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.sup.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.

Claims

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.sup.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.sup.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 71 C. for 14 days; adding 200 l of 4N H.sub.2SO.sub.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.sup.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.sup.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 110.sup.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 110.sup.5 cfu/cm.sup.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.sup.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.

Description

DESCRIPTION OF THE FIGURES

[0125] FIG. 1: Growth of molds on plates prepared from milk fermented with a starter culture alone (reference, first column), together with FreshQ4 (second column), together with Holdbac YM-C Plus (third column) or together with Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 71 C. for 19 days (top row) and 27 days (bottom row). The target contaminants were added in concentrations of 500 spores/spot: (A) P. carneum, (B) P. paneum and (C) P. roqueforti.

[0126] FIG. 2: Growth of molds on plates prepared from milk fermented with a starter culture alone (reference, first column), together with FreshQ4 (second column), together with Holdbac YM-C Plus (third column) or together with Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 251 C. for 6 days (top row) and 11 days (bottom row). The target contaminants were added in concentrations of 500 spores/spot: (A) P. carneum, (B) P. paneum and (C) P. roqueforti.

[0127] FIG. 3: Growth of molds on plates prepared from milk fermented with a starter culture alone (reference, first column), together with FreshQ4 (second column), together with Holdbac YM-C Plus (third column) or together with Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 71 C. for 19 days (top row) and 27 days (bottom row). The target contaminants were added in concentrations of 500 spores/spot: (A) P. brevicompactum, (B) P. crustosum and (C) P. solitum.

[0128] FIG. 4: Growth of molds on plates prepared from milk fermented with a starter culture alone (reference, first column), together with FreshQ4 (second column), together with Holdbac YM-C Plus (third column) or together with Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 251 C. for 6 days (top row) and 11 days (bottom row). The target contaminants were added in concentrations of 500 spores/spot: (A) P. brevicompactum (DSM32093), (B) P. crustosum and (C) P. solitum (DSM32093).

[0129] FIG. 5: Growth of yeast on plates prepared from milk fermented with a starter culture alone (reference, first column), together with FreshQ4 (second column), together with Holdbac YM-C Plus (third column) or together with Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 71 C. for 11 days (Top row) or at 251 C. for 5 days (bottom row). The target contaminants were added in concentrations of 110.sup.3 cfu/spot (top row), 110.sup.2 cfu/spot (middle row) and 110.sup.1 cfu/spot (bottom row): (A) Torulaspora delbrueckii, (B) Cryptococcus hansenii, (C) Debaryomyces hansenii and (D) Yarrowia lipolytica.

[0130] FIG. 6: Diacetyl levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture alone (Reference), or starter cultures in combination with FreshQ4, Holdbac YM-C Plus or Lb. fermentum CHCC14591. LOD: Limit of detection. LOQ: Limit of quantification.

[0131] FIG. 7: Growth of molds on plates prepared from milk fermented with a starter culture alone (reference, first column), together Lb. rhamosus CHCC15860 (second column), together with Lb. fermentum CHCC14591 (third column) or together with a combination of Lb. rhamnosus CHCC15860 and Lb. fermentum CHCC14591 (fourth column). The plates had been incubated at 251 C. for 5 days. The target contaminants were added in concentrations of 500 spores/spot: (A) P. carneum, (B) P. paneum and (C) P. roqueforti.

[0132] FIG. 8: Growth of molds on plates prepared from milk fermented with (1) a starter culture alone, together with (2) Lb. fermentum CHCC12798, (3) Lb. fermentum CHCC12797, (4) Lb. fermentum CHCC14591, (5) Lb. fermentum CHCC14588, (6) Lb. fermentum CHCC15844, (7) Lb. fermentum CHCC15865, (8) Lb. fermentum CHCC15847, (9) Lb. fermentum CHCC15848, (10) Lb. fermentum CHCC15926, and (11) Lb. fermentum CHCC2008. The plates had been incubated at 251 C. for 5 days (left column of photos) or 7 days (right column of photos). The target contaminants were added in concentrations of 500 spores/spot: (A) P. brevicompactum (DSM32094), (B) P. crustosum and (C) P. solitum (DSM32093).

