Phage-resistant strains

Abstract

The present invention is in the field of dairy technology. It relates to improved phage-resistant strains of the species Streptococcus thermophilus, compositions and fermented products comprising the strains, and methods for producing said fermented products.

Description

[0001] PHAGE-RESISTANT STRAINS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to lactic acid bacteria strains with an improved resistance towards bacteriophages, i.e. improved phage-resistance, compositions such as starter cultures comprising the strains, and fermented products comprising the strains.

[0004] BACKGROUND OF THE INVENTION

[0005] The food industry uses numerous bacteria, in particular lactic acid bacteria (LAB) in the production of fermented food. The choice of LAB impacts the characteristics and features of the fermented food product, such as e.g. taste, texture, and shelf life. In the dairy industry, LAB are used intensively in order to bring about the acidification of milk (by fermentation) but also in order to texturize the product into which they are incorporated.

[0006] Streptococcus thermophilus is one of the most common bacteria used worldwide as a starter in the production of fermented foods, such as cheese and yoghurt. This microorganism is a thermophilic, aerotolerant, Gram-positive coccus, and a member of a heterogeneous group of lactic acid bacteria. The extensive use of S. thermophilus in dairy plants, through cultivation in large vats, has resulted in increased susceptibility to bacteriophage infections. Indeed, phage outbreaks represent the major cause of slow or faulty fermentations, frequently leading to a lower quality of dairy products and suboptimal production costs. Bacteriophages, in short called phages, have been identified for many of the bacterial strains used in the industry of the species such as e.g. Lactococcus sp., Lactobacillus sp., Leuconostoc sp., Pediococcus sp. or Streptococcus sp.

[0007] The lytic development of bacteriophages involves adsorption of the phages to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phages and release of progeny from the host. Cell-mediated mechanisms of interference with any of these events can prevent a phage infection. The ability of bacterial cultures to resist bacteriophage infection during industrial use depends to a large extent on host strain characteristics affecting one or more of the above mechanisms.

[0008] A factor, which may lead to frequent bacteriophage infections in lactic acid bacterial starter cultures, is the fact that fermentation conditions in the food industry including the dairy industry in general are non-sterile. Diverse treatments have been applied to minimize phage infections in the dairy environment. Predominant approaches include chemical and physical methods for equipment sanitation, as well as a culture replacement and strain rotation programs. The latter require strains with identical technological performance, but different phage sensitivities. Thus, isolation and characterization of bacteriophage insensitive mutants (BIMs) of strains used in dairy starter cultures has been widely performed. Several methods for generating BIMs of S. thermophilus have been proposed including insertional mutagenes, the secondary culture method, serial passaging in the presence of high phage titers, chemical mutagenesis, and transformation with an antisense mRNA-generating plasmid. Generally, the acquired resistance is due to the activation of intracellular resistance mechanisms, mainly clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems or restrictionmodification (R-M) systems. Additional phage-resistance systems, such as abortive infection (Abi) and superinfection exclusion (Sie), have also been detected in S. thermophilus. However, those mechanisms probably are not very widespread and therefore, they do not commonly mediate phage-resistance in BIMs of dairy strains.

[0009] It has not yet been possible to eliminate bacteriophage contamination under the current industrial conditions. In order to meet the requirements of the industry, it has become necessary to propose novel phage-resistant strains of Streptococcus thermophilus. Thus, development of new strains that are phage-resistant and which are suitable in methods that provide fermented food products having good texture are desirable.

[0010] SUMMARY OF THE INVENTION

[0011] An objective of the present invention is to provide phage-resistant mutants of strains of Streptococcus thermophilus with improved phage insensitivity and robustness. The present inventors have provided lactic acid bacteria of the species Streptococcus thermophilus, which are more resistant to phage attack than their (mother) strain from which they are derived. These strains also provide similar or improved properties such as reduced acidification time, and increased texture measured as shear stress and / or gel stiffness when used for fermenting milk as compared to fermented milk produced with their mother strain or a starter culture in the prior art.

