anti-phage strains

Abstract

The present invention belongs to the field of dairy technology. The present invention relates to strains of Streptococcus thermophilus species having improved resistance to phages, compositions and fermented products comprising these strains, and methods for producing said fermented products.

Description

Technical Field

[0001] The present invention relates to lactic acid bacteria strains with improved resistance to bacteriophages (i.e., improved bacteriophage resistance), compositions containing such strains such as starter cultures, and fermentation products containing such strains. Background Technology

[0002] The food industry uses many bacteria, particularly lactic acid bacteria (LAB), in the production of fermented foods. The choice of LAB affects the properties and characteristics of fermented food products, such as flavor, texture, and shelf life. In the dairy industry, LAB is widely used to acidify milk (through fermentation) and also to provide texture to products incorporating lactic acid bacteria.

[0003] Streptococcus thermophilus is one of the most common bacteria used worldwide as a starter culture in the production of fermented foods such as cheese and yogurt. This microorganism is a thermophilic, aerobic, Gram-positive coccus and a member of the heterogeneous group of lactic acid bacteria. The widespread use of Streptococcus thermophilus in dairy plants, through its cultivation in large fermentation tanks, makes it more susceptible to bacteriophage infection. In fact, bacteriophage outbreaks are a major cause of slow or abnormal fermentation, often leading to decreased dairy product quality and unsatisfactory production costs. Bacteriophages have been identified from many industrially used bacterial strains, including species of *Lactococcus*, *Lactobacillus*, *Leuconostoc*, *Pediococcus*, and *Streptococcus*.

[0004] The lytic life cycle of a bacteriophage involves phage adsorption 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 phages from the host. Cell-mediated interference mechanisms with any of these events can prevent phage infection. The ability of bacterial cultures to resist phage infection during industrial use depends largely on host strain characteristics that influence one or more of these mechanisms.

[0005] One factor that may contribute to frequent phage infection in lactic acid bacteria starter cultures is the fact that fermentation conditions in the food industry (including the dairy industry) are typically non-sterile. Various measures have been taken to minimize phage infection in dairy environments. Key methods include chemical and physical methods for equipment sterilization, as well as culture replacement and strain rotation protocols. The latter requires strains with similar process performance but different phage susceptibility. Therefore, the isolation and characterization of phage-insensitive mutants (BIMs) of strains used in dairy starter cultures have been extensively pursued. Several methods have been proposed for generating Streptococcus thermophilus BIMs, including insertional mutagenesis, secondary culture methods, continuous passage in the presence of high phage titers, chemical mutagenesis, and transformation with plasmids that produce antisense mRNA. Generally, the acquired resistance is due to the activation of intracellular resistance mechanisms, primarily the CRISPR-Cas system or the restriction-modification (RM) system. Additional phage resistance systems, such as abortion infection (Abi) and superinfection rejection (Sie), have also been detected in Streptococcus thermophilus. However, these mechanisms may not be widely distributed, and therefore, they generally do not mediate phage resistance in dairy strains BIM.

[0006] Under current industrial conditions, it is not possible to eliminate bacteriophage contamination. To meet industrial requirements, it is necessary to develop novel phage-resistant strains of Streptococcus thermophilus. Therefore, there is a desire to develop new strains that are phage-resistant and suitable for methods of providing fermented food products with good texture. Summary of the Invention

[0007] The object of this invention is to provide phage-resistant mutants of *Streptococcus thermophilus* strains, exhibiting improved phage insensitivity and robustness. The inventors of this invention have provided lactic acid bacteria of *Streptococcus thermophilus* species that are more resistant to phage attack than their derived (mother) strains. When used for milk fermentation, these strains also provide similar or improved properties, such as reduced acidification time and improved texture as measured by shear stress and / or gel hardness, compared to fermented milk produced using their mother strains or prior art starter cultures.

[0008] In a first aspect, this disclosure provides a phage-resistant lactic acid bacteria strain derived from a mother strain of *Streptococcus thermophilus*, wherein the strain (a) is resistant to one or more phages selected from DSM 35003 CHPC869, DSM 35011 CHPC1230 and DSM35012 CHPC1636, the mother strain being sensitive to the one or more phages, and (b) causes faster acidification of the lactic acid in the presence of the one or more phages compared to the mother strain.

