Method for producing a fermented milk product using a streptococcus thermophilus strain having no or only limited nisin-inactivating activity of salt

Abstract

This invention relates to a novel method for producing fermented dairy products, preferably cheese. Furthermore, this invention relates to a novel bacterial culture, starter culture, and kit for this method.

Description

Technical Field

[0001] This invention relates to a novel method for producing fermented dairy products, preferably cheese. Furthermore, this invention relates to a novel bacterial culture, starter culture, and kit of parts for this method. Background Technology

[0002] Cheese is a fermented dairy product. For example, cheese can be made through the acidification of milk and the coagulation of casein (the so-called curdling process). The curd and whey formed during cheese making can be easily separated. The curd can then be pressed into cheese. After pressing, a maturation process can be carried out to bring the cheese to the desired degree of maturity.

[0003] There are many types of cheese. So-called "hard" cheeses are popular for their taste, texture, nutritional value, and long shelf life. Examples of hard cheeses include Cheddar, Emmental, Grana Padano, Gruyere, Minolette, Parmigiano, Parmesan, and Pecorino.

[0004] To produce the highest quality cheese, cheese makers employ various biochemical mechanisms. Many cheese producers use starter cultures containing lactic acid bacteria (LAB) to acidify the milk and create the desired texture. *Streptococcus thermophilus* is a commonly used LAB in many starter cultures. Other bacterial cultures, called helper cultures, are used for cheese maturation and / or flavor development. Cheeses such as Cheddar, Gouda, Gruyère, Parmesan, Provolone, and Swiss also require salting to develop the desired flavor. Milk coagulation is typically accomplished using a coagulant. Coagulants include animal-derived coagulants (such as rennet or chymosin) as well as microbially produced coagulants or chymosin produced during fermentation.

[0005] In addition to the lactic acid bacteria required in the starter culture, some unwanted bacteria may also be present during cheese making. These unwanted bacteria, including so-called non-starter lactic acid bacteria (NSLABs), can cause problems for cheese producers because their presence can lead to defects such as slits and cracks in the cheese.

[0006] A major challenge in cheese production is the risk of spoilage from Gram-positive bacteria such as Clostridium, Staphylococcus, Bacillus, and Listeria. Clostridium, in particular, can cause a notorious defect known as "delayed foaming" or "butyric acid bloating." Clostridium spores, such as Clostridium butyricum, can withstand heat treatment of milk, and their subsequent growth leads to butyric acid fermentation, producing gases (H2, CO2) and unpleasant odors in the cheese matrix. This gas production can cause excessively large cavities within the cheese, potentially leading to bloating and even bursting.

[0007] Nisin is known to effectively inhibit the growth of Gram-positive bacteria, especially during the production of processed and spread cheeses. Therefore, nisin has been successfully used in food production to prevent food spoilage caused by Gram-positive bacteria such as Clostridium, Staphylococcus, Bacillus, and Listeria. It is worth noting that if nisin is produced in situ from the starter culture, the final dairy product does not need to be labeled with its nisin content.

[0008] EP1273237 describes a method for producing a fermented product, wherein a starter culture is used in the fermentation step, the starter culture comprising a lactococcus lactis strain producing lactic acid cocci and one or more lactococcus lactis-resistant non-lactococcus strains. For example, using a starter culture from DAIRYSAFE TM Cheddar cheese is produced using a nisin-producing starter culture consisting of TC17 (a diacetyl variant of Lactococcus lactis), 13M (an nisin-immune Lactococcus lactis), and 30% nisin-resistant Streptococcus thermophilus. The nisin content in cheese prepared using this starter culture is approximately 200 IU per gram of cheese.

[0009] However, thermophilic streptococcal strains, especially those resistant to lactococci, inactivate lactococci during fermentation and acidification, resulting in low lactococcal concentrations in the early stages of cheese aging. Adding salt during fermentation and aging enhances and accelerates lactococcal inactivation, particularly by thermophilic strains, for example, in the production of salted cheeses. Low lactococcal concentrations, combined with the presence of NSLABs during aging, ultimately lead to the aforementioned cheese defects, such as cracks and fissures.

[0010] Meanwhile, acidification is always necessary during the production of fermented dairy products such as cheese. A high acidification rate can be achieved by adding a higher concentration of Streptococcus thermophilus cells to accelerate the acidification process. Therefore, adding a high concentration of Streptococcus thermophilus cells is desirable. However, excessively high concentrations of Streptococcus thermophilus during fermentation can lead to low nisin levels at the end of fermentation and before the start of cheese maturation, thus adversely affecting the flavor, appearance, and especially the shelf life of the cheese.

[0011] Garde et al. (International Journal of Food Microbiology, Vol. 96 (2004), pages 165-172) reported that during cheese maturation, nisin produced by *Lactococcus lactis* subsp. *lactococcus* lyses the nisin-sensitive *Streptococcus thermophilus* strain INIA 463. However, *Streptococcus thermophilus* strain INIA 463 developed nisin resistance less than 2 hours after exposure to sub-minimum inhibitory concentrations (1–3 IU / ml) of nisin in skim milk. Only intracellular nonspecific nisin degradation activity was detected, which was also present in unexposed cultures. It is speculated that nisin resistance in *Streptococcus thermophilus* may be related to changes in the cell wall. Summary of the Invention

[0012] Therefore, providing an efficient and sufficiently rapid method for producing savory fermented dairy products (especially savory cheeses) while maintaining a sufficiently high nisin concentration at the end of fermentation and / or before maturation would be an advancement in the art. The method involves using a starter culture containing a *Streptococcus thermophilus* strain whose inactivation of nisin is weakened upon the addition of salt, as is required, for example, in the production of savory cheese products. This document describes the identification, screening, and generation of *Streptococcus thermophilus* strains whose nisin inactivation activity is reduced or eliminated due to the presence of salt (especially in the case of salt addition during cheese production).

[0013] Surprisingly, we have now found a way to identify, screen, and generate these thermophilic streptococcal strains that exhibit no or low salt-associated nisin inactivation activity, which can be used to produce savory fermented dairy products, particularly savory cheeses.

[0014] Even more surprisingly, we identified an endogenous Streptococcus thermophilus protein called TraX, which belongs to the acetyltransferase family and is known to be involved in the acetylation of F fimbriae in Escherichia coli. TraX plays a crucial role in inducing nisin immunity in strains such as Streptococcus thermophilus. The traX gene was identified in over 120 sequenced Streptococcus thermophilus strains. Therefore, contrary to the views proposed in the prior art, the inactivation of nisin in the presence of salt / after the addition of salt is not primarily caused by strain lysis (e.g., lysis of Streptococcus thermophilus), but depends on the activity of the TraX enzyme expressed in the presence of nisin.

[0015] In particular, strains lacking the TraX protein, such as traX gene knockout mutants or strains with abnormal traX expression, lose (partially) their inhibitory activity against nisin when salt is added during the production of savory fermented dairy products (such as savory cheese). These strains are especially important in the production of cheddar cheese.

[0016] The present invention also relates to the identification and characterization of thermophilic streptococcal strains that do not exhibit or only exhibit limited nisin inactivation activity upon the addition of salt (or in the presence of salt), in contrast to the fact that many known thermophilic streptococcal strains exhibit intracellular nisin release inactivation activity in the presence of salt (e.g., under conditions that occur during the production of savory fermented dairy products, particularly savory cheeses).

[0017] The present invention also relates to a method for converting a Streptococcus thermophilus strain that exhibits degradation of nisin when salt is added during the production of fermented dairy products (e.g., savory cheese) into a strain that exhibits reduced or no degradation activity of nisin when salt is added during the production of said fermented dairy products, wherein said conversion includes gene modification of endogenous TraX expression, as detailed below.

[0018] Therefore, the present invention relates to an improved method for the rapid production of savory fermented dairy products (including, but not limited to, savory cheeses) in the presence of thermophilic streptococcal strains identified herein that do not exhibit or only exhibit limited salt-associated nisin inactivation activity, wherein sufficiently high nisin concentrations are maintained at the end of fermentation and / or prior to cheese aging, and wherein the use of said strains does not negatively affect the desired savory flavor, appearance, shelf life, and / or reduce spoilage during or after cheese aging (particularly for savory cheeses).

[0019] Specifically, the present invention relates to a bacterial culture mixture for producing savory fermented dairy products, the method being carried out in the presence of nisin and salt, preferably with a salt concentration of 1% to 5% (w / w) of the final concentration, the mixture comprising:

[0020] (i) One or more strains of Streptococcus thermophilus,

[0021] (ii) A starter culture comprising one or more strains of lactococcus and / or lactobacillus, preferably lactococcus and / or lactobacillus strains that produce nisin, more preferably strains selected from subsp. latifolium and / or subsp. lactococcus lactis.

[0022] (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactobacillus and / or lactococcal strains;

[0023] (iv) Optionally, one or more non-bacterial cryoprotectants and / or non-bacterial additives;

[0024] The one or more *Streptococcus thermophilus* strains exhibit limited salt-associated nisin inactivation activity, assessed by means of the following ability: degradation of 70% or less of 50 U / ml nisin within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride; wherein the nisin is added to 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution containing approximately 5% (v / v) of such *Streptococcus thermophilus* strains, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.3; preferably, degradation of less than 50% of 50 U / ml nisin within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride. U / ml nisin, wherein the nisin was added to a culture of approximately 5% (v / v) of such thermophilic streptococcal strains inoculated into approximately 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.3.

[0025] More specifically, the present invention relates to a bacterial culture mixture as defined herein, comprising one or more Streptococcus thermophilus strains, wherein salt-associated nisin inactivation activity is eliminated, the activity being evaluated by means of its ability to degrade less than 50% of 50 U / ml nisin within 90 minutes at about 35°C in the presence of 5% (w / v) sodium chloride, wherein the nisin is added to a culture of about 5% (v / v) of such Streptococcus thermophilus strains inoculated in about 200 ml of about 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with about 15 ppm sodium formate, and grown to pH 5.3 at about 38°C; and wherein the strain is modified in the following manner:

[0026] (1) The presence of a nonfunctional TraX enzyme in terms of lactic acid degradation when expressing acetyltransferase TraX, particularly when expressing the endogenous gene of the TraX protein according to SEQ ID NO: 2 or 3, but the TraX enzyme is nonfunctional in terms of lactic acid degradation.

[0027] (2) The endogenous gene sequence expressing the acetyltransferase TraX, for example, based on a mutation in a polynucleotide of SEQ ID NO: 1, wherein the mutation is selected from gene knockout or nonsense mutation;

[0028] (3) No translational modifications of active TraX RNA were detected.

[0029] In one embodiment, the present invention relates to a bacterial culture mixture as defined herein, comprising one or more Streptococcus thermophilus strains having limited salt-associated nisin inactivation activity and containing and expressing the acetyltransferase TraX, particularly the TraX protein according to SEQ ID NO: 2 or 3.

[0030] According to one aspect of the present invention, the present invention relates to a method for producing fermented dairy products, the method comprising:

[0031] (a) Fermenting a milk base in the presence of a bacterial culture or a mixture of bacterial cultures as defined herein to produce a fermented milk base;

[0032] (b) Contact the fermented milk base with nisin;

[0033] (c) Optionally, the fermented milk base is brought into contact with a coagulant;

[0034] (d) Contact at least a portion of the fermented milk base with salt;

[0035] The bacterial culture contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0036] In some embodiments, the present invention provides a fermentation agent, bacterial culture mixture, or kit that comprises or consists of the following components:

[0037] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity, preferably, wherein the salt is sodium chloride, preferably, wherein the nisin is nisin A; and

[0038] (ii) Optionally, one or more nisin-producing bacterial strains, preferably wherein the nisin is nisin A, preferably wherein the bacterial strain is a *Lactococcus* species, such as *Lactococcus lactis* or *Lactococcus faecium* strains; and

[0039] (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0040] In some embodiments, the present invention provides a fermented dairy product, preferably cheese, more preferably cheddar cheese, comprising:

[0041] (i) Salt, preferably sodium chloride;

[0042] (ii) One or more Streptococcus thermophilus strains and / or residues of one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity, preferably, wherein the salt is sodium chloride, preferably, wherein the nisin is nisin A; and

[0043] (iii) Nisin, preferably nisin A.

[0044] Unless otherwise defined or the context clearly requires, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0045] In this specification and its appended claims, the terms “comprising” and “including”, as well as variations of various tenses, are to be interpreted inclusively. That is, these terms are intended to indicate that, where the context permits, other elements or components not expressly listed may be included.

[0046] In this text, the articles “a” and “a (species)” are used to refer to one or more (i.e., one or at least one) of the grammatical objects mentioned in the article. For example, “an element” can refer to one element or multiple elements. When referring to the singular form of a pronoun (such as a compound, additive, etc.), the plural form should be included. Therefore, when referring to a specific part, such as “strain”, unless otherwise specified, it usually refers to “at least one (species)” of that strain, such as “at least one strain”.

[0047] When referring to a compound that has multiple isomers (e.g., D and L enantiomers), in principle the compound includes all its enantiomers, diastereomers, and cis-trans isomers, which can be used in the specific form of the present invention; in particular, when referring to such a compound, it includes the natural isomers.

[0048] Unless otherwise expressly stated, the various embodiments of the invention described herein can be combined in various ways.

