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Title:
MESOPHILIC DAIRY PRODUCTS WITH ENHANCED FLAVOR
Document Type and Number:
WIPO Patent Application WO/2012/136833
Kind Code:
A1
Abstract:
The present invention relates to a composition suitable for preparing a dairy product comprising at least one mesophilic starter culture, such as a Lactococcus starter culture, and a Lactobacillus rhamnosus strain capable of imparting onto the dairy product an enhanced creamy flavor without affecting the rheology negatively, the fermentation time or the post-acidification of the dairy product. The present invention further relates to mesophilic processes for preparing dairy products, such as fresh cheese or sour cream, which has a high content of diacetyl. A Lactobacillus rhamnosus strain useful for preparing such dairy product is also part of the present invention.

Inventors:
ODINOT JEAN-MARIE (FR)
PIROIS-BLIN SABRINA (FR)
LISBERG MAIKE (DK)
OEREGAARD GUNNAR (DK)
FOLKENBERG DITTE MARIE (DK)
Application Number:
EP2012/056387
Publication Date:
October 11, 2012
Filing Date:
April 09, 2012
Export Citation:
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Assignee:
CHR HANSEN AS (DK)
ODINOT JEAN-MARIE (FR)
PIROIS-BLIN SABRINA (FR)
LISBERG MAIKE (DK)
OEREGAARD GUNNAR (DK)
FOLKENBERG DITTE MARIE (DK)
International Classes:
A23C9/123; A23C19/032; A23L29/00; C12N1/20; C12P7/18; C12P7/26; C12R1/225
Foreign References:
US4867992A1989-09-19
US5236833A1993-08-17
US4678673A1987-07-07
Other References:
MUHAMMAD RAMZAN ET AL: "Evaluation of volatile flavouring compounds in Cheddar cheese, manufactured by using Lactobacillus rhamnosus as an adjunct culture", vol. 16, no. 2, 1 January 2010 (2010-01-01), pages 188 - 195, XP009151277, ISSN: 1453-1399, Retrieved from the Internet [retrieved on 20110817]
CICHOSZ GRAIYNA ET AL: "Aroma compounds in gouda cheese produced with addition of probiotic strains", POLISH JOURNAL OF NATURAL SCIENCES,, vol. 21, no. 2, 1 January 2006 (2006-01-01), pages 987 - 997, XP009151267
CHAMPAGNE ET AL: "Fresh-cheesemilk formulation fermented by a combination of freeze-dried citrate-positive cultures and exopolysaccharide-producing lactobacilli with liquid lactococcal starters", FOOD RESEARCH INTERNATIONAL, ELSEVIER APPLIED SCIENCE, BARKING, GB, vol. 39, no. 6, 1 July 2006 (2006-07-01), pages 651 - 659, XP005365860, ISSN: 0963-9969, DOI: 10.1016/J.FOODRES.2006.01.002
KOCAOGLU-VURMA N A ET AL: "Microbiological, Chemical, and Sensory Characteristics of Swiss Cheese Manufactured with Adjunct Lactobacillus Strains Using a Low Cooking Temperature", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 91, no. 8, 1 August 2008 (2008-08-01), pages 2947 - 2959, XP026954967, ISSN: 0022-0302, [retrieved on 20080801]
DEGHEIDI M A ET AL: "Utilization of probiotic bacteria in UF white soft cheese", EGYPTIAN JOURNAL OF DAIRY SCIENCE, EGYPTIAN SOCIETY OF DAIRY SCIENCE, CAIRO, EG, vol. 37, no. 1, 1 January 2009 (2009-01-01), pages 73 - 84, XP009151276, ISSN: 0378-2700
DATABASE GNPD [online] 1 September 2010 (2010-09-01), ANONYMOUS: "Organic Cream Cheese", XP002657009, retrieved from www.gnpd.com Database accession no. 1405368
DATABASE GNPD [online] 1 October 2010 (2010-10-01), ANONYMOUS: "Pear & Vanilla Curd", XP002657010, retrieved from www.gnpd.com Database accession no. 1408338
MEDINA DE FIGUEROA R MEDINA ET AL: "Flavour compound production and citrate metabolism in Lactobacillus rhamnosus ATCC 7469", MILCHWISSENSCHAFT, VV GMBH VOLKSWIRTSCHAFTLICHER VERLAG. MUNCHEN, DE, vol. 53, no. 11, 1 January 1998 (1998-01-01), pages 617 - 619, XP009151269, ISSN: 0026-3788
B. D. JYOTI ET AL: "Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates", WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, 6 February 2003 (2003-02-06), pages 509 - 514, XP055004586, Retrieved from the Internet [retrieved on 20110811]
JYOTI, B.D.; SURESH, A.K.; VENKATESH, K.V.: "Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates", WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, vol. 19, 2003, pages 509 - 514, XP055004586
Attorney, Agent or Firm:
UEXKÜLL & STOLBERG (Beselerstr. 4, Hamburg, 22607, DE)
Download PDF:
Claims:
CLAIMS