[0012] In a first aspect the present disclosure provides a phage-resistant lactic acid bacteria strain derived from a mother strain of the species Streptococcus thermophilus, wherein the strain

[0013] (a) is resistant towards one or more phages against which the mother strain is sensitive selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM 35012 CHPC1636, and

[0014] (b) provides a faster acidification of a milk base in the presence of said one or more phages as compared to the mother strain.

[0015] In a second aspect the disclosure provides a composition comprising the strain. In a third aspect the disclosure provides a method for producing fermented products comprising the steps:

[0016] (a) adding the strain or the composition to a milk base; and

[0017] (b) fermenting the milk base at a temperature between about 22°C and about 45°C until a pH of 4.6 or less than 4.6 is reached.

[0018] In a fourth aspect the disclosure provides a fermented product comprising the strain, the composition, or is obtained by the method.

[0019] In a fifth aspect the disclosure provides a food or feed product comprising the strain, the composition, or is obtained by the method.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0022] Lactic acid bacteria strains

[0023] In the context of present disclosure, the term "lactic acid bacteria" or "LAB" is used to refer to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram-positive, low-GC, acid tolerant, non- sporulating, non-respiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of dairy product. As used herein, the term "lactic acid bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium animalis and Leuconostoc spp.

[0024] The term "mutant" should be understood as a strain derived from a strain of the present disclosure, for example by means of e.g. genetic engineering, radiation and / or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same or improved properties in particular in relation to the effects on phage-resistance, texturizing properties, and fermentation or acidification time, as the deposited strain. Respective mutants represent embodiments of the present disclosure. The term "mutant" in particular refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening / selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening / selection steps) are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain.

[0025] The term "variant" should be understood as a strain which is functionally equivalent to a strain of the present disclosure, having substantially the same or improved properties in particular in relation to the effects relating to phage-resistance, texturizing properties, and fermentation or acidification time, as the deposited strain. Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0026] As used herein, the term "bacteriophage" has its conventional meaning as understood in the art ie. a virus that selectively infects one or more bacteria. Many bacteriophages are specific to a particular genus or species or strain of bacteria. The term "bacteriophage" is synonymous with the term "phage". Bacteriophages may include, but are not limited to, bacteriophages that belong to any of the following virus families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae. The bacteriophage may be a lytic bacteriophage or a lysogenic bacteriophage. A lytic bacteriophage is one that follows the lytic pathway through completion of the lytic cycle, rather than entering the lysogenic pathway. A lytic bacteriophage undergoes viral replication leading to lysis of the cell membrane, destruction of the cell, and release of progeny bacteriophage particles capable of infecting other cells. A lysogenic bacteriophage is one capable of entering the lysogenic pathway, in which the bacteriophage becomes a dormant, passive part of the cell's genome through prior to completion of its lytic cycle.

[0027] In the present context, the term "phage-resistance" or "phage-resistant" refers to the ability of the lactic acid bacterium strain to propagate (at optimal growth temperature) in a milk base which contains 1000 phages per mL, i.e. the bacterium is able to reach a cell density above 10E8 cfu / mL after 48 hours when inoculated at a concentration of 10E5 cfu / mL. cfu is "cell forming units".

[0028] The term "improved resistance to a bacteriophage" denotes that the bacteria strain when tested in e.g. a plaque assay, such as the assay described as "Determination of phageresistance by the agar overlay method" or the "Heap Lawrence assay" have an improved phage-resistance to at least one phage e.g. expressed as the difference in pfu / mL (plaque forming unit per mL) obtainable with said at least one bacteriophage on the given strain, compared to the pfu / mL obtainable with the same bacteriophage on the mother strain. A strain with improved resistance to a bacteriophage preferably show a reduction of pfu / mL of a factor at least 50, such as at least 100, e.g. 500, preferably at least 1000, more preferably at least a factor 10.000 or more.

