Anti-mold compositions comprising chitosan

Abstract

The present application is directed to anti-mold compositions comprising chitosan polymers having average molecular weights (Mws) of about 20 kDa, about 170 kDa and about 200 kDa. The present application further includes a method of making and using the compositions and products comprising the compositions. The compositions of the application advantageously inhibit the growth of mold species without inhibiting the growth of Saccharomyces yeast.

Description

ANTI-MOLD COMPOSITIONS COMPRISING CHITOSANCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority from U.S. provisional patent application no. 63 / 606,298 filed on December 5, 2023, the contents of which are incorporated by reference in their entirety.FIELD

[0002] The present application relates to anti-mold compositions comprising chitosan that can be used, for example, in food products, particularly baked goods.INTRODUCTION

[0003] The growth of spoilage microorganisms in products causes the spoilage of food, beverages, cosmetics, and personal care items, and leads to unsanitary medical devices and pharmaceuticals that pose serious risks for patients. Baked food products, for example, suffer from having a short shelf life due to the ease at which they can grow mold (Denkova et al., Production of wheat bread without preservatives using sourdough starters, Biotech. Biotechn. Equp., 2014 Sep, 28(5), 889-898). Mold is especially a problem in reduced calorie baked good products due to the high-water activity they have. In addition to causing health risks to the human consumer, mold spoilage represents wasted resources and energy, and lost revenue to producers.

[0004] Preservatives reduce or eliminate microbial spoilage and are thus vital ingredients to a wide variety of products. However, in yeast-raised baked food products, for example, preservatives may also inhibit the baker’s yeast (Saccharomyces cerevisae) used for leavening. This effect may result in manufacturing problems and increased costs due to the need to use greater amounts of yeast to offset yeast inhibition. The most common preservative used today is propionic acid and its salts (sodium propionate and calcium propionate). Propionate, especially calcium propionate is used in yeast- leavened baked foods because it has no effect against the species of baker’syeast. However, propionate has been associated with health risks like insulin resistance and obesity (Tirosh et al., The short-chain fatty acid propionate increases glucagon and FABP4 production, impairing insulin action in mice and humans, Science Translational Medicine, Apr 2019, 11 , 489). Other preservatives like natamycin and nisin are not applicable in baked goods since they will kill the baker’s yeast (Kalli nteri et al., Efficacy of nisin and / or natamycin to improve the shelf-life of Galotyri cheese, Food Microbiol., 2013 Dec;36(2): 176-81).

[0005] Natural preservative agents are growing in demand due to increased consumer awareness of the potential risks associated with artificial preservatives. Consumer choice increasingly deviates from products listing artificial preservatives as an ingredient.

[0006] Chitosan, is a natural polysaccharide composed of repeating N- deacetyl-glucosamine and N-Acetyl-glucosamine monomers, produced in the cell wall of, crustaceans, fungi, and insects. Chitosan and various fractions thereof have been shown to be effective antimicrobial agents (see for example Applicant’s own PCT Patent Application publication no. WO 2019046950). However, no specific antimicrobial product comprising chitosan has been developed for yeast-based products.

[0007] Therefore, there is an unmet need for natural anti-mold compositions which avoid the negative impact against the strains of Saccharomyces yeast and that can be used, for example, in dough-based food products.SUMMARY

[0008] The present application includes compositions that use chitosan to inhibit the growth of mold species while minimizing the inhibition of the growth of Saccharomyces yeast. The compositions of the present application can be used for example, in food products, particularly baked goods.

[0009] The present application includes an anti-mold composition comprising or consisting essentially of: a chitosan polymer having an average molecular weight (Mw) of about 20 kDa; a chitosan polymer having an average Mw of about 170 kDa; and a chitosan polymer having an average Mw of about 200 kDa, wherein the composition comprises substantially greater amounts in percent by weight of the chitosan polymer having an average Mw of about 200 kDa.

[0010] The present application also includes a method of making a composition of the application, wherein the method comprises: a) combining the chitosan polymer having an average molecular weight (Mw) of about 20 kDa, the chitosan polymer having an average Mw of about 170 kDa, and the chitosan polymer having an average Mw of about 200 kDa.

[0011] The present application also includes a product comprising a composition of the application, in particular, a food product, such as a doughbased food product.

[0012] Also included in the present application is a method of inhibiting mold growth in a product comprising incorporating an effective amount of a composition of the application into the product as well as a method of preserving a product comprising incorporating an effective amount of a composition of the application, into the product.

[0013] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.DESCRIPTION OF VARIOUS EMBODIMENTSI. Definitions

[0014] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0015] The term “composition of the application” or “composition of the present application” and the like as used herein refers to a composition comprising the chitosan polymers of the application.

[0016] The term “average molecular weight” or “Mw” as used herein refers to the weight average molecular weight as measured in kiloDaltons (kDa) using Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC).

[0017] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0018] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a microorganism” should be understood to present certain aspects with one microorganism species or two or more additional microorganisms.

[0019] In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0020] In understanding the scope of the present application, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components,groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.

[0021] The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0022] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0023] The term "suitable" as used herein means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, and the identity of the molecule(s) to be transformed, but the selection would be well within the skill of a person trained in the art.