[0133] FIG. 9: Diacetyl levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture alone (Reference), or starter cultures in combination with FreshQ4, Holdbac YM-C Plus or Lb. fermentum strains. LOD: Limit of detection. LOQ: Limit of quantification.

[0134] FIG. 10: pH development in fermented milk products over time when stored at (A) 71 C. and (B) 251 C. for 28 days. The products are fermented with starter culture only, Reference, or starter culture in combination with FreshQ4, Holdbac YM-C Plus or Lb. fermentum strains.

[0135] FIG. 11: pH development in fermented milk products over time when stored at (A) 71 C. or (B) 251 C. for 21 days. The products are fermented with starter culture only (Reference, ) or starter culture in combination with FreshQ4 (), Holdbac YM-C Plus () or Lb. fermentum CHCC14591 ().

[0136] FIG. 12: Acetaldehyde levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture alone (Reference), or starter cultures in combination with Lb. fermentum strains. LOD: Limit of detection. LOQ: Limit of quantification.

[0137] FIG. 13: Acetaldehyde levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture alone (Reference), or starter cultures in combination with Lb. fermentum CHCC14591. LOD: Limit of detection. LOQ: Limit of quantification.

[0138] FIG. 14: Acidification curves of four commercial starter cultures, FD-DVS YF-L812, F-DVS YF-L901, F-DVS YoFlex Mild 2.0 and F-DVS CH-1, grown in milk (1% fat and 4.5% protein) at 43 C.

[0139] FIG. 15: Post-acidification curves of yoghurt fermented with one of four commercial starter cultures, FD-DVS YF-L812, F-DVS YF-L901, F-DVS YoFlex Mild 2.0 and F-DVS CH-1 after storage at 6 C. for up to 43 days.

[0140] FIG. 16: Growth of molds on plates prepared from milk fermented with (1) a starter culture alone, FD-DVS YF-L812, together with (2) Lb. fermentum CHCC12798, (3) Lb. fermentum CHCC12797, (4) Lb. fermentum CHCC14591, (5) Lb. fermentum CHCC14588, (6) Lb. fermentum CHCC15844, (7) Lb. fermentum CHCC15865, (8) Lb. fermentum CHCC15847, (9) Lb. fermentum CHCC15926, and (10) Lb. fermentum CHCC2008. The plates had been incubated at 251 C. for 4 days (left column of photos) or 8 days (right column of photos). The target contaminant, P. brevicompactum (DSM32094), was added in concentrations of 500 spores/spot.

[0141] FIG. 17: Growth of molds on plates prepared from milk fermented with (1) a starter culture alone, F-DVS CH-1, together with (2) Lb. fermentum CHCC12798, (3) Lb. fermentum CHCC12797, (4) Lb. fermentum CHCC14591, (5) Lb. fermentum CHCC14588, (6) Lb. fermentum CHCC15844, (7) Lb. fermentum CHCC15865, (8) Lb. fermentum CHCC15847, (9) Lb. fermentum CHCC15926, and (10) Lb. fermentum CHCC2008. The plates had been incubated at 251 C. for 4 days (left column of photos) or 8 days (right column of photos). The target contaminant, P. brevicompactum (DSM32094), was added in concentrations of 500 spores/spot.

[0142] FIG. 18: Growth of molds on plates prepared from milk fermented with (1) a starter culture alone, FD-DVS YF-L812, together with (2) Lb. fermentum CHCC12798, (3) Lb. fermentum CHCC12797, (4) Lb. fermentum CHCC14591, (5) Lb. fermentum CHCC14588, (6) Lb. fermentum CHCC15844, (7) Lb. fermentum CHCC15865, (8) Lb. fermentum CHCC15847, (9) Lb. fermentum CHCC15926, and (10) Lb. fermentum CHCC2008. The plates had been incubated at 251 C. for 4 days (left column of photos) or 8 days (right column of photos). The target contaminant, P. solitum (DSM32093), was added in concentrations of 500 spores/spot.

