Bacterial compositions for controlling fungal spoilage and uses thereof

Abstract

For economic and environmental reasons, there is always a need for new or improved methods that are effective in controlling yeast and mold contamination.SOLUTION: The present invention relates to a direct vat set starter culture composition comprising lactic acid bacteria comprising a manganese transporter for fermenting a foodstuff and inhibiting or delaying the growth of fungi in said foodstuff, wherein said composition comprises a maximum 600ppm of manganese.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a highly concentrated starter culture composition and its preparation for controlling fungal spoilage without relying on preservatives, such as chemical preservatives. Thus, the present invention contributes to the demand for less processed, preservative-free foods while providing an effective solution for managing yeast and mold growth. The present invention also relates to food products comprising said composition. [Background technology]

[0002] A major problem in the food industry is spoilage caused by unwanted microorganisms. According to the Food and Agriculture Organization (FAO), one-quarter of calories intended for human consumption are not ultimately consumed by humans. In an era of food shortages, with over 800 million people suffering from hunger, the issue of food waste has become a priority for global policymakers and food manufacturers. In addition to the negative social and economic impacts on society, wasted food has many associated environmental impacts, such as unnecessary greenhouse gas emissions and inefficient use of scarce resources such as water and land.

[0003] Yeast and mold are highly efficient at spoiling food and are a problem for most food manufacturers. Spoilage caused by yeast and mold is clearly visible as mold spots or discoloration on the food's surface, allowing the food to be disposed of before consumption. Yeasts tend to grow within food and beverage matrices in the form of plankton, ferment sugars, and thrive under anaerobic conditions. In contrast, molds tend to grow on the surface of products in the form of visible hyphae composed of cells.

[0004] The use of antifungal microbial agents for food biopreservation is known, see, for example, Salas et al. “Antifungal microbial agents for food biopreservation—a review.” Microorganisms 5.3(2017):37.

[0005] In the dairy sector in particular, 29 million tonnes of dairy products are wasted every year in Europe. One of the main challenges in keeping dairy products fresh is managing contamination by ubiquitous naturally occurring yeasts and moulds, especially when the cold chain from production to the consumer's table is broken. Summary of the Invention [Problem to be solved by the invention]

[0006] For economic and environmental reasons, there is a constant need for new or improved methods that are effective in controlling yeast and mold contamination. [Means for solving the problem]

[0007] Manganese is essential for human health and has therefore been considered an essential trace element. Manganese is necessary for the function of many cellular enzymes, such as manganese superoxide dismutase and pyruvate carboxylase, and can help activate many other enzymes, such as kinases, decarboxylases, transferases, and hydrolases, making it vital for the proper functioning of both humans and animals.

[0008] Manganese can be found naturally in many food sources, including leafy green vegetables, nuts, grains, and animal products. Typical ranges of manganese concentrations in common foods include, for example, 0.4-40 ppm in grain products, 0.1-4 ppm in meat, poultry, fish, and eggs, 0.4-7 ppm in vegetable products, and 0.03 ppm in homogenized milk.

[0009] In addition to being a dietary supplement, manganese is sometimes added to fermented products as an active ingredient to enhance the growth of bifidobacteria in milk (see, for example, WO 2017 / 021754, Gervais-Danone, France).

[0010] Recently, it has been discovered that manganese is an important growth-limiting substance for fungal growth in food. Therefore, manganese-scavenging agents, such as manganese-scavenging bacteria, can be applied to food to compete with fungi for free manganese. This depletes this nutrient, resulting in the inhibition or delay of fungal growth. Such bacteria can be used as starter cultures for fermented foods. The present invention relates to the preparation of such antifungal bacteria for industrial use.

[0011] The present invention is based in part on the surprising discovery that when high levels of manganese are used in the preparation of starter cultures during upscaling processes, which is a common practice in the art, the bacteria are less effective at inhibiting or retarding fungal growth when later applied to food. Therefore, bacteria during the upscaling process should not be exposed to high levels of manganese, as such levels adversely affect their antifungal activity. In other words, the inventors have discovered that the manganese level contained in a bacterial composition is closely related to its antifungal activity.

[0012] The present invention relates to a concentrated biomass composition comprising one or more strains of lactic acid bacteria that inhibit the growth of yeast or mold. The biomass composition can be prepared by culturing the bacteria in a growth medium and concentrating the bacteria.

[0013] Commercial starter cultures are usually sold as frozen or freeze-dried (FD) cultures. High-concentration cultures are of great commercial interest because they can be inoculated directly into milk without an intermediate transfer. High-concentration cultures are sometimes called direct vat set (DVS) cultures.

[0014] Commercially available high-concentration DVS starter cultures are sometimes available in powder form as freeze-dried or lyophilized cultures. In this form, the starter can be shipped without refrigeration.

[0015] Lactic acid bacteria are typically supplied to the food industry, such as the dairy industry, as frozen or freeze-dried cultures for bulk starter growth or as so-called "direct vat set" (DVS) cultures intended for direct inoculation of fermentation vessels, without the need for bulk starter preparation, or as vats for the production of dairy products such as fermented milk products and cheese.

[0016] Direct vat set starter cultures are highly concentrated biomass (typically 10 10 ~10 12 cfu / g). Advantages include reduced risk of phage attack, flexibility of use, availability of mixed strain and species cultures, and no need for growth facilities. Freeze-dried cultures are typically stored at -18°C, while frozen cultures must be cooled on dry ice during transport and stored at -45°C.

[0017] A typical production process for starter cultures involves the following steps: (a) inoculum handling, (b) medium preparation, (c) culture growth in fermentors under pH control, (d) concentration, (e) freezing, (f) drying, and (g) packaging and storage. The steps in starter culture production are critical to achieving the desired identity, purity, and quality of the culture product. Cultures used as direct inoculum are prepared under sterile conditions, and transfer is minimized.

[0018] The growth medium for producing cultures may contain selected dairy ingredients, and various nutrients such as yeast extract, vitamins, and minerals may be supplemented to the growth medium. The culture growth medium is heated to an ultra-high temperature and cooled to 30°C or 40°C for mesophilic or thermophilic cultures, respectively. After inoculation of the culture, growth is optimized by maintaining the pH at 6.0-6.3 for mesophilic cultures and 5.5-6.0 for thermophilic cultures by adding alkali such as NaOH or NH4OH.

[0019] Processing parameters such as temperature, agitation rate, and headspace gas in the fermenter are adjusted to produce a cell suspension much more concentrated than that produced by bulk starter. After fermentation, which is typically a batch fermentation in vessels with a capacity of 10,000 to 40,000 L, the contents are cooled and the biomass is harvested, resulting in an additional 10- to 20-fold increase in cell concentration. A separator device is typically used to separate the aqueous liquid and collect the bacteria.

[0020] Higher yields of biomass are always needed for cost-effectiveness and efficiency. To use DVS, it is desirable to concentrate the bacterial culture as much as possible while simultaneously achieving the highest possible cell recovery.

[0021] To contain sufficient bacteria, commercially relevant high-concentration cultures generally have high levels of manganese. It is known in the art that manganese enhances bacterial growth, particularly lactic acid bacteria. This is an important economic consideration for starter culture manufacturers, for whom biomass yield is a major concern (Raccach, M. "Manganese and lactic acid bacteria." Journal of food protection 48.10 (1985):895-898).

[0022] EP 2119766 discloses that manganese can increase the growth yield of lactic acid bacteria.

[0023] European Patent No. 0130228 discloses that manganese salts can be used to accelerate the fermentation of lactic acid bacteria. Manganese has, for example, been conventionally added in the form of food-grade manganese salts in amounts suitable for enhancing cell growth.

[0024] The final enrichment culture often contains high levels of manganese. Examples of food-grade manganese salts that can be used include manganese chloride, manganese oxide, manganese sulfate, manganese citrate, manganese glycerophosphate, manganese gluconate, etc. Manganese salts can be added to the fermentation medium prior to or simultaneously with inoculation with the bacteria.

[0025] The present invention generally relates to a composition comprising lactic acid bacteria intended for addition to products such as food. The bacterial composition can be used as a starter culture composition for food. The composition may be added to food or may be added to ferment food and simultaneously control fungal growth. The composition is characterized by containing low or reduced levels of manganese. As used in this context, the term "starter culture" refers to a culture of one or more bacteria capable of acidifying food.

[0026] In a first aspect, the present application provides a starter culture composition comprising lactic acid bacteria and low levels of manganese (e.g., up to 600 ppm manganese). This composition can be used to capture free manganese otherwise available for yeast(s) or mold(s) in a product. The inventors have shown that common yeasts and molds are inhibited by the composition as disclosed.