[0024] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0025] The term “to inhibit the growth of mold species” and variations thereof as used herein means any detectable inhibition of the growth of or killing of the mold species in the presence of one or compositions of the applicationcompared to otherwise the same conditions except in the absence of the one or more compositions of the application.

[0026] As used herein, the term “effective amount” means an amount of one or more compositions of the application that is effective to achieve the desired result. For example in the context of inhibiting mold growth while minimizing inhibition of Saccharomyces yeast growth, an effective amount is an amount that, for example, increases said inhibition of mold while decreasing inhibition of said yeast growth, compared to the inhibitions under otherwise the same conditions except without administration of the one or more compositions.II. Compositions of the Application

[0027] The present application includes an anti-mold composition comprising: a chitosan polymer having an average molecular weight (Mw) of about 20 kDa; a chitosan polymer having an average Mw of about 170 kDa; and a chitosan polymer having an average Mw of about 200 kDa, wherein the composition comprises substantially greater amounts in percent by weight of the chitosan polymer having an average Mw of about 200 kDa.

[0028] In some embodiments, the term “substantially greater amounts in percent by weight” means that the referenced chitosan polymer is present in the composition in amounts that are about 5 percentage points (w / w) to about 100 percentage points (w / w), or about 7 percentage points (w / w) to about 50 percentage points (w / w), or about 10 percentage points (w / w) to about 30 percentage points (w / w) greater than other chitosan polymers in the composition. For example, if the chitosan polymer having an average Mw of about 200 kDa is present in the composition in about 40 percentage points (w / w) greater than the other two polymers, then the composition may compriseabout 70% (w / w) by weight of the polymer present in greater amounts and about 15% (w / w) of each the remaining two polymers. The amounts of the other two polymers does not need to be the same, therefore the chitosan polymer having an average Mw of about 200 kDa may present in the composition in percentage points for each of the remaining two polymers that are different. Therefore, for example, if the chitosan polymer having an average Mw of about 200 kDa is present in the composition in about 60 percentage points (w / w) greater than the first of the two remaining polymers and in about 65 percentage points greater than the second of the two remaining polymers, then the composition may comprise about 75% (w / w) by weight of the polymer present in greater amounts and about 15% (w / w) of the first of the remaining polymers and about 10% (w / w) of the second of the two remaining polymers.

[0029] In some embodiments, the anti-mold composition consists essentially of chitosan polymers having an average molecular weight (Mw) of about 20 kDa; about 170 kDa; and about 200 kDa.

[0030] In some embodiments, the anti-mold composition comprises chitosan polymers having an average molecular weight (Mw) of about 20 kDa; about 170 kDa; and about 200 kDa as the only chitosan polymers in the composition.

[0031] In some embodiments, the chitosan polymers in the composition of the application consist essentially of chitosan polymers having an average molecular weight (Mw) of about 20 kDa; about 170 kDa; and about 200 kDa.

[0032] In some embodiments, the chitosan polymers in the composition of the application consists of chitosan polymers having an average molecular weight (Mw) of about 20 kDa; about 170 kDa; and about 200 kDa.

[0033] In some embodiments, the anti-mold composition comprises: about 5 % w / w to about 25 % w / w of the chitosan polymer having an average molecular weight (Mw) of about 20 kDa;about 5 % w / w to about 20 % w / w of the chitosan polymer having an average Mw of about 170 kDa; and about 65 % w / w to about 90 % w / w chitosan polymer having an average Mw of about 200 kDa, wherein the percentage is by total weight of chitosan polymers in the composition.

[0034] In some embodiments, the chitosan polymer having an average Mw of about 20 kDa is present in an amount of about 7 % w / w to about 23 % w / w by total weight of the chitosan in the composition. In some embodiments, the chitosan polymer having an average Mw of about 20 kDa is present in an amount of about 10 % w / w to about 20 % w / w, about 12 % w / w, about 15 % w / w, or about 18% w / w by total weight of the chitosan in the composition, and values therebetween. In some embodiments, the chitosan polymer having an average Mw of about 20 kDa is present in an amount of about 15 % w / w by total weight of the chitosan in the composition.

[0035] In some embodiments, the chitosan polymer having an average Mw of about 170 kDa is present in an amount of about 7 % w / w to about 18 % w / w by total weight of the chitosan in the composition. In some embodiments, the chitosan polymer having an average Mw of about 170 kDa is present in an amount of about 7 % w / w, about 9 % w / w, about 12 % w / w, about 15 % w / w, or about 18 % w / w by total weight of the chitosan in the composition, and values therebetween. In some embodiments, the chitosan polymer having an average Mw of about 170 kDa is present in an amount of about 9 % w / w by total weight of the chitosan in the composition.

[0036] In some embodiments, the chitosan polymer having an average Mw of about 200 kDa is present in an amount of about 70 % w / w to about 90 % w / w by total weight of the chitosan in the composition. In some embodiments, the chitosan polymer having an average Mw of about 200 kDa is present in an amount of about 72 % w / w, about 74 % w / w, about 76 % w / w, about 78 % w / w,about 80 % w / w, about 84 % w / w, or about 88 % w / w by total weight of the chitosan in the composition, and values therebetween. In some embodiments, the chitosan polymer having an average Mw of about 200 kDa is present in an amount of about 76 % w / w by total weight of the chitosan in the composition.