[0143] FIG. 19: Growth of molds on plates prepared from milk fermented with (1) a starter culture alone, F-DVS CH-1, together with (2) Lb. fermentum CHCC12798, (3) Lb. fermentum CHCC12797, (4) Lb. fermentum CHCC14591, (5) Lb. fermentum CHCC14588, (6) Lb. fermentum CHCC15844, (7) Lb. fermentum CHCC15865, (8) Lb. fermentum CHCC15847, (9) Lb. fermentum CHCC15926, and (10) Lb. fermentum CHCC2008. The plates had been incubated at 251 C. for 4 days (left column of photos) or 8 days (right column of photos). The target contaminant, P. solitum (DSM32093), was added in concentrations of 500 spores/spot.

[0144] FIG. 20: Diacetyl levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture, FD DVS YF-L812 or F-DVS CH-1, alone (Reference), or starter cultures in combination one of the nine Lb. fermentum strains. LOD: Limit of detection. LOQ: Limit of quantification.

[0145] FIG. 21: Acetaldehyde levels after storage at 71 C. for 14 days in fermented milk products fermented with starter culture, FD DVS YF-L812 or F-DVS CH-1, alone (Reference), or starter cultures in combination one of the nine Lb. fermentum strains. LOD: Limit of detection. LOQ: Limit of quantification.

EXAMPLE 1

[0146] Semi-Quantitative Analysis of the Inhibitory Effect of Lb. Fermentum CHCC14591 Against Different Yeast and Mold Contaminants and Diacetyl Production

[0147] For the semi-quantitative analysis of the inhibitory effect of Lb. fermentum CHCC14591, an agar-assay was used, resembling the manufacturing process and product of yoghurt:

[0148] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS Mild 2.0) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. One bottle was inoculated with Lb. fermentum CHCC14591 in total concentration of 210.sup.7 CFU/g, two bottles were inoculated with either of two commercial bioprotective cultures (FreshQ4 and Holdbac YM-C Plus) in recommended dosages (100 U/T and 20 DCU/100 L for FreshQ4 and Holdbac YM-C Plus, respectively), and one bottle was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. Then the fermented milk was warmed to a temperature of 40 C. and added 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60 C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.

[0149] Spore suspension of six different molds were spotted in concentration of 500 spores/spot; Penicillium brevicompactum (DSM32094), P. crustosum, P. solitum (DSM32093), P. carneum, P. paneum and P. roqueforti. Three molds were spotted on each plate. Four yeasts including Torulaspora delbrueckii, Cryptococcus hansenii, Debaryomyces hansenii and Yarrowia lipolytica were spotted in concentrations of 10.sup.4, 10.sup.3 and 10.sup.2 CFU/spot. Plates were incubated at 71 C. and 251 C. and regularly examined for the growth of molds and yeast.

[0150] On day 14 samples were analysed for diacetyl by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0151] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0152] HS-software: HSControl v.2.00, Perkin Elmer. [0153] GC: Autosystem XL, Perkin Elmer. [0154] GC-software: Turbochrom navigator, Perkin Elmer. [0155] Column: HP-FFAP 25 m0.20 mm0.33 pin, Agilent Technologies

[0156] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0157] Results of the agar-assay are presented in FIGS. 1-5, showing that all of the tested molds grew very well on the agar plates made from milk fermented only with the starter culture (reference). However, when Lb. fermentum CHCC14591 was present during milk fermentation the resulting plates inhibited growth of all of the Penicillium species tested. The level of inhibition was comparable or even higher than the inhibition observed for the two commercial bioprotective cultures. FIG. 3 shows that all of the tested yeasts grew on the agar plates made from milk fermented only with the starter culture (reference). When Lb. fermentum CHCC14591 was present during milk fermentation the resulting plates prevented growth of C. fragiola and Y. lipolytica added in all concentrations. Growth of T. delbrueckii and D. hansenii was inhibited in the lower concentrations when Lb. fermentum CHCC14591 was present during milk fermentation. The level of inhibition was comparable or even higher than the inhibition observed for the two commercial bioprotective cultures.

