Lactobacillus fermentum bacteria with antifungal activity

Abstract

The present invention relates to a bacterium of the species Lactobacillus fermentum having the ability to inhibit the growth of the fungus Penicillium solitum deposited under the accession No.: DSM32093 or the growth of the fungus Penicillium brevicompactum deposited under the accession No.: DSM32094 by at least 50%. The inhibitory activity can be determined in an assay comprising: (1) preparing a fermented milk product by: (a) inoculating a milk with the Lactobacillus fermentum in a concentration of at least 10 7 CFU/g and with a starter culture, (b) fermenting until a p H of 4.6 is reached, and (c) solidifying the fermented milk by the addition of agar; (2) generating at least one spot of the P. solitum or the P. brevicompactum on the agar solidified fermented milk with a concentration of 500 spores/spot and incubating the 1 same for 7 days at 25° C.; (3) determining the percent inhibition by determining the largest diameter of the colony formed by growth of the P. solitum or P. brevicompactum and expressing the diameter as percent of the largest diameter formed under the same conditions but in the absence of the Lactobacillus fermentum strain. The invention further relates to compositions comprising the bacterium, methods for producing fermented milk products using the bacterium and the products thus obtained.

1 - 23 . (canceled) 
     
     
         24 . A method of producing a fermented milk product, comprising:
 adding a  Lactobacillus fermentum  bacterium to milk or to a milk product to obtain a mixture, and   fermenting the mixture at a temperature between about 22° C. and about 43° C. until a pH of less than 4.6 is reached,   wherein the  Lactobacillus fermentum  bacterium has the ability to inhibit the growth of the fungus  Penicillium solitum  deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM32093 or the fungus  Penicillium brevicompactum  deposited with the DSMZ under accession number DSM32094 by at least 50%.   
     
     
         25 . The method of  claim 24 , wherein the ability of the  Lactobacillus fermentum  bacterium to inhibit the growth of the  P. solitum  or  P. brevicompactum  fungus by at least 50% is determined in an assay comprising:
 preparing a fermented milk product by:
 (a) inoculating a milk with the  Lactobacillus fermentum  at a concentration of at least 10 7  CFU/g and with a starter culture, 
 (b) fermenting the inoculated milk until a pH of 4.6 is reached, and 
 (c) solidifying the fermented milk by adding agar; 
   generating at least one spot of the  P. solitum  or  P. brevicompactum  fungus on the agar-solidified fermented milk with a concentration of 500 spores/spot and incubating the same for 7 days at 25° C.;   determining the percent inhibition by determining the largest diameter of the colony formed by growth of the  P. solitum  or  P. brevicompactum  fungus and expressing the diameter as percent of the largest diameter formed under the same conditions but in the absence of the  Lactobacillus fermentum.      
     
     
         26 . The method of  claim 24 , wherein the  Lactobacillus fermentum  secretes diacetyl in a concentration range of 0 to 5 ppm. 
     
     
         27 . The method of  claim 28 , wherein the ability of the  Lactobacillus fermentum  bacterium to secrete diacetyl is determined in an assay comprising:
 preparing a fermented milk product by:
 (a) inoculating a milk with the  Lactobacillus fermentum  at a concentration of at least 10 7  CFU/g and with a starter culture, 
 (b) fermenting the inoculated milk until a pH of 4.6 is reached, and 
 (c) storing the fermented milk product at a temperature of 7±1° C. for 14 days; 
   adding 200 μl of 4N H 2 SO 4  to 1 g of the stored fermented milk product and determining the concentration of diacetyl by static head space gas chromatography.   
     
     
         28 . The method of  claim 24 , wherein the  Lactobacillus fermentum  bacterium is selected from:
 (a) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32084;   (b) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32085;   (c) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32086;   (d) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32087;   (e) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32088;   (f) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32089;   (g) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32090;   (h) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32091;   (i) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32096;   (j) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM22584; or   (k) a mutant strain obtained from one of (a) to (j), wherein the mutant strain has the ability to inhibit the growth of the fungus  Penicillium solitum  deposited with the DSMZ under accession number DSM32093 or the fungus  Penicillium brevicompactum  deposited with the DSMZ under accession number DSM32094 by at least 50% as determined in an assay comprising:   preparing a fermented milk product by:
 (a) inoculating a milk with the  Lactobacillus fermentum  mutant strain at a concentration of at least 10 7  CFU/g and with a starter culture, 
 (b) fermenting the inoculated milk until a pH of 4.6 is reached, and 
 (c) solidifying the fermented milk by adding agar; 
   generating at least one spot of the  P. solitum  or  P. brevicompactum  fungus on the agar-solidified fermented milk with a concentration of 500 spores/spot and incubating the same for 7 days at 25° C.;   determining the percent inhibition by determining the largest diameter of the colony formed by growth of the  P. solitum  or  P. brevicompactum  fungus and expressing the diameter as percent of the largest diameter formed under the same conditions but in the absence of the  Lactobacillus fermentum.      
     
