Protective culture combination

Abstract

The present invention relates to novel strains of Lactiplantibacillus plantarum and preparations and compositions, such as fermentation broths, protective cultures, finished food or feed products, that contain strains of the species Lacticaseibacillus rhamnosus in combination with strains of the species Lactiplantibacillus plantarum. The present invention further relates to methods for controlling the growth of contaminants, such as yeasts or molds, by using these novel strains and combinations.

Description

[Technical field]

[0001] The present invention relates to novel strains of Lactiplantibacillus plantarum and preparations and compositions, such as fermentation broths, protective cultures, finished food or feed products, that contain strains of the species Lacticaseibacillus rhamnosus in combination with strains of the species Lactiplantibacillus plantarum. The present invention further relates to methods for controlling the growth of contaminants, such as yeasts or molds, by using these novel strains and combinations. [Background technology]

[0002] Yeasts and molds play a major role in the spoilage of various types of dairy products such as yogurt, sour cream, and fresh and ripened cheeses, and can therefore lead to significant economic losses. Chemical preservatives such as organic acids and their salts (e.g. sorbates and propionates) are used to preserve and extend the shelf life of dairy products. The drawbacks of using chemical preservatives are the possible adverse effects on labelling requirements (often as E numbers) and organoleptic properties of the foodstuffs.

[0003] Lactic acid bacteria are known to produce a variety of antimicrobial compounds, such as organic acids, hydrogen peroxide, diacetyl and bacteriocins. Lactic acid bacteria and products produced by lactic acid bacteria have been suggested as alternative types of additives to improve the keeping quality of food and animal feed. Summary of the Invention [Means for solving the problem]

[0004] Although bioprotective cultures that inhibit yeasts and molds are commercially available and are used in fresh dairy products, various types of cheese and plant-based milk substitutes to preserve the shelf life of the products, there is a demand by the market for bioprotective cultures with improved antifungal activity. [Brief description of the drawings]

[0005] [Figure 1] Figure 1 shows the levels of yeasts (Candida kefir and Candida lusitaniae) in yoghurts produced with and without antifungal cultures stored at 5°C without protective culture (◆), Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 (■). [Diagram 2] Figure 2 shows the levels of yeasts (Candida kefir and Candida lusitaniae) in yoghurts produced with and without antifungal cultures stored at 10°C. No protective culture (◆), Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 (■). [Diagram 3] Figure 3 shows the shelf life of yogurt produced with and without antifungal cultures without visible mold growth stored at 5° C. Samples were inoculated with a pool of three mold strains (Penicillium crustosum, Penicillium commune and Penicillium citrinum). [Figure 4]Figure 4 shows the shelf life of yogurt produced with and without antifungal cultures without visible mold growth stored at 10° C. Samples were inoculated with a pool of three mold strains (Penicillium crustosum, Penicillium commune and Penicillium citrinum). [Diagram 5] Figure 5 shows the levels of yeasts (Candida kefir and Candida lusitaniae) in sour creams produced with and without antifungal cultures stored at 10°C. No protective culture (◆), Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 (■). [Figure 6] FIG. 6 shows the levels of yeast (Kluyveromyces marxianus) in sour creams produced with and without antifungal cultures stored at 10°C. No protective culture (◆), Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 (■). [Figure 7] Figure 7 shows the shelf life of sour cream produced with and without antifungal cultures without visible mold growth stored at 5° C. Samples were inoculated with a pool of three mold strains (Penicillium crustosum, Penicillium commune and Penicillium citrinum). [Figure 8]Figure 8 shows the shelf life of sour cream produced with and without antifungal cultures without visible mold growth stored at 10° C. Samples were inoculated with a pool of three mold strains (Penicillium crustosum, Penicillium commune and Penicillium citrinum). [Figure 9] Figure 9 shows the antifungal activity against Geotrichum silvicola. Inhibitory effect of the combination of Lactiplantibacillus plantarum DSM34014 and Lacticaseibacillus rhamnosus DSM33650 against the growth of Geotrichum silvicola 1730-J at storage temperatures of 6°C and 12°C. [Figure 10] A: Inhibitory activity of the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 against Geotrichum silvicola 1730-J compared to the standard, incubated at 6°C for 28 days; B: Inhibitory activity of the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 against Geotrichum silvicola 1730-J compared to the standard, incubated at 12°C for 13 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] It is an object of embodiments of the present invention to provide preparations of bacterial strains with improved antibacterial properties, such as antifungal activity against yeasts and / or molds.

