Bioprotective Lacticaceibacillus rhamnosus with reduced post-acidification

Abstract

This application is in the field of dairy products. The present invention relates to Lacticaseibacillus rhamnosus DSM 34195 or a variant thereof and its use in the production of food, feed or pharmaceutical products, in particular fermented dairy products. Lacticaseibacillus rhamnosus DSM 34195 reduces post-acidification in products and has antifungal activity. The present invention also provides a composition comprising Lacticaseibacillus rhamnosus DSM 34195 or a variant thereof.

Description

[Technical Field]

[0001] The present invention relates to the bacterium Lacticaseibacillus rhamnosus, which has low post-acidification in fermented dairy products and antifungal activity. [Background technology]

[0002] Lactic acid bacteria (LAB) have been used for decades to extend the shelf life of foods. During the fermentation process, lactic acid and other organic compounds are produced by LAB, which lower the pH of the food and consequently inhibit the growth of undesirable microorganisms such as yeasts and molds.

[0003] Bioprotection is defined as the use of natural or controlled antimicrobial compounds to extend the shelf life and enhance the safety of foods. In dairy products, spoilage caused by mold and yeast cells is one of the major problems that negatively impacts shelf life. Over the past decade, considerable efforts have been devoted to exploring the bioprotective capabilities of LAB, identifying new strains with bioprotective properties from various food sources, and elucidating the mechanisms of the observed antifungal activity. Numerous metabolites produced by LAB have been identified with antifungal activity.

[0004] Further research has revealed that competitive exclusion of limited resources by other organisms is the primary mechanism by which lactic acid bacteria inhibit fungal growth. In particular, depletion of the essential trace element manganese is a major bioprotective mechanism for lactic acid bacteria in dairy products. It has also been shown that manganese removal is an active mechanism, requiring energy to maintain a high manganese gradient (Siedler et al. "Competitive exclusion is a major bioprotective mechanism of lactobacilli against fungal spoilage in fermented milk products." Applied and Environmental Microbiology 86.7 (2020)).

[0005] At the same time, we found that the high antifungal activity of biodefense strains is generally accompanied by high activity, which leads to post-acidification, i.e., continued acidification after fermentation is completed. Because the production of bioprotective compounds in LAB typically exhibits growth-related kinetics, it is expected to cease when growth is inhibited (Lv et al. "Modeling the production of nisin by Lactococcus lactis in fed-batch culture." Applied microbiology and biotechnology 68.3 (2005): 322-326). Because milk acidification is typically associated with growth (Dandoy et al. "The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS." Microbial cell factories. Vol. 10, No. S1. BioMed Central, 2011), strains with reduced post-acidification are expected to also exhibit reduced bioprotective activity.

[0006] International Publication No. WO2021239574 discloses Lactobacillus rhamnosus DSM 33515, a bioprotectant strain described as exhibiting a combination of reduced post-acidification and high bioprotective efficacy. Summary of the Invention [Problem to be solved by the invention]

[0007] For economic reasons, improved solutions are constantly being sought that can effectively inhibit microbial spoilage and contamination while reducing post-acidification. [Means for solving the problem]

[0008] Accordingly, the present application provides a bacterium of the species Lacticaceibacillus rhamnosus deposited as DSM 34195, or a mutant Lacticaceibacillus rhamnosus of the deposited bacterium. The mutant may have substantially the same or improved bioprotective effect against yeasts and molds and reduced post-acidification compared to DSM 34195. The mutant is obtained by using DSM 34195 as a starting material. The mutant is different from DSM 33515 and DSM 23035. The mutant may have maintained or improved properties compared to DSM 34195, or may have improved properties compared to DSM 33515. The properties discussed in this paragraph and in the present disclosure are antifungal effect or activity (in other words, bioprotective effect against yeasts and molds) and reduced post-acidification.

[0009] The present invention further provides a composition comprising the bacteria of the species Lacticaceus rhamnosus as described above. According to one embodiment, the composition further comprises a cryoprotectant, a lyoprotectant, an antioxidant, a nutrient, a bulking agent, a flavoring agent, or a mixture thereof. According to one embodiment, the concentration of the bacteria in the composition is at least 10 9 Colony forming units (CFU) / g, at least 10 10 CFU / g, or at least 10 11 CFU / g. According to a preferred embodiment, the composition is frozen or lyophilized. According to one embodiment, the composition further comprises a starter culture.

[0010] The present invention further provides a method for producing a fermented dairy product, comprising adding the above-described Lacticaceus rhamnosus bacteria or a composition containing the same to milk or a dairy product, and fermenting the mixture at a temperature of about 22°C to about 43°C, for example, 22°C to 43°C, until the pH reaches 4.6 or less than 4.6.

[0011] The present invention provides a fermented milk product comprising the bacteria of the Lacticaceibacillus rhamnosus species described above. Preferably, the fermented milk product is obtained by the method described above. According to a further embodiment, the fermented milk product maintains a pH above 3.8 when stored at 25°C for at least 28 days. According to another embodiment, the bacteria of the Lacticaceibacillus rhamnosus species are present in a concentration of at least 10 ... 7 Present at a concentration of CFU / g.

[0012] Furthermore, the present invention provides a food, feed, or pharmaceutical product containing the Lacticaceus rhamnosus bacterium or a composition containing the same. According to a preferred embodiment, the food, feed, or pharmaceutical product is obtained by the method described above. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows post-acidification of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation doses in a 96-well low plate assay.

[0014] [Figure 2] Figure 2 shows mold inhibition of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation doses in a 96-well low plate assay.

[0015] [Figure 3] Figure 3 shows yeast inhibition of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation doses in a 96-well low plate assay.

[0016] [Figure 4] Figure 4 shows the growth of yeasts prepared from milk fermented with starter culture alone (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or with a combination of starter culture and L. rhamnosus bacteria DSM 34195 (three different doses).

[0017] [Figure 5] Figure 5 shows the growth of six different moulds added as contaminants to agar plates made from milk fermented with starter culture alone (reference), or with a combination of starter culture and L. rhamnosus bacterium DSM 33515, or with a combination of starter culture and L. rhamnosus bacterium DSM 34195 (at three different doses).

[0018] [Figure 6] Figure 6 shows the growth of three different molds added as contaminants to agar plates made from milk (with or without 10% sucrose) fermented with starter culture alone (YF-812 or Advance 2.0) (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or with a combination of starter culture and L. rhamnosus bacteria DSM 34195 (at three different doses).

