Umami taste modifiers, and food and beverage products including umami taste modifiers

Abstract

Taste modifier compositions are provided that include one or more umami taste-enhancing peptides. The taste modifier compositions may be added to food or beverage products to modify, enhance, or add umami taste to the food or beverage product.

Description

UMAMI TASTE MODIFIERS, AND FOOD AND BEVERAGE PRODUCTS INCLUDING UMAMI TASTE MODIFIERSTechnical Field

[0001] This invention relates generally to umami taste modifiers and the inclusion of such umami taste modifiers in food and beverage products. More particularly, this disclosure generally relates to umami-modifier peptides and the inclusion of such peptides in food and beverage products.Reference to a Sequence Listing[0002) The Sequence Listing associated with this application is filed in electronic form via Patent Center and is hereby incorporated by reference in its entirety. The name of the electronic file containing the Sequence Listing is 1410-163201 -US_SL. The size of the.xml file is 21 KB and the file was created on December 29, 2025.Background

[0003] According to the World Health Organization (WHO), a large number of people around the world are consuming too much sodium. The mean daily intake of sodium by adults globally is reportedly double the WHO's recommended intake of less than 2,000 mg / day.Efforts have been made by food companies to provide lower sodium alternatives, but those efforts have generally met with reduced consumer acceptance of the food products.[0004) Improving consumer acceptance may require maintaining the flavor profile delivered by sodium chloride so that the reduced sodium product has the same flavor as a non¬ reduced sodium formulation. However, commercially available options for adding a salty taste to food products generally are characterized by a limited salty flavor impact and often deliver off flavors, including bitter or metallic flavors, that further limit consumer acceptance of the food products. For example, potassium chloride has been used as a salt substitute, but it is often perceived as adding bitter flavor.

[0005] It has also been found that inclusion of sodium chloride in food products influences other aspects of the flavor of the food, it has been found that removal or reduction of salt can result in alteration of other flavors in the food. For example, perception of sweet, sour, and bitter flavors may increase or decrease when salt content is reduced in a food product.

[0006] Attempts have also been made to increase umami taste in reduced sodium food products to help mitigate flavor changes. However, commercially available umami-enhancing ingredients, such as yeast extract, often deliver meaty or yeasty flavors that are uncharacteristic of the food or beverage products. Commercially available umami- enhancing ingredients also often add other undesirable flavors, such as sour, bitter, or astringent flavors to the food or beverage products, which results in reduced consumer acceptance. Monosodium glutamate also is known for adding umami taste but has negative consumer perception.Detailed Description

[0007] Described herein are umami-enhancing taste modifier compositions that can be added to food or beverage products. The taste modifier compositions include one or more umami taste-enhancing peptides that, either alone or in combination, are effective to modify umami taste to the food or beverage products. In some approaches, the umami taste modifier compositions modify, add, and / or enhance an umami taste in the food or beverage product. Umami is often described as a savory or meaty taste. There are five main compounds that conventionally have been used to provide umami taste to food products, namely glutamate, theanine, inosinate, guanylate, and succinate. These compounds have been found in soy -based ingredients (e.g., soy sauce, miso paste), kimchi (vegetables fermented with Lactobacillus), seaweed (e.g., dashi (seaweed broth)), mushrooms, seafood, meat, tomatoes, green tea, and aged cheeses, which are characterized by a strong umami taste. Umami is considered a rich, full-bodied flavor that enhances the taste of foods. Umami is commonly found in foods like meat, cheese, soy sauce, tomatoes, mushrooms, and fermented products. It plays an important role in deepening the overall flavor profile of dishes.

[0008] Umami is one of the five recognized main taste characteristics in food and beverage products, along with sweet, sour, bitter, and salty. In addition to those five characteristics,9Attorney Docket 1410-163201-PCkokumi and astringency are also sometimes classified as taste characteristics. Generally speaking, a salty taste is typically associated with the presence of salts, particularly sodium chloride (NaCl). It provides a sensation of savoriness and can enhance the overall flavor profile of a dish. Saltiness is often perceived as a basic taste that balances and complements other flavors. Foods such as chips, pretzels, and salted nuts are common sources of salty taste.Sweetness is a taste typically associated with sugars, for example, sucrose, glucose, and fructose, as well as artificial sweeteners. It is often perceived as pleasurable and is commonly found in desserts, fruits, and sugary beverages. Sourness is generally caused by acids, such as citric acid (found in citrus fruits), acetic acid (in vinegar), and lactic acid (in fermented dairy products). The sour taste is sharp and tangy and can range from mildly tart to intensely acidic. It is a flavor commonly found in fruits like lemons, green apples, and in fermented products like yogurt or pickles. Acids can also act to improve the stability in some foods and beverages by lowering the pH of the system. Bitterness is a taste often associated with compounds like alkaloids (e.g., caffeine or quinine) or certain plant-based substances. It is usually perceived as unpleasant or sharp, and many bitter foods are avoided due to their strong flavor. Bitter tastes are found in foods such as dark chocolate, coffee, kale, and certain herbs. While it can be off- putting in large quantities, bitterness can also add complexity and balance to some dishes. Kokumi is described as providing a background richness and complexity to foods, and particularly savory foods. Astringency is described as providing a puckering and drying feeling in the mouth.

[0009] As used herein, the term "taste modifier" refers to a food ingredient that has the effect of modifying (either by increasing or decreasing) a perceived taste of at least one of the five main taste characteristics in food or beverage prod ucts when the taste modifier is added to the food or beverage product. Also described herein are food and beverage compositions comprising the taste modifier, as well as methods of making a food or beverage composition comprising the taste modifier.

[0010] At least in some approaches, by modulating the perceived taste of the food or beverage product, one or more other ingredients may be reduced or removed from the food or beverage product without having a deleterious effect on the taste characteristic of the food or3Attorney Docket 1410-163201-PCbeverage product. In another approach, an existing taste characteristic may be increased in the food or beverage product by addition of the taste modifier.

[0011] In some approaches, the umami taste modifiers described herein are umamienhancing taste modifiers. As used herein, the term ''enhancing" means that the umamienhancing modifiers add an umami taste or increase the intensity of an umami taste in the food or beverage product.

[0012] The umami-enhancing modifiers described herein may he added alone or in combination with one or more of the other umami-enhancing modifiers to add, modify, enhance, and / or increase the intensity of umami taste to food or beverage products. The umami-enhancing taste modifiers may also be added alone or in combination with one or more other taste modifying ingredients to food or beverage products.|0013| In one approach, by including one or more such umami-enhancing modifiers, in addition to enhancing the umami taste of a food or beverage product, the amount of sodium in the food or beverage product may be reduced or eliminated. For example, the addition of one or more of the umami-enhancing modifiers in an amount of at least about 0.00002 wt.%, at least about 0.00003 wt.%, at least about 0.00004 wt.%; at least about 0.00005 wt.%, at least about 0.00006 wt.%, at least about 0.00007 wt.%, at least about 0.00008 wt.%, at least about 0.00009 wt.%, at least about 0.0001 wt.%, at least about 0.0002 wt.%, at least about 0.0003 wt.%, at least about 0.0004 wt.%, at least about 0.0005 wt.%, at least about 0.0006 wt.%, at least about 0.0007 wt.%, at least about 0.0008 wt.%, at least about 0.0009 wt.%, at least about 0.001 wt.%, at least about 0.002 wt.%, at least about 0.003 wt.%, at least about 0.004 wt.%, at least about 0.005 wt.%, at least about 0.006 wt.%, at least about 0.007 wt.%, at least about 0.008 wt.%, at least about 0.009 wt.%, at least about 0.01 wt.%, at least about 0.05 wt.%, at least about 0.1 wt.%, at least about 0.5 wt.%, or at least about 1.0 wt.% of the food or beverage product may allow for reduction of sodium, such as by at least about 10 percent, at least about 20 percent, or at least about 30 percent.

[0014] In another approach, the umami-enhancing modifier can be added in an amount effective to add umami taste to the food or beverage product but without adding off fla vors,Attorney Docket 1410-163201-PCsuch as bitterness, that will reduce consumer acceptance of the food or beverage product. To achieve this, the umami-enhancing modifier has an umami modulation threshold concentration that is less than its intrinsic taste threshold concentration (e.g., bitter, astringent, or sour taste) in S given food or beverage product. The umami-enhancing effect of the umami-enhancing modifier detectable by taste in a given food or beverage product when the umami-enhancing modifier is added is achieved at a lower amount of the umami -enhancing modifier than the amount of the umami-enhancing modifier at which a bitter, astringent, or sour taste of the umami-enhancing modifier is detectable in the food or beverage product. Further, the lower the umami-enhancing threshold value, the more potent the umami effect in a food or beverage product, meaning that the umami-enhancing modifier may be included in a small quantity and still provide the desired umami-enhancing effect.

[0015] In one particular approach, the umami-enhancing peptides include one or more umami-enhancing peptides having the following amino acid sequences: RDMPIQA (SEQ ID NO. 5); QEFVLGFVRGFFP (SEQ ID NO. 6); PGFI (SEQ ID NO. 9); and KAVFYPQ (SEQ ID NO.21). Any one of these umami-enhancing peptides may be added to food or beverage products alone, in combination with one or more of the other of these four umami-enhancing peptides, and / or in combination with other umami-enhancing peptides or umami-enhancing modifiers. When used in combination, the umami-enhancing peptides may be combined in any ratio to provide the desired flavor profile in the food or beverage product.

[0016] In some aspects, the one or more umami-enhancing peptides are added to a food or beverage product as a taste-modifier composition in liquid (e.g., as a solution or other aqueous mixture) or in powder form.

[0017] In some approaches, the one or more umami-enhancing peptides may be provided in a composition that includes other peptides, such as dairy-based peptides, but in which the one or more umami-enhancing peptides are included at a significantly higher amount than the other peptides. For example, the one or more umami-enhancing peptides may be individually included in an amount of at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times the amount of other individual peptides in a composition. In some approaches, the one or moreAttorney Docket 1410-163201-PCumami-enhancing peptides may be individually included in an amount of at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times the amount of other individual dairy-based peptides and / or dairy-based aroma compounds in a composition.[0018) In some approaches, the umami-enhancing peptides are added to the food and beverage products in substantially pure or isolated form. The umami-enhancing peptides may be isolated from other dairy-based proteins and / or peptides, such as other proteins and peptides found in dairy products like milk, cream, and natural cheese, isolated from other dairy-based compounds, or isolated from other peptides (such as other peptides found in dairy products). For example, each of the umami-enhancing peptides may be provided in isolated form, meaning that one of the umami-enhancing peptides is the primary peptide (e.g., at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, or at least 95 wt.%) of the total peptides present) in a composition. Alternatively, a combination of the umamienhancing peptides may be the primary peptide component (e.g., at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, or at least 95 wt.% of the total peptides present) in a composition. As used herein, the term "isolated" with respect io an umamienhancing peptide composition means that the umami-enhancing peptide composition includes one or more of the umami-enhancing peptides in an amount of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% by dry weight of the umami-enhancing peptides.

[0019] In certain implementations, the umami-enhancing peptides are added to a food or beverage product as a taste-enhancing composition further comprising one or more carriers, which may also be a bulking agent, solvent, or other liquid. For example, the flavor-enhancing composition may be in a dry form such as a powder or particulate. In other approaches, the taste-modifier composition may be in liquid form, such as in the form of a fermentation extract or cultured milk. In yet other approaches, the taste-modifier composition may be in solid or semi-solid form, such as in the form of a flavor ingredient, concentrated cheese flavor, or enzyme-modified cheese. Suitable carriers include any food grade carrier that does not adversely impact the flavor of the food or beverage product. Exemplary food grade carriers include one or more of gum arabic, dextrose, sucrose, fructose, lactose, proteins, cellulose, silica.calcium silicate, tapioca flour, rice flour, salt, potassium chloride, starch, and / or maltodextrin, such as corn maltodextrin, potato maltodextrin, tapioca starch, modified tapioca starch, rice flour, and tapioca maltodextrin. For instance, the umami-enhancing peptides may be included in a flavor-enhancing composition in an amount of at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, by dry weight of the flavor-enhancing composition. In some approaches, the carrier may be the major component of the flavor¬ enhancing composition.

