Study On The In Vitro Gastric Digestion Mechanism Of Modified Soybean Protein Isolate Induced By Lipoxygenase Catalyzed Linoleic Acid Oxidation

Protein is the most important nutritional and functional ingredient in food,which is of significance to human health.Among plant protein,soybean protein is nowadays widely utilized as functional ingredients in the food industry due to its high nutrition and desirable ability to improve physicochemical properties.After ingestion,soybean protein digestion is catalyzed by pepsin in the stomach,and gastric digests are subsequently emptied into the small intestine,with vital implications for energy supply,nutrient delivery,and other functional properties.And previous studies have proved that protein gastric digestibility is closely related to the food matrices structure(texture,rheology)and the pepsin diffusivity.However,the protein oxidation may inevitably occur during soybean preservation and transportation due to a relatively high lipoxygenases(LOX)activity in soybean,causing protein structural changes such as cross-linking and aggregation and affecting the protein nutrient substances release as well as bioavailability during digestion.Therefore,this article first investigated the effect of oxidation on the protein structure and functional properties of soybean protein isolates(SPI)in a model of the LOX-catalyzed linoleic acid(LA)oxidation system.And then evaluated the interaction of food matrix structure(gel,emulsion,and emulsion gel)on in vitro gastric digestion and pepsin diffusion.The main contents and results are as follows:(1)Protein oxidation results in a structural modification which affects its digestion.The objective of this work was to investigate the influence of LOX-catalyzed LA oxidation on the structure and in vitro gastric digestion of soybean protein isolate(SPI).Fluorescence recovery after photobleaching(FRAP)was used to evaluate the relationship between pepsin diffusion and gastric digestion.Results indicated that oxidation induced carbonyl formation and loss of free sulfhydryl.Increased surface hydrophobicity and zeta-potential verified the protein unfolding and thus resulted in small particle size and low fluorescence intensity.Fourier transform infrared spectroscopy(FTIR)showed that oxidation caused the increases in?-sheets mostly at the expense of?-helix and random coils.Fluorescein isothiocyanate(FITC)-pepsin in SPI solution modified with 3 mL LA showed a faster diffusion rate with 80.51?m²/s as well as a higher degree of hydrolysis(DH)value of 9.11%,showing that pepsin diffusivity might play an important role in protein gastric digestion.(2)The effects of oxidation on the gastric digestion properties of SPI in a model of LOX-catalyzed LA oxidation system and the multiscale structural characterization of SPI hydrolysate were investigated.Results indicated that the feature of SPI hydrolysate is dependent upon the degree of oxidation.Pepsin hydrolysis caused a red shift in fluorescence intensity and a reduction in surface hydrophobicity and diminished the particle size of SPI hydrolysate during gastric digestion.Compared with the control,mild oxidation was beneficial to protein unfolding and gastric digestibility,as manifested by minimal molecular weight(MW)distribution>50kDa(32.34%)and smaller peptide fragments under scanning electron microscopy.However,severe oxidation brought about a 39.47%loss of free amino acids.It was interesting to find that glycinin was more vulnerable to pepsin hydrolysis after oxidation as compared to the native SPI.Overall,the moderately oxidized SPI appeared to be digested to a greater extent.(3)The model of LOX-catalyzed LA oxidation was selected as representative of a lipid peroxidation system to investigate the effects of oxidative modification on SPI gel properties and in vitro gastric digestion.FRAP technique was applied to evaluate pepsin diffusion in the gel.The results showed that oxidative modification increased the gel hardness as well as brought about a compact and three-dimensional network structure,which consequently decreased the water mobility as manifest by lowering the relaxation time of T_2b and T₂₁ from0.55 and 3.22 ms for the control to 0.32 and 2.42 ms for the 7 LA+LOX group,respectively.It was interesting to note that pepsin diffusion was significantly correlated(P<0.05)with T_2b and DH,indicating that water mobility might be a factor related to FITC-pepsin diffusion,which would ultimately influence the gel gastric digestion.Compared with native SPI,moderate oxidation can improve the digestibility of SPI gel by the summed effects of pepsin diffusion limitation,microstructure variation,and hydrolysis degree.(4)The effects of oxidation on emulsion properties and in vitro gastric digestion of SPI emulsions were investigated.Results showed that oxidation induced parts of SPI aggregation,which decreased the emulsification of SPI as manifest by a large oil droplet under CLSM.Rheological analysis indicated that all emulsions are non-Newtonian fluids.Pepsin hydrolysis caused a reduction of turbidity and particle size,and increased the pH value of SPI emulsions during gastric digestion.After gastric digestion,protein hydrolysates adsorbed on the oil-water interface,enhanced the stability of emulsion,presented a small size of droplets under CLSM,and reduced the content of free amino acids.In addition,the particle size of the oil droplets gradually increased with strengthening the oxidation degree,which would increase the spatial hindrance and decrease the pepsin diffusivity from 82.81?m²/s(control group)to 67.52?m²/s.Therefore,the protein structure changes induced by oxidation can affect the adsorption of interfacial proteins and alter the particle size of oil droplets,thus changing the diffusion and accessibility of pepsin,which would ultimately influence the gel gastric digestion.(5)This study has focused on the structural changes and in vitro gastric digestion behavior of oxidized SPI emulsion gels.Results showed that the corn oil droplet was embedded into the protein gel network and exhibited an obvious porous structure under CLSM.Compared with the control group,the gel hardness and apparent viscosity were increased in the 1 LA+LOX group due to the insertion of a large number of droplets into the gel network.On the contrary,several droplets embedded into the gel network of the 3 LA+LOX group caused a reduction of gel hardness and apparent viscosity as well as an increasing in the T₂ relaxation time,and make it easier to be chewed.FRAP results showed that the presence of gel network and oil droplets increased the steric inhibition impact,which might decrease the diffusion rate of pepsin,ranging from 40.49 to 56.55?m²/s.In addition,the FITC-Pepsin fluorescent intensity gradually decreased from the surface to 3 mm depth under 37?water bath and shaking.Besides,the real-time disintegration results showed that the fluorescence intensity of SPI gel decreased by 65%after pepsin digestion.As for SPI emulsion gels,the green fluorescence intensity of protein decreased,and the oil was gradually exposed to the surface of the gel,leading to the red fluorescence increased.And there was no significant structural collapse,indicating that the main mechanism of pepsin digestion of protein gel was surface erosion.