EA048110B1 - METHOD FOR PRODUCING A FERMENTED DAIRY PRODUCT WITH INCREASED LEVEL OF PROBIOTICS - Google Patents

Description

Field of invention

The invention relates to compositions and methods for producing fermented dairy products with an increased amount of probiotic bacteria.

Prior art

Probiotic strains such as Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis are widely used in fermented dairy products.

These probiotic strains, when inoculated into milk as single strains, grow very slowly. In addition, some strains are not able to acidify to a pH below 6.0 within 24 h, see e.g. Fig. 1, which shows that BB-12® and LGG® (BB-12® and LGG® are registered trademarks of Chr. Hansen A/S), respectively, do not grow/acidify properly when inoculated without yoghurt culture. In combination with yoghurt culture, the probiotic strain(s) may grow slightly better than a single strain, but rarely grow more than 1 log. Due to the taste and aroma desired by consumers, typical yoghurt fermentation stops at a pH of 4.60-4.55. In addition, for safety reasons, it is important to achieve a low pH, e.g. a pH below approximately 5.5, e.g. pH 4.60-4.55. At this point (pH 4.60-4.55) the yogurt species Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) are dominant over the probiotic strain(s). Overall, the final probiotic yogurt contains approximately 5 E+08 to 1 E+09 CFU (colony forming unit)/ml ST and LB, and 2-3 E+07 to 1 E+08 CFU/ml probiotic strain.

In addition, over the course of shelf life (i.e., storage for 50-60 days, which is the typical shelf life of fresh fermented products in North America and some other regions of the world), the probiotic cell count in a typical probiotic yogurt decreases. For example, over the course of shelf life, the cell count of Bifidobacterium - BB-12® typically decreases by 0.5-1 log over 50-60 days, depending on the yogurt culture, milk base, culture, and storage conditions. The cell count of LA-5® (LA-5® is a registered trademark of Chr. Hansen A/S) typically decreases by 1-2 log over 50-60 days of shelf life.

Certain markets or product types require higher probiotic levels than are possible with mixtures of traditional yoghurt culture and probiotic strain(s) (2-3 E+07 - 1 E+08 CFU (colony forming units)/ml), particularly where cell counts are maintained throughout the shelf life of the products. Examples of such products include:

1) fermented probiotic portion drinks, which must contain a documented level of probiotics (1E+09 CFU/serving) in 65 ml of product after 60 days of storage;

2) probiotic yogurt in which documented levels of probiotics (1E+09 CFU/serving) are present for more than 50-60 days;

3) probiotic yogurt with a very high cell count (10-20E+09 CFU/serving); and

4) Freeze-dried yoghurt pearls (pellets) and drops (wafers). Probiotic yoghurt intended for this application must contain a very high cell count of the probiotic strain (5E+08 CFU/g) to ensure an effective dose (1E+09 CFU/serving at the end of shelf life) after processing, freezing and freeze-drying.

A specially developed culture and combination of strains can support the growth of, for example, Bifidobacterium - BB-12® up to 1-2E+08 CFU/ml (see, for example, WO 2008/148561). Higher numbers of some probiotic strains can also be achieved by increased inoculation rates (5-10 times higher - 0.05-0.1%), but this route is expensive and almost never used.

WO 2017/125600 shows that co-cultivation of lactose(-)sucrose(+) S. thermophilus (ST) in combination with L. paracasei CRL 431 under specific conditions resulted in a higher number of L. paracasei CRL 431 cells compared to co-cultivation of lactose(+) S. thermophilus (ST) in combination with L. paracasei CRL 431.

There is a need for other compositions and methods for producing fermented dairy products with a high number of viable probiotic cells, such as Bifidobacterium animalis subsp. lactis - BB-12®, Lactobacillus acidophilus - LA-5® or Lactobacillus rhamnosus - LGG®, in particular wherein the number of viable cells increases during the shelf life of the fermented dairy product, which is typically 60 days, preferably at 4°C.

Brief summary of the invention

The present invention is based on unexpected experimental data that when using:

a) a starter culture containing at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one

- 1 048110 a lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus; and

b) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are present in the composition in an amount measured such that it/they are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3 (e.g. about 0.41% sucrose, wherein % is weight per volume of the total amount of the milk base (% w/v), and where the milk base contains about 2% by weight (w/v) fat and about 4.1% (w/v) protein, the starter culture in (a) is preferably added as a frozen concentrated culture in an amount of about 0.01% weight per volume of the total amount of the milk base (% w/v), and the fermentation temperature is about 38°C) during fermentation of the milk base in the presence of a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, the amount of probiotic bacteria present in this fermented dairy product is increased compared to the amount of probiotic bacteria in a fermented dairy product fermented with:

only probiotic bacteria (as stated above, probiotic strains, when inoculated into milk as single strains, grow very slowly, see also Fig. 1), or a starter culture comprising at least one strain of Streptococcus thermophilus that is not lactose-deficient and at least one strain of Lactobacillus that is not lactose-deficient, such as a strain of Lactobacillus delbrueckii spp. bulgaricus (a traditional lactose (+) yogurt culture), or a starter culture comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, such as a lactose-deficient Lactobacillus delbrueckii spp. bulgaricus, in the presence of sucrose in an amount measured such that it is depleted at a pH of the fermented milk of less than 4.9, such as about 4.55 (for example, 0.9% sucrose, where % is a percentage by weight per volume (% w/v) based on a milk base, when the milk base contains about 2% (w/v) fat and about 4.1% (w/v) protein, the starter culture is preferably added as a frozen concentrated culture in an amount of 0.01% (w/v) of the total amount of the milk base, and the fermentation temperature is 38°C).

Furthermore, there is an improved or increased survival of the probiotic cells over time, for example over a shelf life of at least 60 days (storage) at about 4°C. The increase in the number of viable probiotic bacteria present in the fermented dairy product is maintained over time, for example immediately after the completion of the fermentation, preferably for more than 1 day after the completion of the fermentation, for example more than 15 days or more than 45 days, even for more than 60 days after the completion of the fermentation. Accordingly, the total number of cells of viable probiotic strains in the presence of the starter culture according to the invention, as defined in (a) above, increases compared to the total number of cells of viable probiotic strains in the absence of the starter culture according to the invention, as defined in (a) above, and this increase is maintained over time, for example after 60 days of storage (shelf life), preferably at about 4°C.

Accordingly, according to the present invention, a method for producing a fermented milk product is provided, comprising the following steps:

1) adding to the milk base:

a) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain;

2) and fermenting this milk base for a period of time until a target pH is reached (preferably from about 4.8 to about 4.0, more preferably from about 4.6 to about 4.3, even more preferably about 4.55), to obtain a fermented milk product.

Furthermore, according to the present invention, there is provided a fermented milk product obtained by the method of the invention and a food or feed product containing at least

- 2 048110 one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, and a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, preferably wherein the probiotic Lactobacillus strain is not a Lactobacillus paracasei strain, even more preferably wherein the probiotic Lactobacillus strain is not L. paracasei strain CRL 431 deposited as ATCC 55544 or L. paracasei strain CHCC 2115 deposited as DSM 19465, wherein the food or feed product comprises more than 1.3E+08 CFU of probiotic bacteria/g of fermented milk product (CFU/g), preferably more than 2E+08 CFU/g, even more preferably more than 5E+08 CFU/g of probiotic strain after fermentation, preferably after at least 1 day of storage at about 4°C.

In addition, according to the present invention, compositions for producing a fermented milk product are proposed, comprising:

a) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, such as a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus; and

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3.

Furthermore, according to the present invention, there is provided the use of the composition of the present invention for increasing the number of viable probiotic cells in a fermented dairy product or for improving the survival of probiotic cells over time, preferably for 60 days, preferably at 4°C, compared to a fermented dairy product fermented using a composition containing

a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus which is not lactose-deficient and at least one strain of Lactobacillus which is not lactose-deficient, preferably a strain of L. delbrueckii subsp. bulgaricus which is not lactose-deficient; and/or

b) 1) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, and

2) one or more non-lactose carbohydrates capable of being metabolized by lactic acid bacteria as defined in (1), wherein said non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3.

Brief description of graphic materials

Fig. 1. Acidification profile of Bifidobacterium - BB-12® (strain Bifidobacterium animalis subst. lactis BB-12® - deposited as DSM 15954) (BB-12®, A, solid line) and L. rhamnosus - LGG® (strain Lactobacillus rhamnosus - LGG® -deposited as ATCC 53103) (LGG®, A, dotted line) inoculated into milk base at 0.01% and incubated at 38°C.

Fig. 2. Acidification profile of a combination of Acidifix® 1.0 (Acidifix® is a registered trademark of Chr. Hansen A/S) and bifidobacteria -BB-12® (Bifidobacterium animalis strain subst. lactis - BB-12® - deposited as DSM 15954) (Acidifix® 1.0, BB-12®, dotted line), Acidifix® 1.0, BB-12® and LA-5® (Lactobacillus acidophilus strain - LA-5® - deposited as DSM 13241) (Acidifix® 1.0, BB-12® and LA-5®, solid line) or Acidifix® 1.0, BB-12® and L. rhamnosus LGG® (Lactobacillus rhamnosus strain - LGG® - deposited as ATCC 53103) (Acidifix® 1.0, BB12® and LGG®, dotted line) inoculated into a milk base and incubated at 38°C.

Fig. 3 Acidification profiles of Acidifix® 1.0 plus 0.01% BB-12® in milk with 0.41% (B) and 0.90% sucrose (D). YoFlex® Mild® 1.0 plus 0.01% BB-12® (E) was used as control (YoFlex® Mild is a registered trademark of Chr. Hansen A/S). % Sucrose is expressed as w/v based on milk base.

Detailed disclosure of the invention

Method for obtaining a fermented milk product

The present invention relates to a method for producing a fermented milk product comprising the following steps:

1) adding to the milk base:

- 3 048110

a) a starter culture of lactic acid bacteria (LAB) comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is(are) added in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain;

2) fermenting the given milk base for some period of time until the target or desired pH is reached, thereby obtaining a fermented milk product.

In the context of the present invention, in any of its embodiments, the expression fermented dairy product means a food or feed product, wherein the production of this food or feed product includes fermentation of a milk base with a lactic acid bacterium. The term fermented dairy product as used herein includes, but is not limited to, products such as thermophilically fermented dairy products, such as yoghurt, drinking yoghurt, broken-curd yoghurt, thermostat-processed yoghurt or a yoghurt-like drink. For example, yoghurt may be filtered to remove most of the whey, resulting in a thicker consistency than unfiltered yoghurt (filtered yoghurt or high solids yoghurt).

In the context of the present invention, in any of its embodiments, the term milk should be understood in the context of the present invention as a secretion of the mammary glands obtained by milking any animal, such as cows, sheep, goats, buffalo or camels. In a preferred embodiment, the milk is cow's milk. According to the present invention, the milk may have been processed, and the term milk includes whole milk, skimmed milk, low-fat milk, full-fat milk, low-lactose milk (e.g. ultra-filtered (UF) milk, provided that lactose is not broken down by the enzyme lactase into glucose and galactose) or condensed milk. Skimmed milk is a low-fat or skimmed milk product. Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat. Full-fat milk often contains 2% fat or more. It is understood that the term milk covers milk from different sources. Mammals that produce milk include, but are not limited to, cow, sheep, goat, buffalo, camel, llama, mare, and deer.

The term dairy base may refer to any dairy substance that can be fermented according to the present invention. Thus, useful dairy bases include, but are not limited to, fractions and solutions/suspensions of any milk or milk-like product containing protein, such as whole or low-fat milk, skimmed milk, buttermilk, reconstituted milk powder, evaporated milk, milk powder, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate or cream. Obviously, the dairy base may be derived from any mammal, for example, being essentially pure mammalian milk or reconstituted milk powder.

In a preferred embodiment of the invention, the milk base to which the starter culture (1a), the non-lactose carbohydrate (1b) and the probiotic strain(s) (1c) are added in step (1) of the method according to the invention has a lactose content of 30.0 mg/ml to 70 mg/ml, preferably 35 mg/ml to 65 mg/ml, more preferably 40 mg/ml to 60 mg/ml and most preferably 50 mg/ml to 60 mg/ml. The level of lactose is not significant. Lactose may be added to the milk base, but only a portion will be fermented by the probiotics.

Preferably, the milk base comprises at least about 2.5% by weight protein, preferably about 2.9 to about 4.5% by weight protein, even more preferably about 4 to about 4.5% by weight protein, such as about 4.1% by weight protein. These amounts of protein in the milk base result in a good broken-curd yoghurt or drinking yoghurt. Preferably, the milk base comprises about 0 to about 3.8% by weight fat, such as about 0.5 to about 3.25% by weight fat. More preferably, the milk base comprises about 2% by weight fat. In a preferred embodiment, the milk base comprises about 2% by weight fat and about 4.1% by weight protein.

