FIELD OF THE INVENTION

The present invention lies in the field of microbiology and relates to methods for controlling of fungal spoilage. The invention also relates to food products and preparations.

BACKGROUND OF THE INVENTION

A major problem in the food industry is spoilage by unwanted microorganisms. According to the Food and Agriculture Organization (FAO), one in every four calories intended for human consumption is ultimately not consumed by humans. In a time of food shortages, with more than 800 million people suffering from hunger, the topic of food waste has become a prioritized issue for global policy makers and food manufacturers. In addition to the negative social and economic impacts for society, wasted food also inflicts a host of related environmental impacts, including unnecessary greenhouse gas emissions and inefficient uses of scarce resources such as water and land.

Yeasts and molds are highly efficient at causing foods to spoil and are a problem for most food manufacturers. Spoilage due to yeasts and molds is clearly visible as patches of mold or discoloration on the surface of the food product, allowing it to be disposed of prior to consumption. Yeasts tend to grow within food and drink matrices in planktonic form and they tend to ferment sugars, growing well under anaerobic conditions. In contrast, molds tend to grow on the surface of products in the shape of a visible mycelium made up of cells.

In particular in the dairy sector, 29 million tons of dairy products go to waste every year in Europe. One of the main challenges in keeping dairy products fresh is to manage contamination by yeast and mold, which are naturally present everywhere, especially if there are disruptions in the cold chain from production to the consumer's table.

For economic and environmental reasons, there is a constant need for novel or improved methods which are effective for controlling yeast and mold contamination.

SUMMARY OF THE INVENTION

The inventors of the present invention have sought to find effective methods to manage fungal contamination and surprisingly identified manganese as important growth constraints for its growth. The present invention provides a novel method of inhibiting fungal growth by limiting the free manganese which is available for yeast(s) or mold(s). The present invention is in part based on the surprising finding that by reducing the level of free manganese in the food product, for example by removing free manganese using manganese scavenging agents, the contamination of yeast(s) and/or mold(s) can be reduced or delayed. The inventors have furthermore shown that common yeasts and molds are responsive to the methods described herein.

Manganese has been considered to be vital to human health and therefore an essential trace element. Manganese is essential to the proper functioning of both humans and animals, as it is required for the functioning of many cellular enzymes, such as manganese superoxide dismutase, pyruvate carboxylase, and it can serve to activate many others like kinases, decarboxylases, transferases and hydrolases.

Manganese can be found naturally in many food sources including leafy vegetables, nuts, grains and animal products. Typical ranges of manganese concentrations in common foods are 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.

Besides being dietary supplements, manganese is sometimes added in fermented products as an active ingredient to enhance growth of Bifidobacteria in milk (see e.g. WO2017/021754, Compagnie Gervais Danone, France). However, the inventors have discovered that this may have adverse effect and it would be advantageous to limit manganese concentration in food products in order to prevent or delay growth of unwanted microorganisms.

To combat the problem of microbial spoilage, the present invention provides in a first aspect a method of inhibiting or delaying growth of fungus/fungi in a product comprising the step of reducing free manganese present in said product. Free manganese concentration can be reduced by the methods described in this invention. In a preferred embodiment, one or more manganese scavenging agents are added to reduce free manganese. Free manganese concentration is preferably reduced to below about 0.01 ppm, such as below about 0.008 ppm or below about 0.003 ppm. Using the method, a product in which unwanted yeast and or mold can hardly thrive can be obtained. The product is characterized by a free manganese concentration of below about 0.01 ppm, such as 0.009 ppm, 0.008 ppm, 0.007 ppm, 0.006, 0.005 ppm or lower. The method further comprises the step of measuring the free manganese concentration in the product and obtaining a value of below about 0.01 ppm.

In particular, the present invention provides a method of inhibiting or delaying yeast and/or mold growth in a fermented food product prepared from milk such as yogurt or cheese. The method is characterized by the step of reducing manganese concentration in the food product in order to deprive the yeast and/or mold of manganese and thereby delaying or inhibiting their growth in the food product.

In one preferred embodiment, the present invention provides a method of inhibiting or delaying growth of Torulaspora spp., Cryptococcus spp. and Rhodoturola spp. in a product comprising the step of reducing free manganese present in said product.

In a second aspect, the present invention provides a method preparing a product such as a food product, comprising reducing free manganese present in said product. Free manganese concentration can be reduced by the methods described in this invention or other methods known to a skilled person in the art. In a preferred embodiment, one or more manganese scavenging agents are added to reduce free manganese. Free manganese concentration is preferably reduced to below about 0.01 ppm, such as below about 0.005 ppm or below about 0.003 ppm. Using the method, a product comprising free manganese concentration below about 0.01 ppm can be obtained.

In a third aspect, the present invention provides products such as food products obtained by the methods described herein. In one embodiment, the present invention provides a method of providing a product, comprising the steps of reducing free manganese in the product and obtaining the product, wherein free manganese concentration is below about 0.01 ppm in the product.

In a further aspect, the present invention provides the use of one or more manganese scavenging agents to inhibit or delay fungal growth as well as to produce food products. Manganese scavenging agents have the effect of making less free manganese available in a product for yeast and mold, thus inhibiting or delaying their growth.

In another aspect, the present invention provides manganese scavenging agents, selections and uses thereof for manganese uptake.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 and 2 show the growth of 2 different Debaryomyces hansenii in chemically defined medium at pH 6.5 (open circles) or 4.5 (black squares) with different manganese concentrations. The growth was measured after 6 days of incubation at 17°C and determined by absorbance at 600 nm.

Figure 3 and 4 show the growth of 2 different Debaryomyces hansenii in the aqueous phase of fermented milk in the presence of manganese scavengers and with different manganese concentrations added. Absorbance at 600 nm was measured after 7 days of incubation at 17°C. Growth after manganese was added to aqueous phase is shown in Figure 3 for Debaryomyces hansenii (strain 1) and Figure 4 for Debaryomyces hansenii (strain 2) (open circles), compared to reference where no additional manganese is added (square). Average and standard deviation of technical replicates n=6 (A) and n=3 (B) are shown.

Figure 5 shows the growth of Debaryomyces hansenii (strain 2) in fermented milk prepared with and without different scavenging agents, after addition of different manganese concentrations ranging from 6 ppm (top row), to 0.000006 ppm (bottom row).

Figure 6 shows the growth of 3 different yeasts on plates prepared from milk fermented with a starter culture alone (reference, top row) or together with manganese scavenger (bottom row). Different manganese concentrations were added as indicated above the pictures. Three target contaminants (column A: Debaryomyces hansenii (strain 2), column B: Cryptococcus fragicola, column C: Debaryomyces hansenii (strain 1) were added in three different concentrations: 1 x 10 ³ cfu/spot (top row on plate), 1 x 10 ² cfu/spot (middle row on plate) and 1 x 10 ¹ cfu/spot (bottom row on plate).

Figure 7 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or together with manganese scavenger (bottom row). Different manganese concentrations were added as indicated above the pictures. Three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added in concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Figure 8 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or together with manganese scavenger (bottom row). Different manganese concentrations were added as indicated above the pictures. Three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added in concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

Figure 9 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added in concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days. Figure 10 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added in concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

Figure 11 shows an example phylogenetic tree of MntH manganese transporters of selected Lactobacillus species.

