EA041084B1 - THERMOPHILIC STREPTOCOCCI FOR USE IN THE PREPARATION OF FERMENTED PRODUCTS - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION

The present invention relates to new strains of Streptococcus thermophilus, compositions containing these strains, and methods for obtaining these compositions.

State of the art of the present invention

Streptococcus thermophilus (also referred to as Streptococcus salivarius subsp. thermophilus and hereinafter S.thermophilus) is a Gram-positive lactic acid bacteria species widely used for the preparation of fermented dairy products. S.thermophilus is often used in conjunction with Lactobacillus delbrueckii subsp. bulgaricus (also called L. bulgaricus) as a starter culture for yogurt and other fermented milks. Yoghurts and other fermented dairy products are ubiquitous and essential to a healthy diet as a source of essential nutrients. However, unflavored fermented dairy products have a characteristic sour taste, so flavored and sweetened fermented dairy products are more popular. Increasing consumer awareness of the benefits of a low-sugar diet, along with a greater consumer focus on natural foods, has led to the need to develop reduced-sugar fermented dairy products that use natural sweeteners (such as stevia). The use of high sugar producing strains of S. thermophilus and L. bulgaricus has also been proposed as a means of increasing the sweetness of fermented dairy products.

WO 2013/160413 discloses mutant strains of S. thermophilus with sweetening properties due to glucose secretion. These strains are characterized by a mutation in the glucokinase (glcK) gene and are described as suitable for the preparation of fermented dairy products. However, it is also disclosed that for such strains, the fermentation process lasts 2-5 hours longer, and at least 20 hours are required for fermentation. Sorensen et al. (Appl Environ Microbiol. 2016 Apr 22) also disclose glucose-secreting strains of S. thermophilus St1-GS-1; St1-GS-2; St2-GS-1; ST2-GS-2. Strains St1-GS-2 and St2-GS-2 are not able to acidify milk, and strains mutated by glucokinase (i.e. St1-GS-1 and St2-GS-1) are characterized by a delay in the onset of acidification by 2-3 hours compared to parental strains.

The essence of the invention

The present invention is based on the unexpected discovery that the new strains of Streptococcus thermophilus CNCM I-5030 and CNCM I-4992 have sweetening properties and are also exceptionally useful in the preparation of fermented dairy products due to their ability to acidify milk while reducing subsequent acidification. fermented dairy products. Accordingly, the present invention provides the Streptococcus thermophilus strains deposited with CNCM under CNCM I-5030 and CNCM I-4992. The present invention also provides compositions containing S. thermophilus CNCM I-5030 and/or CNCM I-4992, and methods for their preparation.

Detailed description of the present invention

As used in the present invention, the term stable composition should be understood as a composition that does not contain sediment and/or in which no serum separation is observed.

As used in the present invention, the term x% (wt%) is equivalent to x g per 100 g.

As used in the present invention, the terms dairy composition, milk-based composition or dairy product should be understood as a product or composition primarily containing or consisting of milk or milk components and optionally other ingredients.

As used in the present invention, the term fermented milk product should be understood as a product or composition that results from the acidifying fermentation of a milk-based composition with a starter culture of fermenting microorganisms, in particular bacteria, preferably lactic acid bacteria. As used in the present invention, the term fermented milk should be understood as a product or composition obtained from milk by acidifying it with at least one lactic acid bacterium. Accordingly, as used in the present invention, the fermented milk product can be a fermented milk, such as yogurt (for example, non-stirred yogurt, stirred yogurt or drinking yogurt) or fresh cheese, such as white cheese or Petit-suisse (Petit-suisse) . It can also be strained fermented milk such as strained yogurt (like concentrated or Greek yogurt).

The terms fermented milk and yogurt are used in the present invention in their usual meaning in the field of the dairy industry, i. means products suitable for human consumption and obtained by acidifying lactic fermentation of milk substances. These products may contain secondary ingredients such as fruits, vegetables, sugar, and the like. The expression fermented milk can be used to refer to fermented milk other than yogurt, such as kefir, koumiss, lassi, dahi, Leben, Filmjolk, Villi, acidophilus milk.

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As used in the present invention, the term yogurt should be understood as fermented milk resulting from acidifying lactic acid fermentation carried out by thermophilic lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (also called Streptococcus salivarius subsp. thermophilus), while the bacteria must be alive in the finished product in a minimum number of CFU. In some countries, regulations allow other lactic acid bacteria to be added to yogurt as well, such as, but not limited to, strains of Bifidobacterium and/or Lactobacillus acidophilus and/or Lactobacillus casei. These additional strains of lactic acid bacteria are intended to impart various qualities to the finished products, for example, they provide organoleptic properties, favor the balance of the intestinal microflora or modulate the immune system.

As used in the present invention, the term strained fermented milk composition should be understood as a lactic acid composition that has been subjected to a process for separating acid whey after fermentation.

As used in the present invention, the term viscous should be understood as a solid or semi-solid product that can be consumed with a spoon or other utensil.

As used in the present invention, the term fermentation, or fermentation, should be understood as the metabolism of substances by microorganisms, such as bacteria, yeast or other microorganisms.

As used in the present invention, the term CFU is an abbreviation for the term colony forming unit.

As used in the present invention, the term CNCM 1st 4-digit number should refer to a strain stored in the National Collection of Cultures of Microorganisms (CNCM) 25 rue du Doeur Roux, Paris, France in accordance with the Budapest Treaty, and having an access number, corresponding to the specified 4-digit number, for example CNCM 1-5030.

