Controlling flavor potential and bacteriophage risk of lactococcus lactis in cheddar cheese

Abstract

The present invention relates to a novel bacterium of Lactococcus lactis, which exhibits temperature induced cell lysis in the absence of phage release. These bacteria may provide improved flavor profile, for example, in cheese production. The invention also relates to a method of selecting such a Lactococcus lactis strain and its use for producing cheese, curing cheese, eliminating the bitterness of cheese and / or increasing the amount of free amino acids in cheese. Methods of making food products as well as food products comprising such Lactococcus lactis strains are also described. Finally, the invention relates to a method for producing the strain of the invention.

Description

Field of the Invention

[0001] The present invention belongs to the field of biotechnology and relates to new Lactococcus lactis bacteria which show temperature-induced cell lysis in the absence of phage release. These bacteria can provide improved flavor characteristics, for example, in cheese production. The present invention also relates to methods for selecting such Lactococcus lactis strains and their use for producing cheese, ripening cheese, eliminating cheese bitterness and / or increasing the amount of free amino acids in cheese. Methods for making food products and foods comprising such Lactococcus lactis strains are also described. Finally, the present invention relates to methods for making the strains of the present invention. Background of the Invention

[0003] The flavor of cheese, especially Cheddar cheese, is closely related to the cell lysis of bacteria in starter culture. Therefore, controlling the lysis of starter is crucial for controlling and accelerating the ripening of Cheddar cheese.

[0004] Without being bound by theory, bacteria produce a large amount of intracellular enzymes, such as but not limited to peptidases, lipases and various amino acid decomposition enzymes. These intracellular components are released into the cheese after bacterial lysis, and facilitate the local flavor and the ripening process of cheese. For example, the proteolysis of known casein, free amino acids are converted into aromatic compounds and the lipolysis of milk fat, which are all indispensable in the cheese ripening process. When starter cracking occurs easily, proteolysis increases, particularly the level of free amino acids increases, and bitterness decreases, resulting in better local flavor.

[0005] Bacterial lysis can occur via two main pathways.

[0006] First, endogenous peptidoglycan hydrolase (PGH) causes autolysis. In bacteria, autolysis is strictly regulated by cellular mechanisms because overactive PGH can threaten cell integrity. This means that autolysis causes measurable cell lysis only due to uncontrolled activity. This can occur under conditions where peptidoglycan synthesis is slowed down relative to PGH activity. For example, this process can be very effective for Lactobacillus helveticus. However, the resulting flavor of the cheese made with Lactobacillus helveticus is not popular in many markets. In this case, for mature cheddar cheese, the traditional flavor characteristics provided by Lactococcus lactis, which usually does not show effective autolysis, are preferred.

[0007] Secondly, bacteriophages (i.e., viruses that infect and replicate in bacteria) can also cause bacterial cell wall lysis. Many bacterial strains carry one or more prophages in their genome. Prophages are latent forms of phages in which viral genes are inserted into circular bacterial DNA. Prophage induction usually occurs through mutagens or environmental stresses. These stresses can be, but are not limited to, heat (e.g., heat in cheesemaking), salt concentration, or pH. Once induced, phages enter a lytic cycle. After new phage DNA and protein synthesis, they assemble into virions. At the end of the cycle, lytic proteins (lysins) are produced that cause cell lysis, thereby releasing phage virions.

[0008] In general, the lytic activity of phage lysins is expected to be more efficient than autolysis caused by PGH. However, the presence and induction of prophages and lytic cycles are believed to necessarily lead to the release of undesirable phages, which may then also infect other strains, for example, in the same seed culture. In fact, one of the main sources of phage contamination in cheese production comes from lysogenic strains.

[0009] Although phage contamination is not necessarily a problem in traditional cheese production, in industrial cheese production (where consistency of production takes precedence over flavor development), strains known to lyse and / or contain at least part of a prophage cluster are not selected because the risk associated with phage release is too high.

[0010] This results in inferior flavor since strains showing efficient cell lysis are not selected. Sometimes, flavor can be enhanced by adding other strains. However, this results in additional costs.

[0011] There is a pressing industrial need for bacterial strains that exhibit efficient cell lysis in the absence of toxic phage release, leading to the release of flavor-contributing intracellular components. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Illustration of the time (min)-temperature (°C) curves for incubation of 43 different strains of Lactococcus lactis.

[0014] Figure 2 : Lysis of experimental Lactococcus lactis strains measured by aminopeptidase activity specific for Gly-Pro dipeptide after 24 h of growth according to a temperature profile simulating cheddar cheese production.

[0015] Figure 3A and 3B : PCR analysis of samples from heat-induced Lactococcus lactis strains.

[0016] Figure 4: Bacterial cell lysis measured by aminopeptidase activity specific for Gly-Pro dipeptide in the cheese serum phase during cheese ripening. Mean values ​​of replicate cheese making experiments are shown.

[0017] Figure 5 : Total amount of free amino acids during cheese ripening. Mean values ​​of replicate cheese making trials are shown.

[0018] Figure 6 : Selected flavour attributes of cheeses after 180 days (bitter) or 360 days (sweetness, umami, brothy, fruity) ripening as assessed by a trained descriptive sensory panel (external). Mean values ​​of replicate cheese manufacturing trials are shown.

[0019] preservation

[0020] At the applicant's request, only samples of the deposited microorganisms mentioned in the table below may be provided to the expert until the date of grant of the patent.

[0021] Table 1. Depositary institutions that have acquired the status of international depositary institutions under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, 38124 Braunschweig, Germany.

[0022] strains Accession number Date of deposit Lactococcus lactis DSM 34326 July 13, 2022 Lactococcus lactis DSM 34327 July 13, 2022 Lactococcus lactis DSM 34329 July 13, 2022 Lactococcus lactis DSM 34331 July 13, 2022 SUMMARY OF THE INVENTION

[0024] The inventors surprisingly found that there are Lactococcus lactis bacteria that show efficient lysis in the absence of an inducible prophage and without releasing phage particles. In other words, for these strains, it was surprisingly found that triggered lysis still occurs in the absence of prophage induction.

[0025] Typically, to provide the desired phage robustness of the starter culture, strains that exhibit efficient lysis and / or contain at least a portion of the prophage cluster are not selected. Efficient lysis is considered an indication of an inducible prophage. In addition, it is believed that the presence of a prophage must also result in the release of phage particles, making the presence of at least a portion of the prophage also a criterion for not being selected.

[0026] In contrast, strains that do not display an inducible prophage and do not release intact phage material would not be considered advantageous by the skilled person from a cheese ripening perspective, as these strains would not be expected to lyse efficiently.