[0049] As used herein, the term "milk" is intended to encompass milk or milk products of mammalian and plant origin, or mixtures thereof. Preferably, the milk is derived from mammals, including but not limited to cows, sheep, goats, buffalo, camels, alpacas, horses, or reindeer. Plant-derived milk includes, but is not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp plants, sesame seeds, and sunflower seeds. Cow's milk is preferred. Furthermore, the term "milk" refers not only to whole milk but also to skim milk or any liquid derivative or reconstituted milk thereof.

[0050] As used herein, the term "milk base" refers to a base composition that contains, or is derived from, milk or milk components as defined herein. This milk base can be used as a raw material for the fermentation production of fermented dairy products. For example, the milk base may contain (fresh) skim milk or whole milk, or reconstituted milk, or be composed of the latter. Optionally, the milk base may also be in concentrate or powder form, or reconstituted from the aforementioned forms. Reconstituted milk, as referred to herein, means liquid milk obtained by adding liquid (e.g., water) to skim milk powder, skim milk concentrate, whole milk powder, or whole milk concentrate. Furthermore, the milk base may or may not have undergone a heat (pre)treatment operation at least as effective as pasteurization, including pasteurization or sterilization. In a preferred embodiment, the milk base has been heat (pre)treated, such as pasteurized or sterilized. The milk base may be derived from plant or mammalian sources. Preferably, the milk base is derived from mammalian sources, such as cow's milk, sheep's milk, goat's milk, buffalo milk, camel milk, alpaca milk, mare's milk, or deer milk, or any combination thereof. Preferably, the milk base is derived from bovine sources. Suitable plant-based milk sources include, but are not limited to, soy milk, pea milk, peanut milk, barley milk, rice milk, oat milk, quinoa milk, almond milk, cashew milk, and coconut milk, with soy milk, oat milk, or almond milk being preferred. Most preferably, the milk base is derived from bovine sources.

[0051] The term "nisin" as used herein refers to nisin polypeptide, also known as nisin peptide, nisinase, or polypeptide, peptide, or enzyme with "nisin" activity; all these terms are used interchangeably herein. In the literature, nisin is described as a polypeptide with bacteriocinogenic activity, preferably containing 31 to 35 amino acids, and preferably having a three-dimensional structure comprising five lanethionine rings. In the literature, the lanethionine ring is understood as a structure consisting of two alanine residues linked by a thioether bridge (also known as a sulfide bridge), with the following structural formula: HOOC-CH(NH2)-CH2-S-CH2-CH(NH2)-COOH. Naturally occurring nisin polypeptides are preferred, i.e., non-genetically modified nisin polypeptides. Suitable natural nisin variants include all currently known natural nisin variants: nisin A, nisin Z, nisin Q, nisin U, nisin U2, nisin F, nisin H, nisin O, nisin J, nisin P, nisin G, and nisin E, see, for example, Sevilleno et al. (International Journal of Molecular Science, vol. 24, 2023, pages 1-20). More preferably are nisin polypeptides produced and / or derived from *Lactococcus lactis*, preferably nisin A, nisin Z, and nisin Q, and / or nisin U, and more preferably, the nisin is a natural nisin as defined herein. Therefore, preferred nisin peptides include nisin peptides selected from nisin A, nisin Z, nisin Q and nisin U, more preferably nisin peptides selected from nisin A, nisin Z and nisin Q, and most preferably nisin A (see, for example, Cheigh et al., Biotechnology Letters (2005) 27: 1641-1648; Fukao et al., Biosci. Biotechnol. Biochem. (2008), 72 (7), 1750-1755; Wirawan et al., Applied and Environmental Microbiology, Feb. 2006, p.1148-1156). Particularly preferred is nisin A, with E number E234, also known as CAS No. 1414-45-5, which is preferably produced and / or derived from Lactococcus lactis.According to all forms of the invention, nisin in contact with the milk base and bacteria (including bacterial culture mixtures as defined herein) can be added to the fermentation medium or provided by in situ generation during or after fermentation, particularly by in situ generation of one or more lactococcal strains in the starter culture. Therefore, it is considered that nisin is dispersed in the milk base rather than added to the surface of the fermented dairy product.

[0052] The term "IMCU" as used in this article should be understood as the International Unit for Milk Coagulation. 1 IMCU is approximately equal to 0.126 nmol of bovine rennet B (e.g., in Maxiren). ® Or CHY-MAX ® (This refers to rennet products sold under a trade name). The strength of milk coagulating enzymes (such as rennet) is determined according to the standard established by the International Dairy Federation (IDF) in ISO 11815|IDF Standard 157A:1997, measured in milk coagulation activity (IMCU per milliliter or per gram). This standard was jointly developed by Subcommittee SC5 (Milk and Dairy Products) under Technical Committee 34 (Food Products) of ISO / TC34 and the International Dairy Federation (IDF).

[0053] The term "chymosin" as used in this article generally refers to aspartic protease, with EC number 3.4.23.4, conforming to the enzyme nomenclature published by the International Union of Biochemistry and Molecular Biology (IUBMB) in 1992. Chymosin is naturally produced by the chief cells of the stomach in young mammals. It is the main enzymatic component of rennet. Calf rennet is extracted from the inner wall of the abomasum (the fourth and last chamber of the stomach) of unweaned calves.

[0054] As used herein, any reference to %w / v, such as 12%w / v reconstituted skim milk (RSM), refers to the weight in grams per 100 ml of solution. For example, 12%w / v RSM is equivalent to 12 grams of skim milk powder dissolved in 100 ml of water.

[0055] According to one embodiment, the present invention relates to a method for producing fermented milk base by fermenting a milk base in the presence of a bacterial culture or a mixture of bacterial cultures, wherein the bacterial culture or the mixture of bacterial cultures contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0056] The present invention also relates to a fermentation agent, bacterial culture mixture or kit containing a thermophilic streptococcal strain that has little or no salt-associated nisin inactivation activity.

[0057] The terms “inactivated nisin,” “inactivation of nisin,” “nisin deactivation,” “nisin degradation,” “nisin degradation,” and “nisin inactivation” used herein are interchangeable. In this document, nisin inactivation activity is preferably understood as the direct or indirect, partial or complete inactivation of nisin by thermophilic streptococcal strains, for example, by modifying and / or degrading nisin in a solution containing nisin and thermophilic streptococcal strains, thereby inactivating the antimicrobial function of nisin. Specifically, the nisin inactivation is initiated in the presence of salt added during the production of fermented dairy products (e.g., particularly in the production of savory cheeses such as cheddar cheese). The amount of salt added / present in this process depends on the type of product, as is understood by those skilled in the art.

[0058] In this document, “salt-associated” (or interchangeably “salt-dependent” or “salt-induced”) nisin inactivation activity should be understood as the ability of *Streptococcus thermophilus* strains to directly or indirectly, partially or completely inactivate nisin in a solution containing nisin, salt, and the *Streptococcus thermophilus* strain in the presence of salt. Examples of *Streptococcus thermophilus* strains capable of inactivating nisin include those that naturally produce an effective number of extracellular nisin-degrading enzymes. Examples of *Streptococcus thermophilus* strains capable of inactivating nisin include those that, in the presence of salt (e.g., due to lysis), release intracellular nisin-degrading enzymes or other nonspecific nisin-degrading activities. Therefore, for the avoidance of doubt, *Streptococcus thermophilus* INIA 463 is not considered a *Streptococcus thermophilus* strain that lacks or has only limited salt-associated nisin inactivation activity. In other words, according to the present invention, thermophilic streptococcal strains that do not have or only have limited salt-associated nisin inactivation activity are not suitable as thermophilic streptococcus INIA 463.

[0059] Therefore, the present invention relates to a newly discovered Streptococcus thermophilus strain that does not have or only has limited salt-associated nisin inactivation activity as defined herein and measured by the determinations described herein, and in particular, wherein the strain is different from and not constitutes Streptococcus thermophilus INIA463.

[0060] The term "salt-associated" particularly includes halide salts, such as bromide, chloride, fluoride, or iodide salts, especially mixtures of these salts. Preferably, the salt is a chloride or bromide salt, more preferably a chloride salt. In one form, the salt is an alkali metal or alkaline earth metal salt, such as a sodium, potassium, calcium, or magnesium salt. More preferably, the salt is a sodium or potassium salt, even more preferably a sodium salt. Still more preferably, it is an alkali metal or alkaline earth metal halide salt, preferably sodium chloride, sodium bromide, potassium chloride, potassium bromide, calcium chloride, or calcium bromide. Even more preferably, the salt is sodium chloride or calcium chloride. Therefore, even more preferably, the salt-associated nisin inactivation activity is the sodium chloride-associated nisin inactivation activity or the calcium chloride-associated nisin inactivation activity. Therefore, even more preferably, the salt-associated or salt-induced nisin inactivation activity mentioned in the embodiments of the present invention can be interchanged with the sodium chloride-associated or sodium chloride-induced nisin inactivation activity or the calcium chloride-associated nisin inactivation activity. Most preferably, the salt is sodium chloride. Therefore, most preferably, the salt-associated nisin inactivation activity is sodium chloride-associated or sodium chloride-induced nisin inactivation activity. Therefore, most preferably, the salt-associated nisin inactivation activity mentioned in the embodiments of the present invention can be interchanged with the sodium chloride-associated nisin inactivation activity, and the salt mentioned can be interchanged with the sodium chloride mentioned.

[0061] As used herein, "salt" includes any salt used in the preparation of food products, particularly fermented dairy products such as cheese. Depending on the product, salt may be added at any stage of the process. For the production of savory cheeses, preferred salts include sodium chloride and / or potassium chloride. The final concentration of salt ranges from 1% to 5% (w / w). Those skilled in the art will understand the type and amount of salt that should be added for a specific (savory) cheese product.

[0062] This document defines nisin with respect to its inactivating activity. Preferably, the nisin is nisin A, and the inactivating activity mentioned herein preferably refers to the inactivating activity of nisin A. Depending on the fermented dairy product (especially cheese product), those skilled in the art know how much nisin should be added or contained during fermentation to effectively inhibit the growth of Gram-positive bacteria, especially NSLABs.

[0063] This invention provides *Streptococcus thermophilus* strains that can directly or indirectly, partially or completely inactivate nisin in a solution containing nisin and the *Streptococcus thermophilus* strain. As illustrated in the examples, salt-associated nisin-inactivated *Streptococcus thermophilus* strains, particularly sodium chloride-associated nisin-inactivated *Streptococcus thermophilus* strains, and preferably nisin A-inactivated *Streptococcus thermophilus* strains, can be conveniently characterized and / or classified by screening tests, as shown in the examples.

[0064] The degree of salt-associated nisin inactivation activity (SAND) of a certain thermophilic streptococcal strain can be determined by testing its ability to degrade approximately 50 U / ml of nisin within approximately 90 minutes at approximately 35°C in the presence of approximately 5% (w / v) sodium chloride. This nisin was added to 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution inoculated with approximately 5% (v / v) of this thermophilic streptococcal strain culture, supplemented with approximately 15 ppmw (parts per million by weight) of sodium formate, and grown at approximately 38°C to pH 5.3. This test simulates the conditions in the production process of savory fermented dairy products (e.g., savory cheese).

[0065] Suitablely, a thermophilic streptococcal strain that can degrade 70% or more of added nisin (particularly added nisin A) can be characterized as a thermophilic streptococcal strain with a clear (positive) salt-associated nisin inactivation activity (P-SAND), i.e., capable of causing nisin inactivation in the presence or addition of a salt as defined herein. In other words, if a thermophilic streptococcal strain can degrade 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or even 100%) of 50 U / ml nisin at approximately 35°C in the presence of approximately 5% (w / v) sodium chloride within approximately 90 minutes, then the strain is considered to have definite salt-associated or salt-induced nisin inactivation activity, wherein the nisin is added to approximately 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution inoculated with approximately 5% (v / v) of such a thermophilic streptococcal strain culture, supplemented with approximately 15 ppmw sodium formate, and grown at approximately 38°C to pH 5.3.

[0066] Suitablely, a thermophilic streptococcal strain that can degrade more than 50% but less than 70% of added nisin (especially added nisin A) can be characterized as a thermophilic streptococcal strain with limited salt-associated nisin inactivation activity (L-SAND), i.e., capable of causing reduced or limited nisin inactivation in the presence or addition of salts as defined herein. In other words, if a thermophilic streptococcal strain can degrade less than 70% (e.g., 69%, 65%, 60%, 55%, 50%, but not less than 50%) of 50 U / ml nisin within about 90 minutes at about 35°C in the presence of about 5% (w / v) sodium chloride, the strain is considered to have limited salt-associated nisin inactivation activity, wherein the nisin is added to a culture of about 5% (v / v) of such thermophilic streptococcal strain inoculated in about 200 ml of about 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with about 15 ppmw sodium formate, and grown at about 38°C to pH 5.3.

[0067] Suitablely, a thermophilic streptococcal strain can be characterized as a non-salt-associated nisin inactivation (N-SAND) strain if it can degrade less than 50%, for example 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or even 0%, of added nisin, i.e., with no inactivation activity whatsoever, preferably with 10% or less of the added nisin as defined herein. In other words, if a thermophilic streptococcal strain can degrade less than 50% (e.g., less than 40%, 30%, 20%, more preferably less than 10%) of 50 U / ml nisin within about 90 minutes at about 35°C in the presence of about 5% (w / v) sodium chloride, then the strain is considered to have salt-associated nisin inactivation activity, wherein the nisin is added to a culture of about 5% (v / v) of such thermophilic streptococcal strain inoculated in about 200 ml of about 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with about 15 ppmw sodium formate, and grown at about 38°C to pH 5.3.