[0009] In a second aspect, this disclosure provides compositions comprising the strain.

[0010] In a third aspect, this disclosure provides a method for producing fermented products, comprising the following steps:

[0011] (a) Adding the strain or the composition to a milk base; and

[0012] (b) Ferment the milk base at a temperature between about 22°C and about 45°C until the pH reaches 4.6 or below.

[0013] In a fourth aspect, this disclosure provides fermentation products that contain the strain, the composition, or are obtained by the method.

[0014] In a fifth aspect, this disclosure provides food or feed products that contain the strain, the composition, or are obtained by the method. Detailed Implementation

[0015] Unless otherwise stated herein or obviously contradicted by the context, the terms “a / an” and “the,” and similar pronouns, used in the context of describing the invention (especially in the context of the claims below), shall be interpreted to include both the singular and plural. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, the description of value ranges herein is intended only as a concise way of referring to each individual value falling within the range, and each individual value is incorporated into this specification as if it were recited individually herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and not to limit the scope of the invention. Nothing in this specification should be construed as essential to carrying out the invention by any unclaimed element.

[0016] Lactic acid bacteria strains

[0017] In the context of this disclosure, the term "lactic acid bacteria" or "LAB" is used to refer to food-grade bacteria that produce lactic acid as a major metabolic end product of carbohydrate fermentation. These bacteria are associated by their shared metabolic and physiological characteristics and are typically Gram-positive, low-GC, acid-resistant, non-spore-forming, non-respiratory, and rod-shaped or cocci. During the fermentation stage, these bacteria consume lactose, leading to lactic acid formation, lowering the pH, and resulting in the formation of protein curds. Therefore, these bacteria are responsible for the acidification of milk and the texture of dairy products. As used herein, the term "lactic acid bacteria" encompasses, but is not limited to, bacteria belonging to the genera *Lactobacillus*, *Bifidobacterium*, *Streptococcus*, and *Lactococcus*, 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.

[0018] The term "mutant" should be understood as a strain derived from the strain disclosed herein, for example, through genetic engineering, radiation, and / or chemical treatment. Preferably, the mutant is a functionally equivalent mutant, for example, a mutant that has substantially the same or improved properties as the preserved strain, particularly in relation to its effects on phage resistance, textural properties, and fermentation or acidification time. The corresponding mutants constitute embodiments of the present disclosure. The term "mutant" specifically refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenesis treatment, including treatment with a chemical mutagen (such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG)) or UV light treatment, or refers to a spontaneously generated mutant. The mutant may have undergone several mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), but currently it is preferred to perform no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps). In the currently preferred mutants, compared with the parent strain, less than 5%, or less than 1%, or even less than 0.1% of the nucleotides in the bacterial genome have been replaced or deleted by other nucleotides.

[0019] The term "variant" should be understood as a strain functionally equivalent to the strain disclosed herein, having substantially the same or improved characteristics compared to the preserved strain, particularly in relation to its effects on phage resistance, textural properties, and fermentation or acidification time. Such variants can be identified using appropriate screening techniques and are part of this invention.

[0020] As used herein, the term "bacteriophage" has its conventional meaning as understood in the art, namely, a virus that selectively infects one or more bacteria. Many bacteriophages are specific to a particular genus, species, or strain of bacteria. The terms "bacteriophage" and "phage" are synonymous and refer to bacteriophages throughout this document. A bacteriophage may include, but is not limited to, bacteriophages belonging to any of the following viral families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae. The bacteriophage may be a lytic bacteriophage or a lysogenic bacteriophage. Lysogenic phages follow the lysis pathway, rather than the lysogenic pathway, by completing their lysis cycle. Lysogenic phages undergo viral replication, leading to cell membrane lysis, cell destruction, and the release of progeny phage particles capable of infecting other cells. Lysogenic phages, on the other hand, enter the lysogenic pathway, becoming a dormant, inert part of the cell's genome before their lysis cycle is complete.

[0021] In this context, the term "phage resistance" or "phage resistance" refers to the ability of a lactic acid bacteria strain to multiply in a milk medium containing 1000 phages per mL (at optimal growth temperature), meaning that the bacteria, after inoculation at a concentration of 10E5 cfu / mL, can reach a cell density higher than 10E8 cfu / mL after 48 hours. Cfu stands for "colony-forming unit".