[0027] Unexpectedly, the inventors have discovered that certain strains can express prophage-derived lysins and efficiently lyse even when the prophage is not intact and therefore would not be expected to release phage particles.

[0028] In other words, within the group of bacteria that did not display inducible prophages, there was an unexpectedly very small subset of strains that still displayed efficient lysis via phage-derived lysins when triggered by exogenous stress. These strains fully met the requirements of the cheddar cheese segment.

[0029] Thus, in a first aspect, the present invention relates to Lactococcus lactis bacteria capable of temperature-induced lysis but without an intact prophage cluster.

[0030] In a second aspect, the present invention relates to a composition and / or a starter culture comprising the Lactococcus lactis bacterium of the first aspect.

[0031] In a third aspect, the present invention relates to a method for selecting a Lactococcus lactis bacterium by determining that the Lactococcus lactis bacterium is capable of temperature-induced lysis and that the Lactococcus lactis bacterium contains an incomplete prophage cluster.

[0032] In a fourth aspect, the present invention relates to the use of the Lactococcus lactis bacterium of the first aspect, the composition and / or the starter culture of the second aspect or the Lactococcus lactis bacterium selected by the method of the third aspect for:

[0033] (i) the production of cheddar-type cheese,

[0034] (ii) Ripened cheese (preferably cheddar cheese)

[0035] (iii) removing the bitter taste of cheese (preferably cheddar-type cheese), and / or

[0036] (iv) increasing the amount of free amino acids in cheese, preferably cheddar-type cheese.

[0037] In a fifth aspect, the present invention provides a method for producing a food or feed product, the method comprising at least one stage using at least one Lactococcus lactis bacterial strain as defined in the first aspect, a composition and / or a starter culture as defined in the second aspect or a Lactococcus lactis bacterium selected by the method of the third aspect.

[0038] In a sixth aspect, the present invention provides a food or feed product comprising at least one Lactococcus lactis bacterial strain as defined in the first aspect, a composition and / or a starter culture as defined in the second aspect or a Lactococcus lactis bacterium selected by the method of the third aspect.

[0039] In a seventh aspect, the present invention provides a method for making the Lactococcus lactis strain of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0041] definition

[0042] All definitions of the terms herein are based on what a skilled person would understand regarding the technical background relevant to this document.

[0043] In the context of the present invention, in any of its embodiments, the expression "lactic acid bacteria" ("LAB") refers to food-grade bacteria that produce lactic acid as the major metabolic end product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are Gram-positive, low GC, acid-tolerant, non-spore-forming rods or cocci. During the fermentation phase, the consumption of carbohydrates by these bacteria leads to the formation of lactic acid, thereby lowering the pH and causing the formation of protein coagulum. Therefore, these bacteria are responsible for the acidification of milk and the texture of dairy products. The Lactococcus lactis strains of the present invention are classified as lactic acid bacteria.

[0044] The LAB strains (e.g., Lactococcus lactis strains) of the present invention may be isolated strains, e.g., isolated strains from naturally occurring sources, or may be non-naturally occurring strains, e.g., strains obtained by recombination. Recombinant strains differ from naturally occurring strains by at least the presence of a nucleic acid construct for transformation or transfection of the parent strain.

[0045] "Bacteriophage" in this specification and claims means a virus that infects and replicates in bacteria. "Bacteriophage" and "phage" are used interchangeably in this specification and claims.

[0046] "Prophage cluster" in this specification and claims means a gene cluster encoding a phage integrated into a bacterial chromosome. "Incomplete prophage cluster" lacks any part of a complete prophage cluster, such as an entire gene or any part of a gene.

[0047] "Prophage induction" in this specification and claims means that the phage enters the "lytic cycle". During the lytic cycle, new phage DNA and proteins are synthesized and then assembled into virus particles. At the end of the cycle, lytic proteins (lysins) are produced, resulting in cell lysis and release of phage virus particles.

[0048] "Holin / lysin cassette" in the present application and claims means the genes encoding perforin and lysin proteins, respectively. The holin / lysin cassette is part of the prophage cluster.

[0049] Perforins are a group of different small proteins produced by bacteriophages to trigger and control the degradation of the host cell wall at the end of the lytic cycle. Lysins, also known as endolysins or murein hydrolases, are hydrolases produced by bacteriophages to lyse the host cell wall at the end of the lytic cycle.

[0050] Without being bound by theory, perforins form pores in the cell membrane, allowing lysins to reach and degrade peptidoglycan, a component of the bacterial cell wall.

[0051] "Lysis" in this specification and claims means bacterial cell wall lysis, preferably temperature-induced lysis. As further described below and in the examples, bacterial cell wall lysis can be measured by aminopeptidase activity. In the context of the present invention, a strain exhibits temperature-induced lysis if it exhibits an aminopeptidase activity of at least 0.25 nmole / min / ml under the conditions described below and illustrated in Example 3 herein.

[0052] "Release of phage particles" in the present specification and claims refers to the release of assembled phage virions. As further described below and in the examples, the release of phage particles can be measured, for example, by a PCR assay that detects phage DNA in the supernatant.

[0053] In the context of the present invention, the terms "strain derived from...", "derivative strain" or "mutant" are to be understood as strains derived from the strains of the present invention by means of, for example, genetic engineering, radiation and / or chemical treatment and / or selection, adaptation, screening, etc. Preferably, the derivative strain is a functionally equivalent mutant, for example, a strain having substantially the same or improved properties as the parent strain in terms of water holding capacity. Such derivative strains are part of the present invention. In particular, the term "derivative strain" or "mutant" refers to a strain obtained by subjecting the strain of the present invention to any commonly used mutagenic treatment (including treatment with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light), or to a spontaneously occurring mutant.

[0054] The mutant may have been subjected to several mutagenesis treatments (a single treatment is understood to be a mutagenesis step followed by a screening / selection step), but it is currently preferred that no more than 20, no more than 10 or no more than 5 treatments are performed. In preferred derivative strains of the present invention, less than 1% or less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been altered (e.g. by substitution, insertion, deletion or a combination thereof) compared to the parent strain.

[0055] The terms "fermented milk" and "dairy product" are used interchangeably herein. In the context of the present invention, in any embodiment thereof, the expression "fermented dairy product" means a food or feed product, wherein the preparation of the food or feed product involves fermenting a dairy substrate with lactic acid bacteria. "Fermented dairy products" as used herein include, but are not limited to, products such as thermophilic fermented dairy products or mesophilic fermented dairy products. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 35°C, for example at a temperature of about 35°C to about 45°C. The term "mesophilic fermentation" herein refers to fermentation at a temperature of about 22°C to about 35°C.

