WO2025061902A1

WO2025061902A1 - Process for producing a fermented milk product with aid of strains of streptococcus thermophilus having no or limited salt-accompanied nisin-deactivating activity

Info

Publication number: WO2025061902A1
Application number: PCT/EP2024/076358
Authority: WO
Inventors: Irma Mathea Hendrina VAN RIJSWIJCK; Gwenda SCHAAD; Noël VAN PEIJ; Lourdes Mariela Serrano Davalos; Nikola Dragan POPOVIĆ; Nemanja Slobodan STANISAVLJEVIĆ
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-09-20
Filing date: 2024-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-20

Abstract

The present invention relates to a new process for producing a fermented milk product, preferably cheese. In addition, the present invention relates to a new bacterial culture, starter culture and kit of parts for use in such a process.

Description

PROCESS FOR PRODUCING A FERMENTED MILK PRODUCT WITH AID OF STRAINS OF STREPTOCOCCUS THERMOPHILUS HAVING NO OR LIMITED SALT-ACCOMPANIED NISIN-DEACTIVATING ACTIVITY

Cheese is a fermented milk product. Cheese can for example be produced by acidification of the milk and by coagulation of the milk casein in a so-called curdling process. The curd and whey that are formed during a cheese-making process can conveniently be separated. Hereafter the curd can be pressed into a cheese. The pressing can be followed by a ripening process to give the cheese its desired age.

Different types of cheese exist. So-called "hard" cheeses are popular because of their taste, texture, nutrition and long shelf-life. Examples of hard cheeses include Cheddar, Emmental, Grana Padano, Gruyere, Mimolette, Parmigiano, Parmesan and Pecorino.

To create the optimal cheese, cheese makers are using different biochemical mechanisms. Many cheese producers use starter cultures comprising lactic acid bacteria (LAB) to acidify the milk and to develop a desired texture. A LAB that is used in many starter cultures for this purpose is Streptococcus thermophilus. Other bacterial cultures, called adjuncts, can be used in the process for ripening and/or flavor development. Cheeses like Cheddar, Gouda, Gruyere, Parmesan, Provolone and Swiss, are also salted to create the desired flavor. The coagulation of the milk is generally carried out with the help of a coagulant. Both animal-derived coagulants such as rennet or chymosin as well as microbially produced coagulants or fermentatively produced chymosins are used.

In addition to the desired LAB in the starter culture, also undesired bacteria can be present during the cheese making process. These undesired bacteria, including the so-called non-starter lactic acid bacteria (NSLABs), are a nuisance to the cheese producers as their presence can result in cheese defects such as slits and cracks.

One of the major problems in the manufacture of cheese is the risk of spoilage by Gram-positive bacteria such as Clostridium, Staphylococcus, Bacillus, and Listeria. Particularly, the growth of Clostridia causes a notorious defect known as "late-blow" or "butyric blowing". Spores of Clostridia such as Clostridium tyrobutyricum can survive heat treatment of the milk and subsequent growth leads to butyric acid fermentation with concomitant gas (H₂, CO₂) production in the cheese-matrix and foul smelling. The production of gas results in excessively large cavities in the cheese and may result in swelling or even in explosion of the cheese.

It is known that nisin effectively inhibits the growth of Gram-positive bacteria in general and particularly in the production of processed and spread cheese. Nisin is therefore successfully being applied in the production of food to prevent spoilage by Gram-positive bacteria such as Clostridium, Staphylococcus, Bacillus, and Listeria. Interestingly, if the nisin is in situ produced by the starter culture, no labelling is required in the final dairy product.

EP1273237 describes a process for producing a fermented product, wherein in the fermentation step a starter culture is used comprising a nisin-producing strain of Lactococcus lactis and one or more nisin -resista nt non-Lactococcus lactis bacterial strain(s). Exemplified is the use of a nisin-producing starter culture consisting of DAIRYSAFE™ TC17 (L. lactis biovar diacetylactis) and 13M (nisin-immune L. cremoris) with 30% of nisin-resistant S. thermophilus for production of a Cheddar type cheese. The nisin production in the cheeses prepared with the nisin-producing starter culture is around 200 IU nisin per gram of cheese.

However, Streptococcus thermophilus strains and especially nisin-resistant Streptococcus thermophilus strains can deactivate nisin during the fermentation and acidification, resulting in low nisin concentrations at the start of the cheese ripening process. The nisin deactivation, especially by Streptococcus thermophilus strains, is enhanced and accelerated by the addition of salt during the fermentation and ripening process, as it is the case in production of e.g. salted cheese. The low nisin concentrations combined with the presence of NSLABs during ripening can subsequently result in the previous mentioned undesired cheese defects such as slits and cracks. At the same time, there is always a need for acidification speed in processes for the production of fermented milk products such as cheese. A higher acidification speed can be achieved by adding a higher concentration of Streptococcus thermophilus cells to speed up the acidification process. Hence, the addition of a high concentration of Streptococcus thermophilus cells is desirable. However, the presence of high concentrations of Streptococcus thermophilus during the fermentation process, resulting in low levels of nisin at the end of fermentation and before the start of a cheese ripening process, will have an increased negative impact on the flavour, appearance and especially on the shelf-life of the cheese product.

Garde et al. (International Journal of Food Microbiology, Vol. 96 (2004), pages 165 - 172) reported that the nisin-sensitive Streptococcus thermophilus I N I A 463 strain was lysed during the cheese ripening by the nisin produced by the adjunct Lc. lactis subsp. lactis strain, but that said S. thermophilus strain INIA 463 became nisin-resistant after exposure in skim milk to subminimal inhibitory concentrations of nisin (1 - 3 III/ ml) for less than 2 hours. Only intracellular unspecific nisin-degrading activity, also present in non-exposed culture, was found. It was speculated that the nisin resistance in S. thermophilus might be related to changes in the cell wall.

Thus, it would be an advancement in the art to provide an efficient, sufficiently fast process for producing a salted fermented milk product, especially salted cheese, whilst maintaining sufficient high concentrations of nisin at the end of fermentation and/or before ripening, wherein said process includes the use of starter cultures comprising strains of S. thermophilus showing reduced nisin deactivation upon the addition of salt, such as required in the production of salted cheese products. The identification, screening and generation of S. thermophilus strains with reduced to no nisin-deactivating activity induced by the presence of salt, particularly salt adding during the process of cheese manufacturing, is described herein.

Surprisingly, we now found a way of identifying, screening and generating such S. thermophilus strains showing no or low salt-accompanied nisin-deactivating activity to be used in the production of salted fermented milk products, particularly in the production of salted cheese.

Further surprisingly, we identified an endogenous S. thermophilus protein named TraX, a member of acetyltransferase family and known to be involved in acetylation of F pilin in E. coli, said TraX playing a crucial role in the induction of nisin-immunity in strains such as Streptococcus thermophilus. The traX gene could be identified in more than 120 sequenced Streptococcus thermophilus strains. Thus, and in contrast to what has been proposed in the prior art, deactivation of nisin in the presence of/ after the addition of salt is not mainly caused by lysis of the strains, such as lyses of Streptococcus thermophilus, but depending on enzymatic activity of TraX, being expressed in the presence of nisin.

Particularly, strains lacking a TraX protein, such as knock-out mutants for traX gene or strains wherein the expression of traX is not functioning, lost (part of) their nisin-deactivating activity after addition of salt during production of salted fermented milk product such as salted cheese. Such strains are particularly useful in the production of Cheddar cheese.

The present invention is furthermore directed to the identification and characterization of Streptococcus thermophilus strains showing upon the addition of (or in the presence of) salt no or limited nisin-deactivating activity in contrast to many known Streptococcus thermophilus strains showing release of intracellular nisin-deactivating activity in the presence of salt, such as conditions occurring in the production process of a salted fermented milk product, particularly salted cheese.

The present invention is furthermore directed to a process of converting an Streptococcus thermophilus strain showing degradation of nisin in the presence of salt added during the production process of a fermented milk product, such as e.g. in the production of salted cheese, into a strain that has reduced or no activity towards degradation of nisin in the presence of salt added during the production process of said fermented milk product, wherein said conversion comprises genetic modification in the expression of endogenous TraX as described furthermore herein.

Thus, the present invention is directed to an improved process for speedy production of salted fermented milk products, including but not limited to salted cheese, in the presence of the herein identified strains of Streptococcus thermophilus showing no or limited salt-accompanied nisin-deactivating activity, wherein a sufficiently high concentration of nisin is maintained at the end of fermentation and/or before ripening of the cheese, and wherein the use of said strains has no negative impact on the desired salty flavour, appearance, shelf-life and/or reduced spoilage during or after cheese ripening, particularly ripening of salted cheese. Particularly, the present invention is directed to a bacterial culture blend for production of salted fermented milk product, said process being performed in the presence of nisin and salt, preferably with a salt concentration of between 1 and 5 % final concentration (w/w), said blend comprising:

(i) one or more strains of Streptococcus thermophilus,

(ii) a starter culture comprising one or more strains of Lactococcus and/or Lactobacillus, preferably nisin-producing strains of Lactobacoccus and/or Lactobacillus, more preferably selected from strain(s) of Lactococcus lactis ssp. cremoris and/or Lactococcus lactis ssp. lactis;

(iii) optionally, one or more bacterial strains that are not Streptococcus thermophilus strain(s) or strains of Lactobacillus and/or Lactococcus bacteria;

(iv) optionally one or more non-bacterial cryoprotectants and/or non-bacterial additives. wherein the one or more strains of Streptococcus thermophilus are limited in the salt-accompanied nisin-deactivation activity as tested via its capability of degrading 70% or less of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride, wherein the nisin being added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3 ; preferably degrading less than 50% of 50U/ ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride, wherein the nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3.

More particularly, the present invention is related to a bacterial culture blend as defined herein comprising one or more strains of Streptococcus thermophilus wherein the salt-accompanied nisin-deactivation activity is nullified as tested via its capability of degrading less than 50% of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride, wherein the nisin is added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3; and wherein said strain in modified by:

(1) presence of a non-functional TraX enzyme with regards to nisin degradation upon expression of the endogenous gene expressing acetyltransferase TraX, particularly expressing TraX protein according to SEQ ID NO:2 or 3, but but the TraX enzyme is not functional in nisin degradation;

(2) mutation in the endogenous gene sequence expressing acetyltransferase TraX, e.g. a polynucleotide according to SEQ ID NO:1, wherein the mutation is selected from a knock-out of the gene or nonsense-mutations;

(3) translational modifications wherein no active TraX RNA is detected.

In one aspect, the present invention relates to a bacterial culture blend as defiend herein, comprising one or more strains of Streptococcus thermophilus with limited salt-accompanied nisin-deactivation activity and comprising and expressing acetyltransferase TraX, particularly TraX protein according to SEQ ID NO:2 or 3.

According to one aspect, the present invention is directed to a process for producing a fermented milk product, said process comprising:

(a) fermenting a milk-base in the presence of a bacterial culture or a blend of bacterial cultures as defined herein to produce a fermented milk-base;

(b) contacting the fermented milk-base with nisin;

(c) optionally contacting the fermented milk-base with a coagulant;

(d) contacting at least part of the fermented milk-base with a salt; wherein the bacterial culture comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or a limited salt-accompanied nisin-deactivating activity.

In some embodiments, the present invention provides a starter culture, bacterial culture blend or kit of parts comprising or consisting of:

(i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin- deactivating activity, preferably wherein the salt is sodium chloride, preferably wherein the nisin is nisin A; and (ii) optionally one or more nisin-producing bacterial strains, preferably wherein the nisin is nisin A, preferably wherein the bacterial strains are Lactococcus species such as Lactococcus lactis or Lactococcus cremoris strain(s); and

(iii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus and/or Lactococcus bacterial strains.

In some embodiments, the present invention provides a fermented milk product, preferably a cheese, more preferably a Cheddar cheese, comprising:

(i) a salt, preferably sodium chloride;

(ii) one or more Streptococcus thermophilus strain(s) and/or residues of one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin deactivating activity, preferably wherein the salt is sodium chloride, preferably wherein the nisin is nisin A; and

(iii) nisin, preferably nisin A.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art.

Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. a "strain", this means "at least one" of that strain, e.g. "at least one strain", unless specified otherwise.

When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

The term "milk" as used herein is intended to encompass milks from mammals and plant sources or mixtures thereof. Preferably, the milk is from a mammal source including but are not limited to cow, sheep, goat, buffalo, camel, llama, horse or reindeer. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. Bovine milk is preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof or reconstituted milk.

The term "milk-base" as used herein refers to a base composition, comprising milk according to the definition given herein or milk ingredients, or derived from milk or milk ingredients. The milk-base can be used as a raw material for the fermentation to produce a fermented milk product. The milk-base may for example comprise or consist of (fresh) skimmed or non-skimmed milk, or reconstituted milk. Optionally, the milk-base may be concentrated or in the form of a powder or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to a thermal (pre-)processing operation which is at least as efficient as pasteurization, including pasteurization or sterilization. In one preferred embodiment the milkbase has been subjected to thermal (pre-)processing, such as pasteurization or sterilization. The milk-base can be derived from a plant-based source or a mammal source. Preferably, the milk-base is derived from a mammal source, such as cow, sheep, goat, buffalo, camel, llama, horse or deer milk or any combination thereof. Preferably the milk-base is from a bovine source. Suitable plant-based sources of milk include but are not limited to soy, pea, peanut, barley, rice, oat, quinoa, almond, cashew, and coconut milk, with preference for soy, oat or almond. Most preferably, the milk-base is from a bovine source.

The term "nisin" as used herein refers to a nisin polypeptide, also referred to herein as a nisin peptide, nisin enzyme, or as a polypeptide, peptide or enzyme having "nisin" activity, all terms used interchangeably herein. Nisin is described in literature as a polypeptide having bacteriocin activity, preferably comprising in the range from 31 to 35 amino acids, preferably comprising a three- dimensional structure including five lanthionine rings. A lanthionine ring is understood in literature to refer to a structure comprising two alanine residues coupled via a thio-ether bridge (also referred to as a sulfide bridge), which can be represented as follows: HOOC-CH(NH2)-CH2-S-CH2-CH(NH2)-COOH). Preferred are natural nisin polypeptides, i.e., nisin polypeptides that were not created by genetic modifications. Examples of suitable natural nisin variants include all currently known natural variants of nisin: nisin A, nisin Z, nisin Q, nisin U, nisin U2, nisin F, nisin H, nisin 0, nisin J, nisin P, nisin G, and E nisin, see e.g., Sevillano et al. (International Journal of Molecular Science, vol. 24, 2023, pages 1-20). More preferred are nisin polypeptides produced and/or derived from Lactococcus lactis, preferably nisin A, nisin Z, and nisin Q, and/or nisin U, more preferably wherein the nisin is a natural nisin as defined herein. Preferred nisin polypeptides thus include the nisin polypeptides selected from the group consisting of nisin A, nisin Z, nisin Q, and nisin U, more preferably wherein the nisin polypeptide is selected from the group consisting of nisin A, nisin Z, and nisin Q, most preferred is nisin A (see e.g. 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, preferably produced and/or derived from Lactococcus lactis. According to all aspects of the present invention, the nisin that is brought into contact with the milk base, the bacteria including the bacterial culture blend as defined herein, is either added to the fermentation medium or is provided via in-situ generation during or after the fermentation, particularly in-situ generation by one or more Lactococcus strains in the starter culture, thus the nisin is said to be dispersed into the milk base in contrast to addition of nisin on the surface of a fermented milk product.

As used herein, the term "IMCU" is understood to refer to International Milk Clotting Units. One IMCU equals about 0.126 nmol of bovine chymosin B (e.g., chymosin products marketed as Maxiren® or CHY-MAX®). The strength of a milk clotting enzyme (such as chymosin enzyme) is determined as the milk clotting activity (IMCU per ml or per gram) in accordance with the standard as set by the International Dairy Federation (IDF) in ISO 118151 IDF standard 157A:1997 as prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). As used herein, "chymosin" typically indicates an aspartic protease, EC 3.4.23.4 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB. Chymosin is naturally produced by gastric chief cells in juvenile mammals. Chymosin is the main enzymatic component in rennet. Calf rennet is obtained of the lining of the abomasum (the fourth and final, chamber of the stomach) of young, unweaned calves.

As used herein, any references to %w/v, such as for example references to 12% w/v reconstituted skim milk (RSM), refer to weight in grams present per volume of 100 ml solution, for example 12% w/v RSM corresponds to 12 grams of skim milk powder dissolved per 100 ml water.

According to one aspect, the present invention is directed to a process of fermenting of a milk-base in the presence of a bacterial culture or a blend of bacterial cultures to produce a fermented milk-base, wherein the bacterial culture or bacterial culture blend comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or limited salt- accompanied nisin-deactivating activity.

The present invention is furthermore directed to a starter culture, bacterial culture blend, or kit of parts comprising a Streptococcus thermophilus strain that has no or limited salt-accompanied nisin-deactivating activity.

The terms "deactivating nisin", "deactivation of nisin", "nisin-deactivating", "inactivating nisin", "inactivation of nisin", "degradation of nisin" and "nisin- degrading" referred to herein are used interchangeably herein. By a nisin- deactivating activity is herein preferably understood that the Streptococcus thermophilus strain, directly or indirectly, partly or completely, deactivates nisin e.g. the antibacterial functionality of nisin for example by nisin modification and/or degradation in a solution comprising both nisin and the Streptococcus thermophilus strain. Particularly, said nisin deactivation is initiated upon the presence of salt that is added in the production process of a fermented milk product, such as particularly in the production of salted cheese, such as e.g. in the production of Cheddar cheese. The amount of salt added/present in such process depends on the kind of product and is within the knowledge of the person skilled in the art.

By a "salt-accompanied", also referred to herein interchangeably as "salt- associated" or salt-induced" nisin-deactivating activity is herein understood that in the presence of a salt, the Streptococcus thermophilus strain, directly or indirectly, partly or completely, deactivates nisin in a solution comprising nisin, salt and the Streptococcus thermophilus strain. Examples of nisin-deactivating Streptococcus thermophilus strains include those Streptococcus thermophilus strains that already naturally produce an effective amount of extracellular nisi n- degrading enzymes. Examples of salt-accompanied nisin-deactivating Streptococcus thermophilus strains include those Streptococcus thermophilus strains that, in the presence of salt, for example due to lysis, release intracellular nisin-degrading enzymes or release other unspecific nisin- degrading activity. Hence, for the avoidance of doubt, the above-mentioned Streptococcus thermophilus INIA 463 is not considered a Streptococcus thermophilus strain having no or a limited salt-accompanied nisin-deactivating activity. That is, the Streptococcus thermophilus strain having no or a limited salt-accompanied nisin-deactivating activity according to the aspects of the invention is suitably not Streptococcus thermophilus INIA 463.

Thus, the present invention is directed to a newly identified strain of Streptococcus thermophilus that has no or limited salt-accompanied nisin- deactivating activity as defined and as measured by an assay described herein, particularly wherein the strain is not identical and does not consist of Streptococcus thermophilus INIA 463.

The term "salt-accompanied" includes particularly a halide salt, for example a bromide, chloride, fluoride or iodine salt, particularly mixtures of such salts. Preferably, the salt is a chloride or bromide salt, most preferably a chloride salt. In one aspect, the salt is an alkali metal or alkaline earth metal salt, such as for example a sodium, potassium, calcium or magnesium salt. More preferably, the salt is a sodium or potassium salt, most preferably a sodium salt. Still more preferred are alkali metal or alkaline earth metal halide salts, 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. Thus, even more preferably the salt-accompanied nisin- deactivating activity is a sodium chloride-accompanied nisin-deactivating activity or a calcium chloride-accompanied nisin-deactivating activity. Hence, even more preferably the references in the embodiments of this invention to a salt-accompanied or salt-induced nisin-deactivating activity can be interchanged with references to a sodium chloride-accompanied or sodium chloride induded nisin-deactivating activity or a calcium chloride-accompanied nisin-deactivating activity. Most preferably, the salt is sodium chloride. Thus, most preferably, the salt-accompanied nisin-deactivating activity is a sodium chloride-accompanied or sodium chloride-induced nisin-deactivating activity. Hence, most preferably the references in the embodiments of this invention to a salt-accompanied nisin-deactivating activity can be interchanged with references to a sodium chloride-accompanied nisin-deactivating activity and the references to a salt can be interchanged with references to sodium chloride.

As used herein, the "salt" includes any salt accepted in preparation of food, particularly fermented milk products such as e.g. cheese. Depending on the product, the salt is added at any time in the process. Particularly for production of salted cheese, the preferred salt includes sodium and/or potassium chloride. The salt might be present in the range of 1% to 5% final concentration (w/w).

The skilled person knows the type and amount of salt to be added for the respective (salted) cheese product.

Preferences for the nisin in respect of the nisin-deactivating activity are as defined herein. Most preferably, the nisin is nisin A, and most preferably the references herein to nisin-deactivating activity are references to nisin A- deactivating activity. Depending on the fermented milk product, particularly cheese product, the skilled person knows how much nisin to be added or to be present during the fermentation process to effectively inhibiting the growth of Gram-positive bacteria, particularly effectively inhibiting the growth of NSLABs.

The present invention provides Streptococcus thermophilus strains that directly or indirectly, partly or completely, deactivate nisin in a solution comprising both nisin and the Streptococcus thermophilus strain. As illustrated by the examples, salt-accompanied nisin-deactivating Streptococcus thermophilus strains, respectively sodium chloride-accompanied nisin-deactivating Streptococcus thermophilus strains, preferably nisin A-deactivating Streptococcus thermophilus strains, can conveniently be characterized and/or classified by means of a screening assay, as exemplified in the examples.

The extent of salt-accompanied nisin-deactivating activity (SAND) of a certain Streptococcus thermophilus strain can be determined by testing its capability, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C, an amount of about 50 U/ml nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts by million by weight (ppmw) sodium formate and grown at about 38°C until pH 5.3. Said test mimics the situation during production of salted fermented milk products such as e.g. salted cheese.

Suitably, a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having positive salt-accompanied nisi n- deactivating activity (P-SAND), i.e. resulting in nisin-deactivation upon presence or addition of salt as defined herein, if it is capable of degrading 70% or more of the added nisin, particularly added nisin A. That is, a Streptococcus thermophilus strain is considered to have positive salt-accompanied or salt- induced nisin-deactivating activity if such a strain is capable, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C 70% or more, such as e.g. 75%, 80%, 85%, 90%, 95%, 98% or even 100% of about 50 U/ml nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) RSM supplemented with about 15 parts by million by weight (ppmw) sodium formate and grown at about 38°C until pH 5.3.

Suitably, a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having limited salt-accompanied nisin- deactivating activity (L-SAND), i.e. resulting in reduced or limited nisin- deactivation upon presence or addition of salt as defined herein, if it is capable of degrading 50% or more but less than 70% of the added nisin, particularly added nisin A. That is, a Streptococcus thermophilus strain is considered to have limited salt-accompanied nisin-deactivating activity if such a strain is capable, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C less than 70%, such as e.g. 69%, 65%, 60%, 55%, 50%, but no less than 50%, of 50 U/ml nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) RSM supplemented with 15 ppmw sodium formate and grown at about 38°C until pH 5.3.

Suitably, a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having no salt-accompanied nisin- deactivating activity (N-SAND) ), i.e. resulting in no or very little nisin- deactivation upon presence or addition of salt as defined herein, if it is capable of degrading less than 50%, such as e.g. 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or even 0%, i.e. no deactivating activity at all, preferably with a nisin deactivating activity of 10% or less of the added nisin as defined herein. That is, a Streptococcus thermophilus strain is considered to have no salt- accompanied nisin-deactivating activity if such a strain is capable, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C less than 50%, such as e.g. less than 40%, 30%, 20%, and most preferably less than 10%, of 50 U/ml nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) RSM supplemented with about 15 ppmw sodium formate and grown at about 38°C until pH 5.3.

Preferably, the Streptococcus thermophilus strain has no salt-accompanied nisin-deactivating activity (N-SAND), most preferably the Streptococcus thermophilus strain has no sodium chloride-accompanied nisin-deactivating activity, wherein preferably the nisin is nisin A.