1. A composition suitable for preparing a dairy product comprising a mesophilic starter culture and a Lactobacillus rhamnosus strain.

2. The composition according to claim 1, wherein the mesophilic starter culture comprises at least one Lactococcus lactis strain.

3. The composition according to claim 2, wherein the mesophilic starter culture comprises a Lactococcus lactis subs p. cremoris strain and a Lactococcus lactis subs p. lactis strain.

4. The composition according to any of the preceding claims, wherein the Lactobacillus rhamnosus strain is Lactobacillus rhamnosus CHCC12697 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession no. DSM24616 or a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material.

5. Use of a composition according to any of the preceding claims for preparing a dairy product preferably a mesophilic dairy product.

6. Use according to claim 5, wherein the dairy product is a fresh cheese, such as quark or tvarog, or a sour cream.

7. Use according to any of claims 5 and 6, wherein said dairy product comprises at least 0.75 ppm of diacetyl.

8. Use according to claim 7, wherein said dairy product comprises at least 1.5 ppm of diacetyl.

9. A method for producing a dairy product, comprising :

a) inoculating a milk substrate with the composition according to any of claims 1 to 4;

b) fermenting said milk substrate at a temperature between about 22°C to about

35°C;

c) optionally adding further microorganisms and/or additives to said milk substrate;

d) optionally post-treating said milk substrate; and

e) optionally packaging the dairy product.

10. A dairy product obtainable by the method according to claim 9.

11. The dairy product according to claim 10, wherein the dairy product is a fresh cheese, such as quark or tvarog, or a sour cream.

12. The dairy product according to any of claims 10 and 11, wherein said dairy product comprises at least 0.75 ppm of diacetyl.

13. The dairy product according to claim 12, wherein said dairy product comprises at least 1.5 ppm of diacetyl.

14. A Lactobacillus rhamnosus CHCC12697 strain that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession no. DSM24616 or a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material.

Description:
MESOPHILIC DAIRY PRODUCTS WITH ENHANCED FLAVOR

FIELD OF THE INVENTION

The present invention relates to a composition suitable for preparing a dairy product comprising at least one mesophilic starter culture and a bacterial strain of the genus Lactobacillus capable of imparting onto the dairy product an enhanced creamy flavor. The present invention further relates to processes for preparing a dairy product, such as a fresh cheese or a sour cream. A Lactobacillus rhamnosus strain useful for preparing such dairy product is also part of the present invention.

BACKGROUND ART

Dairy products, such as fresh cheese, butter milk and sour cream, prepared with mesophilic starter cultures are in popular demand with consumers. For such products LD cultures (such as CHN-22 obtainable from Chr. Hansen A/S, Denmark, containing Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc) are used. This type of culture produces C0 2 and flavor.

Today, a lot of customers demand a high flavor performance from the culture but they do not accept C0 2 production which can lead to blowing, i.e. pressure increase in the packaging of the dairy product.

Dairy products can be produced by mesophilic processes without C0 2 by O-cultures (such as MO-3 obtainable from Chr. Hansen A/S, Denmark, containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris) but O-cultures have homofermentative metabolism and do not produce flavor compounds (diacetyl, ethanol, acetate).

Thus, there is a need for a mesophilic culture which produces aroma, low post acidification and high texture but no C0 2 .

Diacetyl is a high value product and it is used in the dairy industry as a buttery flavor- producing compound added to such products as margarines and oil-based products.