[0056] In the context of the present application, the term "milk" is widely used in its common sense to refer to a liquid produced by the mammary glands of animals (e.g., cows, sheep, goats, buffaloes, camels, etc.) or produced using a plant substrate. The term "milk substrate" or "milk substrate" can be any milk material that can be fermented according to the present invention. In a preferred embodiment, the milk is cow's milk. According to the present invention, the milk may have been processed, and therefore the term "milk" includes whole milk, skim milk, fat-free milk, low-fat milk, full fat milk, low-lactose milk or concentrated milk. Fat-free milk is a non-fat milk product or a skim milk product. Low-fat milk is generally defined as milk containing about 1% to about 2% fat. Whole milk generally contains 2% or more fat. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk-like products containing proteins, such as whole milk or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, cream, or plant-based milk. The milk substrate may be derived from any mammal, such as substantially pure mammalian milk or reconstituted milk powder. Plant sources of milk include, but are not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp, sesame seeds, and sunflower seeds. Preferably, the plant-based milk is soy milk, which may preferably be supplemented with glucose, such as 0.5-5% glucose, preferably 0.5-2% glucose, more preferably about 2% glucose.

[0057] As used herein, the term "dairy product" refers to a food product produced from milk.

[0058] Prior to fermentation, the dairy base may be homogenized and pasteurized according to methods known in the art.

[0059] In the context of the present invention, "homogenization" as used in any embodiment thereof means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, homogenization can be performed to break down the milk fat into smaller sizes so that it can no longer separate from the milk. This can be achieved by forcing the milk through small holes under high pressure.

[0060] In the context of the present invention, "pasteurization" as used in any embodiment thereof means treating a milk substrate to reduce or eliminate the presence of living organisms (e.g., microorganisms). Preferably, pasteurization is achieved by maintaining the milk substrate at a specified temperature for a specified period of time. The specified temperature is typically achieved by heating. The temperature and duration may be selected so as to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may then be performed.

[0061] In the context of the present invention, in any of its embodiments, "fermentation" means the conversion of carbohydrates into acids or alcohols or a mixture of the two by the action of microorganisms (LAB). Fermentation processes for the production of foods (such as dairy products) are well known and the skilled person will know how to choose appropriate process conditions, such as temperature, oxygen, amount of microorganisms and process time. In any embodiment of the present invention, the fermentation conditions are selected to support the implementation of the present invention, for example, to obtain a food, preferably a food having an improved water holding capacity compared to a food produced by a method not involving the use of at least one EPS structure and / or the strain producing said structure as described according to the first aspect of the present invention or not involving the use of the composition as described according to the second aspect of the present invention.

[0062] "Cheddar-type cheese" refers to a range of natural cheeses consumed worldwide, classified as hard to semi-hard cheeses that are aged. The cheddar-type cheese making process is characterized by the lumping of cheese particles and the use of dry salting of ground curds, which produces a ground curd interface that is typically still visible in aged cheeses. Cheddar cheese is aged for periods ranging from 2 weeks to greater than 1 year, depending on the type, and can be further distinguished by a dense and crumbly texture that includes a variety of flavor characteristics, among which brothy, nutty, sulfurous, and fruity are frequently encountered. Examples of cheddar-type cheeses are mild to mature cheddar, Monterey Jack, Colby, and Territorial.

[0063] As used herein, the term "about" (or "approximately") means the indicated value ± 1% of its value, or the term "about" means the indicated value ± 2% of its value, or the term "about" means the indicated value ± 5% of its value, the term "about" means the indicated value ± 10% of its value, or the term "about" means the indicated value ± 20% of its value, or the term "about" means the indicated value ± 30% of its value; preferably, the term "about" means the exact indicated value (±0%).

[0064] Throughout the description and claims, the word "comprise" and variations of the word (e.g., "comprising", "having", "including", "containing") are generally non-limiting and therefore do not exclude other features, which may be, for example, technical features, ingredients, substrates, components or steps. However, whenever the word "comprise" is used herein, this also includes special embodiments in which the word is understood to be limiting; in this particular embodiment, the word "comprise" has the meaning of the term "consisting of".

[0065] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the appended claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0066] Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0067] Lactococcus lactis bacterial strain of the present invention

[0068] In a first aspect, the present invention relates to Lactococcus lactis bacteria capable of temperature-induced lysis but lacking intact prophage clusters.

[0069] More particularly, wherein temperature-induced lysis is determined by the Lactococcus lactis bacterium exhibiting an aminopeptidase activity of at least 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2 or 2.5 nmole / min / ml as measured by the activity of an aminopeptidase specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulated for cheddar cheese making, see Example 3 for further details.

[0070] Preferably, the aminopeptidase activity is at least 0.25 nmole / min / ml.

[0071] In another embodiment, the strain of the invention achieves aminopeptidase activity after 24 hours of growth according to a temperature profile simulating cheddar cheese making by adding 1% glucose and inoculating 2% of an overnight culture grown in B-milk or adding 1% glucose and directly inoculating 1% of a culture stock solution.

[0072] In a typical temperature profile for cheddar cheese making, the following temperatures are used, see Figure 1 :

[0073] Time (min) Temperature(℃) 0 32 70 32 100 40 160 40 225 37 285 30 525 24 1245 20 1600 20

[0074] However, the temperature profile is not limited to this particular example. The skilled person knows which temperature profiles are suitable for producing cheddar-type cheese.

[0075] In addition, Lactococcus lactis bacteria do not contain a complete prophage cluster. An incomplete prophage cluster lacks some parts of the prophage cluster. For example, the prophage cluster may lack the entire gene or part of the gene. For example, an example of a potentially missing but essential gene is a gene encoding a structural protein as a phage tail protein or a phage capsid protein. If a functional phage tail cannot be synthesized or assembled, or a phage capsid (containing phage DNA) cannot be synthesized or assembled, then a functional phage particle cannot be constructed, which makes it impossible for phage attack to become after cell lysis. The complete lack or mutation of these structural genes such as point mutations or deletions (causing gene truncation) clearly shows that the production of complete phage particles has been abolished.

[0076] Based on the genome sequence of a particular strain, BLAST searches are performed using known temperate phage or prophage sequences found in the chromosomes of strains of the same species, or by using a specific phage DNA search program (e.g., PHASTER program) (Arndt, D. et al., 2016). Phage-related DNA clusters can be identified. For example, the lack or truncation of a specific essential gene combined with the presence of a perforin / lysin gene cluster enables the selection of candidate strains that are not expected to produce complete phage particles after induction. The PHASTER program, for example, does directly indicate the presence of complete or incomplete phage particles in a particular bacterial chromosome. Those that are presumed to indicate the presence of incomplete phage particles can be further screened to determine the presence of a perforin / lysin box. By testing the release of phage particles after induction of lysis, it is possible to measure whether lysis does occur without releasing such complete phages.

