Fungistatic compositions and uses thereof

Abstract

The present disclosure describes methods and compositions for inhibiting fungal growth in food and cosmetic products, as well as food and cosmetic products treated with anti-fungal compositions. The anti-fungal composition can include hydrolyzed protein, antimicrobial peptides, and organic acids.

Description

[0001] FUNGISTATIC COMPOSITIONS AND USES THEREOF

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 633,458, filed on April 12, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 37942-0036W01_SL_ST26.xml. The XMLfile, created on April 9, 2025, is 3,682 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This disclosure generally relates to methods and compositions for inhibiting fungal growth in food and cosmetic products.

[0008] BACKGROUND

[0009] Food spoilage due to the presence of microorganisms including fungi is a major issue for the food industry, leading to significant food waste and substantial economic losses for manufacturers and consumers. Fungal contamination can occur at various stages of the food chain (e.g., postharvest, during processing, or storage), and can lead to issues ranging from visual deterioration to noticeable odor, flavor, or texture changes. Such issues can result in lower quality, shorter shelf life, more waste, more consumer complaints, and economic loss. In some instances, fungal growth in food can negatively impact health due to, for example, mycotoxin production by some molds.

[0010] Similarly, cosmetic and beauty products can harbor microorganisms including fungi, which can cause products to become unpleasant or unsafe for consumers. Preservatives, including antimicrobial ingredients, can be added to product formulations to maintain the microbiological safety of the products by inhibiting or limiting the growth of and reducing the amount of microbial contaminants.

[0011] Currently, fungicides and chemical preservatives are used to inhibit or limit fungal growth in food and cosmetic products; however, they can have limited efficacy. Therefore, new methods and compositions for inhibiting fungal growth in food and cosmetic products are needed. SUMMARY

[0012] This disclosure is related to a method of making an anti-fungal composition, the method comprising: combining a solution comprising bulk protein with an organic acid or a salt thereof to form a mixture; hydrolyzing the bulk protein before or after combining the bulk protein with the organic acid or the salt thereof; pasteurizing the solution; and drum drying the mixture to yield a dried mixture, wherein the dried mixture comprises one or more antimicrobial peptides, thereby making the anti-fungal composition. In some embodiments, the bulk protein is hydrolyzed before combining with the organic acid or the salt thereof. In some embodiments, the bulk protein is bulk milk protein, bulk legume protein, bulk soy protein, or bulk egg protein. In some embodiments, the organic acid or the salt thereof comprises a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof. In some embodiments, the organic acid or the salt thereof comprises propionic acid , succinic acid, acetic acid, a salt thereof, or combinations thereof. In some embodiments, the organic acid or the salt thereof comprises propionic acid or the salt thereof. In some embodiments, the one or more antimicrobial peptides comprise lactophoricin, LL-37, and / or alpha S-l casein. In some embodiments, the composition comprises two or all three of lactophoricin, LL-37, and alpha S-l casein. In some embodiments, the drum-drying uses a feed rate from about 20 milliliters per minute (ml / min) to about 40 ml / min; a drum speed of about 1 revolution per minute (rpm) to about 10 rpm; a steam pressure of about 20 psi to about 40 psi; and / or a scrapper temperature from about 100°C to about 125°C. In some embodiments, the composition comprises about 5% to about 95% of particles in the form of flakes. In some embodiments, the method described herein further comprises dissolving the dried mixture in a solvent. In one aspect, this disclosure is related to antifungal compositions made by the methods as described herein.

[0013] This disclosure is also related to an anti-fungal composition comprising hydrolyzed bulk protein, one or more organic acid or a salt thereof, and / or one or more antimicrobial peptides, wherein the one or more antimicrobial peptide is about 0.0001% to about 1% by weight of the antifungal composition. In some embodiments, the hydrolyzed bulk protein is about 10% to about 90% by weight of the anti-fungal composition. In some embodiments, the one or more organic acids or a salt thereof is about 0.1% to about 50% by weight of the anti-fungal composition. In some embodiments, the hydrolyzed bulk protein comprises hydrolyzed bulk milk protein, hydrolyzed bulk legume protein, hydrolyzed bulk soy protein, or hydrolyzed bulk egg protein. In some embodiments, the one or more organic acids or the salt thereof comprise a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof. In some embodiments, the one or more organic acids or the salt thereof comprise propionic acid, succinic acid, acetic acid, a salt thereof, or combinations thereof. In some embodiments, the organic acids or the salt thereof comprises propionic acid or a salt thereof. In some embodiments, the one or more antimicrobial peptides comprise lactophoricin, LL-37, and / or alpha S-l casein. In some embodiments, the one or more antimicrobial peptides comprise two or all three of lactophoricin, LL-37, and alpha S-l casein.

[0014] This disclosure is further related to a method of inhibiting fungal growth in a food or cosmetic product, the method comprising: contacting the food or cosmetic product with an antifungal composition comprising hydrolyzed bulk protein at a percentage range of about 10% to about 90% by weight, one or more organic acids or a salt thereof at a percentage range of about 0.1% to about 35% by weight, and one or more antimicrobial peptides at a percentage range of about 0.0001% to about 1% by weight. In some embodiments, the food product is selected from bakery products, meat and poultry products, dairy products, snacks, and pet foods. In some embodiments, the cosmetic product is selected from a cream, a lotion, an oil, a mask, a lipstick, and / or a solution. In some embodiments, the method as described herein further comprises combining the anti-fungal composition with a solvent to yield a composition, and contacting the food or cosmetic product with the composition. In some embodiments, the method as described herein further comprises packaging the food and / or cosmetic product. In some embodiments, the hydrolyzed bulk protein is hydrolyzed bulk milk protein, hydrolyzed bulk legume protein, hydrolyzed bulk soy protein, or hydrolyzed bulk egg protein. In some embodiments, the one or more organic acids or the salt thereof comprise a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof. In some embodiments, the one or more organic acids or the salt thereof comprise propionic acid, succinic acid, acetic acid, a salt thereof, or combinations thereof. In some embodiments, the one or more antimicrobial peptides comprise lactophoricin, LL-37, alpha S-l casein, or combinations thereof. In some embodiments, the bulk protein is bulk milk protein, and the organic acid or the salt thereof is propionic acid or a propionic acid salt. In some embodiments, the organic acid or the salt thereof is part of an organic acid powder. In some embodiments, the organic acid or the salt thereof is about 10 wt% to about 80 wt% of the organic acid powder.