[0022] The term "improved phage resistance" indicates that, when tested in, for example, plaque assays, such as assays described as "phage resistance determination by agar covering method" or "Heap Lawrence assay," the bacterial strain exhibits improved phage resistance to at least one phage, expressed, for example, as the difference in pfu / mL (plaque-forming units per mL) achievable on a given strain with said at least one phage compared to pfu / mL achievable on the parent strain with the same phage. Strains exhibiting improved phage resistance preferably show a reduction in pfu / mL of at least 50-fold, such as at least 100-fold, for example 500-fold, preferably at least 1000-fold, more preferably at least 10,000-fold or more.

[0023] In one embodiment, this disclosure relates to a strain that is resistant to one or more phages or a group or more groups of phages. In one embodiment, this disclosure relates to a strain that is resistant to the same phage to which DSM 34995 or DSM34996 is resistant. In this context, the term "phage robust" may be used interchangeably with the term "phage resistant".

[0024] In one embodiment, this disclosure relates to a phage-resistant lactic acid bacteria strain derived from a mother strain of *Streptococcus thermophilus*, wherein the strain (a) is resistant to one or more phages selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM35012 CHPC1636, the mother strain being sensitive to the one or more phages, and (b) causes faster acidification of the lactic acid in the presence of the one or more phages compared to the mother strain.

[0025] In one embodiment, this disclosure relates to a strain that, when measured 10 hours and / or 20 hours after the start of acidification, is capable of acidifying the lactose containing the one or more phages to a pH below 5.

[0026] pH can be measured using a pH electrode or by using a pH indicator such as bromophenol purple or bromophenol green.

[0027] In one embodiment, this disclosure relates to the strain wherein the milk base is B-milk made from reconstituted skim milk with a dry matter content of 9.5% and which has been boiled at 99°C for 30 min.

[0028] In one embodiment, this disclosure relates to the strain, wherein the acidification is carried out at a temperature in the range of 35°C-40°C, 36°C-38°C, or at a temperature of 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0029] In one embodiment, this disclosure relates to a strain that, compared to the parent strain, causes faster acidification of the milk base in the presence of one or more bacteriophages.

[0030] In one embodiment, this disclosure relates to the strain, wherein the one or more phages are selected from DSM 35003CHPC869, DSM 35011 CHPC1230 and DSM 35012 CHPC1636.

[0031] In one embodiment, this disclosure relates to the strain, wherein the strain provides improved texture in fermentation products made with said strain.

[0032] In one embodiment, this disclosure relates to the strain, wherein the parent strain is DSM 34993 or DSM 34991.

[0033] In one embodiment, this disclosure relates to the strain, wherein the strain is DSM 34995, DSM 34996, or a mutant or variant thereof.

[0034] Composition

[0035] Lactic acid bacteria (LAB) are most commonly added to milk bases in the form of compositions, preferably as starter cultures. Alternatively, LABs can be added to milk bases alone or as a set of components. As used in this context, the term "starter culture" refers to a composition or culture of one or more food-grade microorganisms (particularly lactic acid bacteria) responsible for acidifying the milk base. Starter cultures are available in various forms, such as fresh, frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried, or liquid, but most commonly in frozen or freeze-dried form. These compositions can be obtained as "direct-inoculation" (DVS) cultures and are intended for direct inoculation of fermentation vessels or fermenters to produce fermented products, particularly fermented food products such as fermented milk products. The corresponding starter cultures are commercially available from numerous suppliers, including Chr. Hansen. One or more strains in the composition must be thermophilic streptococci. It may also include strains from other genera such as Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium. Of particular interest are the following strains: Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lacticaseibacillus casei, Lacticaseibacillus paracasei, and / or Bifidobacterium animalis.

[0036] In one embodiment, this disclosure relates to a composition comprising the strain.

[0037] In one embodiment, this disclosure relates to a composition, which, as a mixture or as a set of components, comprises: i) the strain; and ii) one or more additional lactic acid bacteria belonging to a different genus than Streptococcus.

[0038] In one embodiment, this disclosure relates to the composition wherein the one or more additional lactic acid bacteria belong to the genera of Lactobacillus, Lactobacillus mucosae, Lactobacillus casei, Lactobacillus assemblica, Lactobacillus lactis, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp., and / or Bifidobacterium spp.

