Process for producing a savoury taste product

Abstract

The present invention relates to a method for producing a savoury taste product from an organic starting material, the method comprising a step of providing an organic starting material, preferably mushroom, chicken, lentil and / or beans, a step of fermenting the organic starting material in presence of a fungi composition, preferably Aspergillus sojae and Aspergillus oryzae and a step of enzymatic hydrolysis.

Description

[0001] Process for producing a savoury taste product

[0002] Technical field

[0003] The present invention relates to a process for producing a savoury taste product.

[0004] Background

[0005] The food industry remains a leading contributor to greenhouse gas emissions, with food waste alone generating emissions equivalent to four times those produced by global air traffic. A shift toward resource optimised practices requires upcycling of industry side streams, circular food manufacturing approaches that minimize waste and provide value added products. Today, looking for solutions that address environmental impact at multiple levels while delivering on price, taste and consumer demands is of fundamental importance to move beyond extractivist food production paradigms.

[0006] Flavouring substances and savoury taste products are compounds or products that may be added to a food product in order to supplement or enhance its intrinsic flavour. The concept of flavour enhancement originated in Asia, where chefs added seaweed to soup stocks in order to provide a richer flavour to certain foods.

[0007] Flavour and flavouring substances affect the five basic tastes associated with human physiology thereby showcasing great potential to affect consumption patterns of a food or beverage product. For example, foods containing high umami have proved useful to decrease consumers’ salt and sugar intake. Additionally, umami has been shown to mask the perception of bitter compounds in commercial food products thereby affecting the consumption of these.

[0008] Humans sense tastes through sensory organs called taste buds or gustatory calyculi, concentrated on the upper surface of the tongue. Five basic tastes are described, these five tastes are bitter, salty, sour, sweet, and umami.

[0009] Umami represents the fifth basic taste and can be perceived through the binding of free amino acids, (e.g. glutamate) and free nucleotides (e.g inosinate, adenylate) to umami taste receptors. As our Umami receptors present double binding sites, both for glutamate and nucleotides, the combined binding of these compounds determines the so called “umami synergy”. Synergy happens when glutamate or aspartate act in combination with inosinate, guanylate, 5’-xanthosine monophosphate (XMP), or adenylate simultaneously bind to umami receptors on the tongue. Being highly nonlinear, umami synergy implies that small amounts of one component, e.g., inosinate, can enhance the sensory perception of glutamate manifold (e.g. up to 8 times enhancement).

[0010] People taste umami when compounds such as glutamates and nucleotides bind to taste receptors on the tongue. These compounds are shown to be widely present in meat broths and fermented products.

[0011] In addition to the 5 tastes mentioned above, sensations like kokumi and Maillard reaction products may be providing improved perception of a food product to be consumed.

[0012] While umami may impart savoury flavour or meatiness to a food product, kokumi may be a sense of richness, body and complexity. Thus, kokumi may be evaluated scientifically in sensory tests, the attributes of kokumi may be thickness (which may relate to the concentration, the amplitude, and / or the strength, however, preferable it does not relate to viscosity), mouthfulness (which may relate to the spread of sensation throughout the whole mouth), continuity (which may relate to the long-lasting sensory effects including an increase in duration of aftertaste), roundedness (which may relate to the smoothness, balance, and / or harmony), depth (which may relate to the richness, and / or complexity), and punch (which may relate to the impact, and / or the quick increase).

[0013] Kokumi may include active peptides, which may be distributed in many kinds of food products, and may induce a rich and long-lasting mouthfeel of the food product. The discovered kokumi peptides mainly comprise glutamyl peptides, leucyl peptides and other peptides without specific features. Derivatives of amino acids and peptides including sulphur-containing amino acids, A / -acyl-Tyr derivatives, A / -acetylated amino acids and Maillard reaction products (MRPs) may also work as kokumi compounds, enhancers. Kokumi may be quantified by High Resolution Mass Spectrometry (HRMS) performances of Amadori products and / or amino acids.

[0014] Generally, these kokumi compounds (or kokumi flavour) may be obtained after a starting material has been subjected to maturation, fermentation, aging, curing, drying, or cooking, like slow cooking. The Maillard reaction products may be obtained from a chemical reaction that occurs between amino acids and reducing sugars in the presence of heat. This reaction may be responsible for the browning of foods during cooking and is crucial in the development of flavour and aroma in a variety of cooked foods.

[0015] The Maillard reaction products (MRPs) may contribute to characteristic flavours, aromas, and brown colour of cooked foods. These products include a wide range of compounds such as melanoidins, which are large, brown, high-molecular-weight polymers, as well as smaller molecules like aldehydes, ketones, and heterocyclic compounds. The specific MRPs formed may depend on the types of amino acids and sugars involved in the reaction, as well as the cooking conditions.

[0016] There is a need in the industry for an improved process for producing savoury taste products according to national environmental responsibility standards (e.g. the United Nations Sustainable Development Goals. In particular there is a need for a more efficient, simple, reliable, and controllable process, which is more environmentally friendly, more productive, which has lower operation costs, lower water consumption, short process time and less energy consumption, resulting in products with great taste, without synthetic additives (clean label) and / or yeast based additives, such as yeast extracts, and wherein valuable nutritional components are preserved.

[0017] Summary

[0018] The present invention relates to a process for producing a savoury taste product based on an organic starting material, a fungi fermentation, and an enzymatic hydrolysis, a fungi composition and a flavouring substance as such.

[0019] In particular the present invention moreover relates to a process for producing a savoury taste product based on an organic starting material, koji fermentation, and an enzymatic hydrolysis.

[0020] One aspect of the present invention relates to a method for producing a savoury taste product from an organic starting material, wherein the process comprises the steps of: a) providing the organic starting material; b) optionally pre-treatment of the organic starting material, thereby providing a pretreated organic starting material; c) fermentation of the organic starting material in the presence of a fungi composition, preferably wherein the fungi composition comprises Aspergillus Sojae and Aspergillus Oryzae, thereby providing a fermented material; d) optionally contacting the fermented material with one or more carbohydrase e) hydrolysis of the fermented material in the presence of an enzyme composition comprising at least one protease, and at least one glutaminase, thereby providing a hydrolysed, fermented material; and f) optionally a step of recovering, concentrating, dehydrating and / or purifying the hydrolysed, fermented material thereby providing the savoury taste product.

[0021] Thus, an objective of the present invention is to provide a method for producing a savoury taste product, a process for producing a flavouring substance, an enzyme composition, and a flavouring substance as such, that solve the above-mentioned problems of the prior art with efficiency, reliability, control of the process, productivity, costs, avoidance of synthetic additives, water consumption, process time, energy consumption and / or loss of valuable nutritional components.

[0022] Another aspect of the invention relates to a process for producing a flavouring substance, such as a natural flavouring substance, from an organic starting material, the process comprises the steps of:

[0023] (i) Providing the organic starting material;

[0024] (ii) Adding a fungi composition to the organic starting material;

[0025] (iii) Allowing the fungi composition to ferment the starting material providing a fermented material; (iv) Adding an enzyme composition to the fermented material;

[0026] (v) Allowing the enzyme composition to hydrolyse the fermented material providing the flavouring substance.

[0027] Another aspect of the present invention relates to a fungi composition comprising the fungi mould, and / or the fungi spore in combination with a fungi substrate.

[0028] Yet another aspect of the present invention relates to an enzyme composition comprising a carbohydrase in combination with a protease (in particular an endoprotease, an exo-protease and / or a peptidase), a glutaminase, and / or a nuclease.

[0029] Still another aspect of the present invention relates to an enzyme composition comprising a protease (in particular an endo-protease, an exo-protease and / or a peptidase), a glutaminase, and a nuclease.

[0030] Yet an aspect of the present invention relates to a flavouring substance, such as a natural flavouring substance, comprising the combination of one or more reaction product obtained from fermenting an organic starter material with a fungi composition according to the present invention, and one or more reaction product obtained from the hydrolysis of the fermented product obtained from fermenting an organic starter material with a fungi composition (the fermented material) using an enzyme composition according to the present invention.

[0031] A further aspect of the present invention relates to a process for producing a flavouring preparation comprising a step of fermenting an organic starting material using a fungi composition resulting in a fermented material and hydrolysing the fermented material using with an enzyme composition, providing the flavouring substance.

[0032] Yet an aspect of the present invention relates to a food product comprising the flavouring substance obtained by the process according to the present invention, or the flavouring substance according to the present invention.

[0033] The present invention will now be described in more detail in the following. Description of Figures

[0034] Figure 1. Experimental design and workflow of producing lentil, bean and mushroom products with and without Koji.

[0035] Figure 2. Levels of selected aroma compounds belonging to (a) fatty acid and lipid oxidation products, (b) Maillard reaction products, (c) phenylpropanoids and (d) sesquiterpenes in bean products made with live or inactivated Kojis, as well as those without Koji. Presented are means of three replicates with error bars representing standard errors. Significance of differences between the ‘with Koji’ and ‘without Koji’ was calculated using Student's t-test: *P < 0.05; differences between all three treatments was assessed using Tukey’s all pairwise comparison test, different letters represent significantly different values.

[0036] Figure 3. Levels of aroma compounds in lentil products across four categories: (a) fatty acid and lipid oxidation products, (b) Maillard reaction products, (c) phenylpropanoids, and (d) sesquiterpenes. Comparisons are shown for products made with live Koji, inactivated Koji, and without Koji. Data represent the means of three replicates, with error bars indicating standard errors. Significant differences between the "with Koji" and "without Koji" groups were determined using Student's t-test (*P < 0.05). Differences among all three treatments were assessed using Tukey’s all-pairwise comparison test; distinct letters indicate significantly different values.

[0037] Figure 4. Koji addition increases free amino acid content in savoury taste products according to the present disclosure. Presented are levels of selected free amino acids in (a) lentil and (b) bean products made with live or inactivated Koji, compared to ‘no Koji’ treatments (means of three biological replicated ± SE). Significance of differences between all three treatments was assessed using Tukey’s all pairwise comparison test, different letters represent significantly different values.

[0038] Figure 5. Koji addition modifies 5’-ribonucleotide content in savoury taste products according to the present disclosure. Presented are levels of UMP, GMP and IMP in (a) lentil and (b) bean products made with live or inactivated Koji, compared to ‘no Koji’ treatments (means of three biological replicated ± SE). Significance of differences between all three treatments was assessed using Tukey’s all pairwise comparison test, different letters represent significantly different values.

[0039] Figure 6. Koji addition increases sugar content in savoury taste products according to the present disclosure. Presented are levels of glucose, fructose and sucrose in (a) lentil and (b) bean products made with live or inactivated Koji, compared to ‘no Koji’ treatments (means of three biological replicated ± SE). Significance of differences between all three treatments was assessed using Tukey’s all pairwise comparison test, different letters represent significantly different values.

[0040] Figure 7. Koji addition increases the perceived aroma intensity of savoury taste products according to the present disclosure. Presented are scores of aroma descriptors in savoury taste products according to the present disclosure made with or without Koji (means of three biological replicated ± SE). Each descriptor is accompanied by associated aroma compounds. Significance of differences between treatments was calculated using Student's t-test: *P < 0.05.

[0041] Figure 8. Koji addition increases the perceived flavor intensity of savoury taste products according to the present disclosure. Presented are scores of flavor descriptors in lentil-based savoury taste products according to the present disclosure made with or without Koji (means of three biological replicated ± SE). Each descriptor is accompanied by associated volatile compounds. Significance of differences between treatments was calculated using Student's t-test: *P < 0.05.

[0042] Figure 9. Koji addition increases the perceived basic taste and aftertaste intensity of savoury taste products according to the present disclosure. Presented are scores of basic taste and aftertaste descriptors in lentil-based savoury taste products according to the present disclosure made with or without Koji (means of three biological replicated ± SE). Significance of differences between treatments was calculated using Student's t- test: *P < 0.05.

[0043] Figure 10. Simplified overview of the platform of the present disclosure. Detailed description

[0044] Over the past two decades consumer expectations for food products have evolved. People are increasingly drawn to products that are natural / healthier, organic, and transparent with regards to both ingredients labels and environmental impact. These demands speak to an overall increased consumer health awareness, coupled with a desire to make food choices that contribute to a cleaner planet. As consumers prompt food industry stakeholders to innovate, businesses struggle to adapt their modus operandi to manufacturing practices with positive ecological footprint. In this context, savoury taste products such as the solutions of the present disclosure play a central offering upcycled, clean label food ingredients made with 100% real food materials.

[0045] The present savoury taste products stand out due to their upcycled origins, environmentally conscious production process, and 100% real foods-based flavours. Utilizing raw materials that would otherwise be lost (e.g industry side streams, and manufacturing byproducts), customers and consumers alike can claim more responsible resource use since CO2 emissions / kg of products are disclosed. In contrast, mainstream ingredient production such as yeast extracts, hydrolysed vegetable proteins and / or other natural flavourings, do not disclose CO2e associated with their ingredients.

[0046] The present invention combines a solid state fermentation process with enzymatic hydrolysis. The aim of this process is to upcycle a variety of industry side streams and byproducts into new food ingredients (also known as value added), in particular savoury taste products and flavouring substances. These ingredients present flavour profiles (both taste and aroma) that are similar to those observed in traditional fermented products like miso and soy sauce but, contrary to traditional ferments, are produced within a shorter time frame. The process allows for preparation of up cycled, clean label products. Moreover, the process allows for preparation of gluten free products.

[0047] The method provides for savoury flavouring solutions with increased savory notes, increasing consumer satiation and reducing the need for meat addition, food additives and the need to add salt / sugar when using the solutions to prepare food or a food product. Definitions

[0048] As used herein, the term “flavour” refers to respectively aroma, which is the sensation when sniffing, and taste which is the sensation when eating.

[0049] As used herein, the term ‘flavouring’ refers to products: (i) not intended to be consumed as such, which are added to food and food products in order to impart or modify odour and / or taste. Different categories of products include: flavouring substances, flavouring preparations, thermal process flavourings, smoke flavourings, flavour precursors or other flavourings or mixtures thereof.

[0050] As used herein, the term ‘flavouring substance’ refers both to a chemical substance with flavouring properties and to a savoury taste product or a savoury taste composition. Flavouring substances are defined chemical substances, which include flavouring substances obtained by chemical synthesis or isolated using chemical processes, and natural flavouring substances.

[0051] In certain aspects of the present application, the term ‘flavouring substance’ is to be understood as a natural flavouring substance.

[0052] As used herein, the term ‘natural flavouring substance’ refers to 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. Natural flavouring substances correspond to substances that are naturally present and have been identified in nature.

[0053] As used herein, the term ‘flavouring preparation’ refers to a product, other than a flavouring substance, obtained from: (i) food by appropriate physical, enzymatic or microbiological processes either in the raw state of the material or after processing for human consumption by one or more of the traditional food preparation processes, and / or (ii) material of vegetable, animal or microbiological origin, other than food, by appropriate physical, enzymatic or microbiological processes, the material being taken as such or prepared by one or more of the traditional food preparation processes. Flavouring preparations are flavourings other than defined chemical substances obtained from materials of vegetable, animal or microbiological origin, by appropriate physical, enzymatic or microbiological processes, either in the raw state of the material or after processing for human consumption. Flavouring preparations produced from food do not need to undergo an evaluation or an approval procedure for use in and on foods unless there is doubt about their safety.

[0054] Flavour

[0055] Flavour is a multifaceted trait encompassing two primary components: aroma, driven by low-molecular-weight volatile compounds, and basic taste, which includes sour, umami, salty, sweet, and bitter, derived from the perception of non-volatile larger molecules.

[0056] The analysis of food flavour typically employs two complementary approaches: sensory evaluation and analytical chemistry. Sensory methods rely on human perception to evaluate flavour characteristics, while analytical chemistry uses instrumental techniques to identify and quantify flavour-related compounds. For aroma profiling, gas chromatography (GC) is the predominant technique, whereas taste-related compounds are commonly analyzed using liquid chromatography (LC).

[0057] Flavour development is influenced by compounds inherent in the raw materials and those formed during processing. Processing methods, such as enzymatic and non- enzymatic reactions during smoking, heating, fermenting, drying, and toasting, play a pivotal role in creating new flavor-active compounds.

[0058] The unique flavour complexity of the flavouring savoury solutions of the present disclosure results from a carefully controlled solid state fermentation process involving koji, coupled with an enzymatic hydrolysis technique using a specific blend enzymes, such as proteases, carbohydrases, and / or nuclease.

[0059] Koji fermentation not only generates nuanced aroma and taste compounds but also naturally increases the presence of sugars derived from the enzymatic breakdown of carbohydrates. These sugars enhance flavour without the need for added sweeteners, providing a balanced sweetness that contributes to the depth and appeal of the ingredient profile. The combined effects of solid state fermentation and enzymatic action create a rich, multi-dimensional taste that complements a wide range of culinary applications without the need for flavour additives, achieve a depth of flavour through koji fermentation and enzymatic hydrolysis rather than through overpowering agents. The koji and enzymatic process allows for a balanced flavour profile without the addition of external enhancers, making the present solutions a clean-label, allergen-free, and GMO-free option which provides a naturally balanced, authentic flavour, free from excessive salt, sugar, or overpowering yeast content.

[0060] The inventors of the present invention surprisingly found an effective process for producing a savoury taste product which showed to be high in umami and may use industrial side streams of organic materials which may be used as organic starting materials which may be subjected to fungi fermentation and enzymatic treatment in the formation of a savoury taste product.

[0061] In general, the inventors of the present invention have provided a savoury taste product produced by combining fungi fermentation and enzymatic hydrolysis, e.g. from a combination of endo- and exopeptidase and glutaminase, to achieve a high degree of hydrolysis but at the same time avoiding a high degree of bitterness by having the endopeptidase cleave the negatively charged terminal amino acid produced by the exopeptidase as well as a high amount of free glutamic acid from the glutaminase which may decrease the perception of bitterness even further. The savoury taste product may also have a high content of free sugars and volatile aroma compounds resulting in a product rich in both taste and aroma compounds.

[0062] Hence, a preferred embodiment of the present invention relates to a process for producing a flavouring preparation comprising a step of fermenting an organic starting material using a fungi composition resulting in a fermented material and hydrolysing the fermented material using with an enzyme composition, providing the savoury taste product.

[0063] One aspect of the present invention relates to a method for producing a savoury taste product from an organic starting material, the process comprises the steps of: a) providing the organic starting material; b) optionally pre-treatment of the organic starting material, thereby providing a pretreated organic starting material; c) fermentation of the organic starting material in the presence of a fungi composition, preferably wherein the fungi composition comprises Aspergillus Sojae and Aspergillus Oryzae, thereby providing a fermented material; d) optionally contacting the fermented material with one or more carbohydrase e) hydrolysis of the fermented material in the presence of an enzyme composition comprising at least one protease, and at least one glutaminase, thereby providing a hydrolysed, fermented material; and f) optionally a step of recovering, concentrating, dehydrating and / or purifying the hydrolysed, fermented material thereby providing the savoury taste product.

[0064] In embodiments where step b) is included, step c) is a step of fermenting the pretreated organic starting material.

[0065] It will be clear that hydrolysis of the fermented material in step e) can refer to hydrolysis of the fermented material obtained in step c), or of the fermented material obtained in step d) when this step is present.

[0066] Another embodiment of the present invention relates to a process for producing a flavouring substance from an organic starting material, the process comprises the steps of:

[0067] (i) Providing the organic starting material;

[0068] (ii) Adding a fungi composition to the organic starting material; (iii) Allowing the fungi composition to ferment the starting material providing a fermented material;

[0069] (iv) Adding an enzyme composition to the fermented material;

[0070] (v) Allowing the enzyme composition to hydrolyse the fermented material providing the flavouring substance.

[0071] The flavouring substance may be further treated, concentrated, or purified.

[0072] The process according to the present invention for producing a flavouring substance from an organic starting material, may comprise the steps of:

[0073] (i) Providing the organic starting material;

[0074] (ii) Adding a fungi composition to the organic starting material;

[0075] (iii) Allowing the fungi composition to ferment the starting material providing a fermented material;

[0076] (iv) Adding an enzyme composition to the fermented material;

[0077] (v) Allowing the enzyme composition to hydrolyse the fermented material providing a hydrolysed material.

[0078] The hydrolysed material may be the flavouring composition, or the hydrolysed material may be further treated, concentrated, or purified providing the flavouring substance.

[0079] A preferred embodiment of the present invention relates to a process for producing a flavouring preparation comprising a step of fermenting an organic starting material using a fungi composition resulting in a fermented material and hydrolysing the fermented material using with an enzyme composition, providing the flavouring substance and / or the savoury taste product. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0080] Organic starting material

[0081] The organic starting material according to the present invention is a carbon based material. The organic starting material may be a food stream, preferably, a food grade side stream or a food byproduct stream.

