Production of an umami ingredient

Abstract

The present invention relates to a method for producing an umami enhancing ingredient and to an umami enhancing ingredient. The method comprises suspending the high protein plant seed in water, cyclically switching the aqueous suspension between a heating step and a cooling step, which is repeated at least twice; disrupting the high protein plant seed; adding hydrolytic enzymes to the heat processed aqueous suspension and maintaining the heat processed aqueous suspension at the hydrolysis temperature to provide a hydrolysed plant protein product; and drying the hydrolysed plant protein product to provide the umami enhancing ingredient.

Description

[0001] PRODUCTION OF AN UMAMI INGREDIENT

[0002] Field of the invention

[0003] The present invention relates to a method for producing an umami enhancing ingredient and to an umami enhancing ingredient. The method involves processing a plant seed to release glutamate and nucleotides to thereby obtain a glutamate containing product where the umami effect is increased by the content of nucleotides.

[0004] Background

[0005] Ensuring the availability of adequate food supplies is one of the key issues of our time. With a rising world population, we will need to feed nine to ten billion people in 2050. Additionally, we need to reduce the environmental impact of our food production to prevent a climate disaster. Currently, about 30% of the global greenhouse gas (GHG) emissions stem from the agricultural sector. While meat currently represents only 15% of the total global human diet, approximately 80% of the agricultural land is used for animal grazing or the production of feed and fodder for animals.

[0006] While awareness of these challenges is on the rise, a notable underconsumption of plant-based foods persists among mainstream European consumers. Overcoming barriers such as pricing, availability, and taste is pivotal in transitioning consumers to a more plant-centric diet. Taste curiosity is a main driver of the first wave of alternative proteins - furthermore, consumers identify taste as the top reason why they do not continue purchasing meat alternatives after initially trying them. Meats typically provide a flavour profile strong in the taste known as “umami”, and as taste emerges as a significant driver for a shift towards a more plant-based diet, studies indicate a preference featuring umami taste also for plant-based alternatives.

[0007] Glutamic acid is a key contributor to the umami taste, and non-meat foods naturally abundant in umami compounds encompass for example tomatoes, mushrooms, soy sauce, and aged cheeses. However, incorporating these raw materials into the production of convenience food products poses challenges due to their expense and complexity. In response to the increasing consumer preference for natural and sustainable ingredients in their food, producers are making efforts to meet this demand and are looking for other plant-based and clean-label sources of umami.

[0008] Affordable sources of umami, such as yeast extract or monosodium glutamate (MSG), exist but suffer from a negative public image due to the public perception regarding their health implications. Furthermore, the inclusion of these alternatives commonly necessitates their declaration in the ingredients list, in some cases as a food additive or with unfamiliar wordings, e.g. with an E number for products marketed within the European Union or with food additives for products marketed within the US. This poses a challenge as consumers increasingly demand clean label products, i.e. that no food additives are used and having a recognisable ingredient list, and moreover consumers are becoming weary of feeling disconnected from the foods and beverages they consume, a disconnection largely attributed to the processing of food products.

[0009] Nucleotides are known to contribute a synergistic effect to the umami taste when combined with glutamate as exemplified with accompanying cheese with ham, beef with tomato, and oysters with champagne. This is also a well-known principle in yeast extract processing used as umami ingredient by the food industry.

[0010] On this basis, there is an ongoing requirement for food ingredients that contribute umami taste to the end product, while also permitting the food to be promoted as animal-free and adhering to clean label standards, especially also for organic standards, e.g. with respect to processing, agriculture and / or farming, along with the need for methods to create such an ingredient, including from other organisms than yeast. Especially organic production may increase consumer accept as such methods allow far less food additives and does not allow any kind of use of genetically modified organisms (GMO). The use of food additives and GMO is often linked to ultra processed foods, which consumers are taking an increasingly negative stand against. Further organic production, agriculture, and farming aim to protect environmental sustainability. Thus, organically produced food ingredients can be added to organic food products and food products that strive not to be identified as ultra processed. Moreover, organically produced food ingredients may support environmental sustainability.. It is an aim of the present invention to provide an umami enhancing ingredient, which contains glutamate and nucleotides. It is especially an aim to provide an umami enhancing ingredient based on plants without the need for material from animals and also without food additives, and genetically modified organisms (GMOs).

[0011] Summary

[0012] The present invention relates to a method for producing an umami enhancing ingredient, the method comprising the steps of: providing a high protein plant seed comprising having a protein content of at least 15 g / 100 g of weight of the seeds; suspending the high protein plant seed in water at an amount of high protein plant seeds to water in the range of 50 g / L to 350 g / L to provide an aqueous suspension; cyclically switching the aqueous suspension between a heating step and a cooling step to provide a heat processed aqueous suspension, where the heating step is at a heating temperature in the range of 80°C to the boiling point of the aqueous suspension and a heating duration in the range of 5 minutes to 1 hour, and the cooling step is at a cooling temperature in the range of 15°C to 35°C and a cooling duration in the range of 5 minutes to 25 minutes, where the heating step is repeated at least twice; disrupting the high protein plant seed; adjusting the temperature of the heat processed aqueous suspension to a hydrolysis temperature in the range of 25°C to 70°C; adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension and maintaining the heat processed aqueous suspension at the hydrolysis temperature for a hydrolytic duration of at least 1 hour to provide a hydrolysed plant protein product; and drying the hydrolysed plant protein product to provide the umami enhancing ingredient.

[0013] The present method employs a high protein plant seed. In the present context, any seed from any plant having a protein content of at least 15 g / 100 g of weight of the seeds, e.g. at least 18 g / 100 g of weight of the seeds, or at least 20 g / 100 g of weight of the seeds. However, the seeds should be from an edible part of a plant. Certain plants may include seeds, which require treatment to be edible, and in such cases, the relevant treatment may be included in the method. For other plant seeds, e.g. various beans, the heating step of the present method can inactivate problematic compounds, e.g. the heating can denature lectins in beans. It is preferred that if the seeds, as obtained from the plant, have an outer shell or coating or the like, the outer shell or coating is removed, e.g. the seeds are “dehulled” or “decorticated”, etc. Exemplary seeds for the present method include legume seeds, sunflower seeds, pumpkin seeds, seeds from flax, e.g. linseeds, hemp seeds, and chia seeds. The high protein plant seed may comprise only one or at least one of legume seeds, sunflower seeds, pumpkin seeds, seeds from flax, e.g. linseeds, hemp seeds, or chia seeds. The present method employs a high protein plant seed. In the present context, it is preferred that the high protein plant seed used in the method is in a dry and mature form. For example, when the high protein plant seed is a legume fruit it is preferably a dry legume fruit, e.g. separated from its pod, unless noted otherwise.

[0014] It is especially preferred that the high protein plant seeds are legume seeds, e.g. seeds from alfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soy, or peanuts. For example, the legume seeds may be dry beans (Phaseolus spp. including several species now in Vigna), such as kidney bean, haricot bean, pinto bean, navy bean (Phaseolus vulgaris), lima bean, butter bean (P. lunatus), azuki bean, adzuki bean (Vigna angulacis), mung bean, golden gram, green gram ( \ / . cadiata), black gram, urad (\ / . mungo), scarlet runner bean (P. coccineus), ricebean (\ / . umbellata), moth bean (\ / . acontifolia), tepary bean (P. acutifolius), dry broad beans (Vicia faba), such as horse bean (Vicia faba equina), broad bean ( Vicia faba), field bean (Vicia faba)', dry peas (Pisum spp.), such as garden pea (Pisum sativum var sativum), protein pea (Pisum sativum vac arvense)', chickpea (Cicec acietinum), dry cowpea (\ / . unguiculata), pigeon pea (Cajanus cajan), lentil (Lens culinaris), peanut (Acachis hypogaea), lupins (Lupinus spp.), and soy (Glycine max). In an example, the high protein plant seeds are a mixture of seeds from different plants. For example, the high protein plant seeds may be a mixture of legume seeds, or the high protein plant seeds may be a mixture of legume seeds and other seeds, or the high protein plant seeds may be from different non-legume plants. The plant sources for the high protein plant seeds may for example be selected to match a specific flavour profile. In a specific example, the high protein plant seeds used in the method do not comprise soybeans. Legumes generally contain trypsin inhibitors, and the trypsin inhibitor of soy has an inhibitory activity about 10 times greater than trypsin inhibitors of other legumes. The present inventors have surprisingly found that including soy as a high protein plant seed in the present method has a detrimental effect on the hydrolytic enzymes, especially the exopeptidase and the endopeptidase, and therefore soy should be avoided as a high protein plant seed. Without the wish to be bound by any theory, it is believed that the trypsin inhibitor of legumes may also inhibit other protease enzymes, which is particularly relevant for the stronger inhibitor of soybeans, and for this reason it is especially advantageous to not include soybeans as the high protein plant seed.

