Phage resistant lactic acid bacteria

Abstract

The present invention relates to a bacteriophage-resistant lactic acid bacterium, a composition comprising the bacterium, and a fermented food product obtained with the bacterium or the composition comprising the bacterium.

Description

Field of the Invention

[0001] The present invention relates to bacteriophage-resistant lactic acid bacteria, a composition containing the bacteria, and a fermented food produced using the bacteria. Background of the Invention

[0003] The food industry uses a large number of bacteria, especially lactic acid bacteria, in the production of fermented foods. The choice of lactic acid bacteria affects the properties of fermented foods, such as taste, texture and shelf life.

[0004] Production failure due to phage infection is considered to be one of the major problems in industrial applications of bacterial culture. Bacteriophages have been found for many bacterial strains used in industry, such as several species of lactic acid bacteria (e.g., Lactococcus sp., Lactobacillus sp., Leuconostoc sp., Pediococcus sp., or Streptococcus sp.).

[0005] In the food industry, lactic acid bacteria starter cultures are widely used for food fermentation. One factor that contributes to the frequent occurrence of bacteriophage infection in lactic acid bacteria starter cultures is that fermentation conditions in the food industry, including the dairy industry, are often non-sterile. Therefore, under these industrial conditions, it has not been possible to eliminate bacteriophage contamination.

[0006] The lysis process of phage includes adsorption of phage to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phage and release of progeny from the host. Interference with the cell-mediated mechanisms of any of these events can prevent phage infection. During industrial use, the ability of bacterial cultures to resist phage infection depends largely on the characteristics of the host strain that affect one or more of the above mechanisms.

[0007] In order to meet the requirements of the industry, it is necessary to come up with new phage-resistant strains of lactic acid bacteria, in particular Streptococcus thermophilus. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention relates to a lactic acid bacterium having enhanced phage resistance to DSM 34256 and / or DSM 34257 compared to its parent strain.

[0010] In a second aspect, the present invention relates to a composition comprising the lactic acid bacteria of the present invention.

[0011] In a third aspect, the present invention relates to a method for producing a fermented food, which comprises fermenting a substrate with the lactic acid bacteria of the present invention or the composition of the present invention.

[0012] In a fourth aspect, the present invention relates to a fermented food comprising the lactic acid bacteria of the present invention or a fermented food obtainable by the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] Before outlining the present invention in more detail, a set of terms and conventions are defined:

[0015] The term "Genesis" means the website: www.ncbi.nlm.nih.gov / taxonomy As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or propagated. It includes multiple identical bacteria.

[0016] In this context, the term "mutant" or "mutant strain" should be understood as derived strains or strains that can be derived from the strain of the present invention by, for example, genetic engineering, radiation and / or chemical treatment. Mutants are preferably functionally equivalent mutants, such as mutants with substantially the same or improved properties (such as texture, shear stress, viscosity, gel hardness, oral coating sense, flavor, post-acidification, acidification speed and / or phage robustness) of the strain from which it originates. Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenic treatment (including chemical mutagens, UV light treatments such as ethane methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG)), or spontaneously generated mutants. Mutants may have undergone several mutagenic treatments (single treatment should be understood as a mutagenic step, followed by screening / selection steps), but it is currently preferred to perform no more than 20 times or no more than 10 times or no more than 5 treatments (or screening / selection steps). In currently preferred mutants, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome are replaced by another nucleotide, or are deleted, compared to the parent strain. As is clear to those skilled in the art, the mutant of the present invention may also be the parent strain.

[0017] In this context, the term "variant" or "variant" is understood to be a strain that is functionally equivalent to the strain of the present invention, for example having substantially the same or improved properties or characteristics (e.g., texture, acidification rate, viscosity, gel hardness, mouth coating, flavor, post-acidification and / or phage robustness). Such variants that can be identified using appropriate screening techniques are part of the present invention.

[0018] For strains of the genus Lactobacillus, the term "CFU" refers to colony forming units, determined by growth (formation of colonies) on MRS agar plates incubated at 37° C. for 3 days under anaerobic conditions. MRS agar has the following composition (g / l):

[0019] Bacto peptone No. 3: 10.0

[0020] Bacto beef extract: 10.0

[0021] Bacto yeast extract: 5.0

[0022] Dextrose: 20.0

[0023] Sorbitan monooleate complex: 1.0

[0024] Ammonium citrate: 2.0

[0025] Sodium acetate: 5.0

[0026] Magnesium sulfate: 0.1

[0027] Manganese sulfate: 0.05

[0028] Potassium hydrogen phosphate: 2.0

[0029] Bacto agar: 15.0

[0030] Milli-Q water: 1000ml.

[0031] Adjust the pH to 5.4 or 6.5: The pH of L. rhamnosus, L. casei and L. paracasei was adjusted to 6.5. For all other Lactobacillus species, the pH was adjusted to 5.4. In particular, the pH of L. delbrueckii subsp. bulgaricus, L. acidophilus and L. helveticus was adjusted to 5.4. The pH of L. rhamnosus, L. casei and L. paracasei was adjusted to 6.5.