[0007] These preparations of bacterial strains with improved antimicrobial properties allow for the preparation of clean label products that may stay fresh for longer periods while retaining good taste, which has the added benefit of reducing food waste.

[0008] The present invention relates in one broad aspect to a bacterial preparation combining at least two different strains, one of which is a strain of the species Lacticaseibacillus rhamnosus and the other of which is a strain of the species Lactiplantibacillus plantarum.

[0009] Thus, in a first aspect, the present invention relates to a bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum.

[0010] In a second aspect, the present invention relates to a bacterial strain of Lactiplantibacillus plantarum or a variant thereof deposited on September 8, 2021 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34014.

[0011] In a third aspect, the present invention relates to a food or drink comprising the bacterial preparation of the present invention.

[0012] In a further aspect, the present invention relates to the use of the bacterial preparation of the invention or the bacterial strain of the invention for inhibiting the growth of yeasts and / or molds.

[0013] In a further aspect, the present invention relates to a method for inhibiting the growth of yeasts and / or molds in food or drink, comprising adding the bacterial preparation of the invention or the bacterial strain of the invention to the food or drink.

[0014] In a further aspect, the present invention relates to the use of the bacterial preparation of the invention or the bacterial strain of the invention for preparing a food or drink.

[0015] In a further aspect, the present invention relates to a method for preparing a food or beverage comprising the step of adding the bacterial preparation of the invention or the bacterial strain of the invention to a food substrate in a process for preparing the food or beverage.

[0016] The inventors of the present invention have found that a particular combination of at least two different strains, namely a strain of the species Lactiplantibacillus plantarum and a strain of the species Lacticaseibacillus rhamnosus, provides a surprisingly good antimicrobial effect against undesirable yeast and mould contamination.

[0017] The bacterial preparation according to the invention contains one or more strains of the species Lacticaseibacillus rhamnosus. In some embodiments, the one or more Lacticaseibacillus rhamnosus is or comprises (a) the Lacticaseibacillus rhamnosus strain deposited at the DSMZ under accession number DSM33650 or a variant thereof.

[0018] The bacterial preparation according to the invention further comprises one or more Lactiplantibacillus plantarum strains. In some embodiments, the bacterial preparation according to the invention comprises Lactiplantibacillus plantarum deposited in the DSMZ under accession number DSM34014 or a variant thereof.

[0019] In some embodiments, the bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum is substantially free of any other bacterial species. In some embodiments, the bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum comprises or consists of two, three, four or five different strains of these two different species. In some embodiments, a bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum comprises or consists of one specific strain from each of these two different species. In some embodiments, a bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum comprises or consists of these two different species. In some embodiments, the bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum comprises one, two, three or four further different species in addition to the two different species.In some embodiments, the bacterial preparation consists essentially of a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum, such as one particular strain of the species Lacticaseibacillus rhamnosus in combination with one particular strain of the species Lactiplantibacillus plantarum.

[0020] In some embodiments, a bacterial preparation according to the invention is concentrated.

[0021] In some embodiments, the bacterial preparations according to the invention are lyophilized or frozen.

[0022] In some embodiments, the preparation according to the invention is a protected culture of Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum.

[0023] In some embodiments, the preparations according to the invention have a concentration of at least about 1×10 5 CFU / g or ml, at least about 1 x 10 6 CFU / g or ml, at least 10 7 CFU / g or ml, at least 5 x 10 7 CFU / g or ml, etc., at least 10 8 CFU / g or ml, etc., at least 10 9 CFU / g or ml, etc., at least 10 10 CFU / g or ml, at least 5 x 10 10 CFU / g or ml, etc., at least 10 11 CFU / g or ml, at least 5 x 10 11 CFU / g or ml, etc., at least 10 12CFU / g or ml, at least 5 x 10 12 CFU / g or ml, etc., at least 10 13 CFU / g or ml, at least 5 x 10 13 Contains live bacteria of Lacticaseibacillus rhamnosus present in amounts of composition such as CFU / g or ml.