[0015] The seeds for use in the present method should, in addition to having a high protein content, also have a high content of glutamic acid, e.g. the seeds should have at least 3000 mg glutamic acid / 100 g of weight of the seeds, e.g. at least 4000 mg glutamic acid / 100 g of weight of the seeds, at least 5000 mg glutamic acid / 100 g of weight of the seeds, or at least 6000 mg glutamic acid / 100 g of weight of the seeds. For example, the high protein plant seeds may be analysed for their glutamate content, e.g. their total glutamate content, in the following procedure: Samples undergo oxidation with hydrogen peroxide and formic acid at a cold temperature, followed by acid hydrolysis using aqueous hydrochloric acid. This process oxidises methionine and cysteine, preventing loss during hydrolysis and breaking peptide bonds in the sample. After hydrolysis, pH adjustment and filtration are carried out. Amino acids are separated in an amino acid analyser, and detection is achieved through postcolumn derivatisation with ninhydrin reagent at 440 and 570 nm. The quantification utilises a 1 -point calibration. The method Reference is ISO 13903:2005. The present method comprises the step of adding hydrolytic enzymes, which include a glutaminase. A glutaminase is a hydrolase enzyme that generates glutamate from glutamine by hydrolysing the amide of glutamine to yield glutamate and ammonium. In general terms, the content of glutamate and glutamine in a plant may be considered together and be expressed as a glutamate content.

[0016] Certain compounds, e.g. amino acids or nucleotides, may exist in an acidic and a basic form. For example, glutamate may also exist as glutamic acid. In the present context, such compounds may be referred to with either name, e.g. “glutamate” or “glutamic acid”, and also include the opposite, but not explicitly mentioned form.

[0017] It is preferred that the high protein plant seeds are in a ripe form meaning that the seeds can be sown and grown to a crop. However, ripe seeds may also have been treated to remove their ability to germinate and still be considered “ripe” in the present context. Without being bound by any theory, the present inventors believe that ripe plant seeds, including ripe high protein plant seeds, contain a significant proportion of nucleotides, including both DNA and RNA, for the plant seed to serve a basis for a new plant to be grown from the seeds. Thereby, ripe high protein plant seeds provide a starting material for producing a food product containing both glutamate and nucleotides. Further without being bound by theory, the present inventors believe that the step of cyclically switching the aqueous suspension between the heating step and the cooling step weakens the cell walls of the cells in the high protein plant seeds to thereby aid in the release of nucleotides from the cells in addition to the protein. The heating duration in the cyclical switching is in the range of 5 minutes to 1 hour, but it may also in the range of 5 minutes to 25 minutes It is especially believed that it is the increase in the temperature, i.e. increasing the temperature from ambient temperature or from the cooling temperature to the heating temperature, that contributes the weakening effect, and therefore the heating step is repeated at least twice. It is further believed that the addition of plant cell disruptive pectinase enzymes and starch degrading enzymes such as amylases combined with an exopeptidase, an endopeptidase a glutaminase, and optionally also a deaminase, increases the degradation and in turn improves the release of inosinate equivalents, especially ribonucleotides, to increase the amount of IMP equivalents in the umami enhancing ingredient. However, the heating step may advantageously be repeated more than twice to thereby further increase the release of nucleotides. Thus, in examples, the heating step is repeated at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In general, the heating step may be repeated up to 10 times, as further repetitions provide limited extra effect.

[0018] Certain high protein plant seeds, e.g. legume seeds, may be soaked before subjected the high protein plant seeds to the heating step. In general, when high protein plant seeds are soaked, the high protein plant seeds are suspended in water, e.g. at an amount of high protein plant seeds, e.g. dry high protein plant seeds, to water in the range of 50 g / L to 350 g / L, e.g. 50 g / L to 250 g / L, to provide an aqueous suspension, and the high protein plant seeds are allowed to soak in the aqueous suspension for a soaking duration. The soaking may be done at ambient temperature. In the present context, ambient temperature is a temperature in the range of 10°C to 40°C. The soaking duration may be at least 1 hour and up to 24 hours, although the soaking duration may also be longer.

[0019] The method comprises the step of disrupting the high protein plant seeds. The high protein plant seeds may be disrupted at any stage, although it is preferred that the disruption is performed after the cyclic switching between the heating step and the cooling step. Thereby, the degrading effect on the cells of the high protein plant seeds of the cyclic switching between the heating step and the cooling step is maximised. The disruption may be any processing intended to reduce the size of parts or particles of the high protein plant seeds, and typical disruptive processing involves cutting, pressing, chopping, milling, grinding, crushing, grating, shredding etc. In particular the disruption aims to degrade or disrupt the cell walls of the high protein plant seeds to make the contents of the cells accessible. The disruption of the high protein plant seeds generally provides particles having a size in the range of 10 pm to 500 pm or more, e.g. up to 1 mm.

[0020] The method involves adding hydrolytic enzymes to the heat processed aqueous suspension, and other enzymes may be used in other steps in the method. In general, the method may involve the use of any hydrolytic enzyme that can modify a component present in the high protein plant seed, e.g. to provide a more useful hydrolysate of the component. For example, the hydrolytic enzymes may further comprise at least one of an asparaginase, a glycosidase, a pectin lyase, a polygalacturonase, a deaminase, and a nuclease. Thus, the method uses an endopeptidase to reduce proteins to fragments, and the fragments are degraded further to peptide fragments with the use of an exopeptidase, and a glutaminase converts glutamine to glutamate. In addition, the hydrolytic enzymes may include enzymes that can catalyse the hydrolysis of specific amino acids, e.g. an asparaginase to hydrolyse asparagine to aspartate. It is also possible to use any kind of deaminase to remove an amino group from a molecule. Deaminases can be classified according to the substrate from which the amino group is removed. In an example, the hydrolytic enzymes comprise at least an exopeptidase, an endopeptidase, a glutaminase and a deaminase. For example, the deaminase may be specific for a specific nucleotide or nucleoside or a specific amino acid. Further hydrolytic enzymes include glycosidases to hydrolyse glycosidic bonds in sugars, e.g. polysaccharides or oligosaccharides. For example, a glycosidase may be an amylase, a pectinase, a pectin lyase, a polygalacturonase, etc. Thus, enzymes having the intended function may be selected freely, and several commercial suppliers of enzymes exist. For example, relevant enzymes are available from Novozymes A / S (Bagsvaerd, Denmark), Chr. Hansen A / S (Horsholm, Denmark), Novonesis A / S (Bagsvaerd, Denmark), Tailorzyme ApS (Herlev, Denmark), Biocatalysts (Cardiff, Wales, the UK), DSM (Heerlen, the Netherlands) and many other sources.

[0021] In an embodiment, the hydrolytic enzymes further comprise at least one of an amylase and a pectinase, e.g. both of an amylase and a pectinase. An amylase and / or a pectinase may be added together with other hydrolytic enzymes, or an amylase and / or a pectinase may be added before or after addition of the other hydrolytic enzymes. Thus, the step of adding hydrolytic enzymes may further comprise adding at least one of an amylase and a pectinase, e.g. both of an amylase and a pectinase, to the heat processed aqueous suspension. When an amylase and / or a pectinase are used as hydrolytic enzymes, the hydrolytic duration is preferably at least 5 hours. Including a pectinase or an amylase, especially both of a pectinase and an amylase, provides an improved, e.g. an optimal, viscosity condition in combination with improved hydrolysis of umami compounds. In particular, the present inventors have surprisingly found that these additional hydrolytic enzymes, i.e. an amylase and / or a pectinase, especially both of an amylase and a pectinase, improve the release of inosinate equivalents. Thus, including an amylase and / or a pectinase as hydrolytic enzymes can increase the amount of IMP equivalents beyond 500 IMP equivalents per 100 g of umami enhancing ingredient and provide an umami enhancing ingredient with at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0022] The method includes the step of adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension. This step may also be referred to as the hydrolysis step. In general, the hydrolytic enzymes may be from any sources, although it is preferred that they are not derived from animal sources. It is especially also preferred that the hydrolytic enzymes are not produced from or produced by GMOs. In the present context, an exopeptidase is an enzyme that hydrolyses a protein or a peptide from an end of the protein. The hydrolysis may be from the amino end, e.g. the N-terminus, of the protein or from the carboxyl end, e.g. the C-terminus, of the protein. The exopeptidase may be an enzyme that cleaves any number of amino acids from a protein. For example, the exopeptidase may cleave single amino acids, dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. from the end of a protein. In contrast, the endopeptidase is, in the present context, an enzyme that cleaves a protein at non-terminal sites in the protein. In general, endopeptidases recognise a specific amino acid motif in a protein sequence and cuts the protein sequence at the motif. The motif may be a short sequence of amino acids or a single amino acid. To produce a product rich in glutamate, it is especially preferred that the endopeptidase recognises a glutamate residue or a glutamine residue in a protein sequence and cuts the protein at the glutamate residue or the glutamine residue. An endopeptidase that cuts a protein sequence at a glutamate residue or a glutamine residue is referred to as a glutamyl endopeptidase. Thus, the action of the endopeptidase and the exopeptidase reduces the treated protein to fragments of amino acids. In the present context, the protein cleaved by the endopeptidase and the exopeptidase may be referred to collectively as “fragments” or “protein fragments”, furthermore the fragments provided by the endopeptidase and degraded further by the exopeptidase may be referred to as “peptide fragments”. The fragments may contain any number of amino acids linked together via peptide bonds after the treatment with the hydrolytic enzymes. The proteins may be fully degraded to single amino acids, and in the present context, the umami effect is also considered to occur if the fragments of the degraded peptides contain a terminal glutamic acid residue. For example, a glutamic acid residue may be contained, especially at a terminal location, in a dipeptide, a tripeptide, etc.

