Flavor enhancer

Abstract

The present disclosure relates to a method of preparing an umami flavor enhancer, to an umami flavor enhancer thus obtained, to its use for enhancing the umami flavor of a food product, to a method of enhancing the umami flavor of a flavor composition or food product by of adding the umami flavor enhancer to the flavor composition or food product, and to a flavor composition comprising the umami flavor enhancer and at least one taste ingredient.

Description

[0001] FLAVOR ENHANCER

[0002] The present invention relates to a method of preparing an umami flavor enhancer, to an umami flavor enhancer thus obtained, to its use for enhancing the umami flavor of a food product, to a method of enhancing the umami flavor of a flavor composition or food product by of adding the umami flavor enhancer to the flavor composition or food product, and to a flavor composition comprising the umami flavor enhancer and at least one taste ingredient.

[0003] Umami is a flavor sensation traditionally associated with Asian cuisine. In addition, improved umami taste helps make low salt products more palatable and to increase the richness of the mouthfeel.

[0004] Umami flavor has traditionally been achieved by the addition of glutamic acid, monosodium glutamate (MSG) and / or other glutamates to food products. However, the presence of glutamic acid and / or glutamates in foodstuffs is not universally welcome, and there is an interest in the achievement of umami taste with lower proportions than is normally the case.

[0005] It is possible to achieve umami taste with a reduced proportion of glutamic acid and / or glutamates by including an umami flavor enhancer, i.e. a material that does not provide an umami flavor by itself but enhances the effect of glutamic acid and / or glutamates. For example, WO 2011 / 039340 describes the use of a compensatory proportion of y-amino butyric acid to partially replace monosodium glutamate in a foodstuff.

[0006] Peptides, especially oligopeptides, are key drivers for a savory, meaty taste and richness of mouthfeel. Nowadays, such peptides are typically prepared by creating a mixture of crude peptides using enzymatic hydrolysis of a protein of plant or animal origin, often followed by fractionation and / or isolation to enrich certain peptides from the mixture.

[0007] The composition of the crude peptide mixtures strongly depends on the proteins and their origin, making it difficult or even impossible to predict the composition. Also, depending on the degree of hydrolysis, the resulting peptides may be so small that it is no longer possible to identify the proteins / organisms from which they originate. The resulting peptides may also contribute to off-notes, such as bitterness. Furthermore, hydrolyzed vegetable proteins (HVPs) tend to have a negative connotation nowadays and may need to be labelled in certain countries.

[0008] Furthermore, fractionation and / or isolation may be necessary in order to obtain certain preferred peptides in the desired quantities.

[0009] This renders the known methods complicated, laborious, expensive, and / or less predictable.

[0010] 31424 PCTIt was therefore an objective of the present inventors to provide a method that allows for preparing such peptides in a straight forward, reliable and cost-effective way.

[0011] This problem has been solved by the methods and products of the present invention.

[0012] In a first aspect, the present invention provides a method of preparing an umami flavor enhancer, comprising the step of treating a mixture of glutamine and an amino acid with a glutaminase and a peptidase.

[0013] In a second aspect, the present invention provides an umami flavor enhancer obtained by the method of the invention.

[0014] In a third aspect, the present invention relates to the use of the umami flavor enhancer of the invention for enhancing the umami flavor of a food product.

[0015] In a fourth aspect, the present invention provides a method of enhancing the umami flavor of a flavor composition or food product, comprising the step of adding the umami flavor enhancer of the invention to the flavor composition or food product.

[0016] In a fifth aspect, the present invention provides a flavor composition comprising the umami flavor enhancer of the invention and at least one taste ingredient.

[0017] Thus, the present invention is related to the preparation of an umami flavor enhancer through an enzymatic treatment of free amino acids to synthesize the certain combination of di- and tri-, or other oligopeptides. Thanks to the selectivity of the enzymes and the freedom in selecting the amino acids, a predictable savory meaty oligopeptide / amino acids mixture is obtained, which ensures a cleaner flavor profile of the umami flavor enhancer.

[0018] Further benefits of this invention include cleaner label, high performance, and cost effectiveness.

[0019] The umami flavor enhancer obtained by the method of the invention provides authentic meaty body and lingering umami.

[0020] The umami flavor enhancer of the invention was found to comprise mostly di-peptides, but may also comprise amino acids, tri-peptides and tetra-peptides.

[0021] Surprisingly, it was found that the method of the invention allows for avoiding the formation of larger oligopeptides of the general structure [y-glu]n-arg with n > 3. Thus, the method of the invention allows for highly selective preparation of short-chain oligopeptides.

[0022] 31424 PCTThis finding is particularly surprising in view of the fact that treatment of the same mixture of glutamine and an amino acid with a glutaminase in the absence of a peptidase provided a product comprising significant amounts of larger oligopeptides of the general structure [y-glu]n-arg with n > 3.