[0039] In one embodiment, this disclosure relates to the composition wherein the one or more additional lactic acid bacteria are *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus casei*, *Lactobacillus paracasei*, and / or *Bifidobacterium animalis*.

[0040] In one embodiment, this disclosure relates to a composition comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0041] The compositions disclosed herein may additionally comprise cryoprotectants, lyophilization protectants, antioxidants, nutrients, fillers, flavorings, or mixtures thereof. The compositions preferably comprise one or more of cryoprotectants, lyophilization protectants, antioxidants, and / or nutrients; more preferably, they comprise cryoprotectants, lyophilization protectants, and / or antioxidants; and most preferably, they comprise either a cryoprotectant or a lyophilization protectant, or both. The use of protectants (such as cryoprotectants and lyophilization protectants) is known to those skilled in the art. Suitable cryoprotectants or lyophilization protectants include monosaccharides, disaccharides, trisaccharides, and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, and gum arabic), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, and xylitol), amino acids (such as proline and glutamic acid), complexes (such as skim milk, peptone, gelatin, and yeast extract), and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, and maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, and vitamins (such as B vitamins and vitamin C). The composition may optionally contain additional substances, including fillers (such as lactose and maltodextrin) and / or flavorings.

[0042] In one embodiment, this disclosure relates to the composition, wherein the composition further comprises a cryoprotectant, a lyophilization protectant, an antioxidant, and / or nutrients.

[0043] In one embodiment, this 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.

[0044] In one embodiment, this disclosure provides a composition in the form of a solid frozen or freeze-dried fermenting agent, comprising a concentration of at least 10 9 Colony forming units (cfu) / g starter culture, or a concentration of at least 10 10 CFU / g starter culture, or a concentration of at least 10 11 Lactic acid bacteria in cfu / g starter culture. These starter cultures further contain bacteria of the Streptococcus thermophilus species preserved in DSM 34995, DSM 34996 or Streptococcus thermophilus mutants obtainable from any of the preserved bacteria.

[0045] Methods for producing fermented products

[0046] In the context of this application, the term "milk" is used broadly in its common meaning, referring to a liquid produced by the mammary glands of an animal or by a plant. According to the invention, milk can be processed, and the term "milk" includes whole milk, skim milk, nonfat milk, low-fat milk, whole milk, low-lactose milk, or concentrated milk. Nonfat milk is a fat-free or skim milk product. Low-fat milk is generally defined as milk containing about 1% to about 2% fat. Whole milk typically contains 2% or more fat. The term "milk" is intended to encompass milk from various mammalian and plant sources. Mammal sources of milk include, but are not limited to, dairy cows, sheep, goats, buffalo, camels, llamas, mares, and deer. Plant-derived milk includes, but is not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp, sesame, and sunflower seeds. In the methods and products of the invention, milk derived from cattle is most preferably used as the starting material for fermentation.

[0047] The term "milk" also includes reduced-fat and / or low-lactose milk products. Such products can be prepared using methods well known in the art and are commercially available. Low-lactose milk can be produced according to any method known in the art, including the hydrolysis of lactose to glucose and galactose by lactase, or by nanofiltration, electrodialysis, ion-exchange chromatography, and centrifugation.

[0048] The term "milk-based" is used broadly in this application to refer to a milk-based or milk-component-based composition that can be used as a culture medium for the growth and fermentation of LABs. A milk-based composition includes components derived from milk and any other components that can be used for the purpose of enabling the growth or fermentation of LABs.

[0049] Prior to fermentation, the milk matrix can be homogenized and pasteurized according to methods known in the art. As used herein, "homogenization" means thorough mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it can be done to break down the milk fat into smaller sizes so that it no longer separates from the milk. This can be accomplished by forcing the milk through small pores under high pressure. As used herein, "pasteurization" means treating the milk matrix to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified time. The specified temperature is typically achieved by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step can then be performed.