[0020] In some aspects, the one or more umami-enhancing peptides are added to a food or beverage product in an aqueous solution or mixture. In certain implementations, the umami¬ enhancing peptides, when added to a food or beverage product as a liquid flavor-enhancing composition, may be added together with one or more carriers including but not limited to water, glycerin, propylene glycol, 1,3-propanediol, ethanol, lactic acid, and combinations thereof.

[0021] In some aspects, the one or more umami-enhancing peptides are added to a food or beverage product in the form of a fermentation extract. For example, one or more of the umamienhancing peptides may be developed within a specific product through targeted fermentation or enzymatic treatment aimed at obtaining a certain concentration. In some implementations, concentrated fermented or enzymatically produced "flavors" with high concentrations of the umami-enhancing peptides may be generated from non-dairy sources or dairy sources. For example the one or more umami-enhancing peptides may be generated in the form of an enzyme-modified cheese, enzyme-modified fresh cheese, cultured milk, or the like, and then added as an ingredient into a food or beverage product. The one or more umami-enhancing peptides might also be added to a food or beverage product in the form of a flavor ingredient. The one or more umami-enhancing peptides may be generated from a dairy-based source in some approaches or a plant-based source in other approaches.

[0022] In one approach, the umami-enhancing peptides may be added to a food or beverage product to increase the umami taste of the food or beverage product. The food or beverage product may be any food or beverage product that already has an umami taste or any food or beverage product and might benefit from having an enhanced umami taste.7Attorney Docket 1410-163201-PC

[0023] Suitable food products to which the umami-enhancing peptides described herein may be added include but are not limited to: process cheese (e.g., in slice, loaf, or sauce form), cream cheese (e.g,, dairy cream cheese, plant-based cream cheese, flavored cream cheese (e.g., chive & onion, jalapeno, etc.), unflavored cream cheese (e.g., plain, etc.), plant-based cheese, shredded cheese, natural cheese, dry meals (e.g., macaroni and cheese powder, nee (e.g., rice mix, etc.), stuffing mix, pasta (e.g., pasta mix, etc.), sauces (e.g., cheese sauce, cocktail sauce, sweet and sour sauce, barbeque sauce, soy sauce, tartar sauce, hot sauce, steak sauce, pasta sauce (e.g., tomato sauce, alfredo sauce, etc.)), and condiments (e.g,, tomato ketchup, mustard, mayonnaise, aioli, salad dressing, dairy or non-dairy creamy dressings (e.g., ranch dressing, green goddess, Caeser, etc.), oil-based dressings (e.g., vinaigrette, Italian dressing, French, etc.)), gravy (ready-to-use or dry), frozen meals and / or sides (e.g., frozen potato products such as French fries, potato tots, beans, etc.), soups, beans or other meals, and dry seasonings (e.g., taco seasoning, etc.) and foods containing such dry seasonings (e.g., potato chips, crackers, etc.). All of the food products mentioned herein refer to either a dairy-based or plant-based version of the food product unless specifically stated otherwise. In some approaches, the food or beverage product to which the one or more umami-enhancing peptides are added are not dairy products. In some approaches, the food or beverage product to which the one or more umami-enhancing peptides are added are not cheese products.

[0024] In some approaches, the food products are dairy-based products. As used herein, the term "dairy-based" refers to a product or ingredient that contains one or more ingredients originating from dairy milk or cream. For example, dairy-originating ingredients include whey protein, milkfat, cream, milk, milk solids (e.g., non-fat dry milk), butter, milk-based yogurt, cheese curd, cheese, and the like, as well as food ingredients that include non-animal based dairy proteins, such as whey or fermented whey proteins that may be obtained from non¬ animal sources.

[0025] In some approaches, the food products are plant-based products. As used herein, the term "plant-based" refers to a product or ingredient that is free of animal-based ingredients, such as proteins (e.g., dairy proteins like casein and whey) and fats (such as milk fats). The plant-based products may instead include a plant-based protein. Any suitable plant -based8Attorney Docket 1410-163201-PCprotein may be used in the plant-based product. In some aspects, the plant-based protein comprises one or more of faba bean protein (also known as fava bean protein), soy protein, lentil protein, potato protein, chickpea protein, canola protein, and pea protein. It has been found that some plant-based proteins may impart an off flavor to the resulting plant-based product. Therefore, inclusion of one or more umami-modifier peptides may be particularly beneficial for plant-based food products.

[0026] In certain aspects, the urn a mi-enhancing peptides may be added to a food or beverage product in which it is desired to reduce the sodium content thereof. At least in some approaches, the addition of the umami-enhancing peptides adds umami taste to the food or beverage product, which may counteract, ameliorate, or otherwise mitigate a reduction of the sodium amount in the food or beverage product.

[0027] In one approach, the umami-enhancing peptides include one or more of the following peptides: RDMPIQA (SEQ ID NO. 5); QEPVLGPVRGPFP (SEQ ID NO. 6); PGPI (SEQ ID NO. 9); and KAVPYPQ (SEQ ID NO. 21). Advantageously, each of these umami-enhancing peptides has an intrinsic taste threshold (bitter, astringent, or sour flavor) of more than 50 pmol / L in water. Each of these four umami-enhancing peptides has an umami modulating threshold concentration in a model broth (including sodium chloride, monosodium L- glutamate, maltodextrin, yeast extract, and water) that is lower than its intrinsic taste threshold concentration for sour or bitter taste in water, which means that these four umami-enhancing peptides can be added to a food or beverage product in an amount that provides umamienhancing effect without also adding a bitter or sour flavor to the food or beverage product. This characteristic makes these four peptides particularly advantageous as an umami¬ enhancing ingredient.

[0028] To enhance the umami taste of a given food or beverage product any one of the following 15 umami taste enhancing peptides may be added to the food or beverage product in an umami taste enhancing composition: (1) RDMPIQA (SEQ ID NO. 5); (2) QEPVLGPVRGPFP (SEQ ID NO. 6); (3) PGPI (SEQ ID NO. 9); (4) KAVPYPQ (SEQ ID NO. 2'1); (5) RDMPIQA (SEQ ID NO. 5) and QEPVLGPVRGPFP (SEQ ID NO. 6); (6) RDMPIQA (SEQ ID NO. 5) and PGPI (SEQ ID NO. 9); (7) RDMPIQA (SEQ ID NO. 5) and KAVPYPQ (SEQ ID NO. 21); (8)9Attorney Docket 1410-163201-PCQEPVLGPVRGPFP (SEQ ID NO. 6) and PGPI (SEQ ID NO. 9); (9) QEPVLGPVRGPFP (SEQ ID NO. 6) and KAVPYPQ (SEQ ID NO. 21); (10) PGPI (SEQ ID NO. 9) and KAVPYPQ (SEQ ID NO.21); (11) RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), and PGPI (SEQ ID NO. 9); (12) RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), and KAVPYPQ (SEQ ID NO. 21); (13) RDMPIQA (SEQ ID NO. 5), PGPI (SEQ ID NO. 9), and KAVPYPQ (SEQ ID NO. 21); (14) QEPVLGPVRGPFP (SEQ ID NO. 6), PGPI (SEQ ID NO. 9), KAVPYPQ (SEQ ID NO. 21); and (15) RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), PGPI (SEQ ID NO. 9), and KAVPYPQ (SEQ ID NO. 21). It will be appreciated that any one of these 15 umami-enhancing peptides may further include one or more other flavor-enhancing (e.g., a salty -enhancing, bitter-enhancing, umami-enhancing, kokumi-enhancing, sweet- enhancing, and / or astringent-enhancing) peptides and / or umami-enhancing compounds known in the art.

[0029] The umami taste enhancing compositions may include any amount of the peptide(s), alone or in combination, necessary to provide an umami-enhancing effect when the umami taste enhancing composition is added to a food or beverage product. For example, the umami taste enhancing compositions may include one or more of the umami-enhancing peptides in an amount, by dry weight, of at least 0.1 wt.%, at least 0.5 wt.%, at least 1 wt.%, at least 2 wt.%, at least 5 wt.%, at least 10 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, at least 35 w t.%, at least 40 w t.%, at least 45 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, or at least 90 wt.% of the taste modifier composition. In another approach, the taste modifier composition (e.g., umami taste enhancing composition) may include one or more of the umami-enhancing peptides in an amount, by total weight, of at least 0.1 wt.%, at least 0.5 wt.%, at least 1 wt.%, at least 2 wt.%, at least 5 wt.%, at least 10 wt%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, or at least 90 wt.% of the composition. The umami taste enhancing composition may then be added in any desired dilution factor or ratio to provide the food or beverage product with an umamienhancing amount of one or more of the peptides.

[0030] Table 1 provides the umami enhancing taste threshold for each peptide (i.e., the minimum concentration to modulate umami taste) in model broth. The umami enhancing taste10Attorney Docket 1410-163201-PCthreshold in model broth for each peptide may provide a starting point when formulating a food or beverage product containing one or more of the umami enhancing peptides. However, the umami enhancing taste threshold for one or more of the peptides may be lower in certain food and beverage products than in the model broth. 'Therefore, the amount of one or more of the peptides may be lower than the umami enhancing threshold provided in Table 1 For those food and beverage products, an amount of 0.25x umami enhancing threshold is provided as a minimum amount for each of the umami enhancing peptides.Table 1: Taste Thresholds for Umami-Modifying PeptidesUmami Taste Amount Umami Taste- Intrinsic Taste 0.25x Umami Present in Threshold in Taste- Peptides Cheddar Threshold in Water Enhancing Cheese in Model Broth Threshold in (wt.%) Model Broth RDMPIQA (SEQ ID 34.54 mg / kg 49.4 mg / L 119.7 mg / L 0.001225 wt.% NO. 5) (sour)(0.003453605 (0.0049 wt.%)wt.%) (0.01197%)QEPVLGPVRGPFP 16.23 mg / kg 24.4 mg / L 179.1 mg / L 0.0006 wt.% (SEQ ID NO. 6) (bitter)(0.00'1623361 (0.0024 wt.%)wt.%) (0.01791 wt.%)PGPI (SEQ ID NO. 0.16 mg / kg 7.0 mg / L 26.0 mg / L 0.000175 wt.% 9) (bitter)(1.60633E-05 (0.0007 wt.%)wt.%) (0.0026 wt.%)KAVPYPQ (SEQ ID 16.33 mg / kg 54.5 mg / L 81.6 mg / L 0.001375 wt.% NO. 21) (bitter)(0.001633456 (0.0055 wt.%)wt.%) (0.00816%)

[0031] In some embodiments, one or more of the above-mentioned umami taste-enhancing compositions may be added to a food or beverage product in an amount effective to provide an umami enhancing effect. The umami taste-enhancing peptides may be added alone or in any combination in an amount effective to provide a desired umami enhancing taste to a food orAttorney Docket 1410-163201-PCbeverage product. In some approaches, the one or more umami taste-enhancing peptides are included in an amount lower than an amount that provides an undesirable taste (e.g., below an amount that provides an intrinsic taste of bitter or sour). The taste thresholds identified in Table 1 are based on inclusion of the peptides in model broth. However, amounts lower than the umami taste enhancing threshold in model broth may still provide a desirable umami taste-enhancing effect in other food or beverages. Also, in some food or beverages, an undesirable intrinsic taste may be partially or completely masked by other ingredients in the product.Additionally, off-flavor masking ingredients (e.g., bitter blocking or masking ingredients) may be added to the food or beverage products. Therefore, it is contemplated that each of the umami-enhancing peptides may be added in higher amounts than their intrinsic taste threshold.