Figure 12 shows the growth of a Debaryomyces hansenii strain (strain 1 or strain 2) in an aqueous phase filtered with a 1.2 mΐti filter (aq filtered), preferably an aqueous phase of fermented milk, with different EDTA concentrations, with and without other manganese scavengers, preferably manganese scavenger 1, and with and without 6 ppm of manganese. Absorbance at 600 nm was measured after 7 days of incubation at 17°C, compared to reference. Samples made in triplicate.

Figure 13 shows the growth of a Debaryomyces hansenii strain (strain 1 or strain 2) in an aqueous phase filtered with a 0.2 mΐti filter (aq sterile), preferably an aqueous phase of fermented milk, with different EDTA concentrations, with and without other manganese scavengers, preferably manganese scavenger 1, and with and without 0.6 ppm of manganese. Absorbance at 600 nm was measured after 7 days of incubation at 17°C, compared to reference. Samples made in triplicate.

Figure 14 shows the growth of a Debaryomyces hansenii strain (strain 1 or strain 2) in yogurt with and without different manganese scavengers, such as bacteria, preferably lactic acid bacteria, and/or a chemical chelating material such as EDTA, after addition of 6 ppm of manganese. The concentration of EDTA ranges from 14 mg/ml (top row), 7.10 mg/ml, 3.55 mg/ml, 1.78 mg/ml, 0.89 mg/ml, 0.44 mg/ml, 0.22 mg/ml to 0 mg/ml (bottom row).

DETAILED DISCLOSURE OF THE INVENTION

Food loss is a major concern worldwide - approximately one third of all food produced for human consumption is either lost or wasted. The reasons for this massive global food loss are diverse, but microbial spoilage that affects organoleptic product quality (aspect, texture, taste, and aroma), plays a major role. Since fungi can grow in different and even harsh environments, they are the major spoilage microorganisms found at all stages of the food process chain. It is therefore crucial to reduce food losses by controlling fungal contamination. In response to this demand, the present invention provides a novel method of inhibiting or delaying fungal growth in a product. The method is based on the surprising finding that low free manganese concentrations can serve as limiting factor for yeast and/or mold growth. Manganese is present in trace amounts in nature and many of our consumer goods. However, there has not yet been any report suggesting that by manipulating the concentration of free manganese, microbial spoilage can be effectively managed. Based on this unexpected finding, it is envisioned that such spoilage prevention strategy is applicable even beyond food products and extending to other products which are generally prone to microbial contamination, such as feed products, biologic products, health care products, pharmaceutical products and the like.

The present invention provides in a first aspect a method of inhibiting or delaying fungal growth in a product comprising depleting free manganese in said product to a concentration of below about 0.01 ppm.

In general, inhibiting means a decrease, whether partial or whole, in function and activity of cells or microorganisms. As used herein, the terms "to inhibit" and "inhibiting" in relation to yeasts and molds mean that the growth, the number, or the concentration of yeasts and molds is the same or reduced. This can be measured by any methods known in the field of microbiology. Inhibition can be observed by comparing the fungal growth, number or concentration in or on a product with reduced free manganese to a control. The control can be the same product but without reduced free manganese.

The term "to delay" in general means the act of stopping, postponing, hindering, or causing something to occur more slowly than normal. As used herein, "delaying growth of fungi" refers to the act of postponing the growth of fungi. This can be observed by comparing the time needed for the fungi to grow to a given level in two products, one of which with reduced manganese and the other one without (but otherwise the same).

In some embodiments, "delaying growth of fungi" refers to delaying by 7 days, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 days.

A fungus is a member belonging to the kingdom of fungi. Fungal growth can be measured with various methods known to a skilled person in the art. For example, fungal growth can be measured by density or size of colony, cell number, mycelial mass changes, spore production, hyphal growth, colony-forming units (CFU) and the like, depending on the fungus type and the product to which the method is applied. Fungal growth can also be observed by measuring the change in nutrient or metabolite concentrations, such as carbon dioxide release and oxygen uptake. The terms "inhibition of fungal growth" or "inhibiting growth of fungi" refer to the inhibition of fungal cell proliferation. The terms "delay of fungal growth" or "delaying growth of fungi" refer to the slowing down of fungal cell proliferation. This can be observed for example, by measuring the fungal growth and comparing it with a control. Such control may be for example a product without manganese scavengers applied. Methods of determining fungal growth inhibition or delay are known to a skilled person in the art.

"Free manganese" or sometimes "manganese" in accordance with the present invention refers to manganese which is present in a product (i.e. forming part of product, such as within the product or on the surface of a product) that is available to be taken up by fungi, including yeasts and molds. For example, free manganese refers to the manganese that is present in the food matrix of the product.

In one preferred embodiment, the present invention is directed to a method of inhibiting or delaying growth of fungi in a food product, comprising reducing free manganese concentration in a food matrix of the food product. As used herein, the term "food matrix" refers to the food's composition and structure. It is based on the concept that nutrients are contained in a continuous medium.

The term "reduce" or "reducing" generally means lowering the amount of a substance in a given context. As used herein, the term "to reduce free manganese" or "reducing free manganese" means to reduce the amount of manganese present in a product that is available to be taken up by fungi, including yeasts and molds.

For example, this can be carried out by removing manganese present in the product or in a material which is to become part of the product. For example, this can be carried out by subjecting the raw material ion exchange chromatography to remove manganese so that the concentration in the final product is reduced.

Once having access, fungi rapidly colonize, increase in population and take up nutrients from their immediate surroundings. In some embodiments, given that fungi may first come into contact with a product on the surface, it is within the spirit of the present invention that the step of reducing is carried out on parts of the product, for example in the exterior part of the product such as the coating or an outer layer. In such cases, the reducing step nevertheless leads to an overall decrease in the concentration in the product.

Manganese concentration or manganese level as used herein is expressed in parts per million ("ppm") calculated on a weight/weight basis. Reducing free manganese in a product to a concentration below a value means reducing free manganese in the product or parts thereof such that the concentration of free manganese in the entire product by weight is reduced. Methods of determining trace elements such as manganese are known in the art and described for example in Nielsen, S. Suzanne, ed. Food analysis. Vol. 86. Gaithersburg, MD: Aspen Publishers, 1998.

In applying the present methods, one skilled in the part may first determine the manganese level which is present in the products to be treated. Manganese concentration for food products is well studied and can be found in national food composition databases such as Danish Food Composition Databank, Canadian Nutrient Files. In general, manganese is present at a concentration of at least 0.03 ppm for milk, making dairy products susceptible for fungal contamination. Manganese levels have been reported to range from 0.04 to 0.1 ppm in cow milk and up to 0.18 ppm in goat or sheep milk (Muehlhoff et al., Milk and dairy products in human nutrition. Food and Agriculture Organization of the United Nations (FAO), 2013). As for fermented milk products like cheese, the manganese level usually increases due to the concentration process from milk, often up to 10-fold or more. Different levels have been reported for various types of cheeses, for example about 0.06 ppm for ricotta cheese, 0.11 ppm for cream cheese, 0.34 ppm for brie, 0.3 ppm for mozzarella, 0.7 ppm for cottage cheese, 0.68 ppm for gouda and 0.74 ppm for cheddar cheese (Smit, L. E., et al. The nutritional content of South African cheeses. ARC-Animal Improvement Institute, 1998; Gebhardt, Susan, et al. "USDA national nutrient database for standard reference, release 12." United States Department of Agriculture, Agricultural Research Service, 1998).

