Isolated mutant Lactobacillus curvatus strains, compositions and methods for inhibiting the amount of spoilage or pathogenic bacteria in a food product.
Patent Information
- Application Number
- BR112020003009
- Authority / Receiving Office
- BR · BR
- Patent Type
- Patents
- Current Assignee / Owner
- Publication Date
- 2026-09-08
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Abstract
Description
1 / 28 Isolated mutant Lactobacillus curvatus strains, compositions and methods for inhibiting the amount of spoilage or pathogenic bacteria in a food product. FIELD OF THE INVENTION
[0001] The present invention relates to the Lactobacillus curvatus strain deposited as DSM 18775, used as a biopreservative culture in a wide range of meat products due to its bacteriocin production.
[0002] The present invention relates to Lactobacillus curvatus strains with a prolonged latency phase of at least 24 hours at 30°C relative to DSM 18775. In a currently preferred embodiment, the strains are mutants of DSM 18775, such as the Lactobacillus curvatus strain deposited as DSM 32590 (TpG3) and the Lactobacillus curvatus strain deposited as DSM 32591 (TpG57).
[0003] In addition, the invention relates to a method for inhibiting Listeria in a food product comprising the addition of bacteria of a Lactobacillus curvatus strain according to the invention to a food product at a concentration of at least 105 CFU / g. BACKGROUND OF THE INVENTION
[0004] Lactobacillus curvatus is probably the most common species of lactobacillus reported in fermented meat products and is known for its potential use in food preservation due to the production of antimicrobial components or peptides. Most of these inhibitory peptides are a type of bacteriocin belonging to Class II lantibiotics, and some are listed under the name curvacin and others under the collective name sakasin P or G or sakasin X, T, or P. The mode of action is Petition 870260074878, dated 07 / 28 / 2026, page 5 / 74 2 / 28 mainly due to alteration of the cell membrane permeability barrier and thus to the dissipation or collapse of the ion transport force system into and out of the cell of spoilage bacteria. Bacteriocins function primarily against other closely related Gram-positive species, but cell extracts isolated from some strains of Lactobacillus curvatus have also been shown to have an antimicrobial effect against Gram-negative bacteria.
[0005] The strain of Lactobacillus curvatus deposited as DSM 18775 produces a bacteriocin that exhibits a bactericidal mode of action relative to other Gram-positive strains, making this strain important for protection against spoilage bacteria in the meat industry. SUMMARY OF THE INVENTION
[0006] Under ideal conditions, a culture for meat protection should demonstrate limited growth at the temperature of its application and sufficient bacteriocin production to limit the growth of spoilage bacteria, such as Listeria spp. In addition, the culture should not produce off-flavors or demonstrate a high rate of acidification when spread on (cooked) meat products.
[0007] The present invention relates to strains of Lactobacillus curvatus that have a prolonged latency phase at 30°C for at least 24 hours relative to DSM 18775.
[0008] The currently preferred strains of the invention are mutants of the Lactobacillus curvatus DSM18775 strain. The development of two mutants is described in detail in the examples. After eight weeks of cell transfer every two days and a total of four UV treatments, a screening of approximately 4,000 isolates resulted in a slow-growing mutant, TpG3, with a phase of Petition 870260074878, dated 07 / 28 / 2026, page 6 / 74 3 / 28 prolonged latency of 24 hours in relation to the parent strain, when incubated at 30°C (Figure 1). The production of the antimicrobial agent, bacteriocin, is provided for the TpG3 mutant in Table 1.
[0009] An additional round of Adaptive Laboratory Evolution (ALE) with the TpG3 mutant was performed over a 2-week period at 15°C with cell transfer every two days, including UV treatment. This resulted in the isolation of another TpG57 isolate, with a substantially prolonged latency phase of more than 48 hours compared to wild-type Lactobacillus curvatus DSM 18775. Bacteriocin production was verified by plate assays with Micrococcus luteus DSM 1790 and demonstrated to be comparable to the antimicrobial bacteriocin production of TpG3.
[0010] It was surprisingly found that these two mutants do not grow during the latency phase, but were nevertheless metabolically active and even had an antilisterial effect that was about 64 times greater compared to the parent strain, when grown under the same conditions at 20°C.
[0011] Application assays demonstrated that both mutants were able to inhibit Listeria growth to the same extent as DSM 18775, and that ham samples inoculated with a cell count of 1.0E+05 cfu / g of TpG3 and TpG57 were significantly better in terms of odor and taste (no acidic taste) than ham samples inoculated with a cell count of 1.0E+07 cfu / g of TpG3 and TpG57 and samples inoculated with DSM 18775 (Table 2). The TpG3 and TpG57 mutant strains showed a slower pH drop and a higher pH value at the end of the shelf life (Figure 2).
[0012] Thus, the Lactobacillus curvatus strain deposited as DSM 32590 (TpG3) and the deposited Lactobacillus curvatus strain Petition 870260074878, dated 07 / 28 / 2026, p. 7 / 74 4 / 28 such as DSM 32591 (TpG57) may be even more useful for the biopreservation of food products than the DSM 18775 strain from which they were derived. DETAILED DESCRIPTION OF THE INVENTION
[0013] Bacterial contamination of food products is known to be responsible for the transmission of foodborne illnesses. This problem is particularly important in meats and dairy products that are not reheated by consumers before ingestion and that are stored for long periods in refrigerators at 2-10°C. This storage period is often referred to as shelf life. As used in this document, the term shelf life means the period of time during which a food product remains saleable to retail consumers. In traditional meat processing, the shelf life of meat and its by-products is approximately 30 to 40 days after an animal is slaughtered. Refrigeration of the meat during this period is expected to largely stop and / or retard the growth of pathogenic bacteria and, to a lesser extent, destroy the bacteria.After about 30 to 40 days, however, refrigeration is no longer able to effectively control the proliferation of spoilage bacteria below acceptable levels.