[0082] In one embodiment the organic starting material is one or more material selected from vegetable, meat, poultry, legume, crustacean, and mushroom. In one embodiment the organic starting material is a vegetable, such as leek, onion or parsley, and / or a root vegetable, such as carrot. In one embodiment the organic starting material is mushroom, such as an Agaricus bisporus mushroom. In one embodiment the organic starting material is poultry such as chicken. In one embodiment the organic starting material is lentils and / or beans. In one embodiment the organic starting material is lentils, such as red lentils, such as green lentils. In one embodiment the organic starting material is beans, such as white beans. In one embodiment the organic starting material is a crustacean such as crab, such as shore crab. In one embodiment the organic starting material is fruit, such as apple. In one embodiment the organic starting material is a grain, such as corn or rice. In one embodiment the organic starting material is a meat, such as beef. In one embodiment the organic starting material is a fish or shrimp.

[0083] The organic starting material according to the present invention may be a food stream, preferably, a food grade side stream or a food byproduct stream.

[0084] In an embodiment of the present invention the food stream (or the food side stream) may be selected from a fungal product, a vegetable product, a meat product, a fish product, a seaweed product, legume product, cereal product or a combination hereof. The meat product may preferably be a beef product or a poultry product.

[0085] In a further embodiment of the present invention side stream may include more than one side stream and wherein the one or more side stream may be processed simultaneously. In an embodiment of the present invention the organic starting material comprises (consists essentially of) one food stream (or the food side stream) may be selected from a fungal product, a vegetable product, a meat product (such as a beef product or a poultry product), a fish product, or a seaweed product.

[0086] In an embodiment of the present invention the flavouring substance according to the present invention comprises at least 30% (w / w) organic starting material as described herein (preferably a single organic starting material), such as at least 40% (w / w), e.g. at least 50% (w / w), such as at least 60% (w / w), e.g. at least 70% (w / w), such as at least 80% (w / w), e.g. at least 90% (w / w), such as at least 95% (w / w), e.g. at least 98% (w / w), such as at least 99% (w / w), e.g. 100% (w / w).

[0087] The flavouring substance may comprise a combination of two or more organic starting materials. Preferably, the two or more organic starting materials may be selected from a fungal product, a vegetable product, a meat product (such as a beef product or a poultry product), a fish product, or a seaweed product.

[0088] In an embodiment of the present invention the flavouring substance may comprise a mixture of at least 2 organic starting materials, such as at least 3 organic starting materials, e.g. at least 4 organic starting materials, such as at least 5 organic starting materials according to the present invention.

[0089] In an embodiment of the present invention the organic starting material may have a moisture content above 15% (w / w), e.g. above 25% (w / w), such as above 50% (w / w), e.g. above 60% (w / w), such as above 70% (w / w), e.g. above 75% (w / w), such as above 80% (w / w), e.g. above 85% (w / w), such as above 90% (w / w).

[0090] In a further embodiment of the present invention the organic starting material provided in step (i) may be a dried organic starting material or a fresh organic starting material. Preferably, the organic starting material may be a fresh organic starting material.

[0091] One challenge of working with fresh organic starting materials may be that the organic starting materials may be provided with large variations in moisture content and thus large variations in dry matter content, e.g. from one supplier to another, how the organic starting material is handled, time from harvest to shipment and season variations throughout the year, may all be factors that may affect the moisture content and dry matter content of the organic starting materials and thereby compromising the end result of the fermentation or the enzymatic treatment of the organic starting material. The benefit of working with fresh raw materials is that no further energy is used to stabilise the foods through e.g. concentrating and drying.

[0092] In an embodiment of the present invention the dry matter content of the organic starting material, in particular the fresh organic starting material, may be used for calculating the amount fungi necessary in the fungi composition for performing the fermentation of the organic starting material.

[0093] In a further embodiment of the present invention the dry matter content of the fermented material or the dry matter content of the organic starting material, in particular the fresh organic starting material, may be used for calculating the amount enzyme(s) to be used in the enzymatic treatment for hydrolysing the organic starting material of the present invention.

[0094] The dry matter content of the organic starting material, in particular the fresh organic starting material, may be considered representing the source of the flavour components, or flavour enhancing components, ending up in the flavouring substance and the savoury taste product. Thus, the inventors of the present invention found that the dry matter content of the fresh organic starting material may be used for calculating the amount of fungi to be added with the fungi composition and / or the amount of enzyme to be added with the enzymatic composition, for the production of the savoury taste product according to the present invention.

[0095] In an embodiment of the present invention the moisture content and / or the dry matter content of the organic starting material according to the present invention may be monitored to ensure and / or improve the success of the fermentation and / or the hydrolysis.

[0096] The moisture content may be determined by using a Titrator Compact V10S or by using a Mettler-Toledo HB43 Halogen device. In an embodiment of the present invention the fresh organic starting material may not have been subjected to drying or partial drying. In one embodiment the fresh organic starting material may have been subjected to freezing.

[0097] Preferably the fresh organic starting material may relate to an organic starting material comprising a moisture content above 15% (w / w), e.g. above 25% (w / w), such as above 50% (w / w), e.g. above 60% (w / w), such as above 70% (w / w), e.g. above 75% (w / w), such as above 80% (w / w), e.g. above 85% (w / w), such as above 90% (w / w).

[0098] In an embodiment of the present invention the fresh organic starting material may relate to an organic starting material comprising an intrinsic water of the organic starting material resulting in large amounts of water being conserved, and which may be used for the process according to the present invention.

[0099] Pre-treatment

[0100] Pre-treatment includes standard preparation of the given organic starting material, such as cleaning from dirt by rinsing, soaking and / or tumbling the organic starting material in water, as well as cutting and cooking. Moreover, pre-treatment may include salting.

[0101] In one embodiment the method includes step (b).

[0102] In one embodiment pre-treatment includes thawing, rehydration, boiling, steaming and / or roasting.

[0103] In one embodiment pre-treatment includes mincing and / or grinding, such as mincing with a bowl cutter. In one embodiment pre-treatment includes combination of the organic starting material with water. In one embodiment pre-treatment includes combination of the organic starting material with salt, such as 5 wt% salt, such as 10 wt% salt, such as 12 wt% salt, such as 15 wt% salt. In one embodiment pre-treatment includes mixing, such as mixing with a stick blender.

[0104] In an embodiment of the present invention the organic starting material may be a minced organic starting material, or the organic starting material may be subjected to mincing before being subjected to fungi fermentation and / or before being subjected to enzymatic hydrolysis. Mincing may be a preparation process in which the organic starting material may be divided, preferably finely divided, into finer particles of the organic starting material. Mincing according to the present invention may be performed by a conventional meat grinder or an industrial cutter. In an embodiment of the present invention mincing may be performed by a thermomixer (TM6), preferably at highest speed.

[0105] The mincing of the organic starting material may provide a homogeneous minced organic starting material, or a homogenous minced fermented material.

[0106] During mincing it may be desirable to provide a minced organic starting material having a particle size that may be small enough to allow sufficient access of the fungi composition during fermentation or the enzyme composition during hydrolysis, and on the other hand not so small that there is a risk of dusting, fouling of equipment and not so small that solid liquid separation would be difficult.

[0107] The (homogeneous) minced organic starting material, and / or the fermented material (homogeneous fermented material), may be subjected to pasteurisation. This step of pasteurisation may not only decrease the chances of unwanted microbial growth or decrease the presence of pathogenic microorganisms, but it may also contribute to partial degradation of the cell walls of the organic starting material, and / or the fermented material.

[0108] Preferably, the process according to the present invention may include a reduction in the amount of water added to the organic starting material, where addition of water may be less than 40% (w / w) added water relative to the dry matter content of the fungi product, such as less than 30% (w / w), e.g. less than 20% (w / w), such as less than 15% (w / w), e g. less than 10% (w / w), such as less than 5% (w / w), e.g. less than 2% (w / w), such as 0% (w / w) added water.

[0109] The added water may be different from the moisture content naturally present in the organic starting material, e.g. intrinsic water, which may be the water bound in the matrix of the organic staring material. Some of this water bound in the matrix of the organic starting material may be liberated when the organic starting material may be minced and may be used for suspending the solid part of the organic starting material and / or the fungi composition or the enzyme composition added during the process. Preferably the organic starting material may be provided by an initial step of cleaning the organic starting material from dirt. The initial cleaning may involve thorough cleaning, by rinsing, soaking and / or tumbling the organic starting material in water. Preferably, the soaking and / or tumbling of the organic starting material may be done in cold water.

[0110] In an embodiment of the present invention the cold water may have a temperature below 20°C, such as below 15°C, e.g. below 12°C, such as below 10°C, e.g. below 8°C, such as below 6°C, e.g. in the range of 4-20°C, such as in the range of 6-15°C, e.g. in the range of 8-10°C.

[0111] Following the initial step of cleaning the organic starting material in water, the water may be drained of the organic starting material providing a cleaned organic starting material. This, draining of water from the organic starting material may not be considered drying of the organic starting material but may be merely a decanting of the cleaning water from the organic starting material.

[0112] In an embodiment of the present invention the organic starting material and / or the fermented material may comprise a salt. Preferably, the salt may be added to the organic starting material and / or the fermented material. Preferably, the salt may be sodium chloride (NaCI).

[0113] The concentration of the salt added to the organic starting material, and / or the fermented material may be below 12% (w / w), e.g. such as below 10% (w / w), such as below 8% (w / w), e.g. below 6% (w / w), such as below 4% (w / w), e.g. below 3.5% (w / w), such as below 3% (w / w), e.g. below 2.5% (w / w), such as about 2% (w / w), e.g. in the range of 0.5-12% (w / w), e.g. in the range of 1-10% (w / w), e.g. in the range of 2.0-8% (w / w), e.g. in the range of 3-6% (w / w), e.g. in the range of 4-5% (w / w).

[0114] Pasteurisation

[0115] In certain aspects of the present invention, it may be desirable to pasteurise the organic starting material prior to fermentation and enzymatic hydrolysis. In other aspects, it may not be desirable to pasteurise the organic starting material prior to fermentation and enzymatic hydrolysis. In some embodiments the step of pre-treatment includes a step of pasteurising the organic starting material. In one embodiment the pre-treated organic starting material is heated to around 60 °C, prior to step (c).

[0116] In one embodiment the pre-treated organic starting material is heated to around 60 °C, with presence of the fungi composition prior to step (c).

[0117] In one embodiment the pre-treated organic starting material is heated to around 60 °C, such as around 65 °C, such as around 70 °C, such as around 75 °C, such as around 80°C, such as around 85°C, such as around 90°C, such as around 95 °C, such as around 100 °C, prior to step (c).

[0118] In one embodiment the pre-treated organic starting material is heated to around 60 °C, such as around 65 °C, such as around 70 °C, such as around 75 °C, such as around 80°C, such as around 85°C, such as around 90°C, such as around 95 °C, such as around 100 °C, and kept at the temperature for 10 minutes prior to step (c).

[0119] In one embodiment the pre-treated organic starting material is heated to around 80 °C, and kept for 10 minutes prior to step (c). In one embodiment the pre-treated organic starting material is cooled to around 60 °C, prior to step (c). In one embodiment the temperature is maintained between 58-62 °C, such as around 60 °C, during step (c). In one embodiment the temperature is maintained between 58-62 °C, such as around 60 °C, during step (c), optional step (d) and / or step (e).

[0120] Pasteurisation may be performed at a temperature in the range of 80-120°C, such as in the range of 95-110°C, e.g. at about 100°C, for a period of 2-15 minutes, such as for a period of 5-12 minutes, e.g. for about 10 minutes. Pasteurisation may be performed with agitation, preferably with moderate agitation.

[0121] After pasteurisation, the organic starting material, and / or the fermented material, may be rapidly cooled to a temperature in the range of 55-62°C preferably to a temperature about 60°C, preferably, by continuous stirring in an ice-cold water bath.

[0122] Fermentation

[0123] The method of the present invention may include koji fermentation. Koji produces a range of enzymes, such as proteases and amylases, that break down plant material into simpler, more digestible forms. This enzymatic activity enhances the presence of free amino acids and sugars, amplifying umami and sweetness in the end product. Furthermore, it prepares the substrate for subsequent fermentation stages, facilitating the formation of additional taste- and aroma-active compounds.

[0124] Koji fermentation provides for increased levels of amino acids, imparting mainly umami, sweet, bitter and slight sour. Fermentation with live Koji cultures provides for markedly higher levels of free amino acids compared to reference methods without fermentation with live koji, and inactivated koji. More specifically higher levels of glutamic acid, tyrosine, tryptophan, phenylalanine, alanine, isoleucine, valine, glucose, sucrose and fructose levels are significantly higher when live Koji is used Specifically, levels of glucose, sucrose, fructose, arginine, glutamic acid, tyrosine, tryptophan, alanine, isoleucine, valine and threonine are higher when live Koji is introduced.

[0125] In one embodiment the fungi composition is a solid state fungi composition, such as koji composition.

[0126] In one embodiment, the fungi composition is a koji composition. In one embodiment, the fungi composition comprises Aspergillus oryzae, Aspergillus sojae, and rice. In one embodiment, the fungi composition comprises Aspergillus oryzae, Aspergillus sojae, rice and lentils and / or chickpea.

[0127] In one embodiment the fungi composition comprises a substrate, such as an organic substrate, and at least two fungi selected from Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Penicillium species, Rhizopus oligosporus, Trichoderma species or Candida milled.

[0128] In one embodiment the method further comprises a step of obtaining the fungi composition, and inoculating the substrate with at least two spores of said fungi composition.

[0129] In one embodiment the method further comprises the step of culturing the fungi spores for 40 to 48 hours, at a temperature between 28 °C and 32 °C, with a humidity of 70 to 80%, such as a humidity of 75%. In one embodiment the substrate is rice, broken rice, beans, broken beans, pumpkin seeds, broken pumpkin seeds, sesame seeds, broken sesame seeds, sunflower seeds, broken sunflower seeds, cereal brans such as wheat bran, sorghum bran, rye bran, oats bran, barley bran, millet bran, maize bran, broken cereal brans, lentils, broken lentils, chickpeas, broken chickpeas, corn, broken corn, quinoa, broken quinoa or spelt, broken spelt or any combination thereof. In one embodiment the substrate is rice. In one embodiment the substrate is broken rice. In one embodiment the substrate is lentil. In one embodiment the substrate is broken lentils. In one embodiment the substrate is chickpeas. In one embodiment the substrate is broken chickpeas. In one embodiment the substrate is lentil and or chickpeas, such as broken lentils and / or broken chickpeas in combination with rice, such as broken rice.

[0130] In one embodiment the fungi spores are Aspergillus Sojae and Aspergillus Oryzae spores. In one embodiment the fungi spores are a blend of Aspergillus Sojae and Aspergillus Oryzae spores. In one embodiment the fungi spores is a Aspergillus Sojae and Aspergillus Oryzae hybrid spores. In one embodiment the fungi spores are Aspergillus Sojae and Aspergillus Oryzae spores in a ratio in the range 4:1 to 1 :4, such as around 4:1 , 3:1 , 2:1 , 1:1, 1 :2, 1 :3 or 1 :4. In one embodiment the fungi spores are Aspergillus Sojae and Aspergillus Oryzae spores, with a ratio of Aspergillus Sojae to Aspergillus Oryzae of around 1:1.

[0131] In one embodiment the fungi composition is Hi-Sojae. In one embodiment the fungi composition is non-GMO.

[0132] In one embodiment the method further comprises the step of inactivating the fungi composition prior to use in step (c).

[0133] The fungi composition may provide a fungal fermentation where the fungi present in the fungi composition via various metabolic processes converts sugars into various end products like alcohol, organic acids, and gases. Additionally, the fungi composition may provide a fungal fermentation where the fungi present in the fungi composition via various metabolic processes coverts proteins into various products like peptides, free amino acids and aromatic compounds. Fungal moulds and fungal spores may relate to two different components of the life cycle of a fungi. The fungi composition may be provided in the form of a fungi mould, or a fungi spore.

[0134] A fungal mould may be a visible, often fuzzy or powdery, mass of fungal mycelium, being the network of thread-like structures (hyphae) that make up the main body of a fungus. These hyphae may be grow and spread across a substrate to extract nutrients.

[0135] Fungal moulds may be visible to the naked eye, especially when they form colonies on surfaces organic materials.

[0136] Fungal moulds may grow by extending their hyphae and absorbing nutrients from the substrate and may reproduce through various means, including both sexual and asexual reproduction. Fungal moulds may produce spores as part of their reproductive process.

[0137] The fungal spores may be a single, specialized cell produced by the fungi. The fungal spores may be a reproductive structure designed for dispersal and with the potential to develop into a new individual fungal mould under suitable conditions.

[0138] Fungal spores may be microscopic, single cells that may have diverse forms depending on the type of fungus and its reproductive strategy.

[0139] Fungal spores may typically not be visible to the naked eye and a microscope may be required for observation.

[0140] Fungal spores are primarily involved in reproduction and dispersal. They are often released into the environment, where they can be carried by air currents, water, or other means to new locations. When a fungal spore lands in a suitable environment with the necessary resources (such as water, nutrients, and appropriate temperature). Fungal spores may germinate and develop into a new fungal organism or new fungal mould.

[0141] The fungi composition may comprise one or more fungi traditionally used in food production and processing. In particular the fungi composition according to the present invention may comprise one or more fungi that contribute to the development of various flavours, textures, and / or preservation.

[0142] In an embodiment of the present invention the fungi composition may comprise a fungi selected from:

[0143] - Aspergillus oryzae (traditionally used in the fermentation of soy sauce, miso, and sake. This fungus is also employed in the production of certain enzymes used in food processing);

[0144] - Aspergillus sojae (traditionally used to make the ferment of soy sauce, miso, or mirin. Soy sauce condiment may be produced by fermenting soybeans with A. sojae along with water and salt);

[0145] - Aspergillus niger (traditionally used to facilitate the industrial production of substances like citric acid and gluconic acid);

[0146] - Penicillium species (traditionally used in the production of several types of cheeses, including blue cheeses like Roquefort and Gorgonzola);

[0147] - Rhizopus oligosporus (traditionally used in the fermentation process of soybeans to produce tempeh);

[0148] Trichoderma species (traditionally used in the production of certain types of enzymes like cellulases and hemicellulases for food processing);

[0149] - Candida milleri (traditionally used in dairy fermentation, particularly in the production of some types of cheese; or

[0150] - A combination hereof.

[0151] In particular, the fungi composition may comprise at least one fungi selected from Aspergillus oryzae, Aspergillus sojae, or a combination of Aspergillus oryzae, and Aspergillus sojae.

[0152] A preferred embodiment of the present invention may relate to a fungi composition comprising the fungi mould, and / or the fungi spore in combination with a fungi substrate.

[0153] The combination of the fungi mould, and / or the fungi spore with the fungi substrate may be allowed to ferment allowing the fungi mould, and / or the fungi spore to grow and to develop. The fungi composition produced may be added to the organic starting material as defined in step (i).

[0154] The fungi substrate may be a substrate which may support the growth and / or development of the fungi mould, and / or the fungi spore.

[0155] In an embodiment of the present invention the fungi substrate may be rice, broken rice, beans, broken beans, hemp (like hemp seeds), pumpkin (like pumpkin seeds), sesame (like sesame seeds), sunflower (like sunflower seeds), cereal (like cereal brans), or any combination hereof.

[0156] The cereal may be selected from wheat, sorghum, rye, oats, barley, millet, or maize. In particular, the cereal bran may be selected from wheat bran, sorghum bran, rye bran, oats bran, barley bran, millet bran, or maize bran.

[0157] Preferred fungi substrates may be broken rice, broken rice in combination with beans, rice in combination with hemp seeds, rice in combination with pumpkin seeds, rice in combination with sesame seeds, rice in combination with sunflower seeds, or sorghum in combination with wheat bran.

[0158] The fungi substrate may be hydrated before the fungi may be added to the fungi substrate resulting in the fungi composition. The fungi substrate may be hydrated by soaking the fungi substrate in an aqueous medium, e.g. water.

[0159] Hydration of the fungi substrate may include a water to fungi substrate ratio in the range of 1 :1 to 5:1 (water:fungi substrate), such as in the range of 1.25:1 to 3:1 (waterfungi substrate), e.g. in the range of 1.5:1 to 2.5:1 (waterfungi substrate), such as in the range of 1.75:1 to 2:1 (waterfungi substrate).

[0160] Preferably, the temperature of the water and fungi substrate during hydration may be in the range of 1-20 °C, such as in the range of 2-15 °C, e.g. in the range of 3-10 °C, such as in the range of 4-6 °C. Such as in the range of 5-10 °C, such as in the range of 10- 15°C, such as in the range of 10-12 °C, such as in the range of 15-20°C. The period for subjecting the fungi substrate to hydration, before adding the fungi, may be more than 1 hour, such as more than 2 hours, e.g. more than 4 hours, such as more than 6 hours, e.g. more than 12 hours, such as more than 16 hours, e.g. more than 20 hours, such as more than 24 hours, e.g. more than 30 hours, such as in the range of 1- 35 hours, e.g. in the range of 6-30 hours, such as in the range of 12-24 hours, e.g. in the range of 16-20 hours.

[0161] In an embodiment of the present invention the fungi substrate may, after being hydrated, be washed, e.g. with water to promote the loss of excess starch. The washing of the hydrated fungi substrate may be performed for about 1 minute using running water.

[0162] The fungi substrate may be cooked before adding the fungi. The cooking may be performed by steaming at a temperature between 95-105°C, such as between 97- 103°C, e.g. between 98-101 °C, e.g. by placing the washed fungi substrate on a perforated baking tray

[0163] The cooking may be performed for a period of 2-60 minutes, such as for a period of 5- 45 minutes, e.g. for a period of 10-30 minutes, such as for a period of 15-20 minutes.