[0023] The glutaminase catalyses the conversion of glutamine to glutamate, releasing ammonia as a by-product. The glutaminase may also be referred to as an amidohydrolase enzyme. Glutaminases exist in the liver, although glutaminases of bacterial origin also exist. It is preferred to use a glutaminase of a non-animal origin. Without the wish to be bound by any theory, it is believed that, while free glutamate is a contributor to umami taste perception, the presence of glutamate within a peptide chain can still interact with umami receptors.

[0024] The present method comprises a hydrolysis step of adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension and allowing the hydrolytic enzymes to react over a hydrolytic duration at a hydrolysis temperature. The present method employs a hydrolysis temperature, and the hydrolytic enzymes may be used at the same hydrolysis temperature, or the hydrolysis temperature may be adjusted to more appropriately match the optimal temperature of a specific hydrolytic enzyme. For example, the hydrolytic enzymes may be added together, and the hydrolysis temperature may be set to match an optimal temperature of the endopeptidase followed by adjusting the hydrolysis temperature to match an optimal temperature of the exopeptidase and finally, the hydrolysis temperature may be adjusted to match an optimal temperature of the glutaminase. The present method involves a hydrolytic duration, which is at least 1 hour, e.g. the hydrolytic duration may be in the range of 1 hour to 48 hours. However, it is also contemplated that the hydrolytic duration may be shorter than 1 hour, e.g. the hydrolytic duration may be at least 10 minutes, at least 20 minutes, or at least 30 minutes. The hydrolysis of each of the hydrolytic enzymes, the exopeptidase, the endopeptidase and the glutaminase may be allowed to run to completion, e.g. when all the substrate of the respective hydrolytic enzyme has been used up, or the hydrolysis may be terminated. The hydrolysis may be terminated by proceeding to the step of drying the hydrolysed plant protein product, or the hydrolytic enzymes may be inactivated. Inactivation of the hydrolytic enzymes may involve increasing the temperature to an inactivating temperature, e.g. an inactivating temperature as appropriate for the specific hydrolytic enzyme to be inactivated. For example, the hydrolytic enzyme may generally be inactivated by heating to 95°C or more, e.g. for 15 minutes. When the hydrolytic enzymes have been inactivated, the hydrolysed plant protein product may be stored before drying the hydrolysed plant protein product.

[0025] Each of the hydrolytic enzymes, e.g. the exopeptidase, the endopeptidase and the glutaminase, may be used with a hydrolytic duration appropriate for the respective enzyme, especially when it is intended to control the extent of the hydrolysis of the respective enzyme. In general, the hydrolytic duration is counted from the addition of the specific hydrolytic enzyme to the heat processed aqueous suspension and to the inactivation of the specific hydrolytic enzyme or to the initiation of the step of drying the hydrolysed plant protein product.

[0026] The hydrolytic enzymes may be added at the same time, or the hydrolytic enzymes may be added sequentially. When the hydrolytic enzymes are added sequentially, they may be added in any order. However, it is preferred to add the endopeptidase before the exopeptidase and / or the glutaminase. In the present context, the endopeptidase cleaves peptide bonds within protein molecules, thereby initiating the breakdown of large protein structures into fragments of smaller peptides. It is preferred that the endopeptidase is a glutamyl endopeptidase that cuts the protein at a glutamate residue or a glutamine residue to thereby provide fragments with a terminal glutamate residue or terminal glutamine residues. Following the action of the endopeptidase, the exopeptidase further degrades the fragments into peptide fragments, e.g. as single amino acids, dipeptides, tripeptides, tetrapeptides or pentapeptides, etc. certain of which contain glutamate and / or glutamine. The endopeptidase and the exopeptidase may be added together, or the exopeptidase may be added after the endopeptidase.

[0027] In an exemplary sequential process, the hydrolytic enzymes are added in the following order:

[0028] - adding an endopeptidase,

[0029] - adding an exopeptidase,

[0030] - adding a glutaminase.

[0031] Additional hydrolytic enzymes may also be added, e.g. after adding the exopeptidase, although hydrolytic enzymes capable of breaking down the cell wall of the high protein plant seeds may also be added before adding the endopeptidase. In a specific example, the proteases are inactivated before adding further hydrolytic enzymes to minimize degradation of the further hydrolytic enzymes by the proteases.

[0032] In a specific example, the heat processed aqueous suspension is adjusted to a hydrolysis temperature of the endopeptidase, and the endopeptidase is added and allowed to react for a hydrolytic duration of the endopeptidase, before adding the exopeptidase. The exopeptidase may be added before or after adjusting the temperature of the heat processed aqueous suspension to a hydrolysis temperature of the exopeptidase. The exopeptidase is allowed to react for a hydrolytic duration of the exopeptidase, before adding the glutaminase. The glutaminase may be added before or after adjusting the temperature of the heat processed aqueous suspension to a hydrolysis temperature of the glutaminase, and the glutaminase is then allowed to react for a hydrolytic duration of the glutaminase. For example, the heat processed aqueous suspension is maintained at the hydrolysis temperature for a hydrolytic duration in the range of 1 hour to 24 hours after each added hydrolytic enzyme. Additionally, peptides containing glutamate may also show the ability to interact with other taste compounds similarly to what free glutamate is capable of, and especially nucleotides are known to provide a synergistic effect to the umami taste when combined with glutamate. Exemplary nucleotides include 5’-ribonucleotides such as inosine 5’-monophosphate (IMP), 5’- guanosine monophosphate (GMP), adenosine 5’-monophosphate (AMP), xanthosine 5’-monophosphate (XMP), which also contribute to umami taste. This interaction between peptides containing glutamate and nucleotides is believed to provide an equally strong umami perception compared to free glutamate and nucleotides. Different nucleotides have different taste intensities and may therefore also have different synergistic effects together with glutamate on the umami taste, and the synergistic effect of a nucleotide may be compared to the synergistic effect of inosinate, which is conventionally, and also in the present context, set to a value of 1 . Thus, the synergistic effect of nucleotides is expressed in terms of “inosinate equivalents” or “IMP equivalents”. GMP has an IMP equivalent of 2.3, IMP has an IMP equivalent of 1 , XMP has an IMP equivalent of 0.61 , and AMP has an IMP equivalent of 0.18. It is therefore preferred to convert nucleotides to a higher ranking nucleotide, e.g. GMP, if possible, or IMP. For example, AMP may be converted to IMP using a deaminase thereby greatly increasing the umami synergy effect. It is preferred that the hydrolytic enzymes include a deaminase, e.g. Flavorpro® 954MDP from Biocatalysts. IMP equivalents are, in the context of the present disclosure, expressed without a unit and it is understood that the amount of the “equivalents” is a relative number compared to the amount of the specific molecule compared to an amount of IMP. Thus, when an IMP equivalent is provided by IMP, 1 IMP equivalent corresponds to 1 mg of IMP, when an IMP equivalent is provided by GMP, 1 IMP equivalent corresponds to 2.3 mg of IMP, when an IMP equivalent is provided by XMP, 1 IMP equivalent corresponds to 0.61 mg of IMP, and when an IMP equivalent is provided by AMP, 1 IMP equivalent corresponds to 0.18 mg of IMP. The IMP equivalents may also be represented by more than one nucleotide and the “IMP equivalents” value is calculated from the mass of each of these and the specific IMP equivalent value for the corresponding nucleotides. However, it is also possible to express the IMP equivalents in a unit of mass, e.g. mg. In this case, 1 mg IMP equivalent corresponds to the synergistic effect available from 1 mg IMP. Thus, for GMP, 1 mg IMP equivalent corresponds to 1 / 2.3 mg or 0.43 mg GMP, for IMP, 1 mg MP equivalent corresponds to 1 / 1 mg or 1 mg IMP, for XMP, 1 mg MP equivalent corresponds to 1 / 0.61 mg or 1 .64 mg XMP, and for AMP, and 1 mg MP equivalent corresponds to 1 / 0.18 mg or 5.56 mg AMP.