[0068] Preferably, the *Streptococcus thermophilus* strain does not have salt-associated nisin inactivation activity (N-SAND), more preferably, the *Streptococcus thermophilus* strain does not have sodium chloride-associated nisin inactivation activity, and preferably, the nisin is nisin A.

[0069] Examples of preferred *Streptococcus thermophilus* strains that do not have or only have limited salt-associated nisin inactivation activity, particularly those that do not have or only have limited sodium chloride-associated nisin inactivation activity (wherein nisin is preferably nisin A) include strains CBS 150251 and CBS 150252, both deposited on July 20, 2023, at the Westdike Institute for Fungal Biodiversity (CBS) in Utrecht, Netherlands, and variants thereof that do not have or only have limited salt-associated nisin inactivation activity.

[0070] In some forms, the present invention provides a method for producing fermented dairy products, particularly savory fermented dairy products, the method comprising:

[0071] (a) Fermenting and coagulating a milk base in the presence of a bacterial culture (mixture), in the presence of nisin and in the presence of a coagulant to produce a coagulated fermented milk base;

[0072] (b) Contact at least a portion of the coagulated fermented milk base with salt;

[0073] The bacterial culture (mixture) contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0074] Specifically, the fermented dairy product is a savory fermented dairy product, more specifically, a savory cheese. This invention provides a method for producing such a savory fermented dairy product, the method comprising:

[0075] (a) Fermenting and coagulating a milk base in the presence of a bacterial culture (mixture), in the presence of nisin and in the presence of a coagulant to produce a coagulated fermented milk base;

[0076] (b) Contact at least a portion of the coagulated fermented milk base with salt;

[0077] The bacterial culture (mixture) contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0078] In step (a), the milk base is fermented in the presence of a bacterial culture (mixture) to produce a fermented milk base as defined herein.

[0079] The bacterial culture contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity. More preferably, the bacterial culture (mixture) is a bacterial culture as described above and / or as follows.

[0080] Preferably, the milk base in step (a) is fermented in the presence of a bacterial culture mixture, which preferably contains or consists of the following substances:

[0081] (i) one or more Streptococcus thermophilus strains, wherein at least one Streptococcus thermophilus strain preferably has no or only limited salt-associated nisin inactivation activity; and

[0082] (ii) Optionally, one or more bacterial strains that are not Streptococcus thermophilus.

[0083] Therefore, in some embodiments of the present invention, therein relates to a method according to the invention, the method comprising fermenting a milk-based material as defined herein in the presence of a bacterial culture mixture, wherein the bacterial culture mixture comprises or consists of the following substances:

[0084] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0085] (ii) One or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus strains, more preferably nisin-producing Lactococcus faecium strains and / or nisin-producing Lactococcus lactis strains; and

[0086] (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0087] Furthermore, in some embodiments of the present invention, therein relates to a method according to the invention, the method comprising fermenting a milk-based material as defined herein in the presence of a bacterial culture mixture, wherein the bacterial culture mixture comprises or consists of the following substances:

[0088] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0089] (ii) Optionally, one or more nisin-producing bacterial strains, preferably nisin-producing *Lactococcus lactis* strains, more preferably nisin-producing *Lactococcus faecium* strains and / or nisin-producing *Lactococcus lactis* strains; and

[0090] (iii) One or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0091] Preferred embodiments of these implementation schemes are described above and below.

[0092] Specifically, the method described herein includes fermenting a milk base material as defined herein in the presence of a bacterial culture or a mixture of bacterial cultures until the pH value is equal to or less than about 6.0, more preferably equal to or less than about 5.8, 5.5, or 5.3. For practical application, the pH value in step (a) of the above method is preferably equal to or less than about 6.8, for example, about 6.7, 6.4, or lower, particularly in the range of 6.8 to 3.0, preferably 6.8 to 3.5, more preferably 6.8 to 4.0, and most preferably between 6.8 and 4.2. If the fermented dairy product is cheese, the pH value in step (a) of the above method is more preferably in the range of 6.8, 6.7, 6.4 to 4.0, 4.5, 5.0, 5.1.

[0093] Preferably, step (a) of the above method is carried out at a temperature of at least about 28°C, for example about 30°C, 32°C or higher, and more preferably at a temperature of about 47°C or lower, for example about 45°C, 42°C, 40°C or lower, particularly in the range of 28°C to 47°C.

[0094] In some embodiments, the method defined herein more preferably includes step (a) of the above method comprising multiple fermentation stages, preferably including one or more mesophilic fermentation stages and / or one or more thermophilic fermentation stages. More preferably, the method further includes step (a) of the method comprising:

[0095] (a1) The first mesophilic fermentation stage is preferably carried out in the temperature range of 30°C to 38°C, more preferably in the temperature range of 30°C to 37°C, and most preferably in the temperature range of 30°C to 36°C.

[0096] (a2) The second thermophilic fermentation stage is preferably carried out in a temperature range of 36°C to 45°C, more preferably in a temperature range of 37°C to 45°C, and most preferably in a temperature range of 38°C to 45°C.

[0097] (a3) Optionally, the third mesophilic fermentation stage is preferably carried out in a temperature range of 30°C to 38°C, more preferably in a temperature range of 30°C to 37°C, and most preferably in a temperature range of 30°C to 36°C.

[0098] The advantage of the aforementioned staged fermentation is that the second stage of thermophilic fermentation can promote the fermentation of the aforementioned thermophilic streptococcal strains. Furthermore, another advantage of the aforementioned staged fermentation is that the first and / or third stage of mesophilic fermentation can promote the fermentation of mesophilic lactococcal bacteria strains (e.g., lactococcal lactis strains).

[0099] Preferably, step (a) is carried out in a fermenter.

[0100] The time required to perform step (a) can vary considerably. However, preferably, the time for step (a) is between 10 and 360 minutes, more preferably between 10, 20, or 30 minutes and 60, 90, 120, or 360 minutes. Shorter time allows for faster production, which is ideal for cheese producers.

[0101] More preferably, steps (b) and / or (c) as described below are performed in step (a). That is, preferably, steps (a), (b), and (c) together constitute a single step comprising fermenting and coagulating the milk base in the presence of a bacterial culture (mixture), in the presence of nisin, and in the presence of a coagulant to produce a coagulated fermented milk base or a composition thereof. The advantage of performing steps (a), (b), and / or (c) simultaneously in the same container is that it provides a very rapid process.

[0102] Step (b) involves contacting or comprising a fermented milk base with nisin. The fermented milk base may be the entire fermented milk base prepared in step (a). Alternatively, only a portion of the fermented milk base prepared in step (a) may be contacted with nisin.

[0103] In the method according to the invention, step (b) may be performed after step (a) or simultaneously with step (a). Preferably, step (b) is performed simultaneously with step (a), or even as part of step (a), and in each case, preferably together in the same container.

[0104] Contact between the fermented milk substrate and nisin can be carried out ex-situ and / or in-situ. That is, as an example of ex-situ contact, nisin can be added to the fermented milk substrate from the outside. As an example of in-situ contact, nisin can be generated in situ, for example, through the bacterial culture (mixture) shown in step (a).

[0105] Step (b) can be performed by adding and / or generating nisin before, during, and / or after fermentation of the milk-based substrate. Preferably, step (b) includes adding nisin and / or generating nisin in situ or consisting thereof during and / or after fermentation in step (a).

[0106] If step (b) includes the addition of nisin, it is preferable to add nisin during and / or after fermentation as described in step (a). Preferably, the concentration of nisin added is higher than the MIC value of the target strain. Here, the MIC value is understood as the minimum inhibitory concentration (MIC). That is, preferably, the concentration of nisin added is higher than the minimum nisin concentration (in μg / mL) required to inhibit the growth of the target NSLAB. In practice, it is preferred that the amount of nisin added is equal to or greater than 1 U / mL, more preferably equal to or greater than 5 U / mL, even more preferably equal to or greater than 10 U / mL, even more preferably equal to or greater than 50 U / mL, most preferably equal to or greater than 100 U / mL, or even equal to or greater than 150 U / mL, or even equal to or greater than 200 U / mL, with a final concentration up to 600 U / mL.

[0107] Step (b) preferably includes or consists of in situ generation of nisin. If step (b) includes in situ generation of nisin, then step (b) preferably includes in situ generation of nisin during and / or after fermentation in step (a). Most preferably, if step (b) includes in situ generation of nisin, then nisin is generated in situ during fermentation in step (a) (i.e., simultaneously with fermentation). If step (b) includes in situ generation of nisin, then nisin is preferably generated in situ using one or more nisin-producing strains. The preference for such nisin-producing strains has been described above and below. Preferably, the concentration of nisin generated is higher than the MIC value of the target NSLAB. In practice, the preferred concentration of nisin produced is equal to or greater than 1 U / ml, more preferably equal to or greater than 5 U / ml, even more preferably equal to or greater than 10 U / ml, even more preferably equal to or greater than 50 U / ml, most preferably equal to or greater than 100 U / ml, or even equal to or greater than 150 U / ml, or even equal to or greater than 200 U / ml, with the final concentration reaching up to 600 U / ml.

[0108] Preferably, in the fermentation process of step (a), the generation of nisin utilizes: one or more nisin-producing bacterial strains, preferably one or more nisin-producing *Lactococcus lactis* strains, more preferably one or more nisin-producing *Lactococcus lactis* strains (also referred to herein as *Lactococcus lactis* subsp. *lactosporum* strains) and / or one or more nisin-producing *Lactococcus lactis* strains (also referred to herein as *Lactococcus lactis* subsp. *lactosporum* strains), even more preferably one or more nisin-producing *Lactococcus lactis* subsp. *lactosporum* strains, and most preferably one or more nisin A-producing *Lactococcus lactis* subsp. *lactosporum* strains. Preferably, such nisin-producing bacterial strains are added alone in step (a), or more preferably, as part of a bacterial culture mixture that also contains *Streptococcus thermophilus* strains.

[0109] Preferably, step (b) is performed in a temperature range of 28°C to 47°C, more preferably in a temperature range of 30°C to 45°C, for example in a temperature range of 32°C to 42°C, even more preferably in a temperature below 40°C, for example, most preferably in a temperature of 38°C or lower, i.e., particularly in a temperature range of 28°C to 38°C.

[0110] As described above, the fermentation process may advantageously include one or more mesophilic fermentation stages. Another advantage of the aforementioned staged fermentation, as previously mentioned, is that the first and / or third mesophilic fermentation stages can stimulate the fermentation of mesophilic nisin-producing strains (e.g., nisin-producing *Lactococcus lactis* strains). If the fermentation process in step (a) includes both thermophilic and mesophilic fermentation stages, it may be advantageous to add one or more mesophilic nisin-producing strains (e.g., nisin-producing *Lactococcus lactis* strains) before and / or during one of the mesophilic fermentation stages, and these strains may optionally be added separately from thermophilic streptococcal strains.

[0111] The duration of step (b) can vary considerably. However, preferably, the duration of step (b) is between 10 minutes and 360 minutes, for example, in the range of 20 or 30 minutes to 120, 90, or 60 minutes. Shorter durations facilitate faster production, which is ideal for cheese producers. Most preferably, step (b), as described herein, is performed during step (a). The advantage of performing steps (a) and (b) simultaneously in the same container is that it provides a very rapid process.

[0112] Step (c) may optionally involve contacting the fermented milk base with a coagulant (e.g., adding one or more coagulants) or comprising thereof. Preferably, the method according to the invention includes step (c), and is a method for producing fermented dairy products (preferably cheese), the method comprising:

[0113] (a) Fermenting a milk base in the presence of a bacterial culture (mixture) to produce a fermented milk base;

[0114] (b) Contacting the fermented milk base with nisin, for example, by adding nisin to the fermented milk base or by generating nisin in situ;

[0115] (c) Contact the fermented milk base with a coagulant, for example, by adding the coagulant to the fermented milk base;

[0116] (d) Contact at least a portion of the fermented milk base with salt, for example, by adding salt to the fermented milk base, particularly to the curd, or by immersing the cheese in a salt bath;

[0117] The bacterial culture (mixture) contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0118] In step (c), the contact between the fermented milk base and the coagulant can easily yield a coagulated fermented milk base. This coagulated fermented milk base is suitable to contain curd and whey.

[0119] Step (c) can be carried out by adding a coagulant before, during, or after the fermentation of the milk base in step (a). Preferably, step (c) is carried out by adding a coagulant during and / or after fermentation in step (a). Most preferably, step (c) as described herein is carried out during step (a). The advantage of carrying out steps (a) and (c) simultaneously in the same container is that it provides a faster process.

[0120] Furthermore, step (c) can be performed by adding a coagulant before, during, or after contact with nisin in step (b). If the nisin is generated in situ (as described below), step (c) preferably includes adding a coagulant during and / or after contact with nisin in step (b). Similarly, if the nisin is added out of situ (as described below), step (c) preferably includes adding a coagulant during and / or after contact with nisin in step (b). Most preferably, step (c) described herein is performed during step (b). The advantage of performing steps (b) and (c) simultaneously in the same container is that it provides a faster process.

[0121] Most preferably, step (c) described herein is performed simultaneously with steps (a) and (b) in the same container. The advantage of performing steps (a), (b), and (c) simultaneously in the same container (i.e., as a single step) is that it provides a very rapid process.

