Phage-resistant lactic acid bacteria

Abstract

The present invention relates to a phage-resistant lactic acid bacterium, a composition containing the bacterium, and a fermented food obtained using the bacterium or the composition containing the bacterium.

Description

Technical Field

[0001] The present invention relates to a phage-resistant lactic acid bacterium, a composition containing the bacterium, and a fermented food produced using the bacterium.

Background Art

[0002] In the food industry, many bacteria, particularly lactic acid bacteria, are used in the production of fermented foods. The selection of lactic acid bacteria affects characteristics such as the taste, texture, and shelf life of fermented foods.

[0003] Production failures due to bacteriophage infection of bacteria are considered to be one of the major problems in the industrial use of bacterial cultures. Bacteriophages have been found in many bacterial strains used in the industry, such as species of Lactococcus sp., Lactobacillus sp., Leuconostoc sp., Pediococcus sp., or Streptococcus sp.

[0004] In the food industry, lactic acid bacteria starter cultures are widely used in food fermentation. One of the factors for frequent bacteriophage infection in lactic acid bacteria starter cultures is the fact that the fermentation conditions in the food industry, including the dairy industry, are generally non-sterile. Therefore, it is still impossible to eliminate bacteriophage contamination under these industrial conditions.

[0005] The lytic development of bacteriophages includes adsorption of the phage to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phages, and release of progeny from the host. Phage infection can be prevented by a cell-mediated mechanism that interferes with any of these events. The ability of a bacterial culture to resist bacteriophage infection during industrial use depends largely on the characteristics of the host strain that affect one or more of the above mechanisms.

Summary of the Invention

Problems to be Solved by the Invention

[0006] To meet the requirements of the industry, it has become necessary to propose novel phage-resistant strains of lactic acid bacteria, particularly Streptococcus thermophilus.

Means for Solving the Problems

[0007] In a first aspect, the present invention relates to lactic acid bacteria having increased phage resistance to DSM34256 and / or DSM34257 as compared to their parental strains.

[0008] In a second aspect, the present invention relates to a composition comprising the lactic acid bacteria of the present invention.

[0009] In a third aspect, the present invention relates to a method for producing a fermented food, which comprises fermenting a substrate using the lactic acid bacteria of the present invention or the composition of the present invention.

[0010] In a fourth aspect, the present invention relates to a fermented food comprising the lactic acid bacteria of the present invention or obtained by the method of the present invention.

Modes for Carrying Out the Invention

[0011] Before explaining the present invention in more detail, a series of terms and rules are defined.

[0012] The term "genus" means the genus defined at the website www.ncbi.nlm.nih.gov / taxonomy. The "strain" of bacteria used herein refers to bacteria that do not genetically change even when they grow or multiply. A multiplicity of the same bacteria is also included.

[0013] In this context, the terms "mutant" or "mutant strain" should be understood as strains derived from the strain (or parent strain) of the present invention, or strains that can be derived, for example, by genetic engineering, radiation and / or chemical treatment. Mutants are preferably functionally equivalent mutants, for example mutants having substantially the same or improved properties (e.g., texture, shear stress, viscosity, gel hardness, mouthfeel, flavor, post-acidification, acidification rate, and / or phage robustness) as the parent strain. 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 conventional mutagenesis treatment, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), treatment with ultraviolet light, or naturally occurring mutants. Mutants may have been subjected to multiple mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), but currently it is preferred that 20 or fewer, 10 or fewer, or 5 or fewer treatments (or screening / selection steps) are performed. In currently preferred mutants, less than 5%, or less than 1%, or less than 0.1% of the nucleotides in the bacterial genome have shifted to or been deleted from another nucleotide compared to the parent strain. As will be apparent to those skilled in the art, a mutant of the present invention can also be the parent strain.

[0014] In this context, the terms "variant" or "variant strain" should be understood as strains that are functionally equivalent to the strain of the present invention, for example strains having substantially the same or improved properties or characteristics (e.g., texture, acidification rate, viscosity, gel hardness, mouthfeel, flavor, post-acidification and / or phage robustness). Such variants, which can be identified using appropriate screening techniques, are part of the present invention.

[0015] Regarding strains of the genus Lactobacillus, the term "CFU" means colony forming units measured by growth (colony formation) on MRS agar medium cultured at 37°C for 3 days under anaerobic conditions. The composition of MRS agar is as follows (g / l): Bacto Proteose Peptone No. 3: 10.0 Bacto Beef Extract: 10.0 Bacto Yeast Extract: 5.0 Dextrose: 20.0 Sorbitan Monooleate Complex: 1.0 Ammonium Citrate: 2.0 Sodium Acetate: 5.0 Magnesium Sulfate: 0.1 Manganese Sulfate: 0.05 Dipotassium Phosphate: 2.0 Bacto Agar: 15.0 Milli-Q water: 1000 ml.

