The Anti-digestion Mechanism Of Starch Induced By Protein In Rice

Rice(Oryza sativa L.),which is primarily composed of starch,protein,and lipids,is one of the vital staple foods for most people in the world.Starch is the main carbohydrate in rice,its digestive behavior is the key of affecting blood sugar response.Protein is the component with the highest content in rice except starch,it is an endogenous factor that affects the digestibility of starch in rice.The effect and mechanism of endogenous proteins on starch digestibility during the digestion of rice flour are still unclear.In addition,proteins are hydrolyzed by proteases such as pepsin and pancreatin in the gastrointestinal tract and release peptides and free amino acids.Therefore,protein hydrolysates may also have an effect on the digestion of rice starch.Based on this,this study explored the effect and reasons of endogenous proteins,protein hydrolysates,and free amino acids released by protein on rice starch digestion,and clarified the anti-digestion mechanism of starch induced by protein in rice.Firstly,the effect of endogenous proteins on rice starch digestibility was investigated.The proteins in the three kinds of rice flour(japonica rice,glutinous rice,and indica rice)were removed by using alkaline protease.After proteins were removed,the hydrolysis rates of rice flour were increased.X-ray diffraction(XRD)and Fourier transform infrared spectroscopy(FT-IR)analysis found that after the proteins were removed,the relative crystallinity decreased from 22.4%,25.8%,and 23.2%to 19.4%,19.6%,and 18.9%,respectively.The absorption peak at 3280 cm^-1in the FT-IR spectrum shifted to the high wave number(3290 cm^-1).These data indicated that the proteins in rice flour combined with rice starch by hydrogen bonds,and improved the long-and short-range order of rice starch,making the starch structure compact and ordered.Scanning electron microscopy and confocal laser scanning microscopy showed that endogenous proteins had a physical barrier effect to hinder the contact between digestive enzymes and rice starch,thus reducing the digestion rate of starch in rice flour.Then the effect of rice protein hydrolysates on starch digestibility were investigated.Japonica rice protein hydrolysates were prepared by pepsin and pancreatin.After the protein hydrolysates were added,the digestibility of japonica rice starch was significantly decreased.The effect of protein hydrolysates on starch digestion was more obvious than that of protein,and it was speculated that the free amino acids in protein hydrolysates inhibited the activity of digestive enzymes.The results of FT-IR showed that the absorption peak associated with O-H stretching vibration in gelatinized starch shifted to low wave number,indicating that hydrogen bonds were formed between starch and protein hydrolysates.Further XRD analysis found that the intensity of the V-shaped peak increased.It was proved that some hydrophobic peptides or free amino acids released by protein formed complexes with starch.DSC analysis showed that the thermal stability of raw and gelatinized rice starch was enhanced,and the enthalpy value increased from 4.67 and 1.01 J/g to 4.73 and 2.82 J/g.The data showed that in the presence of protein hydrolysates,the crystal structure of starch was compact,thus the resistance of starch to enzymatic hydrolysis was improved.The effect of all kinds of amino acids released by endogenous proteins on porcine pancreatic?-amylase activity was further determined and the mechanism was preliminarily explored.The results showed that the amino acids inhibited the activity of porcine pancreatic?-amylase,and the inhibition rate was between 0.2%and 13.5%.Glutamic(Glu),aspartic(Asp),and lysine(Lys)had significant inhibitory effect on the activity of porcine pancreatic?-amylase,with the inhibition rates of 13.5%,11.9%,and 10.6%.The inhibition rates of different concentrations(0.01,0.1,0.5,1,3,5,and 10 mg/m L)of Glu on porcine pancreatic?-amylase activity were 12.03%,23.3%,26.2%,28.8%,37.8%,43.6%,and 54.1%.The data showed that the inhibitory effect on porcine pancreatic?-amylase activity gradually enhanced with the increase of Glu concentration.The analysis of Lineweaver-Buck double reciprocal curve,Dixon equation,and Cornish-Bowden equation indicated that the inhibition of Glu on porcine pancreatic?-amylase activity was a mixed type of inhibition.It indicated that Glu could combine with free porcine pancreatic?-amylase or with the intermediate formed by starch and porcine pancreatic?-amylase,thus slowing down the digestion of rice starch.Finally,the possible interaction between starch and free amino acids released by protein and its influence on the digestibility of starch were further explored.After Glu,Asp,and Lys were added into starch,the total content of SDS and RS increased by 21.7%,15.8%,and 20%,respectively,indicating that amino acids promoted the formation of SDS and RS.XRD and FT-IR analysis showed that after Glu,Asp,and Lys were added,the relative crystallinity of rice starch increased by 6.1%,4.9%,and 4.5%,respectively.The value of 1047 cm^-1/1022cm^-1increased from 0.561 to 0.678,0.641,and 0.583,showing that amino acids significantly increased the long-and short-range order of starch.DSC analysis showed that after amino acids were complexed with rice starch,the crystal melting peak shifted to the right.The gelatinization temperature was increased from 60.76 to 67.57,67.11,and 69.37°C,respectively.The results revealed that the hydrogen bonds between amino acids and starch restricted the flow of starch chain and hindered the melting of starch crystal.Therefore,the difficulty of enzymatic digestion of starch increased.