[0050] In the methods disclosed herein, "fermentation" refers to the conversion of carbohydrates into alcohols or acids through the action of microorganisms. Preferably, the fermentation in the methods of the present invention includes the conversion of lactose into lactic acid. Fermentation processes for the production of fermented milk products are well known, and those skilled in the art know how to select suitable process conditions, such as temperature, oxygen, the amount and characteristics of one or more microorganisms, and process time. Obviously, selecting fermentation conditions that are conducive to achieving the present invention, i.e., obtaining fermented milk products, is beneficial.

[0051] In one embodiment, this disclosure relates to a method for producing a fermented product, the method comprising the following steps:

[0052] (a) Adding the strain or the composition to a milk base; and

[0053] (b) Ferment the milk base at a temperature between about 22°C and about 45°C until the pH reaches 4.6 or below.

[0054] The milk base can be any milk base suitable for producing the desired fermented product. In one embodiment, this disclosure relates to a milk base of mammalian and / or plant origin. The amount of mammalian milk base can range from 0 to 100%. The percentage ratio of mammalian milk base to plant milk base can 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.

[0055] In one embodiment, this disclosure relates to a method in which the acidification time for producing the fermentation product is shortened compared to the acidification time for producing the fermentation product made from the mother strain.

[0056] In one embodiment, this disclosure relates to a method in which a fermented product has improved texture compared to a fermented food product made with the mother strain.

[0057] Texture can be determined by shear stress and / or gel hardness, as is known to those skilled in the art.

[0058] Rheological properties can be measured using an ASC rheometer (model DSR101) from Anton Paar. The method employs a rotational step, subjecting the sample to rotational deformation from 0.273 s⁻¹ to 300 s⁻¹ and back to 0.273 s⁻¹, during which the corresponding shear stress is measured. Preferably, the shear stress is measured at a shear rate of 30.2 s⁻¹. In one embodiment, this disclosure relates to a method in which the texture is determined by shear stress. In another embodiment, this disclosure relates to a method in which the shear stress is measured at a shear rate of 30.2 s⁻¹.

[0059] Gel firmness is a sensory descriptor of the texture of fermented milk. It is correlated with instrumental measurements (such as the positive compressibility area measured by a texture analyzer and the complex modulus (G) measured by a rheometer). The gel hardness can be measured using a texture analyzer (TA.XT PlusC, Stable Micro Systems) or using a small-scale compression test (Hamilton robot and Mettler Toledo precision balance). In one embodiment, this disclosure relates to a method in which the texture measurement is gel hardness.

[0060] Fermented products

[0061] The term "fermented milk product" means a food or feed product whose preparation involves fermenting a milk base with lactic acid bacteria. As used herein, "fermented milk product" includes, but is not limited to, products such as thermophilic fermented milk products (e.g., yogurt), thermophilic fermented milk products (e.g., sour cream and buttermilk), and fermented whey and cheese products.

[0062] Fermentation is carried out to produce food or feed products. The terms "fermented dairy product," "food," or "feed" product refer to products obtainable by the fermentation method of this invention, and include cheese, yogurt, fruit yogurt, yogurt beverages, whey-free yogurt (Greek yogurt, Labneh yogurt), quark cheese, fromagefrais, and cream cheese. The term "food" further encompasses other fermented foods, including fermented meat products such as fermented sausages, and fermented fish products.

[0063] The term "cheese" should be understood to encompass any type of cheese, including hard, semi-hard, and soft cheeses, such as the following types: Pasta filata, Cottage, Feta, Cheddar, Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheese, brine cheese, Camembert, and Brie. As used herein, cheeses with a NaCl concentration of less than 1.7% (w / w) are referred to as "low-salt cheeses."

[0064] In the context of this application, the term "yogurt" refers to a product containing *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. *lactis*, and optionally other microorganisms such as *Lactobacillus delbrueckii* subsp. *lactis*, *Bifidobacterium animalis* subsp. *lactis*, *Lactococcus lactis*, *Lactobacillus acidophilus*, and *Lactobacillus paracasei*, or any microorganisms derived therefrom. It includes lactic acid bacteria strains other than *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. *lactis* to impart various properties to the finished product, such as properties that promote gut microbiota balance. As used herein, the term "yogurt" encompasses set yogurt, stirred yogurt, drinkable yogurt, Petit Suisse, heat-treated yogurt, whey-free or Greek yogurt characterized by high protein levels, and yogurt-like products.