[0024] In some embodiments, the preparations according to the invention have a concentration of at least about 1×10 5 CFU / g or ml, at least about 1 x 10 6 CFU / g or ml, at least 10 7 CFU / g or ml, at least 5 x 10 7 CFU / g or ml, etc., at least 10 8 CFU / g or ml, etc., at least 10 9 CFU / g or ml, etc., at least 10 10 CFU / g or ml, at least 5 x 10 10 CFU / g or ml, etc., at least 10 11 CFU / g or ml, at least 5 x 10 11 CFU / g or ml, etc., at least 10 12 CFU / g or ml, at least 5 x 10 12 CFU / g or ml, etc., at least 10 13 CFU / g or ml, at least 5 x 10 13 Contains live bacteria of Lactiplantibacillus plantarum present in amounts of composition such as CFU / g or ml.

[0025] In some embodiments, the composition according to the invention is a finished food or feed product, wherein said live bacteria of Lacticaseibacillus rhamnosus are at least 10 5 CFU / g or ml, at least 5 × 10 5 CFU / g or ml, etc., at least 10 6CFU / g or ml, at least 5 x 10 6 CFU / g or ml, etc., at least 10 7 CFU / g or ml, at least 5 x 10 7 CFU / g or ml, etc., at least 10 8 CFU / g or ml, at least 5 x 10 8 CFU / g or ml, etc., at least 10 9 CFU / g or ml, at least 5 x 10 9 CFU / g or ml, etc., at least 10 10 CFU / g or ml, at least 5 x 10 10 CFU / g or ml, etc., at least 10 11 CFU / g or ml, at least 5 x 10 11 CFU / g or ml, etc., at least 10 12 CFU / g or ml, at least 5 x 10 12 CFU / g or ml, etc., at least 10 13 CFU / g or ml, at least 5 x 10 13 It is present in an amount such as CFU / g or ml of composition.

[0026] In some embodiments, the composition according to the invention is a finished food or feed product, wherein said live bacteria of Lactiplantibacillus plantarum are at least 10 5 CFU / g or ml, at least 5 × 10 5 CFU / g or ml, etc., at least 10 6 CFU / g or ml, at least 5 x 10 6 CFU / g or ml, etc., at least 10 7 CFU / g or ml, at least 5 x 10 7 CFU / g or ml, etc., at least 10 8 CFU / g or ml, at least 5 x 10 8 CFU / g or ml, etc., at least 10 9 CFU / g or ml, at least 5 x 10 9 CFU / g or ml, etc., at least 1010 CFU / g or ml, at least 5 x 10 10 CFU / g or ml, etc., at least 10 11 CFU / g or ml, at least 5 x 10 11 CFU / g or ml, etc., at least 10 12 CFU / g or ml, at least 5 x 10 12 CFU / g or ml, etc., at least 10 13 CFU / g or ml, at least 5 x 10 13 It is present in an amount of CFU / g or ml of composition.

[0027] In an aspect, a food or drink obtained according to the method or use provided herein is provided. In some embodiments, the food or drink is a fermented food or drink. In some embodiments, the fermented food or drink is yogurt, yogurt drink, cheese, fresh cheese, cream cheese, cottage cheese, semi-soft or soft cheese, semi-hard or hard cheese, white cheese such as white brine cheese, sour milk product or sour cream, or fermented plant-derived milk substitute food or drink. In some embodiments, the food or drink is a plant-derived yogurt substitute, a plant-derived cream substitute, a plant-derived mature cream substitute, a plant-derived butter substitute, a plant-derived fat spread, a plant-derived cheese substitute, a plant-derived fromage frais substitute, a plant-derived dairy beverage substitute, a plant-derived processed cheese substitute, a plant-derived cream dessert substitute, a plant-derived cottage cheese substitute, or a plant-derived kefir substitute.

[0028] The term "food" is used in a broad sense and includes feed, foodstuffs, food ingredients, dietary supplements and functional foods. As used herein, the term "food" is used in a broad sense and includes food for humans and food for animals (i.e. feed or pet food). In a preferred embodiment, the food is for human consumption.

[0029] In some embodiments, the bacterial strains or bacterial compositions described herein, e.g., starter cultures, may be or be added to food ingredients, dietary supplements, or functional foods. As used herein, the term "functional food" refers to food that can not only provide nutritional benefits and / or taste satisfaction, but can also deliver additional beneficial effects to the consumer. Although there is no legal definition of functional food, most stakeholders interested in this area acknowledge that there are foods that are marketed as having certain health benefits.