[0122] Preferably, the coagulant contains or is composed of a protease, preferably an aspartic protease. In this document, the terms protease, proteinase, and peptidase are used interchangeably. Preferably, the coagulant contains or is composed of a protease produced by microorganisms or mammals, particularly an aspartic protease produced by microorganisms or mammals. Most preferred coagulants are those containing or composed of mucorpepsin, chymosin, or their precursors. Examples of suitable commercially available coagulants include Maxiren. ® Maxiren ® XDS, Fromase ® CHY-MAX ® and Hannilase ® .

[0123] Preferably, the amount of coagulant added should be such that the concentration of the coagulant reaches the following range: equal to or greater than 1 IMCU / L milk, more preferably equal to or greater than 5 IMCU / L milk, more preferably equal to or greater than 10 IMCU / L milk, more preferably equal to or greater than 15 IMCU / L milk, more preferably equal to or greater than 20 IMCU / L milk, more preferably equal to or greater than 25 IMCU / L milk, most preferably equal to or greater than 30 IMCU / L milk, up to equal to or less than 100 IMCU / L milk, more preferably equal to or less than 80 IMCU / L milk, more preferably equal to or less than 75 IMCU / L milk, more preferably equal to or less than 70 IMCU / L milk, more preferably equal to or less than 65 IMCU / L milk, most preferably equal to or less than 60 IMCU / L milk.

[0124] Step (c) is preferably carried out in a temperature range of 28°C to 47°C, more preferably in a temperature range of 30°C to 45°C, for example 32°C to 42°C, and most preferably at a temperature of 40°C or lower, for example 32°C to 40°C.

[0125] The time required for step (c) can vary considerably. However, preferably, the time for step (c) is within the following range: equal to or greater than 10 minutes, more preferably equal to or greater than 20 minutes, most preferably equal to or greater than 30 minutes, up to equal to or less than 360 minutes, more preferably equal to or less than 120 minutes, even more preferably equal to or less than 90 minutes, and most preferably equal to or less than 60 minutes. Shorter processing times facilitate faster production, which is ideal for cheese producers.

[0126] Step (d) includes contacting at least a portion of the fermented milk base with salt or having it as a component therein.

[0127] Preferably, the salt is a halide salt, such as a bromide salt, chloride salt, fluoride salt, or iodide salt. More preferably, the salt is a chloride salt or bromide salt, and most preferably a chloride salt. Preferably, the salt is an alkali metal or alkaline earth metal salt, such as a sodium salt, potassium salt, calcium salt, or magnesium salt. More preferably, the salt is a sodium salt or potassium salt, and most preferably a sodium salt. More preferably, the salt is an alkali metal or alkaline earth metal halide salt, preferably sodium chloride, sodium bromide, potassium chloride, potassium bromide, calcium chloride, or calcium bromide. Even more preferably, the salt is sodium chloride or calcium chloride. Most preferably, the salt is sodium chloride.

[0128] Step (d) can be performed by adding salt before, during, or after the fermentation of the milk base. Preferably, step (d) is performed by adding salt during or after the fermentation in step (a). More preferably, step (d) is performed by adding salt after the fermentation in step (a). In a preferred embodiment, step (d) is performed after step (a), after step (b), and (if any) after step (c).

[0129] If the method according to the invention comprises steps (a), (b), and (c), preferably, these steps (a), (b), and (c) together constitute a single step, also referred to herein as a curdling step, which produces a coagulated fermented milk base that conveniently contains curd and whey. Optional steps such as cutting, stirring, and / or cooking can be conveniently performed after this curdling step, after which the whey can be conveniently separated from the curd. The curd can advantageously be ground and then salted.

[0130] Therefore, the method preferably includes one or more separation steps, wherein a portion (preferably curd) of the coagulated fermented milk base is separated from another portion (preferably whey). Here, curd is preferably understood as the coagulated portion of the milk. This coagulated portion of the milk preferably contains aggregated milk proteins, more preferably aggregated casein.

[0131] Therefore, preferably, step (d) includes separating the (optionally coagulated) fermented milk base into curd and whey, and then contacting the separated curd (i.e. the curd portion of the fermented milk base) with salt.

[0132] Therefore, preferably, this method is a method for producing fermented dairy products, comprising:

[0133] (a) Fermenting a milk base in the presence of a bacterial culture (mixture) to produce a fermented milk base;

[0134] (b) Contact the fermented milk base with nisin;

[0135] (c) Bring the fermented milk base into contact with the coagulant;

[0136] (d) Separate the fermented milk base into curd and whey, and contact the curd with salt, for example, by adding salt to the curd;

[0137] The bacterial culture (mixture) contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0138] The curd is preferably milled before and / or after contact with salt. Other preferred conditions are as described above and below.

[0139] More preferably, the present invention provides a method for producing fermented dairy products (preferably savory fermented dairy products), the method comprising:

[0140] - A milk base is fermented and coagulated in the presence of a bacterial culture (mixture), in the presence of nisin, and in the presence of a coagulant to produce a coagulated fermented milk base; and

[0141] - Separate the coagulated fermented milk base into curd and whey, and contact the curd with salt, for example, by adding salt as defined herein to the curd;

[0142] The bacterial culture (mixture) contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0143] The curd is preferably milled before and / or after contact with salt. Other preferred conditions are as described above and below.

[0144] Preferably, the amount of salt added is equal to or greater than 0.10% (w / w), more preferably at least about 0.25% (w / w), even more preferably at least about 0.50% (w / w), even more preferably at least about 1.0% (w / w), and most preferably at least about 2.0% (w / w), for example, in the range of 0.25% (w / w) to 7% (w / w), 0.50% to 6% (w / w), 1.0% to 5.5% (w / w), 1.0% to 5% (w / w), or 2.0% to 4% (w / w); all of the above are based on the total weight of the (partial) fermented dairy product in contact with it. Preferably, the amount of salt added is 1% to 5% (w / w). All values ​​refer to the final concentration.

[0145] Preferably, the salt added in step (d) is in the form of an aqueous solution, preferably an aqueous solution of sodium chloride and / or calcium chloride.

[0146] The advantage of the method according to the invention is that the level of nisin can be maintained even in methods involving the addition of salt. Nisin can effectively inhibit the growth of non-fermenting lactic acid bacteria (NSLAB).

[0147] Preferably, the method further includes a step of recycling fermented dairy products.

[0148] The fermented dairy product produced by this method is preferably cheese, and more preferably savory cheese. Other preferred conditions for the fermented dairy product produced by this method are described below. Most preferably, the fermented dairy product produced by this method is cheddar cheese.

[0149] Preferably, the bacterial culture comprises, constitutes, or belongs to a starter culture, a bacterial culture mixture, or a kit. The starter culture described herein is preferably understood to be a mixture comprising two, three, four, five, six, or even more different bacteria used to inoculate food ingredients (e.g., milk) to induce a predetermined change in the food ingredient.

[0150] In one embodiment, the present invention suitably provides a fermentation agent, bacterial culture mixture, or kit comprising or consisting of:

[0151] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0152] (ii) One or more nisin-producing bacterial strains, preferably nisin-producing *Lactococcus lactis* strains, more preferably nisin-producing *Lactococcus lactis* subsp. *milk fat* strains and / or nisin-producing *Lactococcus lactis* subsp. *lactolaccos* strains; and

[0153] (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0154] In another embodiment, the present invention suitably provides a fermentation agent, bacterial culture mixture, or kit comprising or consisting of:

[0155] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0156] (ii) Optionally, one or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus strains, more preferably nisin-producing Lactococcus lactis subsp. milk fat strains and / or nisin-producing Lactococcus lactis subsp. lactis strains; and

[0157] (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0158] Preferably, the fermentation agent, bacterial culture mixture, or kit comprises or consists of the following:

[0159] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0160] (ii) One or more nisin-producing bacterial strains, preferably nisin-producing *Lactococcus lactis* strains, more preferably nisin-producing *Lactococcus lactis* subsp. *milk fat* strains and / or nisin-producing *Lactococcus lactis* subsp. *lactolaccos* strains; and

[0161] (iii) One or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus and / or lactococcal bacterial strains.

[0162] In these implementations, the preferred conditions for one or more Streptococcus thermophilus strains under (i) are as described above.

[0163] The starter culture, bacterial culture mixture, or kit described above preferably does not contain *Streptococcus thermophilus* strains with defined salt-associated nisin inactivation activity (P-SAND). More preferably, the starter culture, bacterial culture mixture, or kit (based on its total weight) contains equal to or less than 5.0% w / w, even more preferably equal to or less than 2.0% w / w, even more preferably equal to or less than 1.0% w / w, even more preferably equal to or less than 0.5% w / w, and most preferably does not contain *Streptococcus thermophilus* strains with defined salt-associated nisin inactivation activity (P-SAND). Characterization may be suitably performed as described herein. If the starter culture, bacterial culture mixture, or kit contains a Streptococcus thermophilus strain with defined salt-associated nisin inactivation activity (P-SAND), the weight percentage of the Streptococcus thermophilus strain with defined salt-associated nisin inactivation activity is preferably equal to or less than 20% w / w, more preferably equal to or less than 10% w / w, even more preferably equal to or less than 5.0% w / w, even more preferably equal to or less than 1.0% w / w, even more preferably equal to or less than 0.5% w / w, and most preferably equal to or less than 0.1% w / w.

[0164] Preferably, each *Streptococcus thermophilus* strain contained in or included in the starter culture, bacterial culture mixture, or kit is a *Streptococcus thermophilus* strain that has little or no salt-associated nisin inactivation activity. More preferably, each *Streptococcus thermophilus* strain contained in or included in the starter culture, bacterial culture mixture, or kit is a *Streptococcus thermophilus* strain that has little or no salt-associated nisin inactivation activity. Most preferably, the starter culture, bacterial culture mixture, or kit contains only *Streptococcus thermophilus* strains that have little or no salt-associated nisin inactivation activity, and most preferably, strains that have no salt-associated nisin inactivation activity. Characterization can be suitably performed according to the methods described above.

[0165] If present in the above embodiments, the one or more nisin-producing bacterial strains are preferably nisin-producing Lactococcus strains, more preferably nisin-producing Lactococcus lactis strains, even more preferably one or more nisin-producing Lactococcus lactis subsp. milk fat strains and / or one or more nisin-producing Lactococcus lactis subsp. lactis strains, and most preferably one or more nisin-producing Lactococcus lactis subsp. lactis strains. If a combination of *Lactococcus lactis* subsp. *milk fat* and *Lactococcus lactis* subsp. *lactolaccos* strains producing lactococcus lactis are used, the ratio of *Lactococcus lactis* subsp. *milk fat* to *Lactococcus lactis* subsp. *lactolaccos* strains is equal to or greater than 1:100, more preferably equal to or greater than 1:10, even more preferably equal to or greater than 1:5, most preferably equal to or greater than 1:2 to equal to or less than 100:1, more preferably equal to or less than 10:1, even more preferably equal to or less than 5:1, and most preferably equal to or less than 2:1. More preferably, if a combination of *Lactococcus lactis* subsp. *milk fat* and *Lactococcus lactis* subsp. *lactolaccos* strains producing lactococcus lactis are used, the ratio of *Lactococcus lactis* subsp. *lactolaccos* strains to *Lactococcus lactis* subsp. *lactolaccos* strains is preferably equal to or greater than 25:75 to equal to or less than 50:50.

[0166] The nisin-producing bacterial strains can produce nisin A and / or nisin Z and / or nisin Q and / or nisin U and / or any other type of nisin and / or any combination thereof. Preferably, the nisin-producing strains produce nisin A, nisin Z, or a combination of nisin A and nisin Z. Most preferably, the nisin-producing bacterial strains are nisin A-producing bacterial strains, more preferably Lactococcus lactis strains producing nisin A, even more preferably Lactococcus lactis strains producing nisin A, further more preferably Lactococcus lactis subsp. milk fat strains producing nisin A and / or Lactococcus lactis subsp. lactis strains producing nisin A, and most preferably Lactococcus lactis subsp. lactis strains producing nisin A.

[0167] Preferably, the nisin-producing strain is capable of producing nisin per milliliter (milk) equal to or greater than 5 IU, 25 IU, 100 IU, 400 IU, 600 IU, or 800 IU, in a preferred order, preferably under the process conditions described in the first embodiment and / or the process conditions described below. The number of nisin-producing strains is preferably such that the nisin content in any fermentation product is sufficient to prevent bacterial spoilage.

[0168] In principle, any *Lactococcus lactis* strain can be converted into a nisin-producing strain. Preferably, suitable *Lactococcus lactis* strains acquire nisin-producing characteristics by conjugating transposons containing genetic information for nisin production. A preferred transposon containing genetic information for nisin production is Tn5276. Hugenholtz et al. described donor strains suitable for conjugation with Tn5276, which were deposited at the Central Culture Collection of Microorganisms in Barn, Netherlands, on June 28, 2001 (accession number CBS109540) and April 3, 1991 (accession number CBS 181.91), respectively. These strains and methods are described in more detail in European Patent EP1273237, which is incorporated herein by reference.

[0169] Alternatively, recombinant DNA techniques known to those skilled in the art can be used to confer the nisin-producing characteristic of *Lactococcus lactis* onto suitable *Lactococcus lactis* strains. However, given the low public acceptance of GMOs (genetically modified organisms) in food, the use of such GMO *Lactococcus lactis* strains is not currently recommended.

[0170] The most preferred nisin-producing strain according to (ii) above is a strain of *Lactococcus lactis* subsp. *lactobacter* diacetyllactobacter, preferably containing the Tn5276 transposon mentioned above. This strain is included in Dairysafe. TM In this context, it is produced by CSK food enrichment company in Leeuwarden, Netherlands, under the name Dairysafe. TM The name TC17 is being sold.

