Extraction Of Oil And Crocins From Gardenia Fruit And Construction Of Pickering Emulsion System

The fruit of Gardenia（Gardenia jasminoides Ellis）is used as a traditional medicine in many Asian countries.Crocins are the main components of gardenia fruits,and geniposide and genipin in gardenia fruits are two strong anti-inflammatory agents.In this study,gardenia fruit oil and crocins were extracted from dried gardenia fruit powder（GFP）and the extraction processes were optimized to explore the performance of extracts in repairing oxidative damage of cell components.Furthermore,gardenia fruit oil and crocin I were made into two model emulsions to explore their physical and chemical stability.The main contents and results were described as follows:（1）The gardenia fruit oil was extracted with n-hexane,ethyl acetate,or ethanol respectively combined with ultrasonic.The yield of the three crude oils and by-products extracted from ethanol,fatty acid composition,biologically active ingredients,and free radical scavenging ability were compared.Under the same condition that the solid-liquid ration was 1:8,the temperature was 40℃,the extraction time was 20 minutes and ultrasonic intensity was 200 W,the oil yield extracted with ethanol was 13.26%,slightly lower than hexane 14.86%and ethyl acetate 13.84%.The by-product ethanol colloid phase（ECP）was stratified at 4°C from ethanol extraction,and the yield was 9.68%.The total tocopherol content in the crude oil of Gardenia fruit extracted by ethanol was 1.62μg/mg,which was 1.27times as that obtained by n-hexane.The total sterol content was 22.63μg/mg in the crude oil extracted by ethanol,which was 1.29 times as that obtained by ethyl acetate.The main aromatic components in the crude oil extracted by ethanol were volatile vanillic acid,syringic acid,and syringol.Different from the three crude oils,γ-linolenic acid（1.50%）and trans-linoleic acid（1.14%）were detected in ECP.The IC₅₀ values of DPPH clearance in ECP were 0.61 mg/m L and ABTS were 0.40mg/m L,respectively,which were significantly lower than the test results in crude oil（7.62-10.31 mg/m L）.In addition,crocetin and eight sugar derivatives were preliminarily identified in the ECP by the method UPLC-Triple-TOF/MS.The experimental results showed that gardenia fruit extracted with ethanol had higher economic value,because it had sweet fragrance,rich types and higher contents of omega-6 fats,higher contents of active substances such as tocopherol and sterol,and strong free radical scavenging ability related to crocetin and its derivatives in the by-product.（2）Through the screening of NADESs with different components,it was found that when the molar ratio of Ch Cl-Pro was 1:2.5 and the water content was 35%（v/v）,the yield of crocins was higher.PLS regression model analysis showed that the conductivity,p H and polarity of NADESs were positively correlated with the yield of crocins,while the surface tension and viscosity were negatively correlated with the yield of crocins.On this basis,the single factor experimental conditions were optimized.The optimal extraction conditions of crocins were 2 min,solid-liquid ratio of 1:40 g/m L,temperature of 35℃and power of 400 W.SEM and extraction kinetics model were established to analyze the superiority of extacted by Ch Cl-Pro assisted with ultrasonic（DESU）among the four extraction methods from microscopic and quantitative perspectives.In the SEM figure,the damage degree of GFP structure after Et OHS,DESS,Et OHU and DESU extraction was successively enhanced.The entire ultrasonic extraction process confirmed to the second-order kinetic model.The extraction rate constant K of DESU was 4.06,significantly higher than that of DESS and Et OHU,which were 1.51 and 1.41 respectively.These results indicate that ultrasound combined with NADESs is a synergistic and efficient way to extract crocins.Infrared spectroscopy（FTIR）and ultra-performance liquid chromatography-mass spectrometry（UPLC-MS）confirmed that the main substance in the powder was crocins after extraction,collection of macroporous resin AB-8,rotary evaporation and lyophilization.Furthermore,it was found that DESU extract has a strong AGE inhibition(IC₅₀=6.22μg/m L),which is significantly（p<0.05）higher than the positive control AH(IC₅₀=203.2μg/m L)and pure crocetin(IC₅₀=7.83μg/m L).Thus,it can effectively inhibit protein non-enzymatic glycosylation reaction.In addition,the DNA damage repair degree of 100μg/m L DESU extract freeze-dried powder was 20.18%,which was significantly higher than that of other experimental groups.This may be due to the extremely high DNA repair activity of the pure crocinⅠ.At a concentration of 100μg/m L,the repair degree reached 70.07%.