[0037] In some embodiments the weight ratio of the chitosan polymer having an average molecular weight (Mw) of about 20 kDa:chitosan polymer having an average Mw of about 170 kDa:chitosan polymer having an average Mw of about 200 kDa is about 1 .5-2.5:0.5-1 .5:8-9 or about 1 .7:1 :8.3.

[0038] The compositions of the application can be used in products to inhibit the growth of various mold genera know in the art. In some embodiments, the composition of the application inhibits the growth of one or more of the mold genera selected from Penicillium, Aspergillus, Rhizopus, Rhizoctonia, Phytophthora, Fusarium, Alternaria and Mucor.

[0039] In some embodiments, the composition of the application inhibits the growth of one or more of the mold species selected from Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora infestans, Rhizoctonia solani, Rhizopus oryzae, Rhizopus stolonifera, Penicillium chrysogenum and Aspergillus niger.

[0040] In some embodiments, the growth of the mold species is inhibited by at least 20% compared to otherwise same conditions, except in the absence of the composition of the application. In some embodiments, the growth of the mold species is inhibited by at least 30%, by at least 40%, or by at least 50%, and values therebetween, compared to otherwise same conditions, except in the absence of the composition of the application.

[0041] In some embodiments, the present application includes an antimold composition comprising: about 15 % w / w chitosan polymer having an average Mw of about 20 kDa;about 9 % w / w chitosan polymer having an average Mw of about 170 kDa; and about 76 % w / w chitosan polymer having an average Mw of about 200 kDa, wherein the percentage is by total weight of chitosan polymers in the composition.

[0042] In some embodiments, the present application includes an antimold composition comprising chitosan polymers, wherein the chitosan polymers consist of: about 15 % w / w chitosan polymer having an average Mw of about 20 kDa; about 9 % w / w chitosan polymer having an average Mw of about 170 kDa; and about 76 % w / w chitosan polymer having an average Mw of about 200 kDa, wherein the percentage is by total weight of chitosan polymers in the composition.

[0043] In some embodiments, different Mw fractions of chitosan are isolated using chromatography, and / or by chemically, physically, or enzymatically altering the molecular weight of a chitosan polymer isolated from natural sources to obtain the desired Mw fraction.

[0044] In some embodiments, the chitosan polymer is from a fungus, crustacean or an insect. In some embodiments, the chitosan polymer is from a fungus. In some embodiments, the chitosan polymer is from a mushroom.

[0045] In some embodiments, the composition of the application is in the form of a powder, liquid solution or suspension. In some embodiments, the composition of the application is in the form of a powder. In some embodiments, the composition of the application is in the form of a liquidsolution or suspension. In some embodiments, the solvent for the solution or suspension is water. In some embodiments, the whole composition is dissolved into a solution.

[0046] In some embodiments, the composition of the application is formulated for inclusion in a product. In some embodiments, further ingredients are added to facilitate this inclusion as would be known to a person skilled in the art. Exemplary ingredients which may be included in the product include but are not limited to other preservatives, antioxidant ingredients, colors, dyes, fillers and other excipients known in the art.

[0047] In some embodiments, the further ingredients in the compositions of the application are organic acids such as, but not limited to, citric, acetic and / or lactic acid. In some embodiments, the organic acids are present in the composition in an amount of about 1 % to about 10% by weight of the composition.

[0048] In some embodiments, the further ingredients in the compositions of the application are other antimicrobial compounds such as, but not limited to, natamycin, nisin, sorbate and / or benzoate, and / or antioxidants, such as but limited to, rosemary extract and / or EDTA. In some embodiments the other antimicrobial compounds and / or antioxidants are present in the composition in an amount of about 100 ppm to about 1000 ppm.

[0049] In some embodiments, the product is a product that is susceptible to mold growth. In some embodiments, the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device ora pharmaceutical product. Pharmaceutical products include both prescription and over-the-counter pharmaceutical products. The product may be in any form, including liquid, solid, semi-solid and combinations thereof.

[0050] In some embodiments, the product is a food product. In some embodiments, the food product is a dough-based food product. In some embodiments, the dough-based food product is a yeast-raised dough productor a sourdough product (yeast and bacteria-raised baked product). Examples of dough-based food products include but are not limited to bread, buns, pizza, cake, doughnuts, muffins, pastry, croissants, brioches, pannetones, pies, tarts, quiches, cookies, scones, crackers, pretzels, bagels, tortillas, pitas, naan and the like. In some embodiments, the dough-based food product is bread.

[0051] In some embodiments, other ingredients can suitably be added in the food products including but not limited to fat components, salt, sweeteners, dairy products, egg products, emulsifiers, flavorings and the like.

[0052] In some embodiments, the food product further comprises yeast of the genus Saccharomyces. In some embodiments, the Saccharomyces species include S. cerevisiae, S. paradoxus, S. mikatae, S. jurei, S. kudriavzevii, S. arboricola, S. eubayanus and / or S. uvarum. As such, in some embodiments, the food product further comprises Saccharomyces yeast. In some embodiments, the yeast is S. cerevisiae. The yeast can be added to the product in any suitable form known to a person skilled in the art.

