| Rice bran protein(RBP)is a superior protein with high nutritional value.In 2018,the annual output of rice bran in China reached 141,300 tons.The source of RBP is huge and has great research value and development potential.However,as far as the current situation is concerned,RBP not only has problems that are difficult to extract,but also tends to have a darker color in the extraction process,which greatly limits its commercial value.Browning reaction is often the main source of colored matter.The browning reaction mainly includes enzymatic browning and non-enzymatic browning.At present,most of the research on RBP browning focuses on enzymatic browning,while the research on non-enzymatic browning of RBP only focuses on the selection of inhibitors.The reaction mechanism of RBP non-enzymatic browning caused by reducing sugar has not yet been clear.RBP is composed of four graded proteins.The basic information and expression of graded proteins determine the life activities of RBP.It is important to explore the color and glycosylation of graded proteins to further clarify the non-enzymatic browning of protein products.Therefore,in this experiment,fresh rice bran was used as raw material,and petroleum ether was degreased and then extracted by ultrasonic assisted alkali method.The color change of protein was observed,and the graded protein was further extracted.The browning of graded protein was observed and glycosylation analysis was carried out to explore The relationship between glycosylation modification and color change on graded proteins and browning of protein products.The single factor method and response surface optimization were used to determine the process parameters of RBP extraction.The effects of ultrasonic time,ultrasonic power and ratio of material to liquid on RBP extraction rate were compared.The single factor experiment showed that the ultrasonic power was 120W and the ultrasonic time was 30min.The highest extraction rate can be achieved when the ratio of material to liquid is 1:9;and the optimal extraction conditions of ultrasonic assisted alkali extraction RBP can be obtained by using the response surface to optimize the extraction conditions:ultrasonic power 116.91W,ratio of material to liquid 1:9.33 Ultrasound time 28.99min.This process was modified during the actual operation:the ultrasonic power was set to 120 W,the ratio of material to liquid was 1:9,and the ultrasonic action was applied for 30 min.At this time,the protein extraction rate can reach 71.3%,which is very close to the predicted value.The extracted RBP was freeze-dried and found to have a darker protein color.The RBP obtained by being dried at different temperatures was darker and varied greatly:the color of the dried protein at 80? and 90? was significantly deeper than 50?,60?,70?.Four graded proteins were sequentially extracted by Osborne fractionation method,and the basic components of the graded proteins were compared and analyzed.After thawing and comparing the color of the graded protein,it was found that the albumin and gluten were less bright and they are brownish,which was significantly darker than the globulin and prolamin;the four graded proteins showed lighter reddish brown and yellow after decolorization,but the color of albumin and gluten was still significantly deeper than globulin and prolamin.Infrared chromatography of the graded protein revealed a functional group in which the carbohydrate binds to the protein,and the occurrence of glycosylation modification can be inferred.The grading protein was subjected to periodic acid Schiff staining,and it was found that the graded protein had a purple-red region at 10.77 KDa.Besides it was found that the graded protein had a glycosylation modification.The bands of the four graded proteins at 10.77KDa were identified by mass spectrometry of protein and carbohydrate.The results of protein identification showed that the main components of the four proteins were different:albumin mainly contains 63KDa globulin,novel protease,glucose and ribitol.Hydrogenase,1-cysteine peroxide reductase,glyceraldehyde-3-phosphate dehydrogenase,nucleoside diphosphate kinase,histones with molecular weight H4,alpha-amylase,trypsin inhibitor,RA5 seed allergy Protein,RA5B allergen and other proteins;globulin is mainly caused by 63KDa globulin,novel protease,Os08g0127900 protein,Os03g0197300 protein,?-amylase,16 kDa oleosin,Os06g0221300 protein,Os03g0277500 protein,H4 histone,RA5 seed allergy Protein,RA5B allergen,trypsin inhibitor,non-specific lipid transfer protein fragment,17D prolamin and Os08g0180500 protein,OSJNBa0041A02.15 protein,Os03g0747600 protein composition;prolamin contains Os05g0331800 protein,17D prolamin,Os05g0329400 Protein,Os05g0328800 protein,Os05g0328632 protein,13 kDa prolamin,18kDa oleosin,RA5 seed allergen,RA5B allergen,8-2 germ protein,?-amylase,Os10g0558400 protein and Os04g0617500 protein;gluten mainly includes A1 gluten,A2 gluten,A3 gluten,novel protease,63KDa globulin,B5 gluten,17D prolamin,B1 gluten,B2 gluten,Os05g0328632 protein,Os05g0331800 protein,16kDa oleosin,Os03g0197300 protein,Os02g0249000 gluten,Os02g0453600 gluten,13 kDa prolamin,RA5 seed allergen,RA5B allergen,BAG domain protein,Os06g0728600 protein,EF hand family protein.The results of mass spectrometry analysis showed that there were hexose modifications in albumin;fucose modification in globulin;modification of hexose and heptose in prolamin;and glycosylation in gluten At most,the occurrence of glycosylation modification such as hexose,heptose,and fucose can be seen.In summary,there are glycosylation phenomena in graded proteins,and it is presumed that glycosylation of some protein fragments in albumin and gluten is related to browning of proteins according to the color difference between graded proteins.In order to make better use of RBP in the food industry,ultrasonic assisted alkali method was used to optimize the extraction of RBP and the properties of graded proteins were studied for the phenomenon of protein browning.The classification protein extraction and color change were further described and further glycosylation was carried out.The analysis provides a theoretical basis for the processing and utilization of RBP and its fractionated protein,and provides a reference for the study of cereal protein. |
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