EA043250B1 - INHIBITION OF FUNGI GROWTH DUE TO MANGANESE DEFINITION - Google Patents

Description

Field of invention

The invention belongs to the field of microbiology and relates to methods for controlling fungal spoilage. The invention also relates to food products and methods for their preparation.

Prior Art

A major problem in the food industry is spoilage by unwanted microorganisms. According to the Food and Agriculture Organization of the United Nations (FAO), one in four calories intended for human consumption is ultimately not consumed by them. At a time of food insecurity, when more than 800 million people suffer from hunger, the issue of food waste has become a priority issue for global government officials and food producers. In addition to negative social and economic impacts on society, tainted food also leads to a range of associated environmental impacts, including unnecessary greenhouse gas emissions and inefficient use of scarce resources such as water and land.

Yeasts and molds are very important in causing food spoilage and are a problem for most food manufacturers. Spoilage due to yeasts and molds is clearly visible as mold spots or discoloration on the surface of the food, allowing it to be removed prior to consumption. Yeasts tend to grow inside food and drink matrices in planktonic form, and they tend to ferment sugars and grow well under anaerobic conditions. Conversely, molds tend to grow on the surface of foods in the form of a visible mycelium composed of cells.

In particular, in the dairy sector, 29 million tons of dairy products are wasted every year in Europe. One of the major challenges in keeping dairy products fresh is dealing with yeast and mold contamination that is found everywhere in nature, especially when the cold chain from production to the consumer's table is disrupted.

For economic and environmental reasons, there is a continuing need for new or improved methods that are effective for controlling yeast and mold contamination.

Brief summary of the invention

The present inventors attempted to find effective ways to control fungal contamination and surprisingly identified manganese as a significant inhibitor of fungal growth. The present invention provides a novel method for inhibiting fungal growth by limiting the free manganese available to yeasts or molds. The present invention is based in part on the surprising discovery that by reducing the level of free manganese in a food, for example by removing free manganese using manganese scavenging agents, yeast and/or mold contamination can be reduced or delayed. In addition, the inventors have shown that conventional yeasts and molds are sensitive to the methods described here.

Manganese is considered vital to human health and thus an essential micronutrient. Manganese is indispensable for the proper functioning of both humans and animals, since it is essential for the functioning of many cellular enzymes such as manganese superoxide dismutase, pyruvate carboxylase, and it can serve to activate many other enzymes such as kinases, decarboxylases, transferases, and hydrolases.

Manganese can be found naturally in many food sources, including leafy vegetables, nuts, grains, and animal products. Typical concentration ranges for manganese in common foods are, for example, 0.4-40 ^ppm in cereals, 0.1-4 ^ppm in meat, poultry, fish and eggs, 0.4-7 ^ppm in vegetable products.

In addition to food additives, manganese is sometimes added to fermented foods as an active ingredient to increase the growth of bifidobacteria in milk (see eg WO2017/021754, Compagnie Gervais Danone, France). However, the inventors have found that this can have an adverse effect, and it would be beneficial to limit the concentration of manganese in foods to prevent or retard the growth of undesirable microorganisms.

To combat the problem of microbial spoilage, the present invention provides in a first aspect a method for inhibiting or delaying the growth of fungus(s) in a product, comprising the step of reducing the level of free manganese present in said product. The concentration of free manganese can be reduced using the methods described in this invention. In a preferred embodiment, one or more manganese scavenging agents are added to reduce the level of free manganese. The free manganese concentration is preferably reduced to less than about 0.01 ^ppm , such as less than about 0.008 ppm or less than about 0.003 ^ppm . Using this method, it is possible to obtain a product in which unwanted yeasts and/or molds are unlikely to grow successfully. This product has a free manganese concentration of less than about 0.01 ^ppm , such as 0.009 ^ppm , 0.008 ^ppm , 0.007 ^ppm , 0.006, 0.005 ^ppm or less. This method further includes the step of measuring the concentration of free manganese in the product and obtaining a value of less than about

0.01 ^ppm .

In particular, the present invention provides a method for inhibiting or inhibiting the growth of yeasts and/or molds in a fermented food product made from milk, such as yogurt or cheese. The method is characterized by the step of reducing the concentration of manganese in the food in order to deprive yeasts and/or molds of manganese and thus delay or suppress their growth in the food.

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

In a second aspect, the present invention provides a process for preparing a product, such as a food product, comprising reducing the level of free manganese present in said product. The concentration of free manganese can be reduced using the methods described in this invention, or using other methods known to experts in this field of technology. In a preferred embodiment, one or more manganese scavenging agents are added to reduce free manganese levels. The free manganese concentration is preferably reduced to less than about 0.01 ^ppm , such as less than about 0.005 ^ppm or less than about 0.003 ^ppm. Using this method, a product containing a concentration of free manganese of less than about 0.01 ^ppm can be obtained.

In a third aspect, the present invention provides products, such as foodstuffs, obtained by the methods described herein. In one of the embodiments, the present invention relates to a method for obtaining a product, including the steps of reducing the content of free manganese in the product and obtaining a product in which the concentration of free manganese is less than about 0.01 million ^-1 .

In yet another aspect, the present invention provides the use of one or more manganese scavenging agents to inhibit or retard the growth of fungi, as well as for food preparation. Manganese scavenging agents have the effect of reducing the level of free manganese available in the product to yeasts and molds, thus inhibiting or delaying their growth.

In yet another aspect, the present invention provides manganese scavenging agents, their selection, and their use for manganese scavenging.

Brief description of graphic materials

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

Fig. 3 and 4 show the growth of 2 different Debaryomyces hansenii in the aqueous phase of fermented milk in the presence of manganese scavenging agents and with the addition of various concentrations of manganese. Absorbance at 600 nm was measured after 7 days of incubation at 17°C. Growth after addition of manganese to the aqueous phase is shown in FIG. 3 for Debaryomyces hansenii (strain 1) and FIG. 4 for Debaryomyces hansenii (strain 2) (open circles) compared with no additional manganese added (square). Shown are the mean and standard deviation for technical repetitions n=6 (A) and n=3 (B).

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

Fig. 6 shows the growth of 3 different yeast species on plates prepared using milk fermented with starter culture alone (control, top row) or together with a manganese scavenging agent (bottom row). Various concentrations of manganese were added as shown in the graphics above. Three target contaminants (column A: Debaryomyces hansenii (strain 2), column B: Cryptococcus fragicola, column C: Debaryomyces hansenii (strain 1) were added at three different concentrations: 1x103 cfu/spot (top row on plate), ^1x102 cfu/ spot (middle row on plate) and 1x101 CFU/spot (bottom row on plate).

Fig. 7 shows the growth of 3 different mold species in plates prepared using milk fermented with starter culture alone (control, top row) or together with a manganese scavenging agent (bottom row). Various concentrations of manganese were added as shown in the graphics above. Three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added at concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Fig. 8 shows the growth of 3 different types of molds on plates prepared using milk fermented with starter culture only (control, top row) or

- 2 043250 together with an agent that absorbs manganese (bottom row). Various concentrations of manganese were added as shown in the graphics above. The three target contaminants (A: Penicillium brevicompactum, B: Penicillium crustosum, and C: Penicillium solitum) were added at concentrations

500 spores/spot. The plates were incubated at 22±1°C for 8 days.

Fig. 9 shows the growth of 3 different mold species in plates prepared using milk fermented with starter culture alone (control, top row) or together with a manganese scavenging agent (bottom row). Next, various concentrations of manganese were added, as shown above. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added at concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Fig. 10 shows the growth of 3 different mold strains on plates prepared with milk fermented with starter culture alone (control, top row) or additionally with a manganese scavenging agent (bottom row). Additionally, various concentrations of manganese were added, as presented above. Three target contaminants (A: Penicillium carneum, B: Penicillium paneum and C: Penicillium roqueforti) were added at concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

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

In FIG. 12 shows the growth of a strain of Debaryomyces hansenii (strain 1 or strain 2) in an aqueous phase filtered through a 1.2 µm filter (water filtration), preferably using a fermented milk aqueous phase, with various concentrations of EDTA (ethylenediaminetetraacetic acid), with other agents, absorbing manganese, and without them, preferably with 1 agent, absorbing manganese, and 6 million ^-1 manganese and without him. The absorbance at 600 nm was measured after 7 days of incubation at 17°C compared to the reference sample. Samples were analyzed in triplicate.

In FIG. 13 shows the growth of a strain of Debaryomyces hansenii (strain 1 or strain 2) in an aqueous phase filtered through a 0.2 μm filter (aqueous sterile filtration), preferably using a fermented milk aqueous phase with various concentrations of EDTA, with other manganese scavenging agents, preferably with 1 manganese scavenging agent and with and without 6 ^ppm manganese. The absorbance at 600 nm was measured after 7 days of incubation at 17°C compared to the reference sample. Samples were analyzed in triplicate.

In FIG. 14 shows the growth of a strain of Debaryomyces hansenii (strain 1 or strain 2) in yogurt with and without various manganese scavenging agents such as bacteria, preferably lactic acid bacteria, and/or a chemical chelating agent such as EDTA after addition of 6 ^{ppm 1} manganese. EDTA concentration 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 description of the invention

Food loss is a major cause of concern worldwide - approximately one third of all food produced for human consumption is either lost or wasted. The causes of this massive global food loss are varied, but the main role is played by bacterial spoilage, which affects the quality of organoleptic products (aspect, texture, taste and aroma). Because fungi can grow in a variety of, and even harsh, environmental conditions, they are the main spoilage microorganisms found throughout the food chain. Therefore, it is essential to reduce food waste by controlling fungal contamination.

In response to this need, the present invention provides a new method for inhibiting or delaying the growth of fungi in a product. This method is based on the unexpected finding that low concentrations of free manganese can act as a limiting factor for the growth of yeasts and/or molds. Manganese is present in trace amounts in nature and in many of our consumer products. However, so far there have been no reports indicating that by manipulating the concentration of free manganese, bacterial spoilage can be effectively controlled. Based on this surprising finding, it is believed that this spoilage prevention strategy is applicable even outside of food and extends to other products that are commonly subject to microbial contamination, such as feed products, biological products, medical products, pharmaceutical products, and the like.

In a first aspect of the present invention, there is provided a method for inhibiting or delaying the growth of fungi in a product, comprising reducing the level of free manganese in said product to a concentration of less than about 0.01 ^ppm .

In general, inhibition means the reduction, partial or complete, of the function and activity of cells or microorganisms. As used herein, suppress and suppress against yeasts and molds mean that the growth, amount or concentration of yeasts and molds are the same or reduced. The latter can be measured using any

- 3 043250 methods known in the field of microbiology. Suppression can be observed by comparing the fungal growth, amount or concentration in/on the reduced free manganese product with a control. The control may be the same product but without the reduction in free manganese.

The term slowing down generally means the act of stopping, delaying, preventing or causing something to happen more slowly than usual. The term fungal growth retardation as used herein refers to delaying the growth of fungi. This can be observed by comparing the time required for fungi to grow to a given level in two products, one with reduced manganese and the other without (but otherwise the same).