[0164] Preferably, cooking may be performed to provide a proper balance between the cooking of the starches and the percentage of water inside the fungi substrate. Preferably, the water inside the fungi substrate does not exceed 60%, such as does not exceed 50%, e.g. does not exceed 40%, does not exceed 30%.

[0165] After cooking of the fungi substrate, the fungi substrate may be cooled (preferably, to a temperature in the range of 15-45°C, such as in the range of 25-35°C) and then inoculated with one or more fungi providing the fungi composition according to the present invention.

[0166] In an embodiment of the present invention the fungi provided to the fungi substrate resulting in the fungi composition, may be one or more spores.

[0167] Preferably, the fungi substrate may be inoculated with a concentration of fungi in the range of 0.01-2% (w / w) on a dry matter basis, such as in the range of 0.05-1.5% (w / w) on a dry matter basis, e.g. in the range of 0.075-1% (w / w) on a dry matter basis, such as in the range of 0.1-0.75% (w / w) on a dry matter basis, such as in the range of 0.2- 0.5% (w / w) on a dry matter basis, e.g. about 0.3% (w / w) on a dry matter basis.

[0168] In an embodiment of the present invention the fungi substrate may be inoculated with a combination of two or more fungi. Preferably, the fungi substrate may be inoculated with a combination of Aspergillus oryzae and Aspergillus sojae.

[0169] Preferably, the fungi substrate may be inoculated with a combination of Aspergillus oryzae and Aspergillus sojae, wherein the Aspergillus oryzae may be provided in a concentration in the range of 0.01-1% (w / w) on a dry matter basis, such as in the range of 0.05-0.5% (w / w) on a dry matter basis, e.g. about 0.1% (w / w) on a dry matter basis, and the Aspergillus sojae may be provided in a concentration in the range of 0.01-1% (w / w) on a dry matter basis, such as in the range of 0.05-0.5% (w / w) on a dry matter basis, e.g. about 0.2% (w / w) on a dry matter basis. Preferably, the Aspergillus sojae may be provided in higher concentration than the Aspergillus oryzae.

[0170] Preferably, the fungi may be inoculated for a period of at least 10 hour, such as for at least 15 hours, e.g. for at least 20 hours, such as for at least 25 hours, e.g. for at least 30 hours, such as for at least 35 hours, e.g. for at least 40 hours, such as for at least 45 hours, e.g. for at least 50 hours, such as for a period of 10-60 hours, such as for a period of 20-55 hours, e.g. for a period of 30-50 hours, such as for a period of 40-48 hours.

[0171] The fungi may be inoculated at a temperature in the range of 15-45°C, such as at a temperature in the range of 25-40°C, e.g. at a temperature in the range of 28-34°C.

[0172] The incubation of the fungi in the fungi substrate may be provided with a relative humidity in the range of 60-90%, such as in the range of 70-85%.

[0173] In an embodiment of the present invention carbon dioxide (CO2) may be added to the fungi composition during incubation to improve growth of the fungi. Preferably, the content of carbon dioxide (CO2) added may be in the range of 0.1-10% in the incubation chamber, such as in the range of 0.5-5%, e.g. in the range of 1-3%, such as in the range of 1.5-2.5%. Preferably, the organic starting material may comprise a concentration of fungi in the range of 0.01-2% (w / w) on a dry matter basis, such as in the range of 0.05-1.5% (w / w) on a dry matter basis, e.g. in the range of 0.075-1% (w / w) on a dry matter basis, such as in the range of 0.1-0.75% (w / w) on a dry matter basis, such as in the range of 0.2- 0.5% (w / w) on a dry matter basis, e.g. about 0.3% (w / w) on a dry matter basis.

[0174] In an embodiment of the present invention the fungi composition may comprise one, two or three of the following:

[0175] - a protein content in the range of 3-40% (w / w) on a dry-matter content, such as in the range of 2-38% (w / w), e.g. in the range of 5-35% (w / w), such as in the range of 7-30% (w / w), e.g. in the range of 5-25% (w / w);

[0176] - a carbohydrate content in the range of 25-80% (w / w) on a dry-matter content, such as in the range of 30-75% (w / w), e.g. in the range of 35-70% (w / w), such as in the range of 40-65% (w / w), e.g. in the range of 50-60% (w / w);

[0177] - a lipid content in the range of 0.1-10% (w / w) on a dry-matter content, such as in the range of 0.25-9% (w / w), e.g. in the range of 0.5-8% (w / w), such as in the range of 1-6% (w / w), e.g. in the range of 2-4% (w / w).

[0178] Enzymatic hydrolysis

[0179] The method of the present invention moreover includes enzymatic hydrolysis.

[0180] Carbohydrase

[0181] The method of the present invention may comprise a step of carbohydrase hydrolysis. This is particularly relevant, for fibrous organic starting materials, such as vegetables. A carbohydrase may hydrolyze (1 ,3)- or (1 ,4)-linkages in beta-D-glucans. A carbohydrase may include xylanase activity, cellulase activity and / or hemicellulase activity.

[0182] In one embodiment the method comprises step (d).

[0183] In one embodiment the organic starting material is a fibrous material, such as a plant material. In one embodiment the one or more carbohydrase is or comprises a broad spectrum carbohydrase.

[0184] In one embodiment the one or more carbohydrase is or comprises an endo-beta- glucanase.

[0185] In one embodiment the one or more carbohydrase has activity as xylanase, cellulase and / or hemicellulase activity.

[0186] In one embodiment the one or more carbohydrase is or comprises VinoTaste Pro, BAN 480 L, BAN, Viscozyme L, Funagmyl 800L, or Sweetzyme IT or combinations thereof. In one embodiment the one or more carbohydrase is or comprises Viscozyme L.

[0187] In one embodiment the concentration of carbohydrase is in the range 0.05 wt% to 5 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt%.

[0188] In one embodiment the concentration of carbohydrase is in the range 0.05 wt% to 10 wt% of the dry matter, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 2.5 wt%, such as around 3 wt%, such as around 5 wt%, such as around 7.5 wt%, such as around 10 wt% dry matter.

[0189] In one embodiment the concentration of carbohydrase is in the range 0.05 wt% to 2 wt% of the wet weight, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt% wet weight.

[0190] In one embodiment step (d) includes pH adjustment. In one embodiment pH adjustment is performed prior to introduction of the one or more carbohydrase.

[0191] In cases where the pH is not already around 4.5, pH adjustment may be performed. In one embodiment pH is adjusted to acidic pH, such as pH 4.5 to 4.7. In one embodiment pH is adjusted by introduction of an acid, such as citric acid.

[0192] In one embodiment step (d) runs for 4 to 16 hours, or such as for 4 to 21 hours, or such as for 4 hours, or such as for 4 to 8 hours, or such as for 8 to 12 hours, or such as for 12 to 16 hours, or such as for 16 to 21 hours.

[0193] Preferably the carbohydrase may comprise at least one broad spectrum carbohydrase. In an embodiment of the present invention the carbohydrase may be selected from one or more enzymes having arabanase activity, cellulase activity, beta-glucanase activity, hemicellulose activity, xylanase activity, or a combination hereof.

[0194] Preferably, the carbohydrase may be selected from the group of enzymes selected from VinoTaste Pro, BAN 480 L, BAN, Viscozyme L, Funagmyl 800L, Sweetzyme IT extra and a combination hereof.

[0195] One effect observed by the carbohydrase may be the ability to release compounds found in the matrix of the organic starting material, while simultaneously increasing protein accessibility and e.g. increasing the efficiency of protein-degrading enzymes, the efficiency of the nucleic acid degrading enzymes, as well as increasing the efficiency of enzymes present in the hydrolysis according to the present invention.

[0196] In an embodiment of the present invention the matrix of the organic starting material may be opened by other methods, improving accessibility and / or effectivity of other enzymes in the enzyme composition. However, subjecting the fermented material to an enzyme composition and the enzymatic treatment provided by this enzyme composition, may be preferred.

[0197] The content of the carbohydrase in the enzyme composition may be in the range of 10- 50% (w / w) of the total enzyme content of the enzyme composition, such as in the range of 15-40% (w / w), e.g. in the range of 20-30, such as about 25% (w / w).

[0198] The content of the carbohydrase in the fermented material to hydrolyse the fermented material may be in the range of 0.01-5% (w / w) on a dry matter basis, such as in the range of 0.025-2.5% (w / w) on a dry matter basis, e.g. in the range of 0.05-2% (w / w) on a dry matter basis, such as in the range of 0.075-1.5% (w / w) on a dry matter basis, e.g. in the range of 0.1 -1.0% (w / w) on a dry matter basis, such as in the range of 0.125- 0.5% (w / w) on a dry matter basis, e.g. in the range of 0.2-0.25% (w / w) on a dry matter basis.

[0199] Enzyme composition

[0200] The method of the present invention moreover comprises enzymatic hydrolysis by an enzyme composition. The enzyme composition can be added in one or more steps, and may comprise two or more enzymes, wherein at least one enzyme is a protease, and at least one enzyme is a glutaminase.

[0201] In one embodiment the enzyme composition of step (e) is added in one step. In one embodiment the enzyme composition of step (e) is added in two individual steps (a first protease hydrolysis step and a second, glutaminase hydrolysis step).

[0202] In one embodiment the enzyme composition of step (e) is added in three individual steps (a first protease hydrolysis step, a second, glutaminase hydrolysis step.

[0203] Protease

[0204] In one embodiment the at least one protease and the at least one glutaminase of step (e) are added in one step.

[0205] In one embodiment the at least one protease and the at least one glutaminase of step (e) are added in two individual steps, respectively a step e1 and a step e2.

[0206] In one embodiment the at least one protease is added in a protease hydrolysis step (e1), and the at least one glutaminase is added in a glutaminase hydrolysis step (e2).

[0207] In one embodiment the at least one protease is added in a first protease hydrolysis step (e1), and the at least one glutaminase is added in a later glutaminase hydrolysis step (e2).

[0208] In one embodiment the at least one protease is added in a first protease hydrolysis step (e1), and the at least one glutaminase is added in a later glutaminase hydrolysis step (e2). In one embodiment the at least one protease is an endo-protease and / or an exoprotease.

[0209] In one embodiment the at least one protease is an endo-protease, such as a serine endo-peptidase and / or a blend of endo- and exo-peptidases.

[0210] In one embodiment the at least one protease is a protease selected from Umamizyme™ Pulse Protana UBoost, Protana Prime, Peptidase R, Glutaminase N 100, Protease HF"Amano"150SD, ProteAX, Flavourzyme WOOL, carboxypeptidase, aminopeptidase, SEBDigest F35 L / F35 P, SEBDigest F59 P, SEBDigest B7 P, Alcalase Pure 2.4 L, Savinase, Novo-Pro D, Neutrase, Alcalase 2.4 L FG, trypsin, THERMOASE PC10FNA, RROTIN SD-NY10, RROTIN SD-AY10, chymotrypsin, thermolysin, Protizyme FAP cone, papain, Protease M"Amano"SD, Protease AN"Amano"100SD, pronase or combinations thereof. In one embodiment the at least one protease is Alcalase 2.4 L FG and Flavourzyme 1000 L.

[0211] In one embodiment the protease hydrolysis step, runs for 4 to 50 hours, such as 4 to 8 hours, such as 8 to 12 hours, such as 12 to 16 hours, such as 16 to 50 hours, such as 18 to 50 hours, such as 16 to 19 hours, such as 16-24 hours, such as 19 to 25 hours, such as 25 to 30 hours, such as 35 to 40 hours, such as 40 to 45 hours, such as 45 to 50 hours, such as 46 to 50 hours, such as 50 to 55 hours, such as 55 to 60 hours, such as 60 to 65 hours, such as 65 to 70 hours, such as 64-69 hours, such as 66 to 69 hours.

[0212] In one embodiment the concentration of protease is in the range 0.05 wt% to 5 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% protease.

[0213] In one embodiment the concentration of protease is in the range 0.05 wt% to 5 wt% of the dry matter, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% dry matter. In one embodiment the concentration of protease is in the range 0.05 wt% to 2 wt% of the wet weight, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt% wet weight.

[0214] In one embodiment the method further comprises pH adjustments prior to step (e), such as pH adjustment with a base, such as sodium carbonate or pH adjustment with an acid, such as citric acid.

[0215] In one embodiment step (e) includes one or more pH adjustment. In one embodiment step (e) includes one or more pH adjustment, such as one pH adjustment prior to the protease hydrolysis step (e1), and one prior to the glutaminase hydrolysis step (e2).

[0216] In one embodiment pH is adjusted to a pH in the range 8.0 to 8.6, such as 8.0 to 8.5, such as 8.0 to 8.2, such as 8.2 to 8.4, such as 8.4 to 8.6, prior to addition of the at least one protease in step (e). In one embodiment pH is adjusted to a pH in the range 4.5 to 6.5, such as 4.5 to 5.0, such as 5.0 to 5.5, such as 5.5 6.0, such as 6.0 to 6.5, prior to addition of the at least one protease in step (e).

[0217] In one embodiment the method further comprises pH re-adjustment during step (e), such as one pH re-adjustment daily, such as two pH re-adjustments daily, such as two pH re-adjustment over two days, such as three pH re-adjustments over two days, such as four pH re-adjustments over two days, such as four pH re-adjustments over three days, such as five pH re-adjustments over three days.

[0218] In one embodiment the protease of step (e) is added in one or more dosages, such as in one dosage, such as in two dosages, such as in three dosages.

[0219] The enzyme composition may comprise a protease, preferably, the protease may be selected from an endo-protease, an exo-protease or a combination of an endoprotease and an exo-protease.

[0220] When the endo-protease and the exo-protease may be added sequentially the endoprotease may preferably be added before the exo-protease. One disadvantage of treating organic starting material, or the fermented material, with enzymes as described in the present context, may be that bitter tasting flavour compounds may be developed, which may be expected to be caused by exposing hydrophobic amino acids at the terminal end of a peptide.

[0221] To avoid these bitter compounds an exo-protease enzyme may be added to the organic material. By cleaving the terminal, hydrophobic amino acid, the exo-protease may reduce the bitterness of the product and make it more appealing and interesting for consumption. The exo-protease may further increase the overall degree of hydrolysis and thereby provide a further contribution to the umami composition of the final products, e.g. the savoury taste product.

[0222] In an embodiment of the present invention the exo-protease enzyme may be selected from Umamizyme™ Pulse Protana UBoost, Protana Prime, Peptidase R, Glutaminase N 100, Protease HF"Amano"150SD, ProteAX, Flavourzyme 1000L, carboxypeptidase, aminopeptidase or a combination hereof.

[0223] In a further embodiment of the present invention the endo-protease enzyme may be selected from SEBDigest F35 L / F35 P, SEBDigest F59 P, SEBDigest B7 P, Alcalase Pure 2.4 L, Savinase, Novo-Pro D, Neutrase, Alcalase 2.4 L FG, trypsin, THERMOASE PC10FNA, RROTIN SD-NY10, RROTIN SD-AY10, chymotrypsin, thermolysin or a combination hereof.

[0224] An embodiment of the present invention the protease may be selected from an enzyme exhibiting both exo-protease activity and endo-protease activity, such enzyme may be Flavourzyme WOOL, Protizyme FAP cone, papain, Protease M"Amano"SD, Protease AN"Amano"100SD, pronase or a combination hereof.

[0225] The content of the protease in the enzyme composition may be in the range of 10-50% (w / w) of the total enzyme content of the enzyme composition, such as in the range of 15-40% (w / w), e.g. in the range of 20-30, such as about 25% (w / w). In the event the enzyme composition does not comprise a carbohydrase the content of the protease in the enzyme composition may be in the range of 10-100% (w / w) of the total enzyme content of the enzyme composition, such as in the range of 20-75% (w / w), e.g. in the range of 30-60 (w / w), such as in the range of 40-50% (w / w). The content of the protease in the fermented material to hydrolyse the fermented material may be in the range of 0.01-5% (w / w) on a dry matter basis, such as in the range of 0.025-2.5% (w / w) on a dry matter basis, e.g. in the range of 0.05-2% (w / w) on a dry matter basis, such as in the range of 0.075-1.5% (w / w) on a dry matter basis, e.g. in the range of 0.1 -1.0% (w / w) on a dry matter basis, such as in the range of 0.125- 0.5% (w / w) on a dry matter basis, e.g. in the range of 0.2-0.25% (w / w) on a dry matter basis.

[0226] Glutaminase

[0227] The enzyme composition comprises a glutaminase.

[0228] In one embodiment the glutaminase of step (e) is a gamma glutamyl transpeptidase and / or a gamma-glutamyl transferase.

[0229] In one embodiment the glutaminase of step (e) is Glutaminase SD-100NA.

[0230] In one embodiment the concentration of glutaminase is in the range 0.01 wt% to 5 wt%, such as around 0.02 wt%, such as around 0.03 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% glutaminase.

[0231] In one embodiment the concentration of glutaminase is in the range 0.001 wt% to 0.5 wt% of the wet weight, such as around 0.001 wt%, such as around 0.005 wt%, such as around 0.01 wt%, such as around 0.02 wt%, such as around 0.03 wt%, such as around 0.04 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.3 wt%, such as around 0.4 wt%, such as around 0.5 wt%, wet weight.

[0232] In one embodiment no pH adjustment is performed before the glutaminase hydrolysis step.

[0233] In one embodiment pH is adjusted to a pH in the range 5.0 to 6.5, such as 5.0 to 5.2, such as 5.2 to 5.4, such as 5.4 to 5.6, such as 5.6 to 5.8, such as 5.8 to 6.0, such as 6.0 to 6.5 prior to addition of the at least one glutaminase in step (e). In one embodiment pH is adjusted to pH in the range pH 5.4 to 5.6 before the glutaminase hydrolysis step (e2).

[0234] In one embodiment the glutaminase hydrolysis step runs for 4 to 16 hours, or such as for 4 to 21 hours, or such as for 4 hours, or such as for 4 to 8 hours, or such as for 8 to 12 hours, or such as for 12 to 16 hours, or such as for 16 to 21 hours.

[0235] In one embodiment the glutaminase is introduced after the protease, in a glutaminase hydrolysis step (e2).

[0236] The glutaminase may be an enzyme that catalyses the conversion of the amino acid glutamine into glutamate and ammonia.

[0237] In an embodiment of the present invention the glutaminase may be a gamma glutamyl transpeptidase or a gamma-glutamyl transferase.

[0238] The gamma-glutamyl transferase may be GLUTAMINASE SD-100NA. This glutaminase may work as a gamma-glutamyl transferase at alkaline pH, e.g. a pH like 8.0 - 9.0.

[0239] The action of the glutaminase, in particular GLUTAMINASE SD-100NA may be in production of kokumi active peptides.

[0240] The content of the glutaminase in the enzyme composition may be in the range of 10- 50% (w / w) of the total enzyme content of the enzyme composition, such as in the range of 15-40% (w / w), e.g. in the range of 20-30, such as about 25% (w / w).

[0241] The content of the glutaminase in the fermented material to hydrolyse the fermented material may be in the range of 0.01-2% (w / w) on a dry matter basis, such as in the range of 0.025-1 .5% (w / w) on a dry matter basis, e.g. in the range of 0.05-1% (w / w) on a dry matter basis, such as in the range of 0.075-0.75% (w / w) on a dry matter basis, such as in the range of 0.1 -0.5% (w / w) on a dry matter basis.

[0242] Nuclease and deaminase

[0243] The method of the present invention may comprise enzymatic hydrolysis with a nuclease and / or a deaminase. In one embodiment the enzyme composition of step (e) further comprises at least one nuclease.

[0244] In one embodiment the enzyme composition of step (e) further comprises at least one deaminase.

[0245] In one embodiment the enzyme composition of step (e) comprises at least one protease, at least one glutaminase and at least one nuclease.

[0246] In one embodiment the enzyme composition of step (e) comprises at least one protease, at least one glutaminase, at least one nuclease and at least one deaminase.

[0247] In one embodiment the at least one glutaminase and the at least one nuclease are added together, after the protease hydrolysis.

[0248] In one embodiment the at least one glutaminase and the at least one nuclease are added together, after the protease hydrolysis step (e1).

[0249] In one embodiment the at least one glutaminase, the at least one nuclease and the at least one deaminase are added together after the protease hydrolysis step (e1).

[0250] In one embodiment the pH is adjusted prior to the introduction of the at least one glutaminase and the at least one nuclease (step (e2)).

[0251] In one embodiment pH is adjusted prior to the introduction of the at least one glutaminase, the at least one nuclease and the at least one deaminase (step (e2)).

[0252] In one embodiment pH is adjusted to pH in the range 5.4 to 6.5, or such as 5.5 to 6 prior to the introduction of the at least one glutaminase and the at least one nuclease (step (e2)).

[0253] In one embodiment pH is adjusted to pH in the range 5.4 to 6.5, or such as 5.5 to 6 prior to the introduction of the at least one glutaminase, the at least one nuclease and the at least one deaminase (step (e2)). In one embodiment the nuclease is an endonuclease, an exonuclease, a restriction enzyme, a RNAse, and / or combinations thereof.

