Food flavouring and colouring compositions comprising microencapsulated myoglobin

Abstract

The present invention relates to a food flavouring and colouring composition comprising microencapsulated myoglobin and a buffer, wherein the myoglobin has an amino acid sequence at least 50% identical to SEQ ID NO:1 and the myoglobin is microencapsulated with an encapsulating agent, preferably a starch. Myoglobin microencapsulation significantly improves sensory characteristics when compared to plant-based products supplemented with non-encapsulated myoglobin. Additionally, the present invention discloses a method for obtaining a food flavouring and colouring composition comprising microencapsulated myoglobin and a buffer, a method for flavouring and colouring foodstuff using the composition comprising microencapsulated myoglobin and a buffer and a flavoured and coloured foodstuff including the composition comprising microencapsulated myoglobin and a buffer.

Description

[0001] FOOD FLAVOURING AND COLOURING COMPOSITIONS COMPRISING MICROENCAPSULATED MYOGLOBIN

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the priority benefit of U.S. Provisional Application No. 63 / 535,324, filed on August 30, 2023, which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present invention relates to the technical field of compositions having microencapsulated proteins and foodstuff products comprising said compositions. Particularly, aspects and embodiments presented herein relate to compositions comprising microencapsulated myoglobin, methods for obtaining said compositions and meat substitutes comprising said compositions. The microencapsulated myoglobin may be added to meat substitutes significantly enhancing the flavouring profile of said meat substitutes with respect to meat substitutes comprising non-microencapsulated myoglobin.

[0006] BACKGROUND

[0007] Attention has considerably shifted from animal-derived products (e.g. cattle and poultry) to plant-based products, which present themselves as alternatives with health and environmental advantages over conventional meat-containing products (e.g. absence of cholesterol and lower levels of saturated fatty acids, or diminished water consumption, respectively). Nonetheless, meat-like colouring and flavouring are still desirable in plantbased foodstuffs to completely attract the meat-eating population.

[0008] Meat colouring and flavouring are related to the amount and chemical state of myoglobin. Myoglobin is a protein with a molecular weight of approximately 17.8 kD that has the function of supplying reversibly bound oxygen to muscle tissue. It functions by allowing molecular oxygen (O2) to reversibly bind the iron atom in the heme porphyrin group cofactor bound to myoglobin, with subsequent oxygen release when muscle tissue is encountered. Ch-bound myoglobin is often referred as oxymyoglobin and provides meat its characteristic red colour. Meat flavouring is also provided by myoglobin, wherein the heme group catalyses the Maillard reaction and lipid oxidation, among other reactions, during the cooking process.

[0009] Heme proteins such as myoglobin have been used to confer meat-like flavour and colour to plant-based products. WO / 2022 / 144434 (Paleo B.V.) describes a meat substitute comprising a recombinant animal myoglobin produced in P. pastoris to provide meat-like colouring and flavouring to plant-based products. WO / 2022 / 187736 (Motif Foodworks Inc.) details meat alternative formulations that include a heme protein, plant-based protein, plantbased fibre, plant-based connective tissue analog, fat, and other ingredients. WO / 2023 / 076307 (Motif Foodworks Inc.) provides compositions comprising a heme protein such as myoglobin and an antioxidant as stabiliser for meat-like colouring and flavouring enhancing in plant-based products. US / 2021 / 0289824 (Impossible Foods Inc.) describes a meat substitute composition comprising 10% or more of a heme protein, preferably leghemoglobin, wherein said meat substitute composition would mimic the taste, texture, or colour of a meat product derived from animal sources.

[0010] Even though the meat-like colour and flavour provided by heme proteins to plantbased products is reasonably well established, specific sensory attributes that contribute to the overall quality of these plant-based alternatives such as umami flavour, greasy mouthfeel and juiciness are still far-off meat products. WO / 2022 / 144434 (Paleo B.V.), WO / 2022 / 187736 (Motif Foodworks Inc.), WO / 2023 / 076307 (Motif Foodworks Inc.), US / 2021 / 0289824 (Impossible Foods Inc.) and US12011016B2 (Impossible Foods Inc.), all disclose compositions with improved meat-like qualities comprising a heme protein and other food-grade ingredients, but the industry is still in need of more stable formulations and better sensory profiles compared to meat products.

[0011] Foodstuff products comprising a heme protein and additional flavouring and texturising ingredients are known in the art. WO / 2023 / 147546 (Motif Foodworks Inc.) discloses oleogel composites with starch for marbled meat analogue production, wherein the analogue may contain a heme protein. WO / 2022 / 187736 (Motif Foodworks Inc.) describes compositions comprising hemeproteins and a hydrocolloid such as modified starch, or a plant-derived fibre such as starch. None of these heme protein flavouring compositions teach the microencapsulation of myoglobin for enhancing the sensory profile of plant-based meat analogues. Various microencapsulation agents have been explored in food ingredients with different aims. WO / 2008 / 083152 (Kraft Foods Holdings Inc.) details microencapsulated polyphenols that are more stable and less astringent for food and beverage compositions using CocoanOX while discouraging the use of starches and inulin. WO / 2013 / 067603 (Clover Corp Ltd.) teaches compositions adapted for encapsulation of oxygen sensitive oils and oxygen sensitive oil-soluble bioactives (proteins) for food and pharmaceutical ingredients for increased stability. US / 2007 / 0172513 (Brunob II B.V.) discloses the microencapsulation of soy protein or casein using starches for increased oxidation resistance. None of these compositions refer to an enhancement of sensory attributes due to microencapsulation of a heme protein and vary widely in the chemical nature of the encapsulating agent.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to food flavouring and colouring compositions comprising a microencapsulated myoglobin and a buffer. The microencapsulation of the myoglobin significantly enhances sensory attributes of foodstuff that includes said microencapsulated myoglobin with respect to non-microencapsulated myoglobin.