[0029] In one embodiment the present disclosure relates to the strain, wherein the strain is resistant to one or more bacteriophages or one or more sets of bacteriophages. In one embodiment the present disclosure relates to the strain, wherein the strain is resistant to the same bacteriophages that DSM 34995 or DSM 34996 is resistant to. In the present context, the term "phage-robust" is interchangeable with the term "phage-resistant".

[0030] In one embodiment the present disclosure relates to a phage-resistant lactic acid bacteria strain derived from a mother strain of the species Streptococcus thermophilus, wherein the strain (a) is resistant towards one or more phages against which the mother strain is sensitive selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM 35012 CHPC1636, and (b) provides a faster acidification of a milk base in the presence of said one or more phages as compared to the mother strain.

[0031] In one embodiment the present disclosure relates to the strain, wherein the strain is able to acidify the milk base comprising the one or more phages to a pH below 5 when measured 10 hours and / or 20 hours after start of acidification. pH may be measured by a pH electrode, or by using a pH indictor such as e.g. bromophenol purple or bromophenol green In one embodiment the present disclosure relates to the strain, wherein the milk base is B- milk made from reconstituted skimmed milk having a dry matter of 9.5% which has been boiled at 99°C for 30 min.

[0032] In one embodiment the present disclosure relates to the strain, wherein the acidification is conducted at a temperature in the range of 35-40°C, 36-38°C, or at a temperature of 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0033] In one embodiment the present disclosure relates to the strain, wherein the strain provides a faster acidification of a milk base in the presence of one or more bacteriophages as compared to the mother strain.

[0034] In one embodiment the present disclosure relates to the strain, wherein the one or more bacteriophages are selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM 35012 CHPC1636.

[0035] In one embodiment the present disclosure relates to the strain, wherein the strain provides increased texture in a fermented product manufactured with said strain.

[0036] In one embodiment the present disclosure relates to the strain, wherein the mother strain is DSM 34993 or DSM 34991.

[0037] In one embodiment the present disclosure relates to the strain, wherein the strain is DSM 34995, DSM 34996, or mutants or variants thereof.

[0038] Compositions

[0039] Lactic Acid Bacteria (LAB) are most commonly added to a milk base in the form of a composition, preferably as a starter culture. Alternatively, the LAB may be added to a milk base individually or as a kit-of part. The term "starter" or "starter culture" as used in the present context refers to compositions or cultures of one or more food-grade microorganisms, in particular to lactic acid bacteria, which are responsible for the acidification of milk base. Starter cultures may be available in various forms such as in fresh, frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried or liquid form, but are most frequently in frozen or freeze-dried form. The compositions may be available as "Direct Vat Set" (DVS) cultures and are intended for direct inoculation of a fermentation vessel or vat for the production of a fermented product, in particular a fermented food product such as a fermented milk product. Respective starter cultures are commercially available from numerous providers including from Chr. Hansen. One or more strains in the composition must be of the species of Streptococcus thermophilus. Strains from other genus such as Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactibacillus, Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium may also be comprised. Strains of the species such as Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lacticaseibacillus easel, Lacticaseibacillus paracasei, and / or Bifidobacterium animalis are of particular interest.

[0040] In one embodiment the present disclosure relates to a composition comprising the strain.

[0041] In one embodiment the present disclosure relates to the composition comprising, either as a mixture or as a kit-of-part, i) the strain; and ii) one or more further lactic acid bacteria of a genus different from Streptococcus.

[0042] In one embodiment the present disclosure relates to the composition, wherein the genus of the one or more further lactic acid bacteria is Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactibacillus, Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium.

[0043] In one embodiment the present disclosure relates to the composition, wherein the one or more further lactic acid bacteria is Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lacticaseibacillus easel, Lacticaseibacillus paracasei, and / or Bifidobacterium animalis.

