Production Of Glutathione By Fermentation In A New Medium Made Of Corn Germ Meal

In this study,corn embryo meal was used as the medium material,and in order to make corn embryo meal meet the carbon source standard,the corn embryo meal was crushed and saccharified.Taking DE value as index,single factor experiment and orthogonal experiment were carried out on the ratio of material to liquid,saccharification time,saccharifying enzyme dosage and saccharification temperature.The optimum saccharification process was obtained,and the devalue of saccharification solution of corn germ meal was above 90.The new culture medium was compared with two kinds of different yeast culture medium,and the medium with the largest number of culture colonies was selected as the medium in this study.Taking glutathione production as the standard,Saccharomyces cerevisiae L5267,Saccharomyces cerevisiae and Baeromyces cerevisiae were compared,and the strains with the highest glutathione production were selected as the test strains in this study.After determining the composition of the medium,ammonium sulfate,cysteine,inositol and potassium dihydrogen phosphate were tested by single factor test and response surface test,and the optimal process conditions were obtained with the yield of glutathione as the standard.Three common extraction methods of glutathione including copper salt method,ion exchange resin and reverse micelle extraction were compared,and the best extraction method of glutathione was selected by single factor and response surface experiments based on extraction rate to obtain the best extraction conditions.After the separation and purification of glutathione,the antioxidant test（ABTS,DPPH,OH^-scavenging test）and in vivo test（anti-fatigue,decanting test）of the standard glutathione were carried out,and the quality of glutathione produced by fermentation was studied by comparison.The results are as follows.（1）After single factor test and orthogonal test,the optimal ratio of solid to liquid was1:4,the optimal saccharization temperature was 53℃,the optimal saccharization time was 49h,and the optimal addition amount of saccharase was 275 u/g.The highest average DE value of the saccharization solution of corn embryo meal was 94,which reached the standard of carbon source of culture medium.（2）Saccharomyces cerevisiae L5267 was selected as the experimental strain in this study by comparing the glutathione production capacity of the three strains.The new medium was selected as the medium for this study through the comparative experiment of medium fermentation ability.Through single factor test and response surface test,the optimal supplemental levels were ammonium 1.435 g/L,inositol 0.108 g/L,cysteine 0.44 g/L and potassium dihydrogen phosphate 0.213 g/L.The results showed that the content of glutathione in the medium was 459.209 mg/L,which was 0.4%lower than the predicted result and12.07%higher than the highest result by single factor.（3）Through single factor test and response surface experiment,the optimal addition amount of AOT was 43.2 g/L,the optimal value of W₀was 24.05,and the optimal addition amount of K⁺was 0.438 mol/L.The verification test showed that the maximum extraction rate of AOT-isooctane reverse micelle reached 90.006%,which was 0.02%lower than the predicted result and 9.6%higher than the single factor experimental result.（4）In the free radical scavenging test,ABTS scavenging capacity was as follows Glutathione standard(IC₅₀=0.0051 mg/m L)>Corn germ meal Glutathione(IC₅₀=0.0068mg/m L)>Ascorbic acid(IC₅₀=0.025 mg/m L);DPPH scavenging capacity was as follows Ascorbic acid(IC₅₀=0.022 mg/m L)>Glutathione standard(IC₅₀=0.028 mg/m L)>Corn germ meal Glutathione(IC₅₀=0.028 mg/m L);OH^-scavenging capacity was as follows Glutathione standard(IC₅₀=0.11 mg/m L)>Corn germ meal Glutathione(IC₅₀=0.15mg/m L)>Ascorbic acid(IC₅₀=0.23 mg/m L).（5）In the anti-fatigue test of mice,the blood dopamine concentration of mice fed with high-dose（0.8 g/kg）GSH standard was up to 0.372 ng/mg,while the blood dopamine concentration of mice fed with high-dose（0.8 g/kg）GSH fermentation sample was up to0.358 ng/mg,indicating that GSH had a certain anti-fatigue effect on mice.（6）In the sobriety experiment,the alcohol concentration decreased by 64.9%and the sobriety time was advanced by 26.3 min in the glutathione standard medium and medium dose（0.2 mg/20g）group.The alcohol concentration of the high-dose（0.3 mg/20g）group was decreased by 75.8%,and the sobriety time was advanced by 38.0 min.Among the glutathione samples produced by fermentation,the alcohol concentration of the high-dose group was reduced by 65.64%,and the time of decanting was advanced by 30.7 min.The glutathione samples had obvious effect of decanting and protecting liver.