[0019] [Figure 7] Figure 7 shows the growth of yeast prepared from milk (containing 10% sucrose) fermented with starter culture alone (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or with a combination of starter culture and L. rhamnosus bacteria DSM 34195 (at three different doses). Figures 7A and 7B show the results using starter cultures YF-L812 and Advance 2.0, respectively.

[0020] [Figure 8]Figure 8 shows the growth of yeast prepared from milk (without sucrose) fermented with starter culture alone (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or with a combination of starter culture and L. rhamnosus bacteria DSM 34195 (at three different doses). Figures 8A and 8B show the results using starter cultures YF-L812 and Advance 2.0, respectively.

[0021] [Figure 9] Figure 9 shows the growth of three types of mold added as contaminants to agar plates made from milk fermented with starter culture alone (YoFlex Mild 1.0 or YoFlex Premium 1.0) (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or with a combination of starter culture and L. rhamnosus bacteria DSM 34195 (at three different doses).

[0022] [Figure 10] Figure 10 shows the growth of yeasts prepared from milk fermented with starter culture alone (F-DVS YoFlex Mild 1.0 or YoFlex Premium 1.0) (reference), or with a combination of starter culture and L. rhamnosus bacteria DSM 33515, or a combination of starter culture and L. rhamnosus bacteria DSM 34195 (at three different doses). Figures 10A and 10B show the growth of Debaryomyces hansenii and Torulaspora delbrueckii, respectively.

[0023] [Figure 11]Figure 11 shows the change in pH over time of fermented milk products stored for 28 days at 25±1°C. The products were fermented with starter culture alone (reference), or with starter culture combined with DSM 33515, or with starter culture combined with DSM 34195. Figures 11A and 11B show the results using starter cultures YF-L812 and Advance 2.0, respectively.

[0024] [Figure 12] Figures 12-14 show the growth of six different molds added as contaminants to agar plates made from Crema acida without L. rhamnosus (reference), or from Crema acida containing L. rhamnosus bacteria DSM 33515 or L. rhamnosus bacteria DSM 34195, and incubated at different temperatures.

[0025] [Figure 13] Figures 12-14 show the growth of six different molds added as contaminants to agar plates made from Crema acida without L. rhamnosus (reference), or from Crema acida containing L. rhamnosus bacteria DSM 33515 or L. rhamnosus bacteria DSM 34195, and incubated at different temperatures.

[0026] [Figure 14] Figures 12-14 show the growth of six different molds added as contaminants to agar plates made from Crema acida without L. rhamnosus (reference), or from Crema acida containing L. rhamnosus bacteria DSM 33515 or L. rhamnosus bacteria DSM 34195, and incubated at different temperatures.

[0027] [Figure 15] Figure 15 shows the growth of D. hansenii yeast in Crema acidostat prepared without L. rhamnosus (reference) or with L. rhamnosus bacteria DSM 33515 or L. rhamnosus DSM 34195.

[0028] [Figure 16] Figures 16-18 show the pH changes of Crema acida products stored for 28 days at 7°C, 12°C, and 25°C. The products contained either no L. rhamnosus (reference), L. rhamnosus bacteria DSM 33515, or L. rhamnosus bacteria DSM 34195.

[0029] [Figure 17] Figures 16-18 show the pH changes of Crema acida products stored for 28 days at 7°C, 12°C, and 25°C. The products contained either no L. rhamnosus (reference), L. rhamnosus bacteria DSM 33515, or L. rhamnosus bacteria DSM 34195.

[0030] [Figure 18] Figures 16-18 show the pH changes of Crema acida products stored for 28 days at 7°C, 12°C, and 25°C. The products contained either no L. rhamnosus (reference), L. rhamnosus bacteria DSM 33515, or L. rhamnosus bacteria DSM 34195. DETAILED DESCRIPTION OF THE INVENTION

[0031] Bioprotective food cultures are available as safe additives for traditional fermented products. These bioprotective cultures are used in combination with conventional starter cultures to co-ferment milk to produce fermented products. They exert bioprotective effects during fermentation, extending the shelf life of fermented products against mold and yeast. Many dairy products, such as yogurt, are halted and cooled at a specific pH. Even after fermentation, bacteria often remain active during storage, resulting in the production of additional lactic acid, a process known as post-acidification. A decrease in the pH of the final product is undesirable because it adversely affects the product's sensory properties. There is a need for the development of new and improved bioprotective cultures that combine reduced post-acidification with high bioprotective efficacy. For example, Lactobacillus rhamnosus DSM 33515 has already been described as a bioprotective strain that combines reduced post-acidification with high bioprotective efficacy. The present application provides an improved bioprotectant strain DSM 34195 and its variants, which have advantages over DSM 33515, as shown in the examples.

[0032] In the present context, the term "mutant" should be understood to mean a strain derived from the strain of the invention, for example by genetic engineering, irradiation, and / or chemical treatment. Preferably, the mutant is a functionally equivalent mutant, e.g., a mutant having substantially the same or improved properties as the deposited strain, in particular with respect to post-acidification inhibitory effect and / or bioprotective effect. Each mutant represents an embodiment of the present application. The term "mutant" particularly refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenesis treatment, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), ultraviolet light, or a spontaneous mutant. Mutants may be subjected to multiple mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), although it is currently preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps) be performed. In currently preferred mutants, less than 5%, or no more than 1%, or no more than 0.1% of the nucleotides in the bacterial genome have been shifted to another nucleotide or deleted compared to the parent strain.

[0033] After extensive screening of 10,000 mutants of DSM 23035, strain DSM 34195 was identified. This strain can be used at low inoculation doses, exhibiting low post-acidification and high antifungal activity. Furthermore, this strain can be combined with a variety of starter cultures and milk bases, demonstrating superior compatibility compared to DSM 33515. Furthermore, DSM 34195 has a clean, mild taste, no off-flavors, and excellent sensory properties.

[0034] Thus, the present application provides Lacticaceus rhamnosus strain DSM 34195 and mutants that maintain the advantageous properties of DSM 34195 when compared under identical conditions.