[0053] In some embodiments, the composition of the application is included in a product in an effective amount. In some embodiments, the effective amount of the composition of the application will depend on the identity of the product. For example, if the product is a food or beverage product an effective amount may be an amount of the composition to deliver or incorporate about 10 ppm to about 500 ppm, about 20 ppm to about 400 ppm, about 30 ppm to about 300 ppm, about 40 ppm to about 250 ppm, or about 50 ppm to about 200 ppm of the composition of the application. In a further example, if the product is a cosmetic or personal care product an effective amount may be an amount of the composition to deliver or incorporate about 50 ppm to about 1000 ppm, about 100 ppm to about 900 ppm, about 200 ppm to about 700 ppm, about 300 ppm to about 600 ppm, or about 400 ppm to about 500 ppm of the composition of the application. In some embodiments, when the product is dough-based food product an effective amount may be an amount of the composition to deliver or incorporate about 50 ppm to about 1000 ppm, about100 ppm to about 900 ppm, about 200 ppm to about 700 ppm, about 300 ppm to about 600 ppm, about 400 ppm to about 500 ppm, or about 800 ppm of the composition of the application.

[0054] In some embodiments, if the product is a food or beverage product an effective amount is about 0.1 wt% to about 10 wt%, about 0.25 wt% to about 5 wt% or about 0.5 wt% to about 3 wt% by total weight of all ingredients used to make the product. In some embodiments, if the product is a flour-based food product the effective amount of the composition of the application is about 0.1 wt% to about 10 wt%, about 0.25 wt% to about 5 wt%, about 0.5 wt% to about 3 wt%, or about 1 .3 wt% to about 1 .5 wt% by total weight of the flour used to make the product (i.e. the baker’s percentage).

[0055] In some embodiments, the compositions of the application are formulated into a product along with organic acids such as, but not limited to, citric, acetic and / or lactic acid. In some embodiments, the organic acids are present in the food product in an amount of about 0.1 wt% to about 10 wt% or 1 wt% to about 5 wt% by weight of all of the ingredients used to make the food product.

[0056] In some embodiments, the composition of the application does not inhibit or shows reduced inhibition of the growth of the Saccharomyces. In some embodiments, the composition of the application does not inhibit or shows reduced inhibition of the growth of the S. cerevisiae. The composition of the application allows to inhibit the growth of the mold species without inhibiting, or with reduced inhibition of, the growth of the Saccharomyces, such as S. cerevisiae. By reduced inhibition it means that the compositions of the application inhibit the growth of the Saccharomyces yeast to a lesser extent than an equivalent or comparable product that does not comprise the composition of the application. This is of great benefit as it allows the composition of the application to be added to food products while avoiding the negative impact against the strains of Saccharomyces yeast. This is particularlybeneficial in dough-based food products, which use S. cerevisiae as a leavening agent.III. Methods of Making Composition of the Application

[0057] The application further includes a method of making the compositions of the application. In some embodiments, the method comprises: combining a chitosan polymer having an average molecular weight (Mw) of about 20 kDa, a chitosan polymer having an average Mw of about 170 kDa, and a chitosan polymer having an average Mw of about 200 kDa.

[0058] The chitosan polymers having different Mws can be obtained by any method known in the art. In some embodiments, the chitosan polymers having different Mws are obtained by dissolving chitosan from a natural source in weak acidic solution, for example a solution of an organic acid such as acetic acid or lactic acid, and exposing for certain periods of time to a chemical which will depolymerize the chitosan. In some embodiments, alkali bases such as NaOH or KOH are used to chemically depolymerize the chitosan since the reaction occurs at a pace that allows the reaction to be stopped once the desired molecular weight has been achieved. Using an alkali base also has another benefit of deacetylating the chitosan simultaneously while depolymerization occurs. In some embodiments, strong acids such as hydrochloric acid, or highly oxidizing chemicals like hydrogen peroxide, are used to depolymerize chitosan quickly. In some embodiments, the chitosan is depolymerized using enzymes. These methods are used to isolate molecular weight fractions either the same size or smaller than the largest molecular weight present in the natural chitosan. For example, if the original natural chitosan is 200 kDa the isolated fractions are equal to or smaller than 200 kDa.

[0059] In some embodiments the chitosan polymers having different Mws are obtained by dissolving chitosan from a natural source in weak acidic solution, for example a solution of an organic acid such as acetic acid or lactic acid. In some embodiments, the chitosan is dissolved in the acidic solution inamounts to allow the solution to be fluid enough to be pumped into a chromatography column. The different Mw fractions of chitosan are then isolated from the column. In some embodiments, the different Mw fractions of chitosan are isolated using other physical methods. In some embodiments, the isolated fractions of different Mws are precipitated by adjusting the pH of a solution of each fraction to >7 using a base, such as sodium or potassium hydroxide (NaOH or KOH). In some embodiments, the precipitated chitosan is collected by filtration or centrifugation and dried into a powder.

[0060] In some embodiments, the polydispersity index (PDI) of the chitosan Mw fractions is about 2 to about 3.

[0061] In some embodiments, molecular weights are analyzed by HPLC with size exclusion chromatography or gel permeation chromatography (GPC).

[0062] In some embodiments, the different Mw polymers of chitosan are combined as powders. In some embodiments, the different Mw polymers of chitosan are combined as solutions or are combined in a solution or suspension.IV. Methods of Using and Products Containing Compositions of the Application

[0063] In some embodiments, the compositions of the application are used as anti-mold agents or preservatives in products in need of such agents. In some embodiments, these products include, but are not limited to food, beverages, cosmetics, personal care items, medical devices and pharmaceuticals. As noted above, pharmaceuticals include prescription and over-the-counter pharmaceuticals. The product may be in any form, including liquid, semi-solid, solid and combinations thereof.