[0015] In some embodiments, the present disclosure provides methods and compositions for inhibiting or limiting fungal growth in food and cosmetic compositions.

[0016] In some embodiments, the present disclosure provides methods of making an anti-fungal composition, the method comprising: combining a solution comprising bulk protein with an organic acid or organic acid salt to form a mixture; hydrolyzing the bulk protein before or after combining the bulk protein with the organic acid; pasteurizing the solution, and drum drying the mixture to yield a dried mixture, wherein the dried mixture comprises one or more antimicrobial peptides, thereby making an anti-fungal composition. In some embodiments, the bulk protein is hydrolyzed before combining with the organic acid. In some embodiments, the bulk protein is bulk milk protein, bulk legume protein, bulk soy protein, or bulk egg protein. In some embodiments, the organic acid or organic acid salt is selected from a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, or combination thereof. In some embodiments, the organic acid or organic acid salt comprises propionic acid, succinic acid, and acetic acid, or a salt thereof, preferably wherein the saturated aliphatic acid is propionic acid, or a salt thereof. In some embodiments, the one or more antimicrobial peptides is selected from lactophoricin, LL-37, and alpha S-l casein. In some embodiments, the anti-fungal composition comprises two or all three of lactophoricin, LL-37, and alpha S-l casein. In some embodiments, the drum-drying uses a feed rate from about 20 milliliters per minute (ml / min) to about 40 ml / min; a drum speed of about 1 revolution per minute (rpm) to about 10 rpm; a steam pressure of about 20 psi to about 40 psi; a scrapper temperature from about 100°C to about 125°C. In some embodiments, the anti-fungal composition comprises about 5% to about 95% of particles in the form of flakes. In some embodiments, the disclosed methods of making the anti-fungal composition further comprising dissolving the dried mixture in a solvent. In some embodiments, the anti-fungal composition is made by the methods disclosed above.

[0017] In some embodiment, this disclosure provides methods of inhibiting fungal growth in a food or cosmetic product, the method comprising: contacting the food or cosmetic product with the antifungal composition disclosed above. In some embodiments, the food product is selected from bakery products, meat and poultry products, dairy products, snacks, and pet foods. In some embodiments, the cosmetic product is selected from a cream and a lotion.

[0018] In some embodiments, the methods of inhibiting fungal growth in a food or cosmetic product further comprising combining the anti-fungal composition with a solvent to yield a composition, and contacting the food or cosmetic product with the composition. In some embodiments, the methods of inhibiting fungal growth in a food or cosmetic product further comprising packaging the food or cosmetic product. In some embodiments, the bulk protein is bulk milk protein, and the organic acid is propionic acid. In some embodiments, the organic acid or organic acid salt is part of an organic acid powder, and wherein the organic acid powder contains between 10-80 wt% organic acid or organic acid salt.

[0019] As used herein, the term "about" means ± 10%, unless otherwise specified.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0021] DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a flow chart depicting an exemplary process for preparing a dried anti-fungal composition including protein hydrolyzed skim milk powder (PHSM) and natural propionic acid salt (NPAS).

[0023] FIG. 2 is a flow chart depicting an alternate exemplary process for preparing a dried antifungal composition including protein hydrolyzed skim milk powder (PHSM) and natural propionic acid salt (NPAS).

[0024] FIG. 3A is a scanning electron microscope (SEM) image of spray-dried protein hydrolyzed skim milk (PHSM) combined with natural organic acid salt high in propionate (NPA) described in Example 1.

[0025] FIG. 3B is a scanning electron microscope (SEM) image of drum-dried protein hydrolyzed skim milk (PHSM) combined with natural organic acid salt high in propionate (NPAS) described in Example 1.

[0026] FIG. 4A is an image of a control of a fungal inhibition assay with Aspergillus Flavus in which no fungicide was used to treat the petri dish as described in Example 1.

[0027] FIG. 4B is an image of a fungal inhibition assay in which spray-dried protein hydrolyzed skim milk (PHSM) combined with natural organic acid salt high in propionate (NPAS) was used to treat the petri dish as described in Example 1.

[0028] FIG. 4C is an image of a fungal inhibition assay in which drum-dried protein hydrolyzed skim milk (PHSM) combined with natural organic acid salt high in propionate (NPAS) was used to treat a petri dish as described in Example 1.

[0029] DETAILED DESCRIPTION

[0030] The present disclosure describes an unexpected synergistic effect on inhibiting the growth of fungi in food and cosmetic products using a mixture of hydrolyzed protein powder, antimicrobial peptides, and organic acids that has been drum dried.

[0031] In many food and cosmetic products, a level of about 0.5 wt% organic acid or organic acid salt is typically used to inhibit or limit the growth of many fungi. See, for example, United States EPA, Pesticides and Toxic Substances (7508W), September 1991 found at epa.gov / pesticides / chem_search / reg_actions / reregistration / fs_G-71_l-Sep-91.pdf. The industry standard for establishing whether a compound inhibits growth of a microorganism is a 3-point inoculation of the compound into a culture plate followed by 3 days of incubation under suitable temperature and humidity conditions. After 3 days, the diameter and size of the fungal colonies indicates whether the compound is effective at inhibiting growth of the fungi.

[0032] The compositions and methods described herein can be used in virtually any food (e.g., human or animal) or cosmetic product that has a water activity (Aw) that supports the growth of fungi (e.g., food or cosmetic products having a water activity of at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, or at least 0.95). For example, the compositions and methods described herein can be used in any number of food and cosmetic products including, without limitation, bakery products (e.g., bread), dairy products, pet foods and treats, meat, and poultry products (e.g., meat snacks), plant-based foods (e.g., non-dairy products, plant-based burgers), and cosmetic products (e.g., creams and lotions). Suitable food and cosmetic products for use with the compositions and methods described herein can be fresh, refrigerated, or frozen.

[0033] Anti-fungal Composition

[0034] As demonstrated herein, a drum-dried anti-fungal composition including hydrolyzed protein powder (e.g., hydrolyzed bulk protein powder), an organic acid or an organic acid salt, and an antimicrobial peptide shows improved fungal inhibitory properties compared to other anti-fungal compositions. In some cases, the anti-fungal composition is a powder mixture. In some cases, the anti-fungal composition is combined directly with a food or cosmetic product to yield a treated food or cosmetic product. In other cases, the anti-fungal composition is combined with a solvent to yield an anti-fungal solution, and the antifungal solution is applied to a food or cosmetic product to yield a treated food or cosmetic product resistant to fungal growth.

