Construction Of Methyl Gallate-hydroxypropyl-β-cyclodextrin-in-liposomes And Its Application In The Preservation Of Agaricus Bisporus

Tyrosinase is a multi-subunit copper-containing oxidase that widely exists in plants,animals,and microorganisms.It is a key rate-limiting enzyme in the melanin synthesis pathway.In the food industry,enzymatic oxidation of phenolic compounds catalyzed by tyrosinase is the main reason for browning of fresh fruits,vegetables,and beverages.Controlling the catalytic activity of tyrosinase has become an effective method to prevent browning,maintain nutritional value,and extend the shelf life of fresh foods.Methyl gallate（MG）is a polyphenolic compound containing three hydroxyl groups,which has various biological effects such as antioxidant,antibacterial and antitumor.It has been found that methyl gallate has a strong inhibitory effect on mushroom tyrosinase,but its inhibition kinetics and molecular mechanism are still unclear.In addition,methyl gallate has a strong odor,poor water solubility,low stability,and is susceptible to environmental factors,thereby limiting its wide application in the fields of food,cosmetics,and medicine.Liposomes can encapsulate hydrophobic and hydrophilic substances in the lipid layer and the inner aqueous phase,respectively.However,due to the limited space provided by the lipid bilayer and the presence of hydrophobic substances in the lipid phase,the stability of liposomes can be affected.Therefore,a novel cyclodextrin-in-liposomes（CDL）bilayer encapsulation system by combining cyclodextrin inclusion complexes containing hydrophobic substances with liposomes can overcome these drawbacks.The results obtained will help to broaden the application scope of methyl gallate,which may provide new ideas for the construction of delivery systems with antioxidant,anti-browning fast-acting and long-lasting active ingredients,and also provide guidance for the development and utilization of new natural preservatives.The main research contents and results are as follows:（1）Study on the molecular mechanism of the inhibitory effect of methyl gallate on tyrosinase.The inhibitory effect and the underlying mechanism of methyl gallate on tyrosinase were systematically studied by enzyme kinetics,fluorescence spectroscopy,copper-ion chelation,fourier transform infrared spectroscopy（FT-IR）,circular dichroism spectroscopy（CD）and molecular docking.The results showed that methyl gallate could effectively inhibit the activities of monophenolase and diphenolase of tyrosinase with IC₅₀values of 0.51±0.07 and 0.46±0.01 mmol/L,respectively.Methyl gallate was a reversible competitive inhibitor of tyrosinase.The mechanisms by which methyl gallate inhibited tyrosinase activity might be achieved by changing the hydrophobic environment of amino acid residues in tyrosinase,preventing the process of substrate catalyzed by tyrosinase,reducing the amount of substrate binding to tyrosinase and chelating with copper ions at the active site of tyrosinase.In addition,methyl gallate was inserted into the active site of tyrosinase by hydrogen bonding and van der Waals forces,resulting in the loss ofα-helical structure and the change of conformation of tyrosinase,thus inhibiting the enzyme activity.（2）Preparation,characterization and bioactivities of cyclodextrin inclusion complexes of methyl gallate.Firstly,the optimum cyclodextrin for methyl gallate encapsulation was screened,and then the inclusion complex of methyl gallate with cyclodextrin was prepared by saturated aqueous solution method,and the preparation process was also optimized.The surface structure and internal conformation of cyclodextrin inclusion complexes of methyl gallate were characterized by scanning electron microscopy（SEM）,X-ray diffraction method（XDR）,thermogravimetric analysis（TG）,FT-IR and molecular docking methods.The antioxidant activities of1,1-diphenyl-2-picryhydrazinyl（DPPH）free radical scavenging assay,2,2’-azinobis-（3-ethylbenzthiazoline-6-sulphonate）diammonium salt（ABTS）free radical scavenging assay,ferric reducing antioxidant power（FRAP）assay and tyrosinase inhibitory activities of methyl gallate before and after inclusion were compared.The results showed thatβ-cyclodextrin（β-CD）,2-hydroxypropyl-β-cyclodextrin（HP-β-CD）,hydroxyethyl-β-cyclodextrin（HE-β-CD）and sulfobutyl ether-β-cyclodextrin（SE-β-CD）all had certain solution-enhancing effects on methyl gallate,and the most effective cyclodextrin was HP-β-CD.The optimum conditions for the preparation of methyl gallate-2-hydroxypropyl-β-cyclodextrin inclusion complex（MG-HP-β-CD）were as follows:molar ratio of methyl gallate to HP-β-CD was 1:0.5,the inclusion temperature was 50℃,and the inclusion time was 3 h.Under the optimum conditions,the inclusion rate of MG-HP-β-CD could reach 80.15±1.04%and the stoichiometric ratio was 1:1.The SEM,XDR,TG,FT-IR and molecular docking simulation showed that methyl gallate entered the hydrophobic cavity of HP-β-CD,significantly increased the water solubility and thermal stability,and formed three hydrogen bonds with HP-β-CD to stabilize the inclusion structure.The inclusion complex of MG-HP-β-CD had good DPPH(IC₅₀=28.30±0.66μg/m L)and ABTS(IC₅₀=28.94±0.91μg/m L)free radical scavenging ability,ABTS ferric reducing antioxidant power（FRAP value was 12.78±0.18 mmol AAE/g）and anti-tyrosinase activity(IC₅₀=0.48±0.01 m M).（3）Preparation and characterization of methyl gallate-2-hydroxypropyl-β-cyclodextrin-in-liposomes（MG-HP-β-CDL）with its preservative effect on Agaricus bisporus.The MG-HP-β-CDL were prepared by reversed-phase evaporation method.The preparation process was optimized,and the structure of the MG-HP-β-CDL was characterized by particle size potential,transmission electron microscopy（TEM）,TG and FT-IR spectroscopy.In addition,the effects of storage temperature and light on the stability of MG-HP-β-CDL were studied,and the activities of MG-HP-β-CDL before and after storage were analyzed by DPPH,ABTS,FRAP antioxidant assays and antityrosinase experiments.Finally,the potential application value of MG-HP-β-CDL in fruit and vegetable preservation was further investigated using A.bisporu as a model.The results showed that the optimum conditions for preparation of the MG-HP-β-CDL were as follows:the ratio of methyl gallate to soybean lecithin was1:15,the ratio of cholesterol to soybean lecithin was 1:8,and the ratio of water to organic solvent was 1:3.Under the optimal conditions,the encapsulation rate of MG-HP-β-CDL was 64.17±1.16%,the average particle size was 355.40±7.98 nm,the PDI was 0.369±0.019,and the Zeta potential was-7.45±0.43.The MG-HP-β-CDL could maintain good stability under the condition of avoiding light and low temperature.Liposomes encapsulation could significantly reduce the loss of activity of methyl gallate during storage.MG-HP-β-CDL still showed good antioxidant activity and tyrosinase inhibitory effect after being stored at 4℃for two weeks.It was also observed that MG-HP-β-CDL could effectively reduce the activities of PPO and POD to retard the surface browning of A.bisporus and thus maintained their storage quality.