[0030] When referring to strains used in accordance with the present invention, it is assumed that these strains are living. As used herein, the term "living" refers to bacterial cells that have the ability to survive, grow or multiply, and have an active metabolism.

[0031] As used herein, the term "protective culture of Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum" refers to a composition that contains live Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum of both species (either pure cultures or culture concentrates), which is not a finished food or drink suitable for consumption, but is added to food or drink for the purpose of reducing the risk from pathogenic or toxin-producing microorganisms. Compositions such as fermentation media and concentrated preparations thereof are included within this definition, where Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum have been or are grown in a suitable medium.

[0032] The European Food and Feed Cultures Association (EFFCA) has characterized protective cultures as follows: The term "protective culture" has been applied to microbial food cultures (MFCs) that exhibit metabolic activity that contributes to inhibiting or controlling the growth of undesirable microorganisms in food. These undesirable microorganisms can be pathogenic or toxin-producing bacteria and fungi, but may also include species that cause spoilage.

[0033] Protective cultures are considered an integral part of starter cultures, which are a traditional tool in food technology used to produce fermented foods such as cheese, yogurt, certain sausages, wine, etc. It is a general property of fermented foods that they have a longer shelf life than non-fermented raw materials (e.g. cheese has a much longer shelf life than milk). This property is the result of the active metabolism of the fermenting cultures, which perform their action through a complex system of competition for nutrients and binding sites and by the production of inhibitory metabolites such as organic acids, hydrogen peroxide, diacetyl, reuterin and bacteriocins.

[0034] Depending on the particular culture used, the culture usually also produces a number of properties of organoleptic and nutritional value to food and drink. Thus, the same starter culture species used in fermentation processes have also been applied to foods to take advantage of their "bioprotective" capabilities, with or without organoleptic impact. To these starter cultures the term protective cultures has been applied.

[0035] Their use is not limited to "typical" fermented foods, but also plays an important role when their metabolic activity occurs in foods with neutral pH and high water activity, which tend to increase the risk of growth of food pathogens. The application of "protective cultures" is a complementary measure to improve food hygiene and should not be allowed to compromise any standards of good manufacturing practice that ensure a high level of food safety.

[0036] Protective cultures are not additives but an integral part of starter cultures. It is clear that these cultures, like all starter cultures, express their protective beneficial capacity as a result of their metabolic activity in or on the food. (EFFCA, December 2011)

[0037] Protective cultures are added together with starter cultures to food or feed substrates prior to fermentation of the food or feed. Thus, the protective cultures are able to grow and / or are metabolically active through the fermentation step. Starter cultures are necessary for producing fermented foods such as yogurt, cheese and sour cream, and contribute to the desired product changes in taste, texture and flavour development. In addition to this, protective cultures are added to foods to limit the growth of pathogenic or spoilage bacteria, thus reducing the risk of food poisoning and preserving shelf life.

[0038] As used herein, the term "final food or feed product" refers to a composition in the form of a food or feed that is suitable for consumption, such as for human or animal consumption.

[0039] As used herein, the term "preparation" refers to any composition or portion thereof derived from a medium conditioned by growth of bacteria of the species Lacticaseibacillus rhamnosus or Lactiplantibacillus plantarum, or an extract thereof, including an extract containing live bacterial cells of the species Lacticaseibacillus rhamnosus or Lactiplantibacillus plantarum, or one or more non-living bacterial cell components, such as lipid, protein or nucleic acid components, derived from bacteria of the species Lacticaseibacillus rhamnosus or Lactiplantibacillus plantarum. Both liquid and dry powder preparations are included within this definition of a preparation in which the water has essentially been removed.

[0040] The term "contaminant" as used herein refers to any unwanted and unintentional growth of any microorganism, such as bacteria, yeast, or fungi such as mold. In some cases, contaminants may cause illness. However, in many cases, contaminants simply degrade and deteriorate the product, which may be perceived and / or give rise to an unpleasant and undesirable taste or mouthfeel.