[0171] If present in the above embodiments, the one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing strains are preferably lactic acid-resistant lactic acid bacteria strains, and preferably do not have or only have limited lactic acid inactivating activity and / or preferably do not have or only have limited salt-associated lactic acid inactivating activity.

[0172] In this document, strains that are resistant to nisin, do not produce nisin, and do not inactivate nisin are also referred to as "nisin-neutral strains." In the above embodiments, the one or more bacterial strains that are not thermophilic streptococcal strains or nisin-producing bacterial strains are preferably nisin-neutral strains.

[0173] The nisin resistance, nisin immunity and / or (deficiency) nisin inactivation activity, (deficiency) salt-associated nisin inactivation activity and / or nisin neutrality described herein are preferably applicable to all types of nisin, including nisin A and / or nisin Z and / or nisin Q and / or nisin U and / or any other type of nisin and / or any combination thereof. More preferably, any nisin resistance, nisin immunity and / or (lack) nisin inactivation activity, (lack) salt-associated nisin inactivation activity and / or nisin neutrality applicable in this invention applies at least to the type of nisin present in the first form of the method and / or the type of nisin produced by any nisin-producing strain in the second form of the starter culture, bacterial culture mixture or kit, preferably nisin A.

[0174] Preferably, "one or more bacterial strains that are not thermophilic streptococcal strains or lactococcal-producing strains" are selected from the group consisting of strains of the genera *Lactobacillus*, *Leuconostoc*, *Propionibacterium*, *Pediococcus*, *Arthrobacter*, *Corynebacterium*, *Staphylococcus*, and other streptococcal genera other than thermophilic streptococci. Additionally, non-lactococcal lactococci may also be present, such as the non-lactococcal lactococcus diacetyl subsp. *lactococcus*.

[0175] More preferably, "one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing strains" are, preferably, lactic acid-resistant, preferably non-lactate-inactivated strains, selected from the group consisting of: preferably lactic acid-neutral *Lactobacillus delbrueckii* subsp. delbrueckii, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus* (also known as *Lactaseibacillus rhamnosus*), *Lactobacillus paracasei* (also known as *Lactaseibacillus paracasei*), *Lactobacillus casei* (also known as *Lactaseibacillus casei*), and *Lactobacillus helveticus*. Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus plantarum (also known as Lactobacillus plantarum), Lactobacillus sanfrancisco, Lactobacillus johnsonii, Lactobacillus pontis, Lactobacillus bavaricus, Lactobacillus curvatus, Lactobacillus sacei, Leuconostoc mesenteroides, Leuconoctoc lactis, Leuconostoc ssp., Pediococcus pentosaveus, Pediococcus avidilactici, Staphylococcus xylose Propionibacterium xylosus, Propionibacterium freudenreichii, and Propionibacterium freudenreichii ssp.(shermanii) and their combinations.

[0176] More preferably, “one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing strains” are, preferably, lactic acid-resistant, preferably non-lactate-inactivated strains, selected from the group consisting of: preferably lactic acid-neutral Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus nodensis, Brevibacterium linens, Kluyveromyces lactis, and combinations thereof. More preferably, “one or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing strains” are preferred to be lactic acid-resistant, and more preferably non-lactate-inactivated strains selected from the group consisting of: preferred lactic acid-neutral lactobacillus strains, more preferably Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus and / or Lactobacillus norovirus.

[0177] Preferably, the fermentation agent, bacterial culture mixture, or kit contains a mixture of or consists of the following components:

[0178] (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0179] (ii) One or more nisin-producing bacterial strains, preferably nisin-producing *Lactococcus lactis* strains, more preferably nisin-producing *Lactococcus lactis* strains, even more preferably nisin-producing *Lactococcus lactis* subsp. *milk fat* strains and / or nisin-producing *Lactococcus lactis* subsp. *lactolaccus* strains; and

[0180] (iii) One or more bacterial strains that are not thermophilic streptococcal strains or lactic acid-producing streptococcal strains, preferably lactobacillus strains, more preferably Lactobacillus casei, Lactobacillus paracasei and / or Lactobacillus helveticus strains.

[0181] The dosage of each component in the above-mentioned fermentation agent, bacterial culture mixture, or kit may vary.

[0182] Component (i), namely “one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity,” is preferably present in an amount equal to or greater than 1% w / w by weight, more preferably equal to or greater than 5% w / w, even more preferably equal to or greater than 10% w / w, even more preferably equal to or greater than 15% w / w, even more preferably equal to or greater than 20% w / w, most preferably equal to or greater than 25% w / w, and preferably present in an amount equal to or less than 99% w / w by weight, more preferably equal to or less than 95% w / w, even more preferably equal to or less than 90% w / w, even more preferably equal to or less than 85% w / w, even more preferably equal to or less than 80% w / w, even more preferably equal to or less than 75% w / w, even more preferably equal to or less than 70% w / w, and most preferably equal to or less than 65% w / w.

[0183] If present, component (ii), namely "one or more nisin-producing bacterial strains", is preferably present in an amount equal to or greater than 1% w / w by weight, more preferably equal to or greater than 5% w / w, even more preferably equal to or greater than 10% w / w, even more preferably equal to or greater than 15% w / w, even more preferably equal to or greater than 20% w / w, and most preferably equal to or greater than 25% w / w, based on the total weight of all bacterial strains in the starter culture, bacterial culture mixture, or kit; and preferably present in an amount equal to or less than 99% w / w by weight, more preferably equal to or less than 95% w / w, even more preferably equal to or less than 90% w / w, even more preferably equal to or less than 85% w / w, even more preferably equal to or less than 80% w / w, even more preferably equal to or less than 75% w / w, even more preferably equal to or less than 70% w / w, and most preferably equal to or less than 65% w / w.

[0184] If present, component (iii), namely "one or more bacterial strains that are not thermophilic streptococcal strains or not lactococcal producing strains", is preferably present in an amount equal to or greater than 1% w / w by weight, more preferably equal to or greater than 5% w / w, even more preferably equal to or greater than 10% w / w, even more preferably equal to or greater than 15% w / w, even more preferably equal to or greater than 20% w / w, and most preferably equal to or greater than 25% w / w, based on the total weight of all bacterial strains in the starter culture, bacterial culture mixture, or kit; and preferably present in an amount equal to or less than 99% w / w by weight, more preferably equal to or less than 95% w / w, even more preferably equal to or less than 90% w / w, even more preferably equal to or less than 85% w / w, even more preferably equal to or less than 80% w / w, even more preferably equal to or less than 75% w / w, even more preferably equal to or less than 70% w / w, and most preferably equal to or less than 65% w / w.

[0185] Most preferably, all three components (i), (ii) and (iii) are present, and more preferably, the weight ratio of all three components (i), (ii) and (iii) is about 1:1:1, about 2:1:1, about 1:2:1 or about 1:1:2, and most preferably, the weight ratio is about 1:1:1.

[0186] If present, the strains described in items (i), (ii), and (iii) are preferably present in frozen or lyophilized form. More preferably, the starter culture, bacterial culture mixture, or kit is a frozen or lyophilized starter culture or a frozen or lyophilized bacterial culture mixture.

[0187] In addition to components (i), (ii), and (iii) described above, the starter culture, bacterial culture mixture, or kit may or may not contain an additional component (iv), which may contain or consist of one or more non-bacterial cryoprotectants and / or non-bacterial additives, such as sodium formate. More preferably, the starter culture, bacterial culture mixture, or kit may optionally contain or consist of one or more other cryoprotectants besides sodium formate. Furthermore, the starter culture, bacterial culture mixture, or kit may or may not contain an additional component (v), which may contain or consist of a solvent, such as water or milk.

[0188] In another embodiment, the present invention provides a fermented dairy product, more preferably cheese, even more preferably savory cheese, and most preferably cheddar cheese, comprising:

[0189] (i) Salt, preferably sodium chloride or calcium chloride, with sodium chloride being the most preferred.

[0190] (ii) One or more Streptococcus thermophilus strains and / or residues of one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and

[0191] (iii) Nisin, preferably nisin A.

[0192] Preferably, the fermented dairy product is cheese. Preferably, the fermented dairy product is a hard cheese. Preferably, the fermented dairy product is a savory cheese. Most preferably, the fermented dairy product is selected from Cheddar, Emmental, Grana Padano, Gruyère, Mimolette, Parmesan-Regano, Parmesan-style cheese, Pecorino, Gouda, Provolone, and Swiss cheese. Most preferably, the fermented dairy product is Cheddar cheese.

[0193] Preferred conditions for one or more *Streptococcus thermophilus* strains have been described above. Furthermore, fermented dairy products preferably contain one or more nisin-producing bacterial strains, preferably *Lactococcus lactis* strains, more preferably *Lactococcus lactis* strains, even more preferably *Lactococcus lactis* subsp. *milk fat* strains and / or *Lactococcus lactis* subsp. *lactolaccus* strains, and / or residues of any of the above strains. These nisin-producing bacterial strains are preferably used for the production of nisin.

[0194] The inventors unexpectedly discovered that the presence of the endogenous hypothetical acetyltransferase TraX plays a crucial role in the salt-induced inactivation of nisin in *Streptococcus thermophilus*. To assess the expression status of the traX gene (i.e., to determine the presence of the Trax peptide), and in conjunction with the detection method defined herein, the ability of *Streptococcus thermophilus* strains to degrade approximately 50 U / ml nisin within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride was tested. This nisin was added to approximately 200 ml of a culture of this *Streptococcus thermophilus* strain inoculated with approximately 5% (v / v) of approximately 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.3.

[0195] Therefore, under non-salt-induced nisin degradation activity (N-SAND), Streptococcus thermophilus strains were able to degrade less than 50% of 50 U / mL nisin under the above and further conditions, i.e., no gene product or TraX protein was detected.

[0196] Under low-salt-induced nisin degradation activity (L-SAND), a Streptococcus thermophilus strain was able to degrade at least 50% to at least 70% of 50 U / mL nisin under the above and further conditions. This strain expresses the traX gene, and the traX gene product or TraX protein can be detected.

[0197] In one embodiment, the present invention relates to a method for converting a strain exhibiting a P-SAND phenotype into an L-SAND or N-SAND phenotype (preferably N-SAND phenotype), wherein the method comprises:

[0198] (1) Provides a thermophilic streptococcal strain with well-defined salt-associated nisin inactivation activity, wherein the strain is capable of degrading 70% or more of 50 U / ml nisin within 90 minutes at about 35°C in the presence of 5% (w / v) sodium chloride under the conditions defined herein.

[0199] (2) Modify the genome of the strain to inactivate the endogenous gene encoding the acetyltransferase TraX, for example, the protein according to SEQ ID NO: 2 or 3, for example, by introducing a knockout of the gene.

[0200] (3) Select strains that can degrade less than 50% of 50 U / ml nisin within 90 minutes at about 35°C in the presence of 5% (w / v) sodium chloride, and do not show any TraX RNA or any translated active TraX protein.

[0201] Modifications to the cellular genome are determined by comparing the DNA sequence of the (mutated) bacterial cell with that of a parent or reference cell (e.g., according to the traX of SEQ ID NO: 1). DNA sequencing and genome sequencing can be performed using standard methods known to those skilled in the art, such as Sanger sequencing technology and / or next-generation sequencing technologies, such as Illumina GA2, Roche 454, Nanopore, etc., as detailed in Elaine R. Mardis's review (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9: 387-402 (doi:10.1146 / annurev.genom.9.081307.164359).

[0202] The TraX deficiency described herein can be determined by the following methods: a salt-induced nisin inactivation assay as described herein, or any assay suitable for determining the activity of the peptides defined herein, which may be used by those skilled in the art, such as transcriptome analysis, Northern blotting, RT-PCR, Q-PCR, and / or Western blotting. Specifically, the quantification of mRNA present in cells can be achieved, for example, by Northern blotting (see Molecular Cloning: A Laboratory Manual, Sambrook et al., New York: Cold Spring Harbour Press, 1989). The quantification of the peptides described herein in cells can be achieved, for example, by Western blotting. Differences in mRNA levels can also be quantified by DNA array analysis or RNA sequencing (Eisen, MB and Brown, PO DNAarrays for analysis of gene expression. Methods Enzymol. 1999, 303:179-205).

[0203] Mutant bacterial cells may contain one or more modifications. Modification of bacterial cells can be achieved in the following ways:

[0204] a) (Classical) mutagenesis of bacterial cells; and / or

[0205] b) Recombinant gene manipulation techniques or genome editing techniques on bacterial cells; and / or

[0206] c) Expose bacterial cells to inhibitory compounds or compositions.

[0207] This document defines modification of the genome of a filamentous fungal host cell (mutant) as any event that results in an alteration of the TraX sequence in the cell's genome. In a preferred embodiment, the genome of the mutant microbial host cell described in this disclosure is modified.

[0208] The method according to the invention can effectively reduce cracks and fissures in such cheeses.

[0209] On July 20, 2023, in accordance with the provisions of the Budapest Treaty, Streptococcus thermophilus strain CBS 150251 was deposited at the Westdike Institute for Fungal Biodiversity (formerly known as CBS, Centralbureau voor Schimmelcultures), 8 Uppsalaland, Utrecht 3508 AD, Netherlands.