Free manganese in a product is preferably reduced to a concentration below about 0.01 ppm, such as below about 0.009 ppm, below about 0.008 ppm, below about 0.007 ppm, below about 0.006 ppm, below about 0.005 ppm, below about 0.004 ppm, below about 0.003 ppm, below about 0.002 ppm, below about 0.001 ppm, below about 0.0009 ppm, below about 0.0008 ppm, below about 0.0007 ppm, below about 0.0006 ppm, below about 0.0005 ppm, below about 0.0004 ppm, below about 0.0003 ppm or lower.

As used herein, the term "about" indicates that values slightly outside the cited values, i.e., plus or minus 0.1% to 10%. Thus, concentrations slightly outside the cited ranges are also encompassed by the scope of the present inventions.

In one embodiment, the present invention provides a method of inhibiting or delaying growth of fungi in a product, preferably a food product, comprising the steps of reducing free manganese in the product, and

obtaining the product wherein free manganese concentration is below about 0.01 ppm in the product. The present method further comprises the step of measuring the concentration of free manganese. This can be performed after the reducing step so to determine whether the concentration of free manganese is reduced. In one embodiment, the present invention provides a method of inhibiting of delaying growth of fungi in a food product, comprising reducing free manganese in the product to a concentration of below about 0.01 ppm in the product, and measuring the free manganese in the product, and optionally obtaining a value of below 0.01 ppm.

In one embodiment, the present invention provides a method of inhibiting or delaying growth of fungi in a product, comprising the steps of reducing free manganese in the product to a concentration of below about 0.01 ppm in the product,

measuring the concentration of the free manganese in the product and obtaining a value of below 0.01 ppm.

Methods of measuring of manganese at low concentration are well known to a person skilled in the art. Such methods include atomic absorption spectroscopy, atomic emission spectroscopy, mass spectrometry, neutron activation analysis and x-ray fluorimetry (see e.g., Williams et al. "Toxicological profile for manganese." (2012)).

Preferably, manganese concentration is measured according the standard procedure as described in "Foodstuffs - Determination of trace elements - Pressure digestion" in European Standard EN13805:2014 published by European Committee for Standardization or as described in "Water guality - Determination of selected elements by inductively coupled plasma optical emission spectrometry (ICP-OES)” in ISO 11885:2007 published by International Organization for Standardization.

Fungus

The inventors of the present invention have surprisingly discovered that both yeast and mold can be inhibited by manganese depletion. In one preferred embodiment, the present invention provides a method of inhibiting or delaying growth of yeast in a product, preferably a food product, comprising the step of reducing free manganese in the product. In another preferred embodiment, the present invention provides a method of inhibiting or delaying growth of mold in a product, preferably a food product, comprising the step of free manganese in the product.

In one embodiment, the method is used to inhibit the growth of yeast, 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 fungi 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.

In one embodiment, the method is used to inhibit the growth of mold. Preferably, the fungus is a mold selected from the group consisting of Aspergillus spp., Cladosporium spp., Didymella spp. or Penicillium spp. More preferably, the fungus is a mold selected from the group consisting of Penicillium brevicompactum, Penicillium crustosum, Penicillium solitum, Penicillium carneum, Penicillium paneum, and Penicillium rogueforti.

Removal of Manganese

Methods of removing manganese are known in the art. Manganese is a common contaminant in many mine waters, groundwater, and freshwaters. In waste water treatment, manganese ions can be chemically removed from effluents by oxidation to Mn0 ₂ , adsorption, or precipitation as a carbonate.

Alternatively, manganese removal can involve biological processes as alternatives to chemical routes. The role of microbial activity in the remediation of manganese-contaminated waters has been described in various literatures, e.g. Burger et al. Manganese removal during bench- scale biofiltration. Water Research. 2008;42(19):4733-4742; Johnson et al. Rapid manganese removal from mine waters using an aerated packed-bed bioreactor. Journal of Environmental Quality. 2005;34(3):987-993; Tekerlekopoulou et al. "Removal of ammonium, iron and manganese from potable water in biofiltration units: a review." Journal of Chemical Technology and Biotechnology 88.5 (2013): 751-773; Patil et al. "A review of technologies for manganese removal from wastewaters." Journal of Environmental Chemical Engineering 4.1 (2016): 468-487. In one embodiment, the step of reducing free manganese in the product comprises using ion-exchange chromatography. This is especially applicable if the product is in liquid or substantially liquid.

In one preferred embodiment, the step of reducing free manganese in the product is carried out by adding a manganese scavenging agent. As used herein, the terms "manganese scavenging agent" or "manganese scavenger" refers to a material that is capable of make manganese unavailable for yeast or mold. The material can be a chemical material, such as a chemical chelating material selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA), diaminocyclohexanetetraacetic acid (DCTA), nitrilotriacetic acid (NTA), l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or diethylenetriaminepentaacetic acid (DTPA), preferably the chemical chelating material is ethylenediaminetetraacetic acid (EDTA). The material can also be a biological material, such as bacteria.

In some preferred embodiment the manganese scavenging agent is one or more bacteria strains. In such cases, it should be noted that when measuring free manganese, such free manganese does not include the manganese which is found intracellularly. Rather, free manganese refers to the manganese that is found extracellularly, i.e. in the cell-free parts of the product, since they would be available to be taken up by fungi. Thus, in such cases, concentration of free manganese should be measured taking only extracellular manganese into account. This can be done for example by removing cells (such as starter cultures) by centrifugation and obtaining cell-free supernatant, followed by measuring the manganese in the cell-free supernatant. As used herein, the term "bacteria strain" has its common meaning in the field of microbiology and refers to a genetic variant of a bacterium.

In one embodiment, the present invention provides a method of inhibiting or delaying growth of fungi in a product, comprising the steps of selecting one or more bacteria strains as a manganese scavenging agent, and reducing free manganese in the product to a concentration of below about 0.03 ppm in the product by adding the manganese scavenging agent.

According to preferred embodiments of the present invention, the method comprises selecting a bacteria strain having manganese uptake activities as a manganese scavenging agent. The selection is based on whether the bacteria strain has manganese transport systems.

Manganese is involved in many crucial biological processes and is ubiquitously found in all organisms. Manganese also contributes to protection against oxidative stress and can also contribute to the catalytic detoxification of reactive oxygen species. Many bacteria have developed sophisticated acquisition system to scavenge essential metals from the environment, using low and high affinity transport systems for chelated or free metals. Manganese which is taken up by bacteria forms a large complex of nondialyzable polyphosphate-protein aggregates in the protein which may reach very high intracellular concentrations. Transport systems for manganese have been studied and are for example described in Kehres et al., "Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria." FEMS microbiology reviews 27.2-3 (2003): 263-290.

In one embodiment, a bacteria strain having manganese uptake activities comprises bacterial Mn ²⁺ transporters. Mn ²⁺ transporters may be an ABC transporter (for example SitABCD and YfeABCD) or a proton-dependent Nramp-related transport system belonging to the family designated as TC#3.A.1.15 and TC#2.A.55 in the transporter classification system given by the Transport Classification Database (M. Saier; U of CA, San Diego, Saier MH, Reddy VS, Tamang DG, Vastermark A. (2014)). The TC system is a classification system for transport proteins which is analogous to the Enzyme Commission (EC) system for classification of enzymes. The transporter classification (TC) system is an approved system of nomenclature for transport protein classification by the International Union of Biochemistry and Molecular Biology. TCDB is freely accessible at http://www.tcdb.ora which provides several different methods for accessing the data, including step-by-step access to hierarchical classification, direct search by sequence or TC number and full-text searching.