（3）Pro-crocin composite particles were made up of cross-linking crocinⅠwith BSA or cross-linking crocinⅠwith casein.The p H stability,binding capacity and emulsifying properties of the composite particles were anylized in the present study.On this basis,the protein milk beverage system was simulated.It was found that when p H was 7.4,PDI value was about 0.5 and the BC composite particle size range was 335.47-459.3nm,which was relatively small,uniform and disperssed well;The Zeta potential value was-10.85～-7.13 m V,which has a relatively strong chargeability.Compared with CC composite particles,the morphology of CC composite particles is also more stable and uniform at p H7.4.ANS fluorescence assay confirmed that crocinⅠcould bind to BSA and reduce the surface hydrophobicity of the protein.Casein had a weak dose response to crocinⅠ,indicating limited binding sites between them.The peaks of amide I and amide II at 1648 and 1535cm^-1 indicated the characteristic structure of BSA and casein.The blue and red shifts of the characteristic peaks of BC and CC composite particle in amideⅡindicated the covalent binding of BSA、casein with crocin I.The three-phase contact angles of BC₁ and BC₂ composite particles were93.6°and 86.5°,respectively,which were close to 90°,indicating good interfacial amphiphilicity.Complex particles with different concentrations of crocinⅠwere used to stabilize protein milk beverage systems with different oil phase ratios.Turbiscan stability analysis showed that the 5%oil phase emulsion system with BC₁ composite particles had the highest stability,while the 5%oil phase milk beverage system stabilized by CC composite particles all had high physical stabilities.（4）HIPEs with an internal phase volume fraction of 75%were obtained by increasing the concentration of BC composite particles by 15 times.Apparent image,laser confocal image and Turbiscan stability analysis revealed that BC₀ and BC₃ could not stabilize the high internal phase emulsion,resulting in structural collapse.BC-1.5 high internal phase emulsion had the highest physical stability.In rheological experiments,BC-1,BC-1.5 and BC-2 all showed typical shear thinning behavior.The thixotropic recovery percentage of BC-1.5 was 112.19%,which was higher than that of BC-2（110.76%）and BC-1（79.87%）,indicating that BC-1.5 had viscoelastic properties during a long relaxation period and no irreversible damage was caused by strong shear force.The linear viscoelastic region of the emulsion was in the range of0.1-1.0%determined by stress scanning.In the frequency scanning,the frequency dependence（time stability）n’and n’’of BC-1.5 were higher than those of the other two high internal phase emulsions,further indicating that BC-1.5 has a stronger network crosslinking structure.The chromaticity stability of the emulsion was verified by high intensity UV irradiation.It was found that the highest retention rate of crocinⅠwas 82.2%and in BC-2,but it was not significantly different from the control system of BC₂ composite particles of equal concentration.BC-1.5 and BC-1were significantly different from the control,proving that the internal phase gardenia oil can effectively inhibit the auto-oxidation of glycosides in crocinⅠ.In addition,a*value（redness）of BC-1.5 was not significantly different and theΔE value had been small during the irradiation process,which proved that BC-1.5 has a high light irradiation stability.The concentration of lipid peroxides and MDA in HIPE samples with high crocinⅠconcentration（BC-2）accumulated slowly at the early stage of storage,but they were higher than that of BC-1.5（3.16 g Fe Cl₃/m L,31.79 nmol/m L）at the later stage of storage,which showed that BC-1.5 has high interface structure strength and can better resist lipid oxidation in the later stage of product storage.