[0044] In one embodiment the present disclosure relates to a composition comprising the species Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

[0045] The composition of the present disclosure may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as croprotectants and lyoprotectantare known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0046] In one embodiment the present disclosure relates to the composition, wherein the composition further comprises cryoprotectants, lyoprotectants, antioxidants and / or nutrients. In one embodiment the present disclosure relates to the composition, wherein the composition is in fresh, frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried or liquid form.

[0047] In one embodiment the present disclosure provides compositions in the form of a solid frozen or freeze-dried starter culture comprising lactic acid bacteria in a concentration of at least 109colony forming units (cfu) per g of starter culture or in a concentration of at least IO10cfu / g of starter culture or in a concentration of at least 1011cfu / g of starter culture. These starter cultures further comprise bacteria of the species Streptococcus thermophilus deposited as DSM 34995, DSM 34996 or a mutant Streptococcus thermophilus obtainable from any of the deposited bacteria.

[0048] Method for producing fermented products

[0049] In the context of the present application, the term "milk" is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals or by plants. In accordance with the present invention the milk may have been processed and the term "milk" includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-reduced milk, or concentrated milk. Fat-free milk is non-fat or skim, 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. The term "milk" is intended to encompass milks from different mammal and plant sources. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, lama, mare and deer. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. In the methods and products of the present invention, milk derived from cows is most preferably used as a starting material for the fermentation.

[0050] The term "milk" also includes fat-reduced and / or lactose-reduced milk products. Respective products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatograph and centrifugation.

[0051] The term "milk base" is broadly used in the present application to refer to a composition based on milk or milk components which can be used as a medium for growth and fermentation of LAB. The milk base comprises components derived from milk and any other component that can be used for the purpose of growing or fermenting LAB. Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art. "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices. "Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0052] "Fermentation" in the methods of the present disclosure means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid. Fermentation processes to be used in production of fermented milk products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected to support the achievement of the present invention, i.e. to obtain a fermented milk product.

[0053] In one embodiment the present disclosure relates to a method for producing fermented products comprising the steps:

[0054] (a) adding the strain or the composition to a milk base; and

[0055] (b) fermenting the milk base at a temperature between about 22°C and about 45°C until a pH of 4.6 or less than 4.6 is reached.

[0056] The milk base may be any milk base suitable for producing a desired fermented product. In one embodiment the present disclosure relates to a milk base, wherein the milk base is of mammalian an / or vegetable origin. The amount of mammalian milk base may be in the range of 0-100%. The amount in percentage of mammalian to vegetable milk base may be 0: 100; 1 :99; 5:95; 10:90; 20:80; 30;70; 40:60; 50:50; 60:40; 70:30; 80:20; 90: 10; 95:5; 99: 1; or 100:0.

[0057] In one embodiment the present disclosure relates to the method, wherein the acidification time for producing the fermented product is reduced as compared to the acidification time for producing a fermented product made with the mother strain. In one embodiment the present disclosure relates to the method a, wherein the fermented product has an increased texture as compared to a fermented food product made with the mother strain.

[0058] Texture may be measured as shear stress and / or as gel firmness as known by the skilled artisan.

[0059] Rheological property may be measured with an ASC rheometer, model DSR101, from Anton Paar. The method is using a rotational step, which is based on a rotational deformation of the sample from 0.273 s-1 to 300 s-1 and then back to 0.273 s-1 where the corresponding shear stress is measured. Preferably the shear stress is measured at the shear rate 30.2 s-1. In one embodiment the disclosure relates to the method, wherein the texture is measured as shear stress. In one embodiment the disclosure relates to the method, wherein the shear stress is measured at the shear rate 30.2 s-1.

[0060] Gel firmness is a sensory descriptor of the fermented milk texture. It correlates with instrumental measurements such as positive compression area measured by texture analyzer and to complex modulus (G*) measured by rheometer. Gel firmness may be measured with a texture analyzer (TA. XT PlusC, Stable Micro Systems) or using a small-scale compression test (Hamilton robot & Mettler Toledo precision balance). In one embodiment the disclosure relates to the method, wherein the texture is measured as gel firmness.