[0158] The effect on diacetyl production is illustrated in FIG. 6, showing that addition of Lb. fermentum CHCC14591 during milk fermentation produce minimal amounts of diacetyl when compared to commercially available alternatives.

EXAMPLE 2

[0159] Semi-Quantitative Analysis of the Inhibitory Effect of Lb. Fermentum CHCC14591 in Combination with Lb. Rhamnosus CHCC15860 Against Different Mold Contaminants

[0160] For the semi-quantitative analysis of the inhibitory effect of a combination of Lb. fermentum CHCC14591 and Lb. rhamnosus CHCC15860, an agar-assay was used, resembling the manufacturing process and product of yoghurt:

[0161] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS Mild 2.0) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. One bottle was inoculated with Lb. rhamnosus CHCC15860 in total concentration of 110.sup.7 CFU/g, one bottle was inoculated with Lb. fermentum CHCC14591 in total concentration of 110.sup.7 CFU/g, one bottle was inoculated with Lb. fermentum CHCC14591 and Lb. rhamnosus CHCC15860 each in concentration of 510.sup.6 CFU/g, and one bottle was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. Then the fermented milk was warmed to a temperature of 40 C. and added 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60 C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.

[0162] Spore suspension of three different molds were spotted in concentration of 500 spores/spot; P. carneum, P. paneum and P. roqueforti. Three molds were spotted on each plate. Plates were incubated at 251 C. and regularly examined for the growth of molds.

[0163] Results of the agar-assay are presented in FIG. 7, showing that all of the tested molds grew very well on the agar plates made from milk fermented only with the starter culture (reference). However, when Lb. rhamnosus 15860 or the Lb. fermentum CHCC14591 were present during milk fermentation the resulting plates inhibited growth of the three Penicillium species tested. Furthermore, a synergistic inhibitory effect was found when Lb. rhamnosus CHCC15860 and Lb. fermentum CHCC14591 were used in combination compared to the inhibitory effect of each strain used alone.

EXAMPLE 3

[0164] Semi-Quantitative Analysis of the Inhibitory Effect of Ten Lb. Fermentum Strains Against Different Mold Contaminants

[0165] For the semi-quantitative analysis of the inhibitory effect of ten Lb. fermentum strains, an agar-assay was used, resembling the manufacturing process and product of yoghurt:

[0166] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS YF-L901) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. Ten bottles were inoculated with the Lb. fermentum strains in concentrations of 110.sup.7 CFU/g and one bottle was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. Then the fermented milk was warmed to a temperature of 40 C. and added 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60 C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.

[0167] The tested Lb. fermentum strains were: Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15848, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008.

[0168] Spore suspension of six different molds were spotted in concentration of 500 spores/spot; Penicillium brevicompactum (DSM32094), P. crustosum, P. solitum (DSM32093), P. carneum, P. paneum and P. roqueforti. Three molds were spotted on each plate. Plates were incubated at 251 C. and regularly examined for the growth of molds.

[0169] On day 14 samples were analysed for diacetyl by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0170] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0171] HS-software: HSControl v.2.00, Perkin Elmer. [0172] GC: Autosystem XL, Perkin Elmer. [0173] GC-software: Turbochrom navigator, Perkin Elmer. [0174] Column: HP-FFAP 25 m0.20 mm0.33 pin, Agilent Technologies

[0175] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0176] To monitor the effect on post acidification, the eleven fermented milk samples (starter culture alone and starter culture in combination with the ten Lb. fermentum strains) were stored at 71 C. and 251 C. for 28 days and pH was measured on day 1, 7, 14, 21 and 28.

[0177] Results of the agar-assay are presented in FIG. 8, showing that all of the tested molds grew very well on the agar plates made from milk fermented only with the starter culture (reference). For P. brevicompactum (DSM32094) and P. solitum (DSM32093) a large delay in the growth was observed for all of the Lb. fermentum strains when present during milk fermentation. For the remaining molds tested a varying delay in the growth was observed when the Lb. fermentum strains were present during milk fermentation. Bacteria of the strain Lb. fermentum CHCC14591 achieved significant inhibition of essentially all molds tested in this assay.

