Enzymatic Extraction And Stabilization Of Flavonoids From Ginkgo Biloba Leaves And Product Development

Ginkgo biloba leaf has a variety of pharmacological functions,so it’s widely used in functional foods,cosmetics and medicine.Its pharmacological activity is mainly derived from flavonoids in leaves,flavonoid is a kind of natural polyhydroxyl phenols with unstable structure.In the process of producing and preparing related products,flavonoids are easily destroyed under unfavorable processing conditions.Therefore,it is necessary to improve the stability of flavonoids in products to make full use of their physiological functions.There are many kinds of flavonoids in Ginkgo biloba leaf extract,in this study,the flavonoid extract of Ginkgo biloba leaf was firstly extracted with the help of cellulase,then Quercetin（Que）,Kaempferol（Kae）and Isorhamnetin（Iso）,the three flavonoids with the highest content and representative were selected as research objects,the self-assembly capability of bovine serum albumin（BSA）was utilized to combine with flavonoids to form stable complexes.To explore the effects of BSA on the stability and antioxidant properties of three flavonoids under natural light,heating and different p H.The interaction mechanism between BSA and three flavonoids was analyzed by multispectral,thermodynamic and molecular docking techniques.Finally,based on the above theoretical research,a kind of health capsule of Ginkgo biloba leaf flavonoids was prepared.The main research contents and results of this study are as follows:1.Using commercially available Ginkgo biloba leaf tea as raw material and cellulase assisted extraction of total flavonoids content in Ginkgo biloba leaf as index.By single factor and orthogonal experiments,the conditions for enzymatic assisted extraction of total flavonoids from ginkgo biloba were explored.The optimal extraction parameters were obtained:enzyme concentration was 0.6%,enzymatic hydrolysis p H was 4.8,the temperature was 50℃,the time was 75 min,the solid-liquid ratio was1:20,at this time,the total flavonoids content of Ginkgo biloba leaves was 0.7963mg/g.2.Effects of BSA on the stability and antioxidant activity of Que,Kae and Iso under natural light,heating and different p H.The results showed that three flavonoids would degrade under natural light（2 h,4 h,6 h,8 h,10 h）,heating（25℃,65℃,75℃,90℃）and different p H（3,4,5,6,7.4,8）.After combining with BSA（0,10,15,25μM）,the stability of the three flavonoids was enhanced,the retention rate was significantly improved,and the final concentration of BSA at 20μM showed the best protective effect.Under natural light,the protective effect of BSA increased with the extension of light time.Under heat and different p H treatments,BSA has the best protection on flavonoids at 65℃and p H 7.4,respectively.Besides,the antioxidant capacity of flavonoids and their complexes decreased under natural light（4 h）,heating（25℃,65℃,75℃,90℃）and different p H（5,6,7.4,8）,while the antioxidant capacity of the complex was still higher than that of free flavonoid or BSA,but less than the sum of the two values.This is because the hydroxyl groups will bind to the active site of BSA through non-covalent interaction during the formation of the complex,and BSA produces a masking effect on flavonoids,which inhibits their antioxidant activity temporarily.However,in vitro digestion experiments,the release and antioxidant capacity of flavonoids in the complex is significantly higher than that of free flavonoids,therefore,masking effect inhibits but does not destroy the active groups of flavonoids^[1,2],and protect the stability of flavonoids during processing.3.Fluorescence quenching,synchronous fluorescence,three-dimensional fluorescence,UV-visible absorption spectrum,circular dichroism,differential scanning calorimetry and molecular docking techniques were used to analyze the interaction mechanism between three flavonoids and BSA.Fluorescence quenching results showed that three flavonoids could cause endogenous fluorescence quenching of BSA,and the quenching constant decreased with increasing temperature,which indicates that the quenching mechanism was static quenching.The results of synchronous fluorescence,three-dimensional fluorescence and UV-visible absorption spectra showed that three flavonoids both affected the secondary structure of BSA and the microenvironment around amino acids.The greater quenching degree of Trp residues suggested that the binding site was closer to the Trp residues.The results of circular dichroism spectrum showed that the contents ofα-helix decreased and the contents ofβ-folding,β-turn and random coil increased in the secondary structure of BSA after binding with flavonoids.The results of differential scanning calorimetry showed that the thermal denaturation temperature of the complex was higher than that of free flavonoids,which indicates that the formation of complex could improve the thermal stability of flavonoids.Molecular docking results showed that the three flavonoids were mainly bonded to the active site of BSA by hydrophobic interaction and hydrogen bonds.4.Based on the sensory score of the product,through single factor and orthogonal experiment,the best formula of Ginkgo biloba leaf flavonoid health capsule was obtained as follows:adding 60%extracting solution,0.2%of citric acid and 8%of white granulated sugar,at this time,the product has a sensory score of 86.79.