Stabilization of hypoallergenic, hyperdigestible previously reduced proteins

Abstract

Disulfide proteins showed mitigated allergenicity and increased digestibility by pepsin following reduction by thioredoxin. The sulfhydryl groups newly formed on reduction by thioredoxin (at 4° C.) or dithiothreitol (DTT) (at 55° C.) were blocked with a physiological disulfide, such as cystamine or oxidized glutathione (GSSG) to obtain stable forms of the disarmed allergen. When derivatized with cystamine, BLG was separated from its oxidized and reduced forms on non-reducing SDS-PAGE and appeared to lack sulfhydryl groups. Although less effective GSSG, gave similar results. Allergenicity of the two derivatives was compared with that of the oxidized, reduced and reoxidized forms of BLG by skin testing dogs from a colony sensitized to cow's milk. Both the cystamine and GSSG derivatized BLG showed decreased allergenicity and increased sensitivity to pepsin as compared to controls. The reoxidized form resembled the derivatives in having lower allergenicity. The thioredoxin- and DTT-reduced forms showed hypoallergenic, hyperdigestible properties, most effectively when the reduced proteins were heated at 55° C. Whole milk subjected to these procedures showed results similar to those obtained with pure BLG. Other proteins are similarly stabilized. Stable forms of such disarmed, hypoallergenic and hyperdigestible disulfide protein allergens or just hypoallergenic or just hyperdigestible protein allergens are useful in foods as well as clinical preparations.

What is claimed is:  
     
         1 . A method of decreasing the allergenicity of an allergenic protein comprising: 
 (a) treating a protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin reductase and NADPH or an amount of dithiothreitol effective for and at a temperature effective for reducing at least one of the disulfide bonds in said protein;    (b) reacting the protein from step (a) with an amount of a physiological disulfide effective for preventing reoxidation of at least one of the reduced disulfide bonds in said protein from step (a); and    (c) administering the reacted protein in step (b) to an animal, wherein said allergenic symptoms exhibited by said animal administered the protein from step (b) are decreased as compared to a control.    
     
     
         2 . The method of  claim 1  wherein the allergenic protein is selected from the group consisting of cow's milk, egg, soy, rice, wheat, barley, peanut and pollen proteins.  
     
     
         3 . The method of  claim 1  wherein the physiological disulfide is cystamine or oxidized glutathione.  
     
     
         4 . The method of  claim 3  wherein the amount of cystamine is about 0.4 μmoles to about 40 μmoles per mg of protein.  
     
     
         5 . The method of  claim 3  wherein the amount of oxidized glutathione is about 0.4 μmoles to about 40 μmoles per mg of protein.  
     
     
         6 . A method of decreasing the allergenicity of an allergenic food comprising: 
 (a) contacting an allergenic food having at least one allergenic protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin reductase and NADPH or effective for reducing at least one of said disulfide bonds in said protein;    (b) treating the food from step (a) with an amount of a physiological disulfide effective for preventing reoxidation of at least one of the reduced disulfide bonds in said protein in said food in step (a); and    (c) administering the treated food in step (b) to an animal allergic to said food, thereby decreasing the allergenic symptoms exhibited by said animal as compared to a control.    
     
     
         7 . The method of  claim 6  wherein said food contains milk or peanut.  
     
     
         8 . The method of  claim 6  wherein the physiological disulfide is cystamine or oxidized glutathione  
     
     
         9 . The method of  claim 8  wherein the amount of cystamine is about 0.4 μmoles to about 40 μmoles per mg of protein in said food.  
     
     
         10 . The method of  claim 8  wherein the amount of oxidized glutathione is about 0.4 μmoles to about 40 μmoles per mg of protein in said food.  
     
     
         11 . A method of decreasing the allergenicity of an allergenic protein comprising: 
 (a) heating a protein containing disulfide bonds at a temperature from about 40° C. to about 70° C. with an amount of dithiothreitol effective for reducing said protein; and    (b) administering the heated protein in step (a) to an animal, wherein said allergenic symptoms exhibited by said animal administered the protein from step (b) are decreased as compared to a control.    
     