Preferred examples of Streptococcus thermophilus strains having no or limited salt-accompanied nisin-deactivating activity, respectively, no or limited sodium chloride-accompanied nisin-deactivating activity, wherein the nisin is preferably nisin A, include strain CBS 150251 and strain CBS 150252, both deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht (NL) and variants thereof having no or limited salt-accompanied nisin-deactivating activity.

In some aspects, the invention provides a process for producing a fermented milk product, particularly a salted fermented milk product, comprising

(a) fermenting and coagulating a milk-base in the presence of a bacterial culture (blend), in the presence of nisin, and in the presence of a coagulant to produce a coagulated fermented milk-base; and

(b) contacting at least part of the coagulated fermented milk-base with a salt; wherein the bacterial culture (blend) comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or a limited salt- accompanied nisin-deactivating activity.

Particularly, the fermented milk product is a salted fermented milk product, more particularly a salted cheese, and the invention provides a process for producing such salted fermented milk product, said process comprising:

In step (a) a milk-base is fermented in the presence of a bacterial culture (blend) to produce a fermented milk-base as defined herein.

The bacterial culture comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or limited salt-accompanied nisin- deactivating activity. More preferably, the bacterial culture (blend) is a bacterial culture as described herein above and/or herein below.

Preferably, the milk-base in step (a) is fermented in the presence of a bacterial culture blend, such bacterial culture blend preferably comprises or consists of:

(i) one or more Streptococcus thermophilus strain(s), wherein at least one Streptococcus thermophilus strain, and preferably each Streptococcus thermophilus strain, has no or limited salt-accompanied nisin-deactivating activity; and

(ii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s).

Thus, the present invention is in some embodiments directed to a process according to the present invention comprising fermentation of a milk-base as defined herein in the presence of a bacterial culture blend, wherein such bacterial culture blend comprises or consists of:

(i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin- deactivating activity; and

(ii) one or more nisin-producing bacterial strains, preferably nisin producing Lactococcus strain(s), more preferably nisin producing Lactococcus cremoris strain(s) and/or nisin producing Lactococcus lactis strain(s); and

Furthermore, the present invention is in some embodiments directed to a process according to the present invention comprising fermentation of a milkbase as defined herein in the presence of a bacterial culture blend, wherein such bacterial culture blend comprises or consists of: (i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisi n- deactivating activity; and

(ii) optionally one or more nisin-producing bacterial strains, preferably nisin producing Lactococcus strain(s), more preferably nisin producing Lactococcus cremoris strain(s) and/or a nisin producing Lactococcus lactis strain(s); and

(iii) one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus and/or Lactococcus bacterial strains.

Preferences for these embodiments are as described herein above and below.

Particularly, the process as described herein comprises a step of fermentation of a milk-base as defined herein in the presence of a bacterial culture or of a bacterial culture blend until a pH of equal to or less than about 6.0, more preferably equal to or less than about 5.8, 5.5, 5.3 is reached. For practical purposes, the pH during step (a) of the process as described above preferably varies in the range from equal to or less than about 6.8, such as e.g. about 6.7, 6.4 or less, such as particularly in a 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 milk product is a cheese, the pH during step (a) in the process as described above more preferably varies in the range from 6.8, 6.7, 6.4 to 4.0, 4.5, 5.0, 5.1.

Preferably step (a) in the process as described above is carried out at temperatures of at least about 28°C, such as e.g. at about 30, 32°C or more, preferably wherein the temperature is about 47°C or less, such as e.g. about 45, 42, 40°C or less, particularly in the range of 28°C to 47° C.

In some embodiments, the process as defined herein, most preferably step (a) of the process as described above, comprises multiple fermentation stages, preferably including one or more mesophilic fermentation stages and/or one or more thermophilic fermentation stages. More preferably such process, most preferably step (a) of such a process, comprises:

(al) a first mesophilic fermentation stage, preferably carried out at a temperature in the range from 30°C to 38°C, more preferably in the range from 30°C to 37° C, and most preferably in the range from 30°C to 36° C; (a2) a second thermophilic fermentation stage, preferably carried out at a temperature in the range from 36°C to 45°C, more preferably in the range from 37°C to 45° C, and most preferably in the range from 38°C to 45° C; and

(a3) optionally, a third mesophilic fermentation stage, preferably carried out at a temperature in the range from 30°C to 38°C, more preferably in the range from 30°C to 37° C, and most preferably in the range from 30°C to 36° C.

The above staged fermentation has the advantage that the second thermophilic fermentation stage can stimulate the fermentation of the above-mentioned Streptococcus thermophilus strain(s). The above-described staged fermentation can further have the advantage that the first and/or third mesophilic fermentation stage can stimulate the fermentation of mesophilic nisi n- producing bacterial strains, such as for example a nisin producing Lactococcus lactis strain.

Preferably step (a) is carried out in a fermentation vessel.

The time period during which step (a) is carried out may vary widely. However, preferably step (a) is carried out in a range of 10 to 360 minutes, more preferably in a range of from 10, 20 or 30 minutes to 60, 90, 120 or 360 minutes. The shorter periods have the advantage that the process becomes a more speedy process which is desired from a cheese producer perspective.

Most preferably steps (b) and/or (c) as described herein below are carried out during step (a). That is, most preferably steps (a), (b) and (c) together form one step comprising or consisting of fermenting and coagulating a milk-base in the presence of a bacterial culture (blend), in the presence of nisin, and in the presence of a coagulant to produce a coagulated fermented milk-base. Carrying out steps (a), (b) and/or (c) simultaneously in one vessel has the advantage of providing a very speedy process.

Step (b) comprises or consists of contacting the fermented milk-base with nisin. The fermented milk-base can suitably be the whole of the fermented milk-base prepared in step (a). Alternatively, only a part of the fermented milk-base of step (a) can be contacted with nisin.

In the process according to the invention step (b) can be carried out after step (a) or simultaneous with step (a). Preferably step (b) is carried out simultaneously with step (a) or even as part of step (a), in each case preferably together in one vessel. The contacting of the fermented milk-base with the nisin can be carried out ex- situ and/or in-situ. That is, as an example of ex-situ contacting, the nisin can be added from an external source to the fermented milk-base. As an example of in- situ contacting, the nisin can be generated in-situ, for example by the bacterial culture (blend) as present in step (a).

Step (b) can be carried out by addition and/or generation of nisin before, during and/or after fermentation of the milk-base. Preferably step (b) comprises or consists of addition of nisin and/or in-situ generation of nisin, during and/or after fermentation in step (a).

If step (b) comprises the addition of nisin, the nisin is preferably added during and/or after the fermentation according to (a). Preferably the nisin is added in a concentration higher than the MIC value of the target strain. By the MIC value is herein understood the minimum inhibitory concentration (MIC). That is, preferably the nisin is added in a concentration higher than the lowest concentration (in pg/ mL) of nisin that inhibits the growth of the targeted NSLAB. In practice, the nisin is preferably added in an amount of equal to or more than 1 U/ml, more preferably equal to or more than 5 U/ml, even more preferably equal to or more than 10 U/ml, still more preferably equal to or more than 50 U/ ml, and most preferably equal to or more than 100 U/ ml of nisin or even equal to or more than 150 U/ ml or even equal to or more than 200 U/ ml and up to 600 U/ml in the final concentration.

Preferably step (b) comprises or consists of the in-situ generation of nisin. If step (b) comprises the in-situ generation of nisin, step (b) preferably comprises the in-situ generation of nisin during and/or after the fermentation in step (a). Most preferably, if step (b) comprises the in-situ generation of nisin, the nisin is generated in-situ during, i.e. simultaneous with, the fermentation in step (a). If step (b) comprises the in-situ generation of nisin, the nisin is preferably generated in-situ with the help of one or more nisin-producing bacterial strains. Preferences for such nisin-producing bacterial strains are described herein above and herein below. Preferably the nisin is generated in a concentration higher than the MIC value of the target NSLAB. In practice, the nisin is preferably generated in an amount of equal to or more than 1 U/ml, more preferably equal to or more than 5 U/ml, even more preferably equal to or more than 10 U/m, still more preferably equal to or more than 50 U/ml, and most preferably equal to or more than 100 U/ ml of nisin or even equal to or more than 150 U/ ml or even equal to or more than 200 U/ ml and up to 600 U/ml in the final concentration.

This nisin is most preferably generated during the fermentation in step (a) by means of one or more nisin-producing bacterial strains, preferably one or more nisin-producing Lactococcus strain(s), more preferably one or more nisin- producing Lactococcus lactis strains (also referred to herein as Lactococcus lactis ssp. lactis strains) and/or one or more nisin-producing Lactococcus cremoris strains (also referred to herein as Lactococcus lactis ssp. cremoris strains), still more preferably one or more nisin-producing Lactococcus lactis ssp. lactis strains, and most preferably one or more nisin A - producing Lactococcus lactis ssp. lactis strains. Preferably such nisin-producing bacterial strain is added in step (a), either separately or, more preferably, as part of a bacterial culture blend also comprising the Streptococcus thermophilus strain. Preferably step (b) is carried out at temperatures in the range between 28°C to 47° C, more preferably between 30°C to 45°C, such as in the range of 32°C to 42°C, even more preferably less than 40°C, such as most preferably at a temperature of 38°C or less, i.e. in the range of particularly 28°C to 38°C.

As indicated above, the fermentation may advantageously comprise one or more mesophilic fermentation stages. As explained, this above-described staged fermentation has the further advantage that the first and/or third mesophilic fermentation stage can stimulate the fermentation of mesophilic nisin- producing bacterial strains, such as for example a nisin producing Lactococcus lactis strain. If the fermentation in step (a) comprises thermophilic and mesophilic fermentation stages, it can be advantageous to add a mesophilic nisin-producing bacterial strain(s), such as for example a nisin- producing Lactococcus lactis strain, before and/or during one of the mesophilic fermentation stages, optionally separately from the Streptococcus thermophilus strain.

The time period during which step (b) is carried out may vary widely. However, preferably step (b) is carried out during a period in the range of 10 minutes to 360 minutes, such as 20 or 30 minutes to 120, 90, or 60 minutes. The shorter periods have the advantage that the process becomes a more speedy process which is desired from a cheese producer perspective. Most preferably step (b) as described herein is carried out during step (a). Carrying out steps (a) and (b) simultaneously in one vessel has the advantage of providing a very speedy process. Step (c) comprises or consists of optionally contacting of the fermented milkbase with a coagulant, such as e.g. adding one or more coagulants. Preferably the process according to the invention comprises a step (c) and is a process for producing a fermented milk product, preferably a cheese, comprising:

(a) fermenting a milk-base in the presence of a bacterial culture (blend) to produce a fermented milk-base;

(b) contacting the fermented milk-base with nisin, e.g. wherein the nisin is added to the fermented milk-base or wherein the nisin is present via in-situ generation;

(c) contacting the fermented milk-base with a coagulant, e.g. wherein the coagulant is added to the fermented milk-base;

(d) contacting at least part of the fermented milk-base with a salt, e.g. wherein salt is added to the fermented milk-base, particularly added in the curd or wherein the cheese is submerged in a salt-bath; wherein the bacterial culture (blend) comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or limited salt- accompanied nisin-deactivating activity.

The contacting of the fermented milk-base with a coagulant in step (c) can conveniently yield a coagulated fermented milk-base. Such a coagulated fermented milk-base suitably comprises curd and whey.

Step (c) can be carried out by addition of the coagulant before, during or after fermentation of the milk-base in step (a). Preferably step (c) is carried out by addition of the coagulant during and/or after fermentation in step (a). Most preferably step (c) as described herein is carried out during step (a). Carrying out steps (a) and (c) simultaneously in one vessel has the advantage of providing a more speedy process.

Further, step (c) can be carried out by addition of the coagulant before, during or after the contacting with nisin in step (b). If the nisin is generated in-situ, as explained below, step (c) preferably comprises addition of the coagulant during and/or after the contacting with nisin in step (b). Also, if the nisin is added ex- situ, as explained below, step (c) preferably comprises addition of the coagulant, during and/or after the contacting with nisin in step (b). Most preferably step (c) as described herein is carried out during step (b). Carrying out steps (b) and (c) simultaneously in one vessel has the advantage of providing a more speedy process.