[0035] For post-acidification, the comparison can be performed, for example, as follows: -Preparing a homogenized milk base consisting of 2.8% protein, 1.2% fat, and 10% sucrose, heating the milk base at 95±1°C for 5 minutes and immediately cooling it; - Inoculate the milk base with starter culture YF-L812 at 500U per 2500L. - L. rhamnosus DSM 34195 and the comparison mutant strains were added to a milk base at a total concentration of 1x10 7 Inoculated at CFU / g Fermentation was carried out at 43°C until a pH of 4.60±0.1 was reached, followed by cooling to 25±1°C, after which the samples were refrigerated at 5-7°C, and -Samples are stored at 25±1°C for 28 days and the pH is measured and measured on the 1st, 7th, 14th, 21st and 28th days.

[0036] For antifungal effects, comparisons can be made, for example, as follows: -Preparing a homogenized milk base consisting of 2.8% protein, 1.2% fat, and 10% sucrose, heating the milk base at 95±1°C for 5 minutes and immediately cooling it; - Inoculate the milk base with starter culture YF-L812 at 500U per 2500L. - L. rhamnosus DSM 34195 and the comparison mutant strains were added to a milk base at a total concentration of 1x10 7 Inoculated at CFU / g Ferment at 43°C until a pH of 4.60 ± 0.1 is reached, then cooled in a cooling chamber. - Fermented milk samples were inoculated with 50 cfu / g of D. hansenii CHCC16374, and -Samples are stored at 7°C for 28 days and total yeast counts are determined on days 1, 7, 14, 21 and 28 of storage.

[0037] The Lacticaceus rhamnosus strains of the present application have the particular advantage of exhibiting antifungal activity while reducing the risk of post-acidification, thus improving the storage stability of foods produced using these bacteria, especially at temperatures above refrigeration.

[0038] In the context of the present application, "mold" refers to a fungus that grows in the form of multicellular filaments called mycelia. The term "inhibit" with respect to mold refers to, for example, a reduction in mold growth or sporulation, or a reduction in the number or concentration of mold in and / or on a food product containing the bacteria of the present application, compared to a food product that does not contain the bacteria. The degree of inhibition by the Lacticaceibacillus rhamnosus strains of the present application is preferably measured by growth on agar-coagulated fermented milk in the presence and absence of Lacticaceibacillus rhamnosus. Examples of molds include the genus Penicillium, such as Penicillium solitum, Penicillium brevicompactum, Penicillium crustosum, Penicillium roqueforti, Penicillium paneum, and Penicillium carneum.

[0039] Yeast is a fungus that grows as a single cell. The Lacticaceibacillus rhamnosus strain of the present application, i.e., the strain deposited under DSM 34195, and mutants thereof that maintain their advantageous properties, can inhibit mold growth and further inhibit yeast growth. With respect to yeast growth, the term "inhibit" also refers to a reduction in yeast growth or a reduction in the number or concentration of yeast in and / or on a food product containing the bacteria of the present application, as compared to a food product that does not contain such bacteria. The degree of inhibition by the Lacticaceibacillus rhamnosus strain of the present application is preferably measured by growth on agar-coagulated fermented milk in the presence and absence of Lacticaceibacillus rhamnosus. Examples of yeasts include the genus Debaryomyces and Torulaspora, such as Debaryomyces hansenii and Torulaspora delbrueckii.

[0040] In the context of describing the present invention (particularly in the context of the claims which follow), the use of the terms "a," "an," and "the" and similar referents shall be construed to include both the singular and the plural, unless otherwise stated herein or clearly contradicted by context.

[0041] Each composition may contain a number of bacteria, including LAB. The term "lactic acid bacteria" or "LAB" refers to food-grade bacteria that produce lactic acid as the primary metabolic end product of carbohydrate fermentation. These bacteria are related by common metabolic and physiological characteristics and are typically Gram-positive, low-GC, acid-tolerant, non-spore-forming, non-respiring bacilli or cocci. During the fermentation stage, these bacteria consume lactose to produce lactic acid, lowering the pH and forming a protein coagulum. Thus, these bacteria are responsible for the acidification of milk and the texture of dairy products. The term "lactic acid bacteria" as used herein includes, but is not limited to, bacteria belonging to the following genera: Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium breve, and Leuconostoc spp.

[0042] Thus, preferred compositions of the present application are characterized in that they further comprise at least one further bacterium selected from one or more of the following genera and species: Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium breve, and Leuconostoc spp.

[0043] According to a particularly preferred embodiment, the composition of the present application comprises bacteria of the species Lacticaceibacillus rhamnosus deposited under DSM 34195, or a mutant of Lacticaceibacillus rhamnosus obtained from the deposited bacteria, and one or more further bacteria. According to one embodiment, several different strains of Lacticaceibacillus rhamnosus bacteria are combined.

[0044] The compositions of the present application may further comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, bulking agents, flavorings, or mixtures thereof. The compositions may be in frozen or lyophilized form. Preferably, the compositions comprise one or more of cryoprotectants, lyoprotectants, antioxidants, and / or nutrients, more preferably cryoprotectants, lyoprotectants, and / or antioxidants, and most preferably cryoprotectants or lyoprotectants, or both. The use of protective agents such as cryoprotectants and lyoprotectants is known to those skilled in the art. Suitable cryoprotectants or lyoprotectants include monosaccharides, disaccharides, trisaccharides, and polysaccharides (e.g., glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, gum arabic (acacia), etc.), polyols (e.g., erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol), amino acids (e.g., proline, glutamic acid), complex substances (e.g., skim milk powder, peptone, gelatin, yeast extract), and inorganic compounds (e.g., sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, maltose, etc. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (e.g., B vitamins, vitamin C), etc. The composition may optionally contain other substances such as bulking agents (lactose, maltodextrin, etc.) and / or flavoring agents.

[0045] LAB are most commonly added to milk in the form of starter cultures. As used in this context, the terms "starter" or "starter culture" refer to a culture of food-grade microorganisms, particularly lactic acid bacteria, responsible for the acidification of the milk base. Starter cultures can be fresh but are most often frozen or lyophilized. These products, also known as "direct vat set" (DVS) cultures, are prepared for direct inoculation of fermentation vessels or vats for the production of dairy products, such as fermented milk products and cheese. Commercially available starter cultures are available from a number of sources. For example, Premium 1.0, YF-L812, Mild 1.0, and Advance 2.0 from Chr. Hansen contain a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Preferably, the starter culture comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0046] Thus, according to one aspect, the present application provides at least 10 9 Colony forming units (CFU) / g, at least 10 10 CFU / g, or at least 10 11 Compositions are provided in the form of solid frozen or lyophilized starter cultures containing lactic acid bacteria at a concentration of CFU / g, wherein the starter cultures further contain bacteria of the species Lacticaceibacillus rhamnosus deposited as DSM 34195, or mutants of Lacticaceibacillus rhamnosus obtained from the deposited bacteria.