[0064] As such, the application includes a product comprising a composition of the application. In some embodiments, the product is a food, beverage, cosmetic, personal care item, medical device or pharmaceutical.

[0065] In some embodiments, the product comprises further ingredients known to a person skilled in the art. Exemplary ingredients which the productmay comprise include but are not limited to other preservatives, antioxidant ingredients, colors, dyes, fillers and other excipients known in the art.

[0066] In some embodiments, the product is the food product. In some embodiments, the food product is a dough-based food product. In some embodiments, the dough-based food product is a yeast-raised dough product or a sourdough product (yeast and bacteria-raised baked product). Examples of dough-based food products include but are not limited to bread, buns, pizza, cake, doughnuts, muffins, pastry, croissants, brioches, pannetones, pies, tarts, quiches, cookies, scones, crackers, pretzels, bagels, tortillas, pitas, naan and the like. In some embodiments, the dough-based food product is bread.

[0067] In some embodiments, the food product comprises further ingredients known to a person skilled in the art. Exemplary ingredients which the food product may comprise include but are not limited to flour, water, fat components, salt, sweeteners, dairy products, egg products, emulsifiers, flavorings and the like.

[0068] In some embodiments, the compositions of the application are incorporated as an ingredient or a component during preparation of the product.

[0069] In some embodiments, the application includes a dough-based food product comprising a composition of the application. In some embodiments, the dough-based food product is bread.

[0070] In some embodiments, the application includes a method of inhibiting mold growth in a product comprising incorporating an effective amount of a composition of the application into the product.

[0071] In some embodiments, the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.

[0072] In some embodiments, the product is the food product. In some embodiments, the food product is a dough-based food product. Exemplary dough-based food products are as defined above.

[0073] In some embodiments, the food product further comprises yeast of the genus Saccharomyces. Exemplary Saccharomyces strains include S. cerevisiae, S. paradoxus, S. mikatae, S. jurei, S. kudriavzevii, S. arboricola, S. eubayanus and / or S. uvarum. In some embodiments, the yeast is S. cerevisiae.

[0074] In some embodiments, the method of inhibiting mold growth in the product does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae. This is of great benefit as this method allows to inhibit the growth of mold in the product and avoid the negative impact against the strains of Saccharomyces yeast. This method is particularly beneficial in dough-based food products, such as breads and the like.

[0075] In some embodiments, the application includes a method of preserving a product comprising incorporating an effective amount of a composition of the application into the product.

[0076] In some embodiments, the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.

[0077] In some embodiments, the product is a food product. In some embodiments, the food product is a dough-based food product. Exemplary dough-based food products are as defined above.

[0078] In some embodiments, the food product further comprises yeast of the genus Saccharomyces. Exemplary Saccharomyces strains are as defined above. In some embodiments, the yeast is S. cerevisiae.

[0079] In some embodiments, the method of preserving the product does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae. This is of great benefit as this method allows to preserve the product and avoid the negative impact against the strains of Saccharomyces yeast. This method is particularly beneficial in dough-based food products, such as breads and the like. By reduced inhibition it means that the compositions of the application inhibit the growth of the Saccharomyces yeast to a lesser extent than anequivalent or comparable product that does not comprise the composition of the application.

[0080] In some embodiments, the application includes a use of a composition of the application to inhibit mold growth in a product or to preserve a product.

[0081] In some embodiments, the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.

[0082] In some embodiments, the product is a food product. In some embodiments, the food product is a dough-based food product. Exemplary dough-based food products are as defined above.

[0083] In some embodiments, the food product further comprises yeast of the genus Saccharomyces. Exemplary Saccharomyces strains are as defined above. In some embodiments, the yeast is S. cerevisiae.

[0084] In some embodiments, the application includes a method of inhibiting mold growth in a dough-based food product or preserving the doughbased food product, the method comprising incorporating an effective amount of a composition of the application into the product.

[0085] In some embodiments, the dough-based food product is bread.

[0086] In some embodiments, the dough-based food product further comprises yeast of the genus Saccharomyces. In some embodiments, the yeast is S. cerevisiae.

[0087] In some embodiments, the method of inhibiting mold growth in a dough-based food product or preserving the dough-based food product does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae.

[0088] In some embodiments, the application includes a use of a composition of the application to inhibit mold growth in a dough-based food product or to preserve the dough-based food product.

[0089] In some embodiments, the dough-based food product is bread.

[0090] In some embodiments, the dough-based food product further comprises yeast of the genus Saccharomyces. Exemplary Saccharomyces strains are as defined above. In some embodiments, the yeast is S. cerevisiae.

[0091] In some embodiments, the use of the composition of the application to inhibit mold growth in the dough-based food product or to preserve the dough-based food product does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae.