Most preferably step (c) as described herein is carried out simultaneously with steps (a) and (b) in one vessel. Carrying out steps (a), (b) and (c) simultaneously in one vessel (i.e. as one step) has the advantage of providing a very speedy process.

Preferably the coagulant comprises or consists of a protease, preferably an aspartic protease. The terms protease, proteinase and peptidase are used interchangeably herein. Preferably the coagulant comprises or consists of a protease produced by a microorganism or a mammal, respectively an aspartic protease produced by a microorganism or a mammal. Most preferred coagulants are coagulants comprising or consisting of mucorpepsin, chymosin or a precursor thereof. Examples of suitable commercially available coagulants include Maxiren®, Maxiren®XDS, Fromase®, CHY-MAX® and Hannilase®.

Preferably the coagulant is added in an amount to achieve a concentration in the range from equal to or more than 1 IMCU/L milk, more preferably from equal to or more than 5 IMCU/L milk, even more preferably from equal to or more than 10 IMCU/L milk, yet more preferably from equal to or more than 15 IMCU/L milk, still more preferably from equal to or more than 20 IMCU/L, even still more preferably from equal to or more than 25 IMCU/L and most preferably from equal to or more than 30 IMCU/L milk, to equal to or less than 100 IMCU/L milk, more preferably to equal to or less than 80 IMCU/L milk, even more preferably to equal to or less than 75 IMCU/L milk, yet more preferably to equal to or less than 70 IMCU/L milk, still more preferably to equal to or less than 65 IMCU/L milk, and most preferably to equal to or less than 60 IMCU/L milk.

Preferably step (c) is carried at temperatures in the range from 28°C to 47° C, more preferably in the range of from 30°C to 45°C, such as from 32°C to 42°C, most preferably at a temperature of 40°C or less, such as e.g. between 32°C to 40° C.

The time period during which step (c) is carried out may vary widely. However, preferably step (c) is carried out during a period in the range from equal to or more than 10 minutes, more preferably from equal to or more than 20 minutes, and most preferably from equal to or more than 30 minutes, to equal to or less than 360 minutes, more preferably to equal to or less than 120 minutes, even more preferably to equal to or less than 90 minutes and most preferably to equal to or less than 60 minutes. The shorter periods have the advantage that the process becomes a speedier or accelerated process which is desired from a cheese producer perspective.

Step (d) comprises or consists of contacting at least part of the fermented milkbase with a salt.

Preferably the salt is a halide salt, for example a bromide, chloride, fluoride or iodine salt. More preferably the salt is a chloride or bromide salt, most preferably a chloride salt. Preferably the salt is an alkali metal or alkaline earth metal salt, such as for example a sodium, potassium, calcium or magnesium salt. More preferably the salt is a sodium or potassium salt, most preferably a sodium salt. Still more preferred are alkali metal or alkaline earth metal halide salts, 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.

Step (d) can be carried out by addition of the salt before, during or after fermentation of the milk-base. Preferably step (d) is carried out by addition of the salt during or after fermentation step (a). More preferably step (d) is carried out by addition of the salt after fermentation step (a). In one preferred embodiment step (d) is carried out after step (a), after step (b) and, if present, after step (c).

If the process according to the invention comprises a step (a), (b) and (c), preferably such steps (a), (b) and (c) together form one step, also referred to herein as a curdling step, yielding a coagulated fermented milk-base, which coagulated fermented milk-base can conveniently comprise curd and whey. Conveniently such curdling may be followed by optional steps of cutting, stirring and/or cooking, whereafter the whey can conveniently be separated from the curd. The curd can advantageously be milled, whereafter it may be subjected to salting.

The process may thus preferably comprise one or more separation steps where one part of a coagulated fermented milk-base, preferably the curd, is separated from another part, preferably the whey. By a curd is herein preferably understood the coagulated fraction of the milk. Such a coagulated fraction of milk preferably comprises aggregated milk proteins, more preferably aggregated casein protein. Hence, preferably step (d) comprises the separation of the, optionally coagulated, fermented milk-base in a curd and a whey, whereafter the separated curd (that is, the curd part of the fermented milk-base) is contacted with a salt.

Thus, preferably the process is a process for producing a fermented milk product, comprising:

(b) contacting the fermented milk-base with nisin;

(c) contacting the fermented milk-base with a coagulant; and

(d) separating the fermented milk-base in a curd and a whey and contacting the curd with a salt, e.g. adding salt to the curd; wherein the bacterial culture (blend) comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or limited salt- accompanied nisin-deactivating activity.

The curd may preferably be milled before and/or after being contacted with the salt. Further preferences are as described herein above and below.

More preferably the invention provides a process for producing a, preferably salted, fermented milk product, comprising:

- fermenting and coagulating a milk-base in the presence of a bacterial culture (blend), in the presence of nisin, and in the presence of a coagulant to produce a coagulated fermented milk-base; and

- separating the coagulated fermented milk-base in a curd and a whey and contacting the curd with a salt, e.g. adding salt as defined herein to the curd; wherein the bacterial culture (blend) comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or a limited salt- accompanied nisin-deactivating activity.

Preferably the salt is added in a weight amount of equal to or more than 0.10% (w/w), more preferably at least about 0.25% (w/w), even more preferably at least about 0.50% (w/w), yet more preferably at least about 1.0% (w/w), and most preferably at least about 2.0% (w/w), such as a 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); based on the total weight of the (part of) the fermented milk product with which it is contacted. Preferably, between 1 to 5% (w/w) salt is added. All numbers refer to the final concentration.

Most preferably the salt (added) in step (d) is present as an aqueous solution, preferably an aqueous solution of sodium chloride and/or an aqueous solution of calcium chloride.

The process according to the invention advantageously allows one to maintain nisin levels in a process comprising salt addition. The nisin can advantageously avoid the growth of non-starter lactic acid bacteria (NSLAB).

Preferably the process further includes a step of retrieving a fermented milk product.

The fermented milk product produced in the process is preferably a cheese, preferably a salted cheese. Further preferences for the fermented milk product are as explained herein below. Most preferably the fermented milk product produced in the process is a Cheddar cheese.

Preferably, the bacterial culture comprises, consists of or is part of a starter culture, bacterial culture blend or kit of parts. By a starter culture is herein preferably understood a composition comprising a mixture of, two, three, four, five, six or even more different bacteria, which bacteria are to be used for the inoculation of a food material, such as for example milk, to initiate a predetermined change in such food material.

In one embodiment, the invention suitably provides a starter culture, bacterial culture blend or kit of parts comprising or consisting of:

(i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisi n- deactivating activity; and

(ii) one or more nisin-producing bacterial strains, preferably nisin producing Lactococcus strain(s), more preferably nisin-producing Lactococcus lactis ssp. cremoris strain(s) and/or nisin-producing Lactococcus lactis ssp. lactis strain(s); and

(iii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus and/or Lactococcus bacterial strains. In another embodiment, the invention suitably provides a starter culture, bacterial culture blend or kit of parts comprising or consisting of:

(ii) optionally, one or more nisin-producing bacterial strains, preferably nisin- producing Lactococcus strain(s), more preferably nisin-producing Lactococcus lactis ssp. cremoris strain(s) and/or nisin-producing Lactococcus lactis ssp. lactis strain(s); and

Preferably the starter culture, bacterial culture blend or kit of parts comprises or consists of:

(ii) one or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus strain(s), more preferably nisin-producing Lactococcus lactis ssp. cremoris strain(s) and/or a nisin-producing Lactococcus lactis ssp. lactis strain(s); and

Preferences for the one or more Streptococcus thermophilus strain(s) under (i) in these embodiments are as described herein above.

The starter culture, the bacterial culture blend or kit of parts of the above embodiments preferably does not comprise Streptococcus thermophilus strain(s) that have a positive salt-accompanied nisin-deactivating activity (P- SAND). More preferably the starter culture, the bacterial culture blend or kit of parts, 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, yet more preferably equal to or less than 1.0 % w/w, still more preferably equal to or less than 0.5% w/w and most preferably no Streptococcus thermophilus strain(s) that have a positive salt-accompanied nisin-deactivating activity (P-SAND). Characterization can be suitably carried out as described herein. If the starter culture, bacterial culture blend or kit of parts contains a Streptococcus thermophilus strain that has positive salt-accompanied nisin-deactivating activity (P-SAND), such Streptococcus thermophilus strain having positive salt-accompanied nisin- deactivating activity is preferably present in a weight percentage of 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, still more preferably equal to or less than 1.0% w/w, yet more preferably equal to or less than 0.5% w/w, and most preferably equal to or less than 0.1% w/w based on the total weight of Streptococcus thermophilus strains present.

Preferably, each Streptococcus thermophilus strain contained or comprised in the starter culture, bacterial culture blend or kit of parts is a Streptococcus thermophilus strain having no or limited salt-accompanied nisin-deactivating activity. More preferably each Streptococcus thermophilus strain contained or comprised in the starter culture, bacterial culture blend or kit of parts is a Streptococcus thermophilus strain having no salt-accompanied nisin- deactivating activity. Most preferably the starter culture, bacterial culture blend or kit of parts contains only Streptococcus thermophilus strains that have no or limited salt-accompanied nisin-deactivating activity, most preferably no salt- accompanied nisin-deactivating activity. Characterization can be suitably carried out as described above.

If present in the embodiments above, 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 ssp. cremoris strain(s) and/or one or more nisin producing Lactococcus lactis ssp. lactis strain(s), most preferably one or more nisin-producing Lactococcus lactis ssp. lactis strains. If a combination of a nisin-producing Lactococcus lactis ssp. cremoris strain and a nisin producing Lactococcus lactis ssp. lactis strain is applied, the ratio of the Lactococcus lactis ssp. cremoris strain to the Lactococcus lactis ssp. lactis strain preferably lies in the range from equal to or more than 1:100, more preferably equal to or more than 1:10, still more preferably equal to or more than 1:5 and most preferably equal to or more than 1:2 to equal to or less than 100:1, more preferably equal to or less than 10:1, still 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 a nisin- producing Lactococcus lactis ssp. cremoris strain and a nisin producing Lactococcus lactis ssp. lactis strain is applied, the ratio of the Lactococcus lactis ssp. cremoris strain to the Lactococcus lactis ssp. lactis strain preferably lies in the range from equal to or more than 25:75 to equal to or less than 50:50.

The nisin-producing bacterial strain may 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 strain(s) produces nisin A, nisin Z or a combination of nisin A and nisin Z. Most preferably the nisin-producing bacterial strain(s) is/are nisin A producing bacterial strains, more preferably nisin A- producing Lactococcus strains, even more preferably nisin A-producing Lactococcus lactis strains, still more preferably nisin A -producing Lactococcus lactis ssp. cremoris strain(s) and/or nisin A-producing Lactococcus lactis ssp. lactis strain(s), most preferably nisin A-producing Lactococcus lactis ssp. lactis strains.

The nisin-producing bacterial strain(s) preferably is/are capable of producing, in order of preference, equal to or more than 5, equal to or more than 25, equal to or more than 100, equal to or more than 400, equal to or more than 600 or equal to or more than 800 IU nisin per ml (of milk), preferably under the conditions of the process according to the first aspect and/or the process conditions as described herein below. The amount of the nisin-producing bacterial strains is preferably such that the nisin level in any fermented product is sufficient to prevent spoilage by bacteria.

In principle any Lactococcus lactis strain can be converted into a nisin- producing bacterial strain. Preferably the nisin-producing property is conferred to the appropriate Lactococcus lactis strains by conjugations of transposons containing the genetic information for nisin production. A preferred transposon containing the genetic information for nisin production is Tn5276. Suitable donor strains for conjugation of Tn5276 are described by Hugenholtz et al. and were deposited 28 June 2001 with deposit number CBS 109540, and 3 April 1991 with deposit number CBS 181.91, respectively, at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands. The strains and method are described in more detail in European patent EP1273237 and are herein incorporated by reference.

Alternatively, the properties of nisin production may be conferred to the appropriate Lactococcus lactis strains by means of recombinant DNA technology as known per se to the skilled person. However, the use of such GMO Lactococcus lactis strains is presently not preferred in view of the poor public' acceptance of GMO’s in food products.