[0027] Archibald et al. (1984) investigated the uptake of manganese by L. plantarum 14917 (Archibald et al. "Manganese acquisition by Lactobacillus plantarum." Journal of Bacteriology 158.1 (1984): 1-8). As disclosed, the high manganese content of L. plantarum 14917 was produced by a specific high-affinity and high-rate uptake system. However, this study did not relate to the inhibition of the growth of contaminating yeasts and / or molds in food. Also, no study was conducted on the preparation of highly concentrated direct vat set starter cultures, such as frozen direct vat set (F-DVS) or freeze-dried vat set (FD-DVS).

[0028] In one aspect, the present invention provides a direct vat set starter culture composition comprising one or more antifungal lactic acid bacteria for fermenting a food product and inhibiting or retarding fungal growth in said food product, wherein the composition comprises up to 600 ppm manganese and the concentration of the lactic acid bacteria is at least 1E+10 colony forming units / g.

[0029] In some embodiments, provided herein are frozen direct vat set (F-DVS) or frozen dry vat set (FD-DVS) starter culture compositions comprising lactic acid bacteria comprising a manganese transporter for fermenting a food product and inhibiting or retarding fungal growth in the food product, wherein the composition comprises up to 600 ppm manganese and the concentration of the lactic acid bacteria is at least 1E+10 colony forming units / g.

[0030] Preferably, the concentration of bacteria in the starter culture composition is at least 1E+10 colony forming units (CFU) / g.

[0031] To combat the problem of microbial spoilage, the present invention provides, in a further aspect, a bacterial starter composition for inhibiting or retarding fungal growth in food, the bacterial starter composition containing at least 1E+10 CFU / g, and the composition containing up to 600 ppm manganese. Preferably, the bacterial composition is a freeze-dried direct vat set (FD-DVS) or frozen direct vat set (F-DVS). The composition preferably contains one or more lactic acid bacteria strains.

[0032] Also provided herein is a starter culture composition for inhibiting or retarding fungal growth in dairy-based products, or a starter culture composition for fermenting a dairy-based food product and inhibiting or retarding fungal growth in said food product, wherein the composition comprises lactic acid bacteria, wherein the composition comprises up to 600 ppm manganese, and wherein the concentration of colony forming units / g of the lactic acid bacteria is at least 1E+10, and preferably wherein the lactic acid bacteria ferment a milk, wine, tea, plant and / or meat matrix.

[0033] The composition, preferably freeze-dried DVS (FD-DVS) or frozen DVS (F-DVS), may comprise up to 550 ppm manganese, up to 500 ppm manganese, up to 450 ppm manganese, up to 400 ppm manganese, up to 350 ppm manganese, up to 300 ppm manganese, up to 250 ppm manganese, up to 200 ppm manganese, up to 150 ppm manganese, up to 100 ppm manganese, up to 70 ppm manganese, up to 50 ppm manganese, up to 40 ppm manganese.

[0034] The composition may contain 10 to 600 ppm manganese, 30 to 600 ppm manganese, 35 to 600 ppm manganese, 40 to 600 ppm manganese, 45 to 600 ppm manganese, 50 to 600 ppm manganese, 60 to 550 ppm, 100 to 500 ppm manganese, 150 to 450 ppm manganese, 190 to 400 ppm manganese, 200 to 350 ppm manganese, or 250 to 300 ppm manganese. Preferably, the composition may contain 40 to 250 ppm manganese, and more preferably, the composition may contain 45 to 200 ppm manganese.

[0035] The composition may comprise lactic acid bacteria having colony forming units / g of bacterial cells of 1E+10 to 5E+12, preferably 2.0E+10 to 6.5E+11, more preferably 6.0E+10 to 6.4E+11, and even more preferably 1.3E+11 to 5.6E+11.

[0036] In a preferred embodiment, the presently disclosed compositions may be freeze-dried vat sets (FD-DVS) or frozen direct vat sets (F-DVS), and in particular, the compositions may be freeze-dried direct vat sets (FD-DVS) containing additives or cryoprotectants that are free or substantially free of manganese, or the compositions may be freeze-dried direct vat sets (F-DVS) containing additives or cryoprotectants that are free or substantially free of manganese. Preferably, the compositions are freeze-dried direct vat sets (FD-DVS) and may further comprise an additive (or cryoprotectant) selected from the group consisting of sodium caseinate, inositol, monosodium glutamate, sodium ascorbate, sucrose, maltodextrin, inosine monophosphate (IMP), inosine, polysorbate 80, glutamic acid, lysine, sodium glutamate, malt extract, whey powder, yeast extract, gluten, collagen, gelatin, elastin, keratin, albumin, carboxylate, or a mixture thereof. Preferably, the additive (or antifreeze) is manganese-free or substantially manganese-free.

[0037] The composition may further contain cryoprotectants and / or conventional additives, including nutrients such as yeast extract, sugars, and vitamins (e.g., vitamins A, C, D, K, or vitamins of the vitamin B family). Suitable cryoprotectants that may be added to the compositions of the present invention are ingredients that improve the cold tolerance of microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, and fructose. Other additives include, for example, carbohydrates, flavors, minerals, and enzymes (e.g., rennet, lactase, and / or phospholipase).

[0038] In one embodiment, the composition may be a frozen direct vat vat set (F-DVS) containing a manganese-free or substantially manganese-free additive or antifreeze at a concentration of 10-40 wt. % antifreeze per weight of F-DVS form, or 20-35 wt. % antifreeze per weight of F-DVS form, for example, 31 wt. % antifreeze per weight of F-DVS form.

[0039] In a preferred embodiment, the composition is a freeze-dry batch set (FD-DVS) containing a carbohydrate as an additive, and preferably, the additive (or cryoprotectant) does not contain manganese. Suitable examples include those selected from the group consisting of pentoses (e.g., ribose, xylose), hexoses (e.g., fructose, mannose, sorbitol), disaccharides (e.g., ducrose, drehalose, melibiose, lactulose), oligosaccharides (e.g., raffinose), oligofructose (e.g., actilite, fribrulose), polysaccharides (e.g., maltodextrin, xanthan gum, pectin, alginate, microcrystalline cellulose, dextran, polyethylene glycol, and sugar alcohols (sorbitol, mannitol)). Preferred carbohydrates are disaccharides, preferably trehalose, sucrose, and / or maltodextrin.

[0040] In a preferred embodiment, the composition, when in a frozen state, may comprise 2% to 70% additive (or antifreeze), measured as weight of additive per weight of DVS form, more preferably 3% to 50% additive (or antifreeze), measured as weight of additive per weight of DVS form, even more preferably 4% to 40% additive (or antifreeze), measured as weight of additive per weight of DVS form, and most preferably 10% to 35% additive (or antifreeze), measured as weight of additive per weight of DVS form. Preferably, the additive is manganese-free or substantially manganese-free.

[0041] In the context of the present invention, an additive or cryoprotectant is manganese-free or substantially manganese-free if manganese is present in the additive or cryoprotectant at a concentration of less than 10 ppm. Furthermore, in the context of the present invention, "additive," "cryoprotectant," or "cryoprotectant agent" are used interchangeably.

[0042] Addition of the additive (or cryoprotectant) to the isolated live bacteria (biomass) after fermentation may be carried out, for example, by mixing a solid cryoprotectant with the biomass at a suitable temperature for 30 minutes. If the cryoprotectant is, for example, sucrose, the suitable temperature may be room temperature. Alternatively, a sterile solution of the additive (or cryoprotectant) may be mixed with the biomass. In the case of sucrose, a suitable sterile solution may be made from a 50% (w / w) sucrose solution. In the case of trehalose, a suitable sterile solution may be made from a 40% (w / w) solution.

[0043] In a preferred embodiment, the composition is a freeze-dried DVS containing 10-600 ppm manganese, 30-600 ppm manganese, 35-600 ppm manganese, 40-600 ppm manganese, 45-600 ppm manganese, 50-600 ppm manganese, 60-550 ppm manganese, 100-500 ppm manganese, 150-450 ppm manganese, 190-400 ppm manganese, 200-350 ppm manganese, or 250-300 ppm manganese. Preferably, the composition is a freeze-dried DVS containing 40-250 ppm manganese, and more preferably, the composition is a freeze-dried DVS containing 45-200 ppm manganese.Furthermore, the preferred freeze-dried DVS composition may contain Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus alimentarius, Lactobacillus acidilactici, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salimentarius, Lactobacillus salivari ... Lactobacillus rhamnosus, and Lactobacillus kefiri, preferably Lactobacillus paracasei and / or Lactobacillus rhamnosus. rhamnosus), wherein the lactic acid bacteria have at least 1E+10 CFU / g, including 2E+10, 3E+10, 4E+10, 5E+10, 6E+10, 7E+10, 8E+10, 9E+10 CFU / g, for example, 2.0E+10 to 6.5E+11, preferably 6.0E+10 to 6.4E+11, more preferably 1.3E+11 to 5.6E+11; and / or the freeze-dried DVS has a manganese-free or substantially manganese-free additive or cryoprotectant at a concentration of 10 to 30% dry weight of cryoprotectant per weight of FD-DVS form or 20 to 30% dry weight of cryoprotectant per weight of FD-DVS form, for example, 27% dry weight of cryoprotectant per weight of FD-DVS form.

