The Distribution,Changes During Storage And Processing,and Enzymatic Removal Of Ginkgotoxin In Ginkgo Seeds

Ginkgo seeds can serve as not only food but also drugs,which is authorized by the national health and nutrition committee of China.Except for nutritional components including starch,lipids,proteins,saccharides,vitamins and trace elements,ginkgo seeds contain functional components such as flavonoids and terpene trilactones（TTLs）.It has been reported that ginkgo seeds have significant effects on anti-tumor,anti-oxidation,anti-hyperlipidemia and anti-bacterial.However,overconsumption of ginkgo seeds will result in emesis,seizure or even death owing to the presence of 4’-O-methylpyridoxine（MPN）and 4’-O-methylpyridoxine-5’-glucoside（MPNG）which were collectively known as ginkgotoxin.Due to this,the development of ginkgo processing industry is greatly limited and a large amount of ginkgo resources has been wasted.So far,domestic and foreign scholars have developed none of practical removal technology of ginkgotoxin.To change the situation abovementioned,in this paper,we first determined the content of ginkgotoxin in different parts of ginkgo seeds and investigated the changes of ginkgotoxin during storage and processing.Then,we developed and optimized the enzymatic removal technology of ginkgotoxin.Next,the safety evaluation and main components determination in ginkgo seeds powder before and after enzymatic detoxification were conducted.Finally,the enzymatic detoxification mechanism of ginkgotoxin was analyzed.The main contents and results of this paper are as follows:（1）The determination of ginkgotoxin in different parts of ginkgo seeds and investigation concerning changes of ginkgotoxin during storage and processing.Instead of ultrapure water,guanidine hydrochloride solution（3 M）was employed to extract ginkgotoxin from ginkgo samples.The content of ginkgotoxin in different parts of ginkgo seeds with six species including Ma-ling,Xiao-yuan-zi,Da-long-yan,Mei-he,Dong-ting-huang and Fu-shou were determined.The results suggested that ginkgotoxin existed in all parts of ginkgo seeds,and compasion of the average content of ginkgotoxin indicated the order:plantule>sarcotesta>endopleura>endosperm>mesosperm.The total content of ginkgotoxin in all parts of ginkgo seeds was between 2714.07μg/g and 4830.44μg/g,and from the largest to smallest,they are Mei-he,Ma-ling,Xiao-yuan-zi,Da-long-yan,Dong-ting-huang and Fu-shou.According to the the relative content of MPNG and MPN in each part,it is inferred that MPNG is the natural accumulation form of ginkgotoxin in mature ginkgo seeds.MPNG was easier to be converted to MPN in ginkgo seeds without shells,and its convertion rate during cold storage（4℃）is higher than that in freezing storage（-20℃）.After 100 d of storage,the TMPN content（the total content of MPNG and MPN）in ginkgo seeds with shells under cold and freezing storage decreased by29.36%and 12.57%,while the TMPN content in ginkgo seeds without shells under cold and freezing storage decreased by 50.63%and 46.14%,respectively.The toxicity of MPN is 4 times than that of MPNG.Freezing storage with shells is determined to be the optimal storage method by taking storage period and toxicity of ginkgo seeds into consideration.Boiled treatment was effective in redcuing the toxicity by dissolving ginkgotoxin from ginkgo seeds to water,whereas frying,microwave and roasting have little influences on the content of ginkgotoxin without destroying the edibility of ginkgo seeds.（2）The study on enzymatic removal technology of ginkgotoxin from ginkgo seeds.Glucose oxidase,tyrosinase,horseradish peroxidase and aflatoxin-detoxifizyme had no degradation effects on standard solution of MPN.Through setting up the control experiment using boiling and inhibitor/metal ion to inactivate enzymes,laccase-ABTS system was proved to have ability of degrading MPN standard.It was demonstrated that degradation of ginkgotoxin in ginkgo seeds could also be realized by laccase-ABTS system.Consideing that ABTS could not be applied in food,syringaldehyde was selected to be a substitute referring to GB 2760-2014.The single-factor experiments and response surface methodology were finally adopted to determine the optimal enzymolysis conditions consisting of 0.032 U/μg（initial enzyme activity/TMPN content）of enzyme dosage,0.8 m M of syringaldehyde concentration,40℃of enzymolysis temperature,1/60 g/m L of solid/liquid ratio and 3 h of enzymolysis time.Under these conditions,MPNG and MPN in ginkgo seeds could be degraded by laccase-ABTS system at the same time,and the degradation of TMPN could reach up to 100%.（3）The assessment of nutritional-functional components and safety of ginkgo seeds powder after enzymatic detoxification.The content of nutritional-functional components of ginkgo seeds powder before and after detoxification were determined and the acute oral toxicity tests in mice were used to evaluate the safety of detoxified ginko seeds powder（DGSP）.The results were presented as follows:compared to undetoxified ginko seeds powder（UGSP）,the content of lipids in DGSP decreased by 17.00%,while the content of starch,proteins,total sugars and reducing sugars content had no significant changes.The content of free amino acids in DGSP was improved by 13.96%.As for vitamins,the content of ascorbic acid in DGSP increased by 16.81%than that in UGSP,whilst the contents ofα-tocopherol,pyridoxamine and nicotinamide in DGSP decreased by 100%,66.96%and 18.29%,respectively.Niacin was only detected in DGSP.Niether of retinol,riboflavin,pyridoxine and pyridoxal could be detected in DGSP and UGSP.The content of various microelements in DGSP was higher than that in UGSP,and it should be noted that the content of different kinds of elements were within the safe range.Flavonoids,TTLs,ginkgolic acids and total phenols are classied into functional components in ginkgo seeds,whose contents after detoxification decreased by 27.50%,49.79%,14.19%and 17.91%,respectively.The results of acute oral toxicity tests in mice suggested that LD₅₀ of ginkgo seeds powder detoxified by laccase-syringaldehyde system was more than 10000 mg/kg·bw,which indidated that DGSP was practically non-toxic.（4）The primary analysis of ginkgotoxin removal mechanism by laccase-syringaldehyde system.The UV-VIS full wavelength scanning,FTIR,HPLC-MS and GC-MS were adopted to identify the enzymolysis products of MPN and syringaldehyde.The results showed that the enzymolysis products of MPN had no UV-VIS characteristic absorption wavelength below 250nm and above 350 nm,but could not be determined in the wavelength range of 250-350 nm.The UV characteristic absorption wavelength of enzymolysis products of syringaldehyde is 274 nm.No new infrared characteristic absorption peak occurred on the infrared spectrogram when the infrared characteristic absorption peak of MPN pyridine ring weakened with the reaction carring on.According to the results of HPLC-MS,seven kinds of new substances were observed from the TIC spectrogram of MPN enzymatic hydrolysis system,and one of them could be identified to the oxidation products of syringaldehyde with a relative molecular mass of 168.05.Combined with the results of GC-MS,it was speculated that the above oxidation products of syringaldehyde might be 2,6-dimethoxybenzo-1,4-quinone.Moreover,the other six kinds of new substances had possibility to be the enzymolysis products of MPN,whose relative molecular mass was166.09,178.07,181.03（two kinds）and 183.10（two kinds）,and the actual chemical structure remained to be identified.