[0014] The strains of the present invention are particularly useful for storage, fermentation, maturation or curing of the food product, which occurs at a temperature of 2°C to 45°C, such as at a temperature of 10°C to 30°C, such as at a temperature of 10°C to 25°C. The storage, fermentation, maturation or curing of the food product can last from 8 hours to several days / weeks.
[0015] Lactic acid bacterial strain(s) added to food to inhibit pathogens, as well as spoilage bacteria and / or extend shelf life without altering the sensory properties of the product, Petition 870260074878, dated 07 / 28 / 2026, p. 8 / 74 5 / 28 is / are referred to as bioprotective cultures. Protective cultures are not intended to alter the sensory properties of the product. Their use or that of their metabolic products (organic acids, hydrogen peroxide, enzymes and bacteriocins) is often referred to as bioprotection. In the present context, the term bioprotective culture and composition of the invention are used interchangeably, unless the context indicates otherwise.
[0016] An important aspect in evaluating the use of a strain as a bioprotective culture is the strain's ability to work in the food product for which it is intended. In this respect, it is not only important that the strain be able to inhibit any undesirable foodborne pathogenic or spoilage bacteria in the product under relevant storage conditions, but also that it does not produce any undesirable sensory effects (undesirable changes in taste, odor or color).
[0017] The manufacture of a fermented food product is often controlled and carried out by a starter culture. The starter culture is responsible for the development of a non-limiting group of quality parameters, such as acidification, reduction of water binding and water activity, overall appearance, color, texture, odor, aroma, taste, flavor, and other sensory and technological parameters. Thus, minimal, or preferably no, influence on the quality parameters of the bioprotective culture should be provided.
[0018] Starter cultures for meat fermentation are commonly composed of one or more lactic acid bacteria. Typically, one starter culture will proliferate during the fermentation process. During the fermentation process, lactic acid bacteria primarily produce lactic acid, whereby the pH drops to the desired value, depending on the culture and the Petition 870260074878, dated 07 / 28 / 2026, page 9 / 74 6 / 28 processing conditions (temperature, type / sugar content, etc.) and, more importantly, the sensory properties of the product are distinctly altered to the desired aroma and flavor profile characteristics for the product.
[0019] To reduce the concentration of spoilage and pathogenic bacteria, it is desirable that this reduction can be achieved without significantly altering the quality of the final food product; that is, the food producer can apply the bioprotective culture to their current or preferred recipe without altering the recipe or processing conditions in any way. To obtain the desired effect, a culture of a bacteriocin-producing species can be applied to a food material as a bioprotective culture, which is separate from the starter culture.In the present context, a bioprotective culture is a culture that is added to or associated with a starter culture, but is not part of the starter culture; that is, the bioprotective culture is an additional culture that does not attempt to produce the fermented food product, but to provide an extra technological advantage; in this case, a killing, inactivating, or inhibiting effect directed at pathogenic or spoilage bacteria. In the present context, bioprotective culture and bacteriocin-producing species are used interchangeably, unless the context indicates otherwise.
[0020] The present invention provides a method for inhibiting the quantity of pathogenic and spoilage bacteria in a food product. In the present context, the term "inhibit quantity" refers to an inhibition of the quantity of pathogenic and / or spoilage bacteria. Inhibition can be provided by killing, inactivating, or inhibiting the growth of spoilage or pathogenic bacteria. In one embodiment of the present invention, 100% of the pathogenic and / or spoilage bacteria are killed, inactivated, or inhibited, such Petition 870260074878, dated 07 / 28 / 2026, page 10 / 74 7 / 28 such as at least 90%, for example, at least 75%, such as at least 50%, for example at least 40%, such as at least 30%, for example at least 25%, such as at least 20%, for example at least 10%, such as at least 5%, for example at least 1%. In a preferred embodiment, the bacteria already present are eliminated or their quantity reduced to an amount below the qualitative detection limit.
[0021] The term spoilage bacteria, as used in this document, refers to any type of bacteria that acts to spoil food. Spoilage bacteria can grow and proliferate to such an extent that a food product is rendered unsuitable or undesirable for human or animal consumption. Bacteria are able to proliferate on food surfaces, such as meat surfaces, assimilating sugars and proteins on such surfaces. By metabolizing these components, spoilage bacteria create byproducts, including carbon dioxide, methane, nitrogen compounds, butyric acid, propionic acid, lactic acid, formic acid, sulfur compounds, and other undesirable gases and acids. The production of these byproducts alters the color of meat surfaces, changing the meat from a red color to a brown, gray, or green color. Gaseous byproducts generated by spoilage bacteria also impart an undesirable odor to spoiled meat.Changes in the color and odor of meat, due to the growth of spoilage bacteria on the surface of a meat product, often render that food product unsaleable to consumers.