A most preferred nisin-producing bacterial strain pursuant to (ii) above is a nisin-producing Lactococcus lactis sub sp. lactis biovar. diacetylactis strain, preferably containing the Tn5276 transposon mentioned above. Such a strain is contained in Dairysafe ™, which is commercially available from CSK food enrichment, Leeuwarden, The Netherlands as Dairysafe ™ TC17.

If present in the embodiments above, the one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, are preferably nisin-resistant lactic acid strains and have preferably no or limited nisin-deactivating activity and/or preferably no or limited salt- accompanied nisin-deactivating activity.

Strains that are nisin-resistant, non-nisin-producing and non-nisin-deactivating are herein also referred to as "nisin-neutral". The one or more bacterial strains in the above embodiments that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, are preferably nisin-neutral.

The nisin-resistance, nisin-immunity and/or (lack of) nisin-deactivating activity, (lack of) salt-accompanied nisin-deactivating activity and/or nisin-neutrality, as referred to herein, preferably applies 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 of) nisin-deactivating activity, (lack of) salt-accompanied nisin-deactivating activity and/or nisin-neutrality as applied within the context of this invention at least applies to the type of nisin present in the process of the first aspect and/or produced by any nisin-producing strain in starter culture, bacterial culture blend or kit of parts of the second aspect, preferably nisin A.

Preferably the "one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains" are selected from the group consisting of Lactobacillus, Leuconostoc, Propionibacterium, Pediococcus, Arthrobacter, Corynebacterium, Staphylococcus and Streptococcus strains other than Streptococcus thermophilus. In addition, non-nisin-producing Lactococci may be present, such as for example a non-nisin producing Lactococcus lactis diacetylactis.

More preferably "the one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains" are, preferably nisin- resistant, preferably non-nisin-deactivating, strains selected from the group consisting of, preferably nisin-neutral, Lactobacillus delbrueckii ssp. delb rueckii, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus (also referred to as Lacticaseibacillus rhamnosus), Lactobacillus paracasei (also referred to as Lacticaseibacillus paracasei), Lactobacillus casei (also referred to as Lacticaseibacillus casei), Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus plantarum (also referred to as Lactiplantibacillus plantarum), Lactobacillus sanfrancisco, Lactobacillus johnsonii, Lactobacillus pontis, Lactobacillus bavaricus, Lactobacillus curvatus, Lactobacillus sacei, Leuconostoc mesenteroides, Leuconoctoc lactis, Leuconostoc ssp., Pediococcus pentosaveus, Pediococcus avidilactici, Staphylococcus xylosus, Propionibacterium freudenreichii, Propionibacterium freudenreichii ssp. shermanii, and combinations thereof.

Still more preferably "the one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains" are, preferably nisin-resistant, preferably non-nisin-deactivating, strains selected from the group consisting of, preferably nisin neutral, Lactobacillus helveticus, Lactobacillus rhamnosus (also referred to as Lacticaseibacillus rhamnosus), Lactobacillus paracasei (also referred to as Lacticaseibacillus paracasei), Lactobacillus casei (also referred to as Lacticaseibacillus casei), Lactobacillus johnsonii, Lactobacillus delbrueckii ssp bulgaricus and Lactobacillus nodensis, Brevibacterium linens, Kluyveromyces lactis, and combinations thereof. Most preferably, "the one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains" are, preferably nisin- resistant, preferably non-nisin-deactivating, strains selected from the group consisting of, preferably nisin neutral, Lactobacillus strains, most preferably Lactobacillus helveticus, Lactobacillus rhamnosus (also referred to as Lacticaseibacillus rhamnosus), Lactobacillus paracasei (also referred to as Lacticaseibacillus paracasei), Lactobacillus casei (also referred to as Lacticaseibacillus casei), Lactobacillus delbrueckii ssp bulgaricus and/or Lactobacillus nodensis.

Preferably the starter culture, bacterial culture blend or kit of parts comprises or consists of a mix of: (i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisi n- deactivating activity; and

(ii) one or more nisin-producing bacterial strains, preferably nisin-producing Lactococcus strain(s), more preferably nisin-producing Lactococcus lactis strain(s), even more preferably nisin-producing Lactococcus lactis ssp. cremoris strain(s) and/or a nisin-producing Lactococcus lactis ssp. lactis strain(s); and

(iii) one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus bacterial strains, more preferably Lactobacillus casei (also referred to as Lacticaseibacillus casei), Lactobacillus paracasei (also referred to as Lacticaseibacillus paracasei) and/or Lactobacillus helveticus strains.

The amount of each component in the above starter culture, bacterial culture blend or kit of parts may vary.

The component (i), i.e. the "one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt- accompanied nisin-deactivating activity", is/are preferably present in a weight percentage of equal to or more than 1% w/w, more preferably equal to or more than 5% w/w, still more preferably equal to or more than 10% w/w, even more preferably equal to or more than 15% w/w, yet more preferably equal to or more than 20% w/w and most preferably equal to or more than 25 % w/w and preferably a weight percentage of equal to or less than 99% w/w, more preferably equal to or less than 95% w/w, still more preferably equal to or less than 90 % w/w, even more preferably equal to or less than 85% w/w, yet more preferably equal to or less than 80% w/w, still even more preferably equal to or less than 75% w/w, yet even more preferably equal to or less than 70% w/w and most preferably equal to or less than 65% w/w, based on the total weight of all the bacterial strains in the starter culture, bacterial culture blend or kit of parts.

If present, the component (ii), i.e. the "one or more nisin-producing bacterial strains", is/are preferably present in a weight percentage of equal to or more than 1% w/w, more preferably equal to or more than 5% w/w, still more preferably equal to or more than 10% w/w, even more preferably equal to or more than 15% w/w, yet more preferably equal to or more than 20% w/w and most preferably equal to or more than 25 % w/w and preferably a weight percentage of equal to or less than 99% w/w, more preferably equal to or less than 95% w/w, still more preferably equal to or less than 90 % w/w, even more preferably equal to or less than 85% w/w, yet more preferably equal to or less than 80% w/w, still even more preferably equal to or less than 75% w/w, yet even more preferably equal to or less than 70% w/w and most preferably equal to or less than 65% w/w, based on the total weight of all the bacterial strains in the starter culture, bacterial culture blend or kit of parts.

If present, the component (iii), i.e. the "one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains", is/are preferably present in a weight percentage of equal to or more than 1% w/w, more preferably equal to or more than 5% w/w, still more preferably equal to or more than 10% w/w, even more preferably equal to or more than 15% w/w, yet more preferably equal to or more than 20% w/w and most preferably equal to or more than 25 % w/w and preferably a weight percentage of equal to or less than 99% w/w, more preferably equal to or less than 95% w/w, still more preferably equal to or less than 90 % w/w, even more preferably equal to or less than 85% w/w, yet more preferably equal to or less than 80% w/w, still even more preferably equal to or less than 75% w/w, yet even more preferably equal to or less than 70% w/w and most preferably equal to or less than 65% w/w, based on the total weight of all the bacterial strains in the starter culture, bacterial culture blend or kit of parts.

Most preferably all three components (i), (ii) and (iii) are present, and more preferably all three components (i), (ii) and (iii) are present in a weight ratio of one to another of about 1:1:1, about 2:1:1, about 1 :2;1 or about 1:1:2, most preferably a weight ratio of one to another of about 1:1:1.

To the extent present, the strains under (i), (ii) and (iii), are preferably present in a frozen or freeze-dried form. More preferably the starter culture, bacterial culture blend or kit of parts is a frozen or freeze-dried starter culture or a frozen or freeze-dried bacterial culture blend.

In addition to the components (i), (ii) and (iii) as described above, the starter culture, bacterial culture blend or kit of parts may or may not comprise or consist of an additional component (iv), comprising or consisting of one or more non-bacterial cryoprotectants and/or non-bacterial additives, such as for example sodium formate. More preferably, the starter culture, bacterial culture blend or kit of parts comprises or consists in addition of sodium formate and optionally one or more other cyro protectants. Furthermore, the starter culture, bacterial culture blend or kit of parts may or may not comprise or consist of an additional component (v), comprising or consisting of a solvent, such as water or milk.

In a further aspect, the invention provides a fermented milk product, more preferably a cheese, still more preferably a salted cheese, and most preferably a Cheddar cheese, comprising:

(i) a salt, preferably sodium chloride salt or calcium chloride salt, most preferably sodium chloride salt.

(ii) one or more Streptococcus thermophilus strain(s) and/or residues of one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin-deactivating activity; and

(iii) nisin, preferably nisin A.

Preferably the fermented milk product is a cheese. Preferably the fermented milk product is a hard cheese. Preferably the fermented product is a salted cheese. Most preferably the fermented milk product is a cheese chosen from the group consisting of Cheddar, Emmental, Grana Padano, Gruyere, Mimolette, Parmigiano, Parmesan and Pecorino, Gouda, Provolone and Swiss. Most preferably the fermented milk product is Cheddar cheese.

Preferences for the one or more Streptococcus thermophilus strain(s) are as described herein above. In addition, the fermented milk product may preferably comprise one or more nisin-producing bacterial strains, preferably nisin- producing Lactococcus strain(s), more preferably nisin-producing Lactococcus lactis strain(s), even more preferably nisin-producing Lactococcus lactis ssp. cremoris strain(s) and/or a nisin-producing Lactococcus lactis ssp. lactis strain(s) and/or residues of any of these. Such nisin-producing bacterial strains may advantageously have been used to produce the nisin.

The inventors surprisingly found that the presence of the endogenous putative acetyltransferase TraX plays a crucial role in the salt-induced nisin deactivation activity of Streptococcus thermophilus. The evaluation of the status of expression of the traX gene, i.e. determination whether or not the Trax polypeptide is present, together with the assay as defined herein to test the capability, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C, an amount of about 50 U/ml nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3.

Thus, in the case of a non salt-induced nising degradation activity (N-SAND), the S. thermophilus strain is capable of degrading less than 50% of 50 U/mL nisin under the conditions as described above and furthermore, i.e. no gene product or TraX protein could be detected.

In the case of low-salt induced nising degradation activity (L-SAND), the S. thermophilus strain is capable of degrading 50% to less than 70% of 50 U/mL nisin under the conditions as described above and furthermore, the strain expresses traX gene , i.e. the traX gene product or TraX protein could be detected.

The present invention is in one aspect related to a process of converting a strain showing P-SAND phenotype into an L-SAND or N-SAND phenotype, preferably an N-SAND phenotype, wherein said process comprises:

(1) providing a strain of Streptococcus thermophilus with positive salt- accompanied nisin deactivating activity, wherein said strain is capable of degrading 70% or more of 50U/ ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride under conditions as defined herein;

(2) modification in the genome of said strain to inactivate the endogenous gene encoding acetyltransferase TraX, such as e.g. a protein according to SEQ ID NO:2 or 3, such as e.g. via introduction of a knock-out of said gene,

(3) selecting strains capable of degrading less than 50% of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride that do not show any TraX RNA or any translated active TraX protein.

A modification in the genome of a cell can be determined by comparing the DNA sequence of a (mutant) bacterial cell to the sequence of the parent or reference cell, such as traX according to SEQ ID NO:1. Sequencing of DNA and genome sequencing can be done using standard methods known to the person skilled in the art, for example using Sanger sequencing technology and/or next generation sequencing technologies such as Illumina GA2, Roche 454, Nanopore, etc. as reviewed in Elaine R. Mardis (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9: 387-402.

(doi:10.1146/annurev.genom.9.081307.164359).

Deficiency in the production of TraX as described herein can be measured using described salt-induced nisin deactivation assay or any assay suitable to the measurement of the polypeptide activity as defined herein available to the skilled person, transcriptional profiling, Northern blotting RT-PCR, Q-PCR and/or Western blotting. In particular, quantifying the amount of mRNA present in a cell may for example be achieved by northern blotting (in Molecular Cloning: A Laboratory Manual, Sambrook et al., New York: Cold Spring Harbour Press, 1989). Quantifying the amount of polypeptide as described herein present in a cell may for example be achieved by western blotting. The difference in mRNA amount may also be quantified by DNA array analysis or RNAseq (Eisen, M.B. and Brown, P.O. DNA arrays for analysis of gene expression. Methods Enzymol. 1999, 303:179- 205).