[0032] For Streptococcus thermophilus, the term "CFU" refers to colony forming units, determined by growth (formation of colonies) on M17 agar plates incubated at 37°C under anaerobic conditions for 3 days. M17 agar has the following composition (g / l):

[0033] Tryptone: 2.5 g

[0034] Meat digest: 2.5g

[0035] Soybean powder papain digest: 5.0g

[0036] Yeast extract: 2.5g

[0037] Meat extract: 5.0g

[0038] Lactose: 5.0g

[0039] Sodium glycerophosphate: 19.0g

[0040] Magnesium sulfate heptahydrate: 0.25g

[0041] Ascorbic acid: 0.5 g

[0042] Agar: 15.0 g

[0043] Milli-Q water: 1000ml.

[0044] Adjust pH to final pH 7.1 ± 0.2 (25°C)

[0045] Phage-resistant lactic acid bacteria.

[0046] Phage-resistant mutants can be produced by exposing a phage-sensitive parent strain to the desired phage or phages, for example by culturing the phage-sensitive parent strain at a concentration of the desired phage sufficient to induce selection pressure on the strain. Methods for producing phage-resistant mutants have been described previously. The phage-resistant mutants of the present invention can be obtained by the methods described in the examples. In a first aspect, the present invention relates to lactic acid bacteria having enhanced phage resistance to DSM 34256 and / or DSM 34257 compared to their parent strains.

[0047] In addition, it may be desirable to increase the texture provided by such a strain. In this case, a phage-resistant mutant with enhanced textural properties should be selected. The phage-resistant mutant of the present invention with enhanced textural properties (such as viscosity) can be obtained by the methods described in the examples. In one embodiment, the present invention relates to a lactic acid bacterium of the present invention, wherein the bacterium has increased viscosity compared to its parent strain.

[0048] It will be appreciated that phage resistance can be obtained by the method using strains derived from a number of species. However, a preferred species in the present invention is Streptococcus thermophilus. In one embodiment, the present invention relates to lactic acid bacteria of the present invention, wherein the bacteria belong to the species of Streptococcus thermophilus.

[0049] Any thermophilic Streptococcus strain can be used as the parent strain. The parent strain of the present invention is preferably the thermophilic Streptococcus strain DSM 34236 or mutants and variants thereof. In one embodiment, the present invention relates to the lactic acid bacteria of the present invention, wherein the parent strain is DSM 34236.

[0050] Preferred mutants derived from the parent strain may be any mutant having improved phage resistance and optionally increased viscosity compared to its parent strain.In one embodiment the invention relates to the lactic acid bacterium of the invention, wherein the bacterium is selected from DSM 34235 and mutants and variants thereof.

[0051] As used herein, the term "lactic acid bacteria", abbreviated as "LAB", means Gram-positive, microaerophilic or anaerobic bacteria that ferment sugars by producing acids, including lactic acid (which is the main acid produced), acetic acid and propionic acid. The most useful industrial lactic acid bacteria belong to the "Lactobacillales", which include species of the genus Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Brevibacterium, Enterococcus and Propionibacterium. In addition, lactic acid-producing bacteria belong to the group of strict anaerobic bacteria, and bifidobacteria, i.e., bifidobacterium, are generally included in the lactic acid bacteria group. These are often used as food cultures alone or in combination with other lactic acid bacteria. Lactic acid bacteria (including bacteria of the species of lactobacillus and thermophilic streptococci) are usually supplied to the dairy industry as frozen or lyophilized cultures (to obtain a large number of starter cultures) or so-called "direct yogurt starter cultures (DirectVat Set)" (DVS) cultures, intended to be directly inoculated into a fermentation vessel or cylinder to produce dairy products, such as fermented dairy products. Such cultures are generally referred to as "starter cultures" or "starters".

[0052] The term "increased phage resistance" means that the strain has improved phage resistance to at least one phage when tested in a plaque assay (e.g., "Determination of phage resistance by agar overlay method" as described in the Examples), expressed as the difference in pfu / mL (plaque forming units per mL) obtainable on a given strain using the at least one phage compared to the pfu / mL obtained with the same phage on the parent strain. Strains with improved phage resistance preferably show a reduction in pfu / mL of at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold or more.

[0053] Composition

[0054] As described herein, the lactic acid bacteria of the present invention may be used in a composition, more particularly in a starter culture for the preparation of a fermented food or feed product. In one aspect, the present invention relates to a composition comprising the lactic acid bacteria of the present invention.

[0055] Since it is normal to apply mixed cultures of lactic acid bacteria in the production of lactic acid fermentation processes, the composition will in some embodiments comprise a plurality of strains belonging to the same species or to different species.

[0056] In one embodiment, the composition of the invention, as a mixture or as a kit of parts further comprises one or more strains belonging to the genus Lactobacillus.

[0057] In one embodiment, the one or more strains belonging to the genus Lactobacillus may be selected from L. delbrueckii, L. delbrueckii subsp bulgaricus, L. delbrueckii subsp. lactis.

[0058] In one embodiment, the lactobacillus strain of the present invention is Lactobacillus delbrueckii subsp. bulgaricus. Lactobacillus delbrueckii is a lactic acid bacteria frequently used in commercial milk fermentation, where the organism is typically used as part of a mixed starter. In one embodiment, at least one lactic acid bacteria strain in the composition of the present invention is Lactobacillus delbrueckii subsp. bulgaricus.