[0035] Hydrolyzed Protein Powder

[0036] Hydrolyzed protein powder is a protein source, often bulk protein, that has been treated with proteolytic enzymes or acids to break down the protein into smaller peptides. The hydrolyzed protein powder can be formed by contacting an appropriate bulk protein powder with an appropriate proteolytic enzyme for an appropriate length of time to yield a mixture of hydrolyzed proteins, which can be dried to yield a hydrolyzed protein powder.

[0037] As defined herein, appropriate bulk protein powders induce little or no inflammatory response in humans or animals. Non-limiting examples of bulk protein powder include bulk vegetable protein powder (e.g., bulk legume protein powder, such as bulk lentil protein powder or bulk soy protein powder), bulk milk protein powder (e.g., skim milk protein powder), and bulk egg protein powder.

[0038] Appropriate proteolytic enzymes can be used to hydrolyze or break the proteins in the bulk protein into smaller peptides. Non-limiting examples of proteolytic enzymes include pepsin, trypsin, chymotrypsin, papain (also called papaya proteinase I), alkaline protease, bromelain, pancreatin, proteinase K, and alcalase. An appropriate amount of proteolytic enzyme can be contacted with a bulk protein powder. For example, the amount of proteolytic enzyme can be based on the amount of bulk protein powder, ranging from about 0.01% to about 10% (e.g., about 0.02% to about 9%, about 0.05% to about 6%, about 0.06% to about 5%, about 0.07% to about 4%, about 0.08% to about 3%, about 0.09% to about 2%, about 0.1% to about 1%, about 0.2% to about 0.9%, about 0.3% to about 0.8%, about 0.4% to about 0.7%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%), of the weight of the bulk protein powder. In some cases, the amount of proteolytic enzyme contacted with the bulk protein powder can be about 0.1% to aboutl% of the weight of the bulk protein powder.

[0039] In some cases, bulk protein powders are mixed with water to make a solution before being contacted with a proteolytic enzyme. In one example, an aqueous solution of about 20 wt% bulk protein powder (e.g., skim milk protein powder) in water is exposed to a proteolytic enzyme. See, for example, 102 of FIG. 1 or FIG. 2. In some cases, bulk protein powders, or solutions thereof, can be contacted with an organic acid or salt thereof (e.g., propionic acid salt) to make a second solution of from about 35 wt% (percentage by weight, wt%) to about 45 wt% (e.g., about 36 wt% to about 44 wt%, about 37 wt% to about 43 wt%, about 38 wt% to about 42 wt% , about 39 wt% to about 41 wt%, about 30 wt%, about 32 wt%, about 35 wt%, about 37 wt%, about 39 wt%, about 40 wt%, about 42 wt%, about 45 wt%, about 47 wt%, or about 50 wt%) bulk protein powder solids after being contacted with a proteolytic enzyme. In some cases, bulk protein powders, or solutions thereof, can be contacted with organic acid or salt thereof (e.g., propionic acid salt) to make a second solution of from about 35 wt% to about 45 wt% bulk protein powder solids before being contacted with a proteolytic enzyme. See, for example, 202 and 204 of FIG. 2. After adding the organic acid or salt thereof (e.g., propionic acid salt), the resulting solution can have a pH of from about 6 to about 7 (e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5), and the solution can have bulk protein powder solids from about 35 wt% to about 45 wt% (e.g., about 36 wt% to about 44 wt%, about 37 wt% to about 43 wt%, about 38 wt% to about 42 wt% , about 39 wt% to about 41 wt%, about 30 wt%, about 32 wt%, about 35 wt%, about 37 wt%, about 39 wt%, about 40 wt%, about 42 wt%, about 45 wt%, about 47 wt%, or about 50 wt%).

[0040] Hydrolyzed protein powder (e.g., hydrolyzed bulk protein powder) is present in the antifungal composition from about 10 wt% to about 90 wt% (e.g., from about 20 wt% to about 80 wt%, from about 30 wt% to about 70 wt%, from about 40wt% to about 60 wt%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% by weight (wt%)). In some cases, the hydrolyzed protein powder is present in the anti-fungal composition at about 15 wt%. In some cases, the hydrolyzed protein powder is present in the anti-fungal composition at about 20 wt%.

[0041] Organic Acids or Salts Thereof And Powders Containing the Same

[0042] Representative categories of organic acids are shown below, with representative examples of each.

[0043] 1) Saturated aliphatic acids (e.g., propionic acid, formic acid, acetic acid, or succinic acid)

[0044] 2) Unsaturated aliphatic acids (e.g., sorbic acid)

[0045] 3) Aromatic acids (e.g., benzoic acid)

[0046] 4) Polycarboxylic acids (e.g., oxalic acid)

[0047] 5) Hydroxy acids (e.g., lactic acid)

[0048] 6) Keto acids (e.g., pyruvic acid)

[0049] 7) Amino acids (e.g., glycine)

[0050] Table 1 provides representative organic acids that can be used in the compositions described herein. Some of the organic acids listed below are generally recognized as safe (GRAS) and have been used as anti-microbials. Some are also intermediates or products produced when fermenting sugar.

[0051] Table 1. Representative Organic Acids.

[0052] Salts of these and other organic acids can include, without limitation, metal cations (e.g., alkali metal cations, alkaline earth metal cations, transition metal cations) such as potassium, lithium, sodium, calcium, zinc, magnesium, or non-metal cations such as ammonium.

[0053] Organic acids and salts thereof can be found in an organic acid salt powder or a natural organic acid salt powder. An organic acid salt powder or a natural organic acid salt powder can contain between about 10 wt% to about 80 wt% of organic acid salt.

[0054] The amount of organic acid salt powder (e.g., NPAS) in an anti-fungal composition is from about 0.1 wt% to about 50 wt% (e.g., from about 1 wt% to about 45 wt%, from about 5% to about 40%, from about 10% to about 35%, from about 15% to about 30%, from about 20% to about 25%, from about 0.1% to about 20%, from about 25% to about 50%, about 0.1%, about 0.5%, about 0.8%, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight (wt%)). The organic acid salt powder can contain one organic acid salt (e.g., propionic acid salt) or many organic acid salts (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more organic acid salts). In some cases, the amount of organic acid salt powder in an antifungal composition is about from 25 wt% to about 50 wt%. The amount of organic acid salt powder (e.g., NPAS) in a food or cosmetic is from about 0.01% to about 10% (e.g., about 0.05% to about 8%, about 0.1% to about 6%, about 0.5% to about 4%, about 1% to about 3%, about 1.5% to about 2%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%). In some cases, the amount of organic acid salt powder in an anti-fungal composition, or combined with a food or cosmetic product is about 1.5 wt%.