[0014] In some aspects of the invention, the myoglobin is a natural or recombinantly produced myoglobin. Preferably, the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NOT.

[0015] In some embodiments of the invention, the myoglobin concentration is at least 10% w / w, preferably between 10 and 50% w / w in the composition, preferably between 15 and 25% w / w in the composition.

[0016] In some embodiments of the invention the myoglobin is microencapsulated with an encapsulating agent selected from, but is not limited to, a starch, maltodextrin, inulin, alginate and combinations thereof. In some embodiments, the encapsulating agent is a starch. In some other embodiments, the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

[0017] In some embodiments of the invention, the starch concentration in the composition is between 10 and 50% w / w, preferably between 15 and 25% w / w.

[0018] In some embodiments of the invention, the mass ratio of myoglobin with respect to starch is between 1: 1 to 1:3, preferably 1: 1, 1:2 or 1 :3.

[0019] In some other embodiments, the composition comprises a buffer, such as sodium phosphate buffer. In some embodiments, the buffer is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM. In some other embodiments, the buffer includes other additives, a salt such as sodium chloride, and food flavouring and stabilisers that comprise but are not limited to ascorbic acid, cysteine, glucose, ribose, glutamic acid, thiamine, among other ingredients. Preferably, the ascorbic acid concentration is 0.25% w / w. In some preferred embodiments, the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and 1 mM thiamine. In a more preferred embodiment, the buffer comprises sodium phosphate 50 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and 1 mM thiamine. In another preferred embodiment, the buffer comprises sodium phosphate 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and 1 mM thiamine.

[0020] In yet another embodiment of the invention, the microencapsulated myoglobin has a mean particle size distribution between 1 and 100 pm, preferably with a mean particle size distribution between 10 and 100 pm and more preferably a mean particle size distribution between 10 and 50 pm. In some of these embodiments, the percentage of microencapsulated myoglobin with respect to total myoglobin in the composition is at least 20%, preferably between 20 to 80%, more preferably 20 to 60% and even more preferably 20 to 40%.

[0021] The present invention additionally discloses methods for obtaining a food flavouring and colouring composition. In some embodiments, the methods comprise: i) dissolving myoglobin in a buffer for obtaining a solution; ii) dispersing an encapsulating agent in the solution of the myoglobin and the buffer for obtaining a mixture and, iii) drying the mixture comprising the dispersed encapsulating agent, the buffer and myoglobin, for obtaining the composition comprising microencapsulated myoglobin. In some embodiments, additional components such as a salt, stabilisers and flavouring enhancers are added between the microencapsulating agent dispersion and the drying steps. In yet another embodiment, the drying step involves, but is not limited to, a spray-drying step.

[0022] In some aspects of the invention, the myoglobin dissolved in the buffer is a natural or recombinantly produced myoglobin. Preferably, the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NO: 1.

[0023] In some embodiments of the invention, the myoglobin is dissolved to a final concentration of at least 10% w / w, preferably between 10 and 50% w / w, preferably between 15 and 25% w / w.

[0024] In some embodiments of the invention, the encapsulating agent dispersed is selected from, but is not limited to, a starch, maltodextrin, inulin, alginate and combinations thereof. In some embodiments, the encapsulating agent is a starch. In some other embodiments, the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

[0025] In some embodiments of the invention, the starch concentration in the solution comprising myoglobin and buffer is between 10 and 50% w / w, preferably between 15 and 25% w / w.

[0026] In some embodiments of the invention, the mass ratio of myoglobin with respect to starch is between 1: 1 to 1:3, preferably 1: 1, 1:2 or 1 :3.

[0027] In some other embodiments, the buffer used to dissolve the myoglobin is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM. In some other embodiments, the buffer includes other additives, a salt such as sodium chloride, and food flavouring and stabilisers that comprise but are not limited to ascorbic acid, cysteine, glucose, ribose, glutamic acid, thiamine, among other ingredients. Preferably, the ascorbic acid concentration is 0.25% w / w. In some preferred embodiments, the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and 1 mM thiamine. In a more preferred embodiment, the buffer comprises sodium phosphate 50 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine. In another preferred embodiment, the buffer comprises sodium phosphate 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

[0028] In yet another embodiment of the invention, the percentage of microencapsulated myoglobin with respect to total myoglobin in the composition is at least 20%, preferably between 20 to 80%, more preferably 20 to 60% and even more preferably 20 to 40%.

[0029] In some embodiments of the invention, the drying of the mixture is performed by spray-drying with a spray-dryer. Preferably, the spray-dryer has an inlet temperature between 140 to 145°C.

[0030] The present invention also includes a method for flavouring and colouring foodstuff comprising the steps of i) providing a food flavouring and colouring composition including a microencapsulated myoglobin and a buffer; and ii) adding the food flavouring and colouring composition including a microencapsulated myoglobin and a buffer to the foodstuff. The food flavouring and colouring composition provided for the method of the present invention includes any of the above embodiments disclosed for the food flavouring and colouring composition of the present invention. In some embodiments, the food flavouring and colouring composition of the present invention is added to the foodstuff, wherein the foodstuff is preferably meat substitutes or plant-based meat analogues (also called plant-based meat matrices). The plant-based meat analogues include but are not limited to common plant-based matrices such as soy protein, pea protein, textured soy protein, textured pea protein, chia fibre, methylcellulose, among others, and mixtures thereof.