[0033] The sequential addition of enzymes (especially endopeptidases, exopeptidases, and glutaminase) allows for precise control over the enzymatic reactions, maximising substrate conversion while minimising the formation of undesirable by-products. By strategically incorporating glutaminase at the final stage of the process, glutamine is efficiently converted to glutamate, enhancing the umami flavour of the hydrolysed plant protein product.

[0034] In an embodiment, the step of adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension further comprises or is followed by adding an asparaginase, which catalyses the removal of -NH2 from the amide group of asparagine to convert it to aspartic acid / aspartate. Thus, the asparaginase is also a hydrolytic enzyme in the present context. An asparaginase may be added to the heat processed aqueous suspension at any time relative to the addition hydrolytic enzymes, e.g. in any order compared to the sequential addition of the endopeptidase, the exopeptidase and the glutaminase, although it is preferred that the asparaginase is added after the activity of exopeptidase. The asparaginase is preferably added to the heat processed aqueous suspension together with a glutaminase. A hydrolytic duration and a hydrolysis temperature may also be defined for the asparagine, and the hydrolytic duration and / or the hydrolysis temperature may be the same or different from the hydrolytic durations and the hydrolysis temperatures of the other hydrolytic enzymes.

[0035] Asparaginase catalyses the hydrolysis of asparagine, an amino acid abundant in many foods, into aspartic acid and ammonia. Asparagine is a precursor to acrylamide, a potential carcinogen formed during high- temperature cooking processes such as frying, baking, and roasting. By selectively targeting and hydrolysing asparagine, asparaginase effectively reduces the substrate available for acrylamide formation, thus mitigating the formation of acrylamide compounds. This is especially relevant when spray drying is used to dry the hydrolysed plant protein product. Thus, in an example, asparaginase is added, especially together with glutaminase, to the heat processed aqueous suspension, and after allowing the asparagine to react, i.e. over a hydrolytic duration, the hydrolysed plant protein product is spray dried. Thereby, formation of acryl amide in the umami enhancing ingredient is minimised, while still providing desirable Maillard products.

[0036] In an embodiment, the step of adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension further comprises adding a pectin lyase and / or a polygalacturonase. The pectin lyase and / or polygalacturonase may be added to the heat processed aqueous suspension at any time relative to the addition hydrolytic enzymes, e.g. in any order compared to the sequential addition of the exopeptidase, endopeptidase and glutaminase. A hydrolytic duration and a hydrolysis temperature may also be defined for the pectin lyase and / or the polygalacturonase, and the hydrolytic durations and / or the hydrolysis temperatures may be the same or different from the hydrolytic durations and the hydrolysis temperatures of the other hydrolytic enzymes. Pectin lyase and polygalacturonase are enzymes that work synergistically to degrade pectin, a structural component of plant cell walls. Pectin lyase cleaves the glycosidic bonds within pectin molecules, while polygalacturonase breaks down the backbone of pectin into smaller oligosaccharides. Without the wish to be bound by any theory, it is believed that the enzymatic action of pectin lyase and polygalacturonase weakens the integrity of the plant cell wall, creating openings that allow access to intracellular components, especially nucleotides, e.g. RNA and DNA. As a result, these enzymes facilitate the extraction of nucleotides from plant tissues without compromising nucleotide integrity. Thereby, the nucleotides of the high protein plant seeds can contribute synergistically to the umami effect of the umami enhancing ingredient.

[0037] In an embodiment, the step of sequentially adding hydrolytic enzymes comprising at least an endopeptidase, an exopeptidase and a glutaminase to the heat processed aqueous suspension, further comprises adjusting the temperature of the heat processed aqueous solution to hydrolysis temperatures specific for the endopeptidase, the exopeptidase and the glutaminase. Thus, the method may further comprise the steps of:

[0038] - adjusting the temperature of the heat processed aqueous suspension to an endopeptidase hydrolysis temperature in the range of 25°C to 70°C;

[0039] - adding an endopeptidase;

[0040] - adjusting the temperature of the heat processed aqueous suspension to an exopeptidase hydrolysis temperature in the range of 25°C to 70°C;

[0041] - adding an exopeptidase;

[0042] - adjusting the temperature of the heat processed aqueous suspension to a glutaminase hydrolysis temperature in the range of 25°C to 70°C;

[0043] - adding a glutaminase.

[0044] The respective hydrolytic enzymes may also be added prior to adjusting the temperature to the respective hydrolysis temperatures.

[0045] In a certain embodiment the hydrolysis temperatures specific for the endopeptidase, the exopeptidase and the glutaminase are in the range of 25°C to 50°C.

[0046] In a certain embodiment the hydrolysis temperatures specific for the endopeptidase are preferably in the range of 40°C to 70°C, particularly in the range of 50°C to 65°C.

[0047] In a certain embodiment, the exopeptidase hydrolysis temperature is preferably in the range of 30°C to 50°C, particularly in the range of 35°C to 45°C.

[0048] In a certain embodiment, the glutaminase hydrolysis temperature is preferably in the range of 30°C to 50°C, particularly in the range of 35°C to 45°C.

[0049] In another embodiment, the endopeptidase, the exopeptidase and the glutaminase hydrolysis temperature are in the range of 20°C to 30°C, e.g. 23°C.

[0050] Enzymatic reactions may produce undesirable by-products or off- flavours at elevated temperatures. By maintaining hydrolysis temperatures in the range of 20°C to 30°C, e.g. at 23°C, the formation of such by-products is minimised, resulting in a cleaner and more desirable final product. Further, enzyme reactions conducted at lower temperatures can reduce the risk of microbial growth or contamination, improving the safety of the hydrolysed plant protein product.

[0051] The method includes the step of drying the hydrolysed plant protein product to provide the umami enhancing ingredient. Any drying procedure may be used in the present method. The drying procedure may provide the hydrolysed plant protein product as a powder, or the hydrolysed plant protein product may be in the form of a paste. For example, the hydrolysed plant protein product may be dried by freeze drying, vacuum drying, microwave drying, drum drying, infrared drying, or spray drying, or a combination thereof. It is preferred that the hydrolysed plant protein product is spray dried to provide the umami enhancing ingredient. The umami enhancing ingredient may be dried to any selected moisture content, but the moisture content is preferably up to 10%, e.g. in the range of 2-8%, so that the water activity is up to 0.3, e.g. in the range of 0.1 to 0.3, or 0.15 to 0.25. In the present context, percentages of components of the umami enhancing ingredient are expressed relative to the weight of the umami enhancing ingredient after drying. The water activity is generally considered to be the water pressure above the relevant component relative to liquid water at the same temperature.

[0052] It is especially preferred that the hydrolysed plant protein product is spray dried, e.g. to a moisture content of up to 10%, e.g. in the range of 2-8%, e.g. to a water activity in the range of 0.15 to 0.25.

[0053] In a specific embodiment, the hydrolysed plant protein product is spray dried such that the umami enhancing ingredient comprises a moisture content in the range of 3.5-7%, and a water activity in the range of 0.18 to 0.23.