[0055] Additional organic acids or salts thereof, or organic acid salt powders containing the same, can be added to the anti-fungal composition. For example, an anti-fungal composition can also include a second organic acid or salt thereof, a third organic acid or salt thereof, a fourth organic acid or salt thereof, etc. The total amount of additional organic acids or salts thereof that can be added to the anti-fungal composition can range from about 0.001 wt% to about 25%, e.g., about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, or about 1 wt% up to about 1 wt%, 2 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, or ranges therebetween with any of those values as endpoints). In some cases, the amount of additional organic acid salt powder in an anti-fungal composition or combined with a food or cosmetic product is about 25 wt%. In some cases, the amount of additional organic acid salt powder in an anti-fungal composition, or combined with a food or cosmetic product is about 1.5 wt%.

[0056] The ratio (by weight) of hydrolyzed protein powder to organic acid salt powder can be, e.g., from 9:1 to 1:9 (e.g., 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9).

[0057] In some instances, an organic acid or salt thereof produced during the fermentation process (e.g., propionic acid or salt thereof can be produced when microorganisms are grown in the presence of sugar (e.g., dextrose) or other sources such as wheat flour, tapioca, oats, or milk), is referred to as a natural organic acid or salt thereof (e.g., natural calcium propionate). In other instances, an organic acid or salt thereof can be added exogenously, referred to as chemically derived organic acid or a chemically derived organic acid salt (e.g., chemically derived calcium propionate or chemically derived calcium propionate salt).

[0058] Antimicrobial Peptides

[0059] Antimicrobial peptides can kill fungi, Gram-negative and Gram-positive bacteria, enveloped viruses, and cancerous cells. Each antimicrobial peptide can have specific targets upon which it acts. Antimicrobial peptides are generally between 12 and 50 amino acids in length and include two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (e.g., greater than 50%) of hydrophobic residues. The secondary structures of antimicrobial peptides are one of 4 themes, including 1) a-helical, 2) p-stranded due to the presence of 2 or more disulfide bonds, 3) p-hairpin or loop due to the presence of a single disulfide bond, cyclization of the peptide chain, or a single disulfide bond and cyclization of the peptide chain, and 4) extended. The peptides can contain hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. The amphipathic nature of antimicrobial peptides often facilitates insertion into the membrane lipid bilayer of a target cell. These peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets.

[0060] Non-limiting example of antimicrobial peptides that can be present in the anti-fungal composition include lactophoricin, LL-37 or a peptide cleaved from LL-37 (e.g., a peptide including amino acids 17 to 37 of LL-37), and a peptide cleaved from alpha S-l casein (e.g., a peptide containing amino acids 92 to 101 of alpha S-l casein).

[0061] Lactophoricin (LPcin-l) is a cationic amphipathic peptide with 23-amino acid residues, and corresponds to the carboxy terminal 113-135 region of component-3 of proteose peptone (PP3), often associated with bovine milk. Lactophoricin inhibits the growth of both gram-negative and gram-positive bacteria. See, for example, Hancock. Lancet Infect. Dis. Vol. 1: 3, pp. 156-164 (2021) and Ordonez et al. Antimicrob Agents Chemother. 2014 Apr; 58(4): 2240-2248.

[0062] LL-37 is a 37-amino-acid peptide that is produced when the C-terminus of cathelicidin antimicrobial peptide (CAMP) is cleaved by proteinase 2-mediated cleavage. Specifically, a major antimicrobial region of the peptide spans amino acids 17 to 37. See, for example, Neshani et al., Gene Reports. Vol. 17. December (2019) and Kim et al. J. Microbiol. Biotechnol. 2018; 28(8): 1299- 1309.

[0063] Alpha S-l casein is a protein found in milk. Casein proteins may be cleaved at several amino acids to produce a series of different antimicrobial peptides. See, for example, Hayes et al. Appl Environ Microbiol. 2006 Mar; 72(3): 2260-2264. For example, an antimicrobial peptide can be derived from or comprise amino acids at position 92 to 101 of the alpha S-l casein protein.

[0064] Anti-microbial peptides can be present in the anti-fungal composition in an amount that is effective to inhibit or slow fungal growth. The amount of anti-microbial peptides in an anti-fungal composition is from about 0.0001 wt% to about 1 wt% (e.g., about 0.0005 wt% to about 0.5 wt%, about 0.001 wt% to about 0.1 wt%, about 0.005 wt% to about 0.05 wt%, about 0.01 wt% to about 0.03 wt%, about 0.001 wt%, about 0.005 wt%, about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, about 0.5 wt% , or about 1 wt%). For example, anti-microbial peptides can be present from about 1 microgram (pg) per gram (g) of anti-fungal composition (pg / g) to about 500 pg / g (e.g., from about 1 pg / g to about 100 pg / g, from about 10 pg / g to about 150 pg / g, from about 25 pg / g to about 125 pg / g, from about 50 pg / g to about 250 pg / g, or from about 150 pg / g to about 500 pg / g, e.g., from about 1, 5, 10, 25, or 50 pg / g up to about 50, 100, 125, 150, or 250 pg / g, or any range therebetween with any of the preceding values as end points).

[0065] Any of the anti-fungal compositions described herein or any of the methods using the same can include any of the antimicrobial peptides described in Table 2, or any functional anti-fungal derivative thereof.

[0066] Table 2. Exemplary Antimicrobial Peptides.

[0067] Methods of Preparing Anti-Fungal Compositions

[0068] Also described herein are methods of preparing any of the anti-fungal compositions described herein. The methods of making anti-fungal compositions include combining a solution comprising bulk protein with an organic acid or a salt thereof to form a mixture; hydrolyzing the bulk protein before or after combining the bulk protein with the organic acid or a salt thereof; pasteurizing the solution; and drum drying the mixture to yield a dried mixture, wherein the dried mixture comprises one or more antimicrobial peptides, thereby making the anti-fungal composition. For example, an anti-fungal composition can be prepared as described in Example 1, FIG. 1, or FIG. 2.