[0059] In one embodiment of the composition of the present invention, the Lactobacillus bulgaricus strain is selected from DSM 19251; DSM 19252; DSM 22586; DSM 26419; DSM 28910; DSM 32092; DSM 33515 and mutants and variants thereof.

[0060] In one embodiment, the composition and / or mixture or kit comprises a thermophilic Streptococcus strain DSM 34235 or a mutant and variant thereof and a strain belonging to a Lactobacillus species. Therefore, in a preferred embodiment, the composition and / or mixture or kit comprises a thermophilic Streptococcus strain DSM 34235 or a mutant or variant thereof and a strain belonging to a Lactobacillus subspecies of bulgaricus. Therefore, in a preferred embodiment, the composition and / or mixture or kit comprises a thermophilic Streptococcus strain DSM 34235 or a mutant or variant thereof and a strain belonging to a species of Lactobacillus delbrueckii subspecies selected from the group consisting of DSM 19251; DSM 19252; DSM 22586; DSM 26419; DSM 28910; DSM 32092; DSM 33515; and mutants and variants thereof.

[0061] In addition to the lactic acid bacteria of the present invention, the composition can also include a thermophilic Streptococcus strain. In one embodiment, the composition also includes one or more thermophilic Streptococcus strains as a mixture or as a complete kit. In one embodiment, the composition of the present invention includes one or more thermophilic Streptococcus strains selected from the following thermophilic Streptococcus strains as a mixture or as a complete kit: DSM 17876; DSM 18111; DSM 19242; DSM 21408; DSM 22585; DSM 22587; DSM 22588; DSM 22589; DSM 22935; DSM 24023; DSM 25012; DSM 26562; DSM 32503; DSM 32826; DSM 33869; and mutants and variants thereof.

[0062] In one embodiment, the composition and / or mixture or kit comprises Streptococcus thermophilus strain DSM 34235 or its mutants and variants and a strain belonging to the species of Streptococcus. Therefore, in a preferred embodiment, the composition and / or mixture or kit comprises Streptococcus thermophilus strain DSM 34235 or its mutants or variants and a strain belonging to the species of Streptococcus thermophilus. Thus, in a preferred embodiment, the composition and / or mixture or kit of parts comprises the Streptococcus thermophilus strain DSM 34235 or a mutant or variant thereof and a strain belonging to the species of Streptococcus thermophilus selected from the group consisting of: DSM 17876; DSM 18111; DSM 19242; DSM 21408; DSM 22585; DSM 22587; DSM 22588; DSM 22589; DSM 22935; DSM 24023; DSM 25012; DSM 26562; DSM 32503; DSM 32826; DSM 33869; and mutants and variants thereof.

[0063] In one embodiment, the composition of the present invention, as a mixture or as a kit of parts, further comprises one or more strains belonging to the species of Lactococcus. In one embodiment, the one or more strains belonging to the genus Lactococcus can be selected from the group consisting of: Lactococcus lactis (L. lactis); Lactococcus lactis subsplactis; Lactococcus lactis subsp. cremoris. In one embodiment, the composition of the present invention, as a mixture or as a kit of parts, further comprises one or more strains of Lactococcus selected from DSM 21404; DSM 33192; DSM 21405; and mutants and variants thereof.

[0064] In one embodiment, the composition of the invention further comprises, as a mixture or as a kit of parts, one or more strains belonging to the species of the genus Pediococcus.

[0065] In one embodiment, the one or more strains belonging to the genus Pediococcus may be strains of Pediococcus acidilactis. In one embodiment, the composition of the invention as a mixture or as a kit of parts further comprises one or more strains of Pediococcus acidilactis selected from DSM 28307 and mutants and variants thereof.

[0066] The composition of the present invention may include probiotics. The probiotic strains may be added before or after fermentation. If added before fermentation, the probiotic strains also act as fermentation bacteria. The term "probiotic" refers to live bacteria administered to a consumer in sufficient amounts to achieve a health-promoting effect in the consumer. Probiotics are able to survive gastrointestinal conditions after ingestion and colonize in the intestinal tract of the consumer.

[0067] It should be understood that the Lactobacillus taxonomy has been updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be used herein unless otherwise indicated. For the purposes of the present invention, the following table lists the new and old names of some Lactobacillus species relevant to the present invention.

[0068] Table 1. New and old names of some Lactobacillus species relevant to the present invention.

[0069]

[0070] In one embodiment of the present invention, the probiotic strain is selected from the group consisting of: Lactobacillus bacteria, such as Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus johnsonii, Bifidobacterium bacteria, such as Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis lactic acid subsp. dentate, Bifidobacterium catenella, Bifidobacterium angularis, Bifidobacterium large, Bifidobacterium pseudocatenella and Bifidobacterium infantis, etc.

[0071] In one embodiment of the invention, the probiotic lactobacillus strain is selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus johnsonii. In one embodiment of the invention, the probiotic strain is Lactobacillus acidophilus deposited under DSM 13241.