[0061] Fermented products

[0062] The expression "fermented milk product" means a food or feed product wherein the preparation of the food or feed product involves fermentation of a milk base with a lactic acid bacteria. "Fermented milk product" as used herein includes but is not limited to products such as thermophilic fermented milk products, e.g. yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey and cheese products.

[0063] Fermentation is carried out to produce food products or feed products. The terms "fermented milk product", "food" or "feed" product refer to products obtainable by the fermentation methods of the present invention and include cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais and cream cheese. The term food further encompasses other fermented food products, including fermented meat, such as fermented sausages, and fermented fish products.

[0064] The term "cheese" is understood to encompass any cheese, including hard, semi-hard and soft cheeses, such as cheeses of the following types: Pasta filata, Cottage, Feta, Cheddar, Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheeses, brine cheeses, Camembert and Brie. As used herein, a cheese which has a NaCI concentration below 1.7% (w / w) is referred to as a "low-salt cheese".

[0065] In the context of the present application, the term "yoghurt" refers to products comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived therefrom. The lactic acid strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, are included to give the finished product various properties, such as the property of promoting the equilibrium of the flora. As used herein, the term "yoghurt" encompasses set yoghurt, stirred yoghurt, drinking yoghurt, Petit Suisse, heat treated yoghurt, strained or Greek style yoghurt characterized by a high protein level and yoghurt-like products.

[0066] The term "stirred-type product" or "stirred product" specifically refers to a fermented milk product which sustains a mechanical treatment after fermentation, resulting in a destructuration and liquefaction of the coagulum formed under the fermentation stage. The mechanical treatment is typically but not exclusively obtained by stirring, pumping, filtrating or homogenizing the gel, or by mixing it with other ingredients. Stirred-type products typically but not exclusively have a milk solid non-fat content of 9 to 15%. In one embodiment the disclosure relates to a food product, wherein the food product is a stirred-type product, preferably wherein the food product is a stirred yogurt.

[0067] The term "set-type product" or "set product" includes a product based on milk which has been inoculated with a starter culture, e.g. a starter culture, and packaged next to the inoculating step and then fermented in the package. In one embodiment the disclosure relates to a food product, wherein the food product is a set-type product, preferably wherein the food product is a set yogurt.

[0068] The term "drinkable product" includes beverages such as "drinking yoghurt" and similar. The term "drinking yoghurt" typically covers a milk product produced by fermentation by the combination of Lactobacillus species and Streptococcus thermophilus. Drinking yoghurt typically has a milk solid non-fat content of 8% or more. Furthermore, the live culture count for drinking yoghurt drinks is typically at least 10E6 cell forming units (cfu) pr mL.

[0069] In particular, term "yoghurt” encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus') which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million CFU / g. Yoghurt may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavoring(s), fruit, cereals, or nutritional substances, especially vitamins, minerals and fibers, as well as stabilizers and thickeners. Optionally the yoghurt meets the specifications for fermented milks and yoghurts of the AFNOR NF 04-600 standard and / or the codex StanA-IIa-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m / m) of the finished product.

[0070] In one embodiment the present disclosure relates to a fermented product comprising the strain, the composition, or is obtained by the method according to the disclosure.

[0071] The strain may be present in a concentration of at least 107, 108, 109, 1010, 1011, or 1012CFU / g fermented product. The strain may be present in a concentration of about 107, 108, 109, 1010, 1011, or 1012cfu / g fermented product. In one embodiment the present disclosure relates to the fermented product, wherein the strain is present in a concentration of at least 107cfu / g.

[0072] In one embodiment the present disclosure relates to the fermented product, wherein the product is a dairy product.

[0073] In one embodiment the present disclosure relates to the fermented product, wherein the dairy product is yogurt, buttermilk, kefir, or cheese such as fresh cheese or pasta filata.

[0074] In one embodiment the present disclosure relates to a food or feed product comprising the strain, the composition, or is obtained by the method.