A mutant bacterial cell might contain one or more modifications. The modification in the bacterial cell can either be effected by: a) subjecting a bacterial cell to (classical) mutagenesis; and/or b) subjecting a bacterial cell to recombinant genetic manipulation techniques or genome editing techniques; and/or c) subjecting a bacterial cell to an inhibiting compound or composition.

Modification of a genome of a (mutant) filamentous fungal host cell is herein defined as any event resulting in a change in a TraX sequence in the genome of the cell. In a preferred embodiment, the mutant microbial host cell according to the disclosure has a modification in its genome.

The process according to the invention advantageously allows one to reduce cracks and slits in such cheeses.

The Streptococcus thermophilus bacterial strain CBS 150251 was deposited on 20 July 2023 at the Westerdijk Fungal Biodiversity Institute (CBS, formally named the Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3508 AD Utrecht, The Netherlands under the provisions of the Budapest Treaty.

The Streptococcus thermophilus bacterial strain CBS 150252 was deposited on 20 July 2023 at the Westerdijk Fungal Biodiversity Institute (CBS, formally named the Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3508 AD Utrecht, The Netherlands under the provisions of the Budapest Treaty.

The present invention is particularly related to the following embodiments:

(1) A process for producing a fermented milk product, comprising:

(a) fermenting a milk-base in the presence of a bacterial culture to produce a fermented milk-base; (b) contacting the fermented milk-base with nisin;

(c) optionally contacting the fermented milk-base with a coagulant;

(d) contacting at least part of the fermented milk-base with a salt; wherein the bacterial culture comprises a Streptococcus thermophilus strain, which Streptococcus thermophilus strain has no or limited salt-accompanied nisin-deactivating activity.

(2) Process as of embodiment (1), wherein (a) comprises fermenting the milkbase in the presence of the bacterial culture until a pH of equal to or less than 6.0, more preferably equal to or less than 5.3 is reached.

(3) Process according to embodiment (1) or (2), wherein (a) and (b), and optionally (c), are carried out simultanously.

(4) Process according to embodiment (l), (2) or (3), wherein (b) comprises the addition of nisin during or after the fermentation according to (a).

(5) Process according to embodiment (l), (2), (3) or (4), wherein (b) comprises the in-situ generation of nisin during or after the fermentation according to (a), and wherein preferably the nisin is generated in-situ with the help of one or more nisin-producing bacterial strains.

(6) Process according to embodiment (l), (2), (3), (4) or (5), wherein the coagulant in (c) comprises or consists of a protease, preferably a microbial or mammal produced aspartic protease.

(7) Process according to embodiment (l), (2), (3), (4), (5) or (6), wherein:: wherein

- the process comprises (a), (b) and (c) and wherein (a), (b) and (c) together form one step comprising or consisting of 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 a coagulated fermented milk-base; and

- wherein the process comprises a subsequent step comprising or consisting of separating the fermented milk-base in a curd and a whey and contacting the curd with a salt.

(8) Process according to embodiment (l), (2), (3), (4), (5), (6) or (7), wherein the salt comprises or consists of sodium chloride.

(9) Process as of embodiment (l), (2), (3), (4), (5), (6), (7) or (8), wherein the bacterial culture is part of a bacterial culture blend which comprises or consists of: (i) one or more Streptococcus thermophilus strain(s), wherein at least one Streptococcus thermophilus strain, and preferably each Streptococcus thermophilus strain, has no or limited salt-accompanied nisin-deactivating activity; and (ii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s).

(10) Process as of embodiment (1), (2), (3), (4), (5), (6), (7), (8) or (9), wherein the bacterial culture comprises or consists of: (i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin-deactivating activity; and (ii) one or more nisin-producing bacterial strains, preferably nisin producing Lactococcus lactis strains, more preferably nisin producing Lactococcus lactis ssp. cremoris strain(s) and/or a nisin producing Lactococcus lactis ssp. lactis strain(s); and (iii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus and/or Lactococcus bacterial strains.

(11) Process as of embodiment (l), (2), (3), (4), (5), (6), (7), (8), (9) or (10), wherein the Streptococcus thermophilus strain having no or limited salt-accompanied nisin-deactivating activity comprises or consists of strain CBS 150251 deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht, the Netherlands and/or strain CBS 150252 deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht and/or any variant thereof having no or limited salt-accompanied nisin-deactivating activity.

(12) Process as of embodiment (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) or (11), wherein the fermented milk product is a cheese, preferably a Cheddar cheese.

(13) A starter culture, bacterial culture blend or kit of parts comprising or consisiting of: (i) one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin-deactivating activity; and (ii) one or more nisin-producing bacterial strains, preferably nisin producing Lactococcus lactis strains, more preferably nisin producing Lactococcus lactis ssp. cremoris strain(s) and/or a nisin producing Lactococcus lactis ssp. lactis strain(s); and (iii) optionally one or more bacterial strains that are not Streptococcus thermophilus strain(s) or nisin-producing bacterial strains, preferably Lactobacillus and/or Lactococcus bacterial strains; and (iv) optionally one or more non-bacterial cryoprotectants and/or non- bacterial additives. (14) The starter culture, bacterial culture blend or kit of parts of embodiment (13), wherein components (i) and/or (ii) and optionally components (iii) and/or (iv) are provided as separate frozen or freeze-dried pellets.

(15) The starter culture, bacterial culture blend or kit of parts of embodiment (13) or (14), wherein the component (i) comprises or consists of strain CBS 150251 deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht, the Netherlands and/or strain CBS 150252 deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht and/or any variant thereof having no or limited salt-accompanied nisin-deactivating activity.

(16) A fermented milk product, preferably a cheese, more preferably a Cheddar cheese, comprising: (i) a salt, preferably sodium chloride salt or calcium chloride salt; (ii) one or more Streptococcus thermophilus strain(s) and/or residues of one or more Streptococcus thermophilus strain(s), wherein each Streptococcus thermophilus strain has no or limited salt-accompanied nisin-deactivating activity; and (iii) nisin, preferably nisin A.

Figures

Figure 1: illustration of a nisin deactivation assay.

Figure 2: exemplary calibration curve for nisin quantitation, where average halo sizes in millimeters (mm) of the calibrations are plotted against the natural logarithm of the nisin concentration (Ln (Units nisin / ml)).

The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application are hereby incorporated by reference.

Examples

Example 1: General methods and materials

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998). Strains and cultivation methods. A list of strains used in these examples can be found in the Table 1 below. A M17 broth is prepared according to manufactures instructions (BD Difco™) and supplemented with 1% w/v lactose (1 gram/100 ml) and further referred to as LM17b. A M17 agar is prepared according manufactures instructions (BD Difco™) and supplemented with 0.5% w/v lactose (0.5 gram/100ml) and further referred to as LM17a. Overnight cultures of all S. thermophilus strains are prepared by inoculating 1% v/v inoculum in LM17b and incubated for 18 hours at 37° C.

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

Unless explicitly stated otherwise herein, a percentage of w/v herein refers to an amount of gram per 100 ml. When frozen concentrate is used in experiments it means commercial product which is used in the dosage as stated in the experiment. Table 1. List of strains. For more details, see text.

Screening assay. The salt-accompanied nisin-deactivating activity of Streptococcus thermophilus strains, respectively sodium chloride-accompanied nisin-deactivating activity of Streptococcus thermophilus strains, can be characterized by screening the Streptococcus thermophilus strain with the following screening method, which is comprising steps A-G, where steps A-B describe the culture and sample generation, steps C-E describe the nisin quantification of a sample and step F describes the characterization of the strains based on Step A-E. An illustration is provided in Figure 1. Streptococcus thermophilus strains can be characterized as having positive salt-accompanied nisin-deactivating activity (P-SAND), limited salt-accompanied nisin-deactivating activity (L-SAND) or no salt-accompanied nisin-deactivating activity (N-SAND) by carrying out the following steps:

A) Culture generation, wherein

- separate and pure cultures for each of the specific Streptococcus thermophilus strains to be tested are cultivated in LM17 broth (Difco™ M17 broth supplemented with 1% (w/v) Lactose) using 1 % v/v inoculum and incubating for 18 hours at 37° C; and

- the so obtained culture is used as inoculum to inoculate at 5% (v/v) an amount of 200ml of 12% (w/v) reconstituted skim milk (RSM) supplemented with 15 parts by million by weight (ppmw) sodium formate in a sterile 250ml Schott bottle (the "RSM bottle"), for each strain separately, and grow the culture at 38°C until pH 5.3 is reached to generate a "12% RSM culture@pH 5.3";

B) Sample generation, wherein

- upon reaching a pH of 5.3, both nisin to a final concentration of 50 U/ ml and sodium chloride salt to a final concentration of 5% (w/v) are added to the "12% RSM culture@pH 5.3", followed by a further incubation for 90 minutes at 35°C to generate a "incubated culture"; and

- subsequently, a sample of 50 ml is taken from the "incubated culture" for nisin quantitation (called "t=1 culture samples") from the RSM bottle and stored in a 50 ml Greiner centrifuge tube for follow-up sample treatment and testing;

C) Nisin-stock solution preparation, wherein

- a 10000 unit per milliliter (U/ ml) nisin stock-solution (Nisin - Sigma Aldrich, N5764) in a 0.05 % (v/v) acetic acid solution is made, filtered through 0.22 pm pore size filter and stored at 4°C; and - this stock-solution is used to spike individual 5 milliliter (ml) samples of the "Blank filtrate" (Step D) with a defined amount to generate a "calibration sample" for the calibration curve in concentrations of nisin to be able to generate an accurate calibration curve;

D) Culture and calibration sample treatment, wherein

- uninoculated sterile 12% RSM solution, as described above, is used as zero nisin reference sample and as sample background ("Blank Filtrate") to be able to generate the nisin calibration curve; and therefore, a reference of 50 ml of sterile 12% RSM solution sample is treated in the same way as all individual fermentation samples from t=1 ("t=1 culture samples") as described herein; and

- the pH of all the "t=1 culture samples" and the sterile "12% RSM solution" sample is lowered until 2.0 +/- 0.05 using hydrogen chloride (HCl) and centrifuged at 10000 rpm for 10 minutes at 4°C in a first centrifugation; and

- the supernatant of the first centrifugation is transferred to a new Greiner centrifuge tube and pH was adjusted to 4.0 +/- 0.05 with sodium hydroxide and centrifuged at 10000 rpm for 10 minutes at 4°C in a second centrifugation; and

- the supernatant of the second centrifugation is filtered through a 0.45 micrometer (pm) filter (for example Millipore) and put into a new tube, where the obtained supernatant of the second centrifugation for each individual "t=1 culture samples" is the final solution for nisin measurement to be used for the well diffusion assay; and where the obtained supernatant of the second centrifugation for the sterile "12% RSM solution" is the 0 U/ml nisin blank sample solution and is the "Blank filtrate" which is used to generate the nisin defined concentration samples for the calibration curve; and

- subsequently the 10000 U/ ml nisin stock-solution is used to spike individual 5 ml samples of the "Blank filtrate" with a defined amount ranging between 1 and 100 Units/ ml nisin (at least 5 concentrations (for example 100, 50 ,20, 5, 1.25

U/ ml) tested at regular interval between 1-100 U/ ml) to generate a "calibration sample" for the calibration curve in concentrations of nisin to be able to generate an accurate calibration curve; and

E) Halo-based screening on agar with indicator strain, wherein

- an LM17 agar plate (Difco™ M17 agar supplemented with 1% lactose (w/v)) was heated to liquify the agar solution and cool down to 46°C and the agar is inoculated with a 1% (v/v) exponential growing culture of Lactococcus lactis ssp. cremoris Indicator Strain HP (NCDO 607, ATCC19257, DSM 20069, this is a nisin sensitive strain that can be used to measure nisin concentrations, since a halo will appear) and immediately poured onto a NUNC™ square BioAssay Dish (cat. Nr: 166508) ensuring equal level of the agar and the agar was cooled down to 20°C and dried in a laminar flow cabinet; and

- holes are punched in the agar at a regular mutual distance using a sterile glass pipette and in each hole, 150 microliter (pl) of a "calibration sample", or a blank sample (0 U/ ml) or a "t=1 culture sample" is added for measurement; and

- the plate is incubated for 18 hours at 30°C; and

- subsequently the horizontal and vertical diameters of any visually detectable halos are recorded using a digital slide Caliper (Mitutoyo 150mm Digital Caliper, 500-181-30) and

- the average of the horizontal and vertical diameter of each halo for each "calibration sample" is plotted on the x-axis against the natural logarithm of the nisin concentration of that "calibration sample" on the y-axis; and

- a line of best-line fit is drawn through all measured datapoints for halo-size versus nisin concentration to obtain the "calibration curve".