[0044] In a preferred embodiment, the present invention may provide a composition for inhibiting or retarding fungal growth in food, or a composition for inhibiting or retarding fungal growth in a milk-based product, or a composition for fermenting a milk-based food product to inhibit or retard fungal growth in said food, wherein the lactic acid bacterium comprises a manganese transporter having at least 55%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1 to 3.

[0045] In a preferred embodiment, the present invention may provide a composition for inhibiting or retarding fungal growth in food, or a composition for inhibiting or retarding fungal growth in a milk-based product, or a composition for fermenting a milk-based food product to inhibit or retard fungal growth in said food, wherein the lactic acid bacteria do not contain superoxide dismutase, preferably do not contain manganese superoxide dismutase.

[0046] Superoxide dismutases such as manganese superoxide dismutase have been studied and are described, for example, in Kehres et al., "Emerging themes in manganese transport, biochemistry, and pathogenesis in bacteria," FEMS microbiology reviews 27, 2-3 (2003): 263-290, Culotta VC, "Superoxide dismutase, oxidative stress, and cell metabolism," Curr. Top. Cell Regul. 36, 117-132 (2000), or Whittaker JW, "Manganese superoxide dismutase," Met. Ions Biol. Syst. 37, 587-611 (2000), among others.

[0047] In the context of the present invention, the term "does not contain" means that the genome of one or more strains does not present a gene encoding a superoxide dismutase, or, even if the genome of one or more strains presents a gene encoding a superoxide dismutase, this gene is not expressed by one or more strains.

[0048] In a preferred embodiment, the present invention relates to a composition for inhibiting or retarding fungal growth in food, or a composition for inhibiting or retarding fungal growth in a milk-based product, or a composition for fermenting a milk-based food product and thereby inhibiting or retarding fungal growth in said food, wherein the lactic acid bacteria are Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus alimentarius, Lactobacillus In some embodiments, the composition may be selected from the group consisting of Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, and Lactobacillus kefiri.

[0049] In a preferred embodiment, the present invention relates to a composition for inhibiting or retarding the growth of fungi in food, or a composition for inhibiting or retarding the growth of fungi in a milk-based product, or a composition for fermenting a milk-based food to inhibit or retard the growth of fungi in said food, wherein the fungi are yeasts and / or molds, preferably the fungi are yeasts selected from the group consisting of Torulaspora spp., Cryptococcus spp., Saccharomyces spp., Yarrowia spp., Debaryomyces spp., Candida spp., and Rhodoturola, and preferably the Debaromyces spp. is Debaromyces hansenii. hansenii, and / or the fungus is a mold selected from the group consisting of Aspergillus spp., Cladosporium spp., Didymella spp., or Penicillium spp., preferably the Penicillium spp. is Penicillium crustosum, Penicillium paneum, Penicillium carneum, or Penicillium roqueforti.

[0050] In a further aspect, the present invention also provides a food product comprising the composition disclosed herein. In one embodiment, the food product may be a fermented food product, preferably a thermophilic or mesophilic fermented food product, more preferably the food product may be yogurt or cheese.

[0051] In a third aspect, the present invention also provides the use of the composition disclosed herein as an inhibitor of fungal growth in food, preferably wherein the food is a fermented food, more preferably a thermophilic or mesophilic fermented food, more preferably yogurt or cheese. [Brief explanation of the drawings]

[0052] [Figure 1] FIG. 1 shows yogurt (1.5% fat) produced using a starter culture (reference), or using a starter culture and L. rhamnosus strain 1 (1E+7 CFU / g) in an F-DVS form, the F-DVS form having about 30 ppm manganese, or about 195 ppm manganese, or about 625 ppm manganese, or using a starter culture and L. paracasei and L. rhamnosus strain 3 in an FD-DVS form, the F-DVS form having about 275 ppm manganese. Yogurt was spiked with 500 spores each of Penicillium (P.) crustosum (X), P. carneum (Y), and P. roqueforti (Z) and stored at 22°C for 7 days.

[0053] [Figure 2] Figure 2 shows the growth of Torulaspora (T.) delbrueckii when 50 CFU / g of T. delbrueckii was used to inoculate yogurt (1.5% fat) produced with a starter culture (reference) or with a starter culture and the F-DVS form of L. rhamnosus strain 1 (1E+7 CFU / g), the F-DVS form having about 30 ppm manganese, about 195 ppm manganese, or about 625 ppm manganese. The yogurt was stored at 7°C for 23 days.

[0054] [Figure 3] Figure 3 shows the growth of D. hansenii when 50 CFU / g of D. hansenii was used to inoculate yogurt (1.5% fat) produced with a starter culture (reference) or with a starter culture and the F-DVS form of L. rhamnosus strain 1 (1E+7 CFU / g), the F-DVS form having about 30 ppm manganese, about 195 ppm manganese, or about 625 ppm manganese. The yogurt was stored at 7°C for 23 days.

[0055] [Figure 4] FIG. 4 shows the results of the FD-DVS produced using either a starter culture (reference), or a starter culture and L. rhamnosus strain 2 in F-DVS form, the F-DVS form having about 45 ppm manganese, or a starter culture and L. rhamnosus strains 1 and 2 in F-DVS form, the F-DVS form having about 65 ppm manganese, or a starter culture and FD-DVS having about 845 ppm manganese. Growth of D. hansenii when 50 CFU / g of D. hansenii was used to inoculate yogurt (1.5% fat) produced with benchmark composition A in VS form, or with benchmark composition B in FD-DVS form with a starter culture and about 630 ppm manganese, or with benchmark composition C in FD-DVS form with a starter culture and about 870 ppm manganese. The yogurts were stored at 7°C for 32 days.

[0056] [Figure 5]FIG. 5 shows yogurt (1.5% fat) produced using a starter culture (reference), or using a starter culture and L. rhamnosus strain 2 (1E+7 CFU / g) in F-DVS form, the F-DVS form having about 45 ppm manganese, or using L. rhamnosus strains 1 and 2 (1E+7 CFU / g) in F-DVS form, the F-DVS form having about 65 ppm manganese, or using benchmark composition A (1E+7 CFU / g) in FD-DVS form having about 845 ppm manganese, or using benchmark composition B (1E+7 CFU / g) in FD-DVS form having about 630 ppm manganese, or using benchmark composition C (1E+7 CFU / g) in FD-DVS form having about 870 ppm manganese. Yogurt was spiked with P. carneum, P. paneum, and P. roqueforti (500 spores each) and stored at 7°C for 24 days (top panel) or at 25°C for 6 days (bottom panel). The composition of Penicillium species on the plates is the same as in Figure 1, except that P. paneum was used instead of P. carneum.

[0057] [Figure 6]Figure 6 shows the growth of D. hansenii when 50 CFU / g of D. hansenii was used to inoculate yogurt (1.5% fat) produced using a starter culture (reference), or a starter culture and the FD-DVS form of L. paracasei and L. rhamnosus strain 3, with the FD-DVS form having approximately 275 ppm manganese, or a starter culture and the FD-DVS form of L. rhamnosus strain 2 (1E+7 CFU / g), with the FD-DVS form having approximately 200 ppm manganese, with or without the addition of skim milk powder (SMP) to the cryoprotectant (standard cryoprotectant) used to obtain the FD-DVS form. The yogurt was stored at 7°C for 27 days.

[0058] [Figure 7] Figure 7 shows yogurt (1.5% fat) produced using a starter culture and the FD-DVS form of L. rhamnosus strain 2 (1E+7 CFU / g), with the FD-DVS form having approximately 200 ppm manganese, and various concentrations of manganese (1, 5, 10, 20, and 40 ppm) added to the cryoprotectant used to obtain the FD-DVS form. The yogurt was spiked with P. crustosum, P. carneum, and P. roqueforti (500 spores each) and stored at 22°C for 12 days. The Penicillium species composition on the plate is the same as in Figure 1.