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

The term homogenization as used in the context of the present invention in any of its embodiments means vigorous mixing to produce a soluble suspension.

- 4 048110 penzies or emulsions. When homogenization is carried out before fermentation, it can be carried out in such a way as to break down the milk fat into smaller sizes so that it no longer separates from the milk. This can be done by forcing the milk through small holes under high pressure.

The term pasteurization as used in the context of the present invention in any of its embodiments means the treatment of the milk base to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a precisely determined temperature for a precisely determined period of time. This precisely determined temperature is typically achieved by heating. The temperature and duration of time may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may then follow. For example, the milk base may be treated by heating at 92°C for 3 minutes, cooled to 38°C and then inoculated as described in step (1) of the method of the present invention.

Step 1 of the method of the present invention comprises adding to the milk base:

a) a lactic acid bacteria (LAB) starter culture comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, such as a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus.

Preferably, the starter culture comprises two lactose-deficient strains of Streptococcus thermophilus that are capable of metabolizing a non-lactose carbohydrate and one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus.

The addition of strains to the milk base may also be referred to in the context of the present invention as inoculation.

In the context of the present invention, in any of its embodiments, the expression lactic acid bacteria (LAB) means food-grade bacteria that produce lactic acid as the main metabolic end product of carbohydrate fermentation. These bacteria are related in their common metabolic and physiological characteristics, and are typically Gram-positive, low GC, acid-tolerant, non-spore-forming, non-respiring rod-shaped bacilli or cocci. During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, lowering the pH and leading to the formation of a protein curd. These bacteria are thus responsible for the souring of milk and for the consistency of the dairy product. The most industrially useful lactic acid bacteria are within the order Lactobacillales, which includes Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Pediococcus species and Propionibacterium species. They are often used as food cultures alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including bacteria of the species Lactobacillus and Streptococcus, are commonly supplied to the dairy industry as either frozen (F-DVS) or freeze-dried (FDDVS) cultures for propagation of bulk starter, or as so-called direct inoculation (DVS) cultures intended for direct inoculation into a fermentation vessel or tank for the production of a dairy product such as a fermented milk product. Such cultures of lactic acid bacteria are commonly referred to as starter cultures or cultures. A typical yogurt starter culture contains Streptococcus thermophilus (also referred to herein as ST or St) and Lactobacillus delbrueckii subsp. bulgaricus (also referred to herein as LB or Lb), and in most countries yogurt is defined by law as a fermented dairy product produced using a starter culture containing the two specified strains.

The lactic acid bacteria (LAB) starter culture according to the present invention in any of its embodiments comprises or, alternatively, consists of at least one lactose-deficient Streptococcus thermophilus strain capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient Lactobacillus strain capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strain. The starter cultures are responsible for the acidification of the milk base. The starter cultures can be fresh, frozen or lyophilized.

For the production of a fermented milk product, the starter culture can be added in any amount. Typically, the starter culture is added in an amount to achieve a concentration of 0.001 to 3%, such as 0.05%, 0.01%, 0.015%, 0.02%, 1%, 2%, 3%, preferably 0.001 to 0.025%, where % is the weight per volume of the total milk base (% w/v), such as 0.0015 to 0.15% w/v, such as 0.01 to 0.015% w/v or 0.01 to 0.02% w/v, or 0.01 to 0.025% w/v of the total milk base. Preferably, the starter culture is added as a frozen concentrated culture in an amount of 0.01%

- 5 048 110 w/v to 0.04% w/v of the total milk base, such as from 0.01% w/v to 0.02% w/v. Frozen concentrated cultures typically contain from 6E+10 to 1.5E+11 CFU/g. Alternatively, the starter culture is added as a lyophilized culture in an amount of from 0.001% to 0.0025% w/v of the total milk base. More preferably, the starter culture is added as a frozen concentrated culture in an amount to achieve a concentration of about 0.01% weight per volume (% w/v) of the total milk, preferably wherein the milk has a fat content of about 2% by weight and a protein content of about 4.1% by weight.

In a preferred embodiment, the starter culture is added to the milk base in an amount of from about 1E+06 to about 1E+08 CFU/ml of the milk base (total bacteria, i.e. at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus which is capable of metabolizing a non-lactose carbohydrate), preferably in an amount of from about 5E+06 to about 1E+07 CFU/ml of the milk base, such as from about 6E+06 CFU/ml to about 1.5E+07 CFU/ml, even more preferably in an amount of from about 1.2 to about 1.3E+07 CFU/ml, preferably when the milk base has a fat content of about 2% by weight and about 4.1% by weight of protein.

As disclosed in WO 2005/003327, it is advantageous to add certain cryoprotectants to the starter culture. Thus, the starter culture of step (1a) of the method of the present invention may comprise one or more cryoprotectants selected from the group consisting of inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uranosine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, orotidine, thymidine, inosine and a derivative of any such compounds.

The terms lactose metabolism deficiency and lactose-deficient are used in the context of the present invention in any of its embodiments to characterize LAB that either partially or completely lose the ability to utilize lactose as a source for cell growth or maintenance of cell viability. The corresponding LAB are capable of metabolizing one or more carbohydrates selected from sucrose, galactose and/or glucose, or any other fermentable carbohydrate. Since these carbohydrates are not naturally present in milk in sufficient quantities to support fermentation by lactose-deficient mutants, it is necessary to add these carbohydrates to the milk. Lactose-deficient and partially lactose-deficient LAB can be distinguished by white colonies on a medium containing lactose and X-Gal. Lactose-deficient LAB and methods for producing them have been generally described, exemplified, and deposited in previous published patent applications, including WO 2013/160413, PCT/EP2015/063767, and PCT/EP2015/063742, which describe methods for producing LAB deficient in lactose metabolism and specific strains obtained by these methods.

The term capable of metabolizing one or more carbohydrates other than lactose present in milk is used in the context of the present invention in any of its embodiments to describe the metabolic activity of lactose-deficient LAB that causes the production of lactic acid as the main metabolic end product of carbohydrate fermentation using a carbohydrate other than lactose.

In a particular embodiment of the invention, the lactose-deficient strain(s) is(are) capable of metabolizing one or more non-lactose carbohydrates selected from the group consisting of sucrose, galactose and glucose, preferably sucrose. In a particular embodiment of the invention, the lactose-deficient strain(s) is(are) capable of metabolizing galactose.

In a preferred embodiment, at least one lactose-deficient Streptococcus thermophilus strain that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient Lactobacillus strain that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient Lactobacillus delbrueckii spp. bulgaricus strain, which is contained in the starter culture that is added to the milk base in step a of the present invention, are capable of metabolizing the same non-lactose carbohydrate, which is preferably sucrose. In other embodiments, at least one lactose-deficient Streptococcus thermophilus strain that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient Lactobacillus strain that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient Lactobacillus delbrueckii spp. bulgaricus, which are contained in the starter culture, which is added to the milk base in step a of the present invention, are capable of metabolizing various non-lactose carbohydrates, preferably wherein these non-lactose carbohydrates are not glucose. For example, at least one lactose-deficient strain of Streptococcus thermophilus is capable of metabolizing sucrose, and at least one lactose-deficient strain of Lactobacillus is capable of metabolizing galactose, or vice versa.

Preferably, the lactose-deficient strain of Streptococcus thermophilus is selected from the group consisting of

- 6 048110 of the following:

(a) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28952;

(2) a strain derived from DSM 28952, wherein this derivative strain is further characterized by the ability to form white colonies on a medium containing lactose and X-Gal;

(b) (1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28953;

(2) a strain derived from DSM 28953, wherein this derivative strain is further distinguished by the ability to form white colonies on a medium containing lactose and X-Gal;

(c) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32599;

(2) a strain derived from DSM 32599, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal; and (d) (1) the strain deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32600; and (2) a strain derived from DSM 32600, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

Preferably, the lactose-deficient strain of Lactobacillus present in the starter culture is a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus. More preferably, the lactose-deficient strain of Lactobacillus delbrueckii subsp. Bulgaricus is selected from the group consisting of the following:

(1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28910; and (2) a strain derived from DSM28910, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

In the context of the present invention, in any of its embodiments, the phrase strain derived from or strain that can be derived from (strains derived from them) or mutant means strains that have been obtained from other strains (e.g. the above-mentioned deposited strains), for example by genetic engineering, radiation and/or chemical treatment. Strains derived from them or mutants may also be spontaneously occurring mutants. It is preferred that strains derived from them or mutants are functionally equivalent mutants, for example mutants that have substantially the same or improved properties, compared to their parent strain. For example, a derived strain or mutant is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal. In particular, the phrases strains, derived therefrom or mutants refer to strains obtained by subjecting a strain of the invention (e.g. the above-mentioned deposited strains) to any conventionally used mutagenic treatment, including treatment with a chemical mutagen such as ethane methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV (ultraviolet) light, or to a spontaneously occurring mutant. The mutant may have been subjected to several mutagenic treatments (one treatment should be understood as one mutagenesis step, followed by a screening/selection step), but it is currently 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 currently preferred mutant, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced by a different nucleotide or have been deleted compared to the parent strain.

In a preferred embodiment of the present invention, at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolizing a non-lactose carbohydrate and/or at least one lactose-deficient strain of Lactobacillus which is capable of metabolizing a non-lactose carbohydrate, preferably L. delbrueckii subsp. bulgaricus, is(are) proteolytic strain(s), preferably highly proteolytic strain(s).

In the context of the present invention, in any of its embodiments, a LAB is a proteolytic LAB if it contains an active cell wall proteinase. A cell wall proteinase hydrolyzes milk proteins such as casein and thus improves the quality of milk as a medium for rapid growth of LAB having amino acid auxotrophies. Cell wall proteinases have been identified and characterized in detail in numerous LAB, including PrtP of L. lactis, PrtS of S. thermophilus and PrtB of Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgariciis). Proteolytic LAB can thus be identified by the presence of a gene encoding a cell wall proteinase. Additionally, proteolytic LAB can be identified by assaying casein labeled with the fluorescent substrate fluorescein isothiocyanate, or FITC casein, which determines the increase in fluorescence caused by growth of a given strain for 6 hours in medium containing fluorescently labeled casein compared to control samples.

- 7 048110 without cells of this strain. Full details of this analysis are given, for example, in Example 1 of WO 2017/125600.

Preferably, at least one lactose-deficient strain of Streptococcus thermophilus, which is capable of metabolizing a non-lactose carbohydrate, is added to the milk base in step (1a) of the method according to the present invention in an amount of from 1E+04 to 1E+10 CFU (colony forming unit)/ml of the milk base, preferably from 1E+05 to 1E+10 CFU/ml or from 1E+06 to 1E+10 CFU/ml, or from 1E+07 to 1E+09 CFU/ml, preferably when the milk base has a fat content of about 2% by weight of fat and about 4.1% by weight of protein. More preferably, at least one lactose-deficient strain of Streptococcus thermophilus, which is capable of metabolizing a non-lactose carbohydrate, preferably sucrose, is added to the milk base in step (1a) of the method of the present invention in an amount of 1E+06 - 1E+08 CFU/ml of the milk base, preferably when the milk base has a fat content of about 2% by weight fat and about 4.1% by weight protein.

Preferably, at least one Lactobacillus strain that is capable of metabolizing a non-lactose carbohydrate, preferably L. delbrueckii spp. bulgaricus, is added to the milk base in step (1a) of the process according to the present invention in an amount of from 1E+04 to 1E+10 CFU/ml of the milk base, preferably from 1E+05 to 1E+10 CFU/ml or from 1E+06 to 1E+10 CFU/ml or from 1E+07 to 1E+09 CFU/ml, preferably when the milk base has a fat content of about 2% by weight fat and about 4.1% by weight protein. More preferably, at least one Lactobacillus strain that is capable of metabolizing a non-lactose carbohydrate, preferably L. delbrueckii spp. bulgaricus, is added to the milk base in step (1a) of the method of the present invention in an amount of 1E+06 - 1E+08 CFU/ml of milk base, preferably when the milk base has a fat content of about 2% by weight of fat and about 4.1% by weight of protein.

As described above, in a preferred embodiment of the present invention, at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus which is capable of metabolizing a non-lactose carbohydrate are added to a milk base (inoculation dose) which preferably has a fat content of about 2% by weight fat and about 4.1% by weight protein in a total amount of from about 1E+06 to about 1E+08 CFU/ml of the milk base, preferably in a total amount of from about 5E+06 to about 1E+07 CFU/ml of the milk base, such as from about 6E+06 CFU/ml to about 1.5E+07 CFU/ml, even more preferably in a total amount of from about 1.2E+07 CFU/ml to about 1.3E+07 CFU/ml.

