LACTOBACILLUS FERMENTUM BACTERIA REDUCING THE CONCENTRATION OF ACETALDEHYDE

Abstract

The present invention relates to a bacterium of the species Lactobacillus fermentum wherein the bacterium has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product by at least 50%. The invention further relates to compositions comprising the bacterium, methods for producing fermented milk products using the bacterium and the products thus obtained.

Claims

1-15. (canceled)

16. A method of producing a fermented milk product comprising adding a Lactobacillus fermentum bacterium to milk or to a milk product to form 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 reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product by at least 50%.

17. The method of claim 16, wherein the Lactobacillus fermentum bacterium has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product by at least 75%, as 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 71 C. for 14 days; adding 200 l of 4N H.sub.2SO.sub.4 to 1 g of the fermented milk product and determining the concentration of acetaldehyde by static head space gas chromatography.

18. The method of claim 16, wherein the starter culture comprises lactic acid bacteria (LAB) able to produce acetaldehyde at a concentration of 3 ppm or more.

19. The method of claim 16, wherein the starter culture comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

20. The method of claim 16, wherein the Lactobacillus fermentum bacterium is selected from: (a) the Lactobacillus fermentum strain deposited deposited with the German Collection of Microorganisms and Cell Cultures (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; and (k) a mutant strain obtained from one of (a) to (j), wherein the mutant strain has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product by at least 50%.

21. The method of claim 16, 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 deposited with the DSMZ as DSM32092; (b) Lactobacillus rhamnosus bacterium of strain CHCC5366 deposited with the DSMZ as DSM23035; (c) Lactobacillus rhamnosus bacterium of strain CHCC12697 deposited with the DSMZ as DSM24616; (d) Lactobacillus paracasei bacterium of strain CHCC12777 deposited with the DSMZ DSM24651; and (e) Lactobacillus paracasei bacterium of strain CHCC14676 deposited with the DSMZ as DSM25612.

22. The method of claim 16, wherein the method comprises fermenting the mixture (a) such that the concentration of the Lactobacillus fermentum bacteria 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 bacteria of claim 1 is at least 110.sup.5 cfu/cm.sup.2 on the surface of the fermented milk product.

23. A fermented milk product obtained by the method of claim 16.

24. A method of producing a food, feed, or pharmaceutical product, comprising adding a fermented milk product according to claim 23 to a food, feed, or pharmaceutical product.

25. A food, feed, or pharmaceutical product obtained by a method of claim 24.

26. A Lactobacillus fermentum bacterium having the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product by at least 50%.

27. The Lactobacillus fermentum bacterium of claim 26, 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%.

28. A composition comprising the Lactobacillus fermentum bacterium of claim 26, optionally further comprising a cryoprotective agent.

29. The composition of claim 28, wherein the composition further comprises at least one further bacterium selected from: (a) Lactobacillus rhamnosus bacterium of strain CHCC15860 deposited with the DSMZ as DSM32092; (b) Lactobacillus rhamnosus bacterium of strain CHCC5366 deposited with the DSMZ as DSM23035; (c) Lactobacillus rhamnosus bacterium of strain CHCC12697 deposited with the DSMZ as DSM24616; (d) Lactobacillus paracasei bacterium of strain CHCC12777 deposited with the DSMZ DSM24651; and (e) Lactobacillus paracasei bacterium of strain CHCC14676 deposited with the DSMZ as DSM25612.

30. A composition according to claim 28, 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.

31. A food, feed, or pharmaceutical product comprising a Lactobacillus fermentum bacterium according to claim 26.

32. The food, feed, or pharmaceutical product according to claim 31. wherein the Lactobacillus fermentum bacteria is present at a concentration of at least 10.sup.7 CFU/g.

33. The food, feed, or pharmaceutical product of claim 32, 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

[0072] FIG. 1: 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.

[0073] FIG. 2: 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.

[0074] FIG. 3: 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.