[0022] In addition to controlling spoilage bacteria, another significant concern in the food processing industry is controlling the growth of foodborne pathogenic bacteria. As used in this document, the term bacteria Petition 870260074878, dated 07 / 28 / 2026, page 11 / 74 8 / 28 Foodborne pathogenic bacteria refers to any foodborne intoxicating bacteria capable of causing disease or illness in animals or humans. The term foodborne pathogenic bacteria will be understood to include bacteria that infect the food product (e.g., meat) and therefore cause disease or illness, as well as bacteria that produce toxins that cause disease or illness. The foodborne pathogenic bacteria to be inhibited may be one or more of the following bacteria: Aeromonas caviae; Aeromonas hydrophila; Aeromonas sobria; Bacillus cereus; Campylobacter jejuni; Citrobacter spp.; Clostridium botulinum; Clostridium perfringens; Enterobacter spp.; Enterococcus spp.; Enteroinvasive strains of Escherichia coli; Enteropathogenic strains of Escherichia coli; Enterotoxigenic strains of Escherichia coli; Escherichia coli O157:H7; Klebsiella ssp.; Listeria monocytogenes; Plesiomonas shigelloides; Salmonella ssp.; Shigella ssp.Staphylococcus aureus; Streptococcus spp.; Vibrio cholerae; Yersinia enterocolitica. More preferably, the pathogenic bacteria are Listeria monocytogenes.
[0023] The present invention provides a method for inhibiting the amount of spoilage or pathogenic bacteria in a food product, comprising adding bacteria of a Lactobacillus curvatus strain according to the invention or a composition comprising a Lactobacillus curvatus strain according to the invention to a food product at a concentration of at least 105 CFU / g.
[0024] A bacterial strain, as used in this document, refers to a bacterium that remains genetically unchanged when grown or multiplied.
[0025] In the context of the present invention, the term strain with a prolonged latency phase of at least 24 hours at 30°C in Petition 870260074878, dated 07 / 28 / 2026, p. 12 / 74 9 / 28 in relation to DSM 18775 means that when the strain is grown under identical conditions to those of DSM 18775, no visible growth is observed for the strain, which has a prolonged lag phase after 24 hours under conditions where good growth is observed for DSM 18775. This can be tested on MRS agar plates, as described in Example 1 and illustrated in Figure 1. In some embodiments of the present invention, the prolonged lag phase is even longer, such as at least 48 hours, at least 72 hours, or at least 96 hours.
[0026] In the present context, the term mutant refers to a bacterial strain derived from the deposited strain, by means of, for example, genetic engineering, radiation and / or chemical treatment, which is a functionally equivalent mutant, that is, a mutant that possesses substantially the same or improved properties with respect to the effect on urate concentration in an individual's blood as the parent strain. The term mutant refers to a strain obtained by subjecting the Lactobacillus curvatus strain deposited as DSM 32590 or its mutant strain deposited as DSM 32591 to conventional mutagenization treatment, including treatment with a chemical mutagen, such as ethane methane sulfonate (EMS) or N-methyl-N-nitro-N-nitroguanidine (NTG), UV light followed by a screening / selection step, as well as a spontaneously occurring mutant.In a currently preferred functionally equivalent mutant, less than 5%, or less than 1%, or even less than 0.1% of the nucleotides in the bacterial genome were displaced with another nucleotide, or deleted, compared to the strain deposited as Lactobacillus curvatus DSM 18775.
[0027] The Lactobacillus curvatus strains of the present invention may be useful for the bioprotection of fermented as well as non-fermented food products. Petition 870260074878, dated 07 / 28 / 2026, p. 13 / 74 10 / 28
[0028] Examples of fermented food products include, but are not limited to, dairy products such as various cheese products, fermented meat products such as sausages, for example spreadable and dried sausages, ham, fermented fish and fermented vegetables.
[0029] The term food product, as used in this document, refers to any food susceptible to bacterial growth and proliferation of pathogenic or spoilage bacteria. These food products include, but are not limited to, meats, dairy products, vegetables, fruits, ready-to-eat products, and grains.
[0030] As used in this document, the term meat refers to any meat product or meat by-product (including processed ones) from an animal that is consumed by humans or animals, including, without limitation, beef, lamb, pork, poultry, fish and crustacean shellfish. Examples of fermented meat products are sausages, for example, spreadable and dry sausages, ham and fermented fish.
[0031] The term dairy products is intended to include any food product produced with milk or milk products, including, but not limited to, milk, yogurt, ice cream, cheese, butter and cream.
[0032] Examples of fruits are grapes, such as edible grapes.
[0033] Examples of ready-to-eat (RTE) products are RTE salads, for example, fresh cut green salads, arugula salad, carrot sticks, corn, salad toppings, chicken sticks, seafood, mixed salads, pasta salads, etc. and couscous, rice, hummus, etc.
[0034] The present invention also provides a composition comprising bacteria of a Lactobacillus curvatus strain according to the invention. Preferably, the Lactobacillus strain Petition 870260074878, dated 07 / 28 / 2026, p. 14 / 74 11 / 28 curvatus is present at a concentration of at least 105 CFU / g.
[0035] In the culture used in example 4 of the present patent application, only one bacteriocin-producing strain is present. In some embodiments, the composition comprises bacteria of the Lactobacillus curvatus strain as the only bacteria present in the composition.
[0036] However, more than one bacteriocin-producing strain is considered useful for some applications.
[0037] Examples of bacteriocin-producing bacteria of the class IIa are: Carnobacterium maltaromaticum, Carnobacterium pisicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc carnosum, Leuconostoc gelidium, Pediococcus acidilactici, Pediococcus pentosaceus.
[0038] Within the scope of the present invention is a composition comprising at least two strains producing class IIa bacteriocins. If more than one strain is used, the strains will preferably produce different class IIa bacteriocin(s) and / or act on different targets.
[0039] The compositions of the invention may comprise bacteria of the Lactobacillus curvatus strain and bacteria of one or more of the species Pediococcus acidilactici, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum, Staphylococcus carnosus.