As used herein, reference to a bacterial strain or species should include functionally equivalent bacteria derived therefrom, such as, but not limited to, mutants, variants, or genetically transformed bacteria. These mutants or genetically transformed strains may be strains in which one or more endogenous genes of the parental strain have been mutated, for example, to modify some of their metabolic properties (for example, their ability to ferment sugars, their resistance to acidification, their survival during transport in the gastrointestinal tract, their properties after acidification, or their production of metabolites). It can also be strains obtained by genetically transforming a parental strain by introducing one or more target genes, for example, to confer additional physiological properties to said genetically transformed strains or to enable them to express proteins of therapeutic or prophylactic purpose, thus allowing introduce these proteins through the indicated strains. These mutants or genetically transformed strains can be obtained from the parent strain by conventional methods of random or site-directed mutagenesis and genetic transformation of bacteria, or by a method known as genome shuffling. In the present text, strains, mutants, and variants derived from a parent species or strain are considered to be encompassed by reference to said parental species or strain, e.g., the phrases Streptococcus thermophilus and CNCM 15030 should be understood to include strains, mutants, and variants derived from them. . Accordingly, as used in the present invention, reference to a bacterial strain by number should be understood to include variants thereof that are at least 95% identical (see Stackebrandt & Goebel, 1994, Int. J. Syst. Bacterial. 44:846 -849). In a most preferred embodiment, said variant has at least 97% identity with the 16S rRNA sequence of the particular strain indicated, more preferably at least 98% identity, more preferably at least 99% identity or more.

As used in the present invention, the term substantially pure in relation to the bacterial strain refers to the percentage of the specified bacterial strain relative to the total content of microorganisms. Substantially pure may be at least about 99.99%, at least about 99.90%, at least about 99.50%, at least about 99.00%, at least about 95.00% , at least about 90.00%, at least about 85.00%, or at least about 75.00%.

As used in the present invention, the term lactic acid bacterium refers to a Gram-positive, acid-fast, usually non-spore-forming and non-oxygen-consuming bacterium, either rod-shaped or coccoid, which is capable of fermenting sugars into lactic acid.

The present invention relates to new strains of Streptococcus thermophilus, compositions comprising the specified strain, and methods for obtaining these compositions.

Streptococcus thermophilus.

In a first aspect, the present invention relates to strains of Streptococcus thermophilus. In lane

- 2 041084 In the second embodiment, the present invention relates to a strain of Streptococcus thermophilus CNCM I-5030. This strain was deposited with the National Collection of Cultures of Microorganisms (CNCM) (Pasteur Institute, 25 Rue du Docteur Roux, Paris, France) in accordance with the Treaty of Budapest on November 26, 2015 under the number CNCM 1-5030. The deposit was made in accordance with the Budapest Treaty on the Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, as stated in the application, the applicant requires that a sample of the deposited microorganisms be provided only to an independent examiner, before the date when a patent can be obtained. In one embodiment, the present invention relates to an isolated strain of Streptococcus thermophilus CNCM I-5030, preferably said isolate being substantially pure.

In a second embodiment, the present invention relates to a strain of Streptococcus thermophilus CNCM I-4992. This strain was deposited with the National Collection of Cultures of Microorganisms (CNCM) (Institut Pasteur, 25 Rue du Docteur Roux, Paris, France) in accordance with the Treaty of Budapest on July 1, 2015 under the number CNCM 1-4992. The deposit was made in accordance with the Budapest Treaty on the Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, as stated in the application, the applicant requires that a sample of the deposited microorganisms be provided only to an independent examiner, before the date when a patent can be obtained. In one embodiment, the present invention relates to an isolated strain of Streptococcus thermophilus CNCM 1-4992, preferably said isolate being substantially pure.

Compositions of the present invention.

In a second aspect, the present invention provides compositions containing Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992. Preferably the composition contains at least 106, even more preferably at least 10 ⁷ and most preferably at least 10 ⁸ colony forming units (CFU) of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per gram of composition according to embodiments inventions.

In embodiments, the composition contains from 105 to 10 ¹² colony forming units (cfu) of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per gram of composition in accordance with embodiments of the invention. In other embodiments, the composition contains from 106 to 10 ¹¹ colony forming units (CFU) of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per gram of composition in accordance with embodiments of the invention.

The bacterium, as provided in the present invention, is suitable for use in edible compositions, accordingly, in one embodiment, the present invention relates to a composition suitable for human consumption, preferably orally. Accordingly, the composition contains or consists of an edible substance. It is particularly preferred that the compositions according to embodiments of the invention are substantially free of pathogenic or toxicogenic substances. The composition according to embodiments of the invention may be a drug or a pharmaceutical composition. In a most preferred embodiment, the composition according to the invention may be a non-therapeutic composition, preferably a nutraceutical composition, a nutritional composition and/or a nutritional composition. Most preferably, the food composition is a fermented food composition, preferably a fermented milk composition. Other compositions in accordance with embodiments of the invention also contain nutritional supplements, food ingredients, food formulas, infant formula, first level milk formulas and second level milk formulas.