[0254] In one embodiment the nuclease is selected from E”Amano”7 Nuclease, Ambion™ RNase, Deamizyme T “Amano”, Amano’s Deamizyme 50000G Nuclease NP-1 , Deaminase D-100, Complex protease EF 108 and / or combinations thereof.

[0255] In one embodiment the nuclease is E”Amano”7 Nuclease.

[0256] In one embodiment the deaminase is Deamizyme T“Amano”50.

[0257] In one embodiment the temperature during the hydrolysis in step (e) is in the range of 58 to 62 °C, such as in the range 58 to 60 °C, such as in the range 60 to 62 °C.

[0258] In a further embodiment of the present invention the enzyme composition comprises at least one enzyme, such as at least 2 enzymes, e.g. at least 3 enzymes, such as at least 4 enzymes, e.g. at least 5 enzymes.

[0259] Preferably, the at least one enzyme may be an industrial enzyme.

[0260] An industrial enzyme may be enzymes produced by living a organism, typically microorganisms like bacteria, fungi, and yeasts, (however, some industrial enzymes may be produced by plants or animals). Industrial enzymes may be used on a large scale in various industrial processes to catalyse various chemical reactions.

[0261] The enzyme composition may involve a single enzyme or a combination of various enzymes. The decision on whether the enzyme composition comprises a single enzyme or a combination of enzymes may depend on e.g. the usage, the product to be produced and / or the organic starting material.

[0262] The enzyme treatment according to the present invention may be a one step enzymatic treatment or a sequential enzymatic treatment. The sequential enzymatic treatment may include at least a first enzymatic treatment and a second enzymatic treatment, which are performed sequentially, preferably, starting with the first enzymatic treatment followed by the second enzymatic treatment. In the event the enzymatic treatment in step (v) may be a sequential enzymatic treatment the enzyme composition may comprise two or more enzyme compositions, which may be sequentially added to the fermented material.

[0263] It may however be preferred that the enzyme composition may be a single enzyme composition comprising one or more enzyme, such as two or more enzymes, e.g. 3 or more enzyme, such as 4 or more enzymes, e.g. 5 or more enzymes, as described herein.

[0264] The single enzyme composition may preferably be used in a one step enzymatic treatment of the fermented material.

[0265] Preferably, the enzyme treatment according to the present invention may be a one step enzymatic treatment, performed by adding the enzyme composition comprises at least one enzyme to the to the fermented material.

[0266] In an embodiment of the present invention the addition of the enzyme composition may be a one step enzymatic treatment.

[0267] Even additional enzymatic composition may be added to the fermented material, the one step enzymatic treatment may relate to a process where no additional pH adjustments may be performed after the initial addition of the enzyme composition.

[0268] In an embodiment of the present invention the fermented material may be subjected to a pH adjustment before addition of the enzymatic composition.

[0269] Preferably, the pH-value of the fermented material may be adjusted to a pH-value in the range of pH 5-10, such as in the range of pH 6-9.5, e.g. in the range of pH 7-9, such as in the range of pH 8-8.5, before the enzymatic composition may be added to the fermented material.

[0270] When the enzymatic treatment is a sequential enzymatic treatment (e.g. a 2-step enzymatic treatment, a 3-step enzymatic treatment, a 4-step enzymatic treatment or the like) at least one of the enzymatic treatments may be performed at a pH value in the range of pH 5-10, such as in the range of pH 6-9.5, e.g. in the range of pH 7-9, such as in the range of pH 8-8.5.

[0271] In an embodiment of the present invention the enzyme composition comprises at least 2 enzymes, such as at least 3 enzymes, e.g. at least 4 enzymes, wherein the at least 2 enzymes, at least 3 enzymes, at least 4 enzymes may be selected from the group consisting of a protease, a carbohydrase, a glutaminase, and a nuclease.

[0272] The protease may comprise an endo-protease, an exo-protease, or a combination of an endo-protease and an exo-protease.

[0273] Preferably, the enzymatic composition comprises at least 2, such as 3, e.g. 4 enzymes, wherein the at least 2, e.g. the at least 3, such as at least 4 enzymes may be selected from the group consisting of a protease (e.g. an endo-protease, an exo-protease, or a combination of an endo-protease and an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0274] The enzymatic composition according to the present invention may comprise at least a protease, in particular an endo-protease, and / or an exo-protease.

[0275] The enzymatic composition according to the present invention may comprise at least a protease (in particular an endo-protease, an exo-protease), and a carbohydrase.

[0276] Preferably, the enzymatic composition comprises a protease (in particular an endo- protease, an exo-protease), a glutaminase, and a nuclease.

[0277] In an embodiment of the present invention the enzymatic composition comprises at least a protease (in particular an endo-protease, an exo-protease), a carbohydrase, and a glutaminase.

[0278] In a further embodiment of the present invention the enzymatic composition comprises at least a protease (in particular an endo-protease, an exo-protease), a carbohydrase, and a nuclease. In yet an embodiment of the present invention the enzymatic composition comprises a protease (in particular an endo-protease, an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0279] In yet an embodiment of the present invention the enzymatic composition consists essentially of a protease (in particular an endo-protease, an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0280] The enzymatic composition according to the present invention may comprise at least a carbohydrase, a glutaminase, and a nuclease.

[0281] The enzymatic composition according to the present invention may comprise a carbohydrase, a glutaminase, or a nuclease.

[0282] The enzymatic composition according to the present invention may comprise:

[0283] - a protease (in particular an endo-protease, an exo-protease), and

[0284] - a glutaminase, and / or a nuclease.

[0285] In an embodiment of the present invention the enzymatic composition comprises a protease (in particular an endo-protease, an exo-protease), a glutaminase, and a nuclease.

[0286] In a further embodiment of the present invention relates to an enzyme composition comprising a protease (in particular an endo-protease, an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0287] A preferred embodiment of the present invention relates to an enzyme composition comprising a carbohydrase in combination with a protease, a glutaminase, and / or a nuclease.

[0288] Preferably, the enzyme composition comprising the combination of a carbohydrase, a protease, and a nuclease.

[0289] Even more preferably, the enzyme composition comprising the combination of a carbohydrase, a protease, a glutaminase, and a nuclease. The enzyme composition may comprise a nuclease.

[0290] The nuclease may be cleaved the phosphodiester bonds between nucleotides in a nucleic acid, in particular the nuclease may be hydrolysing DNA and / or RNA. This hydrolysis may occur in a sequence-specific manner, where the nuclease targets and cuts at particular sequences of nucleotides.

[0291] The nuclease may be an endonucleases, an exonucleases, a restriction Enzymes, a RNAse, or a combination hereof.

[0292] Preferably the nuclease may be selected from the group of nucleases consisting of Ambion™ RNase, Deamizyme T “Amano”, Nuclease E“Amano”7, Amano’s Deamizyme 50000G Nuclease NP-1 , Deaminase D-100, Complex protease EF 108, or a combination hereof. The nucleases Nuclease E”amano”7, Deamizyme 50000G or a combination hereof may be most preferred.

[0293] The content of the nuclease in the enzyme composition may be in the range of 10-50% (w / w) of the total enzyme content of the enzyme composition, such as in the range of 15-40% (w / w), e.g. in the range of 20-30, such as about 25% (w / w).

[0294] The content of the nuclease in the fermented material to hydrolyse the fermented material may be in the range of 0.01-2% (w / w) on a dry matter basis, such as in the range of 0.025-1 .5% (w / w) on a dry matter basis, e.g. in the range of 0.05-1% (w / w) on a dry matter basis, such as in the range of 0.075-0.75% (w / w) on a dry matter basis, such as in the range of 0.1 -0.5% (w / w) on a dry matter basis.

[0295] In an embodiment of the present invention the fermented material may comprise an enzyme composition comprising a concentration of enzyme which is in the range of 0.04-8% (w / w) on a dry matter basis, such as in the range of 0.1-6% (w / w) on a dry matter basis, e g. in the range of 0.2-4% (w / w) on a dry matter basis, such as in the range of 0.3-3% (w / w) on a dry matter basis, such as in the range of 0.4-2.0% (w / w) on a dry matter basis.

[0296] In an embodiment of the present invention the temperature during the hydrolysis in step (v) may be in the range of 58-62°C. Preferably, the temperature during the hydrolyses performed in step (v) when the enzymatic composition may be added to the fermented material may be in the range of 40-75°C, e.g. in the range of 50-70°C, such as in the range of 55-65°C, e.g. in the range of 58-62°C, such as in the range of 60-61°C, e.g. in the range of 60.1-60.9°C.

[0297] The temperature provided during the hydrolysing step (step (v)) of the present invention may be preferred since it may allow the enzyme activity to be maintained at an optimum level, favouring the denaturation of proteins and browning process, inhibiting the growth pathogenic bacteria and mould.

[0298] Preferably, the temperature during the enzymatic treatment may be maintained constant, or substantially constant, throughout the hydrolysis in step (v). The substantially constant may relate to a deviation from the stated temperature of less than 5%, such as less than 4%, e.g. less than 3%, such as less than 2%, e.g. less than 1%.

[0299] In an embodiment of the present invention the enzyme composition may be allowed to hydrolyse the fermented material for a time period in the range of 24-105 hour, such as in the range of 40-100 hours, e.g. in the range of 50-90 hours, such as in the range of 60-80 hours.

[0300] Preferably, the enzyme composition may be allowed to hydrolyse the fermented material for a time period in the range of 10-120 hours, such as in the range of 30-110 hours, e.g. in the range of 50-105 hours, such as in the range of 70-100 hours, e.g. in the range of 80-95 hours, such as in the range of 85-90 hours, e.g. for about 87 hour.

[0301] In an embodiment of the present invention, the time interval may preferably be set to allow almost total denaturation of the proteins and starches present in the fermented material.

[0302] The time necessary to provide sufficient denaturation of the proteins and starches present in the fermented material may depend on the organic starting material, where e.g. vegetable starting materials, may require longer hydrolysing times than animal starting materials. The hydrolysis of the fermented material may be performed at a pH value in a range of pH 3-9, such as in the range of pH 3.5-8.5, e.g. in the range of pH 4-8, such as in the range of pH 4.5-7.5, e.g. in the range of pH 5-7, such as in the range of pH 5.5-6.8, e.g. in the range of pH 6.0-6.5, such as in the range of pH 6.1-6.4, e.g. in the range of pH 6.2-6.3.

[0303] The pH value of the enzyme composition may be adjusted to optimize the hydrolysis of the fermented material and / or the optimal production of the desired products.

[0304] The pH-adjusted organic material, like the pH-adjusted fungi product and the enzyme composition may be subjected to stirring during the enzymatic treatment.

[0305] To further improve hydrolysis of the fermented material, improve the speed of the process and / or improve the efficiency of the hydrolysis, the fermented material may be subjected to agitation during the hydrolysis (e.g. step (v)).

[0306] Preferably, the fermented material may during the hydrolysis be subjected to agitation at a speed in the range of 10-3000 RPM, such as in the range of 20-2000 RPM, 25- 1000 RPM, such as in the range of 30-750 RPM, e.g. in the range of 40-500 RPM, such as in the range of 50-250 RPM, e.g. in the range of 60-125 RPM, such as in the range of 70-100 RPM, e.g. in the range of 80-90 RPM.

[0307] The term “consists essentially of” may relate to a limitation of the scope of a claim to the features or steps specified, and to those features or steps, not mentioned and that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0308] A preferred embodiment of the present invention relates to an enzyme composition comprising a protease (in particular an endo-protease, an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0309] Post-treatment

[0310] The hydrolysed, fermented material may be post-treated, to provide the final savoury taste product.

[0311] In one embodiment the method comprises step (f).

[0312] In one embodiment pH is adjusted back to the initial pH of the pre-treated organic starting material. In one embodiment the hydrolysed, fermented material is separated into a solid fraction and a liquid fraction. In one embodiment the solid fraction is pressed in a hydraulic sheet press.

[0313] The solid fraction may be dried. In particular the solid fraction provided by plant-based organic starting material may be dried. In one embodiment the solid fraction is dried in a tunnel drier.

[0314] In one embodiment the hydrolysed, fermented material is filtered, such as filtered with a vibra-screen fitted filter, such as with a 300 micron filter, such as with a 40 micron filters, thereby providing a filtered, fermented material.

[0315] In one embodiment the method further comprises addition of salt to the liquid fraction.

[0316] In one embodiment the liquid fraction is heated to 85 °C for 5 minutes, such as for 10 minutes, such as for 15 minutes, such as for 25 minutes or more.

[0317] In one embodiment the salt content in savoury taste product is in the range 5 wt% to 15 wt%, such as in the range 9.8 wt% to 13.5 wt%, such as around 9.8 wt%, such as around 10.0 wt%, such as around 10.5 wt%, such as around 11.4 wt%, such as around 12.1 wt%, such as around 13.4 wt%.

[0318] In one embodiment the method further comprises a step of concentration using a dehydration technology. In one embodiment the filtered, fermented material is concentrated through dehydration technology until a brix between 55-65°, to provide a concentrated filtered material.

[0319] In one embodiment the hydrolysed, fermented material is concentrated through dehydration technology until a brix between 55-65°, to provide a concentrated, fermented material

[0320] In one embodiment the method further comprises a step of spray drying. In one embodiment spray drying includes introduction of a carrier such as maltodextrin.

[0321] In one embodiment the carrier is introduced in a ratio of dry matter and carrier in the range 2:1 to 1 :2, such as 2:1 , 1.5:1, 1 :1 , 1 :1.5. In one embodiment the hydrolysed, fermented material is combined with maltodextrin and dehydrated into a powder using spray drying technology.

[0322] In one embodiment the filtered, fermented material is combined with maltodextrin and dehydrated into a powder using spray drying technology.

[0323] In one embodiment the savoury taste product is a powder.

[0324] Product

[0325] The savoury taste product is a versatile product, with use in many food products. The savoury taste product also provides flavour, including saltiness, and thus the savoury taste product allows for a reduction in salt in the final products.

[0326] In one embodiment the savoury taste product is a concentrate, an extract, a paste, a syrup, a broth, a stock, a bouillon, a flavouring substance, a flavouring preparation, a natural flavouring and / or a base for food products such as soup, a sauce, meat alternatives, processed meat.

[0327] A preferred embodiment of the present invention relates to a food product comprising the flavouring substance obtained by the process according to the present invention, or the flavouring substance according to the present invention.

[0328] The food product according to the present invention may be a snack, a soup, a sauce, a ready to eat meal, or an artificial meat product.

[0329] The hydrolysed organic starting material may be used directly as a flavouring substance and a savoury taste product or the hydrolysed organic starting material may be further treated.

[0330] In an embodiment of the present invention further treatment may involve a separation process where a solid fraction of the hydrolysed organic starting material may be separated from a liquid fraction.

[0331] Preferably, the separation process may include decanting, filtration, centrifugation, pressing or a combination hereof. The liquid fraction may be subjected to a moisture reducing or concentrating process. This moisture reducing or concentrating process may result in a concentrated flavouring substance or a savoury taste product. In an embodiment of the present invention the liquid fraction may be dried, e.g. spray dried, to a powdered flavouring substance or savoury taste product.

[0332] The solid fraction of the hydrolysed organic starting material may comprise a wet and viscous paste comprising the dry matter and may be dried into a powdered flavouring preparation.

[0333] In an embodiment of the present invention the powdered flavouring preparation may be re-integrated in the dried (e.g. spray dried) liquid fraction providing the flavouring preparation.

[0334] Taste

[0335] The method of the present invention provides for savoury taste products with increased umami, increased saltiness perception, increased sweetness and increased Maillard reaction flavours, compared to reference methods not comprising a step of fermentation by a fungi composition, in particular not comprising koji fermentation.

[0336] The increased saltiness perception, allows for less salt addition to food products comprising the savoury taste product of the present invention.

[0337] In one embodiment the method provides a savoury taste product with enhanced taste properties, including flavour and aroma, and savoury notes, such as enhanced umami, enhanced sweetness, enhanced saltiness and enhanced Maillard reaction flavours, compared to a reference savoury taste product, wherein the reference savoury taste product is obtained from the same organic material, without fermentation in the presence of the fungi composition (step (c)).

[0338] In one embodiment the method provides a savoury taste product with more umami, as well as more sweetness, compared to the reference savoury taste product. In one embodiment the method provides a savoury taste product with more saltiness, compared to the reference savoury taste product.

[0339] In one embodiment the method provides a savoury taste product with higher saltiness perception, compared to the reference savoury taste product.

[0340] In one embodiment the method provides a savoury taste product with enhanced aroma, such as enhanced overall intensity, fishy, dark toasted, miso, meaty, mushrooms and cocoa aroma, compared to the reference savoury taste product.

[0341] In one embodiment the method provides a savoury taste product with enhanced flavour, such as enhanced yeasty, caramel and smoky flavours compared to the reference savoury taste product.

[0342] In one embodiment the method provides a savoury taste product with enhanced meaty, mushroom-like, miso-like, and / or yeasty aromas, compared to the reference savoury taste product.

[0343] In one embodiment the method provides a savoury taste product with enhanced flavour intensity, compared to the reference savoury taste product.

[0344] In one embodiment the method provides a savoury taste product with enhanced aftertaste intensity, compared to the reference savoury taste product.

[0345] In one embodiment the method provides a savoury taste product with enhanced flavour longevity and mouthfeel, compared to the reference savoury taste product.

[0346] In one embodiment the method provides a savoury taste product with enhanced cocoa and toasted aromas, compared to the reference savoury taste product.

[0347] In one embodiment the method provides a savoury taste product with reduced off- flavours and / or masked off-flavours, compared to the reference savoury taste product.

[0348] In one embodiment the method provides a savoury taste product with increased amounts of amino acids and / or fatty acid and lipid oxidation products, such as 3- methyl- 1 -butanol, 2-methyl-1 -butanol and 1-octen-3-ol, compared to the reference savoury taste product.

[0349] In one embodiment the method provides a savoury taste product with increased amounts of amino acids and / or fatty acid and lipid oxidation products, such as a 1.3 fold increase in fatty acid and lipid oxidation products, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase, in fatty acid and lipid oxidation products, compared to the reference savoury taste product.

[0350] In one embodiment the method provides a savoury taste product with increased amounts of Maillard reaction products, such as pyrazine, ethyl-pyrazine, 5-methyl-2- furanmethanol and 3-ethyl-2,5-dimethyl-pyrazine, compared to the reference savoury taste product.

[0351] In one embodiment the method provides a savoury taste product with increased amounts of Maillard reaction products, such as a 1.3 fold increase in Maillard reaction products, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in Maillard reaction products, compared to the reference savoury taste product.

[0352] In one embodiment the method provides a savoury taste product with increased amounts of phenylpropanoids, such as benzyl chloride, 1-ethenyl-4-ethyl-benzene, 2- methoxyphenol and phenylethyl alcohol, compared to a reference savoury taste product.

[0353] In one embodiment the method provides a savoury taste product with increased amounts of phenylpropanoids, such as a 1.3 fold increase in phenylpropanoids, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in phenylpropanoids, compared to the reference savoury taste product.

[0354] In one embodiment the method provides a savoury taste product with increased amounts of sesquiterpenes such as bicyclosesquiphellandrene, germacrene D and cis- Calamenene, compared to a reference savoury taste product.

[0355] In one embodiment the method provides a savoury taste product with increased amounts of sesquiterpenes, such as a 1.3 fold increase in sesquiterpines, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in sesquiterpines, compared to the reference savoury taste product.

[0356] In one embodiment the method provides a savoury taste product with increased amounts of 5’-ribonucleotides, such as Uridine Monophosphate (UMP), compared to a reference savoury taste product.

[0357] In one embodiment the method provides a savoury taste product with increased amounts of Uridine Monophosphate, such as a 1.3 fold increase in Uridine Monophosphate, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in Uridine Monophosphate, compared to the reference savoury taste product.

[0358] In one embodiment the method provides a savoury taste product with increased amounts of free amino acids, such as glutamic acid compared to a reference savoury taste product.

[0359] In one embodiment the method provides a savoury taste product with increased amounts of glutamic acid, such as a 1.3 fold increase in glutamic acid, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in glutamic acid, compared to the reference savoury taste product.

[0360] In one embodiment the savoury taste product prepared by the method of the present invention comprises at least 0.6 mM glutamic acid, such as at least in the range 0.6 to 1 mm, such as at least 1.0 to 2.0 mM, such as at least 2.0 to 3.0 mM, such as at least 3.0 to 4.3 Mm, such as at least 4.3 to 7.0 mM, such as at least 7.0 to 10.0 mM, such as at least 10.0 to 15.0 mM, such as at least 15.0 to 20.0 mM, such as at least 20.0 to 25.0 mM, such as at least 25.0 to 30.0 mM, such as at least 30.0 to 35.0 mM, such as at least 35.0 to 40.0 mM, such as at least 15.4 mM, such as at least 25 mM glutamic acid.

[0361] In one embodiment the method provides a savoury taste product with increased amounts of sugars, such as glucose, fructose and sucrose.

[0362] In one embodiment the method provides a savoury taste product with increased amounts of sugars, such as glucose, fructose and sucrose, such as a 1.3 fold increase, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in sugars, such as glucose, fructose and sucrose, compared to the reference savoury taste product.