In one embodiment, the method comprises selecting a bacteria strain comprising a protein belong to the family designated as TC#3.A.1.15 (manganese chelate uptake transporter (MZT) family) as manganese scavenging agent.

For example, the manganese scavenging agent is a bacteria strain comprising a manganese chelate uptake transporter designated as TC#3.A.1.15.2, TC#3.A.1.15.6, TC#3.A.1.15.8, TC#3. A.1.15.14 or functional variants thereof.

While the ABC transporter is mainly active at higher pH, proton driven transporters may be more active under acidic conditions. This makes them particularly useful as manganese scavenging agents in fermented food products. Thus, in one embodiment, a bacteria strain comprising a protein belong to the family designated as TC#2.A.55 (the metal ion (Mn ²⁺ -iron) transporter (Nramp) family) is selected.

The step of selecting one or more bacteria strains as manganese scavenging agent comprises determining whether one or more bacteria strain comprises a manganese transporter designated as TC#2.A.55 or functional variants thereof.

More preferably, the transporter belongs to the subfamily designated as TC#2.A.55.2 or the subfamily designated as TC#2.A.55.3, as manganese scavenging agent.

For example, the manganese scavenging agent is a bacteria strain comprising a metal ion (Mn ²⁺ -iron) transporter (Nramp) 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 or functional variants thereof as manganese scavenging agent.

Most preferably, the method comprises selecting a bacteria strain comprising a protein designated as TC#2.A.55.2.6 or functional variants thereof as manganese scavenging agent.

Preferably, the manganese scavenging agent is 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 term "functional variant" is a protein variant having a substantially similar biological activity, i.e. manganese uptake activities.

As used herein, a "variant" refers to a variant form of a protein which 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.

The invention additionally provides polypeptide sequences of manganese transporters for selecting suitable manganese scavengers to carry out the present invention.

In one preferred embodiment, a manganese scavenging agent is a bacteria strain comprising a polypeptide having the sequence of SEQ ID NO:

1(MASEDKKSKREHIIHFEDTPSKSLDEVNGSVEVPHNAGFWKTLAAYTGPGILVAV GYMDPGNWITSI

AGGASFKYSLLSVILISSLIAMLLQAMAARLGIVTGRDLAQMTRDHTSKAMGGFLWV ITELAIMATDIAEI

IGSAIALKLLFNMPLIVGIIITTADVLILLLLMRLGFRKIEAVVATLVLVILLVFAY EVILAQPNVPELLKGYLP

HADIVTNKSMLYLSLGIVGATVMPHDLFLGSSISQTRKIDRTKHEEVKKAIKFSTID SNLQLTMAFIVNSL

LLILGAALFFGTSSSVGRFVDLFNALSNSQIVGAIASPMLSMLFAVALLASGQSSTI TGTLAGQIIMEGFIH

LKMPLWAQRLLTRLMSVTPVLIFAIYYHGNEAKIENLLTFSQVFLSIALPFAVIPLV LYTSDKKIMGEFANR

AWVKWTAWFISGVLIILNLYLIAQTLGFVK) or functional variants thereof.

In other preferred embodiments, a manganese scavenging agent is a bacteria strain comprising 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 with the sequence of SEQ ID NO: 1.

Table 1 shows exemplary sequences which encodes functional variants of SEQ ID NO: 1 and their sequence identity with SEQ ID NO: 1. Table 1

In one preferred embodiment, a manganese scavenging agent is a bacteria strain comprising a polypeptide having the sequence of SEQ ID NO : 2

(MARPDERLTVQREKRSLDDINRSVQVPSVYESSFFQKFLAYSGPGALVAVGYMDPG NWLTALEGGSRY HYALLSVLLMSILVAMFMQTLAIKLGVVARLDLAQAIAAFIPNWSRICLWLINEAAMMAT DMTGVVGTAI ALKLLFGLPLMWGMLLTIADVLVVLLFLRFGIRRIELIVLVSILTVGIIFGIEVARADPS IGGIAGGFVPHTDI LTNHGMLLLSLGIMGATIMPH NIYLHSSLAQSRKYDEHIPAQVTEALRFGKWDSNVHLVAAFLINALLLIL GAALFYGVGGHVTAFQGAYNGLKNPMIVGGLASPLMSTLFAFALLITGLISSIASTLAGQ IVMEGYLNIRM PLWERRLLTRLVTLIPIMVIGFMIGFSEHNFEQVIVYAQVSLSIALPFTLFPLVALTNRR DLMGIHVNSQLV RWVGYFLTGVITVLNIQLAISVFV) or functional variants thereof.

In other preferred embodiments, a manganese scavenging agent is a bacteria strain comprising 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 with the sequence of SEQ ID NO : 2. Table 2 shows exemplary seq uences which encode functional variants of SEQ ID NO : 2 and their sequence identity with SEQ ID NO : 2. Table 2

In one preferred embodiment, a manganese scavenging agent is a bacteria strain comprising a polypeptide having the sequence of SEQ ID NO : 3 (MSDDHKKRHPIKLIQYANGPSLEEINGTVEVPHGKGFWRTLFAYSGPGALVAVGYMDPG NWSTSITG GQNFQYLLISVILMSSLIAMLLQYMAAKLGIVSQMDLAQAIRARTSKKLGIVLWILTELA IMATDIAEVIG AAIALYLLFHIPLVIAVLVTVLDVLVLLLLTKIGFRKIEAIVVALILVILLVFVYQVALS DPNMGALLKGFIPTG ETFASSPSINGMSPIQGALGIIGATVMPHNLYLHSAISQTRKIDYKNPDDVAQAVKFSAW DSNIQLSFAF VVNCLLLVMGVAVFKSGAVKDPSFFGLFQALSDSSTLSNGVLIAVAKSGILSILFAVALL ASGQNSTITGT LTGQVIMEGFVHMKMPLWARRLVTRIISVIPVIVCVMLTARDTPIQQHEALNTLMNNSQV FLAFALPFSM LPLLMFTNSKVEMGDRFKNTGWVKVLGWISVLGLTGLNLKGLPDSIAGFFGDHPTATQTN MANIIAIVLI VAILALLAWTIWDLYKGNQRYEAHLAAVADEKEAKADVDEQ) or functional variants thereof.

In other preferred embodiments, a manganese scavenging agent is a bacteria strain comprising 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 with the sequence of SEQ ID NO: 3.

Table 3 shows exemplary sequences which encode functional variants of SEQ ID NO: 3 and their sequence identity with SEQ ID NO: 3.

Table 3

For purposes of the present invention, the degree of "sequence identity" between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48 : 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. _r 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, 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 and is calculated as follows:

(Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide 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. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the - nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in

Alignment).

In one embodiment, the selecting step comprises determining whether the bacteria strain 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 with the sequences of any one of SEQ ID NO: 1-3. The determination can be based on sequencing the bacteria strain or a blast search in known sequence databases.

The manganese scavengers used in the Examples sections in the present invention have manganese transporter as encoded SEQ ID NO: 1-3 or functional variants thereof.

In other embodiments, the present invention provides a method of inhibiting or delaying growth of fungi in a product, comprising the steps of: selecting one or more bacteria strains as manganese scavenging agent, and reducing free manganese in the product to a concentration of below about 0.01 ppm in the product by adding the manganese scavenging agent,

wherein the selecting step comprises measuring a manganese uptake activity of one or more bacteria strains.