[0065] The term "stirred product" or "stirred product" specifically refers to a fermented milk product that has undergone mechanical processing after fermentation, resulting in the disruption and liquefaction of the curd structure formed during fermentation. This mechanical processing is typically (but not limited to) agitating, pumping, filtering, or homogenizing the gel, or by mixing it with other ingredients. Stirred products typically (but are not limited to) have a non-fat milk solids content of 9% to 15%. In one embodiment, this disclosure relates to a food product that is a stirred product, preferably a stirred yogurt.

[0066] The terms "set product" or "set product" include products based on milk inoculated with a starter culture (e.g., a starter culture), packaged immediately after the inoculation step, and then fermented in the package. In one embodiment, this disclosure relates to a food product, wherein the food product is a set product, preferably wherein the food product is set yogurt.

[0067] The term "drinking product" includes beverages such as "drinking yogurt" and the like. The term "drinking yogurt" typically encompasses dairy products produced through a combination of fermentation by Lactobacillus species and Streptococcus thermophilus. Drinking yogurt typically has a nonfat milk solids content of 8% or more. Furthermore, the viable cell count of drinking yogurt beverages is typically at least 10E6 colony-forming units (cfu) / mL.

[0068] Specifically, the term "yogurt" encompasses, but is not limited to, yogurt as defined under French and European regulations, such as coagulated dairy products obtained solely through lactic acid fermentation using specific thermophilic lactic acid bacteria (i.e., *Lactobacillus delbrueckii* subsp. bulgaricus and *Streptococcus thermophilus*), which are simultaneously cultured and remain viable in the final product at a concentration of at least 10 million CFU / g. Yogurt may optionally contain added dairy ingredients (e.g., cream) or other components (such as sugar or sweeteners, one or more flavorings, fruits, grains, or nutrients, especially vitamins, minerals, and fiber), as well as stabilizers and thickeners. Optionally, yogurt meets the specifications of AFNOR NF 04-600 and / or Codex Stan A-IIa-1975 regarding fermented milk and yogurt. To meet AFNOR NF 04-600, the product must not be heated after fermentation, and dairy ingredients must constitute at least 70% (m / m) of the finished product.

[0069] In one embodiment, this disclosure relates to a fermentation product comprising the strain, the composition, or obtained by the method according to this disclosure.

[0070] This strain can be at least 10 7 10 8 10 9 10 10 10 11 Or 10 12 The strain exists at a concentration of approximately 10 CFU / g fermentation product. 7 10 8 10 9 10 10 10 11 Or 10 12 The fermentation product is present at a concentration of at least 10 cfu / g. In one embodiment, this disclosure relates to the fermentation product, wherein the strain is present at a concentration of at least 10 cfu / g. 7 A concentration of cfu / g is present.

[0071] In one embodiment, this disclosure relates to the fermented product, wherein the product is a dairy product.

[0072] In one embodiment, this disclosure relates to the fermented product, wherein the dairy product is yogurt, buttermilk, kefir, or cheese, such as fresh cheese or pastafilata cheese.

[0073] In one embodiment, this disclosure relates to a food or feed product that contains the strain, the composition, or is obtained by the method.

[0074] Preservation and expert solutions

[0075] The applicant requests that, prior to the patent grant date, only the following deposited microbial samples be provided to experts, subject to the current regulations of the industrial property offices of the contracting parties to the Budapest Treaty.

[0076] Table 1. In accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for use in patent proceedings, the deposit has been completed at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), 7B Inhofenstrasse, Braunschweig, Germany (postcode D-38124).

[0077]

[0078] Example

[0079] Example 1

[0080] Production of antiphage strain DSM 34995

[0081] After overnight incubation of 0.1 mL of DSM 34993 M17-2% lactose culture with 0.1 mL of DSM 35003 CHPC869 containing 1.0E09 phage particles per mL on a plate at 37°C, phage-resistant mutants were generated from the parent strain DSM 34993 on M17-2% lactose agar plates with a 10 mM MgCl2 / CaCl2 concentration. For this purpose, the phage and host strain were mixed with 2.5 mL of top agar solution (melted agar maintained at 46–50°C, with an agar concentration half that of the standard concentration (0.75%)) and poured onto the bottom M17-2% lactose agar. Both the bottom and top agars contained 10 mM MgCl2 / CaCl2. After the top agar solidified, incubation was performed under the conditions described above.