Heterolactic acid bacteria form diacetyl/acetoin as a by-product along with lactate as the main product. The cells form active acetaldehyde from pyruvate and thiamine pyrophosphate by pyruvate oxidase. The active acetaldehyde condenses with another molecule of pyruvate and forms alpha-acetolactate synthase. Formation of diacetyl in Lactobacillus rhamnosus is not well understood - in Lactococcus lactis subsp. lactis biovar. diacetylactis it has been suggested that alpha-acetolactate is oxidized to diacetyl by an alpha acetolactate oxidase (Jyoti et al 2003). Acetoin is formed directly by decarboxylation of alpha-acetolactate. Acetoin formation may also occur by the irreversible diacetyl reductase of diacetyl into acetoin.

Lactobacillus rhamnosus is a heterolactic acid bacterium which can be used to produce flavor compounds like diacetyl and acetoin (Jyoti et al. 2003). The level of diacetyl produced depends on the strain as well as the substrate on which it is grown.

US patent No. 4,867,992 and US patent No. 5,236,833 relate to processes for production of diacetyl by fermenting a coffee substrate and a pectin substrate, respectively, with a lactic acid producing bacteria.

The preparation, concentration and addition of diacetyl and/or acetoin to food products are connected with substantial costs.

US 4,678,673 is directed to oilseed products fermented with a Lactobacillus rhamnosus strain which produces diacetyl and acetoin. The fermented oilseed products have a buttery or dairylike flavor. There is no mention of use of Lactobacillus rhamnosus in dairy products.

Thus, there exists a need for a mesophilic process of preparing dairy products with improved flavor and high texture.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a composition and a method for preparing a mesophilic dairy product improved with an enhanced creamy flavor imparted by the presence of a strain of Lactobacillus rhamnosus.

It is another object of the present invention to provide a novel Lactobacillus rhamnosus strain with improved properties in relation to being able to give an enhanced creamy flavor to a mesophilic dairy product, such as a sour cream, a buttermilk or a fresh cheese.

Additional objects will become apparent hereinafter and still others will be obvious to one skilled in the art to which the invention pertains.

As can be seen in the working examples herein, the described Lactobacillus rhamnosus strain CHCC12697 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession no. DSM24616 produces diacetyl thereby giving enhanced creamy flavor to a mesophilic dairy product, without significantly affecting the rheology and the post-acidification of the mesophilic dairy product. The presence of the described Lactobacillus rhamnosus strain CHCC12697 in a mesophilic fermented milk product can also be seen to result in increased levels of acetate and acetaldehyde.

Accordingly, a first aspect of the present invention relates to a composition for preparing a dairy product comprising a mesophilic starter culture and a Lactobacillus rhamnosus strain.

In a much preferred embodiment the Lactobacillus rhamnosus strain is a Lactobacillus rhamnosus CHCC12697 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession no. DSM24616 or a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material.

A second aspect of the present invention relates to use of a composition according to the first aspect of the present invention for preparing a dairy product.

A third aspect of the present invention is directed to a method for producing a dairy product, said method comprising the steps:

a) inoculating a milk substrate with the composition according to the first aspect of the present invention;

b) fermenting said milk substrate at a temperature between about 22°C to about 35°C; c) optionally adding further microorganisms and/or additives to said milk substrate;

d) optionally post-treating said milk substrate; and

e) optionally packaging the dairy product.

A fourth aspect of the invention relates to a dairy product obtainable by the method according to the third aspect of the invention.

A fifth aspect of the present invention relates to a Lactobacillus rhamnosus CHCC12697 strain that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession no. DSM24616 or a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows acetaldehyde in ppm in mesophilic fermented milk product with and without Lactobacillus rhamnosus CHCC12697. Means and 95.0 percent LSD (least significant difference) intervals are calculated from 4 replicates.

FIGURE 2 shows diacetyl in ppm in mesophilic fermented milk product with and without Lactobacillus rhamnosus CHCC12697. Means and 95.0 percent LSD intervals are calculated from 4 replicates. FIGURE 3 shows acetate in ppm in mesophilic fermented milk product with and without Lactobacillus rhamnosus CHCC12697. Means and 95.0 percent LSD intervals are calculated from 4 replicates.

FIGURE 4 depicts the fermentation profile (pH evolution versus time) at 26°C for MO-3 alone (None, cross) and MO-3 together with Lactobacillus rhamnosus CHCC12697 (+CHCC12697, square).

FIGURE 5 depicts the fermentation profile (pH evolution versus time) at 26°C for Sico-200-0 alone (None, cross) and Sico-200-0 together with Lactobacillus rhamnosus CHCC12697 (+CHCC12697, square).