The composition may also contain additional strains of Bifidobacterium and/or lactic acid bacteria; usually 2, 3, 4 or more additional strains. Examples of Bifidobacterium that can be used include, but are not limited to, Bifidobacterium animalis (eg Bifidobacterium animalis subsp. animalis or Bifidobacterium animalis subsp. lactis); Bifidobacterium longum; Bifidobacterium breve; Bifidobacterium bifidum. Examples of lactic acid bacteria that can be used include, but are not limited to, Lactobacilli (e.g. Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbruckei, in particular L. delbrueckii subsp. bulgaricus or lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus johnsonii , Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus rhamnosus); Lactococci (eg Lactococcus lactis, usually Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris). Preferably, the composition further comprises Lactobacillus and/or Streptococcus. For the preparation of yogurt, the composition typically contains Lactobacillus bulgaricus (also referred to as Lactobacillus delbrueckii subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms such as, but not limited to, probiotic species or other species that may provide the desired organoleptic or other properties of the composition, for example additional strains of Lactococcus lactis.

Accordingly, in one embodiment, the present invention relates to a composition comprising Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 and further comprising at least one strain of Lactobacillus bulgaricus, and optionally one or more strains of Lactococcus lactis and/or Bifidobacterium.

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Dairy compositions.

The strains of the present invention are well suited for the preparation of fermented compositions. Accordingly, in one embodiment, the present invention relates to a dairy composition, preferably a lactic acid composition. The milk composition of the present invention contains milk, preferably fermented milk. Preferably the composition contains at least about 30% (wt.%) milk, more preferably at least about 50% (wt.%) milk, and even more preferably at least about 70% (wt.%) milk. In embodiments, the composition contains from 30 to 100% (wt.%) milk. In embodiments, the composition contains from 50 to 100% (wt.%) milk. In embodiments, the composition contains from 70 to 100% (wt.%) milk. Preferably said milk is vegetable and/or animal milk, more preferably soy, almond, oat, hemp, wheat, coconut, rice, goat, sheep, camel, mare or cow's milk and most preferably cow's milk. Preferably, said milk is subjected to a heat treatment, usually pasteurization, to ensure sterility. Preferably, said heat treatment is carried out prior to the preparation of the fermented milk composition.

Preferably said milk contains skimmed, partially skimmed or full fat milk. Preferably said milk may be in liquid, powder and/or concentrated form. In one embodiment, said milk further comprises milk components, preferably selected from the group consisting of cream, casein, caseinate (e.g., calcium or sodium caseinate), whey proteins, in particular in the form of a concentrate (WPC), milk proteins, in particular, in the form of a concentrate (MPC), milk protein hydrolysates and mixtures thereof. In one embodiment, said mixture further comprises vegetable and/or fruit juices. In one embodiment, said milk may be fortified with additional milk components or other nutrients such as, but not limited to, vitamins, minerals, trace elements, or other micronutrients.

Preferably, the dairy composition contains more than about 0.3 g per 100 g (by weight) of free lactic acid, more preferably more than about 0.7 g or 0.6 g per 100 g (by weight) of free lactic acid. In embodiments, the composition contains from 0.3 to 0.7 g per 100 g (by weight) of free lactic acid.

Preferably, the protein content of the milk composition is at least equivalent to the protein content of the milk from which it is derived, preferably at least about 2.5%, more preferably at least about 3 or 3.5% (wt.%) . Preferably the composition has a pH equal to or less than 5, preferably from about 3 to 4.5, and more preferably from about 3.5 to 4.5.

Preferably the dairy composition has a viscosity below 200 mPa-s, more preferably below 100 mPa-s and most preferably below 60 mPa-s at 10°C and a shear rate of 64 s ^-1 . In embodiments, the composition has a viscosity range of 1 to 200 mPa-s, 1 to 100 mPa-s, or 1 to 60 mPa-s at 10° C. and a shear rate of 64 s ^-1 . In embodiments, the composition has a viscosity range of 10 to 200 mPa-s, 10 to 100 mPa-s, or 10 to 60 mPa-s at 10° C. and a shear rate of 64 s ^-1 . In embodiments, the composition has a viscosity range of 30 to 200 mPa-s, 30 to 100 mPa-s, or 30 to 60 mPa-s at 10° C. at a shear rate of 64 s ^-1 .

The fermented milk composition according to embodiments of the invention is preferably a product selected from the group consisting of yogurt, unstirred yogurt, stirred yogurt, liquid yogurt, drinking yogurt, frozen yogurt, kefir, buttermilk, cottage cheese, sour cream, fresh cheese and cheese. In one embodiment, the composition according to embodiments of the invention is a drinkable composition, more preferably a fermented milk drink such as, but not limited to, a yoghurt drink, kefir, and the like. In an alternative embodiment, the composition according to embodiments of the invention is a viscous composition, such as unstirred or stirred yogurt, or equivalent.

In one embodiment, the fermented milk composition is a strained fermented milk composition. The filtered fermented milk composition preferably has the following composition (wt.%):

8.5 to 11.0% milk protein, 0.0 to 8.0% fat, e.g. 0.0 to 3.5% or 3.5 to 8.0%, 0.00 to 4 .20% lactose, for example from 2.80 to 4.20%.

The pH value of the strained fermented milk composition may be, for example, from 3.80 to 4.65.

Preferably, the composition according to embodiments of the invention can be stored, transported and/or distributed at a temperature of from 1 to 10°C for at least

- 4 041084 at least about 30 days, at least about 60 days, or at least about 90 days from the date of packaging, while remaining fit for consumption.

In embodiments, the dairy compositions of the present invention contain at least 105 CFU/g, more preferably at least 106 CFU/g, such as at least 10 ⁷ CFU/g, such as at least 10 ⁸ CFU/g, such as , at least 109 CFU/g, for example, at least 10 ¹⁰ CFU/g, for example, at least 10 ¹¹ CFU/g Streptococcus thermophilus CNCM I5030 and/or CNCM I-4992 per 1 g of milk composition. In embodiments, the compositions of the present invention contain 10 ⁵ to 10 ¹² or 106 to 10 ¹⁰ colony forming units (cfu) of Streptococcus thermophilus CNCM I-503 0 and/or CNCM I-4992 per gram of composition.