[0031] In some embodiments, the food flavouring and colouring composition is mixed, sprinkled on, or integrated with the meat substitutes or plant-based meat matrices at any moment of the foodstuff preparation process, but preferably is done before cooking. In some embodiments, the cooking may be done by any known cooking method such as baking, frying, stir-frying, roasting, grilling, boiling, steaming, among others, or a combination thereof, with the corresponding cooking equipment like ovens or pans, among others.

[0032] The present invention also discloses a flavoured and coloured foodstuff comprising a meat substitute or a plant-based meat analogue, and a food flavouring and colouring composition including a microencapsulated myoglobin and a buffer. The food flavouring and colouring composition comprised in the foodstuff of the present invention includes any of the above embodiments disclosed for the food flavouring and colouring composition of the present invention. In a preferred embodiment, the flavoured and coloured foodstuff of the present invention is obtained by any of the above embodiments disclosed for the method for flavouring and colouring foodstuff of the present invention.

[0033] In some embodiments, the improved sensory effects of the invention, including but not limited to enhanced meat-like flavour and colour, are observed in meat analogues or substitutes or plant-based meat analogues. Plant-based meat analogues or matrices are not to be limited to the ones illustrated in the examples and may be designed and selected by those skilled in the art. Some common plant-based matrices include soy protein, pea protein, textured soy protein, textured pea protein, chia fibre, methylcellulose, among others, and mixtures thereof.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 : shows the powder form of different compositions of microencapsulated myoglobin with 1. Maltodextrin (Malto), Alginate (Alg), Inuline (Inu), starch (Alm) and a buffer comprising sodium phosphate 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

[0036] FIG. 2: shows plant-based burgers comprising the different compositions of microencapsulated myoglobin of the present invention, wherein the different compositions correspond to 1: INU Fl, 2: ALM Fl, 3: Control, 4: Yeast Extract 1, and 5: Yeast extract 2. Panels of this figure are as follows: FIG. 2A: pre-cooked burgers, FIG. 2B: difference between inulin (1) and starch (2) prior to cooking, with starch appearing slightly redder, FIG. 2C: burgers at 3 minutes of cooking, FIG. 2D: burgers at 6 minutes of cooking releasing juices, FIG. 2E: burgers at 12 minutes of cooking, beginning to brown and showing more juice, and FIG. 2F: burgers ready after approximately 18 minutes of baking.

[0037] FIG. 3: shows different embodiments of burgers comprising the compositions with microencapsulated myoglobin of the present invention with 1: INU Fl, 2: ALM Fl, 3: Control, 4: Yeast Extract 1, and 5: Yeast extract 2.

[0038] FIG. 4: shows the microcapsule morphology of the microencapsulated myoglobin in the ALM Fl composition.

[0039] FIG. 5 : shows mean particle size (in pm) distribution as determined by laser scattering for ALM Fl.

[0040] FIG. 6: shows the controlled release experiment of microencapsulated myoglobin. FIG. 6A: release of myoglobin monitored by SDS-PAGE and Coomassie brilliant blue stain over time. FIG. 6B: sigmoidal plot of myoglobin release over time.

[0041] FIG. 7: depicts different embodiments of burgers comprising the ALM Fl composition. FIG. 7A is the control plant-based matrix with no myoglobin; FIG. 7B is the plant-based burger supplemented with non-encapsulated myoglobin (0.465% w / w final concentration of myoglobin); FIG. 7C is the plant-based matrix supplemented with 1.5% w / w ALM Fl composition (0.2325% w / w final concentration of myoglobin); FIG. 7D is the plant-based matrix supplemented with 3.0% w / w ALM Fl composition (0.465% w / w final concentration of myoglobin); FIG. 7E is a commercially available plant-based burger that does not contain a heme protein in its formulation; and FIG. 7F is a commercially available plant-based burger that does contain a heme protein, particularly leghemoglobin, in its formulation.

[0042] FIG 8: shows the sensory analysis using a Sensory Quantitative Descriptive Analysis (QDA) for plant-based burgers comprising the composition with microencapsulated myoglobin with respect to a composition having non-microencapsulated myoglobin.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The terminology used herein is for the purpose of describing particular aspects of the invention only and is not intended to be limiting. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0045] “Myoglobin” as detailed in the description and referred to herein corresponds to a protein with a molecular weight of approximately 17.8 kD that functions to supply reversibly bound oxygen to muscle tissue. Myoglobin contains a heme porphyrin group that binds oxygen, contributing to the red colour of meat and playing a key role in meat flavour through the Maillard reaction and lipid oxidation. Myoglobin is also defined according to the sequence identity to SEQ ID NO: 1.

[0046] “Sequence identity” as used herein refers to the relationship between two or more amino acid sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s.

[0047] “Recombinant myoglobin”, also referred to as “recombinantly produced myoglobin” is defined herein as a myoglobin produced through genetic engineering techniques in host organisms and their derived publicly available strains such as, but not limited to, Escherichia coli. Pichia pastoris, among others. Recombinant myoglobin used in the invention may have an amino acid sequence 50 to 100% identical to SEQ ID NO: 1.

[0048] “Microencapsulation” as used herein refers to a process by which active ingredients, such as myoglobin, are encased within a coating material to form small particles. Common microencapsulation techniques include emulsification, gelation, extrusion, spray drying, and lyophilization. This process usually enhances the stability and bioavailability of the encapsulated ingredient.