[0023] A comparison of the products from the two processes, i.e. the one of the invention vs. the one from the alternative process without peptidase (the latter containing oligopeptides of the general structure [y-glu]n-arg with n > 3), revealed that the umami flavor enhancers of the invention were more powerful and showed fewer off-notes.

[0024] Furthermore, the method employing both glutaminase and peptidase also proved to be more selective for the formation of gamma-linkages.

[0025] Thus, the method of the invention allows for a highly selective preparation of the desired peptide mixtures, avoiding the drawbacks of the conventional processes involving random cleavage of plant proteins.

[0026] Furthermore, the method of the invention was found to provide an optimal balance of free glutamate and y-glutamyl peptides.

[0027] The umami flavor enhancer of the invention is suitable for all culinary products, including plant-based products (e.g. meat analogs and cheese alternatives), and snacks. For example, when added to a beef broth base, the product clearly enhances the meaty taste and aroma, as well as lingering meatiness, bringing it towards a more intense, more fatty, richness overall profile. The umami flavor enhancer contributes to give more umami body to the base and to reduce its boney I metallic aspects.

[0028] Glutamine (symbol Gin or Q) is a charge-neutral, polar amino acid. Dietary sources of glutamine include especially protein-rich foods like beef, chicken, fish, dairy products, eggs, vegetables like beans, beets, cabbage, spinach, carrots, parsley, vegetable juices and also wheat, papaya, Brussels sprouts, celery, kale and fermented foods like miso.

[0029] Unless expressly defined otherwise, technical and scientific terms used in this disclosure have the meanings that are commonly understood by people skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them

[0030] 31424 PCTbelow, unless specified otherwise. The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0031]

[0032] When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively.

[0033] When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0034] The term “and / or” means either “and” or “or” unless indicated otherwise.

[0035] 'Conversion" refers to the enzymatic transformation of a substrate to the corresponding product. Percent conversion" or "conversion" refers to the percentage of substrate that is converted to product 31424 PCTwithin a period of time under the specified conditions. Thus, "enzymatic activity" or "activity" of an enzyme (e.g. a glutaminase) can be expressed as the "percent conversion" of the substrate to the product.

[0036] An isolated polypeptide refers to a polypeptide that is substantially separated from other substances with which it is naturally associated, such as proteins, lipids, and polynucleotides. The term comprises polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g. in host cells or in vitro synthesis). Generally, enzyme polypeptides may be present in the cell, in the cell culture medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the enzyme polypeptide (e.g. the glutaminase) may be an isolated polypeptide.

[0037] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism that can be isolated from sources in nature and which has not been intentionally modified by manual procedures.

[0038] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g. glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids. Preferably, the amino acids have L configuration.

[0039] "Recombinant" or "engineered" or "non-naturally occurring" when used with reference to, for example, a cell, nucleic acid or polypeptide, refers to a material or material corresponding to the native or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic material and / or by manipulation using recombinant techniques.

[0040] "Suitable reaction conditions" in the context of the biocatalytic processes of the present disclosure refer to those conditions (e.g. enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, co-solvent, etc.) in the biocatalytic reaction system, under which the enzyme(s) in the present disclosure can convert a substrate to a desired product compound. Exemplary "suitable reaction conditions" are provided in the present disclosure and illustrated by examples.

[0041] "Thermostable" means that an enzyme that retains similar activity (e.g. greater than 50%) after being exposed to an elevated temperature (e.g. 30-85 °C) for a period of time (e.g. 0.5-24 h).

[0042] 31424 PCTMost amino acids are suitable for use in the method of the invention. For example, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, taurine, valine or mixtures thereof may be used.

[0043] The mixture of glutamine and the amino acid(s) may be obtained by any process typically used in the art.

[0044] Both enzymes used in the method of the invention, i.e. both the glutaminase and the peptidase, are commercial food enzymes. Also, both of them possess gamma glutamyl transpeptidase (gammaglutamyl transferase, GGT, EC 2.3.2.2) activity.

[0045] In the present invention, the glutaminase and / or the peptidase may be used in isolated form or as part of a bacterium or culture medium or other source containing the respective enzyme(s).

[0046] The glutaminase and the peptidase may be added at the same time or consecutively. It is also possible to add one or both enzymes stepwise, i.e. in two or more batches. For example, about half of the glutaminase may be added at the beginning of the treatment and the other half after about 24 hours.

[0047] Glutaminase is an amidohydrolase enzyme that typically generates glutamate from glutamine. Preferably, an enzyme having an EC number of 3.5.1.2 according to the IUBMB Enzyme Nomenclature is used. Alternative names include glutaminase I; L-glutaminase; glutamine aminohydrolase. Microbial sources of this enzyme are typically Bacillus amyloliquefaciens, Bacillus licheniformis.

[0048] A peptidase (also called a protease, proteinase, or proteolytic enzyme) is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds.

[0049] Preferably, the umami flavor enhancer obtained by the method of the invention is natural.