Manganese uptake activities can be measured using routine methods known in the art, see e.g. Kehres et al. "The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen." Molecular microbiology 36.5 (2000): 1085-1100.

For fermented food products such as fermented milk products, a manganese scavenging agent is preferably a lactobacillus species. Different manganese transporter families are present in Lactobacillus and oftentimes multiple homologs of these are present as well. In Figure 11, the inventors provide a tree providing an overview of the phylogeny of the manganese transporter MntH family within Lactobacillus species. As shown, manganese transporters can be found across the Lactobacillus species. Apart from Lactobacillus spp., the MntH transporter family can be found in other bacteria as well. The tree was constructed by alignment of Lactobacillus MntH protein sequences using mafft v.7 while phylogeny was inferred by neighbor-joining clustering.

In preferred embodiments, the manganese scavenging agent is a bacteria strain selected from the group consisting of L. rhamnosus, L. salivarius, L. casei, L. paracasei, L. fermentum, L. sakei, L. reuteri, L. piantarum, L. brevis, L. kefiri, L alimentarius and Pedicoccus acidilactici.

On the other hand, such transporter appears to be absent in L. helveticus, L. acidophilus, L. gasseri, and L. delbrueckii subsp. bulgaricus, making them less suitable for removing free manganese.

According to a preferred embodiment of the present invention, the method comprises a selecting step of determining that the one or more bacteria strains is free of a superoxide dismutase, preferably free of a manganese superoxide dismutase.

Superoxide dismutases, such as manganese superoxide dismutase, have been studied and are for example described in Kehres et al., "Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria." FEMS microbiology reviews 27.2-3 (2003): 263- 290; Culotta V.C "Superoxide dismutase, oxidative stress, and cell metabolism" Curr. Top. Cell Regul. 36, 117-132 (2000) or Whittaker J.W "Manganese superoxide dismutase" Met. Ions Biol. Syst. 37, 587-611 (2000), among others.

In the context of the present invention, the term "free of" means that genome of the one or more bacteria strains do not present a gene coding for a superoxide dismutase, or even if the genome of the one or more bacteria strains presents a gene coding for a superoxide dismutase, this gene is not express by the one or more bacteria strains.

Products

In some embodiments, the product is a food product, cosmetic product, health care product or a pharmaceutical product. "Food" and "food product" have the common meaning of these terms. "Food product" refers to any food or feed products suitable for consumption by humans or animals. Food products can be fresh or perishable food products as well as stored or processed food products. Food products include, but are not limited to, fruits and vegetables including derived products, grain and grain-derived products, dairy products, meat, poultry and seafood. More preferably, the food product is a meat product or dairy products, such as yogurt, tvarog, sour cream, cheese and the like. However, it should be noted that within the context of the present invention, the term "product" and "food product" in the present invention does refer to water as such. Although manganese is essential to human nutrition, in water it is generally regarded as unhealthy for humans according to United States Environmental Protection Agency (EPA). Therefore, the treatment of drinking water or waste water to remove excess manganese is sometimes carried out for decontamination and health purposes, which is not related to the spirit of the present invention.

The present invention is especially applicable for food products having intermediate to high water activity. Water activities (a _w ) determine viability and functionality of microorganisms. Water activity or a _w is the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water. In the field of food science, the standard state is most often defined as the partial vapor pressure of pure water at the same temperature. Using this particular definition, pure distilled water has a water activity of exactly 1.

The main food categories prone to fungal spoilage are dairy products having intermediate to high water activity, such as yogurt, cream, butter, cheese and the like. However, it is also envisioned that the present invention is suitable for food products having lower water activities, such processed meat, cereals, nuts, spices, dried milk, dried meats and fermented meats.

In preferred embodiment, the product where the methods disclosed in the present invention can be applied is a product having a water activity (a _w ) of less than 0.98, such as less than about 0.97, less than about 0.96, less than about 0.95, less than about 0.94, less than about 0.93, less than about 0.92, less than about 0.91, less than about 0.90, less than about 0.89, less than about 0.88, less than about 0.87, less than about 0.86, less than about 0.85, less than about 0.84, less than about 0.83, less than about 0.82, , less than about 0.81, less than about 0.80, less than about 0.79, less than about 0.78, less than about 0.77, less than about 0.76, less than about 0.75, less than about 0.74, less than about 0.73, less than about 0.72, less than about 0.71, less than about 0.70 or lower.

In some embodiments, the product is one having a water activity (a _w ) of about 0.70 to about 0.98, such as about 0.75 to about 0.97, such as about 0.80 to about 0.96, such as about 0.85 to about 0.95.

Methods for measuring water activity are known in the art, for example, as described in Fontana Jr, Anthony J. "Measurement of water activity, moisture sorption isotherms, and moisture content of foods." Water activity in foods: Fundamentals and applications (2007): 155-173. In one embodiment, the steps described herein are carried out to inhibit or delay growth of fungi in fermented food products. Fermented food products are foods produced or preserved by the action of microorganisms. Fermentation means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Fermentation typically refers to the fermentation of sugar to alcohol using yeast. However, it may also involve the conversion of lactose to lactic acid. For example, fermentation may be used to make foods such as yogurt, cheese, salami, sauerkraut, kimchi, pickle and the like.

In one embodiment, the food product is a product of lactic acid fermentation, i.e. prepared by lactic acid bacteria (LAB) fermentation. "Lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid. The food product typically has a pH of about 3.5 to about 6.5, such as about 4 to about 6, such as about 4.5 to about 5.5, such as about 5.

The present invention is particularly useful in inhibiting or delaying growth of fungi in dairy products. In such products, contamination with yeast and molds are common and limits the shelf life of such products. "Dairy product" includes, in addition to milk, products derived from milk, such as cream, ice cream, butter, cheese and yogurt, as well as secondary products such as lactoserum and casein and any prepared food containing milk or milk constituents as the main ingredient, such as formula milk. In one preferred embodiment, the dairy product is a fermented dairy product. The term "milk" is understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein/fat solutions made of plant materials, e.g. soy milk.

Concentration of manganese varies in milk, depending on the animal from which it is produced, the feed, as well as the season. In general, manganese is present at a concentration of at least 0.03 ppm in dairy products, for example at least 0.08 ppm for skimmed milk, and at least 0.1 ppm for whole milk. With the present finding of the inventors, reducing the manganese amount in such products or products prepared therefrom would render them more resistant to spoilage.

In one embodiment, the food product is a product prepared by fermentation with thermophiles, i.e. thermophilic fermented food product. The term "thermophile" refers to microorganisms that thrive best at temperatures above 43°C. The industrially most useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 35°C, such as between about 35°C and about 45°C. "Thermophilic fermented food product" refers to fermented food products prepared by thermophilic fermentation of a thermophilic starter culture. Include in such products are for example yogurt, skyr, labneh, lassi, ayran and doogh.

In one embodiment, the food product is a product prepared by fermentation with mesophiles, i.e. mesophilic fermented food product. The term "mesophile" refers to microorganisms that thrive best at moderate temperatures (15°C-40°C). The industrially most useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" herein refers to fermentation at a temperature between about 22°C and about 35°C. "Mesophilic fermented food product," which refers to fermented food products prepared by mesophilic fermentation of a mesophilic starter culture. Included in such products are for example buttermilk, sour milk, cultured milk, smetana, sour cream and fresh cheese, such as quark, tvarog and cream cheese.