[0016] The pH is adjusted to 5.4 or 6.5: For L. rhamnosus, L. casei, and L. paracasei, the pH is adjusted to 6.5. For all other Lactobacillus species, the pH is adjusted to 5.4. In particular, for L. delbrueckii subsp. Bulgaricus, L. acidophilus, and L. helveticus, the pH is adjusted to 5.4. For L. rhamnosus, L. casei, and L. paracasei, the pH is adjusted to 6.5.

[0017] Regarding S. thermophilus, the term "CFU" means colony forming units measured by growth (colony formation) on M17 agar medium cultured at 37°C for 3 days under aerobic conditions. The composition of M17 agar is as follows (g / l): Tryptone: 2.5 g Pepsin digest of meat: 2.5 g Papain digest of soybean meal: 5.0 g Yeast extract: 2.5 g Meat extract: 5.0 g Lactose: 5.0 g Sodium glycerophosphate: 19.0 g Magnesium sulfate, 7H 2 0: 0.25 g Ascorbic acid: 0.5 g Agar: 15.0 g Milli-Q water: 1000 ml. The pH is adjusted to a final pH of 7.1 ± 0.2 (25 °C).

[0018] Phage-resistant lactic acid bacteria Phage-resistant mutants can be generated by exposing a phage-sensitive parental strain to a desired phage, for example, by growing the phage-sensitive parental strain in the presence of a desired phage at a concentration sufficient to induce a selection pressure on said strain. Methods for generating 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 one aspect, the present invention relates to lactic acid bacteria having increased phage resistance to DSM34256 and / or DSM34257 compared to their parental strain.

[0019] Furthermore, it may be desirable to enhance the texture provided by such strains. In that case, selection of phage-resistant mutants with enhanced texture-enhancing properties should be applied. The phage-resistant mutants of the present invention with enhanced texture-enhancing properties such as viscosity can be obtained by the methods described in the Examples. According to one embodiment, the present invention relates to the lactic acid bacteria of the present invention, the strain of which has a higher viscosity compared to its parental strain.

[0020] It will be understood that phage resistance can be obtained by this method using strains from many species. However, in the present invention, the preferred species is Streptococcus thermophilus. According to one embodiment, the present invention relates to the lactic acid bacteria of the present invention, which belong to the Streptococcus thermophilus species.

[0021] Any Streptococcus thermophilus strain can be used as the parent strain. The parent strain of the present invention is preferably the Streptococcus thermophilus strain DSM34236 or a variant or mutant thereof. According to one embodiment, the present invention relates to the lactic acid bacteria of the present invention, wherein the parent strain is DSM34236.

[0022] Preferred mutants obtained from the parent strain can be any mutant with improved phage resistance and optionally increased viscosity compared to the parent strain. According to one embodiment, the present invention relates to the lactic acid bacteria of the present invention, which are selected from the group consisting of DSM34235, and variants and mutants thereof.

[0023] As used herein, the term "lactic acid bacterium" is abbreviated as "LAB" and refers to Gram-positive, microaerophilic or anaerobic bacteria that ferment sugars while producing acids such as lactic acid, acetic acid, and propionic acid, which are the mainly produced acids. The most industrially useful lactic acid bacteria belong to the "Lactobacillales order", which includes the genera Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp., and Propionibacterium spp. Furthermore, lactic acid-producing bacteria belonging to the group of Bifidobacterium, which are strict anaerobic bacteria, i.e., Bifidobacterium spp. bacteria, are generally included in the group of lactic acid bacteria. These are frequently used as food cultures either alone or in combination with other lactic acid bacteria. Lactic acid bacteria (including bacteria of the genera Lactobacillus and Streptococcus thermophilus) are usually supplied to the dairy industry as frozen cultures or freeze-dried cultures for bulk starter propagation, or as so-called "direct vat set" (DVS) cultures intended for direct inoculation into fermentation vessels or vats for the production of dairy products such as fermented dairy products. Such cultures are generally referred to as "starter cultures" or "starters".

[0024] The term "improvement in bacteriophage resistance" or "improvement in phage resistance" means that the phage resistance to at least one bacteriophage, as represented by the difference in pfu / mL (plaque-forming units per mL) obtained with the at least one bacteriophage in a given strain compared to the pfu / mL obtained with the same bacteriophage in the parental strain in a plaque assay such as the "measurement of phage resistance by the agar overlay method" described in the examples, is improved. A strain with improved resistance to bacteriophage preferably has a decrease 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.

[0025] Composition The lactic acid bacteria of the present invention described herein are useful for preparing compositions, particularly starter cultures for fermented foods or feed products. In one aspect, the present invention relates to a composition comprising the lactic acid bacteria of the present invention.

[0026] In the production of lactic acid bacteria fermentation processes, it is common to apply mixed cultures of lactic acid bacteria. According to some embodiments, the composition will therefore contain a number of strains belonging to the same species or to different species.