[0049] “Encapsulating agents” as used herein are substances used to form the coating around the active ingredient in microencapsulation. In this invention, encapsulating agents include starch, maltodextrin, inulin, and alginate, chosen for their ability to stabilise myoglobin and enhance its sensory attributes in food products. In the context of the present invention, the terms “microencapsulating agents” and “encapsulating agents” are used interchangeably. “Particle size distribution” as used herein and understood by those of ordinary skill in the art, corresponds to the range and mean size of particles in the composition, being size understood as the diameter of assumed or theoretical spherical particles. Preferred ranges are between 1 and 100 pm, which influence the physical properties of the final product. Techniques used for particle size distribution determination of the present invention, with illustrative purposes only and with no intentions of limiting the scope of the invention, are scanning electron microscopy (SEM) and laser diffraction.

[0050] “Foodstuff applications” is referred to as the use of microencapsulated myoglobin in various food products, particularly plant-based meat substitutes to enhance their nutritional value and / or sensory attributes. “Plant-based meat substitutes” or “Plant-based meat analogues” are used interchangeably.

[0051] “Buffer” as used for the present invention is defined as compounds that maintain the pH stability of the microencapsulated compositions. Sodium phosphate buffer is an example used in the invention to ensure the stability and functionality of myoglobin, preferably with a pH range between 5.8 and 6.2.

[0052] “Food flavoring and stabilisers” or “additives” are used interchangeably in the context of the present invention for describing further ingredients added to foodstuff or to the compositions to enhance the flavour and stability of food products. Non-limiting examples include ascorbic acid, cysteine, and ribose, which contribute to the sensory profile and preservation of microencapsulated myoglobin.

[0053] “Starch” is herein defined as a natural polymer or polysaccharide comprising long chains of glucose molecules. Natural polymers for starches include but are not limited to pectin, cellulose, alginate, protein-containing starches, and other similar starches. Acronyms used to refer to different type of starches include:

[0054] Rapidly digestible starch (RDS): The RDS form of starch exists in cooked foods, such as potatoes and bread. This type of starch breaks down relatively easily and the body rapidly converts it to glucose.

[0055] Slowly digestible starch (SDS): This starch has a more complex structure, meaning that the body breaks it down more slowly. The SDS form exists in cereal grains. Resistant starch (RS): This starch cannot be easily digested by the body meaning that it can pass through the digestive system largely untouched, similar to dietary fibre.

[0056] “RS” type of starches may be further classified into 1) RSI, which exists in grains, seeds, and beans; 2) RS2 from raw potatoes and unripe bananas; 3) RS3 from foods that undergo cooking then cooling, such as rice and cornflakes; and 4) RS4, which is in bread.

[0057] “Foodstuff’ definition in the context of the invention refers to any substance that can be used or prepared for use as food, encompassing both conventional animal-based products and plant-based alternatives. In the context of this patent, foodstuff includes plant-based mimics of red meat, poultry, and fish, which are designed to offer similar sensory and nutritional qualities to their animal-derived counterparts.

[0058] In line with the definition of “foodstuff’, a “plant-based alternative”, which is used interchangeably with “plant-based analogue” and “plant-based matrix”, refers to food products that are formulated to replicate the taste, texture, and nutritional profile of traditional animal -derived food products using plant-derived ingredients. These alternatives aim to provide health and environmental benefits over conventional meat-containing products, often containing lower levels of saturated fats and no cholesterol, aiming to offer desirable meat-like qualities such as flavour and colour.

[0059] Compositions comprising microencapsulated myoglobin

[0060] In certain embodiments, the microencapsulated myoglobin comprises an animal- sourced myoglobin obtained from, but not limited to, Bos iaurus. Sus scrofa. Eqqus ferns. Bison bison, Cervus elaphus, Thunnus thymus, among others. In a preferred embodiment, the microencapsulated myoglobin is recombinantly produced for plant-based products or foodstuff, wherein said recombinant myoglobin has an amino acid residue sequence 50 to 100% identical to SEQ ID NO: 1. Preferably, the recombinant myoglobin has at least 60% sequence identity and more preferably at least 70% sequence identity to SEQ ID NO: 1. SEQ ID NO: 1 shows the amino acid sequence of a recombinant bovine myoglobin, which is identical to the natural bovine myoglobin (Bos iaurus)' . In certain embodiments, the myoglobin is a recombinant myoglobin with an amino acid sequence identical or at least 50% identical with the amino acid sequence of the natural myoglobin obtained from, but not limited to, Bos taurus. Sus scrofa. Eqqus ferus. Bison bison, Cervus elaphus, Thunnus ihynnus. among others.

[0061] In other preferred embodiments, the composition comprises myoglobin and an appropriate buffer, wherein the encapsulating agent is dispersed. The myoglobin concentration in some embodiments is at least 10%, preferably between 10% and 50% w / w, preferably between 15% and 30% w / w and more preferably between 15 and 25% w / w.

[0062] The preferred buffer in certain embodiments of the invention is a food-grade or food- suitable buffering system in a pH range between 5.8 and 6.2. The examples enclosed herein teach the use of a 50 mM sodium phosphate buffer with a pH between 5.8 and 6.2, but a skilled in the art should recognise that the counterion in the buffer and buffer concentration are adjustable as long as the buffer is suitable for food applications and has a pH between 5.8 and 6.2. A buffer solution comprising a bicarbonate buffer system can be used to achieve the desired pH range. Other buffering agents that can be used include BES (N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES 2-[(2 -Hydroxy- l,l-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid), glycine, triethanolamine. Other buffers that may be used include, potassium hydrogen phthalate, HEPES (4-(2-hydroxyethyl)piperazine-l -ethane sulfonic acid), PIPES (piperazine- l,4-bis(2 -ethanesulfonic acid)), ACES (N-(2-acetamido)-2-aminoethanesufonic acid), and TRIS (tris(hydroxymethyl)aminomethane).