[0178] The effect on diacetyl production is illustrated in FIG. 9, showing that each of the anti-fungal strains Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15848, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008 secretes either none or very little diacetyl.

[0179] The effects on post-acidification are illustrated in FIG. 10 and show that each of the Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15848, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008 does not contribute to post-acidification or even reduce post-acidification compared to reference yoghurt.

[0180] These findings were unexpected and are highly significant, as prior art antifungal food-grade bacteria were observed to contribute to the secretion of volatile compounds and to increase the post-acidification effects caused by the starter culture.

EXAMPLE 4

[0181] Effect of One Lb. Fermentum Strain on Post-Acidification

[0182] One Lb. fermentum strain (CHCC14591) was tested for the effect on post-acidification.

[0183] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS Mild 2.0) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. One bottle was inoculated with Lb. fermentum CHCC14591 in total concentration of 210.sup.7 CFU/g, two bottles were inoculated with either of two commercial bioprotective cultures (FreshQ4 and Holdbac YM-C Plus) in recommended dosages (100 U/T and 20 DCU/100 L for FreshQ4 and Holdbac YM-C Plus, respectively), and one bottle was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice.

[0184] To monitor the effect on post acidification, the four fermented milk samples (starter-only, FreshQ4, Holdbac YM-C Plus and Lb. fermentum CHCC14591) were stored at 71 C. and 251 C. for 21 days and pH was measured on day 1, 7, 14 and 21.

[0185] The effect on post-acidification is illustrated in FIG. 11, showing that addition of Lb. fermentum CHCC14591 during milk fermentation prevents post-acidification of the fermented milk product. The starter culture alone gives slight post-acidification and the two commercial bioprotective cultures both contribute to post-acidification.

EXAMPLE 5

[0186] Effect of the Ten Lb. Fermentum Strains on Acetaldehyde Content

[0187] Ten Lb. fermentum strains were tested for their ability to lower acetaldehyde content.

[0188] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS YF-L901 Yo-Flex) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. Ten bottles were inoculated with the Lb. fermentum strains in concentrations of 110.sup.7 CFU/g and one bottle was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. The bottles were stored at 71 C. for 14 days.

[0189] On day 14 samples were analyzed for acetaldehyde by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0190] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0191] HS-software: HSControl v.2.00, Perkin Elmer. [0192] GC: Autosystem XL, Perkin Elmer. [0193] GC-software: Turbochrom navigator, Perkin Elmer. [0194] Column: HP-FFAP 25 m0.20 mm0.33 i, Agilent Technologies

[0195] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0196] The results are illustrated in FIG. 12 and show that each of the strains Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15848, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008 has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product.

EXAMPLE 6

[0197] Effect of One Lb. Fermentum Strain on Acetaldehyde Content

[0198] One Lb. fermentum strain was tested for the ability to lower acetaldehyde content.

[0199] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. A commercial starter culture (F-DVS Mild 2.0) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into two 200 ml bottles. One bottle was inoculated with the Lb. fermentum strains in concentrations of 110.sup.7 CFU/g and one bottle was used as a reference and only inoculated with the starter culture. Both bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.600.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. The bottles were stored at 71 C. for 14 days.

[0200] On day 14 samples were analyzed for acetaldehyde by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0201] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0202] HS-software: HSControl v.2.00, Perkin Elmer. [0203] GC: Autosystem XL, Perkin Elmer. [0204] GC-software: Turbochrom navigator, Perkin Elmer. [0205] Column: HP-FFAP 25 m0.20 mm0.33 i, Agilent Technologies

[0206] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0207] The results are illustrated in FIG. 13 and show that Lb. fermentum CHCC14591 has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product.