[0027] According to one embodiment, the composition of the present invention further comprises one or more strains belonging to the genus Lactobacillus as a mixture or as a kit of parts.

[0028] According to one embodiment, one or more strains belonging to the genus Lactobacillus can be selected from the group consisting of L. delbrueckii, L. delbrueckii subsp bulgaricus, and L. delbrueckii subsp. lactis.

[0029] According to one embodiment, the Lactobacillus strain of the present invention is L. bulgaricus. L. bulgaricus is a lactic acid bacterium frequently used in commercial milk fermentation, and this microorganism is usually used as part of a mixed starter culture. According to one embodiment, in the composition of the present invention, at least one Lactobacillus strain is Lactobacillus delbrueckii subsp. bulgaricus.

[0030] According to one embodiment of the composition of the present invention, the Lactobacillus bulgaricus strain is selected from the group consisting of DSM19251, DSM19252, DSM22586, DSM 26419, DSM 28910, DSM 32092, DSM 33515, and variants and mutants thereof.

[0031] According to one embodiment, the composition and / or mixture or kit of parts comprises a S. thermophilus strain DSM34235 or a variant or mutant thereof, and a strain belonging to the Lactobacillus species. Thus, according to a preferred embodiment, the composition and / or mixture or kit of parts comprises the S. thermophilus DSM34235 strain or a mutant or variant thereof, and a strain belonging to Lactobacillus delbrueckii subsp. bulgaricus. Thus, according to a preferred embodiment, the composition and / or mixture or kit of parts is a S. thermophilus strain DSM34235 or a variant or mutant thereof, and a strain belonging to the Lactobacillus bulgaricus strain species, which is selected from the group consisting of DSM19251, DSM19252, DSM22586, DSM 26419, DSM 28910, DSM 32092, DSM 33515, and variants and mutants thereof.

[0032] In addition to the lactic acid bacteria of the present invention, the composition may further comprise additional strains of Streptococcus thermophilus. According to one embodiment, the composition further comprises one or more Streptococcus thermophilus strains as a mixture or kit of parts. According to one embodiment, the composition of the present invention, as a mixture or kit of parts, comprises one or more Streptococcus thermophilus strains selected from the group consisting of DSM17876, DSM18111, DSM19242, DSM21408, DSM22585, DSM22587, DSM22588, DSM22589, DSM22935, DSM24023, DSM25012, DSM26562, DSM32503, DSM32826, and DSM33869, and variants and mutants thereof.

[0033] According to one embodiment, the composition and / or mixture or kit of parts comprises a Streptococcus thermophilus strain DSM34235 or a variant or mutant thereof, and a strain belonging to the genus Streptococcus. Thus, according to a preferred embodiment, the composition and / or mixture or kit of parts comprises a Streptococcus thermophilus strain DSM34235 or a variant or mutant thereof, and a strain belonging to the species Streptococcus thermophilus. Thus, according to a preferred embodiment, the composition and / or mixture or kit of parts comprises a Streptococcus thermophilus strain DSM34235 or a variant or mutant thereof, and a strain belonging to the species Streptococcus thermophilus, which is a strain selected from the group consisting of DSM17876, DSM18111, DSM19242, DSM21408, DSM22585, DSM22587, DSM22588, DSM22589, DSM22935, DSM24023, DSM25012, DSM26562, DSM32503, DSM32826, and DSM33869, and variants and mutants thereof.

[0034] According to one embodiment, the composition of the present invention further comprises, as a mixture or kit-of-parts, one or more strains belonging to the genus Lactococcus. According to one embodiment, the one or more strains belonging to the genus Lactococcus can be selected from the group consisting of L. Lactis, L. lactis subsp lactis, and L. lactis subsp. cremoris. According to one embodiment, the composition of the present invention further comprises, as a mixture or kit-of-parts, one or more Lactococcus strains selected from the group consisting of DSM21404, DSM33192, DSM21405, and their variants and mutants.

[0035] According to one embodiment, the composition of the present invention further comprises, as a mixture or kit-of-parts, one or more strains belonging to the genus Pediococcus.

[0036] According to one embodiment, the one or more strains belonging to the genus Pediococcus may be Pediococcus acidilactis strains. According to one embodiment, the composition of the present invention further comprises, as a mixture or kit-of-parts, one or more Pediococcus acidilactis strains selected from the group consisting of DSM 28307, and its variants and mutants.

[0037] The composition of the present invention may contain probiotic bacteria. The probiotic bacterial strain may be added before or after fermentation. When added before fermentation, the probiotic bacterial strain also acts as a fermenting bacterium. The term "probiotic bacteria" refers to live bacteria that are administered to a consumer in an appropriate amount for the purpose of achieving a health-promoting effect on the consumer. Probiotic bacteria can survive under gastrointestinal conditions after ingestion and can colonize the consumer's intestine.