Preferably the composition is a packaged product that contains at least 106, more preferably at least 10 ⁷ and most preferably at least 10 ⁸ colony forming units (CFU) Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per 1 g compositions in accordance with embodiments of the invention after storage, transportation and/or distribution at a temperature of from 1 to 10°C for at least about 30 days, at least about 60 days, or at least about 90 days from the date of packaging.

In embodiments, the composition is a packaged product that contains 10 ⁵ to 10 ¹² or 106 to 10 ¹⁰ colony forming units (cfu) of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per gram of composition according to embodiments of the invention after storage, transport and/or distribution at a temperature of from 1 to 10°C for at least about 30 days, at least about 60 days, or at least about 90 days from the date of packaging.

In embodiments, the dairy composition further comprises an adjuvant. Adjuvants are known to the person skilled in the art. They are typically used to change the taste, mouthfeel and/or texture of a dairy composition, such as a fermented milk composition. They can also be used to administer certain supplements such as nutrients. They typically include sweeteners, flavors, color modifiers, cereals and/or fruits. Fruit auxiliaries are, for example, suspensions or fruit preparations. Flavors include, for example, fruit flavors, vanilla flavors, caramel flavors, coffee flavors, chocolate flavors.

Fruit preparations usually contain fruits. As used herein, the term fruit refers to any form of fruit, including, for example, whole fruit, fruit pieces, purees, concentrates, juices, and the like.

The auxiliary preparation or suspension usually contains a stabilizing agent having at least one stabilizer. The stabilizing agent may include at least two stabilizers. Such stabilizers are known to the person skilled in the art. They generally help to avoid phase separation of solids, eg fruits or fruit extracts, and/or help to avoid syneresis. They typically provide some viscosity to the composition, eg a viscosity (Bostwick viscosity at 20° C.) of 1 to 20 cm/min, preferably 4 to 12 cm/min.

The stabilizing system or stabilizer may be, for example, starch, pectin, guar, xanthan, carrageenan, locust bean gum, or a mixture thereof. The amount of the stabilizing system is usually from 0.5 to 5% by weight.

The adjuvant may typically contain organoleptic modifiers. Such ingredients are known to the person skilled in the art.

Sensory modifiers can be, for example, non-sugar sweeteners, colorants, cereals and/or cereal extracts.

Examples of sweeteners are ingredients called high intensity sweeteners such as sucralose, acesulfame potassium, aspartame, saccharin.

Examples of fruits include, for example, strawberry, peach, apricot, mango, apple, pear, raspberry, blueberry, blackberry, passion fruit, cherry, and a mixture or combination thereof, such as peach-passion fruit.

Fruit can be provided, for example, in the form of frozen fruit cubes, for example, in the form of 10 mm fruit cubes, for example, in the form of individual quick frozen (Individual Quick Frozen) fruit cubes, for example, in the form of strawberry, peach, apricot, mango fruit cubes , apples, pears or mixtures thereof, aseptic fruit cubes, for example in the form of 10 mm fruit cubes, for example in the form of strawberry, peach, apricot, mango, apple or pear fruit cubes or mixtures thereof, fruit purees, for example fruit purees, concentrated 2 to 5 times, preferably 3 times, e.g. aseptic fruit purees, e.g. strawberry, peach, apricot, mango, raspberry, blueberry or apple fruit puree or mixtures thereof, individual aseptic fruit purees, e.g. as single strawberry, raspberry, peach, apricot, blueberry or apple aseptic fruit puree or mixture thereof, frozen whole fruit, e.g. frozen flax (Individual

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Quick Frozen) whole fruits, such as whole frozen blueberries, raspberries or blackberries, or mixtures thereof, mixtures thereof.

The ingredients and/or components of the adjuvant and their amounts can generally be such that the composition has a Brix from 1 to 65 Brix, such as from 1 to 10 Brix, or from 10 to 15 Brix, or from 15 to 20 Brix, or from 20 to 25 brix or 25 to 30 brix or 30 to 35 brix or 35 to 40 brix or 40 to 45 brix or 45 to 50 brix or 50 to 55 brix or 55 up to 60 brix, or from 55 to 60 brix, or from 60 to 65 brix.

For example, a fruit preparation may contain fruit in an amount of 30 to 80% by weight, such as 50 to 70% by weight.

The auxiliary preparation may contain water. It is mentioned that part of the water may come from the ingredients used to prepare the fruit preparation, for example from fruits or fruit extracts or from a phosphoric acid solution.

The fruit preparation may contain pH modifying agents such as citric acid. The fruit preparation has a pH of 2.5 to 5, preferably 2.8 to 4.2.

Typically, the fruit preparation can be added in an amount of 5-35% by weight, based on the total amount of the composition. In embodiments, the composition of the present invention contains up to about 30 wt.% of the specified auxiliary drug, for example up to 10, 15, 20, 25 wt.%. In one of the embodiments, the implementation of the composition in accordance with the embodiments of the invention contains from 1 to 30 wt.% of the specified auxiliary drug. In alternative embodiments, the implementation of the composition in accordance with the embodiments of the invention contains from 1 to 25 wt.% of the specified adjuvant drug. In other alternative embodiments, the implementation of the composition in accordance with the embodiments of the invention contains from 1 to 20 wt.% of the specified adjuvant drug. In additional embodiments, the implementation of the composition in accordance with embodiments of the invention contains from 1 to 15 wt.% of the specified adjuvant drug. In other additional embodiments, the implementation of the composition in accordance with embodiments of the invention contains from 1 to 10 wt.% of the specified adjuvant drug.