An example of readings for diameters of calibration samples for the agar halo assay carried out for the below examples is shown below in Table 2, with the data points and best-fit line as deduced calibration curve illustrated in Figure 2.

Table 2. Calibration curve used in the below examples. For more details, see text.

Subsequently the nisin concentration of the "t=1 culture samples" can be deduced from the "calibration curve" using the halo size. This is illustrated in Table 3 below for the strains used in the examples. The nisin concentration subsequently can be used for characterization in step F). F) Strain characterization, wherein

- the specific strains are characterized according to the criteria as described herein above on the basis of the nisin concentration att=1, namely:

- a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having positive salt-accompanied nisin-deactivating activity (P-SAND) if it is capable of degrading equal to or more than 70% of the added 50 U/ ml nisin. That is, a Streptococcus thermophilus strain is considered to have positive salt-accompanied nisin-deactivating activity if the strain at t=1 has degraded equal to or more than 70% of the 50 U/ml nisin added;

- a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having limited salt-accompanied nisin-deactivating activity (L-SAND) if it is capable of degrading equal to or more than 50%, but less than 70%, of the added 50 U/ml nisin. That is, a Streptococcus thermophilus strain is considered to have limited salt-accompanied nisin-deactivating activity if the strain at t=1 has degraded 50%, but less than 70%, of the U/ml nisin added; and

- a Streptococcus thermophilus strain can be characterized as a Streptococcus thermophilus strain having no salt-accompanied nisin-deactivating activity (N- SAND) if it is capable of degrading less than 50%, more preferably less than 40%, still more preferably less than 30%, yet more preferably less than 20% and most preferably less than 10%, of the added 50 U/ml nisin. That is, a Streptococcus thermophilus strain is considered to have no salt-accompanied nisin- deactivating activity if the strain at t=l has degraded less than 50%, more preferably less than 40%, still more preferably less than 30%, yet more preferably less than 20% and most preferably less than 10%, of the U/ ml nisin added.

RNA extraction, purification and expression analysis (RNAseq). For RN A extraction experiments, a strain was grown in presence of 150 U/ml nisin and absence of nisin for 16 hours at 37° C. Cells were concentrated by centrifugation of which RNA was extracted using the EXTRACTIVE TOTAL RNA KIT (Qiagen) protocol with slight modifications. Briefly, bacterial cells (approximately 10¹⁰) were washed three times in phosphate-buffered saline (PBS) buffer (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 minutes, samples were resuspended in RTL buffer (4 M guanidinium thiocyanate, 50 mM TRIS-HCl, 25 mM EDTA, 3% (v/v) Triton X-100 and 1% 2-mercaptoethanol) and sonicated in three cycles of 10 seconds with amplitude of 8 dB. After this step, RNA isolation was performed according to the manufacturer's instructions (Qiagen). The purity of RNA was checked using NanoDrop (ThermoFisher Scientific, Waltham, MA, USA), and RNA integrity was checked both on 2% agarose gel and using an Agilent 5400.

Samples were sequenced (RNAseq) on the Illumina NovaSeq 6000 platform in 150 paired end mode (2Gb raw output per sample) according to the manufacturer's instructions. Raw Illumina reads were analyzed using FastQC (vO.11.9), while adapter removal and trimming of low-quality reads were performed using Trimmomatic (v0.39). The following parameters were used for trimming: [SLIDINGWINDOW:4:20 AVGQUAL:28 MINLEN:60], Trimmed reads were rechecked using FastQC. Trimmed reads were then aligned to the provided S. thermophilus ST01 sequenced genome using Bowtie 2 (v2.4.4). Quantification of gene expression levels from the aligned reads was performed using featurecounts (v2.0.3). The genomic feature used for the reading assignment was gene. Differential analysis of expressed genes was conducted in RStudio (v4.3.2) using DESeq2 (v1.42.0). The count data underwent variance stabilization and normalization with the rlog function.

To identify differentially expressed genes as a result of growth in the presence and 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 was constructed using the log2 fold change values on the x-axis and the -loglO of the adjusted p-values (false discovery rate, FDR) on the y-axis. Genes meeting the significance criteria were highlighted on the volcano plot. Previously determined significant genes were plotted on a bar graph using GraphPad Prism (v10.0.0).

Example 2: Characterization of Streptococcus thermophilus strains

The screening method described above in Example 1 was used to characterize the tested Streptococcus thermophilus strains as P-SAND, L-SAND or N-SAND. The results are shown in Table 3. In the assay, 50 U/ ml nisin and 5% (w/v) sodium chloride salt were added. After 90 minutes of incubation at 35°C only 9 and 7 units/ ml of remaining nisin were detected when ST01 and ST02 were used, respectively. Both strains were therefore characterized as having positive salt- accompanied nisin deactivating activity (P-SAND), since these strains had degraded more than 70% of the nisin, the nisin level was below 15 U/ ml. However, ST03, ST04, ST05 and ST06 could be characterized as having limited salt-accompanied nisin deactivating activity (L-SAND) since for those strains after 90 minutes, respectively 25, 22, 24, 19 U/ ml nisin remained in the screening test. The highest nisin concentration was found when using strain ST09 characterized as N-SAND.

Table 3. Results of the screening assay and characterization of the salt- accompanied nisin-deactivation activity of the individual Streptococcus thermophilus strains used in the examples. Nisin concentrations ("nisin cone.") were measured at t=l sample and expressed in Units/ml. For more details, see text.

This result clearly indicates that, in the presence of salt, nisin degradation is lower and remaining nisin levels are higher after fermentation using L-SAND Streptococcus thermophilus strains (see e.g., strains ST03, ST04, ST05, ST06 and ST08).

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

The enzyme responsible for nisin deactivation by Streptococcus thermophilus has been described as 'nisinase' or 'nisin inactivating enzyme'. Nevertheless, up to date neither the gene encoding the functional enzyme nor the molecular structure of the enzyme has been described.

To allow the identification of the gene encoding the 'nisinase enzyme', the P- SAND strain ST01 with the ability of degrading 70% or more of nisin at t=1 was grown in the presence or absence of 150 U/ ml nisin for 16 hours at 37°C. RNA was extracted from the cells according to the protocol described herein. The expression pattern with and without the addition of nisin revealed the highest upregulation in the presence of nisin for a gene encoding TraX (EMBL AZA18053.1).

Characterization of strains characterized as missing (expression of) traX gene shows as being a N-SAND strain, i.e. lack of the presence of (a functional) TraX protein when the strain is grown on nisin. This N-SAND type is in contrast to e.g. to the P-SAND strain, with upregulation of said gene and thus presence of the functional TraX protein in the presence of nisin.

To test and confirm the function of TraX, a traX deletion mutant strain was made via deletion of the full length traX gene (SEQ ID NO:1) in Streptococcus thermophilus ST01 (P-SAND) using standard genetic engineering methods. Deletion of the gene traX was confirmed by whole genome sequencing, resulting in Streptococcus thermophilus STOIAtraX strain (ST09). Surprisingly, the phenotype of strain ST09 was determined as N-SAND (Table 5). In addition, the growth rate, esp. in the presence of nisin, was slowed down (see Table 4).

Characterization of strains characterized as being a N-SAND strain type could mean a missing traX gene, lack of expression/induction of a traX gene of or lack of the presence of (a functional) TraX protein when the strain is grown on nisin. This N-SAND type is in contrast to e.g. to the P-SAND strain, with upregulation of said gene and thus presence of the functional TraX protein in the presence of nisin.

Further experiments were performed to investigate the traX gene function in response to growth on nisin of Streptococcus thermophilus, in particular to evaluate what is needed to convert a strain with positive salt-accompanied nisin deactivation activity, i.e. a P-SAND strain, into an L-SAND or N-SAND strain.

Table 4. Growth as measured by OD600nm when grown for 48 hours at 37°C in LM17b (LM bouillon) in the presence of amounts of U/ml nisin as indicated. For more details, see text.

Table 5. Role of TraX for nisin degradation in S. thermophilus. For more details, see text.

The Streptococcus thermophilus ST01 strain carrying the full length traX gene could grow in presence of 500 U/mL nisin and can therefore be considered nisin-resistant. Upon deletion of the traX gene, the nisin resistance of P-SAND strain ST01 is converted into a nisin-sensitive strain that is barely growing in the presence of nisin. Thus, the deletion of traX in Streptococcus thermophilus ST01, a P-SAND strain, resulted in the N-SAND phenotype of Streptococcus thermophilus ST09, as defined herein. The slow-/ no-growth of ST09, accompanied by the N-SAND phenotype is not a problem for the nisi n- degradation test as described herein nor for the cheese making process using such a strain, since in the initial fermentation phase, the nisin levels are low to zero, allowing growth and acidification by S. thermophilus strains before the addition and/or impact of nisin applies.

Example 4: Characterization (genotypic) of TraX protein

The whole genome sequence of 121 Streptococcus thermophilus strains has been sequenced of which the traX gene was identified using BLASTn search with reference traX nucleotide sequence of Streptococcus thermophilus ST01 strain (SEQ ID NO:1). The traX gene was found in all 121 strains, showing the great conservation of presence of traX within the species.

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

Table 6. TraX protein sequence of the strains used in the examples.

From these results of traX variants and disruption thereof, it is clear that sequencing the traX gene of a strain can serve as a biomarker for selection of starter cultures. The sequencing of traX in a strain can give indications for Positive-/Limited- or No-salt-accompanied nisin-deactivating activity, especially in case of active site mutations and/or insertions, deletions or stop-codons in the traX gene. From the traX deletion results, it is clear that for certain traX mutations (stop-codons in coding sequence, mutated translational start codons, RBS or Shine-Dalgarno mutations) there's a better chance of have a preferred L- SAND or N-SAND phenotype, so genetic screening might be used to pre-select preferred strains or select candidate mutant strains in a screening.

Example 5: Production of salted Cheddar cheese

The performance of various strains with regards to the nisin-level in the production of salted Cheddar cheese was evaluated using nisin-producing starter cultures. A salted Cheddar cheese was produced with a bacterial starter culture comprising only nisin-producing Lactococcus strains as such or nisin- producing Lactococcus strains mixed with a nisin-immune Lactococcus strain (see Table 7).

As a Reference ("Ref. Blend (nis-)") a bacterial starter culture was used consisting of Lactococcus species suitable for Cheddar cheese, which did not produce nisin.

In addition, two nisin-producing bacterial starter cultures were used:

- "Blend A" consisted of two nisin producing Lactococcus strains, namely Lactococcus lactis (LOl) and Lactococcus lactis (L02); and

- "Blend B" consisted of four nisin producing Lactococcus strains, namely Lactococcus lactis (LOl), Lactococcus lactis (L02), Lactococcus cremoris (L03) and Lactococcus cremoris (L04). To prepare the Cheddar cheese, fresh milk was pasteurized at 73°C for 15 seconds and subsequently cooled to 32°C. The specific bacterial starter culture was added to the milk and the milk was fermented with help of the strains at 31- 32°C for 60 minutes. Subsequently, a coagulant (Maxiren® XDS) was added (40 IMCU/L milk). After 20-30 minutes, the coagulum that had been formed (comprising curd and whey) was cut and gently stirred for 10 minutes. Hereafter the temperature was increased to 38°C over a time period of 30 minutes. When the pH of the curd reached a pH of 6.2, the whey was drained from the curd and the temperature was decreased during the subsequent cheddaring phase to approximately 36°C. When the pH in the curd reached 5.2 - 5.3, the curd was milled. Subsequently the curd was salted with sodium chloride to a target salt of approximately 2% (w/w) in the final product and pressed overnight at room temperature (about 20° C).