[0038] It will be understood that the classification of the genus Lactobacillus was updated in 2020. The new classification is disclosed in Zheng et al. 2020 and is incorporated herein by reference unless otherwise specified. For the purposes of the present invention, the following table shows a list of the old and new names of some Lactobacillus species relevant to the present invention.

[0039] [Table 1]

[0040] According to one embodiment of the present invention, the probiotic strain is selected from the group consisting of the following bacteria: genus Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Limosilactobacillus fermentum, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii; genus Bifidobacterium, such as 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, etc.

[0041] According to one embodiment of the present invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, and Lactobacillus johnsonii. According to one embodiment of the present invention, the probiotic strain is Lactobacillus acidophilus (LA-5 (registered trademark)) deposited as DSM13241.

[0042] According to one embodiment of the present invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactobacillus rhamnosus strain, Lactobacillus paracasei strain, and Limosilactobacillus fermentum strain. According to one embodiment of the present invention, the probiotic strain is LGG® deposited as Lactobacillus rhamnosus strain DSM33870 or ATCC 53103. According to one embodiment of the present invention, the probiotic strain is CRL 431 deposited as Lactobacillus paracasei strain DSM19465 or ATCC 55544. According to one embodiment of the present invention, the probiotic strain is Limosilactobacillus fermentum strain DSM22584.

[0043] According to one embodiment of the present invention, the probiotic Bifidobacterium strain is selected from the group consisting of the following bacteria: 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, etc. According to one embodiment of the present invention, the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. lactis. According to one embodiment of the present invention, the Bifidobacterium animalis subsp. lactis strain is BB-12® deposited as DSM33868 or DSM 15954.

[0044] The above-described mixture or kit-of-parts can also be further combined with other lactic acid bacteria such as probiotic bacteria, but is not limited thereto. According to one embodiment, the one or more lactic acid bacteria are selected from the group consisting of Bifidobacterium animalis subsp. lactis (e.g., BB-12®), Lactobacillus acidophilus (e.g., LA-5®), Lactobacillus rhamnosus (e.g., LGG®), and any combination thereof. Which Bifidobacterium, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus to apply depends on their uses and the foods to be produced.

[0045] The expression "kit-of-parts" containing strains means that the strains or cultures of the strains are physically separated but are intended to be used together. Thus, the strains or cultures of S. thermophilus strains and Lactobacillus strains are stored in different boxes or sachets. According to one embodiment, the S. thermophilus strains and Lactobacillus (e.g., Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, and / or Limosilactobacillus reuteri strains) are in the same form, i.e., the frozen form (in the form of pellets or frozen pellets), the powder form (such as dry powder or lyophilized powder).

[0046] According to one embodiment, the present invention relates to a composition of the present invention containing one or more probiotic strains selected from Bifidobacterium, Lactobacillus acidophilus, Lactobacillus casei (former Lactobacillus casei), Lactobacillus paracasei (former Lactobacillus paracasei), Lactobacillus rhamnosus (former Lactobacillus rhamnosus), and Limosilactobacillus fermentum (former Lactobacillus fermentum) as a mixture or kit-of-parts.

[0047] According to one embodiment, the composition of the present invention contains bacteria in a concentrated form with a total viable cell concentration in the range of 10 4 to 10 12 cfu (colony forming units) per gram of the composition, and at least 10 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.

[0048] According to one embodiment, the composition of the present invention contains bacteria in a concentrated form with the concentration of each strain of viable cells in the range of 10 4 to 10 12 cfu (colony forming units) per gram of the composition, and at least 10 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.

[0049] According to one embodiment of the present invention, the composition contains S. thermophilus strains of 10 4 to 10 12 CFU (colony forming units) / g, 10 5 to 10 11 CFU / g, 10 6 to 10 10 CFU / g, or 10 7 to 10 9 CFU / g.

[0050] According to one embodiment, the composition further comprises from 10 4 to 10 12 CFU / g of Lactobacillus strains, 10 5 to 10 11 CFU / g, 10 6 to 10 10 CFU / g, or 10 7 to 10 9 CFU / g of Lactobacillus strains.

[0051] According to one embodiment, the composition comprises from 10 4 to 10 12 CFU / g, 10 5 to 10 11 CFU / g, 10 6 to 10 10 CFU / g, or 10 7 to 10 9 CFU / g of each of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, and / or Limosilactobacillus reuteri strains.

[0052] S. thermophilus, Lactobacillus, and Lactobacillus genera, such as L. bulgaricus, L. acidophilus, L. casei, L. paracasei, and / or L. rhamnosus, and other lactic acid bacteria are commonly used as starter cultures for technical purposes such as fermented dairy products in the manufacture of various foods such as the dairy industry. Thus, according to another preferred embodiment, the composition is a starter culture. The composition may be a starter culture such as a yogurt starter culture.

