METHOD OF PRODUCING A FERMENTED MILK PRODUCT USING LACTOBACILLUS CASEI

Abstract

The present invention relates to a method of producing a fermented milk product comprising adding lactic acid bacteria to milk, wherein the bacteria comprise Lactobacillus casei and at least one further strain of lactic acid bacteria of a species other than Lactobacillus casei, wherein the further strain has a deficiency in lactose metabolism but is capable of metabolizing one or several carbohydrates other than lactose present in the milk.

Claims

1-15. (canceled)

16. A method of producing a fermented milk product, comprising adding lactic acid bacteria to milk, wherein the bacteria comprise Lactobacillus casei and at least one further strain of lactic acid bacteria of a species other than Lactobacillus casei, wherein the further strain has a deficiency in lactose metabolism but is capable of metabolizing one or several carbohydrates other than lactose present in the milk.

17. The method of claim 16, wherein the at least one further strain of lactic acid bacteria having a deficiency in lactose metabolism is capable of metabolizing one or more carbohydrates selected from sucrose, galactose and glucose, and wherein said carbohydrate is added to the milk before fermentation.

18. The method of claim 16, wherein the Lactobacillus casei strain is selected from L. casei CRL 431 deposited as ATCC 55544 and L. casei CHCC2115 deposited as DSM19465.

19. The method of claim 16, wherein the further strain is selected from: (a) a Streptococcus thermophilus strain selected from: (i) the strain deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under Accession No. DSM 28952, and (ii) a strain derived from DSM 28952, wherein the derived strain has the ability to generate white colonies on a medium containing lactose and X-Gal; (b) a Streptococcus thermophilus strain selected from: (i) the strain deposited at DSMZ under Accession No. DSM 28953, and (ii) a strain derived from DSM 28953, wherein the derived strain has the ability to generate white colonies on a medium containing lactose and X-Gal; and (c) a Lactobacillus delbrueckii ssp. bulgaricus strain selected from: (i) the strain deposited at DSMZ under Accession No. DSM 28910, and (ii) a strain derived from DSM 28910, wherein the derived strain has the ability to generate white colonies on a medium containing lactose and X-Gal.

20. The method according to claim 16, wherein the at least one further strain of lactic acid bacteria having a deficiency in lactose metabolism is a proteolytic strain of S. thermophilus, wherein the proteolytic strain exhibits (a) the presence of an active cell wall proteinase and/or (b) an increase in fluorescence caused by growth of the strain for 6 hours in a medium containing fluorescently labeled casein as compared to fluorescence of control samples of control strains.

21. The method of claim 16, wherein the method produces L. casei at a concentration of from 110.sup.8 to 510.sup.9 CFU/g.

22. The method of claim 16, wherein the method produces L. casei at a concentration of from 110.sup.9 to 510.sup.9 CFU/g.

23. The method of claim 16, wherein the method produces L. casei at a concentration of from 110.sup.9 to 310.sup.9 CFU/g.

24. A composition comprising bacteria of the species L. casei and bacteria of at least one further strain of lactic acid bacteria, wherein the at least one further strain of lactic acid bacteria has a deficiency in lactose metabolism.

25. The composition of claim 24, wherein the at least one further strain of lactic acid bacteria is of a species selected from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

26. A food or feed product comprising bacteria of the species L. casei and bacteria of at least one further strain of lactic acid bacteria, wherein the at least one further strain of lactic acid bacteria has a deficiency in lactose metabolism.

27. The food or feed product of claim 26, wherein the at least one further strain of lactic acid bacteria is of a species selected from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

28. The food or feed product of claim 26, comprising L. casei at a concentration of from 110.sup.8 CFU/g to 510.sup.9 CFU/g.

29. The food or feed product of claim 26, comprising L. casei at a concentration of from 110.sup.9 CFU/g to 510.sup.9 CFU/g.

30. The food or feed product of claim 26, comprising L. casei at a concentration of from 110.sup.9 CFU/g to 310.sup.9 CFU/g.

31. The food or feed product according to claim 26, wherein the food or feed product is a fermented milk beverage.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0104] FIG. 1 shows acidification curves of milk using different starter cultures.

[0105] FIG. 2 shows cell counts (CFU/g) of L. casei after fermentation and storage.

[0106] FIG. 3 shows cell counts (CFU/g) of L. casei after fermentation and storage.