[0075] DEPOSIT AND EXPERT SOLUTION

[0076] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0077] Table 1. Deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0078] EXAMPLES

[0079] EXAMPLE 1

[0080] Generation of the phage resistant strain DSM 34995

[0081] Phage-resistant mutants were generated from the mother strain DSM 34993 on M17-2% lactose agar plates with lOmM MgCL / CaCk after plating O.lmL of a M17-2% lactose overnight culture of DSM 34993 together with O.lmL of phage DSM 35003 CHPC869 containing 1.0E09 phage particles per mL and incubation overnight at 37°C. For this, phages and host strain were mixed with 2.5 mL of a top agar solution (molten agar with half (0.75%) of the standard agar concentration kept at 46-50°C), and poured on a bottom M17-2% lactose agar. Both bottom and top agar were comprising 10 mM MgCL / CaCk. When the top agar was solidified incubation occurred with conditions described above.

[0082] Among a number of phage-resistant mutants which appeared on the agar plates after incubation, the strain DSM 34995 was three times colony purified and retested in plaque assay on M17-MgCl2 / CaCl2 lactose agar plates at 37°C test using phage DSM 35003 CHPC869 for phage challenge. Phage-resistance was confirmed by the absence of single plaques in the plaque test.

[0083] Acidification profile in the presence of phage

[0084] Acidification assays with the mother strain DSM 34993 and the phage-resistant mutant DSM 34995 were conducted.

[0085] For the acidification test 200mL B-milk (reconstituted skimmed milk (9.5% dry matter) boiled at 99°C for 30 min.) was inoculated with 1% of the bacterial strain to be tested (from an overnight culture grown in M17-2% lactose incubated at 37°C), and incubated for 23h at 37°C. The pH was followed over time with the CINAC system (Scientific Solutions).

[0086] Bacteriophage DSM 35003 CHPC869 was added to a final concentration of 1.0E06 phage particles per mL. The result from the phage challenge test is shown in table below as pH measured after 0, 10, and 20 hours of fermentation. Table 2. pH measured during fermentation.

[0087] EXAMPLE 2

[0088] Generation of the phage resistant strain DSM 34996

[0089] Phage-resistant mutants were generated in two steps from the mother strain DSM 34991 on M17-2% lactose agar plates with lOmM MgCL / CaCk after plating O.lmL of an M17-2% lactose overnight culture of the mother strain together with O.lmL of phage DSM 35011 CHPC1230 (first round) and DSM 35012 CHPC1636 (second round starting from the phage resistant mutant generated in the first round) containing resp. 1.0E09 phage particles per mL and incubation overnight at 37°C. For this, phages and host strain were mixed with 2.5mL of a top agar solution (molten agar with half (0.75%) of the standard agar concentration kept at 46-50°C), and poured on a bottom M17-2% lactose agar. Both bottom and top agar were comprising lOmM MgCL / CaCk. When the top agar was solidified incubation occurred with conditions described above.

[0090] Among a number of phage-resistant mutants which appeared on the agar plates after incubation, the strain DSM 34996 was three times colony purified and retested in plaque assay on M17-MgCl2 / CaCl2 lactose agar plates at 37°C test using phage DSM 35011 CHPC1230 and DSM 35012 CHPC1636 for phage challenge. Phage-resistance towards the two phages was confirmed by the absence of single plaques in the plaque test.

[0091] Acidification profile in the presence of phages

[0092] Acidification assays with the mother strain DSM 34991 and the phage-resistant mutant DSM 34996 were conducted.

[0093] For the acidification test 200mL B-milk (reconstituted skimmed milk (9.5% dry matter) boiled at 99°C for 30 min.) was inoculated with 1% of the bacterial strains to be tested (from an overnight culture grown in M17-2% lactose incubated at 37°C), and incubated for 23h at 37°C. The pH was followed over time with the CINAC system (Scientific Solutions). Bacteriophages DSM 35011 CHPC1230 and DSM 35012 CHPC1636 were added respectively to a final concentration of 1.0E06 phage particles per mL. The result from the phage challenge test is shown in table below as pH measured after 0, 10, and 20 hours of fermentation.

