Preparation,Safety And Effectiveness Evaluation Of Grape Seed Proanthocyanidins Probiotics Complex

Oxidative stress is one of the main pathological mechanisms leading to the destruction of animal liver cell structure and function,as well as tissue damage and organ disease.It is of great significance to find and develop high-efficiency antioxidant preparations for the prevention and treatment of oxidative stress-induced liver injury diseases.Grape seed proanthocyanidins have the advantages of strong antioxidant,abundant raw materials and low cost.However,the current physical and chemical methods for depolymerization of grape seed procyanidins have many shortcomings,such as environmental pollution,high cost and low yield.Therefore,it is urgent to develop an environmental-friendly and efficient depolymerization method to improve the utilization rate of grape seed procyanidins.In this study,the probiotics complex composed of grape seed procyanidins,Saccharomyces cerevisiae and Lactobacillus fermentum were prepared as the main components,and combined fermentation with optimized fermentation medium to obtain the compound proanthocyanidins with oligomic proanthocyanidins.The Saccharomyces cerevisiae and Lactic acid bacteria were independently isolated and inspected by our laboratory.Saccharomyces cerevisiae H2 and Lactobacillus fermentum O were combined with the crude extract of grape seed proanthocyanidins in fermentation medium.The average degree of polymerization（m DP）of procyanidins decreased from 6.845±0.1 to 4.767±0.27 before and after fermentation.Single factor and response surface optimization were carried out on the inoculation ratio and amount of bacteria in the preparation,components and concentration of fermentation medium and fermentation conditions.The inoculation ratio of Saccharomyces cerevisiae H2,Lactobacillus fermentum O 2:3,and the inoculation amount of bacteria 7%were determined by optimization.The optimized fermentation medium consisted of glucose 26.73 g/L,peptone 10.52 g/L,yeast extract5.06 g/L,dipotassium hydrogen phosphate 2.0 g/L,ammonium citrate 2.0 g/L and magnesium sulfate 0.2 g/L.The optimized fermentation conditions were as follows:temperature 39.6℃,time 37 h,shaking speed 116 rpm,initial p H 5.7.According to the optimized scheme,the m DP of grape seed proanthocyanidin was reduced from 4.767±0.27 to 3.972±0.047 after preparation.Acute oral and subchronic oral toxicity tests in mice proved that the preparation is safe and non-toxic.The preparation significantly improved the scavenging ability of procyanidins on ABTS and DPPH free radicals in vitro.Subsequently,this experiment explored the preventive and therapeutic effects of grape seed procyanidins on CCl₄-induced acute liver injury.The results showed that the probiotics could decrease the pus points on liver surface,decrease liver weight and liver weight ratio,decrease the activities of AST,ALT,Cr and LDH in serum of mice.Probiotics increased the activity of glutathione peroxidase（GSH-PX）and superoxide dismutase（SOD）in liver.In addition,the central venous necrosis area and the proportion of liver injury area in the whole liver tissue were significantly reduced in HE staining sections of mice given grape seed proanthocyanidin probiotics.In this study,the preparation of grape seed procyanidins enhanced the ability of scavenging free radicals,the activity of antioxidant enzyme and the antioxidant capacity of the body.