[0047] In the context of this application, the term "milk" is used broadly in its general sense to refer to the fluid produced by the mammary glands of animals or plants. According to this application, milk may be processed, and the term "milk" includes whole milk, skim milk, nonfat milk, low-fat milk, full-fat milk, lactose-reduced milk, or concentrated milk. Nonfat milk is nonfat milk or a skim milk product. Low-fat milk is typically defined as milk with a fat content of about 1% to about 2%. Full-fat milk often has a fat content of 2% or more. The term "milk" is intended to encompass milk from a variety of mammalian and plant sources. Mammalian sources include, but are not limited to, milk from cows, sheep, goats, buffalo, camels, llamas, mares, and deer. Plant sources include, but are not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp, sesame, and sunflower seeds. The methods and products of the present application most preferably use milk from cows as the starting material for fermentation.

[0048] The term "milk" also includes reduced-fat dairy products and / or reduced-lactose dairy products, each of which can be produced using methods well known in the art and are commercially available. Lactose-reduced milk can be produced by any method known in the art, such as hydrolyzing lactose to glucose and galactose with the enzyme lactase, or by nanofiltration, electrodialysis, ion-exchange chromatography, centrifugation, etc.

[0049] The terms "milk product" or "milk base" are used broadly in this application to refer to a composition based on milk or milk components that can be used as a medium for the growth and fermentation of lactic acid bacteria (LAB). A dairy product or milk base includes milk-derived components and other components that can be used for the growth or fermentation of LAB.

[0050] Prior to fermentation, the dairy substrate can be homogenized and pasteurized according to methods known in the art. As used herein, "homogenizing" refers to intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it is preferably performed to break down the milk fat into fine particles and prevent the milk fat from separating from the milk. This can be achieved by forcing the milk through small openings at high pressure. As used herein, "pasteurizing" refers to treating the dairy substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Pasteurization is preferably achieved by maintaining a predetermined temperature for a predetermined time. The predetermined temperature is usually achieved by heating. The temperature and time can be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step can also be performed afterward.

[0051] The present invention further provides a method for storing the fermented product at a temperature above 7° C., preferably at a temperature between 7° C. and 25° C. The product can be stored for any length of time, but preferably for at least 14 days, and the pH of the fermented milk product is maintained at or above 4.0 during storage.

[0052] The present invention further provides a method for producing a fermented milk product as described above, and a method for producing a food, feed or pharmaceutical product, including a food, feed or pharmaceutical product obtainable by this method.

[0053] Fermentation is carried out for the production of food, feed, or pharmaceutical products. The terms "fermented milk product," "food," or "feed" product refer to products obtained by the fermentation methods of the present application and include cheese, yogurt, fruit yogurt, yogurt drinks, strained yogurt (Greek yogurt, labneh), quark, fromage frais, cream cheese, etc. The term "food" also includes other fermented foods, such as fermented meats, including fermented sausages, and fermented seafood. Examples of fermented milk products include crema acida and sour cream.

[0054] The term "cheese" refers to any cheese, including hard, semi-hard, and soft cheeses. Examples include cottage cheese, tvorog, quark, feta, cheddar, Parmesan, mozzarella, Emmental, d'Ambeau, Gouda, Edam, feta cheeses such as UF feta, soft cheeses, pasta filata, continental cheeses, blue cheeses, brine cheeses such as white brine cheeses, queso fresco, Camembert, Brie, and the like. Those skilled in the art know how to convert coagulum into cheese, and this method is described in, for example, Kosikowski, FV, and VV Mistry, "Cheese and Fermented Milk Foods," 1997, 3rd Ed. FV Kosikowski, LLC, Westport, CT. Cheeses with an NaCl concentration of less than 1.7% (w / w) are referred to herein as "low-salt cheeses."

[0055] In this application, "yogurt" refers to a product containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus paracasei, or microorganisms derived therefrom. Lactic acid bacteria strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are included to impart various properties to the final product, such as promoting a balanced microflora. As used herein, the term "yogurt" includes set yogurt, stirred yogurt, drinking yogurt, petit suisee, heat-treated yogurt, strained or Greek-style yogurt characterized by high protein levels, and yogurt-like products.

[0056] In particular, the term "yogurt" includes, but is not limited to, yogurt as defined in accordance with French and European regulations, such as a coagulated dairy product obtained by lactic acid fermentation using only specific thermophilic lactic acid bacteria (i.e., Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), which are cultured simultaneously and confirmed to be viable in the final product at a concentration of at least 10 million CFU (colony forming units) / g. Yogurt may contain, as needed, dairy ingredients (e.g., cream) and other ingredients, such as sugar or sweeteners, one or more flavorings, fruits, grains, or nutritional ingredients (especially vitamins, minerals, and dietary fiber), as well as stabilizers and thickeners. Optionally, the yogurt meets the specifications for fermented milk and yogurt of the AFNOR NF 04-600 standard and / or the Codex Stan A-IIa-1975 standard. To meet the AFNOR NF 04-600 standard, no heat treatment may be performed after fermentation, and dairy ingredients must account for at least 70% (m / m) of the final product.

[0057] taxonomy Lactobacillus rhamnosus is now known as Lacticaseibacillus rhamnosus, as described by Zheng et al., Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107.

[0058] Deposits and expert solutions The applicant requests that until the date on which the patent is granted, samples of the deposited microorganisms described below shall be provided only to experts in accordance with the regulations established by the industrial property offices of the Contracting States of the Budapest Treaty.

[0059] [Table 1] [Example]

[0060] Example 1: Creation of L. rhamnosus DSM 34195 1.1 Mutant pool A mutant pool was generated from the L. rhamnosus DSM 23035 parent strain using ethyl methanesulfonate (EMS) mutagenesis. A preliminary experiment was conducted to establish the efficacy and bactericidal rate of EMS against this strain. A bactericidal rate of 95% or higher was targeted. Based on this, 15 μL of EMS was added to 1 mL of an overnight culture (OD620 = 3-4) grown in MRS-Difco medium. The culture was incubated at 37°C for 4 hours, and then 10 -2 From 10 -6 The diluted culture medium was then plated on MRS-Difco agar medium and the cell number was counted.