[0210] On July 20, 2023, in accordance with the provisions of the Budapest Treaty, Streptococcus thermophilus strain CBS 150252 was deposited at the Westerdike Institute for Fungal Biodiversity (formerly known as CBS, Centralbureau voor Schimmelcultures) at Uppsalaland 8, Utrecht 3508 AD, Netherlands.

[0211] This invention particularly relates to the following embodiments:

[0212] (1) A method for producing fermented dairy products, comprising:

[0213] (a) Fermenting a milk base in the presence of a bacterial culture to produce a fermented milk base;

[0214] (b) Contact the fermented milk base with nisin;

[0215] (c) Optionally, the fermented milk base is brought into contact with the coagulant;

[0216] (d) Contact at least a portion of the fermented milk base with salt;

[0217] The bacterial culture contains a Streptococcus thermophilus strain that has little or no salt-associated nisin inactivation activity.

[0218] (2) The method according to embodiment (1), wherein (a) includes fermenting the milk base in the presence of a bacterial culture until the pH value is equal to or less than 6.0, more preferably equal to or less than 5.3.

[0219] (3) The method according to implementation scheme (1) or (2), wherein (a) and (b), and optionally (c), are performed simultaneously.

[0220] (4) The method according to implementation scheme (1), (2) or (3), wherein (b) includes adding nisin during or after fermentation according to (a).

[0221] (5) The method according to embodiments (1), (2), (3) or (4), wherein (b) includes the in situ generation of nisin during or after fermentation according to (a), and wherein preferably, the nisin is generated in situ with the assistance of one or more nisin-producing bacterial strains.

[0222] (6) The method according to embodiments (1), (2), (3), (4) or (5), wherein the coagulant in (c) comprises or is composed of a protease, preferably an aspartic protease produced by a microorganism or mammal.

[0223] (7) The method according to embodiments (1), (2), (3), (4), (5) or (6), wherein: the method comprises (a), (b) and (c), and wherein (a), (b) and (c) together constitute a step comprising fermenting and coagulating a milk base in the presence of a bacterial culture, in the presence of nisin and in the presence of a coagulant to produce, or constitute, a coagulated fermented milk base; and

[0224] —The method includes a subsequent step in which the fermented milk base is separated into curd and whey, and the curd is contacted with salt, or constituted thereof.

[0225] (8) The method according to implementation scheme (1), (2), (3), (4), (5), (6) or (7), wherein the salt comprises or is composed of sodium chloride.

[0226] (9) The method according to embodiments (1), (2), (3), (4), (5), (6), (7) or (8), wherein the bacterial culture is part of a bacterial culture mixture comprising or consisting of: (i) one or more Streptococcus thermophilus strains, wherein at least one Streptococcus thermophilus strain, preferably each Streptococcus thermophilus strain, has no or only limited salt-associated nisin inactivation activity; and (ii) optionally, one or more bacterial strains that are not Streptococcus thermophilus strains.

[0227] (10) The method according to embodiments (1), (2), (3), (4), (5), (6), (7), (8) or (9), wherein the bacterial culture comprises or is composed of: (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain does not have or only has limited salt-associated nisin inactivation activity; and (ii) one or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus lactis strains, more preferably nisin-producing Lactococcus lactis subsp. milk fat strains and / or nisin-producing Lactococcus lactis subsp. lactis strains; and (iii) optionally, one or more bacterial strains that are not Streptococcus thermophilus strains or nisin-producing strains, preferably Lactobacillus and / or Lactococcus lactis bacterial strains.

[0228] (11) The method according to implementation schemes (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10), wherein the thermophilic streptococcal strain that does not have or only has limited salt-associated nisin inactivation activity comprises or consists of the following strains: CBS 150251 strain deposited at the Westdike Institute for Fungal Biodiversity Research (CBS) Utrecht, Netherlands on July 20, 2023 and / or CBS 150252 strain deposited at the Westdike Institute for Fungal Biodiversity Research (CBS) Utrecht, Netherlands on July 20, 2023 and / or any variant thereof that does not have or only has limited salt-associated nisin inactivation activity.

[0229] (12) The method according to implementation scheme (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11), wherein the fermented dairy product is cheese, preferably cheddar cheese.

[0230] (13) A starter culture, bacterial culture mixture, or kit comprising or consisting of: (i) one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and (ii) one or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus lactis strains, more preferably nisin-producing Lactococcus lactis subsp. milk fat strains and / or nisin-producing Lactococcus lactis subsp. lactis strains; and (iii) optionally, one or more bacterial strains that are not Streptococcus thermophilus strains or nisin-producing strains, preferably Lactobacillus and / or Lactococcus lactis bacterial strains; and (iv) optionally, one or more non-bacterial cryoprotectants and / or non-bacterial additives.

[0231] (14) The fermentation agent, bacterial culture mixture or kit according to embodiment (13), wherein components (i) and / or (ii) and optional components (iii) and / or (iv) are provided in the form of individual freeze-dried or lyophilized granules.

[0232] (15) The fermenting agent, bacterial culture mixture or kit according to embodiment (13) or (14), wherein component (i) comprises or consists of the following strains: strain CBS 150251 deposited at the Westdike Institute for Fungal Biodiversity Research (CBS) Utrecht, Netherlands on July 20, 2023 and / or strain CBS 150252 deposited at the Westdike Institute for Fungal Biodiversity Research (CBS) Utrecht, Netherlands on July 20, 2023 and / or any variant thereof that does not have or only has limited salt-associated nisin inactivation activity.

[0233] (16) A fermented dairy product, preferably cheese, more preferably cheddar cheese, comprising: (i) salt, preferably sodium chloride or calcium chloride; (ii) one or more Streptococcus thermophilus strains and / or residues of one or more Streptococcus thermophilus strains, wherein each Streptococcus thermophilus strain has little or no salt-associated nisin inactivation activity; and (iii) nisin, preferably nisin A. Attached Figure Description

[0234] Figure 1 Schematic diagram of nisin inactivation test.

[0235] Figure 2 An exemplary calibration curve for nisin quantification, wherein the average halo size (mm) of the calibration is plotted against the natural logarithm of the nisin concentration (Ln (units of nisin / ml)). Detailed Implementation

[0236] The following embodiments are merely examples and are not intended to limit the scope of the invention in any way. All references, patent applications, patents, and published patent applications cited in this application are incorporated herein by reference.

[0237] Example

[0238] Example 1: General Methods and Materials

[0239] All basic molecular biology and DNA manipulation procedures described herein were generally performed in accordance with Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al., Current Protocols in Molecular Biology. Wiley: New York (1998).

[0240] Strains and culture methods The list of strains used in these examples is shown in Table 1 below. M17 broth was prepared according to the manufacturer's instructions (BD Difco). TM Prepared with 1% w / v lactose (1 g / 100 ml), hereinafter referred to as LM17b. M17 agar was prepared according to the manufacturer's instructions (BD Difco). TMThe culture was prepared by adding 0.5% w / v lactose (0.5 g / 100 ml), hereinafter referred to as LM17a. Overnight cultures of all Streptococcus thermophilus strains were prepared by inoculating LM17b with 1% v / v bacterial solution and culturing at 37°C for 18 hours.

[0241] 12% (w / v) reconstituted skim milk (RSM) is prepared by dissolving 120 grams (g) of skim milk powder in 1 liter (L) of water.

[0242] Unless otherwise expressly stated herein, w / v percentages refer to grams per 100 ml of solution. When frozen concentrate is used in experiments, it refers to a commercially available product used in the dosage specified in the experiment.

[0243] Table 1. List of strains. See the main text for more details.

[0244]

[0245] Screening assay The salt-associated nisin inactivation activity (especially sodium chloride-associated nisin inactivation activity) of *Streptococcus thermophilus* strains can be characterized by screening *Streptococcus thermophilus* strains using the following screening method: the method includes steps A through G, wherein steps A through B describe culture and sample generation, steps C through E describe the quantification of nisin in the sample, and step F describes the characterization of strains based on steps A through E. Figure 1 A schematic diagram is provided. By performing the following steps, *Streptococcus thermophilus* strains can be characterized as having explicit salt-associated nisin inactivation activity (P-SAND), limited salt-associated nisin inactivation activity (L-SAND), or no salt-associated nisin inactivation activity (N-SAND):

[0246] A) Culture formation, in which

[0247] - Individual pure cultures of each specific Streptococcus thermophilus strain tested in LM17 broth (Difco TM M17 broth with 1% (w / v) lactose was inoculated at a 1% v / v inoculum and incubated at 37°C for 18 hours; and

[0248] - For each strain, the resulting culture was used as inoculum and inoculated at 5% (v / v) into 200 ml of 12% (w / v) reconstituted skim milk (RSM) supplemented with 15 ppmw sodium formate in a sterile 250 ml Schott flask (“RSM flask”). The culture was then grown at 38°C to pH 5.3 to generate “12% RSM culture at pH 5.3”.

[0249] B) Sample generation, in which

[0250] - When the pH reaches 5.3, add nisin to a final concentration of 50 U / ml and sodium chloride to a final concentration of 5% (w / v) to the "12% RSM culture at pH 5.3", and then continue incubation at 35°C for 90 minutes to obtain the "incubated culture"; and

[0251] - Subsequently, 50 ml of sample (referred to as "t=1 culture sample") was taken from the "incubated culture" of the RSM bottle for nisin quantification and stored in a 50 ml Greiner centrifuge tube for subsequent sample processing and testing.

[0252] C) Preparation of nisin stock solution, wherein...

[0253] - A stock solution of nisin (nisin: Sigma Aldrich, N5764) with a concentration of 10,000 units per milliliter (U / ml) was prepared in 0.05% (v / v) acetic acid solution, filtered through a 0.22 µm pore size filter membrane, and stored at 4°C; and

[0254] - Using this stock solution, a certain concentration of nisin is added to each of the 5 ml "blank filtrate" (step D) samples to generate "calibration samples" for calibration curves, thereby enabling the generation of accurate calibration curves.

[0255] D) Culture and calibration sample processing, where

[0256] - An uninoculated sterile 12% RSM solution as described above was used as the zero-lactamase reference sample and sample background (“blank filtrate”) to generate a nisin calibration curve; therefore, the 50 ml sterile 12% RSM solution reference sample was treated in the same manner as all individual fermentation samples at t=1 (“t=1 culture samples”) described herein; and

[0257] - The pH of all "t=1 culture samples" and sterile "12% RSM solution" samples was lowered to 2.0 ± 0.05 with hydrochloric acid (HCl), and a first centrifugation was performed at 10,000 rpm for 10 minutes at 4°C; and

[0258] - Transfer the supernatant from the first centrifugation to a new Greiner centrifuge tube, adjust the pH to 4.0 ± 0.05 with sodium hydroxide, and then perform a second centrifugation at 10,000 rpm for 10 minutes at 4°C; and

[0259] - Filter the supernatant from the second centrifugation through a 0.45-micron (µm) filter membrane (e.g., a Millipore membrane) and transfer it to a new test tube. For each individual "t=1 culture sample," the supernatant from the second centrifugation is the final solution used for pore diffusion determination of nisin; while for sterile "12% RSM solutions," the supernatant from the second centrifugation is a 0 U / ml nisin blank sample solution, i.e., "blank filtrate," used to generate the nisin concentration determination sample required for calibration curves; and

[0260] - Subsequently, using a 10,000 U / ml nisin stock solution, a certain amount of nisin was added to a 5 ml "blank filtrate" sample. The concentration of nisin ranged from 1 to 100 U / ml (at least 5 concentrations were tested, such as 100, 50, 20, 5, and 1.25 U / ml, with a concentration range of 1 to 100 U / ml), to generate a "calibration sample" for plotting a nisin concentration calibration curve, thereby enabling the generation of an accurate calibration curve;

[0261] E) Perform halo-based screening on agar containing indicator strains, where

[0262] LM17 agar plate (Difco) TM M17 agar (with 1% lactose added, w / v) was heated until liquefied, cooled to 46°C, and then inoculated with 1% (v / v) of *Lactococcus lactis* subsp. *liquid fat* HP indicator strain culture in the exponential growth phase (NCDO607, ATCC19257, DSM 20069; this strain is sensitive to nisin and can be used to determine nisin concentration because a halo appears after inoculation). The agar solution was immediately poured onto NUNC agar. TM On a square bioassay dish (catalog number: 166508), ensure the agar surface is uniform, then cool the agar to 20°C and dry it in a laminar flow cabinet; and

[0263] - Use sterile glass pipettes to punch wells in agar at regular intervals, and add 150 µl of "calibration sample," blank sample (0 U / ml), or "t=1 culture sample" to each well for measurement; and

[0264] - Incubate the petri dish at 30°C for 18 hours; and

[0265] -Then, use a digital caliper (Mitutoyo 150mm digital caliper, 500-181-30) to record the horizontal and vertical diameters of any visible halo;

[0266] - Plot the average horizontal and vertical diameters of each halo in each "calibration sample" on the x-axis, and plot the natural logarithm of the nisin concentration in that "calibration sample" on the y-axis; and

[0267] - By plotting the best-fit line through all measurement data points, the relationship curve between halo size and nisin concentration is obtained, thus yielding the "calibration curve".

[0268] Table 2 below shows examples of calibration sample diameter readings for agar halo measurements performed in the following embodiments. Figure 2 The data points and the best-fit line (i.e., the derived calibration curve) are displayed.

[0269] Table 2. Calibration curves used in the following examples. Please see the main text for further details.