[0094] Table 3. pH measured during fermentation.

[0095] EXAMPLE 3

[0096] Acidification time of compositions comprising the new strains.

[0097] Set yogurt were made with the following blends: Blend 1 comprising DSM 34996.

[0098] Blend 2 comprising DSM 34995.

[0099] F-DVS eXact® Dahi 3 (Chr. Hansen product no. 717445) was used as Benchmark.

[0100] A milk base (3.5% protein, 3.0% fat, 0% sugar) was made from fresh milk standardized with skim milk power. Three fermented products were produced by inoculating the milk base with the two experimental blends and the benchmark respectively according to the following process:

[0101] Preparation of the milk base:

[0102] Mixing 50°C for lhour

[0103] Homogenization 75°C (150 / 50 bar)

[0104] Pasteurization 95°C for 5 minutes

[0105] Fermentation:

[0106] Inoculation 500U / 5000L

[0107] Fermentation temperature 43°C

[0108] Target pH 4.6 Cooling a storage of the fermented product:

[0109] Cooling after fermentation Cooling from 43°C to 10°C over lOh, from

[0110] 10°C to 5°C over lOh before keeping overnight at 5°C. The next day, store then at 13°C.

[0111] Storage Store at 13°C for 14 days

[0112] The acidification time was measured using the axone system (equipment initially developed for Chr. Hansen by Absciss, who is now part of ARDPI), which allows for continuous measurement of pH during fermentation.

[0113] Table 4. Acidification time to target pH 4.6 (minutes).

[0114] Texturizing properties

[0115] Gel firmness was measured with a texture analyzer (TA. XT PlusC, Stable Micro Systems). The test mode was set to compression and the probe used was a 40mm disk. The pre-test, test and post-test speed were set at 5 mm / s, the distance to 15mm and the trigger force to 5grams. The data was measured at day +7, at 13°C, and the sample was stored at 13°C until the day of measurement.

[0116] Table 5. Gel firmness (g)

[0117] Rheological property was measured with an ASC rheometer, model DSR101, from Anton Paar. The method is using a rotational step, which is based on a rotational deformation of the sample from 0.273 s-1 to 300 s-1 and then back to 0.273 s-1. The corresponding shear stress is measured.

[0118] The sample was stored at 13°C until the day of measurement and the shear stress at the shear rate 30.2 s1was measured at day +7, at 13°C.

[0119] Table 6. Shear stress at shear rate 30.2 s1(Pa)

[0120] EXAMPLE 4

[0121] Acidification time provided by the compositions.

[0122] Set yogurt were made with the following blends: Experimental blend 1 comprising DSM 34996.

[0123] Experimental blend 2 comprising DSM 34995.

[0124] F-DVS eXact® Dahi 3 (Chr. Hansen product no. 717445) was used as Benchmark.

[0125] A milk base (3.5% protein, 3.0% fat) was made from low heat milk powder standardized with water and 38% cream. Three fermented milk products were produced by inoculating the milk base with the two experimental blends and the benchmark respectively according to the following process:

[0126] Preparation of the milk base:

[0127] Hyd ration 2 hours at 6°C

[0128] Homogenization 65°C (200 / 50 bar)

[0129] Pasteurization 95°C for 5 minutes

[0130] Cooling until usage 5 °C

[0131] Heating to fermentation temperature 43°C

[0132] Fermentation:

[0133] Inoculation 500U / 5000L

[0134] Fermentation temperature 43°C

[0135] Target pH 4.6

[0136] Cooling and storage of the fermented product:

[0137] Cooling after fermentation Cooling from 43°C to 26°C over 2h, from 26°C to 10°C over 8h, from 10°C to 5°C over lOh, and at 5°C overnight.