In some embodiments, fungal growth retardation refers to a 7 day growth retardation 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 of the fungi kingdom. Fungal growth can be measured using various methods known to the person skilled in the art. For example, fungal growth can be measured by colony density or size, cell number, changes in mycelial mass, spore production, hyphae growth, colony forming units (CFU), and the like. depending on the type of fungus and the product for 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. The terms fungal growth suppression or fungal growth suppression refer to the suppression of fungal cell proliferation. The terms fungal growth retardation or fungal growth retardation refer to the inhibition of fungal cell proliferation. The latter can be observed, for example, by measuring the growth of the fungus and comparing it with a control. Such a control may be, for example, a product without the use of manganese scavenging agents. Methods for determining inhibition or inhibition of fungal growth are known to those skilled in the art.

Free manganese or sometimes manganese in accordance with the present invention refers to manganese that is present in the product (i.e. forms part of the product, for example inside the product or on the surface of the product), which can be taken up by fungi, including yeasts and moulds. For example, free manganese refers to manganese that is present in the food matrix of a product.

In one of the preferred embodiments, the present invention relates to a method for inhibiting or delaying the growth of fungi in a food product, including reducing the concentration of free manganese in the food matrix of the food product. As used herein, the term food matrix refers to the composition and structure of a food product. It is based on the concept that nutrients are enclosed in a continuous medium.

The term reduce or reduce generally means reducing the amount of a substance in this context. The term free manganese reduction as used herein, or free manganese reduction, means a reduction in the amount of manganese present in a product that may be available for uptake by fungi, including yeasts and moulds.

For example, the latter can be done by removing the manganese present in the product or in the material that is to become part of the product. For example, the latter can be done by subjecting the starting material to ion exchange chromatography to remove manganese such that the concentration in the final product is reduced.

Once accessed, the fungi quickly colonize, increase in numbers, and absorb nutrients from their immediate environment. In some embodiments, given that the fungus may first come into contact with the product on the surface, it is in the spirit of the present invention that the reduction step is carried out on parts of the product, for example on an external part of the product, such as a coating or outer layer. However, in such cases the reduction step results in an overall decrease in product concentration.

As used herein, the manganese concentration or manganese level is expressed in parts per million ( ^ppm ) calculated on a mass/mass basis. Reducing the level of free manganese in the product to a concentration below a given value means reducing the level of free manganese in the product or parts thereof in such a way that the concentration of free manganese in the entire product, by weight, decreases. Methods for determining 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.

When applying the methods in accordance with the present invention, a person skilled in the art can first determine the level of manganese that is present in the products to be processed. Manganese concentrations in food are well studied and can be found in national food composition databases such as the Danish Food Composition Database, Canadian Nutrient Database. As a rule, manganese is present in a concentration of at least 0.03 ^ppm for milk, which makes dairy products susceptible to fungal contamination. Manganese levels have been reported to range from 0.04 to 0.1 ^ppm in cow's milk and up to 0.18 ^ppm in goat or sheep's milk (Muehlhoff et al., Milk and dairy products in human nutrition, Food and Agriculture Organization of the United Nations (FAO), 2013). For fermented milk products such as cheese, manganese levels are typically increased by the concentration process from milk, often up to 10 times or more. Various levels have been reported for different types of cheese, for example approximately 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 house cheese, 0.68 ^ppm for gouda and 0.74 ^ppm for cheddar cheese (Smit, LE, 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).

The free manganese in the product is preferably reduced to a concentration of less than about 0.01 ^ppm , such as less than about 0.009 ^ppm , less than about 0.008 ^ppm , less than about 0.007 ^ppm , less than about 0.006 ppm ^{. 1} , less than about 0.005 ^ppm , less than about 0.004 ^ppm , less than about 0.003 ^ppm , less than about 0.002 ^ppm , less than about 0.001 ^ppm , less than about 0.0009 ^{ppm 1} , less than about 0.0008 ^ppm , less than about 0.0007 ^ppm , less than about 0.0006 ^ppm , less than about 0.0005 ^ppm , less than about 0.0004 ^{ppm 1} less than about 0.0003 ^ppm or less.

The term used here roughly indicates that the values are slightly outside the quoted values, that is, plus or minus 0.1% to 10%. Thus, concentrations slightly outside these limits are also covered by the scope of the present invention.

In one embodiment, the present invention provides a method for inhibiting or delaying the growth of fungi in a product, preferably a food product, comprising the steps of reducing the content of free manganese in the product and obtaining a product in which the concentration of free manganese is less than about 0.01 ^ppm in the product. .

The method according to the present invention further includes the step of measuring the concentration of free manganese. The latter can be done after the reduction step to determine if the concentration of free manganese has been reduced. In one embodiment, the present invention provides a method for inhibiting or delaying the growth of fungi in a food product, comprising reducing the level of free manganese in the product to a concentration of less than about 0.01 ^ppm in the product and measuring the free manganese content in the product and optionally reaching a value of less than 0.01 ^ppm .

In one embodiment, the present invention provides a method for inhibiting or delaying the growth of fungi in a product, comprising the steps of:

reducing the content of free manganese in the product to a concentration of less than about 0.01 ^ppm in the product, measuring the concentration of free manganese in the product and achieving a value of less than 0.01 ^ppm .

Methods for measuring low levels of manganese are well known to those skilled in the art. Such methods include atomic absorption spectroscopy, atomic emission spectroscopy, mass spectrometry, neutron activation analysis, and X-ray fluorometry (see, for example, Williams et al. Toxicological profile for manganese. (2012)).

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

Mushroom.

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

In one embodiment, this method is used to 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.

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In one embodiment, this method is used to inhibit the growth of molds. Preferably, the fungus is a mold selected from the group consisting of Aspergillus spp.,

Cladosporium spp., Didymella spp. or Penicillium spp. Preferably, the fungus is a mold selected from the group consisting of Penicillium brevicompactum, Penicillium crustosum, Penicillium solitum, Penicillium carneum, Penicillium paneum and Penicillium roqueforti.

removal of manganese.

Methods for removing manganese are known in the art. Manganese is a common contaminant in many mine waters, ground waters and fresh waters. In wastewater treatment, manganese ions can be chemically removed from wastewater by oxidation to MnO ₂ , adsorption or precipitation as carbonate.

Alternatively, manganese removal may involve biological processes as alternatives to chemical routes. The role of microbial activity in the treatment of manganese-contaminated waters is described in various sources, for example 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 the level of free manganese in the product includes the use of ion exchange chromatography. The latter is particularly applicable if the product is in a liquid or is essentially liquid.

In one preferred embodiment, the step of reducing the level of free manganese in the product is carried out by adding a manganese scavenging agent. The terms manganese scavenging agent or manganese scavenger as used herein refer to a material that is capable of rendering manganese unavailable to yeasts or molds. The material may be a chemical, such as a chemical chelating agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis-(β-aminoethyl ether)-^^Ni,Ni-tetraacetic acid (EGTA), diaminocyclohexanetetraacetic acid (DCTA), nitrilotriacetic acid (NTA), 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or diethylenetriaminepentaacetic acid (DTPA), preferably the chemical chelating agent is is ethylenediaminetetraacetic acid (EDTA). The material may also be biological material such as bacteria.

In one preferred embodiment, the manganese scavenging agent is one or more bacterial strains. In such cases, it should be noted that when measuring free manganese, such free manganese does not include manganese that is found intracellularly. Rather, free manganese refers to manganese that is found extracellularly, that is, in the cell-free parts of the product, as these can be available for uptake by fungi. Thus, in such cases, the concentration of free manganese should be measured, taking into account only extracellular manganese. The latter can be done, for example, by removing cells (such as seed cultures) by centrifugation and obtaining a cell-free supernatant, followed by measurement of manganese in the cell-free supernatant. As used herein, the term bacterial strain has a general meaning in the field of microbiology and refers to a genetic variant of a bacterium.

In one embodiment, the present invention provides a method for inhibiting or delaying the growth of fungi in a product, comprising the steps of selecting one or more bacterial strains as ^the manganese scavenging agent and reducing the level of free manganese in the product to a concentration below about 0.03 ppm ¹ in the product by adding a manganese scavenging agent.

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

Manganese is involved in many important biological processes and is ubiquitous in all organisms. Manganese also contributes to protection against oxidative stress and may also contribute to the catalytic detoxification of reactive oxygen species. Many bacteria have developed sophisticated collection systems to capture essential metals from the environment, using low and high affinity transfer systems for chelated or free metals. Manganese, which is taken up by bacteria, forms a large complex of non-dialyzable polyphosphate protein aggregates in the protein, which can reach very high intracellular concentrations.

Transport systems for manganese 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.

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In one embodiment, the bacterial strain having manganese uptake activities includes bacterial Mn ²⁺ transporters. The Mn ²⁺ transporters may be an ABC transporter (eg, SitABCD and YfeABCD) or a proton-dependent Nramp-associated transport system belonging to the family designated TC#3.A.1.15 and TC#2.A.55 in the carrier classification system given by base vector classification data (M. Saier; U of CA, San Diego, Saier MH, Reddy VS, Tamang DG, Vastermark A. (2014)). The TC system is a classification system for carrier proteins that is similar to the Enzyme Commission (EC) system for classifying enzymes. The Carrier Classification System (TC) is the approved nomenclature system for classifying carrier proteins by the International Union of Biochemistry and Molecular Biology. TCDB is freely available at http://www.tcdb.org, which offers several different ways to access data, including incremental access to hierarchical classification, direct search by vehicle sequence or number, and full-text search.

In one embodiment, the method includes selecting a bacterial strain containing a protein belonging to the family designated TC#3.A. 1.15 (manganese chelate trapping (MZT) transporter family) as a manganese scavenging agent.

For example, the manganese scavenging agent is a bacterial strain containing a carrier scavenging manganese chelate designated as TC#3.A.1.15.2, TC#3.A.1.15.6, TC#3.A.1.15.8 , ТС#3.А.1.15.14, or its functional variants.

Although the ABC transporter is primarily active at higher pH, proton-driven transporters may be more active in acidic conditions. The latter makes them particularly useful as manganese scavenging agents in fermented foods. Thus, in one of the embodiments, a bacterial strain is selected containing a protein belonging to the family designated as TC#2.A.55 (the family of metal ion transporters (Mn ²⁺ -iron) (Nramp)).

The step of selecting one or more bacterial strains as the manganese scavenging agent includes determining whether the one or more bacterial strains contain a manganese carrier, designated TC#2.A.55, or functional variants thereof.

More preferably, the carrier is of the subfamily designated TC#2.A.55.2 or the subfamily designated TC#2.A.55.3 as a manganese scavenging agent.

For example, the manganese scavenging agent is a bacterial strain containing a metal ion transporter (Mn ⁺ -iron) (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 a manganese scavenging agent.

Most preferably, the method comprises selecting a bacterial strain containing the protein designated TC#2.A.55.2.6 or functional variants thereof as the 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 substantially similar biological activity, ie. manganese uptake activities.

The term variant as used herein refers to a protein form variant having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a particular nucleic acid sequence, or the amino acid sequence of the protein.

