| Ginkgo biloba leaf is rich in bioactive substances such as ginkgo flavonoids and ginkgolides,and the ginkgo tea processed by it has the health function of regulating blood lipids.Because of the presence of toxic ginkgolic acid,the key and difficult point of this study is how to reduce the content of ginkgolic acid during the processing of ginkgo tea by using traditional tea-making steps(withering,fixing,rolling,fermentation,drying,etc.).This study based on reducing the content of ginkgolic acid,and delving into the effects of fixing and fermentation steps on reducing ginkgolic acid,meanwhile explored the effects of different tea-making methods on the quality and the content of bioactive substances of ginkgo tea.The main research findings are as follows:(1)Based on the quantitative detection method of ginkgolic acid of European Pharmacopoeia7.0 edition,a HPLC-DAD analysis method for the simultaneous determination of ginkgolic acid and ginkgol was established.A C8 reverse-phase chromatographic column(250×4.6 mm,5μm)was used for separation.Acetonitrile(containing 0.01%trifluoroacetic acid,v/v)as mobile phase A,water(containing 0.01%trifluoroacetic acid,v/v)as mobile phase B.Gradient elution conditions were:0-30 min,75%-90%phase A;30-35 min,90%-90%phase A;35-36 min,90%-75%phase A;36-46 min,75%-75%phase A.The detection wavelengths of ginkgolic acid and ginkgol were 210 nm and 270 nm,the flow rate was 1.0 m L/min,and the injection volume was 50μL.The linear relationship is good,and the RSD of precision,stability and repeatability of total ginkgol were 0.20%,2.21%and 2.45%respectively in six parallel determinations.The recoveries of low,medium and high groups were 96.58%,97.67%and 101.52%respectively.The limit of detection(LOD)of ginkgol C13:0,C15:1 and C17:1 were 0.61 ppm,0.50 ppm,and 0.06 ppm,respectively.The method is accurate and efficient for the simultaneous detection of ginkgol and ginkgolic acid in ginkgo tea.(2)Ginkgo leaves were processed into fixed and fermented tea(dark tea),fermented tea(black tea),withered tea(white tea)and fixed tea(green tea)by using traditional tea-making methods,the freeze-dried ginkgo leaves as a control.The quality,the content of ginkgol,ginkgolic acid and bioactive substances in various ginkgo teas were explored.For results,compared with the freeze-dried leaves,the content of ginkgolic acid in dark tea was significantly reduced by 8.41%,decreased by 3.72%and 2.49%in black tea and green tea,respectively,and increased by 10.42%in white tea.The content of ginkgol in dark tea significantly increased,but no significant change was found in black tea and green tea.The toxicity of dark tea,black tea,and green tea on Hep G2cells decreased compared with freeze-dried leaves,while white tea has the opposite trend.The cytotoxicity of ginkgo tea was consistent with the content of ginkgolic acid.Ginkgo flavonoids in white tea were retained and antioxidant capacity increased,while dark tea,black tea,and green tea all showed a decrease in ginkgo flavonoids,polyphenols,and antioxidant capacity.Various processing methods had little effect on terpene trilactone.Soluble sugar and free amino acid content in dark tea,black tea,and green tea decreased,while white tea showed an opposite trend.91.54%,89.55%,89.88%,and 68.82%of polyphenol oxidase in dark tea,black tea,green tea,and white tea were inactivated,and the peroxidase activity in the four teas was reduced by 85.75%-87.47%.The electronic nose and color test results of various ginkgo tea infusions showed that dark tea had a stronger aroma and deeper tea infusion,white tea had a weaker aroma and lighter tea infusion,and black tea and green tea were in the middle.Among the four ginkgo teas,dark tea has the lowest ginkgolic acid content,richer aroma and deeper color.(3)Compared pulsed light fixation with high temperature fixation.Specifically,fresh ginkgo leaves were irradiated with pulsed light(0.52 J/cm2/pulse)for 50,100,200,and 300 s or heating at 150°C and 180°C for 300 s.The effects of fixing on ginkgolic acids,ginkgo flavonoids and,lactones,and quality indicators of ginkgo leaves were explored.For results,compared with the unfixed,pulsed light fixation for 200 and 300 s could significantly reduce the total ginkgolic acid content by 21.08%and 34.22%.However,high temperature fixation had no significant effect on ginkgolic acid content.The total flavonoid content was increased by 5.82%of pulsed light fixation for 200 s,but decreased by 3.53%for 300 s,while the total flavonoid content was reduced by 15.70%after 180°C fixation.Bilobalide did not significantly decrease after fixing with pulsed light,but decreased significantly by 23.48%heating at 180°C for 300 s.Both pulsed light and high-temperature fixing can effectively reduce the enzyme activity.High temperature fixation reduces more soluble sugars and free amino acids in ginkgo leaves than pulse light fixation.The results of infrared spectroscopy showed that various fixing methods would not destroy the primary structure of ginkgo leaves.LC-MS was employed to qualitatively analyze pulse light fixation degradation products of ginkgolic acid C13:0,found that it can degrade to 3-tridecylphenol,2-alkylbenzoic acid,or tridecylbenzene.(4)Five strains of Lactobacillus plantarum,Pediococcus pentosaceae,Bacillus subtilis(B.subtilis),Bacillus natto and Saccharomyces cerevisiae(S.cerevisiae)were selected and cultured in a medium containing ginkgolic acid at 35°C for 60 h to determining the growth curve of the strain and concentration change of ginkgolic acid,it was found that B.subtilis and S.cerevisiae can effectively reduce the concentration of ginkgolic acid in the medium.The fresh ginkgo leaves were fixed by pulsed light at 0.52 J/cm~2/pulse for 200 s.Three types of new ginkgo tea are made by B.subtilis fermentation,S.cerevisiae fermentation,or natural fermentation,respectively.The drying ginkgo leaves as the control.The content of ginkgolic acids,ginkgol,ginkgo flavonoids and lactones in three new ginkgo teas fermented by different strains were analyzed,and their quality indicators and sensory evaluation were compared.For results,compared with the control,the ginkgolic acid content of ginkgo tea fermented by B.subtilis was the lowest,decreasing by 29.74%;fermentation by S.cerevisiae reduced by 25.21%;natural fermentation reduced by 24.53%.The content of ginkgol in three new ginkgo teas decreased.Total flavonoids content in ginkgo tea fermented by B.subtilis increased by 14.64%,while there was no significant increase in S.cerevisiae fermentation and natural fermentation.Soluble sugar content in three new ginkgo teas decreased due to fermentation,but the free amino acid content did not decrease significantly.The polyphenol content and antioxidant capacity of ginkgo tea fermented by natural fermentation and B.subtilis fermentation increased significantly.The result of sensory evaluation found that the color,aroma and taste of the tea infusion fermented by B.subtilis were better and more prominent,so the total score was the highest.In short,ginkgo tea fermented by Bacillus subtilis can effectively reduce ginkgolic acid,increase the content of bioactive compounds and antioxidant capacity,and make infusion color,aroma and taste more prominent. |
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