EXAMPLE 7

[0208] Functional Analysis of Commercial Starter Starter Cultures

[0209] The four commercial starter cultures included herein were chosen based on their different acidification profiles. Three were frozen, F-DVS CH-1, F-DVS YoFlex Mild 2.0 and F-DVS YF-L901, and one was freeze dried, FD-DVS YF-L812. To test the difference in acidification profiles, semi fat milk was standardized to 1% fat and 4.5% protein with skim milk powder and heat-treated at 851 C. for 30 min and cooled immediately. One of four different commercial starter cultures (F-DVS CH-1, F-DVS YoFlex Mild 2.0, F-DVS YF-L901 or FD-DVS YF-L812) was inoculated at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. The bottles were incubated in a water bath at 431 C. and fermented under these conditions until pH 4.5 was reached. The pH was measured continually throughout the fermentation. Subsequently, the bottles were stored at 6 C. for 43 for days and pH was measured with intervals of 7 days to determine the level of post-acidification.

[0210] The acidification profiles of the four commercial starter cultures, F-DVS CH-1, F-DVS YoFlex Mild 2.0, F-DVS YF-L901 and FD-DVS YF-L812, are shown in FIG. 14. F-DVS CH-1 showed fast fermentation time reaching pH 4.55 in 4.87 hours. F-DVS YoFlex Mild 2.0 showed intermediate fermentation time reaching pH 4.55 in 5.29 hours. FD-DVS YF-L812 and F-DVS YF-L901 showed slower fermentation reaching pH 4.55 in 6.45 and 5.87 hours, respectively. Post-acidification profiles showed very low levels of post-acidification for FD-DVS YF-L812 and F-DVS YoFlex Mild 2.0 (pH=0.12 and pH=0.11 after storage at 6 C. for 43 days), intermediate levels of post-acidification for F-DVS YF-L901 (pH=0.26 after storage at 6 C. for 43 days and high degree of post-acidification for F-DVS CH-1 (pH=0.55 after storage at 6 C. for 43 days) (FIG. 15).

EXAMPLE 8

[0211] Semi-Quantitative Analysis of the Inhibitory Effect of Nine Lb. Fermentum Strains Against Different Mold Contaminants when Fermented with Two Different Starter Cultures

[0212] For the semi-quantitative analysis of the inhibitory effect of nine Lb. fermentum strains, an agar-assay was used, resembling the manufacturing process and product of yoghurt:

[0213] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. Milk was inoculated with one of two commercial starter cultures (F-DVS CH-1 or FD-DVS YF-L812) at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. Nine bottles inoculated with each starter culture were further inoculated with the Lb. fermentum strains in concentrations of 110.sup.7 CFU/g and one bottle inoculated with each starter culture was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.550.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. Then the fermented milk was warmed to a temperature of 40 C. and added 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60 C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.

[0214] The tested Lb. fermentum strains were: Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008.

[0215] Spore suspensions of two different molds were spotted in concentration of 500 spores/spot; Penicillium brevicompactum (DSM32094) and P. solitum (DSM32093). One mold was spotted on each plate. Plates were incubated at 71 C. and 251 C. and regularly examined for the growth of molds.

[0216] On day 14 samples were analysed for diacetyl by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0217] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0218] HS-software: HSControl v.2.00, Perkin Elmer. [0219] GC: Autosystem XL, Perkin Elmer. [0220] GC-software: Turbochrom navigator, Perkin Elmer. [0221] Column: HP-FFAP 25 m0.20 mm0.33 pin, Agilent Technologies

[0222] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0223] Results of the agar-assay are presented in FIGS. 16-19, showing that both of the tested molds grew very well on the agar plates made from milk fermented only with one of the starter cultures (reference). For both P. brevicompactum (DSM32094) and P. solitum (DSM32093) a large delay in the growth was observed for all of the Lb. fermentum strains, when present during milk fermentation regardless of the starter culture used.

[0224] The effect on diacetyl production is illustrated in FIG. 20, showing that each of the anti-fungal strains Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008 adds no diacetyl to the level produced by the starter culture.

[0225] These findings were unexpected and are highly significant, as prior art antifungal food-grade bacteria were observed to contribute to the secretion of volatile compounds caused by the starter culture.

EXAMPLE 9

[0226] Effect of the Nine Lb. Fermentum Strains on Acetaldehyde Content when Fermented with Two Different Starter Cultures

[0227] Nine Lb. fermentum strains were tested for their ability to lower acetaldehyde content.

