Lactobacillus fermentum bacteria with antifungal activity

Patent metadata

Field	Value
Publication number	US-2018249727-A1
Application number	US-201615755023-A
Country	US
Kind code	A1
Filing date	Aug 30, 2016
Priority date	Aug 31, 2015
Publication date	Sep 6, 2018
Grant date	—

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.

First claim

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 fung

Assignees

• Chr Hansen As

Inventors

• Nielsen Cecilie Lykke Marvig
• Hornbaek Tina
• Rasmussen Pia
• Poulsen Lone
• Eckhardt Thomas
• Oeregaard Gunnar
• Moghadam Elahe Ghanei

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P31/10

  Antimycotics · CPC title

• A23V2002/00

  Food compositions, function of food ingredients or processes for food or foodstuffs · CPC title

• A23L33/135

  Bacteria or derivatives thereof, e.g. probiotics · CPC title

• A23C9/1234 Primary

  characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp. · CPC title