[0063] In an embodiment, the buffer that is used comprises a sodium phosphate buffer present at a concentration of 50 mM (pH 6) supplemented with 10 mM cysteine, 10 mM glucose and 1 mM thiamine. Ascorbic acid is optionally added to this buffer, preferably at a final concentration of 0.25% w / w, with the ascorbic acid acting as an antioxidant. In another embodiment, the buffer that is used comprises a sodium phosphate buffer present at a concentration of 50 mM (pH 6) supplemented with 10 mM cysteine, 10 mM glucose, ImM thiamine, 10 mM ribose, 1 mM lysine and / or 1 mM lactic acid added to the buffer. Ascorbic acid is also optionally added to this buffer, preferably at a concentration of 0.25% w / w. For those of ordinary skill in the art will recognise that these concentrations are not limiting for the scope of the invention considering that they may be adjusted as required achieving a similar effect. Encapsulation is achieved by methods compatible with the food industry such as emulsification, gelation, extrusion, spray drying, and lyophilization. There are also chemical encapsulation techniques such as coacervation, co-crystallization, interfacial polymerization, ionic gelation, polymeric incompatibility, liposome entrapment and molecular inclusion that may be used.

[0064] In some embodiments, the encapsulating agent concentration is between 10% and 30% w / w, preferably between 15% and 25% w / w and more preferably between 15 and 20% w / w. In yet other embodiments, the % w / w concentration ratio between myoglobin and the encapsulating agent may be between 1: 1 and 1:3, preferably 1: 1, 1:2 and 1:3, respectively, with variations as deemed reasonable by those of ordinary skill in the art.

[0065] In some embodiments of the invention, the composition is characterised by the mean particle size of the microencapsulated myoglobin. In some of these embodiments, the composition has a mean particle size between 1 and 100 pm, preferably with a mean particle size distribution between 10 and 100 pm and more preferably a mean particle size distribution between 10 and 50 pm.

[0066] In yet another preferred embodiment, the composition has a relative microencapsulated myoglobin proportion with respect to total myoglobin in the composition of at least 20%, preferably 20 to 80%, more preferably 20 to 60%, and even more preferably 20 to 40%.

[0067] In preferred embodiments, the microencapsulating agent is selected from starch, maltodextrin, inulin, and alginate. More preferably, the encapsulating agent is a starch. Starches may be used because they have a variety of beneficial properties including their relatively high availability, their low cost and multiple characteristics that make them ideally suited for microencapsulation such as high-water retention, high and / or low viscosity that may be adapted for the environment in which they are to be used. Other non-limiting examples of starches corresponding to hydrophobically modified starches such as octenyl succinic anhydride modified starches (OSA-starches), may prove to be useful as surfaceactive food additives. OSA-starches tend to be advantageous because they are highly functional, they can be consistently made and reproduced, they are not expensive, and they tend to be highly versatile for a variety of microencapsulation processes. In a variation, for example, in spray drying microencapsulation, modified starches offer a high oil load, high volatile retention, long shelf life, and they can be efficiently reproduced with high manufacturing efficiency.

[0068] In yet another embodiment, the starches may comprise naturally occurring polymers, which include but are not limited to pectin, cellulose, alginate, protein containing starches, other starches, and mixtures thereof. Types of other starches that can be used include but are not limited to unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and similar starches and combinations thereof. The aforementioned starches may be selected based on their availability and easily adapted by those skilled in the art.

[0069] The starches of the present invention can occur in either of two forms or in a combination of the two forms, with the two forms being amylose and amylopectin. Amylose is a linear or straight-line polymer that is amorphous or solid. Amylopectin form is a branched chain and is crystalline in form. In an embodiment, when a combination of the two forms of starches is used, the amylose contribution to the total starch amount is between 20- 40% by weight and the amylopectin amount is between 60-80% by weight. In a variation, the amount of amylose that is present is 30% by weight.

[0070] In an embodiment, the starch that is used fits into one of three groups of starches: 1) Rapidly digestible starch (RDS); 2) Slowly digestible starch (SDS); 3) Resistant starch (RS). In an embodiment, the starch may be a combination of RDS, SDS, and / or RS. By using combinations of starches, the microencapsulation can be made tuneable so that myoglobin can be released from its microencapsulation as desired.

[0071] In an embodiment, the encapsulating agent may be a trehalose-based bead polymer. Trehalose protects colour and has antioxidant properties. Trehalose might also enhance juiciness, forms fewer crystals, and aids in freeze-preservation.

[0072] In some embodiments, the composition may include flavouring agents or flavouring enhancers. In some embodiments, yeast extract can be used to enhance the flavour of myoglobin in the formulation. Yeast extracts enhance flavours and provide a savoury, umami taste and help with meaty flavour for plant-based products.

[0073] Methods for obtaining a composition comprising microencapsulated myoglobin

[0074] The invention discloses a method for obtaining food flavouring and colouring composition, according to what has been illustrated by the different embodiments of the invention. The method comprises the steps of: i) dissolving a myoglobin in a buffer for obtaining a solution; ii) dispersing an encapsulating agent in the solution of the myoglobin and the buffer for obtaining a mixture; and iii) drying the mixture comprising the dispersed encapsulating agent, the buffer and myoglobin, for obtaining the composition comprising microencapsulated myoglobin.