FIGURE 6 depicts the fermentation profile (pH evolution versus time) at 30°C for MO-3 alone (None, cross) and MO-3 together with Lactobacillus rhamnosus CHCC12697 (+CHCC12697, square).

FIGURE 7 depicts the fermentation profile (pH evolution versus time) at 30°C for Sico-200-0 alone (None, cross) and Sico-200-0 together with Lactobacillus rhamnosus CHCC12697 (+CHCC12697, square).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are frequently used as food cultures alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including bacteria of the species Lactobacillus spp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product or a cheese. Such lactic acid bacterial cultures are in general referred to as "starter cultures" or "starters".

The term "mesophile" herein refers to microorganisms that thrive best at moderate temperatures (15°C-40°C). The industrially most useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" herein refers to fermentation at a temperature between about 22°C and about 35°C. The term "mesophilic dairy product" refers to dairy products prepared by mesophilic fermentation of a mesophilic starter culture and include such dairy products as buttermilk, sour milk, cultured milk, smetana, sour cream and fresh cheese, such as quark, tvarog and cream cheese.

The term "thermophile" herein refers to microorganisms that thrive best at temperatures above 43°C. The industrially most useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 37°C. The term "thermophilic dairy product" refers to dairy products prepared by thermophilic fermentation of a thermophilic starter culture and include such dairy products as yoghurt.

The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein/fat solutions made of plant materials, e.g. soy milk.

The term "milk substrate" may be any raw and/or processed milk material that can be subjected to fermentation according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk-like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder.

Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

"Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.

"Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

"Fermentation" in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of dairy products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected so as to support the achievement of the present invention, i.e. to obtain a dairy product in solid (such as a cheese) or liquid form (such as a fermented milk product).

In the present context, the term "shear stress" determines viscosity. Viscosity (unit is Pa s) is defined as Shear Stress (Pa) / Shear rate (1/s).

Shear stress value is reported as a standard herein at shear rate = 300 1/s. Sensory experiments have shown (data not shown) that the best correlation between rheological measurements and sensory viscosity/mouth thickness are found when using the viscosity measured at shear rate 300 1/s.

In the present context, the term "mutant" should be understood as a strain derived from a strain of the invention by means of e.g. genetic engineering, radiation, UV light, and/or chemical treatment and/or methods that induce changes in the genome. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding diacetyl production, viscosity, gel stiffness, mouth coating, flavor, post acidification, acidification speed, and/or phage robustness) as the mother strain. Such a mutant is a part of the present invention. Especially, the term "mutant" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood as containing one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening/selection steps) are carried out. In a presently preferred mutant, less that 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain.

The use of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Implementation and aspects of the invention

The inventors of the present invention have surprisingly discovered that by inoculating and fermenting a milk substrate with a strain of Lactobacillus rhamnosus it is possible to impart onto the resulting dairy product a pleasant creamy flavor without negatively affecting the texture of the dairy product, the fermentation time and post-acidification.

The enhanced creamy flavor was detected in fermented milk products prepared both by mesophilic (26°C-37°C) and thermophilic (43°C) fermentation processes particularly well in the presence of a Lactobacillus rhamnosus CHCC12697 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession no. DSM24616.

By the term "enhanced creamy flavor" is meant that the content of diacetyl and/or acetoin in the product is increased compared to a product which does not comprise a Lactobacillus rhamnosus strain according to the present invention.

Without wishing to be bound by theory, it is thought that the enhanced creamy flavor imparted to the dairy product by a Lactobacillus rhamnosus strain according to the present invention is due to the enhanced production of diacetyl and/or acetoin by the Lactobacillus rhamnosus strain. The dairy product in a preferred embodiment is a mesophilic dairy product which essentially lacks a creamy flavor when the Lactobacillus rhamnosus strain according to the present invention is not present in the milk or has not been used in the fermentation.

The Lactobacillus rhamnosus strain described herein is useful in a composition for the preparation of a mesophilic dairy product comprising at least one mesophilic starter culture and the Lactobacillus rhamnosus strain.