[0077] Preferably, the temperature-induced lysis is carried out in the absence of phage particle release. The phage is not particularly limited, but temperate phages, especially Lactococcus P335, are relevant. Phage release can be determined, for example, by using PCR to detect the supernatant of the temperature-induced sample, wherein the absence of a PCR band indicates the absence of phage release, see Example 3.

[0078] The skilled person is aware of suitable primers that can be used to detect phages (Muhammed et al., 2018). PCR primers can be designed based on the (incomplete) prophage sequence detected in the chromosome. For example, phages can be detected based on PCR using primers based on truncated prophage sequences. In the context of the present invention, a preferred method is to use primers based on a perforin / lysin box. According to the previous requirements, the box is present in the relevant strain and can then be used to detect the release of phages because the putative phage has a perforin / lysin box in the chromosome.

[0079] The type of phage is not particularly limited, but temperate phages are of particular interest. In particular, the P335 species is of interest, and the skilled person knows methods for designing suitable primers for targets that can be used to detect this species.

[0080] By way of example, the primer pairs shown below were used as they are frequently used to detect P335-type temperate phages.

[0081]

[0082] or

[0083]

[0084] This primer pair detects P335 type bacteriophage CHPC1237, which is used herein as a positive control for the PCR assays in the Examples. Other suitable targets include PDUT and dUPTase.

[0085] Bacterial strain of the present invention can also have the gene of coding autolytic enzyme in its genome.However, as previously discussed, the cracking carried out by autolysis is expected to be much weaker than the cracking triggered by perforin / lysin.Therefore, the existence of autolytic enzyme will not cause the cracking of obvious temperature induction.The example of the gene of coding autolytic enzyme in Lactococcus lactis is acmA, as further described in the following embodiment.

[0086] In a further preferred embodiment, the Lactococcus lactis bacterium contains the complete perforin / lysin cassette.

[0087] Even more preferably, the Lactococcus lactis bacterium is selected from:

[0088] (i) DSM 34326;

[0089] (ii) DSM 34327;

[0090] (iii) DSM 34329;

[0091] (iv) DSM 34331;

[0092] or a mutant or variant thereof having retained or further improved aminopeptidase activity and lacking phage release as determined by PCR.

[0093] Compositions and / or starter cultures of the invention

[0094] In a second aspect, the present invention relates to a composition and / or a starter culture comprising the Lactococcus lactis bacterium of the first aspect.

[0095] Preferably, in any embodiment of the present invention, the composition or starter culture of the present invention comprises at least 1x10 6 CFU (colony forming units) / ml of total LAB strains. It may be preferred that the composition comprises at least 1x10 8 CFU / ml of at least one, preferably two, lactic acid bacteria strains according to the invention.

[0096] In another embodiment, the composition or starter culture of the present invention may further comprise at least one LAB strain according to the present invention and another Lactococcus lactis strain. The other Lactococcus lactis strain is not particularly limited.

[0097] In another embodiment, the composition of the present invention or the starter culture further comprises a Streptococcus thermophilus and / or a Lactobacillus strain. For example, the Lactobacillus strain can be Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus pentosus or Lactobacillus rhamnosus.

[0098] LAB (including bacteria of the species Lactococcus lactis) are typically supplied to the dairy industry as frozen (F-DVS) or freeze-dried (FD-DVS) cultures for batch starter propagation, or as so-called "Direct Vat Set" (DVS) cultures intended for direct inoculation into fermentation vessels or fermentation vats to produce dairy products (e.g., fermented dairy products). Such lactic acid bacteria cultures are commonly referred to as "starter cultures" or "starters". Therefore, the composition of the present invention can be frozen or freeze-dried. In addition, the composition of the present invention can be provided in liquid form. Therefore, in one embodiment, the composition is in frozen, dried, freeze-dried or liquid form.

[0099] For DVS cultures, the culture preferably contains at least 1x10 10 CFU / g, more preferably 5x10 10 CFU / g of at least one, preferably two strains according to the invention.

[0100] Composition of the present invention or starter culture can also additionally include cryoprotectant, lyoprotectant, antioxidant, nutrient, filler, flavoring or its mixture.Composition preferably includes one or more of cryoprotectant, lyoprotectant, antioxidant and / or nutrient, more preferably cryoprotectant, lyoprotectant and / or antioxidant, and most preferably cryoprotectant or lyoprotectant or both.Protective agent such as the use of cryoprotectant and lyoprotectant is well known to those skilled in the art.Suitable cryoprotectant or lyoprotectant include monosaccharide, disaccharide, trisaccharide and polysaccharide (for example glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (arabic gum) etc.), polyol (for example erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol etc.), amino acid (for example proline, glutamic acid), complex substance (for example skim milk, peptone, gelatin, yeast extract) and inorganic compound (for example sodium tripolyphosphate).

[0101] In one embodiment, the composition or starter culture according to the present invention may include one or more cryoprotectants selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uridine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and derivatives of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallic acid esters, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (e.g., vitamin B family, vitamin C). The composition may also optionally include other substances, including fillers (e.g., lactose, maltodextrin) and / or flavoring agents.

[0102] In one embodiment of the invention, the cryoprotectant is an agent or mixture of agents that has a potentiating effect in addition to its cryoprotective properties.

[0103] The expression "enhancing effect" is used to describe the following situation, in which, when the thawed or reconstructed culture is inoculated into the culture medium to be fermented or converted, the cryoprotectant confers an increased metabolic activity (enhancing effect) to the thawed or reconstructed culture. Vitality and metabolic activity are not synonymous concepts. Commercially frozen or freeze-dried cultures can maintain their vitality, although they may have lost a large part of their metabolic activity, for example, when stored (even if the storage period is short), the culture may lose its acidogenic (acidifying) activity. Therefore, vitality and enhancing effect must be evaluated by different assays. Vitality is evaluated by vitality assays (e.g., determining colony forming units), while enhancing effect is evaluated by quantifying the relevant metabolic activity of the thawed or reconstructed culture relative to the vitality of the culture. The term "metabolic activity" refers to the oxygen removing activity of the culture, its acidogenic activity (i.e., the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid), or its metabolite producing activity, for example, the production of aromatic compounds such as acetaldehyde, (α-acetolactate, acetoin, diacetyl and 2,3-butanediol (butanediol)).

