionsand properties of polysaccharides were determined.The RBPSs were obtained at high purities and contained approximately 84.15 ± 0.88% total carbohydrates,6.58 ± 0.61% protein.This indicated that RBPSs were non-starch polysaccharides containing small amounts of protein.3.The intracellular enzymes from G.frondosa and RBPSs were mixed to make enzymatic modification experiment.Basedon the single-factor test,the orthogonaltest(L9(3)4)was designed with NK cell improvement rate as the index.The best conditions were determined to be an enzyme to polysaccharide ratio of 1:5,a reaction temperature of 40 ?,a reaction p H of 4,and a reaction time of 4 h.By optimizing the conditions,the NK cell cytotoxicity of m RBPSs6 was seen to be the highest and increased 12.01±0.08%.A gas chromatographic analysis revealed that m RBPSs-6 consists of rhamnose,arabinose,xylose,mannose,glucose,and galactose at a molar ratio of 7:21:6:5:53:48,which was 8:13:8:5:44:44 before modification.High-performance liquid chromatography results indicated that the molecular weights of the RBPSs were approximately 106 Da,and the weights decreased to 104 to 105Da after modification.Antioxidant activity tests revealed that m RBPSs6 had high capacity for scavenging DPPH radicals and hydroxyl free radicals at 1.0 mg/m L.The results obtained in this study provide theoretical basis for the application of G.frondosaintracellular enzymes in rice bran polysaccharides,and also provide technical support for the further development and application of modified polysaccharides as an auxiliary medicine.