[0053] The composition and / or starter culture can be frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried, or dried in liquid form. Usually, the storage stability of the composition and / or starter culture can be extended by formulating the product with a low water activity. By controlling the water activity (Aw), the effect of water movement on the product can be predicted and controlled. Therefore, the water activity (Aw) of the dry composition herein is preferably in the range of 0.01 to 0.8, more preferably in the range of 0.05 to 0.4.

[0054] The composition may further comprise additional components such as conventional additives including, for example, cryoprotectants and / or yeast extracts, nutrients such as sugars and vitamins.

[0055] The composition of the present invention can be provided in several forms. It may be a powder, pellet, or tablet. It may be in frozen form, dried form, freeze-dried form, or liquid form. Thus, according to one embodiment, the composition is in frozen, dried, freeze-dried, or liquid form. According to one embodiment, the composition of the present invention is in frozen, freeze-dried, or liquid form.

[0056] The composition of the present invention may further contain a cryoprotectant, a lyoprotectant, an antioxidant, a nutrient, a filler, a flavoring agent, or a mixture thereof. The composition preferably contains one or more of a cryoprotectant, a lyoprotectant, an antioxidant and / or a nutrient, more preferably contains a cryoprotectant, a lyoprotectant and / or an antioxidant, and most preferably contains a cryoprotectant or a lyoprotectant, or both. The use of protectants such as cryoprotectants and lyoprotectants is known to those skilled in the art. Suitable cryoprotectants or lyoprotectants include monosaccharides, disaccharides, trisaccharides and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia), etc.), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (such as proline, glutamic acid, etc.), complex substances (such as skim milk, peptone, gelatin, yeast extract, etc.), and inorganic compounds (such as sodium tripolyphosphate, etc.).

[0057] According to one embodiment, the composition according to the present invention can contain one or more cryoprotectants selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine, and derivatives of these compounds. Suitable antioxidants include ascorbic acid, citric acid and its salts, gallic acid, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B group, vitamin C, etc.). The composition may optionally contain additional substances such as fillers (such as lactose, maltodextrin, etc.) and / or flavorings.

[0058] According to one embodiment of the present invention, the cryoprotectant is a drug or a mixture of drugs having a booster effect in addition to cryoprotective ability. The expression "booster effect" is used to describe a situation where the cryoprotectant gives an increase in metabolic activity (booster effect) to the thawed or reconstituted culture when inoculated into the medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or freeze-dried cultures may have lost a significant portion of their metabolic activity, but their viability may be retained. For example, the culture may lose its acid production (acidification) activity even when stored for a shorter period. Therefore, viability and booster effect need to be evaluated by different assays. Viability is evaluated by viability assays such as measurement of colony-forming units, while the booster effect is evaluated by quantifying the relevant metabolic activity of the thawed or reconstituted culture compared to the viability of the culture. The term "metabolic activity" refers to the oxygen removal activity of the culture, its acid production activity, i.e., the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite production activity, such as the production of aromatic compounds such as acetaldehyde, (α-acetolactic acid, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0059] According to one embodiment, the composition of the present invention contains 0.2% to 20% of a cryoprotectant or a mixture of drugs measured as % by weight of the material. However, it is preferred to add the cryoprotectant or the mixture of drugs in amounts in the ranges of 0.2% to 15%, 0.2% to 10%, 0.5% to 7%, and 1% to 6% by weight, including the range of 2% to 5% of the cryoprotectant or the mixture of drugs measured as % by weight of the frozen material. According to a preferred embodiment, the culture contains about 3% of the cryoprotectant or the mixture of drugs measured as % w / w with respect to the weight. An amount of about 3% of the cryoprotectant corresponds to a concentration in the range of 100 mM. It should be recognized that according to each aspect of the embodiments of the present invention, the ranges may also be increments of the recited ranges.

[0060] In this context, the term "from x% to y%" means including the endpoints and is equivalent to the term "from x% (including x%) to y% (including y%)".

[0061] According to a further aspect, the composition of the invention may or may include an ammonium salt (such as an ammonium salt of an organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (such as a growth booster or an acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, such as (substantially) urease-negative bacterial cells. The terms "ammonium salt", "ammonium formate", etc. should be understood as a source of a salt or a combination of ions. For example, the term "source" of "ammonium formate" or "ammonium salt" refers to a compound or a mixture of compounds that provides ammonium formate or ammonium salt when added to a cell culture. According to some embodiments, the source of ammonium releases ammonium into the growth medium, and according to other embodiments, the ammonium source is metabolized to produce ammonium. According to some preferred embodiments, the ammonium source is exogenous. According to some particularly preferred embodiments, ammonium is not provided by a dairy product matrix. Of course, it should be understood that ammonia may be added instead of the ammonium salt. Thus, the term ammonium salt includes ammonia (NH 3 )、NH 4 OH、NH 4 + etc.