[0040] Preferred additional bacteriocin-producing strains are the Lactobacillus curvatus strain deposited as DSM 18775, the Pediococcus acidilactici strain deposited as DSM 28307, the Lactobacillus sakei strain deposited as DSM 14022, the Lactococcus lactis strain deposited as DSM 11037 and Leuconostoc carnosum LC1043 available from the Danish Research Institute. Petition 870260074878, dated 07 / 28 / 2026, p. 15 / 74 12 / 28 of Meat.
[0041] In currently preferred embodiments, the composition comprises a strain of Lactobacillus curvatus of the invention and the strain of Pediococcus acidilactici deposited as DSM 28307, a strain of Lactobacillus curvatus of the invention and Lactobacillus sakei deposited as DSM 14022, a strain of Lactobacillus curvatus of the invention and the strain of Lactococcus lactis deposited as DSM 11037, or a strain of Lactobacillus curvatus of the invention and Leuconostoc carnosum LC1043.
[0042] In a further embodiment, the composition may comprise at least three strains producing class IIa bacteriocin. Preferably, one of them will be a Lactobacillus curvatus strain according to the invention, the Pediococcus acidilactici strain deposited as DSM 28307, and Leuconostoc carnosum LC1043.
[0043] In yet another embodiment, the culture comprises four or more strains producing class IIa bacteriocin. Preferably, one of them will be a Lactobacillus curvatus strain according to the invention, the Pediococcus acidilactici strain deposited as DSM 28307, or both.
[0044] As explained above, the use of lactic acid bacteria to induce fermentation of food products, typically raw salted meat products, is well known, in order to provide the desired alteration in the characteristics of the food matrix during fermentation (e.g., a desired acidification and other determined sensory and technological parameters). During the fermentation process, lactic acid bacteria primarily produce lactic acid, whereby the pH drops to the desired value, depending on the strain(s) and processing conditions (temperature, type / sugar content, etc.), and, most importantly, the sensory properties of the product are altered. Petition 870260074878, dated 07 / 28 / 2026, page 16 / 74 13 / 28 distinct way.
[0045] As used in this document, the term fermentation refers to the process of biochemical changes, for example, acidification in animal and / or vegetable material (i.e., a food matrix), involving the activity of live microbial cells under aerobic and / or anaerobic conditions to obtain a food product with the desired quality.
[0046] The term maturation refers to the ripening, drying, flavor development, enzymatic activity such as lipolysis or proteolysis, leading to the complex development of flavor, particularly for longer-matured products such as salami.
[0047] If desired, the composition may, in addition to at least one bacteriocin-producing strain, comprise at least one fermentation strain that assists in the development of quality parameters of at least one bacteriocin-producing strain, such as acidification, reduction of water binding and water activity, overall appearance, color, texture, odor, aroma, taste, flavor, and other sensory and technological parameters. Examples of such fermentation strains are Staphylococcus carnosus and Staphylococcus xylosus. For meat products with a short maturation period, only bacteriocin-producing strains are considered sufficient to produce a suitable fermented meat product, since bacteriocin-producing strains also contribute to flavor. However, if desired, at least one strain of Staphylococcus or Micrococcaceae may be added for flavor and color reasons.
[0048] In currently preferred embodiments, the composition comprises a strain of Lactobacillus curvatus of the invention, the strain of Pediococcus acidilactici deposited as DSM 28307, and a strain of Staphylococcus carnosus, a strain of Lactobacillus curvatus of Petition 870260074878, dated 07 / 28 / 2026, p. 17 / 74 14 / 28 invention, Lactobacillus sakei deposited as DSM 14022 and Staphylococcus carnosus, a strain of Lactobacillus curvatus of the invention, the strain of Lactococcus lactis deposited as DSM 11037, Staphylococcus carnosus, or a strain of Lactobacillus curvatus of the invention, Leuconostoc carnosum LC1043 and Staphylococcus carnosus.
[0049] As used in this document, the term effective quantity refers to the amount of Lactobacillus curvatus bacteria according to the invention that results in the inhibition of bacterial growth or the reduction in the number of other bacteria in the food product.
[0050] In a preferred embodiment of the present invention, the strain or strains of the bioprotective composition is / are added at a concentration in the range of 10²-10¹⁰ CFU / g of product, for example, in the range of 10²-10⁹ CFU / g of product, such as in the range of 10³-10⁹ CFU / g of product, for example, in the range of 10⁴-10⁹ CFU / g of product, such as in the range of 10²-10⁸ CFU / g of product, for example, in the range of 10²-10⁷ CFU / g of product, such as in the range of 10³-10⁷ CFU / g of product, for example, in the range of 10⁴-10⁷ CFU / g of product, such as in the range of 10⁵-10⁷ CFU / g of product, for example, in the range of 10⁶-10⁷ CFU / g of product, such as in the range of 10³-10⁶ CFU / g of product, for example, in the range of 10³-10⁵ CFU / g of product, such as in the range of 10²-10⁴ CFU / g of product for each of the strains, if more than one strain. In a currently preferred embodiment, the Lactobacillus curvatus strain of the invention is added at a concentration of 10⁵ CFU / g of product.
[0051] The present invention provides a composition of the present invention comprising bacteria of the Lactobacillus curvatus strains of the invention, such as the Lactobacillus curvatus strain deposited as DSM 32590 or a mutant strain thereof, such as Petition 870260074878, dated 07 / 28 / 2026, p. 18 / 74 15 / 28 strain deposited as DSM 32591, in dried, frozen or lyophilized form.
