Construction Of High Yield γ-Oryzanol System Based On Microbial Metabolism Regulation

γ-oryzanol is present in a variety of vegetable oils and has multiple physiologically active functions.It has good effects in terminating lipid peroxidation and eliminating free radicals,and its heat resistance is superior to other antioxidants,which has broad application prospect in inhibiting the oxidation of thermal processing products such as frying oil.At present,γ-oryzanol is mainly derived from plant extraction or chemical synthesis,and there are problems such as difficulties in enrichment or insecurity.The method of enzymatic catalysis has the defects of high cost,complex technology and difficult popularization.In order to obtain a method for bio-concentration ofγ-oryzanol,which is easy to operate,safe,efficient and low in cost,this study intended to use the method of fermentation（by-product of cereals fermented by microorganism）to select the dominant strain and high-quality substrates of producingγ-oryzanol.Constracting high-yield fermentation system ofγ-oryzanol based on regulating metabolic process of the strain from cell membrane permeability,metal ion concentration and substrate induction.The oxidation reaction system of lipid-cholesterol emulsification model was established to evaluate the cholesterol and lipid antioxidant capacity of the extracted oil which was rich inγ-oryzanol,and provides preliminary data and reference for the development and application of deep/gradient fermentation products.The main results were as follows:（1）selection and optimization ofγ-oryzanol fermentation system:Among the four molds（Monascus purpureus,Aspergillus niger,Mocor and Penicillium）,the content ofγ-oryzanol in the fermentation product of Monascus purpureus was the highest and the maximum was 0.84 mg/mL,an increase of 2.65 times compared to none fermentation.In the six fermentation substrate of broken coix seed,broken tartary buckwheat,broken millet,broken oat,broken rice and coix bran,the enrichment ofγ-oryzanol in the broken tartary buckwheat was the most significant（P<0.05）,and its content increased from 0.08 mg/mL to 1.10 mg/mL,an increase of12.75 times while achieved a fermentation product of high color price（1099.21 U/g）.The fermentation parameters for producingγ-oryzanol were optimized by orthogonal experiment:ratio of material to liquid 3:1（w:w）,inoculum of Monascus purpureus8%,fermentation temperature 30°C,the content ofγ-oryzanol after 9 days fermentation was 1.59 mg/mL,which was 1.45 times than that before optimization.This was 18.87 times more than none fermentation.The red kojic color price increased synchronously to 1428.77 U/g,which was 23.07%higher than that before optimization.（2）Construction of high-yieldγ-oryzanol fermentation system:the cell membrane permeability was adjusted by ultrasonic treatment and surfactant addition,indicated that ultrasonic treatment could not promote the enrichment ofγ-oryzanol in the fermentation product,while the additive amount of surfactants（Triton X-100,Tween-80 and SDS）was in the range of 0³‰,which promoted the synthesis ofγ-oryzanol.Metal ions could affect the content ofγ-oryzanol in the fermentation product.Ca²⁺and Mg²⁺had significant incremental effects（P<0.01）onγ-oryzanol in a certain range of addition（0⁴‰）,while Na⁺inhibited its production.Substrate induction showed that the addition of ferulic acid alone did not promote the accumulation ofγ-oryzanol,but the addition of sitosterol promoted the content,the addition of a mixture of ferulic acid and sitosterol（ferulic acid:sitosterol=1:1,w:w）could significantly increaseγ-oryzanol production（P<0.05）.The optimal regulatory parameters of high-yieldγ-oryzanol were obtained by response surface analysis:the additive amount of Triton X-100 was 3.2‰,the additive amount of Ca²⁺was 4.2‰,the additive amount of mixture of ferulic acid and sitosterol（ferulic acid:sitosterol=1:1,w:w）was 0.5‰.Under the conditions,theγ-oryzanol content was 2.61 mg/mL,which was consistent with the predicted result.Theγ-oryzanol content in the fermentation product was 1.64 times that of the control group,which was 31.62 times higher than that none fermentation.（3）Evaluation of anti-cholesterol oxidation and anti-lipid oxidation of extracted oil which was rich inγ-oryzanol:The oil extracted from fermentation products（broken tartary buckwheat fermented by Monascus purpureus）was rich inγ-oryzanol,and its effective content was 18.57 mg/g.After treatment at 200°C for 8 h,the loss rate ofγ-oryzanol was only 17.79%,which had good thermal stability.After 168 h of oxidation reaction,the 7-keto content of fish oil-cholesterol emulsion with 1.0 mL of extracted oil（extracted oil:emulsion=1:20,v:v）was only 5.68μg/mL,the inhibition rate of cholesterol oxidation was as high as 95.19%;which was better than that of olive oil group（93.02%）and linseed oil group（87.65%）.The additive amount was positively correlated with anti-cholesterol oxidation effect.In the three emulsions with 1.0 mL of extracted oil（extracted oil:emulsion=1:20,v:v）,the POV values were 18.52 mmol/kg,21.37 mmol/kg and 23.58 mmol/kg after oxidation for 168 h,respectively.It was significantly lower than the control emulsion（P<0.05）;the anti-lipid oxidation was gradually enhanced with the increase of the additive amount.The order of anti-lipid oxidation of the extracted oil in each emulsion was:fish oil-cholesterol emulsion>olive oil-cholesterol emulsion>linseed oil-cholesterol emulsion.