Typically, such a composition comprises the bacteria in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells, which is in the range of 10 4 to 10 12 cfu (colony forming units) per gram of the composition including at least 10 4 cfu per gram of the composition, such as at least 10 5 cfu/g, e. g. at least 10 6 cfu/g, such as at least 10 7 cfu/g, e.g. at least 10 s cfu/g, such as at least 10 9 cfu/g, e.g. at least 10 10 cfu/g, such as at least 10 11 cfu/g. Thus, the antimicrobial composition of the invention is preferably present in a frozen, dried or freeze-dried form, e.g. as a Direct Vat Set (DVS) culture. However, as used herein the antimicrobial composition may also be a liquid that is obtained after suspension of the frozen, dried or freeze-dried cell concentrates in a liquid medium such as water or PBS buffer. Where the antimicrobial composition of the invention is a suspension, the concentration of viable cells is in the range of 10 4 to 10 12 cfu (colony forming units) per ml of the composition including at least 10 4 cfu per ml of the composition, such as at least 10 5 cfu/ml, e.g. at least 10 6 cfu/ml, such as at least 10 7 cfu/ml, e.g. at least 10 s cfu/ml, such as at least 10 9 cfu/ml, e.g. at least 10 10 cfu/ml, such as at least 10 11 cfu/ml.

The composition may additionally contain as further components cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and vitamins, e.g. vitamin A, C, D, K or vitamins of the vitamin B family. Suitable cryoprotectants that may be added to the compositions of the invention are components that improve the cold tolerance of the microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose and fructose. Other additives to may include, e.g., carbohydrates, flavors, minerals, enzymes (e.g. rennet, lactase and/or phospholipase).

As it is normal in lactic acid bacterial fermentation processes to apply a mixed culture as a starter culture, the composition will in certain embodiments comprise a multiplicity of strains either belonging to the same species or belonging to different species. A typical example of such a useful combination of lactic acid bacteria in a starter culture is a mixture of a Lactobacillus bulgaricus strain and a Streptococcus thermophilus strain. The mesophilic starter culture in a preferred embodiment comprises at least one Lactococcus lactis strain. The mesophilic starter culture may comprise any Lactococcus lactis strain known in the art, such as strains from the Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae or Lactococcus lactis subsp. lactis. In yet another preferred embodiment the mesophilic starter culture comprises a Lactococcus lactis subsp. cremoris strain and a Lactococcus lactis subsp. lactis strain.

The composition can be used for preparing a dairy product, preferably a mesophilic dairy product with enhanced creamy flavor. The mesophilic dairy product may be any dairy product that was prepared by mesophilic fermentation. For example, the mesophilic dairy product of the invention may be buttermilk, sour milk, cultured milk, smetana, sour cream and fresh cheese, such as curd, quark, tvarog and cream cheese. In a preferred embodiment the mesophilic dairy product is a fresh cheese, such as quark, or a sour cream.

In another preferred embodiment the mesophilic dairy product contains at least 0.75 ppm of diacetyl, such as at least 1.0 ppm of diacetyl, such as at least 1.5 ppm of diacetyl. The mesophilic dairy product may contain between about 0.75 ppm and 3.00 ppm of diacetyl, more preferably between about 1.00 and 2.50 ppm of diacetyl and most preferably between about 1.5 ppm and 2 ppm of diacetyl. In a preferred embodiment the mesophilic dairy product contains more than 1.5 ppm of diacetyl. The skilled person will be aware of numerous methods to determine the content of diacetyl in the dairy products of the invention. For example, the content may be determined by suitable chromatographic methods, such as static head space gas chromatography (HSGC).

As said above, an aspect of the invention relates to a method of manufacturing a mesophilic dairy product with a creamy flavor comprising :

a) inoculating a milk substrate with the composition according to the first aspect of the invention;

b) fermenting said milk substrate at a temperature between about 22°C to about

37°C, preferably between about 22°C and 35°C;

c) optionally adding further microorganisms and/or additives to said milk substrate;

d) optionally post-treating said milk substrate; and

e) optionally packaging the dairy product.

As described above, the milk substrate to be used in step a) may be any raw and/or processed milk material that can be subjected to fermentation according to the method of the invention. The milk substrate may be inoculated with the above composition by any suitable method. For example, the milk substrate may be inoculated by direct inoculation into a fermentation vessel.

The milk substrate will then be subjected to mesophilic fermentation at a temperature of between about 22°C and about 37°C, preferably between about 22°C and 35°C, more preferably between about 25°C and about 35°C. In a preferred embodiment, the milk substrate will be fermented at about 26°C. In another preferred embodiment, the milk substrate will be fermented at about 30°C. Fermentation processes to be used in production of dairy products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected so as to obtain a fermented milk product suitable in the production of a mesophilic dairy product with improved flavor and high texture.

