Methods for producing sialyloligosaccharides in a dairy source

Abstract

The present invention provides methods for producing sialyloligosaccharides in situ in dairy sources and cheese processing waste streams, prior to, during, or after processing of the dairy source during the cheese manufacturing process. The methods of the present invention use the catalytic activity of α(2-3) trans-sialidases to exploit the high concentrations of lactose and α(2-3) sialosides which naturally occur in dairy sources and cheese processing waste streams to drive the enzymatic synthesis of α(2-3) sialyllactose. α(2-3) sialyloligosaccharides produced according to these methods are additionally encompassed by the present invention. The invention also provides for recovery of the sialyloligosaccharides produced by these methods. The invention further provides a method for producing α(2-3) sialyllactose. The invention additionally provides a method of enriching for α(2-3) sialyllactose in milk using transgenic mammals that express an α(2-3) trans-sialidase transgene. The invention also provides for recovery of the sialyllactose contained in the milk produced by this transgenic mammal either before or after processing of the milk. Transgenic mammals containing an α(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue are also provided by the invention.

What is claimed is:  
     
         1 . A method for producing sialyloligosaccharides in a dairy source comprising: 
 (i) contacting a catalytic amount of at least one α(2-3) trans-sialidase with a dairy source to form a dairy/trans-sialidase mixture; and    (ii) incubating said dairy/trans-sialidase mixture under conditions suitable for α(2-3) trans-sialidase activity.    
     
     
         2 . The method of  claim 1  further comprising recovering sialyloligosaccharides from said incubated dairy/trans-sialidase mixture.  
     
     
         3 . The method of  claim 1  further comprising the steps of: 
 (iii) processing said dairy/trans-sialidase mixture for cheesemaking to form a cheese processing waste stream; and  
 (iv) recovering sialyloligosaccharides from said cheese processing waste stream.  
 
     
     
         4 . The method of  claim 1  wherein the α(2-3) trans-sialidase is a Kinetoplastid trans-sialidase.  
     
     
         5 . The method of  claim 1  wherein the α(2-3) trans-sialidase is encoded by a gene isolated from a species of the genera selected from the group consisting of Trypanosoma, Endotrypanum and Pneumocystis.  
     
     
         6 . The method of  claim 1  wherein the α(2-3) trans-sialidase is recombinantly produced.  
     
     
         7 . The method of  claim 1  wherein the dairy source comprises a member selected from the group consisting of milk, colostrum, and cheese processing mixture.  
     
     
         8 . The method of  claim 1  wherein the dairy source/trans-sialidase mixture is incubated for at least 1 hour.  
     
     
         9 . The method of  claim 1  wherein the dairy source/trans-sialidase mixture is incubated at a temperature of about 5° C. to about 45° C.  
     
     
         10 . The method of  claim 1  wherein the dairy source/trans-sialidase mixture has a pH of about 6 to about 8.  
     
     
         11 . The method of  claim 3  wherein the cheese processing waste stream comprises a member selected from the group consisting of: whole whey, demineralized whey permeate, a regeneration stream from demineralized whey permeate, whey permeate, crystallized lactose, spray dried lactose, whey powder, edible lactose and lactose.  
     
     
         12 . The method of  claim 2  wherein the recovering step comprises ultrafiltration of the incubated dairy source/trans-sialidase mixture to form an ultrafiltrate.  
     
     
         13 . The method of  claim 3  wherein the recovering step comprises ultrafiltration of the cheese processing waste stream to form an ultrafiltrate.  
     
     
         14 . The method of  claim 12  or  13  wherein the recovering step further comprises contacting said ultrafiltrate with an ion exchange resin.  
     
     
         15 . The method of  claim 14  wherein the ion exchange resin is an anion exchange resin.  
     
     
         16 . The method of  claim 14  wherein the ion exchange resin is a cation exchange resin.  
     
     
         17 . The method of  claim 2  wherein the recovering step comprises: 
 (a) contacting said incubated dairy source/trans-sialidase mixture of step (ii) with a solvent and extracting sialyloligosaccharides with said solvent to form a sialyloligosaccharide-containing solvent;  
 (b) separating said sialyloligosaccharide-containing solvent from said incubated dairy source/trans-sialidase mixture; and  
 (c) isolating said sialyloligosaccharides from said sialyloligosaccharide-containing solvent.  
 
     
     
         18 . The method of  claim 3  wherein the recovering step comprises: 
 (a) contacting said cheese processing waste stream with a solvent and extracting sialyloligosaccharides with said solvent to form a sialyloligosaccharide-containing solvent;  
 (b) separating said sialyloligosaccharide-containing solvent from said cheese processing waste stream; and  
 (c) isolating said sialyloligosaccharides from said sialyloligosaccharide-containing solvent.  
 
     
     
         19 . A method for producing sialyloligosaccharides in a cheese processing waste stream comprising: 
 (i) contacting a catalytic amount of at least one α(2-3) trans-sialidase with a cheese processing waste stream to form a waste stream/trans-sialidase mixture; and    (ii) incubating said waste stream/trans-sialidase mixture under conditions suitable for α(2-3) trans-sialidase activity.    
     