[0050] According to the IOFI definition, “Natural flavoring substances are flavouring substances obtained by physical processes that may result in unavoidable but intentional changes in the chemical structure of the components of the flavouring (e.g. distillation and solvent extraction), or by enzymatic or microbiological processes, from material of plant or animal origin. Such material may be unprocessed, or processed for human consumption by traditional food-preparation processes (e.g.,

[0051] 31424 PCTdrying, torrefaction (roasting) and fermentation. This means substances that have been identified I detected in a natural material of animal or vegetable origin.”

[0052] The US FDA’s definition is as follows: “The term natural flavor or natural flavoring means the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate, or any product of roasting, heating or enzymolysis, which contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products, or fermentation products thereof, whose significant function in food is flavoring rather than nutritional.”

[0053] And the European EFSA’s definition states: “’natural flavouring substance’ shall mean a flavouring substance obtained by appropriate physical, enzymatic or microbiological processes from material of vegetable, animal or microbiological origin either in the raw state or after processing for human consumption by one or more of the traditional food preparation processes listed in Annex II. Natural flavouring substances are naturally present and have been identified in nature”.

[0054] In an embodiment, the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, taurine, valine, and mixtures thereof. It has been found that these amino acids are particularly well suited for the preparation of the desired umami flavor enhancer.

[0055] Preferably the amino acid is selected from the group consisting of alanine, arginine, cysteine, glycine, histidine, valine, and mixtures thereof.

[0056] In a particularly preferred embodiment, the amino acid comprises or consists of arginine. The umami flavor enhancers obtained when including arginine were found to provide the best taste profiles.

[0057] Alternatively or in addition, the amino acid comprises or consists of histidine. The umami flavor enhancers obtained when including histidine were found to be particularly advantageous for use in cheese applications. They also provided some sweetness enhancement, as is shown in the examples below.

[0058] The molar ratio of glutamine to amino acid is preferably adjusted based on the desired flavor profile.

[0059] In an embodiment, the molar ratio of glutamine to amino acid in the mixture is from about 0.05:1 to about 10:1, more preferably from about 0.5:1 to about 5:1, and most preferably about 2:1. It has been found that these ratios provide the best umami flavor enhancers.

[0060] 31424 PCTIn an embodiment, the glutaminase is a glutaminase with gamma glutamyl transferase activity. Gamma glutamyl transferases are enzymes that catalyze the transfer of glutamyl groups between peptides.

[0061] Preferably, the glutaminase is selected from the group consisting of SD-C-100S, SD-C-100NA, and mixtures thereof. Glutaminase SD-C-100S is particularly preferred.

[0062] Glutaminase SD-C-100S is a glutaminase (EC 3.5.1.2) preparation derived from Bacillus amyloliquefaciens and catalyzes the hydrolysis of L-glutamine to L-glutamic acid. This enzyme has an excellent thermal stability, salt resistance and alcohol-resistance.

[0063] In an embodiment, the peptidase is Peptidase R.

[0064] Peptidase R is a commercial proteolytic enzyme preparation extracted from the fungus Rhizopus oligosporus. While this preparation is a mixture, it has been documented to contain aspartic proteases EC 3.4.23, which are acid endopeptidases (cleave internal peptide bonds). Other microbial sources are coming from the Aspergillus or Rhizopus genus. Peptidase R has high peptidase activity in contrast to other fungal proteinase preparations. It helps break down larger peptides and improves the solubility of the product.

[0065] Examples of suitable peptidases include, but are not limited to: Protin NY100, Protin SD-NY10, Protin SD-AY10, Protin SD-PC10F, Thermoase PC10F, Thermoase PC10FNA, Protease P, Newlase F, and mixtures thereof.

[0066] In an embodiment, the molar ratio of the glutaminase to the peptidase is from about 100:1 to about 1:100, more preferably from about 10:1 to about 1:10, still more preferably from about 2:1 to about 1 :2, and most preferably about 1:1. It has been found that these ratios provide the best umami flavor enhancer profiles.

[0067] In an embodiment, the molar ratio of the glutaminase and the peptidase to the glutamine and amino acid is from about 0.0005:1 to about 1:1, more preferably from about 0.05:1 to about 0.1:1, and most preferably about 0.05:1. At these ratios, the preferred organoleptic properties were achieved.

[0068] The method of the invention should be carried out at a suitable pH for the desired enzymatic reactions to take place.

[0069] In an embodiment, the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase at a pH of about 4 to about 10, more preferably of about 5 to about 8, and most preferably

[0070] 31424 PCTof about 6 to about 7. In these pH ranges, the enzymes used in the method of the invention create peptide bonds among the desired amino acids. Furthermore, these pH ranges help achieve the right balance between free glutamate and y-glutamyl-peptide formation

[0071] The method of the invention should be carried out at a suitable temperature for the desired enzymatic reactions to take place within an acceptable amount of time.

[0072] In an embodiment, the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase at a temperature of about 30 °C to about 70 °C, more preferably of about 35 °C to about 50 °C, still more preferably of about 37 °C to about 45 °C, and most preferably of about 40 °C.