[0052] In a preferred embodiment, the present invention provides a composition of the present invention comprising Lactobacillus curvatus bacteria deposited as DSM 32590 and Lactobacillus curvatus bacteria deposited as DSM 32591, which may be in dried, frozen or lyophilized form.
[0053] If the bacteria are to be freeze-dried, they are usually mixed with a cryoprotectant before freeze-drying in order to obtain high viability. The term cryoprotectant is used in the context of the present invention to refer to a substance that is capable of improving survival during freezing and / or drying and improving the storage stability of bacteria. The cryoprotectant used in this document preferably comprises a saccharide.
[0054] A saccharide can be a mono-, di-, oligo-, or polysaccharide or a mixture of at least two saccharides. Useful monosaccharides include glucose (also known as dextrose), fructose, ribose, and galactose, and useful disaccharides include, among others, sucrose, trehalose, maltose, and lactose. The composition may comprise one or more mono- or disaccharides, such as one, two, or three, or even more different saccharides.
[0055] As an example, the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.
[0056] The cryoprotectant may also comprise a peptide, protein, protein hydrolysate, or a mixture thereof. Examples of peptides and proteins to be used are casein, pea protein, whey protein, albumin, soy protein, glutamic acid, or gelatin, and any of their isolates or hydrolysates. Other additives, for example, may also be used. Petition 870260074878, dated 07 / 28 / 2026, page 19 / 74 16 / 28 For example, antioxidants such as ascorbate, sodium citrate, and propyl gallate may also be present.
[0057] The use of the terms "a" and "an" and similarly referring to the terms in the context of the invention description (especially in the context of the following claims) should be interpreted as encompassing both the singular and the plural, unless otherwise indicated in this document or clearly contradicted by the context. The terms "comprising," "having," and "containing" should be interpreted as open-ended terms (i.e., meaning to include, but not limited to), unless otherwise indicated. The recitation of value ranges in this document is simply intended to serve as a shorthand method of individually referring to each separate value that lies within the range, unless otherwise indicated in this document, and each separate value is incorporated into the specification as if it were individually recited in this document.The use of any and all examples or exemplary language provided in this document is intended merely to better clarify the invention and does not represent a limitation of the scope of the invention, unless otherwise claimed. No language in the specification should be interpreted as indicating any unclaimed element as essential to the practice of the invention. FIGURE CAPTIONS Figure 1
[0058] TpG3 growth performance
[0059] Right plate, TpG3 after 24 hours of incubation at 30°C; Left plate, DSM 18775 incubated under the same conditions. Inoculation was performed with an equal number of cells (200 pl: 0.25 OD600; Petri dish diameter 14 cm). Figure 2 Petition 870260074878, dated 07 / 28 / 2026, page 20 / 74 17 / 28
[0060] Evolution of the pH of cooked ham samples (n = 3) during storage at + 7°C.
[0061] The geometric y-axis shows the pH. A pH drop of 0.3 units is generally considered sensorially acceptable. Figure 3
[0062] Total cell count in cooked ham samples (n = 3) during storage at +7°C.
[0063] The geometric y-axis shows the total cell count in cfu / g. The detection limit is 1.0E+02 cfu / g. Figure 4
[0064] Lactic acid bacterial cell count in cooked ham samples (n = 3) during storage at +7°C.
[0065] The geometric y-axis shows the lactic acid bacterial cell count in cfu / g. The detection limit is 1.0E+02 cfu / g. Figure 5
[0066] Listeria spp. cell count in cooked ham samples (n = 3) during storage at +7°C.
[0067] The geometric y-axis shows the cell count of Listeria spp. in CFU / g. The detection limit is 1.0E+02 CFU / g. STORAGE AND SPECIALIZED SOLUTIONS
[0068] The Lactobacillus curvatus TpG3 strain was deposited in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under accession number DSM 32590, filed on August 16, 2017 by Chr. Hansen A / S, Denmark. The filing was made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Petition 870260074878, dated 07 / 28 / 2026, p. 21 / 74 18 / 28
[0069] The Lactobacillus curvatus TpG57 strain was deposited in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under accession number DSM 32591, filed on August 16, 2017 by Chr. Hansen A / S, Denmark. The filing was made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
[0070] The strain of Pediococcus acidilactici HP was filed in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under accession number DSM 28307, with a filing date of January 30, 2014, by Chr. Hansen A / S, Denmark. The filing was made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
[0071] The Lactobacillus sakei BJ-33 strain was deposited in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under accession number DSM 14022, filed on January 31, 2001, by Chr. Hansen A / S, Denmark. The filing was made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
[0072] For the deposited microorganisms identified above, the following additional indications apply: With regard to the respective Patent Offices of the designated states, applicants request that a sample of the aforementioned deposited microorganisms be made available only to a specialist appointed by the applicant until the date the patent is granted or Petition 870260074878, dated 07 / 28 / 2026, page 22 / 74 19 / 28 is the date on which the application was refused or withdrawn or is considered withdrawn. The Lactobacillus curvatus DSL 18775 strain is referenced in European Patent EP2132297. The Lactococcus lactis DSM 11037 strain is referenced in European Patent EP928333. The Pediococcus acidilactici DSM 10313 strain is referenced in European Patent EP1716258. Leuconostoc carnosum LC1043 is available from the Danish Institute for Meat Research or commercially from Chr. Hansen A / S (Denmark). The Listeria innocua Seeliger strain was deposited as ATCC 33090. -The strain of Micrococcus luteus was deposited as DSM 1790.
