| Ginkgo kernel,containing various bioactive ingredients such as ginkgolides and flavonoids,is a medicinal and edible food.Ginkgolide B(GB)is a monomer of ginkgolides with the highest content,and has beneficial pharmacological effects such as anti-inflammatory,liver protection,heart protection,antioxidant activity and lipid metabolism regulation.This study aimed to obtain the lactic acid bacteria(LAB)strains that have the potential of bio-transforming GB.Ginkgo kernel juice rich in GB and total phenols(TP)was prepared through enzymatic hydrolysis to degrade starch and pectin,and used as medium to screen high GB-producing lactic acid bacteria.Furthermore,the precursors and inducers required for the biotransformation of GB were added to promote LAB to product GB.On this basis,cell experiments were conducted to evaluate the vascular endothelial cells protection and lipid metabolism regulation of fermented ginkgo kernel juice in vitro,and the metabolite compositions in ginkgo kernel juice with or without fermentation were analyzed by metabolomics technology.The main results are given as follows:(1)The orthogonal experiment was employed to optimize the enzymatic hydrolysis for the preparation of ginkgo kernel juice.The optimized conditions for the enzymatic hydrolysis were:α-amylase 6 U/m L,glucoamylase 9 U/m L,hydrolysis temperature 60 ℃ and enzymatic hydrolysis time 2 h.The yield of GB and TP were 49.29 ± 0.87 μg/m L and 455.18 ± 4.05 μg/m L in ginkgo kernel juice,1.82 and 1.32 times higher than those in raw juice.(2)The GB-producing lactic acid strain NJBC17 was screened from more than 200 LAB collections.The yield of GB and TP produced by lactic acid bacteria NJBC17 in ginkgo kernel juice fermentation were 70.79 ± 0.63 μg/m L and 440.42±0.01 μg/m L in ginkgo kernel juice,1.30 and 1.07 times higher than those in unfermented ginkgo kernel juice.NJBC17 was identified as Lactobacillus plantarum(L.plantarum)through morphological and molecular biological analysis.(3)The production of GB and TP in ginkgo kernel juice increased under the optimized fermentation conditions,with initial juice p H 6,inoculation 6%,cultivation temperature at 30 ℃and fermentation time 48 h.The single-factor experiment showed that the addition of magnesium chloride(0.02 mg/m L),salicylic acid(1 mmol/L)and pyruvate(0.75%)as precursors and inducers could increase the production of GB and TP furtherly.Co-induced fermentation is performed using the above three substances,the yield of GB and TP in ginkgo kernel juice reached 109.94 ± 5.76 μg/m L and 599.57 ± 7.71 μg/m L,which were 1.55 and 1.36 times higher than those in uninduced fermentation,and 2.02 times and 1.45 times higher than those in unfermentation.(4)p H,viable bacterial count,total sugar,TP and GB contents in ginkgo kernel juice were monitored during the fermentation process.The results showed that p H and total sugar content were gradually decreased as the fermentation went on,while the viable bacterial count increased.The number of viable bacteria in induced group was lower than that in uninduced group,but still reached 8.60 ± 0.03 lg CFU/m L.The GB content increased significantly in the first 4-32 h and kept a stable tendency at the highest level of 109.79 ± 3.71 μg/m L in the co-induced fermented ginkgo juice after 48 h of fermentation.The TP content increased in the first 0-8 h,decreased during 8-24 h and then increased again along with fermentation time,reaching the highest level of 594.05 ± 9.80 μg/m L in the co-induced fermented ginkgo kernel juice.(5)The cell viability of HUVEC was measured by CCK-8 to evaluate the cytoprotective effects of the fermented ginkgo kernel juice against H2O2-induced oxidative damage.Pretreatment of HUVEC cells with the fermented and the co-induced fermented ginkgo kernel juice contained 1.25 μmol/L GB significantly increased the cell viability,which were 1.68 and1.81 times higher than that of in the H2O2-injured cells,respectively.As detected by the reactive oxygen species fluorescence probe,the co-induced fermented ginkgo kernel juice significantly decreased ROS production,which accounted for only 18.38% of H2O2-injured cells.In addition,insulin-induced 3T3-L1 cells could form more obvious lipid droplets.The number of lipid droplets and the lipid content in 3T3-L1 cells treated with fermented and co-induced fermented ginkgo kernel juice were significantly reduced compared with those of untreated,indicating that ginkgo samples inhibited lipid accumulation in 3T3-L1 cells.(6)Metabolomics analysis revealed significant difference among the ginkgo kernel juice group,the fermentation group and the co-induced fermentation group.Terpenoids biosynthesis pathways and lipid metabolism pathways were annotated in the fermentation group and the co-induced fermentation group,including the upregulation of oryzalexin E in the diterpene biosynthesis pathway and the downregulation of 4-deacetyl neosolaniol in the sesquiterpene and triterpene biosynthesis pathway.The differential metabolites in co-induced fermentation group also involved in the metabolism of exogenous substances and the metabolitization of drugs in the cytochrome P450 pathway,and the enrich pathways were associated with the upregulation of hesperetin 7-neohesperidoside and 4-coumaroylshikimate in the flavonoid biosynthesis pathway.In conclusion,the contents of GB and TP in ginkgo kernel juice increased by enzymatic hydrolysis and lactic acid bacteria biotransformation.The ginkgo kernel juice fermented by lactic acid bacteria has vascular endothelial cells protection and lipid metabolism regulation.Therefore,the fermented ginkgo kernel juice are important for promoting the utilization of ginkgo resources. |
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