[0069] Briefly, a mixture including a solution comprising hydrolyzed protein and one or more organic acids in a solvent, e.g., water, e.g., tap water, can be prepared and then dried. In some cases, preparing the mixture includes combining hydrolyzed protein powder, and one or more organic acids. In certain cases, preparing the mixture includes combining hydrolyzed protein powder and one or more organic acids. In some cases, one or more antimicrobial peptides are formed during the drying process and are not added. A hydrolyzed protein powder can be prepared as described herein.

[0070] In some cases, a bulk protein powder (e.g., skim milk protein powder) is contacted with a proteolytic enzyme. For example, a bulk protein powder (e.g., skim milk protein powder) is contacted with a proteolytic enzyme at its optimal temperature and pH of action. In one example, a bulk protein powder is contacted with a proteolytic enzyme and heated from about 25°C to about 65°C (e.g., from about 30°C to about 65°C from about 40°C to about 65°C, from about 50°C to about 65°C, or from about 55°C to about 65°C). In some cases, a bulk protein powder is contacted with a proteolytic enzyme and heated to about 60°C (e.g., about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C). Bulk protein powder contacted with a proteolytic enzyme and heated can be heated for any appropriate about of time needed to break down the proteins into smaller peptides. In one example, a bulk protein powder is contacted with a proteolytic enzyme and heated from about 30 minutes to about 4 hours (e.g., from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1.5 hour to about 2.5 hours, or about 40 minutes, about 1 hour, about 1.5 hours, about 1.6 hours, about 1.7 hours, about 1.8 hours, about 1.9 hours, about 2 hours, about 2.1 hours, about 2.2 hours, about 2.3 hours, about 2.4 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours). In some cases, a bulk protein powder is contacted with a proteolytic enzyme and heated for about 2 hours. In some cases, a bulk protein powder is contacted with a proteolytic enzyme and heated to about 60°C for about 2 hours.

[0071] In another example, a bulk protein powder is firstly mixed with a NPAS powder to yield a solution including about 40 wt% (e.g., about 35 wt%, about 36 wt%, about 37 wt%, about 38 wt%, about 39 wt%, about 40 wt%, about 41 wt%, about 42 wt%, about 43 wt%, about 44 wt%, or about 45 wt%) of organic acid. The mixture of a bulk protein powder and a NPAS powder is then contacted with a proteolytic enzyme and cooled to less than 15°C (e.g., 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C). The mixture of a bulk protein powder and a NPAS powder is contacted with a proteolytic enzyme for about 6 to about 12 hours (e.g., about 6, about 7, about 8, about 9, about 10, about 11, or about 12 hours). In some embodiments, the mixture of a bulk protein powder and a NPAS powder is contacted with a proteolytic enzyme at a temperature of less than 15°C for about 6- 12 hours.

[0072] The extent of protein hydrolysis can be measured using any appropriate technique. Nonlimiting examples of methods to measure the extent of protein hydrolysis can include colorimetric assays, high-performance liquid chromatography (HPLC), gel analysis, and mass spectrometry.

[0073] In some cases, after preparing a solution comprising hydrolyzed protein, the one or more organic acids, or a salt thereof, is added. For example, one or more organic acids is combined with the hydrolyzed protein solution to yield a solution including about 40 wt% (e.g., about 35 wt%, about 36 wt%, about 37 wt%, about 38 wt%, about 39 wt%, about 40 wt%, about 41 wt%, about 42 wt%, about 43 wt%, about 44 wt%, or about 45 wt%) of organic acid. In some cases, propionic acid is added to the hydrolyzed protein solution to yield a solution including about 40 wt% of propionic acid. Alternatively, the organic acid can be added to the protein solution, which is they hydrolyzed.

[0074] In some cases, the solution can be pasteurized before drying. In one example, the solution is held at about 80°C for at least about 15 seconds (e.g., from about 15 seconds to about 60 seconds).

[0075] Any of the compositions described herein can be dried to produce a dried anti-fungal composition (e.g., an anti-fungal powder). Methods of drying can include spray-drying and drumdrying. Drying methods can produce different textures of the anti-fungal composition. For example, see FIGs. 3A and 3B, which provide scanning electron microscope (SEM) images of spray-dried protein hydrolyzed skim milk (PHSM) combined with natural propionic acid (NPA) (3A) and drum- dried protein hydrolyzed skim milk (PHSM) combined with natural propionic acid (NPA) (3B). In some cases, one drying method may produce a more desirable texture than another drying method.

[0076] Spray-drying - Spray drying is a method of forming a dry powder from a liquid or slurry by rapidly drying the liquid or slurry with a hot gas. For example, an inlet air temperature can be from about 150°C to about 175°C and an outlet air temperature can be from about 85°C to about 95°C. An atomizer or spray nozzle (e.g., rotary disk and single-fluid high pressure swirl nozzles) can disperse the liquid or slurry to be dried into a controlled drop size spray. Atomizer wheels are known to provide broader particle size distribution, but both methods allow for consistent distribution of particle size. Alternatively, for some applications, two-fluid or ultrasonic nozzles are used. Depending on the process requirements, drop sizes from 10 pm to 500 pm can be achieved with the appropriate choices. The most common applications are in the 100 pm to 200 pm diameter range. The dry powder is often free-flowing. Any of the compositions described herein can be spray-dried.

[0077] Drum-drying - Drum drying removes moisture from pastes, purees, and liquids with rotating, heated drums. A thin layer of a paste, puree, or liquid is applied to the outer surface of revolving drums that are internally heated (e.g., by steam). After most of a revolution around the drum from the point of application (e.g., about three quarters of a revolution), the thin layer of a dried product is scrapped off the drum and is often flaky in appearance. The dried product can be further processed into uniform flakes (small, flat, thin pieces) or ground into a fine powder. Any of the antifungal compositions described herein can be drum-dried.

[0078] When drum-drying a composition, there are a variety of parameters that can be optimized. For example, optimizable parameters include a reed rate, a drum speed, a steam pressure, a scrapper temperature, a drum diameter, and a drum length. In some cases, drum-drying uses a feed rate from about 20 milliliters per minute (mL / min) to about 40 mL / min. In some cases, drum-drying uses a drum speed of about 1 revolution per minute (rpm) to about 10 rpm. In some cases, drum-drying uses a steam pressure of about 20 pounds per square inch (psi) to about 40 psi. In some cases, drumdrying uses a scrapper temperature from about 100°C to about 125°C. In some cases, drum-drying uses a commercially available drum dryer, e.g., up to 24-30 feet long.

