The Mechanism Of Melatonin Change During Wine Grape And Mulberry Fruit Development And Fermentation Process

Melatonin and its isomer were the newfound functional components in fermented beverage such as wine in recent years,and it plays many important physiological roles in human body.However,the methods of melatonin and its isomer detection in fruits and wine were various,the mechanism and effect factors of melatonin and isomer change in fruits and wine were still unknown now.So we selected mulberry and grape fruits and wine as the experimental materials in our research,and the melatonin and its isomer detection method was established.The mechanism of melatonin and its isomer change from fruit development to wine fermentation was systemically studied.In addition,the interaction relationship between melatonin,melatonin isomer and Saccharomyces cerevisiae were deeply discussed,the essential conditions of melatonin and its isomer biosynthesis by Saccharomyces cerevisiae were also studied.The main results are as follows:1 The extraction process for melatonin and its isomer has been optimized,the curate and reliable detected method with HPLC-ESI-MS has been established in our research.The limits of detection(LODs)and limits of quantification(LOQs)of our method were 0.01 and 0.1 ng/mL,respectively.The recovery rate was more than 85%and the relative standard deviation(RSD)was less than 10%.2 Mulberry and grape fruit and its wine both contain melatonin,but the level in wine was much higher than fruits.The highest conteng of melatonin in wine and grape fruit were 24.48 ng/mL and 8.18 ng/g FW.The content of melatonin in grape and its wine was more than mulberry about 2-fold.We did not find melatonin isomer in all fruits,but it was detected in wine sample which suggested melatonin isomer was formed in fermentation process.The content of melatonin and its isomer in red varieties was higher than that in white varieties in both fruits and wine.The results showed no significant influence of vintage on melatonin and its isomer,but the origin has significant impact.3 Melatonin in mulberry with high content in young fruit period(about 10 days after fruit-set)decreased gradually in the development but there was a peak in wine grape developmenton 60th day.The TDC maybe the key gene of the melatonin biosynthetic in fruit and its relative quantitative expression was correlated with melatonin change in fruit.The melatonin level had a negative correlation with the change of MDA which implied melatonin participated in the antioxidant system of fruit.We found the melatonin level in matured grape fruit "Cabernet Sauvignon" Immediately increased about 2.44 fold compared with control under the UV-C treatment after 1 hour and The TDC gene was also quickly up-regulated at the same time which suggested UV-C could induced the formation of melatonin in fruit.4 The melatonin level rose rapidly in the early days of mermentation(day 1-3)and then gradually decreased until the end in both mulberry and grape wine.And in contrast,the melatonin isomer was gradually accumulating with high level(day 7-10)until the end of fermentation.5 The scAANAT was the key gene of the melatonin biosynthesis in Saccharomyces cerevisiae and its relative quantitative expression was correlated with melatonin change in fermentaion.Melatonin and its isomer were not biosynthesized In YPD meidium but in MSM medium by Saccharomyces cerevisiae.however the content was significant lower than that in real wine.The melatonin and its isomer biosynthesis in commercial yeasts(yeast A and F)was stronger than that in wild type(yeast B).In addition,exogerous melatonin(100 ng/mL)could improve the antioxidant ability of Saccharomyces cerevisiae in MSM medium.6 In YPD medium,both yeast strain A and B did not generate melatonin under exogenous light and tryptophan treatment,but the isomer can be produced in MSM and YPD medium treated with different environmetal adversity stress.In addition,high copper stress can induce the formation of melatonin in both A and B more than 5-fold than control in MSM medium.And the content of melatonin isomer can increase significantly under the treatment of low pH more than 76%compared with control treatment in MSM medium.These results suggest that melatonin was mainly come from the fermentation process in wine and it was produced only under adversity stress by Saccharomyces cerevisiae.The melatonin and its isomer has different biosynthesis mechiasm in Saccharomyces cerevisiae.