Preferably, the composition according to embodiments of the invention is provided in a sealed or sealed container containing about 50, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 , 145, 150, 200, 300, 320 or 500 g or approximately 1 oz, 2 oz, 3 oz, 4 oz, 5 oz, 6 oz or 12 oz of product by weight.

In embodiments, a composition according to embodiments of the invention is provided in a sealed or sealed container containing about 50 to 500 g, 60 to 500 g, 70 to 500 g, 75 to 500 g, 80 to 500 g, 85 to 500 g, 90 to 500 g, 95 to 500 g, 100 to 500 g, 105 to 500 g, 110 to 500 g, 115 to 500 g, 120 to 500 g, 125 to 500 g , 130 to 500 g, 135 to 500 g, 140 to 500 g, 145 to 500 g, 150 to 500 g, 200 to 500 g, 300 to 500 g, 320 to 500 g or 500 g product by weight. In embodiments, a composition according to embodiments of the invention is provided in a sealed or sealed container containing about 1 to 12 ounces, 2 to 12 ounces, 3 to 12 ounces, 4 to 12 ounces, 5 to 12 ounces, 6 to 12 ounces or 12 ounces of product by weight.

Compositions of inoculums.

The bacterium, as described in the present invention, can be used as a starter culture in the preparation of food compositions such as fermented milk products. Accordingly, in one embodiment, the present invention relates to an inoculum containing Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992, which is suitable for the preparation of fermented milk products. The inoculum of the present invention is suitable for direct inoculation of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 into a milk-containing composition to produce the fermented milk products of the present invention, wherein a culture step prior to said direct inoculation is generally not necessary.

Usually the inoculum additionally contains an auxiliary substance or carriers, the choice of which is within the competence of a person skilled in the art, but may contain buffers or culture media. The inoculum may optionally contain additional components such as cryoprotectants, preservatives and/or additives, including nutrients such as yeast extracts, cysteine, sugars and vitamins.

Usually the inoculum is intended for the preparation of fermented milk products, in accordance with one embodiment, the implementation of the inoculum of the present invention can be provided for a dairy composition in an amount of up to about 500 mg/L.

Typically, the inoculum is fresh, frozen, dry or lyophilized. The inoculum may be in liquid, dry, lyophilized or solid form. It is particularly preferred that the inoculum is in liquid form. The inoculum may be thawed and/or dispersed in a liquid (eg water) prior to inoculation into the milk composition.

In embodiments, the inoculum contains at least 109 cfu, such as at least

- 6 041084 least 10 ¹⁰ CFU, for example at least 10 ¹¹ CFU Streptococcus thermophilus CNCM I-5030 and/or

CNCM I-4992 per 1 g of inoculum composition. In embodiments, the inoculum contains from 109 to

10 ¹² colony forming units (CFU) or more preferably 10 ¹⁰ to 10 ¹² colony forming units (CFU) Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 per 1 g of inoculum.

Typically, an inoculum containing Streptococcus thermophilus CNCM I-5030 or CNCM I-4992 is essentially pure.

In another embodiment, the present invention relates to a mixture or set of inoculums of the present invention together with a Bifidobacterium and/or lactic acid bacteria inoculum.

Examples of Bifidobacterium that can be used include Bifidobacterium animalis (eg Bifidobacterium animalis subsp. animalis or Bifidobacterium animalis subsp. lactis); Bifidobacterium longum; Bifidobacterium breve; Bifidobacterium bifidum, but not limited to. Examples of lactic acid bacteria that can be used include Lactobacilli (e.g. Lactobacillus acidophilus, Lactobacillus buchnerii, Lactobacillus delbrueckii, in particular L. delbrueckii subsp. bulgaricus or lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus brevis , Lactobacillus rhamnosus); Lactococci (for example, Lactococcus lactis, usually Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris), but not limited to. Preferably the mixture of inoculum additionally contains Lactobacillus and/or Streptococcus. For the preparation of yogurt, the inoculum mixture typically includes Lactobacillus bulgaricus (also referred to as Lactobacillus delbruckeii subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms such as, but not limited to, probiotic species or other species that may provide the desired organoleptic or other properties of the composition. such as Lactococcus lactis.

Accordingly, in one embodiment, the present invention provides an inoculum mixture comprising a Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 inoculum and further comprising at least one Lactobacillus bulgaricus inoculum and optionally one or more additional Lactococcus lactis inoculum and/or or Bifidobacterium.

Methods for obtaining fermented milk products.

The bacterium, as provided in the present invention, can be used in the preparation of fermented milk products.

Accordingly, in a third aspect, the present invention also relates to the intended use of Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 for the preparation of a food composition.

The present invention provides a method for producing fermented milk products, including inoculation into a composition based on milk strain S.thermophilus CNCM I-5030 and/or CNCM I4992 rhamnosus CNCM I-4993 and fermentation.

Accordingly, in one embodiment, the present invention provides a method comprising the following steps:

i) obtaining a mixture containing

a) milk

b) Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992, ii) fermenting said mixture to obtain a fermented milk product.

Preferably, the fermented milk products are prepared using milk that has undergone heat treatment at least equivalent to pasteurization. Preferably said heat treatment is carried out prior to preparation of the composition.