Further microorganisms and/or additives may be added to the milk substrate before, during or after fermentation of the milk substrate in step (b). Microorganisms that may be added to the milk substrates will contribute in an advantageous manner to the properties of the mesophilic dairy product. For example, the microorganism may improve or support the diacetyl production, the viscosity, gel stiffness, mouth coating, flavor, post acidification, and/or acidification speed in the mesophilic dairy product. Optionally, other ingredients may be added to the milk substrate, such as colors, stabilizers, e.g., pectin, starch, modified starch, CMC, etc.; or polyunsaturated fatty acids, e.g. omega-3 fatty acids. Such ingredients may be added at any point during the production process, e.g. before or after fermentation.

The milk substrate may further be post-treated by any means necessary to create the desired mesophilic dairy product. For example, further components, such as cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and vitamins, may be added to the milk substrate. Further, the milk substrate may e.g. be homogenized or treated with heat, i.e. pasteurized.

The dairy product may be packaged in any suitable manner known in the art. For example, the dairy product may be packaged in a sealed container having a volume in the range of e.g. 25 to 1500 ml. The product may be packaged at any point during the production process, e.g. packaged next to the inoculating step and then fermented in the package.

A dairy product obtainable by the method above is also part of the present invention. In a preferred embodiment the dairy product is a dairy product selected from the group consisting of a fresh cheese, such as quark, and a sour cream.

In another preferred embodiment the dairy product contains at least 0.75 ppm of diacetyl, such as at least 1.0 ppm of diacetyl, such as at least 1.5 ppm of diacetyl.

The fifth aspect of the invention relates to the Lactobacillus rhamnosus CHCC12697 strain that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession no. DSM24616. Apart from this strain, the invention also pertains to mutants that have been derived from it, i.e. they have been obtained by using the deposited strain CHCC12697 as a starting material. The mutant strain may be derived from CHCC12697, e.g., by means of genetic engineering, radiation, UV light, chemical treatment and/or methods that induce changes in the genome. A mutant according to the invention will essentially have the same characteristics as the mother strain in terms of the production levels of acetate, acetaldehyde, diacetyl and/or acetoin. It is preferred that the mutant produces essentially at least 80% or more, at least 90% or more, at least 95% or more, or even up to 100% or more of acetate, acetaldehyde, diacetyl and/or acetoin compared with its mother strain.

It is clear for the skilled person that by using the deposited strain as starting material, the skilled reader can by conventional mutagenesis or re-isolation techniques routinely obtain further mutants or derivatives thereof that retain the herein described relevant features and advantages. Accordingly, the term "a mutant thereof" of the first aspect relates to mutant strains obtained by using the deposited strain as starting material.

Embodiments of the present invention are described below, by way of non-limiting examples. EXAMPLES

Example 1: Screening for thermophilic Lactobacillus spp. with high acetoin levels in acidified boiled-milk.

A selection of 176 Lactobacillus sp. strains was examined for ability to acidify boiled milk at 30°C, 37°C, 40°C and 43°C for approximately 24 hours. The acidified boiled milk from 37°C incubations was examined directly after acidification for volatile organic compounds (VOC) by means of head space gas chromatography. A strain, named CHCC12697, was found to produce high levels of acetoin (135 ppm) and also fairly high levels of acetaldehyde (8 ppm) (data not shown). This strain was observed to acidify boiled milk at both a mesophilic temperature (30-37°C) as well as at thermophilic temperatures (40-43°C). The strain was also observed to grow to high OD in MRS (de Man, Rogosa and Sharpe) broth supplemented with 2% glucose or 2% lactose or 2% fructose or 2% galactose at 40 and 43°C. The strain was not resistant to antibiotics. Partial 16S rRNA gene sequencing showed that the strain was a Lactobacillus rhamnosus strain.

Example 2: Effect of Lactobacillus rhamnosus CHCC12697 strain in mesophilic process

Objective:

In this study we evaluated the benefits of using Lactobacillus rhamnosus CHCC12697 in mesophilic fermented milk products (e.g. sour cream). The purpose of using Lactobacillus rhamnosus CHCC12697 is to improve the aroma (diacetyl production), the texture and the post acidification in the end product. This strain is suggested to be added together with an CD- culture (Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris) because we would avoid any blowing problems (C0 2 production).

