Mechanism Of Pine Nut Oil Oleogels Promoted By Protein-Polyphenol Conjugates

Solid fat not only could give food a good flavor and taste,but also has functional properties during food processing such as crisping and film coating.However,natural or artificial solid fat contains more saturated fatty acids or trans fatty acids.Excessive intake of solid fat will be the major factor related to hyperlipidemia,then triggering the process of atherosclerosis and increase the risk of cardiovascular disease.Oleogel has attracted more and more attention,it can be tailored using specific triglycerides and oleogelators that can be designed to reduce the content of saturated fatty acid and lessen appetite or carry the bioactive substance to be absorbed in the digestive tract.Nutrient rich in pine nut,and the content of pine nut oil is as high as 60%-69%.More than 90%of them are unsaturated fatty acids,including linolenic acid,linoleic acid,arachidonic acid and a unique fatty acid-pinolenic acid.But higher unsaturated fatty acid content makes it more susceptible to oxidative deterioration occurs.Therefore,this article aims to design a pine nut oil oleogel with solid lipid properties and can improve its own oxidative stability.In this thesis,pine nut oil was used as the base oil and the hydrogel was used as a template to prepare pine nut oil oleogel by indirect method.First,the hydrogel was constructed based on the covalent reaction of soy protein(SPI)and tannic acid(TA),investigating the effects of the variation of environmental pH value and TA concentration on gelation properties of protein hydrogel.Furthermore,modern analysis methods such as fluorescence spectrometer,Fourier infrared spectrometer(FTIR),circular dichrograph(CD),rheometer,thermogravimetric analyzer,texture analyzer,scanning electron microscope,and other modern analytical methods were used to explore the effects of covalent reactions on the spatial structure of proteins,physical and chemical properties,gelation,and microstructure.Then the SPI-TA hydrogel was exploited as a template to prepare piece oleogel(PO),spread oleogel(SO)by solvent exchange method,and oleogel(AO)by aerogel adsorption method.Optimize the preparation process of different types of pine nut oleogel,evaluate its rheology and thermal stability,and estimate the oxidation stability and in vitro gastrointestinal digestion characteristics of oleogels.Finally,pine nut oil oleogel was used as a carrier loaded with quercetin,studying the possibility of oleogel to deliver hydrophobic active substances,and probe the bioavailability of quercetin during in vitro gastrointestinal digestion.Caco-2 cells model was further used to investigate the cellular transport behavior of quercetin in QE-AO and QE-oil after digestion.The main conclusions are as follows:(1)Preparation and characterization of SPI-TA covalent conjugatesThe pH value and TA concentration significantly affect the physical and chemical properties and spatial structure of SPI-TA conjugates.The covalent binding rate raised with the increase of pH and tannic acid concentration.The covalent binding rate was the highest(95.06±0.32%)when the TA concentration was 117 ?mol/g SPI and pH 11.The occurrence of the covalent reaction between SPI and TA was confirmed by SDS-PAGE.The results of FTIR showed that pH and TA concentration have a greater impaction on the characteristic groups of the amide I band of the conjugate,which manifested in the transition from ?-helix to ?-turn and random coiling of secondary structures.The change of pH value reduces the fluorescence peak of SPI-TA conjuages to a higher degree than the change of TA concentration.Also,the thermal stability of the SPI-TA conjugate is significantly higher than SPI,and its antioxidant activity was enhanced.The results of in vitro digestion characteristics showed that after modification by TA,the degree of protein digestion in the small intestine was inhibited with the concentration of TA increases,and the degree of hydrolysis decreases.(2)Preparation and characterization of SPI-TA covalented hydrogelThe pH value significantly affects the gelation properties of the SPI-TA covalent hydrogel.The hardness and elasticity of hydrogel increased with the increase of pH value and TA concentration.When the pH is 11 and the TA concentration is 117 ?mol/g SPI,the hardness of the hydrogel reaches the highest(43.33±5.90 g),and the elasticity is 6.12±0.68 mm.But the higher concentration of TA will result in a decrease in the textural properties.The storage modulus(G')of the hydrogel is much greater than the loss modulus(G"),and the gel structure is stable.G'&G" changed little with frequency and temperature,and the rheology is confirmed it exhibited as the typical gel-like behavior of gels.Scanning electron microscopy showed that the gel was porous,the pore size was uniform,and the porosity was significantly less than SPI,but the characteristic value of gel color changed in a dose-dependent manner with the increase of pH and tannin concentration.The hydrogel prepared at a TA concentration of 117 ?mol/g SPI and pH 11 has an inhibitory effect on the growth of E.coli and S.auteus.The results showed that the covalent reaction of SPI and TA can be used to prepare protein-based hydrogels with a certain hardness and conform to the characteristics of typical gels.