[0072]

[0073] In one embodiment of the invention, the probiotic lactobacillus strain is selected from the group consisting of a Lactobacillus rhamnosus strain, a Lactobacillus paracasei strain and a Lactobacillus mucilaginosus strain. In one embodiment of the invention, the probiotic strain is a Lactobacillus rhamnosus strain DSM 33870 or a strain deposited under ATCC 53103. In one embodiment of the invention, the probiotic strain is Lactobacillus paracasei strain DSM 19465 or CRL 431 deposited as ATCC 55544. In one embodiment of the invention, the probiotic strain is Lactobacillus mucimulans strain DSM 22584.

[0074] In one embodiment of the invention, the probiotic bifidobacterium strain is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis. In one embodiment of the invention, the probiotic bifidobacterium strain is Bifidobacterium animalis subsp. lactis. In one embodiment of the invention, Bifidobacterium animalis subsp. lactis is DSM 33868 or deposited as DSM 15954

[0075] The above mixture or kit can be further combined with other lactic acid bacteria, such as but not limited to probiotics. In one embodiment, one or more lactic acid bacteria are selected from the group consisting of: Bifidobacterium such as Bifidobacterium animalis subsp. lactis (e.g. ), Lactobacillus acidophilus (e.g.

[0076] ), Lactobacillus rhamnosus (e.g. and any combination thereof. Which Bifidobacterium, Lactobacillus acidophilus and / or Lactobacillus rhamnosus is applied depends on their application and the food to be produced.

[0077] The expression "kit" comprising a strain means that the strain or strain culture is physically separated, but is intended to be used together. Therefore, the strain or culture of the thermophilic streptococcus strain and the lactobacillus strain are in different boxes or sealed pouches. In one embodiment, the thermophilic streptococcus strain and the lactobacillus, for example Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and / or Lactobacillus mucilaginosus reuteri strain are in the same form, i.e. in frozen form, in pellet or frozen pellet form, in powder form, for example dry or lyophilized powder.

[0078] In one embodiment, the invention relates to a composition according to the invention comprising, as a mixture or as a kit of parts, one or more probiotic strains selected from Bifidobacterium, Lactobacillus acidophilus, Lactobacillus casei (formerly Lactobacillus casei), Lactobacillus paracasei (formerly Lactobacillus paracasei), Lactobacillus rhamnosus (formerly Lactobacillus rhamnosus) and Lactobacillus mucofermentans (formerly Lactobacillus fermentum).

[0079] In one embodiment, the composition of the invention comprises bacteria in concentrated form, wherein the total concentration of viable cells is 10 4 Up to 10 12 The range of cfu (colony forming units) includes at least 10 per gram of composition. 4 cfu, for example at least 10 5 cfu / g, for example at least 10 6 cfu / g, for example at least 10 7 cfu / g, for example at least 10 8 cfu / g, for example at least 10 9 cfu / g, for example at least 10 10 cfu / g, for example at least 10 11 cfu / g.

[0080] In one embodiment, the composition of the present invention comprises bacteria in concentrated form, wherein the concentration of each viable cell strain is 10 4 Up to 10 12 The range of cfu (colony forming units) includes at least 10 per gram of composition. 4 cfu, for example at least 10 5 cfu / g, for example at least 10 6 cfu / g, for example at least 10 7 cfu / g, for example at least 10 8 cfu / g, for example at least 10 9cfu / g, for example at least 10 10 cfu / g, for example at least 10 11 cfu / g.

[0081] In one embodiment of the present invention, the composition comprises 10 4 Up to 10 12 CFU (colony forming unit) / g of Streptococcus thermophilus strain, 10 5 Up to 10 11 CFU / g, 10 6 Up to 10 10 CFU / g or 10 7 Up to 10 9 CFU / g of Streptococcus thermophilus strains.

[0082] In one embodiment, the composition further comprises 10 4 Up to 10 12 CFU / g of Lactobacillus strains, 10 5 Up to 10 11 CFU / g, 10 6 Up to 10 10 CFU / g or 10 7 Up to 10 9 CFU / g of Lactobacillus strains.

[0083] In one embodiment, the composition comprises 10 4 Up to 10 12 CFU / g, 10 5 Up to 10 11 CFU / g, 10 6 Up to 10 10 CFU / g, or 10 7 Up to 10 9 CFU / g of each of the Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and / or Lactobacillus reuteri strains.

[0084] Thermophilus streptococci, lactobacillus and lactobacillus, for example Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and / or Lactobacillus rhamnosus and other lactic acid bacteria are usually used as fermentation agents for technical purposes in various food productions, for example in the dairy industry, for example for fermented dairy products. Therefore, in another preferred embodiment, the composition is a fermentation agent. The composition can be a fermentation agent, for example a yogurt fermentation agent.

[0085] The composition and / or leavening agent can be in the form of freezing, spray drying, freeze drying, vacuum drying, air drying, tray drying or liquid. Usually, by preparing a product with low water activity, the storage stability of the composition and / or leavening agent can be extended. By controlling the water activity (Aw), the influence of moisture migration on the product can be predicted and regulated. Therefore, it may be preferred that the water activity (Aw) of the dry composition herein is in the range of 0.01-0.8, preferably in the range of 0.05-0.4.