[0075] In certain embodiments, the myoglobin used in step (i) comprises an animal-sourced myoglobin, but a recombinantly produced myoglobin is preferred for plant-based products, wherein said recombinant myoglobin has an amino acid residue sequence 50 to 100% identical to SEQ ID NO: 1 . Preferably, the recombinant myoglobin has at least 60% sequence identity and more preferably at least 70% sequence identity to SEQ ID NO: 1. Myoglobin is dissolved in the buffer reaching a final concentration of at least 10%, preferably between 10% and 50% w / w and more preferably between 15% and 25% w / w.

[0076] In a preferred embodiment, the preferred buffer for step (i) is a food-grade or food- suitable buffering system in a pH range between 5.8 and 6.2. In a more preferred embodiment, the buffer that is used comprises a sodium phosphate buffer with a pH ranging between 5.8 and 6.2. In more preferred embodiments, the sodium phosphate buffer is present at a concentration of 50 mM and a pH ranging between 5.8 and 6.2. In some embodiments, the buffer is further supplemented with ascorbic acid, preferably 0.25% w / w. In some embodiments, the buffer is supplemented with 10 mM cysteine, 10 mM glucose, and 1 mM thiamine. In yet other embodiments, the buffer is supplemented with 10 mM cysteine, 10 mM glucose, 1 mM thiamine, 10 mM ribose, 1 mM lysine, and / or 1 mM lactic acid added to the buffer. In one of the embodiments, the encapsulating agent dispersion of step (ii) is performed by agitation, preferably the encapsulating agent dispersion is performed for 30 minutes at 550 rpm. In some embodiments, the encapsulating agent is selected from a starch, maltodextrin, inulin, alginate, and combinations thereof, but preferably is a starch. In some other preferred embodiments, the method additionally includes adding to the buffer one or more additives selected from ascorbic acid, cysteine, glucose, glutamic acid, thiamine, or sodium chloride. Preferably, the ascorbic acid addition is performed for 5 minutes at 550 rpm.

[0077] In certain embodiments, the starch used as encapsulating agent in step (ii) is selected from naturally occurring polymers which include but are not limited to pectin, cellulose, alginate, protein-containing starches, other starches, and mixtures thereof. Types of other starches that can be used include but are not limited to unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and similar starches and combinations thereof. In an embodiment, the encapsulating agent may be a mixture of two or more types of starches, selected based on the desired release profile and sensory attributes of the final food product.

[0078] In an embodiment, when a combination of the two forms of starches is used, the amylose contribution to the total starch amount is between 20-40% by weight and the amylopectin amount is between 60-80% by weight. In a variation, the amount of amylose that is present is 30% by weight.

[0079] In an embodiment, the starch that is used fits into one of three groups of starches: 1) Rapidly digestible starch (RDS); 2) Slowly digestible starch (SDS); 3) Resistant starch (RS). In an embodiment, the starch may be a combination of RDS, SDS, and / or RS.

[0080] In some embodiments of step (iii), the drying method is selected from lyophilization, fluidized bed drying, freeze spray-drying and spray-drying. Preferably, the selected drying method is spray-drying. In a more preferred embodiment, the drying method is performed by spray-drying wherein inlet temperature of the spray-dryer is set between 100 and 400°C, more preferably between 140 and 145°C.

[0081] In certain embodiments of the invention, the method is adjusted to achieve a mean particle size between 1 and 100 pm and more preferably a mean particle size distribution between 10 and 100 pm. In other preferred embodiments, the method has myoglobin microencapsulation yield with respect to total myoglobin in the composition is between 20 and 100%, preferably 20 to 80%, preferably 20 to 60%, and even more preferably 20 to 40%.

[0082] EXAMPLES Testing of encapsulating agents in plant-based matrices concentrations

[0083] Myoglobin is microencapsulated using starch as the encapsulating agent. Recombinant bovine myoglobin (SEQ ID NO: 1) was dissolved to a final concentration of 30% w / w in a buffer comprising sodium phosphate 50 mM at pH 6.0, supplemented with 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, 1 mM thiamine, 50 to 140 mM sodium chloride and 0.25% w / w ascorbic acid.

[0084] One to seven grams of an encapsulating agent selected from the group comprising maltodextrin (Malto), alginate (Alg), inuline (Inu) or starch (Alm) were dispersed in 100 to 200 ml of the solution comprising the buffer and dissolved myoglobin using a paddle agitator at 550 rpm for 30 minutes.

[0085] Following the dispersion of the encapsulating agent, sodium chloride, and ascorbic acid were added until reaching a final concentration of 50 to 140 mM NaCl and 0.25% w / w, respectively. The resulting mix was agitated under paddle agitation at 550 rpm for 5 minutes.

[0086] The obtained solution was dried using spray-drying with the spray-dryer having the following parameters: peristaltic pump at 5%, vacuum at 95%, inlet temperature = 145 °C. The obtained compositions having microencapsulated myoglobin in powder format are shown in FIG. 1. Concentration of the different microencapsulated myoglobin compositions are detailed in Table 1.

[0087] Table 1: Overview of different formulations of microencapsulated myoglobin.

[0088] Microencapsulated myoglobin formulation effects on plant-based meat analogues Formulations have been tested using a plant-based matrix (containing pea protein with vegetable oil and carboxymethylcellulose). Plant-based matrices with no myoglobin formulation added presented herein are known by a person skilled in the art and by no means limit the scope of the invention.