[0032] In some aspects, a food or beverage composition includes at least about 0.0049 wt.%, at least about 0.006 wt.%, at least about 0.007 wt.%,. at least about 0.008 wt.%, at least about 0.009 wt.%, at least about 0.01 wt.%, at least about 0.02 wt.%, at least about 0.03 wt.%, at least about 0.04 wt.%, at least about 0.05 wt.%, at least about 0.06 wt.%, at least about 0.07 wt.%, at least about 0.08 wt.%, at least about 0.09 wt.%, at least about 0.1 wt.%, at least about 0.5 wt.%, and at least about 1.0 wt.% of the RDMPIQA peptide (SEQ ID NO. 5) to have an umami enhancing effect. In some approaches, the RDMPIQA peptide (SEQ ID NO. 5) may be included in a food or beverage product in an amount of at least its taste threshold value (i.e., 0.0049 wt.%) in model broth but below its intrinsic taste value in water (i.e., 0.01197 wt.% with a sour taste). For example, a food or beverage composition may include about 0.0049 wt.% to less than 0.01197 wt.%, about 0.006 wt.% to less than 0.01197 wt.%, about 0.007 wt.% to less than 0.01197 wt.%, about 0.008 wt.% to less than 0.01197 wt.%, about 0.009 wt.% to less than 0.01197 wt.%, or about 0.01 wt.% to less than 0.01197 wt.% of RDMPIQA peptide (SEQ ID NO. 5) to have an umami enhancing effect.

[0033] In other food and beverage systems, the RDMPIQA peptide (SEQ ID NO. 5) may be effective to provide an umami enhancing effect at a lower concentration than its umami taste threshold in model broth. For example, the RDMPIQA peptide (SEQ ID NO. 5) may be included in a food or beverage product in an amount of at least 0.25x its taste threshold in model broth. The RDMPIQA peptide (SEQ ID NO. 5) may be included in a food or beverage product in anAttorney Docket 1410-163201-PCamount of at least 0.001225 wt.%, at least 0.0002 wt.%, at least 0.0003 wt.%. at least 0.0004 wt.%, at least 0.0005 wt.%, at least 0.0006 wt.%, at least 0.0007 wt.%, at least 0.0008 wt.%, at least 0.0009 wt.%, at least 0.001 wt.%, at least 0.002 wt.%, at least 0.003 wt.%, or at least 0.004 wt.% to provide an umami enhancing effect. In another approach, a food or beverage composition includes about 0.001225 wt.% to less than 0.01197 wt.%, about 0.002 wt.% to less than 0.01197 wt.%, about 0.003 wt.% to less than 0.01197 wt.%, about 0.004 wt.% to less than 0.01197 wt.%, about 0.005 wt.% to less than 0.01197 wt.%, about 0.006 wt.% to less than 0.01197 wt.%, about 0.007 wt.% to less than 0.01197 wt.%, about 0.008 wt.% to less than 0.01197 wt.%, about 0.009 wt.% to less than 0.01197 wt.%, or about 0.01 wt.% to less than 0.01197 wt.% of RDMPIQA peptide (SEQ ID NO. 5) to provide an umami enhancing effect.

[0034] In some aspects, a food or beverage composition includes at least about 0.0024 wt.%, at least about 0.003 wt.%, at least about 0.004 wt.%, at least about 0.005 wt.%, at least about 0.006 wt.%, at least about 0.007 wt.%, at least about 0.008 wt.%, at least about 0.009 wt.%, at least about 0.01 wt.%, at least about 0.05 wt.%, at least about 0.1 wt.%, at least about 0.5 wt.%, or at least about 1.0 wt.% of the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) to have an umami enhancing effect. In some approaches, the QEPVLGPVRGPFP (SEQ ID NO. 6) may be included in a food or beverage product in an amount of at least its taste threshold value (i.e., 0.0024 wt.%) in model broth but below its intrinsic taste value in water (i.e., 0.01791 wt.% with a bitter taste). For example, a food or beverage composition may include about 0.0024 wt.% to less than 0.01791 wt.%, about 0.003 wt.% to less than 0.01791 wt.%, about 0.004 wt.% to less than 0.01791 wt.%, about 0.005 wt.% to less than 0.01791 wt.%, about 0.006 wt.% to less than 0.01791 wt.%, about 0.007 wt.% to less than 0.01791 wt.%, about 0.008 wt.% to less than 0.01791 wt.%, about 0.009 wt.% to less than 0.01791 wt.%, or about 0.01 wt.% to less than 0.01791 wt.% of QEPVLGPVRGPFP (SEQ ID NO. 6) to have an umami enhancing effect.

[0035] In other food and beverage systems, the QEPVLGPVRGPFP (SEQ ID NO. 6) may be effective to provide an umami enhancing effect at a lower concentration than Its umami taste threshold in model broth. For example, the QEPVLGPVRGPFP (SEQ ID NO. 6) may be included in a food or beverage product in an amount of at least 0.25x its taste threshold in model broth. The QEPVLGPVRGPFP (SEQ ID NO. 6) may be included in a food or beverage product in anAttorney Docket 1410-163201-PCamount of at least 0.0006 wt.%, at least 0.0007 't.%, at least 0.0008 wt.%, at least 0.0009 wt.%, at least 0.001 wt.%, or at least 0.002 wt.% to provide an umami enhancing effect. In another approach, a food or beverage composition includes about 0.0006 wt.% to less than 0.01791 wt.%, about 0.0007 wt.% to less than 0.01791 wt.%, about 0.0008 wt.% to less than 0.01791 wt.%, about 0.0009 wt.% to less than 0.01791 wt.%, about 0.001 wt.% to less than 0.01791 wt.%, about 0.002 wt.% to less than 0.01791 wt.%, about 0.003 wt.% to less than 0.01791 wt.%, about 0.004 wt.% to less than 0.01791 wt.%, about 0.005 wt.% to less than 0.01791 wt.%, about 0.006 wt.% to less than 0.01791 wt.%, about 0.007 wt.% to less than 0.01791 wt.%, about 0.008 wt.% to less than 0.01791 wt.%, about 0.009 wt.% to less than 0.01791 wt.%, or about 0.01 wt.% to less than 0.01791 wt.% of QEPVLGPVRGPFP (SEQ ID NO. 6) to provide an umami enhancing effect.

[0036] In some aspects, a food or beverage composition includes at least about 0.0007 w t.%, at least about 0.0008 wt.%, at least about 0.0009 wt.%, at least about 0.001 wt.%, at least about 0.002 wt.%, at least about 0.003 wt.%, at least about 0.004 wt.%, at least about 0.005 wt.%, at least about 0.01 wt.%, at least about 0.05 wt.%, at least about 0.1 wt.%, at least about 0.5 wt.%, or at least about 1.0 wt.% of PGP1 peptide (SEQ ID NO. 9) to have an umami enhancing effect. In some approaches, the PGPI peptide (SEQ ID NO. 9) may be included in a food or beverage product in an amount of at least its taste threshold value (i.e., 0.0007 wt.%) in model broth but below its intrinsic taste value in water (i.e., 0.0026 wt. % with a bitter taste). For example, a food or beverage composition may include about 0.0007 wt.% to less than 0.0026 wt.%, about 0.0008 wt.% to less than 0.0026 wt.%, about 0.0009 wt.% to less than 0.0026 wt.%, about 0.001 wt.% to less than 0.0026 wt.%, or about 0.002 wt.% to less than 0.0026 wt.% of the PGPI peptide (SEQ ID NO. 9) to provide an umami enhancing effect.

[0037] In some food and beverage systems, the PGPI peptide (SEQ ID. NO, 9) may be effective to provide an umami enhancing effect at a lower concentration than its umami taste threshold in model broth. For example, the PGPI peptide (SEQ ID NO. 9) may be included in a food or beverage product in an amount of at least 0.25x its taste threshold in model broth. The PGPI peptide (SEQ ID NO. 9) may be included in a food or beverage product in an amount of at least 0.000175 wt.%, at least 0.0002 wt.%, at least 0.0003 wt.%, at least 0.0004 wt.%, at least 0.0005 wt.%, or at least 0.0006 wt.%. In another approach, the PGPI peptide (SEQ ID NO. 9) may beincluded in a food or beverage product in an amount of at least 0.000175 wt.% to less than 0.0026 wt.%, at least 0.0002 wt.% to less than 0.0026 wt.%, at least 0.0003 wt.% to less than 0.0026 wt.%, at least 0.0004 wt.% to less than 0.0026 wt.%, at least 0.0005 wt.% to less than 0.0026 wt.%, at least 0.0006 wt.% to less than 0.0026 wt.%, at least 0.0007 wt.% to less than 0.0026 wt.%, at least 0.0008 wt.% to less than 0.0026 wt.%, at least 0.0009 wt.% to less than 0.0026 w't.%, at least 0.001 wt.% to less than 0.0026 wt.%, or at least 0.002 wt.% to less than 0.0026 wt. % to provide an umami enhancing effect.

[0038] In some aspects, a food or beverage composition includes at least about 0.0055 wt.%, at least about 0.006 wt.%, at least about 0.007 wt.%, at least about 0.008 wt.%, at least about 0.009 wt.%, at least about 0.01 wt.%, at least about 0.05 wt.%, at least about 0.1 wt.%, at least about 0.5 wt.%, or at least about 1.0 wt.% of the KAVPYPQ peptide (SEQ ID NO. 21) to have an umami enhancing effect. In some approaches, the KAVPYPQ peptide (SEQ ID NO. 21) may be included in a food or beverage product in an amount of at least its taste threshold value (i.e., 0.0055 wt.%) in model broth but below its intrinsic taste value in water (i.e., 0.00816 wt.% with a bitter taste). For example, a food or beverage composition may include about 0.0055 wt.% to less than 0.00816 wt.%, about 0.006 wt.% to less than 0.00816 wt.%, about 0.007 wt.% to less than 0.00816 wt.%, or about 0.008 wt.% to less than 0.00816 wt.%, of the KAVPYPQ peptide (SEQ ID NO, 21) to provide an umami enhancing effect.