[0059] [Figure 8]Figure 8 shows the growth of D. hansenii when 50 CFU / g of D. hansenii was used to inoculate yogurt (1.5% fat) produced using a starter culture and the FD-DVS form of L. rhamnosus strain 2 (1E+7 CFU / g), the FD-DVS form having approximately 200 ppm manganese, and various concentrations of manganese (1 and 40 ppm) were added to the cryoprotectant used to obtain the FD-DVS form. The yogurt was stored at 7°C for 27 days. DETAILED DESCRIPTION OF THE INVENTION

[0060] Food waste is a major concern worldwide. Approximately one-third of all food produced for human consumption is thrown away (lost) or wasted. The reasons for this large-scale global food loss are varied, but microbial spoilage, which affects organoleptic product quality (appearance, texture, taste, and aroma), plays a major role. Fungi are the primary spoilage microorganisms found at all stages of the food processing chain because they can grow in a variety of, even harsh, environments. Therefore, it is important to reduce food waste by controlling fungal contamination.

[0061] In response to this need, the present invention provides a novel composition for inhibiting or retarding fungal growth in foods. Manganese is present in trace amounts in nature and in many consumer products. It has recently been discovered that low levels of free manganese can act as a limiting factor for yeast and / or mold growth. Therefore, manipulating the concentration of free manganese in a given product can effectively control microbial spoilage. This spoilage prevention strategy is applicable not only to foods, but also to other products commonly susceptible to microbial contamination, such as feed products, biological products, health care products, and pharmaceuticals.

[0062] Many bacteria have developed sophisticated acquisition systems for capturing essential metals from the environment. Therefore, manganese-capturing bacteria can be applied to incorporate free manganese into products. Furthermore, the present inventors have recently discovered that lower manganese levels are preferred when preparing bacteria for the upscaling process.

[0063] In a first aspect, the present invention provides a starter culture composition for inhibiting or retarding fungal growth in a milk-based food product, or a starter culture composition for fermenting a milk-based food product and inhibiting or retarding fungal growth in said food product, wherein the composition comprises lactic acid bacteria, the composition comprises up to 600 ppm manganese, and the concentration of lactic acid bacteria colony forming units / g is at least 1E+10, and preferably the lactic acid bacteria ferment a milk, wine, tea, plant and / or meat matrix.

[0064] Generally, inhibition refers to a partial or total reduction in the function and activity of a cell or microorganism. As used herein, the terms "to inhibit" and "inhibiting" with respect to yeast and mold mean that the growth, number, or concentration of yeast and mold remains the same or is reduced. This can be measured by any method known in the art of microbiology. Inhibition can be observed by comparing the growth, number, or concentration of fungi in or on a product with a control. The control can be the same product but does not contain the composition.

[0065] Generally, the term "delay" refers to the act of stopping, postponing, hindering, or causing something to occur more slowly than normal. As used herein, "delaying fungal growth" refers to the act of delaying fungal growth. This can be observed by comparing the time required for fungal growth to a given level between two products, one containing a composition as disclosed and the other not.

[0066] In some embodiments, "inhibiting or slowing fungal growth" refers to a delay of 7 days, e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days.

[0067] The term "antifungal" in the present application is to be understood as the ability to inhibit the retardation of fungal growth in food products such as dairy-based products.

[0068] As used herein, the term "food matrix" refers to the composition and structure of a food, which is based on the concept that nutrients are contained in a continuous medium.

[0069] As used herein, the term "meat matrix" refers to the composition and structure of meat, which is based on the concept that nutrients are contained in a continuous medium.

[0070] manganese

[0071] Manganese is involved in many important biological processes and is found ubiquitously in all organisms. Manganese can also contribute to protection against oxidative stress and catalytic detoxification of reactive oxygen species. Many bacteria express sophisticated acquisition systems to capture essential metals from the environment, using low- and high-affinity transport systems for chelated or free metals. Manganese taken up by bacteria can form large, non-dialyzable polyphosphate-protein aggregate complexes within proteins, reaching very high intracellular concentrations.

[0072] "Manganese" according to the present application refers to manganese present in the composition for inhibiting or retarding fungal growth in food. In the context of the present invention, "manganese" includes manganese found intracellularly and extracellularly.

[0073] As used herein, the term "strain" has its general meaning in the field of microbiology and refers to a genetic variant of a bacterium.

[0074] As used herein, manganese concentration or manganese level is expressed in parts per million ("ppm") calculated on a weight / weight basis. Concentrating manganese in a product or composition below a certain value means having manganese in the product or a portion thereof such that the weight concentration of manganese in the entire product or composition is below a given value. Methods for measuring trace elements such as manganese are known in the art and are described, for example, in Nielsen, S. Suzanne, ed. Food analysis. Vol. 86. Gaithersburg, MD: Aspen Publishers, 1998.

[0075] Methods for measuring low concentrations of manganese are well known to those skilled in the art, including atomic absorption spectroscopy, atomic emission spectroscopy, mass spectrometry, neutron activation analysis, and x-ray fluorescence analysis (see, e.g., Williams et al. "Toxicological profile for manganese." (2012)).

[0076] Preferably, the manganese concentration is measured according to standard procedures such as those described in European Standard EN 13805:2014, "Foodstuffs - Determination of trace elements - Pressure digestion", published by the European Organization for Standardization, or in ISO 11885:2007, "Water quality - Determination of selected elements by inductively coupled plasma optical emission spectrometry (ICP-OES)", published by the International Organization for Standardization.

[0077] The level of manganese present in the final composition, either the F-DVS or FD-DVS product, is one of the main parameters affecting the antifungal activity of the strain, with high levels of Mn conferring lower antifungal activity and low levels of Mn conferring higher antifungal activity.

[0078] Lactobacillus plantarum Cadmium and manganese uptake genes from Lactobacillus plantarum ATCC 14917 have been previously studied. Here, Hao et al. described two cadmium uptake systems in Lactobacillus plantarum ATCC 14917. One is the Mn 2+ one with low affinity, but unrelated to Mn depletion, and the other with high affinity, 2+ It is induced by depletion of Mn 2+ In the latter case, Mn 2+ and Cd 2+ are competitive inhibitors of each other, and Cd 2+ Its affinity for Mn 2+ (Hao et al. "Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum." Applied and Environmental Microbiology 65.11(1999):4746-4752, and Hao et al. "Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants." Applied and Environmental Microbiology 65.11(1999):4741-4745). These papers are unrelated to high-biomass cell cultures for direct inoculation. They do not teach or suggest the findings of the present application.

[0079] One skilled in the art can adjust the manganese level in the medium to obtain a final product containing the desired manganese level. For example, if the manganese level in the growth medium is low, the final composition will have a low level of manganese, as manganese is expected to be retained in the concentration process. On the other hand, if the manganese level in the growth medium is high, the final composition will have a correspondingly high level of manganese.

[0080] fungi

[0081] Fungi are members of the fungal kingdom. Fungal growth can be measured by various methods known to those skilled in the art. For example, fungal growth can be measured by colony density or size, cell number, mycelium mass change, spore production, mycelial growth, colony forming units (CFU), etc., depending on the type of fungus and the product to which the method is applied. Fungal growth can also be observed by measuring changes in nutrient or metabolite concentrations, such as carbon dioxide release and oxygen uptake.

[0082] The terms "inhibition of fungal growth" or "inhibiting fungal growth" refer to the inhibition of fungal cell proliferation.

[0083] The terms "fungal growth retardation" or "retarding fungal growth" refer to a slowing of fungal cell proliferation. This can be observed, for example, by measuring fungal growth and comparing it to a control. Such a control can be, for example, a product prepared without the presently disclosed composition. Methods for determining inhibition or retardation of fungal growth are known to those skilled in the art.

[0084] In one embodiment, the presently disclosed compositions inhibit the growth of yeasts such as Candida spp., Meyerozyma spp., Kluyveromyces spp., Pichia spp., Galactomyces spp., Trichosporon spp., Sporidiobolus spp., Torulaspora spp., Cryptococcus spp., Sacharomyces spp., Yarrowia spp., Debaryomyces spp., and Rhodoturola spp. Preferably, the fungus is a yeast selected from the group consisting of Torulaspora spp., Cryptococcus spp., Sacharomyces spp., Yarrowia spp., Debaryomyces spp., Candida spp., and Rhodoturola spp. More preferably, the fungus is a yeast selected from the group consisting of Torulaspora delbrueckii, Cryptococcus fragicola, Sacharomyces cerevisiae, Yarrowia lipolytica, Debaryomyces hansenii, and Rhodoturola mucilaginosa.