[0086] The composition may contain other components, such as cryoprotectants and / or conventional additives, including nutrients, such as yeast extract, sugars and vitamins.

[0087] The composition of the present invention can be provided in various forms. It can be a powder, a pill or a tablet. It can be a frozen form, a dried form, a lyophilized form or a liquid form. Therefore, in one embodiment, the composition is a frozen, dried, lyophilized or liquid form. In one embodiment, the composition of the present invention, wherein the composition is a frozen, lyophilized or liquid form.

[0088] Composition of the present invention can also comprise cryoprotectant, lyoprotectant, antioxidant, nutrient, filler, spices or its mixture.Composition preferably comprises one or more in cryoprotectant, lyoprotectant, antioxidant and / or nutrient, more preferably cryoprotectant, lyoprotectant and / or antioxidant, and most preferably cryoprotectant or lyoprotectant, or both.Protective agent such as cryoprotectant and lyoprotectant known to those skilled in the art is used.Suitable cryoprotectant or lyoprotectant include monosaccharide, disaccharide, trisaccharide and polysaccharide (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia gum) etc.), polyol (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol etc.), amino acid (such as proline, glutamic acid), mixed substance (such as skim milk, peptone, gelatin, yeast extract) and inorganic compound (such as sodium tripolyphosphate).

[0089] In one embodiment, the composition according to the present invention may include one or more cryoprotectants selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uridine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and derivatives of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include carbohydrates, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B family, vitamin C). The composition may optionally include additional substances, including fillers (such as lactose, maltodextrin) and / or spices.

[0090] In one embodiment of the invention, a cryoprotectant is a mixture of an agent or agents, which, in addition to its cryoprotective properties, has an enhancing effect. The term "enhancing effect" is used to describe a situation in which, when a cryoprotectant is inoculated into a culture medium to be fermented or converted, the cryoprotectant imparts an increased metabolic activity (enhancing effect) to the culture thawed or re-dissolved. Vitality and metabolic activity are not synonymous concepts. Commercial frozen cultures or freeze-dried cultures can maintain their vitality, but they may have lost a large portion of metabolic activity, for example, even if the storage time is short, the culture may lose its acidogenic (acidifying) activity. Therefore, vigor and enhancing effect must be evaluated by different assays. Vitality is assessed by vigor assays (such as assaying colony forming units), while enhancing effect is assessed by quantifying the relevant metabolic activity of thawing or re-dissolving cultures relative to the vigor of the culture. The term "metabolic activity" refers to the oxygen-scavenging activity of the culture, its acidogenic activity, i.e. the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite-producing activity, e.g. the production of aromatic compounds such as acetaldehyde, (α-acetolactate, acetoin, diacetyl and 2,3-butanediol (butanediol)).

[0091] In one embodiment, the composition of the present invention contains or comprises 0.2% to 20% cryoprotectant or mixture of agents in the %w / w of the material. However, it is preferred to add cryoprotectant or mixture of agents in an amount in the range of 0.2% to 15%, 0.2% to 10%, 0.5% to 7% and 1% to 6% by weight, including cryoprotectant or mixture of agents in the range of 2% to 5% by weight in the %w / w of the frozen material. In a preferred embodiment, the culture comprises about 3% cryoprotectant or mixture of agents, in the %w / w of the material by weight. An amount of about 3% cryoprotectant corresponds to a concentration in the range of 100mM. It should be appreciated that for each aspect of the embodiments of the present invention, the range may be an increment of the range.

[0092] In the present context, the term "from x% to y%" is intended to include the endpoint and is therefore equivalent to the term "from x% to y%, including x% and y%".

[0093] In another aspect, the composition of the invention contains or comprises an ammonium salt (e.g., an ammonium salt of an organic acid (e.g., ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as an enhancer (e.g., a growth enhancer or an acidification enhancer) of bacterial cells, e.g., cells belonging to the species of Streptococcus thermophilus, e.g., (substantially) urease-negative bacterial cells. The terms "ammonium salt," "ammonium formate," and the like are to be understood as a source of salts or a combination of ions. The term "source," e.g., "ammonium formate" or "ammonium salt," refers to a compound or mixture of compounds that provides ammonium formate or an ammonium salt when added to a cell culture. In some embodiments, the ammonium source releases ammonium into the growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, the ammonium is not provided by the milk matrix. It should of course be understood that ammonia may be added instead of an ammonium salt. Thus, the term ammonium salt includes ammonia (NH3), NH4OH, NH4 + wait.

[0094] In one embodiment, the composition of the present invention may comprise a thickener and / or stabilizer, such as pectin (eg, HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginate and guar gum.

[0095] In one embodiment, wherein the microorganism produces polysaccharides (such as EPS), polysaccharides cause high / viscous texture in acidified dairy products, and the acidified dairy products produced are substantially free of or completely free of any thickeners and / or stabilizers, such as pectins (e.g., HM pectins, LM pectins), gelatin, CMC, soy fiber / soy polymers, starches, modified starches, carrageenan, alginates, and guar gum. Substantially free should be understood as the product comprising 0% to 20% (w / w) (e.g., from 0% to 10%, from 0% to 5%, or from 0% to 2%, or from 0% to 1%) of thickeners and / or stabilizers.

