FIELD OF INVENTION

The invention relates to a method for treatment of milk which is to be used in the preparation of cheese, the method comprises adding a bacterial culture to the milk. It further relates to the resulting milk, and to the milk's use in a cheese process.

BACKGROUND OF INVENTION

Raw milk received for cheese production, especially in an industrial cheese plant, has to be stored until it can be used for cheese production, mainly due to bottlenecks in the cheese plant. During the storage, wherein the milk is kept cold, the mineral balance of the milk is displaced, minerals lost, and it therefore looses some of its original ability to coagulate and undergo syneresis, two very important properties in cheese making (Lane, C. N., Sousa, MJ., and McSweeney, P.L.H. (2001)).

In order to restore these properties to the milk, especially milk to be used for soft cheese such as camembert, the milk normally undergoes a processing, a so-called ^λΛ cold-maturation" step, which purpose is to prepare the milk for cheese making.

Cold maturation consists of physical and biological maturation that aims at obtaining five objectives, generally believed to make the milk more suitable for cheese making (Pernoud S. and Mayer H. L (2008)):

1. Physical maturation: Re-equilibrate the mineral balance of the milk to restore the milks ability to coagulate and undergo syneresis (e.g. by adding CaCI2 and storing the milk at temperatures between 10 and 15 C).

2. Biological maturation: Lower the pH of the milk from about pH 6.7 to a level suitable for renneting (normally in the range of pH 6.2 - 6.4 for e.g. Camembert).

3. Reduce the red-ox potential to favour the growth of strains inhibited by oxygen.

4. Produce small peptides and amino acids in surplus to support growth of lactic acid bacteria from proteolytic degradation of casein.

5. Release bacterial enzymes to enhance the ripening of the cheese.

In cold maturation the milk normally undergoes a mild heat treatment (thermisation, e.g. 62 °C for 20 seconds) or pasteurisation (e.g. 72C for 15 seconds) to remove psycrotroph bacteria such as Listeria species. CaCI2 is added and the milk is kept at 10-15 ⁰ C for 14 to 18 hrs to restore the calcium balance of the milk (physical maturation). In addition, the milk is added a lactic acid bacterial culture to achieve biological maturation as described above. Normally, lactic acid bacteria cultures that acidify milk well are used to obtain biological maturation as described above.

Following physical and biological maturation, the milk is normally pasteurised (e.g., 72 ⁰ C for 20 seconds) to kill and lyse the culture used for the biological maturation thereby releasing bacterial enzymes that may assist in ripening.

Even if cold maturation brings many advantages to the cheese making process, its present day implementation may also be a source of variation in yield and quality if not perfectly controlled by the cheese maker.

First, the pH of renneting is to a wide extent governed by the pH reached in cold maturation. It is well known, that the level of pH during renneting will have an important influence on coagulation and syneresis and therefore the moisture content and yield of soft cheese (Mietton, B., Gaucheron, F. and Salaϋn-Michel, F. (2004)).

Furthermore, the moisture content of the soft cheese has a direct influence on yield and texture and an indirect influence on ripening.

SUMMARY OF INVENTION

Surprisingly, the present inventors have found that lactic acid bacteria cultures that only acidify milk slowly are less sensitive to variations in process temperature and process times during cold maturation. And that this may result in a cheese product with uniform yield and quality from batch to batch, ensuring more profitable cheese making. Experiments wherein a protease negative culture of lactic acid bacteria has been added to milk have surprisingly revealed that the resulting milk results in standardized milks which can be used for cheese production, and the present inventors contemplate that addition to milk of other slow acidifying lactic acid bacteria will result in a standardized milk.

In accordance with this surprising finding, the present inventors have invented a novel method for maturation and storage of the milk to be used for cheese production, the method results in more standardized milk for cheese making, which may result in a cheese product with uniform yield and quality from batch to batch.