[0104] In one embodiment, the composition or starter culture of the present invention contains or comprises 0.2% to 20% of a cryoprotectant or mixture of agents (measured in % w / w of the material). However, it is preferred to add a cryoprotectant or mixture of agents in an amount in the range of 0.2% to 15%, 0.2% to 10%, 0.5% to 7% and 1% to 6% by weight, including a cryoprotectant or mixture of agents in the range of 2% to 5% (measured in % w / w of the frozen material by weight). In a preferred embodiment, the culture comprises about 3% of a cryoprotectant or mixture of agents (measured in % w / w of the material by weight). An amount of about 3% cryoprotectant corresponds to a concentration in the range of 100 mM. It should be appreciated that for each aspect of the embodiments of the present invention, the range can be an increment of the described range.

[0105] In another aspect, the composition or starter culture of the present invention contains or comprises an ammonium salt (e.g., an ammonium salt of an organic acid (e.g., ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as an enhancer (e.g., a growth enhancer or an acidification enhancer) for bacterial cells, such as cells belonging to the species Lactococcus lactis. The terms "ammonium salt," "ammonium formate," and the like should be understood as a source of salts or a combination of ions. The term "source," such as "ammonium formate" or "ammonium salt," refers to a compound or mixture of compounds that provides ammonium formate or an ammonium salt when added to a cell culture. In some embodiments, the source of ammonium releases ammonium into the growth medium, while in other embodiments, the source of ammonium is metabolized to produce ammonium. In some preferred embodiments, the source of ammonium is exogenous. In some particularly preferred embodiments, the ammonium is not provided by the dairy matrix. Of course, it should be understood that ammonia can be added instead of an ammonium salt. Thus, the term ammonium salt includes ammonia (NH3), NH4OH, NH4 + wait.

[0106] In one embodiment, the composition of the present invention may comprise a thickener and / or stabilizer, such as pectin (eg, HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginate and guar gum.

[0107] Method for selecting Lactococcus lactis of the present invention

[0108] In a third aspect, the present invention relates to a method for selecting Lactococcus lactis bacteria by determining that the Lactococcus lactis bacteria are capable of temperature-induced lysis and that the Lactococcus lactis bacteria contain an incomplete prophage cluster.

[0109] As explained above, the skilled person would not consider a strain that does not display an inducible prophage and does not release intact phage material to be advantageous from a cheese ripening point of view.

[0110] Unexpectedly, the inventors have discovered that certain strains can express prophage-derived lysins and efficiently lyse even when the prophage is not intact and therefore would not be expected to release phage particles.

[0111] In other words, within the group of bacteria that did not display inducible prophages, there was an unexpectedly small subset of strains that, when triggered by exogenous stress, still displayed efficient lysis via phage-derived lysins. These strains fully met the requirements of the cheddar cheese market segment.

[0112] Therefore, in a preferred embodiment, the method according to the first aspect is to select a Lactococcus lactis bacterium.

[0113] In an embodiment, the method comprises:

[0114] (1) Determining that Lactococcus lactis bacteria are capable of temperature-induced lysis,

[0115] wherein the Lactococcus lactis bacterium exhibits an aminopeptidase activity of at least 0.25 nmole / min / ml as measured by aminopeptidase activity specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulating cheddar cheese production, and

[0116] (2) Determined that Lactococcus lactis bacteria contain incomplete prophage clusters.

[0117] In a further preferred embodiment, the method further comprises the step of determining that the Lactococcus lactis bacteria do not release phage particles after induction of temperature-induced lysis.

[0118] In a preferred embodiment, the lack of phage particle release is determined by detecting phage in the supernatant of a temperature-induced sample using PCR, wherein the absence of a PCR band indicates the lack of phage release.

[0119] As discussed in the first aspect of the invention, the skilled person is aware of suitable primers for detecting the presence of phage particles.

[0120] PCR primers can be designed based on the (incomplete) prophage sequence detected in the chromosome. For example, phage can be detected based on PCR using primers based on truncated prophage sequences. In the context of the present invention, a preferred method is to use primers based on a perforin / lysin box. According to previous requirements, the box is present in the relevant strain, and can then be used to detect the release of phage, because the putative phage has a perforin / lysin box in the chromosome.

[0121] The type of phage is not particularly limited, but temperate phages are of particular interest. In particular, the P335 species is of interest, and the skilled person knows methods for designing suitable primers for targets that can be used to detect this species.

[0122] By way of example, the primer pairs listed below were used as they are commonly used to detect P335-type temperate phages.

[0123]

[0124] or

[0125]

[0126] Other suitable targets include PDUT and dUPT enzymes.

[0127] In a particularly preferred embodiment, the following primer pair is used to detect P335 type phage:

[0128]

[0129] This primer pair detects the P335-type bacteriophage CHPC1237, which is used herein as a positive control for the PCR assays in the Examples.

[0130] Use of the lactococcus lactis of the present invention for producing cheese, ripening cheese, eliminating the bitterness of cheese and / or increasing the amount of free amino acids in cheese

[0131] In a fourth aspect, the present invention relates to the use of the Lactococcus lactis bacterial strain of the first aspect, the composition and / or starter culture of the second aspect, or the Lactococcus lactis bacteria selected by the method of the third aspect for:

[0132] (i) production of cheese,

[0133] (ii) ripened cheese, preferably cheese,

[0134] (iii) remove the bitter taste of cheese, and / or

[0135] (iv) Increase the amount of free amino acids in cheese.

[0136] In a specific embodiment of the fourth aspect, the present invention provides

[0137] (i) DSM 34326

[0138] (ii) DSM 34327

[0139] (iii) DSM 34329

[0140] (iv) DSM 34331,

[0141] or its mutants or variants for the following uses:

[0142] (i) production of cheese,

[0143] (ii) Ripened cheese,

[0144] (iii) remove the bitter taste of cheese, and / or

[0145] (iv) increasing the amount of free amino acids in cheese;

[0146] Said mutants or variants have retained or further improved aminopeptidase activity and lack phage release as determined by PCR.

[0147] In a preferred embodiment, the cheese is a cheddar-type cheese, see the preceding definition. More particularly, the cheese is selected from mild to mature cheddar, Monterey Jack, Colby and Territory. In a further preferred embodiment, the cheese is a continental cheese. Continental cheese is a semi-hard yellow cheese, such as Gouda, Edam, Tilsit and Maasdam.

[0148] Method for producing a food or feed product and a food or feed product

[0149] In a sixth aspect, the present invention provides a food or feed product comprising at least one Lactococcus lactis bacterial strain as defined in the first aspect, a composition and / or starter culture as defined in the second aspect or a Lactococcus lactis bacterium selected by the method of the third aspect.

[0150] In one embodiment, the present invention relates to a method for producing a food or feed product, which method comprises at least one stage using the lactic acid bacteria strain Lactococcus lactis DSM 34326 or a mutant or variant thereof.