[0089] Formulations INU Fl and ALM Fl were tested at 3% w / w final concentration in the plant-based matrix and are shown in FIG. 2 with the following key:

[0090] 1. INU Fl

[0091] 2. ALM Fl

[0092] 3. Control (plant-based matrix only)

[0093] 4. Yeast extract 1, Bionis (at 1% in plant-based matrix, enhances the flavour). 5. Yeast Extract 2 , (at 1 % in plant-based mixture) . Panels of FIG. 2 depict the following: FIG. 2A shows the pre-cooked burgers, FIG. 2B shows the difference between inulin and starch prior to cooking, FIG. 2C shows the burgers at 3 minutes of cooking, FIG. 2D shows the burgers at 6 minutes of cooking, FIG. 2E shows the burgers at 12 minutes of cooking, and FIG. 2F shows burgers that are ready after approximately 18 minutes of cooking (baking). Burgers were flipped every three minutes during baking (for the approximate 18 -minute cooking period).

[0094] During the cooking period, the following observations were noted: at 3 minutes, juice was visible, and a hint of aroma was noticeable; by 6 minutes, the presence of juice had increased, and the aroma became more distinct; at 12 minutes, certain parts of the burgers showed a golden colour and the aroma intensified resembling that of cooked meat.

[0095] The colour of the burgers with the microencapsulated myoglobin compositions were of note as there were differences between compositions comprising starch and inulin. Prior to cooking, the burger with the starch microencapsulated myoglobin composition appeared more red when compared to the inulin microencapsulated myoglobin composition, resembling raw meat. Upon cooking, a colour shift occurred closer to that of cooked meat.

[0096] FIG. 3 shows the burgers after cooking. Microencapsulated myoglobin compositions provide a better meat-like colour than all other ingredients tested. The microencapsulated myoglobin compositions of the present invention have a superior meat flavour relative to the other tested compositions.

[0097] The following sensory opinions were collected after tasting the burgers: a. Meat-like flavour is good but it still needs improvement to fully resemble animal meat. b. Burger with ALM Fl composition (starch) was more pleasant than the version with INU Fl composition (inulin). c. Meat-like flavour was enjoyable with low acidity. d. Yeast extract 1 was considered pleasant and interesting as a possible flavour enhancer. e. Yeast extract 2 didn't contribute significantly to flavour as it had a slight mushroom -like taste. f. Approximately 20 minutes after cooling, burgers with ALM Fl and Inu Fl compositions took on a dark brown colour.

[0098] Example 2: Characterisation of the starch microencapsulated myoglobin composition

[0099] Particle morphology and mean size

[0100] The microcapsules and particle mean size distribution in the starch microencapsulated myoglobin composition ALM Fl was characterised.

[0101] FIG. 4 depicts the particle morphology of the starch microencapsulated myoglobin composition (ALM Fl) determined by Scanning Electron Microscopy (SEM). Observed morphology corresponds to raisin-like microcapsules and supports the encapsulation state of the invention.

[0102] FIG. 5 shows the mean particle size determination of the starch microencapsulated myoglobin composition by laser diffraction. Table 2 shows three independent determinations resulting in a mean particle size of 11.54 pm.

[0103] Table 2: Particle size determination by laser diffraction. SD corresponds to the standard deviation.

[0104] Microencapsulation efficiency of myoglobin

[0105] Microencapsulation efficiency was measured in controlled release experiments wherein myoglobin was released in PBS 20 mM pH=5,6 at 20°C. Myoglobin release was monitored by SDS-PAGE and Coomassie blue stain, as per standard protocols known in the art as shown in FIG. 6A. FIG. 6B shows the released amount of myoglobin at each chosen time point.

[0106] The controlled release curve shows a sigmoidal kinetics curve as expected for an encapsulated myoglobin. According to these experiments, 24,9 to 36,5% of myoglobin in the formulation was determined to be encapsulated.

[0107] Example 3 : Sensory profde of starch microencapsulated myoglobin compositions compared to non-encapsulated myoglobin compositions.

[0108] ALM Fl composition was tested for its improved sensory qualities over nonencapsulated myoglobin. Preparation protocol of plant-based burgers corresponds to the one disclosed for example 1.

[0109] FIG. 7 shows the plant-based burgers after the cooking process. According to the key of the figure: FIG. 7A is the control plant-based matrix with no myoglobin; FIG. 7B is the plant-based burger supplemented with non-encapsulated myoglobin (0.465% w / w final concentration of myoglobin); FIG. 7C is the plant-based matrix supplemented with 1.5% w / w ALM Fl composition (0.2325% w / w final concentration of myoglobin); FIG. 7D is the plant-based matrix supplemented with 3.0% w / w ALM Fl composition (0.465% w / w final concentration of myoglobin); FIG. 7E is a commercially available plant-based burger that does not contain a heme protein in its formulation; and FIG. 7F is a commercially available plant-based burger that does contain a heme protein, particularly leghemoglobin, in its formulation.

[0110] FIG. 8 shows the sensory attributes including flavour, texture, colour and overall quality were studied by a trained sensory panel (n=4) using a Sensory Quantitative Descriptive Analysis (QDA). The key is enclosed in the Figure. Starch microencapsulated myoglobin compositions at 1.5 and 3.0% both show improved characteristics, particularly in the greasy mouthfeel, juiciness, umami flavour and overall quality when compared to nonmicroencapsulated myoglobin formulations and commercially available competitors.

[0111] The examples described herein support the improved sensory attributes of microencapsulated myoglobin over non-encapsulated myoglobin and other nonencapsulated heme protein formulations.

Claims

CLAIMS1. A food flavouring and colouring composition comprising a microencapsulated myoglobin and a buffer.

2. The composition of claim 1, wherein the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NO: 1.

3. The composition of claim 1 or 2, wherein myoglobin concentration is between 10 and 50% w / w in the composition.

4. The composition of any one of claims 1 to 3, wherein the myoglobin is microencapsulated with an encapsulating agent selected from a starch, maltodextrin, inulin, alginate, and combinations thereof.