The method comprises addition of a slow acidifying lactic acid bacteria culture that exhibits limited acidification in the milk. By using such a culture, it has turned out that the following benefits are obtained:

The cold maturation acidification of the milk by the culture is less sensitive to fluctuations in process temperatures, which may reduce batch to batch variations in a cheese product. The acidification of the milk by the culture is less sensitive to fluctuations in process times, which may reduce batch to batch variations in a cheese product.

DETAILED DISCLOSURE

In a first aspect, the present invention relates to a method of treatment of milk (e.g. to be used for production of cheese), said method comprises adding to the milk a culture of bacteria which: is not able to acidify the milk, or acidifies the milk slowly, and/or is not able to grow in milk, or grows slowly in milk.

The invention also relates to a method of treatment of milk (e.g. to be used for production of cheese), said method comprises adding to the milk a culture of bacteria which are (substantially) protease negative and/or (substantially) incapable of degrading milk proteins (e.g. casein).

The present invention further relates to a method of treatment of milk (e.g. to be used for production of cheese), said method comprises adding to the milk a culture of bacteria, which culture is characterized by that it lowers the pH of the milk less than 0.25 pH units per hour at 30 degrees C, such as less than 0.20 pH units, less than 0.15 pH units, or less than 0.10 pH units, when inoculated at a quantity of 10E6 cfu (cell forming units) per ml milk (esp RSM or laboratory milk).

In an embodiment of the methods of the invention, the bacteria (in the culture) are selected from the group consisting of: bacteria which are (substantially) protease negative, - bacteria which are (substantially) incapable of degrading milk proteins (e.g. casein), bacteria which are (substantially) incapable of degrading lactose, lactate dehydrogenase (Ldh) defective bacteria (cf. EP0928333B1) bacteria which are thymidine auxotrophic mutants (thyA) (cf. EP1102837B1), and bacteria which are defective in pyruvate formate-lyase (PfI) (cf. EP0928333B1).

It should be understood that the culture may comprise one or more strains (or bacteria belonging to such a strain) of the group defined above, optionally together with other strains, as long as the culture as a whole is not able to grow in milk, grows slowly in milk, or is (substantially) protease negative, or is (substantially) incapable of degrading milk proteins, lactose, etc..

It is presently preferred that the culture of bacteria is a culture of lactic acid bacteria, such as a culture of one or more strains, selected from the group consisting of Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacteήum spp., Enterococcus spp. and Propionibacterium spp. However, the culture may comprise one or more strains of other species than these.

The culture should lower the pH of the milk less than 0.25 pH units per hour at 30 degrees C, such as less than 0.20 pH units, less than 0.15 pH units, or less than 0.10 pH units, when inoculated at a quantity of 10E6 cfu (cell forming units) per ml milk (esp. in RSM or in laboratory milk).

Interesting cultures to be used is a culture of a strain (or comprising a strain) selected from the group consisting of DN224 (DSM11037), DIM223 (DSM11036), DN221 (DSM11034), DIN227 (DSM11040), MBP71 (DSM12891), DN105 (DSM12289), or mutants or variants of any of these strains.

In a presently preferred embodiment, the method of the invention further comprises: a) no microorganisms, which are able to degrade (a substantial part of) milk proteins, are added to the milk, or are allowed to act on the milk proteins; and/or b) the milk is not subjected to any (substantial) degradation of milk proteins.

Thus, the present invention relates to a method of treatment of milk, said method comprises adding to the milk a culture of bacteria which are (substantially) protease negative and/or (substantially) incapable of degrading milk proteins (e.g. casein).

In an other embodiment, milk is treated in a way such that less than 30 % (such as less than 20%, less than 10%, or less than 5%) of the milk proteins (e.g. casein) are degraded, or less than 30% % (such as less than 20%, less than 10%, or less than 5%) of the lactose is degraded, such as when in the milk is stored 24 hours at 15 degrees C after inoculation with 10E6 cfu/ml milk.

The method may further comprise: a) heat treatment of the milk (such as to a temperature in the range 40-80 degrees C, e.g. pasteurization or thermisation); b) cooling the milk (such as to a temperature in the range 5-15 degrees C); and c) storing the milk (such as for 1-48 hours); and d) optionally heat treatment of the milk (e.g. pasteurization or thermisation). In this embodiment, the culture should added after step a).