[0075] FIG. 4: 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.

[0076] FIG. 5: 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

[0077] Effect of the Ten Lb. Fermentum Strains on Acetaldehyde Content

[0078] Ten Lb. fermentum strains were tested for their ability to lower acetaldehyde content.

[0079] 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.

[0080] 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:

[0081] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

[0082] HS-software: HSControl v.2.00, Perkin Elmer.

[0083] GC: Autosystem XL, Perkin Elmer.

[0084] GC-software: Turbochrom navigator, Perkin Elmer.

[0085] Column: HP-FFAP 25 m0.20 mm0.33 in, Agilent Technologies

[0086] 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.

[0087] The results are illustrated in FIG. 1 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 2

[0088] Effect of One Lb. Fermentum Strain on Acetaldehyde Content

[0089] One Lb. fermentum strain was tested for the ability to lower acetaldehyde content.

[0090] 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 YoFlex 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.

[0091] 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:

[0092] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

[0093] HS-software: HSControl v.2.00, Perkin Elmer.

[0094] GC: Autosystem XL, Perkin Elmer.

[0095] GC-software: Turbochrom navigator, Perkin Elmer.

[0096] Column: HP-FFAP 25 m0.20 mm0.33 in, Agilent Technologies

[0097] 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.

[0098] The results are illustrated in FIG. 2 and show that Lb. fermentum 14591 has the ability to reduce the concentration of acetaldehyde produced by a starter culture during fermentation in a fermented milk product.

EXAMPLE 3

Functional Analysis of Commercial Starter Starter Cultures

[0099] The three 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.

[0100] The acidification profiles of the three 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. 3. 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, respectively), 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. 4).

EXAMPLE 4

[0101] Effect of the Nine Lb. Fermentum Strains on Acetaldehyde Content when Fermented with Two Different Starter Cultures

[0102] Nine Lb. fermentum strains were tested for their ability to lower acetaldehyde content.

[0103] 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.

[0104] 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.

[0105] 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:

[0106] HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

[0107] HS-software: HSControl v.2.00, Perkin Elmer.

[0108] GC: Autosystem XL, Perkin Elmer.

[0109] GC-software: Turbochrom navigator, Perkin Elmer.

[0110] Column: HP-FFAP 25 m0.20 mm0.33 in, Agilent Technologies

[0111] 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.

[0112] The results are illustrated in FIG. 5 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

[0113] 1. Tamime, A. Y., and H. C. Deeth. 1980. Yoghurt: technology and biochemistry. J. Food Prot. 43:939-977. [0114] 2. A. C. S. D. Chaves, M. Fernandez, A. L. S. Lerayer, I. Mierau, M. Kleerebezem, and J. Hugenholtz, 2002, Metabolic Engineering of Acetaldehyde Production by Streptococcus thermophilus [0115] 3. Lees, G. J., and G. R. Jago. 1976. Formation of acetaldehyde from threonine by lactic acid bacteria. J. Dairy Res. 43:75-83.

Deposits and Expert Solution

[0116] 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.

[0117] 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 Jul. 16, 2015 under the accession No.: 32084.

[0118] 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 Jul. 16, 2015 under the accession No.: 32085.

[0119] 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 Jul. 16, 2015 under the accession No.: 32086.

[0120] 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 Jul. 16, 2015 under the accession No.: 32087.

[0121] 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 Jul. 16, 2015 under the accession No.: 32088.

[0122] 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 Jul. 16, 2015 under the accession No.: 32089.

[0123] 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 Jul. 16, 2015 under the accession No.: 32090.

[0124] 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 Jul. 16, 2015 under the accession No.: 32091.

[0125] 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 Jul. 22, 2015 under the accession No.: 32096.

[0126] 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 May 19, 2009 under the accession No.: 22584.

[0127] 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 Jul. 16, 2015 under the accession No.: 32092.

[0128] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.