[0054] In spray drying, a liquid containing the product to be dried is sprayed into air heated to a high temperature, e.g. in the range of 160°C to 400°C or more, to transform the liquid or semi-liquid formulations into a powdered form, thereby removing water from the hydrolysed plant protein product to increase the glutamate concentration by a factor in the range of 3-8, preferably 6, thereby providing an increased umami perception of the umami enhancing ingredient. Surprisingly, it has been found that spray drying the hydrolysed plant protein product provides an additional cheese-like taste to the umami enhancing ingredient, which is especially noticeable together with the increased umami perception provided by the concentrating effect of the spray drying process. The cheese taste is generally regarded as desirable as the umami enhancing ingredient obtains a flavour profile closer to food of animal origin. Without the wish to be bound by any theory, it is believed that the hydrolysed plant protein product provides both amino acids and sugars which, when treated at elevated temperatures, undergo Maillard reactions where the amino group of amino acids and the carbonyl group of reducing sugars form a diverse array of flavour and aroma compounds contributing to the cheesy taste of the spray dried umami enhancing ingredient. Thus, for example the method may cause a Maillard reaction between a peptide fragment and a sugar of the high protein plant seed. In the present context, the product of the reaction between a peptide fragment and a sugar of the high protein plant seed according to the Maillard reaction may be referred to as a Maillard reaction product. Thereby, the umami enhancing ingredient may further comprise a Maillard reaction product between the peptide fragments and a sugar of the high protein plant seed.

[0055] In an embodiment, the method for producing an umami enhancing ingredient further comprises the steps of: adjusting the temperature of the heat processed aqueous suspension to a fermentation temperature in the range of 15°C to 40°C; inoculating the heat processed aqueous suspension with a glutamate producing microorganism and maintaining the heat processed aqueous suspension at the fermentation temperature for a growth duration of at least 1 hour. For example, the growth duration may be in the range of 1 hour to 72 hours, e.g. 1 hour to 48 hours. For example, the method for producing an umami enhancing ingredient may further comprise the steps of: heating the temperature of the enzymatically pretreated aqueous suspension to the range of 80°C to the boiling point and a heating duration in the range of 5 minutes to 25 minutes and adjusting the temperature of the heat processed and partially enzymatically pretreated aqueous suspension to a fermentation temperature in the range of 15°C to 40°C; inoculating the heat processed enzymatically pretreated aqueous suspension with a glutamate producing microorganism seed culture and maintaining the heat processed aqueous suspension at the fermentation temperature under aerobic conditions for a growth duration of at least 1 hour. For example, the growth duration may be in the range of 1 hour to 72 hours, e.g. 1 hour to 48 hours.

[0056] The present method may thus employ a fermentation step. The heat processed aqueous suspension may be inoculated with any glutamate producing microorganism. The glutamate producing microorganism may generally be selected freely, but it is preferred that the glutamate producing microorganism is a high proliferating glutamate producing microorganism. Glutamate producing microorganisms encompass various bacteria, fungi, and yeast strains capable of fermenting substrates to yield glutamate as a metabolic by-product. These microorganisms often exhibit robust growth and glutamate production under controlled fermentation conditions, making them valuable for glutamate production. In addition, some glutamate microorganisms are also known to produce ribonucleotides, and the use of ribonucleotide producing microorganisms is preferred in the present method. It is preferred that glutamate producing microorganism is not a yeast. Correspondingly, it is preferred that umami enhancing ingredient does not comprise yeast and / or a yeast extract.

[0057] It is preferred that the glutamate producing microorganism is selected from the group consisting of Corynebacterium spp., Brevibacterium spp., Bacillus spp., Arthrobacter spp., Microbacterium spp., Lactobacillus spp, and Pseudomonas spp. It is especially preferred that the glutamate producing microorganism is Corynebacterium glutamicum. C. glutamicum is well-known to possess a high intrinsic capacity for glutamate production, a capacity for ribonucleotide production, and a rapid growth rate under optimal fermentation conditions, allowing for shorter fermentation cycles and higher throughput. As such, C. glutamicum is commonly grown on substrates based on molasses and starch hydrolysates and the like. However, the present inventors have now surprisingly found that C. glutamicum growth has been shown to be especially high on the heat processed aqueous suspension itself, especially an enzymatically pre-processed aqueous suspension, i.e. a substrate based on the high protein plant seeds, without the addition of a nitrogen or glucose source, e.g. molasses or starch. Thus, in an example, the glutamate producing microorganism, especially C. glutamicum, is grown on the heat processed aqueous suspension, e.g. the heat processed enzymatically pre-processed, or in part enzymatically pre-processed, aqueous suspension, without adding additional sources of nitrogen and / or carbon. In particular, the glutamate producing microorganism, especially C. glutamicum, may be grown on the heat processed aqueous suspension without adding molasses and / or starch. Without the wish to be bound by any theory, it is believed that the weakening of the cell walls of the cells in the high protein plant seeds obtained by cyclically switching the aqueous suspension between a heating step and a cooling step to provide a heat processed aqueous suspension further enhances the solubility and accessibility of nutrients within the high protein plant seed and likely also serves to denature or inactivate potentially inhibitory compounds for the organism, which may hinder microbial proliferation on the untreated seed material. As a result, the cyclical heat and cold treated heat processed aqueous suspension becomes a nutrient-rich and microbiologically permissive environment, primed for growth of microorganisms, especially C. glutamicum, capable of utilising the available nutrients for growth and metabolism. Additionally, combining the cyclic heat and cold treatment with pre-processing step using enzymes including pectin lyase, amylase, and peptidase subtilisin is believed to substantially freeing up native additional sources of nitrogen and / or carbon.

[0058] Relevant species of Brevibacterium are B. divaricatum now, B. flavum, B. lactofermentum, B. saccharolyticum, B. immariophilum, B. roseum, B. lactofermentumnov, and B. thiogenetalis. Relevant species of Bacillus are B. megaterium, B. circulans, B. cereus var. mycoides. Other relevant microorganisms are Arthrobacter globiformis, Microbacterium ammoniaphilum, Lactobacillus plantarum, and Pseudomonas reptilivora. The fermentation step may be included before or after the hydrolysis step. Likewise, a hydrolysis step may be included before the fermentation step, and another hydrolysis step may be included after the fermentation step. In particular, the fermentation step may be included during the enzymatic hydrolysis step. Regardless of the placement of the fermentation in the order of the steps in the process, the combination of the enzymatic hydrolysis step with the fermentation with the glutamate producing microorganism even further increases the amount of glutamate produced in the umami enhancing ingredient, and the fermentation may also provide additional nucleotides for an even improved synergistic effect on the umami taste.

[0059] In an embodiment the hydrolytic enzymes comprise a glycosidase. In general, a glycosidase is an enzyme that catalyses the hydrolysis of glycosidic bonds in polysaccharides and oligosaccharides. The glycosidase may for example break down glycosidic bonds in cellulose, hemicellulose, or starch. In a preferred embodiment, the glycosidase is an amylase.

[0060] In an example, the method further comprises adding a hydrolytic enzyme capable of degrading the cells, e.g. the cell walls, of the glutamate producing microorganism. The hydrolytic enzyme capable of degrading the cells of the glutamate producing microorganism can evidently be added after fermenting the heat processed aqueous suspension with a glutamate producing microorganism.

[0061] However, it is also possible to ferment the heat processed aqueous suspension after adding a hydrolytic enzyme capable of degrading the cells, e.g. the cell walls. By adding a glycosidase to the heat processed aqueous suspension, starch from the suspension is hydrolysed releasing fermentable sugars, along with the endo- and exopeptidases releasing free amino acids from the proteins. By hydrolysing both starch and proteins prior to fermentation, the release of fermentable sugars and nitrogen sources is increased which further improves the growth capabilities of the glutamate producing microorganism during fermentation. Subsequently providing an increased glutamate concentration of the hydrolysed plant protein product after fermentation due to improved growth conditions of the glutamate producing microorganism. When the hydrolytic enzymes include a glycosidase, it is preferred that the glycosidase, and optionally also the other hydrolytic enzymes, is added before the fermentation step.

[0062] The hydrolysis step generally ensures the release of components contained in the cells of the high protein plant seeds to the hydrolysed plant protein product, and the components may include nucleotides. The nucleotides may be in the form of nucleic acids like DNA, e.g. chromosomal DNA, and also RNA. The present method may employ a nuclease, e.g. an endonuclease and / or an exonuclease, to hydrolyse nucleic acids, e.g. polymers of RNA and / or DNA, from the high protein plant seed, e.g. in the hydrolysed plant protein product into single ribonucleotides. An endonuclease is an enzyme that cuts phosphodiester bonds within polymers of nucleic acids at specific internal sites, and an exonuclease is an enzyme that sequentially removes nucleotides from the ends of polymers of nucleic acids.

[0063] The treatment with a nuclease, e.g. an endonuclease and / or an exonuclease, is preferably combined with adding a deaminase specific for converting AMP to IMP to thereby further increase the umami synergistic effect. It is even more preferable to employ a nuclease and a deaminase specific for converting AMP to IMP when the heat processed aqueous suspension has been inoculated, and also fermented, with a glutamate producing microorganism.