(3)Preparation of pine nut oil oleogel with SPI-TA hydrogel by the indirect methodThe optimum preparation processes of PO were:the material-to-liquid ratio of hydrogel to acetone solution is 1:15,the material-to-liquid ratio of after dehydration gel to pine nut oil and acetone solution is 1:8,and the replacement time is 6 h.The oil content of PO oil gel is77.85±0.76%.The optimum technological conditions of SO were:shear time 5 min,centrifugal force 4000×g,at this time,the oil content of SO oil gel is 78.55±0.62%.AO oleogel could reach equilibrium after SPI-TA aerogel adsorbs pine nut oil for 1 h,and the highest oil content is 80.28±0.85%.The microstructures of three oleogels were different.The pores of the AO oleogel are significantly smaller than those of the PO oleogel,and the pore sizes of the PO were uneven.The SO oleogel was consisted of pine nut oil surrounding by the composition.The oil loss rate of oleogels was shown as AO<PO<SO,in contrast,the thermal stability of oleogels was AO>PO>SO.The measurement of rheological properties showed that G'>G" of the oleogels showed similar gel performance,but G' and G" raised with the increase of frequency,so it is speculated that PO,SO,AO belong to a weak gel structure.(4)After 16 days of accelerated storage,the peroxide values of AO,PO,and SO oleogel were 0.02±0.001 meq/kg,0.21±0.05 meq/kg and 0.48 ± 0.03 meq/kg,which were much lower than the pine nut oil(2.61 ±0.06 meq/kg).It showed that the oxidative stability of pine nut oil is significantly improved after gelation.At the same time,the protein carbonyl content in the oleogel increases,and the free amino content decreased.The fluorescence spectrum showed that the fluorescence intensity of the protein oxidation product increases with the storage time.The lipolysis rates of AO,PO,and SO within 20 min of digestion in the small intestine were 0.06 min-1,0.06 min-1,and 0.07 min-1,respectively,which were lower than the pine nut oil lipolysis rate(0.13 min-1).However,the oil flocculated at the end of digestion.The lipolysis degree of oleogel was higher than that of pine nut oil,with AO>PO>SO>pine nut oil.The laser confocal image shows that the aggregates after gastric digestion are broken down in the small intestine digestion stage,and most of the lipid droplets in the digested product were lipolysis when the small intestine digestion ends.Compared with before and after digestion,the content of unsaturated fatty acids in the micellar phase of the oleogel was the highest,which increased by 2.66%,while the content of unsaturated fatty acids in the micellar phase of pine nut oil decreased by 6.25%,indicating that the oleogel system promoted the digestion and absorption of unsaturated fatty acids in the pine nut oil.(5)In vitro digestion characteristics of pine nut oil oleogel loaded with quercetinBased on the in vitro digestion characteristics of oleogel,AO was used as a carrier to load quercetin(QE)during its preparation process to prepare QE-AO oleogel,and quercetin-pine nut oil(QE-oil)was compared at the same time as a control.Investigating its feasibility and in vitro digestion characteristics.The adsorption time of the aerogel after loading QE is 2 h.Currently,the quercetin loading of QE-AO is 0.41%.QE-AO releases free fatty acids(FFAs)at a rate of 0.08 min-1 within 20 min of in vitro small intestine digestion,which is less than the release rate of FFAs in QE-oil(0.24 min-1);but after digestion,the content of FFAs in QE-AO is higher than QE-oil.After intestinal digestion,the particle size of QE-AO was bimodal,with a peak less than 300 nm,indicating the formation of micellar particles.The absolute value of Zeta-potential increased with the progress of digestion.QE-AO was 23.93±0.42 mV at the end of intestinal digestion,and QE-oil was 16.91±0.34 mV,indicating that the hydrolysis of the protein matrix in QE-AO contributes to emulsification,resulting in a higher chance of contact between pine nut oil and lipase than QE-oil.The bioavailability of QE in the QE-AO digestion process was improved by 11.08%compared with QE-oil,and the fluorescence signal of QE in its micellar phase was stronger than that of QE in the micellar phase of QE-oil.The QE secretion rate of QE-AO system in Caco-2 monolayer was higher than QE-oil,and the apparent permeability coefficient value increased by 1.68 times.The above conclusions provide a theoretical basis for the construction of oleogel as a delivery system for hydrophobic active substances.