The ratio of the bacterial cell number of at least one lactose-deficient Streptococcus thermophilus strain which is capable of metabolizing a non-lactose carbohydrate (ST) and at least one lactose-deficient Lactobacillus strain which is capable of metabolizing a non-lactose carbohydrate, preferably L. delbrueckii subsp. bulgaricus (LB) (ST:LB) in the starter culture or in the milk base at the beginning of the fermentation can be easily determined by a person of ordinary skill in the art. In a particular embodiment, this ratio is in the range of from 99:1 to 1:99, such as from 95:5 to 5:95, from 80:20 to 20:80 or from 70:30 to 30:70, or from 60:40 to 40:60, or 50:50 (ST:LB). The preferred ratio is in the range of 90:10 to 99:1 (ST:LB).

b) a non-lactose carbohydrate capable of being metabolized by lactic acid bacteria as defined in (a).

In the context of the present invention, in any of its embodiments, the term non-lactose carbohydrate means any carbohydrate that is not lactose and that the lactose-deficient LAB of the invention is capable of metabolizing. In a particular embodiment of the invention, the non-lactose carbohydrate is selected from the group consisting of sucrose, galactose and glucose. Preferably, the non-lactose carbohydrate is not glucose. Even more preferably, the non-lactose carbohydrate is sucrose.

The non-lactose carbohydrate(s) is/are added to the milk base in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of 4.9 to 5.5, such as 5.0 to 5.4, preferably at a pH of the fermented milk product of about 5.3. The acidification profile of the milk base can be monitored by standard means known to the person skilled in the art, such as, for example, real-time pH measurement equipment.

In the context of the present invention in any of its embodiments, the term depletion in relation to the non-lactose carbohydrate(s) means that the concentration of the given non-lactose carbohydrate(s) is zero or so low that the starter culture as determined in step (1a) is no longer able to grow, or so low that the starter culture as determined in step (1a) is no longer able to acidify the milk base further. It is noteworthy that the growth rate/profile and acidification are directly correlated. The indication of no growth of the yoghurt starter culture is shown in the acidification profile. As soon as the fermentable carbohydrate(s) (e.g. sucrose) is/are depleted, there is a stop in the acidification curve. From this point onwards, the slope/shape of the curve changes, indicating that only the other part (the probiotic) of the culture mixture is growing. The absence of growth of the starter culture of step (a) of the method according to the invention can also be determined, for example, by planting on a plate

- 8 048110 ku strains of ST (Streptococcus thermophilus). In a particular embodiment of the present invention, at the end of fermentation, the concentration of non-lactose carbohydrate at which it is depleted may be in the range of less than 100 mg/g, such as less than 30 mg/g, including the range of 25 mg/g to 0.01 mg/g or the range of 5 mg/g to 0.01 mg/g.

In this context, at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably at a pH of the fermented milk product of about 5.3, the fermentation due to the metabolism of the starter culture ceases. According to the present invention, the fermentation of the starter culture is thus terminated by the depletion of one or more non-lactose carbohydrates. However, since the milk base further comprises probiotic strains which are able to metabolise carbohydrates present in the composition, such as lactose, the fermentation due to the metabolism of the probiotic strains would continue. Indeed, in the context of the present invention, the fermentation of the milk base due to the metabolism of probiotic strains, preferably a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, see below, is desirable and will preferably take place according to step (2) of the process according to the present invention.

Accordingly, the fermentation of the milk base due to the metabolism of the starter culture (catabolism of the non-lactose carbohydrate(s)) will cease at a pH of 4.9 to 5.5, such as 5.0 to 5.4, preferably at about 5.3, since the non-lactose carbohydrate(s) has been exhausted and the starter culture is essentially no longer able to grow/acidify the milk base. However, since the milk base contains other strains, i.e. a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, which is/are able to metabolize one or more carbohydrates still present in the milk base, such as lactose, the fermentation of the milk base will continue, see below.

The amount of non-lactose carbohydrate(s) to be added to the milk base depends on a number of parameters, including the lactic acid bacteria strains used in the starter culture, the composition of the milk base, the fermentation temperature and the desired target pH, which in the present case is from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3. The amount of non-lactose carbohydrate(s) to be added to the milk base can be determined by experimentation, and it is within the skill of the skilled person to carry out such experimentation. Accordingly, the skilled person is able to calculate the amount of non-lactose carbohydrate(s), preferably sucrose, to be added to the milk base in step (1b) of the method according to the present invention, such that the starter culture added in step (1a) stops growing because the non-lactose carbohydrate(s) has been(are) exhausted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably at a pH of the fermented milk product of about 5.3.

The amount of non-lactose carbohydrate(s) can thus be easily determined based on the LAB used and the desired acidification (target pH from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3), mainly caused by a starter culture comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing the non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing the non-lactose carbohydrate, preferably L. delbrueckii subsp. bulgaricus. In most cases, sucrose, galactose and/or glucose, preferably sucrose, are added to milk in an amount resulting in a concentration in the range of 0.4 g/l to 10 g/l or in the range of 1 g/l to 8 g/l, or in the range of 2 g/l to 6 g/l.

In a preferred embodiment, the non-lactose carbohydrate, which is preferably sucrose, is added to the milk base in step (1b) of the process of the present invention in an amount of less than 0.9%, where % is the weight per volume of the total amount of the milk base (% w/v), preferably in an amount of less than 0.7%, even more preferably in an amount of less than 0.5%, such as 0.41%, preferably where the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, the starter culture in step (1a) is preferably added as a frozen concentrated culture in an amount of 0.01% w/v (such as about 1.2-1.3E+07 CFU/ml) of the total amount of milk, and the fermentation temperature is about 38°C.

For example, when the amount of the starter culture added in step (1a) is 0.01% w/v (e.g. about 1.2-1.3E+07 CFU/ml), the non-lactose carbohydrate(s) added in step (1b), preferably sucrose, is added in an amount of less than 0.9%, preferably in an amount of less than 0.7%, even more preferably in an amount of less than 0.5%, preferably from 0.5% to 0.41%, most preferably about 0.41%, wherein % is weight per volume (w/v) based on the milk base (% w/v), preferably where the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, and the fermentation temperature is about 38°C.

c) a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bi strain

- 9 048110 fidobacterium.

In the context of the present invention, in any of its embodiments, the term probiotic bacteria or probiotic strain refers to viable bacteria that are administered to a consumer in adequate amounts to achieve a health-promoting effect in the consumer. Probiotic bacteria are capable of surviving in the gastrointestinal tract after ingestion and colonizing the intestines of the consumer.

In a particular embodiment of the present invention, the probiotic strain according to the present invention is selected from the group consisting of bacteria of the genus Lactobacillus such as Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus johnsonii, the genus Bifidobacterium such as Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis, and the like.

In a preferred embodiment, the probiotic strain of Lactobacillus is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus johnsonii.

In a specific embodiment of the present invention, the probiotic strain of Lactobacillus is selected from the group consisting of a strain of Lactobacillus rhamnosus, a strain of Lactobacillus acidophilus and a strain of Lactobacillus paracasei.

In a preferred embodiment of the present invention, the probiotic strain is the Lactobacillus rhamnosus LGG® strain deposited as ATCC 53103. In another preferred embodiment of the present invention, the probiotic strain is the Lactobacillus acidophilus LA-5® strain deposited as DSM 13241. In a specific embodiment of the present invention, the probiotic strain is the Lactobacillus paracasei strain CRL431 deposited as ATCC 55544, which is commercially available. In a preferred embodiment, the Lactobacillus probiotic strain is not the L. paracasei strain CRL431 deposited as ATCC 55544 or the L. paracasei strain CHCC 2115 deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 27.06.2007 under accession number DSM 19465.

In a specific embodiment of the present invention, the probiotic strain of Bifidobacterium is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis.

In a specific embodiment of the present invention, the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis BB-12®, also referred to as BB-12®, deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, 30.09.2003 under accession number DSM 15954. This Bifidobacterium BB-12® is a well-known probiotic bacterium available from Chr. Hansen A/S, Horsholm, DK. In the case of BB-12®, the available clinical evidence shows that a daily dose of viable probiotic bacteria of at least 1E+09 - 1E+10 CFU is required. Accordingly, it is desirable to have a high level, such as 1E+08 CFU or more, of probiotic bacteria per gram of fermented milk product (e.g. fermented milk yogurt product).

In a preferred embodiment, step (1c) comprises adding to the milk base a Bifidobacterium strain, preferably a Bifidobacterium strain selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis, even more preferably adding to the milk base Bifidobacterium animalis subsp. lactis BB-12®, deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, 30.09.2003 under accession number DSM 15954.

For example, step (1c) may comprise adding to the milk base a probiotic strain belonging to the genus Bifidobacterium, preferably belonging to the species Bifidobacterium animalis, even more preferably Bifidobacterium animalis subsp. lactis BB-12®, as described above, and a probiotic strain belonging to the genus Lactobacillus, for example Lactobacillus rhamnosus and/or Lactobacillus acidophilus, preferably wherein the probiotic strain belonging to the genus Lactobacillus is not a strain of L. paracasei, even more preferably wherein the probiotic strain of Lactobacillus is not the strain of L. paracasei CRL 431, deposited as ATCC 55544, or the strain of L. paracasei CHCC 2115, deposited as DSM 19465.

Even more preferably, the composition according to the invention comprises a probiotic strain belonging to the species Bifidobacterium animalis, preferably the strain Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954, and a probiotic strain belonging to the species Lactobacillus rhamnosus, preferably the strain LGG®, deposited as ATCC53103, and/or a probiotic strain belonging to the species Lactobacillus acidophilus, preferably the strain LA-5®, deposited

- 10 048110 similar to DSM 13241.

Preferably, the probiotic strain Bifidobacterium is added to the milk base in step (1c) of the method according to the present invention in an amount of from 1E+06 to 1E+08 CFU/ml of the milk base, preferably from 5E+06 to 5E+07 CFU/ml, more preferably about 1.2E+07 CFU/ml of the milk base.

Preferably, the probiotic strain is added to the milk base in step (1c) of the method according to the invention in an amount of from 0.001 to 2%, where % is the weight per volume of the total amount of milk base (% w/v), such as 0.005%, 0.01%, 0.015%, 0.02%, preferably from 0.001 to 0.025% by weight per volume of the total amount of milk base, such as from 0.0015 to 0.15%, such as from 0.01 to 0.015% or from 0.01 to 0.02%, or from 0.01 to 0.025% by weight per volume of the total amount of milk base. Preferably, this probiotic strain is added to the milk base in an amount to achieve a concentration of about 0.01% by weight per volume of the total amount of the milk base, preferably where this probiotic strain is added as a frozen concentrated culture, preferably where the milk base has a fat content of about 2% by weight and a protein content of about 4.1% by weight. If the probiotic strain is added to the milk base in step (1c) of the method according to the present invention in an amount of about 0.001% by weight per volume of the total amount of the milk base, this probiotic strain is preferably added as a lyophilized concentrated culture.

In a preferred embodiment, the BB-12® cell count in milk when inoculated with 0.01% F-DVS is about 1.2E+07 CFU/mL. In a preferred embodiment, the LA-5® cell count in milk when inoculated with 0.01% F-DVS is about 7E+06 CFU/mL. In a preferred embodiment, the LGG® cell count in milk when inoculated with 0.001% FD-DVS is about 7E+06 CFU/mL.

Accordingly, in a preferred embodiment, step (1) of the method of the present invention comprises adding to the dairy base:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus, preferably in an amount of about 1.2-1.3+07 CFU/ml;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subs. lactis BB-12®, deposited as DSM 15954, preferably in an amount of about 1.2E+07 CFU/ml;

or:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus, preferably in an amount of about 1.2-1.3+07 CFU/ml;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis strain lactis BB-12®, deposited as DSM 15954, preferably in an amount of about 1.2E+07 CFU/ml and Lactobacillus rhamnosus strain LGG®, deposited as ATCC 53103, preferably in an amount of about 7E+06 CFU/ml;

or:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus that is capable of metabolizing a non-lactose carbohydrate, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus, preferably in an amount of about 1.2-1.3+07 CFU/ml;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subst. lactis BB-12®, deposited as DSM 15954, and Lactobacillus acidophilus strain LA-5®, deposited as DSM 13241, preferably wherein BB-12® is added to

- 11 048110 in an amount of approximately 1.2E+07 CFU/ml, and LA-5® is added in an amount of approximately 7E+06 CFU/ml.