[0363] In one embodiment the method provides a savoury taste product with a Brix value of at least 5°, such at least 10°, such at least 20°. In one embodiment the method provides a savoury taste product with a Brix value in the range 20° to 45°, such as 23° to 35°, such as 23.0° to 25.0°, such as 25.0° to 28.0°, such as 24.0° to 27.0°, such as 25.6°, such as 28.0° to 33.0°, such as 28.0° to 31.0°, such as 29.0° to 32.0°, such as 30.0° to 35.0°.

[0364] In one embodiment the method provides a savoury taste product comprising at least 5 mM glucose, such as at least 10 mM glucose, such as at least 25 mM glucose, such as at least 50 mM glucose, such as at least 75 mM glucose, such as at least 100 mM glucose, such as at least 130 mM glucose, such as at least 200 mM glucose, such as at least 300 mM glucose, such as at least 400 mM glucose, such as at least 500 mM glucose, such as at least 600 mM glucose, such as at least 700 mM glucose.

[0365] In one embodiment the method provides a savoury taste product comprising at least 5 mM fructose, such as at least 25 mM fructose, such as at least 50 mM fructose, such as at least 75 mM fructose, such as at least 100 mM fructose, such as at least 200 mM fructose, such as at least 300 mM fructose.

[0366] In one embodiment the method provides a savoury taste product comprising at least 5 mM sucrose, such as at least 10 mM sucrose, such as at least 20 mM sucrose, such as at least 30 mM sucrose, such as at least 40 mM sucrose, such as at least 50 mM sucrose, such as at least 100 mM sucrose, such as at least 200 mM sucrose, such as at least 300 mM sucrose, such as at least 400 mM sucrose, such as at least 500 mM sucrose, such as at least 600 mM sucrose, such as at least 700 mM sucrose.

[0367] In one embodiment the savoury taste product is produced without introduction of yeast and / or yeast extracts.

[0368] The flavouring preparation according to the present invention may include flavour compounds, aroma compounds, natural flavour compounds, flavour enhancers, and / or flavour extracts. The flavouring preparation, as well as the savoury taste product of the present invention may: provide part of the water required for plant-based products' production (through hydration of plant-based protein e.g. soy, pea protein, fava, lentils);

[0369] - allow for meat content reduction in hybrid food products;

[0370] - increase the overall umami content of the final food product;

[0371] - increase salt perception in final product thereby decreasing the need of additional salt;

[0372] - increase sugar / sweet perception in final product thereby decreasing the need of additional sugar to a food product;

[0373] - mask off flavours associated with plant-based products (beany flavours as well as grassy and / or bitter flavours);

[0374] - fit for organic and non-organic end products;

[0375] - add colour to plant-based products to resemble meat like products;

[0376] - add natural sugars to the product to which it is added increasing opportunities for Maillard-derived flavours through browning, roasting, pan frying etc.;

[0377] - provide complex flavour profile increasing the diversity of food applications; provide long after-taste; provide shortened ingredient list of the food product to which it is added;

[0378] - facilitate the removal of additives in food products;

[0379] - increase consumption of food products containing natural flavouring preparations;

[0380] - promote consumption of healthier products, which are not ultra-processed.

[0381] Thus, inventors of the present invention surprisingly found that process according to the present invention exceeds more conventional processes by:

[0382] - enhanced umami flavour;

[0383] - reduced production time;

[0384] - presence of browning flavours;

[0385] - Fit for both organic and non-organic production lines;

[0386] - reduced water consumption; repurposing of industrial food side stream;

[0387] - compliance with SDGs number 9 (“Industry, Innovation and Infrastructure”);

[0388] 12(“Responsible Consumption and Production”), 13(“Climate Action”);

[0389] - reduced energy use; - avoidance of synthetic additives;

[0390] - allowing for clean label products;

[0391] - inactivation of enzymes used in the hydrolysis process;

[0392] - reduced operation and capital costs;

[0393] - using the inherent water content of the organic material, e.g. the fungi product, for the enzymatic treatments according to the present invention; and / or

[0394] - preserving valuable and / or nutritional compounds, such as e.g. free amino acids and / or peptides and nucleotides.

[0395] Preferably, the flavouring substance may be a savoury flavouring substance, such as a natural savoury flavouring substance, and / or a savoury taste product.

[0396] In an embodiment of the present invention the savoury taste product may be enhancing the umami flavour, the kokumi flavour, and / or the Maillard flavour (Maillard reaction products).

[0397] The process according to the present invention may involve production of the umami flavour, the kokumi flavour, and / or the Maillard flavour individually or production of two of them or all three.

[0398] A preferred embodiment of the present invention relates to a flavouring substance comprising the combination of one or more reaction product obtained from fermenting an organic starter material with a fungi composition according to the present invention, and one or more reaction product obtained from the hydrolysis of the fermented product obtained from fermenting an organic starter material with a fungi composition (the fermented material) using an enzyme composition according to the present invention.

[0399] The one or more reaction products obtained from fermenting an organic starter material with the fungi composition may include free amino acids, in particular glutamic acid, aspartic acids; small peptides; free sugars; and volatile aroma compounds such as alcohols, aldehydes, ketones, and esters. In particular the one or more reaction products obtained from fermenting an organic starter material with the fungi composition may include glutamic acid, nucleic acids, aspartic acid, inosinate, guanylate, 5’-xanthosine monophosphate (XMP), adenylate, kojic acid, fumaric acid, malic acid, glutamine, leucine, tryptophane, mannitol and / or xylitol.

[0400] The one or more reaction products obtained from hydrolysing the fermented material with the enzyme composition may include free amino acids, in particular glutamic acid, aspartic acids; small peptides (the ones related to kokumi, for example gamma glutamyl tripeptides) other kokumi compounds such as glutathione); free sugars; and flavour nucleotide especially IMP, GMP, AMP, CMP and UMP.

[0401] In an embodiment of the present invention the flavouring substance comprises glutamic acid, aspartic acid and / or nucleic acids, wherein the content of the glutamic acid, aspartic acid and / or nucleic acids in the flavouring composition may be at least 50% higher than the content of the glutamic acid, aspartic acid and / or nucleic acids of the starting material before being processed according to the present invention, such as at least 100% higher, e.g. at least 150% higher, such as at least 200% higher, e.g. at least 250% higher, such as at least 300% higher, e.g. at least 350% higher, such as at least 400% higher, e.g. at least 450% higher, such as at least 500% higher, e.g. at least 550% higher.

[0402] The content of the glutamic acid, aspartic acid and / or nucleic acids of the starting material before being processed may be determined either by analysing the starting material (before starting the process) or by a search in a governmental database, e.g. the Frida food data provided by the National Food Institute, Technical University of Denmark, Research Group for Nutrition, Sustainability and Health Promotion.

[0403] Items

[0404] 1 . A method for producing a savoury taste product from an organic starting material, the method comprising the steps of: a) providing the organic starting material; b) optionally pre-treatment of the organic starting material, thereby providing a pre-treated organic starting material; c) fermentation of the organic starting material of step a) or of step b) in the presence of a fungi composition, preferably wherein the fungi composition comprises Aspergillus Sojae and Aspergillus Oryzae, thereby providing a fermented material; d) optionally contacting the fermented material with one or more carbohydrase e) hydrolysis of the fermented material in the presence of an enzyme composition comprising at least one protease, and at least one glutaminase, thereby providing a hydrolysed, fermented material; and f) optionally a step of recovering, concentrating, dehydrating and / or purifying the hydrolysed, fermented material thereby providing the savoury taste product.

[0405] 2. The method according to item 1 , wherein the savoury taste product is a concentrate, an extract, a pate, a syrup, a broth, a stock, a bouillon, a flavouring substance, a flavouring preparation, a natural flavouring and / or a base for food products such as soup, a sauce, meat alternatives, processed meat .

[0406] 3. The method according to any one of the preceding items, wherein the savoury taste product has enhanced taste properties, including flavour and aroma, and savory notes, such as enhanced umami, enhanced sweetness, enhanced saltiness and enhanced Maillard reaction flavours, compared to a reference savoury taste product, wherein the reference savoury taste product is obtained from the same organic material, without fermentation in the presence of the fungi composition (step (c)).

[0407] 4. The method according to any one of the preceding items, wherein the savoury taste product has more umami, as well as more sweetness, compared to the reference savoury taste product. 5. The method according to any one of the preceding items, wherein the savoury taste product has more saltiness, compared to the reference savoury taste product.

[0408] 6. The method according to any one of the preceding items, wherein savoury taste product has enhanced aroma, such as enhanced overall intensity, fishy, dark toasted, miso, meaty, mushrooms and cocoa aroma, compared to the reference savoury taste product.

[0409] 7. The method according to any one of the preceding items, wherein the savoury taste product has enhanced flavour, such as enhanced yeasty, caramel and smoky flavours compared to the reference savoury taste product.

[0410] 8. The method according to any one of the preceding items, wherein the savoury taste product has enhanced meaty, mushroom-like, miso-like, and / or yeasty aromas, compared to the reference savoury taste product.

[0411] 9. The method according to any one of the preceding items, wherein the savoury taste product has enhanced flavour intensity, compared to the reference savoury taste product.

[0412] 10. The method according to any one of the preceding items, wherein savoury taste product has enhanced aftertaste intensity, compared to the reference savoury taste product.

[0413] 11. The method according to any one of the preceding items, wherein the savoury taste product has enhanced flavour longevity and mouthfeel, compared to the reference savoury taste product.

[0414] 12. The method according to any one of the preceding items, wherein the savoury taste product has enhanced cocoa and toasted aromas, compared to the reference savoury taste product. 13. The method according to any one of the preceding items, wherein the savoury taste product has reduced off-flavours and / or masked off-flavours, compared to the reference savoury taste product.

[0415] 14. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of amino acids and / or fatty acid and lipid oxidation products, such as 3-methyl-1-butanol, 2-methyl-1 -butanol and 1- octen-3-ol, compared to the reference savoury taste product.

[0416] 15. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of amino acids and / or fatty acid and lipid oxidation products, such as a 1 .3 fold increase in fatty acid and lipid oxidation products, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase, in fatty acid and lipid oxidation products, compared to the reference savoury taste product.

[0417] 16. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of Maillard reaction products, such as pyrazine, ethyl-pyrazine, 5-methyl-2-furanmethanol and 3-ethyl-2,5-dimethyl- pyrazine, compared to the reference savoury taste product.

[0418] 17. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of Maillard reaction products, such as a 1.3 fold increase in Maillard reaction products, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in Maillard reaction products, compared to the reference savoury taste product.

[0419] 18. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of phenylpropanoids, such as benzyl chloride, 1-ethenyl-4-ethyl-benzene, 2-methoxyphenol and phenylethyl alcohol, compared to a reference savoury taste product.

[0420] 19. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of phenylpropanoids, such as a 1.3 fold increase in phenylpropanoids, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in phenylpropanoids, compared to the reference savoury taste product.

[0421] 20. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of sesquiterpenes such as bicyclosesquiphellandrene, germacrene D and cis-Calamenene, compared to a reference savoury taste product.

[0422] 21. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of sesquiterpenes, such as a 1.3 fold increase in sesquiterpines, such as a 1 .5 fold increase, such as a two-fold increase, such as a three-fold increase in sesquiterpines, compared to the reference savoury taste product.

[0423] 22. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of 5’-ribonucleotides, such as Uridine Monophosphate (UMP), compared to a reference savoury taste product.

[0424] 23. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of Uridine Monophosphate, such as a 1.3 fold increase in Uridine Monophosphate, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in Uridine Monophosphate, compared to the reference savoury taste product.

[0425] 24. The method according to any one of the preceding items, wherein savoury taste product has increased amounts of free amino acids, such as glutamic acid compared to a reference savoury taste product.

[0426] 25. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of glutamic acid, such as a 1.3 fold increase in glutamic acid, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in glutamic acid, compared to the reference savoury taste product. 26. The method according to any one of the preceding items, wherein the savoury taste product comprises at least 0.6 mM glutamic acid, such as at least in the range 0.6 to 1 mm, such as at least 1.0 to 2.0 mM, such as at least 2.0 to 3.0 mM, such as at least 3.0 to 4.3 Mm, such as at least 4.3 to 7.0 mM, such as at least 7.0 to 10.0 mM, such as at least 10.0 to 15.0 mM, such as at least 15.0 to 20.0 mM, such as at least 20.0 to 25.0 mM, such as at least 25.0 to 30.0 mM, such as at least 30.0 to 35.0 mM, such as at least 35.0 to 40.0 mM, such as at least 15.4 mM, such as at least 25 mM glutamic acid.

[0427] 27. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of sugars, such as glucose, fructose and sucrose.

[0428] 28. The method according to any one of the preceding items, wherein the savoury taste product has increased amounts of sugars, such as glucose, fructose and sucrose, such as a 1.3 fold increase, such as a 1.5 fold increase, such as a two-fold increase, such as a three-fold increase in sugars, such as glucose, fructose and sucrose, compared to the reference savoury taste product.

[0429] 29. The method according to any one of the preceding items, wherein the savoury taste product has a Brix value of at least 5°, such at least 10°, such at least 20°.

[0430] 30. The method according to any one of the preceding items, wherein savoury taste product has a Brix value in the range 20° to 45°, such as 23° to 35°, such as 23.0° to 25.0°, such as 25.0° to 28.0°, such as 24.0° to 27.0°, such as 25.6°, such as 28.0° to 33.0°, such as 28.0° to 31 .0°, such as 29.0° to 32.0°, such as 30.0° to 35.0°.

[0431] 31. The method according to any one of the preceding items, wherein the savoury taste product comprises at least 5 mM glucose, such as at least 10 mM glucose, such as at least 25 mM glucose, such as at least 50 mM glucose, such as at least 75 mM glucose, such as at least 100 mM glucose, such as at least 130 mM glucose, such as at least 200 mM glucose, such as at least 300 mM glucose, such as at least 400 mM glucose, such as at least 500 mM glucose, such as at least 600 mM glucose, such as at least 700 mM glucose. The method according to any one of the preceding items, wherein savoury taste product comprises at least 5 mlVI fructose, such as at least 25 mM fructose, such as at least 50 mM fructose, such as at least 75 mM fructose, such as at least 100 mM fructose, such as at least 200 mM fructose, such as at least 300 mM fructose. The method according to any one of the preceding items, wherein the savoury taste product comprises at least 5 mM sucrose, such as at least 10 mM sucrose, such as at least 20 mM sucrose, such as at least 30 mM sucrose, such as at least 40 mM sucrose, such as at least 50 mM sucrose, such as at least 100 mM sucrose, such as at least 200 mM sucrose, such as at least 300 mM sucrose, such as at least 400 mM sucrose, such as at least 500 mM sucrose, such as at least 600 mM sucrose, such as at least 700 mM sucrose. The method according to any one of the preceding items, wherein the savoury taste product is produced without introduction of yeast, such as yeast extracts. The method according to any one of the preceding items, wherein the organic starting material is one or more material selected from vegetable, meat, poultry, legume, crustacean, and mushroom. The method according to any one of the preceding items, wherein the organic starting material is a vegetable, such as leek, onion or parsley, and / or a root vegetable, such as carrot. The method according to any one of the preceding items, wherein the organic starting material is mushroom, such as a Agaricus bisporus mushroom. The method according to any one of the preceding items, wherein the organic starting material is poultry such as chicken. The method according to any one of the preceding items, wherein the organic starting material is lentils and / or beans. 40. The method according to any one of the preceding items, wherein the organic starting material is lentils, such as red lentils, such as green lentils.

[0432] 41. The method according to any one of the preceding items, wherein the organic starting material is beans, such as white beans.

[0433] 42. The method according to any one of the preceding items, wherein the organic starting material is a crustacean such as crab, such as shore crab.

[0434] 43. The method according to any one of the preceding items, wherein the organic starting material is fruit, such as apple.

[0435] 44. The method according to any one of the preceding items, wherein the organic starting material is a grain, such as corn or rice.

[0436] 45. The method according to any one of the preceding items, wherein the organic starting material is a meat, such as beef.

[0437] 46. The method according to any one of the preceding items, wherein the organic starting material is a fish or shrimp.

[0438] 47. The method according to any one of the preceding items, wherein the method comprises step (b).

[0439] 48. The method according to any one of the preceding items, wherein pre-treatment includes thawing, rehydration, boiling, steaming and / or roasting.

[0440] 49. The method according to any one of the preceding items, wherein pre-treatment includes mincing and / or grinding, such as mincing with a bowl cutter.

[0441] 50. The method according to any one of the preceding items, wherein pre-treatment includes combination of the organic starting material with water. 51. The method according to any one of the preceding items, wherein pre-treatment includes combination of the organic starting material with salt, such as 5 wt% salt, such as 10 wt% salt, such as 12 wt% salt, such as 15 wt% salt.

[0442] 52. The method according to any one of the preceding items, wherein pre-treatment includes mixing, such as mixing with a stick blender.

[0443] 53. The method according to any one of the preceding items, wherein the pretreated organic starting material is heated to around 60 °C, prior to step (c).

[0444] 54. The method according to any one of the preceding items, wherein the pretreated organic starting material is heated to around 60 °C, with presence of the fungi composition prior to step (c).

[0445] 55. The method according to any one of the preceding items, wherein the pretreated organic starting material is heated to around 60 °C, such as around 65 °C, such as around 70 °C, such as around 75 °C, such as around 80°C, such as around 85°C, such as around 90°C, such as around 95 °C, such as around 100 °C, prior to step (c).

[0446] 56. The method according to any one of the preceding items, wherein the pretreated organic starting material is heated to around 60 °C, such as around 65 °C, such as around 70 °C, such as around 75 °C, such as around 80°C, such as around 85°C, such as around 90°C, such as around 95 °C, such as around 100 °C, and kept at the temperature for 10 minutes prior to step (c).

[0447] 57. The method according to any one of the preceding items, wherein the pretreated organic starting material is heated to around 80 °C, and kept for 10 minutes prior to step (c).

[0448] 58. The method according to 53 to 57, wherein the pre-treated organic starting material is cooled to around 60 °C, prior to step (c).

[0449] 59. The method according to any one of the preceding items, wherein the temperature is maintained between 58-62 °C, such as around 60 °C, during step (c). 60. The method according to any one of the preceding items, wherein the temperature is maintained between 58-62 °C, such as around 60 °C, during step (c), optional step (d) and step (e).

[0450] 61. The method according to any one of the preceding items, wherein the fungi composition is a solid state fungi composition.

[0451] 62. The method according to any one of the preceding items, wherein the fungi composition comprises a substrate, such as an organic substrate, and at least two fungi selected from Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Penicillium species, Rhizopus oligosporus, Trichoderma species or Candida milleri.

[0452] 63. The method according to any one of the preceding items, wherein the method further comprises a step of obtaining the fungi composition, including inoculating the substrate with at least two fungi spores.

[0453] 64. The method according to any one of the preceding items, wherein the method further comprises the step of culturing the fungi spores for 40 to 48 hours, at a temperature between 28 °C and 32 °C, with a humidity of 70 to 80%, such as a humidity of 75%.

[0454] 65. The method according to items 62 to 64, wherein the substrate is rice, broken rice, beans, broken beans, pumpkin seeds, broken pumpkin seeds, sesame seeds, broken sesame seeds, sunflower seeds, broken sunflower seeds, cereal brans such as wheat bran, sorghum bran, rye bran, oats bran, barley bran, millet bran, maize bran, broken cereal brans, lentils, broken lentils, chickpeas, broken chickpeas, corn, broken corn, quinoa, broken quinoa or spelt, broken spelt or any combination thereof.

[0455] 66. The method according to items 62 to 64, wherein the substrate is rice.

[0456] 67. The method according to items 62 to 65, wherein the fungi spores are Aspergillus Sojae and Aspergillus Oryzae. 68. The method according to items 62 to 66, wherein the fungi spores are a blend of Aspergillus Sojae and Aspergillus Oryzae.

[0457] 69. The method according to items 62 to 67, wherein the fungi spores is a Aspergillus Sojae and Aspergillus Oryzae hybrid.

[0458] 70. The method according to items 62 to 68, wherein the fungi spores are Aspergillus Sojae and Aspergillus Oryzae in a ratio in the range 4:1 to 1 :4, such as around 4:1 , 3:1 , 2:1, 1 :1 , 1:2, 1 :3 or 1 :4.

[0459] 71. The method according to items 62 to 69, wherein the fungi spores are Aspergillus Sojae and Aspergillus Oryzae, with a ratio of Aspergillus Sojae to Aspergillus Oryzae of around 1 :1.

[0460] 72. The method according to any one of the preceding items, wherein the method further comprises the step of inactivating the fungi composition prior to use in step (c).

[0461] 73. The method according to any one of the preceding items, wherein the method comprises step (d).

[0462] 74. The method according to item 73, wherein the organic starting material is a fibrous material, such as a plant material.

[0463] 75. The method according to any one of the preceding items, wherein the one or more carbohydrase is a broad spectrum carbohydrase.

[0464] 76. The method according to any one of the preceding items, wherein the one or more carbohydrase is a beta-glucanase, such as an endo-beta-glucanase.

