Preparation,digestion And Absorption Characteristics Of Highly Soluble Rice Bran Protein Based On Non-thermal Treatment

Rice bran,a by-product of rice processing,is a kind of renewable resource with large quantity and wide range.Rice bran protein is a kind of high-quality cereal protein,which is nutrient-rich and hypoallergenic.However,poor extraction rate and solubility lead to insufficient use of rice bran proteins,so that the rice bran protein could not be used and developed on a large scale.Nowadays,new non-thermal processing technology has been paid attention to by scientific researchers because of the more stringent requirements of food nutrition,safety and function.Thus,it is crucial for the continued usage of rice bran resources to use non-thermal processing technology to increase the solubility of rice bran protein in order to prepare high-solubility rice bran protein and investigate its nutritional value.In this paper,rice bran protein was extracted from fresh rice bran,and firstly,the extraction process was optimized to determine the best process conditions;secondly,based on non-thermal treatment to improve the solubility of rice bran protein,the high-solubility rice bran protein was prepared,and the relationship between solubility and structural changes of protein was investigated;finally,an in vitro digestion and absorption model was constructed to investigate the digestion and absorption characteristics of highly soluble rice bran protein.The main content and results of the research are as follows:（1）The rice bran protein was extracted from fresh rice bran by combining ultra-micro-grinding and ultrasonic cell crushing.The effects of single factors such as material-liquid ratio,ultrasonic power,ultrasonic time,and extraction time on protein extraction rate were investigated;and the optimal extraction process of rice bran protein was determined by combining response surface optimization experiment as follows:material-liquid ratio 1:57（g/m L）,extraction time 4 h,ultrasonic time 9 min,and ultrasonic power 130 W.（2）High hydrostatic pressure（HHP）and ultrasonic（US）non-thermal processing techniques were used to enhance the solubility of rice bran protein and to investigate the change pattern between solubility and structure.When the HHP treatment and US treatment were controlled at 200 Mpa-40 min and 350 W-40 min,respectively,the solubility of rice bran protein increased from 15.10%to 26.68%and 35.75%,respectively.The subunit composition,free sulfhydryl content,secondary and tertiary structure,surface hydrophobicity,and surface microstructure of rice bran protein were used to investigate the structural changes that occurred prior to and following non-thermal treatment.The sodium dodecyl sulfate-polyacrylamide（SDS-PAGE）gel electrophoresis maps showed that the non-thermal treatment improved the solubility of rice bran proteins mainly by directing changes in the physical level of the macromolecular proteins,with no significant effect on their subunit composition.The free sulfhydryl content illustrated that moderate HHP treatment and US treatment broke the disulfide bonds of rice bran proteins,which increased their free sulfhydryl content by 35.54%and 42.46%,respectively;however,excessive non-thermal treatment led to the repolymerization of rice bran proteins,which decreased their free sulfhydryl content.Fourier transform infrared spectroscopy（FT-IR）maps and secondary structure analysis showed that the non-thermal treatment altered the secondary structure of rice bran protein and depolymerized the aggregated protein bodies.The UV-visible and endogenous fluorescence spectrograms showed that the non-thermal treatment caused changes in the tertiary structure of rice bran proteins,with the chromogenic groups tending to the hydrophilic environment and the hydrophilic groups being exposed.The surface hydrophobicity（H₀）indicated that the hydrophobic concentration region of rice bran protein was disrupted by non-thermal treatment and hydrophobic units were formed.Scanning electron microscopy（SEM）results showed that the non-thermal treatment transformed the compact block rice bran protein into a loose and porous sheet-like structure.（3）The solubility of rice bran protein was further improved by non-thermal treatment combined with restriction enzymatic hydrolysis（RE）,and its solubility mechanism was investigated in conjunction with its structural changes.By comparing the effects of HHP,US,RE,HHP-US,US-HHP,HHP-RE,and US-RE modifications on the solubility of rice bran protein,it was found that the US-RE modifications were more significant（P<0.05）in improving the solubility of rice bran protein.The effects of hydrolysis degrees DH1 to DH6on the solubility of rice bran protein were investigated based on the enzymatic time-hydrolysis degree curves on the basis of non-thermal treatment.The results showed that the solubility of rice bran protein was greatest at 4%hydrolysis degree,which increased from 15.10%to76.37%,while a slight decrease occurred when the hydrolysis degree increased to 6%.By measuring the subunit composition,free sulfhydryl content,secondary and tertiary structures,surface hydrophobicity,and surface microstructure,the structural characterization of rice bran protein before and after US-RE modification was carried out.SDS-PAGE plots revealed that US-RE was responsible for the disappearance of theα-subunit bands in rice bran protein,and as the hydrolysis degree increased from DH1 to DH5,the remaining subunit bands’colors became lighter.At DH6,rice bran protein’s basic polypeptide chain bands largely vanished.Secondly,on the basis of non-thermal treatment,restriction enzymatic hydrolysis further broke the disulfide and hydrogen bonds of rice bran protein,and the hydrophobic concentration region was further disrupted,indicating further depolymerization of the proteome;however,excessive hydrolysis would cause aggregation between peptides due to hydrophobic interactions.Meanwhile,the UV characteristic peak andλ_max of rice bran protein were red-shifted,UV absorption intensity and fluorescence intensity were enhanced,and hydrophilic groups were exposed after US-RE treatment,which led to the increase of solubility.In addition,on the basis of non-thermal treatment,the restriction enzymatic hydrolysis further enhanced the solubility of rice bran protein by destroying the lamellar structure and creating a loose structure with small fragments and large pores on the surface.（4）Simulating in vitro gastrointestinal digestion and absorption allowed researchers to investigate the digestive and absorption properties of highly soluble rice bran protein.It was found that the digestive properties of highly soluble rice bran protein were enhanced compared with those before modification,and the digestibility increased from 68.3%to 76.5%.Caco-2cell viability was measured by CCK-8（cell counting kit-8）to determine the sample concentration,and the protein digest at a concentration of 0.5 mg/m L was finally selected to simulate small intestine absorption.At the same time,the bioavailability of highly soluble rice bran protein was found to be better than that before modification by using the Caco-2 cell monolayer model to simulate small intestinal absorption,and its absorption rate increased from17.6%to 83.7%,and the amino acid composition before and after absorption was approximately similar.In addition,non-thermal treatment combined with restricted enzymatic hydrolysis and in vitro digestion and absorption did not significantly affect the nutritional value of rice bran protein.