[0062] According to one embodiment, the composition of the invention may include a thickening agent and / or a stabilizer such as pectin (such as HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymer, starch, modified starch, carrageenan, alginate, and guar gum.

[0063] According to one embodiment, where the microorganism produces a polysaccharide (such as EPS) and imparts a high viscosity / gummy texture to the acidified dairy product, the acidified dairy product is produced substantially free or completely free of the addition of thickeners and / or stabilizers such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymer, starch, modified starch, carrageenan, alginate, and guar gum. Substantially free means that the product should be understood to contain 0% to 20% (w / w) (e.g., 0% to 10%, 0% to 5%, or 0% to 2%, or 0% to 1%) of the thickener and / or stabilizer.

[0064] As described above, the interaction between microbial species is equally important not only in terms of growth but also in the development of rheological properties (such as texture) and flavor.

[0065] Method for producing a fermented product One aspect of the present invention relates to a method for producing a fermented food, which includes adding the starter culture composition described herein to a food raw material and maintaining the inoculated raw material under conditions where lactic acid bacteria are metabolically active.

[0066] Useful starting food materials, i.e., substrates, include milk, vegetable materials, meat products, fruit juices, fruit juices, doughs, batters, and all materials conventionally subjected to lactic acid bacteria fermentation.

[0067] Fermentation products obtained by this method include, as representative examples, cheeses including fresh cheese products, and dairy products such as buttermilk.

[0068] Yet another important use of the lactic acid bacteria according to the present invention is to use the bacterial culture as a so-called probiotic. As used herein, the term "probiotic" is understood to mean a microbial culture that, when ingested by a human or animal in the form of live cells, improves the health state, for example, by suppressing harmful microorganisms in the gastrointestinal tract, by strengthening the immune system, or by contributing to the digestion of nutrients.

[0069] The term "milk" is understood as a milk secretion obtained by milking mammals such as cows, sheep, goats, water buffalo, camels, etc. According to a preferred embodiment, the milk is cow's milk. The term "milk" also includes protein / fat solutions made from plant materials. Suitable plant materials are legumes, nuts, or grains. Preferred legumes include soybeans, peas, beans, lupinus, and lentils. Preferred nuts include coconuts, almonds, cashew nuts, and walnuts. Preferred grains include oats, wheat, rye, and millet. Milk can be obtained from mammals, plant materials, or any combination thereof.

[0070] The term "milk substrate" can be either raw milk and / or processed milk materials that can be fermented according to the method of the present invention. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of milk or milk-like products containing proteins such as whole milk or low-fat milk, skim milk, buttermilk, reconstituted powdered milk, condensed milk, dried milk, whey, whey permeate, lactose, mother liquor by crystallization of lactose, whey protein concentrate, or cream. Clearly, the milk substrate can be derived from any mammal, such as substantially pure mammalian milk or reconstituted milk powder, or the milk substrate can be wholly or partly derived from a plant material, i.e., plant milk, as described above. Preferably, at least a part of the protein in the milk substrate is (i) a protein naturally present in mammalian milk such as casein or whey protein, or (ii) a protein naturally present in plant milk. However, a part of the protein may be a protein not naturally present in milk.

[0071] Before fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

[0072] As used herein, "Homogenizing" means vigorously mixing to obtain a soluble suspension or emulsion. When homogenization is performed before fermentation, it may be done to break down the milk fat into small sizes so that the milk fat does not separate from the milk. This can be achieved by forcing the milk through small openings at high pressure.

[0073] As used herein, "Pasteurizing" means treating the milk substrate to reduce or remove the presence of living organisms such as microorganisms. Pasteurization is preferably achieved by maintaining a specified temperature for a specified time. The specified temperature is usually achieved by heating. The temperature and duration can be selected to kill or inactivate certain bacteria such as harmful bacteria. Subsequently, a rapid cooling step may follow.

[0074] "Fermentation" in the method of the present invention means converting carbohydrates into alcohol or acid by the action of microorganisms. Preferably, fermentation in the method of the present invention includes converting lactose into lactic acid.

[0075] The fermentation process used in the production of fermented dairy products is well-known, and those skilled in the art will know how to select appropriate process conditions such as temperature, oxygen, the amount and characteristics of microorganisms, and process time. Obviously, the fermentation conditions are selected to assist in the achievement of the present invention, that is, to obtain a fermented product (fermented dairy product) such as a dairy product or a dairy product-like product in solid or liquid form.

[0076] In this context, the term "starter culture" is a culture that is a preparation (composition) of one or more bacterial strains (such as lactic acid bacterial strains) to assist in initiating the fermentation process in the production of fermented products such as various foods, feeds, beverages, etc.