The invention further provides manganese transporter polypeptide sequences for selecting suitable manganese scavenging agents for the practice of the present invention.

In one preferred embodiment, the manganese scavenging agent is a bacterial strain containing a polypeptide having the sequence according to SEQ ID NO: 1 (MASEDKKSKREHIIHFEDTPSKSLDEVNGSVEVPHNAGFWKTLAAYTGPGILVAVGYM DPGNWITSIAGGASFKYSLLSVILISSLIAMLLQAMAARLGIVTGRDLAQMTRDHTSKAM GGFLWVITELAIMATDIAEII GSAIALKLLFNMPLIVGIIITTADVLILLLLMRLGFRKIEAV VATLVLVILLVFAYEVILAQPNVPELLKGYLPHADIVTNKSMLYLSLGIVGATVMPHDLF LGSSISQTRKIDRTKHEEVKKAIKFSTIDSNLTMAFIVNSLLLILGAALFFGTSSSVGRF VDLFNALSNSQIVGAIASPMLSMLFAVALLASGQSSTITGTLAGQIIMEGFI HLKMPLWA QRLLTRLMSVTPVLIFAIYYHGNEAKIENLLTFSQVFLSIALPFAVIPLVLYTSDKKIMGEF ANRAWVKWTAWFISGVLIILNLYLIAQTLGFVK) or its functional variants.

In other preferred embodiments, the manganese scavenging agent is a bacterial strain containing a polypeptide having at least 55% sequence identity, such as at least 60, 65, 70, 75, 80, 85, 90, 95 , 96, 97, 98, 99 or 100% identity with the sequence according to SEQ ID NO: 1.

In table. 1 shows examples of sequences that encode functional variants of SEQ ID NO: 1 and their sequence identity with SEQ ID NO: 1.

Table 1

Origin SEQ ID NO Squirrel ID % identi PARTICULARS Subsequence Lactobacillus casei 4 WP 013245 308.1 99.8 MASEDKKSKREHIIHFEDTPSKSLDEVN GSVEVPHNAGFWKTLAAYTVPGILVAV GYMDPGNWITSIAGGASFKYSLLSVILIS SLIAMLLQAMAARLGIVTGRDLAQMTR DHT SK AMGGFLW VITEL AIMATDIAEII GSAIALKLLFNMPLIVGIIITTADVLILLL LMRLGFRKIEAVVATLVLVILLVFAYEV ILAQ PNVPELLKGYLPHADIVTNKSMLY LSLGIVGATVMPHDLFLGSSISQTRKIDR TKHEEVKKAIKF STID SNLQLTMAFIVN SLLLILGAALFFGTSSSVGRFVDLFNALS NSQIVGAIASPMLSMLFAVALLASGQSS TITGTLAGQIIMEGFIHLKMPLWAQRLL TRLMSVTPVLIFAIYYHGNEAKIENLLTF SQVFLSIALPFAVIPLVLYTSDKKIMGEF ANRAWVKWTAWFISGVLIILNLYLIAQT LGFVK Lactobacillus brevis 5 WP011667 125.1 76.5 MKNHETDTKTKHHMIESTGSGQKSLDE VNGTVEVPQNAGFWRTLMAYTGPGALI AVGYMDPGNWITSIAGGAQYKYTLLTV VLLS SLVAMLLQ AMSARLGIVTTGKDLA QLTREHTGKRTGFALWnTELAIMATDI AEIIGSAIALKLLFGFPLIVGIIITAMDVL VLLVLMKLGFRKIEAIVATLVAVILFVF LY EVILAQPHMGEVLKGYLPS STVVTN HGMLYLSLGIVGATVMPHDLYLGS SISQ TRSFDRI<NRI<SVAQAH<FTTIDSNIQLTL AFVVNSLLLILGAALFFGTNSDLGRFVD LFNALSDSQIVGAIASPMLSMLFALALL S SGQS STITGTLAGQIIMEGFINLKMPLW AQRLITRLLSVTPVIIFAIIYHGN EAKIED LLTFSQVFLSIALPFAMIPLVIFTSSKKLM GEFANRTWSKILGWIIAVILIILNIYLILN TLHIVQ Pediococcus acidilactici 6 WP_070366 435.1 76.4 MSKKLDEVDNKSLDE1NGS1KVPKNAG FFKTLMAYTGPGILIAVGYMDPGNWITS IAGGAQFK YTLLS VVLIS SLIAMLLQ AM SARLGIVTTGKDLAQLTRERTSKRVGFM LWVVAELAIMATDIAEIIGSGIALELLFH IPLnGILITAADVLILLLLMRLGFRKIEAI VATLVMVILIVFAYEVFLSDPSISGIIKG Y VPAPVILQNNSMLYLSLGIVGATVMP

- 8 043250

HDLYLGS SISQTREIDRRDRKNVAQAIR FSTIDSNMQLFLAFIVNSLLLILGAALFY GTDSSLGRFVDLFNALSDNQIVGAIASP MLSMLFAVALLASGQSSTITGTLSGQII MEGFIRLRVPLWVQRLVTRLLSVAPVLI FAIYYHGDEAKIENLLTFSQVFLSVALPF AVIPLVMYTS SKKLMGEF ANRQWVKW CAW IATIILILLNIYLILQTLGIVK Lactobacillus plantarum 7 YP_0048883 16.1 74.3 MKSAKTKDHAKMKAAEEKAIHSTGAD SKSLDEVNGSVRVPKDASFWRTLIAYT GPGALVAVGYMDPGNWITSIAGGSQYK YALLSVTLLSSLTAMLLQAMAARLGTVT GKDLAQLTRERTSKGMGIFLWIITELAI MATDVAEIIGSGIALKLLFGFPLIVGILIT TADVLILLLLMKLGFRKIEAIVATLVAVI LFVFLYEVI ISQPNIPEMLKGYVPTSRIVS NRSMLFLALGIVGATVMPHNLYLGS SIS QTRQVDRSDEI<EVAI<AVI<FTTIDSNIQL SVAFVVNSLLLILGAALFFGTKGDLGRF VDLYNALGDSKVVGSIASPLLSMLFAIA LLSSGQSSTITGTLSGQIIMEGFIRLKMPL WAQRLLTRLISVTPVLAFAIYYHGNEAK IEDLLTMSQVFLSIALPFAMIPLVMFTSN RALMGNFTNRVWVKWTAWIVTVILIIL NIYLILQTVGLVK Lactobacillus sakei 8 WP_089535 161.1 67.9 MHYADGS SLEEINNT VAIPKNAGF WKT LMAFMGPGALVAVGYMDPGNWITSIA GGAQFAYTLISVILVSNLIAMLLQAMAA RLGIVTTGMDLAQMTRAKTGKKMGIFL WIVTELAIMATDIAEIIGSAIALELIFNIPL LWGVLITAFDVLLLLLLMKLGFRKIEAI VATLVAVILFVFLYEVILAQPNMGDVV RGFVPSPRIMTDKKMLFLALGIVGATV MPHNLYLHS SIAQ ARQYDRDDVAEKRK AIKFTVIDSNIQLTIAFVVNCLLLILGAA MFYGTNSDLGRFVDLFNALQNKEIVGSI ASPMLSLLFAVALLASGQNSTITGTLSG QIVMEGFVRMKIPLWARRVITRGLSILP VIIFTVYYHGNEAQVENLLIYSQVFLSIA LPVSMIPLTLFTSDEKIMGPFVNRPWVK YTAWFVTIVLTLLNIYLILQTVGLAA Lactobacillus alimentarius 9 WP_057737 113.1 68.3 MSSKNKKHESLIHYANGPSLEEINDTVE IPKDAGFFKTLLAYSGPGALVAVGYMD PGNWVTSIAGGAQFKYKLLS VILIS SLIA MLLQYMSAKLGIVTGRDLAQLTRDRTS RVGGFILWIITELAIMATDIAEIIGSAIAL KLLFNIPVLWGVIITAFDVLLLLVLMKL GFRKIEAIVATLIMVILLVFLYEVILAKP DV GQMMVGFIPEPKILQNQSMLYLSLGI VGATVMPHNLYLHS SISQ ARKYDRDDP KSIHQAVRFSTWDSNIQLTLAFVVNTLL LLLGAALFYGTSSDLGRFVDLFNALQDP KVAGAVASPVLSILFAVALLASGQNSTI TGTLSGQIVMEGFIHMKMKLWARRVIT RLMSIIPVITFAIIYHGNEAKIESLLTFS Q VFLSVALPFSIFPLIKFTSNKKLMGEFVN NKLVEYIGYFVAIVLTILNIWLIYTTFVP TA Lactobacillus floricola 10 WP_056974 015.1 65.1 MTKEETKLFHYADGPSLEEINGTVAVP KKGGFWKTLFAFSGPGALVAVGYMDP GNWVTSIAGGAQYQYTLLS VILIS SLIA MLLQAMSARLGIASGLDLAQATAKHSP KWLRYTLWIITELAIMATDIAEIVGAAIA LKLLFNLPLIVGIFLTTLDVMLLLLLMK

- 9 043250

LLLGAALF YGAQTDLGTF SELYNALQN PQVAGVIASPILSVLFAVALLASGQNSTI TGTLSGQIVMEGFIHLKMPAR RVIT RLIS V IP VLIF A11YH SNE A КIEDLL VF SQ VFLSIALPVSIIPLVMFTANKKIMGPFVN KKWVTITS SLVAIILTGLNIFLILQTLGW VQ Lactobacillus brevis AND WP011667 396.1 57.6 MTDNVSAKSVQGDLTNGPSLAEINGSV RVPKEKGFVRNLLAFSGPGALVAVGYM DPGNWVTSIGGGAQ YGYLLMS VILMS S LIAMLLQYMAAKLGIVTQMDLARATR AHTGKRIGAVLWVMTELAIMATDIAEV IGGAIALKLLFGVPLILGVSLTVLDVLLL LLLTRLGFRKIEALCLILVILVV FAYE VVIAQPSMGQAVASFVPQAEIMRPGQL TMALGIVGATVMPHNLYLHS SIAQTRK FDRQDPAEMARAVKFTAWD SNIQLFGA FIINCLLLLGAAMFFGKDAGALGTFGQ LYDALQDNRLAGAVASPVLSTLFAVAL LASGQNSTITGTLTGQVIMEGFINMRLPI WVRRLVTRLISVAPVUVTILYGSE QAL DRLLVNSQVFLSIALPFSMIPLTIFTSSKR IMGTRWVNRWWVTALAWGCTAILTVL NIQIVWATMTTLF

In one preferred embodiment, the manganese scavenging agent is a bacterial strain containing a polypeptide having the sequence according to SEQ ID NO: 2 (MARPDERLTVQREKRSLDDINRSVQVPSVYESSFFQKFLAYSGPGALVAVGYMDPGN

WLTALEGGSRYHYALLSVLLMSILVAMFMQTLAIKLGVVARLDLAQAIAAFIPNWSRIC LWLINEAAMMATDMTGVVGTAIALKLLFGLPLMWGMLLTIADVLVVLLFLRFGIRRIEL

IVLVSILTVGIIFGIEVARADPSIGGIAGGFVPHTDILTNHGMLLLSLGIMGATIMPHNIYLH SSLAQSRKYDEHIPAQVTEALRFGKWDSNVHLVAAFLINALLLILGAALFYGVGGHVTA FQGAYNGLKNPMIVGGLASPLMSTLFAFALLITGLISSIASTLAGQIVMEGYLNIRMPLW ERRLLTRLVTLIPIMVIGFMIGFSEHN FEQVIVYAQVSLSIALPFTLFPLVALTNRRDLMGI HVNSQLVRWVGYFLTGVITVLNIQLAISVFV) or its functional variants.