EXAMPLE 1

[0107] FITC Assay: Protocol for measuring Proteinase activity in lactic acid bacteria (LAB), employing Hamilton liquid handling unit.

[0108] The cell-wall associated proteinase(s) contribute to rapid growth of LAB in milk. The proteinases are also responsible for the first step in degradation of casein and thereby development of flavor. The assay is based on the fluorescent substrate fluorescein isothiocyanate labeled casein (FITC).

[0109] Materials [0110] Costar Deep well plate2.0 ml [0111] Microtiter plates: Nunc, black plates, (Cat. No. 237105), Nunc (product no. 167008), [0112] MJR PCR plate (V-shaped, product no. HSP-9665) [0113] Hamilton liquid handling robot [0114] Hamilton disposable sterile tips [0115] Calcium chloride dihydrate (Merck Cat. No. 1.02382.1000) [0116] MES hydrate, minimum 99.5% titration (Sigma M8250-1 kg) [0117] Tris, (Sigma Cat. No. 11503) [0118] Samples in Costar deep well plate (2.0 ml) [0119] Enspire, 2300 multilabel reader, PerkinElmer [0120] Control strains: LH-Emfour, batch no. 2995517, material no. 501638 [0121] LH-32 batch no. 2990465, material no. 616703 added as thawed DVS (50 l) to grow up o/n. When using a new batch of control compare proteolytic activity to old batch before using. [0122] 500 mM Tris-HCL, pH 8.5: [0123] 60.55 g Tris-HCl. Add Milli-Q water to 800 ml, adjust pH to 8.5 with 1M HCl. [0124] Fill with Milli-Q water to 1000 ml. [0125] FITC, FITC-labeled casein (Sigma C3777) [0126] 5 mg/ml FITC, prepared in sterile filtered Milli-Q water. [0127] MES 100 mM+ CaCl.sub.2), pH 6.5 [0128] 19.52 g MES and 7.35 g CaCl.sub.2). Add water to 800 ml, adjust pH to 6.5, fill water to 1000 ml, sterile filter. [0129] 5% TCA [0130] 5 g Trichloric acetic acid is dissolved in Milli-Q water to a total volume of 100 ml.

[0131] Cultivation medium: BD5-3-broth

[0132] Methods [0133] Wash cells [0134] The strains are grown overnight in the appropriate dilution, a 10% inoculation from WS-Plate in deep well plate with 1800 l BD5-3 broth. Lactococcus sp. are incubated at 30 C.; Lactobacillus sp. and Streptococcus sp. are incubated at 37 C. Wash 3 with cold MES buffer pH 6.5 (centrifuge deep well plate after each wash, 4000 rpm for 4 min at 10 C.), measure OD620, and from there take samples for proteinase activity on FITC substrate and incubate the plate at 37 C. for 6 h. [0135] FITC protocol [0136] Dispense 8 l of FITC solution to a MJR PCR microtiter plate (V-shaped wells in order to minimize evaporation during incubation). [0137] Dispense 40 l of washed cell suspension to the MJR PCR microtiter plate & seal the plates. [0138] Incubate MJR PCR microtiter plate for 0-6 h at 37 C. (Lb's & St's). [0139] Dispense 115 l of 5% w/v TCA to MJR PCR microtiter plate. [0140] Incubate the MRJ PCR microtiter plate for 1 h at room temperature or 30 min on ice bath. [0141] Centrifuge the MJR PCR microtiter plate (3700 rpm for 10 min, 10 C.). [0142] Dispense 125 l of Tris-HCl 0.5 M (pH 8.5) to the Nunc Black microtiter plate. [0143] Aspirate 45 l from MJR PCR microtiter plate to Nunc Black microtiter plate. [0144] Read the fluorescence at Exitation 497 nmEmission 515 nm, on Enspire, 2300 multilabel reader, PerkinElmer. [0145] T0 Samples without incubation [0146] T6 Samples were incubated for 6 h at 37 C., immediately after wash with MES-buffer. [0147] Data treatment: Subtract T0 from T6, make graphs, then divide by OD of the washed cells, make new graphs. Check whether very high activity is due to a low OD. Check whether controls are in normal range.

EXAMPLE 2

[0148] Milk was fermented using one of the following two different L. casei strains of Chr. Hansen alone or in combination with the following Streptococcus thermophilus strain of Chr. Hansen: [0149] L. casei CRL 431 deposited as ATCC 55544; [0150] L. casei 01, CHCC2115 deposited as DSM19465; and [0151] S. thermophilus, CHCC17861 deposited as S. thermophilus DSM 28952.