[0096] As mentioned earlier, interactions between microbial species are crucial – not only in terms of growth, but also in terms of rheological properties (such as texture) and taste development.

[0097] Methods for producing fermented products.

[0098] One aspect of the present invention relates to a method of producing a fermented food product comprising adding a starter culture composition as described herein to a food starting material and maintaining the starting material so inoculated under conditions in which the lactic acid bacteria are metabolically active.

[0099] Useful food starting materials, ie substrates, include any material which is normally subjected to fermentation by lactic acid bacteria, such as milk, vegetable material, meat products, fruit juice, must, dough and batters.

[0100] The fermented products obtained by this method include dairy products such as cheese and buttermilk including fresh cheese products as typical examples.

[0101] Another important application of the lactic acid bacteria according to the invention is the use of bacterial cultures as so-called probiotics. In this context, the term "probiotics" refers to microbial cultures that, when ingested by humans or animals in the form of living cells, improve health by inhibiting harmful microorganisms in the gastrointestinal tract, by boosting the immune system or by promoting the digestion of nutrients.

[0102] The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo or camel. In a preferred embodiment, the milk is bovine milk. The term milk also includes protein / fat solutions made from plant materials. Suitable plant materials may be legumes, nuts or cereals. Preferred legumes include soya, peas, beans, lupines and lentils. Preferred nuts include coconut, almonds, cashews and walnuts. Preferred cereals include oats, wheat, rye and millet. The milk may be from a mammal, a plant material or any combination thereof.

[0103] The term "milk matrix" may be any raw milk and / or processed milk material that can be fermented according to the method of the present invention. Thus, useful milk matrices include (but are not limited to) solutions / suspensions of any milk or milky products containing proteins, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dry milk, whey, whey permeate, lactose, mother liquor obtained by crystallization of lactose, whey protein concentrate or milk fat. Obviously, the milk matrix may be derived from any mammal, such as essentially pure mammalian milk, or reconstituted milk powder, or the milk matrix may be derived in whole or in part from plant materials, i.e., vegetable milk as described above. Preferably, at least part of the protein in the milk matrix is ​​(i) a protein naturally occurring in mammalian milk, such as casein or whey protein, or (ii) a protein naturally occurring in vegetable milk. However, part of the protein may be a protein that is not naturally present in milk.

[0104] Prior to fermentation, the dairy base may be homogenized and pasteurized according to methods known in the art.

[0105] As used herein, "homogenization" means mixing thoroughly to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, this can break down the milk fat into smaller sizes so that it can no longer separate from the milk. This can be achieved by forcing the milk through small holes under high pressure.

[0106] As used herein, "pasteurization" refers to treating a milk base to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. The specified temperature is typically achieved by heating. The temperature and duration may be selected so as to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step may then be performed.

[0107] "Fermentation" in the method of the present invention refers to the conversion of carbohydrates into alcohols or acids by the action of microorganisms. Preferably, the fermentation in the method of the present invention comprises the conversion of lactose into lactic acid.

[0108] Fermentation processes for producing fermented dairy products are well known, and a person skilled in the art will understand how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganisms and process time. Obviously, fermentation conditions are selected that support the realization of the present invention, i.e. obtaining a fermented product, such as a dairy product or dairy product analog (fermented dairy product) in solid or liquid form.

[0109] In the present context, the term "starter culture" is a culture which is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to assist in the start of the fermentation process in the preparation of fermented products such as various foods, feeds and beverages.

[0110] In the present context, a "yogurt starter" is a bacterial culture comprising one or more lactobacilli selected from the group consisting of Lactobacillus delbrueckii strains and / or Lactobacillus acidophilus strains and one or more Streptococcus thermophilus strains. Accordingly, "yogurt" refers to a fermented dairy product obtained by inoculating and fermenting a milk base with a composition comprising a lactobacillus strain, such as Lactobacillus bulgaricus and / or Lactobacillus acidophilus and a Streptococcus thermophilus strain.

[0111] It may be preferred that the matrix is ​​a milk matrix. The milk matrix may be derived from animals, preferably mammals, or from plants. However, the milk matrix does not have to be purely animal-derived, it may also contain a matrix of plant origin. The fermented product may be a food, which may also be a dairy product.

[0112] Depending on the product to be produced, the base may be a dairy base. A dairy base is particularly preferred when a fermented dairy product such as yogurt, buttermilk or kefir is the final product.

[0113] The milk base may be a product of animal or plant origin. Thus, in one embodiment, the fermented product is a food product, such as a dairy product. The dairy product may be selected from the group consisting of fermented dairy products, such as but not limited to yogurt, buttermilk and kefir or cheeses, such as but not limited to fresh cheese or pasta filata.

[0114] Likewise, enzymes selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminases, aspartic proteases, lactases, chymosins, rennets, and mixtures thereof may be added to the matrix, such as a milk matrix, before, during and / or after fermentation.

[0115] In a first aspect, the present invention relates to a method for producing a fermented food, which comprises fermenting a substrate or a composition of the present invention with the lactic acid bacteria of the present invention.