[0064] The hydrolysis step can be considered to open up the high protein plant seed cells, and when the cell walls of the glutamate producing microorganism is also degraded enzymatically, the hydrolysis of the cell walls of the glutamate producing microorganism opens up the glutamate producing microorganism cell envelope making the contents of the cells available, especially RNA and DNA. Thereby, fermentation of the heat processed aqueous suspension with a glutamate producing microorganism and degradation of the cell walls with a glutamate producing microorganism provides a high content of glutamate and ribonucleotides via overproduction and together with the plant seed also acts as a source of RNA to be hydrolysed into single ribonucleotides, which together greatly enhances the umami taste of the umami enhancing ingredient due to the synergy between glutamate and nucleotides. Thus, the endonuclease and the exonuclease break down RNA molecules, and also DNA molecules, into shorter fragments, releasing nucleotides, e.g. ribonucleotides, which act synergistically with free glutamate and / or peptide fragments containing glutamate to provide an increased umami taste intensity. With the bacterial fermentation, the final concentration of the umami compounds may be greater than what is native to the plant seed alone.

[0065] In a preferred embodiment, the nuclease enzyme is an exonuclease, particularly a 5’-phosphodiesterase. 5’-phosphodiesterase is an enzyme involved in DNA and RNA degradation pathways. It catalyses the removal of terminal phosphates from DNA and RNA molecules, specifically targeting the 5’-phosphate releasing 5’-monophosphates. Especially preferred 5’- ribonucleotides for the increased umami taste intensity are inosine 5’- monophosphate, 5’-guanosine monophosphate, adenosine 5’- monophosphate, xanthosine 5’-monophosphate, even more preferred is guanosine 5’-monophosphate.

[0066] In another aspect, the invention relates to an umami enhancing ingredient. The umami enhancing ingredient is obtainable in the method of the invention. The umami enhancing ingredient comprises a protein hydrolysate, e.g. a fermented protein hydrolysate, of a high protein plant seed having a protein content of at least 15 g / 100 g of weight of the seeds, which protein hydrolysate comprises peptide fragments containing a terminal glutamic acid residue so as to provide the umami enhancing ingredient with at least 1000 mg of glutamate per 100 g of umami enhancing ingredient. The content of glutamate in the umami enhancing ingredient may be determined using any method, as desired. For example, the content of amino acids, including glutamate, in the umami enhancing ingredient or in the heat processed aqueous suspension, may be determined using a ninhydrin-based assay, where coloured complexes representative of amino acids are detected photometrically at 440 nm and / or 570 nm, e.g. according to the following procedure. Thus, a sample, e.g. the umami enhancing ingredient or the heat processed aqueous suspension, is extracted with sulphosalicylic acid to precipitate any protein from the sample. The sample is then brought to volume with loading buffer and transferred to a vial. Amino acids are separated in an amino acid analyser and the detection is carried out using post column derivatisation with ninhydrin reagent and detection at 440 and 570 nm. For quantification, a 1 -point calibration may be used. For quality assurance, a standard may be analysed together with the samples. In general, amino acids are detected according to the standard ISO 13903:2005.

[0067] The peptide fragments are derived from the protein of the high protein plant seed and as such generally contains amino acids linked to each other via a peptide bond between two adjacent amino acids. The same applies for the biomass from the bacteria, e.g. the glutamate producing high proliferating bacteria, used in the fermentation. The peptide fragments may for example have a length in the range of 1 amino acid to 20 amino acids including the terminal glutamate residue. For example, the peptide fragments may contain glutamate residues, dipeptides with a terminal glutamate residue, tripeptides with a terminal glutamate residue, tetrapeptides with a terminal glutamate residue, pentapeptides with a terminal glutamate residue, etc. In general, the content of glutamate is expressed as the weight of glutamate relative to the umami enhancing ingredient, regardless of the number of other amino acids contained in the peptide fragments having the terminal glutamate residues. The content of glutamate is preferably expressed relative to the dry weight of the umami enhancing ingredient. The content of glutamate relative to the weight, e.g. the dry weight, of the umami enhancing ingredient may be at least 1500 mg per 100 g, at least 2000 mg per 100 g, at least 2500 mg per 100 g, at least 3000 mg per 100 g, at least 3500 mg per 100 g, at least 4000 mg per 100 g, at least 5000 mg per 100 g, at least 6000 mg per 100 g, at least 7000 mg per 100 g, or at least 10000 mg per 100 g.

[0068] The umami enhancing ingredient preferably also contains nucleotides. The nucleotides may be ribonucleotides, deoxyribonucleotides, or a combination of ribonucleotides, and deoxyribonucleotides. The nucleotides may be expressed relative to the weight, e.g. the dry weight, of the umami enhancing ingredient, and the content of nucleotides in the umami enhancing ingredient may be determined using any method, as desired. In the present context, the amount of nucleotides in the product can consist of any mixture of ribonucleotides and at any different ratios, and the amount of nucleotides is generally expressed with as IMP equivalents, where GMP has an IMP equivalent of 2.3, IMP has an IMP equivalent of 1 , XMP has an IMP equivalent of 0.61 , and AMP has an IMP equivalent of 0.18.

[0069] For example, the content of nucleotides, including ribonucleotides, in the umami enhancing ingredient may be determined using any appropriate procedure. In general, nucleotides may be detected according to the standard ISO 20638:2015. A nucleotide assay typically detects inosine-5'- monophosphate, cytidine-5'-monophosphate, uridine-5'-monophosphate, and guanosine-5'-monophosphate, adenosine-5'-monophosphate, and may also detect xanthosine 5’-monophosphate. For example, nucleotides may be extracted from a sample, e.g. the umami enhancing ingredient or the heat processed aqueous suspension, in a sodium chloride / EDTA solution and purified using solid-phase extraction on a strong anion exchange column. Chromatographic separation can then be performed using a C18 column. Quantification may be performed using internal standard and detection on UV / DAD. The umami enhancing ingredient may contain at least 100 mg IMP equivalents per 100 g of umami enhancing ingredient, e.g. per 100 g of dry weight of the umami enhancing ingredient. In other examples, the umami enhancing ingredient comprises at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 400 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 1500 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 2000 mg IMP equivalents per 100 g of umami enhancing ingredient at least 2500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 3000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0070] In a specific example, the umami enhancing ingredient comprises at least 1500 mg of glutamate per 100 g umami enhancing ingredient and at least 100 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 400 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 1500 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 2000 mg IMP equivalents per 100 g of umami enhancing ingredient at least 2500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 3000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0071] In another example, the umami enhancing ingredient comprises at least 2500 mg of glutamate per 100 g umami enhancing ingredient and at least 100 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 400 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 1500 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 2000 mg IMP equivalents per 100 g of umami enhancing ingredient at least 2500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 3000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0072] In another example, the umami enhancing ingredient comprises at least 3000 mg of glutamate per 100 g umami enhancing ingredient and at least 100 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 400 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 1500 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 2000 mg IMP equivalents per 100 g of umami enhancing ingredient at least 2500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 3000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0073] In another example, the umami enhancing ingredient comprises at least 5000 mg of glutamate per 100 g umami enhancing ingredient and at least 100 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient, at least 400 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 1500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 2500 mg IMP equivalents per 100 g of umami enhancing ingredient, or at least 3000 mg IMP equivalents per 100 g of umami enhancing ingredient.

[0074] The umami enhancing ingredient may also comprise at least 6000 mg, e.g. up to 7000 mg, of glutamate per 100 g umami enhancing ingredient and at least 2000 mg, e.g. up to 4000 mg, IMP equivalents per 100 g of umami enhancing ingredient. For example, the umami enhancing ingredient may comprise glutamate in the range of 4000 mg to 7000 mg and IMP equivalents in the range of 2000 mg to 4000 mg per 100 g of umami enhancing ingredient.

[0075] The umami enhancing ingredient generally has a low moisture content, e.g. up to 10%, such as in the range of 1 % to 10%. The moisture content of up to 10% generally corresponds to a water activity of up to 0.3. Thereby, the umami enhancing ingredient is inherently stable and does not require cooling for storing. The umami enhancing ingredient generally has a salt content of up to 15%, e.g. in the range of 1 % to 15%. In the present context, the salt content is expressed by weight of the umami enhancing ingredient, and the salt content may be expressed as a dry weight content of the umami enhancing ingredient, and the salt content may be up to 15%, e.g. in the range of 1 % to 15%. By providing a significant umami enhancing effect, the umami enhancing ingredient can be used in such small amounts that the salt contained in the umami enhancing ingredient does not cause concern with respect to the amount of salts in the food to which the umami enhancing ingredient is added.