Preparation of fermented products

The methods disclosed herein are particularly useful to inhibit or delay yeast and/or mold growth in fermented milk product such as thermophilic and mesophilic fermented milk product, for example a yogurt product. The term "fermented milk product" is a term generally defined in accordance with relevant official regulations and the standards are well known in the field. For example, symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are used as starter culture for yogurt, whereas Lactobacillus acidophilus is used to make acidophilus milk. Other mesophilic lactic acid bacteria are used to produce quark or fromage frais.

The expression "fermented milk product" means a food or feed product wherein the preparation of the food or feed product involves fermentation of a milk base with a lactic acid bacterium. "Fermented milk product" as used herein includes but is not limited to products such as thermophilic fermented milk products (e.g. yogurt) and mesophilic fermented milk products (e.g. sour cream and buttermilk, as well as fermented whey, quark and fromage frais). Fermented milk product also includes 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, Tilsiter, Havarti or Emmental, Swiss cheese, and Maasdamer.

The term "yogurt" has its usual meaning and is generally defined in accordance with relevant official regulations and standards are well known in the field. Starter cultures used for making yogurt comprises at least one Lactobacillus delbrueckii subsp. bulgaricus strain and at least one Streptococcus thermophilus strain. Interestingly, the manganese transporter is not present in L delbrueckii subsp. bulgaricus and only displays low expression in Streptococcus thermophilus, the two strains found in the starter culture in yogurt, making them particularly susceptible to fungal spoilage. It is therefore preferable to include other bacteria strain(s) to scavenging free manganese present in yogurt.

Preferably, free manganese in the fermented product is reduced to a concentration of below about 0.005 ppm.

During food processing chemical preservatives have traditionally been used to avoid fungal spoilage. However, in view of a strong societal demand for less processed and preservative- free foods, the invention contributes to provide an effective solution to manage yeast and mold growth by using biological manganese scavenging agents to reduce the manganese concentration.

When using a biological scavenging agent, the skilled person is able to adjust various parameters such as pH, temperature, and amount of manganese scavenging agent or bacteria to achieve the desired results, taking into consideration the examples provided in this invention as well as the properties of the food product such as water activity, nutrients, level of naturally occurring manganese, shelf life, storage conditions, packing, etc.

The product in which free manganese concentration is reduced is preferably packaged to further limit contact with yeast and mold. It is also preferred to store the product under cold temperature (below 15°C) to help extend shelf life.

For fermented food product, manganese scavenging bacteria may be added before, at the start, or during the fermentation. Depending on parameters chosen, the step of reducing manganese level to a preferred level may take several hours, such as at least 5 hours, such as at least 10 hours, such as at least 15 hours, such as at least 20 hours, such as at least 1 day, 2 days, 3 days or more. A skilled person in the art will be able to choose appropriate parameters, depending on the product where inhibition or delay of fungi is desired.

The invention provides a method of preparing a fermented food product, comprising adding a starter culture and a manganese scavenging agent to a food substrate, fermenting the substrate for a period of time until a target pH is reached. The manganese scavenging agent is preferably a lactobacillus bacteria strain.

As used herein, the term "food substrate" base refers to the substrate in which fermentation is to be carried out. To make fermented dairy products, the food substrate is a milk base. "Milk base" is broadly used in the present invention to refer to a composition based on milk or milk components which can be used as a medium for growth and fermentation of a starter culture. "Milk" generally refers to the lacteal secretion obtained by milking of any mammal, such as cows, sheep, goats, buffaloes or camels. Milk base can be obtained from any raw and/or processed milk material as well as from reconstituted milk powder. Milk base can also be plant-based, i.e. prepared from plant material e.g. soy milk. Milk base prepared from milk or milk components from cows is preferred.

Milk bases include, but are not limited to, solutions/suspensions of any milk or milk like products comprising protein, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk.

Milk base may also be lactose-reduced depending on the need of the consumers. Lactose- reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatography and centrifugation.

To ferment the milk base a starter culture is added. The term "starter" or "starter culture" as used in the present context refers to a culture of one or more food-grade microorganisms in particular lactic acid bacteria, which are responsible for the acidification of the milk base.

The manganese scavenging agent can be added before, at the start, or during the fermentation at the same time or at a different time with the starter culture.

After adding the starter culture and the manganese scavenging agents and subjecting the milk base to a suitable condition, the fermentation process begins and continues for a period of time. A person of ordinary skill in the art knows how to select suitable process conditions, such as temperature, oxygen, addition of carbohydrates, amount and characteristics of microorganism(s) and the process time it takes. This process may take from three, four, five, six hours or longer.

These conditions include the setting of a temperature which is suitable for the particular starter culture strains. For example, when the starter culture comprises mesophilic lactic bacteria, the temperature can be set to about 30°C, and if the culture comprises thermophilic lactic acid bacterial strains, the temperature is kept in the range of about 35°C to 50°C, such as 40°C to 45°C. The setting of the fermentation temperature also depends on the enzyme(s) added to the fermentation which can be readily determined by a person of ordinary skill in the art. In a particular embodiment of the invention the fermentation temperature is between 35°C and 45°C, preferably between 37°C and 43°C, and more preferably between 40°C and 43°C. In another embodiment, the fermentation temperature is between 15°C and 35°C, preferably between 20°C and 35°C, and more preferably between 30°C and 35°C.

Fermentation can be terminated using any methods known to in the art. In general, depending on various parameters of the process, the fermentation can be terminated by making the milk base unsuitable for the strain(s) of the starter culture to grow. For example, termination can be carried out by rapid cooling of the fermented milk product when a target pH is reached. It is known that during fermentation acidification occurs, which leads to the formation of a three- dimensional network consisting of clusters and chains of caseins. The term "target pH" means the pH at which the fermentation step ends. The target pH depends on the fermented milk product to be obtained and can be readily determined by a person of ordinary skill in the art.

In a particular embodiment of the invention, fermentation is carried out until at least a pH of 5.2 is reached, such as 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, the fermentation is carried out until a target pH between 4.0 and 5.0 and more preferably between 4.0 and 4.6 is reached. In a preferred embodiment, the fermentation is carried out until target pH below 4.6 is reached.

In a preferred embodiment, fermented food product is selected from the group consisting of quark, cream cheese, fromage frais, greek yogurt, skyr, labneh, butter milk, sour cream, sour milk, cultured milk, kefir, lassi, ayran, twarog, doogh, smetana, yakult and dahi.

In another preferred embodiment, fermented food product is a cheese, including 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, Tilsiter, Havarti or Emmental, Swiss cheese, and Maasdamer.

In a further embodiment, the method further comprises packing the food product to reduce contact with yeast and mold.

Included in the present invention is a food product obtained by the methods described herein.

The product obtained by the present invention is preferably a fermented milk product with a concentration of free manganese reduced to less than 0.01 ppm after being stored for at least two days, for example at least 3 days, at least 4 days, more preferably at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, and at least 14 days. Other features and advantages of the invention will become apparent from reading the following description in conjunction with the accompanying figures. 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" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g. all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about", where appropriate). The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

EXAMPLES

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this 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 the following examples. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will prevail.

Example 1 Growth of yeasts in different manganese concentration

The example demonstrates manganese requirements of Debaryomyces hansenii in minimal medium. Two D. hansenii isolated from spoiled yogurt (strain 1) and quark (strain 2), respectively, were used. The strains were grown in chemically defined medium with different manganese concentrations.