[0073] Embodiments of the present invention are described below by way of non-limiting examples. EXAMPLES Materials and methods: Aloa agar (Oxoid) Lactobacilli MRS Broth™ (Difco) Lactobacilli MRS Agar agar™ (Difco) Brain Heart Infusion (BHI) Agar (Oxoid CM375) BHI, Merck Bacto™ Heart Infusion Broth (BD) Difco Microtiter plates (MTP) NUNC, Denmark M17 Agar (Oxoid) MRS (Oxoid) agar MRS Medium (Merck) EXAMPLE 1 Petition 870260074878, dated 07 / 28 / 2026, p. 23 / 74 20 / 28 Mutants inducing Lactobacillus curvatus DSM 18775 exhibiting a prolonged lag phase at 30°C. DSM 18775 Adaptive Laboratory Evolution (ALE)
[0074] For the ALE experiment, Lactobacillus curvatus cells were cultured in MRS broth (10 ml) and incubated for 2x24 hours at 15°C, at which point a transfer (2%) to fresh MRS broth was performed and continuous incubation at the permissive temperature of 15°C was carried out. This cell transfer was repeated every 48 hours for a period of 8 weeks. Every 2 weeks throughout the incubation period, a sample of the cells was subjected to UV radiation to accelerate mutants known to be temperature-sensitive (TS). Instead of the 2% transfer, a 7 ml aliquot of exponentially growing cells was diluted to 0.25 DO600 in a Petri dish. UV mutagenesis was performed by exposing the cell layer to UV at 70 mJ / cm2 for 10 min. One ml of the UV-treated cells was then transferred to 10 ml of fresh MRS broth and incubated continuously at 15°C until the next transfer.At the end of the eight-week period, a sample was removed and screened for TS variants, as described below. Screening for temperature-sensitive mutants of DSM 18775
[0075] Screening for temperature-sensitive mutants (TS) was performed by spreading aliquots of TS cells onto MRS agar plates at an appropriate dilution to give approximately 150 to 200 colonies per plate after incubation at 15°C for 3 x 24 hours. Single colonies were then transferred to individual MTP wells (96-well plates) containing 100 µl of fresh MRS broth and incubated for a further 3 x 24 hours at 15°C. TS colonies were identified by spreading replicates onto other MTPs with 100 µl of MRS broth and incubating aerobically for 24 hours at 30°C, the temperature at which TS mutants are identified as exhibiting growth with Petition 870260074878, dated 07 / 28 / 2026, page 24 / 74 21 / 28 a longer latency phase than the wild type. Screening for TS mutants was also performed by spreading replicas of individual colonies on MTP in MRS agar, followed by aerobic incubation for 24 hours at 30°C. Finally, the TS colonies were cultured in MRS broth at 20°C. Results
[0076] After eight weeks of transferring Lactobacillus curvatus at 15°C and UV treatment performed 4 times, approximately 4,000 mutants were screened for their inability to grow at 30°C. One of these mutants, TpG3, demonstrated a reduced growth rate compared to the mother strain during a 24-hour period at 30°C. The same reduced growth rate was observed when retesting the strain known as TS in a larger volume of 10 ml of MRS broth for a period of 72 hours at 30°C. On MRS agar plates, no growth was observed after 24 hours of incubation, while good growth was observed in DSM 18775 (Figure 1). EXAMPLE 2 Selection of other Lactobacillus curvatus DSM 18775 mutants with a prolonged latency phase at 30°C. Second-generation mutants derived from TpG3.
[0077] To extend the latency phase even further, the mutant The TpG3 strain of Lactobacillus curvatus DSM 18775 was selected and subjected to continuous ALE at 15°C for a period of 2 weeks with cell transfers (2%) every two days, in addition to a mutagenic UV treatment, as described in Example 1. Among approximately 500 colonies screened, only two mutants were isolated, showing deferred growth at 30°C with a prolonged latency phase of more than 48 hours compared to the parent strain, DSM 18775. It was decided to continue with one of the two mutants, TpG57 (DSM). Petition 870260074878, dated 07 / 28 / 2026, page 25 / 74 22 / 28 32591). EXAMPLE 3 Detection of the antimicrobial product of mutants with a prolonged latency phase at 30°C. Bacteriocin activity test
[0078] The bacteriocin-sensitive indicator strain, Micrococcus luteus DSM 1790, was amplified by incubation at 30°C in BHI broth (BD) under vigorous shaking (125 rpm). To test bacteriocin production, a series of double dilutions of the preculture supernatants (medium used) were made in MTP and an aliquot of these dilutions (50 µl) was applied to the indicator strain, Micrococcus luteus DSM 1790. The optical density [OD] at 600 nm was recorded every 30 minutes for a period of 20 hours at 30°C. Activity is reported in arbitrary units (AU / ml) (Table 1) and defined as the reciprocal of the highest double dilution showing 50% inhibition of the growth of the sensitive strain. Results