In other preferred embodiments, the manganese scavenging agent is a bacterial strain containing a polypeptide having at least 55% sequence identity, such as at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98, 99 or 100% identity with the sequence according to SEQ ID NO: 2.

In table. 2 shows examples of sequences that encode functional variants of SEQ ID NO: 2 and their sequence identity with SEQ ID NO: 2.

table 2

Origin SEQ ID NO Squirrel ID % identi PARTICULARS Subsequence Lactobacillus casei 12 WP_0124917 67.1 99.3 MARPDERLTVQREKRSLDDINRSVQVP S VYES SFFQKFLAYSGPGALVAVGYM DPGNWLTALEGGSRYHYALLSVLLMSI LVAMFMQTLAIKLGVARLDLAQAIA AFIPHWSRICLWLINEAAMMATDMTG VVGTAIALKLLFGLPLMWGMLLTIADV LVVLLFLRFGIRRVELIVLVSILTVGIIFG lEVARAD PSIGGIAGGFVPHTDILTNHG MLLLSLGIMGATIMPHNIYLHSSLAQSR KYDEHIP AQ VTE ALRFGKWD SNVHLV AAFLINALLLILGAALFYGVGGHVTAF QGVYNGLKNPMIVGGLASPLMSTLFAF ALLITGLIS SIASTLAGQIVMEGYLNIRM P LW ERR LLTR L VTLIPIMVIGFMIGF SEH NFEQ VIVYAQVSLSIALPFTLFPLVALT

- 10 043250

NRRDLMGIHVNSQLVRWVGYFLTGVI TVLNIQLAISVFV Lactobacillus rhamnosus 13 WP_0057128 42.1 85.6 GI EVVRARPSMGGIVAGLVPHTEILTNR GMLLLSLGIMGATIMPHNIYLHSSLAQS RRYDEHIPAQVTEALRFGKWDSNVHL VAAFIINALLLILGATLF YGMS SHATAF EGVYNGLKNPAIVGGLASPLMSTLFAF ALLITGLIS SIASTLAGQIVMEGYLNIQIP LW A RRLLTRL VTLIPIMIIGF VMGF SEQ HFEQVI VYAQVALSIALPFTLFPLVALT DRRDLMGQHVNSPVVRWMGYVLTGII TLLNVQLILSVILP Lactobacillus kefiri 14 WP 0547687 93.1 72.4 MSQEPTHKSLDEINQSVEVPSVYETSFL QKFLAYSGPGALVAVGYMDPGNWLTS LSGGSQFRYALLSVLLMSILVAMFMQT LSIKLGVVARLDLAQAIAQKVPKSGRY TLWIINELAMMATDMTGVVGTAIALK LLFGLPLVYGILLTIFDVLLVLLFLRFGI RRIEFIVLAAILIVGVIFGIEVTRATPNIV EIAGGLIPTTHIVTNHEMLIMSLGIVGA TIMPHNVYLHS SL AQ SRRYD YHNPKQ VNEALRFAKWDSNVHLVAAFLINALL LVLGGTLFFHTNSHFSAFQDVYNGLKS SAIVGSLASPLMSTLFAFALLITGMISSI TSTLSGQIVMEGYLHIRLPLWERRLLTR FVTLIPILAIGFLVGFNDHDFEEIIVYAQI ALSIALPFTLFPMVALTSNHDLMGVHT NRRYVT VIGYLLT SIITILNLQF VL ASI Lactobacillus alimentarius 15 WP_0577389 92.1 68.7 MPNKKSLDEINESVKVPSVYDTSFLQK FLAYSGPGALVAVGYMDPGNWLTSLS GGSQYRYDLLSVLLISILVAMFMQTLSI KLGVVARLDLAQAIATKVSKPIRYFLW ILNEIAMMATDLTGVIGTAIALKLLFNL PLVFGILLTVFDVLIVLIFLRFGIRRIEFI VLAAILTVGIIFGIEVFRAQPKLFSIISG V IPSTDLFTNHRKLVLSLGIVGATIMPHNI YLHSSLAQSRRYDHNDPLQVNEALRFA KWDSNVHLIAAFIINALLLVLGGTLFY HMTNQLASLQDVFTGLKSHAIVGTLAS PLMSWLFAFALLITGMISSITSTLSGQIV MEGYLNIRLPLWQRRLLTRFVTLIPILII GFIVHFNEQDFENLIVYAQIILSIALPFTL FPMIF LTNDKKIMGNHVNSKLTTTVGII LASAITILNLQLLFSLI Lactobacillus plantarum 16 YP_0048891 77.1 67.4 MQSHRHQSLEEINQSVAVPDVHQTAF WRKFLAYSGPGALVAVGYMDPGNWL TSLAGGGQFQYRLLAVLALAITVAMFM QGLAIRLGVVARQDLAQAIASKLPRPV RYAAWILNEVAMMATDMTGVIGTAIA LKMLFGLPLLAGILLTIADVLVVLLFLR FGIRRVEVIVLVAILTVGIIFGIEVGRAH VQF GNVLLGLVPTPLIVKNHTALVLSL GILGATIMPHNLYLHSSLAQSRRYDYH NPAQVTEALRFANWDSTVHLIAAFLIN ALLLVLGGTLFFGHTNALASLQAVFDG LKSTTVVGALASPVMSWLFALALLITG LISSITSTLAGQIVMEGYLHIRLPLWQR

- 11 043250

RLLTRAVTLIPILIIGMLVGF SD AAFENL IIYAQVALSIALPFTLLPLVALTNDASL MKAHVNRPAVTWVGYGLAGIITVLNI YLVYSLF Lactobacillus reuteri 17 WP_0036689 01.1 65.8 MERKSLDEINGSVDVPNVYQSAFWQK FLAYSGPGALVAVGYMDPGNWLTSLA GGSQYRYQLLVVLFTAILIAMYMQSLA IKLGVTTRTDLAQAIARRLPTPLRIALW LFNEIAMMATDLTGVVGTAVALNMLF KLPLLIGVLLTIADVLVVLFFLHFGIRRI EFIVLTAILWGAIFAIEVCRAHPEFSAI MDGFVPRSTIFTNHSELLISLGIVGATIM PHNIYLHSSLAQSRRYDEHDPKQVKET LRFANWDSLIHLFAAFIVNALLLILGGT LFFHAASLGSLEDVFFGLKNPQIVGSLA SPLMSWLFAFALLVTGLISSITSTLAGQI VMEGFINIRLPLWKRRLLTRAVTLVPIL IIGFMINFKEEQFEQLIIYAQLSIALPF TLYPL VALTGNKKLMGPHVNSRWQTV LGYILASLVTGLNLLVLV Lactobacillus crustorum 18 WP_0758872 52.1 61.4 FVPS QTIITNQNKLILSLGIIGATIMPHNI YLHSALAQSRRYDYHDSRQVREALRF ANWDSIVHLIAALIINCLLLILGGTIFYD KADQLASLMTVFKGLMNYQWGSLAS SFMSYLFAFALLVTGLISSITSTLSGQIV MEGYLNIRLPLWQRRLLTRIITLIPILVI GFLVHFNEVIFEDLIVYAQIALSVALPF TLFPLVYLTNNAKIMGKHVNKKWQTI LGFVLALIITILN1VL1ATTLSH

In one preferred embodiment, the manganese scavenging agent is a bacterial strain comprising a polypeptide having the sequence according to SEQ ID NO: 3 (MSDDHKKRHPIKLIQYANGPSLEEINGTVEVPHGKGFWRTLFAYSGPGALVAVGYMDP GNWSTSITGGQNFQYLLISVILMSSLIAMLLQYMAAKLGIVSQMDLAQAIRARTSKKLGI VLWILTELAIMATDI AEVIGAAIALYLLFHIPLVIAVLVTVLDVLVLLLLTKIGFRKIEAIV VALILVILLVFVYQVALSDPNMGALLKGFIPTGETFASSPSTNGMSPTQGALGITGATVMP HNLYLHSAISQTRKroYKNPDDVAQAVKFSAWDSNIQLSFAFWNCLLLVMGVAVFKS GAVKDPSFFGLFQALSDSSTLSNGVLIAVAKSGILSILFAV or its functional variants .

In other preferred embodiments, the manganese scavenging agent is a bacterial strain containing a polypeptide having at least 55% sequence identity, such as at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98, 99 or 100% identity according to SEQ ID NO: 3.

In table. 3 shows examples of sequences that encode functional variants of SEQ ID NO: 3 and their sequence identity with SEQ ID NO: 3.

Table 3

Origin SEQ ID NO Squirrel ID % identi PARTICULARS Subsequence Lactobacillus casei 19 WP_003 567390.1 99.8 MSDDHKKRHPIKLIQ YANGP SLEEINGT VE VPHGKGFWRTLFAYSGPGALVAVGYMDP GNWSTSITGGQNFQYLLISVILMSSLIAMLL