[0270]

[0271] Subsequently, the nisin concentration of the "t=1 culture sample" can be derived using the "calibration curve" and halo size. Table 3 below lists the nisin concentrations of the strains used in the examples. The nisin concentration can then be used for characterization in step F).

[0272] F) Strain characterization, among which

[0273] -Based on the above criteria, specific strains are characterized based on the nisin concentration at t=1, i.e.:

[0274] - If a Streptococcus thermophilus strain can degrade 70% or more of the added 50 U / ml nisin, it can be characterized as a Streptococcus thermophilus strain with defined salt-associated nisin inactivation activity (P-SAND). In other words, if a Streptococcus thermophilus strain degrades 70% or more of the added 50 U / ml nisin at t=1, it is considered to have defined salt-associated nisin inactivation activity.

[0275] - If a Streptococcus thermophilus strain can degrade 50% or more, but less than 70% of the added 50 U / ml nisin, it can be characterized as having limited salt-associated nisin inactivation activity (L-SAND). That is, if a Streptococcus thermophilus strain degrades 50% but less than 70% of the added nisin at t=1, the strain is considered to have limited salt-associated nisin inactivation activity; and

[0276] - If a Streptococcus thermophilus strain can degrade less than 50%, more preferably less than 40%, even more preferably less than 30%, even more preferably less than 20%, and most preferably less than 10% of the added 50 U / ml nisin, it can be characterized as a Streptococcus thermophilus strain without salt-associated nisin inactivation activity (N-SAND). That is, if a Streptococcus thermophilus strain degrades less than 50%, more preferably less than 40%, even more preferably less than 30%, even more preferably less than 20%, and most preferably less than 10% of the added U / ml nisin at t=1, it is considered that the strain does not have salt-associated nisin inactivation activity.

[0277] RNA extraction, purification, and expression analysis (RNA sequencing). In the RNA extraction experiment, the bacterial strain was grown at 37°C for 16 hours under conditions containing 150 U / ml nisin and without nisin. The cells were centrifuged and concentrated, and then RNA was extracted using the EXTRACTME TOTAL RNA KIT (Qiagen) kit protocol with slight modifications. In short, bacterial cells (approximately 10...) were... 10 Samples were washed three times with phosphate-buffered saline (PBS, pH 7.0) and resuspended in TE buffer (10 mM Tris-HCl, containing 1 mM EDTA-Na2, pH 8.0) containing proteinase K (30 U / mg) and lysozyme (100,000 U / mg). After incubation at 37°C for 30 min, samples were resuspended in RTL buffer (4 M guanidine thiocyanate, 50 mM Tris-HCl, 25 mM EDTA, 3% (v / v) Triton X-100 and 1% 2-mercaptoethanol) and sonicated at an amplitude of 8 dB for 10 seconds per cycle for a total of 3 cycles. RNA extraction was then performed according to the manufacturer's instructions (Qiagen). RNA purity was assessed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was assessed by 2% agarose gel electrophoresis and an Agilent 5400.

[0278] According to the manufacturer's instructions, samples were sequenced using the Illumina NovaSeq 6000 platform in 150 paired-end sequencing mode (2Gb raw output per sample) (RNAseq). Illumina raw reads were analyzed using FastQC (v0.11.9), and adapters were removed and low-quality reads were pruned using Trimmomatic (v0.39). The pruning parameters were as follows: [SLIDINGWINDOW:4:20 AVGQUAL:28 MINLEN:60]. The pruned reads were then verified using FastQC. Bowtie2 (v2.4.4) was then used to align the pruned reads to the provided Streptococcus thermophilus ST01 sequencing genome. Gene expression levels were quantified using featureCounts (v2.0.3) on the aligned reads. Genomic features used for read assignment were genes. Differential analysis of expressed genes was performed using RStudio (v4.3.2) and DESeq2 (v1.42.0). The rlog function is used to stabilize and normalize the variance of the count data.

[0279] To identify differentially expressed genes during growth in the presence or absence of nisin (see above), a volcano plot was generated using Enhanced Volcano (v1.20.0) to visualize the results of the differential expression analysis. The plot uses the log2 fold change value as the x-axis and the -log10 value of the corrected p-value (false discovery rate, FDR) as the y-axis. Genes meeting the significance criteria are highlighted in the volcano plot. Previously identified significant genes were plotted as bar charts using GraphPad Prism (v10.0.0).

[0280] Example 2: Characteristic analysis of Streptococcus thermophilus strains

[0281] Using the screening method described in Example 1 above, the tested Streptococcus thermophilus strains were characterized as P-SAND, L-SAND, or N-SAND. The results are shown in Table 3. In this assay, 50 U / ml of nisin and 5% (w / v) sodium chloride salt were added. After incubation at 35°C for 90 minutes, only 9 U / ml and 7 U / ml of residual nisin were detected when using strains ST01 and ST02, respectively. Since these two strains degraded more than 70% of the nisin, and the nisin level was below 15 U / ml, they were characterized as having clear salt-associated nisin inactivation activity (P-SAND).

[0282] However, ST03, ST04, ST05, and ST06 could be characterized as having limited salt-associated nisin inactivation activity (L-SAND) because, in screening tests, these strains left only 25, 22, 24, and 19 U / ml of nisin, respectively, after 90 minutes. The highest nisin concentration was observed when using strain ST09, characterized as N-SAND.

[0283] Table 3. Screening results and characterization of the salt-associated nisin inactivation activity of each thermophilic streptococcal strain used in the examples. Nisin concentration (“nisin concentration”) was determined at t=1, in units / ml. See the main text for further details.

[0284]

[0285] This result clearly demonstrates that, in the presence of salt, fermentation with L-SAND Streptococcus thermophilus strains (see, for example, strains ST03, ST04, ST05, ST06, and ST08) results in lower levels of nisin degradation and higher levels of residual nisin.

[0286] Example 3: Genotypic characterization of P-SAND, L-SAND, and N-SAND strains

[0287] The enzyme responsible for inactivating nisin in Streptococcus thermophilus is called "nisinase" or "nisin inactivating enzyme." However, to date, the gene encoding this functional enzyme and its molecular structure have not been elucidated.

[0288] To identify the gene encoding nisinase, P-SAND strain ST01 (capable of degrading 70% or more of nisin at t=1) was grown at 37°C for 16 hours in the presence or absence of 150 U / ml nisin. RNA was then extracted from the cells according to the protocol described herein. Expression patterns with and without nisin showed that the gene encoding TraX (EMBL AZA18053.1) was most significantly upregulated in the presence of nisin.

[0289] The strain lacking (expressing) the traX gene was characterized and found to be an N-SAND strain, meaning it lacked the (functional) TraX protein when grown on nisin. This N-SAND strain contrasted with the P-SAND strain, where the gene expression was upregulated in the presence of nisin, resulting in the production of the functional TraX protein.

[0290] To test and confirm the function of TraX, the full-length traX gene (SEQ ID NO: 1) was knocked out in *Streptococcus thermophilus* ST01 (P-SAND) using standard genetic engineering methods, constructing a traX-deleted mutant strain. Whole-genome sequencing confirmed the deletion of the traX gene, yielding *Streptococcus thermophilus* ST01∆traX strain (ST09). Unexpectedly, strain ST09 was identified as having an N-SAND phenotype (Table 5). Furthermore, its growth rate, especially in the presence of nisin, was significantly reduced (see Table 4).

[0291] Characteristics of N-SAND strains may include: deletion of the traX gene, insufficient expression / induction of the traX gene, or lack of (functional) TraX protein when the strain is grown on nisin. This N-SAND strain type contrasts with the P-SAND strain type, in which the expression of the gene is upregulated in the presence of nisin, resulting in the production of functional TraX protein.

[0292] To investigate the function of the traX gene in *Streptococcus thermophilus* when grown on nisin, further experiments were conducted, particularly to evaluate the conditions required to convert strains with definite salt-associated nisin inactivation activity (i.e., P-SAND strains) into L-SAND or N-SAND strains.

[0293] Table 4. Growth of LM17b (LM broth) at 37°C for 48 hours in the presence of a specified amount (U / ml) of nisin, measured by OD600nm. See the main text for further details.

[0294]

[0295] Table 5. The role of TraX in the degradation of nisin in Streptococcus thermophilus. See the main text for more details.

[0296]

[0297] The *Streptococcus thermophilus* strain ST01, carrying the full-length *traX* gene, can grow in the presence of 500 U / mL nisin, and is therefore considered to be nisin resistant. Upon deletion of the *traX* gene, the nisin resistance of the P-SAND strain ST01 transforms into nisin sensitivity, and it can hardly grow in the presence of nisin. Therefore, the deletion of the *traX* gene in *Streptococcus thermophilus* ST01 (a P-SAND strain) results in the N-SAND phenotype of *Streptococcus thermophilus* ST09 as defined herein. The slow or non-growing growth of ST09, along with the accompanying N-SAND phenotype, does not pose a problem for the nisin degradation assays described herein or for the cheese-making methods using this strain, because the nisin content is extremely low or even zero during the initial fermentation stage, allowing the *Streptococcus thermophilus* strain to grow and acidify before the addition of nisin or before nisin exerts its effects.

[0298] Example 4: Characterization of TraX protein (genotype)

[0299] Whole-genome sequencing was performed on 121 Streptococcus thermophilus strains, and the traX gene was identified using BLASTn search based on the reference traX nucleotide sequence (SEQ ID NO:1) of Streptococcus thermophilus strain ST01. The results showed that all 121 strains contained the traX gene, indicating that the gene is highly conserved within this species.

[0300] For the tested strains ST01 to ST08, two variants listed in Table 6 were observed.

[0301] Table 6. TraX protein sequences of the strains used in the examples.

[0302]

[0303] Based on research findings on traX variants and their functional disruption, it is clear that sequencing the traX gene of a specific strain can serve as a biomarker for screening fermentation agents. Sequencing the traX gene in a strain can reveal whether it possesses definite / limited or salt-free nisin inactivation activity, especially in cases of active site mutations and / or insertions, deletions, or stop codons in the traX gene. Results from traX deletions indicate that certain traX mutations (stop codons in the coding sequence, mutated translation start codons, RBS or Shine-Dalgarno mutations) are more likely to produce the desired L-SAND or N-SAND phenotype. Therefore, gene screening can be used to pre-screen preferred strains or to select candidate mutant strains during screening.

[0304] Example 5: Production of Salty Cheddar Cheese

[0305] The effects of different bacterial strains on nisin levels in the production of savory cheddar cheese were evaluated using nisin-producing starter cultures. Two starter cultures were used in the production of savory cheddar cheese: one consisting solely of a nisin-producing lactococcus strain, and the other a mixture of a nisin-producing lactococcus strain and a nisin-immune lactococcus strain (see Table 7).

[0306] As a reference (“reference mixture (nis-)”), a bacterial starter composed of Lactococcus species suitable for cheddar cheese was used, which does not produce nisin.

[0307] In addition, two bacterial fermentation agents that produce lactic acid nisin were used:

[0308] - "Mixture A" consists of two lactococcal strains that produce nisin, namely Lactococcus lactis (L01) and Lactococcus lactis (L02); and

[0309] - "Mixture B" consists of four lactic acid bacteria strains that produce nisin: Lactococcus lactis (L01), Lactococcus lactis (L02), Lactococcus fattya (L03), and Lactococcus fattya (L04).

[0310] To make cheddar cheese, fresh milk is pasteurized at 73°C for 15 seconds, then cooled to 32°C. A specific bacterial starter is added, and the milk is fermented at 31-32°C for 60 minutes with the help of the bacterial strain. Afterward, a curdling agent (Maxiren) is added. ® XDS (40 IMCU / L milk). After 20-30 minutes, cut the formed curd (composed of curd and whey) and gently stir for 10 minutes. Then, raise the temperature to 38°C over 30 minutes. When the pH of the curd reaches 6.2, drain the whey from the curd and lower the temperature to approximately 36°C during the subsequent cheddar cheese-making stage. When the pH of the curd reaches 5.2-5.3, grind the curd. Then, salt the curd with sodium chloride to achieve a target salt content of approximately 2% (w / w) in the final product and press overnight at room temperature (approximately 20°C).

[0311] After pressing, samples were taken for component analysis. Then, the cheddar cheese blocks were vacuum sealed and aged at 11°C for up to 12 months.

[0312] Table 7. Cheese production without the use of Streptococcus thermophilus strains. Specific items TTH and TTF are in hours, where TTR refers to "time required to reach the target pH of 6.2 (for draining)," and TTF refers to "time until completion, i.e., the time required from the addition of the starter culture to the final pH reaching 5.2–5.3." See the main text for more details.

[0313]

[0314] As shown in Table 7, the time required to reach the target pH of 6.2 (“TTR pH 6.2”) was similar in the reference mixture (“Reference Mixture (nis-)”) without nisin, as well as in mixtures A and B, at approximately 2.8 hours. Table 6 also shows that at pH 5.2, the nisin content in mixtures A and B reached 196 U / g and 248 U / g, respectively, while the nisin content in the reference mixture was 0. The increase in nisin content over time indicates that nisin production continues during the maturation process (see the last few columns of Table 7). Therefore, we can conclude that the addition of salt during cheese making does not impair nisin production by *Lactococcus lactis*, nor does it lead to nisin inactivation.