The minimal medium contains biotin 2 pg/L, calcium pantothenate 400 pg/L, folic acid 2 pg/L, inositol 2 mg/L, nicotinic acid 400 pg/L, p-amino benzoic acid 200 pg/L, pyridoxine 400 pg/L, riboflavin 200 pg/L, thiamine 400 pg/L, Boric acid 500 pg/L, copper sulfate 40 pg/L, potassium iodide 100 pg/L, ferric chloride 200 pg/L, sodium molybdate 200 pg/L, zinc sulfate 400 pg/L, potassium phosphate monobasic 0.5 g/L, potassium phosphate dibasic 0.5 g/L, magnesium sulfate 0.5 g/L, sodium chloride 0.1 g/L, calcium chloride 0.2 g/L, glucose 20 g/L, ammonium sulfate 5 g/L.

Manganese concentration used : 6 ppm, 0.6 ppm, 0.06 ppm, 0.006 ppm, 0.0006 ppm, 0.00006 ppm 0.000006 ppm.

Two different pH were tested : pH 6.5 and 4.5.

The strains were inoculated to 150 pi of the different medium in a 96 well plate. The plates were incubated at 17°C for several days and the growth of the yeast strains was followed by measuring absorbance at 600 nm in a plate reader.

The effect of different pH and different manganese concentrations to the growth of the two different D. hansenii strains are shown in Figure 1 (strain 1) and Figure 2 (strain 2). It can be seen that growth of D. hansenii is inhibited below a concentration of about 0.01 ppm manganese. There was no difference between the two different pH, demonstrating that this mechanism is valid in this pH range. Example 2 Inhibition of yeast in fermented milk products

The example demonstrates manganese requirements of Debaryomyces hansenii in fermented milk products.

Fermented milk products with a starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus ) or additionally with manganese scavenging agents (L. rhamnosus and L.paracasei) were prepared. The fermented milk products were centrifuged (10 min at 5000 rpm) and the supernatant was sterile filtered. The supernatant was transferred to a sterile 96-well plate (150 pi in each well), and manganese was added to the first well to a final concentration of 6 ppm, then a 10-fold serial dilution was performed to result in different manganese concentrations reaching from 6 ppm to 0.000006 ppm. D. hansenii was inoculated to ~20 cells/well and the plates were incubated at 17°C for several days and the growth of the yeast strains was followed by measuring absorbance at 600 nm in a plate reader at day 7.

Figure 3 and 4 show the growth of D. hansenii (strain 1 and strain 2, respectively). Yeast growth after manganese was added to aqueous phase is shown in Figure 3 for strain 1 and Figure 4 for strain 2 (open circles). Average and standard deviation of technical replicates n=6 (A) and n=3 (B) are shown. As comparison, yeast growth is also shown for the reference yogurt aqueous phase where neither manganese scavengers nor additional manganese was added (square). It is noted that the reference has an inherent manganese concentration of 0.03 ppm. For the open circles the x- axis indicates the manganese concentration added, and for the square (the reference yogurt aqueous phase) the x- axis indicates the manganese inherent in the aqueous phase.

This result demonstrates that the growth of the yeast strains in a food matrix depends on manganese. Addition of manganese results in a similar growth to the reference, proving that low manganese concentrations are the major limitation for yeast growth in the fermented milk product. Example 3 Inhibition of Debarvomvces and Rhodoturola

This example shows manganese scavenging activities of the various bacteria and their inhibitory effect against Debaryomyces and Rhodoturola. Inhibitory effect in fermented milk products with low manganese concentration and elevated manganese concentration was evaluated.

Table 4 lists the bacteria added and whether they comprise manganese transporters.

Table 4

The listed bacteria were grown in MRS medium overnight. Preparation: 2 ml milk containing a starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was inoculated with 10 pi of the overnight culture. The milk was fermented at 43°C for about 6 hours until pH 4.5 was reached. The fermented milk was stored in the fridge upon further use. 150 pi of the fermented milk was transferred to individual wells in a 96 well plate in duplicate. To half of the sample manganese was added to obtain an increase of 6 ppm manganese and all the wells were inoculated with about 20 cells of either Debaryomyces hansenii or Rhodoturola mucilaginosa. After 4 days a dilution row was spotted on selective YGC plates to analyze the yeast growth. The yeast growth was was measured by optical inspection, whereas a value of 0 is given for no growth and 5 for complete growth. The average of two biological independent experiments was shown in Table 5 for Debaryomyces and Table 6 for Rhodoturola. Table 5 Inhibition of Debaryomyces

Table 6 Inhibition of Rhodoturola

The results show that the strains that scavenge manganese can be used to inhibiting D. hansenii and R. mucilaginosa, but the inhibition is diminished upon addition of manganese. Example 4 Inhibition of Debarvomvces, Saccharomvces, Rhodoturola, Crvotococcus and Torulasoora

The example evaluates the differences in yeast growth in aqueous phase of fermented milk which is prepared with a starter culture, with and without manganese scavenging agents.

Table 7 lists manganese scavenging agents used : Table 7

This example shows yeast growth under 2 different manganese concentrations. Inhibitory effects towards 6 different yeasts in fermented milk products with low manganese concentration and elevated manganese concentration was evaluated.

Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 90±1°C for 20 min and cooled immediately. A commercial starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus ) was inoculated at 0.02% (v/w) in 3 L buckets. One bucket was inoculated with manganese scavenging agents in total concentration of 100 U/T and one bucket was used as a reference and only inoculated with the starter culture. All buckets were incubated in a water bath at 43± 1°C and fermented at these conditions until pH of 4.60 ± 0.1 was reached. The fermented milk products were divided into 200 ml. bottles and cooled down.

The fermented milk product was then centrifuged (10 min at 5000 rpm) and the supernatant was sterile filtered. The supernatant was transferred to a sterile 96-well plate (150 pi in each well) and to half of the manganese was added to obtain an increase of 6 ppm manganese. Six different yeasts were selected and inoculated about 20 cells per well at grown for 7 days at 17°C. As a control milk fermented with a starter culture alone (reference) was used. The growth was determined by absorbance by measuring at 600 nm.

The average and standard deviation from at least 5 replicates are shown in Table 8 -13. Table 8 Inhibition of Debaryomyces hansenii (Strain 1):

Table 9 Inhibition of Debaryomyces hansenii (Strain 2)

Table 10 Inhibition of Saccharomyces cerevisiae Table 11 Inhibition of Rhodoturola mucilaginosa

Table 12 Inhibition of Cryptococcus fragicola

Table 13 Inhibition of Torulaspora delbrueckii

The results demonstrate that the strains Debaryomyces, Saccharomyces, Rhodoturola, Cryptococcus and Torulaspora can be inhibited by bacteria which scavenge manganese, and the inhibition effect is reduced upon addition of manganese. Example 5 Inhibition of Debarvomvces hansenii

The example evaluates the influence of different manganese concentrations in fermented milk prepared with a starter culture, with and without manganese scavenging agents was tested.

Table 14 lists manganese scavenging agents used : Table 14

Fermented milk product was prepared as in Example 4.

150 pi of the fermented milk was transferred to individual wells in a 96 well plate in duplicate or triplicate. A serial dilution was performed to result in different manganese concentrations added reaching from 6 ppm to 0.000006 ppm. All wells were inoculated with about 20 cells of Debaryomyces hansenii (strain 2) and the plates were incubated at 17°C for 5 days. Afterwards, 10 pi of a 1000-fold dilution, prepared in saline peptone, was spotted on selective YGC plates to analyze the yeast growth.