[0151] In one embodiment, the present invention relates to a method for producing a food or feed product, which method comprises at least one stage using the lactic acid bacteria strain Lactococcus lactis DSM 34327 or a mutant or variant thereof.

[0152] In one embodiment, the present invention relates to a method for producing a food or feed product, which method comprises at least one stage using the lactic acid bacteria strain Lactococcus lactis DSM 34329 or a mutant or variant thereof.

[0153] In one embodiment, the present invention relates to a method for producing a food or feed product, which method comprises at least one stage using the lactic acid bacteria strain Lactococcus lactis DSM 34331 or a mutant or variant thereof.

[0154] Preferably, the food product is a dairy product and the method in any of its embodiments comprises fermenting a dairy base (also referred to as "dairy substrate" in the context of the present invention) with at least one Lactococcus lactis and / or composition or starter culture according to the invention.

[0155] In another embodiment, the food product is a dairy product and the method in any of its embodiments comprises fermenting a plant-based milk base (also referred to as "plant-based milk substrate" in the context of the present invention), such as soy milk, preferably soy milk supplemented with glucose, such as 0.5-5% glucose, preferably 0.5-2% glucose, more preferably about 2% glucose, with at least one Lactococcus lactis strain and / or composition or starter culture according to the present invention.

[0156] In a preferred embodiment, the food product is a cheddar-type cheese, see the previous definition. More particularly, the cheese is selected from mild to mature cheddar, Monterey Jack, Colby and Territory. In a further preferred embodiment, the cheese is a continental cheese. Continental cheese is a semi-hard yellow cheese, such as Gouda, Edam, Tilsit and Maasdam.

[0157] In other embodiments, the food product further comprises other ingredients, such as colorants, flavoring agents, starch, protein, fat, salt, emulsifiers and / or preservatives.

[0158] Methods for producing additional Lactococcus lactis strains

[0159] In a seventh aspect, the present invention provides a method for making the Lactococcus lactis strain of the present invention.

[0160] In particular, the method comprises:

[0161] (a) providing one of the strains defined in claim 4 as a mother strain;

[0162] (b) introducing one or more mutations into the genome of the mother plant; and

[0163] (c) screening mutant strains having retained or further improved aminopeptidase activity,

[0164] Therein, the aminopeptidase activity was measured by the activity of an aminopeptidase specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulating cheddar cheese production.

[0165] The skilled person is aware of methods and techniques for introducing one or more mutations of step b). For example, one or more mutations of step b) can be introduced by chemical treatment or radiation treatment, or by genetic engineering techniques. For example, one or more mutations of step b) can be introduced by site-directed mutagenesis. For example, one or more mutations of step b) can be introduced using CRISPR / Cas9 technology.

[0166] Preferably, the mutation is located outside the prophage cluster, ie in any part of the genome other than the prophage cluster.

[0167] Alternatively, the present invention also provides a method for making a further Lactococcus lactis strain of the present invention using the reverse approach, ie starting from a strain that does produce intact phage particles.

[0168] In particular, the method comprises:

[0169] (a) providing a lysogenic strain that actually produces complete phage particles as a mother strain;

[0170] (b) introducing one or more mutations into the genome of the mother plant; and

[0171] (c) screening mutant strains having retained or further improved aminopeptidase activity,

[0172] Wherein, after 24 hours of growth according to the temperature profile simulated by cheddar cheese production, the aminopeptidase activity was measured by the activity of aminopeptidase with Gly-Pro dipeptide specificity,

[0173] The induced lysis occurs in the absence of phage particle release.

[0174] The skilled person is aware of methods and techniques for introducing one or more mutations of step b). For example, one or more mutations of step b) can be introduced by chemical treatment or radiation treatment, or by genetic engineering techniques. For example, one or more mutations of step b) can be introduced by site-directed mutagenesis. For example, one or more mutations of step b) can be introduced using CRISPR / Cas9 technology.

[0175] In this case, preferably the mutation is located inside the prophage cluster.

[0176] Unless otherwise indicated herein or clearly contradicted by context, the invention includes any combination of the above-described elements, aspects and embodiments in all possible variations. Embodiments of the invention are described below by way of example only. Example

[0177] Example 1: Sequencing and screening of strains with perforin / lysin clusters and incomplete prophage clusters

[0178] according to et al., 2018, described in Chr.Hansen A / S. Total DNA was purified and used to prepare a 250-bp paired-end library for genome sequencing using the Illumina MiSeq system. Sequence reads were quality trimmed (Phred score <25) and assembled into contigs using the de novo assembly algorithm in CLC Genomics Workbench version 10.1.1 (CLC bio, Qiagen Bioinformatics). The resulting genome assembly was filtered by removing contigs with coverage less than 15X and / or less than 20% of the median coverage of the assembly. The consensus sequence of the remaining contigs was exported in FASTA format, which is referred to as a draft genome sequence and used for subsequent sequence analysis.

[0179] To identify strains with the perforin / lysin cluster, a BLAST search was performed using the CLC program and the perforin / lysin cluster from bacteriophage P335 of Lactococcus lactis as query sequence.

[0180] The genomes of strains containing the perforin / lysin cluster were then further searched using the PHASTER program to determine the presence of prophage gene clusters. Strains that appeared to contain incomplete prophage clusters were further analyzed by comparing these prophage clusters to the complete genomes of sobria Lactococcus phages (e.g., P335).

[0181] The candidate strains are DSM 34326, DSM 34327, DSM 34329 and DSM 34331.

[0182] Candidate strains that exhibited only an incomplete prophage DNA cluster and perforin / lysin cassette were further analyzed by heat induction and PCR analysis of phage particle release, see below.

[0183] Reference strain A was used as negative control (non-heat-inducible).

[0184] Reference strain B was used as positive control (lysogenic strain with an inducible prophage).

[0185] Example 2: Screening to confirm the presence of the acmA gene

[0186] To identify strains containing the acmA gene cluster, a BLAST search was performed using the CLC program and the acmA gene (N-acetylmuramidase, locus tag EFV54_01480) from strain Lactococcus lactis IL1403 (locus CP033607 Genbank) as query sequence.

[0187] All candidate strains as well as the negative control possessed the acmA gene. Therefore, temperature-induced lysis was not associated with autolysis.

[0188] Example 3: Screening of strains to confirm their lysis ability

[0189] Using a temperature profile that simulates cheddar cheese production ( Figure 1 The lysis tendency of Lactococcus lactis candidates under cheese making conditions was determined after incubation for 24 hours in reconstituted skim milk (9.5% dry matter) at boiling (99°C x 30min). The milk was inoculated with 1-2% of an overnight culture propagated in reconstituted skim milk (9.5% dry matter) at boiling (99°C x 30min), and additionally 1% glucose was added to stimulate the growth of some strains.

