Method for the prevention or reduction of haze in beverages

Abstract

The invention relates to a method for the prevention or reduction of haze in a beverage by the addition of an prolyl-specific endoprotease and to new beverages obtainable by the method according to the invention. It also relates to new endoproteases. Sequence information of a genomic DNA, cDNA as well as protein sequences.

1 . A method to prevent or reduce haze in a beverage, which method comprises adding a proline-specific endoprotease to the beverage. 
     
     
         2 . The method of  claim 1 , wherein said endoprotease has maximum prolyl-specific activity at a pH corresponding to the pH of the beverage to which it is added. 
     
     
         3 . The method of  claim 1 , wherein the beverage contains at least one protein. 
     
     
         4 . The method of  claim 3 , wherein the beverage contains polyphenols. 
     
     
         5 . The method of  claim 1 , wherein the beverage has a pH value below 7. 
     
     
         6 . The method of  claim 3 , wherein at least 150 milli-units of proline-specific endoprotease activity, as determined using Z-Gly-Pro-pNA as a substrate, is added to the beverage per gram protein in the beverage. 
     
     
         7 . The method of  claim 6 , wherein at least 500 milli-units of proline-specific endoprotease activity, as determined using Z-Gly-Pro-pNA as a substrate, is added to the beverage per gram protein in the beverage. 
     
     
         8 . The method of  claim 7 , wherein at least 1 unit of proline-specific endoprotease activity, as determined using Z-Gly-Pro-pNA as a substrate, is added to the beverage per gram protein in the beverage. 
     
     
         9 . The method of  claim 1 , wherein the beverage is beer. 
     
     
         10 . The method of  claim 1 , wherein the beverage is wine. 
     
     
         11 . The method of  claim 1 , wherein the beverage is fruit juice. 
     
     
         12 . The method of  claim 9 , wherein the proline-specific endoprotease is added to a mash. 
     
     
         13 . The method of  claim 9 , wherein the proline-specific endoprotease is added to a beer before haze is formed. 
     
     
         14 . The method of  claim 9 , wherein the proline-specific endoprotease is added to a fermented beer after haze has been formed. 
     
     
         15 . The method of  claim 10 , wherein the proline-specific endoprotease is added to a fermented wine. 
     
     
         16 . The method of  claim 1 , wherein the proline-specific endoprotease is isolated or purified. 
     
     
         17 . An isolated polypeptide having proline specific endoprotease activity, selected from the group consisting of:
 (a) polypeptide which has an amino acid sequence which has at least 40% overall amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:7 or a fragment thereof;   (b) a polypeptide which is encoded by a polynucleotide which hybridizes under low stringency conditions with the complement of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:6 or a fragment thereof which is at least 80% identical over 60 nucleotides.   
     
     
         18 . The polypeptide of  claim 17  which has an amino acid sequence which has at least 50% identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:7. 
     
     
         19 . The polypeptide of  claim 17 , comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:7. 
     
     
         20 . The polypeptide of  claim 17 , which is encoded by a polynucleotide that hybridizes under low stringency conditions to a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:6. 
     
     
         21 . The polypeptide of  claim 17 , which is obtainable from a fungus, preferably an  Aspergillus , more preferably from  Aspergillus niger.    
     
     
         22 . An isolated polynucleotide comprising a nucleic acid sequence which encodes the polypeptide of  claim 17 . 
     
     
         23 . A nucleic acid construct comprising the polynucleotide of  claim 22  operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host. 
     
     
         24 . A recombinant expression vector comprising the nucleic acid construct of  claim 23 . 
     
     
         25 . Recombinant host cells comprising the nucleic acid construct of  claim 23 . 
     
     
         26 . A method to produce a polypeptide having proline-specific endoprotease activity comprising cultivating the cells of  claim 25  and recovering the polypeptide. 
     
     
         27 . A polypeptide produced by the method of  claim 26 . 
     
     
         28 . The method of  claim 1 , wherein said endoprotease is added as a filtrate obtained from a fermentation broth of cells which are modified to contain a recombinant nucleic acid construct for the production of said endoprotease. 
     
     
         29 . The method of  claim 1 , wherein said endoprotease is selected from the group consisting of:
 (a) a polypeptide which has an amino acid sequence which has at least 40% amino acid sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7 or a fragment thereof; and   (b) a polypeptide which is encoded by a polynucleotide which hybridizes under low stringency conditions with the complement of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 6 or a fragment thereof which is at least 80% identical over 60 nucleotides.   
     
     
         30 . A beverage obtainable by a method of  claim 1 . 
     
     
         31 . The beverage of  claim 30  which is beer. 
     
     
         32 . The beverage of  claim 30  which is wine. 
     
     
         33 . The beverage of  claim 30  which is fruit juice.