[0041] In some embodiments, the contaminant is selected from the group consisting of Penicillium species, Penicillium brevicompactum, Penicillium crustosum, Penicillium chrysogenum, Penicillium commune, Penicillium citrinum, Penicillium solitum, Penicillium glabrum, Penicillium corylophilum, Penicillium roqueforti, Aspergillus species, Aspergillus ochraceus, Aspergillus ocracis ... Fungi in the order Eurotiales such as Aspergillus ochraceus, Aspergillus parasiticus, Aspergillus versicolor, Aspergillus niger, and Eurotium species; fungi in the family Polytrichum such as Fusarium species; Candida species, Candida colliculosa, Candida intermedia, Candida famata, Candida guilliermondii, Candida kefyr, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida lusitaniae, Candida sake, Candida sphaerica, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida zeylanoidesThe fungus or mold is selected from the list consisting of fungi of the family Saccharomycetaceae, such as Saccharomyces zeylanoides, Debaryomyces spp., Debaryomyces hansenii, Kluyveromyces spp., Kluyveromyces marxianus, Rhodotorula spp., Rhodotorula mucilaginosa, Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces selvajii, and Geotrichum spp., Geotrichum candidum, and mold species in the family Mucormycetaceae, such as Mucor spp. and Rhizopus spp.

[0042] In some embodiments, the contaminant is a bacterium different from the species Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum used in the method according to the invention, such as any selected from the list consisting of Enterobacteriaceae, such as Listeria monocytogenes, Pseudomonas spp., Staphylococcus aureus, Bacillus spp., Lactobacillus spp., Leuconostoc spp., Clostridium spp., Citrobacter spp., Enterobacter spp., Escherichia spp., Klebsiella spp., and Salmonella spp.

[0043] The species Lacticaseibacillus rhamnosus was formerly known as Lactobacillus rhamnosus. The species Lactiplantibacillus plantarum may also be called Lactobacillus plantarum and was formerly known as Lactobacillus arabinosus.

[0044] The following deposits have been or are being made pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: - Lacticaseibacillus rhamnosus strain deposited at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] on September 29, 2020 under accession number DSM33650. - Lactiplantibacillus plantarum is a strain deposited at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] on September 08, 2021 under the accession number DSM34014.

[0045] It is required that biological material shall be made available only by furnishing samples to experts designated by the claimant. In respect of the indication that a European patent is sought, samples of the deposited microorganisms shall be made available until the publication of a reference to the grant of a European patent or until the date on which the application is refused or withdrawn or considered withdrawn only by furnishing such samples to experts designated by the claimant requesting samples and recognized by i) the applicant and / or ii) the European Patent Office, whichever is applicable (Rule 32 EPC). EXAMPLES

[0046] Example 1 The antifungal activity of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 was evaluated against yeasts by agar assay. Reference samples without added protective culture and samples with well-known standard protective cultures were included for comparison.

[0047] Yogurt samples were prepared by fermenting full fat milk at 43°C with a starter culture consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (IFF, Denmark). Test samples were additionally inoculated with a commercial standard antifungal culture or a combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650. Yogurt samples were mixed with an agar solution and the mixture was poured into Petri dishes or 12-well culture plates and allowed to dry in a laminar flow bench.

[0048] The yeast strains used in the inhibition tests are shown in Table 1.

[0049] [Table 1]

[0050] Yeast strains were diluted and added to the agar solution. Yogurt samples in 12-well culture plates were covered with agar inoculated with selected yeast strains, aiming for an inoculation concentration of 30 CFU / well. Plates were incubated at 25° C. and yeast growth on samples supplemented with protective cultures was compared to growth on a reference plate without protective culture. Inhibition was judged as follows: No suppression- 0~25%+ 26~50%++ 51~75%+++ 76~100%++++

[0051] [Table 2]

[0052] The combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 showed stronger inhibition against 10 of the 14 tested yeast strains compared to the commercial standard protective culture as shown in Table 2. Five of the 14 strains were not inhibited by the commercial standard protective culture except by the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650. Thus, the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 showed a broader antifungal spectrum compared to the commercial standard protective culture under the conditions tested.