[0076] The umami enhancing ingredient of the present disclosure enhances flavour when added to a dish not inherently having a high umami taste, e.g. a vegetarian or vegan dish, and the product preferably does not comprise any components of animal origin, so that the umami enhancing ingredient can enhance the umami flavour of a purely plant-based dish. Furthermore, the umami enhancing ingredient improves the mouthfeel, and by generally enhancing the flavour, i.e. via providing a strong umami effect can also provide a reduction of the need for salt and sugar in enhancing salt and sweet tastes, respectively. Correspondingly, the umami enhancing ingredient can reduce bitter taste in food.

[0077] The umami enhancing ingredient may for example be used in plantbased foods (e.g. vegan mayonnaise and cheese, spreads, burger patties, cold cut slices, extruded meat analogue products), hybrid food products (cheesereduction, meat-reduction), conventional foods (climate- and expense heavy ingredient cost reduction), etc.

[0078] Any embodiment of the invention may be used in any aspect of the invention, and any advantage for a specific embodiment applies equally when an embodiment is used in a specific aspect.

[0079] Detailed Description

[0080] The present invention relates to a method for producing an umami enhancing ingredient and to an umami enhancing ingredient. The umami enhancing ingredient is obtainable in the method.

[0081] The present method involves adding hydrolytic enzymes to the heat processed aqueous suspension. Relevant hydrolytic enzymes include (with the supplier included for the listed enzymes):

[0082] Proteases peptidase subtilisin, e.g. TailorFood Endocut-07L (Tailorzymes), Alcalase (Novozymes), aminopeptidase, e.g. TailorFood Exocut-TR L (Tailorzymes), Protana Prime (Novozymes), Protamex (Novozymes), Protease AP-30L (Enzyme supplies), Neutral Protease (EnzymesBio), Formea® TL (Novonesis), Neutrase® 0.8 L (Novonesis), Flavourzyme®CONC BG (Novonesis), Promatex® (Novonesis).

[0083] Glutaminases

[0084] Tail254 or Tail297 (Tailorzymes), Protana UBoost (Novozymes) Ribonuclease

[0085] Flavorpro® 848MDP (Biocatalysts).

[0086] Deaminase

[0087] Flavorpro® 954MDP (Biocatalysts) Pectin lyases

[0088] Pectinex® Ultra Passover (Novozymes), Pectinex® Ultra Color (Novozymes)

[0089] Hemi cellulase

[0090] Vertera Release (Novonesis)

[0091] Asparaginases

[0092] Summase ASP L (QES), Summase ASP M (QES), AcrylAway L (Novozymes), Acrylaway BG (Novozymes), PreventAse M (DSM), PreventAse L (DSM)

[0093] Amylases

[0094] TailorFood Liquefact-01 L. Liquozyme® Pro HP, Vertera Liquify, Vertera Sweet, Vertera Sweet H (Novonesis), Deltazym BAA Classic (Weiss Biotech)

[0095] Pectinases

[0096] Natuzym Biomax Q (WeissBioTech GmbH), Natuzym BE+ (WeissBioTech GmbH), Rapidase® (DSM).

[0097] Example

[0098] The present method is illustrated in the following example in which an umami enhancing ingredient is produced.

[0099] Preparing a heat processed aqueous suspension

[0100] Yellow split peas were selected as a high protein plant seed for preparing a heat processed aqueous suspension. The peas were washed in cold water, the water was discarded. 143 g of yellow split peas (dry weight) were suspended in 1 L of tap water and heated up to boiling or 100°C. The boiling temperature was kept for 15 minutes. Subsequently, a temperature cycle of cyclically switching between a heating step, heating the aqueous suspension to boiling, and a cooling step, cooling the aqueous suspension to 30°C was implemented. The boiling temperature was held for 15 minutes each for two heating steps, and the cooling temperature was held for 15 minutes each for two cooling steps. After the second cooling period the temperature was increased to boiling for the last time and the mixture was kept at boiling for 30 minutes.

[0101] Subsequently the mixture was blended using an immersion stick blender with a rotary knife blade for at least 5 minutes or until a homogenous paste was formed.

[0102] In an alternative version, yellow split peas were selected as a high protein plant seed for preparing a heat processed aqueous suspension. The peas were washed in cold water, the water was discarded. 250 g of yellow split peas (dry weight) were suspended in 1 L of tap water and heated up to boiling or 100°C. The boiling temperature was kept for 15 minutes. Subsequently, a temperature cycle of cyclically switching between a heating step, cooling the solution to 30°C for 45 minutes, then heating the aqueous suspension to boiling for 1 hr, and a cooling step, cooling the aqueous suspension to 45°C was implemented.

[0103] Subsequently the mixture was blended using an immersion stick blender with a rotary knife blade for at least 5 minutes or until a homogenous paste was formed.

[0104] Preparing a hydrolysed plant protein product

[0105] Enzymatic treatment: Salt was added to the heat processed aqueous suspension at an amount of 4 wt% of the heat processed aqueous suspension. The heat processed aqueous suspension was heated to boiling for 15 minutes and then cooled down to 23°C. The heat processed aqueous suspension was continuously stirred while 0.6 wt% of peptidase subtilisin (Talorzymes; TailorFood Endocut-07L) was added to the heat processed aqueous suspension. After 30 minutes 0.15 wt% of aminopeptidase (Talorzymes; TailorFood Exocut-TR L) and 0.078 wt% of glutaminase (Tailorzymes; Tail254) were added to the heat processed aqueous suspension and the temperature increased to 55°C and maintained for 23.5 hours.

[0106] In an alternative step, salt was added to the heat processed aqueous suspension at an amount 1.6% wt% of the heat processed aqueous suspension.

[0107] Then 1.02 wt% of aminopeptidase (Talorzymes; TailorFood Exocut- AOL) and 0.05 wt% of glutaminase (Tailorzymes; Tail254) were added to the heat processed aqueous suspension and the temperature increased to 55°C and maintained for 23.5 hours.

[0108] In an alternative, additional enzymes were added to the heat processed aqueous suspension after addition of salt and before addition of aminopeptidase and glutaminase. Cooling down to 45°C, the heat processed aqueous suspension was continuously stirred and added 0.7 wt% of Alcalase subtilisin (Novonesis) and 0.08% of pectin lyase (Pectinex Ultra Colour, Novonesis) and maintained for 1 hr.

[0109] In another alternative additional enzymes were added to the heat processed aqueous suspension after addition of salt and before addition of aminopeptidase and glutaminase. The aqueous suspension was cooled to 60 C adding 0.06 % amylases Liquefact 01 L (Tailorzymes) and 0.1 % Vertera Sweet (Novonesis) maintaining the temperature for 4 hr.

[0110] Then cooling down to 30C a subsequent step adding 0.08% pectin lyase Pectinex Ultra Colour (Novonesis) and 0.7% peptidase subtilisin Alcalase 24L FG (Novonesis). The aquous suspension was left agitating for 12-16 hrs.

[0111] Then the aquous suspension was heated to the boiling point for 15 min.

[0112] In alternative scenarios, the suspension was sonicated for 15 min.

[0113] In an alternative scenario, the suspension was homogenised.

[0114] Then the aqueous suspension was cooled down to 55C and 0.7% peptidase subtilisin Alcalase 24L FG (Novonesis), 1 .02 wt% of aminopeptidase (Talorzymes; TailorFood Exocut-AOL) and 0.05 wt% of glutaminase (Tailorzymes; Tail254) were added and the temperature of 55°C was maintained for 23.5 hour with the aqueous suspension in agitated conditions.

[0115] In an alternative, additional enzymes were then added to the heat processed aqueous suspension after 30 minutes: 0.5 wt% to 1 wt% of ribonuclease 5’-Phosphodiesterase (Biocatalysts; Flavorpro® 848MDP) and 0.25 wt% to 1 wt% of deaminase (Biocatalysts; Flavorpro® 954MDP) were added and the temperature was increased to 55°C and maintained for 23.5 hours.

[0116] Subsequently the enzymes were inactivated by heating the heat processed aqueous suspension to 95°C for 15 minutes. In yet a further alternative, the following additional step was performed: After inactivation of the enzymes, the heat processed aqueous suspension was cooled to 65°C, and 0.5 wt% to 1 wt% of 5’-Phosphodiesterase (Flavorpro® 848MDP) was added while the heat processed aqueous suspension was stirred continuously for 0.5 to 6 hours. After the stirring duration of 0.5 to 6 hours the temperature of the heat processed aqueous suspension was reduced to 50°C, and 0.25 wt% of deaminase (Flavorpro® 954MDP) was added. Subsequently the enzymes were inactivated by heating the medium to 95°C for 30 minutes.