     
         29 . The method of  claim 24 , wherein the method comprises inoculating the milk with the  Lactobacillus fermentum  at a concentration of at least 10 7  CFU/g. 
     
     
         30 . The method of  claim 24 , wherein the method comprises fermenting the mixture
 (a) such that the concentration of the  Lactobacillus fermentum  bacterium is at least 1×10 6  cfu/g in the fermented milk product at the termination of fermentation; and/or   (b) such that the concentration of the  Lactobacillus fermentum  bacterium is at least 1×10 5  cfu/cm 2  on the surface of the fermented milk product.   
     
     
         31 . The method of  claim 37 , wherein the method further comprises adding to the milk or to the milk product at least one further bacterium selected from:
 (a)  Lactobacillus rhamnosus  bacterium of strain CHCC15860 as deposited with the DSMZ under accession No. DSM32092;   (b)  Lactobacillus rhamnosus  bacterium of strain CHCC5366 as deposited with the DSMZ under accession No. DSM23035;   (c)  Lactobacillus rhamnosus  bacterium of strain CHCC12697 as deposited with the DSMZ under accession No. DSM24616;   (d)  Lactobacillus paracasei  bacterium of strain CHCC12777 as deposited with the DSMZ under accession No. DSM24651; and   (e)  Lactobacillus paracasei  bacterium of strain CHCC14676 as deposited with the DSMZ under accession No. DSM25612.   
     
     
         32 . A fermentate obtained by the method of  claim 24 . 
     
     
         33 . The fermentate of  claim 32 , wherein the fermentate is a liquid. 
     
     
         34 . A method of producing a food, feed, or pharmaceutical product, comprising adding a fermentate according to  claim 32  to a food, feed, or pharmaceutical product. 
     
     
         35 . A food, feed, or pharmaceutical product obtained by a method of  claim 34 . 
     
     
         36 . A  Lactobacillus fermentum  bacterium having the ability to inhibit the growth of the fungus  Penicillium solitum  deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM32093 or the fungus  Penicillium brevicompactum  deposited with the DSMZ under accession number DSM32094 by at least 50%. 
     
     
         37 . The  Lactobacillus fermentum  bacterium of  claim 36 , selected from:
 (a) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32084;   (b) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32085;   (c) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32086;   (d) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32087;   (e) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32088;   (f) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32089;   (g) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32090;   (h) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32091;   (i) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM32096;   (j) the  Lactobacillus fermentum  strain deposited with the DSMZ as DSM22584; or   (k) a mutant strain obtained from one of (a) to (j), wherein the mutant strain has the ability to inhibit the growth of the fungus  Penicillium solitum  deposited with the DSMZ under accession number DSM32093 or the fungus  Penicillium brevicompactum  deposited with the DSMZ under accession number DSM32094 by at least 50%.   
     
     
         38 . A composition comprising the  Lactobacillus fermentum  bacterium of  claim 36 , optionally further comprising at least one cryoprotective compound. 
     
     
         39 . The composition of  claim 38 , wherein the composition is a solid frozen or freeze dried starter culture comprising lactic acid bacteria at a concentration of at least 10 9  colony forming units per g of frozen material. 
     
     
         40 . A food, feed, or pharmaceutical product comprising a  Lactobacillus fermentum  bacterium according to  claim 36 . 
     
     
         41 . The food, feed, or pharmaceutical product of  claim 40 , further comprising one or more of:
 (a) one or more further bacterium selected from one or more of the following genera  Lactococcus  spp.,  Streptococcus  spp.,  Lactobacillus  spp.,  Leuconostoc  spp.,  Pseudoleuconostoc  spp.,  Pediococcus  spp.,  Brevibacterium  spp. and  Enterococcus  spp.;   (b)  Lactobacillus rhamnosus  bacterium of strain CHCC15860 deposited with the DSMZ under accession No. DSM32092;   (c)  Lactobacillus rhamnosus  bacterium of strain CHCC5366 deposited with the DSMZ under accession No. DSM23035;   (d)  Lactobacillus rhamnosus  bacterium of strain CHCC12697 deposited with the DSMZ under accession No. DSM24616;   (e)  Lactobacillus paracasei  bacterium of strain CHCC12777 deposited with the DSMZ under accession No. DSM24651; and   (f)  Lactobacillus paracasei  bacterium of strain CHCC14676 deposited with the DSMZ under accession No. DSM25612.