[0077] In this context, a "yoghurt starter culture" is a bacterial culture comprising one or more Lactobacillus selected from Lactobacillus bulgaricus strains and / or Lactobacillus acidophilus strains, and one or more Streptococcus thermophilus strains. Accordingly, "yoghurt" refers to a fermented dairy product obtained by inoculating and fermenting a composition comprising Lactobacillus strains such as Lactobacillus bulgaricus strains and / or Lactobacillus acidophilus strains, and Streptococcus thermophilus strains, in a milk substrate.

[0078] The substrate is preferably a milk substrate. The milk substrate may be of animal, preferably mammalian, or plant origin. However, the milk substrate need not be purely of animal origin and may further contain a plant-derived substrate. The fermented product may be a food product and may also be a dairy product.

[0079] Depending on the product to be produced, the substrate may be a milk substrate. When fermented dairy products such as yoghurt, buttermilk, kefir, etc. are the final products, a milk substrate is particularly preferred.

[0080] The milk substrate may be a product of animal or plant origin. Thus, according to one embodiment, the fermented product is a food product such as a dairy product. The dairy product can be selected from the group consisting of, but not limited to, fermented dairy products such as yoghurt, buttermilk, kefir, etc., or cheeses such as fresh cheese and pasta filata.

[0081] Similarly, enzymes can be added to the substrate, e.g., the milk substrate, before, during, and / or after fermentation, and the enzymes are selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, lactase, chymosin, rennet, and mixtures thereof.

[0082] According to one aspect, the present invention relates to a method for producing a fermented food product, comprising fermenting a substrate using the lactic acid bacteria of the present invention or the composition of the present invention.

[0083] Fermented product As used herein, the term "fermented milk product" refers to a food or feed product that includes the fermentation of a milk substrate by lactic acid bacteria. The "fermented milk products" used herein include, but are not limited to, products such as yogurt and cheese. Other examples of fermented products include buttermilk, kefir, quark, tvorog, clotted cream, sour cream, and the like.

[0084] According to one embodiment, the fermented food of the present invention is yogurt, buttermilk, kefir, quark, tvorog, clotted cream, sour cream, or cheese. Preferably, the fermented milk product is yogurt.

[0085] Examples of cheeses produced by fermentation using Thermophilus bacteria and Lactobacillus delbrueckii subsp. bulgaricus include mozzarella cheese and pizza cheese (Hoier et al. (2010) in The Technology of Cheesemaking, 2nd Ed. Blackwell Publishing, Oxford; 166 - 192).

[0086] The term "stirred type product" refers to a fermented milk product that undergoes mechanical processing after fermentation, and the structure of the curd formed during the fermentation stage is broken and liquefied. The mechanical processing is usually carried out by stirring, pumping, filtering, homogenizing the gel, or mixing with other components, but is not limited thereto. Stirred type products usually have a fat - free milk solids content of 9 - 15%, but are not limited thereto.

[0087] The "set - type product" includes products based on milk that are inoculated with a starter culture, for example, a starter culture, packaged adjacent to the inoculation step, and then fermented within the package.

[0088] According to one embodiment, the fermented product may be in the form of a stirred product, a solidified product, or a drinkable product.

[0089] The fermented product and / or the food itself contain acids and flavors generated during fermentation. However, it may be desirable for the fermented product and / or the dairy product to contain components selected from the group consisting of fruit concentrates, syrups, probiotic strains or cultures, colorants, thickeners, flavoring agents, preservatives, and mixtures thereof. According to one embodiment of the present invention, the fermented food contains components selected from the group consisting of fruit concentrates, syrups, probiotic strains or cultures, colorants, thickeners, flavoring agents, preservatives, and mixtures thereof.

[0090] It will be understood that the aspects and embodiments disclosed in the sections entitled "phage-resistant lactic acid bacteria", "composition", "method for producing a fermented product", and "fermented product" are equally relevant to other parts of this specification. In particular, the disclosure regarding specific strains of the present invention is applicable to all parts of this specification that are obvious to those skilled in the art.

[0091] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (especially in the context of the following claims) are to be construed to include both the singular and the plural forms unless specifically stated otherwise herein or the context clearly dictates otherwise. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless specifically stated otherwise. The recitation of ranges of values herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range unless otherwise specifically stated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of all examples or exemplary language (e.g., "such as") provided herein is merely for the purpose of better illustrating the invention and is not intended to limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0092] Just because a document is listed or described in this specification as having been previously publicly available does not necessarily mean that the document is part of the prior art or common general knowledge.

[0093] Priority, options, and embodiments regarding specific aspects, features, or parameters of the present invention are to be considered as disclosed in combination with priority, options, and embodiments regarding all other aspects, features, and parameters of the present invention unless otherwise stated in the context. This applies particularly to the description of encapsulated microbial cultures and all their features, which can form part of the final composition or product obtained by the methods described herein. Embodiments and features of the present invention are also outlined in the following items.

[0094] Deposit and Solutions by Experts The applicant requests that, until the date of patent grant, samples of the deposited microorganisms listed in the following table be provided only to experts.