[0039] In other food and beverage systems, the KAVPYPQ peptide (SEQ ID NO. 21) may be effective to provide an umami enhancing effect at a lower concentration than its umami taste threshold in model broth. For example, the KAVPYPQ peptide (SEQ ID NO. 21) may be included in a food or beverage product in an amount of at least 0.25x its taste threshold in model broth. The KAVPYPQ peptide (SEQ ID NO. 21) may be included in a food or beverage product in an amount of at least 0.001375 wt. %, at least 0.002 wt. %, at least 0.003 wt. %, at least 0.004 wt.%, or at least 0.005 wt.%. In another approach, the KAVPYPQ peptide (SEQ ID NO. 21) may be included in a food or beverage product in an amount of at least 0.0055 wt.% to less than 0.00816 wt.%, at least 0.006 wt.% to less than 0.00816 wt.%, at least 0.007 wt.% to less than 0.00816 wt.%, or at least 0.008 wt.% to less than 0.00816 wt.% to provide an umami enhancing effect.Attorney Docket 1410-163201-PC{0040] In certain aspects, the addition of one or more of the umami taste-enhancing peptides may advantageously not only enhance the umami taste of the food or beverage product but also may allow for a reduction of sodium in the food or beverage product by at least about 10 wt.%, at least about 20 wt.%, at least about 30 wt.%, without substantial loss of flavor. In some aspects, the amount of salt in the food or beverage product may be reduced by about 10% to about 50%, about 15% to about 45%, about 20% to about 40%, about 25% to about 35%, or about 30%, as compared to a substantially identical food or beverage product to which the one or more umami-enhancing peptides have not been added (with the weight percentages of the ingredients of this substantially identical food or beverage product being identical to the food or beverage product containing the umami-enhancing peptides except for water, bulking agents (e.g., a major bulk contributor in a given food product, such as tomato paste in ketchup), and the like, which is included in the substantially identical food or beverage product in an amount to replace the one or more umami-enhancing peptides). Additionally, one or more of the umami taste -enhancing peptides may be used for the replacement or reduction of monosodium glutamate (MSG), disodium inosinate, disodium guanylate, hydrolyzed vegetable protein, or yeast extract,(0041) Without wishing to be limited to theory, the umami-enhancing peptides can be produced via any suitable means in the art. In one approach, the umami-enhancing peptides may be derived or isolated from one or more cheddar cheeses, such as using an extraction method, in some aspects, the umami-enhancing can be produced using enzymatic treatments for casein proteolysis and selecting enzymes that produce the desired umami-enhancing peptides in high yields.{0042] For example, chemical synthesis may be used, such as solid phase peptide synthesis (SPPS), which is a well-known synthesis procedure. SPPS allows for assembly of a peptide chain by carrying out successive reactions of amino acid derivatives on a solid support. The Fmoc / tBu solid phase peptide synthesis may also be used. In some aspects, LPPS (liquid phase peptide synthesis), fragment condensation, native ligation or recombinant methods may be used for peptide synthesis.Attorney Docket 1410-163201-PC

[0043] In some aspects, when SPPS is used, the peptide assembly is conducted from the C to the N terminus, where the initial amino acid or short-chained peptide is covalently linked to a solid support material (e.g., resin). Next, a solution of the desired amino acid with a protecting group at the N terminus and a second protecting group at the side chain is reacted with the previous molecule in a coupling reaction. It is added in excess and washed away after the reaction. The solid support will ensure easy reagent removal, and the protective groups will ensure selective reaction. After this, the N terminus of the newly added amino acid is deprotected (typically under basic or acidic conditions, depending on the protective group), and the next amino acid can be added. This is repeated until the desired peptide has been assembled. Finally, the peptide is cleaved from the solid support and the amino acid side chains are deprotected to yield the final peptide product. For high peptide purities (as desired for use in food and beverages), the peptides will likely need to be purified (e.g., by RP-HPLC) to remove any traces of protective groups, reagents, or shorter-chained peptides.

[0044] In some aspects, the umami-enhancing peptides described herein may be generated via recombinant techniques. For example, recombinant microorganisms (e.g., known bacterial cultures used in cheddar cheese production) comprising one or more genes whose expression results in synthesis of one or more of the umami-enhancing peptides may be used. In some approaches, the genes used in recombinant peptide / peptide precursor synthesis may be designed.100451 In some aspects, lactic acid bacterial proteases may be used as recombinant enzymes that could generate one or more of the umami-enhancing peptides directly from casein. In one aspect, the RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), PGP1 (SEQ ID NO.9), and KAVPYPQ (SEQ ID NO. 21) peptides can be formed from β-casein. In some aspects, the umami-enhancing peptides may be generated through fermentation and aging of cheddar cheese (but may likewise be generated through fermentation and aging of other cheeses).Without wishing to be limited to theory, the longer natural cheeses have been aged, the greater concentration of the peptides would be expected to be present in such cheeses. In certain aspects, expression of the one or more genes whose expression results in synthesis of one or more of the umami -enhancing peptides may be inducible (i.e., activated under specificIAttorney Docket 1410-163201-PCconditions or in the presence of specific compounds, providing control over when the gene is expressed in the recombinant microorganism).

[0046] Also contemplated herein are genes encoding a peptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO. 5. Also contemplated herein are genes encoding a peptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO. 6. Also contemplated herein are genes encoding a peptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO. 9. Also contemplated herein are genes encoding a peptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO. 21.

[0047] In some approaches, the peptides may include additional amino acids than listed in the peptides of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9, and SEQ ID NO. 21 so long as the additional amino acids do not significantly adversely impact the umami taste enhancing effect of the peptides.[0048) In certain aspects, one or more of the genes for the umami-enhancing peptides described herein may be expressed in a recombinant host to produce the one or more of the umami-enhancing peptides in a reproduceable and efficient manner. The recombinant host may be grown in any suitable media effective to result in production of the one or more of the umami-enhancing peptides. For example, a single recombinant host may be used to generate the umami-enhancing peptide of SEQ ID NO. 5, the umami-enhancing peptide of SEQ ID NO. 6, the umami-enhancing peptide of SEQ ID NO. 9, and the umami-enhancing peptide of SEQ ID NO.21. The recombinant host may also be used to generate other umami-modifying or other tastemodifying peptides. The recombinant host may also be used to generate one or more aroma compounds of interest.

[0049] In another approach, multiple recombinant hosts may be used to generate the umami-enhancing peptides. For example, a first recombinant host may be used to generate the umami-enhancing peptide of SEQ ID NO. 5, a second recombinant host may be used to generate18Attorney Docket 1410-163201-PCthe umami-enhancing peptides of SEQ NO. 6, a third recombinant host may be used to generate the umami- enhancing peptides of SEQ ID NO. 9, and a fourth recombinant host may be used to generate the umami-enhancing peptides of SEQ ID NO. 21. These recombinant hosts may be cultured separately or in mixed culture to generate the respective umami-enhancing peptides. Additional recombinant hosts may also be used to generate one or more aroma compounds of interest.

[0050] Possible recombinant hosts that may be suitable for this purpose include, but are not limited to Escherichia coli, Pichia pastoris, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and mammalian cell lines. In some aspects, a suitable recombinant host is E. coll, which can be gro wn in Terrific broth culture media at 37°C for about 36 hours. Any suitable recombinant host or culture media may be used. After the culturing step, the umami-enhancing peptides thus produced can be recovered from the culture media using any suitable technique in the art. The umami-enhancing peptides may be concentrated or otherwise purified from the culture media by, for example, Ni-NTA affinity chromatography. At least in some approaches, the umami-enhancing peptides are recovered in substantially pure form, meaning that the one or more umami-enhancing peptides are the primary ingredient recovered from the culture media.

[0051] In some approaches, the recombinant host(s) may be used to produce a cultured milk containing one or more of the umami-enhancing peptides. For example, the recombinant host may be cultured in milk, concentrated milk, or other dairy liquid. In yet other approaches, the milk may be a plant-based milk, such as soy, oat, almond, cashew, chickpea protein, coconut, macadamia nut, pea protein, fava protein, or other aqueous liquid containing a plant protein source.

[0052] The following example is intended to illustrate the production and use of the umami-enhancing peptides and not to limit or otherwise restrict the disclosure. Unless indicated otherwise, all parts, ratios, and percentages are based on weight.

[0053] EXAMPLES

[0054] Example 1

[0055] Extraction and Isolation of Taste-Active Compounds from Cheddar Cheese[00561 Sensoproteomics was used to identify candidate taste-modifier peptides from cheddar cheese. As generally described in literature by Sebald et al. (2018), sensoproteomics combines sensory analysis (the study of how humans perceive taste, aroma, and texture) with proteomics (the study of proteins, particularly their functions and interactions). As such, sensoproteomics involves analyzing peptide sequences that may be responsible for influencing the taste of a product. As a result of the procedure followed in this example, seven umami- enhancing peptides were identified, and four of those umami-enhancing peptides were found to have particularly favorable umami-enhancing taste thresholds.

[0057] Activity-guided fractionation was performed on cheddar cheese to identify taste active peptide fractions. The cheddar cheese selected for the experiment had a strong cheddar flavor. Activity-guided fractionation is a technique used in natural product research to isolate and identify bioactive compounds from complex mixtures with the goal of identifying the specific compounds responsible for a particular activity (in this case, taste) by fractionating the mixture and testing each fraction for its activity.

[0058] The activity-guided fractionation involved extracting peptides from cheddar cheese to obtain a crude extract. For extraction, 80 g of cheddar cheese was homogenized with 300 mL 0.1% (v / v) formic acid in water (MilliQ Reference system with Elix 3, Millpore, Molsheim, France) using an Ultra-Turrax. T 25 digital (Ika Labortechnik, Staufen, Germany) at 13000 rpm for 5 min. The mixture was centrifuged (Avanti J-E Centrifuge, Beckmann Coulter, Brea California, US) at 4 °C and 10000 rpm for 30 min. 'The supernatant was collected, and the extraction residue was extracted twice more with 300 mL 0.1 % (v / v) formic acid, respectively. The combined supernatants were lyophilized to yield the crude extract, which was stored at - 20 " C until further use.2.0Attorney Docket 1410-163201-PC

[0059] The crude extract was then subjected to fractionation using Medium Pressure Liquid Chromatography (MPLC) to separate the crude extract into smaller, more manageable fractions, each containing a different combination of compounds.[0060) The MPLC instrument had two detectors, an evaporative light scattering detector (ELSD) and a UV detector. The UV detector was set to 220 nm based on preliminary experiments to optimize signal intensity. While UV detection is limited to UV-active compounds, ELSD is activated by most compounds present in a sample, including the investigated peptides. The detector signals indicate which fractions contain the highest amount of compounds. The information from the ELSD was used to determine how many fractions to pool for sensory analysis and to determine which fractions contain high concentrations of potentially taste-active compounds. However, the most active fractions were selected based only on TDA and cTDA, from which the TD- and cTD-factors were obtained. TD and cTD factors were indicative of the fraction(s) with the strongest taste impression and the strongest taste-modulating activity, respectively (i.e., the highest TD factor corresponds to the most taste¬ active fraction and the cTD factor corresponds to the most taste-modulating fraction).[0061) The crude extract (3 g) was re-dissolved in 0.1% (v / v) formic acid in water (10 L) and fractionated using MPLC (Btichi, Flawil Switzerland), equipped with a reversed-phase (RP) polypropylene cartridge filled with Chromabond Flash RS 120 C18ec bulk material (40–63 μm, 60 A, 26.2 cm x 37.2 mm, Macherey-Nagel, Dueren, Germany) with an injection volume of 2 mL. Separation was performed at a 40 mL / min flow rate using 0.1 % formic acid in water (A) and methanol (B) as solvents. After injection at 0% B, the concentration was held constant for 5 min, before increasing to 100% B within 50 min, and another isocratic step of 15 min. The concentration was then decreased to 0% B within 12 min and held constant for 8 min.

[0062] Fractions F1-F60 were collected in one-minute intervals, and aliquots (1 mL) were dried under nitrogen, dissolved in 100 pL acetonitrile / water (50 / 50, v / v), and used for Liquid Chromatography-Time-of-Flight-Mass Spectrometry ("LC-ToF-MS") analysis followed by MaxQuant evaluation, as described in more detail below. The remainder of the MPLC fractions were pooled for fractions over 3 min each (fraction 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, 37-39, 40-42, 43-45, 46-48, 49-51, 52-54, 55-57, 58-60), freed from2.1Attorney Docket 1410-163201-PCsolvent in vacuo, and lyophilized twice to ensure complete solvent removal before sensory analysis via taste dilution analysis (TDA) and comparative taste dilution analysis (cTDA). The dried powders were stored at -20 °C until further use.

