Modification Of Osborne Method For Fractionation Of Four Wheat Bran Proteins

Wheat bran as the research object, using the modified Osborne method proposed in thispaper for fractionation of four wheat proteins including albumin, globulin, gliadin and gluten,and comparing separation process with the traditional Osborne method. Furthermore, thispaper tested the physical and chemical properties and evaluated the nutritional value of theproducts separated, which provided a new path for the high value recycling of wheat branresources.In order to get over defects existed in the traditional Osborne method, this paperproposed a modified Osborne method, where the innovation was to take advantage of thesolubility differences of the various components of wheat bran. First to realize the separationof total protein and other ingredients in wheat bran by alkaline extraction, followed by using acombined solvent system based on the isoelectric point differences of each protein thatseparated four proteins one by one from the total protein protein, that was albumin, globulin,gliadin and gluten successively. Testing the extraction rate by Kjeldahl method and Bradfordmethod.Best extraction conditions for each protein:(1) Separation of the total protein: using dilute solution of sodium hydroxide to deal withwheat bran, solid-liquid ratio 1:15(m/v, the same below), adjusting pH to 11, temperature50℃, mixing for 1.5h, after centrifugation taking the liquid as the intermediate product for thenext step;(2) Separation of albumin: using hydrochloric acid to neutralize the total protein solution,adjusting pH to 7, temperature 50℃, mixing for 0.5h, after centrifugation taking the solid asthe intermediate product for the next step. Adjusting the liquid pH to 4.0, isoelectric point ofalbumin, with 0.1 mol/L HCl solution, entrifuged to get the solid phase followed by waterwash and dry to obtain albumin;(3) Separation of globulin: using dilute solution of sodium chloride to deal with the solidremained by the last step, sodium chloride concentration 2%, solid-liquid ratio 1:12,temperature 45℃, mixing for 0.5h, after centrifugation taking the solid as the intermediateproduct for the next step. Adjusting the liquid pH to 5.0, isoelectric point of globulin, with 0.1mol/L HCl solution, entrifuged to get the solid phase followed by wash and dry to obtainglobulin;(4) Separation of gliadin: using ethanol as solvent to deal with the solid remained by thelast step, ethanol volume concentration 65%, solid-liquid ratio 1:14, temperature 45℃,mixing for 0.5h, after centrifugation taking the solid as the intermediate product for the nextstep. Adjusting the liquid pH to 5.8, isoelectric point of gliadin, with 0.1 mol/L HCl solution,entrifuged to get the solid phase followed by wash and dry to obtain gliadin;(5) Separation of gluten: using dilute solution of sodium hydroxide to deal with the solidremained by the last step, adjusting pH to 11, temperature 55℃, mixing for 0.5h, aftercentrifugation keeping the liquid. Adjusting the liquid pH to 5.2, isoelectric point of gluten,with 0.1 mol/L HCl solution, entrifuged to get the solid phase followed by wash and dry toobtain gluten.By using the modified Osborne method, 71.2% of the total protein was extracted, whichimproved the yield of traditional Osborne method by 14.8%. Yields of albumin, globulin,gliadin and gluten were 42.67%, 16.48%, 1.94%, 2.18%, respectively. In contrast to thetraditional Osborne method, modified Osborne method was different in the distribution of thefour proteins. Mass ratio of four proteins separated by traditional Osborne and modifiedOsborne were 0.75, 0.68, 1.50 and 1.11 times respectively. Traditional Osborne extractedmore gliadin, while modified Osborne extracted more albumin and globulin. Both methodshad similar protein mass distribution: albumin accounted for more than 65%, globulinaccounted for more than 20%, both gliadin and gluten were less than 10%.The results of physical and chemical properties showed that isoelectric points of albumin,globulin, gliadin and gluten were 4.0, 5.0, 5.8, 5.2, respectively. SEM showed that albuminhad surface with holes, globulin with loose surface, gliadin and gluten molecules were looselyconnected. Globulin possessed both the highest waterbinding capacity(5.00g/g) and the bestholding oil capacity(2.40g/g), while prolamin held the strongest foaming capacity(376%)and stability(71%), and the highest emulsibility(75%).Nutritional evaluation of the four proteins were tested initially. All four proteinscontained eight kinds of amino acids necessary for human body. Proportions of essentialamino acids to total amino acid of albumin, globulin, gliadin and gluten were 27.75%,26.17%, 25.02%, 23.42%, respectively. First limiting amino acid protein of four proteins werelysine, methionine and cysteine, methionine and cysteine, and tryptophan. Tryptophan contentof albumin, globulin, gliadin all exceeded WHO / FAO’s Recommended valueRC = 1. EAAIall bigger than 0.9. SDS-PAGE electrophoresis results showed that the sizes of the fourprotein subunits were mainly between 16 ~ 71 kDa, which were small molecular, and easy todigest by people.