Generally, milk is pasteurized according to the following successive steps:

1) standardization of fatty substances of raw materials in order to obtain a standardized substance,

2) enrichment with the dried substance of the standardized substance obtained in the previous stage, in order to obtain an enriched substance,

3) preheating the enriched substance obtained in the previous step in order to obtain the starting substance,

4) pasteurization and aging of the original substance obtained in the previous stage, in order to obtain a pasteurized and aged substance,

5) an optional step of homogenizing the pasteurized and aged substance obtained in the previous step in order to obtain a pasteurized, aged and optionally homogenized substance,

6) initial cooling of the pasteurized, aged and optionally homogenized material obtained in the previous step, in order to obtain a pasteurized starting material, aged and optionally homogenized, and chilled.

As used in the present invention, the term standardization of fatty substances means the step of bringing the amount of fat present in the original substance to a predetermined level. Dry matter fortification involves the addition of proteins and fatty matter to change the hardness of curdled milk.

As used in the present invention, the term aging means rapid heating and

- 7 041084 maintaining a high temperature of milk, which allows you to destroy the vegetative bacterial microflora, including pathogenic forms. A typical holding time is 4 to 10 minutes, in particular 5 to 8 minutes, and in particular about 6 minutes.

As used in the present invention, the term homogenization means the dispersion of fatty substances in the milk substance to the state of small fat globules. The homogenization is carried out, for example, under a pressure of 100 to 280 bar, in particular 100 to 250 bar, in particular 100 to 200 bar, in particular approximately 200 bar. This homogenization step is optional. In particular, it is absent in the process for the production of products with 0% fatty substances.

Typically, the fermented milk product is obtained by culturing milk at a suitable temperature with suitable microorganisms to bring the pH down, preferably to a pH equal to or below 5, preferably about 3 to 4.7; more preferably from about 3.5 to 4.7. The pH value can be adjusted by controlling the fermentation by the microorganism and stopping it when necessary, for example by cooling.

In accordance with another embodiment of the process for producing a fermented milk product as defined above, the mixture containing milk and Streptococcus thermophilus CNCM I-5030 and/or CNCM I-4992 further comprises at least one, two, three or more strains of Bifidobacterium and/or lactic acid bacteria. Selection of suitable strains of Bifidobacterium is within the skill of the skilled artisan and is typically probiotic lactic acid bacteria. Examples of Bifidobacterium that can be used include, but are not limited to, Bifidobacterium animalis (eg Bifidobacterium animalis subsp. animalis or Bifidobacterium animalis subsp. lactis); Bifidobacterium longum; Bifidobacterium breve; Bifidobacterium bifidum.

The selection of suitable lactic acid bacteria strains is within the skill of the art and are typically thermophilic lactic acid bacteria. Examples of lactic acid bacteria that can be used include, but are not limited to, Lactobacilli (e.g. Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbruckeii, in particular L. delbrueckii subsp. bulgaricus or lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus johnsonii , Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus rhamnosus); Lactococci (eg Lactococcus lactis, usually Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris).

Typically, a mixture or combination of multiple types of lactic acid bacteria can be used, typically a mixture or combination of Lactobacillus and Streptococcus. For the preparation of yogurt, the mixture or combination typically contains Lactobacillus bulgaricus (also referred to as Lactobacillus delbrueckii subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms such as, but not limited to, probiotic species or other species that may provide the desired organoleptic or other properties. compositions, for example Lactococcus lactis.

Accordingly, in one embodiment, the mixture further comprises at least one strain of Lactobacillus bulgaricus and optionally one or more strains of Lactococcus lactis and/or Bifidobacterium.

Suitable temperatures for milk fermentation are typically between about 36°C and 44°C, and this temperature is maintained for a sufficient incubation time to provide the desired pH reduction. For the preparation of a fermented milk product, the temperature at the beginning of fermentation is usually about 36 to 43°C, in particular about 37 to 40°C, the temperature at the end of fermentation is usually about 37 to 44°C, in particular about 38 to 41° WITH. The fermentation time is usually about 6 to 11 hours.

After fermentation, the fermented milk is cooled. Optionally, a stage of intermediate cooling of the fermented milk can be carried out to obtain pre-cooled fermented milk with a temperature of from about 22°C to about 4°C. Typically, the intermediate cooling time is from about 1 hour to about 4 hours, in particular from about 1 hour 30 minutes to about 2 hours. Pre-chilled fermented milk is usually stored for 40 hours or less.

Preferably, the final cooling step of the fermented milk is carried out such that the temperature at the beginning of the final cooling step is less than about 22°C and the temperature at the end of the final cooling step is from about 4°C to about 10°C. Further, the chilled product can be stored, transported and/or distributed at a temperature of from about 1 to about 10°C for at least about 30 days, at least about 60 days, or at least about 90 days.

According to yet another embodiment, the process for producing a fermented milk product as defined above optionally includes a mixing step under a pressure of at least 20 bar, or a dynamic leveling step, to obtain a composition having the desired viscosity, typically up to 20 mPa-s. Mixing or dynamic leveling operations provide the composition with a consistency that usually allows to reduce the viscosity. Such operations are known to the person skilled in the art and can be carried out using conventional suitable equipment. This step is usually carried out at a low temperature, for example between 1 and 20°C. Without wishing to be bound by theory, it is believed that imparting some consistency at low temperature, usually by high pressure agitation or by dynamic leveling, can result in the formation of a liquid gel in the composition, which provides improved stability even at low viscosity up to 20 mPa-s.

