Study On The Biotransformation Of Polymeric Proanthocyanidins From Litchi(Litchi Chinensis Sonn)Pericarp

Litchi pericarp is one of by-products of litchi（Litchi chinensis Sonn.）processed products,which is rich in A-type proanthocyanidins and accounts for 15%of the fresh weight of the whole litchi fruit.With the development of processing industry of litchi,tens of thousands of tons of litchi pericarps are discarded instead of comprehensive utilization every year.Litchi pericarp proanthocyanidins（LPCs）have extremely strong scavenging activity on free radicals.At present,the researches on LPCs mainly focus on oligomeric proanthcyanidins（LOPCs）,while a large number of polymeric proanthcyanidins（LPPCs）are ignored because of their poor bioavailability which affects their exertion of biological activity.Therefore,the tansformation of polymeric proanthocyanidins is of great significance in the comprehensive application of proanthocyanidins.In this paper,LPCs were extracted from litchi pericarp under the optimal method and condition,and the LPPCs were isolated and their molecular structures were preliminarily identified.Then,the biotransformation process of LPPCs were explored,and preliminary identification were conducted to the biotransformation products.Finally,the changes on antioxidant activity and anti-nutritional activity of LPPCs before and after biotransformation were investigated.The details are as followed:1.Optimization of extraction process on LPCs and structural identification of LPPCs.Taking the high yield and high mean of degree of polymerization（m DP）of LPCs as target,ultrasonic-microwave synergistic extraction was selected as the best extraction method for its highest yield of LPCs of 7.54%compared with the other three methods,and the m DP was4.43.And the optimal process obtained by single factor experiments and response surface experiments was as followed:liquid to material ratio was 1∶31 g/m L,microwave power was307 W,extraction temperature was 46℃,extraction time was 17 min.The optimized yield of LPCs reached 8.10%which increased by 7.43%.LOPCs and LPPCs were purified and separated from LPCs extracts.The purity of resulting LPPCs was 81.07%,m DP was 6.12,and accounted for 45.21%weight of LPCs.According to MALDI-TOF-MS analysis,LPPCs were mainly composed of four to six polymers and rich in type A proanthocyanidins.The highest degree of polymerization was 12.LPPCs with different degrees of polymerization had a variety of structures,and the structural diversity decreased with the degree of polymerization increased.2.Establishment and optimization of the biotransformation process on LPPCs.According to the screening model,Lactobacillus plantarum was selected as the suitable microbe to biotransform LPPCs among five Lactobacillus for its growth and metabolism was hardly disturbed by LPPCs and the biotansformation effect on LPPCs was outstanding compared with other microbes.According to the results,the biotansformation rate reached78.36±2.71%;the content of（oligomeric）proanthocyanidins in the biotransformation products reached 302.84±11.46μg GSP/mg DW,which was 1106.37%higher than the control group;the total phenolics content reached 823.59±30.92μg GAE/mg DW,which was the highest among the five Lactobacillus groups,and was 724.32%higher than the control group.Taking the total phenolics content as an index,the biotransformation process on LPPCs was optimized by single factor experiments and orthogonal experiments.After verification,the optimal process were determined as followed:inoculation amount was 2%（v/v）,fermentation time was 48 h,fermentation temperature was 35℃,and the initial p H was 5.5.Finally,the total phenolics content of the biotransformation products increased by 30.88%,up to 1077.93μg GAE/mg DW.3.Structural identification and determination of antioxidant activity and anti-nutritional activity of biotransformation products.According to the result of HPLC-QTOF-MS/MS analysis,there were seven substances identified,which were 4-hydroxycinnamic acid、3,4-dihydroxybenzeneacrylic acid、perillic acid、3-（4-Hydroxy-3-methoxyphenyl）propenoic acid、cinnamic acid、quinic acid、procyanidin A2,of which 4-hydroxycinnamic acid、proanthocyanidin A2 and 3,4-dihydroxyphenylacrylic acid were preliminarily considered as the main components of the biotransformation products.By analyzing the difference on detected substances from the early stage and later stage during the fermentation,it was preliminarily revealed that Lactobacillus plantarum biotransformed LPPCs by producing acid to lower the p H of the system,which promoted the depolymerization of LPPCs to free epicatechin,afzelechin and other flavan-3-ols structure at the early stage,and further degradation and metabolization into different phenolic acids was happened under the action of the microbe enzyme system at the later stage.According to the results of ABTS⁺·and DPPH·free radical scavenging activity experiments,it was found that the IC₅₀ value of biotransformation products for ABTS⁺·free radicals was 4.16±0.28μg/m L,which was significantly lower than（P<0.05）LPPCs and LOPCs and had no significant difference from V_C.However,the scavenging activity of DPPH·free radicals was not significantly different from LPPCs,but it was still significantly higher than（P<0.05）LOPCs.The ORAC value of the biotransformation products was found to be 4 times higher than LPPCs,and significantly higher than（P<0.05）V_C.And the result of liposome oxidation experiment showed that the inhibition rate of CD-POV by biotransformation products was 1.8 times higher than V_E and2.0 times higher LPPCs.The IC₅₀ values of the biotransformation products forα-amylase,pancreatic lipase and pepsin were 381.06±5.75μg/m L,101.53±7.23μg/m L and 283.76±7.73μg/m L respectively,which were higher than LPPCs and LOPCs.In general,after being biotransformed by Lactobacillus plantarum,LPPCs had stronger antioxidant activity while their anti-nutritional activity were weakened.This study established an optionmal process for the biotransformation of litchi pericarp polymeric proanthocyanidins and obtained the high-value bioconversion products with improved antioxidant activity and weakened anti-nutritional activity,which provided a new idea for the comprehensive utilization of polymeric proanthocyanidins and litchi pericarp.