[0465] 77. The method according to any one of the preceding items, wherein the one or more carbohydrase has activity as xylanase, cellulase and / or hemicellulase

[0466] 78. The method according to any one of the preceding items, wherein the one or more carbohydrase is selected from VinoTaste Pro, BAN 480 L, BAN, Viscozyme L, Funagmyl 800L, and Sweetzyme IT or combinations thereof. The method according to any one of the preceding items, wherein the one or more carbohydrase is Viscozyme L. The method according to any one of the preceding items, wherein the concentration of carbohydrase is in the range 0.05 wt% to 5 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt%. The method according to any one of the preceding items, wherein the concentration of carbohydrase is in the range 0.05 wt% to 10 wt% of the dry matter, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 2.5 wt%, such as around 3 wt%, such as around 5 wt%, such as around 7.5 wt%, such as around 10 wt% dry matter. The method according to any one of the preceding items, wherein the concentration of carbohydrase is in the range 0.05 wt% to 2 wt% of the wet weight, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt% wet weight. The method according to any one of the preceding items, wherein step (d) includes pH adjustment. The method according to item 83, wherein the pH adjustment is performed prior to introduction of the one or more carbohydrase. The method according to items 83 to 84, wherein the pH is adjusted to acidic pH, such as pH 4.5 to 4.7. The method according to items 83 to 85, wherein the pH is adjusted by introduction of an acid, such as citric acid. 87. The method according to any one of the preceding items, wherein step (d) runs for 4 to 16 hours, or such as for 4 to 21 hours, or such as for 4 hours, or such as for 4 to 8 hours, or such as for 8 to 12 hours, or such as for 12 to 16 hours, or such as for 16 to 21 hours.

[0467] 88. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) is added in one step.

[0468] 89. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) is added in two individual steps (e1 and e2).

[0469] 90. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) is added in three individual steps (e1, e2 and e3).

[0470] 91. The method according to anyone of the preceding items, wherein the at least one protease and the at least one glutaminase of step (e) are added in one step.

[0471] 92. The method according to anyone of the preceding items, wherein the at least one protease and the at least one glutaminase of step (e) are added in two individual steps, (e1 and e2).

[0472] 93. The method according to anyone of the preceding items, wherein the at least one protease is added in a protease hydrolysis step (e1), and wherein the at least one glutaminase is added in a glutaminase hydrolysis step (e2).

[0473] 94. The method according to anyone of the preceding items, wherein the at least one protease is added in a first protease hydrolysis step (e1), and wherein the at least one glutaminase is added in a later glutaminase hydrolysis step (e2).

[0474] 95. The method according to anyone of the preceding items, wherein the at least one protease is added in a first protease hydrolysis step (e1), and wherein the at least one glutaminase is added in a later glutaminase hydrolysis step (e2). The method according to anyone of the preceding items, wherein the at least one protease is an endo-protease and / or an exo-protease. The method according to anyone of the preceding items, wherein the at least one protease is an endo-protease, such as a serine endo-peptidase and / or a blend of endo- and exo-peptidases. The method according to anyone of the preceding items, wherein the at least one protease is a protease selected from Umamizyme™ Pulse Protana U Boost, Protana Prime, Peptidase R, Glutaminase N 100, Protease HF"Amano"150SD, ProteAX, Flavourzyme WOOL, carboxypeptidase, aminopeptidase, SEBDigest F35 L / F35 P, SEBDigest F59 P, SEBDigest B7 P, Alcalase Pure 2.4 L, Savinase, Novo-Pro D, Neutrase, Alcalase 2.4 L FG, trypsin, THERMOASE PC10FNA, RROTIN SD-NY10, RROTIN SD-AY10, chymotrypsin, thermolysin, Protizyme FAP cone, papain, Protease M"Amano"SD, Protease AN"Amano"100SD, pronase or combinations thereof. The method according to anyone of the preceding items, wherein the at least one protease is Alcalase 2.4 L FG and Flavourzyme 1000 L. . The method according to anyone of the preceding items, wherein the protease hydrolysis step, runs for 4 to 50 hours, such as 4 to 8 hours, such as 8 to 12 hours, such as 12 to 16 hours, such as 16 to 50 hours, such as 18 to 50 hours, such as 16 to 19 hours, such as 16-24 hours, such as 19 to 25 hours, such as 25 to 30 hours, such as 35 to 40 hours, such as 40 to 45 hours, such as 45 to 50 hours, such as 46 to 50 hours, such as 50 to 55 hours, such as 55 to 60 hours, such as 60 to 65 hours, such as 65 to 70 hours, such as 64-69 hours, such as 66 to 69 hours. . The method according to any one of the preceding items, wherein the concentration of protease is in the range 0.05 wt% to 5 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% protease. 102. The method according to any one of the preceding items, wherein the concentration of protease is in the range 0.05 wt% to 5 wt% of the dry matter, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% dry matter.

[0475] 103. The method according to any one of the preceding items, wherein the concentration of protease is in the range 0.05 wt% to 2 wt% of the wet weight, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.22 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt% wet weight.

[0476] 104. The according to anyone of the preceding items, wherein the method further comprises pH adjustments prior to step (e), such as pH adjustment with a base, such as sodium carbonate or pH adjustment with an acid, such as citric acid.

[0477] 105. The method according to any one of the preceding items, wherein step (e) includes one or more pH adjustment.

[0478] 106. The method according to any one of the preceding items, wherein step (e) includes one or more pH adjustment, such as one pH adjustment prior to the protease hydrolysis step (e1), and one prior to the glutaminase hydrolysis step (e2).

[0479] 107. The method according to anyone of the preceding items, wherein the pH is adjusted to a pH in the range 8.0 to 8.6, such as 8.0 to 8.5, such as 8.0 to 8.2, such as 8.2 to 8.4, such as 8.4 to 8.6, prior to addition of the at least one protease in step (e).

[0480] 108. The method according to anyone of the preceding items, wherein the pH is adjusted to a pH in the range 4.5 to 6.5, such as 4.5 to 5.0, such as 5.0 to 5.5, such as 5.5 6.0, such as 6.0 to 6.5, prior to addition of the at least one protease in step (e). . The method according to anyone of the preceding items, wherein the method further comprises pH re-adjustment during step (e), such as one pH readjustment daily, such as two pH re-adjustments daily, such as two pH readjustment over two days, such as three pH re-adjustments over two days, such as four pH re-adjustments over two days, such as four pH re-adjustments over three days, such as five pH re-adjustments over three days. . The method according to anyone of the preceding items, wherein the protease of step (e) is added in one or more dosages, such as in one dosage, such as in two dosages, such as in three dosages. . The method according to anyone of the preceding items, wherein the glutaminase of step (e) is a gamma glutamyl transpeptidase and / or a gammaglutamyl transferase. . The method according to anyone of the preceding items, wherein the glutaminase of step (e) is Glutaminase SD-100NA. . The method according to any one of the preceding items, wherein the concentration of glutaminase is in the range 0.01 wt% to 5 wt%, such as around 0.02 wt%, such as around 0.03 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.25 wt%, such as around 0.3 wt%, such as around 0.5 wt%, such as around 1 wt%, such as around 2 wt%, such as around 3 wt%, such as around 4 wt%, such as around 5 wt% glutaminase. . The method according to any one of the preceding items, wherein the concentration of glutaminase is in the range 0.001 wt% to 0.5 wt% of the wet weight, such as around 0.001 wt%, such as around 0.005 wt%, such as around 0.01 wt%, such as around 0.02 wt%, such as around 0.03 wt%, such as around 0.04 wt%, such as around 0.05 wt%, such as around 0.1 wt%, such as around 0.2 wt%, such as around 0.3 wt%, such as around 0.4 wt%, such as around 0.5 wt%, wet weight. 115. The method according to anyone of the preceding items, wherein no pH adjustment is performed before the glutaminase hydrolysis step.

[0481] 116. The method according to anyone of the preceding items, wherein the pH is adjusted to a pH in the range 5.0 to 6.5, such as 5.0 to 5.2, such as 5.2 to 5.4, such as 5.4 to 5.6, such as 5.6 to 5.8, such as 5.8 to 6.0, such as 6.0 to 6.5 prior to addition of the at least one glutaminase in step (e).

[0482] 117. The method according to item 104, wherein the pH is adjusted to pH in the range pH 5.4 to 5.6 before the glutaminase hydrolysis step (e2).

[0483] 118. The method according to any one of the preceding items, wherein the glutaminase hydrolysis step runs for 4 to 16 hours, or such as for 4 to 21 hours, or such as for 4 hours, or such as for 4 to 8 hours, or such as for 8 to 12 hours, or such as for 12 to 16 hours, or such as for 16 to 21 hours.

[0484] 119. The method according to any one of the preceding items, wherein the glutaminase is introduced after the protease, in a glutaminase hydrolysis step (e2).

[0485] 120. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) further comprises at least one nuclease.

[0486] 121. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) further comprises at least one deaminase.

[0487] 122. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) comprises at least one protease, at least one glutaminase and at least one nuclease.

[0488] 123. The method according to anyone of the preceding items, wherein the enzyme composition of step (e) comprises at least one protease, at least one glutaminase, at least one nuclease and at least one deaminase. 124. The method according to anyone of the preceding items, wherein the at least one glutaminase and the at least one nuclease are added together, after the protease hydrolysis.

[0489] 125. The method according to anyone of the preceding items, wherein the at least one glutaminase and the at least one nuclease are added together, after the protease hydrolysis step (e1).

[0490] 126. The method according to anyone of the preceding items, wherein the at least one glutaminase, the at least one nuclease and the at least one deaminase are added together after the protease hydrolysis step (e1).

[0491] 127. The method according to anyone of the preceding items, wherein the pH is adjusted prior to the introduction of the at least one glutaminase and the at least one nuclease (step (e2)).

[0492] 128. The method according to anyone of the preceding items, wherein the pH is adjusted prior to the introduction of the at least one glutaminase, the at least one nuclease and the at least one deaminase (step (e2)).

[0493] 129. The method according to anyone of the preceding items, wherein the pH is adjusted to pH in the range 5.4 to 6.5, or such as 5.5 to 6 prior to the introduction of the at least one glutaminase and the at least one nuclease (step (e2)).

[0494] 130. The method according to anyone of the preceding items, wherein the pH is adjusted to pH in the range 5.4 to 6.5, or such as 5.5 to 6 prior to the introduction of the at least one glutaminase, the at least one nuclease and the at least one deaminase (step (e2)).

[0495] 131. The method according to anyone of the preceding items, wherein the nuclease is an endonucleases, an exonucleases, a restriction Enzymes, a RNAse, and / or combinations thereof. . The method according to anyone of the preceding items, wherein the nuclease is selected from E”Amano”7 Nuclease, Ambion™ RNase, Deamizyme T “Amano”, Amano’s Deamizyme 50000G Nuclease NP-1 , Deaminase D-100, Complex protease EF 108 and / or combinations thereof. . The method according to anyone of the preceding items, wherein the nuclease is E”Amano”7 Nuclease. . The method according to anyone of the preceding items, wherein the deaminase is Deamizyme T“Amano”50. . The method according to anyone of the preceding items, wherein the temperature during the hydrolysis in step (e) is in the range of 58 to 62 °C, such as in the range 58 to 60 °C, such as in the range 60 to 62 °C. . The method according to any one of the preceding items, wherein the method comprises step (f). . The method according to anyone of the preceding items, wherein the pH is adjusted back to the initial pH of the pre-treated organic starting material. . The method according to anyone of the preceding items, wherein the hydrolysed, fermented material is separated into a solid fraction and a liquid fraction. . The method according to item 138, wherein the solid fraction is pressed in a hydraulic sheet press. . The method according to items 138 to 139, wherein the solid fraction is dried in a tunnel drier. . The method according to anyone of the preceding items, wherein the hydrolysed, fermented material is filtered, such as filtered with a vibra-screen fitted filter, such as with a 300 micron filter, such as with a 40 micron filters, thereby providing a filtered, fermented material. 142. The method according to items 138 or 141 , wherein the method further comprises addition of salt to the liquid fraction.

[0496] 143. The method according to item 138, wherein the liquid fraction is heated to 85 °C for 5 minutes, such as for 10 minutes, such as for 15 minutes, such as for 25 minutes or more.

[0497] 144. The method according to any one of the preceding items, wherein the salt content in savoury taste product is in the range 5 wt% to 15 wt%, such as in the range 9.8 wt% to 13.5 wt%, such as around 9.8 wt%, such as around 10.0 wt%, such as around 10.5 wt%, such as around 11.4 wt%, such as around 12.1 wt%, such as around 13.4 wt%.

[0498] 145. The method according to anyone of the preceding items, wherein the method further comprises a step of concentration using a dehydration technology.

[0499] 146. The method according to item 145, wherein the filtered, fermented material is concentrated through dehydration technology until a brix between 55-65°, to provide a concentrated filtered material.

[0500] 147. The method according to item 145, wherein the hydrolysed, fermented material is concentrated through dehydration technology until a brix between 55-65°, to provide a concentrated, fermented material.

[0501] 148. The method according to item 145, wherein the hydrolysed, fermented material is concentrated through spray drying.

[0502] 149. The method according to item 148, wherein the spray drying includes introduction of a carrier such as maltodextrin.

[0503] 150. The method according to items 148 to 149, wherein the carrier is introduced in a ratio of dry matter and carrier in the range 2:1 to 1 :2, such as 2:1 , 1.5:1 , 1:1 , 1 :1.5. 151. The method according to items 148 to 150, wherein the hydrolysed, fermented material is combined with maltodextrin and dehydrated into a powder using spray drying technology.

[0504] 152. The method according to items 148 to 150, wherein the filtered, fermented material is combined with maltodextrin and dehydrated into a powder using spray drying technology.

[0505] 153. The method according to anyone of the preceding items, wherein the savoury taste product is a powder.

[0506] 154. A process for producing a flavouring substance, such as a natural flavouring substance, from an organic starting material, the process comprises the steps of:

[0507] (i) Providing the organic starting material;

[0508] (ii) Adding a fungi composition to the organic starting material;

[0509] (iii) Allowing the fungi composition to ferment the starting material providing a fermented material;

[0510] (iv) Adding an enzyme composition to the fermented material;

[0511] (v) Allowing the enzyme composition to hydrolyse the fermented material providing the flavouring substance,

[0512] (vi) Optionally purifying, treating or concentrating the flavouring substance.

[0513] 155. The process according to item 1 , wherein the enzyme composition comprises at least 2 enzymes.

[0514] 156. The process according to anyone of the preceding items, wherein the addition of enzyme composition is a one-step enzymatic treatment. 157. The process according to anyone of the preceding items, wherein the enzyme composition comprises at least 2 enzymes, wherein the at least 2 enzymes are selected from the group consisting of a protease, a carbohydrase, a glutaminase, and a nuclease.

[0515] 158. The process according to anyone of the preceding items, wherein the enzyme composition comprises a protease (in particular an endo-protease, an exo-protease), a glutaminase, and a nuclease.

[0516] 159. The process according to anyone of the preceding items, wherein the temperature during the hydrolysis in step (v) may be in the range of 58-62°C.

[0517] 160. The process according to anyone of the preceding items, wherein the enzyme composition is allowed to hydrolyse the fermented material for a time period in the range of 24-105 hour.

[0518] 161. An enzyme composition comprising a protease (in particular an endo- protease, an exo-protease), a carbohydrase, a glutaminase, and a nuclease.

[0519] 162. A flavouring substance comprising obtainable by a process according to anyone of items 1-7.

[0520] 163. A process for producing a flavouring preparation comprising a step of fermenting an organic starting material using a fungi composition resulting in a fermented material and hydrolysing the fermented material using with an enzyme composition, providing the flavouring substance.

[0521] Examples

[0522] Example 1. Savoury taste product from Agaricus Bisporus using a sequential enzymatic treatment (mushroom)

[0523] Production of a flavouring substance from Agaricus Bisporus using a sequential enzymatic treatment A fungi substrate comprising equal amounts of rice and pumpkin seeds is prepared by heat-treating the ingredients by steaming or conventional baking to obtain a sterile and easily digestible fungi substrate and supplemented a combination of fungi comprising a ratio of 1 :2 of Aspergillus Oryzae and Aspergillus Sojae, providing the fungi composition.

[0524] Allowing the fungi composition to growth to reach maturity of the fungi, thus obtaining the fungi composition.

[0525] An organic starting material, a mushroom (Agaricus Bisporus) was provided which was initially thoroughly cleaned by rinsing the mushroom from dirt, briefly rinsing and tumbling them in cold water (5°C-8°C) and excess water was drained off. This step was repeated until no visible trace of dirt identified.

[0526] The cleaned mushrooms were finely minced in a thermomixer (TM6) at maximum speed

[0527] (10 / 10) until a homogeneous minced mushroom was obtained, this process took about 3-4 minutes.

[0528] The homogeneous minced mushroom was then pasteurized in the thermomixer at 100°C for 10 minutes using blade rotation speed of 3 / 10. This process not only decreases the chances of unwanted microbial growth or presence of pathogenic microorganisms but also contributes to partial degradation of the mushroom’s cell walls.

[0529] The heated homogeneous minced mushroom was rapidly cooled to 60°C by continuous stirring in an ice-cold water bath.

[0530] The dry matter content of the homogeneous minced mushroom was determined by measuring the moisture content and subtracting the moisture content from the total weight of the homogeneous minced mushroom.

[0531] The homogeneous minced mushroom was weighed out on a scale (DYMO) and transferred into a previously sanitized condi box. Sanitation is achieved by spraying the condi box with a water solution with 70% ethanol concentration. Mix the fungi composition (comprising a ratio of 1 :2 Aspergillus Oryzae and Aspergillus Sojae) with water, salt and the organic starter material (the mushroom, Agaricus Bisporus) and allowing the mixture to ferment for 45 hours, at 30°C to obtain a fermented material and a moisture content of 75% (w / w), providing a fermented material.

[0532] After fermentation the fermented material is pasteurised.

[0533] Adjust the pH of the fermented material to a pH of 4,5 and add a carbohydrase to the slurry at a concentration of 5% (enzyme:dry matter content w / w) providing a first enzymatic treatment. The carbohydrase added was Viscozyme L.

[0534] The enzymatic treatment using carbohydrase was moved to a Simax screw top bottle (1000 ml) in which a magnet was placed. The bottle was placed in a heated water bath at 60°C and agitation (75 RPM) was provided using a magnetic stirring plate (IntILab) placed underneath the bottle, outside of the water bath.

[0535] The enzymatic treatment using carbohydrase was allowed to run for 1.5 hours providing a partly hydrolysed mushroom.

[0536] Following the enzymatic treatment using carbohydrase the acidic environment of the partly hydrolysed mushroom was adjusted to pH 8 using commercial powdered natrium hydroxide (NaOH).

[0537] The partly hydrolysed mushroom was then supplemented with a protease (in particular an endo-protease and an exo-protease), which is added to the partly hydrolysed mushroom at a concentration each of 5% (w / w) (enzyme:dry mushroom matter weight w / w).

[0538] The temperature of the water bath was increased to 60.5°C and allowed to run for 1 hour before the second enzyme was added to the partly hydrolysed mushroom.

[0539] The enzymatic treatment using the protease was allowed to proceed for about 87 hours and water was continuously refiled to the water bath. Finally, the enzymes were deactivated by placing the bottles directly in the oven at 100°C for 10 minutes and the hydrolysed mushroom obtained was neutralized to pH 6,1 using additional lactic acid, resulting in a flavouring substance.

[0540] The flavouring substance was separated into two different fractions using a fine filter mesh, resulting in a clear liquid fraction (resulting in a liquid flavouring substance) and a wet, viscous fraction consisting of the dry matter of the hydrolysed mushrooms, and some residual liquid (resulting in a solid / paste flavouring substance).

[0541] The clear liquid fraction, the liquid flavouring substance, was further reduced at low temperatures (60°C) to obtain a concentrated flavouring substance. The wet, viscous fraction was dehydrated in a Combi oven (Rational) at 60°C with 0% humidity. The solids are powdered through a thermomix and used as a dry powdered flavouring substance.

[0542] Example 2. Savoury taste product from an Agaricus Bisporus using a one step enzymatic treatment (mushroom)

[0543] Production of a flavouring substance from an Agaricus Bisporus using a one step enzymatic treatment.

[0544] A fungi substrate comprising equal amounts of rice and pumpkin seeds is prepared by heat-treating the ingredients by steaming or conventional baking to obtain a sterile and easily digestible fungi substrate and supplemented a combination of fungi comprising a ratio of 1 :2 of Aspergillus Oryzae and Aspergillus Sojae, providing the fungi composition.

[0545] Allowing the fungi composition to growth to reach maturity of the fungi, thus obtaining the fungi composition.

[0546] An organic starting material, a mushroom (Agaricus Bisporus) was provided which was initially thoroughly cleaned by rinsing the mushroom from dirt, briefly rinsing and tumbling them in cold water (5°C-8°C) and excess water was drained off. This step was repeated until no visible trace of dirt identified. The cleaned mushrooms were finely minced in a thermomixer (TM6) at maximum speed (10 / 10) until a homogeneous minced mushroom was obtained, this process took about 3-4 minutes.