[0073] The reaction time may vary depending on various factors, such as the nature and concentrations of the enzymes and reactants, the pH, and the temperature, for example. Preferably, the reaction time is selected such that a sufficient conversion is achieved.

[0074] Stirring or mixing the reaction mixture during the reaction is advantageous in order to keep the reaction mixture uniform and for keeping the temperature constant throughout the reaction mixture, but high shear blending should be avoided.

[0075] In an embodiment, the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase for a period of about 2 hours to 96 hours, more preferably of about 24 hours to about 72 hours, and most preferably for about 48 hours.

[0076] Analysis of the umami flavor enhancer obtained by the method of the invention, typically done by high resolution LC-MS with normal phase chromatography, revealed a complex mixture of oligopeptides. Typically, mixtures of various di-, tri- and tetra-peptides based on combinations of gamma glutamic acid (y-glu) , arginine (arg) and / or glutamine (gin), and in particular combinations of gamma glutamic acid bound to arginine and / or glutamine, were found in the umami flavor enhancers of the invention.

[0077] Preferably, the umami flavor enhancer comprises at least one, more preferably at least two, and most preferably all three of y-glu-arg, y-glu-y-glu-arg, and y-glu- y-glu-y-glu-arg.

[0078] In an embodiment, the umami flavor enhancer further comprises one or more ofy-glu-glu, y-glu-y-glu-glu, y-glu-y-glu-gln, y-glu-y-gl u-y-glu-gln, and y-glu-y-glu-y-glu-glu.

[0079] 31424 PCTAs mentioned above, the method of the invention allows for highly selective preparation of shortchain oligopeptides and for avoiding the formation of larger oligopeptides of the general structure [y-glu]n-arg with n > 3.

[0080] Therefore, in an embodiment the umami flavor enhancer comprises no or essentially no [y-glu]n-arg with n > 3.

[0081] In particular, it is preferred that the umami flavor enhancer comprises no detectable amounts of [y-glu]n-arg with n > 3. In other words, the umami flavor enhancer of the invention preferably comprises less than 3 fg of [y-glu]n-arg with n > 3.

[0082] For example, an umami flavor enhancer in the form of a spray dried powder, which is obtained by the method of the invention, may comprise about 14% to about 16% of glu (e.g. about 15%), about 4% of y-glu-glu, about 6% to about 8% of arg (e.g. about 7%), and about 4% of y-glu-arg.

[0083] For example, an umami flavor enhancer in liquid form, which is obtained by the method of the invention, may comprise about 8% to about 10% of glu (e.g. about 9%), about 2.5% of y-glu-glu, about 3.5% to about 5% of arg (e.g. about 4.5%), and about 2.5% of y-glu-arg.

[0084] In an embodiment, the umami flavor enhancer comprises y-glu-his. It has been found that umami flavor enhancers comprising y-glu-his are particularly useful for use in cheese and sweet applications.

[0085] In an embodiment, the umami flavor enhancer comprises one or more tripeptides selected from the group consisting of y-glu-val-gly, y-glu-cys-gly, and mixtures thereof.

[0086] The present invention also relates to the umami flavor enhancer obtained by the method of the invention.

[0087] The umami flavor enhancer thus obtained provides an authentic meaty body and lingering umami when combined with glutamic acid and / or glutamate. It also enhances richness and salt perceptions of savory products.

[0088] The umami flavor enhancer of the invention was found to comprise mostly di-peptides, but may also comprise amino acids, tri-peptides and tetra-peptides.

[0089] Preferably, the umami flavor enhancer comprises at least one, more preferably at least two, and most preferably all three of y-glu-arg, y-glu-y-glu-arg, and y-glu- y-glu-y-glu-arg.

[0090] 31424 PCTIn an embodiment, the umami flavor enhancer further comprises one or more ofy-glu-glu, y-glu-y-glu-glu, y-glu-y-glu-gln, y-glu-y-gl u-y-glu-gln, and y-glu-y-glu-y-glu-glu.

[0091] The umami flavor enhancer of the invention may also comprise glutamic acid and / or glutamate. In this case, it will not only enhance the umami flavor of other flavoring components, but may also provide an umami flavor by itself. In other words, it can function both as an umami flavor and an umami flavor enhancer at the same time.

[0092] The umami flavor enhancer of the invention may be provided in any form typically used in the industry.

[0093] For example, the umami flavor enhancer may be provided in a dry format, e.g. as spray dried powder using maltodextrin as a carrier.

[0094] In order to obtain the umami flavor enhancer in a dry format, it can be dried with or without a carrier. Suitable carriers include, but are not limited to, maltodextrin, starches, fibers, gum acacia, and gum Arabic. Suitable drying methods include, but are not limited to, spray-drying, vacuum-drying, oven drying, and freeze drying.

[0095] Optionally, depending on the drying method, the umami flavor enhancer in a dry format may also be ground and / or sieved in order to adjust the particle size of the product.