Materials and Method:

Materials:

O-cultures:

F-DVS MO-3 (Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris) obtainable from Chr. Hansen A/S, Denmark.

F-DVS Sico-200-0 (Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris).

Lactobacillus rhamnosus:

F-DVS Lactobacillus rhamnosus CHCC12697

Acidification :

Tests were carried out in milk with 1.5% fat in 200 ml baby bottles. The milk was heat- treated at 90°C for 20 min. and cooled to fermentation temperatures 26°C and 30°C, respectively. Hereafter, the milk was inoculated according to table 1.

Table 1 : Inoculation scheme

Temperature MO-3 Sico-200-0 CHCC12697

26°C

30°C

lOg/1001

26°C

lOg/1001

30°C

26°C lOg/1001

30°C

26°C lOg/1001

Acidification profile was recorded by measurement of pH over time. The milk was fermented to pH 4.60.

Aroma profile:

The aroma profile was measured on fermented milk at the end of fermentation (pH 4.60) and analyzed by CPG on a gas chromatograph.

The fermented milk samples were analyzed by static head space gas chromatography (HSGC) which is a powerful technique for analyzing volatiles in complex matrices. The setup consisted of a Static Head Space sampler connected to Gas Chromatograph with Flame Ionization Detector (FID). Below is a list of the apparatus (including column) and software used :

HS-autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

HS-software: HSControl v. 2.00, Perkin Elmer.

GC: Autosystem XL, Perkin Elmer.

GC-software: Totalchrom Navigator, Perkin Elmer.

Column : HP-FFAP 25 m x 0.20 mm x 0.33 μπι, Agilent Technologies

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 μί. of 4N H 2 S0 4 to 1 g fermented milk sample.

Results:

Lactobacillus rhamnosus CHCC12697 with MO-3 or Sico-200-0 at 26°C or 30°C (CHCC12697) produces more acetaldehyde than MO-3 and Sico-200-0 alone 26°C or 30°C (None) (Figure 1) .

Lactobacillus rhamnosus CHCC12697 with MO-3 or Sico-200-0 at 26°C or 30°C (CHCC12697) produces more diacetyl than MO-3 and Sico-200-0 alone 26°C or 30°C (None) (Figure 2).

Lactobacillus rhamnosus CHCC12697 with MO-3 or Sico-200-0 at 26°C or 30°C (CHCC12697) produces more acetate than MO-3 and Sico-200-0 alone 26°C or 30°C (None) (Figure 3) . The fermentation profile of Lactobacillus rhamnosus CHCC12697 with MO-3 at a fermentation temperature of 26°C is unaffected (Figure 4). MO-3 together with Lactobacillus rhamnosus CHCC12697 reached the end pH of 4.60 slightly faster.

The fermentation profile of Lactobacillus rhamnosus CHCC12697 with Sico-200-0 at a fermentation temperature of 26°C is unaffected (Figure 5). Sico-200-0 together with Lactobacillus rhamnosus CHCC12697 reached the end pH of 4.60 slightly faster and it is also faster than MO-3 + Lactobacillus rhamnosus CHCC12697 at 26°C (Figure 4).

The fermentation profile of Lactobacillus rhamnosus CHCC12697 with MO-3 at a fermentation temperature of 30°C is unaffected (Figure 6). MO-3 together with Lactobacillus rhamnosus CHCC12697 reached the end pH of 4.60 slightly faster.

The fermentation profile of Lactobacillus rhamnosus CHCC12697 with Sico-200-0 at a fermentation temperature of 30°C is unaffected (Figure 7). Sico-200-0 together with Lactobacillus rhamnosus CHCC 12697 reached the end pH of 4.60 slightly faster and it is also faster than MO-3 + Lactobacillus rhamnosus CHCC12697 at 30°C (figure 6).

DEPOSITS and EXPERT SOLUTION

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.

The Lactobacillus rhamnosus strain CHCC12697 was deposited on 2011-03-01 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig and given the accession No. : DSM24616.

The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

REFERENCES

Jyoti, B.D., Suresh, A.K., and Venkatesh, K.V. (2003) : Diacetyl production and growth of

Lactobacillus rhamnosus on multiple substrates. World Journal of Microbiology & Biotechnology 19: 509-514.

US 4,678,673 (Marshall et al.)

US 4,867,992 (Boniello et al.)

US 5,236,833 (Duboff et al.)