[0117] In yet a further alternative, the following additional step was performed: during the enzymatic hydrolysis: 0.07 %wt ribonuclease (Biocatalysts; Flavorpro® 848MDP) and 0.07 %wt deaminase (Biocatalysts; Flavorpro® 954MDP) were added after 19.5 hrs of enzymatic hydrolysis.

[0118] Subsequently the enzymes were inactivated by heating the medium to 95°C for 30 minutes.

[0119] In yet a further alternative, the following additional step was performed during the enzymatic hydrolysis: 0.07 %wt ribonuclease (Biocatalysts; Flavorpro® 848MDP) was added after 19.5 hrs and 0.07 %wt deaminase (Biocatalysts; Flavorpro® 954MDP) were added after 21.5 hrs of enzymatic hydrolysis.

[0120] Subsequently the enzymes were inactivated by heating the medium to 95°C for 30 minutes.

[0121] Preparing a fermentation of a heat processed aqueous suspension

[0122] C. glutamicum was selected as a glutamate producing microorganism for fermentation of the heat processed aqueous suspension. 25 mL of heat processed aqueous suspension was distributed to 100 mL Erlenmeyer flasks. Glucose was added as a substrate by adding 0.5 mL of a 50% autoclaved glucose solution (Sigma-Aldrich; D-(+)-Glucose; G7021 ). The heat processed aqueous suspension was then inoculated with 0.5 mL of overnight culture of C. glutamicum. The fermentation was provided in a shaking incubator at 32°C and 200 rpm. The inoculated fermentation medium was kept at 32°C and 200 rpm for 24 hours and then heated up to 100°C to inactivate the C. glutamicum.

[0123] In an alternative, a bacterial fermentation step with C. glutamicum selected for fermentation of the heat processed enzymatically treated aqueous suspension. 50 mL of the enzymatically treated aqueous suspension was dispersed into 200 mL baffled shake flasks (filter lid, Duran) and then inoculated with 0.2 mL of overnight culture of C. glutamicum. The overnight culture was prepared grown in LB media supplemented with 2% glucose (Sigma-Aldrich; D-(+)-Glucose; G7021 ) at 30°C and 220 rpm. The fermentation was provided in a shaking incubator at 30°C and 220 rpm. The inoculated fermentation medium was kept at 30°C and 220 rpm for 12-16 hours and then heated up to 100°C to inactivate the C. glutamicum.

[0124] In alternative version, the suspension was sonicated instead of boiling.

[0125] In a further alternative version, the suspension was sonicated and boiled.

[0126] In yet another version the suspension was homogenised instead of boiling.

[0127] In yet a further, version the suspension was homogenised and boiled.

[0128] Preparing an umami enhancing ingredient

[0129] In the final step of the method, the fermentation broth was dried in a spray dryer at a temperature in the range of 150°C to 300°C to provide the umami enhancing ingredient. The umami enhancing ingredient was a dry powder with moisture content of about 6% and a water activity of about 0.21 with a neutral to mild aroma (of peas and corn) and colour (light yellow / golden). The dry powder contained particles having a size of about 10 pm to 200 pm. The appropriate dosage of the thus prepared umami enhancing ingredient to food recipes is 0.1 -5% (w / w). The umami enhancing ingredient had a glutamic acid content of 1453 mg / 100 g of the umami enhancing ingredient, and the content of AMP was about 14 mg / 100 g of the umami enhancing ingredient.

[0130] An alternative umami ingredient had a glutamic acid content of 5000 to 7000 mg / 100 g dry weight and IMP equivalent 1000 to 3000 mg / 100 g.

Claims

P A T E N T C L A I M S1. A method for producing an umami enhancing ingredient, the method comprising the steps of: providing a high protein plant seed comprising having a protein content of at least 15 g / 100 g of weight of the seeds; suspending the high protein plant seed in water at an amount of high protein plant seeds to water in the range of 50 g / L to 350 g / L to provide an aqueous suspension; cyclically switching the aqueous suspension between a heating step and a cooling step to provide a heat processed aqueous suspension, where the heating step is at a heating temperature in the range of 80°C to the boiling point of the aqueous suspension and a heating duration in the range of 5 minutes to 1 hour, and the cooling step is at a cooling temperature in the range of 15°C to 35°C and a cooling duration in the range of 5 minutes to 25 minutes, where the heating step is repeated at least twice; disrupting the high protein plant seed; adjusting the temperature of the heat processed aqueous suspension to a hydrolysis temperature in the range of 25°C to 70°C; adding hydrolytic enzymes comprising at least an exopeptidase, an endopeptidase and a glutaminase to the heat processed aqueous suspension and maintaining the heat processed aqueous suspension at the hydrolysis temperature for a hydrolytic duration of at least 1 hour to provide a hydrolysed plant protein product; and drying the hydrolysed plant protein product to provide the umami enhancing ingredient.

2. The method for producing an umami enhancing ingredient according to claim 1 , wherein the method further comprises the steps of: adjusting the temperature of the heat processed aqueous suspension to a fermentation temperature in the range of 15°C to 40°C; and inoculating the heat processed aqueous suspension with a glutamate producing microorganism and maintaining the heat processed aqueoussuspension at the fermentation temperature for a growth duration of at least 1 hour.

3. The method for producing an umami enhancing ingredient according to claim 2, wherein the glutamate producing microorganism is selected from Cory nebacteri urn spp., Brevibacterium spp., Bacillus spp., Arthrobacter spp., Microbacterium spp., Lactobacillus spp, and Pseudomonas spp.,4. The method for producing an umami enhancing ingredient according to any one of claims 1 to 3, wherein the hydrolytic enzymes further comprise at least one of an asparaginase, a glycosidase, a pectin lyase, a polygalacturonase, deaminase, and a nuclease.

5. The method for producing an umami enhancing ingredient according to any one of claims 1 to 4, wherein the high protein plant seed comprises at least one of legume seeds, sunflower seeds, pumpkin seeds, seeds from flax, e.g. linseeds, hemp seeds, or chia seeds.

6. The method for producing an umami enhancing ingredient according to any one of claims 1 to 5, wherein the high protein plant seed does not comprise soybeans.

7. The method for producing an umami enhancing ingredient according to any one of claims 1 to 6, wherein the hydrolysed plant protein product is spray dried.

8. An umami enhancing ingredient comprising a protein hydrolysate of a high protein plant seed having a protein content of at least 15 g / 100 g of weight of the seeds, which protein hydrolysate comprises peptide fragments containing a terminal glutamic acid residue so as to provide the umami enhancing ingredient with at least 1000 mg of glutamate per 100 g of umami enhancing ingredient.

9. The umami enhancing ingredient according to claim 8, wherein the umami enhancing ingredient contains at least 100 mg inosine 5’- monophosphate equivalents per 100 g of umami enhancing ingredient.

10. The umami enhancing ingredient according to claim 8 or 9, wherein the umami enhancing ingredient further comprises a Maillard reaction product between the peptide fragments and a sugar of the high protein plant seed.11 . The umami enhancing ingredient according to any one of claims 8 to 10, wherein the umami enhancing ingredient does not comprise at least one of yeast and a yeast extract.

12. The umami enhancing ingredient according to any one of claims 8 to 11 , wherein the umami enhancing ingredient comprises at least 1500 mg of glutamate per 100 g umami enhancing ingredient and at least 200 mg IMP equivalents per 100 g of umami enhancing ingredient.

13. The umami enhancing ingredient according to any one of claims 8 to 11 , wherein the umami enhancing ingredient comprises at least 2500 mg of glutamate per 100 g umami enhancing ingredient and at least 300 mg IMP equivalents per 100 g of umami enhancing ingredient.

14. The umami enhancing ingredient according to any one of claims 8 to 11 , wherein the umami enhancing ingredient comprises at least 3000 mg of glutamate per 100 g umami enhancing ingredient and at least 500 mg IMP equivalents per 100 g of umami enhancing ingredient.

15. The umami enhancing ingredient according to any one of claims 8 to 11 , wherein the umami enhancing ingredient comprises at least 5000 mg of glutamate per 100 g umami enhancing ingredient and at least 1000 mg IMP equivalents per 100 g of umami enhancing ingredient.

16. The umami enhancing ingredient according to any one of claims 8 to 11 , wherein the umami enhancing ingredient comprises at least 6000 mg of glutamate per 100 g umami enhancing ingredient and at least 2000 mg IMP equivalents per 100 g of umami enhancing ingredient.