In these preferred embodiments described above, the at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate is preferably selected from the group consisting of the following:

(a) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28952;

(2) a strain derived from DSM 28952, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal;

(b) (1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28953;

(2) a strain derived from DSM 28953, wherein this derivative strain is further distinguished by the ability to form white colonies on a medium containing lactose and X-Gal;

(c) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32599;

(2) a strain derived from DSM 32599, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal; and (d) (1) the strain deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32600; and (2) a strain derived from DSM 32600, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

Preferably, the lactose-deficient strain of Lactobacillus present in the starter culture is a lactose-deficient strain of Lactobacillus delbrueckii spp. bulgaricus. Preferably, the lactose-deficient strain of Lactobacillus delbrueckii spp. bulgaricus is selected from the group consisting of the following:

(1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28910; and (2) a strain derived from DSM 28910, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

As will be appreciated by the skilled person, step (1) of the method according to the present invention comprises adding to the milk base (1a) (starter culture), (1b) (non-lactose carbohydrate(s)) and (1c) (probiotic strain). The order of adding these three elements is not relevant; for example, the starter culture may be added to the milk base first, and then the non-lactose carbohydrate(s), and then the probiotic strain. Or the starter culture and the probiotic strain may be mixed with each other and then added at the same time to the milk base which contains the non-lactose carbohydrate(s). Most preferably, (1) the non-lactose carbohydrate(s) (preferably sucrose) are first added to the milk base and then (2) the starter culture and the probiotic strain are added to the milk base, for example the starter culture and the probiotic strain are added at the same time and at a time after the non-lactose carbohydrate(s) are added to the milk base. Preferably the non-lactose carbohydrate(s) (which is preferably sucrose), if any, are added to the milk base prior to heat treatment (e.g. pasteurization) to ensure the absence of impurities. Typically frozen concentrated yoghurt cultures and probiotic cultures (F-DVS) contain 6E+10 - 1.5E+11 CFU/g. When inoculated at 0.01% w/v. the cell count in the milk before incubation (before fermentation) is preferably from about 6E+06 CFU/ml to about 1.5E+07 CFU/ml. When inoculated at 0.02% w/v, the cell count in the milk before incubation (before fermentation) is preferably from about 1.2E+07 CFU/ml to about 3E+07 CFU/ml.

Step 2 of the method of the present invention comprises fermenting the milk base for a period of time until a target (or desired) pH is reached, thereby producing a fermented milk product.

The term fermentation in the context of the present invention in any of its embodiments means the conversion of carbohydrates into alcohols or acids by the action of a microorganism. For example, fermentation in the context of the starter culture of the present invention includes the conversion of a non-lactose carbohydrate, such as sucrose, into lactic acid.

In the context of step (2) of the method of the present invention, the fermentation comprises:

a first step, wherein the fermentation is mainly due to the conversion of a non-lactose carbohydrate added to the milk base in step (1b), for example sucrose, to lactic acid by means of a LAB starter culture comprising at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolising the non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably a strain of L. delbrueckii subsp. bulgaricus which is capable of metabolising the non-lactose carbohydrate added in step (1a);

a second stage, wherein the fermentation is mainly due to a probiotic strain, as defined in the context of the present invention, added to the milk base in stage (1c), which

- 12 048110 paradise involves the conversion of lactose to lactic acid by a probiotic strain.

In the method of the present invention, during the first fermentation stage, the lactose-deficient strains would metabolize the non-lactose carbohydrate(s) until the non-lactose carbohydrate(s) are exhausted. As indicated above, in combination with the yoghurt culture, the probiotic strain(s) may grow slightly better than the single strain, but still grow significantly slower than the yoghurt species Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB), which would dominate the probiotic strain(s) at this stage.

Since the non-lactose carbohydrate(s) is(are) added in an amount measured so that it(they) is(are) exhausted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3, the first stage of fermentation will end at a milk pH of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3. At this stage, the lactose-deficient strains that dominate the probiotic strain(s) are(are) unable to grow further, since they are essentially unable to metabolize lactose.

However, the probiotic strain(s) selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, added to the milk base in step (1c) of the method according to the present invention, which contains probiotic strains capable of metabolizing lactose, will continue the acidification of the milk base. Accordingly, in the second stage, the fermentation will be mainly due to the metabolic activity of the probiotic strain(s). These probiotic strains will consume the lactose present in the milk base and will continue the acidification until the target (desired) pH is reached. The target (desired) pH may be from about 3.2 to about 4.9, preferably from about 3.6 to about 4.8, more preferably from about 4.0 to about 4.6, such as about 4.0 or about 4.3, or about 4.4, or about 4.5, preferably from about 4.6 to about 4.5, even more preferably about 4.55. In a preferred embodiment, the target (desired) pH is about 4.55.

This second fermentation step (and thus the fermentation step (2) of the present invention) may be terminated by any means known to the skilled person, such as by cooling treatment, or because the milk reaches a pH that makes the probiotic strain(s) unable to grow, or because the lactose in the milk is depleted and the probiotic strains are unable to grow further, etc. For example, the fermentation step (2) of the present invention may be terminated by cooling (e.g., about 4°C), and the fermented milk product is stored chilled (e.g., at about 4°C). Cooling is commonly used as a method to slow down metabolic activity and keep the cultures and probiotics alive.

Fermentation methods to be used in the production of dairy products are well known and the person skilled in the art will know how to select suitable process conditions such as temperature, oxygen, amount and characteristics of the microorganism(s) and process time. Obviously, the fermentation conditions are selected so as to support the achievement of the present invention, for example to obtain a dairy product in solid (for example, filtered yoghurt or yoghurt with a high solids residue) or in liquid form (such as yoghurt, drinking yoghurt, broken yoghurt, thermostat yoghurt and yoghurt-like drink). In the context of the present invention, the fermentation is carried out at a temperature of from about 34°C to about 43°C, for example about 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, preferably at about 38°C, about 40°C or about 43°C.

In a preferred embodiment, the fermented milk product obtained by the method of the present invention comprises 1.3E+08 CFU probiotic cells/g fermented milk product (CFU/g) or more, preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g or more of at least one probiotic strain, such as immediately after fermentation, preferably at a time point of at least 1 day after completion of fermentation (i.e. completion of fermentation step (2) of the present invention), such as 15 days or 30 days or 45 days, more preferably 60 days after completion of fermentation, wherein preferably the food or feed product has been stored at about 4°C after completion (termination) of the fermentation according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight fat and about 4.1% by weight protein, preferably wherein fermentation occurs at about 38°C, and preferably until a pH of about 4.55 is reached.

Fermented milk product

Furthermore, according to the present invention, there is provided a fermented milk product produced, obtained or directly obtained by the method of the present invention.

Advantageously, the fermented milk product of the present invention will contain higher amounts of viable probiotic bacteria (higher number of viable probiotic cells) compared to the amount of viable probiotic bacteria present in a fermented milk product incubated with probiotics alone.

- 13 048110 bacteria or fermented with a starter culture comprising at least one strain of Streptococcus thermophilus that is not lactose-deficient and at least one strain of Lactobacillus, preferably L. delbrueckii spp. bulgaricus, that is not lactose-deficient (e.g. traditional lactose (+) yoghurt culture), or with a starter culture comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably at least one lactose-deficient strain of L. delbrueckii spp. bulgaricus, which is capable of metabolizing a non-lactose carbohydrate in the presence of a non-lactose carbohydrate, preferably sucrose, in an amount measured such that it is depleted at a pH of the fermented milk product of less than 4.9, such as 4.55 (for example, about 0.9% sucrose). In addition, the advantageously fermented milk product of the present invention will have a higher stability of the amount of probiotics over time, such as for at least 60 days of shelf life, preferably at about 4°C (storage at about 4°C).

Therefore, according to the present invention, a food or feed product (fermented milk product) is proposed, comprising at least one lactose-deficient strain of Streptococcus thermophilus, which is capable of metabolizing non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus which is capable of metabolizing a non-lactose carbohydrate, and a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, wherein the food or feed product comprises 1.3E+08 CFU or more probiotic cells/g of fermented milk product (CFU/g), preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g or more of at least one probiotic strain present in the food or feed product, immediately after fermentation (i.e. fermentation step (2) according to the present invention), preferably at a time point of at least 1 day after the completion of fermentation, such as 15 days or 30 days or 45 days or 60 days after the completion fermentation, wherein the food or feed product has been stored at about 4°C after completion (termination) of the fermentation according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, preferably wherein the fermentation occurs at about 38°C, preferably until a pH of about 4.55 is reached. The food or feed product of the present invention thus has a very high amount of probiotics (greater than 1.3E+08 CFU/g, as described above). Adding such high amounts of probiotics to an already fermented milk product would affect properties such as taste and aroma of this fermented milk product. In addition, it would be very expensive, since it would involve adding probiotics in an amount 30-50 times greater than the inoculation value of the milk base before fermentation according to the present invention. Accordingly, the food or feed product of the present invention also exhibits these advantages compared to a food or feed product containing substantially the same amount of probiotics, but where these probiotics were added after fermentation of the milk base.

As stated above, in the context of the method of the present invention, preferably at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolising a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus which is capable of metabolising a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, which are contained in the food or feed product (fermented milk product) of the present invention, are capable of metabolising the same non-lactose carbohydrate, which is preferably sucrose.

The food or feed product (fermented dairy product) of the present invention may contain any number of other components, including fermented milk, food additives, stabilizers, cryoprotectants, flavoring agents, artificial sweeteners, and the like. The food or feed product of the present invention may be any fermented dairy product, including yogurt, such as fruit yogurt, yogurt drink, broken-curd yogurt, incubated yogurt, yogurt-like drink, filtered yogurt, etc. Preferably, the food or feed product of the present invention is yogurt.

In the context of the present invention, in any of its embodiments, the term yogurt refers to products containing Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, and possibly other microorganisms such as Lactobacillus delbrueckii ssp. lactis, Bifidobacterium animaiis ssp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived from them. Strains of lactic acid bacteria other than Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus are included to impart various properties to the final product, such as the property of stimulating the balance of microflora. The term yogurt as used herein

- 14 048110 is used, covers incubated yoghurt, broken-curd yoghurt, drinking yoghurt, curd cheese, incubated yoghurt, filtered yoghurt or Greek yoghurt characterised by a high protein level and yoghurt-like products. In particular, the term yoghurt covers yoghurt as defined by French and European regulations, such as curdled milk products obtained by lactic acid fermentation using only specific thermophilic lactic acid bacteria (i.e. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) which are cultured simultaneously and are present in the final product alive in an amount of at least 10 million CFU (colony forming units)/g, but is not limited to it. Yoghurts may optionally contain added milk raw materials (e.g. cream) or other ingredients such as sugar or sweeteners, one or more flavouring agents, fruits, cereals or nutrients, especially vitamins, minerals and fibres, as well as stabilisers and thickeners. In one alternative embodiment, the yogurt complies with the specifications for fermented milk products and yogurts of the AFNOR standard NF 04-600 and/or the StanA-Ila-1975 codex standard. In order to comply with the AFNOR standard NF 04-600, the product must not be heated after fermentation and milk raw materials must constitute a minimum of 70% (w/w) of the final product.

The fermented milk obtained by the method of the present invention, containing 1.3E+08 CFU probiotic cells/g of fermented milk (CFU/g) or more, preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, for example 6E+08 CFU/g or more of at least one probiotic strain present in the fermented milk as described herein, can also be used as a supplement product, for example, which is added to other edible food products such as cottage cheese, chocolate, juices, meat products and milk powder products for infants in the first months of life.

Preferred lactose-deficient strains of Streptococcus thermophilus have already been defined in the context of the method of the present invention and are equally applicable to this embodiment.

Preferably, the lactose-deficient Lactobacillus strain is a lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strain. The preferred lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strain has already been defined in the context of the method according to the present invention and is equally applicable to this embodiment.

Preferred probiotic strains have already been described in the context of the method of the invention and are equally applicable to this embodiment. Accordingly, preferably the probiotic strain present in the food or feed product of the present invention is one or more of the following probiotic strains:

Bifidobacterium animalis strain lactis BB-12®, deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, on 30.09.2003 under accession number DSM 15954; and/or the strain Lactobacillus rhamnosus LGG®, deposited as ATCC53103; and/or the strain Lactobacillus acidophilus LA-5®, deposited as DSM 13241.