[0228] Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 901 C. for 20 min and cooled immediately. Milk was inoculated with one of two commercial starter cultures (F-DVS CH-1 or FD-DVS YF-L812) at 0.02% (v/w), and the inoculated milk was distributed into 200 ml bottles. Nine bottles were inoculated with the Lb. fermentum strains in concentrations of 110.sup.7 CFU/g and one bottle inoculated with each starter culture was used as a reference and only inoculated with the starter culture. All bottles were incubated in a water bath at 431 C. and fermented at these conditions until pH of 4.550.1 was reached. After fermentation, the bottles were vigorously shaken to break the coagulum and cooled on ice. The bottles were stored at 71 C. for 14 days.

[0229] The tested Lb. fermentum strains were: Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008.

[0230] On day 14 samples were analyzed for acetaldehyde by static head space gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). For that purpose the following equipment was used: [0231] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer. [0232] HS-software: HSControl v.2.00, Perkin Elmer. [0233] GC: Autosystem XL, Perkin Elmer. [0234] GC-software: Turbochrom navigator, Perkin Elmer. [0235] Column: HP-FFAP 25 m0.20 mm0.33 i, Agilent Technologies

[0236] Standards of known concentration were used to determine response factors (calibration), controls were used to control that the used response factors were stable within an analytical series as well as in-between series and over time (months). Concentration of volatiles (ppm) in samples and controls was determined using response factors coming from standards. Samples were prepared by adding 200 l of 4N H.sub.2SO.sub.4 to 1 g yoghurt sample and immediately analyzed by HSGC.

[0237] The results are illustrated in FIG. 21 and show that each of the strains Lb. fermentum CHCC12798, Lb. fermentum CHCC12797, Lb. fermentum CHCC14591, Lb. fermentum CHCC14588, Lb. fermentum CHCC15844, Lb. fermentum CHCC15865, Lb. fermentum CHCC15847, Lb. fermentum CHCC15926, and Lb. fermentum CHCC2008 has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product.

REFERENCES

[0238] EP0221499 [0239] EP0576780 [0240] EP1442113 [0241] U.S. Pat. No. 5,378,458 [0242] EP2 693 885 [0243] EP13717237 [0244] EP13714671 [0245] Gerez et al., Control of spoilage fungi by lactic acid bacteria, Biological Control, vol. 64 (2013): 231-237 [0246] Aunsbjerg et al., Contribution of volatiles to the antifungal effect of Lactobacillus paracasei in defined medium and yoghurt, Int J Food Microbiology, vol. 194 (2015): 46-53 [0247] Kosikowski, F. V. and Mistry, V. V., Cheese and Fermented Milk Foods, 1997, 3rd Ed. F. V. Kosikowski, L.L.C. Westport, Conn.

DEPOSITS AND EXPERT SOLUTION

[0248] The applicant requests that a sample of the deposited micro-organisms stated below may only be made available to an expert, until the date on which the patent is granted.

[0249] The Lactobacillus fermentum strain CHCC12798 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32084.

[0250] The Lactobacillus fermentum strain CHCC12797 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32085.

[0251] The Lactobacillus fermentum strain CHCC14591 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32086.

[0252] The Lactobacillus fermentum strain CHCC14588 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32087.

[0253] The Lactobacillus fermentum strain CHCC15844 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32088.

[0254] The Lactobacillus fermentum strain CHCC15865 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32089.

[0255] The Lactobacillus fermentum strain CHCC15847 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32090.

[0256] The Lactobacillus fermentum strain CHCC15848 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32091.

[0257] The Lactobacillus fermentum strain CHCC15926 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-22 under the accession No.: 32096.

[0258] The Lactobacillus fermentum strain CHCC2008 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2009-05-19 under the accession No.: 22584.

[0259] The Lactobacillus rhamnosus strain CHCC15860 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32092.

[0260] The Penicillium solitum strain CHCC16948 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32093.

[0261] The Penicillium brevicompactum strain CHCC16935 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig deposited on 2015-07-16 under the accession No.: 32094.

[0262] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.