[0096] Alternatively, the umami flavor enhancer may be provided in a liquid format, for example in a pasteurized and aseptically filled form or as a liquid stabilized e.g. with a suitable preservative, such as propylene glycol (e.g. about 15 %) or ethanol (e.g. about 18 %).

[0097] Liquid and dry forms were found to perform on a comparable level, taking into account the respective concentrations.

[0098] The umami flavor enhancer of the invention may be used in all kind of food products, including plantbased products (e.g. meat analogs and cheese alternatives).

[0099] Therefore, the present invention also relates to the use of the umami flavor enhancer of the invention for enhancing the umami flavor of a food product. To this end, the umami flavor enhancer may be combined with one or more flavor constituents of the food product, or it may be added separately.

[0100] In an embodiment, the food product is selected from the group consisting of a bouillon, a broth, a sauce, a dressing, a gravy, a frozen meal, processed meat, a snack, a drink, a plant-based meat or dairy analogue, a dairy product, and a cheese product. It has been found that the umami flavor

[0101] 31424 PCTenhancer of the invention is particularly useful for improving the umami taste and richness of these food products.

[0102] The concentration of the umami flavor enhancer in the food product is typically selected depending on the type of food product and the desired impact of the umami flavor enhancer.

[0103] In an embodiment, the umami flavor enhancer is added to the food product at a concentration of from about 0.1 ppm to about 5000 ppm, more preferably from about 1 ppm to about 1000 ppm, and most preferably from about 10 ppm to about 200 ppm. For example, it may be added in a concentration of about 50 ppm. In particular food product types, for example in meat or dairy alternatives, higher levels of the umami flavor enhancer may be advantageous.

[0104] The present invention also provides a method of enhancing the umami flavor of a flavor composition or food product, comprising the step of adding the umami flavor enhancer of the invention to the flavor composition or food product.

[0105] The present invention also provides a flavor composition comprising the umami flavor enhancer of the invention and at least one taste ingredient.

[0106] Preferably, the flavor composition comprises a taste ingredient selected from the group consisting of monosodium glutamate (MSG), disodium inosinate (IMP), disodium guanylate (GMP), monoammonium glutamate (MAG), an amino acid, a mineral, an organic acid, an aroma composition, a yeast extract, soy sauce, fish sauce, fish powder, a hydrolyzed vegetable protein (HVP), tomato sauce, tomato powder, mushroom powder, and a mixture thereof, or a process flavor derived from any such taste ingredient.

[0107] The present invention is further illustrated by means of the following non-limiting examples:

[0108]

[0109] of Umami Flavor Enhancers at Different

[0110]

[0111] In a FlexyCube reactor system (Systag) equipped with a stirrer, 12.78 g of L-arginine hydrochloride and 22.22 g of L-glutamine were mixed in a total of 111.43 g of H2O (part of which was taken to suspend the enzymes, see below), and the pH was adjusted to 6.5 using sodium hydroxide. The mixture was heated using a hot water bath to 40 °C, 45 °C and 50 °C, respectively, and 0.74 g of Glutaminase SD-C100S (Amano) and 0.74 g of Peptidase R (Amano) were added after suspending

[0112] 31424 PCTin H2O taken from the total water content indicated above. The temperature was kept constant for 38 h, 48 h and 72 h, respectively, while stirring.

[0113] At the end of this time, 2.02 g of a natural reducing sugar syrup was added, and the enzyme was inactivated at 95 °C for 20 minutes. The liquid product was subjected to analysis:

[0114] - The amino acid analysis was done by high performance liquid chromatography with diodearray detection (HPLC-DAD), with post-column derivatization using ninhydrin, and was measured at 440 and 570 nanometers.

[0115] - The dipeptide analysis was done by liquid chromatography tandem mass spectrometry (LC- MS / MS) in positive mode with multiple reaction monitoring (MRM).

[0116] Quantitative analysis was done by comparison to standards.

[0117] The results are shown in the following table:

[0118]

[0119] As can be seen from the above, a higher temperature and / or a longer incubation time did not improve the composition of the product.

[0120] Example 2: Split Addition of Glutaminase

[0121] In order to assess the impact of a split addition of Glutaminase SD-C100S - half of the amount at TO and the other half after 24 hours - one of the experiments of Example 1 (40 °C, 72 h) was repeated with an adjusted timing of the Glutaminase addition and a maximal process time of 48 h, while the Peptidase R was again added at once in the beginning.

[0122] The experiment showed that a similar end value was reached with the modified experimental conditions after 48 hours.

[0123] 31424 PCTExample 3: Preparation of a Powder

[0124] In a double jacket 4 L glass reactor (Buchi) equipped with a stirrer, 281.11 g of L-arginine hydrochloride and 488.89 g of L-glutamine were mixed in 2451.53 g of H2O and the pH was adjusted to 6.5 using sodium hydroxide. The mixture was heated to 40 °C and 16.3 g of Glutaminase SD-C100S (Amano) and 16.30 g of Peptidase R (Amano) were added after suspending in H2O. The temperature was kept constant for 48 h at 40 °C while stirring at 120 rpm. At the end of this time, 44.51 g of natural reducing sugar syrup was added, and the enzyme was inactivated at 95 °C for 20 minutes.