[0082] Among the many phage-resistant mutants that appeared on agar plates after incubation, strain DSM 34995 underwent three colony purifications and was retested in a plaque assay on M17-MgCl2 / CaCl2 lactose agar plates at 37°C, challenged with phage DSM 35003 CHPC869. Phage resistance was confirmed by the absence of a single plaque in the plaque assay.

[0083] Acidification curve in the presence of bacteriophage

[0084] Acidification was determined using the mother strain DSM 34993 and the phage-resistant mutant DSM 34995.

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

[0086] Bacteriophage DSM 35003 CHPC869 was added to a final concentration of 1.0E06 phage particles per mL. The results of the phage challenge experiment are shown in the table below, i.e., the pH measured after 0, 10, and 20 hours of fermentation.

[0087] Table 2. pH measured during fermentation.

[0088]

[0089] Example 2

[0090] Production of antiphage strain DSM 34996

[0091] After overnight incubation of 0.1 mL of the mother strain's M17-2% lactose culture at 37°C with 0.1 mL of phages DSM 35011 CHPC1230 (first round) and DSM 35012 CHPC1636 (second round, starting with the phage-resistant mutant generated in the first round), containing 1.0E09 phage particles per mL, the phage-resistant mutant was generated from the mother strain DSM 34991 in two steps on M17-2% lactose agar plates with 10 mM MgCl2 / CaCl2. For this purpose, the phages and host strain were mixed with 2.5 mL of top agar solution (melted agar maintained at 46–50°C, with an agar concentration half that of the standard concentration (0.75%)) and poured onto the bottom M17-2% lactose agar. Both the bottom and top agars contain 10 mg Cl2 / CaCl2. After the top agar solidifies, it is incubated under the conditions described above.

[0092] Among the many phage-resistant mutants that appeared on agar plates after incubation, strain DSM 34996 was purified three times and retested in a plaque assay on M17-MgCl2 / CaCl2 lactose agar plates at 37°C using phage challenges of phages DSM 35011 CHPC1230 and DSM 35012 CHPC1636. Phage resistance to these two phages was confirmed by the absence of a single plaque in the plaque assay.

[0093] Acidification curve in the presence of bacteriophage

[0094] Acidification was determined using the mother strain DSM 34991 and the phage-resistant mutant DSM 34996.

[0095] For the acidification test, 200 mL of B milk (reconstituted skim milk (9.5% dry matter) boiled at 99°C for 30 min) was inoculated with 1% of the test bacterial strain (from an overnight culture grown in M17-2% lactose at 37°C) and incubated at 37°C for 23 h. pH was monitored over time using the CINAC (Scientific Solutions) system.

[0096] Bacteriophages DSM 35011 CHPC1230 and DSM 35012 CHPC1636 were added to a final concentration of 1.0E06 phage particles per mL. The results of the phage challenge experiment are shown in the table below, i.e., the pH measured after 0, 10, and 20 hours of fermentation.

[0097] Table 3. pH measured during fermentation.

[0098]

[0099] Example 3

[0100] Acidification time of the composition containing the new strain.

[0101] Use the following compound to prepare set-type yogurt:

[0102] Compound 1 containing DSM 34996.

[0103] Compound 2 containing DSM 34995.

[0104] F-DVS eXact® Dahi 3 (Chr. Hansen, product number: 717445) was used as the benchmark.

[0105] A milk base (3.5% protein, 3.0% fat, 0% sugar) was prepared from fresh milk standardized with skim milk powder. Three fermentation products were produced according to the following process by inoculating this milk base with two experimental blends and the standard, respectively:

[0106]

[0107] Acidification time was measured using the axone system (originally developed by Absciss for Chr. Hansen, now part of ARDPI), which continuously measures pH during fermentation.

[0108] Table 4. Time (minutes) required to acidify to the target pH 4.6.

[0109]

[0110] Texture properties

[0111] Gel hardness was measured using a texture analyzer (TA.XT PlusC, Stable Micro Systems). The test mode was set to compression, and a 40 mm disk probe was used. The speed was set to 5 mm / s before, during, and after the test, the distance to 15 mm, and the trigger force to 5 g. Data were measured on day 7 (day +7) at 13°C, and samples were stored at 13°C until the day of measurement.