[0095]

Table 2

[0096] References All references cited in this patent document are hereby incorporated by reference in their entirety into this specification. Hoier et al. (2010) The Technology of Cheesemaking, 2 nd Ed. Blackwell Publishing, Oxford, p.166 - 192. Zheng et al. (2020.04.15) Int J Syst Evol Microbiol. Vol. 70(4). P.2782 = 2858.

Examples

[0097] Example 1 - Preparation of Phage - Resistant Mutants Isolation of phage-resistant strains The phage - resistant mutants were generated from the parental strain DSM34236 after inoculating 0.1 mL of an overnight culture of DSM34236 in M17 - 2% lactose agar containing 10 mM MgCl 2 / CaCl 2 with 0.1 mL of phage DSM34256 containing 1 x 10 9 phage particles per mL and incubating overnight at 37°C. For this, the phage and the host strain were mixed with 2.5 mL of an upper agar solution (molten agar at half the standard agar concentration (0.75%) maintained at 46 - 50°C) and poured onto the lower M17 - 2% lactose agar. Both the lower agar and the upper agar contained 10 mM MgCl 2 / CaCl 2It is composed of. When the upper agar solidified, incubation was carried out under the above conditions.

[0098] Among the numerous phage-resistant mutants that appeared on the agar plate after incubation, the DSM34235 strain was colony purified three times and retested by plaque assay on M17 lactose agar plates at 37°C using phage DSM34256 for phage challenge.

[0099] Phage resistance to DSM34256 was confirmed by the fact that no single plaque was observed in the plaque test. Phage resistance to phage DSM34257 was also observed.

[0100] Example 2 - Viscosity of Phage-Resistant Mutants Pipette test The phage-resistant mutants of the present invention were further selected based on viscosity. Viscosity was measured by pipette inspection for a number of purified phage-resistant mutants. In this inspection, the outflow time from a volumetric pipette is measured. The longer the outflow time, the higher the viscosity. Coagulated milk was made from 200 mL of skim milk inoculated with 1% of the test bacterial strain (cultured overnight at 37°C in skim milk) and cultured at 37°C for 20 hours. The viscosity of the coagulated milk was measured with a 25 mL volumetric pipette, and the time for the coagulated milk to flow out of the pipette was measured three times. The coagulated milk was carefully stirred with a spoon to homogenize it. Next, it was filled into a 25 mL volumetric pipette, and the time until the pipette emptied by gravity was measured. The time for 25 mL of coagulated milk to flow out of the pipette was recorded in seconds.

[0101] As shown in the following table, DSM34235 has a 15% increase in the discharge time measured by pipette inspection and a higher viscosity than the parental strain DSM34236.

[0102]

Table 3

[0103] In another experiment, DSM34235 had a 39% increase in the efflux time measured by pipette test compared to DSM22589, as shown in the table below.

[0104]

Table 4

[0105] This specification describes preferred embodiments of the present invention, including the best mode known to the inventors for carrying out the present invention. Variations of these preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced in a manner different from that specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise specified herein or clearly contradicted by context, any combination of any possible variations of the above elements is included in the present invention.

[0106] Example 3 - Acidification Profile of Phage-Resistant Mutants Acidification assay In the acidification test, 200 mL of skim milk was inoculated with 1% of the test bacterial strain (cultured overnight in M17 - 2% lactose at 37 °C) and cultured at 37 °C for 18 hours. The pH was monitored over time using a CINAC system (Scientific Solutions).

[0107] The acidification profile of the DSM34235 strain is similar to that of its parent strain, indicating that the acidification activity of the DSM34235 strain is equivalent to that of its parent strain.

Claims

1. A lactic acid bacterium having enhanced phage resistance against DSM 34256 and / or DSM 34257 compared to the parent strain.

2. The lactic acid bacterium according to claim 1, wherein the bacterium has an enhanced viscosity compared to its parent strain.

3. The lactic acid bacterium according to claim 1 or 2, wherein the 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 bacterium is selected from the group consisting of DSM 34235, and its mutants and variants.

6. A composition comprising the lactic acid bacterium according to any one of claims 1 to 5.

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

8. The composition according to claim 6 or 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 the group consisting of DSM 19251, DSM 19252, DSM 22586, DSM 26419, DSM 28910, DSM 32092, and DSM 33515.

10. The composition according to any one of claims 6 to 9, comprising, as a mixture or kit-of-parts, one or more Streptococcus thermophilus strains 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.

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

12. A method for producing a fermented food, comprising fermenting a substrate using the lactic acid bacterium 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 comprising the lactic acid bacterium according to any one of claims 1 to 5, or obtained by the method according to claim 12.

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

15. The fermented food according to claim 13 or 14, wherein the food is yogurt, buttermilk, kefir, quark, tvorog, quark, sour cream, or cheese.

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

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