     
         20 . The method of  claim 19  further comprising recovering sialyloligosaccharides from said incubated waste stream/trans-sialidase mixture.  
     
     
         21 . The method of  claim 19  wherein said α(2-3) trans-sialidase is a Kinetoplastid trans-sialidase.  
     
     
         22 . The method of  claim 19  wherein said α(2-3) trans-sialidase is encoded by a gene isolated from a species of the genus Trypanosoma, Endotrypanum, or Pneumocystis.  
     
     
         23 . The method of  claim 19  wherein said α(2-3) trans-sialidase is recombinantly produced.  
     
     
         24 . The method of  claim 19  wherein the waste stream/trans-sialidase mixture is incubated for at least 1 hour.  
     
     
         25 . The method of  claim 19  wherein the waste stream/trans-sialidase mixture is incubated at a temperature of about 5° C. to about 45° C.  
     
     
         26 . The method of  claim 19  wherein the waste stream/trans-sialidase mixture has a pH of about 5 to about 8.  
     
     
         27 . The method of  claim 19  wherein the cheese processing waste stream comprises a member selected from the group consisting of: whole whey, demineralized whey permeate, the regeneration stream from demineralized whey permeate, whey permeate, and whey powder.  
     
     
         28 . The method of  claim 20  wherein the recovering step comprises ultrafiltration of the incubated waste stream/trans-sialidase mixture to form an ultrafiltrate.  
     
     
         29 . The method of  claim 28  wherein the recovering step further comprises contacting said ultrafiltrate with an ion exchange resin.  
     
     
         30 . The method of  claim 29  wherein the ion exchange resin is an anion exchange resin.  
     
     
         31 . The method of  claim 29  wherein the ion exchange resin is a cation exchange resin.  
     
     
         32 . The method of  claim 20  wherein the recovering step comprises: 
 (a) contacting said incubated waste stream/trans-sialidase mixture of step (ii) with a solvent and extracting said sialyloligosaccharides with said solvent to form a sialyloligosaccharide-containing solvent;  
 (b) separating said sialyloligosaccharide-containing solvent from said incubated waste stream/trans-sialidase mixture; and  
 (c) isolating said sialyloligosaccharides from said sialyloligosaccharide-containing solvent.  
 
     
     
         33 . The method of claim or  17 ,  18  or 32 wherein said solvent is selected from the group consisting of water, C[1-5] alcohol and a mixture thereof.  
     
     
         34 . The method of  claim 3  or  19  wherein said cheese processing waste stream is the mother liquor obtained by crystallizing lactose from cheese whey.  
     
     
         35 . The method of  claim 1  or  19  wherein exogenous α(2-3) sialyloligosaccharides are added during said incubating step.  
     
     
         36 . A method for producing α(2-3) sialyllactose comprising: 
 (i) contacting a catalytic amount of at least one α(2-3) trans-sialidase with lactose and an α(2-3) sialyloligosaccharide, in the absence of CMP-sialyltransferase, to form a mixture; and  
 (ii) incubating said mixture under conditions suitable for α(2-3) trans-sialidase activity.  
 
     
     
         37 . A transgenic mammal comprising an α(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue.  
     
     
         38 . The transgenic mammal of  claim 37  wherein said regulatory sequence is derived from a gene encoding a milk specific protein.  
     
     
         39 . The method of  claim 38  in wherein said regulatory sequence is derived from a gene encoding a protein selected from the group consisting of: whey acidic protein, β-lactoglobulin, α-lactalbumin, αs1-casein and β-casein.  
     
     
         40 . The transgenic mammal of  claim 37  wherein the α(2-3) trans-sialidase encoding sequence encodes a kinetoplastid trans-sialidase.  
     
     
         41 . The transgenic mammal of  claim 37  wherein said α(2-3) trans-sialidase encoding sequence hybridizes under high stringency conditions to an α(2-3) trans-sialidase gene selected from the group consisting of  Trypanosoma cruzi, Trypanosoma brucei , Endotrypanum spp. and  Pneumocystis carinii.    
     
     
         42 . The transgenic mammal of  claim 37  in which the transgenic mammal is a cow, sheep, pig or goat.  
     
     
         43 . A method for enriching for α(2-3) sialyllactose in milk comprising: 
 (i) introducing a transgene comprising an α(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue into the germline of a mammal to produce a transgenic mammal;  
 (ii) selecting a transgenic mammal demonstrating α(2-3) trans-sialidase activity; and  
 (iii) obtaining milk from the selected transgenic mammal.  
 
     
     
         44 . The method of  claim 43  further comprising recovering α(2-3) sialyllactose from said milk.  
     
     
         45 . An α(2-3) sialyllactose formed by the process comprising contacting a catalytic amount of at least one α(2-3) trans-sialidase with a dairy source to form a dairy/trans-sialidase mixture; and incubating said dairy/trans-sialidase mixture under conditions suitable for α(2-3) trans-sialidase activity.  
     
     
         46 . An α(2-3) sialyllactose formed by the process comprising contacting a catalytic amount of at least one α(2-3) trans-sialidase with a cheese processing waste stream to form a waste stream/trans-sialidase mixture; and incubating said waste stream/trans-sialidase mixture under conditions suitable for α(2-3) trans-sialidase activity.