[0061] 1.2 Selection of DSM 34195 Mutants were selected from the mutant pool by high-throughput screening, which included hit-picking of mutants from agar plates, growth of mutants in broth, and selection of mutants with reduced antifungal activity after milk acidification and post-acidification.

[0062] Specifically, MRS-Difco agar medium (pH 6.5) was dispensed into low-profile square bioassay dishes (Corning 431301). The mutant pool was plated onto the agar medium using sterile glass beads and incubated anaerobically at 37°C for 2 days. Approximately 10,000 colonies were picked into a 200 μl 96-well microtiter plate containing MRS-Difco medium using a QPix2 colony picker (Genetix). The microtiter plate was incubated overnight at 37°C under anaerobically controlled conditions, and then 1% of the overnight culture inoculum was inoculated into milk in a 2 ml 96-well microtiter plate. A homogenized milk base consisting of 2.8% protein, 1.2% fat, and 10% sucrose was heated at 95±1°C for 5 minutes and immediately cooled. Milk acidification was monitored by pH colorimetry as described by Poulsen et al., "High-throughput screening for texturing Lactococcus strains." (FEMS microbiology letters 366.2 (2019): fnz001). The mutant strains were grown in milk supplemented with starter culture YF-L812 and a pH indicator at 40°C for 5–6 h until the pH reached 4.55, compared with control wells containing the wild-type strain. YF-L812 is a commercially available culture from Chr. Hansen A / S containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0063] Based on the milk acidification data, the mutant with the lowest post-acidification was identified. The selected mutant was then transferred to a 200 μl 96-well microtiter plate and tested for antifungal activity. As a preculture, the mutant was grown overnight at 37°C in BD5-1 medium. Then, 10 μl of the preculture was inoculated into a 96-well microtiter plate with 2 ml of B milk (ISO 26323:2009) supplemented with 2% sucrose and starter culture YF-812. In the control well containing the wild-type strain, the milk was incubated at 40°C for approximately 6 hours until the pH reached 4.55. The microtiter plate was then stored at 4°C overnight. Then, 150 μl of the fermented milk was transferred to a 200 μl 96-well microtiter plate and inoculated with Debaryomyces hansenii CHCC16374 (Chr. Hansen Culture Collection) at approximately 40 cells / ml. The 200 μl microtiter plate was stored at 17°C for 5 days, after which serial dilutions were spotted onto YGC plates and the yeast counts were determined on a scale from 0 (no yeast growth) to 5 (confluent growth). The remaining fermented milk sample was filled into a 2 ml microtiter plate and stored at room temperature for 14 days, after which acidification was measured. As a result, the L. rhamnosus DSM 34195 mutant strain was selected.

[0064] Example 2: Antifungal effect during storage and post-acidification in a 96-well plate assay 2.1 Post-acidification of L. rhamnosus DSM 34195 and L. rhamnosus DSM 33515 Homogenized milk base containing 2.8% protein, 1.2% fat, and 10% sucrose was heat-treated at 95 ± 1°C for 5 min and immediately cooled. To follow the progress of acidification, a pH indicator was added to the milk and the color change was measured as described in Section 1.2, but the color value was not converted to pH.

[0065] A commercial starter culture containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (F-DVS YF-L812) was inoculated at 0.02% (v / w) and 270 μL was dispensed into each well. Then, 30 μL of L. rhamnosus DSM 34195 or DSM 33515 was inoculated into each well. The final inoculum concentration was adjusted to four levels (5 x 10). 6 CFU / g, 1 x 10 7 CFU / g, 2 x 10 7 CFU / g, and 3 x 10 7 The assay was performed in 12 replicates using multiple 96-well plates. Reference wells ("blank") containing only the starter culture were also prepared. The 96-well plates were incubated at 43±1°C until the pH reached a value equivalent to 4.55±0.5. The pH was monitored using an added pH indicator. The plates were scanned from the bottom and the color value was monitored until the pH reached a value equivalent to 4.55. Additional plates were prepared to monitor the post-acidification condition and antifungal effect.

[0066] Post-acidification was monitored by placing the plates on a scanner and scanning them continuously every 2 hours for 21 days at 25 ± 1°C. The change in color value (ΔpH equivalent) during incubation was used as an indicator of post-acidification and is shown in Figure 1.

[0067] The results shown in Figure 1 indicate that post-acidification was more advanced with increasing inoculum doses of L. rhamnosus DSM 34195 and DSM 33515. In particular, when compared at the same inoculum dose, post-acidification was suppressed with L. rhamnosus DSM 34195 compared to DSM 33515 at all inoculum doses tested.

[0068] 2.2 Antifungal Effect of L. rhamnosus DSM 34195 and L. rhamnosus DSM 33515 against Penicillium roqueforti A spore suspension of P. roqueforti DSM 33518 was applied to the surface of fermented yogurt in a 96-well low-well plate at a concentration of 50 spores per well. The plates were then incubated at 7 ± 1°C for 52 days, and the plate surface was scanned with a scanner. The resulting color change on the surface was used as an index of P. roqueforti inhibition. The time to the onset of growth was estimated from the color value and indicates the inhibitory effect on P. roqueforti.

[0069] The results, shown in Figure 2, indicate that L. rhamnosus DSM 34195 inhibits P. roqueforti at all doses tested, whereas L. rhamnosus DSM 33515 inhibits P. roqueforti at 5 x 10 6 CFU / g and 1 x 10 7 CFU / g showed low inhibition, 2 x 10 7 CFU / g showed almost complete inhibition, 3 x 10 7 CFU / g shows complete inhibition.

[0070] 2.3 Antifungal effects of L. rhamnosus DSM 34195 and L. rhamnosus DSM 33515 against Debaryomyces hansenii A cell suspension of D. hansenii CHCC16374 was applied to the surface of fermented yogurt in a 96-well plate at a concentration of 50 CFU / g per well. The plate was then incubated at 7±1°C for 52 days, and the plate surface was scanned with a scanner. The resulting change in surface color was used as an index of yeast inhibition. Figure 3 shows the time to the onset of yeast growth estimated from the color value and the inhibitory effect of D. hansenii.