Accordingly, in a preferred embodiment, the food or feed product (fermented dairy product) of the present invention comprises 1.3E+08 CFU or more probiotic bacteria/g fermented dairy product (CFU/g), preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g or more of at least one of the above probiotic strains, preferably Bifidobacterium animalis subf. lactis BB-12®, DSM 15954, immediately after fermentation, preferably at a time point of at least 1 day after completion of the fermentation according to step (2) of the present invention, such as 15 days or 30 days or 45 days or 60 days after completion of the fermentation, wherein the food or feed product has been stored at about 4°C after completion of the fermentation according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, preferably wherein the fermentation has occurred at about 38°C, and preferably until a pH of about 4.55 has been reached.

It is noteworthy that the food or feed product (fermented dairy product) of the present invention comprises 1.3E+08 CFU/g or more, preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 5.7E+08 CFU/g or more, of at least one probiotic strain present in the product at 60 days after completion of fermentation (60 days storage), where the food or feed product has been stored at about 4°C after completion of fermentation according to step (2) of the method of the present invention, and preferably where the dairy base comprises about 2% by weight of fat and about 4.1% by weight of protein, preferably where the fermentation has taken place at about 38°C, and preferably until a pH of about 4.55 has been reached. Accordingly, the food or feed product of the present invention (fermented milk)

- 15 048110 lactic acid product) exhibits higher stability (increased numbers of viable probiotic bacteria are maintained over time) during 60 days of storage (at approximately 4°C) than a food or feed product that has been fermented using the same milk base, under the same fermentation conditions, with the same initial number of probiotic bacteria, but with one of the following:

there is no starter culture, i.e. a milk base incubated only with probiotic bacteria;

a starter culture comprising at least one strain of Streptococcus thermophilus that is not lactose-deficient (lac+) and at least one strain of Lactobacillus that is not lactose-deficient, preferably (lac+) L. delbrueckii subsp. bulgaricus (e.g. traditional lactose (+) yoghurt culture);

a starter culture comprising at least one lactose-deficient (lac-) strain of Streptococcus thermophilus which is capable of metabolising a non-lactose carbohydrate and at least one lactose-deficient (lac-) strain of Lactobacillus, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus which is capable of metabolising a non-lactose carbohydrate, in the presence of non-lactose carbohydrate(s), preferably sucrose, in an amount measured such that it(they) is(are) depleted when the pH of the fermented milk is less than 4.9, such as 4.55.

Composition

According to the present invention, a composition (hereinafter referred to as the composition of the invention) is proposed for producing a fermented milk product, comprising

a) a lactic acid bacteria (LAB) starter culture comprising or, alternatively, consisting of at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably a strain of L. delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate; and

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein said non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3.

As stated above, in the context of the method of the present invention, preferably at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus which is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, which are contained in the food or feed product (fermented milk product) of the present invention, are capable of metabolizing the same non-lactose carbohydrate, which is preferably sucrose. In other embodiments, at least one lactose-deficient Streptococcus thermophilus strain that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient Lactobacillus strain that is capable of metabolizing a non-lactose carbohydrate, preferably a lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strain, which are contained in the starter culture that is added to the milk base in step a of the present invention, are capable of metabolizing different non-lactose carbohydrates, preferably wherein the non-lactose carbohydrate is not glucose. For example, at least one lactose-deficient Streptococcus thermophilus strain is capable of metabolizing sucrose and at least one lactose-deficient Lactobacillus strain is capable of metabolizing galactose, or vice versa.

In a particular embodiment, the composition comprises two or more lactose-deficient strains of Streptococcus thermophilus and one lactose-deficient strain of Lactobacillus, preferably one lactose-deficient strain of L. delbrueckii subsp. bulgaricus.

The starter culture of the composition according to the present invention has been described in detail earlier in the description of the starter culture added in step (1a) of the method according to the present invention. Accordingly, the starter culture (a) contained in the composition according to the present invention corresponds to the starter culture added to the milk base in step (1a) of the method according to the present invention, which has been described in detail above, and is equally applicable to the composition according to the present invention.

Furthermore, the non-lactose carbohydrate capable of being metabolized by lactic acid bacteria of the starter culture contained in the composition of the present invention (b) has been described in detail in the context of the method of the present invention (step 1b).

Preferred lactose-deficient strains of Streptococcus thermophilus have already been defined in the context of the method of the present invention and are equally applicable to this embodiment.

Preferably, the lactose-deficient Lactobacillus strain is the lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strain. Preferred lactose-deficient Lacto strains

- 16 048110 bacillus delbrueckii subsp. bulgaricus have already been defined in the context of the method of the present invention and are equally applicable to this embodiment.

In a preferred embodiment, the composition of the present invention further comprises at least one probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain.

The probiotic strain preferably contained in the composition of the present invention has been described in detail in the context of the method of the present invention (step 1c). Accordingly, the probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain preferably contained in the composition of the present invention corresponds to the probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain added to the milk base of step (1c) of the method of the present invention, which has been described in detail above, and is equally applicable to the composition of the present invention.

Accordingly, in a preferred embodiment of the present invention, the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are present in the composition in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subs. lactis BB-12®, deposited as DSM 15954. Or the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate;

b) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are present in the composition in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3;

c) Bifidobacterium animalis subs. lactis BB-12®, deposited as DSM 15954; and

d) the strain Lactobacillus rhamnosus LGG®, deposited as ATCC 53103. Or the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate;

b) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are present in the composition in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3;

c) Bifidobacterium animalis subs. lactis BB-12®, deposited as DSM 15954; and

d) the strain Lactobacillus acidophilus LA-5®, deposited as DSM 13241. The amounts of strains present in the starter culture and/or the amounts of probiotic strain(s) have been described above in the context of the method of the present invention and are equally applicable to the composition of the present invention.

In a preferred embodiment, the composition of the invention comprises from 1E+04 to 1E+09 CFU of the Streptococcus thermophilus strain/g of the composition or more, preferably from 1E+05 to 1E+07 CFU/g or from 1E+06 to 1E+07 CFU/g of the Streptococcus thermophilus strain. More preferably, the composition of the invention comprises about 6-7E+08 CFU/g or less of the Streptococcus thermophilus strain.

In a preferred embodiment, the composition of the invention comprises from 1E+04 to 1E+09 CFU of the strain Lactobacillus delbrueckii spp. bulgaricus/g of the composition, preferably from 1E+05 to 1E+07 CFU/g or from 1E+06 to 1E+07 CFU/g of the strain Lactobacillus delbrueckii spp. bulgaricus. More preferably, the composition of the invention comprises about 1E+07 CFU of the strain Lactobacillus delbrueckii spp. bulgaricus/g of the composition.

In a preferred embodiment, the composition of the invention comprises a total CFU count of at least 1E+10 CFU/g (i.e. considering the count of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and probiotic strains, if present).

As disclosed in WO 2005/003327, it is useful to add certain cryoprotectants to the starter culture. Thus, the starter culture contained in the composition of the present invention (a) may contain one or more cryoprotectants selected from the group,

- 17 048110 consisting of inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uranosine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compounds.

In addition, the starter cultures can be provided as frozen or dried starter cultures, in addition to liquid starter cultures. Thus, the composition of the present invention can be in frozen, lyophilized or liquid form.

Application of the present invention

According to the present invention, there is further provided the use of the composition of the present invention for increasing the number of viable probiotic cells of at least one of the probiotic strains present in a fermented dairy product, compared to a fermented dairy product fermented using a composition comprising:

a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus which is not lactose-deficient and at least one strain of Lactobacillus, preferably L. delbrueckii subsp. bulgaricus, which is not lactose-deficient; or

b) 1) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii subsp. bulgaricus, that is capable of metabolizing a non-lactose carbohydrate, and

2) one or more non-lactose carbohydrates capable of being metabolized by lactic acid bacteria as defined in (1), wherein the non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; or

c) a milk base incubated with at least one probiotic bacterium (i.e. in the absence of a starter culture as described above).

Accordingly, the composition of the present invention can be used to increase the viable cell number of at least one of the probiotic strains present in a fermented dairy product, wherein the food or feed product comprises 1.3E+08 CFU or more probiotic bacteria/g of fermented dairy product (CFU/g), preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g or more of at least one probiotic strain present in the food or feed product, immediately after fermentation, preferably at a time point which is at least 1 day after completion of the fermentation according to step (2) of the method of the present invention, such as 15 days or 30 days or 45 days or 60 days after completion of the fermentation, wherein the food or feed product has been stored at about 4°C after the fermentation has ceased according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, preferably wherein the fermentation has occurred at about 38°C, and preferably until a pH of about 4.55 has been reached.

Preferably, the composition of the present invention is used to increase the number of viable cells (increase or improve survival) of at least one probiotic strain selected from the following:

Bifidobacterium animalis strain lactis BB-12®, deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, 30.09.2003 under accession number DSM 15954; and/or the strain Lactobacillus rhamnosus LGG®, deposited as ATCC 53103; and/or the strain Lactobacillus acidophilus LA-5®, deposited as DSM 13241.

Accordingly, in a preferred embodiment, the composition of the present invention is used to increase the viable cell number (increase or improve the survival) of at least one of the above-described probiotic strains present in a fermented dairy product as described above, wherein the fermented dairy product comprises 1.3E+08 CFU or more viable cells of probiotic bacteria/g of fermented dairy product (CFU/g), preferably 2E+08 CFU/g or more, or 3E+08 CFU/g or more, or 4E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g or more of at least one of the above-described probiotic strains, preferably Bifidobacterium animalis subf. lactis BB-12®, DSM 15954 - immediately after fermentation, preferably at a time that is at least 1 day after completion of fermentation according to step (2) of the method of the present invention, such as 15 days or 30 days or 45 days or 60 days after completion of fermentation, where the food or feed product has been stored

- 18 048 110 at about 4°C after the fermentation has ceased according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein, preferably wherein the fermentation has occurred at about 38°C, and preferably until a pH of about 4.55 has been reached.

Accordingly, according to the present invention, there is provided a method for increasing the number of viable probiotic cells of at least one of the probiotic strains present in a fermented dairy product, using the composition of the present invention, as described in detail above.

The term for increasing or improving the survival of viable probiotic cells over time, as used herein, means that the number of viable probiotic cells in a product fermented with a starter culture of the present invention is maintained at a higher level over time than the number of probiotic cells in a fermented product that was fermented using the same dairy base, under the same fermentation conditions, with the same initial number of probiotic cells, but with one of the following:

there is no starter culture, i.e. a milk base incubated with only probiotic bacteria (as shown in Fig. 1, probiotics do not grow easily in milk or grow very slowly);

a starter culture comprising at least one strain of Streptococcus thermophilus that is not lactose-deficient (lac+) and at least one strain of Lactobacillus that is not lactose-deficient, preferably (lac+) L. delbrueckii subsp. bulgaricus (e.g. traditional lactose (+) yoghurt culture);

a starter culture comprising at least one lactose-deficient (lac-) strain of Streptococcus thermophilus which is capable of metabolising a non-lactose carbohydrate and at least one lactose-deficient (lac-) strain of Lactobacillus, preferably at least one lactose-deficient strain of L. delbrueckii subsp. bulgaricus which is capable of metabolising a non-lactose carbohydrate, in the presence of a non-lactose carbohydrate, preferably sucrose, in an amount measured so that it is depleted when the pH of the fermented milk is less than 4.9, such as 4.55.

In this context, the phrase over time means for at least 1 day after completion of the fermentation according to step (2) of the method of the present invention, such as 15 days or 30 days or 45 days or 60 days after completion of the fermentation, wherein the food or feed product has been stored at about 4°C after completion of the fermentation according to step (2) of the method of the present invention, preferably wherein the milk base comprises about 2% by weight of fat and about 4.1% by weight of protein.

The term approximately (or about) as used herein means the stated value plus/minus 1% of its value, or the term approximately means the stated value plus/minus 2% of its value, or the term approximately means the stated value plus/minus 5% of its value, the term approximately means the stated value plus/minus 10% of its value, or the term approximately means the stated value plus/minus 20% of its value, or the term approximately means the stated value plus/minus 30% of its value; preferably the term approximately means the exact stated value (plus/minus 0%).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention can be practiced using methods and materials similar or equivalent to those described herein. Additional objects, advantages, and characteristics of the invention will become apparent to those skilled in the art upon review of this description or may be learned by practicing the present invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the present invention.

Throughout this description and claims, the word contain and variations of this word (e.g., comprising, having, including, containing) are typically not limiting and thus do not exclude other characteristics, which may, for example, be technical characteristics, additives, components or steps. However, whenever the word contain is used herein, it also includes a special embodiment in which this word is to be understood as limiting; in this particular embodiment, the word contain has the meaning of the term consists of.