Alternatively, according to another embodiment, the process for producing a fermented milk product as defined above optionally includes the step of removing acid whey to obtain a strained fermented milk composition. In this step, the acid whey composition is separated from curdled milk resulting from protein coagulation due to acidification during fermentation. Thus, a fermented milk product is obtained, usually containing a coagulate of proteins, called a strained fermented milk composition, and an acid whey by-product.

Such separation steps are known to the person skilled in the art, for example in methods for making Greek yoghurts. Separation can be carried out, for example, by reverse osmosis, ultrafiltration or centrifugation. The separation step can be carried out, for example, at a temperature of from 30 to 45°C.

According to another embodiment, the process for producing a fermented milk product as defined above optionally includes the step of adding an auxiliary preparation as described above, before or after fermentation, said auxiliary preparation usually includes a preparation of fruits and/or cereals and/or additives, such as flavorings and/or colorants.

The invention will be further illustrated by the following non-limiting figures and an example.

Description of figures

In FIG. 1 shows the acidification kinetics of milk for S. thermophilus CNCM I-4992 strain, determined according to Example 1. Time in minutes is plotted on the x-axis, pH values are plotted on the y-axis, FIG. 2 shows milk acidification kinetics for S. thermophilus CNCM I-5030 strain, determined according to Example 1. Time in minutes is plotted on the x-axis, pH values are plotted on the y-axis, FIG. 3 is a graph showing fermented milk acidification over time, S.thermophilus viability and texturing properties for a panel of over 40 S.thermophilus strains according to Example 2. Fermented milk acidification over time plotted along the x-axis as an increase in Dornik acidity of fermented milk in storage for 28 days. The viability of S.thermophilus is plotted on the y-axis and is defined as the reduction in CFU over 28 days. Each tested strain corresponds to a separate round dot on the graph, while strains differ from each other in the viscosity of the fermented milk, for strains indicated by a dot in a square, the measured viscosity is <0.25 Pa-s' ¹ , for strains indicated simply by a dot, the viscosity is 0.25-0.5 Pa-s' ¹ , for strains indicated by a dot in a pentagon, the measured viscosity is 0.5-0.75 Pa-s' ¹ , and for strains indicated simply by a dot in a triangle, the measured viscosity is 0.75-1.1 Pa-s' ¹ in Fig. 4 - acidity according to Dornik for the tested fermented milk in accordance with example 3. The acidity was measured at 3 time points; day 0 (white box), day 14 (black box), and day 28 (grey box), in FIG. 5 - change in the acidity of the test and control fermented milk for 28 days in accordance with example 4, in fig. 6 shows acidification kinetics of test (dark grey) and control (light grey) fermented milk, determined according to example 4. Time in minutes is plotted on the x-axis, pH values are plotted on the y-axis.

Examples

More than 40 strains of S. thermophilus were screened to identify strains that are firstly suitable for the preparation of fermented dairy products, and which also provide such products with increased sweetness by influencing the acidification process of the product during storage and improving texture. Two strains of S.thermophilus were selected based on their high viability, good ability to acidify milk, as well as improve texture (higher viscosity), along with allowing them to acidify more slowly during storage. The properties of strains to give products increased sweetness was determined using a tasting panel evaluating two types of fermented dairy product.

Example 1. Milk acidification by strains CNCM I-5030 and CNCM I-4992 and their viability.

Reconstituted milk was obtained by mixing 137.5 g of skimmed milk powder (Arla) per 1 liter of permuted water and pasteurized at 95°C for 45 min, 0.5% Vitalarmor 950 (Armor Proteins) was added for strain CNCM I-5030. The bacterial strains were provided in frozen form by Danone. Strains (1%) were inoculated into reconstituted milk from a culture obtained from

- 9 041084 milk enriched with 0.2% yeast extract, obtained in turn by inoculation of 1% of the strain from the first stage of cultivation in M17 medium, inoculated with a frozen bacterial strain. Fermentation was carried out at 37° C. and monitored with a CINAC pH probe. The resulting fermented milk was cooled and stored at 10°C to determine the viability of the strain and subsequent acidification of the fermented milk during storage for 28 days.

The S.thermophilus strains CNCM I-1630 and CNCM I-2130 used to prepare commercially available fermented dairy products were used as references for CNCM I5030 and CNCM I-4992, respectively, in determining the suitability of these strains for the preparation of fermented dairy products.

As can be seen from the milk souring curves shown in Fig. 1 and 2, Streptococcus thermophilus strains CNCM I-5030 and CNCM I-4992 are able to ferment milk perfectly. According to this indicator, strains CNCM I-5030 and CNCM I-4992 are comparable with commercially used strains CNCM I-1630 and CNCM I-2130, respectively.

Example 2 Characteristics of fermented milk produced using CNCM I-5030 and CNCM I-4992.

Further acidification during storage of fermented dairy products affects their taste and is associated with the continued production of lactic acid by bacterial cultures during the shelf life of the products. To characterize the ability of S. thermophilus strains to such acidification, fermented milk products were prepared using the strains indicated in Example 1. The change in Dornik acidity in fermented milk was determined over 28 days of storage at 10°C. The viability of S.thermophilus strains was also determined by measuring the number of bacteria in colony forming units (CFU). The texturing properties of the strains were determined by measuring the viscosity. Viscosity was measured by gradually increasing the shear stress using a rheometer with two coaxial cylinders (RM 180, Mettler). Portable cylinder (rotating) n°1 was used together with stationary cylinder (stationary) n°1. For 10 s at 10° C., the test product was exposed to a shear rate of 64 s ^-1 .