[0116] Fermented products.

[0117] As used herein, the term "fermented dairy product" refers to a food or feed product, wherein the preparation of the food or feed product involves fermentation of a dairy base with lactic acid bacteria. As used herein, "fermented dairy product" includes, but is not limited to, products such as yogurt and cheese. Other examples of fermented products may be, for example, buttermilk, kefir, quark, yogurt, crème brûlée and sour cream.

[0118] In one embodiment of the fermented food product of the present invention, the product is yogurt, buttermilk, kefir, quark, sour cheese, crème fraîche, sour cream or cheese. Preferably, the fermented dairy product is yogurt.

[0119] Examples of cheeses prepared by fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus include mozzarella cheese and pizza cheese (Hoier et al. (2010) in The Technology of Cheesemaking, 2 nd Ed. Blackwell Publishing, Oxford; 166-192).

[0120] The term "stirred product" refers specifically to fermented dairy products which, after fermentation, have been subjected to a mechanical treatment resulting in the breaking up and liquefaction of the coagulum formed during the fermentation phase. The mechanical treatment is usually, but not exclusively, stirring, pumping, filtering or homogenizing the gel or obtaining it by mixing it with other ingredients. Stirred products usually, but not always, have a non-fat milk solids content of 9% to 15%.

[0121] The term "set product" includes milk-based products which have been inoculated with a starter culture, such as a fermentation culture, and which have been packaged after the inoculation step and which have then been fermented in the packaging.

[0122] In one embodiment, the fermented product may be in the form of a stirred product, a set product, or a drinkable product.

[0123] Although the fermented product and / or food itself contains the acid and flavor produced during fermentation, it may be desirable for the fermented product and / or dairy product to contain an ingredient selected from the group consisting of: fruit concentrate, syrup, probiotic strain or culture, colorant, thickener, flavoring agent, preservative, and mixtures thereof. In one embodiment of the invention, the fermented food contains an ingredient selected from the group consisting of: fruit concentrate, syrup, probiotic strain or culture, colorant, thickener, flavoring agent, preservative, and mixtures thereof.

[0124] It should be understood that aspects and embodiments disclosed in the sections called "phage-resistant lactic acid bacteria", "compositions", "methods for producing fermentation products" and "fermentation products" may also be relevant to other parts of the specification. In particular, the disclosure about specific strains of the present invention is applicable to all parts of this specification, which is clear to those skilled in the art.

[0125] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a, an" and "the" and similar references should be interpreted as covering the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including, but not limited to"). Unless otherwise indicated herein, the description of the numerical range herein is intended only as a shorthand method for referring to each individual numerical value within the range individually, and each individual value is incorporated into the specification as if it were described individually herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise required. Any language in the specification should not be interpreted as indicating that any unrequired element is essential to the practice of the present invention.

[0126] Any listing or discussion in this specification of an apparently prior-published document should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0127] Unless the context indicates otherwise, preferences, options and embodiments for a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences, options and embodiments of all other aspects, features and parameters of the invention. This is especially true for the description of fat-encapsulated microbial cultures and all their features, which can easily become part of the final composition or final product obtained by the methods described herein. Embodiments and features of the invention are also summarized in the following items.

[0128] Preservation and Expert Solutions

[0129] The applicant requests that, prior to the date of grant of the patent, samples of the deposited microorganisms described in the table below may only be provided to experts.

[0130] Table 2. Deposit made with a depository institution that has been granted the status of an international depository institution under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institut DSMZ-German Collection of Microorganisms, Inhofenstrasse 7B, 38124 Braunschweig, Germany.

[0131] Preserved microorganisms Accession Number Date of deposit Streptococcus thermophilus DSM 34236 April 7, 2022 Streptococcus thermophilus DSM 34235 April 7, 2022 Pac-type phage DSM 34256 April 7, 2022 cos-type bacteriophage DSM 34257 April 7, 2022

[0132] References

[0133] All references cited in this patent document are hereby incorporated by reference in their entirety.

[0134] Hoier et al.(2010)The Technology of Cheesemaking,2 nd Ed.BlackwellPublishing,Oxford,p.166-192.

[0135] Zheng et al. (2020.04.15) Int J Syst Evol Microbiol. Vol. 70(4), p. 2782-2858. Example

[0136] Example 1 - Preparation of phage-resistant mutants.

[0137] Isolation of phage-resistant strains

[0138] In 0.1 mL of DSM 34236 M17-2% lactose overnight culture and 0.1 mL of 1 × 10 9 After plating DSM 34256 phage particles and incubating overnight at 37°C, phage resistant mutants were generated from the parent strain DSM 34236 on M17-2% lactose agar plates containing 10 mM MgCl2 / CaCl2. For this purpose, phage and host strain were mixed with 2.5 mL of top agar solution (molten agar with half (0.75%) standard agar concentration maintained at 46°C-50°C) and M17-2% lactose agar was poured to the bottom. Both the bottom and top agars contained 10 mM MgCl2 / CaCl2. When the top agar solidified, incubation was carried out under the above conditions.