[0071] The results, shown in Figure 3, demonstrate that L. rhamnosus DSM 34195 completely inhibited D. hansenii at all doses tested, whereas DSM 33515 inhibited D. hansenii at 5x10 6 The inhibition rate increased with increasing dose, reaching 2x10 CFU / g. 7 Complete inhibition of D. hansenii in CFU / g.

[0072] Example 3: Comparison of the antifungal effect of L. rhamnosus DSM 34195 with that of L. rhamnosus DSM 33515 The inhibitory effect of L. rhamnosus DSM 34195 on molds was demonstrated using semi-quantitative agar cultures, and its inhibitory effect on yeasts was verified using challenge tests tracking the growth (Log(CFU / g)) of various target contaminants in fermented milk samples (yogurt).

[0073] Homogenized milk base consisting of 2.8% protein, 1.2% fat, and 10% sucrose was heated at 95±1°C for 5 minutes and immediately cooled. Commercially available starter culture (F-DVS YF-L812) was inoculated at 500U per 2500L, and the inoculated milk was dispensed into 200ml bottles. Some bottles contained 5x10 L of L. rhamnosus DSM 34195. 6 CFU / g, 1x10 7 CFU / g, 2x10 7 CFU / g, 3x10 7 The other bottles were inoculated with L. rhamnosus DSM 33515 at a total concentration of 3 x 10 7 The inoculation was performed at CFU / g. Bottles inoculated with the starter culture alone served as controls. All bottles were incubated in a water bath at 43 ± 1°C and fermented under these conditions until a pH of 4.55 ± 0.1 was reached. After fermentation, the bottles were vigorously agitated to break up the coagulum and immediately cooled in a cooling chamber.

[0074] For the yeast test, fermented milk samples were inoculated with the target contaminant, D. hansenii CHCC16374, at 50 cfu / g. The inoculated samples were stored at 7°C for 24 days, and yeast growth was assessed as total yeast counts five times during the storage period.

[0075] For the mold test, 200 ml of fermented milk was heated to 40 °C, 40 ml of 5% sterile agar solution was dissolved in it, and the mixture was cooled to 60 °C. The fermented milk and agar solution were poured into sterile Petri dishes and allowed to dry on an LAF bench for 30 minutes. Spore suspensions of the following six mold species were spotted onto agar plates at a concentration of 500 spores per spot (three strains per plate, as shown in Figure 5): P. brevicompactum DSM 32094, P. crustosum DSM 33517, P. solitum DSM 32093, P. carneum DSM 33520, P. panum DSM 33519, and P. roqueforti DSM 33518. Three types of mold were spotted on each plate, and the target contaminant was added at a concentration of 500 spores per spot. The plates were incubated at 22±1°C for 8 days and periodically observed for mold growth.

[0076] The effectiveness of low inoculation rates was confirmed again in a yeast challenge test, the growth curves of which (Log(CFU / g)) are shown in Figure 4. The starter culture and L. rhamnosus DSM 33515 were mixed at 3x10 7 When fermented products inoculated with D. hansenii at CFU / g were inoculated with D. hansenii, growth inhibition of D. hansenii was observed. Surprisingly, when inoculated with L. rhamnosus DSM 34195, growth inhibition of D. hansenii was observed at all inoculation levels tested (5x10 6 CFU / g, 1x10 7 CFU / g, 2x10 7 CFU / g, and 3x10 7 CFU / g), an even higher inhibitory effect was observed.

[0077] The results of the agar assay are shown in Figure 5 and show that all tested molds grew very well on agar plates made from milk fermented with only the starter culture (reference). However, when L. rhamnosus DSM 34195 was added during milk fermentation, the resulting medium inhibited the growth of the six tested Penicillium species, with 5x10 6CFU / g to 3x10 7 The inhibitory effect was clearly observed at all doses up to CFU / g. Surprisingly, the lowest dose of L. rhamnosus DSM 34195, 5 x 10 6 The efficacy when administered in CFU / g is 3x10 7 It was similar to L. rhamnosus DSM 33515 when administered as CFU / g.

[0078] Conclusion: The results show that DSM 34195 has a higher anti-yeast and anti-mold effect than DSM 33515.

[0079] Example 4: Antifungal effect of L. rhamnosus DSM 34195 in combination with different starter cultures in different milk bases 4.1 Combining different starter cultures with different milk bases The inhibitory effect of L. rhamnosus DSM 34195 on molds was demonstrated using semi-quantitative agar plates, and its inhibitory effect on yeasts was verified using challenge tests tracking the growth (Log(cfu / g)) of various target contaminants in fermented milk (yogurt).

[0080] Two milk bases containing 2.8% protein, 1.2% fat, and 10% sucrose, with or without, were homogenized and heat-treated at 95 ± 1°C for 5 minutes, followed by immediate cooling. Commercially available starter cultures (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (F-DVS YF-L812 or YoFlex Advance 2.0, Chr. Hansen A / S, Denmark)) were inoculated at a rate of 500 U per 2500 L, and the inoculated milk was dispensed into 200 ml bottles. Some bottles contained 1 x 10 L of L. rhamnosus DSM 34195. 7 CFU / g and 3x10 7 In other bottles, L. rhamnosus DSM 33515 was inoculated at a total concentration of 3 x 10 7 The inoculation was performed at CFU / g. Bottles inoculated with the starter culture alone served as controls. All bottles were incubated in a water bath at 43 ± 1°C and fermented until a pH of 4.55 ± 0.1 was reached. After fermentation, the bottles were vigorously agitated to break up the coagulum and immediately cooled in a cooling room.

[0081] For the mold test, 200 ml of fermented milk was heated to 40°C and 40 ml of a 5% sterile agar solution that had been melted and cooled to 60°C was added. The fermented milk and agar solution was poured into sterile Petri dishes and allowed to dry on an LAF bench for 30 minutes. Spore suspensions of three mold species were spotted onto the agar medium at a concentration of 500 spores per spot: P. brevicompactum DSM 32094, P. crustosum DSM 33517, and P. solitum DSM 32093. Each of the three mold species was spotted onto a single plate, and the target contaminant was added at a concentration of 500 spores per spot. The plates were incubated at 22 ± 1°C for 11 days and periodically observed for mold growth.

[0082] For the yeast test, fermented milk samples were inoculated with the target contaminant, D. hansenii CHCC16374, at 50 cfu / g. The inoculated samples were stored at 7°C for 27 days, and yeast growth was assessed as total yeast counts six times during the storage period.