[0085] In one embodiment, the presently disclosed composition inhibits the growth of mold. Preferably, the fungus is selected from the group consisting of Aspergillus spp., Cladosporium spp., Didymella spp., or Penicillium spp. More preferably, the fungus is selected from the group consisting of Penicillium brevicompactum, Penicillium crustosum, Penicillium solitum, Penicillium carneum, Penicillium paneum, and Penicillium roqueforti.

[0086] Lactic acid bacteria (LAB)

[0087] "Lactic acid bacteria" refers to Gram-positive, microaerobic or anaerobic bacteria that ferment sugars to produce acids, with lactic acid being the primary acid produced. Foods typically have a pH of about 3.5 to about 6.5, e.g., about 4 to about 6, e.g., about 4.5 to about 5.5, e.g., about 5.

[0088] In a preferred embodiment, the presently disclosed compositions may include lactic acid bacteria having manganese transport systems that have been studied and described, for example, in Kehres et al., "Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria," FEMS microbiology reviews 27.2-3 (2003):263-290.

[0089] Lactic acid bacteria strains useful in the present application have manganese uptake activity. By routine experimentation, one skilled in the art can select bacteria with manganese uptake activity. Such bacteria can be, for example, bacteria with Mn uptake activity. 2+ It may contain transporters. 2+ Transporters may be ABC transporters (e.g., SitABCD and YfeABCD) or proton-dependent Nramp-related transport systems belonging to the families designated as TC#3.A.1.15 and TC#2.A.55 in the transporter classification system provided by the Transport Classification Database (M. Saier; University of California, San Diego, Saier MH, Reddy VS, Tamang DG, Vastermark A. (2014)). The TC system is a classification system for transport proteins similar to the Enzyme Commission (EC) system for enzyme classification. The Transporter Classification (TC) system is a nomenclature system for transport protein classification approved by the International Union of Biochemistry and Molecular Biology. The TCDB is freely accessible at http: / / www.tcdb.org and offers several different ways to access data, including step-by-step access to hierarchical classifications, direct searching by sequence or TC number, and full-text searches.

[0090] In a preferred embodiment, the lactic acid bacteria strain may comprise a protein or a functional variant thereof belonging to the family designated as TC#3.A.1.15 (manganese chelate uptake transporter (MZT) family) (i.e., TC#3.A.1.15.6, TC#3.A.1.15.8, TC#3.A.1.15.14).

[0091] ABC transporters are primarily active at higher pH, while proton-driven transporters may be more active under acidic conditions. Thus, in one embodiment, the presently disclosed compositions contain a family of transporters designated as TC#2.A.55 (metal ions (Mn 2+ The present invention may also include a strain containing a protein or a functional variant thereof belonging to the (iron) transporter (Nramp) family. More preferably, the transporter belongs to the subfamily designated TC#2.A.55.2 or TC#2.A.55.3.

[0092] For example, the compositions disclosed herein may contain a metal ion (Mn) designated as TC#2.A.55.3.1, TC#2.A.55.3.2, TC#2.A.55.3.2, TC#2.A.55.3.3, TC#2.A.55.3.4, TC#2.A.55.3.5, TC#2.A.55.3.6, TC#2.A.55.3.7, TC#2.A.55.3.8, or TC#2.A.55.3.9. 2+ The present invention may include lactic acid bacteria having an iron) transporter (Nramp) or a functional variant thereof, preferably TC#2.A.55.2.6 or a functional variant thereof.

[0093] The term "functional variant" refers to a protein variant that has substantially the same biological activity, i.e., manganese uptake activity.

[0094] As used herein, "variant" refers to a variant form of a protein that shares at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a particular nucleic acid or amino acid sequence of the protein.

[0095] The present disclosure further provides polypeptide sequences of manganese transporters that can be present in lactate for practicing the invention.

[0096] In a preferred embodiment, the lactic acid bacteria strain has the sequence of SEQ ID NO: 1 (MASEDKKSKREHIIHFEDTPSKSLDEVNGSVEVPHNAGFWKTLAAYTGPGILVAVGYMDPGNWITSIAGGASFKYSLLSVILISSLIAMLLQAMAARLGIVTGRDLAQMTRDHTSKAMGGFLWVITELAIMATDIAEIIGSAIALKLLFNMPLIVGIIITTADVLILLLLMRLGFRKIEAVVATLVLVILLVFAYEVILAQPNVPELLKGYLPHADIVTNKSMLYLSL GIVGATVMPHDLFLGSSISQTRKIDRTKHEEVKKAIKFSTIDSNLQLTMAFIVNSLLLILGAALFFGTSSSVGRFVDLFNALSNSQIVGAIASPMLSMLFAVALLASGQSSTITGTLAGQIIMEGFIHLKMPLWAQRLLTRLMSVTPVLIFAIYYHGNEAKIENLLTFSQVFLSIALPFAVIPLVLYTSDKKIMGEFANRAWVKWTAWFISGVLIILNLYLIAQTLGFVK) or a functional variant thereof.

[0097] In other embodiments, the lactic acid bacteria strain comprises a polypeptide having at least 55%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:1.

[0098] Table 1 shows exemplary sequences encoding functional variants of SEQ ID NO:1 and their sequence identity with SEQ ID NO:1. [Table 1-1] [Table 1-2]

[0099] In a preferred embodiment, the lactic acid bacteria strain has the sequence of SEQ ID NO: 2 (MARPDERLTVQREKRSLDDINRSVQVPSVYESSFFQKFLAYSGPGALVAVGYMDPGNWLTALEGGSRYHYALLSVLLMSILVAMFMQTLAIKLGVVARLDLAQAIAAFIPNWSRICLWLINEAAMMATDMTGVVGTAIALKLLFGLPLMWGMLLTIADVLVVLLFLRFGIRRIELIVLVSILTVGIIFGIEVARADPSIGGIAGGFVPHTDILTNHGMLLLSLG IMGATIMPHNIYLHSSLAQSRKYDEHIPAQVTEALRFGKWDSNVHLVAAFLINALLLILGAALFYGVGGHVTAFQGAYNGLKNPMIVGGLASPLMSTLFAFALLITGLISSIASTLAGQIVMEGYLNIRMPLWERRLLTRLVTLIPIMVIGFMIGFSEHNFEQVIVYAQVSLSIALPFTLFPLVALTNRRDLMGIHVNSQLVRWVGYFLTGVITVLNIQLAISVFV) or a functional variant thereof.

[0100] In other embodiments, the lactic acid bacteria strain comprises a polypeptide having at least 55%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:2.

[0101] Table 2 shows exemplary sequences that encode functional variants of SEQ ID NO:2 and their sequence identity with SEQ ID NO:2. [Table 2-1] [Table 2-2]

[0102] In a preferred embodiment, the lactic acid bacteria strain comprises a polypeptide having the sequence SEQ ID NO: 3 () or a functional variant thereof.

[0103] In other embodiments, the lactic acid bacteria strain comprises a polypeptide having at least 55%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:3.

[0104] Table 3 shows exemplary sequences encoding functional variants of SEQ ID NO:3 and their sequence identity with SEQ ID NO:3. [Table 3-1] [Table 3-2]

[0105] For purposes of the present invention, the degree of "sequence identity" between two amino acid sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 3.0.0 or later (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the nobrief option) is used as the percent identity, calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0106] For purposes of the present invention, the degree of "sequence identity" between two deoxyribonucleic acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleic acids × 100) / (length of alignment−total number of gaps in the alignment).

[0107] In one embodiment, the lactic acid bacteria strain comprises a manganese transporter having at least 55%, for example at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1 to 3. Determination can be based on sequencing the strain or a blast search in a database of known sequences.

[0108] The lactic acid bacteria used in the Examples section of the present disclosure have the encoded manganese transporters as SEQ ID NOs: 1 to 3 or functional variants thereof.

[0109] In a preferred embodiment, the lactic acid bacteria are Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus rhamnosus, and Lactobacillus kefiri.

[0110] Direct Bat Set (DVS)

[0111] In one aspect, the present invention provides a frozen direct vat set (F-DVS) starter culture composition comprising lactic acid bacteria for fermenting a food product and inhibiting or retarding fungal growth in said food product, wherein the composition comprises up to 600 ppm manganese and the concentration of the lactic acid bacteria is at least 1E+10 colony forming units / g.

[0112] In another embodiment, the present invention provides a freeze-dry batch set (FD-DVS) starter culture composition comprising lactic acid bacteria for fermenting a food product and inhibiting or retarding fungal growth in said food product, wherein the composition comprises up to 600 ppm manganese and the concentration of the lactic acid bacteria is at least 1E+10 colony forming units / g.