[0138] Storage Store at 13°C for 7 days

[0139] The acidification time was measured using a colorimetric method which allows the continuous measurement of color / pH during fermentation as described in W02005 / 068982. Table 7. Acidification time to target pH 4.6 (minutes).

[0140] Texturizing properties

[0141] The gel firmness of the fermented products were measured at 13°C on day+7 using a small- scale compression test (Hamilton robot & Mettler Toledo precision balance). Table 8. Gel firmness (g)

[0142] (Original in Electronic Form)

[0143] (This sheet is not part of and does not count as a sheet of the international application)

[0144] (Original in Electronic Form)

[0145] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0146] (This sheet is not part of and does not count as a sheet of the international application)

[0147] FOR RECEIVING OFFICE USE ONLY

[0148] FOR INTERNATIONAL BUREAU USE ONLY -5 This form was re international Bur -5-1 Authorized officer

Claims

CLAIMS1. A phage-resistant lactic acid bacteria strain derived from a mother strain of the species Streptococcus thermophilus, wherein the strain (a) is resistant towards one or more phages against which the mother strain is sensitive selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM 35012 CHPC1636, and (b) provides a faster acidification of a milk base in the presence of said one or more phages as compared to the mother strain.

2. The strain according to claim 1, wherein the strain is able to acidify the milk base comprising the one or more phages to a pH below 5 when measured 10 hours and / or 20 hours after start of acidification.

3. The strain according to claim 1-2, wherein the milk base is B-milk made from reconstituted skimmed milk having a dry matter of 9.5% which has been boiled at 99°C for 30 min.

4. The strain according to any one of claims 1-3, wherein the strain provides increased texture in a fermented product manufactured with said strain.

5. The strain according to any one of claims 1-4, wherein the mother strain is DSM 34993 or DSM 34991.

6. The strain according to any one of claims 1-5, wherein the strain is DSM 34995, DSM 34996, or mutants or variants thereof.

7. A composition comprising the strain according to any one of claims 1-6.

8. The composition according to claim 7 comprising, either as a mixture or as a kit-of- part, i) the strain according to any one of claims 1-6; and ii) one or more further lactic acid bacteria of a genus different from Streptococcus.

9. The composition according to claim 8, wherein the genus of the one or more further lactic acid bacteria is Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactibacillus , Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium.

10. The composition according to claim 8-9, wherein the one or more further lactic acid bacteria is Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lacticaseibacillus easel, Lacticaseibacillus paracasei, and / or Bifidobacterium animalis.

11. The composition according to any one of claims 7-10, wherein the composition further comprises cryoprotectants, lyoprotectants, antioxidants and / or nutrients.

12. The composition according to any one of claims 7-11, wherein the composition is in frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried or liquid form.

13. A method for producing a fermented product comprising the steps:(a) adding the strain according to any of claims 1-6 or the composition according to any of claims 7-12 to a milk base; and(b) fermenting the milk base at a temperature between about 22°C and about 45°C until a pH of 4.6 or less than 4.6 is reached.

14. The method according to claim 13, wherein the acidification time for producing the fermented product is reduced as compared to the acidification time for producing a fermented product made with the mother strain.

15. The method according to any one of claims 13-14, wherein the fermented product has an increased texture as compared to a fermented food product made with the mother strain.

16. A fermented product comprising the strain according to any of claims 1-6, the composition according to any one of claims 7-12, or is obtained by the method according to any one of claims 13-15.

17. The fermented product according to claim 16, wherein the strain according to any one of claims 1-6 is present in a concentration of at least 107CFU / g.

18. The fermented product according to any one of claims 16-17, wherein the product is a dairy product.

19. The fermented product according to any one of claims 18, wherein the dairy product is yogurt, buttermilk, kefir, or cheese such as fresh cheese or pasta filata.

20. A food or feed product comprising the strain according to any of claims 1-6, the composition according to any one of claims 7-12, or is obtained by the method according to any one of claims 13-15.

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