Method of improving the efficacy of lactic acid bacterial starter cultures and improved starter culture compositions

Abstract

Methods of enhancing the growth rate and/or controlling the metabolic activity of lactic acid bacteria and of improving the shelf life and/or the quality of an edible product using lactic acid bacterial organisms which are defective in their pyruvate metabolism. There is also provided starter culture compositions comprising such defective lactic acid bacteria as helper organisms and lactic acid bacterial starter culture strains. Useful helper organisms are Lactococcus strains which are defective with respect to pyruvate formate lyase (Pfl) and/or lactate dehydrogenase (Ldh) activity. The helper organisms may overexpress a gene coding for a NAD + regenerating enzyme such as NADH oxidase encoded by nox gene.

1 - 17 . (cancelled)  
     
     
         18 . A method of improving the shelf life and/or the quality of an edible product comprising adding to the product a lactic acid bacterial strain that is defective in its pyruvate metabolism.  
     
     
         19 . A method according to  claim 18  wherein the lactic acid bacterial strain essentially does not produce lactic acid.  
     
     
         20 . A method according to  claim 18  wherein the lactic acid bacterial strain is defective in its ability to express at least one enzyme selected from the group consisting or pyruvate formate lyase, pyruvate dehydrogenase, lactate dehydrogenase, acetolactate synthetase, second acetolactate synthetase, acetolactate decarboxylase and diacetyl reductase.  
     
     
         21 . A method according to  claim 18  wherein the edible product is selected from the group consisting of milk, flour dough, meat, wine and a plant material.  
     
     
         22 . A method according to  claim 21  wherein the edible product is non-pasteurized milk.  
     
     
         23 . A method according to  claim 18  wherein the lactic acid bacterial strain is added to the product at its site of production.  
     
     
         24 . A method according to  claim 23  wherein the lactic acid bacterial strain is added to raw milk following milking.  
     
     
         25 . A starter culture composition comprising a lactic acid bacterium and a lactic acid bacterial helper organism that is defective in its pyruvate metabolism, said helper organism being capable of enhancing the growth rate and/or controlling the metabolic activity of the lactic acid bacterium.  
     
     
         26 . A composition according to  claim 25  wherein the helper organism essentially does not produce lactic acid.  
     
     
         27 . A composition according to  claim 25  wherein the helper organism is defective in its ability to produce at least one enzyme selected from the group consisting of pyruvate formate lyase, pyruvate dehydrogenase, lactate dehydrogenase, acetolactate synthetase, second acetolactate synthetase, acetolactate decarboxylase and diacetyl reductase.  
     
     
         28 . A composition according to  claim 25  wherein the helper organism is  Lactococcus lactis  subs.  lactis  strain DN223 deposited under the accession No. DSM 11036.  
     
     
         29 . A composition according to  claim 25  wherein the helper organism is  Lactococcus lactis  subs.  lactis  strain DN224 deposited under the accession No. DSM 11037.  
     
     
         30 . A composition according to  claim 25  wherein a gene coding for an enzyme that is capable of regenerating NAD +  is overexpressed in the helper organism.  
     
     
         31 . A composition according to  claim 30  wherein the enzyme catalyses the reduction of O 2  to H 2 O or H 2 O 2 .  
     
     
         32 . A composition according to  claim 31  wherein the enzyme is NADH:H 2 O oxidase including the enzyme having the sequence SEQ ID NO:2.  
     
     
         33 . A composition according to  claim 30  wherein the helper organism is an Ldh −  strain.  
     
     
         34 . A composition according to  claim 25  that comprises two or more different lactic acid bacterial strains.  
     
     
         35 . An isolated or non-naturally occurring lactic acid bacterium that is defective in its ability to express lactic acid dehydrogenase and in which a gene encoding a protein capable of regenerating NAD +  is overexpressed, wherein the gene capable of regenerating NAD +  that is overexpressed codes for an enzyme catalysing the reduction of O 2  to H 2 O or H 2 O 2 .  
     
     
         36 . A bacterium according to  claim 35  which is defective in its ability to express at least one further enzyme selected from the group consisting of pyruvate formate lyase, pyruvate dehydrogenase, acetolactate synthetase, second acetolactate synthetase, acetolactate decarboxylase and diacetyl reductase.  
     
     
         37 . A bacterium according to  claim 37  wherein the gene capable of regenerating NAD +  that is overexpressed codes for an enzyme catalyzing the reduction of O 2  to H 2 O or H 2 O 2 .  
     
     
         38 . A bacterium according to  claim 35  wherein the enzyme is NADH:H 2 O oxidase including the enzyme having the sequence SEQ ID NO:2.  
     
     
         39 . An isolated DNA fragment derived from a lactic acid bacterium comprising a gene coding for a polypeptide having NADH:H 2 O oxidase activity.  
     
     
         40 . A DNA fragment according to  claim 39  which is selected from the group consisting of the sequence shown in SEQ NO ID:1 and a variant or derivative hereof which is at least 50% identical with said sequence.  
     
     
         41 . A recombinant DNA molecule comprising the DNA fragment of  claim 39 .  
     
     
         42 . A recombinant DNA molecule comprising the DNA fragment of  claim 40 .  
     
     
         43 . The bacterium of  claim 35  which is from the group consisting of bacteria of the genera  Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Propionibacterium,  and  Bifidobacterium.    
     
     
         44 . The bacterium of  claim 35  which is a strain of  Lactococcus lactis, Lactobacillus bulgaricus, Streptococcus thermophilus,  or  Lactobacillus delbruecki, Leuconostoc mesenteroides.    
     
     
         45 . The bacterium of  claim 35  which is a strain of  Lactococcus lactis.