[0113] In some embodiments, the lactic acid bacteria are Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salimentarius, Lactobacillus salivari ... rhamnosus, and Lactobacillus kefiri. Preferably, the lactic acid bacterium is Lactobacillus paracasei or Lactobacillus rhamnosus.

[0114] Methods for preparing freeze-dried or frozen bacterial cultures are known in the art, for example as disclosed in US9848615.

[0115] Commercially relevant high-concentration frozen cultures generally have a bacterial concentration of at least 1E+9 CFU / g. In preferred embodiments, the bacteria in the compositions according to the invention have a concentration of at least 1E+10 CFU / g, including 2E+10, 3E+10, 4E+10, 5E+10, 6E+10, 7E+10, 8E+10, 9E+10 CFU / g. In other embodiments, the bacteria in the compositions have a concentration of at least 1E+11 CFU / g, including 2E+10, 3E+10, 4E+10, 5E+10, 6E+10, 7E+10, 8E+10, 9E+10 CFU / g. Longer fermentation times may be required to achieve this.

[0116] product

[0117] In some embodiments, the product is a food product, a cosmetic, a health care product, or a pharmaceutical product. "Foodstuff" and "food" have the common meaning of these terms. "Food" refers to a food or feed product suitable for human or animal consumption. Food can be fresh or perishable food, as well as preserved or processed food. Food includes, but is not limited to, fruits and vegetables (including derived products), grains and grain-derived products, dairy products, meat, poultry, and seafood. More preferably, the food is a meat or dairy product, such as yogurt, quark, sour cream, or cheese. Food can also be a plant-based product or a ready-to-eat product, such as a salad.

[0118] In one embodiment, the compositions described herein can be added to non-fermented or fermented foods. Non-fermented products generally have a higher pH than fermented foods. Fermented foods are foods produced or preserved by the action of microorganisms. Fermentation refers to the conversion of carbohydrates into alcohol or acid by the action of microorganisms. Fermentation usually refers to the fermentation of sugars into alcohol using yeast. However, fermentation may also involve the conversion of lactose to lactic acid. For example, fermentation may be used to produce foods such as yogurt, cheese, salami, sauerkraut, kimchi, and pickles.

[0119] The present invention is particularly useful for inhibiting or retarding fungal growth in dairy products. Yeast and mold contamination is common in such products, limiting their shelf life. "Dairy products" include milk, as well as milk-derived products such as cream, ice cream, butter, cheese, and yogurt, as well as secondary products such as whey and casein, and any prepared food containing milk or milk components as a primary ingredient, such as formula. In one preferred embodiment, the dairy product is a fermented dairy product. The term "milk" is understood to mean the lacteal secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo, or camel. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein / fat solutions made from plant materials, such as soy milk.

[0120] In one embodiment, the food product is a product prepared by fermentation with thermophilic bacteria, i.e., a thermophilic fermented food. The term "thermophilic bacteria" refers to microorganisms that grow best at temperatures above 43°C. The most industrially useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. As used herein, the term "thermophilic fermentation" refers to fermentation at temperatures above about 35°C, e.g., from about 35°C to about 45°C. "Thermophilic fermented food" refers to a fermented food prepared by thermophilic fermentation of a thermophilic starter culture. Examples of such products include yogurt, sukir, labneh, lassi, ayran, and doo.

[0121] In one embodiment, the food product is a product prepared by fermentation with mesophilic bacteria, i.e., a mesophilic fermented food. The term "mesophilic bacteria" refers to microorganisms that grow best at moderate temperatures (15°C to 40°C). The most industrially useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" herein refers to fermentation at temperatures between about 22°C and about 35°C. A "mesophilic fermented food" refers to a fermented food prepared by mesophilic fermentation of a mesophilic starter culture. Such products include, for example, buttermilk, sour milk, cultured milk, smetana, sour cream, and fresh cheeses such as quark, quark, and cream cheese.

[0122] Preparation of fermented products

[0123] The compositions herein are particularly useful for inhibiting or retarding yeast and / or mold growth in thermophilic and mesophilic fermented dairy products, such as yogurt. The term "fermented dairy product" is a commonly defined term according to relevant official regulations, and standards are well known in the art. For example, a co-culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is used as a starter culture for yogurt, while Lactobacillus acidophilus is used to make acidophilus milk. Other mesophilic lactic acid bacteria are used to produce quark or fromage frais.

[0124] The term "fermented dairy product" refers to a food or feed product, the preparation of which involves the fermentation of a milk base by lactic acid bacteria. As used herein, "fermented dairy product" includes, but is not limited to, thermophilic fermented dairy products (e.g., yogurt) and mesophilic fermented dairy products (e.g., sour cream and buttermilk, as well as fermented whey, quark, and fromage frais). Fermented dairy products include continental-type cheeses, fresh cheeses, soft cheeses, cheddar, mascarpone, pasta filata, mozzarella, provolone, white brine cheese, pizza cheese, feta, brie, camembert, cottage cheese, Edam, Gouda, Tilsiter, Havarti or Emmental, Swiss cheese, and Maasdamer cheese.

[0125] The term "yogurt" has its ordinary meaning and is generally defined in accordance with relevant official regulations, standards well known in the art. The starter culture used to make yogurt contains at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus. Interestingly, manganese transporters are absent in L. delbrueckii subsp. bulgaricus and are expressed at low levels only in Streptococcus thermophilus (these two strains are found in yogurt starter cultures), making them particularly susceptible to fungal spoilage. Therefore, it is preferable to include other bacterial strains to capture free manganese present in the yogurt.

[0126] During food processing, chemical preservatives have traditionally been used to avoid fungal spoilage. However, in view of the strong societal demand for less processed and preservative-free foods, the present invention contributes to providing an effective solution for controlling yeast and mold growth by using a composition for fermenting food and inhibiting or retarding fungal growth in said food, comprising up to 600 ppm manganese and lactic acid bacteria with at least 2E+10 colony forming units / g of bacterial cells.

[0127] For the presently disclosed compositions, one of skill in the art can adjust various parameters such as pH, temperature, and amount of said composition to achieve the desired result, taking into account the examples provided in this disclosure, as well as food product characteristics such as water activity, nutrients, levels of naturally occurring manganese, shelf life, storage conditions, packaging, etc.

[0128] The presently disclosed compositions may be added before, at the beginning of, or during fermentation of a given product, and the product may be further packaged to further limit contact with yeast and mold, and / or stored at low temperatures (below 15°C) to help extend shelf life.

[0129] The presently disclosed compositions may be added before, at the beginning of, or during fermentation of a given fermented dairy product. To produce a fermented dairy product, the food substrate is milk-based. "Milk-based" is 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 a starter culture. "Milk" generally refers to the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffalo, or camels. The milk base can be obtained from any raw and / or processed dairy material, as well as from reconstituted milk powder. The milk base can also be plant-based, i.e., prepared from plant materials, such as soy milk. A milk base prepared from cow's milk or dairy components is preferred.

[0130] Dairy bases include, but are not limited to, solutions / suspensions of any milk or dairy-like product containing protein, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dry milk, etc.

[0131] The milk base can also be lactose reduced depending on the consumer's needs. Low-lactose milk can be produced according to any method known in the art, including hydrolysis of lactose to glucose and galactose by the enzyme lactase, or by nanofiltration, electrodialysis, ion exchange chromatography, and centrifugation.

[0132] Food-grade microorganisms are added to ferment the milk base.

[0133] After adding the starter culture, the presently disclosed composition, and exposing the milk base to suitable conditions, the fermentation process begins and continues for a period of time. Those skilled in the art know how to select suitable process conditions, such as temperature, oxygen, carbohydrate addition, amount and characteristics of microorganism(s), and the duration of the process. This process may take 3, 4, 5, 6, or more hours.

[0134] These conditions include setting the temperature appropriate for the particular starter culture strain. For example, if the starter culture contains mesophilic lactic acid bacteria, the temperature can be set to about 30°C, and if the culture contains a thermophilic lactic acid bacteria strain, the temperature is maintained in the range of about 35°C to 50°C, e.g., 40°C to 45°C. Setting the fermentation temperature also depends on the enzyme(s) added to the fermentation, which can be easily determined by one skilled in the art. In certain embodiments of the present invention, the fermentation temperature is 35°C to 45°C, preferably 37°C to 43°C, and more preferably 40°C to 43°C. In another embodiment, the fermentation temperature is 15°C to 35°C, preferably 20°C to 35°C, and more preferably 30°C to 35°C.