In yet an embodiment, the milk is kept at a temperature below 20 degrees C, such as at a temperature in the range 5-20 degrees C, or in the range 5-15 degrees C, such as from 1-48 hours (e.g. from 4-24 hours or from 5-20 hours). It is presently preferred that the culture is added to the milk in a final concentration of 10E3 to 10E12 CFU pr ml milk, such as from 10E5 to 10E10 cfu/ml, or from 10E7 to 10E9 cfu/ml.

In an other aspect, the invention relates to a milk, e.g. for use in the production of cheese, which is obtainable by a method of the invention, or a milk which is treated using a method of the invention.

In a further aspect, the invention relates to a method for preparing cheese, wherein a milk of the present invention or a milk obtainable by a method of the invention, is contacted with a a) a culture of lactic acid bacteria, such as a culture of protease positive bacteria; and b) a coagulant, such as a protease (e.g. rennet, a chymosin or a microbial coagulant). The method may comprise further cheese making steps. Such steps are known to the person skilled in the art.

In a last aspect, the invention relates to a cheese obtainable by the method of the invention, such as a soft cheese, e.g. camembert.

DEFINITIONS

In the present context, the term "milk" refers to 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, and especially raw cow's milk. However, it should be understood that the term milk also comprises compositions comprising milk, and milk compositions that have been treated, e.g. chemically, enzymatically, and/or mechanically.

In the context of the present invention, "microorganism" may include any bacterium, or fungus being able to ferment the milk substrate. Lactic acid bacteria are preferred microorganisms.

The microorganisms used for most fermented milk products are selected from the group of bacteria generally referred to as lactic acid bacteria. 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. A strain which might be used in the present invention is a strain which has lost the capability of de novo synthesising essentially compounds (also referred to in the art as an "auxotrophic strain"). Therefore, in preferred embodiments, the bacterial strain is a strain being auxothrophic in respect of a compound which is not present in the milk and which is required by the strain for growth.

As used herein, the term "culture" refers to any sample or item that contains one or more microorganisms. "Pure cultures" are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to "mixed cultures," which are cultures in which more than one genus and/or species of microorganism are present. In some embodiments of the present invention, pure cultures find use, but normally a culture as used in present invention contains more than one strain.

In the context of the present invention, "laboratory milk" is a reconstituted skim milk (RSM) with 9.5% dry-matter on a weight basis that has been subjected to temperatures of 99C for 30 minutes before use.

In the context of the present invention, a "slow acidifying" bacteria culture (or a culture which acidifies the milk slowly) is a culture which has a maximum rate of acidification of 0.25 pH units per hour at 30 degrees C when inoculated at a quantity of 10^6 cfu (10E6 cell forming units) per ml laboratory milk. (For instance, if the culture consists of more than one strain, the culture as a whole should have the max rate of acidification of 0.25 pH per hour at 3OC as when inoculated 10E6 cfu/ml milk as defined above). In the context of the present invention, any other culture than a slow acidifying culture is defined as a "fast acidifying" culture.

The term "grows slowly" in the present context means that the cell count in the milk doubles in more than two hours (such as more than 4, 6, 10, or 24 hours) at 30 degrees C when inoculated at a quantity of 10 ^A 6 cfu (10E6 cell forming units) per ml (laboratory) milk.

By the term "substantially" is understood that the enzymatic activity (or the degrading activity) is less than 10% of the type strain of the same species or of the mother strain from which the mutant is obtained, measured under identical conditions.

"Soft cheese" is defined as any Rennet coagulated cheese that contains about 70 - 74 % moisture on a non fat solids basis and is produced without scalding and pressing.

In the present context, the term "mutant" should be understood as a strain derived, or a strain which can be derived, from a strain of the invention (or the mother strain) by means of e.g. genetic engineering, radiation and/or chemical treatment. 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 acidification speed) 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 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.