[0092] In some embodiments, the effective amount of the composition of the application will depend on the identity of the product. For example, if the product is a food or beverage product an effective amount may be an amount of the composition to deliver or incorporate about 10 ppm to about 500 ppm, about 20 ppm to about 400 ppm, about 30 ppm to about 300 ppm, about 40 ppm to about 250 ppm, or about 50 ppm to about 200 ppm of the composition of the application. In a further example, if the product is a cosmetic or personal care product an effective amount may be an amount of the composition to deliver or incorporate about 50 ppm to about 1000 ppm, about 100 ppm to about 900 ppm, about 200 ppm to about 700 ppm, about 300 ppm to about 600 ppm, or about 400 ppm to about 500 ppm of the composition of the application. In some embodiments, when the product is dough-based food product an effective amount may be an amount of the composition to deliver or incorporate about 50 ppm to about 1000 ppm, about 100 ppm to about 900 ppm, about 200 ppm to about 700 ppm, about 300 ppm to about 600 ppm, about 400 ppm to about 500 ppm, or about 800 ppm of the composition of the application.

[0093] In some embodiments, if the product is a food or beverage product an effective amount is about 0.1 wt% to about 10 wt%, about 0.25 wt% to about 5 wt% or about 0.5 wt% to about 3 wt% by total weight of all ingredients used to make the product. In some embodiments, if the product is a flour-based food product the effective amount of the composition of the application is about0.1 wt% to about 10 wt%, about 0.25 wt% to about 5 wt%, about 0.5 wt% to about 3 wt%, or about 1 .3 wt% to about 1 .5 wt% by total weight of the flour used to make the product (i.e. the baker’s percentage).V. EXAMPLESExample 1: Preparation of chitosan polymers having different Mws

[0094] The chitosan molecular weight fractions were separated by chemical depolymerization using a sodium hydroxide solution. 10g of chitosan was dissolved in 250mL of 1 % (v / v) glacial acetic acid solution at 35°C for 5 hours. 10mL of 0.3M sodium hydroxide was added into the chitosan solution and the solution heated to 60°C while being stirred with a magnetic stir bar. The starting Mw was measured using GPC according to the method outlined below and depending on the starting Mw measurement the chitosan was exposed for varying amounts of time to achieve the desired Mw. Once the sufficient period of time has elapsed the reaction was stopped by adding sodium hydroxide to raise the pH above 7 in order to precipitate the chitosan and the precipitate was collected using centrifugation, rinsed with water, and dried using lyophilization. This process was repeated until each desired Mw was obtained. A general trend for the rate of the reaction was that there was a rapid period of depolymerization where the Mw decreased from approximately 200kDa to approximately 100kDa in 1 hour, and then a slow period where the molecular weight decreased from approximately 100kDa to 20kDa in over 12 hours. In this example the following molecular weights were produced: 20, 170, and 200kDa.

[0095] The chitosan Mw fractions were analyzed by HPLC with size exclusion chromatography or gel permeation chromatography, and incorporating an oven that has four detectors: refractive index (Rl), ultra-violet (UV), right angle and low angle light scattering (RALS / LALS), and four-capillary differential viscometer (VIS). Chitosan samples were dissolved in the mobile phase of 0.1 M acetic acid / 0.3M NaNOa for 4-6 hours on a rocker at roomtemperature. A concentration of chitosan at ~5-10mg / mL was used for the samples. Columns can be used in a series and with a large range of separation efficiency. The isolated chitosan Mw’s used for antimicrobial testing had a PDI between 2 and 3. Example 2: Anti-mold compositions

[0096] Composition (a) with chitosan having molecular weight of about 20, 170, and 200 kDa and composition (b) with chitosan having an Mw of about 200 kDa were prepared by accurately weighing out each Mw fraction of chitosan on an analytical balance. Once the individual portions were measured, they were carefully transferred into a clean beaker, and mixed by stirring for 5minutes to ensure complete even distribution.

[0097] The following anti-mold formulations were prepared:Exemplary Composition (a)Mw (kDa) % (w / w)20 15.00170 9.00200 76.00100.00 Comparative Composition (b)Mw (kDa) % (w / w)200 100.00100.00Comparative Composition C-MFC (based on teachings in WO 2019 / 046950)Mw (kDa) % (w / w)20 6.7030 7.0040 6.7060 7.00150 7.00170 7.00200 60.00100.00Comparative Composition Badawy (based on teachings in Badawy et al., Antimicrobial Activity of Different Molecular Weight Chitosans Produced from Shrimp Shells Against Different Plant Pathogens, Current Bioactive Compounds, 2015, 11 , 3):Mw (kDa) % (w / w)22 20.00203 20.00214 20.70300 20.00387 20.00100.00Comparative Composition Chen (based on teachings in Chen et al., CN 106719667):Mw (kDa) % (w / w)5 16.7030 83.30100.00

[0098] The effect of the chitosan compositions on the growth of a variety of mold species was assessed (ECso mg / ml) in comparison to prior art samples which include optimal Mw’s fold each mold species (Badawy et al., Antimicrobial Activity of Different Molecular Weight Chitosans Produced from Shrimp Shells Against Different Plant Pathogens, Current Bioactive Compounds, 2015, 11 , 3). The results are summarized in Table 1.Table 1

[0099] The chitosan exemplary composition (a) showed the best growth inhibition of all tested mold species as compared to all comparative compositions and further, aside from comparative composition (b) which comprised only 200 kDa MW chitosan polymer, the exemplary formulation showed the least inhibition of the growth of the S. cerevisiae.Example 3: Recipes for baked food products (a) White Bread8 loaves Batch (g) Wt % IngredientsAll-purpose flour 400 56.2%Sugar 28 3.9%Salt 8 1.1 %Yeast 9.6 1 .3%Canola oil 16 2.2%Acetic Acid 7% 8 1.1 %Chitosan composition (a) 5.6 0.8%Water 237 33.3%Total 712.2 100.0%