[0079] In some cases, the drum-dried composition or dried mixture comprises about 5 wt% to about 95 wt% (e.g., about 10 wt% to about 90 wt% , about 15 wt% to about 85 wt%, about 20 wt% to about 80 wt%, about 25 wt% to about 75 wt%, about 30 wt% to about 70 wt%, about 35 wt% to about 65 wt%, about 40 wt% to about 60 wt%, about 45 wt% to about 55 wt%, about 5 wt%, about 10 wt%, about 20 wt%, about 30 wt%, about 40 wt%, about 50 wt%, about 60 wt%, about 70 wt%, about 80 wt%, about 90 wt%, or about 95 wt%) of particles that are substantially flaky. In some cases, the drum-dried composition or dried mixture comprises about 5% to about 95% of the total number of particles that are substantially flaky. In some cases, the drum-dried particles have a substantially planar face with a minimum width between about 20 pm and about 140 pm (e.g., about 20 pm, about 40 pm, about 60 pm, about 80 pm, about 100 pm, about 120 pm, or about 140 pm). In some cases, the drum-dried composition or dried mixture comprises about 5% to about 95% of particles that are substantially flaky and wherein the particles have a substantially planar face with a minimum width between about 20 pm and about 140 pm.

[0080] In accordance with the present embodiments, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The embodiments will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

[0081] Methods of Inhibiting Fungal Growth

[0082] Also described herein are methods of inhibiting fungal growth. The methods described herein can be used for inhibiting fungal growth in food or cosmetic products. In some cases, the methods include contacting the food or cosmetic product with an anti-fungal composition containing hydrolyzed bulk protein at a percentage range of about 10% to about 90% by weight, one or more organic acid or a salt thereof at a percentage range of about 0.1% to about 50% by weight, and one or more antimicrobial peptide at a percentage range of about 0.0001% to about 1% by weight. In some cases, the methods further include combining the anti-fungal composition with a solvent to yield a composition, which is used to contact the food or cosmetic product. In some cases, the methods described herein further include packaging the food or cosmetic product using the methods known in arts.

[0083] In some cases, the anti-fungal composition is combined directly with food or cosmetic products to yield treated food or cosmetic products that are resistant to fungal growth (e.g., no fungal growth for about 3 days to about 10 days). In other cases, the anti-fungal composition is combined with a solvent to yield an anti-fungal solution, and the antifungal solution is applied to a food or cosmetic product to yield treated food or cosmetic products.

[0084] As described herein, the food product that can be combined with the anti-fungal composition include bakery products, meat and poultry products, dairy products, snacks, and / or pet foods. The cosmetic product described herein can be a cream, a lotion, an oil, a mask, a lipstick, an / or a solution.

[0085] In some cases, the methods described herein can be used for inhibiting fungal growth in a agar (e.g., petri dish agar plate, a food product, or a cosmetic product). Appropriate fungal species can be used for the fungistatic screening. Nonlimiting examples of fungal species include Aspergillus flavus, Aspergillus flavus Bi, Aspergillus flavus Bi, B2, Aspergillus parasiticus, Aspergillus niger, Penicillium crustosum, Penicillium expansum, and Penicillium digitatum. In some cases, Aspergillus flavus Bi, B2are used for the fungistatic screening. It would be appreciated that the methods described herein are fungistatic (mold inhibition). As disclosed herein, fungistatic (mold inhibition) screening was conducted by recording the number of days of inhibition and measuring the diameter of the colony. In some cases, the methods described herein can inhibit fungal growth in a product (e.g., CYA agar, a food product, or a cosmetic product) for about 3-10 days (e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days) after inoculation of fungal species (e.g., Aspergillus flavus Bi, B2). As described herein, a longer duration of inhibition can be a condition of minimal or no fungal growth for a longer time (e.g., about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, compared to the colonies that are not treated with the methods described herein). As described herein, the longer duration of inhibition can indicate stronger static effects (e.g., compared to the colonies that are not treated with the methods described herein). As described herein, a smaller diameter of the fungal colony (e.g., compared to the colonies that are not treated with the methods described herein) signifies greater inhibition (e.g., compared to the colonies that are not treated with the methods described herein).

[0086] EXAMPLES

[0087] Example 1 - Preparing an organic acid salt powder high in propionate (e.g., natural propionic acid salt, NPAS)

[0088] An NPAS was generated by fermenting a medium composed of carbon, nitrogen, and nutrient source with a strain of propionibacterium (e.g., Propionibacterium acidipropionici) and neutralize the broth with a base to pH of 5.5 - 10.0. The resulting fermentate that is high in propionate is dried either with or without biomass using drying process known in the industry. The NPAS has the properties described in Table 3.

[0089] Table 3. Exemplary NPAS properties. Example 2— Preparing Anti-fungal Compositions

[0090] Czapek Yeast Autolysate Agar (CYA Agar) was used to culture various species and strains of fungi in the presence or absence of a composition for inhibiting fungal growth. A 3-point inoculation was used and cultures were grown at 25°C.

[0091] A fungal sample of Aspergillus flavus aflatoxin producer (Bi, B2) isolated from a commercial dairy product was tested with various compositions for their ability to inhibit fungal growth.

[0092] Materials

[0093] Natural propionic acid (NPA) or natural propionic acid salt (NPAS) as described in Example 1 was produced through fermentation of a carbon source with propionic acid bacteria (e.g., Propion! bacterium acidipropionici) .

[0094] Commercially available skim milk powder (SMP) was purchased.

[0095] Protein hydrolyzed skim milk powder (PHSM) was made through treating a ~20% weight / weight (w / w) solution of SMP with proteolytic enzymes such as papain, alkaline protease, or alcalase, and spray dried; alkaline protease was used in the present Example.

[0096] A spray dried combination of PHSM and NPA was made through combining PHSM and NPA into a solution and spray-drying the solution.

[0097] A drum dried combination of PHSM and NPA was made through combining PHSM and NPA into a solution and drum-drying the solution.

[0098] Aspergillus flavus aflatoxin producer (Bl, B2) isolated from a commercial dairy product was used in this experiment. The strains were initially growth on Czapek Yeast Autolysate Agar (CYA) at 25°C for 7 days.