     
         12 . The method of  claim 11  wherein the allergenic protein is selected from the group consisting of cow's milk, egg, soy, rice, wheat, barley, peanut and pollen proteins.  
     
     
         13 . A method of decreasing the allergenicity of an allergenic protein comprising: 
 (a) reducing a protein containing disulfide bonds with an amount of dithiothreitol effective for and at a temperature effective for reducing said protein;    (b) allowing the protein from step (a) to reoxidize; and    (c) administering the treated protein in step (b) to an animal, wherein said allergenic symptoms exhibited by said animal administered the protein from step (b) are decreased as compared to a control.    
     
     
         14 . A method of increasing the digestibility by pepsin of a protein comprising: 
 (a) reducing a protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin reductase and NADPH or an amount of dithiothreitol effective for and at a temperature effective for increasing the digestibility of said protein;    (b) reacting the protein from step (a) with an amount of a physiological disulfide effective for preventing reoxidation of at least one of the reduced disulfide bonds in said protein from step (a); and    (c) contacting the reacted protein in step (b) with pepsin, wherein the digestibility of the reacted protein is increased as compared to a control.    
     
     
         15 . The method of  claim 14  wherein the protein is selected from the group consisting of beef, cow's milk, egg, soy, rice, wheat, barley, peanut and pollen proteins.  
     
     
         16 . The method of  claim 14  wherein the physiological disulfide is cystamine or oxidized glutathione.  
     
     
         17 . A method of increasing the digestibility by pepsin of a food comprising: 
 (a) contacting a food having at least one protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin and NADPH effective for reducing at least one of said disulfide bonds in said protein;    (b) treating the food from step (a) with an amount of a physiological disulfide effective for preventing reoxidation of at least one of the reduced disulfide bonds in said protein in said food in step (a); and    (c) administering the treated food in step (b) to an animal, thereby increasing the digestibility of the food as measured by the symptoms exhibited by said animal as compared to a control.    
     
     
         18 . The method of  claim 17  wherein said food contains cow's milk, egg, soy, rice, wheat, barley or peanut.  
     
     
         19 . The method of  claim 17  wherein the physiological disulfide is cystamine or oxidized glutathione.  
     
     
         20 . A method of decreasing the allergenicity of an allergenic protein comprising: 
 (a) reducing a protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin reductase and NADPH or an amount of dithiothreitol or an amount of lipoic acid for reducing said protein;    (b) reacting the protein from step (a) with an amount of a physiological disulfide effective for stabilizing said protein from step (a); and    (c) administering the reacted protein in step (b) to an animal, wherein said allergenic symptoms exhibited by said animal administered the protein from step (b) are decreased as compared to a control.    
     
     
         21 . The method of  claim 20  wherein the allergenic protein is selected from the group consisting of cow's milk, egg, soy, rice, wheat, barley, peanut and pollen proteins.  
     
     
         22 . The method of  claim 20  wherein the physiological disulfide is cystamine or oxidized glutathione.  
     
     
         23 . A method of decreasing the allergenicity of an allergenic protein comprising: 
 (a) treating a protein containing disulfide bonds with an amount of thioredoxin, nicotinamide adenine dinucleotide phosphate-thioredoxin reductase and NADPH effective for reducing said protein;    (b) allowing the protein from step (a) to reoxidize; and    (c) administering the treated protein in step (b) to an animal, wherein said allergenic symptoms exhibited by said animal administered the protein from step (b) are decreased as compared to a control.    
     
     
         24 . A method of increasing the digestibility by pepsin of a food comprising: 
 (a) contacting a food having at least one protein containing disulfide bonds with an amount of lipoic acid effective for increasing the digestibility of the food;    (b) treating the food from step (a) with an amount of a physiological disulfide effective for maintaining the digestibility of said food in step (a); and    (c) administering the treated food in step (b) to an animal, thereby increasing the digestibility of the food as measured by the symptoms exhibited by said animal as compared to a control.    
     
     
         25 . The method of  claim 24  wherein said food contains cow's milk, egg, soy, rice, wheat, barley or peanut.  
     
     
         26 . The method of  claim 24  wherein the physiological disulfide is cystamine or oxidized glutathione