[0100] Into a mixing bowl, the oil, water (at 50-50°C), chitosan composition (a) and acetic acid were added. Yeast, sugar and 100g of the flour was then added and mixing was performed at high speed for 2 mins. Next salt, 300g of the flour was added and mixing was performed at slow speed for 2 minutes. When all ingredients came together to form a dough, the dough was kneaded at medium speed (speed 5 of Cuisantart mixer) for 8 minutes. The dough was divided into 8x85g balls, shaped, and placed in a pan, which was covered with plastic wrap (oil-greased). The dough was allowed to rise for 15mins, the was baked at 400°F for 13 minutes. The resulting bread was allowed to cool on rack before being packed in zipper bags of transparent plastic.(b) Wheat Tortilla8 tortillas Batch (g) Wt %IngredientsAll-purpose flour 180 53.4%Salt 3 0.9%Baking powder 2.4 0.7%Canola oil 36 10.7%Acetic Acid 7% 8 2.4%Chitosan composition (a) 2.52 0.7%Water 105 31 .2%Total 337 100.0%

[0101] Oil, water, chitosan composition (a) and acetic acid were added to a mixing bowl. In another bowl, dry ingredients, including flour, salt, and baking powder were added. The dry ingredients were added to the oil, water and chitosan mixture and mixing was performed at slow speed until combined into a dough. The mixing speed was increased to medium speed, followed by kneading for 5 minutes. The dough was allowed to rest for 45 minutes to 1 hourwrapped in plastic wrap. The dough was divided into 8x40g balls, pressed by a tortilla press or rolled by rolling pin and then allowed to rest for 2 minutes. The pressed dough was then cooked in pan on a stovetop for 45 seconds to 1 minute, then flipped over and cooked for another 15-20 seconds. 8 tortillas were stacked in a zipper bag of transparent plastic and allowed to cool before packing in individual zipper bags of transparent plastic.Example 4: Chemical / Physical parameters of baked Products

[0102] Certain chemical / physical parameters were measured & calculated, depending on the type of baked goods product (see Table 2 below). Sensory was evaluated in the in-pair or blind testing vs Control, mainly focusing on off-taste or off-aroma.Table 2Product ParametersWhite Bread - pH: calculated from pH of crumb slurry (10g crumb + 40g water)- Loaf dimensions- WeightWheat - pH calculated from pH of slurry (10g tortilla + 40g water) TortillaExample 5: Storage conditions of baked products

[0103] Storage conditions simulated those of commercial products (see Table 3 below). Qualitative visual checking for any signs of spoilages, like molds, yeasts, roping was performed (see Tables 4 and 5 below). Duration of testing was for 200% of target shelf-life or when all samples are spoiled (whichever is first).Table 3Product Temperature Humidity StorageWhite Ambient Ambient Zipper bags in transparent plasticBread toteWheat Ambient Ambient Zipper bags in transparent plasticTortilla toteTable 4: BreadTable 5: Wheat TortillaExample 6: Comparison of Mold Spoilage in Baked Goods

[0104] Baked products were prepared using the above recipes with and without (control) a chitosan composition of the application. A summary of the number of days for the product to show the presence of mold is shown in Table 6. As can be seen, the products containing the chitosan compositions remained without mold for significantly longer that those products that did not contain thechitosan compositions. For bread, in particular, this time different was as much as 20 days.Table 6: Summary of the Mold Growth Analysis of Baked Good Products Comprising a Composition of the ApplicationDays to Mold Presence0 10 15 20 30ControlBreadChitosan 1 4% (baker's %)ControlWheat TortillaChitosan 1 4% (baker's %)- - No visual mold+ = 1-2 spots of visual mold++ - 10-25% coverage with mold(Baker’s % means based on the amount of flour in the product)

Claims

CLAIMS:1 . An anti-mold composition comprising: a chitosan polymer having an average molecular weight (Mw) of about 20 kDa; a chitosan polymer having an average Mw of about 170 kDa; and a chitosan polymer having an average Mw of about 200 kDa, wherein the composition comprises substantially greater amounts in percent by weight of the chitosan polymer having an average Mw of about 200 kDa.

2. An anti-mold composition comprising chitosan polymers, wherein the chitosan polymers consist essentially of: a chitosan polymer having an average molecular weight (Mw) of about 20 kDa; a chitosan polymer having an average Mw of about 170 kDa; and a chitosan polymer having an average Mw of about 200 kDa.

3. The anti-mold composition of claim 1 , wherein the composition comprises: about 5 % w / w to about 25 % w / w chitosan polymer having an average molecular weight (Mw) of about 20 kDa; about 5 % w / w to about 20 % w / w chitosan polymer having an average Mw of about 170 kDa; and about 65 % w / w to about 90 % w / w chitosan polymer having an average Mw of about 200 kDa wherein the percentage is by total weight of chitosan polymers in the composition.

4. The anti-mold composition of claim 3, wherein the chitosan polymer having an average Mw of about 20 kDa is present in an amount of about 7 % w / w to about 23 % w / w by total weight of the chitosan in the composition.