[0125] 3203.2 g reactor liquids were spray dried (SD) in a spray dry tower (SiccaDania SD900) using 1146.8 g of maltodextrin. Both the liquid before spray drying and the resulting powder after spray drying were characterized by the analytical methods described in Example 1. The results are shown in in the following table:

[0126]

[0127] The thus obtained powder was used in a seasoning for potato crisps.

[0128] To this end, plain potato crisps were warmed up in an oven (80 °C) for half an hour until the crisps were warm and crunchy, and then coated with either one of the following two seasonings by dusting-on:

[0129] - Seasoning 1 : 5% RTE salt flavor (salt, monosodium glutamate, ribotide, sugar, organic acids, vegetable fat, Maltodextrin, vegan cheese aroma)

[0130] - Seasoning 2: 5% RTE salt flavor (salt, monosodium glutamate, ribotide, sugar, organic acids, vegetable fat, Maltodextrin, vegan cheese aroma) + 0.02% RTE of the umami flavor enhancer

[0131] The two potato crisp samples were then assessed in a blind tasting by six technical experts.

[0132] 31424 PCTSeasoning 1 was found to provide a salty, umami, upfront cheesiness flavor profile.

[0133] Seasoning 2 was found to provide a salty, umami, upfront cheesiness flavor profile, as well as an increased body, mouthfeel, richness and cheesy dairy mouthfeel.

[0134] The umami flavor enhancer was also subjected to sensory testing.

[0135] To this end, the umami flavor enhancer was added at 0.02% RTE to a beef broth and served at 60 °C to a panel of nine trained panelists, comparing it to the beef broth without the umami flavor enhancer in a blind test.

[0136] For each sample, panelists rated the perceived intensity of descriptors using a standard 0- 100 point intensity scale. 3 repetitions were conducted. Samples were presented one at a time with 3-digit blinding codes following a balanced design.

[0137] The following sensory attributes were used to describe the aroma and taste of the 2 samples: Boney (Odour, Taste), Meaty (Odour, Taste, Lingering), Fatty (Taste), Salty (Taste), Umami (Taste), as well as overall intensity.

[0138] The results are visualized in Figure 1.

[0139] As can be seen, the addition of the umami flavor enhancer in the base significantly increased meaty (Odour, Taste, lingering), fatty, umami and overall intensity, while boney (Taste) was decreased.

[0140] In additional experiments, it was found that the Peptidase and / or Glutaminase could also be replaced by the respective vegan enzymes and / or enzymes produced on gluten-free substrates, providing comparable umami flavor enhancers.

[0141] Example 4: Preparation and Assessment of 3 Umami Flavor Enhancers

[0142] Three umami flavor enhancers (F, G and H, respectively) were prepared according to the amounts and steps described below:

[0143]

[0144] 31424 PCT

[0145]

[0146] Evaluation:

[0147] 0.2 g of comparative sample F and 0.3 g of salt were dissolved in 99.5 g of water to prepare a taste solution. This taste solution was tasted at 0.4 wt% (in water) by five trained panelists in comparison to a blank control (consisting of 0.2 % of the respective untreated amino acid mixture (8.6 g arginine + 14.6 g glutamine), 0.3% of salt and 99.5% water). Compared to the blank control, comparative sample F gave a clean meaty taste with upfront saltiness, umami, and juiciness; but the effect was less pronounced than with umami flavor enhancers G and H.

[0148] 0.2 g of umami flavor enhancer G and 0.3 g of salt were dissolved in 99.5 g of water to prepare a taste solution. This taste solution was tasted at 0.4 wt% (in water) by five trained panelists in comparison to a blank control (consisting of 0.2 % of the respective untreated amino acid mixture (4.3 g arginine + 14.6 g glutamine + 2.0 g cysteine + 2.0 g alanine), 0.3% of salt and 99.5% water). Umami flavor enhancer G gave a more umami, salty, eggy and meaty flavor.

[0149] 0.2 g of umami flavor enhancer H and 0.3 g of salt were dissolved in 99.5 g of water to prepare a taste solution. This taste solution was tasted at 0.4 wt% (in water) by five trained panelists in comparison to a blank control (consisting of 0.2 % of the respective untreated amino acid mixture (8.6 g arginine + 14.6 g glutamine), 0.3% of salt and 99.5% water). Umami flavor enhancer H gave a slight upfront saltiness, more middle meatiness, and umami body.