[0139] Among the many phage-resistant mutants that appeared on agar plates after incubation, strain DSM 34235 was colony purified three times and retested in a plaque assay using phage challenge with phage DSM 34256 on M17 lactose agar plates at 37°C.

[0140] No single plaque was observed in the plaque assay, confirming phage resistance to DSM 34256. Phage resistance to phage DSM 34257 was also observed.

[0141] Example 2 - Viscosity of phage resistant mutants.

[0142] Pipette test

[0143] The phage-resistant mutants of the present invention are further selected based on viscosity. The viscosity of many purified phage-resistant mutants was measured by a pipette test. In this test, the time to flow out of a pipette of a certain capacity was measured. The longer the flow time, the higher the viscosity. 200 mL of skim milk was inoculated with 1% of the bacterial strain to be tested (from an overnight culture grown in 37°C skim milk) to make coagulated milk and incubated at 37°C for 20 hours. The viscosity of the coagulated milk was measured with a 25 mL capacity pipette, in which the flow time of the coagulated milk from the pipette was measured three times. The coagulated milk was carefully stirred with a spoon to homogenize it. Then a 25 mL capacity pipette was filled and the time to empty the pipette under gravity was measured. The time required to empty 25 mL of coagulated milk from the pipette was in seconds.

[0144] DSM 34235 provided a higher viscosity than its parent strain DSM 34236 as it increased the flow time by 15% as measured by the pipette test shown in the table below.

[0145] Table 3. Viscosity of DSM 34235, its parent strain DSM 34236 and prior art strains.

[0146]

[0147] In another experiment, DSM 34235 showed a 39% increase in efflux time as measured by the pipette test compared to DSM 22589, as evident in the table below.

[0148] Table 4. Viscosity of DSM 34235 and prior art strains.

[0149]

[0150] Preferred embodiments of the present invention are described herein, including the best mode of implementing the present invention known to the inventor. After reading the foregoing description, changes to those preferred embodiments may be apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will appropriately adopt these changes, and the inventor expects to implement the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents allowed by applicable law of the subject matter described in the appended claims. In addition, unless otherwise specified herein or clearly contradictory to the context, any combination of the above-mentioned elements with all possible changes thereof is included in the present invention.

[0151] Example 3 - Acidification curves of phage resistant mutants.

[0152] Acidification assay

[0153] In the acidification test, 200 mL skim milk was inoculated with 1% of the test strain (from an overnight culture grown in M17-2% lactose incubated at 37° C.) and incubated for 18 h at 37° C. The pH was followed over time using the CINAC system (Scientific Solutions).

[0154] The acidification curve of strain DSM 34235 was similar to that of its parent strain, indicating that the acidification activity of strain DSM 34235 was equivalent to that of its parent strain.

Claims

1. A lactic acid bacterium having enhanced phage resistance to DSM 34256 and / or DSM 34257 compared to its parent strain. 2 . The lactic acid bacterium according to claim 1 , wherein the lactic acid bacterium has increased viscosity compared to its parent strain.

3. The lactic acid bacterium according to any one of claims 1 to 2, wherein the lactic acid bacterium belongs to the species Streptococcus thermophilus.

4. The lactic acid bacterium according to any one of claims 1 to 3, wherein the parent strain is DSM 34236.

5. The lactic acid bacterium according to any one of claims 1 to 4, wherein the lactic acid bacterium is selected from DSM 34235 and mutants and variants thereof. A composition comprising the lactic acid bacteria according to any one of claims 1 to 5.

7. The composition according to claim 6, further comprising one or more strains belonging to the species of the genus Lactobacillus as a mixture or as a kit of parts.

8. The composition according to any one of claims 6-7, wherein at least one Lactobacillus strain is Lactobacillus delbrueckii subsp. bulgaricus).

9. The composition according to any one of claims 6 to 8, wherein the Lactobacillus bulgaricus strain is selected from: DSM 19251; DSM 19252; DSM 22586; DSM 26419; DSM 28910; DSM 32092; and DSM 33515.

10. A composition according to any one of claims 6 to 9, comprising as a mixture or as a kit of parts one or more selected from DSM 17876; DSM 18111; DSM 19242; DSM 21408; DSM 22585; DSM 22587; DSM 22588; DSM 22589;DSM 22935;DSM 24023;DSM 25012;DSM 26562;DSM 32503; DSM 32826 and DSM 33869 of Streptococcus thermophilus.

11. A composition according to any one of claims 6 to 10, comprising as a mixture or as a kit of parts one or more probiotic strains selected from the group consisting of Bifidobacterium, Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus and Limosilactobacillus fermentum.

12. A method for producing a fermented food, comprising fermenting a substrate with the lactic acid bacteria according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 11.

13. A fermented food product, comprising the lactic acid bacteria according to any one of claims 1 to 5 or obtainable by the method according to claim 12.

14. The fermented food product of claim 13, wherein the product is a dairy product.

15. A fermented food product according to any one of claims 13-14, wherein the product is yogurt, buttermilk, kefir, quark, sour cheese, crème fraiche, sour cream or cheese.

16. The fermented food product according to claim 15, wherein the yogurt is in the form of a stirred product, a set product or a drinkable product.

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