- 12 043250

Q YMAAKLGIVSQMDLAQ AIRART SKKLGI VLWILTELAIMATDIAEVIGAAIALYLLFHIP LVIAVLVTVLDVLVLLLLTKIGFRKIEAIVV ALILVILLVFVYQVALSDPNMGALLKGFIPT GETFASSPSINGMSPIQGALGIIGATVMPHN LYLHSAISQTRKIDHKNPDDVAQAVKFSA WDSNIQLSFAFVVNCLL TQTNMANI IAIVLIVAILALLAWTIWDLYK GNQRYEAHLAAVADEKEAKADVDEQ Lactobacillus rhamnosus 20 WP005 686822.1 95 MSDDHKKKHSMKLIQ YANGP SLEEINGT V EVPHGKGFWRTLFAYSGPGALVAVGYMD PGNWSTSITGGQNFQ YLLIS VILMS SLIAML LQYMAAKLGTVSQMDLAQATRARTSKKLG IVLWILTELAIMATDIAEVIGAAIALYLLFHI PLVIAVLVTVLDVLVLLLTKIGFRKIEAIV VALILVILLVFV YQVALSDPNMGALLKGFI PTGETF AS SPS VNGMSPIQGALGIIGATVMP HNLYLHSAISQTRKIDHKDPEDVAQAVKFS AWDSNIQLTFAFVVNCLLLVMGVAVFKSG AVKDPSFFGLFQALSDSSTLSNGVLIAVAK SGILSILFAVALLASGQNSTITGTLTGQVIM EGFIHMKMPLWARRLVTRVISVIPVIVM LTARETPIQQHEALNTLMNNSQVFLAFALP FSMLPLLMFTNSKVEMGDRFKNTGWVKV LGWVSVIGLTYLNLKGLPDSIAGFFGDNPT AAQTNIANMIAYVLIAAVLALLAWTIWDL YKGNKRYEAHLEAVADEEEAKANDDVQ Lactobacillus plantarum 21 YP_0048 90566.1 76.3 MSEKTNTPNRKHKLIEYANGPSLEEINGTIE VPKNLNFWKTLFAYSGPGALVAVGYMDP GNWSTSITGGQNYQYMLMSVILISSLIAML LQYMAAKLGIVSQMDLAQAIRARTSKSLGI VLWILTELAIMATDIAEVIGAAIALYLLFNIP LVIAVFITVLDVLVLLLLTKIGFRKIEAIVVC LILVILFVFVYQVALSNPD WGGVIKGLVPT ADTF STSRS VNGMTPLSGALGIIGATVMPH NLYLHSAISQTRKIDHNDEEDVARTVKFAA WDSNIQLSFAFVVNSLLLIMGVAVFKSGAV KDPSFFGLYEALSNTSMLSNGILISVAKSGA LSALFA1ALLASGQNSTITGTLTGQV1MEGF VHMRMPLWLRRLVTRLISVIPVLICVLLTS GKS AIDEHTALNN LMNNSQVFLAFALPF S MLPLLMMTDSAAEMGKRFKNSLWIKGLG WLSVIGLTFLNLLGLPDSILGFFGDNPSAGE QTF SKILAYLLIA AIL ALL V WT VFDLQRGN KRYVEQQLAAAAKEANK Pediococcus acidilactici 22 WP_065 124048.1 76.2 MSNEIKNPKKRRKLISYANGRSLEEINGTV KVPKNTGFWKTLFMYSGPGALVAVGYMD PGNWSTSITGGQNFQYMLMSIILIS SLIAML LQYMAAKLGIVS QMDL AQ AIRART SRALG IVLWILTELAIMATDIAEVIGAAIALYLLFHI PLVVAVFITVFDVLLLLLTKIGFRKIEAIVV CLIMVILVVFVYQVAL SHPSWGAVFGGLIP TTKAFATTPTVGGMTPLSGSLGIIGATVMP HNLYLHSAVSQTRKTNHDDEEDVARTVRF STWDSNIQLSFAFVVNALLLVMGVAVFKT GAVQDP SFGLFHALNDT STLSNGILIGVA KTGILSTLFAVALLASGQNSTITGTLTGQVI MEGFVHMRMPLWARRLITRLISVVPVLICV

- 13 043250

MLTSGKGTIQEHEALNNLMNNSQVFLAFA LPF SMVPLLMMTD SRVEMGDRFKNS WIVR ILGWISVIFLTYLNLTGLPDSIAAFFGENASA AEISMAHDIAYALIVAVLALLAWTVIELYK GNI<RYEIELAEI<ANAI<EAA Lactobacillus salivarius 23 YP_5357 97.1 72.7 MVNNENNHKKHKMIQYANGKSLEEANGT VEIPKGKGFWKTLFAYSGPGALVAVGYMD PGNWSTSITGGQNFQYLLMSVILLSSLIAML LQYMAAKLGIVSQMDLAQAIRARTSKALG IVLWILTELAIMATDIAEVIGAAIALYLLFDI PLIIAVFITVFDVLLLLLTKVGFRKIEAIVV CLIFVILFVFV YQVALSNPDWGGVFKGLIPT SETFAKHPVVHDMSPLNGALGIIGATVMPH NLYLHSAISQTRKFDRNNEDDIANAVRFTA WDSNIQLGLAFVVNSLLLIMGVAVFKSGA VEDPSFFGLYQALSDTSVMSNGLLAAAAR TGILSTLFAVALLASGQNSTITGTLTGQVIM EGFIHLRMPLWARRLITRLLSVIPVLICVAL TSGKSTIEEHE ALNNLMNNSQVFLAFALPF SMLPLVIMTGSKVEMGERFKNRLWINILG WISVISLTYLNMIGLPQNLEPFFPADKVGLA HTVAYILIVLIIALLIWTLVELHLGNKRFAA EQAI<I<HNI< Lactobacillus fermentum 24 WP_012 391805.1 69.6 MDNTKNQHRKLRLIEHANGKSLEEINGTV EVPHGKGFFRTLFAYSGPGALVAVGYMDP GNWSTSITGGQSFQYTLMTTILISSLIAMLL Q YMAAKLG1V SQMDLAQ AIRARTGK ALG VILWLMTELAIMATDIAEVIGAAIALNLLFH IPLVLAVFITVLDVLVLLLTKIGFRKIEAIV ACLILVILAVFAYQ VALSHPDWAGVFKGLL PTKEAIAKEPVVGGISPLTGSLGIIGATVMP HNLYLHSAISQTRKIDHTNAEDIKQTVRFT AWD SNIQLTL AFF VN ALLLIMGVA VFKNG AVQDSSFFGLYDALNNTDMLSNGLLIAVA KSGVLSTLFAIALLASGQNSTITGTLTGQVI MEGFVHMKMPLWARRLITRLLSVVPVLVC VAMTAHESTIDQHASLNILMENSQVFLAFA LPFSMLPLLIMTNSDTEMGQFKNSLWVRV LGWISVIGLTFLNLYNLPQ TYEGFGIWSKG LSDVLAWISIVVIVVLLAWTCFELIRGDRRL AAEREKHTWEK Lactobacillus amylolyticus 25 WP_080 881380.1 63.7 MC SRI<VLLTI<QI<GI<HYLIR YANGK S LSEIN GTIEIPKKRTFWRMLWAYTGPGALVAVGY MDPGNWATSITGGQSFQYILMSTILISSLMA MLLQYMAAKLGIVTQMDLAQAIRLRTGK ALGIVLWLMTELAIMATDIAEVIGAAIALN LLFDIPLVPAVFITVLDVLLLLLARIGFRKI EAVV SCLILVILLVFVYEVLLSNPDWSKAF VGLVPSAKIIQTHPVVGGISPLTGTLGIIGAT VMPHNLYLHSAISQTRKINHHNLQLIRDAV KYTALDSNIQLSLAFLVNALLLIMGAAVFK SGAVRDSSFFGLYQALDNAKMLSDPLLVH VARTGILSTLFAVALLASGQNSTITGTLTGQ VIMEGYIHLKMPLWARRLVTRLLSVIPVL L CVSFTMNDSVMQQHFALNMLMENSQVFL AFALPFSVLPLLIMTNNKAEMGEFKNKPL WHYLGWACALVLTFLNLYNLPSQFVNFKF ASKEVSTIIAYFVIVVIAALLLWTCIEIYIGD RKVKIHHSGFDAKEKELKEEGQK

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) built into 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. The possible parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62). The result of the Needle program, denoted as the most

- 14 043250 long identity (obtained using the nobrief option) is used as the percentage identity and calculated as follows:

(identical remnants of 100)/(alignment length - total number of gaps in the alignment).

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, supra) built into the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 , above), preferably version 3.0.0 or later. The possible parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL replacement matrix (EMBOSS version of NCBI NUC4.4). The result of the Needle program, denoted as the longest identity (obtained using the nobrief option), is used as the percentage identity and calculated as follows:

(identical deoxyribonucleotide 100)/(length of alignment-total number of gaps in alignment).

In one embodiment, the selection step includes determining if the bacterial strain contains a manganese transporter having 3 sequence identity such as at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity with any of the sequences of SEQ ID NO: 1-3. The determination may be based on sequencing of the bacterial strain or searching for blast in databases with known sequences.

The manganese scavenging agents used in the Examples section of this invention have a manganese transporter coded according to SEQ ID NOs: 1-3 or functional variants thereof.

In other embodiments, the present invention provides a method for inhibiting or delaying the growth of fungi in a product, comprising the steps of selecting one or more bacterial strains as ^the manganese scavenging agent and reducing the level of free manganese in the product to a concentration of less than about 0.01 ppm ¹ in the product by adding a manganese scavenging agent, the selection step comprising measuring the manganese scavenging activity of one or more bacterial strains.

Manganese uptake activity can be measured using conventional methods known in the art, see, for example, 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 foods such as dairy products, the manganese scavenging agent is preferably a species of lactobacilli. Lactobacillus has different families of manganese transporters, and their many homologues are often also present. In FIG. 11, the inventors present a tree reflecting an overview of the phylogeny of the manganese transporter family MntH in Lactobacillus species. As presented, manganese transporters can be found among Lactobacillus species. In addition to Lactobacillus spp. the MntH transporter family can also be found in other bacteria. The tree was constructed by aligning the Lactobacillus MntH protein sequences using mafft v.7, while the phylogeny was inferred by clustering by neighbor pairing.

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

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

In accordance with a preferred embodiment of the present invention, the method includes the step of selecting to determine that one or more than one bacterial strain does not contain superoxide dismutase, preferably manganese superoxide dismutase.

Superoxide dismutases, such as manganese superoxide dismutase, have been studied and described, among others, 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 V.C Superoxide dismutase, oxidative stress, and cell metabolism Curr. top. Cellregul. 36, 117-132 (2000) or Whittaker J.W Manganese superoxide dismutase Met. Ions Biol. Syst. 37, 587-611 (2000).

In the context of the present invention, the term not containing means that the genome of one or more than one bacterial strain does not carry a gene encoding superoxide dismutase, or even if the genome of one or more than one bacterial strain carries a gene encoding superoxide dismutase, then this gene is not expressed by one or more than one bacterial strain.

- 15 043250

Products.

In some embodiments, the product is a food product, cosmetic product, medical product, or pharmaceutical product. Food product and food product have the general meaning of these terms. Food product means any food or feed product suitable for human or animal consumption. Food can be fresh or perishable food, as well as stored or processed food. Food products include, but are not limited to, fruits and vegetables, including derivatives, cereals and cereal-derived products, dairy products, meat, poultry, and seafood. More preferably, the food product is a meat product or a dairy product such as yogurt, curd, sour cream, cheese, and the like.

However, it should be noted that in the context of the present invention, the term product and food product in the present invention refers to the water itself. Although manganese is essential to human nutrition, in water it is generally considered harmful to human health according to the US Environmental Protection Agency (EPA). Therefore, the treatment of drinking water or waste water to remove excess manganese is sometimes carried out for disinfection and sanitation, which is not the essence of the present invention.

The present invention is particularly applicable to food products having intermediate to high water activity. Water activities (a _w ) determine the viability and functionality of microorganisms. Water activity or a _w is the partial pressure of water vapor in a substance divided by the standard state of the partial pressure of water vapor. In the field of food science, the standard state is most commonly 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 1.

The main food categories prone to fungal spoilage are dairy products with intermediate or high water activity, such as yogurt, cream, butter, cheese, etc. However, the present invention is also expected to be suitable for foodstuffs having lower water activity, such as processed meats, grains, nuts, spices, milk powder, dried meats, and fermented meats.

