Study On Processes Of Extraction And Purification Of Ergothioneine From Pleurotus Eryngii And Its Antioxidant Activity

(College of Food Science, South China Agricultural University, Guangzhou 510642, China)Abstact : Ergothioneine, named 2-mercaptohistidine trimethylbetaine, is a new powerful natural antioxidant, which has great potential application value in the field of food additives,health products, cosmetics and so on. The contents of ergothioneine in common edible mushrooms, such as Pleurotus eryngii and Flammulina velutipes, are much higher than those in other plants. Due to the advantages of easy cultivation, short growth period and natural safety, mushrooms are the best source for obtaining ergothioneine. This research was conducted to study the processes of extraction and purification of ergothioneine from Pleurotus eryngii and its antioxidant activity. The results could provide a theoretical basis for the exploration and utilization of ergothioneine in Pleurotus eryngii. The main contents and results are as follows:(1) The optimal pretreatment of Pleurotus eryngii, extraction solvent and extraction method were selected by comparing extract rate of ergothioneine. The results showed that Pleurotus eryngii dried by vacuum freezing method preserved higher content of ergothioneine compared with those processed by hot air drying at 50℃, and thus were applied as the material in the follow-up study. In view of the ergothioneine yield and environmentally-friendly, 70% ethanol was chosen as the extraction solvent from 10 studied solvents due to its higher extract rate of ergothioneine(2.63 mg/g.DW). The highest content of ergothioneine was obtained by microwave-ultrasonic assisted extraction compared with other four extract methods.(2) The optimal processes of microwave-ultrasonic assisted extraction were investigated by conducting single factor experiments and response surface analytical method. The resulted showed that solid-liquid ratio, ethanol concentration and ultrasonic power were three improtant single factors which could significantly influence the ergothioneine yield of microwave-ultrasonic assisted extraction in seven studied single factors. The optimum extraction conditions of microwave-ultrasonic assisted extraction were the the solid-liquid ratio of 1:48, ethanol consentration of 54%, ultrasonic power of440 W, microwave power of 500 W, microwave temperature of 70 ℃, microwave time of 5min and ultrasonic time of 5 min, and the ergothioneine content was 3.7195 mg/g.DW.(3)Alcohol precipitation and Glucan gel column chromatography were used to purifythe ecrtact of ergothioneine. It turned out that the total sugar deposition rate was 70.52%,the deposition rate of protein was 82.28% and the retention ratio of ergothioneine was94.19% by adding 4 times 95 % ethanol to ergothioneine extracting solution and then leaved it for 24 hours in 4℃. After concentration and filtration of extracting solution, the experiments of separation and purification were carried out by Sephadex G-10. The purification conditions were as followings: using the distilled water as the eluant, the sample volume was 2 m L, and at the flow rate of 1.5 m L/min, and the recovery rate was87.32%.(4)The antioxidant activity of ergothioneine was detected by detection of hydroxyl radical scavenging activity, DPPH radical scavenging activity, ABTS radical scavenging activity, FRAP antioxidant capacity and the antioxidation and color protection function for fresh fish. Results indicated that in the range of concentration of 0.044~0.262 mmol/L, the hydroxyl radical scavenging activity of ergothioneine was observably stronger than the glutathione(P < 0.05). In the range of concentration of 0.436 ~ 1.047 mmol/L, its DPPH radical scavenging activity of ergothioneine was higher than that of glutathione but markedly lower than ascorbic acid(P < 0.05). The maximum value of ABTS radical scavenging activity of ergothioneine was 89.28 % which was dramatically lower than glutathione and ascorbic acid(P < 0.05). The FRAP antioxidant capacity of ergothioneine was relative weak. The add of ergothioneine to fresh fish could contribute to color retention,maintain the p H of the fish, inhibition of protein oxidation and the spoilage of fish. The above results of the add of ergothioneine to fish was better than TBH. The ergothioneine from Pleurotus eryngii had some ability to inhibit fat oxidation, but the effect was weaker than TBH.(5) The antioxidation stability of purified ergothioneine extracts from Pleurotus eryngii was studied by detecting the DPPH free radical scavenging rate in different condition. The results showed that the antioxidant activity of ergothioneine was relatively stable in the condition of 37 ℃, 90 ℃, Fe3+, Ca2+, Physiological p H and alkali conditions.The antioxidant activity of ergothioneine was instability in the condition of 4℃, K+, Mg2+,oxidizing agent, reducing agent and acidic medium.