[0135] Fermentation can be terminated using any method known in the art. Generally, fermentation can be terminated by making the milk base unsuitable for the growth of the starter culture strain(s), depending on various process parameters. For example, it can be terminated by rapidly cooling the fermented milk product when the target pH is reached. It is known that acidification occurs during fermentation, leading to the formation of a three-dimensional network of casein clusters and chains. The term "target pH" refers to the pH at which the fermentation process is terminated. The target pH depends on the resulting fermented milk product and can be easily determined by one skilled in the art.

[0136] In certain embodiments of the invention, fermentation is carried out until a pH of at least 5.2 is reached, for example, until a pH of 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, or 3.7 is reached. Preferably, fermentation is carried out until a target pH of 4.0 to 5.0, more preferably 4.0 to 4.6, is reached. In a preferred embodiment, fermentation is carried out until a target pH of less than 4.6 is reached.

[0137] In a preferred embodiment, the fermented food is selected from the group consisting of quark, cream cheese, fromage frais, Greek yogurt, sukir, labneh, buttermilk, sour cream, sour milk, fermented milk, kefir, lassi, ayran, quark (twarog), doo, smetana, Yakult, and dahi.

[0138] In another embodiment, the fermented food product is a cheese such as continental-type cheese, fresh cheese, soft cheese, cheddar, mascarpone, pasta filata, mozzarella, provolone, white brine cheese, pizza cheese, feta, brie, camembert, cottage cheese, Edam, Gouda, Tilsitter, Havarti or Emmental, Swiss cheese, and Maasdam.

[0139] Other features and advantages of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the claims below) shall be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referencing each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated into the specification as if set forth individually herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Unless otherwise specified, all precise values ​​provided herein are representative of the corresponding approximations (e.g., all precise exemplary values ​​provided for a particular factor or measurement can be considered to provide the corresponding approximate measurement, modified by "about" where appropriate). The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the invention and does not impose limitations on the scope of the invention unless specifically recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0140] The invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein, as these embodiments are intended as illustrations of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and examples below. Such modifications are also intended to be included within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

[0141] Manganese determination

[0142] For each composition disclosed herein, manganese concentration was measured using inductively coupled plasma mass spectrometry (ICP-MS), particularly after microwave-assisted digestion. After growth, the cell culture was harvested and subjected to standard procedures to obtain either the F-DVS or FD-DVS form. Additionally, CFU / g was also measured. Those skilled in the art know how to perform inductively coupled plasma mass spectrometry and how to measure CFU / g.

[0143] The results obtained are shown in Table 4.

[0144] [Table 4]

[0145] Preparation of fermented milk samples

[0146] The fermented milk samples used in this disclosure were prepared as follows: Homogenized milk, specifically low-fat (1.5% w / v) homogenized milk, was heat-treated at 90±1°C for 20 minutes and immediately cooled. The manganese concentration already present in the homogenized milk was previously determined to be approximately 0.03 ppm. Commercially available starter cultures (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) were used to prepare the fermented milk samples. subsp. bulgaricus was inoculated into 3 L buckets or 200 ml bottles at 0.02% (v / w). The first bucket was inoculated with a composition containing an L. rhamnosus strain (Strain 1) and approximately 30 ppm manganese at a total concentration of 1E+7 CFU / g. The second bucket was inoculated with a composition containing an L. rhamnosus strain (Strain 1) and approximately 195 ppm manganese at a total concentration of 1E+7 CFU / g. The third bucket was inoculated with an L. rhamnosus strain (Strain 1) and approximately 625 ppm manganese. The first bucket was inoculated with a composition containing 1E+7 CFU / g of manganese at a total concentration of 1E+7 CFU / g. The fourth bucket was inoculated with a composition containing an L. rhamnosus strain (strain 3) and an L. paracasei strain and approximately 275 ppm manganese at a total concentration of 1E+7 CFU / g. The fourth bucket was used as a reference and was inoculated with only the starter culture. All buckets were incubated in a water bath at 43±1°C and fermented under these conditions until a pH of 4.60±0.1 was reached. The fermented milk product was divided into 200 mL bottles and cooled.

[0147] All portions of the fermented milk samples were warmed to a temperature of 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 then poured into sterile Petri dishes, and the plates were allowed to dry on the LAF bench for 30 minutes. These plates were used for mold challenge and yeast challenge, and growth was assessed by scoring.

[0148] Mold stress test

[0149] In this disclosure, mold challenge tests were performed as follows: various target contaminants (P. crustosum, P. roqueforti, and P. paneum or P. carneum) were added at a concentration of 500 spores per spot. Plates were incubated at selected temperatures and times and periodically inspected for mold growth.

[0150] Stress test using yeast

[0151] In the present disclosure, yeast challenge tests were performed as follows: The growth or growth scores of various target contaminants (T. delbrueckii, D. hansenii, C. fragiolas, Y. lipolytica) were tested by inoculating fermented milk samples, such as yogurt, with approximately 50 CFU / g of each target contaminant.

[0152] Example 1: Inhibition of mold in fermented dairy products containing compositions containing lactic acid bacteria and various manganese concentrations

[0153] This example demonstrates the influence of manganese on the inhibitory effects of various molds. An agar assay similar to the manufacturing process and production of fermented dairy products was used. Lactobacillus rhamnosus and Lactobacillus paracasei strains were used.

[0154] Figure 1 shows the growth of three different molds (P. crustosum, P. carneum, and P. roqueforti) on plates prepared from milk fermented with a starter culture (reference) or with a composition further comprising lactic acid bacteria and various manganese concentrations, i.e., lactic acid bacteria and about 30 ppm manganese, about 195 ppm manganese, about 275 ppm manganese, or about 625 ppm manganese. The three target contaminants were added at a concentration of 500 spores / spot. The plates were incubated at 22±1°C for 7 days.

[0155] Figure 1 demonstrates that the inhibition of the test molds is more pronounced when compositions containing lactic acid bacteria and low concentrations of manganese, i.e., about 30, about 195 ppm, or about 275 ppm, are used. A composition containing lactic acid bacteria and about 625 ppm manganese is also able to inhibit the growth of P. crustosum (X) and P. carneum (Y), but not P. roqueforti (Z). Furthermore, without such a composition, the test molds proliferate and cause spoilage of the fermented milk product.

[0156] Figure 7 shows the growth of three different molds on plates prepared from milk fermented with a starter culture (reference) or with a freeze-dried (FD-DVS) DVS composition additionally containing lactic acid bacteria; the FD-DVS form had approximately 200 ppm manganese, and the cryoprotectant used was further supplemented with various concentrations of manganese (1, 5, 10, 20, and 40 ppm). Yogurt was spiked with P. crustosum, P. carneum, and P. roqueforti (500 spores each) and stored at 22°C for 12 days.

[0157] Figure 7 demonstrates that growth of the test mold is impaired when subjected to manganese-deficient conditions versus manganese-rich conditions. Thus, when manganese is added to the cryoprotectant, the manganese promotes growth of the test mold, causing food spoilage. Thus, Figure 7 surprisingly demonstrates the need for compositions (such as skim milk powder) in the presently disclosed FD-DVS form from which manganese has been removed, particularly cryoprotectants having manganese in their composition.

[0158] In conclusion, all test molds grew very well on agar plates made from milk fermented only with a starter culture (reference). Therefore, without using a composition containing lactic acid bacteria and manganese, the test molds may proliferate and cause spoilage of the fermented milk product. Furthermore, Figure 1 demonstrates that when fermented milk products are treated with a composition containing lactic acid bacteria and various concentrations of manganese, various test molds can be inhibited, with this effect being more pronounced when lower concentrations of manganese are used. Therefore, spoilage of fermented milk products can be avoided by using a composition containing lactic acid bacteria and low levels of manganese, e.g., less than 600 ppm manganese, more preferably less than 275 ppm, and even more preferably less than 200 ppm, e.g., about 30 to about 200 ppm or about 45 ppm to about 200 ppm manganese.

[0159] Example 2: Comparison of the effects of lactic acid bacteria and various manganese concentrations on the development of lactic acid bacteria compared to the prior art Mold inhibition in fermented dairy products

[0160] A comparison of the compositions of the present invention with the prior art was also carried out.

[0161] FIG. 5 shows the growth of three different molds (P. crustosum, P. paneum, and P. roqueforti) on plates prepared from milk fermented with a starter culture alone (reference), or with a composition further comprising lactic acid bacteria (strain 2 or 1+2) in combination with low levels of manganese (e.g., 45 or 65 ppm manganese), or with benchmark compositions (A, B, or C). Benchmark composition A is Holdbac® XPM with 845 ppm manganese. Benchmark composition B is Holdbac® YM-BPlus with 630 ppm manganese. Benchmark composition C is Holdbac® YM-C with 870 ppm manganese.