In a preferred embodiment, the product to which the methods of the present invention can be applied is a product having water activity. (a _w ) 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 even less.

In some embodiments, the product is a product having a water activity (a _w ) from about 0.70 to about 0.98, such as from about 0.75 to about 0.97, such as from about 0.80 to about 0, 96, such as from 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 the growth of fungi in fermented foods. Fermented foods are products produced or preserved by the action of microorganisms. Fermentation means the conversion of carbohydrates into alcohols or acids by the action of a microorganism. Fermentation generally 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 can be used to prepare foods such as yogurt, cheese, salami, sauerkraut, kimchi, pickles, and the like.

In one embodiment, the food product is a lactic acid fermentation product, i. e. obtained by fermentation with lactic acid bacteria (LAB). Lactic acid bacterium refers to a Gram-positive microaerophilic or anaerobic bacterium that ferments sugars to form acids, including lactic acid as the predominantly produced acid. The food 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.

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The present invention is particularly useful for inhibiting or delaying the growth of fungi in dairy products. In such products, yeast and mold contamination is common and limits the shelf life of such products. A dairy product includes, in addition to milk, products derived from milk such as cream, ice cream, butter, cheese and yogurt, as well as by-products such as whey and casein, and any prepared foods containing milk or milk components as main ingredient such as milk formula. In one preferred embodiment, the dairy product is a fermented dairy product. The term milk is understood as the milk secretion obtained by milking any mammal such as cows, sheep, goats, buffalo or camels. 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.

The concentration of manganese in milk varies depending on the animal from which it is produced, the feed, and the season. Typically, manganese is present in dairy products at a concentration of at least 0.03 ^ppm , such as at least 0.08 ^ppm for skim milk and at least 0.1 ^ppm for whole milk. In accordance with the discovery of the authors of the present invention, reducing the amount of manganese in such products or products made from them, makes them more resistant to spoilage.

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

In one embodiment, the food product is a product prepared by fermentation with mesophiles, i. mesophilic fermented food product. The term mesophile refers to microorganisms that thrive best at moderate temperatures (15°C to 40°C). The mesophilic bacteria most useful for industry include Lactococcus spp. and Leuconostoc spp. As used herein, the term mesophilic fermentation refers to fermentation at a temperature of from about 22°C to about 35°C. Mesophilic fermented food product, which refers to fermented food products prepared by the mesophilic fermentation of a mesophilic starter culture. Such products include, for example, buttermilk, sour milk, sour milk, sour cream, sour heavy cream, and fresh cheese such as quark, cottage cheese, and cream cheese.

Preparation of fermented foods.

The methods described herein are particularly useful for inhibiting or delaying the growth of yeasts and/or molds in a fermented milk product, such as a thermophilic and mesophilic fermented milk product, such as a yoghurt product. The term fermented milk product is a term usually defined according to official regulations and standards are well known in the art. For example, symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is used as a starter culture for yogurt, while Lactobacillus acidophilus is used to produce acidophilus. Other mesophilic lactic acid bacteria are used to produce quark or low fat soft cheese.

The expression fermented milk product means a food or feed product, where the preparation of the food or feed product involves the fermentation of a milk base with a lactic acid bacterium. As used herein, the term fermented milk product includes, but is not limited to, products such as thermophilic fermented milk products (e.g. yogurt) and mesophilic fermented milk products (e.g. sour milk and buttermilk, as well as fermented whey, quark and low fat soft cheese). The fermented milk product also includes cheese such as continental type cheese, fresh cheese, soft cheese, cheddar, mascarpone, draft cheese, mozzarella, provolone, white pickled cheese, pizza cheese, feta, brie, camembert, house cheese, edam, gouda, tilsiter, havarti or emmental, swiss cheese and maasdam.

The term yogurt has its usual meaning and is usually defined according to official regulations and standards are well known in the art. Yogurt starter cultures include at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus. Interestingly, the manganese transporter is absent in L. delbrueckii subsp. bulgaricus and shows low expression only in Streptococcus thermophilus, two strains found in starter culture in yogurt, making them particularly susceptible to fungal spoilage. Therefore, it is preferable to include another(s) bacterial(s) strain(s) to absorb the free manganese present in the yogurt.

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

During food processing, chemical preservatives have traditionally been used to prevent fungal spoilage. However, in view of the high societal demand for less processed and preservative-free foods, the invention contributes to providing an effective solution to control the growth of yeasts and molds by using biological manganese scavenging agents to reduce manganese concentrations.

When using a biological manganese scavenging agent, one skilled in the art can adjust various parameters such as pH, temperature, and the amount of manganese scavenging agent or bacteria to achieve the desired results, taking into account the examples provided in the present invention, as well as food properties such as like water activity, nutrients, natural manganese levels, shelf life, storage conditions, packaging, etc.

The product, in which the concentration of free manganese is reduced, is preferably packaged to further limit contact with yeasts and molds. It is also preferable to store the product at a cold temperature (below 15°C) to help increase shelf life.

For a fermented food product, manganese absorbing bacteria can be added before, at the beginning or during fermentation. Depending on the parameters chosen, the step of reducing the manganese level to the preferred level may be 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. The person skilled in the art can select suitable parameters depending on the product in which the inhibition or inhibition of fungal growth is desired.

The invention provides a process for preparing a fermented food product, comprising adding a starter culture and a manganese scavenging agent to a food substrate, fermenting the substrate for a time until the desired pH is reached. Preferably, the manganese scavenging agent is a strain of lactic acid bacteria.

As used herein, the term food substrate refers to the substrate in which the fermentation is to take place.

For the production of fermented milk products, the food substrate is a milk base. Milk base is widely used in the present invention to refer to a composition based on milk or dairy components that can be used as a medium for the growth and fermentation of a starter culture. Milk generally refers to the milk 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 milk material, as well as from reconstituted milk powder. The milk base may also be vegetable, ie. made from plant material such as soy milk. Preferred is a milk base made from milk or milk components obtained from cows.

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

The milk base can also be lactose-reduced depending on the needs of the customers. Lactose-reduced milk can be obtained by any method known in the art, including hydrolysis of lactose by the enzyme lactase to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatography, and centrifugation.

To start the milk base, a starter culture is added. The terms starter or starter culture used in the context of the present invention refers to the culture of one or more food microorganism, in particular lactic acid bacteria, which are responsible for acidifying the milk base.

The manganese scavenging agent can be added before, at the start of, or during fermentation at the same time as the starter culture or at other times.

After adding the starter culture and manganese scavenging agents and exposing the milk base to suitable conditions, the fermentation process begins and continues for a period of time. One skilled in the art knows how to select suitable process conditions such as temperature, oxygen, carbohydrate addition, the amount and characteristics of the microorganism(s), and the process time it takes. This process can take three, four, five, six hours or more.

These conditions include establishing a temperature that is appropriate for particular strains of the starter culture. For example, when the starter culture contains mesophilic lactic bacteria, then the temperature can be set at about 30°C, and if the culture

- 18 043250 contains thermophilic lactic acid bacterial strains, then the temperature is maintained in the range from about 35°C to 50°C, for example from 40°C to 45°C. The setting of the fermentation temperature also depends on the enzyme(s) added for fermentation, which(e) can be easily determined(s) by a person skilled in the art. In a specific embodiment of the invention, the fermentation temperature is from 35°C to 45°C, preferably from 37°C to 43°C, and more preferably from 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.

The fermentation may be stopped using any means known in the art. In general, depending on various process parameters, fermentation can be stopped by making the milk base unsuitable for growth of the starter culture strain(s). For example, stopping can be done by rapidly cooling the fermented milk product when the desired pH is reached. It is known that acidification occurs during fermentation, which leads to the formation of a three-dimensional network consisting of clusters and chains of caseins. The term desired pH refers to the pH at which the fermentation step is terminated. The desired pH depends on the resulting fermented milk product and can be easily determined by a person skilled in the art.

In a specific embodiment of the invention, fermentation is carried out until at least a pH of 5.2 is reached, for example, until a pH of 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5 is reached, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8 or 3.7. Preferably, the fermentation is carried out until the desired pH is reached between 4.0 and 5.0, and more preferably between 4.0 and 4.6. In a preferred embodiment, the fermentation is carried out until the desired pH is below 4.6.

In a preferred embodiment, the fermentable food product is selected from the group consisting of quark, cream cheese, low fat soft cheese, Greek yogurt, skyr, labneh, buttermilk, sour cream, sour milk, sour milk, kefir, lassi, ayran, cottage cheese, duka, sour cream , yakulta and dahi.

In another preferred embodiment, the fermented food product is a cheese, including continental type cheese, fresh cheese, soft cheese, cheddar, mascarpone, draw cheese, mozzarella, provolone, white pickled cheese, pizza cheese, feta, brie, camembert, cottage cheese , edam, gouda, tilsiter, havarti or emmental, swiss cheese and maasdam.

In yet another embodiment, the method further includes packaging the food product to reduce contact with yeast and molds.

The present invention includes a food product obtained using the methods described here.

The product obtained in accordance with the present invention is preferably a fermented milk product with a concentration of free manganese reduced to less than 0.01 ^ppm after storage 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 the following description in conjunction with the accompanying drawings. Unless otherwise defined, all terms used herein have the same meaning as generally understood by a person skilled in the art. The use of the terms a and an and the and the like in the context of the description of the invention (especially in the context of the following claims) is to be construed to include both the singular and the plural, unless otherwise indicated or clearly contradicted by the context. Unless otherwise noted, the terms containing, having, including, and containing are to be understood as open terms (ie, meaning including, but not limited to). The listing of value ranges here is simply intended to serve as a shorthand way of referring separately to each individual value falling within the range, unless otherwise noted, and each individual value is included in the description as if it were individually stated here. All of the methods described herein may be performed in any suitable order, unless otherwise indicated here, or unless otherwise clearly contradicts the context. Unless otherwise noted, all exact values provided herein are representative of their respective approximate values (e.g., all exact approximate values provided for a particular factor or measurement may also be considered to provide the corresponding approximate measurement, modified approximately where appropriate) . The use of any and all examples, or exemplary language (eg, such as) provided herein is intended only to better illuminate the invention and does not limit the scope of the invention unless otherwise stated. Nothing in the specification is to be construed as indicating any element not claimed to be essential to the practice of the invention.

Examples

The invention described and claimed herein should not be limited in scope to the specific aspects disclosed herein, as these aspects are intended to illustrate certain 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. It is intended that such modifications also fall within the scope of the claims. In case of conflict, the description of the present invention, including definitions, takes precedence.

EXAMPLE 1 Growth of yeast at various concentrations of manganese.

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

The minimal medium contains biotin 2 µg/l, calcium pantothenate 400 µg/l, folic acid 2 µg/l, inositol 2 mg/l, nicotinic acid 400 µg/l, para-aminobenzoic acid 200 µg/l, pyridoxine 400 µg/l , rioflavin 200 mcg/l, thiamine 400 mcg/l, boric acid 500 mcg/l, copper sulfate 40 mcg/l, potassium iodide 100 mcg/l, ferric chloride 200 mcg/l, sodium molybdate 200 mcg/l, zinc sulfate 400 µg/l, dibasic potassium phosphate 0.5 g/l, dibasic potassium phosphate 0.5 g/l, magnesium sulfate 0.5 g/l, sodium chloride 0.1 g/l, calcium chloride 0.2 g/l l, glucose 20 g/l, ammonium sulfate 5 g/l.