[0083] The results of the agar assay are shown in Figure 6 and show that all tested molds grew very well on agar plates made from milk fermented with starter cultures alone, as well as on milk base (reference). However, when L. rhamnosus DSM 34195 was added during milk fermentation, the resulting medium inhibited the growth of the three Penicillium species tested, with clear inhibitory effects observed in the starter culture, milk base, and at all doses. At 3x10 L. rhamnosus DSM 34195 and L. rhamnosus DSM 33515, the growth of the three Penicillium species was significantly inhibited. 7 When coexisted at CFU / g, the inhibitory effect of L. rhamnosus DSM 34195 was equal to or greater than that of L. rhamnosus DSM 33515.

[0084] Figures 7 and 8 show the results of two starter cultures, YF-L812 or YoFlex Advance 2.0, and 3x10 7 CFU / g of L. rhamnosus DSM 33515 or 1x10 7 CFU / g and 3x10 7Growth curves of D. hansenii in fermented products with and without 10% sugar using L. rhamnosus DSM 34195 added at CFU / g are shown. L. rhamnosus DSM 34195 consistently exhibited high inhibitory effects on D. hansenii growth at all inoculation levels in combination with two starter cultures and two milk bases. The inhibitory effects were less consistent with L. rhamnosus DSM 33515, especially in fermented products using YF-L812 as the starter culture (7A).

[0085] 4.2 Combination with different starter cultures Improved antifungal and antiyeast effects were also observed in the production of fermented milk products using different starter cultures. A milk base consisting of 2.8% protein, 1.2% fat, and 10% sucrose was used. The milk base was homogenized, heated at 95 ± 1°C for 5 minutes, and then poured into 3-liter buckets and immediately cooled. At the start of fermentation, the milk buckets were inoculated with 500 U per 2500 liters of commercial starter cultures containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (F-DVS YoFlex Mild 1.0 or YoFlex Premium 1.0, Chr. Hansen A / S, Denmark). L. rhamnosus DSM 34195 or L. rhamnosus DSM 33515 were added at a total concentration of 1 x 10, respectively. 7 CFU / g and 3x10 7 The samples were inoculated at CFU / g. Two buckets served as controls and were inoculated with only one of the starter cultures. All buckets were incubated in a water bath at 43 ± 1°C and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, the buckets were vigorously agitated to break up the coagulum, cooled to 25 ± 1°C, dispensed into 250 ml cups, and immediately refrigerated at 5–7°C.

[0086] For mold testing, 200 ml of fermented milk was warmed to 40°C and 40 ml of a 5% sterile agar solution that had been melted and cooled to 60°C was added. This solution of fermented milk and agar was then poured into sterile Petri dishes, and the plates were allowed to dry on the LAF bench for 30 minutes. Agar plates were spotted with a spore suspension of three mold species: P. carneum DSM 33520, P. paneum DSM 33519, and P. roqueforti DSM 33518, at a concentration of 500 spores / spot. Each of the three mold species was spotted on a single plate, and the target contaminant was added at a concentration of 500 spores / spot. Plates were incubated at 7 ± 1°C for 34 days and periodically inspected for mold growth.

[0087] For the yeast challenge test, fermented milk samples were inoculated with the target contaminant, Debaryomyces hansenii CHCC16374 or Torulaspora delbrueckii CHCC17090 (Chr. Hansen Culture Collection), at 50 cfu / g. The inoculated samples were stored at 7°C for 28 days, and yeast growth was assessed as total yeast counts five times during the storage period.

[0088] The results of the agar tests are shown in Figure 9, which shows that all tested molds grew very well on agar media made from milk fermented with starter cultures alone (reference). However, when L. rhamnosus DSM 34195 was added during milk fermentation, the resulting medium inhibited the growth of the three tested Penicillium species, and this inhibitory effect was clearly observed. 7 The efficacy of L. rhamnosus DSM 34195 inoculated at CFU / g was 3x10 7 was even higher than that of L. rhamnosus DSM 33515 inoculated at CFU / g.

[0089] Figure 10 shows the growth curves of D. hansenii (10A) and T. delbreuckii (10B) stored at 7°C for 28 days. The growth of the two target contaminants was most significantly inhibited by L. rhamnosus DSM 34195 compared to L. rhamnosus DSM 33515. This was due to the 3x10 7 CFU / g compared to 1x10 7 The inhibitory effect was also significant at inoculation levels of CFU / g. Higher inhibitory effects were observed when F-DVS YoFlex Mild 1.0 or YoFlex Premium 1.0 were used as starter cultures.

[0090] Conclusion: L. rhamnosus DSM 34195 exhibits higher and more consistent anti-yeast and anti-mold efficacy than L. rhamnosus DSM 33515 when combined with different starter cultures and used in different milk bases.

[0091] Example 5: Low post-acidification of L. rhamnosus DSM 34195 in fermented milk products This example compares L. rhamnosus DSM 34195 and L. rhamnosus DSM 33515 to demonstrate the effect of post-acidification. In this experiment, a homogenized milk base containing 2.8% protein, 1.2% fat, and 10% sucrose was heat-treated at 95±1°C for 5 minutes and immediately cooled. A commercial starter culture (F-DVS YF-L812 or F-DVS YoFlex Advance 2.0) was inoculated at a rate of 500 U per 2500 L, and the inoculated milk was dispensed into 3 L buckets. One bucket contained L. rhamnosus DSM 33515 at a total concentration of 3x10 7 CFU / g, and the other bucket was inoculated with L. rhamnosus DSM 34195 at a total concentration of 1x10 7One bucket was inoculated with 100 CFU / g of fermented yeast, and the other bucket was inoculated with the starter culture alone as a control. All buckets were incubated in a water bath at 43±1°C and fermented under these conditions until the pH reached 4.60±0.1. After fermentation, the buckets were vigorously agitated to break up the coagulum, cooled to 25±1°C in cold water, dispensed into 200ml bottles, and immediately refrigerated at 5-7°C.

[0092] To monitor the effect on post-acidification, 2 × 3 fermented milk samples (starter only, starter + DSM 34915, starter + DSM 33515) were stored at 25 ± 1°C for 28 days and pH was measured on days 1, 7, 14, 21 and 28.

[0093] Figure 11 shows the effect on post-acidification when combined with F-DVS YF-L812 (11A) or F-DVS YoFlex Advance 2.0 (11B). The addition of L. rhamnosus DSM 33515 enhanced post-acidification more than L. rhamnosus DSM 34195.