[0162] FIG. 5 demonstrates that a composition comprising lactic acid bacteria and a low concentration of manganese, for example, a manganese concentration of less than about 600 ppm, preferably a manganese concentration of about 40-600 ppm, or a manganese concentration of about 45-600 ppm, more preferably a manganese concentration of 40-70 ppm, serves to prevent food spoilage regardless of the conditions used to store the fermented milk sample (e.g., 24 days at 7±1°C vs. 11 days at 25±1°C).

[0163] Example 3: Inhibition of yeast in fermented dairy products containing compositions containing lactic acid bacteria and various manganese concentrations

[0164] This example demonstrates the growth challenge faced by various yeasts, such as Torulaspora or Debaryomyces, when ingested in a fermented milk product fermented with a starter culture alone (reference) or additionally with a composition comprising lactic acid bacteria (e.g., L. rhamnosus strain 1, L. rhamnosus strain 2, L. rhamnosus strain 3, L. paracasei) and about 30 ppm manganese, or about 195 ppm manganese, or about 275 ppm manganese, or about 625 ppm manganese. The growth challenge was maintained at 7±1°C for 23 days (Figures 2-3) or 27 days (Figure 6).

[0165] Figures 2 and 3 show the growth impairment of various yeasts (Torulaspora and Debaryomyces) when inoculated into fermented dairy products fermented with starter cultures or with compositions further comprising lactic acid bacteria and about 30 ppm manganese, about 195 ppm manganese, or about 625 ppm manganese. This impairment was more dramatic and prolonged with compositions containing about 30 ppm manganese (Figures 2-3). However, compositions with about 195 ppm manganese or about 625 ppm manganese still caused growth impairment of the test yeasts, but to a lesser extent, thus demonstrating that compositions containing lactic acid bacteria and low concentrations of manganese affect fungal growth and, consequently, food spoilage.

[0166] Figure 6 further demonstrates that a composition containing lactic acid bacteria and approximately 275 ppm manganese significantly reduces the growth of yeasts such as Debaryomyces. The cryoprotectant used is composed of sodium caseinate, inositol, monosodium glutamate, sodium ascorbate, and water, preferably excluding manganese. All values ​​are expressed as w(ingredients) / w(cryoprotectant solution)%. For example, sodium caseinate: 5.55, inositol: 3.75, monosodium glutamate: 3.75, sodium ascorbate: 5.65, and water: 81.3, preferably excluding manganese. All values ​​are expressed as w(ingredients) / w(cryoprotectant solution)%.

[0167] Figure 8 further shows the growth of Debaryomyces on fermented milk products inoculated with a freeze-dried DVS composition containing either a starter culture alone (reference) or additionally containing lactic acid bacteria (e.g., L. rhamnosus strain 2), the FD-DVS form having approximately 200 ppm manganese and further supplemented with various concentrations of manganese (1 and 40 ppm). Figure 8 thus surprisingly demonstrates the need for compositions (such as skim milk powder) in the FD-DVS form from which manganese has been removed, particularly cryoprotectants having manganese in their composition.

[0168] Example 4: Inhibition of yeast in fermented dairy products containing compositions containing lactic acid bacteria and various manganese concentrations versus the prior art

[0169] A comparison of the compositions disclosed herein with the prior art was also performed.

[0170] Figure 4 shows the growth of Debaryomyces in fermented dairy products containing either starter culture alone (reference) or a composition comprising lactic acid bacteria (L. rhamnosus strain 2 or L. rhamnosus strain 1+2) in combination with low levels of manganese (e.g., about 45 or about 65 ppm), or a benchmark composition (A, B, or C). Benchmark composition A is Holdbac® XPM with 843 ppm manganese. Benchmark composition B is Holdbac® YM-BPlus with 630 ppm manganese. Benchmark composition C is Holdbac® YM-C with 870 ppm manganese.

[0171] Figure 4 demonstrates that growth of Debaryomyces is significantly impaired on fermented dairy products when subjected to compositions containing lactic acid bacteria and low concentrations of manganese (e.g., 45 ppm or 65 ppm) versus benchmark compositions containing greater than 600 ppm manganese.

Claims

1. 1. A direct vat set starter culture composition comprising lactic acid bacteria containing a manganese transporter for fermenting a food product and inhibiting or retarding fungal growth in said food product, comprising: the composition comprises up to 600 ppm manganese; the concentration of the lactic acid bacteria is at least 1E+10 colony forming units / g; Optionally, the composition, wherein said lactic acid bacteria do not contain superoxide dismutase, preferably do not contain manganese superoxide dismutase.

2. 2. The composition of claim 1, comprising a maximum of 400 ppm manganese, preferably a maximum of 300 ppm manganese, more preferably a maximum of 250 ppm, and even more preferably a maximum of 200 ppm.

3. 3. The composition of claim 1 or 2, comprising 30 to 600 ppm, preferably 35 to 600 ppm, or 40 to 400 ppm, or 40 to 300 ppm, or 40 to 250 ppm, more preferably 40 to 200 ppm or 45 to 200 ppm of manganese.

4. 4. The composition according to any one of claims 1 to 3, wherein the concentration of the lactic acid bacteria is from 2.0E+10 to 6.5E+11, preferably from 6.0E+10 to 6.4E+11, more preferably from 1.3E+11 to 5.6E+11 colony forming units / g.

5. The composition of any one of claims 1 to 4, wherein the composition is a frozen direct vat set (F-DVS) or a frozen dry vat set (FD-DVS).

6. 6. A composition according to any one of claims 1 to 5, wherein the composition is a freeze-dried direct vat set (FD-DVS) composition comprising 2% to 70% additive, preferably 3% to 50% additive, more preferably 4% to 40% additive, even more preferably 10% to 30% additive or 20-30% additive, measured as dry weight of additive per weight of FD-DVS form, preferably wherein the additive is manganese-free or substantially manganese-free; or wherein the composition is a frozen direct vat set (F-DVS) comprising 2% to 70% additive, preferably 3% to 50% additive, more preferably 4% to 40% additive, even more preferably 10-40% additive or 20-35% additive, measured as weight of additive per weight of F-DVS form, preferably wherein the additive is manganese-free or substantially manganese-free.

7. 7. The composition of any one of claims 1 to 6, wherein the soluble fiber is selected from the group consisting of sodium caseinate, inositol, monosodium glutamate, sodium ascorbate, sucrose, maltodextrin, inosine monophosphate (IMP), inosine, polysorbate 80, glutamic acid, lysine, sodium glutamate, malt extract, whey powder, yeast extract, gluten, collagen, gelatin, elastin, keratin, albumin, and mixtures thereof.

8. The composition of any one of claims 1 to 7, comprising sodium caseinate, inositol, monosodium glutamate, and sodium ascorbate.

9. 9. The composition of any one of claims 1 to 8, wherein the lactic acid bacterium comprises a manganese transporter having at least 55%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1 to 3.

10. The composition according to any one of claims 1 to 9, wherein the lactic acid bacteria do not contain superoxide dismutase, preferably do not contain manganese superoxide dismutase.

11. The lactic acid bacteria may be selected from the group consisting of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus alimentarius, Pediococcus acidilactici, Lactobacillus rhamnosus, and Lactobacillus kefiri. The composition according to any one of claims 1 to 10, wherein the compound is selected from the group consisting of:

12. The fungus is a yeast and / or a mold, preferably the fungus is a yeast selected from the group consisting of Torulaspora spp., Cryptococcus spp., Saccharomyces spp., Yarrowia spp., Debaryomyces spp., Candida spp., and Rhodoturola, preferably the Debaromyces spp. is Debaromyces hansenii, and / or the fungus is a fungus selected from the group consisting of Aspergillus spp., Cladosporium spp., Didymella spp., or Penicillium spp. spp.), and preferably the Penicillium spp. is Penicillium crustosum, Penicillium paneum, Penicillium carneum, or Penicillium roqueforti.

13. A food product comprising the composition according to any one of claims 1 to 12.

14. The food product according to any one of claims 1 to 13, wherein the food product is a fermented food, preferably a thermophilic or mesophilic fermented food, more preferably a yogurt or cheese.

15. 13. Use of a composition according to any one of claims 1 to 12 for inhibiting fungal growth in food, preferably wherein said food is a fermented food, more preferably a thermophilic or mesophilic fermented food, more preferably yogurt or cheese.

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