[0112] Table 5. Gel hardness (g)

[0113]

[0114] Rheological properties were measured using an ASC rheometer (model DSR101) from Anton Paar. The method employed a rotational step, subjecting the sample to rotational deformation from 0.273 s⁻¹ to 300 s⁻¹ and back to 0.273 s⁻¹. The corresponding shear stresses were measured.

[0115] The samples were stored at 13°C until the day of measurement, and the shear rate was measured at 13°C for 30.2 s on day 7 (day +7). -1 Shear stress below.

[0116] Table 6. Shear rate 30.2 s -1 Shear stress (Pa)

[0117]

[0118] Example 4

[0119] The acidification time imparted by the composition.

[0120] Use the following compound to prepare set-type yogurt:

[0121] Experimental compound 1 containing DSM 34996.

[0122] Experimental compound 2 containing DSM 34995.

[0123] F-DVS eXact® Dahi 3 (Chr. Hansen, product number: 717445) was used as the benchmark.

[0124] A milk base (3.5% protein, 3.0% fat) was prepared from low-heat milk powder standardized with water and 38% butter. Three fermented milk products were produced according to the following process by inoculating this milk base with two experimental blends and the standard, respectively:

[0125]

[0126] Acidification time was measured using a colorimetric method, which allows for continuous measurement of color / pH during fermentation as described in WO 2005 / 068982.

[0127] Table 7. Time (minutes) required to acidify to the target pH 4.6.

[0128]

[0129] Texture properties

[0130] On day +7, the gel hardness of the fermentation product was measured using a small-scale compression test (Hamilton robot and Mettler Toledo precision balance) at 13°C.

[0131] Table 8. Gel hardness (g)

[0132]

[0133]

[0134]

[0135] . PCT / RO / 134 form

Claims

1. A phage-resistant lactic acid bacteria strain derived from a mother strain of *Streptococcus thermophilus*, wherein the strain (a) is resistant to one or more phages selected from DSM 35003 CHPC869, DSM 35011 CHPC1230, and DSM 35012 CHPC1636, the mother strain is sensitive to the one or more phages, and (b) acidifies the lactic acid more rapidly in the presence of the one or more phages compared to the mother strain.

2. The strain according to claim 1, wherein, when measured 10 hours and / or 20 hours after the start of acidification, the strain is capable of acidifying the lactose containing one or more of the phages to a pH below 5.

3. The strain according to claims 1-2, wherein the milk base is a B-milk made from reconstituted skim milk with a dry matter content of 9.5%, the reconstituted skim milk having been boiled at 99°C for 30 minutes.

4. The strain according to any one of claims 1-3, wherein the strain provides improved texture in fermentation products made with the 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 a mutant or variant thereof.

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

8. The composition according to claim 7, which, as a mixture or as a set of components, comprises i) the strain according to any one of claims 1-6; and ii) one or more additional lactic acid bacteria belonging to a different genus than Streptococcus.

9. The composition according to claim 8, wherein the one or more additional lactic acid bacteria belong to the genera of Lactobacillus, Lactobacillus mucosae, Lactobacillus casei, Lactobacillus assemblica, Lactobacillus lactis, Lactobacillus spp. ...ococcus spp., Leuconostoc spp., Pediococcus spp., and / or Bifidobacterium spp.

10. The composition according to claims 8-9, wherein the one or more additional lactic acid bacteria are *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus casei*, *Lactobacillus paracasei*, and / or *Bifidobacterium animalis*.

11. The composition according to any one of claims 7-10, wherein the composition further comprises a cryoprotectant, a lyophilization protectant, an antioxidant, and / or nutrients.

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

13. A method for producing a fermented product, the method comprising the following steps: (a) adding the strain according to any one of claims 1-6 or the composition according to any one of claims 7-12 to the emulsion base; and (b) Ferment the milk base at a temperature between about 22°C and about 45°C until the pH reaches 4.6 or below.

14. The method of claim 13, wherein the acidification time for producing the fermentation product is shortened compared to the acidification time for producing the fermentation product made from the mother strain.

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

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

17. The fermentation product according to claim 16, wherein the strain according to any one of claims 1-6 is at least 10 7 The concentration of CFU / g is present.

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 pastafilata cheese.

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

Images