[0063] The most active (i.e., more-taste -active) fractions were then identified by a trained human sensory panel for flavor characteristics by Taste Dilution Analysis (TDA). Generally, TDA is a sensory technique used to evaluate the strength and detection threshold of specific flavors or taste compounds in food or beverages. TD was used as a screening tool to identify the most taste-active MPLC fractions. When looking for umami taste-active compounds, the focus was on the MPLC fractions that have the most intense umami taste in the TDA. After identification of the taste-active fractions, further purification can be used to isolate the specific compound(s) (e.g., peptides) responsible for this activity.

[0064] TDA was performed both in water (to determine ''intrinsic taste") and in a model broth system (to determine "taste-modulating activity", cTDA). Duo-trio tests in increasing concentrations were used to determine the taste dilution (TD) factors for each fraction. A duo¬ trio test is a sensory test that determines if there is a difference between two samples by presenting a reference sample and two coded samples. The tasting panel was presented with a reference sample and two coded samples, one of which matched the reference sample. The tasting panel then identified the coded sample that was different from the reference sample.

[0065] The panelists were asked to taste each fraction in several dilution steps and the taste dilution (TD) factor was calculated, which is the dilution factor at which the panelists can only just taste a difference between the sample and a blank (water). In this test, four panelists were used, and the TD-factors represented an average of the four individual TD-factors. A high TD factor is re esentative of the fact that this specific fraction is particularly taste-active. After the TD factors 'were determined, the most taste-active fractions (i.e., the fractions with the highest TD factors) were identified.

[0066] For TDA in water, each fraction was separately diluted in water beginning at natural cheese concentration and then diluted 1:2 in stepwise fashion to a total of 15 dilution steps. The natural cheese concentration was calculated from the amount of crude extract which wasseparated via MPLC. Since at this point it was not known which compounds were present in the fractions and in which concentrations, this was only a qualitative comparison between the MPLC fractions to find out which fractions contain the taste-active compounds of interest.

[0067] The panelists started with the lowest concentrations. The panelists rinsed their mouths with water before each sample and wore a nose clip for all taste sensory tests to exclude cross-modal interactions by odor-active compounds. The results are presented in Table 2. Table 2 includes the most taste-active fractions that were identified. Table 3 below represents the full set of TD-factors for all of the tested fractions, from which the fractions shown in Table 2 were selected as the most taste-active fractions. In Table 3, the underlined TD-factors represent the TD-factors of the fractions selected as the most taste-active fractions for Table 2.

[0068] Table 2 – TDA of Most Taste-Active MPLC Fractions in WaterFraction | Number of Postulated | Taste QualityPeptides4–6 | 1 | Salt, umami, sour7–9 | 2 | Sour13–15 | 7 | Sour, salty28-30 | 143 | Astringent, umami31–33 | 231 | Astringent, bitter

[0069] Table 3 - TDA of all Tested MPLC Fractions in Water and cTDA in Model Broth Fraction Taste quality in cTD- TasteNumber of factor water factor in modulating postulated peptides In model activity in based on MaxQuant water broth model broth (score > 50)1-3 2 bitter 4 unspecific taste 04-6 431 salty, umami, 27 salty, umami, 1sour sweet7-9 215 sour 27 sour, sally 710-12 10 sour, astringent 16 umami 513-15 23 sour, salty 10 salty, sour 716-18 2 unspecific taste 2 unspecific taste 1419-21 2 unspecific taste umami 822-24 5 astringent 7 salty 4025-27 4 astringent 3 salty 5928-30 32 astringent, 6 salty 143umami31-33 13 astringent, hitler 6 umami 23134-36 2 astringent 8 salty 9837-39 3 astringent 8 unspecific taste 9740-42 8 astringent, 32 umami, sweet 223burning43-45 5 bitter 32 umami 9146-48 5 bitter, astringent 8 unspecific taste 7049-51 9 astringent 8 salty 5052-54 10 sour, salty 32 salty 21255-57 8 unspecific taste 5 unspecific taste 1058-60 3 astringent 16 unspecific taste 0

[0070] For cTDA (comparative taste-dilution analysis) in the model broth system, a model broth was prepared by mixing 2.9 g NaCl, 1.9 g monosodium L-glutamate, 6.4 g maltodextrin, and 2.1 g yeast extract in water (1 L). Similarly to TDA, each fraction was separately diluted in model broth beginning at the natural cheese concentration and then diluted 1:2 in stepwise fashion to a total of 12 dilution steps. Sensory evaluation was conducted as detailed for TDA, and the comparative taste dilution (cTD)-factors were calculated for each fraction to identify the most active fractions (i.e. highest taste-modulating activity).

[0071] The results of TDA revealed that fractions 4—6 (TD factor = 430), 7-9 (TD factor = 215), 13–15 (TD factor = 23), 28–30 (TD factor = 32) and 31–33 (TD factor = 13) had the highest24Attorney Docket 1410-163201-PCintrinsic taste activity, where fraction 4-6 was perceived as salty, umami and sour, fraction 7-9 was sour, fraction 13-15 was sour and salty, fraction 28-30 was astringent and umami, and fraction 31-33 was astringent and bitter. In addition, cTDA revealed that fractions 4-6 (cTD factor = 27), 7–9 (cTD factor = 27), 40-42 (cTD factor = 32), 43-45 (cTD factor = 32) and 52-54 (cTD factor = 32) were the most taste-modulating for salty, umami and sweet (fraction 4-6), sour and salty (fraction 7-9), umami and sweet (fraction 40-42), salty and umami (fraction 43-45) and salty (fraction 52–54) taste.

[0072] 'The fractions were further investigated using LC-ToF-MS analysis and MaxQuant evaluation, which revealed the presence of 1 (fraction 4-6), 2 (fraction 7-9), 7 (fraction 13-15), 143 (fraction 28-30), 231 (fraction 31–33), 223 (fraction 40-42), 212 (fraction 43-45) and 66 (fraction 52-54) candidate taste -active peptides in the fractions, respectively, comprising 266 peptide sequences.|0073| MaxQuant software (Max Planck Institute of Biochemistry in Martinsried, Germany) was used to determine peptide sequences using the methodology of Jürgen Cox et al., " MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein quantification," Nat. Biotechnol., 26(12); 1367-72 (2008). In certain aspects, the MaxQuant software creates a list of all possible peptides that could theoretically be formed from the specified proteins. In this case, bovine alpha s1-, alpha s2-, beta- and kappa-casein were specified, since they are the most abundant proteins in cheese. The protein sequences can be found in the database UniProt. MaxQuant simulates the peptide fragmentation pattern, which is highly predictable, and compares these obtained theoretical mass spectra with the measured LC-ToF-MS data. The output is a list of peptides that were found in the measured LC-ToF-MS mass spectra, and the score states how close the match was between the theoretical and the measured spectra. Therefore, a high score makes it more likely that the peptide is contained in the original sample. The cut-off value that was used to predict whether a peptide is contained in a sample was a score of 50, and, if the score is over 100, the peptide is assumed to be almost certainly contained in the sample.{0074J The source protein sequences were obtained from the UniProt database. The most abundant milk proteins — bovine αs1- (ID P02662), αs2- (ID P02663), β- (ID P02666), and κ- (ID25Attorney Docket 1410-163201-PCP02668) casein— were imported into the MaxQuant software for in silico unspecific digestion and comparison with the LC-ToF-MS data. MaxQuant analysis was run with LC-ToF-MS data, once for an intact sample of water-soluble extract (from classic extraction) in water, and also for aliquots of each MPLC fraction obtained after fractionation of the water-soluble extract.[0075| The analysis of the cheddar cheese via MaxQuant software revealed the presence of 819 candidate taste-active peptides, with 438 of these peptides having a score of 50 or higher, and 126 of these peptides having a score of 100 or higher. In this case, the MaxQuant software found 819 peptide sequences m all MPLC fractions with the specified parameters (chain length of 2 to 25 amino acids, origin protein sequences of bovine αs1-, αs2-, β- and K-casein, unspecific digestion, contaminants not included). The phrase "candidate taste-active peptides" in reference to the 819 peptides revealed by the Max Quant software means that these 819 peptides could be found in sufficient amounts for detection based on the search parameters and may be taste¬ active. In some aspects, the connection of the peptides to "taste" may be obtained by analyzing the peptides within the fractions with the highest (comparative) taste dilution factor, which are expected to include the most relevant taste-active peptides.{0076J Using targeted LC-MS / MS analysis, the identity of peptides postulated by the MaxQuant software was confirmed based on the alignment of multiple reaction monitoring (MRM) transitions characteristic for each peptide. The amino acid sequences of the 266 candidate peptides were entered into Skyline Software (a free and open-source tool), followed by MRM computation. This software uses the predictable fragmentation patterns of the peptides, which are characteristic for each input peptide sequence, to create LC-MS / MS methods for targeted peptide analysis (MacLean et al., 2010). The MRM transitions were exported to multiple LC-MS / MS methods with a maximum of 150 MRM transitions per method and 5 ms dwell time at unit resolution for QI and Q3. If a peptide exists in a sample, the MRM transitions align in one chromatographic signal at the same retention time. To ensure reliable peptide identification, peptides were only considered as confirmed when at least five MRM transitions aligned in one chromatographic signal.{0077J Then LC-MS / MS analysis was performed using the mass transitions computed by Skyline and a standardized chromatography procedure. The LC-MS / MS system comprised a26Attorney Docket 1410-163201-PCShimadzu Nexera X2 ultra-high-performance liquid chromatography (UHPLC) system (Shimadzu, Kyoto, Japan), connected to a 5500 QTrap mass spectrometer (AB Sciex, Darmstadt, Germany). Targeted analysis was then performed via liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) following the method adapted from Sebald et al., 2018. Samples were cooled in an autosampler at 10 °C and 5 pL aliquots were injected into the LC- MS / MS system for analysis. The UHPLC was equipped with a Kinetex C 8 column ('1.7 p, 100 A, 100 x 2.1 mm, Phenomenex, Aschaffenburg, Germany) heated at 40 °C in a column oven, and was operated using 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B) as solvents. At a flow rate of 0.4 mL / min, the separation gradient was as follows: 5% B held constant for 1 min, increased to 100% B within 11 min, held constant for 3 min, decreased to 5% B within 1 min and finally held constant for 2 min. The analytes were ionized in positive electrospray ionization (ESI) and detected in MRM mode. The ionization voltage was 5500 V and the source temperature 450 °C with nitrogen as curtain gas at 35 psi and heater gas at 65 psi, and air as nebulizer gas at 55 psi.

[0078] To prepare the samples for targeted peptide analysis, a portion of the crude cheese extract (0.3 g) was re-dissolved in water (1 mL), as described above. ’The mixture was vortexed and shaken for 1 hour at room temperature, then centrifuged and membrane-filtered (0.45.urn) to remove any insoluble residues prior to injection into the LC-MS / MS system.

[0079] Targeted LC-MS / MS analysis of the crude extract from cheddar cheese revealed that for 204 of the 266 candidate taste-active peptides, the MRM transitions aligned in one chromatographic signal. Therefore, it was concluded that these peptides were contained in the cheddar cheese crude extract. The remaining peptides showed no alignment of the MRM transitions and were excluded in further steps.

[0080] The list of candidate peptides was further reduced to 184 peptides by identifying the peptides having a signal to noise ratio of > 10 in the recorded targeted LC-MS / MS data. The signal to noise ratios (S / N) were calculated from the targeted LC-MS / MS data as the peak area of the peptide divided by the area of the noise. As peak areas can be determined for each MRM transition, characteristic mass transitions were selected for each peptide to determine the S / N. Generally, the signal-to-noise ratio facilitates distinguishing meaningful data (signal) from27Attorney Docket 1410-163201-PCbackground interference or irrelevant data (noise) and helps identify and quantify taste-active peptides in a complex mixture, such as cheese. This filter ensured a sufficiently high abundance of the peptides in the cheddar cheese for a contribution to the cheese taste.