[0079] The results provided in Table 1 show the inhibition of the bacteriocin-sensitive strain Micrococcus luteus (DSM1790), using the medium from the Lactobacillus curvatus strains DSM 32590 and DSM 18775 and the positive control Pediococcus acidilactici DSM 10313 cultured for 20 hours at 20°C. DO600 is the cell density of Micrococcus after incubation with various dilutions of the medium used (bacteriocin). Table 1 DO600 after 20 hours of growth Dilution factor of the medium used DSM 32590 DSM 18775 DSM 10313 Undiluted 0 0 0 1 / 2 0 0.216 0 1 / 4 0 0.483 0 Petition 870260074878, dated 07 / 28 / 2026, page 26 / 74 23 / 28 1 / 8 0 0.612 0 1 / 16 0 0.713 0 1 / 32 0 0.734 0 1 / 64 0 0.744 0 1 / 128 0.256 0.744 0.309 1 / 256 0.554 0.743 0.679 1 / 512 0.677 0.732 0.703 1 / 1024 0.677 0.732 0.707 No Bacteriocin 0.698 0.732 0.698 Sample Activity (UA / ml) 2560 40 2560
[0080] The bacteriocin production assay demonstrated that the TS strain produced substantially more bacteriocin than the parent strain DSM 18775 over a 20-hour period at 20°C. For DSM 32590 and DSM 10313 (a positive bacteriocin control strain), 50% inhibition was recorded at a 1 / 128-fold dilution in a sample without bacteriocin and without medium added to the Micrococcus luteus DSM 1790 inoculate. In Table 1, DSM 18775 shows 50% inhibition at a 1 / 2-fold dilution, with a total of 40 UA / ml [2 / 0.05]. The UA / ml for TpG3 was recorded at 2,560 UA / ml [128 / 0.05]. Thus, TpG3 activity is recorded as 64 times higher than the parent strain [UA-TpG3 / UA-DSM 18775 = 2560 / 40 = 64]. EXAMPLE 4 TpG3 and TpG57 challenge test in cooked ham
[0081] TpG3 and TpG57 were tested to verify both strains in relation to their ability to inhibit Listeria spp. as well as sensory attributes and benefits in the shelf life of cooked ham. Preparation of Listeria innocua pre-culture
[0082] The L. innocua strain used to prepare the spray culture for inoculation was stored at -50°C. Three days before the start of the challenge test, a cryodroplet of the L. innocua strain was placed in a tube containing 9 ml of BHI medium and was Petition 870260074878, dated 07 / 28 / 2026, page 27 / 74 24 / 28 incubated at 37°C overnight. The following day, 0.1 ml of the overnight culture was transferred to 9 ml of fresh BHI medium and incubated at 37°C overnight. The next morning, the pre-culture was stored at 4°C until use. Meanwhile, the L. innocua cell count of the final pre-culture was enumerated on ALOA agar. The plate was incubated at 37°C overnight and enumerated for calculation of the cell count of the spray solution to be used. Preparation of Lactobacillus curvatus precultures
[0083] Pre-cultures of TpG3 and TpG57, respectively, were prepared in 150 ml of MRS medium and incubated at 30°C overnight (without agitation). The active cell count of the overnight cultures was enumerated using a flow cytometer and an appropriate dilution was made in salt-peptone solution to obtain a spray solution. Samples
[0084] 100 g of sliced cooked ham were packed in a vacuum bag for sample preparation. All bags were inoculated by spraying the surface of the cooked ham slices with 100 cfu / g of Listeria innocua and with one of the test strains (except for the control batch) at a cell count of 1.0E+07 cfu / g or 1.0E+05 cfu / g, as follows: Batch 1 - Control Batch 2 - Lactobacillus curvatus DSM 18775 (1.0E+07 cfu / g) Batch 3 - TpG3 (1.0E+07 cfu / g) Batch 4 - TpG57 (1.0E+07 cfu / g) Batch 5 - TpG3 (1.0E+05 cfu / g) Batch 6 - TpG57 (1.0E+05 cfu / g)
[0085] Next, the vacuum bags were packed in a modified atmosphere (70% N2 / 30% CO2) and stored at +7°C for at least 28 days. Analyses Petition 870260074878, dated 07 / 28 / 2026, page 28 / 74 25 / 28
[0086] Enumeration and pH measurement of each batch of Listeria spp. were enumerated weekly in triplicate. Both total cell count and lactic acid bacteria enumeration were performed every other week, also in triplicate. Cell counts of the test strains were enumerated on MRS agar, and Listeria cell counts were enumerated on ALOA agar according to the corresponding ISO methods (total plate count: ISO 4833-2:2013, lactic acid bacteria: ISO 15214:1998, Listeria spp.: ISO 11290-2:2017). To measure the pH value of the cooked ham, the sample material was ground and homogenized using a household mixer (Moulinette from Moulinex). In addition, a sensory evaluation focusing on the olfactory and gustatory aspects of the samples was performed weekly, in addition to and to support the microbiological analyses. Results
[0087] The results of the pH evolution of the cooked ham samples, the total cell count, the lactic acid bacterial cell count and the Listeria spp. cell count are given in Figures 3 to 6.