In the present context, the term ^" "variant" should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties e.g. regarding acidification speed). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

The use of the terms "a" and "an" and "the" and similar referents 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.

EXAMPLES

Example 1: reduced sensitivity towards process delays in a laboratory system

Two different frozen Direct Vat Set (F-DVS) cultures (obtainable from Chr. Hansen, Hørsholm, Denmark): Culture A: Bulk Item 699759 F-DVS SICO-Ol-O-PRT- and Culture B: Bulk Item 693091 F-DVS SICO-14-O were Inoculated 10 g per 100 kg milk in re-constituted skim milk (9.5 % DM) and heat treated milk (99C for 30 minutes), cooled to fermentation temperature 1OC, 15C and 2OC, respectively. Culture A represents the slow acidifying culture. Culture B represents the fast acidifying culture.

The results shown in figure 1 below, wherein the thin solid horizontal line represents pH 6.4 our target for soft cheese. At 20 ⁰ C this target is reached after 5.25 hours for Culture B (dashed bold line), and 7.0 hours for Culture A (solid bold line).

As seen from the curve, a delay of one hour at 20 C will translate into a pH drop of about 0.15 pH units for culture B and only about 0.05 pH units for culture A.

We therefore conclude that the acidification of the protease negative, slow acidifying culture will be more robust towards delays in production than the protease positive, fast acidifying culture.

Example 2: Sensitivity towards temperature fluctuations

Two different frozen direct vat set (F-DVS) cultures. Both obtainable from Chr Hansen, Hørsholm, Denmark: Culture A: Bulk Item 699759 F-DVS SICO-01-O-PRT-, and Culture B: Bulk Item 693091 F-DVS SICO-14-0, were Inoculated 10 g per 100 kg milk in re-constituted skim milk (9.5 % DM) and heat treated milk (99C for 30 minutes), cooled to fermentation temperature 1OC, 15C and 2OC, respectively.

Culture A represents the slow acidifying culture. Culture B represents the normal acidifying culture.

Figure 2 below shows the pH after 12 hours at 1OC, 15C, and 2OC, respectively. The horizontal line represents pH 6.4 our target for soft cheese. The blue line is a calculated fit to the data.

If pH 6.4 is sought, it will be reached in 12 hours at 14 ⁰ C with culture B (filled Triangles), and in 12 hours at 15.5 °C with culture A (Filled Circles).

A variation in temperature of 13 to 15 °C will translate into a pH variation of about 0.3 pH units for culture B.

A variation in temperature of 14.5 to 16.5 ⁰ C will translate into a pH variation of about 0.125 pH units for culture A.

We therefore conclude that the acidification of the slow acidifying culture is more robust towards temperature fluctuations than the fast acidifying culture. Example 3: reduced batch to batch variation due to process delays in soft cheese Culture A: slow acidification culture 699759 F-DVS SICO-Ol-O-PRT-, Culture B: traditional fast acidifying cold maturation culture 501691 F-DVS FLORA-DANICA, both obtainable from Chr Hansen, Hørsholm, Denmark

For each culture A and B: Pasteurized milk is inocultated with the culture (72C for 15s), cooled to 15C with 5 g culture per 100 liter milk, and fermented at 15C for until pH 6.4 is reached, note the time (TO) needed.

At TO half of the milk is collected for pasteurization (72C for 15s) and camembert is produced according to a standard soft cheese make (see below).

The other half of the milk is allowed to continue the fermentation at TO + 1 hr, then pasteurized (72C for 15s) the milk and to produce camembert according to a standard soft cheese make (see below).

The experiment is repeated a sufficient number of times, e.g. six times for each culture. For each experiment the weight, moisture, and flavor difference between the cheeses made from TO milk and those from TO+lhr milk is calculated.

The magnitudes of these differences are compared for each culture, using Analysis of Variance.