[0190] After 24 hours, the aliquot of 1.9g acidified milk was mixed with 95μl of 1M sodium citrate (pH 8.0), and centrifuged. Supernatant was filtered to remove whole bacterial cells, and used for subsequent cell lysis analysis, which was carried out using the activity of the aminopeptidase with Gly-Pro dipeptide specificity as a marker enzyme. Triplicate aliquots of 60μL 0.5M Tris-HCl (pH 7.5) and 30μL supernatants were added to black microtiter plates, then 10μL substrate solutions (5mM Gly-Pro-7-amino-4-methylcoumarin (AMC)) were added. After rotating the plate, immediately by incubating at 37°C for 1 hour, with the fluorescence (Wallac 1420Victor2, Perkin Elmer, Waltham, MA, the U.S.) at intervals of 5min, the enzymatic cleavage of AMC was followed. The initial release rate of AMC was calculated from the linear regression slope of the samples and the AMC standard curve. The activity or "lysis index" of the strains was expressed as nanomoles of AMC released per milliliter of supernatant per minute at pH 7.5 and 37°C and is summarized in Figure 2 middle.

[0191] From the perspective of cheese ripening potential, strains with a lysis index higher than 0.25 nmole / min / ml were considered to be of concern.

[0192] Reference strain A was used as negative control (non-heat-inducible) and reference strain B was used as positive control (lysogenic strain with inducible prophage).

[0193] Example 4: PCR of phage release

[0194] according to Figure 1 Some strains were heat induced and then tested by PCR for the ability to release phage particles.

[0195] Candidate strains tested: DSM 34326, DSM 34327, DSM 34329, DSM 34331.

[0196] These strains were previously selected by genomic analysis. All strains have a perforin-lysin cluster similar to that of bacteriophage P335. This is a prerequisite for inducing lysis due to activation of the lysis cassette from the prophage integrated in the host chromosome.

[0197] Reference strain A was used as negative control (non-heat-inducible).

[0198] Reference strain B was used as positive control (lysogenic strain with an inducible prophage).

[0199] For PCR detection of bacteriophages, the following primer pairs were used:

[0200]

[0201] Genomic analysis showed that strains DSM 34327, DSM 34329, DSM 34331 and DSM 34326 and CHCC6052 have the target sequence for both primers. If phage particles are released, the PCR assay should be positive.

[0202] For PCR analysis, the samples were treated with DNase to remove "free" chromosomal DNA. Phage DNA encapsulated in the phage capsid is not digested. As a control for the PCR itself, DNA of the P335-type phage CHPC1237 was also added. For PCR, 1 μl of undiluted and 10-fold diluted heat-induced samples were added. The results of the PCR assay are shown in Figure 3A and 3B Shown in.

[0203] No PCR bands were detected for strains DSM 34326, DSM 34327, DSM 34329 and DSM 34331. Therefore, no free phage particles were released. These strains showed heat-induced lysis without producing free phage particles.

[0204] Reference Example 1: Flavor Potential

[0205] A 'proof of concept' trial was conducted using strains with different heat induction potentials and cheese ripening was monitored over a 12 month period. Four strains were selected based on their 'high', 'medium' and 'low' lysis indices and tested as single strain cultures to exclude strain interaction effects in the experimental design.

[0206] Following a traditional cheddar cheese recipe, the cheese milk was inoculated with 1% (w / w) of a culture grown overnight in B milk, and the cheese was then ripened at 9°C.

[0207] Bacterial cell lysis was analyzed by measuring the activity of intracellular Gly-Pro dipeptidyl aminopeptidase in cheese serum after 1 day, 90 days, 180 days and 360 days of ripening. Grated cheese (2.0 g) was rotated at 4 ° C for 2 h in 8.0 mL 50 mM Tris-HCl (pH 7.5) containing 1% (w / v) BSA. The liquid phase was collected and centrifuged (15,000 x g, 10 min, 4 ° C). The supernatant below the fat layer was filtered (0.20 μm) to remove the entire bacterial cells and diluted in cold 50 mM Tris-HCl / 1% (w / v) BSA as needed. Aliquots of 60 μL 0.5M Tris-HCl (pH 7.5) and 30 μL supernatant in triplicate were added to a black microtiter plate, followed by the addition of 10 μL 5mM Gly-Pro-AMC. The plates were then treated as described in Example 1. The activity was expressed as nanomoles of AMC released per gram of cheese per minute at pH 7.5 and 37°C.

[0208] The results showed that the aminopeptidase activity measured in the slurry phase was significantly higher in cheeses made with either the "high" or "medium" lysis strains. Similarly, in the present study, the aminopeptidase activity in cheeses made with either the "high" or "medium" lysis strains increased until 180 days of ripening, after which the enzyme activity leveled off, but remained high ( Figure 4 ).

[0209] In addition, the concentration of free amino acids (FAA) in cheese was analyzed by LC-MS after 1 day, 90 days, 180 days and 360 days of ripening. All analyses were performed in duplicate and after the last sampling time point.

[0210] Cheeses made with either the “high” or “medium” lysis strains contained significantly higher concentrations of free amino acids, which continued to increase linearly throughout the ripening period ( Figure 5 ).

[0211] Finally, the Food Science Department, Sensory Service Center, North Carolina State University (NCSU), conducted a comprehensive descriptive sensory analysis of 180-day and 360-day-old cheeses in accordance with the approval of the NCSU Institutional Review Board for Human Subjects. The cheese was cut into 4-ounce soufflé cups with lids and a 3-digit code on the cups for descriptive sensory analysis. The cheese was tempered at 10°C for 1 hour and served to humans at this temperature for flavor profile analysis, with spring water and unsalted crackers used to clean the palate. The descriptive analysis of flavor used a universal intensity scale of 0-15 points, as well as a Spectrum TM Methods and previously established sensory language for cheddar cheese flavor. Texture used a product-specific scale of 0-15 and an established vocabulary. Flavor and texture were evaluated at different sessions. Paper ballots were used. A descriptive sensory panel (N=6; 6 females, aged 22-46 years) with more than 200 hours of experience in descriptive analysis of cheddar cheese flavor evaluated the cheeses. TM Descriptive analysis training, where panelists were presented with reference solutions of sweet, sour, salty, and bitter to learn to consistently use the universal intensity scale. TM After the scale was developed, panelists learned to use the same intensity scale to identify and measure flavor descriptors by presenting and discussing flavor definitions, references, and various cheeses. Various cheeses were also discussed and evaluated during the training to allow panelists to consistently differentiate and replicate samples. Analysis of data collected during the training session confirmed that the panel's results were consistent and that terminology was not redundant, consistent with the language previously developed. Each panelist evaluated each product in duplicate.