[0152] The S. thermophilus strain has a deficiency in lactose metabolism.

[0153] When used in combination the following ratios were used: [0154] (1) 50:50 [0155] (2) 90:90; and [0156] (3) 10:90.

[0157] S. thermophilus with a deficiency in lactose metabolism alone and each of the two L. casei strains alone were used as controls.

[0158] Standard milk was used for the trials (3.8% protein, 1.5% fat). Sucrose (0.4% w/v) was added to the milk to allow fast acidification of the S. thermophilus with a deficiency in lactose metabolism to pH 5.50. Acidification was performed at 39 C. and at 37 C.

[0159] Development of pH was determined during fermentation. Development of cell count was determined on days 1, 7 and 14 after fermentation.

[0160] Three repetitions were performed to confirm the data.

[0161] Typical results are illustrated in FIGS. 1 and 2. As shown in FIG. 1, L. casei 01 alone has a very long lag phase and reaches pH 4.5 only after 40 h of fermentation. However, when combined with S. thermophilus with a deficiency in lactose metabolism in different ratios the lag phase is significantly reduced (8 h for L. casei 01 alone to achieve pH 6.3; whereas only 1 h when combined with S. thermophilus with a deficiency in lactose metabolism). S. thermophilus with a deficiency in lactose metabolism alone reduced the pH value to a final pH of 5.1 after sucrose depletion. However, when combined with L. casei 01, the acidification continues due to the presence of L. casei 01, which is capable to use the lactose present in the milk.

[0162] The same results were obtained using L. casei CRL 431 and in trials performed at 37 C. (data not shown).

[0163] L. casei 01 cell counts are shown in FIG. 2. FIG. 2 shows that the co-culture of L. casei 01 and S. thermophilus with a deficiency in lactose metabolism does not reduce the L. casei cell counts, in comparison to single strain fermentation.

[0164] As a consequence, the combination of L. casei 01 with a Lac () ST boosts the L. casei 01 without disturbing its growth (no competition). The combination allows a high reduction of the lag phase while maintaining high cell counts.

EXAMPLE 3

[0165] Milk was fermented using the following L. casei strain of Chr. Hansen alone or in combination with the following Streptococcus thermophilus strains of Chr. Hansen:

[0166] L. casei CRL 431 deposited as ATCC 55544; in combination with

1) S. thermophilus, CHCC17861 deposited as S. thermophilus DSM 28952. The strain has a deficiency in lactose metabolism (Lac()); or
2) S. thermophilus, CHCC11977 deposited as DSM22935, with no deficiency in lactose metabolism (mother strain of CHCC17861) (Lac(+)).

[0167] When used in combination the following ratio was used: [0168] (1) 50:50

[0169] The milk base used for the present experiment is Skimmed Milk Powder (SMP) reconstituted at 9.6%. The milk base was subjected to heat treatment at 99 C. for 15 minutes. Sucrose (0.3% w/v) was added to the milk to allow fast acidification of the S. thermophilus with a deficiency in lactose metabolism to pH 5.50. Acidification was performed at 39 C.

[0170] Development of cell count was determined 40 hours after fermentation.

[0171] One repetition of the experiment was performed.

[0172] As will appear from FIG. 3 the L. casei cell count was significantly reduced, when the L. casei strain was grown together with a conventional S. thermophilus strain (no deficiency in lactose metabolism (Lac(+)) as compared to when the L. casei is grown alone. Also, the L. casei cell count was significantly increased, when the L. casei strain was grown together with a S. thermophilus strain with a deficiency in lactose metabolism (Lac()) as compared to when the L. casei is grown alone.

Deposits and Expert Solution

[0173] The Applicant requests that a sample of the deposited microorganism should be made available only to an expert approved by the Applicant.

[0174] Streptococcus thermophilus strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014 Jun. 12 under the accession no. DSM 28952.

[0175] Streptococcus thermophilus strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014 Jun. 12 under the accession no. DSM 28953.

[0176] Streptococcus thermophilus strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2009 Sep. 8 under the accession no. DSM22935.

[0177] Lactobacillus delbrueckii subsp. bulgaricus strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014 Jun. 12 under the accession no. DSM28910.

[0178] Lactobacillus casei strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2007 Jun. 27 under the accession no. DSM19465.

[0179] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.