The results are shown in FIG. 3, no change in Dornik degrees (ie no further acidification) is preferred. The number of S.thermophilus bacteria was determined by measuring the number of CFU at the beginning and at the end of the storage period, it is preferable to maintain high CFU values and the absence of acidification during storage. As shown in FIG. 3, the test strains CNCM I-5030 and CNCM I-4992 exhibit acceptable acidification, good texturing properties, and good strain viability.

Example 3

Starter cultures of fermented dairy products typically contain a combination of S. thermophilus and L. bulgaricus strains. Accordingly, in order to characterize the acidification process of these strains of fermented dairy products, strain CNCM I-4992 was tested in combination with 3 different strains of L. bulgaricus. An edible fermented milk product was prepared by fermenting a pasteurized milk mixture (13.75% skimmed cow's milk powder in permuted water, pasteurized at 95° C. for 45 min) with a yoghurt starter culture (L. delbrueckiir S. thermophilus). Fermentation was carried out at 37° C. and monitored with a CINAC probe. The fermentation was stopped by rapid cooling when reaching pH 4.7. The change in the degree of acidity of Dornik in fermented milk was determined within 28 days of storage at 10°C. As shown in FIG. 4, further acidification of the fermented milk was negligible.

Example 4 Testing of a fermented dairy product.

A combination of strains CNCM I-5030 and CNCM I-4992 and strain L. bulgaricus (CNCM I-2787) (test product) was used as a starter culture for preparing the test fermented dairy product. Two strains of S. thermophilus and L. bulgaricus (control) were used as a reference yogurt (Vitalinea™) starter culture. The starter cultures provided as frozen granules were thawed before use at 38° C., liquid starter cultures were added to the milk mixture to 0.02% by volume. An edible fermented dairy product was prepared from a homogenized and pasteurized milk mixture of 95% milk base (milk, skimmed milk powder and cream) and 5% sugar, which was fermented with test and control starter cultures. Fermentation was carried out at 38°C to pH 4.6 (6-7 hours). The resulting fermented milk was stirred with a mixer (Ystral), cooled to 20°C and stored at 10°C for 30 days. The change in the degree of acidity of Dornik was determined at the end of the storage period. In FIG. 6 shows acidification curves for fermented milk made with test (dark gray) and control (light gray) starter cultures, showing that the acidification kinetics of control starter culture and test starter culture are comparable and both are acceptable. As seen in FIG. 5, the test product containing strains according to the invention acidifies more slowly.

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Claims (13)

Characteristics of the test and reference products such as sweetness, acidity, body and overall milky notes were evaluated by a panel of volunteers. The products were tested on days 14 and 28 of storage at 10°C. As a result, fermented dairy products made using the test starter culture were found to be sweeter than fermented dairy products made using the reference starter culture. Example 5 Evaluation of the sweetness of a fermented milk product. For soft cheese (fresh cheese) enriched with calcium, it was confirmed that products prepared using the test starter culture according to Example 4 (CNCM I5030 + CNCM I-4992 + L. bulgaricus) have increased sweetness compared to products prepared prepared using a control starter culture. An edible fermented dairy product was prepared by fermenting a pasteurized milk mixture: 93.85% milk base (milk, skimmed milk powder and cream), 1.5% starch, 4.5% sugar and 0.15% calcium with test and control starter cultures. Fermentation was carried out at 38°C to pH 4.6 (6-7 hours). The two products were compared by a panel of volunteers who evaluated serum (visual), color, firmness, acidity, fruitiness, thick mouth and unpleasant sensations. No significant differences were found between test and control products in terms of whey (visually determined), color, firmness, sweetness, acidity, fruitiness, thick mouth and unpleasant sensations. However, increased sweetness was noted for the test product, as well as differences in density. CLAIM 1. Streptococcus thermophilus strain deposited with CNCM under CNCM number I-5030 for the preparation of fermented foods. 2. Streptococcus thermophilus strain deposited with CNCM under CNCM number I-4992 for the preparation of fermented foods. 3. A composition for the preparation of fermented food products containing at least 105 cfu/g of the strain of Streptococcus thermophilus deposited with CNCM under the number CNCM I5030 or the strain of Streptococcus thermophilus deposited with CNCM under the number CNCM I-4992. 4. Composition according to claim 3, characterized in that the fermented food product is a dairy composition. 5. Composition according to claim 3 or 4, additionally containing at least one strain of Bifidobacterium or lactic acid bacteria. 6. Composition according to claim 3, characterized in that said composition is an inoculum. 7. Composition according to claim 5 or 6, characterized in that said composition is fresh, frozen, dry or lyophilized. 8. The composition of claim 5, wherein said lactic acid bacteria strain comprises at least one Lactobacillus strain. 9. The composition according to claim 8, further comprising one or more strains selected from the group consisting of Lactococcus lactis and Bifidobacterium. 10. A method for producing a fermented milk product, including: i) obtaining a mixture containing a) milk b) S.thermophilus CNCM I-5030 and/or CNCM I-4992, ii) fermenting said mixture to obtain a fermented milk product. 11. The method of claim 10, wherein the mixture further comprises at least one strain of Bifidobacterium or lactic acid bacteria. 12. The method of claim 11 wherein said lactic acid bacteria strain comprises at least one Lactobacillus strain. 13. The method of claim 11, wherein said lactic acid bacteria strain comprises at least one strain of Lactococcus lactis. -