[0088] The results of the sensory evaluation of the cooked ham samples (n = 2) during storage at +7°C are presented in Table 2. Petition 870260074878, dated 07 / 28 / 2026, page 29 / 74 26 / 28 Table 2 Battering of the Strain Day 0 Day 7 Day 14 Day 21 Day 28 Flavor Aroma / Odor Flavor Aroma / Odor Flavor Aroma / Odor Flavor Aroma / Odor Flavor Aroma / Odor 1 Control ok ok ok Ok Still good 1 ok / 2 bad old Old old old 2 DSM18775 ok ok ok Ok Still good Slightly sour slightly sour Still good sour sour 3 TpG3 (1.0E+07 cfu / g) Ok ok ok ok / more intense ok Ok ok slightly sour sour sour 4 TpG57 (1.0E+07 cfu / g) Ok ok ok Slightly sour ok Not good bitter slightly sour / fresh slightly sour / dry slightly sour / fresh 5 TpG3 (1.0E+05 cfu / g) Ok ok ok / less flavor Ok ok ok / more flavor + flavor / juicy Ok Slightly sour / fresh ok 6 TpG57 (1.0E + 0.5 cfu / g) Ok ok Slightly sour Ok ok Ok ok ok / slightly sour slightly sour slightly sour Petition 870260074878, dated 07 / 28 / 2026, page 30 / 74 27 / 28 Conclusion
[0089] In all samples where TpG3 or TpG57 were applied, the lactic acid bacteria flora was homogeneous and the corresponding strain was identified by its morphology with high probability. All batches reached a cell count of 1.0E+08 - 1.0E+09 cfu / g after 28 days at 7°C, as did batches with an inoculation level of 1.0E+05 cfu / g, as well as the control batch. Regarding the inhibitory effect on Listeria spp., no significant differences were observed between the batches. It is worth noting that batches with a lower inoculation level of 1.0E+05 cfu / g showed an equal inhibitory effect on the growth of Listeria spp., as did batches with an inoculation cell count of 1.0E+07 cfu / g. The Listeria spp. cell count, from all batches in which TpG3 or TpG57 was applied, remained around the detection limit of 1.0E+02 cfu / g throughout the challenge test, while the Listeria spp. cell count...The concentration of the control batch increased to approximately 1,05,0E+06 cfu / g after 28 days (Figure 5).
[0090] The pH drop in batches with the lowest inoculation level of 1.0E+05 cfu / g was slower compared to batches with a high cell count inoculation of 1.0E+07 cfu / g, which reached a slightly higher pH value after 28 days. Perhaps due to the slower acidification and the slightly higher final pH value of the batches inoculated with 1.0E+05 cfu / g, only a smaller sensory deviation was observed, respectively, with no negative impact on the sensory characteristics of the product throughout the challenge test (Figure 2). In sensory terms, the TpG3 batch with an initial cell count of 1.0E+05 cfu / g showed the best results, followed by the TpG57 batch with an initial cell count of 1.0E+05 Petition 870260074878, dated 07 / 28 / 2026, page 31 / 74 28 / 28 cfu / g, and also the batch with TpG3 with a cell count of 1.0E+07 cfu / g was sensorially acceptable up to day 28, the end of the study (Table 2).
[0091] In summary, when applying the mutants with an initial cell count of 1.0E+05 cfu / g, the pH drop in the batches was slower and reached a slightly higher pH value after 28 days, resulting in less negative impact on the sensory characteristics of the product. These batches were sensorially much better than those with a higher inoculation cell count, as well as the batch inoculated with DSM 18775 and the control batch, and yet the inhibitory effect on Listeria spp. growth was as good as that of the batches inoculated with 1.0E+07 cfu / g. Petition 870260074878, dated 07 / 28 / 2026, page 32 / 74
Claims
1 / 2 CLAIMS 1. Isolated mutant Lactobacillus curvatus strain, characterized in that it is a mutant of the Lactobacillus curvatus strain deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM 18775, wherein said isolated mutant strain is the strain deposited in the DSMZ under accession number DSM 32590 and has a prolonged latency phase of at least 24 hours at 30°C on MRS agar plates relative to the DSM 18775 strain when evaluated under identical conditions.
2. Isolated mutant Lactobacillus curvatus strain, characterized in that it is a mutant of the strain deposited under DSM accession number 32590, as defined in claim 1, wherein said isolated mutant strain is the strain deposited under DSM accession number 32591 and has a prolonged latency phase of at least 48 hours at 30°C on MRS agar plates relative to the DSM 18775 strain when evaluated under identical conditions.
3. Strain, according to claim 1 or 2, characterized in that it is a bacteriocin-producing strain.
4. Composition, characterized by the fact that it comprises bacteria of the Lactobacillus curvatus mutant strain DSM 32590 and bacteria of the Lactobacillus curvatus mutant strain DSM 32591.
5. Composition, according to claim 4, characterized in that mutant Lactobacillus curvatus strains are present in the composition at a concentration of at least 105 CFU / g each.
6. Composition, according to claim 4, characterized in that the bacteria of the mutant strains Lactobacillus curvatus are the only bacteria present in the composition.
7. Composition, according to claim 4, Petition 870260074878, dated 07 / 28 / 2026, p. 33 / 74 2 / 2 characterized in that the composition further comprises bacteria of one or more of the species selected from Pediococcus acidilactici, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum and Staphylococcus carnosus.
8. Composition, according to claim 7, characterized in that the bacteria of one or more of the species selected from Pediococcus acidilactici, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum and Staphylococcus carnosus is selected from Pediococcus acidilactici deposited in the DSMZ under accession number DSM 28307, Lactobacillus sakei deposited in the DSMZ under accession number DSM 14022, Lactococcus lactis deposited in the DSMZ under accession number DSM 11037 and Leuconostoc carnosum LC1043.
9. A method for inhibiting the amount of spoilage or pathogenic bacteria in a food product, characterized in that it comprises adding bacteria of a mutant Lactobacillus curvatus strain, as defined in claim 1 or 2, to a food product at a concentration of at least 105 CFU / g.
10. Method according to claim 9, characterized in that the pathogenic bacterium is Listeria.
11. A method for inhibiting the amount of spoilage or pathogenic bacteria in a food product, characterized in that it comprises adding a composition, as defined in claim 4, to a food product in an amount sufficient to provide mutant strains of Lactobacillus curvatus at a concentration of at least 105 CFU / g each.
12. Method according to claim 11, characterized in that the pathogenic bacterium is Listeria. Petition 870260074878, dated 07 / 28 / 2026, p. 34 / 74