[0150] 31424 PCTExample 5: Preparation and Evaluation of Umami Flavor Enhancers Comprising v-Glu-His

[0151] In a FlexyCube reactor system (Systag) equipped with a stirrer, L-histidine monohydrochloride and L-glutamine were mixed at quantities given in the table below in a total of 111.43 g of H2O (part of which was taken to suspend the enzymes, see below), and the pH was adjusted to 6.5 or 8.5 using sodium hydroxide. The mixture was heated to 40 °C using a hot water bath, and Glutaminase SD-100NA and optionally also Peptidase R (samples J and K) were added after suspending it in H2O taken from the total water content indicated above. The temperature was kept constant over the entire reaction time, while stirring.

[0152]

[0153] At the end of the reaction time, 2.02 g of a natural reducing sugar syrup was added, and the pH was re-adjusted to 6.5 using hydrochloric acid for enzyme reactions performed at a pH of 8.5 (samples K and L). The enzyme was inactivated at 95 °C for 20 minutes.

[0154] The liquid product was subjected to analysis as described in Example 1. Shorter reaction times and higher pH were found to favor the formation of higher levels of y-glutamyl histidine dipeptides, which are particularly beneficial for applications in cheese.

[0155] For tasting the reaction product was stabilized by adding 15 % propylene glycol.

[0156] Evaluation in vegan cheese sauce:

[0157] The three samples J, K and L were evaluated in a vegan cheese sauce base with 3% chickpea protein, 1% of a vegan Cheddar flavor and 0.15% of a Cheddar topnote. The vegan cheese sauce was thoroughly mixed and samples J, Kand L, respectively, were added at 100 ppm.

[0158] Sample Kwas observed to enhance salt and acidity, increase body and mouthfeel, increase umami, and add complexity and richness. It also increased the Cheddar cheese notes in the base.

[0159] Sample J was found to enhance acidity and add complexity. It also slightly increased astringency.

[0160] 31424 PCTComparative sample L enhanced saltiness and increased mouthfeel. However, it was described as less complex than sample K.

[0161] Evaluation of sweetness enhancement:

[0162] Sample K was evaluated by three expert tasters at a dosage of 5 ppm in a tasting base containing 5% sucrose, 0.05% citric acid, and 0.003% of a stevia-based ingredient.

[0163] It exhibited a very good sweetness enhancement and stevia modulation effect.

[0164] Example 6: Preparation of an Umami Flavor Enhancer Comprising a Tripeptide

[0165] In a FlexyCube reactor system (Systag) equipped with a stirrer, L-valine, L-glycine and L-glutamine were mixed in the quantities below in a total of 111.43 g of H2O (part of which was taken to suspend the enzymes, see below).

[0166]

[0167] The mixture was heated using a hot water bath to 40 °C, and the pH was adjusted using sodium hydroxide. 0.74 g of GLUTAMINASE SD-100NA and 0.74 g of Peptidase R were added in the first reaction after suspending in H2O taken from the total water content indicated above. The temperature was kept constant for 48 h, while stirring. At the end of this time, 2.02 g of a natural reducing sugar syrup was added, and the enzyme was inactivated at 95 °C for 20 minutes. The liquid reaction product was subjected to analysis for tripeptide formation.

[0168] Samples M and O were found to contain about 80 ppm of y-glu-val-gly, while sample N contained about 100 ppm of y-glu-val-gly.

[0169] 31424 PCTExample 7: Preparation of an Umami Flavor Enhancer Using Different Peptidases

[0170] In a FlexyCube reactor system (Systag) equipped with a stirrer, 12.78 g of L-arginine hydrochloride and 22.22 g of L-glutamine were mixed in a total of 111.43 g of H2O (part of which was taken to suspend the enzymes, see below). The mixture was heated to 40 °C using a hot water bath, and the pH was adjusted to 6.5 using sodium hydroxide. GLUTAMINASE SD-100NA and the peptidase to be tested were added after suspending it in H2O taken from the total water content indicated above.

[0171]

[0172] The temperature was kept constant for 48 h, while stirring. At the end of this time, 2.02 g of a natural reducing sugar syrup was added, and the enzymes were inactivated at 95 °C for 20 minutes.

[0173] The liquid reaction products were subjected to analysis for di-peptides or amino acids with methods described before.

[0174] Analysis of the resulting products (see Figure 2) did not reveal any significant differences between Samples P through V for glutamic acid, arginine, y-glu-glu , y-glu-arg, and y-glu-gln.

[0175] Therefore, it was concluded that both Newlase F and Protease P are also suitable enzymes for the formation of these desired umami flavor enhancers.

[0176] Example 8: Evaluation of Umami Flavor Enhancer in Non-Alcoholic Beer

[0177] In order to assess the impact of the umami flavor enhancer of the invention on richness and mouthfeel, Sample Kof Example 5 was tasted in alcohol-free beer.

[0178] To this end, Sample Kwas added to a commercially available non-alcoholic beer (Feldschldsschen Alkoholfrei) at a concentration of 5 ppm, 20 ppm, and 100 ppm, respectively. These test products were evaluated by four tasters and compared to the original commercial product.

[0179] The test products containing the umami flavor enhancer of the invention were found to exhibit a smoother mouthfeel.