[0212] Data were analyzed using general linear model analysis of variance with Fisher's least significant difference (LSD) as a post hoc test (SAS version 9.1, Cary, NC).

[0213] Most importantly, sensory analysis showed that bitterness disappeared faster and positive flavor attributes (sweetness, umami, brothy, and fruity) were more intense and diverse in cheeses made with either the "high" or "medium" lysis strains ( Figure 6 ). The sensory results were in good agreement with the enzymatic and chemical measurements of cheese ripeness described above.

[0214] References

[0215] Y., Stuer-Lauridsen, B., Bjerre, K., Jensen, M.G., Johansen, E., Bennedsen, M., Brockmann, E., Nielsen, B. (2018). Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection. Applied and Environmental Microbiology, 84(19), e01108-18.

[0216] Arndt, D., Grant, J., Marcu, A., Sajed, T., Pon, A., Liang, Y., Wishart, D.S. (2016). PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res. 44(W1): W16-21.

[0217] Muhammed, M.K., Krych, L., Nielsen, D.S., Vogensen, F.K. (2017). A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages. PLoS One, 12(3): e0174223.

Claims

1. A Lactococcus lactis bacterium capable of temperature-induced lysis, said Lactococcus lactis bacterium not containing an intact prophage cluster, wherein the temperature-induced lysis is determined by the Lactococcus lactis bacterium exhibiting an aminopeptidase activity of at least 0.25 nmole / min / ml as measured by aminopeptidase activity specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulating cheddar cheese making.

2. The Lactococcus lactis bacterium according to claim 1, wherein the induced lysis occurs in the absence of phage particle release, wherein the lack of phage particle release is determined using PCR to detect phage in the supernatant of the temperature-induced sample, wherein the absence of a PCR band is indicative of a lack of phage release, wherein a suitable primer pair for detecting temperate phage is used, optionally wherein the primer pair detects the perforin / lysin cassette of the phage, The following primer pairs are further optionally used: PDUTF29:AAGCGTGGCATTGCATT PDUTR29:CAGGCTCTTTGAGATGTTCA.

3. The Lactococcus lactis bacterium according to claim 1 or 2, wherein the Lactococcus lactis bacterium contains a complete perforin / lysin cassette.

4. The Lactococcus lactis bacterium according to any one of claims 1 to 3, wherein the bacterium is selected from: (i) DSM 34326; (ii) DSM 34327; (iii) DSM 34329; (iv) DSM 34331, or a mutant or variant thereof having retained or further improved aminopeptidase activity and lacking phage release as determined by PCR.

5. A composition comprising at least one Lactococcus lactis bacterial strain as described in any one of claims 1 to 4.

6. A composition according to claim 5, further comprising at least one strain of Streptococcus thermophilus and / or at least one other strain of Lactococcus lactis and / or at least one strain of Lactobacillus, optionally wherein the strain of Lactobacillus is selected from the group consisting of Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus pentosus or Lactobacillus rhamnosus.

7. A starter culture consisting of at least one Lactococcus lactis bacterial strain as defined in any one of claims 1 to 4, a Streptococcus thermophilus strain, optionally a Lactobacillus helveticus strain and / or another Lactococcus lactis strain.

8. A method for selecting Lactococcus lactis bacteria, the method comprising: (1) determining that the Lactococcus lactis bacteria are capable of temperature-induced lysis, wherein the Lactococcus lactis bacterium exhibits an aminopeptidase activity of at least 0.25 nmole / min / ml as measured by aminopeptidase activity specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulating cheddar cheese making, and (2) Determining that the Lactococcus lactis bacteria contain an incomplete prophage cluster.

9. The method according to claim 8, further comprising the step of determining that the Lactococcus lactis bacteria do not release phage particles after inducing temperature-induced lysis, wherein the lack of phage particle release is determined using PCR to detect phage in the supernatant of the temperature-induced sample, wherein the lack of a PCR band indicates the lack of phage release, Optionally use the following primer pairs: PDUTF29:AAGCGTGGCATTGCATT PDUTR29:CAGGCTCTTTGAGATGTTCA.

10. The method according to claim 8 or 9, further comprising the step of determining that the Lactococcus lactis bacterium contains a complete perforin / lysin cassette.

11. Use of a Lactococcus lactis bacterial strain as defined in any one of claims 1 to 4, a composition as defined in claim 5 or 6, a starter culture according to claim 7 or a Lactococcus lactis bacterium selected by the method according to any one of claims 8 to 10 for: (i) the production of cheddar-type cheese, (ii) ripened cheese, preferably cheddar-type cheese, (iii) removing the bitter taste of cheese, preferably cheddar-type cheese, and / or (iv) increasing the amount of free amino acids in cheese, preferably cheddar-type cheese.

12. The use according to claim 11, wherein the cheddar-type cheese is selected from the group consisting of cheddar cheese, Monterey Jack cheese, Colby cheese, Territory cheese, and American cheddar cheese.

13. A method for producing a food or feed product, said method comprising at least one stage of using at least one Lactococcus lactis bacterial strain as defined in any one of claims 1 to 4, a composition as defined in claim 5 or 6, a starter culture according to claim 7 or a Lactococcus lactis bacterium selected by a method according to any one of claims 8 to 10.

14. A food or feed product comprising a Lactococcus lactis bacterial strain as defined in any one of claims 1 to 4, a composition as defined in claim 5 or 6, a starter culture according to claim 7 or a Lactococcus lactis bacterium selected by a method according to any one of claims 8 to 10.

15. The food or feed product according to claim 14, wherein the food or feed product is a dairy product comprising fermented plant-based milk and / or mammal-based milk.

16. A method for producing a further Lactococcus lactis bacterial strain as defined in any one of claims 1 to 3, the method comprising: (a) providing one of the strains defined in claim 4 as a mother strain; (b) introducing one or more mutations in the genome outside the prophage cluster of the parent strain; and (c) screening mutant strains having retained or further improved aminopeptidase activity, The aminopeptidase activity was measured by the activity of an aminopeptidase specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulated for cheddar cheese production.

17. A method for producing a further Lactococcus lactis bacterial strain as defined in any one of claims 1 to 3, the method comprising: (a) providing a lysogenic strain that actually produces complete phage particles as a mother strain; (b) introducing one or more mutations into the prophage cluster of the genome of the parent strain; and (c) screening mutant strains having retained or further improved aminopeptidase activity, The aminopeptidase activity was measured by the activity of an aminopeptidase specific for Gly-Pro dipeptide after 24 hours of growth according to a temperature profile simulated for cheddar cheese production. The induced lysis occurs in the absence of release of phage particles.

Images