[0099] The process flow to make either drum dried or spray dried combinations is illustrated in FIGs. 1 and 2. FIG. 1 shows a short process for preparing a spray-dried or drum-dried anti-fungal composition containing PHSM and NPA. This process includes heating a solution of SMP in tap water to 60°C with proteolytic enzymes for a 2-hour incubation to generate PHSM before combining with NPA and drying. FIG. 2 shows a long (alternate) process for preparing a spray-dried or drum-dried anti-fungal composition including PHSM and NPA. This process uses a long incubation (e.g., 6-12 hours) at a temperature of <15°C of a SMP and NPA solution with proteolytic enzymes and drying. The drying parameters are provided in Table 4. Table 4. Drying parameters

[0100] Methods

[0101] Standard methods were used for determining moisture and pH of the materials. Organic acid profile (propionic, acetic, and succinic) was determined using a reversed phase HPLC method. The particle morphology was visualized using scanning electron microscopy.

[0102] Analysis of Compositions

[0103] Table 5 below provides properties of the anti-fungal compositions studied. Both moisture and pH were as expected for powder materials, but the organic acid profile differed based on the material.

[0104] Table 5. Exemplary Anti-fungal Composition Properties

[0105] The presence of antimicrobial peptides in the dried powder was analyzed by mapping peptide signal matches (PSM) to curated databases containing known amino acid sequences of antimicrobial peptides (Table 6). (The powder was resuspended into a liquid and run through Liquid Chromatography coupled with two mass spectrometry.) PSM were identified using a stringent false- discovery rate of 0.01 and databases were queried using the search engine Mascot with a minimum match scoring of 50. The Database of Antimicrobial Activity and Structure of Peptides (DBAASP) is an NIH-supported server that provides users with experimentally verified structure and antimicrobial activity data on peptide query matches. The DBAASP was chosen to obtain antimicrobial peptide data because it is equipped with an application programmable interface, manually curated by subject matter experts, and is frequently updated while most other databases are outdated. Thus, DBAASP provides access to the latest experimental data on antimicrobial activity and toxicity.

[0106] Antimicrobial peptides that are unique to the dried combinations include lactophoricin, LL- 37, and alpha S-l casein. A drum dried composition of PHSM and NPA had all the 3 peptides whereas spray-dried combination had only one antimicrobial peptide (as shown in Table 6 below). It should be noted that the PHSM should have had peptides, but the analysis indicated that there were no peptides present. It is possible that peptides were below the detection threshold of the instrument.

[0107] Table 6. Antimicrobial Peptides of Exemplary Anti-Fungal Compositions

[0108] Visualizing particles under scanning electron microscopy indicated that morphology is standard for drying technologies (FIGS. 3A and 3B). Spray dried powder particles were spherical whereas drum dried powder particles had varying sizes and shapes and were more flaky (e.g., substantially flattened particles compared to a sphere) in appearance than the spray dried powder particles. Exemplary percentage ranges of ingredients in anti-fungal compositions are shown in Table 7.

[0109] Table 7. Percentage Ranges of Ingredients in Exemplary Anti-Fungal Compositions

[0110] Example 3— Inhibiting Fungal Growth Using Anti-fungal Compositions

[0111] Anti-fungal (mold inhibition) screening was conducted by recording the number of days of inhibition (higher days of inhibition, higher the static effect) and measuring the diameter of the colony (smaller the diameter, higher the inhibition). CYA was used to grow different molds such as Aspergillus flavus, Aspergillus flavus Bi, Aspergillus flavus Bi, B2, Aspergillus parasiticus, Aspergillus niger, Penicillium crustosum, Penicillium expansum, and Penicillium digitatum. Each treatment was added to CYA recipe and sterilized. A sterile loop with glycerol was used to pick each mold and inoculate it into a new plate. Media was incubated at 25°C, 75% relative humidity (RH), and monitored after 3 days of incubation to record any growth.

[0112] The following compositions were used in the experiments described herein:

[0113] 1. Control, i.e., No anti-fungal composition

[0114] 2. Natural propionic acid (NPA) powder

[0115] 3. Skim milk powder or non-fat dry milk powder (SMP)

[0116] 4. Protein hydrolyzed skim milk (PHSM) powder

[0117] 5. Dry blend of skim milk powder and NPA powder

[0118] 6. Spray-dried combination of PHSM + NPA

[0119] 7. Drum-dried combination of PHSM + NPA

[0120] In terms of mold inhibition, it took about 3 days from inoculation for the mold to grow. The results showed there was no mold inhibition with control (no preservative), NPA, SMP, PHSM, and dry blend of NPA and PHSM. The diameter of the mold colonies was the same for all these ingredients (e.g., 4 cm except for drum-dried and spray-dried combinations of PHSM+NPA). Spray- dried combinations had a diameter of 3.5 cm and drum-dried combinations had a diameter of 2.0 cm (FIGs. 4A-4C, Table 8). This indicates that the peptides and organic acid present in the combinations worked synergistically to limit mold growth, and drum-dried combination has superior effect.

[0121] Table 8. Results of Mold Inhibition Assay

[0122] References:

[0123] 1. Nath A, Eren BA, Csighy A, Pasztorne-Huszar K, Kisko G, Abranko L, et al. Production of Liquid Milk Protein Concentrate with Antioxidant Capacity, Angiotensin Converting Enzyme Inhibitory Activity, Antibacterial Activity, and Hypoallergenic Property by Membrane Filtration and Enzymatic Modification of Proteins. Processes. 2020;8(7):871.

[0124] 2. Korhonen H, Pihlanto A. Food-derived bioactive peptides-opportunities for designing future foods. Curr Pharm Des. 2003;9(16):1297-308.

[0125] 3. Garnier L, Mounier J, Le S, Pawtowski A, Pinon N, Camier B, et al. Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application. Food Microbiol. 2019;81:97-107.

[0126] 4. Kitts DD, Weiler K. Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Curr Pharm Des. 2003;9(16):1309-23.

[0127] 5. El-Zahar KM, Sitohy MZ, Choiset Y, Metro F, Haertle T, Chobert JM. Antimicrobial activity of ovine whey protein and their peptic hydrolysates. Milchwissenschaft-milk Science International. 2004;59:653-6.

[0128] 6. Coban HB. Organic acids as antimicrobial food agents: applications and microbial productions. Bioprocess Biosyst Eng. 2020;43(4):569-91.

[0129] 7. Razavi-Rohani SM, Griffiths MW. Antifungal effects of sorbic acid and propionic acid at different pH and NaCI conditions. Journal of Food Safety. 1999;19:109-20.

[0130] 8. Kim JS, Joeng JH, Kim Y. Design, Characterization, and Antimicrobial Activity of a Novel Antimicrobial Peptide Derived from Bovine Lactophoricin. J Microbiol Biotechnol. 2017;27(4):759-67.