The use of terms and similar reference objects in the context of describing the present invention (especially in the context of the following claims) are to be construed as including both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values herein is merely intended to serve as a shorthand way of individually referring to each distinct value falling within the range unless otherwise indicated herein, and each distinct value is incorporated into this specification as if it were individually recited herein. All methods described herein may

- 19 048110 can be carried out in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. It is intended that the use of any and all examples or typical language (such as, for example, as) provided herein is merely intended to better illuminate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any unreported element as essential to the practice of the invention.

Preferred embodiments

1. A method for producing a fermented milk product, comprising the following stages:

1) adding to the milk base:

a) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii spp. bulgaricus, that is capable of metabolizing a non-lactose carbohydrate, preferably wherein this starter culture is added in an amount of 1.2-1.3E+07 CFU of the Streptococcus thermophilus strain and the Lactobacillus strain/ml of the milk base, preferably wherein the ratio of at least one lactose-deficient strain of Streptococcus thermophilus (ST) to at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii spp. bulgaricus (LB) in the starter culture is from 1:99 to 99:1 (ST:LB), for example 50:50, more preferably from 90:10 to 99:1 (ST:LB);

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain;

2) fermenting the given milk base for some period of time until the target pH is reached, resulting in a fermented milk product.

2. The method according to claim 1, wherein at least one lactose-deficient strain of Streptococcus thermophilus and at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii subsp. bulgaricus, are capable of metabolizing the same non-lactose carbohydrate.

3. The method according to any one of claims 1, 2, wherein the non-lactose carbohydrate(s) is(are) selected from the group consisting of sucrose, galactose and glucose, preferably where the non-lactose carbohydrate is not glucose, even more preferably where the non-lactose carbohydrate is sucrose.

4. The method according to any one of claims 1 to 3, wherein the target pH of step (2) is from about 4.8 to about 4.0, preferably from about 4.6 to about 4.55, even more preferably about 4.55.

5. The method according to any one of claims 1 to 4, wherein the lactose-deficient strain of Streptococcus thermophilus is selected from the group consisting of the following:

(a) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28952;

(2) a strain derived from DSM 28952, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal;

(b) (1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28953;

(2) a strain derived from DSM 28953, wherein this derivative strain is further distinguished by the ability to form white colonies on a medium containing lactose and X-Gal;

(c) (1) strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32599;

(2) a strain derived from DSM 32599, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal; and (d) (1) the strain deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32600; and (2) a strain derived from DSM 32600, wherein the derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

6. The method according to any one of claims 1 to 5, wherein the lactose-deficient strain of Lactobacillus is a strain of L. delbrueckii subsp. bulgaricus selected from the group consisting of the following:

(1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28910; and (2) a strain derived from DSM 28910, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal.

7. The method according to any one of claims 1 to 6, wherein the probiotic strain is not a Lactobacillus paracasei strain, even more preferably wherein the probiotic Lactobacillus strain is not a strain

- 20 048110

CRL 431 L. paracasei, deposited as ATCC 55544, or strain CHCC 2115 L. paracasei, deposited as DSM 19465.

8. The method according to any one of claims 1 to 7, wherein the probiotic strain of Lactobacillus is selected from the group consisting of a strain of Lactobacillus rhamnosus, a strain of Lactobacillus paracasei and a strain of Lactobacillus acidophilus, and/or wherein the probiotic strain of Bifidobacterium is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis.

9. The method according to any one of claims 1 to 8, wherein the probiotic strain is selected from the group consisting of Lactobacillus rhamnosus LGG® strain deposited as ATCC 53103, Lactobacillus paracasei strain CRL 431 deposited as ATCC 55544, Lactobacillus acidophilus LA-5® strain deposited as DSM 13241, and Bifidobacterium animalis subsp. lactis BB-12® deposited as DSM 15954.

10. The method according to any one of claims 1 to 9, wherein the probiotic strain added to the milk base in step (1c) comprises a Bifidobacterium strain, preferably a probiotic Bifidobacterium strain selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis, even more preferably Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954.

11. The method according to any one of claims 1 to 10, wherein step (1) comprises adding to the milk base:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate, preferably wherein at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate are added in an amount of 1.2-1.3E+07 CFU;

b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subl. lactis BB-12®, deposited as DSM 15954, preferably in an amount of about 1.2E+07 CFU/ml of the milk base.

12. The method according to any one of claims 1 to 10, wherein step (1) comprises adding to the milk base:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate, preferably wherein at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate are added in an amount of 1.2-1.3E+07 CFU;

b) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954, and Lactobacillus rhamnosus strain LGG®, deposited as ATCC 53103, preferably wherein Bifidobacterium animalis subsp. lactis BB-12® is added in an amount of about 1.2E+07 CFU/ml, and Lactobacillus rhamnosus strain LGG® is added in an amount of about 7E+06 CFU/ml.

13. The method according to any one of claims 1 to 10, wherein step (1) comprises adding to the milk base:

a) at least one lactose-deficient strain of Streptococcus thermophilus, which is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, which is capable of metabolizing a non-lactose carbohydrate, preferably wherein at least one lactose-deficient strain of Streptococcus thermophilus, which is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, which is capable of metabolizing a non-lactose carbohydrate, are added in an amount of 1.2-1.3E+07 CFU;

b) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and

c) Bifidobacterium animalis subst. lactis - BB-12® - deposited as DSM 15954, and the strain Lactobacillus acidophilus LA-5®, deposited as DSM 13241, preferably where Bifidobacterium ani

- 21 048110 malis subsp. lactis ВВ-12® is added in an amount of approximately 1.2E+07 CFU/ml, and the strain Lactobacillus acidophilus LA-5® is added in an amount of approximately 7E+06 CFU/ml.

14. The method according to any one of claims 11 to 13, wherein at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 5, and wherein at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 6.

15. The method according to any one of claims 1 to 14, wherein from 1E+04 to 1E+10 CFU (colony forming units)/ml of the milk base of the Streptococcus thermophilus strain, preferably from 1E+05 to 1E+10 CFU/ml or from 1E+06 to 1E+10 CFU/ml, or from 1E+07 to 1E+09 CFU/ml, preferably from about 1E+06 to about 1E+08 CFU/ml of the Streptococcus thermophilus strain, is added to the milk base in step (1a).

16. The method according to any one of claims 1 to 15, wherein from 1E+04 to 1E+10 CFU/ml of the milk base of the strain Lactobacillus delbrueckii subs. bulgaricus, preferably from 1E+05 to 1E+10 CFU/ml or from 1E+06 to 1E+10 CFU/ml, or from 1E+07 to 1E+09 CFU/ml, preferably from about 1E+06 to about 1E+08 CFU/ml of the strain Lactobacillus delbrueckii subs. bulgaricus, is added to the milk base in step (1a).

17. The method according to any one of claims 1 to 16, wherein from about 1E+06 to about 1E+08 CFU/ml of the milk base of the probiotic strain, preferably from about 5E+06 to about 5E+07 CFU/ml, more preferably from about 1 to 1.5E+07 CFU/ml, such as about 1.2E+07 CFU/ml, or about 7E+06 CFU/ml of the probiotic strain, is added to the milk base in step (1c).

18. A method according to any one of claims 1 to 17, wherein the non-lactose carbohydrate, preferably sucrose, is added to the milk base in step (1b) in an amount of less than 0.9%, preferably in an amount of less than 0.7%, even more preferably in an amount of less than 0.5%, such as 0.41%, where % is the weight per volume (% w/v) of the milk base.

19. A fermented milk product obtained by the method according to paragraphs 1-18.

20. A food or feed product containing at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus which is capable of metabolizing a non-lactose carbohydrate, and a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, preferably wherein the Lactobacillus strain is not a Lactobacillus paracasei strain, even more preferably wherein the Lactobacillus strain is not the L. paracasei strain CRL431 deposited as ATCC 55544 or the L. paracasei strain CHCC 2115 deposited as DSM 19465, wherein the food or feed product comprises 1.3E+08 CFU viable cells of probiotic bacteria/g fermented milk product (CFU/g) or more, preferably 2E+08 CFU/g or more, even more preferably 5E+08 CFU/g or more, such as 6E+08 CFU/g of at least one of the probiotic strains present in the food or feed product immediately after fermentation, preferably at a time that is at least 1 day after completion of fermentation, such as 15 days or 30 days or 45 days or 60 days after completion of fermentation, wherein the food or feed product has been stored at about 4°C after completion of fermentation.

21. A food or feed product according to claim 20, wherein the probiotic strain of Lactobacillus is selected from the group consisting of a strain of Lactobacillus rhamnosus, a strain of Lactobacillus paracasei and a strain of Lactobacillus acidophilus, and wherein the probiotic strain of Bifidobacterium is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis.

22. A food or feed product according to any one of claims 20 to 21, wherein the food or feed product is a fermented milk product, preferably a fermented milk drink, more preferably yoghurt.

23. The food or feed product according to any one of claims 20 to 22, wherein the lactose-deficient strain of Streptococcus thermophilus is a strain as defined in claim 5; and/or wherein the lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus is a strain as defined in claim 6; and/or wherein the probiotic strain of Lactobacillus is selected from the group consisting of the strain Lactobacillus rhamnosus LGG® deposited as ATCC 53103, the strain CRL 431 of Lactobacillus paracasei deposited as ATCC 55544, the strain Lactobacillus acidophilus LA-5® deposited as DSM 13241, and Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954.

24. A composition for producing a fermented milk product, comprising:

a) a starter culture of lactic acid bacteria comprising at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii subsp. bulgaricus, that is capable of metabolizing a non-lactose carbohydrate; and

b) one or more non-lactose carbohydrates capable of being metabolized by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is(are) present in the

- 22 048110 items in a quantity measured in such a way that it(they) is(are) depleted at a pH of the fermented milk product from 4.9 to 5.5, for example from 5.0 to 5.4, preferably about 5.3.

25. The composition according to claim 24, wherein at least one lactose-deficient strain of Streptococcus thermophilus and at least one lactose-deficient strain of Lactobacillus, preferably L. delbrueckii subsp. bulgaricus, is capable of metabolizing the same non-lactose carbohydrate.

26. The composition according to any one of claims 24-25, wherein the non-lactose carbohydrate is selected from the group consisting of sucrose, galactose and glucose, preferably wherein the non-lactose carbohydrate is not glucose, even more preferably wherein the non-lactose carbohydrate is sucrose.

27. The composition according to any one of claims 24 to 26, wherein the lactose-deficient strain of Streptococcus thermophilus is a strain as defined in claim 5; and/or wherein the lactose-deficient strain of Lactobacillus is L. delbrueckii subsp. bulgaricus, as defined in claim 6.

28. The composition according to any one of claims 24 to 27, wherein the composition further comprises a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, preferably wherein the Lactobacillus strain is not a Lactobacillus paracasei strain, even more preferably wherein the Lactobacillus strain is not the L. paracasei strain CRL 431 deposited as ATCC 55544 or the L. paracasei strain CHCC 2115 deposited as DSM 19465.

29. The composition according to any one of claims 24 to 28, wherein the probiotic strain of Lactobacillus is selected from the group consisting of a strain of Lactobacillus rhamnosus, a strain of Lactobacillus paracasei and a strain of Lactobacillus acidophilus, and wherein the probiotic strain of Bifidobacterium is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium infantis.

30. The composition according to any one of claims 28, 29, wherein the probiotic strain of Lactobacillus is selected from the group consisting of the strain Lactobacillus rhamnosus LGG®, deposited as ATCC 53103, the strain CRL 431 Lactobacillus paracasei, deposited as ATCC 55544, the strain Lactobacillus acidophilus LA-5®, deposited as DSM 13241, and Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954.

31. A composition according to any one of claims 28-30, wherein the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate; and

b) Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954.

32. A composition according to any one of claims 28-30, wherein the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate;

b) Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954; and

c) Lactobacillus rhamnosus strain LGG®, deposited as ATCC 53103.

33. A composition according to any one of claims 28-30, wherein the composition comprises:

a) at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate;

b) Bifidobacterium animalis subsp. lactis BB-12®, deposited as DSM 15954; and

c) Lactobacillus acidophilus strain LA-5®, deposited as DSM 13241.

34. The composition according to any one of claims 31 to 33, wherein at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 5, and wherein at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 6.

35. The composition of any one of claims 24 to 34, wherein the composition comprises from about 6-7E+08 CFU (colony forming units)/g of the Streptococcus thermophilus strain or less.

36. The composition according to any one of claims 24 to 35, wherein the composition comprises approximately 1E+07 CFU/g of the strain Lactobacillus delbrueckii subsp. bulgaricus.