Used manganese concentration: 6 ^ppm , 0.6 ^ppm , 0.06 ^ppm , 0.006 ^ppm , 0.0006 ^ppm , 0.00006 ^ppm , 0.000006 ^ppm .

Two different pH values were tested: pH 6.5 and 4.5.

The strains were inoculated into 150 μl of different media in a 96-well plate. The plates were incubated at 17° C. for several days and the growth of the yeast strains was monitored by measuring absorbance at 600 nm in a plate reader.

The effect of different pH and different concentrations of manganese on the growth of two different strains of D. hansenii are shown in FIG. 1 (strain 1) and FIG. 2 (strain 2). As can be seen, the growth of D. hansenii is inhibited below a concentration of about 0.01 ^ppm manganese. There is no difference between the two different pH, demonstrating that this mechanism is effective in this pH range.

Example 2 Yeast suppression in fermented milk products.

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

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

Fig. 3 and 4 show the growth of D. hansenii (strain 1 and strain 2, respectively). Yeast growth after the addition of manganese to the aqueous phase is shown in FIG. 3 for strain 1 and FIG. 4 for strain 2 (light circles). Shown are the mean and standard deviation for technical repetitions n=6 (A) and n=3 (B). For comparison, yeast growth is also shown in the comparative aqueous yogurt phase to which neither manganese scavenging agents nor additional manganese was added (square). It is noted that in the sample used for comparison, the concentration of manganese is 0.03 ^ppm . For light circles, the x-axis indicates the concentration of added manganese, and for squares (the aqueous phase of the yoghurt used for comparison), the x-axis indicates manganese characteristic of the aqueous phase.

This result demonstrates that the growth of yeast strains in the food matrix is manganese dependent. The addition of manganese results in growth close to that of the reference sample, ensuring that low manganese concentrations represent a major limitation for yeast growth in the fermented milk product.

Example 3 Suppression of Debaryomyces and Rhodoturola.

This example shows the manganese uptake activities for various bacteria and their inhibitory effect on Debaryomyces and Rhodoturola. The inhibitory effect was evaluated in fermented milk products with a low concentration of manganese and an increased concentration of manganese.

In table. Table 4 lists the added bacteria and whether they include manganese transporters.

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Table 4

No. Kinds Contains manganese carrier 1 Lactobacillus rhamnosus Yes 2 Lactobacillus rhamnosus Yes 3 Lactobacillus rhamnosus Yes 4 Lactobacillus rhamnosus (ATCC 7469) Yes 5 Lactobacillus salivarius Yes 6 Lactobacillus casei Yes 7 Lactobacillus paracasei Yes 8 Lactobacillus fermentum Yes 9 Lactobacillus sakei (ATCC 15521) Yes 10 Lactobacillus reuteri (ATCC 23272) Yes eleven Lactobacillus plantarum Yes 12 Lactobacillus delbrueckii no 13 Lactobacillus helveticus no 14 Lactobacillus gasseri (ATCC 33323) no 15 Lactobacillus brevis Yes 16 Lactobacillus kefiri (ATCC 35411) Yes 17 Lactobacillus alimentarius Yes 18 Pediococcus acidilactici (DSM20284) Yes 19 Control (starter culture only)

The listed bacteria were grown in MRS medium (deMan, Rogosa and Sharpe) overnight.

Preparation: 2 ml of milk containing a starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was inoculated with 10 µl of an overnight culture. The milk was fermented at 43°C for approximately 6 hours until a pH of 4.5 was reached. The fermented milk was stored in the refrigerator until further use. 150 μl of fermented milk was transferred to individual wells in a 96-well plate in duplicate. Manganese was added to half of the sample to reach 6 ^ppm manganese and all wells were inoculated with approximately 20 Debaryomyces hansenii or Rhodoturola mucilaginosa cells. After 4 days, the dilution series was applied to YGC selective plates for yeast growth assay. Yeast growth was measured by optical observation, where a value of 0 is assigned for no growth and 5 for full growth. The average value for two biological independent experiments is presented in table. 5 for Debaryomyces and tab. 6 for Rhodoturola.

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Table 5

Suppression of Debaryomyces

absorbent agent manganese Yeast Growth (e.p.) Yeast growth (e.p.) with 6 ppm ^manganese 1 Lactobacillus rhamnosus 0 5 2 Lactobacillus rhamnosus 0 5 3 Lactobacillus rhamnosus 0 5 4 Lactobacillus rhamnosus 0 4.5 5 Lactobacillus salivarius 3.75 5 6 Lactobacillus casei 3.75 5 7 Lactobacillus paracasei 0 5 8 Lactobacillus fermentum 0 5 9 Lactobacillus sakei 0.5 5 10 Lactobacillus reuteri 4 5 AND Lactobacillus plantarum 0.5 5 12 Lactobacillus delbrueckii 4.5 4.5 13 Lactobacillus helveticus 5 3 14 Lactobacillus gasseri 5 5 15 Lactobacillus brevis 3.5 5 16 Lactobacillus kefiri 1.5 5 17 Lactobacillus alimentarius 3.5 5 18 Pediococcus acidilactici 4 5 19 Control (starter culture only) 5 5

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Table 6

Suppression of Rhodoturola

Manganese scavenging agent(s) yeast growth (e.p.) Yeast growth (e.p.) with 6 ppm ^manganese 1 Lactobacillus rhamnosus 1 4.5 2 Lactobacillus rhamnosus 1.5 4.5 3 Lactobacillus rhamnosus 1 4.5 4 Lactobacillus rhamnosus 1 4.5 5 Lactobacillus salivarius 3.5 5 6 Lactobacillus casei 3.5 5 7 Lactobacillus paracasei 2 5 8 Lactobacillus fermentum 3 5 9 Lactobacillus sakei 1 4.5 10 Lactobacillus reuteri 4 5 eleven Lactobacillus plantarum 2.5 4 12 Lactobacillus delbrueckii 4 4 13 Lactobacillus helveticus 4.5 5 14 Lactobacillus gasseri 4.5 4.5 15 Lactobacillus brevis 3.5 4.5 16 Lactobacillus kefiri 3.5 4.5 17 Lactobacillus alimentarius 5 5 18 Pediococcus acidilactici 5 5 19 Control (starter culture only) 5 5

The results demonstrate that strains that absorb manganese can be used to suppress D. hansenii and R. mucilaginosa, but the suppression is attenuated when manganese is added. Example 4 Suppression of Debaryomyces, Saccharomyces, Rhodoturola, Cryptococcus and Torulaspora.

This example evaluates differences in yeast growth in the aqueous phase of fermented milk prepared with and without starter cultures with manganese scavenging agents.

In table. 7 lists the manganese scavenging agents used.

Table 7

No. Absorbing agent(s) manganese Manganese carrier 1 L. rhamnosus L. paracasei Yes 2 L. rhamnosus Yes 3 L. rhamnosus Yes

This example shows yeast growth at 2 different manganese concentrations. The inhibitory effects were evaluated against 6 different yeasts in fermented milk products with low manganese concentration and increased manganese concentrations.

Homogenized reduced fat milk (1.5% w/v) was heat treated at 90±1°C for 20 minutes and immediately cooled. A commercially available starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was inoculated at 0.02% (v/w) into 3 L containers. One container was inoculated with manganese scavenging agents at a total concentration of 100 U/T, and one container was used as a control, inoculating it exclusively with the starter culture. All containers 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. Dairy products were aliquoted into 200 ml vials and cooled.

The fermented milk product was then centrifuged (10 min at 5000 rpm) and the supernatant subjected to sterile filtration. The supernatant was transferred to a sterile 96-well plate (150 µl in each well) and manganese was added to half of the samples until an increase in manganese of up to 6 ppm was reached ^.

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1. Six different yeasts were selected and inoculated with approximately 20 cells per well to grow for 7 days at 17°C. Milk fermented only with starter culture was used as a control (control). Growth was determined by measuring absorbance at 600 nm.

The mean value and standard deviation for at least 5 repetitions are presented in table.

8-13.

Table 8

Suppression of Debaryomyces hansenii (strain 1)______

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.14±0.01 0.48 ± 0.07 2 L. rhamnosus 0.18±0.01 0.46 ± 0.05 3 L. rhamnosus 0.27±0.01 0.62 ± 0.08 Control Absence 0.40±0.03

Table 9

Suppression of Debaryomyces hansenii (strain 2)

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.01±0.01 0.43 ± 0.06 2 L. rhamnosus 0.01±0.00 0.44 ± 0.03 3 L. rhamnosus 0.10±0.02 0.51±0.06 Control Absence 0.33 ± 0.04

Table 10

Suppression of Saccharomyces cerevisiae

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.51±0.06 0.63 ± 0.09 Control Absence 0.62±0.02

Table 11

Suppression of Rhodoturola mucilaginosa

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.15±0.01 0.19±0.01 2 L. rhamnosus 0.13±0.01 0.23±0.02 3 L. rhamnosus 0.17±0.01 0.22±0.02 Control Absence 0.24±0.01

Table 12

Suppression of Cryptococcus fragicola____________

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.06±0.01 0.08±0.01 2 L. rhamnosus 0.06±0.01 0.08±0.01 3 L. rhamnosus 0.07±0.00 0.09±0.01 Control Absence 0.10±0.01

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Table 13

Suppression of Torulaspora delbrueckii

Manganese scavenging agent(s) Yeast growth (absorbance at 600 nm) Yeast growth (absorbance at 600 nm) with 6 ppm ^manganese 1 L. rhamnosus L. paracasei 0.11±0.00 0.16±0.02 Control Absence 0.20±0.02

The results demonstrate that strains of Debaryomyces, Saccharomyces, Rhodoturola, Cryptococcus and Torulaspora can be inhibited by bacteria that absorb manganese, and the inhibitory effect is reduced after the addition of manganese.

Example 5 Suppression of Debaryomyces hansenii.

This example evaluates the effect of various concentrations of manganese in fermented milk prepared with starter culture, with and without manganese scavenging agents.

In table. 14 lists the manganese scavenging agents used.

Table 14

No. Manganese scavenging agent(s) Contains manganese carrier 1 L. rhamnosus L. paracasei Yes 2 L. rhamnosus Yes 3 L. rhamnosus Yes

The fermented milk product was prepared as in example 4.

150 μl of fermented milk was transferred to separate wells of a 96-well plate in two or three parallels. A serial dilution was carried out, which resulted in the addition of various manganese concentrations ranging from 6 ^ppm to 0.000006 ^ppm . All wells were inoculated with approximately 20 Debaryomyces hansenii cells (strain 2) and the plates were incubated at 17° C. for 5 days. Thereafter, 10 μl of a 1000-fold dilution prepared in saline peptone was applied to YGC selective plates for yeast growth assay.