[0315] Next, the method for making cheddar cheese is the same as described above, except that this time a lactic acid-producing bacterial starter is used, which also contains the following thermophilic streptococcal strains (see Table 8):

[0316] - "Mixture 1" consists of the following components: (i) 80% of a reference mixture containing strains of *Lactococcus lactis* (L01), *Lactococcus lactis* (L02), and *Lactococcus lactis* (L03), all of which are lactococcal strains producing nisin; and (ii) 20% of L-SAND *Streptococcus thermophilus* strain (ST06); and

[0317] - "Mixture 2" consists of the following components: (i) 80% of a reference mixture containing strains of Lactococcus lactis (L01), Lactococcus lactis (L02) and Lactococcus fat (L03), all of which are lactococcal strains that produce nisin; and (ii) 20% of P-SAND Streptococcus thermophilus strain (ST07).

[0318] When thermophilic streptococci are present during acidification, the time to completion (TTF), i.e. the time required from the addition of the starter culture to the final pH reaching 5.2–5.3, is faster than when only lactococci are added.

[0319] Table 8. Cheese production with (1 / 2) / without (A / B) Streptococcus thermophilus strains. Specific TTH and TTF are in hours, where TTR refers to "time required to reach the target pH of 6.2 (for draining)," and TTF refers to "time until completion, i.e., time required from the addition of the starter culture to the final pH reaching 5.2–5.3." See the main text for further details.

[0320]

[0321] nd = Undetermined

[0322] As expected, at pH 5.3, the nisin content was significantly higher when using the L-SAND strain than when using the P-SAND strain on day 1 after TTF (Table 8). This clearly demonstrates the positive effect of using the L-SAND strain in cheese making, not only increasing the fermentation rate but also increasing the nisin content in the cheese matrix. The effect of the N-SAND strain was even more significant than that of the L-SAND strain (see Example 6 in this article). From 30 days after aging until the final inspection at 150 days after aging, the cheese made from "Mix 1" containing the L-SAND strain had fewer cracks and fissures compared to "Mix 2" containing the P-SAND strain.

[0323] Example 6: Reduction of NSLAB during cheese production (challenge test)

[0324] The quantity and gas-producing capacity of non-fermenting lactic acid bacteria (NSLAB) in the cheese matrix are closely related to the severity of cheese cracks and fissures. Cracks and fissures in cheese are undesirable to consumers, and in severe cases, can even cause the cheese to burst due to gas production, rendering it inedible. Most NSLABs are sensitive to low concentrations of nisin (≥5 U / ml). Therefore, the presence of nisin during milk processing can reduce the number of live NSLABs during milk acidification and cheese maturation, thereby reducing the formation of cracks and fissures.

[0325] A nisin inactivation test was performed on milk contaminated with 1.5E+05 CFU / ml Companilactobacillus nodensis as an indicator strain and representing NSLABs (as described in Example 1). Strains ST01 (P-SAND) and ST09 (N-SAND) were inoculated into the contaminated milk and incubated at 38°C until the pH reached 5.3 (t=0). Then, 50 U / ml nisin and 5% (w / v) sodium chloride solution were added, and the mixture was incubated at 35°C for 1.5 hours. A sample was then taken at t=1, and incubation continued at 32°C for 18 hours, followed by another sample at t=2.

[0326] Using LBS agar (BD Difco) TM NSLAB was counted (prepared according to the manufacturer's instructions) to show the effect of the presence of P-SAND or N-SAND Streptococcus thermophilus strains on NSLAB inactivation (Table 9), due to the different degradation levels of nisin by the two strains. In the presence of N-SAND Streptococcus thermophilus strains, the reduction in NSLAB was 1.2 log CFU / ml at t=1 and >4.2 log CFU / ml at t=2. In the presence of P-SAND Streptococcus thermophilus ST01 strain, the reduction in NSLAB at both time points was significantly reduced, specifically 0.2 log CFU / ml at t=1 and 1.3 log CFU / ml at t=2.

[0327] Table 9. Viable cell counts of NSLAB on LBS agar (expressed as colony-forming units per milliliter (CFU / ml)) at the different steps of the determination described in Example 1. See the main text for further details.

[0328]

[0329] Using N-SAND type Streptococcus thermophilus strains mixed with lactococcus lactis producing lactococci results in less lactococcus degradation. This means that compared to using P-SAND type Streptococcus thermophilus, the concentration of lactococcus lactis after salting is higher and lasts longer. The same effect can be expected if lactococcus lactis is added early in cheese making, rather than through in-situ production with lactococcus lactis producing lactococci. By using N-SAND type Streptococcus thermophilus strains, the effective concentration of lactococcus lactis is higher due to reduced degradation. This means that the viable count of lactococcus lactis-sensitive NSLAB cells will decrease due to the (elevated) presence of lactococcus lactis, thus preventing cracks and fissures during cheese aging.

[0330] On the other hand, when cheese is made using P-SAND type Streptococcus thermophilus strains mixed with nisin-producing lactic acid bacteria, the nisin content after salting is approximately 5 to 0 units / g. Lower nisin concentrations lead to reduced NSLAB inactivation, thus increasing the potential for cracks and fissures in the cheese. This is consistent with the results observed when using N-SAND type Streptococcus thermophilus strains, where nisin content is higher than with P-SAND type Streptococcus thermophilus strains (see Examples 3 and 4).

[0331] When using L-SAND type thermophilic streptococcal strains in combination with nisin-producing lactococci, a benefit of reduced nisin degradation is expected during salting and cheese making compared to P-SAND type strains. Therefore, using N-SAND and / or L-SAND type thermophilic streptococcal strains in combination with nisin-producing lactic acid bacteria during cheese making, or adding nisin early in the cheese-making process instead of in-situ production via nisin-producing lactic acid bacteria, offers significant advantages.

[0332]

[0333]

Claims

1. A bacterial culture mixture for producing savory fermented dairy products, the method being carried out in the presence of nisin and salt, preferably with a salt concentration of 1% to 5% (w / w) of the final concentration, the mixture comprising: (i) One or more strains of Streptococcus thermophilus, (ii) A starter culture comprising one or more strains of lactococcus and / or lactobacillus, preferably lactococcus and / or lactobacillus strains that produce nisin, more preferably strains selected from subsp. latifolium and / or subsp. lactococcus lactis. (iii) Optionally, one or more bacterial strains that are not thermophilic streptococcal strains or lactobacillus and / or lactococcal strains; (iv) Optionally, one or more non-bacterial cryoprotectants and / or non-bacterial additives; in, The one or more *Streptococcus thermophilus* strains exhibit limited salt-associated nisin inactivation activity, assessed by means of the ability to degrade 70% or less of 50 U / ml nisin within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride, wherein the nisin is added to 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution inoculated with approximately 5% (v / v) of such *Streptococcus thermophilus* strains, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.3; preferably, less than 50% of 50 U / ml nisin is degraded within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride. U / ml nisin, wherein the nisin was added to a culture of approximately 5% (v / v) of such thermophilic streptococcal strains inoculated into approximately 200 ml of approximately 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.

3.

2. The bacterial culture mixture according to claim 1, comprising one or more strains of Streptococcus thermophilus, wherein, The salt-induced inactivation of nisin was eliminated, and this activity was evaluated by testing its ability to degrade less than 50% of 50 U / ml nisin within 90 minutes at approximately 35°C in the presence of 5% (w / v) sodium chloride, wherein the nisin was added to 200 ml of a culture of approximately 5% (v / v) of this type of thermophilic streptococcus strain inoculated with approximately 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with approximately 15 ppm sodium formate, and grown at approximately 38°C to pH 5.3; and wherein the strain was modified as follows: (1) The presence of a nonfunctional TraX enzyme in terms of lactic acid degradation when expressing acetyltransferase TraX, particularly when expressing the endogenous gene of the TraX protein according to SEQ ID NO: 2 or 3. (2) The endogenous gene sequence expressing the acetyltransferase TraX, for example, based on a mutation in a polynucleotide of SEQ ID NO: 1, wherein the mutation is selected from gene knockout or nonsense mutation; (3) No translational modifications of active TraX RNA were detected.

3. The bacterial culture mixture according to claim 1, comprising one or more Streptococcus thermophilus strains having limited salt-associated nisin inactivation activity and containing and expressing the acetyltransferase TraX, particularly the TraX protein according to SEQ ID NO: 2 or 3.

4. The bacterial culture mixture according to claim 3, wherein, The one or more Streptococcus thermophilus strains are selected from strain CBS 150251 and / or strain CBS 150252, both of which were deposited on July 20, 2023 at the Westdike Institute for Fungal Biodiversity (CBS) in Utrecht, Netherlands, and / or any variant thereof that does not have or only has limited salt-associated nisin inactivation activity.

5. The bacterial culture mixture according to any one of claims 1 to 4, wherein component (i) and / or (ii) and optional component (iii) and / or (iv) are provided in the form of individual freeze-dried or lyophilized granules.

6. A method for producing a Streptococcus thermophilus strain, the Streptococcus thermophilus strain lacking salt-associated nisin inactivation activity and used for the production of savory fermented dairy products, the method comprising: (1) A thermophilic streptococcal strain is provided that has a defined salt-associated nisin-inactivating activity, which is evaluated by testing its ability to degrade 70% or less of 50 U / ml nisin within 90 minutes at about 35°C in the presence of 5% (w / v) sodium chloride; wherein the nisin is added to a culture of about 5% (v / v) of such thermophilic streptococcal strain inoculated in about 200 ml of about 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with about 15 ppm sodium formate, and grown at about 38°C to pH 5.3; (2) Modify the genome of the strain to inactivate the endogenous gene encoding the acetyltransferase TraX, particularly the polypeptide according to SEQ ID NO: 2 or 3, preferably by introducing a knockout of the gene. (3) Select strains that can degrade less than 50% of 50 U / ml nisin within 90 minutes in the presence of 5% (w / v) sodium chloride at about 35°C and do not show any TraX RNA or TraX activity, wherein the nisin is added to a culture of such thermophilic streptococci inoculated with about 5% (v / v) of about 12% (w / v) reconstituted skim milk (RSM) in 200 ml of the culture, supplemented with about 15 ppm sodium formate, and grown at about 38°C to pH 5.

3.

7. A method for producing a savory fermented dairy product, preferably a hard or semi-hard cheese, using a bacterial mixture according to any one of claims 1 to 5, the method comprising: (a) Fermenting the milk base with the bacterial mixture, preferably fermenting to a pH value equal to or less than 6.0, more preferably equal to or less than 5.3; as well as (b) Providing nisin, preferably nisin A, to the fermented milk base; (c) Optionally, one or more coagulants are provided to the fermented milk base, preferably wherein the coagulant is selected from proteases, more preferably from aspartic proteases produced by microorganisms or mammals; and (d) Provide salt, especially sodium chloride, preferably in an amount of 1% to 5% (w / w).

8. The method according to claim 7, wherein steps (a), (b) and optional step (c) are performed simultaneously.

9. The method according to claim 7 or 8, wherein step (b) comprises adding nisin during or after fermentation in step (a).

10. The method according to any one of claims 7 to 9, wherein step (b) comprises the in situ generation of nisin during or after the fermentation of step (a), and wherein preferably, the nisin is generated in situ with the aid of one or more nisin-producing bacterial strains from the fermenting agent.

11. The method according to any one of claims 7 to 10, further comprising: (e) Separate the fermented and, if necessary, coagulated milk base into curd and whey; and (f) Add salt to the curd, wherein the salt is preferably sodium chloride.

12. The method according to any one of claims 7 to 11, wherein the savory fermented dairy product is cheese, preferably hard or semi-hard cheese, more preferably cheddar cheese.

13. Use of the bacterial mixture of any one of claims 1 to 4 in a cheese-making process, wherein the number of cracks is small, which means that the product can be sold at a good price.

14. A savory fermented dairy product, preferably cheese, more preferably cheddar cheese, comprising: (i) Salt, preferably sodium chloride or calcium chloride, more preferably with a final concentration of 1% to 5% (w / w); (ii) A bacterial mixture according to any one of claims 1 to 5, (iii) Nisin, preferably nisin A, more preferably a final concentration of 2 to 600 U / g.

15. A method for identifying a thermophilic streptococcal strain exhibiting reduced nisin degradation in the presence of salt, used in a bacterial mixture containing a lactococcal strain producing nisin for the production of a savory fermented dairy product, the method comprising: (i) Provide a library of Streptococcus thermophilus strains, (ii) A method for testing nisin degradation at a fixed salt concentration, the method measuring the ability of a Streptococcus thermophilus strain to degrade a certain amount of 50 U / ml nisin in the presence of about 5% (w / v) sodium chloride at about 35°C for about 90 minutes, the nisin being added to a culture of about 5% (v / v) of such Streptococcus thermophilus strain inoculated in about 200 ml of about 12% (w / v) reconstituted skim milk (RSM) solution, supplemented with about 15 ppm sodium formate, and grown at about 38°C to pH 5.3; (iii) Test the ability of this strain to degrade salt-associated nisin in the production of savory fermented dairy products, especially savory cheese. Strains showing a nisin inactivation rate of less than 50% under the conditions of step (ii) are classified as salt-free nisin-inactivating strains (N-SAND); strains showing a nisin inactivation rate of 50% to less than 70% under the conditions of step (ii) are classified as limited salt-associated nisin-inactivating strains (L-SAND); and strains showing a nisin inactivation rate of 70% to 100% under the conditions of step (ii) are classified as definitive salt-associated nisin-inactivating strains (P-SAND). (iii) Test the expression and presence of the endogenous acetyltransferase TraX in this strain, where the absence of TraX indicates that the strain is classified as N-SAND; (iv) Optionally, the strain of step (iii) may be tested in the presence of lactic acid bacteria that produce lactic acid.

Images