[0180] 31424 PCTIn a further study, Sample K was also added to Heineken 0.0, another commercially available alcohol-free beer, and assessed by three tasters. It was found that the umami flavor enhancer of the invention had a positive impact on the drinkability of the beer, adding some complexity.

[0181] 31424 PCT

Claims

Claims1. Method of preparing an umami flavor enhancer, comprising the step of treating a mixture of glutamine and an amino acid with a glutaminase and a peptidase.

2. The method according to claim 1 , wherein the flavor enhancer is natural.

3. The method according to claim 1 or 2, wherein the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, taurine, valine, and mixtures thereof, wherein more preferably the amino acid is selected from the group consisting of alanine, arginine, cysteine, glycine, histidine, valine, and mixtures thereof, and wherein most preferably the amino acid comprises or consists of arginine and / or histidine.

4. The method according to any one of claims 1 to 3, wherein the molar ratio of glutamine to amino acid is from about 0.05: 1 to about 10: 1 , more preferably from about 0.5: 1 to about 5: 1 , and most preferably about 2:1.

5. The method according to any one of claims 1 to 4, wherein the glutaminase is a glutaminase with gamma glutamyl transferase activity, wherein preferably the glutaminase is selected from the group consisting of SD-C-100S, SD-C-100NA, and mixtures thereof.

6. The method according to any one of claims 1 to 5, wherein the peptidase is Peptidase R.

7. The method according to any one of claims 1 to 6, wherein the molar ratio of the glutaminase to the peptidase is from about 100: 1 to about 1 : 100, more preferably from about 10:1 to about 1:10, still more preferably from about 2:1 to about 1:2, and most preferably about 1:1.

8. The method according to any one of claims 1 to 7, wherein the molar ratio of the glutaminase and the peptidase to the glutamine and amino acid is from about 0.0005:1 to about 1:1, more preferably from about 0.05:1 to about 0.1:1, and most preferably about 0.05:1.

9. The method according to any one of claims 1 to 8, wherein the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase at a pH of about 4 to about 10, more preferably of about 5 to about 8, and most preferably of about 6 to about 7.31424 PCT10. The method according to any one of claims 1 to 9, wherein the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase at a temperature of about 30 °C to about 70 °C, more preferably of about 35 °C to about 50 °C, still more preferably of about 37 °C to about 45 °C, and most preferably of about 40 °C.

11. The method according to any one of claims 1 to 10, wherein the mixture of glutamine and amino acid is treated with the glutaminase and the peptidase for a period of about 2 hours to 96 hours, more preferably of about 24 hours to about 72 hours, and most preferably for about 48 hours.

12. The method according to any one of claims 1 to 11, wherein the umami flavor enhancer comprises y-glu-arg, y-glu-y-glu-arg, and y-glu- y-glu-y-glu-arg.

13. The method according to claim 12, wherein the umami flavor enhancer further comprises one or more of y-glu-glu, y-glu-y-glu-glu, y-glu-y-glu-gln, y-glu-y-glu-y-glu-gln, and y-glu-y-glu-y-glu- glu.

14. The method according to any one of claims 1 to 13, wherein the umami flavor enhancer comprises no or essentially no [y-glu]n-arg with n > 3.The method according to any one of claims 1 to 14, wherein the umami flavor enhancer comprises y-gl u-his.

16. The method according to any one of claims 1 to 15, wherein the umami flavor enhancer comprises one or more tripeptides selected from the group consisting of y-glu-val-gly, y-glu- cys-gly, and mixtures thereof.

17. The umami flavor enhancer obtained by the method according to any one of claims 1 to 16.

18. Use of the umami flavor enhancer according to claim 17 for enhancing the umami flavor of a food product, wherein the food product is preferably selected from the group consisting of a bouillon, a broth, a sauce, a dressing, a gravy, a frozen meal, processed meat, a snack, a drink, a plant-based meat or dairy analogue, a dairy product, and a cheese product.

19. The use of claim 18, wherein the umami flavor enhancer is added to the food product at a concentration of from about 0.1 ppm to about 5000 ppm, more preferably from about 1 ppm to about 1000 ppm, and most preferably from about 10 ppm to about 200 ppm.31424 PCT20. Method of enhancing the umami flavor of a flavor composition or food product, comprising the step of adding the umami flavor enhancer of claim 17 to the flavor composition or food product.

21. Flavor composition comprising the umami flavor enhancer of claim 17 and at least one taste ingredient, wherein the taste ingredient is preferably selected from the group consisting of monosodium glutamate (MSG), disodium inosinate (IMP), disodium guanylate (GMP), monoammonium glutamate (MAG), an amino acid, a mineral, an organic acid, an aroma composition, a yeast extract, soy sauce, fish sauce, fish powder, a hydrolyzed vegetable protein (HVP), tomato sauce, tomato powder, mushroom powder, and a mixture thereof, or a process flavor derived from any such taste ingredient.31424 PCT

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