In FIG. 5 shows the growth of Debaryomyces hansenii in fermented milk prepared with manganese scavenging agents (No. 1, No. 2, and No. 3) and without manganese scavenging agents (REF), after adding various concentrations of manganese: 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 manganese scavenging agent(s) No. 1 and No. 2 was suppressed after adding 0.6 ^ppm (compare row 3 with row 2); the addition of 0.006 ^ppm resulted in some yeast growth in the presence of manganese scavenging agent No. 3.

Example 6 Yeast suppression in a fermented milk product with different levels of manganese.

The influence of manganese on the inhibitory effect against various types of mold fungi was evaluated. Agar analysis was used, similar to the method of preparation and production of fermented milk products. L. rhamnosus and L. paracasei were used together as manganese scavengers.

Preparation of fermented milk samples.

Homogenized reduced fat milk (1.5% w/v) was heat treated at 90±1° C. for 20 minutes and immediately cooled. A commercially available starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was inoculated at 0.02% (v/w) into 3 L containers. One container was inoculated with manganese scavenging agents at a total concentration of 100 U/T and the container was used as a control, inoculating it exclusively with the starter culture. All containers 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. Dairy products were aliquoted into 200 ml vials and cooled. The previously determined concentration of manganese already present in the control product was approximately 0.03 ^ppm and in the product with manganese absorbing strains below the detection threshold of 0.003 ^ppm .

Addition of manganese.

Various concentrations of manganese were added to fermented milk products with and without a manganese scavenging agent to achieve manganese addition levels (0.0006 and 6 ^ppm manganese in controls and 0.000006, 0.00006, 0.0006, 0.006, 0.06, 0.6 and 6 ^ppm manganese).

All fermented milk samples were heated to 40°C and 40 ml of 5% sterile agar solution was added, which was melted and cooled to 60°C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a laminar flow hood for 30 minutes.

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Load test using yeast.

Three target sources of contamination, including two Debaryomyces hansenii (strain 1 and strain

2) and one Cryptococcus fragicola was applied at concentrations of 10 ³ , 10 ² and 10 CFU/spot. The plates were incubated at 7±1° C. and examined regularly for yeast growth.

Results.

The results of yeast analysis on agar are shown in FIG. 6 showing the growth of 3 different yeasts on plates prepared with milk fermented with starter culture alone (control, top row) or together with manganese scavenging agents (bottom row). Various concentrations of manganese were added as shown in the graphics above. Three target contamination sources (column A: Debaryomyces hansenii (strain 2), column B: Cryptococcus fragicola, column C: Debaryomyces hansenii (strain 1) were added at three different concentrations: 1x10 ³ cfu/spot (top row on plate), 1x10 ² cfu/spot (middle row on plate) and 1x101 cfu/spot (bottom row on plate).

As can be seen in FIG. 6, the tested yeasts grew well on agar plates prepared from milk fermented with starter culture only (control). However, when manganese scavenging agents are present during milk fermentation, the overgrowth of all yeasts is retarded.

At manganese levels up to 0.0006 ^ppm , the manganese scavenging agent retained high inhibitory activity against all three yeast species. At manganese levels from 0.006 ^ppm to 0.6 ^ppm , the inhibitory activity of the manganese scavenging agent against C. fragicola was reduced. A manganese concentration of 6 ^ppm appears to inhibit the growth of C. fragicola. D. hansenii (strain 1) was suppressed by the manganese scavenging agent at manganese levels up to 0.006 ^ppm . At manganese levels of 0.06 ^ppm and above, the manganese scavenging agent loses its inhibitory effect on D. hansenii (strain 1). D. hansenii (strain 2) was inhibited by the manganese scavenging agent at manganese levels up to 0.6 ^ppm and activity was lost at 6 ^ppm manganese.

Example 7 - Inhibition of molds in a fermented milk product with different levels of manganese.

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

Load test using fungi.

Fig. 7 shows the growth of 3 different mold strains in plates prepared with milk fermented with starter culture alone (control, top row) or additionally with a manganese scavenging agent (bottom row). Additionally, various concentrations of manganese were added, as shown above above the figure. Three target contamination sources (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added at concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Fig. 8 shows the growth of 3 different mold strains on plates prepared with milk fermented with starter culture alone (control, top row) or together with a manganese scavenging agent (bottom row). Additionally, various concentrations of manganese were added, as shown above above the figure. Three target contamination sources (A: Penicillium brevicompactum, B: Penicillium crustosum and C: Penicillium solitum) were added at concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

Fig. 9 shows the growth of 3 different mold strains in plates prepared with milk fermented with starter culture alone (control, top row) or additionally with a manganese scavenging agent (bottom row). Additionally, various concentrations of manganese were added, as shown above above the figure. Three target contamination sources (A: Penicillium carneum, B: Penicillium rapeite and C: Penicillium roqueforti) were added at concentrations of 500 spores/spot. The plates were incubated at 7±1°C for 25 days.

Fig. 10 shows the growth of 3 different mold strains on plates prepared with milk fermented with starter culture alone (control, top row) or additionally with a manganese scavenging agent (bottom row). Additionally, various concentrations of manganese were added as shown above in the figure. Three target contamination sources (A: Penicillium carneum, B: Penicillium rapeite and C: Penicillium roqueforti) were added at concentrations of 500 spores/spot. The plates were incubated at 22±1°C for 8 days.

All mold strains tested grew very well on agar plates prepared from milk fermented with starter culture only (control). However, when manganese scavenging agents were present during milk fermentation, growth of all mold strains tested was suppressed. The suppressive effect was stronger at low temperature (FIGS. 7 and 9).

The manganese scavenging agent retained high inhibitory activity against all mold strains at additional manganese levels up to 0.006 ^ppm . At manganese levels of 0.06 ^ppm and above, the inhibitory effect of manganese scavenging agents decreased. The manganese scavengers lost most of their inhibitory effect at 6 ^ppm for susceptible molds (FIGS. 7 and 8) and at 0.06 ^ppm for non-susceptible mold strains (FIGS. 9 and 10).

The higher added manganese concentration, which reduces the inhibitory effect found in this assay compared to the concentrations found in the aqueous phase, is a consequence of the continued consumption of the live and metabolically active manganese scavenging agent in the fermented milk product.

Example 8 Inhibition of Debaromyces hansenii in the aqueous phase and at various concentrations of a chemical chelating agent.

Example 8 used two manganese concentrations of 6 ^ppm and 0.6 ^ppm . A concentration of 6 ^ppm has been shown to be inhibitory/toxic to yeast growth, while 0.6 ^ppm has been used as a standard concentration sufficient to eliminate manganese deficiency caused by one or more bacterial strains active in as manganese scavenging agents.

Fig. 12 and 13 demonstrate that a chemical chelating agent such as EDTA has an inhibitory effect on the growth of Debaromyces hansenii in the aqueous phase.

In FIG. 12 shows that Debaromyces hansenii cells stop growing when they encounter a concentration of 0.05 mg/ml EDTA. Thus, EDTA shows an inhibitory effect at a concentration of 0.05 mg/ml.

In FIG. 13 shows that at a concentration of 0.05 mg/ml EDTA and in the absence of manganese (0.6 ^ppm ), Debaromyces hansenii stopped growing; thus, EDTA has an inhibitory effect on Debaromyces hansenii under these conditions. The addition of 0.6 ^ppm manganese and 0.05 mg/ml EDTA restores the growth of Debaromyces hansenii, and the action of EDTA is terminated with an excess of manganese.

Example 9 Inhibition of Debaryomyces hansenii in yoghurt.

In yogurt, it is possible to reproduce suppression with manganese scavenging agents selected from one or more bacterial strains (manganese scavenging agent 1) by adding up to 0.89 mg/ml of EDTA, which is a manganese scavenging agent, to yogurt. At this concentration, the yogurt used as a control showed suppression close to that found when Manganese Scavenging Agent 1 was added. The negative controls in the bottom row of FIG. 14 (no EDTA added) showed normal suppression in the presence of manganese scavenging agent 1 and restored yeast growth with manganese addition.

Example 9 shows that a chemical chelating agent such as EDTA has the same effect as manganese scavenging agents selected from one or more of the bacteria disclosed here (FIG. 14). Therefore, Example 9 demonstrates that EDTA has the same effect as one or more of the bacterial strains disclosed herein as manganese scavengers.

Manganese scavenging agents, both a chemical chelating agent and/or biological material such as one or more bacterial strains found in a given product such as food, result in manganese depletion for spoilage fungi, thus suppressing mushroom growth.

Claims (13)

1. A method for suppressing or delaying the growth of fungi in a fermented milk product, comprising the step of reducing the level of free manganese in the product to a concentration of less than 0.01 ^ppm in the product, where the step of reducing the level of free manganese in the product includes adding one or more than one agent, absorbing manganese, where the agent absorbing manganese is one or more than one bacterial strain containing a carrier of manganese and/or a chemical chelating agent. 2. The method according to claim 1, further comprising the step of measuring the concentration of free manganese in the product and achieving a value of less than 0.01 ^ppm . 3. Method according to any one of claims 1, 2, wherein the fungi are yeasts or moulds. 4. The method according to any one of claims 1 to 3, wherein the fungi are yeasts selected from the group consisting of Torulaspora spp., Cryptococcus spp., Saccharomyces spp., Yarrowia spp., Debaryomyces spp., Candida spp. and Rhodoturola spp., or where the fungi are molds selected from the group consisting of Aspergillus spp., Cladosporium spp., Didymella spp. or Penicillium spp. 5. Process according to any one of claims 1 to 4, wherein the product is a thermophilic or mesophilic fermented milk product. 6. The method according to any one of claims 1 to 5, wherein the level of free manganese in the product is reduced to a concentration of less than 0.005 ^ppm . - 27 043250 7. The method according to any one of claims 1 to 6, wherein the step of reducing the level of free manganese in the product comprises the use of ion exchange chromatography. 8. The method according to any one of claims 1 to 7, wherein the chemical chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'tetraacetic acid, diaminocyclohexanetetraacetic acid , nitrilotriacetic acid, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or diethylenetriaminepentaacetic acid, preferably the chemical chelating agent is ethylenediaminetetraacetic acid. 9. The method according to any one of claims 1 to 8, further comprising the step of selecting one or more bacterial strains as the manganese scavenging agent. 10. The method according to any one of claims 1 to 9, wherein the step of selecting comprises determining whether one or more bacterial strains contain a manganese transporter having at least 55% sequence identity, such as at least 60, 65, 70 , 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity, with any of the sequences of SEQ ID NOs: 1-3. 11. The method according to any one of claims 1 to 10, wherein the step of selecting comprises determining that one or more than one bacterial strain does not contain superoxide dismutase, preferably does not contain manganese superoxide dismutase. 12. The method according to any one of claims 1 to 11, wherein the step of selecting comprises measuring the manganese uptake activity of one or more bacterial strains. 13. The method according to any one of claims 1 to 12, wherein the step of reducing the level of free manganese comprises adding, as a manganese scavenging agent, one or more lactic acid bacteria, optionally 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.