After pressing a sample was taken for compositional analysis after which the Cheddar cheese blocks were vacuum sealed and ripened at 11°C for up to 12 months.

Table ?. Cheese production without Streptococcus thermophilus strains. Features TTH and TTF are given in hours, wherein TTR means "Time to Reach the target pH of 6.2 for draining" and TTF means "Time to Finish, i.e. time from a starter addition to final pH 5.2-5.3". For more details, see text.

As illustrated in Table 7, the "Time To Reach" the target pH of pH 6.2 ("TTR pH 6.2") was similar and about 2.8 hours for the Reference Blend without nisin ("Ref. Blend (nis-)") as well as for Blend A and Blend B. Table 6 also showed that nisin levels reached 196 U/g and 248 U/g at pH 5.2 for Blend A and Blend B, respectively, whilst the nisin level for the Reference Blend was 0. The levels of nisin increased over time meaning the production of nisin continued during the ripening process (see the last columns of Table 7). Therefore, we can conclude that the salt addition, as such, in a cheese making process does not impair production by nisin-producing Lactococcus sp. and it does not cause nisin deactivation. In a next step, Cheddar cheese making process is repeated as above with the exception that this time a nisin-producing bacterial starter culture is used also comprising Streptococcus thermophilus strains as follows (see Table 8):

- "Blend 1" consisting of: (i) 80% of a Reference Blend, which Reference Blend contains the strains Lactococcus lactis (L01) and Lactococcus lactis (L02) and Lactococcus cremoris (L03), i.e. all nisin-producing Lactococcus strains; and (ii) 20% of a L-SAND Streptococcus thermophilus strain (ST06); and

- "Blend 2" consisting of: (i) 80% of a Reference Blend, which Reference Blend contains the strains Lactococcus lactis (L01) and Lactococcus lactis (L02) and Lactococcus cremoris (L03), i.e. all nisin-producing Lactococcus strains; and (ii) 20% of a P-SAND Streptococcus thermophilus strain (ST07).

The Time To Finish (TTF), i.e. time from starter addition to final pH S.2-5.3, is faster when a Streptococcus thermophilus strain is present during the acidification than when only Lactococcus sp is added.

Table 8. Cheese production with (1/2) / without (A/B) Streptococcus thermophilus strains. Features TTH and TTF are given in hours, wherein TTR means "Time to Reach the target pH of 6.2 for draining" and TTF means "Time to Finish, i.e. time from a starter addition to final pH 5.2-5.3". For more details, see text.

n.d. = not determined

As expected, the nisin levels after TTF at pH 5.3 and after day 1 are much higher when the L-SAND strain was used as compared to P-SAND strain usage (Table 8) This clearly shows the positive impact of using a L-SAND strain during cheese making, showing an increased speed combined with elevated nisin levels within the cheese matrix. The effect of an N-SAND strain was even more pronounced than that of the L-SAND strains (see Example 6 herein). The cheeses made with "Blend 1" containing the L-SAND strain contained less slits and cracks compared to "Blend 2" containing the P-SAND strain from as early as 30 days of ripening up to the final examination after 150 days of ripening.

Example 6: NSLAB reduction during cheese production (challenge test)

The amount and gas producing NSLAB (Non-Starter Lactic Acid Bacteria) in the cheese matrix is correlated with the severity of slit and crack defects in the cheese. Slit and crack defects in the cheese are unwanted by customers and can be as severe that cheeses explode due to gas production and are lost for normal cheese consumption. Most NSLABs are sensitive to low concentrations of nisin (> 5 U nisin/ ml). As a result, the presence of nisin during milk processing will reduce the number of viable NSLABs during the acidification of the milk and ripening of the cheese, thereby also reducing the slit and crack defects.

Milk contaminated with 1.5E+05 CFU/ ml Companilactobacillus nodensis as indicator strain and representative for NSLABs was used for the nisin inactivation test (as described in Example 1). Strain ST01 (P-SAND) and strain ST09 (N-SAND) were separately inoculated in contaminated milk and incubated at 38°C until pH 5.3 was reached (t=0). Subsequently 50 U/ ml nisin and 5% (w/v) sodium chloride was added and incubated for 1.5 hours at 35°C. After which sample t=1 was taken and incubated for another 18 hours at 32°C after which sample t=2 was taken.

The NSLABs were enumerated using LBS agar (BD Difco™, prepared according to manufacturer's instructions) to show the impact of the presence of a P-SAND or N-SAND Streptococcus thermophilus strain on the inactivation of NSLABs (Table 9), due to the differential level of nisin degradation by the 2 strains. The reduction of NSLAB was 1.2 log CFU/ ml reduction at t=1 and >4.2 log CFU/ ml reduction at t=2 when the N-SAND Streptococcus thermophilus strain was present. When the P-SAND Streptococcus thermophilus ST01 strain was present the reduction of NSLAB at the two timepoints are much lower, specifically 0.2 log CFU/ ml reduction at t=1 and 1.3 log CFU/ ml reduction at t=2.

Table 9. Number of viable counts of NSLAB on LBS agar in Colony Forming Units per ml (CFU/ml) during the different steps of the assay described in Example 1. For more details, see text.

Using an N-SAND type Streptococcus thermophilus strain in a blend with nisin producing Lactococcus sp, the nisin degradation is more limited to low, meaning the concentration of nisin will be higher and remain present for a longer time after salting when compared to the use of a P-SAND type S. thermophilus. The same effect is to be expected when nisin is added during the early cheese making process instead of in situ production by nisin producing Lactococcus sp. The effective higher nisin concentration (due to reduced nisin degradation) means nisin-sensitive NSLABs viable counts will go down due to the (elevated) presence of nisin which will prevent slit and crack defects during cheese ripening from use of an N-SAND type s. thermophilus strain.

On the other hand, the amount of nisin is about 5 to 0 units/g after salting when using a P-SAND type Streptococcus thermophilus strain in the blend with nisin producing Lactococcus sp for making cheese. A lower nisin concentration will show a decreased inactivation of NSLABs, resulting in increased potential to develop slits and cracks. This is in line with the higher nisin levels that were found when using the Streptococcus thermophilus strain N-SAND type compared to Streptococcus thermophilus P-SAND type strain (see Example 3 and 4).

For L-SAND type Streptococcus thermophilus strains in a blend with nisin producing Lactococcus sp, a benefit on reduced nisin degradation in the salting and cheese making process is expected when compared to P-SAND type strains. Therefore, there is clear benefit of using an N-SAND and/or L-SAND type S. thermophilus strain in a blend with nisin producing Lactococcus sp in the cheese making process or when nisin is added during the early cheese making process instead of in situ production by nisin producing Lactococcus sp. (Original in Electronic Form)

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Claims

1. A bacterial culture blend for production of salted fermented milk product, said process being performed in the presence of nisin and salt, preferably with a salt concentration of between 1 and 5 % final concentration (w/w), said blend comprising:

(i) one or more strains of Streptococcus thermophilus,

2. A bacterial culture blend according to claim 1 comprising one or more strains of Streptococcus thermophilus wherein the salt-accompanied nisin- deactivation activity is nullified as tested via its capability of degrading less than 50% of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride, wherein the nisin is added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3; and wherein said strain in modified by:

(3) translational modifications wherein no active TraX RNA is detected.

3. A bacterial culture blend according to claim 1, comprising one or more strains of Streptococcus thermophilus with limited salt-accompanied nisin- deactivation activity and comprising and expressing acetyltransferase TraX, particularly TraX protein according to SEQ ID NO:2 or 3.

4. A bacterial culture blend according to claim 3, wherein the one or more strains of Streptococcus thermophilus selected from strain CBS 150251 and/or strain CBS 150252, both deposited on 20 July 2023 with the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht, the Netherlands and/or any variant thereof having no or limited salt-accompanied nisin-deactivating activity.

5. A bacterial culture blend according to any one of claims 1 to 4, wherein components (i) and/or (ii) and optionally components (iii) and/or (iv) are provided as separate frozen or freeze-dried pellets.

6. A process for generating a strain of Streptococcus thermophilus having no salt-accompanied nisin deactivating activity to be used in the production of a salted fermented milk product, said process comprising:

(1) providing a strain of Streptococcus thermophilus with positive salt- accompanied nisin deactivating activity as tested via its capability of degrading 70% or more of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride, wherein the nisin is added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3; (2) modification in the genome of said strain to inactivate the endogenous gene encoding acetyltransferase TraX, particularly polypeptide according to SEQ ID NO:2 or 3, preferably via introduction of a knock-out of said gene,

(3) selecting strains capable of degrading less than 50% of 50U/ml nisin in 90 minutes at about 35°C in the presence of 5% (w/v) sodium chloride that do not show any TraX RNA or TraX activity, wherein the nisin is added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3.

7. A process for production of a salted fermented milk product, preferably hard or semi hard cheese, using a bacterial blend according to any one of claims 1 to 5, said process comprising:

(a) fermenting a milk base with said bacterial blend, preferably with fermentation until a pH of equal to or less than 6.0, more preferably equal to or less than 5.3 is reached; and

(b) providing nisin, preferably nisin A, to the fermented milk base

(c) optionally providing one or more coagulant(s) to the fermented milk base, preferably wherein the coagulant(s) is/are selected from the group consisting of protease, more preferably a microbial or mammal produced aspartic protease; and

(d) providing salt, particularly sodium chloride, preferably in an amount of 1 to 5% (w/w).

8. Process according to claim 7, wherein step (a), (b) and optionally (c) are carried out simultaneously.

9. Process according to claim 7 or 8, wherein step (b) comprises the addition of nisin during or after the fermentation according to step (a).

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

11. Process according to any one of claims 7 to 10, further comprising: (e) separating the fermented and optionally coagulated milk base in a curd and a whey; and

(f) adding salt to the curd, wherein the salt is preferably sodium chloride.

12. Process according to any one of claims 7 to 11, wherein the salted fermented milk product is a cheese, preferably a hard or semi-hard cheese, more preferably a Cheddar cheese.

13. Use of a bacterial blend according to any one of claims 1 to 4 in a cheesemaking process, wherein the number of slits is low, meaning the product can be sold for a good value.

14. A salted fermented milk product, preferably a cheese, more preferably a Cheddar cheese, comprising:

(i) a salt, preferably sodium chloride salt or calcium chloride salt, more preferably with a final concentration of 1 to 5% (w/w);

(ii) a bacterial blend according to any one of claims 1 to 5,

(iii) nisin, preferably nisin A, more preferably with a final concentration of 2 to 600 U/g.

15. A method for identification of Streptococcus thermophilus strains showing reduced nisin-degradation in the presence of salt to be used in a bacterial blend comprising nisin-producing Lactococcus strains for production of salted fermented milk products, said method comprising:

(i) providing a pool of Streptococcus thermophilus strains,

(ii) providing an assay for testing the nisin degradation at a fixed salt concentration, said assay mesu ring the capability of the Streptococcus thermophilus strain, in the presence of about 5% (w/v) sodium chloride, of degrading in about 90 minutes at about 35°C, a certain amount of 50 U/ml of nisin added to a culture of such Streptococcus thermophilus strain inoculated at about 5% (v/v) in a 200ml solution of about 12% (w/v) reconstituted skim milk (RSM) supplemented with about 15 parts per million by weight (ppm) sodium formate and grown at about 38°C until pH 5.3;

(iii) testing the strains for their capability of salt-accompanied nisin degradation in the production of salted fermented milk products, particularly salted cheese, wherein a strain showing less than 50% nisin deactivation under the conditions of step (ii) is classified as no salt-accompanied nisin-deactivating (N-SAND), a strain showing 50 to less than 70% nisin deactivation under the conditions of step (ii) is classified as limited salt-accompanied nisin-deactivating (L-SAND); and a strain showing 70% to 100% nisin deactivation under the conditions of step (ii) is classified as positive salt-accompanied nisin-deactivating (P-SAND); (iii) testing the strains for their expression and presence of the endogenous acetyltransferase TraX, wherein no presence of TraX indicates a strain classified as N-SAND;

(iv) optionally testing the strains of step (iii) in the presence of a nisin-producing lactic acid bacterium.