[0053] Example 2 Antifungal activity in yogurt The antifungal activity of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 was evaluated against yeasts and molds in yogurt. A reference sample without added protective culture was included for comparison. Yogurt samples were prepared using whole milk with 3.5% fat. Milk was inoculated with a commercially available thermophilic yogurt starter culture (IFF, Denmark). Test samples were additionally inoculated with a blend of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650. Fermentation was carried out at 43°C until the pH reached 4.60 in the samples. The yogurt was cooled, quickly poured into sterile beakers and inoculated with yeast or mould. Yogurt samples were stored at 5 and 10°C and yeast counts were made throughout the 37 and 13 day storage periods, respectively. Samples inoculated with mould were stored at both 5°C and 10°C and visually inspected for mould growth up to 42 days of storage.

[0054] As shown in Figure 1, the growth of pools of Candida kefyr and Candida lusitaniae was inhibited by a combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 at a storage temperature of 5°C, compared to a reference without antifungal culture. The yeasts grew to a level of 1.0E07 CFU / g, while the levels in samples with Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 were less than 1.0E04 CFU / g.

[0055] A delay in the growth of pools of Candida kefyr and Candida lusitaniae was also observed in samples prepared with Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 compared to the reference at a storage temperature of 10°C, as shown in Figure 2.

[0056] The combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 inhibited the growth of a pool of strains of the genus Penicillium compared to the reference at both 5°C and 10°C, as shown in Figures 3 and 4, respectively. While the first growth of mold in the reference occurs after 18 days at 5°C and 8 days at 10°C, in samples prepared with the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650, no growth was observed until 42 days, when the test was terminated.

[0057] Example 3 Antifungal activity in sour cream Sour cream samples were prepared using whole milk fat with 18% fat. Milk was inoculated with a commercially available mesophilic starter culture (IFF, Denmark). Test samples were further inoculated with a blend of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650. Fermentation was carried out at 28°C until the pH reached 4.60 in the samples. The sour cream was cooled, quickly poured into a sterile beaker and inoculated with yeast or mould. Sour cream samples were stored at 10°C and yeast counts were made throughout the 19-day storage period. Samples inoculated with mould were stored at both 5°C and 10°C and visually inspected for mould growth.

[0058] A combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 retarded the growth of pools of Candida kefir and Candida lusitaniae at a storage temperature of 10° C. compared to the reference, as shown in FIG. 5.

[0059] The growth of Kluyveromyces marxianus was retarded by the combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 at a storage temperature of 10°C compared to the reference, as shown in Figure 6.

[0060] Example 4 Antifungal activity against Geotrichum silvicola Yogurt samples were prepared using milk with 1.5% fat. The milk was inoculated with a commercially available thermophilic starter culture (IFF, Denmark). The test samples were further inoculated with a blend of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650. Fermentation was carried out at 43°C until the pH reached 4.70 in the samples, which were mixed in a 2:1 ratio with an agar solution with 2.5% strength adjusted to 45-50°C. The agar was filled into Petri dishes and left in a clean bench to solidify. Strain Geotrichum silvicola 1730-J was diluted to a spore count of 1,000 spores / ml and 20 μl aliquots of the mold spore solution were spotted onto plates (5 plates per sample). The plates were incubated at 12°C for 13 days and at 6°C for 28 days and the area of ​​grown mold colonies was measured. The inhibitory activity of the combination of Lactiplantibacillus plantarum DSM34014 and Lacticaseibacillus rhamnosus DSM33650 was calculated as follows:

number

[0061] In addition, I took a photo of the petri dish.

[0062] A combination of Lactiplantibacillus plantarum DSM 34014 and Lacticaseibacillus rhamnosus DSM 33650 retarded the growth of Geotrichum silvicola 1730-J at both 6°C and 12°C storage temperatures, as shown in Figures 9 and 10.

[0063]

change

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Claims

1. A bacterial preparation comprising a strain of the species Lacticaseibacillus rhamnosus in combination with a strain of the species Lactiplantibacillus plantarum.

2. 2. The bacterial preparation of claim 1, wherein the strain of the species Lactiplantibacillus plantarum is the strain deposited at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] on September 08, 2021 under the accession number DSM 34014 or a mutant thereof.

3. 3. The bacterial preparation according to claim 1 or 2, wherein the strain of the species Lacticaseibacillus rhamnosus is the strain deposited at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] on September 29, 2020 under the accession number DSM 33650 or a mutant thereof.