5. The anti-mold composition of claim 4, wherein the chitosan polymer having an average Mw of about 20 kDa is present in an amount of about 15 % w / w by total weight of the chitosan in the composition.

6. The anti-mold composition of any one of claims 3 to 5, wherein the chitosan polymer having an average Mw of about 170 kDa is present in an amount of about 7 % w / w to about 18 % w / w by total weight of the chitosan in the composition.

7. The anti-mold composition of claim 6, wherein the chitosan polymer having an average Mw of about 170 kDa is present in an amount of about 9 % w / w by total weight of the chitosan in the composition.

8. The anti-mold composition of any one of claims 3 to 7, wherein the chitosan polymer having an average Mw of about 200 kDa is present in an amount of about 70 % w / w to about 90 % w / w by total weight of the chitosan in the composition.

9. The anti-mold composition of claim 8, wherein the chitosan polymer having an average Mw of about 200 kDa is present in an amount of about 76 % w / w by total weight of the chitosan in the composition.

10. The anti-mold composition of any one of claims 1 to 3, wherein the weight ratio of the chitosan polymer having an average molecular weight (Mw) of about 20 kDa:chitosan polymer having an average Mw of about 170 kDa:chitosan polymer having an average Mw of about 200 kDa is about 1.5- 2.5:0.5-1 .5:8-9.

11. The anti-mold composition of any one of claims 1 to 10, wherein the composition inhibits the growth of one or more of the mold genera selected fromPenicillium, Aspergillus, Rhizopus, Rhizoctonia, Phytophthora, Fusarium, Alternaria and Mucor.

12. The anti-mold composition of claim 11 , wherein the composition inhibits the growth of one or more of the mold species selected from Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora infestans, Rhizoctonia solani, Rhizopus oryzae, Rhizopus stolonifera, Penicillium chrysogenum and Aspergillus niger.

13. The anti-mold composition of any one of claims 1 to 12, wherein the growth of the mold species is inhibited by at least 20%.

14. The anti-mold composition of any one of claims 1 to 13, wherein the anti-mold composition comprises or consists essentially of: about 15 % w / w chitosan polymer having an average Mw of about 20 kDa; about 9 % w / w chitosan polymer having an average Mw of about 170 kDa; and about 76 % w / w chitosan polymer having an average Mw of about 200 kDa, wherein the percentage is by total weight of chitosan polymers in the composition.

15. The anti-mold composition of any one of claims 1 to 14, wherein composition is in the form of a powder, liquid solution or suspension.

16. The anti-mold composition of any one of claims 1 to 15, wherein the composition is formulated for inclusion in a product.

17. The anti-mold composition of claim 16, wherein the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.

18. The anti-mold composition of claim 17, wherein the product is a food product.

19. The anti-mold composition of claim 18, wherein the food product is a dough-based food product.

20. The anti-mold composition of claim 19, wherein the dough-based food product is bread.

21. The anti-mold composition of any one of claims 17 to 20, wherein the food product further comprises yeast of the genus Saccharomyces.

22. The anti-mold composition of claim 21 , wherein the yeast is S. cerevisiae.

23. The anti-mold composition of claim 22, wherein the composition does not inhibit or shows reduced inhibition of the growth of the S. cerevisiae.

24. A method of making the composition of any one of claims 1 to 23, wherein the method comprises: a) combining the chitosan polymer having an average molecular weight (Mw) of about 20 kDa, the chitosan polymer having an average Mw of about 170 kDa, and the chitosan polymer having an average Mw of about 200 kDa.

25. A product comprising a composition of any one of claims 1 to 16.

26. The product of claim 25, wherein the product is a food, beverage, cosmetic, personal care item, medical device or pharmaceutical.

27. The product of claim 26, wherein the product is the food product.

28. The product of claim 27, wherein the food product is a dough-based food product.

29. The product of claim 28, wherein the dough-based food product is bread.

30. A dough-based food product comprising a composition of any one of claims 1 to 23.

31. The dough-based food product of claim 30, wherein the dough-based food product is bread.

32. A method of inhibiting mold growth in a product comprising incorporating an effective amount of a composition of any one of claims 1 to 16 into the product.

33. The method of claim 32, wherein the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.

34. The method of claim 33, wherein the product is the food product.

35. The method of claim 34, wherein the food product is a dough-based food product.

36. The method of claim 35, wherein the food product further comprises yeast of the genus Saccharomyces.

37. The method of claim 36, wherein the yeast is S. cerevisiae.

38. The method of claim 37, wherein the method of inhibiting mold growth in the product does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae.

39. A method of preserving a product comprising incorporating an effective amount of a composition of any one of claims 1 to 16, into the product.

40. The method of claim 39, wherein the product is a food product, a beverage product, a cosmetic product, personal care items, a medical device or a pharmaceutical product.41 . The method of claim 40, wherein the product is the food product.

42. The method of claim 41 , wherein the food product is a dough-based food product.

43. The method of claim 42, wherein the food product further comprises yeast of the genus Saccharomyces.

44. The method of claim 43, wherein the yeast is S. cerevisiae.

45. The method of claim 44, wherein the method does not inhibit, or shows reduced inhibition of, the growth of the S. cerevisiae.

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