[0547] The homogeneous minced mushroom was then pasteurized in the thermomixer at 100°C for 10 minutes using blade rotation speed of 3 / 10. This process not only decreases the chances of unwanted microbial growth or presence of pathogenic microorganisms but also contributes to partial degradation of the mushroom’s cell walls.

[0548] The heated homogeneous minced mushroom was rapidly cooled to 60°C by continuous stirring in an ice-cold water bath.

[0549] The dry matter content of the homogeneous minced mushroom was determined by measuring the moisture content and subtracting the moisture content from the total weight of the homogeneous minced mushroom.

[0550] The homogeneous minced mushroom was weighed out on a scale (DYMO) and transferred into a previously sanitized condi box. Sanitation is achieved by spraying the condi box with a water solution with 70% ethanol concentration.

[0551] Mix the fungi composition (comprising a ratio of 1 :2 of Aspergillus Oryzae and Aspergillus Sojae) with water, salt and the organic starting material (the mushroom, Agaricus Bisporus) and allowing the mixture to ferment for 45 hours, at 30°C to obtain a fermented material and a moisture content of 75% (w / w), providing a fermented material.

[0552] After fermentation the fermented material is pasteurised.

[0553] Adjust the pH of the fermented material to a pH of 8.0 and add an enzyme composition comprising a protease (in particular an endo-protease and an exo-protease), which is added in a concentration of 5% (w / w) (enzyme:dry mushroom matter weight w / w), a nuclease added in a concentration of 5% (w / w) and a glutaminase added in a concentration of 5% (w / w). The enzymatic treatment using the enzyme composition was moved to a Simax screw top bottle (1000 ml) in which a magnet was placed. The bottle was placed in a heated water bath at 61°C and agitation (75 RPM) was provided using a magnetic stirring plate (IntILab) placed underneath the bottle, outside of the water bath.

[0554] The enzymatic treatment using the enzyme composition was allowed to proceed for about 87 hours and water was continuously refiled to the water bath.

[0555] Finally, the enzymes were deactivated by placing the bottles directly in the oven at 100°C for 10 minutes and the hydrolysed mushroom obtained was neutralized to pH 6,1 using additional lactic acid, resulting in a flavouring substance.

[0556] The flavouring substance was separated into two different fractions using a fine filter mesh, resulting in a clear liquid fraction (resulting in a liquid flavouring substance) and a wet, viscous fraction consisting of the dry matter of the hydrolysed mushrooms, and some residual liquid (resulting in a solid / paste flavouring substance).

[0557] The clear liquid fraction, the liquid flavouring substance, was further reduced at low temperatures (60°C) to obtain a concentrated flavouring substance. The wet, viscous fraction was dehydrated in a Combi oven (Rational) at 60°C with 0% humidity. The solids are powdered through a thermomix and used as a dry powdered flavouring substance.

[0558] Example 3. Savoury taste product from chicken using a one step enzymatic treatment

[0559] Production of a flavouring substance from chicken using a one step enzymatic treatment.

[0560] A fungi substrate comprising equal amounts of rice and pumpkin seeds is prepared by heat-treating the ingredients by steaming or conventional baking to obtain a sterile and easily digestible fungi substrate and supplemented a combination of fungi comprising a ratio of 1 :2 of Aspergillus Oryzae and Aspergillus Sojae, providing the fungi composition. Allowing the fungi composition to growth to reach maturity of the fungi, thus obtaining the fungi composition.

[0561] The experiment included chicken products comprising various chicken parts comprising 75% bone cake and 25% mechanically deboned meat.

[0562] Roasting the chicken product at 240°C for 20 minutes in an oven.

[0563] Water is added to the roasted chicken product in a ratio of 1 part roasted chicken product to 3.5 parts water, on a weight by weight basis.

[0564] The temperature of the roasted chicken product was rapidly adjusted to 60°C and under continuous stirring.

[0565] The dry matter content of the roasted chicken product was determined by measuring the moisture content and subtracting the moisture content from the total weight of the roasted chicken product. The moisture content of the chicken product was determined to 67%.

[0566] The roasted chicken product was weighed out on a scale (DYMO) and transferred into a previously sanitized condi box. Sanitation is achieved by spraying the condi box with a water solution with 70% ethanol concentration.

[0567] Mix the fungi composition (comprising a ratio of 1 :2 of Aspergillus Oryzae and Aspergillus Sojae) with water, salt and the roasted chicken product (the organic starter material) and allowing the mixture to ferment for 45 hours, at 30°C to obtain a fermented material and a moisture content of 75% (w / w).

[0568] After fermentation the fermented material is pasteurised.

[0569] Adjust the pH of the fermented material to a pH of 8.0 and add an enzyme composition comprising a protease (in particular an endo-protease and an exo-protease) in a concentration of 5% (w / w) (enzyme:dry mushroom matter weight w / w), a nuclease added in a concentration of 5% (w / w) and a glutaminase added in a concentration of 5% (w / w). The enzymatic treatment using the enzyme composition was moved to a Simax screw top bottle (1000 ml) in which a magnet was placed. The bottle was placed in a heated water bath at 61°C and agitation (75 RPM) was provided using a magnetic stirring plate (IntILab) placed underneath the bottle, outside of the water bath.

[0570] The enzymatic treatment using the enzyme composition was allowed to proceed for about 87 hours and water was continuously refiled to the water bath.

[0571] Finally, the enzymes were deactivated by placing the bottles directly in the oven at 100°C for 10 minutes and the hydrolysed mushroom obtained was neutralized to pH 6,1 using additional lactic acid, resulting in a flavouring substance.

[0572] The flavouring substance was separated into two different fractions using a fine filter mesh, resulting in a clear liquid fraction (resulting in a liquid flavouring substance) and a wet, viscous fraction consisting of the dry matter of the hydrolysed mushrooms, and some residual liquid (resulting in a solid / paste flavouring substance).

[0573] The clear liquid fraction, the liquid flavouring substance, was further reduced at low temperatures (60°C) to obtain a concentrated flavouring substance. The wet, viscous fraction was dehydrated in a Combi oven (Rational) at 60°C with 0% humidity. The solids are powdered through a thermomix and used as a dry powdered flavouring substance.

[0574] Example 4. Impact of Koji addition on flavor properties

[0575] Aim

[0576] The main aim of this work is to assess how the addition of Koji a fungal starter culture (Aspergillus oryzae x A. sojae hybrid) grown on a grain broken rice kernels) impacts the flavor properties of the savoury taste products of the present invention. The assessment was conducted using the following:

[0577] 1) Analytical methods, to measure levels of discreet sets metabolites known to affect flavor perception, namely:

[0578] • Aroma profiles - low molecular weight volatile organic compounds

[0579] • Taste profiles - primary sugars, free amino acids and 5’-ribonucleotides 2) Sensory methods - through trained panelist sensory evaluation

[0580] 3) Biochemical methods to assess the hydrolytic activity of Koji during the processing of the food ingredients.

[0581] Experimental design, materials and methods

[0582] To assess the contribution of Koji to flavor, we generated a simplified experimental system using three plant-based substrates: button mushrooms (Agaricus bisporus), red lentils (Lens culinaris) and white beans (Phaseolus vulgaris).

[0583] Figure 1 describes the overall process workflow and experimental design. Briefly, raw materials were purchased commercially and underwent pretreatment: mushrooms were chopped, beans and lentils were soaked overnight and steamed at 100°C for 20 mins (lentils) or 60 mins (beans), until soft. For lentil and beans, three treatments were generated: ‘with Koji’, ‘without Koji’ and ‘with inactivated Koji’; whereas only the first two variations were generated for the mushroom substrate. Accordingly, substrates were mixed with live or inactivated rice Koji (Aspergillus oryzae x A. sojae hybrid, 15% w / w, inactivation achieved by heating the culture to 85°C, 15 mins), water (70-80% w / w of final volume) and salt (5% w / w). Koji was omitted completely for the ‘without Koji’ treatment.

[0584] To compensate for the difference in raw material composition in the ‘without Koji’ treatments, the main substrate amount was adjusted to maintain an identical ratio between the water and the main substrates (i.e. mushrooms, lentils or beans).

[0585] For the mushroom preparation alone, pH was adjusted to 4.5 with powdered citric acid, after which Viscozyme L (0.29%, Novozymes, Denmark) was added for 4 hrs at 60°C. For all substrates, pH was subsequently adjusted to 8.5, before Alcalase 2.4 (0.3%, Sigma-Aldrich, Denmark) and Flavourzyme (0.29%, Novozymes, Denmark) were added and allowed to operate for 36 hrs, 60°C, with periodic pH adjustments. For the final hydrolysis step, pH was adjusted to 5.5 and preparations were supplemented with E”Amano”7 Nuclease, SD-100NA Glutaminase and T“Amano”50 Deamizyme (all 0.02%, Amano Enzyme, Japan). Enzymes were allowed to operate for 24 hrs at 60°C. All products were then filtered and heat-treated for enzymes inactivation (15 mins, 80°C). 1 . Aroma profiling using Headspace Solid-Phase Micro-Extraction coupled with Gas Chromatography Mass Spectrometry (HS-SPME-GCMS)

[0586] All products generated in the experiments detailed above, as well as active Koji culture, were analyzed for aroma profiles using headspace solid-phase micro-extraction (HS- SPME) coupled with GCMS as in (Weinblum et al., 2021). Samples (1 gr / sample, n=3) placed in 20 ml SPME glass vials (MikroLab, Denmark), containing 1 gr of NaCI and 7 ml of a 20% (w / v) NaCI solution. 1 -Pentanol, 4-methyl- (10 ppm, Sigma-Aldrich, Denmark), was used as the internal standard. Vials were pre-incubated for 15 min, 60°C, in the ACC-6000 autosampler oven (Shimadzu, Japan) to promote volatile release into the headspace. Then, a 10 mm long DVB / CAR / PDMS fiber, assembly 50 / 30 pm (Supelco, USA), was inserted into the vial (22 mm) for 30 min at 60°C. Volatile compounds were desorbed for 10 min at 250°C, and injected into a gas chromatograph (Nexis GC-2030, Shimadzu, Japan), equipped with a HP5MS capillary column (30m length, 0.25 mm I.D., 0.25 pm film thickness, Agilent, USA) and coupled to a GCMS- QP2020 single quadrupole mass spectrometer (Shimadzu, Japan). Helium was used as a carrier gas at a rate of 1 ml / min. Injection temperature was set to 220°C (splitless mode), the interface temperature to 250°C and the ion source to 250°C. Analysis was performed under the following conditions: 1 min of isothermal heating at 40°C, followed by 6°C / min oven temperature ramp to 250°C and then increased to 300°C for 5 mins. Mass acquisition was set to a range of 40-250 m / z, with scanning rate of 9.12 spectra / s. Retention index (Rl) was calculated by running C8-C20 n-alkanes (Sigma-Aldrich, Denmark) under the same conditions listed above.

[0587] Compounds were identified by Wiley 10 with NIST 2014 mass spectral library data using the Mass Hunter software package (version B.08.00, Agilent, USA). Further identification of major compounds was based on a comparison of mass spectra and retention index. Compounds with authentic standards (Sigma-Aldrich, Denmark) were analyzed under similar conditions. Quantitative evaluation was performed using internal standard; peak areas were normalized to that of the ISTD.

[0588] 2. Taste-related profiling using High Performance Liquid Chromatography

[0589] Free amino acid analysis was conducted as in (Vinther Schmidt et al., 2021) using a Ultra High Performance Liquid Chromatograph (UHPLC, LC-2030C, Shimadzu, Japan), equipped with a revered-phase column (Shim-pack XR-ODSII, 3 x 150 mm, 2.2 m particle size, Shimadzu, Japan) and a PDA detector (LC-2060C, Shimadzu, Japan). Amino acids were derivatized with a solution of 10 mM o-phthaldialdehyde (OPA) / 3.15 mM 3-mercapto-propionic acid (MPA), prepared in 0.1 M borate buffer pH 9.5. An additional derivatization step was conducted with 1 mM 9-fluorenylmethyl chloroformate (FMOC) solution. Derivatization reactions were terminated using 0.1 M phosphoric acid. A 3-mobile phase gradient system was applied with mobile phase A (20 mM sodium acetate pH 6), mobile phase B (9:1 w / w acetonitrile: H2O) and mobile phase C (20 mM sodium acetate pH 5, containing 0.5 mmol / L EDTA-2Na). Column oven temperature was set to 40 °C; flow rate of 1 mL / min using the following gradient conditions (Table 1):

[0590] Table 1. Mobile phase conditions during the UHPLC run for amino acids

[0591] Data collection rate was set to 25 Hz and response time was 0.8 s, and recorded for 350 and 266 nm, corresponding to OPA and FMOC, respectively. A standard curve (0-2.5 mM each) was constructed using an amino acid standard mix (Sigma-Aldrich, Denmark). All samples analyzed in triplicates.

[0592] 5’-Ribonucleotide analysis was carried out as in (Poojary et al., 2017). Briefly, samples were treated as described above for amino acid analysis. A two mobile phase gradient system was applied with mobile phase A (KH2PO4 buffer, pH 4.8), and mobile phase B (100% MeOH). Column oven temperature was set to 40 °C; flow rate of 1 mL / min using the following gradient conditions (Table 2):

[0593] Table 2. Mobile phase conditions during the UHPLC run for 5’-ribonucleotides Data collection rate was set to 25 Hz and response time was 0.8 s, and recorded for 254 nm. A standard curve (0-250 pg / ml) was constructed using the five main 5’- ribonucleotides (Adenosine 5'-monophosphate [AMP], Guanosine 5 -monophosphate [GMP], Inosine 5'-monophosphate [IMP], Uridine 5'-monophosphate [UPM], Sigma- Aldrich, Denmark and Xanthosine 5'-monophosphate [XMP], Bioynth, Slovakia). All samples analyzed in triplicates.

[0594] Sugar analysis was carried using a Shim-pack SCR-102H ion-exclusion column (Shimadzu, Japan) and a Refractive Index Detector (RID-20A, Shimadzu, Japan). A single mobile phase was used (0.01 M H2SO4). Column oven temperature was set to 40 °C; flow rate of 0.6 mL / min for 30 mins. Data collection rate was set to 25 Hz and response time was 0.8 s. A standard curve (0-75 mM) was constructed using sucrose, fructose and glucose authentic standards (Sigma-Aldrich, Denmark). All samples analyzed in triplicates.

[0595] 3. Sensory analysis

[0596] A full sensory descriptive analysis was performed on lentil-based ferments with or without Koji. Undiluted samples were allowed to reach room temperature and divided into serving portions for each panelist (10 gr), and kept at 20°C until serving in a KB8182 cabinet (Termaks, Norway).

[0597] Panelists (n=10, 7 females and 3 males) were screened based on the ISO guidelines (ISO 3972:2011) and their ability to verbally convey sensory impressions. The analysis was carried out over three consecutive days, consisting of 6 hrs of training and 1-2 hrs scoring. Training included developing a set of consensus sensory descriptors, perfecting a group of references for each descriptor as well as training the panelists in scoring the samples and aligning their scaling according to attribute intensity. Briefly, 20 descriptors were clustered into sample odor (ortho-nasal perception, 8), basic taste (2), flavor (oral and retro-nasal perception, 5), mouthfeel (2) and aftertaste (2).

[0598] All compounds and concentrations presented were in the ranges allowed by the ISO standard for selection and training of panelists (ISO 13299). Evaluation was conducted in a sensory test facility, with panelists seated in individual booths, each equipped with a source of light and air-flow, for a duration of 1 -2 hrs. Panelists used the aforementioned 20 descriptors to evaluate two samples in three replicates. Descriptors were scored using a continuous 15 cm scale for each feature (left endpoint: zero or lowest intensity / property, right endpoint: maximum intensity / property). Sample order was randomized in each session, according to a Williams’ Latin Square, to counteract for first- order carry-over effects. Evaluations were registered on a digital interface, where the data was collected in FIZZ Network Acquisition ver. 2.4 OE (Biosystemes, France). The general performance of the panelists was checked using Panelcheck software ver. 1.3.2 (Nofima, Norway).

[0599] 4. Koji proteolytic activity assay

[0600] Proteolytic activity in live and inactivated Kojis was assessed using an azocasein-based assay. Azocasein (2%) was prepared in 100 mM Tris-HCI buffer (pH 8.0). Reaction mixtures containing equal volumes of sample azocasein solution were incubated at 55°C for 60 minutes. The reaction was terminated by adding 5% (w / v) TCA. Samples were then centrifuged at 3000 rpm for 5 minutes, and the supernatant was filtered through a 0.45 pm membrane. Absorbance at 440 nm was measured to determine enzymatic activity. For the calibration curve, commercial endoprotease dilutions (0-5 ppm) were prepared and measured.

[0601] 5. Statistical analysis

[0602] One and two-way ANOVA, Student’s T-test and Tukey’s post-hoc test were conducted using JMP ver.pro 16.0 statistical package (SAS Institute, USA).

[0603] Results

[0604] 1 . Aroma profiling of Koji-contaim ng products

[0605] Our approach in assessing the contribution of Koji to the flavor of analyzed products was based on the notion that food flavor is determined by the interaction between raw materials (in this case, different plant-based protein-rich substrates) and the processes imposed by the W2 technology of the present invention (i.e., solid-state fermentation, enzymatic hydrolysis and mild heating). We therefore started by analyzing the aroma profiles of active Koji culture by means of GCMS. The full list of aroma compounds found in Koji and their classification can be found in Table 3, below.

[0606] Table 3. Aroma compounds found in active Koji culture, their chemical classification and sensory descriptions.

[0607] Koji aroma profile was found to be dominated by: i) fatty acid and lipid oxidation products, primarily esters and alcohols, eliciting fruity, sweet notes, ii) phenylpropanoids (phenylalanine derivatives), such as phenylethyl alcohol and benzeneacetaldehyde, bearing floral, spice-like and honey aromas, and iii) terpenoids, mostly sesquiterpenes. These results are consistent with previous reports focused on Koji aroma profiles (Ito et al., 1990; Li et al., 2023). Next, we analyzed mushroom ferments that were produced with or without Koji. Overall, 86 aroma compounds were identified across treatments, with 16 compounds shared between Koji and mushroom products. Next, we focused on compounds found to be either over- or under-represented in products containing Koji compared to those without (Overrepresented with koji: alcohols, aldehydes, furans, ketones, sesquiterpenes and sulfides, underrepresented with koji: Pyrazines).

[0608] Koji addition to the mushroom products significantly increased the levels of aroma compounds of several classes, mainly alcohols, sesquiterpenes and sulfides, in range of x1.2-x50 fold change. In parallel, a decrease in levels of pyrazines was noted, between 25-98% reduction. Notably, out of the 16 compounds shared between the aroma profiles of mushrooms and the Koji itself, 10 were significantly enriched in the ‘with Koji’ treatment. Based on publically available databases and results presented here, the aroma of products prepared with Koji are projected to be a complex blend of fruity notes combined with herbal and floral undertones, as well as mushroom, earthy, waxy and sulfurous aromas.

[0609] Next, we analyzed the aroma profiles of the bean and lentil products made with either live or inactivated Koji, and compared them to the corresponding products prepared without Koji. Overall, GCMS analysis revealed 115 and 109 aroma compounds across treatments in bean and lentil products, respectively. To determine which aroma compounds were affected by the addition of live or inactivated Koji, a pairwise Tukey’s post-hoc statistical analysis was conducted.

[0610] The analysis revealed a clear significant increase in levels of most metabolites: approximately 90% of volatiles in beans and 80% in lentils, were over-accumulated in response to the addition of at least one type of Koji. 70 compounds significantly increased in response to the addition of live Koji in bean products. These metabolites were mainly lipid oxidation products (alcohols, aldehydes and ketones), phenylpropanoids, Maillard reaction products (pyrazines, furans) and sesquiterpenes. A small group of compounds markedly decreased as a result of Koji addition, e.g. sulfides and some pyrazines. Figure 2 shows the level of selected aroma compounds belonging to fatty acid derivatives and lipid oxidation products, Maillard reaction products, phenylpropanoids and sesquiterpenes. 20 aroma compounds were shared between the Koji culture aroma bouquet and the aroma profile of the bean products, and 12 out of those were significantly increased in products made with live Koji compared to those made without it. Interestingly, the addition of inactivated Koji, by large, still increased the levels of most aroma compounds. When considering all three treatments, three main patterns emerge within compounds positively affected by Koji: 1) compounds whose levels increase with Koji addition, regardless of the culture being alive or not (e.g. 1- Octen-3-ol), 2) compounds that only increase in response to live Koji addition (e.g. o- Cymene) and 3) compounds in which the addition of inactive Koji partially increases their concentration, while live Koji promotes their accumulation even further (e.g. pyrazine). The majority of tested compounds presented patterns 1 or 3.

[0611] Aroma profiles of lentil products behaved similarly to those observed for bean products, i.e.: most volatiles (58) detected in this analysis increased with the addition of live Koji. Those volatiles are classified mainly into fatty acid and lipid oxidation products (acids, alcohols, aldehydes and ketones), Maillard reaction products (pyrazines, furans), phenylpropanoids and sesquiterpenes. In parallel, levels of a handful of compounds decreased with the addition of live Koji, mainly apocarotenoids, some pyrazines and several aldehydes. Figure 3 shows the levels of selected compounds clustered under the classes presented in Figure 2. 17 of the compounds found in lentil products could also be detected in live Koji culture, and out of those, 10 were enriched in the ‘with Koji’ treatments products. When considering the overall dataset of compounds increased with Koji addition, including all three treatments, it is apparent that lentil products demonstrate the same trends observed for bean products (see above).

