Study On Lysinoalanine Generation And Inhibition Of Ultrasound Technology In Alkali Extraction Of Rice Residue Protein Isolates

Rice dregs are recognized as an excellent plant protein resource.In industry,rice dregs were mainly used as raw material to prepare rice residue protein isolates?RRPI?with high yield and purity by alkaline extraction and acid precipitation method.Alkaline extraction,however,may also induce some reactions which cause the formation of lysinoalanine?LAL?,especially at higher alkali concentration.The generation of LAL can decrease the digestibility and utilization of protein,which directly or indirectly affect the body function.At present,there is little information on generation,digestibility and absorption characteristics,and inhibition methods of LAL in plant protein.The purpose of this study was thus to examine the influence of alkali extraction conditions on the LAL generation during the alkaline extraction of RRPI.Also,the effects of LAL generation on the functionality properties,structural properties,and digestibility and absorption properties of RRPI under various alkali treatment conditions were investigated.Moreover,the influences of ultrasound treatment on the generation of LAL was explored,and the molecular mechanism of ultrasound in decreasing LAL content in RRPI was investigated.The main conclusions obtained by the study are as follows:?1?The pattern of LAL generation rule during the alkaline extraction of RRPI was studied.It was found that LAL content increased from 256 to 13 079 mg/kg_protein as NaOH concentration increased from 0.03 to 0.09 mol/L,and then decreased to 1 541 mg/kg_proteinrotein at 0.13 mol/L NaOH.The extraction temperature and time were found to have a positive correlation with LAL content.The results of amino acid analysis indicated that alkali treatment could degrade cystine,threonine,lysine content,among which cystine was the most sensitive to alkali treatment.The possible mechanism of production of LAL in the treated RRPI is that cystein or lesser amount of cysteine produce dehydroalanine through?-elimination that is catalyzed by hydroxide group.Then dehydroalanine conjugate with?-NH₂ of lysine to produce LAL.?2?The effects of alkali treatment on the functional and structural characteristics of RRPI were investigated.The results showed that with increasing alkali?NaOH?concentrations,alkali treatment temperature and time,the solubility,foaming capacity and emulsion properties of RRPI increased first and then decreased,while there was no significant change in foaming stability.Alkali treatment could change protein secondary structure by reducing?-helix and?-turn contents and increasing?-sheet and random coil contents.The results of microstructure indicated that alkali treatment could alter the surface morphology and surface roughness of RRPI.There were more microparticles and small fragments on the surface of the protein,and the structure became more loose.It was observed from the nanostructure that the number of protein micropores increases significantly,and the surface roughness R_q and R_a also increased.?3?The effects of alkali treatment on the digestibility and absorption characteristics of RRPI and the intestinal absorption characteristics of LAL were studied.With increase of alkali?NaOH?concentration and treatment temperature,the in-vitro digestibility of RRPI increased first and then decreased.With the extension of alkali treatment time,the in-vitro digestibility decreased.Alkali treatment reduced the release of all amino acids,especially arginine,lysine,phenylalanine,tyrosine,cysteine,and threonine.Only 2.65-9.28%of LAL was released from RRPI after digested by gastro-pancreatic protein digestive enzymes.LAL existed mainly as the long chain peptide chain with a molecular weught range from 1000 Da to 3000 Da.The ex-vivo small intestine perfusion experiment in rats showed that higher alkali concentration of alkali significantly reduced the absorption rate of RRPI.LAL had no specific absorption site in the small intestine of rats,and its absorption rate constant K_a was 6.27×10^-6 min^-1 at the end of jejunum and 4.03×10^-7 min^-1 in the ileum.?4?The ultrasonic pretreatment for controlling the generation of LAL during the alkaline extraction of RRPI was studied.The optimal ultrasonic working modes and ultrasonic treatment conditions for dual frequency power ultrasound and five-frequency countercurrent S-type ultrasound to inhibiting LAL content were studied.Results showed that ultrasound treatment significantly decreased LAL content and improved protein dissolution rate.In addition,the results indicated that five-frequency countercurrent S-type ultrasound was superior to the dual frequency power ultrasound.Following dual-frequency ultrasound?20/40 kHz?pretreatment,LAL content was 11 057 mg/kg_protein,which was 24.37%lower than the non-sonication sample,and the protein dissolution rate was 71.47%,compared with that of other ultrasound operating modes.Under20/40 kHz,the optimal ultrasonic parameters were obtained as follows:ultrasonic power density of 36 W/L,temperature of 38?,and time of 22 min.Validation tests indicated that under the optimal sonication treatment conditions,the LAL content of RRPI was 11 093 mg/kg_protein.?5?The influences of ultrasonic treatment conditions on the c onformational characteristics of RRPI were studied.The results indicated that ultrasonic treatment altered the chemical interactions between protein molecules,the relative content of ionic bonds and hydrophobic interaction force increased,and the content of hydrogen bonds and disulfide bonds decreased.The reduction in disulfide bonds caused the formation of more sulfhydryl groups.Ultrasonic treatment changed the secondary structure of protein by the decreasing the content of?-helix and?-turn content and increasing?-sheet and random coil content.After ultrasonic treatment,particle size distribution curve of the protein moved to the direction of smaller particle size,and the protein particle size decreased.Moreover,the number of small protein particles increased,and the protein particle distribution was more concentration and uniform.The decrease of Zeta potential indicated that the amino acid residues with a negative charge in protein solution decreased,which was not conducive to the generation of LAL.?6?The quantitative association model between LAL content and changes in protein structural characteristics change of protein in the process of ultrasonic treatment of RRPI was established by Pearson correlation coefficient analysis,principal component analysis,and stepwise regression analysis.Pearson correlation coefficient analysis showed that the free sulfhydryl content,Zeta potential value,and total sulfhydryl content negatively correlated with the LAL content of RRPI?P<0.01?;the?-helix positively correlated with LAL content of RRPI?P<0.01?.The principal component analysis indicated that the cumulative contribution rates of the three principal components were 89.705%,reflecting all the change information of the original variables.The first principal component was contributed by random coil content,?-helix,?-sheet,and total sulfhydryl group,the second principal component was particle size and free sulfhydryl group,and the third principal component was Zeta potential.The stepwise multiple regression analysis indicated that free sulfhydryl content and Zeta potential value could be used to predict LAL content of RRPI.