Example 4; reduced batch to batch variation due to temperature differences in soft cheese Culture A: slow acidification culture 699759 F-DVS SICO-Ol-O-PRT-, Culture B: traditional fast acidifying cold maturation culture 501691 F-DVS FLORA-DANICA, both obtainable from Chr Hansen, Hørsholm, Denmark.

For each culture A and B: Inoculate simultaneously with 5 g culture per 100 liter pasteurized milk (72C for 15s), cooled to 14, 15, and 16C, respectively. Ferment until pH 6.4 is reached at 15C, and pasteurize (72C for 15s) each batch of milk simultaneously.

With each batch of milk produce camembert according to a standard soft cheese make (see below).

Repeat a sufficient number of times, e.g. six times for each culture. For each experiment calculate the weight, moisture, and flavor difference between the cheeses made from 15C milk and those from 14C and 16C, respectively.

Compare the magnitude of these differences for each culture, using Analysis of Variance. Standard soft cheese make (Camembert style)

After pasteurization, cool the milk to 33 - 36C and add CaCl2 as needed.

Inoculate with 2000 g F-DVS FLORA DANICA and add 50 U PCAl and 10 U Geo CDA per 10000 liters (all cultures are obtainable from Chr Hansen, Hørsholm, Denmark)

Ferment the milk at the 33 Cto 36 C for 1 to 2 hrs. Add 1.3 to 1.6 liters of CHY-MAX plus per IOOOOL (obtainable from Chr. Hansen, Hørsholm, Denmark). Floatation time should be 7 to 8 minutes, coagulation time 30 - 40 minutes.

Cut curd in 2 x 2 x 2 cm cubes, and after some syneresis, pre-draw 30 - 40 % of the whey.

Put curd into standard camembert moulds after 1 hour and 15 minutes to 1 hour and 25 minutes in the vat. Drain whey at 28 - 20 C. Turn moulds three times, until a pH 4.7 to 4.9 is reached.

Remove the cheeses from the moulds and measure the weight and the moisture content of representative sample cheeses.

Dry salt the remaining cheeses, by rubbing salt on the surfaces.

Dry the cheeses at 14 to 16 C at a relative humidity of 85 to 90% for one day.

Ripen the cheeses at 12C at 95% relative humidity for eight days, then pack the cheeses and cool to 5C.

Sensory evaluation of the cheeses is carried out after 21 days.

Example 5: a slow and a fast acidifying culture

Two different frozen Direct Vat Set (F-DVS) cultures (obtainable from Chr Hansen, Hørsholm, Denmark): Culture A: Bulk Item 699759 F-DVS SICO-01-O-PRT-, and Culture B: Bulk Item 693091 F-DVS SICO-14-0, were Inoculated 5 g per 100 kg milk (10 ^Λ 6 cfu / ml) in re- constituted skim milk (9.5 % DM) and heat treated milk (99C for 30 minutes), cooled to fermentation temperature 3OC.

The results are shown in the figure 3 below. Culture A (solid bold line) represents the slow acidifying culture. Culture B (dashed bold line) represents the fast acidifying culture. The thin dotted line represents the maximum acidification rate of 0.25 pH units per hour (i.e. the maximum slope of the acidification curve) defining a slow acidifying culture in the context of this invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

Pernoud, S. and Mayer H. L. (2008) "Suppression de Ia maturation froide biologique", La Revue

Laitiere Francaise (2008), No 678 p. 26 Lane, C. N., Sousa, MJ., and McSweeney, P.L.H. (2001) "Effect of prematuration conditions on the proteolytic and rheological properties of cheesemilk", Lait 81, pp 415 - 427

Mietton, B., Gaucheron, F. and Salaun-Michel, F. (2004) "Minereux et transformations fromageres", Chapter 16 in "Minereaux et produits laitieres" ed. Gaucheron, F., Lavoiser ISBN

2-7430-0641 WO2000/001799A1, EP1102837B1 (thyA), WO1998/07843A1, EP928333B1 (pyruvate formate- lyase (PfI) defective lactic acid bacterium).

All references cited in this patent document are hereby incorporated herein in their entirety by reference.