[0081] Fold-Change Analysis10082| Next, the fold-change was calculated as an indicator of differences between two samples. Fold-change refers to the relative difference in the concentration (or amount) of a specific peptide between two samples with different sensory properties. Here, fold-change was determined between two cheddar cheese samples (named for purposes of this experiment " Sample A" and " Sample B") with different salty taste intensities and ripening times. Sample A was the cheddar cheese used in the extraction and sensory analysis described above. Sample A had a 12-month ripening time, and Sample B was a cheddar cheese with a ’’-month ripening time. Selection of these samples was based on the assumption that longer ripening times coincide with a higher degree of proteolysis and, subsequently, increased amounts of peptides that impact the salty taste of the cheese. Sample B was also perceived as less salty than Sample A during taste analysis.

[0083] With respect to fold-change, the targeted LC-MS / MS data was used. The mass spectrometer can distinguish between the different peptides. The fold-change metric is used to assess how much the concentration of a particular peptide changes between different conditions, samples, or time points, and is used to identify peptides that are most responsible for the flavor profile. In LC-MS analysis, when a sample is analyzed, different peptides will elute and ionize at different times, generating distinct peaks in a chromatogram, and the area under the peak is proportional to the amount or concentration of that peptide in the sample. For calculation of the fold-change, the peak area ratio is the ratio of the peak area of a target peptide in two samples with different properties. The ratio gives an indication of how much more (or less) of a particular peptide is present in one sample relative to another.

[0084] In the context of taste-active peptides in cheese, fold-change helps to identify which peptides increase or decrease in concentration between different cheese samples, such as cheeses that are aged differently, processed differently, or prepared under different conditions.28Attorney Docket 1410-163201-PCPeptides with a high fold-change (i.e., significantly increased peak area in one sample compared to another could be identified as contributing more to the flavor profile of the cheese and may explain any differences in the sensory profiles of the two cheese samples. These are potentially the most taste-active peptides that play a major role in flavor perception. Peptides with a fold¬ change around 1 might not significantly affect the flavor differences between the samples and may be considered less important in the context of flavor development. Furthermore, a foldchange of less than 1 suggests that the peptide of interest is more abundant in the cheese sample with sensory properties that differ from the desired taste profile.

[0085] For example, if a peptide's peak area in one sample (e.g., cheese from a certain aging process) is twice as large as in another sample (e.g., cheese from a different aging process), the fold-change would be 2 (or a 2-fold increase). Conversely, if the peak area in one sample is half of the other, the fold-change would be 0.5 (or a 2-fold decrease).10086] Fold-change is calculated as follows:Fold-change = (peak area in condition A) / (peak area in condition B).

[0087] Using the fold-change, the number of candidate peptides was further reduced to 25 peptides by picking out the peptides with a fold-change of 4 or above. The chosen fold-change of 4 suggests that there is a relevant difference in peptide concentrations between the two samples. Since the peptides of interest were the peptides that cause the increased salty taste in Sample A compared to Sample B, the focus was on the peptides that had a large difference in concentrations. Setting the cutoff fold-change value at 4 ensured a high likelihood that the observed differences in peptide concentrations were not simply caused by natural fluctuations.

[0088] After reducing the number of taste-modulating candidates to 25, the 25 peptides were custom synthesized by Genscript (New Jersey, USA). Based on quantitative ¹H-NMR spectroscopy, the resulting peptides were 71-96% pure. Furthermore, targeted LC-MS / MS analysis using the same method described above confirmed the identity of the 25 candidate peptides by close agreement of retention times and the signal intensity ratios of the MRM transitions. Therefore, it could be confirmed that all 25 candidate peptides occurred in the investigated cheddar cheese (Sample A).29Attorney Docket 1410-163201-PC

[0089] The reference compounds of the 25 candidate peptides were then separately evaluated by a trained sensory panel to assess the taste properties of each peptide to determine w'hich ones have significant taste activity, such as sweetness, bitterness, umami, etc. In particular, the intrinsic taste thresholds of the candidate peptides in water were assessed to evaluate bitter, astringent and sour taste peptides.

[0090] Samples were prepared of each of the 25 peptides in water at a beginning concentration of 1 mmol / L and diluted stepwise 1:2 with water to a total of up to 24 dilution steps until the panelists could no longer perceive a difference between the peptide solution and water. Before sensory evaluation, the pH value was adjusted to 5.4 using trace amounts of formic acid or sodium hydroxide to represent the pH of the cheese. The samples were evaluated by a trained sensory panel comprised of twelve trained panelists. Each panelist tasted the samples in a set of duo- trio tests in increasing concentrations, identified the sample that deviated from the two blanks (water), and stated which taste quality was detected. The procedure followed here was the same as the procedure described above for IDA and c'TD except that single compounds at known concentrations were used here instead of analyte mixtures with unknown compositions. Similarly to the methodology used for TDA and cTDA, the panel taste threshold was calculated as the geometric mean of the individual taste thresholds for each panelist.

[0091] Further evaluation of the 25 peptides was performed in the model broth system. The peptides were evaluated by the sensory panel for modulation of any one or more taste qualities they perceived in a sample. Samples were prepared of each of the 25 peptides in model broth at a beginning concentration of 1 mmol / L and diluted stepwise 1:2 with model broth to a total of up to 30 dilution steps until the panelists could no longer perceive a difference between the peptide solution and model broth. Before sensory evaluation, the pH value was adjusted to 5.4 using trace amounts of formic acid or sodium hydroxide to match the pH of the starting cheese (Sample A). Again, the samples were evaluated by a trained sensory panel comprised of twelve trained panelists. Each panelist tasted the samples in a set of duo-trio tests in increasing concentrations, identified the sample that deviated from the two blanks (model broth) andstated which taste quality was detected. The panel taste threshold was calculated as the geometric mean of the individual taste thresholds for each panelist.

[0092] The intrinsic taste thresholds (bitter, astringent sour) in water of the 25 candidate compounds are provided in Table 4 below. In this experiment, the taste threshold value means that a bitter, astringent, or sour taste was first identified by the tasting panel at the concentration listed in Table 4. The taste-modulating thresholds (sally, bitter, umami, kokumi, astringent) in the model broth system are also provided in Table 4. Peptides that have a higher taste threshold value for bitter, astringent, or sour taste are generally more advantageous for use as a taste modifier because they can be used in higher amounts to contribute a desired taste (e.g., salty or umami) but without adding an undesirable taste (e.g., bitter, astringent, or sour) at tha t c oncentra ti on.

[0093] Table 4 - Intrinsic Taste Threshold and Taste Modulating Threshold of peptidesPeptides Intrinsic Taste Taste Amount in Threshold in Threshold in Threshold in Starting Water Model Broth Model Broth < Cheddar Intrinsic Cheese Threshold inWaterE1V 20.3 mg / L 7.4 mg / L Yes 0.05 mg / kg (bitter) (salty)(56.6 µmol / L) (20.6 µmol / L)YGLN (SEQ ID NO. 40.6 mg / L 47.4 mg / L No 0.94 mg / kg 2) (bitter) (salty)(87.2 µmol / L) (101.9 µmol / L)NIPPLTQTPV (SEQ 34.9 mg / L 32.7 mg / L. A 4.65 mg / kg ID NO. 3) (bitter) (bitter)(32.4 µmol / L) (30.3 µmol / L)AVPYPQ (SEQ ID 38.6 mg / L 73.4 mg / L No 13.14 mg / kg NO. 4) (astringent) (salty)(57.2 µmol / L) (109 µmol / L)RDMPIQA (SEQ ID 119.7 mg / L 49.4 mg / L Yes 34.54 mg / kgNO. 5) (sour) (umami)3'1Attorney Docket 1410-163201-PCPeptides Intrinsic Taste Taste Amount in Threshold in Threshold in Threshold in Starting Water Model Broth Model Broth < Cheddar Intrinsic Cheese Threshold inWater(144.2 µmol / L) (59.6 µmol / L)QEPVLGPVRGPFP 179.1 mg / L 24.4 mg / L Yes 16.23 mg / kg (SEQ ID NO. 6) (bitter) (umami)(128.6 µmol / L) (17.5 µmol / L)NIPPLTQ (SEQ ID 37.1 mg / L 48.7 mg / L No 1.84 mg / kg NO. 7) (bitter) (kokmni)(47.5 µmol / L) (62.2 µmol / L)HQPHQPLPP (SEQ 125.8 mg / L 18.2 mg / L Yes 7.02 mg / kg ID NO. 8) (sour) (salty)(119.8 µmol / L) (17.3 µmol / L)PGPI (SEQ ID NO. 26.0 mg / L 7.0 mg / L Yes 0.16 mg / kg9) (bitter) (umami)(68.1 µmol / L) (18.2 µmol / L)RDMPIQ (SEQ ID 32.0 mg / L 31.9 mg / L Slightly 5.10 mg / kg NO. 10) (bitter) (salty)(42.2 µmol / L) (42 µmol / L)NAVPITPT (SEQ ID 86.6 mg / L 165.2 mg / L No 4.16 mg / kg NO. 11) (bitter) (salty)(106.7 µmol / L) (203.4 µmol / L)SKVLPVFQ (SEQ 82.7 mg / L 41.3 mg / L Yes 6.30 mg / kg ID NO. 12) (bitter) (95.4 (salty)umol / L)(47.7 pmol / L)GPVRGPEPIIV 37.2 mg / L 8.7 mg / L * 7.96 mg / kg (SEQ ID NO. 13) (biter) (bitter)(32.3 µmol / L) (7.6 µmol / L)EEIVPN (SEQ ID 46.5 mg / L 22.8 mg / L * 2.13 mg / kg NO. 14) (sour) (bitter)(66.4 µmol / L) (32.6 µmol / L)VVPP (SEQ ID NO. 36.3 mg / L 38.9 mg / L No 1.81 mg / kg5) _ (bitter) (salty)32Attorney Docket 1410-163201-PCPeptides Intrinsic Taste Taste Amount in Threshold in Threshold in Threshold in Starting Water Model Broth Model Broth < Cheddar Intrinsic Cheese Threshold inWater(88.3 µmol / L) (94.8 µmol / L)AVRSPAQIL (SEQ 17.2 mg / L 78.2 mg / L No 0.69 mg / kg ID NO. 16) (bitter) (salty)(18 µmol / L) (82 µmol / L)FVAP (SEQ ID NO. 1.3 mg / L 29.5 mg / L No 7.32 mg / kg 17) (bitter) (salty)(2.9 µmol / L) (68.1 µmol / L)SLPQNIPP (SEQ ID 2.2 mg / L 49.7 mg / L No 2.36 mg / kg NO. 18) (astringent) (salty)(2.5 µmol / L) (57.4 µmol / L)VLGPVR (SEQ ID 11.6 mg / L. 9.6 mg / L Slightly 0.39 mg / kg NO. 19) (umami)(astringent)(15.arn oI / L)(18.1 gmol / L)NIPPL (SEQ ID NO. 39.7 mg / L 57.1 mg / L No 2.46 mg / kg20) (bitter) (bitter)(71.9 µmol / L) (103.4 µmol / L)KAVPYPQ (SEQ ID 81.6 mg / L 54.5 mg / L Yes 16.33 mg / kg NO. 21) (bitter) (umami)(101.7 µmol / L) (68 µmol / L)QEPVL (SEQ ID 70.7 mg / L 81.9 mg / L No 2.56 mg / kg NO. 22) (bitter) (astringent)(120.9 µmol / L) (140.1 µmol / L)DMPIQA (SEQ ID 43.1 mg / L 81.3 mg / L No 7.70 mg / kg NO. 23) (bitter) (umami)(64 µmol / L) (120.7 µmol / L)KVLPVPQ (SEQ ID 13.0 mg / L 47.4 mg / L. No 19.09 mg / kg NO. 24) (bitter) (salty)(16.7 µmol / L) (60.8 µmol / L)33Attorney Docket 1410-163201-PCPeptides Intrinsic Taste Taste Amount in Threshold in Threshold in Threshold in Starting Water Model broth Model Broth < Cheddar Intrinsic Cheese Threshold inWaterVLGFVRGPFP (SEQ 20.3 mg / L 86.6 mg / L No 0.73 mg / kg ID NO. 25) (bitter) (umami)(19.6 µmol / L) (83.4 µmol / L)* The taste threshold in model broth was lower than the intrinsic threshold in water butthe taste was a bitter taste.