4. Each particular individual strain in the preparation is independently at least about 1×10 ５ CFU / g, at least about 1 x 10 ６ At least about 1 x 10 CFU / g ７ At least about 1 x 10 CFU / g ８ At least about 1 x 10 CFU / g ９ At least about 1 x 10 CFU / g １０ A bacterial preparation according to any one of claims 1 to 3, present in an amount such as CFU / g.

5. 5. The bacterial preparation according to any one of claims 1 to 4, wherein the at least two strains of the species Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum are present in a ratio between 1:20 to 20:1, such as in the range of 1:15 to 15:1, such as in the range of 1:10 to 10:1, such as in the range of 1:8 to 8:1, such as in the range of 1:6 to 6:1, such as in the range of 1:4 to 4:1, such as in the range of 1:2 to 2:1, such as in the range of 1:1.5 to 1.5:1, such as about 1:

1.

6. The bacterial preparation according to any one of claims 1 to 5, wherein the preparation is concentrated.

7. The bacterial preparation according to any one of claims 1 to 6, wherein the preparation is lyophilized or frozen.

8. A bacterial preparation according to any one of claims 1 to 7, wherein the bacterial preparation inhibits the growth of contaminating microorganisms such as yeasts and / or moulds.

9. A bacterial strain of Lactiplantibacillus plantarum or a mutant thereof deposited on September 8, 2021 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34014.

10. A food or drink comprising a bacterial preparation according to any one of claims 1 to 8 or a bacterial strain according to claim 9.

11. 11. The food or drink of claim 10, wherein the food or drink is a meat product, such as a fermented meat product or a meat substitute product or a fermented food or drink, optionally a fermented milk product or a fermented milk substitute product.

12. The food or beverage according to claim 10 or 11, wherein the food or beverage is yogurt, a yogurt drink, cheese, fresh cheese, cream cheese, cottage cheese, semi-soft or soft cheese, semi-hard or hard cheese, white cheese such as white brine cheese, sour milk products or sour cream.

13. The food or drink according to any one of claims 10 to 12, wherein the food or drink is preserved.

14. Use of a bacterial preparation according to any one of claims 1 to 8 or a bacterial strain according to claim 9 for inhibiting the growth of yeasts and / or molds.

15. A method for inhibiting the growth of yeasts and / or molds in food or drink, comprising adding a bacterial preparation according to any one of claims 1 to 8 or a bacterial strain according to claim 9 to the food or drink.

16. Use of a bacterial preparation according to any one of claims 1 to 8 or a bacterial strain according to claim 9 for preparing a food or drink.

17. 10. A method for preparing a food or beverage comprising the step of adding a bacterial preparation according to any one of claims 1 to 8 or a bacterial strain according to claim 9 to a food substrate in a process for preparing said food or beverage.

18. 20. The method of claim 17, further comprising adding a starter culture to the food substrate and fermenting the food substrate to produce a food or beverage, optionally wherein the fermenting is a thermophilic or mesophilic fermentation.

19. 19. The use according to claim 16 or the method for preparing a food or beverage according to claim 17 or claim 18, wherein the bacterial preparation inhibits the growth of yeast and / or mold in the food or beverage.

20. The food or drink is preserved. The method according to claim 15 for inhibiting the growth of yeast and / or mould in a food or drink, the use according to claim 16 or claim 19 or the method according to any one of claims 17 to 18 for preparing a food or drink.

21. 21. The method of claim 15 or claim 20, the use of any one of claims 16 or 19 or the method for preparing a food or drink according to any one of claims 17 to 18 for inhibiting the growth of yeast and / or mould in a food or drink, wherein the food or drink is a meat product or a meat substitute product such as a fermented meat product, a fermented food or drink, optionally a fermented milk product or a fermented milk substitute product.

22. 22. A method for inhibiting the growth of yeast and / or mould in a food or drink according to any one of claims 15, 20 to 21, the use according to any one of claims 16 or 19 or a method for preparing a food or drink according to any one of claims 17 to 18, wherein the food or drink is a meat product, a plant-based product or a meat substitute product, yoghurt, a yoghurt drink, cheese, fresh cheese, cream cheese, cottage cheese, semi-soft or soft cheese, semi-hard or hard cheese, white brine cheese, sour milk product or sour cream.

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