[0612] 2. Taste profiling of Koji-containing products

[0613] We then went on to study the non-volatile content of the bean and lentil products, namely amino acids (imparting mainly umami, sweet, bitter and slight sour), 5’-ribonucleotides (umami) and primary sugars (sweet), to better understand the effect of Koji addition on basic taste (sweet and umami).

[0614] To that end, free amino acid levels were measured using a UHPLC, findings presented in Figure 4. For lentils (Figure 4a), products made with live Koji cultures contained markedly higher levels of free amino acids compared to the ‘no Koji’ treatment. More specifically: glutamic acid, tyrosine, tryptophan, phenylalanine, alanine, isoleucine and valine levels were significantly higher when live Koji was used. Conversely, levels of serine, lysine and leucine were higher when Koji was omitted. When considering all treatments, most amino acids followed a pattern where the addition of inactivated Koji increases the level of free amino acids, and live Koji increases that even further (e.g. glutamic acid, tyrosine, alanine, phenyalanine, isoleucine, valine). Tryptophan was the only example where the same degree of amino acid accumulation was identical regardless of the type of Koji added. In the case of serine and leucine, addition of inactivated Koji resulted in similar or moderately lower levels to products with no Koji. For beans (Figure 4b), a similar trend was observed when comparing amino acids contents in products made with live Koji vs. the ‘no Koji’ treatments. Specifically, levels of arginine, glutamic acid, tyrosine, tryptophan, alanine, isoleucine, valine and threonine were significantly higher when live Koji was introduced. Similarly to lentils, leucine levels increased when Koji was omitted. Finally, the increase in amino acids was less sensitive to the type of Koji added, i.e. the rise in levels of most amino acids was identical when live or inactivated Koji was used.

[0615] 5’-ribonucleotides (UMP, IMP, AMP, GMP and XMP) were analyzed for lentil and bean products (Figure 5); however only UMP, GMP and IMP were detected across samples. For lentils (Figure 5a) as well as bean products (Figure 5b), Koji addition increased the concentration of UMP. In beans, addition of inactivated Koji resulted in a similar level of UMP observed for live Koji, whereas in lentils inactivated Koji was able to positively increase the level only partially compared to live Koji. GMP was only detected in products without Koji, which may suggest that Koji or its enzymes utilize this metabolite or its precursors to other ends. IMP concentration in lentils was unaffected by treatment type, but in beans only the addition of inactivated Koji slightly increased its levels. Additionally, live Koji addition resulted in lower levels of IMP in beans.

[0616] Finally, we analyzed the primary sugar content of both lentil and bean products across all three treatments (Figure 6). For both types of substrates, Koji addition markedly increased the levels of glucose, sucrose and fructose compared to the ‘no Koji’ treatments. With the exception of glucose in lentils, addition of live Koji resulted in the highest concentration of sugars, followed by inactivated Koji.

[0617] 3. Sensory analysis and association of sensory and chemical data

[0618] We then complemented the flavor chemistry data with sensory analysis, in order to validate that the overall flavor of products made with Koji is indeed different compared to products that does not contain Koji. Moreover, changes in levels of specific metabolite does not immediately translate different organoleptic effects, due to sensory thresholds and various compound interactions. Therefore a sensory analysis is important to understand which flavors are actually perceivable.

[0619] To that end, we ran a full sensory descriptive analysis. A trained panel consisting of 10 highly skilled tasters first generated a set of 20 descriptors clustered into odor (orthonasal perception), basic taste, flavor (oral and retro-nasal perception), mouthfeel and aftertaste. The panel then scored the intensity of the aforementioned descriptors in lentil products made with or without Koji (we will not focus on mouthfeel attributes hereon). The overall intensity of product made with Koji was perceived as significantly higher in the product made with Koji (Figure 7). Furthermore, all aroma parameters were scored higher when Koji was introduced during production, with the exception of ‘Soy’ aroma. The increase in aroma intensity is likely associated to overrepresentation of specific aroma compounds, e.g. ‘Miso’ (increase in 1-Butanol, 3-methyl-), ‘Meaty’ (increase in Dimethyl sulfide and Methional) and sauteed mushrooms (increase in 1- octen-3-ol and 2-furanmethanol).

[0620] For flavor, the panel scored the products containing Koji higher for all descriptors except for ‘Bouillon’ (found to be more intense without Koji), and ‘Baked vegetables’ (scored similarly). The putative association with levels of aroma compounds is presented in Figure 8. For example, high intensity of ‘Yeasty’ aromas could be correlated to higher levels of Furfural and Phenylethyl alcohol) and ‘Smoky’ could be associated to increase in Phenol, 2-methoxy- and Furfural.

[0621] Basic taste attributes ‘Umami’ and ‘Sweet’ were both perceived as more intense in the lentil products made with Koji, although the increase in ‘Umami’ was not as pronounced as in other descriptors (Figure 9). This was evident in the aftertaste module, in which ‘Umami’ was scored similarly for both treatments. The overall aftertaste intensity was, however, still scored higher for the products made with Koji. The increase in ‘Umami’ can be explained in higher levels of Glutamic acid (and perhaps UMP). Similarly, the increase in perceived sweetness is correlated to higher concentrations of fructose, sucrose, glucose and alanine in Koji-containing products.

[0622] 4. Koji proteolytic activity

[0623] To validate the hypothesis that the increase in amino acid content observed in live Koji- containing products is due to the activity of fungal enzymes acting on proteins, the proteolytic activity of live vs. inactivated Koji was measured using the azocasein-based assay. Indeed, live Koji addition results in a x6.2-fold increase in enzymatic activity, which correlates to 0.56% dilution of the commercial enzyme.

[0624] Conclusions

[0625] The addition of live Koji culture was shown here to dramatically alter the flavor of the savoury taste products of the present invention, by increasing the overall intensity and complexity, as demonstrated by analytical and sensory approaches across three different substrates.

[0626] The basic taste attributes of the products were notably modified by Koji, resulting in the the savoury taste products of the present invention being perceived as more umami and sweet. Specifically, Koji addition led to the following observations:

[0627] - Levels of most free amino acids and some 5’-ribonucleotides increased, at least partially due to Koji hydrolytic activity but also due to amino acids readily available in the culture (Figures 5+6). This conclusion is supported by findings that inactive Koji led to a moderate increase in free amino acid levels (likely from amino acids present in the culture), while live Koji produced significantly higher levels, particularly in lentils, but also to some extent in beans. Indeed, Koji was found to possess proteolytic activity here (see Examples), supported by previous research (Zhang et al., 2023). These findings were further substantiated by a sensory analysis, which indicated that Koji-containing products were found to be more umami and sweet, as well as having a more intense aftertaste.

[0628] - Levels of primary sugars increased, likely due to amylolytic activity (Figure 6). Again, and similar to amino acids, the increase in sugar concentration is less dramatic when Koji is inactive, implying that a portion of the sugar concentration is formed due to Koji-related enzymatic hydrolysis of starch, and the rest is already present as a free sugar. Consequentlyt, products made with Koji were found to be sweeter.

[0629] In parallel, Koji addition dramatically alters the aroma profile of the savoury taste products of the present invention. More specifically:

[0630] - Products made with Koji are enriched with aroma compounds readily present in the fungal culture, or produced by their enzymes, e.g.: 1-octen-3-ol, 1-butanol- 3-methyl, phenylethyl alcohol, 2-octen-1-ol, (E), cis-Calamenene and more. These compounds convey mushroom-like, woody, floral, fermented, yeasty and fruity notes.

[0631] - Products made with Koji contain markedly higher levels of Maillard reaction products, such as pyrazines, furans and thiophenes, as well as caramelization products (Figures 2-4, Supplementary tables S2-4). Maillard reaction precursors are chiefly free amino and reducing sugars, both of which are significantly increased when Koji is used in the savoury taste products of the present invention. Therefore, Koji increases the availability of Maillard and caramelization precursors, thereby promoting the subsequent formation of new aroma compounds. These compounds are responsible for nutty, roasted, toasted, meaty and caramel-like aromas.

[0632] - In addition, there is evidence that suggests that biosynthetic enzymes present in the Koji culture act on precursors found in either the substrate or the culture itself, as evident by increase of, e.g. phenylpropanoids, many of which have floral and fruity aromas.

[0633] - As a result, products containing Koji are scored higher for overall aroma intensity, and are perceived as more meaty, mushroom-like, cocoa, caramel and miso-like, which are descriptors that are generally favorable by consumers. Example 5. W2T process

[0634] Preparation of rice koji

[0635] For all products the process starts by preparing the rice koji needed which accounts for 8.3% - 20.3% of the raw material composition. The process starts by soaking upcycled rice in 1 :3 (v / v) cold water for a minimum of 18 hours. After soaking, the rice is rinsed to remove excess starch before being filled into perforated gastro trays lined with tea towels. The rice is then steamed in the oven until the rice is cooked through and contains approximately 30% - 40% water, the rice is then cooled to <40 °C. Once cooled the rice is inoculated with spores of a strain of Aspergillus called “Hi sojae” (purchased from HIGUCHI-MATSUNOSUKE SHOTEN CO. LTD) which is a hybrid between Aspergillus Sjoae and Aspergillus Oryzae. 0.0134% of spores is used to inoculate the rice. After inoculation the rice is again filled into perforated gastro trays lined with tea towels and covered, before being moved to the incubation container. The koji mold is allowed to develop for 40 - 48 hours between 28 °C and 32 °C, with 75% humidity before being ready to use.

[0636] Pre-processing all raw materials are pre-processed according to their original physico-chemical status. Some raw materials e.g. chicken wings involves roasting at high temperatures in an oven to create the desired flavor before grinding. For other raw materials e.g. lentils and rice pre-treatment involves steaming at 100 °C to rehydrate and cook the raw material so that the nutrients are more easily available during the following processing steps. All raw materials with a particle size larger than fine salt are ground as part of the pre-treatment process. Most raw materials are ground in a large bowl cutter individually to decrease the particle size. For all “W2T products, as a final step after all raw materials have been combined with water, the substrate is mixed thoroughly with a large stick blender. The reason for this final blending step is to reduce the particle size further as this again makes the nutrients more available in the following processing steps and to create a homogeneous substrate which can be easily pumped into the bioreactor for further processing.

[0637] Hydrolysis / fermentation

[0638] Once the substrate has been filled into the bioreactor the substrate starts to be heated until it reaches 60 °C. However, some plant based products are heated to 80 °C and kept for 10 minutes before further processing to eliminate a high microbial load. Once the temperature is stabilized at 60 °C, this temperature is maintained until the end of the hydrolysis process. Simultaneously with the heating of the substrate the pH is adjusted in order to provide optimal working conditions for the industrial enzyme solutions.

[0639] For products where the substrate has a high content of fibers which makes the proteins within more inaccessible for the proteolytic enzyme solutions, a carbohydrase step is included in the process which runs for 4 - 16 hours. The enzyme solution used for this step is called Viscozyme L and is a broad spectrum carbohydrase which mainly hydrolyzes various fibers and has no amylase activity. Viscozyme need acidic conditions to work optimally. If pH adjustment is necessary powdered citric acid is used to lower the pH.

[0640] If a Viscozyme L hydrolysis step is not necessary the pH is immediately raised to alkaline pH of 8.0 - 8.5 depending on the product being produced, powdered sodium carbonate is used to raise the pH.

[0641] After the pH has been adjusted a mix of two enzyme solutions are used in equal amounts (w / w); Alcalase 2.4 L FG which is a is a serine endo-peptidase, which means that it hydrolyzes at various points along the amino acid chain of the protein. Alcalase is a highly effective protease. However, when the amino acid bonds are broken, this creates a negative charge on the terminal amino acid, which in high amounts can cause unwanted bitterness in the final product. To eliminate the bitterness and achieve a more effective hydrolysis, a second protease solution is used. Flavourzyme 1000 L is a high quality blend of endo- and exo-peptidases. It provides unique flavor generation and debittering benefits as it breaks down peptides into free amino acids, thereby producing flavor active compounds such as glutamic acid as well as removing the negatively charged terminal amino acid, resulting in a debittering effect. This protease step runs for 18 - 50 hours hydrolysis depending on the product. For longer processing times the pH will often be adjusted daily to provide the enzymes with better working conditions.

[0642] The final hydrolysis treatment starts by lowering the pH to 5.5 - 6.0 depending on the initial pH of the substrate. The aim of this final step is to enhance the umami taste of the final product further by optimizing the compounds created during the first proteolytic treatment. Glutaminase SD-100NA is used to convert glutamine which is almost insipid in flavor, into glutamate which has a strong umami taste. Furthermore, Nuclease E“Amano”7 and Deamizyme T“Amano”50 which hydrolyze RNA to create 5’- nucleotides of which some are known as flavor nucleotides because of their ability to enhance the perception of umami from the glutamate, by a concept known as umami synergy which causes the glutamate to form a stronger bond with the taste receptor on our tongue. This process step runs for 16 - 24 hours and does not require readjustment of the pH throughout the process.

[0643] Once the hydrolysis process is complete the substrate undergoes the final steps of the process. First the substrate is split into a solid fraction and a liquid fraction using a vibra-screen fitted with 300 and 40 micron filters. The solid fraction still contains a high amount of moisture and is pressed in a hydraulic sheet press to extract more liquid in the products with a large solid fraction. Currently the solid fraction is discarded, however in the future we aim to utilize this as a commercial product by having the solid fraction dried in a tunnel drier to create a shelf stable powdered product. The liquid product has an additional quantity of salt added ensuring that the water activity of the liquid is lowered <0.92 to preserve the product. After the additional salt is added the pH is adjusted back to the initial pH of the substrate before hydrolysis. By readjusting the pH of the final product the chemicals used falls into the category of processing aids which means that they do not have to be listed on the ingredients list as long as they do not have a function in the final product. Lastly the liquid fraction is heated to 85 °C for 15 minutes, this final heat treatment has two purposes. Primarily it inactivates the enzymes which are still present in the product, by denaturing the proteins which make up the structure of the enzymes, this step means that the enzymes also fall into the category of processing aids with the same advantages as listed before. Lastly it acts as a final pasteurization step, which together with hot filling the final liquid product into an IBC tank ensures the quality and shelf stability of the final product until bottling.

[0644] Table 4. Product specific process overview

[0645] Table 5. Overview of differences in enzymatic hydrolysis process between products

[0646] Table 6. Product quality overview

[0647] Table 7. Overview of Aroma, flavor, and taste qualities in the savoury taste products of the present invention. Example 6. Salt Reduction Assays

[0648] To explore the feasibility of reducing salt content in food applications using the savoury taste product of the present invention, we designed an experiment involving creamy potato soup and vegetable soup. In these trials, a savoury taste product of the present invention, Mushroom 019 as described in detail in the previous examples, was employed as the umami source.

[0649] Recipe details (ingredients as % of total weight):

[0650] Potato Soup

[0651] Butter: 1.43%, Shallots: 4.29%, Potatoes: 57.14%, Heavy cream: 14.29%, Water: 71.43%.

[0652] Vegetable Soup

[0653] Carrots: 9.76%, Celery: 9.76%, Onions: 19.51 %, Apples: 4.88%, Parsley: 0.49%, Water: 78.05%.

[0654] Both soups were prepared without added salt or other seasonings to serve as a neutral base for testing. A standard approach in salt reduction studies involves determining the "salt exchange rate" — the lowest salt concentration at which an umami-rich product becomes indistinguishable from a reference product containing higher salt levels. To establish this rate, a reference soup was prepared with a final salt concentration of 1%. A series of test samples was then created, each containing progressively less salt (0.88% to 0.3%), with the savoury taste product of the present invention (Mushroom 019) providing both umami flavor and salt content. Table 8 below outlines the specific formulation of each test variant:

[0655] Table 8. Experimental Design of the Salt Reduction Assay

[0656] The formulations (using potato and vegetable soups) were assessed by a professional sensory panel composed of product developers. Each test soup was evaluated in tandem with its respective reference soup to determine whetherthe umami enhancement effectively compensated for reduced salt content.

[0657] References

[0658] Weinblum, N., Cna’ani, A., Yaakov, B., Sadeh, A., Avraham, L., Opatovsky, I., and Tzin, V. (2021). Tomato cultivars resistant or susceptible to spider mites differ in their biosynthesis and metabolic profile of the monoterpenoid pathway. Front. Plant Sci. 12, 630155.

[0659] Vinther Schmidt, C., Olsen, K., and Mouritsen, O.G. (2021). Umami potential of fermented beverages: Sake, wine, champagne, and beer. Food Chem. 360, 128971.

[0660] Poojary, M.M., Orlien, V., Passamonti, P., and Olsen, K. (2017). Improved extraction methods for simultaneous recovery of umami compounds from six different mushrooms. Journal of Food Composition and Analysis 63, 171-183.

Claims

Claims1. A method for producing a savoury taste product from an organic starting material, the method comprising the steps of: a) providing the organic starting material; b) optionally pre-treatment of the organic starting material, thereby providing a pre-treated organic starting material; c) fermentation of the organic starting material or of the pre-treated organic starting material in the presence of a fungi composition, preferably wherein the fungi composition comprises Aspergillus Sojae and Aspergillus Oryzae, thereby providing a fermented material; d) optionally contacting the fermented material with one or more carbohydrase e) hydrolysis of the fermented material in the presence of an enzyme composition comprising at least one protease, and at least one glutaminase, thereby providing a hydrolysed, fermented material; and f) optionally a step of recovering, concentrating, dehydrating and / or purifying the hydrolysed, fermented material thereby providing the savoury taste product.

2. The method according to claim 1 , wherein the organic starting material comprises or consists of mushroom, chicken, lentil and / or beans.

3. The method according to any one of the preceding claims, wherein the method comprises step (b).

4. The method according to any one of the preceding claims, wherein the method comprises heating of the pre-treated organic starting material to around 60 °C, prior to step (c).

5. The method according to any one of the preceding claims, wherein the method comprises heating of the pre-treated organic starting material to around 60 °C, such as around 65 °C, such as around 70 °C, such as around 75 °C, such as around 80°C, such as around 85°C, such as around 90°C, such as around 95 °C, such as around 100 °C, and keeping the pre-treated organic starting material at the temperature for 10 minutes prior to step (c).

6. The method according to claim 5, further comprising cooling the pre-treated organic starting material to around 60 °C, prior to step (c).

7. The method according to any one of the preceding claims, further comprising maintaining the temperature between 58-62 °C, such as around 60 °C, during step (c), optional step (d) and / or step (e).

8. The method according to any one of the preceding claims, wherein the fungi composition is a solid state fungi composition.

9. The method according to any one of the preceding claims, wherein the fungi composition comprises a substrate, selected from rice, broken rice, beans, broken beans, pumpkin seeds, sesame seeds, sunflower seeds, cereal brans, such as wheat bran, sorghum bran, rye bran, oats bran, barley bran, millet bran, maize bran, lentils, broken lentils, chickpeas, broken chickpeas, corn, quinoa, or spelt or any combination thereof.

10. The method according to any one of the preceding claims, wherein the method comprises step (d).

11. The method according to any one of the preceding claims, wherein the one or more carbohydrase is a beta-glucanase, such as an endo-beta-glucanase.

12. The method according to any one of the preceding claims, wherein step (d) runs for 4 to 8 hours, or such as for 8 to 12 hours, or such as for 12 to 16 hours, or such as for 16 to 21 hours13. The method according to any one of the preceding claims, wherein the at least one protease is added in a first protease hydrolysis step (e1), and wherein the at least one glutaminase is added in a second, glutaminase hydrolysis step (e2).

14. The method according to any one of the preceding claims, wherein the at least one protease is an endo-protease, such as a serine endo-peptidase and / or a blend of endo- and exo-peptidases.

15. The method according to any one of the preceding claims, wherein step (e) includes one or more pH adjustment, such as one pH adjustment prior to the protease hydrolysis step (e1), and one prior to the glutaminase hydrolysis step (e2) and / or pH re-adjustment during step (e).

16. The method according to any one of the preceding claims, wherein the glutaminase of step (e) is a gamma glutamyl transpeptidase and / or a gammaglutamyl transferase.

17. The method according to any one of the preceding claims, wherein the enzyme composition of step (e) further comprises at least one nuclease.

18. The method according to any one of the preceding claims, wherein the enzyme composition of step (e) further comprises at least one deaminase.

19. The method according to any one of the preceding claims, wherein the enzyme composition of step (e) comprises at least one protease, at least one glutaminase and at least one nuclease.

20. The method according to any one of the preceding claims, wherein the enzyme composition of step (e) comprises at least one protease, at least one glutaminase, at least one nuclease and at least one deaminase.

21. The method according to any one of the preceding claims, wherein the method comprises step (f).

22. The method according to any one of the preceding claims, wherein the hydrolysed, fermented material is concentrated through dehydration, preferably until a brix between 55-65°, to provide a concentrated, fermented material.

23. The method according to any one of the preceding claims, wherein the hydrolysed, fermented material is concentrated through spray drying, to provide a powdered, fermented material.

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