[0094] The intrinsic taste thresholds ranged between 2.5 µmol / L to 144.2 µmol / L in water. The taste-modulating thresholds ranged between 7.6 and 203.4 µmol / L in the model broth. In this experiment, the taste threshold value means that a salty, bitter, umami, kokurni, or astringent taste was first identified at the concentration listed in Table 4. The peptides having a taste modulating threshold concentration in model broth for salty, umami, or kokumi that was lower than an intrinsic taste threshold concentration in water for bitter, astringent, or sour were deemed the most promising taste modulating peptides.

[0095] The taste threshold values in model broth for each peptide were also compared to the amount of each peptide in the starting cheddar cheese. The amounts of the peptides in the cheddar cheese were generally much lower than the taste threshold values in the model broth. This means that the peptides are present in the starting cheddar cheese in amounts lower than would appear to be necessary to taste them on an individual basis. However, the umami¬ modulating peptides may enhance umami taste in certain food and beverage systems at amounts lower than the taste threshold in model broth.[00961 In Table 4. there are seven peptides that were determined to have umami-modifying activity, namely: RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), PGP1 (SEQ ID NO. 9), VLGPVR (SEQ ID NO. 19), KAVPYPQ (SEQ ID NO. 21), DMPIQA (SEQ ID NO. 23), and VLGPVRGPFP (SEQ ID NO. 25). The umami-modulating thresholds of each of these seven candidate peptides were compared to their respective intrinsic taste thresholds (i.e., bitter,34Attorney Docket 1410-163201-PCastringent, and sour) to identify which of these seven peptides have a taste modulating threshold concentration for umami that is lower than their respective intrinsic taste threshold.

[0097] As a result, it was determined that the following four out of seven umami-modifying peptides have umami-modulating thresholds that are lower than their respective intrinsic taste threshold: RDMPIQA (SEQ ID NO. 5) (taste modulating threshold (umami) 59.6 µmol / L and intrinsic threshold (sour) 144.2 µmol / L); QEPVLGPVRGPFP (SEQ ID NO. 6) (taste modulating threshold (umami) 17.5 µmol / L and intrinsic threshold (bitter) 128.6 µmol / L); PGP1 (SEQ ID NO. 9) (taste modulating threshold (umami) 18.2 µmol / L and intrinsic threshold (bitter) 68.1 µmol / L); and KAVPYPQ (SEQ ID NO. 21) (taste modulating threshold (umami) 68 µmol / L and intrinsic threshold (sour) 101.7 µmol / L).

[0098] 'This means that each of these four candidate peptides exhibits an umami-enhancing taste when it is present in a sample at a concentration that is lower than the concentration of the peptide that is required to exhibit the intrinsic (bitter, sour, or astringent) taste of this peptide in a sample. Such peptides are desirable for use to modulate the umami taste of a food or beverage product. To enhance the umami taste of a given food or beverage product, a taste enhancing composition including one or more of the four umami-enhancing peptides RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), PGPI (SEQ ID NO. 9), and KAVPYPQ (SEQ ID NO.2.1) may be added to the food or beverage product without negatively affecting the overall flavor of the food or beverage product (e.g., by adding bitter, sour, or astringent flavor notes to the food or beverage product).

[0099] On the other hand, the other three candidate peptides had a taste modulating threshold concentration for umami that is higher than or very close to its respective intrinsic taste threshold (bitter, sour, astringent): DMPIQA (SEQ ID NO. 23) (taste modulating threshold (umami) 120.7 µmol / L and intrinsic threshold (bitter) 64 µmol / L); VLGPVRGPFP (SEQ ID NO.25) (taste modulating threshold (umami) 83.4 µmol / L and intrinsic threshold (bitter) 19.6 µmol / L); and VLGPVR (SEQ ID NO. 19) (taste modulating threshold (umami) 15 µmol / L and intrinsic threshold (sour) 18.1 µmol / L). This means that each of these other three umami¬ modulating peptides exhibit a bitter, sour, or astringent taste in a sample at a concentration that is lower than the concentration required of this peptide to enhance the umami taste of a sample.35Attorney Docket 1410-163201-PCSuch peptides are not desirable for use to modulate the umami taste of a food or beverage product. It will be appreciated, however, that such peptides may be added to a food or beverage product and may interact with other substances / ingredients in the food or beverage matrix to favorably change the taste perception of the food or beverage product.

[0100] Example 2

[0101] Evaluation of Umami-Enhancing Peptides in Model Broth Systems

[0102] A human sensory panel was first trained on sodium chloride (NaCl) solutions as a control. Reference lines of NaCl concentrations (30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, and 75 M) were prepared in water and model broth. The model broth (1.9 g monosodium L-glutamate, 6.4 g maltodextrin, and 2.1 g yeast extract in 1 L water) was initially prepared without sodium chloride and spiked with NaCl to obtain the respective NaCl concentration. In multiple sessions, coded samples of NaCl concentrations (samples A-D) were prepared in water and model broth, respectively. Panelists were asked to taste the samples and match each sample to the NaCl reference line in water or model broth, respectively. Those sessions were conducted until each reference concentration was tested as a coded sample at least twice. This methodology was used to train panelists on iso-intensity evaluations and to identify the confidence interval of each NaCl concentration in water and model broth.

[0103] Another set of NaCl concentrations (5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 50 mM, 65 mM, 80 mM, 100 mM, and 150 mM) was prepared in the model broth excluding prior NaCl addition. These samples were prepared as coded samples A- J in order of increasing concentration. Panelists were asked to evaluate the perceived salty intensity on a scale without limitation and without predetermined values ("magnitude estimation" approach). The individual dose-response curves were normalized to 50 mM (which corresponds to the "evaluated salty taste Intensity" value of “5") while preserving the individual rating ratios of each panelist. This methodology was used to identify a dose-response curve for NaCl in the model broth for comparison with further recorded dose-response curves. The panelists were trained on "salty " intensity and were asked to adapt their training for "salty" taste intensity relative to umami taste intensity.36Attorney Docket 1410-163201-PC

[0104] Next, to determine their umami-enhancing potential in the model broth system, stock solutions were prepared of each of the four umami-modifying peptides (RDMPIQA (SEQ ID NO. 5), QEPVLGPVRGPFP (SEQ ID NO. 6), PGPI (SEQ ID NO. 9), and KAVPYPQ (SEQ ID NO. 21 )). The aim of the present experiment was to identify the lowest concentration of peptide at which there is the greatest umami enhancing effect. A model broth was prepared by mixing 2.9 g NaCl (~ 50 mM), 1.9 g monosodium L-glutamate, 6.4 g maltodextrin, and 2.1 g yeast extract in water (1 L), and the pH value was adjusted to 5.4 using trace amounts of formic acid or sodium hydroxide (to provide the approximate pH of cheddar cheese). Each of the four umami-modifying peptides were dissolved in the model broth at a concentration four times (4x) the respective taste threshold of each umami-modifying peptide in model broth shown in Table 4. The stock solutions were then diluted with model broth: 1:2, 1:2.66, 1:4, and 1:8 (i.e., 2x, 1.5x, lx, and 0.5x the taste threshold in model broth, respectively).{00105} The trained sensory panel comprised of twelve trained panelists was presented with a reference sample (prepared model broth) and five coded samples (samples A-E). To maintain a similar intensity scale as established in the salty taste intensity panel training, the umami taste intensity of the model broth with 50 mM NaCl without added peptides was designated as "5". The coded samples were evaluated by the panel. The panelists were asked to taste each sample and assign it a value regarding its umami intensity in direct correlation to the reference sample. The panelists were instructed that a value greater than 5 is representative of a more intense umami taste than the reference, while a value less than 5 is representative of a less intense umami taste than the reference. The umami taste intensity values identified by the panelists of the model broths with the four umami taste-modifying peptides are provided in Tables 5A-5D below. These values are an average of 9-12 separate values determined by the panelists.[00106) Because the model broth (unspiked) has a Baseline Umami Taste Intensity value of 5.00, when a peptide provides an Evaluated Umami Taste Intensity value of greater than 5.00, the amount of sodium in a food or beverage system containing the peptide theoretically could be reduced to the amount needed to provide a value of 5.00 without any loss of umami perception.

[0107] The average n of 9-12. umami taste intensity values for the four umami taste¬ modifying peptides in model broth identified by the panelists are provided in Tables 5A-5C below.

[0108] Table 5A – Umami Taste Intensity of RDMPIQA Peptide (SEQ ID NO. 5) in pH-Adjusted Model BrothRDMPIQA Peptide (SEQ ID NO. 5)Peptide Cone,Peptide Evaluated Umami(x threshold inConcentration Umami Taste Enhancementmodel broth)(pmol / L) Intensitylx 59.6 7.13 43%1.5x 89.4 9.00 80%2x 119.2 7.41 48%4x 238.4 10.42 108%

[0109] As determined in Example 1, the RDMPIQA peptide (SEQ ID NO. 5) had a modulating taste threshold (umami) in model broth of 59.6 µmol / L and an intrinsic taste threshold (sour) in model broth of 144.2 µmol / L. As shown in Table 5 A, the RDMPOQA peptide (SEQ ID NO. 5) clearly enhanced the umami taste at increasing amounts in pH-adjusted model broth. The strongest umami enhancing effect was found at a 1.5x threshold concentration. The impact of RDMPOQA peptide (SEQ ID NO. 5) on umami perception may continue to increase beyond the 4x threshold concentration, as umami enhancement did not level off at the tested amounts. It is possible that the intrinsic sour taste provided by RDMPIQA peptide may also play a role in the enhancement of the umami taste.

[0110] Table 5B - Umami Taste Intensity of QEPVLGPVRGPFP Peptide (SEQ ID NO. 6) in pH-Adjusted Model Broth38Attorney Docket 1410-163201-PCQEPVLGPVRGPFP Peptide (SEQ ID NO. 6)Pep ride C one,Peptide Evaluated Umami(x threshold inConcentration Umami Taste Enhancementmodel broth)(pmol / L) Intensitylx 17.5 7.55 51 %1.5x 26.3 7.95 59%2x 35 10.14 103%4x 70 7.41 48%

[0111] As determined in Example 1, the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) had a modulating taste threshold (umami) in model broth of 17.5 µmol / L and an intrinsic taste threshold (bitter) in model broth of 128.6 µmol / L. As shown in Table 5B, the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) clearly enhanced the umami taste at increasing amounts in pH-adjusted model broth. The strongest umami enhancing effect was found at a 2x threshold concentration, and the impact of QEPVLGPVRGPFP peptide (SEQ ID NO. 6) on umami taste decreased at the 4x threshold concentration. Therefore, the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) may reach peak umami -enhancing effect at the 2x threshold concentration.