Figure 5 shows the growth of Debaryomyces hansenii in fermented milk prepared with scavenging agents (No. 1, No. 2 and No. 3) and without scavenging agents (REF), after addition of different manganese concentrations: 6 ppm (top row), 0.6 ppm (row 2), 0.06 ppm (row 3), 0.006 ppm (row 4), 0.0006 ppm (row 5), 0.00006 ppm (row 6), 0.000006 ppm (row 7) and 0 ppm (bottom row).

As shown, yeast growth in fermented milk with scavenging agent(s) No. 1 and No. 2 was inhibited upon addition of 0.6 ppm (compare row 3 to row 2); addition of 0.006 ppm did result in some yeast growth in the present of scavenging agent No. 3.

Example 6 Inhibition of yeast in fermented milk product with different manganese levels

Influence of manganese on the inhibitory effect against different molds were evaluated. An agar-assay resembling the manufacturing process and production of fermented milk products was used. L. rhamnosus and L. paracasei were used together as manganese scavenging agents.

Preparation of fermented milk samples:

Reduced-fat (1.5% w/v) homogenized milk was heat-treated at 90±1°C for 20 min and cooled immediately. A commercial starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus ) was inoculated at 0.02% (v/w) in 3 L buckets. One bucket was inoculated with manganese scavenging agents in total concentration of 100 U/T and one bucket was used as a reference and only inoculated with the starter culture. All buckets were incubated in a water bath at 43± 1°C and fermented at these conditions until pH of 4.60±0.1 was reached. The fermented milk products were divided into 200 ml. bottles and cooled down. Manganese concentration already present in the reference product was previously determined to be about 0.03 ppm and in the product with the scavenging strains below the detection limit of 0.003 ppm.

Addition of manganese:

Different manganese concentrations were added to fermented milk products with and without manganese scavenger to obtain an addition in the levels of manganese (0, 0.006 and 6 ppm manganese in reference products and 0, 0.000006, 0.00006, 0.0006, 0.006, 0.06, 0.6 and 6 ppm manganese).

All the fermented milk samples were warmed to a temperature of 40°C and added 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60°C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.

Challenge test using yeast:

Three target contaminants, including two Debaryomyces hansenii (strain 1 and strain 2) and one Cryptococcus fragicola were spotted in concentrations of 10 ³ , 10 ² and 10 ¹ CFU/spot. Plates were incubated at 7±1°C and regularly examined for the growth of yeast.

Results:

Results of the yeast agar-assay are presented in Figure 6 showing the growth of 3 different yeasts on plates prepared from milk fermented with a starter culture alone (reference, top row) or together with manganese scavengers (bottom row). Different manganese concentrations were added as indicated above the pictures. Three target contaminants (column A: Debaryomyces hansenii (strain 2), column B: Cryptococcus fragicola, column C: Debaryomyces hansenii (strain 1) were added in three different concentrations: 1 x 10 ³ cfu/spot (top row on plate), 1 x 10 ² cfu/spot (middle row on plate) and 1 x 10 ¹ cfu/spot (bottom row on plate).

As can be seen from Figure 6, the tested yeasts grew well on the agar plates made from milk fermented only with the starter culture (reference). However, when manganese scavengers were present during milk fermentation the outgrowth of all the yeasts was delayed.

At manganese levels at up to 0.0006 ppm, the scavenger kept the high inhibitory activity towards all three yeast. At manganese levels between 0.006 ppm and 0.6 ppm the inhibitory activity of scavenger towards C. fragicola was decreased. Manganese concentration of 6 ppm seemed to inhibit the growth of C. fragicola. D. hansenii (strain 1) was inhibited by manganese scavenger at manganese levels up to 0.006 ppm. At manganese levels of 0.06 ppm and above, manganese scavenger lost the inhibitory activity towards D. hansenii (strain 1). D. hansenii (Strain 2) was inhibited by manganese scavenger at manganese levels up to 0.6 ppm and at 6 ppm manganese the activity was lost.

Example 7 Inhibition of mold in fermented milk product with different manganese levels

Fermented milk product samples with different levels of manganese were prepared as described in Example 6.

Challenge test using mold :

Figure 7 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added in concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Figure 8 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added in concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

Figure 9 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added in concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Figure 10 shows the growth of 3 different molds on plates prepared from milk fermented with starter culture alone (reference, top row) or additionally with manganese scavenger (bottom row). Different manganese concentrations were further added as indicated above the pictures. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added in concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

All the tested molds grew very well on the agar plates made from milk fermented only with the starter culture (reference). However, when manganese scavengers were present during milk fermentation, the outgrowth of all the molds tested was inhibited delayed. The inhibitory effect is stronger at lower temperature (Figure 7 and 9).

The manganese scavenger kept the high inhibitory activity towards all molds at additional manganese levels up to 0.006 ppm. At manganese levels from 0.06 ppm and above the inhibitory activity of manganese scavengers was decreased. Manganese scavengers lost most of the inhibitory activity at 6 ppm towards the sensitive (Figure 7 and 8) and at 0.06 ppm towards the robust molds (Figure 9 and 10).

The higher concentration of manganese added which diminishes the inhibitory effect found in this assay compared to the concentrations found in the aqueous phase is due to the continuous uptake by the living and metabolically active manganese scavenger in the fermented milk product.

Example 8 Inhibition of Debaromvces hansenii in aqueous phase and in different concentrations of a chemical chelating material

Two concentrations of manganese were used, 6 ppm and 0.6 ppm, in example 8. The concentration of 6 ppm has shown to have an inhibitory/toxic effect on yeast growth, while 0.6 ppm was used as a standard concentration, which is enough to repeal the manganese deficiency caused by the one or more bacteria strains acting as manganese scavengers.

Figure 12 and 13 show that a chemical chelating material, such as EDTA, has an inhibitory effect on Debaromyces hansenii growth, in aqueous phase. Figure 12 shows that Debaromyces hansenii cells stop growing when faced with a concentration of 0.05 mg/ml of EDTA. Thus, EDTA shows an inhibitory effect at a concentration of 0.05 mg/ml.

Figure 13 shows that a concentration of 0.05 mg/ml EDTA and in the absence of manganese (0.6 ppm) Debaromyces hansenii stop growing; thus, EDTA has an inhibitory effect on Debaromyces hansenii under these conditions. The addition of 0.6 ppm of manganese and 0.05 mg/ml EDTA restores the growth of Debaromyces hansenii and the effect of EDTA is repealed by the excess of manganese.

Example 9 Inhibition of Debarvomvces hansenii in yogurt In yogurt, it was possible to reproduce the inhibition of the manganese scavengers selected from one or more bacteria strains (scavenging agent 1) by adding up to 0.89 mg/ml EDTA, a manganese scavenger, to the yogurt. At said concentration, the reference yogurt showed inhibition similar to one observed when manganese scavenging agent 1 is added. The negative controls in bottom row of Figure 14 (no EDTA added) show normal inhibition in the presence of manganese scavenger 1 and restored yeast growth with added manganese.

Example 9 shows that a chemical chelating material, such as EDTA, has the same effect as manganese scavengers selected from one or more bacteria herein disclosed (Figure 14). Hence, example 9 demonstrates that EDTA has the same effect as the one or more bacteria strains herein disclosed as manganese scavengers.

Manganese scavengers, either a chemical chelating material and/or a biological material, such as one or more bacteria strains, when present in a given product, such as a food product, lead to manganese depletion for the spoilage fungi, thereby inhibiting fungi growth.