[0094] Example 6: Antifungal effect and lower post-acidification rate of L. rhamnosus DSM 34195 compared to L. rhamnosus DSM 33515 on Crema acida The inhibitory effect of L. rhamnosus DSM 34195 on molds was demonstrated using semi-quantitative agar cultures, while its inhibitory effect on yeasts was verified using challenge tests tracking the growth (Log(CFU / g)) of various target contaminants in fermented milk samples (Crema acida).

[0095] A homogenized milk base consisting of 2.8% protein, 14-31% fat, and 4% carbohydrate was heat-treated at 85±1°C for 15 seconds and immediately cooled after homogenization. The resulting crema acida was chemically acidified with citric acid solution to adjust the pH to 4.6 and then dispensed into 200ml bottles. Some bottles contained L. rhamnosus DSM 34195 at a total concentration of 1x10 7 The other bottles were inoculated with L. rhamnosus DSM 33515 at a total concentration of 3 x 10 CFU / g. 7The bottles were inoculated at CFU / g. Bottles without L. rhamnosus were used as controls. After inoculation, all bottles were stored at 7°C.

[0096] For the yeast test, fermented milk samples were inoculated with the target contaminant, D. hansenii CHCC16374, at 50 cfu / g. The inoculated samples were stored at 7°C for 24 days, and yeast growth was assessed as total yeast counts five times during the storage period.

[0097] For the mold test, 200 ml of fermented milk was heated to 40°C, 40 ml of 5% sterile agar solution was dissolved in it, and the mixture was cooled to 60°C. The fermented milk and agar solution were poured into sterile 6-well plates and allowed to dry on an LAF bench for 30 minutes. Spore suspensions of the following six mold species were spotted onto the plates at a concentration of 500 spores per spot (one mold per well, as shown in Figure 12): P. brevicompactum DSM 32094, P. crustosum DSM 33517, P. solitum DSM 32093, P. carneum DSM 33520, P. panum DSM 33519, and P. roqueforti DSM 33518. The plates were incubated at 12 ± 1°C for 15 days and at 7 ± 1°C for 22 days, and periodically observed for mold growth. Plates were incubated at 22 ± 1°C for 7 days and inoculated with two mold species: P. crustosum DSM 33517 and P. roqueforti DSM 33518.

[0098] The results of the agar tests are shown in Figures 12-14, and show that all the tested molds grew very well on agar plates made from Crema acida without the addition of L. rhamnosus (reference). However, the addition of L. rhamnosus DSM 34195 inhibited the growth of the six Penicillium species tested when incubated at 7°C (Figure 12) or 12°C (Figure 13), and also inhibited the growth of two mold species when incubated at 22°C (Figure 14).

[0099] The antifungal activity was confirmed in a yeast challenge test, and the growth curve (Log(CFU / g)) is shown in Figure 15. The fermented product was inoculated with 3x10 L. rhamnosus DSM 33515.7 Growth inhibition of D. hansenii was observed when inoculated at 1x10 CFU / g, whereas L. rhamnosus DSM 34195 was inoculated at 1x10 7 Even higher levels of inhibition were observed when inoculated at CFU / g.

[0100] Post-acidification was monitored by measuring the pH of samples stored for 28 days at 7°C, 12°C, and 25°C. pH was measured on days 1, 7, 14, 21, and 28. Figures 14A-C show that post-acidification of L. rhamnosus DSM 34195 was lower than that of L. rhamnosus DSM 33515 at the end of the storage period at 7°C (Figure 16), 12°C (Figure 17), and 25°C (Figure 18).

[0101] Conclusion: The results show that DSM 34195 has a higher anti-yeast and anti-mold effect and lower post-acidification than DSM 33515 in Crema acida, even when applied at lower doses.

Claims

1. 1. A bacterium of the species Lacticaseibacillus rhamnosus deposited under DSM 34195, or a variant thereof, wherein the variant is obtained by using DSM 34195 as a starting material, and is different from DSM 33515 and DSM 23035, and has retained or improved properties compared to DSM 34195, and has improved properties compared to DSM 33515, characterized in that the properties are antifungal effect or activity and reduced post-acidification.

2. A composition comprising the bacterium of the species Lacticaceus rhamnosus deposited under DSM 34195 or a mutant thereof according to claim 1.

3. 3. The composition of claim 2, wherein the composition comprises a cryoprotectant, a cryoprotectant, an antioxidant, a nutrient, a filler, a flavoring, or a mixture thereof; preferably, the nutrient is a sugar, an amino acid, a fatty acid, a mineral, a trace element, a vitamin; preferably, the filler is lactose, maltodextrin.

4. The bacterium or mutant thereof is ９ A concentration of colony forming units (CFU) / g, or at least 10 １０ CFU / g or at least 10 １１ 4. The composition of claim 2 or 3, wherein the concentration is CFU / g.

5. The composition of any one of claims 2 to 4, wherein the composition is frozen or lyophilized.

6. The composition of any one of claims 2 to 5, wherein the composition further comprises a starter culture.

7. 7. The composition of claim 6, wherein the starter culture comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

8. A method for producing a fermented dairy product, comprising adding the bacterium of the species Lacticaceus rhamnosus or a mutant thereof according to claim 1, or the composition according to any one of claims 2 to 7, to milk or a dairy product, and fermenting the mixture at a temperature of from about 22°C to about 43°C until the pH reaches 4.6 or less.

9. A fermented milk product comprising the bacterium of the species Lacticaceus rhamnosus deposited under DSM 34195 or a mutant thereof according to claim 1.

10. 10. Fermented milk product according to claim 9, wherein the product is cheese or yogurt or crema acida or sour cream.

11. 11. Fermented milk product according to claim 9 or 10, wherein the fermented milk product is obtainable by the method according to claim 8.

12. The bacterium of the species Lacticaceus rhamnosus or a mutant thereof is at least 10 ７ The fermented milk product according to any one of claims 9 to 11, which is present at a concentration of CFU / g.

13. Fermented milk product according to any one of claims 9 to 12, wherein the product is stored at a temperature above 7°C.

14. A food, feed or pharmaceutical comprising the Lacticaceus rhamnosus species deposited as DSM 34195 according to claim 1 or a mutant thereof, or the composition according to any one of claims 2 to 7.

15. 15. The food, feed or pharmaceutical product of claim 14, wherein the product is obtainable by the method of claim 8.

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