[0112] Table 5C - Umami Taste Intensity of PGPI Peptide (SEQ ID NO. 9) in pH- Adjusted Model BrothPGPI Peptide (SEQ ID NO. 9)Peptide Cone, Peptide Evaluated Umami(x threshold in Concentration Umami Taste Enhancementmodel broth) Intensity(pmol / L)lx 18.2 9.18 84%1.5x 27.3 9.18 84%2x 36.4 11.82 136%4x 72.8 12.55 151%

[0113] As determined in Example 1, the PGPI peptide (SEQ ID NO. 9) had a modulating taste threshold (umami) in model broth of 18.2 µmol / L and an intrinsic taste threshold (bitter) in model broth of 68.1 µmol / L. As shown in Table 5C, the PGPI peptide (SEQ ID NO. 9) clearly enhanced the umami taste at increasing amounts in pH-adjusted model broth. Of the amounts tested, the strongest umami enhancing effect was found at the 4x. threshold concentration, and the impact of PGPI peptide (SEQ ID NO. 9) on umami perception may continue to increase39Attorney Docket 1410-163201-PCbeyond the 4x threshold concentration. However, in certain food or beverage systems, the higher amounts of the peptide may contribute an intrinsic bitter taste.

[0114] Table 5D - Umami Taste Intensity of KAVPYPQ Peptide (SEQ ID NO. 21) in pH- Adjusted 'Model BrothKAVPYPQ Peptide (SEQ ID NO. 21)Pepude Cone,Peptide Evaluated i Umami(x threshold inConcentration Umami Taste | Enhancementmodel broth)(µmol / L) Intensitylx 68 7.18 44%1.5x 102 7.00 40%2x 136 8.05 i 61%4x 272 9.23 85%

[0115] . As determined in Example 1, the KAVPYPQ peptide (SEQ ID NO. 21) had a modulating taste threshold (umami) in model broth of 68 p.mol / L and an intrinsic taste threshold (bitter) in model broth of 101.7 pmol / L. As shown in Table 5D, the KAVPYPQ peptide (SEQ ID NO. 21) clearly enhanced the umami perception at increasing amounts in pH- adjusted model broth. Of the amounts tested, the strongest umami enhancing effect was found at a 4x threshold concentration, and the impact of KAVPYPQ peptide (SEQ ID NO. 21) on umami perception may continue to increase beyond the 4x threshold concentration. However, in certain food or beverage systems, the higher amounts of the peptide may contribute an intrinsic bitter taste.100116] The results demonstrate that each of the four tested peptides provide significant umami-enhancing effect. Because of these umami-enhancing effects, each of the peptides may be added, either alone or in any combinations, to food or beverage products in amounts to either enhance umami taste or as a substitute for or to allow the reduction of other umami-enhancing ingredients, such as yeast extract, monosodium glutamate (MSG), disodium inosinate, hydrolyzed vegetable protein, or disodium guanylate.

[0117] All percentages described herein are percent by weight unless otherwise specified. The description herein is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles of exemplary embodiments. Reference throughout this specification to "one embodiment,” "an embodiment," "some embodiments", "animplementation", “some implementations", "some applications", or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," "in some embodiments", "in some implementations", and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0118] Those skilled in the art will recognize that a wide variety of other modifications, alterations, and combinations can also be made with respect to the above-described embodiments without departing from the scope of the invention, and that such modifications, alterations, and combinations are to be viewed as being within the ambit of the inventive concept.

Claims

CLAIMSWhat is claimed is:

1. A taste modifier composition comprising:a food grade carrier; andone or more of the following umami-enhancing peptides:RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 0.1 wt.%; and / or QEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 0.1 wt.%; and / orPGPI peptide (SEQ ID NO. 9) in an amount of at least 0.1 wt.%; and / or KAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 0.1 wt.%.

2. The taste modifier composition according to claim 1, wherein the one or more umami¬ enhancing peptides comprises the RDMPIQA peptide (SEQ ID NO. 5).

3. The taste modifier composition according to claim 1, wherein the one or more umamienhancing peptides comprises the QEPVLGPVRGPFP peptide (SEQ ID NO. 6).

4. The taste modifier composition according to claim 1, wherein the one or more umami- enhancing peptides comprises the PGPI peptide (SEQ ID NO. 9).

5. The taste modifier composition according to claim 1, wherein the one or more umamienhancing peptides comprises the KAVPYPQ peptide (SEQ ID NO. 21).

6. The taste modifier composition according to claim 1, wherein the umami-enhancing peptides comprise:(i) the RDMPIQA peptide (SEQ ID NO. 5) and the QEPVLGPV RGPFP peptide (SEQ ID NO. 6); or(ii) the RDMPIQA peptide (SEQ ID NO. 5) and the PGPI peptide (SEQ ID NO. 9); or(iii) the RDMPIQA peptide (SEQ ID NO. 5) and the KAVPYPQ peptide (SEQ ID NO. 21); or(iv) the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) and the PGPI peptide (SEQ ID NO.9); or(v) the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) and the KAVPYPQ peptide (SEQ ID NO. 21); or(vi) the PGPI peptide (SEQ ID NO. 9) and the KAVPYPQ peptide (SEQ ID NO. 21); or (vii) the RDMPIQA peptide (SEQ ID NO. 5), the QEPVLGPVRGPFP peptide (SEQ ID NO. 6), and the PGPI peptide (SEQ ID NO. 9); or(viii) the RDMPIQA peptide (SEQ ID NO. 5), the QEPVLGPVRGPFP peptide (SEQ ID NO. 6), and the KAVPYPQ peptide (SEQ ID NO. 21); or(ix) the RDMPIQA peptide (SEQ ID NO. 5), the PGPI peptide (SEQ ID NO. 9), and the KAVPYPQ peptide (SEQ ID NO. 21); or(x) the QEPVLGPVRGPFP peptide (SEQ ID NO. 6), the PGPI peptide (SEQ ID NO. 9), the KAVPYPQ peptide (SEQ ID NO. 21); or(xi) the RDMPIQA peptide (SEQ ID NO. 5), the QEPVLGPVRGPFP peptide (SEQ ID NO. 6). the PGPI peptide (SEQ ID NO. 9), and the KAVPYPQ peptide (SEQ ID NO. 21).

7. The taste modifier composition according to any one of claims 1 to 6, comprising one or more of:the RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 1 wt.%; and / or the QEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 1 wt.%; and / or the PGPI peptide (SEQ ID NO. 9) in an amount of at least 1 wt.%; and / orthe KAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 1 wt.%.

8. The taste modifier composition according to any one of claims 1 to 7. wherein the food grade carrier is one or more of water, propylene glycol, 1,3- propanediol, ethanol. lactic acid, and glycerin, and the taste modifier composition is in liquid form.

9. The taste modifier composition according to any one of claims 1 to 7, wherein the food grade carrier comprises one or more of gum arabic, dextrose, sucrose, fructose, lactose, protein,43Attorney Docket 1410-163201-PCcellulose, silica, calcium silicate, tapioca flour, rice flour, salt, potassium chloride, starch, and / or maltodextrin, and the taste modifier composition is in powder or particulate form.

10. The taste modifier composition according to any one of claims 1 to 7, wherein the taste modifier composition is in the form of a cultured milk or culture medium.

11. An umami taste -enhanced food or beverage product comprising the taste modifier composition of claim 1 as an added ingredient of the umami taste-enhanced food or beverage product, and the taste modifier composition included in an amount effective to provide at least one of the umami-enhancing peptides in one or more of the following amounts:RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 0.001225% by weight of the food or beverage product; and / orQEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 0.0006% by weight of the food or beverage product; and / orPGPI peptide (SEQ ID NO. 9) in an amount of at least 0.000175% by weight of the food or beverage product; and / orKAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 0.001375% by weight of the food or beverage product.

12. The umami taste-enhanced food or beverage product according to claim 11, wherein the taste modifier composition included in an amount effective to provide at least one of the umami-enhancing peptides in one or more of the following amounts:the RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 0.0049% by weight of the food or beverage product; and / orthe QEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 0.0024% by weight of the food or beverage product; and / orthe PGPI peptide (SEQ ID NO. 9) in an amount of at least 0.0007% by weight of the food or beverage product: and / orthe KAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 0.0055% by weight of the food or beverage product.44Attorney Docket 1410-163201-PC13. The umami taste-enhanced food or beverage product according to claim 11, wherein: RDMPIQA peptide (SEQ ID NO. 5) is included in an amount of at least 0.001225% to less than 0.01197% by weight of the food or beverage product; and / orQEPVLGPVRGPFP peptide (SEQ ID NO, 6) is included in an amount of at least 0.00067% to less than 0.01791% by weight of the food or beverage product; and / orPGPI peptide (SEQ ID NO. 9) is included in an amount of at least 0.000175% to less than 0.0026% by weight of the food or beverage product; and / orKAVPYPQ peptide (SEQ ID NO. 21) is included in an amount of at least 0.001375% to less than 0.00816% by weight of the food or beverage product.

14. The umami taste-enhanced food or beverage product according to claim 11 or 13, wherein the umami taste-enhanced food is a dairy-based or plant-based food product selected from process cheese, cream cheese, cheese powder, sauce, condiment, broth, and gravy.

15. A method of enhancing umami taste of a food or beverage product, the method comprising adding to the food or beverage product one or more umami-erihancing peptides:RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 0.001225% by weight of the food or beverage product; and / orQEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 0.0006% by weight of the food or beverage product; and / orPGPI peptide (SEQ ID NO. 9) in an amount of at least 0.000175% by weight of the food or beverage product; and / orKAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 0.001375% by weight of the food or beverage product.

16. The method according to claim 15, wherein the one or more umami-enhancing peptides comprises the RDMPIQA peptide (SEQ ID NO. 5).

17. The method according to claim 15, wherein the one or more umami-enhancing peptides comprises the QEPVLGPVRGPFP peptide (SEQ ID NO. 6).45Attorney Docket 1410-163201-PC18. The method according to claim 15, wherein the one or more umami-enhancing peptides comprises the PGPI peptide (SEQ ID NO. 9).

19. The method according to claim 15, wherein the one or more umami-enhancing peptides comprises the KAVPYPQ peptide (SEQ ID NO. 21).

20. The method according to claim 15, wherein the food product is a dairy-based or plant¬ based food product selected from process cheese, cream cheese, cheese powder, cheese sauce, sauce, condiment, broth, and gravy.

21. The method according to any one of claims 15 to 20, wherein the food or beverage product includes at least one of the umami-enhancing peptides in one or more of the following amounts:the RDMPIQA peptide (SEQ ID NO. 5) in an amount of at least 0.0049% by weight of the food or beverage product; and / orthe QEPVLGPVRGPFP peptide (SEQ ID NO. 6) in an amount of at least 0.0024% by weight of the food or beverage product; and / orthe PGPI peptide (SEQ ID NO. 9) in an amount of at least 0.0007% by weight of the food or beverage product; and / orthe KAVPYPQ peptide (SEQ ID NO. 21) in an amount of at least 0.0055% by weight of the food or beverage product.

22. The method according to any one of claims 15 to 20, wherein:RDMPIQA peptide (SEQ ID NO. 5) is included in an amount of at least 0.001225% to less than 0.01197% by weight of the food or beverage product; and / orQEPVLGPVRGPFP peptide (SEQ ID NO. 6) is included in an amount of at least 0.0006% to less than 0.01791 % by weight of the food or beverage product; and / orPGPI peptide (SEQ ID NO. 9) is included in an amount of at least 0.000175% to less than 0.0026% by weight of the food or beverage product; and / orKAVPYPQ peptide (SEQ ID NO. 21) is included in an amount of at least 0.001375% to less than 0.00816% by weight of the food or beverage product.

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