37. The composition according to any one of claims 24 to 36, wherein the composition comprises from 1E+06 to 1E+08 CFU/g of the probiotic strain, preferably from 5E+06 to 5E+07 CFU/g, more preferably about 1.2E+07 CFU/g of the probiotic strain.

38. A composition according to any one of claims 24 to 37, wherein the non-lactose carbohydrate is present in the composition in an amount of less than 0.9%, preferably in an amount of less than 0.7%, even more preferably in an amount of less than 0.5%, such as about 0.41%, where % is the weight per volume (% w/v) of the milk base.

39. Use of a composition as defined in any one of claims 24 to 38 for increasing the number of probiotic cells in a fermented dairy product compared to a fermented dairy product fermented with a composition comprising

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a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus that is not lactose-deficient and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus that is not lactose-deficient; and/or

b) 1) a starter culture of lactic acid bacteria containing at least one lactose-deficient strain of Streptococcus thermophilus that is capable of metabolizing a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus that is capable of metabolizing a non-lactose carbohydrate, and

2) one or more non-lactose carbohydrates metabolizable by lactic acid bacteria as defined in (1), wherein the non-lactose carbohydrate(s) is/are(s) present in the composition in an amount measured such that it(they) is/are(are) depleted at a pH of the fermented milk product of less than 4.9, preferably wherein the fermented milk product comprises at least 2E+08 CFU viable probiotic cells/g fermented milk product, preferably at least 4E+08 CFU viable probiotic cells/g fermented milk product, even more preferably at least 5.5E+08 CFU, such as 5.7E+08 CFU viable probiotic cells/g fermented milk product after a shelf life of 60 days at 4°C.

Deposits and expert decisions

The applicant requests that the availability of the deposited microorganism referred to in Rule 33 EPC be effected only by the provision of a sample to an independent expert appointed by the applicant (Rule 32(1) EPC).

Streptococcus thermophilus strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 12.06.2014 under accession number DSM 28952.

Strain Streptococcus thermophilus, deposited in DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 12.06.2014 under accession number DSM 28953.

Streptococcus thermophilus strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32599.

Streptococcus thermophilus strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32600.

Lactobacillus delbrueckii strain subsp. bulgaricus, deposited in DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 12.06.2014 under accession number DSM 28910;

Bifidobacterium animalis strain lactis BB-12®, deposited in the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, 30.09.2003 under accession number DSM 15954;

Lactobacillus acidophilus LA-5® strain, deposited in the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg. 1b, D-38124 Braunschweig, on 30.09.2003 under accession no. DSM 13241.

These deposits were made by the applicant CHR. HANSEN A/S in accordance with the Budapest Agreement on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Trademarks

BB-12® is a registered trademark of Chr. Hansen.

LGG® is a registered trademark of Chr. Hansen.

LA-5® is a registered trademark of Chr. Hansen.

YoFlex® is a registered trademark of Chr. Hansen.

Acidifix® is a registered trademark of Chr. Hansen.

Examples

Example 1.

The purpose of this example is to compare the effect of a lactic acid bacteria starter culture on the cell count of the probiotic cultures BB-12®, LGG® and/or LA-5®, wherein this starter culture comprises at least one lactose-deficient strain of Streptococcus thermophilus which is capable of metabolising a non-lactose carbohydrate, and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus which is capable of metabolising a non-lactose carbohydrate, and a non-lactose carbohydrate capable of being metabolised by the lactic acid bacteria of the starter culture, as defined above, wherein this non-lactose carbohydrate is present in the composition in an amount measured such that it is depleted at a pH of the fermented milk product of about 5.3.

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Starter cultures

Acidifix®: a lactose-deficient culture comprising at least one lactose-deficient strain of Streptococcus thermophilus (ST) and at least one lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus (LB), which is commercially available as F-DVS YoFlex® Acidifix® 1.0 from Chr. Hansen A/S. These strains were isolated as described, for example, in Example 1 of EP 2957180.

YoFlex® Mild 1.0: a commercially available lactose-positive yoghurt culture containing lactose-positive strains of Streptococcus thermophilus and lactose-positive strains of Lactobacillus delbrueckii subsp. bulgaricus. The commercially available strain F-DVS (frozen for direct inoculation (DVS), concentrated frozen culture) YoFlex® Mild 1.0 is from Chr. Hansen A/S. F-DVS YoFlex® Mild 1.0 is available from Chr. Hansen A/S, GIN 702897.

Probiotic cultures

LGG®: Lactobacillus rhamnosus strain LGG®, deposited as ATCC 53103.

BB-12®: Bifidobacterium animalis strain subsp. lactis BB-12®, deposited as DSM 15954.

LA-5®: Lactobacillus acidophilus LA-5®, deposited as DSM 13241.

Cultural compositions

The probiotic strains used for the analysis were Bifidobacterium animalis subsp. lactis BB-12®, Lactobacillus acidophilus LA-5® and Lactobacillus rhamnosus LGG®. They were combined with F-DVS YoFlex® Acidifix® 1.0, which consists of the Lac(-) ST and Lac(-) LB strains as described above. The lactose(+) yoghurt culture F-DSV YoFlex® Mild® 1.0 as described above plus BB-12® and F-DVS BB-12® alone without the yoghurt culture were used as controls.

The inoculation matrix (culture combination) is shown in Table 1 below.

Table 1

Inoculation Matrix. % Inoculation refers to the mass per volume (w/v) of the total milk base. % Sucrose is given as (w/v) based on the milk base.

Milk base F-DVS Acidifix® 1.0 F-DVS YF Mild 1.0 F-DVS BB-12® F-DVS LA-5® FDDVS LGG® note A Milk +0.41% sucrose 0.01% 0.01% 0.001% IN Milk +0.41% sucrose 0.01% 0.01% WITH Milk +0.41% sucrose 0.01% 0.01% 0.01% D Milk +0.90% sucrose 0.01% 0.01% E Milk +0.41% sucrose 0.01% 0.01% control F Milk +0.41% sucrose 0.02% control

When inoculated with F-DVS Acidifix® 1.0 (F-DVS YoFlex® Acidifix® 1.0) or F-DVS YF Mild 1.0 (FDVS YoFlex® Mild 1.0) at a content of 0.01%, the number of ST and LB cells upon inoculation (before fermentation) was 1.2-1.3E+07 CFU/ml. The number of BB-12® cells when inoculated at a content of 0.01% FDVS was 1.2E+07 CFU/ml. The number of LA-5® cells when inoculated at a content of 0.01% F-DVS was 7E+06 CFU/ml. The number of LGG® cells when inoculated at a content of 0.001% (FD-DVS) was 7E+06 CFU/ml.

Cultures were analyzed in milk containing 2% w/w fat and supplemented with skim milk powder to standardize to 4.1% w/w protein (milk base). Sucrose was added to 0.41% or 0.90% (where % is (w/v) based on the milk base) to provide acidification to approximately pH 5.3 and 4.55, respectively. The milk base was heat treated at 92°C for 3 min, cooled to 38°C, and inoculated as described. Milk was incubated at 38°C. The acidification profile was monitored using real-time pH measurement equipment (CINAC) over 20-24 h.

Fermentation was stopped at pH 4.55, probiotic yoghurts were cooled to 4°C and kept at 4°C for the shelf life (storage). Cell numbers were determined by plate counts per day 1, 15, 30, 45 and 60.

Theoretically, the highest cell numbers are achieved by culturing a single strain. However, probiotic strains are selected based on their ability to survive in the human gastrointestinal tract, their ability to adhere to the intestinal mucosa, and their specific health benefits. They have different metabolic activity than the lactic acid bacteria used to acidify milk and produce yogurt and other fermented dairy products. Since this is not their primary function, probiotic strains such as

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BB-12® and LGG® are not well adapted to grow in milk and are therefore unable to acidify milk effectively. They are unable to grow and acidify milk to a pH of less than 6.1 and 5.8, respectively, within 24 h, see Fig. 1.

A significantly higher number of probiotic cells was achieved using a combination of probiotics with F-DVS YoFlex® Acidifix® 1.0, i.e. Lac(-) strains ST and LB, see Fig. 2. The culture combinations were inoculated into milk supplemented with just enough sucrose to ensure acidification to a pH of approximately 5.30.

As shown in Fig. 2, there are two phases of acidification. The first phase corresponds to acidification to a pH of about 5.30. This is due to the growth of Lac(-) strains ST and LB (F-DVS YoFlex® Acidifix® 1.0). The second phase of acidification from 5.30 to 4.55 or less is due only to the growth of probiotic strains, e.g. Bifidobacterium BB-12® with or without LA-5® or LGG®. These probiotic strains are able to metabolize the lactose present in the milk and continue the fermentation (acidification) until the desired pH, e.g. 4.55, is reached. At this point, the milk is cooled to stop further acidification.

Table 2 shows the time required for each of the cultures analyzed to reach a pH of 4.55. Fig. 3 shows the acidification profiles of Acidifix® 1.0 plus 0.01% BB-12® in milk with 0.41% (B) and 0.90% sucrose (D). The % sucrose amount is given as (w/v) based on the milk base as described above.

Table 2 Time to pH 4.55

Combination of culture minutes watch A Acidifix® 1.0 (sucrose added for acidification to pH 5.30) plus BB-12® plus LGG® 1000 16.7 IN Acidifix® 1.0 (sucrose added for acidification to pH 5.30) plus BB-12® 1138 19 WITH Acidifix® 1.0 (sucrose added for acidification to pH 5.30) plus BB-12® plus LA-5® 668 And,1 D Acidifix® 1.0 (sucrose added for acidification to pH 4.55) plus BB-12® 528 8,8 E Mild 1.0 (Lac (+) yogurt culture) plus BB-12® 450 7.5 F BB-12® n/a (not available) n/a

The combination of F-DVS YoFlex® Acidifix® 1.0 (i.e. lactose-deficient Streptococcus thermophilus (ST) and lactose-deficient Lactobacillus delbrueckii subs. bulgaricus (LB)), which was developed to arrest acidification at a pH of about 5.30, with probiotics resulted in higher probiotic cell counts, such as 1.4-9.6E+08 CFU viable probiotic bacteria cells/g fermented milk product (CFU/g) of Bifidobacterium BB-12®, 2.4-4.6E+08 CFU/g L. acidophilus LA-5® and 2.7-4.3E+08 CFU/g L. rhamnosus LGG®. Cell counts of all probiotics were higher than those typically observed in probiotic fermented milks, and these counts were more stable over the 60-day shelf life.

In fermentations with Acidifix® 1.0 in milk with limited sucrose levels (0.41%, i.e. designed to stop acidification at pH around 5.30) (combination of culture A, B and C), the BB-12® cell count was almost 1 log higher than that typically achieved with the combination with yoghurt culture (lac+), see Table 3 below.

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Table 3

Cell count (CFU/g fermented milk product) over a shelf life of 60 days determined by selective counting on agar plates

A Acidifix® 1.0+ BB-12® +LGG® Cl (day 1) C15 C30 C45 C60 Bifidobacterium lactis BB-12® 6.00E+08 8.90E+08 9.60E+08 6DE+08 5.70E+08 L. rhamnosus LGG® 4.30E+08 ZD0E+08 2.80E+08 2.7EE+08 2.80E+08 IN Acidifix® 1.0+BB-12® Bifidobacterium lactis BB-12® 5.20E+08 4.30E+08 3.80E+08 3.60E+08 4.40E+08 WITH Acidifix® 1.0 + BB-12® + LA-5® Bifidobacterium lactis BB-12® 2,20E+08 2D0E+08 l,40E+08 2,20E+08 2,20E+08 L. acidophilus LA-5® 2.70E+08 2.40E+08 4.60E+08 2.80E+08 ZD0E+08 D Acidifix® 1.0+BB-12®pH 4.55 Bifidobacterium lactis BB-12® l,30E+08 9.90E+07 9.20E+07 1D0E+08 7.40E+07 E Mild 1.0+BB-12® Bifidobacterium lactis BB-12® l,20E+08 9.20E+07 8.90E+07 7D0E+07 6.60E+07 F BB-12® Bifidobacterium lactis BB-12® l,20E+07

When the milk base was supplemented with 0.9% sucrose, which was calculated to provide acidification to pH 4.55 (variable D), Acidifix® 1.0 plus BB-12® performed similarly to the lactose (+) yogurt culture YoFlex® Mild 1.0 plus BB-12® (variable E). The BB-12 cell counts in both variables, in which lactose-deficient Streptococcus thermophilus (ST) and lactose-deficient Lactobacillus delbrueckii subs. bulgaricus (LB) were allowed to acidify to 4.55, were comparable and varied around 1.21.3+08 CFU/g. The number of BB-12® cells did not increase when inoculated without yogurt culture (variable F), it was about 1.2E+07 CFU/g, which essentially corresponds to the number of cells in the inoculum.