[0131] 9. Wang X, Mishra B, Lushnikova T, Narayana JL, Wang G. Amino Acid Composition Determines Peptide Activity Spectrum and Hot-Spot-Based Design of Merecidin. Adv Biosyst. 2018;2(5).

[0132] 10. Overhage J, Campisano A, Bains M, Torfs EC, Rehm BH, Hancock RE. Human host defense peptide LL-37 prevents bacterial biofilm formation. Infect Immun. 2008;76(9):4176-82.

[0133] 11. Sistla S. Structure-activity relationships of as-casein peptides with multifunctional biological activities. Mol Cell Biochem. 2013;384(l-2):29-38.

[0134] 12. Liu Y, Eichler J, Pischetsrieder M. Virtual screening of a milk peptide database for the identification of food-derived antimicrobial peptides. Mol Nutr Food Res. 2015;59(ll):2243-54.

[0135] OTHER EMBODIMENTS

[0136] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of making an anti-fungal composition, the method comprising: combining a solution comprising bulk protein with an organic acid or a salt thereof to form a mixture; hydrolyzing the bulk protein before or after combining the bulk protein with the organic acid or the salt thereof; pasteurizing the solution; and drum drying the mixture to yield a dried mixture, wherein the dried mixture comprises one or more antimicrobial peptides, thereby making the anti-fungal composition.

2. The method of claim 1, wherein the bulk protein is hydrolyzed before combining with the organic acid or the salt thereof.

3. The method of claim 1, wherein the bulk protein is bulk milk protein, bulk legume protein, bulk soy protein, or bulk egg protein.

4. The method of claims 1 or 2, wherein the organic acid or the salt thereof comprises a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof.

5. The method of claim 4, wherein the organic acid or the salt thereof comprises propionic acid , succinic acid, acetic acid, a salt thereof, or combinations thereof.

6. The method of claim 4, or 5, wherein the organic acid or the salt thereof comprises propionic acid or the salt thereof.

7. The method of claim 1, wherein the one or more antimicrobial peptides comprise lactophoricin,LL-37, and / or alpha S-l casein.

8. The method of claim 6, wherein the composition comprises two or all three of lactophoricin, LL-37, and alpha S-l casein.

9. The method of claim 1, wherein the drum-drying uses a feed rate from about 20 milliliters per minute (ml / min) to about 40 ml / min; a drum speed of about 1 revolution per minute (rpm) to about 10 rpm; a steam pressure of about 20 psi to about 40 psi; and / or a scrapper temperature from about 100°C to about 125°C.

10. The method of claim 1, wherein the composition comprises about 5% to about 95% of particles in the form of flakes.

11. The method of any one of claims 1-9, further comprising dissolving the dried mixture in a solvent.

12. An anti-fungal composition made by the method of any of claims 1-10.

13. An anti-fungal composition comprising hydrolyzed bulk protein, one or more organic acid or a salt thereof, and / or one or more antimicrobial peptides, wherein the one or more antimicrobial peptide is about 0.0001% to about 1% by weight of the anti-fungal composition.

14. The anti-fungal composition of claim 13, wherein the hydrolyzed bulk protein is about 10% to about 90% by weight of the anti-fungal composition.

15. The anti-fungal composition of claim 13, wherein the one or more organic acids or a salt thereof is about 0.1% to about 50% by weight of the anti-fungal composition.

16. The anti-fungal composition of claim 13 or 14, wherein the hydrolyzed bulk protein comprises hydrolyzed bulk milk protein, hydrolyzed bulk legume protein, hydrolyzed bulk soy protein, and / or hydrolyzed bulk egg protein.

17. The anti-fungal composition of claim 13 or 15, wherein the one or more organic acids or the salt thereof comprise a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof.

18. The anti-fungal composition of any one of claims 13, 15, or 17, wherein the one or more organic acids or the salt thereof comprise propionic acid, succinic acid, acetic acid, a salt thereof, or combinations thereof.

19. The anti-fungal composition of claim 18, wherein the organic acids or the salt thereof comprises propionic acid or a salt thereof.

20. The anti-fungal composition of claim 13, wherein the one or more antimicrobial peptides comprise lactophoricin, LL-37, and / or alpha S-l casein.

21. The anti-fungal composition of claim 13, or 20, wherein the one or more antimicrobial peptides comprise two or all three of lactophoricin, LL-37, and alpha S-l casein.

22. A method of inhibiting fungal growth in a food or cosmetic product, the method comprising: contacting the food or cosmetic product with an anti-fungal composition comprising hydrolyzed bulk protein at a percentage range of about 10% to about 90% by weight, one or more organic acids or a salt thereof at a percentage range of about 0.1% to about 35% by weight, and one or more antimicrobial peptides at a percentage range of about 0.0001% to about 1% by weight.

23. The method of claim 22, wherein the food product is selected from bakery products, meat and poultry products, dairy products, snacks, and pet foods.

24. The method of claim 22, wherein the cosmetic product is selected from a cream, a lotion, an oil, a mask, a lipstick, and / or a solution.

25. The method of any one of claims 22-24, further comprising combining the anti-fungal composition with a solvent to yield a composition, and contacting the food or cosmetic product with the composition.

26. The method of any one of claims 22-25, further comprising packaging the food or cosmetic product.

27. The method of any one of claims 22-26, wherein the hydrolyzed bulk protein is hydrolyzed bulk milk protein, hydrolyzed bulk legume protein, hydrolyzed bulk soy protein, or hydrolyzed bulk egg protein.

28. The method of any one of claims 22-27, wherein the one or more organic acids or the salt thereof comprise a saturated aliphatic acid, an unsaturated aliphatic acid, an aromatic acid, a hydroxyl acid, a keto acid, an amino acid, a salt thereof, or combinations thereof.

29. The method of any one of claims 22-28, wherein the one or more organic acids or the salt thereof comprise propionic acid, succinic acid, acetic acid, a salt thereof, or combinations thereof.

30. The method of any one of claims 22-29, wherein the one or more antimicrobial peptides comprise lactophoricin, LL-37, alpha S-l casein, or combinations thereof.

31. The method of any one of claims 1-11 and 22-30, wherein the bulk protein is bulk milk protein, and the organic acid or the salt thereof is propionic acid or a propionic acid salt.

32. The method of any one of claims 1-11 and 22-30, wherein the organic acid or the salt thereof is part of an organic acid powder.

33. The method of claim 32, wherein the organic acid or the salt thereof is about 10 wt% to about80 wt% of the organic acid powder.

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