Improved shelf life survival

Cell counts were also analyzed over 60 days, which is a typical shelf life for fresh fermented dairy products in North America and some other regions of the world. Over the shelf life of a typical probiotic yogurt, Bifidobacterium BB-12 cell counts typically decline by 0.5-1 log per 60 days, depending on the yogurt culture, dairy base, culture, and storage conditions. LA-5® cell counts typically decline by 1-2 log per 60 days of shelf life. When co-cultured with Acidifix® 1.0 (growth limited by pH around 5.30), cell counts of BB-12®, LA-5®, and LGG® probiotics were 0.5-1 log higher than typically observed and also demonstrated excellent stability over the 60 day shelf life.

RST (Original in electronic form)

0-1 0-1-1 Form PCT/RO/134 Indications relating to the deposited microorganism(s) or other biological material (PCT Rule 13bis) Obtained using Filling out the PCT online Version 3.51.000.266e MT/FOR 20141031/0.20.5.20 0-2 International Application No. 0-3 Link to applicant or agent file P6473РС00

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1 1-1 1-2 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description on: page line 53 27 1-3 Deposit identification 1-3-1 Name of the depository DSM Leibniz Institute DSMZ - German Institute collection of microorganisms and cell cultures 1-3-2 Address of the depository institution Inhoffenstr. 7B, D-38124 Braunschweig, Germany 1-3-3 Deposit date June 12, 2014 (12.06.2014) 1-3-4 Access number DSM 28952 1-4 Additional instructions The sample is subject to issue only to the expert 1-5 The countries indicated for which the instructions are given All specified 2 The instructions given below apply to the deposited microorganism(s) or other biological material for which the

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2-1 2-2 link in description on: page line 54 1 2-3 Deposit identification 2-3-1 Name of the depository institution DSM Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures 2-3-2 Address of the depository institution Inhoffenstr. 7B, D-38124 Braunschweig, Germany 2-3-3 Deposit date June 12, 2014 (12.06.2014) 2-3-4 Access number DSM 28953 2-4 Additional instructions The sample is subject to issue only to the expert 2-5 The countries indicated for which the instructions are given All specified 3 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description of: 3-1 page 54 3-2 line 5 3-3 Deposit identification 3-3-1 Name of the depository institution DSM Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures 3-3-2 Address of the depository institution Inhoffenstr. 7B, D-38124 Braunschweig, Germany 3-3-3 Deposit date August 22, 2017 (08/22/2017) 3-3-4 Access number DSM 32599 3-4 Additional instructions The sample is subject to issue only to the expert 3-5 The countries indicated for which the instructions are given All specified 4 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description of: 4-1 page 54 4-2 line 10

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4-3 4-3-1 Deposit Identification Name of the depository institution DSM Leibniz Institute DSMZ - German collection of microorganisms and cellular cultures 4-3-2 Address of the depository institution Inhoffenstr. 7B, D-38124 Braunschweig, Germany 4-3-3 Deposit date August 22, 2017 (08/22/2017) 4-3-4 Access number DSM 32600 4-4 Additional instructions The sample is subject to issue only to the expert 4-5 The countries indicated for which the instructions are given All specified 5 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description of: 5-1 page 54 5-2 line 15 5-3 Deposit identification 5-3-1 Name of the depository institution DSM Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures 5-3-2 Address of the depository institution Inhoffenstr. 7B, D-38124 Braunschweig, Germany 5-3-3 Deposit date June 12, 2014 (12.06.2014) 5-3-4 Access number DSM 28910 5-4 Additional instructions The sample is subject to issue only to the expert 5-5 The countries indicated for which the instructions are given All specified 6 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description of: 6-1 page 54 6-2 line 19 6-3 Deposit identification 6-3-1 Name of the depository institution DSM Leibniz Institute DSMZ - German Collection of Microorganisms and Cells

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Claims (2)

6-3-2 6-3-3 6-3-4 Address of depository institute Date of deposit Culture accession number Inhoffenstr. 7B, D-38124 Braunschweig, Germany September 30, 2003 (09/30/2003) DSM 15954 6-4 Additional instructions The specimen is to be released only to an expert 6-5 Specified countries for which instructions are given All specified 7 The instructions given below apply to the deposited microorganism(s) or other biological material referred to in the description on: 7-1 page 54 7-2 line 21 7-3 Identification of the deposit 7-3-1 Name of the depository institute DSM Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures 7-3-2 Address of the depository institute Inhoffenstr. 7B, D-38124 Braunschweig, Germany 7-3-3 Date of deposit September 30, 2003 (09/30/2003) 7-3-4 Accession number DSM 13241 7-4 Additional indications Sample to be issued only to examiner 7-5 Indicated countries for which indications are made All of those indicated For use by receiving office only 0-4 This form was received with the international application: (yes or no) 0-4-1 Authorized officer For use by the International Bureau only 0-5 This form was received by the International Bureau: 0-5-1 Authorized officer CLAIMS (1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28910; and (2) a strain derived from DSM 28910, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal. 7. The method according to any one of claims 1 to 6, wherein the probiotic strain is selected from the group consisting of the Lactobacillus rhamnosus strain deposited as ATCC 53103, the Lactobacillus acidophilus strain deposited as DSM 13241, and Bifidobacterium animalis subsp. lactis deposited as DSM 15954. 8. The method according to any one of claims 1 to 7, wherein the probiotic strain added to the milk base in step (1c) is Bifidobacterium animalis subsp. lactis, deposited as DSM 15954. 9. The method according to any one of claims 1 to 8, wherein step (1) comprises adding to the milk base: a) at least one strain of Streptococcus thermophilus deficient in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus deficient in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate; b) a non-lactose carbohydrate capable of being metabolised by lactic acid bacteria as defined in (a), wherein said non-lactose carbohydrate is added in an amount measured such that it is depleted at a pH of the fermented milk product of from 4.9 to 5.5, such as from 5.0 to 5.4, preferably about 5.3; and (c) (1) Bifidobacterium animalis subsp. lactis, deposited as DSM 15954; or (2) Bifidobacterium animalis subsp. lactis, deposited as DSM 15954 and the Lactobacillus rhamnosus strain deposited as ATCC 53103; or (3) Bifidobacterium animalis subsp. lactis, deposited as DSM 15954 and the Lactobacillus acidophilus strain deposited as DSM 13241. 10. The method according to claim 9, wherein at least one strain of Streptococcus thermophilus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 5, and wherein at least one strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate is defined as in claim 6. 11. A fermented milk product obtained by the method according to any one of claims 1 to 10, comprising 1.3E+08 CFU (colony forming unit) of viable cells of probiotic bacteria/g of fermented milk product (CFU/g) or more of at least one of the probiotic strains present in the fermented milk product selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, wherein the probiotic Lactobacillus strain is selected from the group consisting of a Lactobacillus rhamnosus strain and a Lactobacillus acidophilus strain, and/or wherein the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis, at a time point that is at least 1 day after the completion of the fermentation as defined in subparagraph (2) of claim 1, wherein the fermented milk product has been stored at about 4°C after - 32 048110 completion of fermentation. 12. A food or feed product which is a fermented milk product comprising at least one strain of Streptococcus thermophilus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate, and a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, wherein the probiotic Lactobacillus strain is selected from the group consisting of a Lactobacillus rhamnosus strain and a Lactobacillus acidophilus strain, and/or wherein the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis, and wherein the food or feed product contains 1.3E+08 CFU (colony forming unit) of viable cells of probiotic bacteria/g fermented milk product (CFU/g) or more of at least one of the probiotic strains present in the food or feed product, at a time point that is at least 1 day after completion of the fermentation as defined in subparagraph (2) of paragraph 1, wherein the food or feed product has been stored at about 4°C after completion of the fermentation. 13. The food or feed product of claim 12, wherein said food or feed product comprises 2E+08 CFU/g or more, preferably 5E+08 CFU/g or more of at least one of the probiotic strains present in the food or feed product, at a time that is at least 1 day after completion of the fermentation as defined in subparagraph (2) of paragraph 1, wherein said food or feed product has been stored at about 4°C after completion of the fermentation. 14. A food or feed product according to claim 12, which is preferably a fermented milk drink, more preferably yoghurt. 15. The food or feed product according to any one of claims 12 to 14, wherein the strain of Streptococcus thermophilus deficient in lactose metabolism is a strain as defined in claim 5; and/or wherein the strain of Lactobacillus delbrueckii subsp. bulgaricus deficient in lactose metabolism is a strain as defined in claim 6; and/or wherein the probiotic strain is selected from the group consisting of the strain of Lactobacillus rhamnosus deposited as ATCC 53103, the strain of Lactobacillus acidophilus deposited as DSM 13241, and Bifidobacterium animalis subsp. lactis deposited as DSM 15954. 16. A composition for producing a fermented milk product, comprising: a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate; and b) one or more non-lactose carbohydrates capable of being metabolized by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 4.9 to 5.5. 17. The composition of claim 16, wherein the non-lactose carbohydrate(s) is(are) present in the composition in an amount measured such that it(they) is(are) depleted at a pH of the fermented milk product of from 5.0 to 5.4, preferably about 5.3. 18. The composition according to claim 16, wherein the strain of Streptococcus thermophilus deficient in lactose metabolism is a strain as defined in claim 5; and/or wherein the strain of Lactobacillus delbrueckii subsp. bulgaricus deficient in lactose metabolism is as defined in claim 6. 19. The composition according to any one of claims 16 to 18, further comprising a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, wherein the probiotic Lactobacillus strain is selected from the group consisting of a Lactobacillus rhamnosus strain and a Lactobacillus acidophilus strain, and/or wherein the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis. 20. Use of a composition as defined in any one of claims 16 to 19 for increasing the number of viable probiotic cells in a fermented dairy product compared to a fermented dairy product fermented with a composition comprising a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus that is not a strain with a deficiency in lactose metabolism and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus that is not a strain with a deficiency in lactose metabolism; and/or b) 1) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate, and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate, and 1) adding to the milk base: a) a starter culture of lactic acid bacteria comprising at least one strain of Streptococcus thermophilus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate, and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism that is capable of metabolizing a non-lactose carbohydrate; b) one or more non-lactose carbohydrates capable of being metabolised by lactic acid bacteria as defined in (a), wherein the non-lactose carbohydrate(s) is/are added in an amount measured such that it/they are/are depleted at a pH of the fermented milk product of from 4.9 to 5.5; and c) a probiotic strain selected from the group consisting of a Lactobacillus strain and a Bifidobacterium strain, wherein the probiotic Lactobacillus strain is selected from the group consisting of a Lactobacillus rhamnosus strain and a Lactobacillus acidophilus strain, and/or wherein the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis; 2) fermenting the given milk base for some period of time until the target pH is reached, resulting in a fermented milk product. 2. The method according to claim 1, wherein the non-lactose carbohydrate(s) is(are) added in an amount measured in such a way that it(they) is(are) depleted at a pH of the fermented milk product from 5.0 to 5.4, pred - 31 048110 respectfully approximately 5.3. 3. The method according to claim 1 or 2, wherein the non-lactose carbohydrate(s) is(are) selected from the group consisting of sucrose, galactose and glucose. 4. The method according to any one of claims 1 to 3, wherein the target pH in step (2) is from about 4.8 to about 4.0, preferably from about 4.6 to about 4.55, more preferably about 4.55. 5. The method according to any one of claims 1 to 4, wherein the strain of Streptococcus thermophilus with a deficiency in lactose metabolism is selected from the group consisting of: (a) (1) the strain deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures), Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28952; (2) a strain derived from DSM 28952, wherein this derivative strain is further distinguished by the ability to form white colonies on a medium containing lactose and X-Gal; (b) (1) the strain deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under accession number DSM 28953; (2) a strain derived from DSM 28953, wherein this derivative strain is further distinguished by the ability to form white colonies on a medium containing lactose and X-Gal; (c) (1) the strain deposited in DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32599; (2) a strain derived from DSM 32599, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal; and (d) (1) the strain deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 22.08.2017 under accession number DSM 32600; and (2) a strain derived from DSM 32600, wherein this derivative strain is further characterized by having the ability to form white colonies on a medium containing lactose and X-Gal. 6. The method according to any one of claims 1 to 5, wherein the strain of Lactobacillus delbrueckii subsp. bulgaricus with a deficiency in lactose metabolism is selected from the group consisting of: 1. A method for producing a fermented milk product, comprising the following stages: 2) one or more non-lactose carbohydrates capable of being metabolized by lactic acid bacteria as defined in (1), wherein the non-lactose carbohydrate(s) is(are) present in the composition -