5. The composition of claim 4, wherein the encapsulating agent is a starch.

6. The composition of claim 5, wherein the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

7. The composition of claim 5 or 6, wherein the starch concentration in the composition is between 10 and 50% w / w.

8. The composition of claim 5 or 6, wherein the mass ratio of myoglobin with respect to starch is between 1: 1 to 1:3.

9. The composition of any one of claims 1 to 8, wherein the buffer is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM.

10. The composition of claim 9, wherein the buffer additionally comprises one or more additives selected from ascorbic acid, cysteine, glucose, glutamic acid, thiamine, or sodium chloride.

11. The composition of claim 10, wherein the ascorbic acid concentration is 0.25% w / w.

12. The composition of any one of claims 9 to 11, wherein the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

13. The composition of any one of claims 1 to 12, wherein the microencapsulated myoglobin has a mean particle size distribution of between 1 and 100 pm.

14. The composition of any one of claims 1 to 13, wherein the composition comprises at least 20% of the total myoglobin microencapsulated.

15. A method for obtaining a food flavouring and colouring composition, wherein the method comprises the steps of: i) dissolving myoglobin in a buffer for obtaining a solution; ii) dispersing an encapsulating agent in the solution of the myoglobin and the buffer for obtaining a mixture; and iii) drying the mixture comprising the dispersed encapsulating agent, the myoglobin and the buffer, for obtaining the composition comprising microencapsulated myoglobin.

16. The method of claim 15, wherein the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NO: 1.

17. The method of claim 15 or 16, wherein the myoglobin is dissolved to a final concentration between 10 and 50% w / w.

18. The method of any one of claims 15 to 17, wherein the encapsulating agent is selected from a starch, maltodextrin, inulin, alginate, and combinations thereof.

19. The method of claim 18, wherein the encapsulating agent is a starch.

20. The method of claim 19, wherein the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

21. The method of claim 19 or 20, wherein the starch concentration in the solution comprising myoglobin and buffer is between 10 and 50% w / w.

22. The method of claim 19 or 20, wherein mass ratio of myoglobin with respect to starch is between 1: 1 to 1:3.

23. The method of any one of claims 15 to 22, wherein the buffer is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM.

24. The method of claim 23, wherein the buffer additionally comprises one or more additives selected from ascorbic acid, cysteine, glucose, glutamic acid, thiamine, or sodium chloride.

25. The method of claim 25, wherein the ascorbic acid concentration is 0.25% w / w.

26. The method of any one of claims 23 to 25, wherein the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

27. The method of any one of claims 15 to 26, wherein the drying of the mixture is performed by spray-drying with a spray-dryer.

28. The method of claim 27, wherein the spray-dryer has an inlet temperature between 140 and 145°C.

29. The method of any one of claims 15 to 28, wherein the composition obtained comprises at least 20% of the total myoglobin microencapsulated.

30. A method for flavouring and colouring foodstuff comprising: i) providing a food flavouring and colouring composition including a microencapsulated myoglobin and a buffer; and ii) adding the food flavouring and colouring composition including a microencapsulated myoglobin and a buffer to the foodstuff.

31. The method of claim 30, wherein the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NO: 1.

32. The method of claim 30 or 31, wherein myoglobin concentration is between 10 and 50% w / w in the composition.

33. The method of any one of claims 30 to 32, wherein the myoglobin is microencapsulated with an encapsulating agent selected from a starch, maltodextrin, inulin, alginate, and combinations thereof.

34. The method of claim 33, wherein the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

35. The method of claim 33 or 34, wherein the starch concentration in the composition is between 10 and 50% w / w.

36. The method of claim 33 or 34, wherein the mass ratio of myoglobin with respect to starch is between 1: 1 to 1:3.

37. The method of any one of claims 30 to 36, wherein the buffer is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM.

38. The method of claim 37, wherein the buffer additionally comprises one or more additives selected from ascorbic acid, cysteine, glucose, glutamic acid, thiamine, or sodium chloride.

39. The method of claim 38, wherein the ascorbic acid concentration is 0.25% w / w.

40. The method of any one of claims 37 to 39, wherein the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

41. The method of claim 30, wherein the mean particle size distribution of the microencapsulated myoglobin is between 1 and 100 pm.

42. The method of any one of claims 30 to 41, wherein the composition comprises at least 20% of the total myoglobin microencapsulated.

43. The method of any one of claims 30 to 42, wherein the foodstuff is a meat substitute or plant-based meat analogues.

44. A flavoured and coloured foodstuff comprising: a meat substitute or a plant-based meat analogue, and a food flavouring and colouring composition including a microencapsulated myoglobin and a buffer.

45. The foodstuff of claim 44, wherein the myoglobin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and any variant having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to SEQ ID NO: 1.

46. The foodstuff of claim 44 or 45, wherein myoglobin concentration is between 10 and 50% w / w in the composition.

47. The foodstuff of any one of claims 44 to 46, wherein the myoglobin is microencapsulated with an encapsulating agent selected from a starch, maltodextrin, inulin, alginate, and combinations thereof.

48. The foodstuff of claim 47, wherein the encapsulating agent is a starch.

49. The foodstuff of claim 48, wherein the starch is selected from the group consisting of unmodified com starch, modified com starch, unmodified tapioca starch, modified tapioca starch, unmodified potato starch, modified potato starch, unmodified rice starch, modified rice starch, unmodified wheat starch, modified wheat starch, alkaline treated starch, bamboo starch, acid treated starch, bleached starch, roasted starch, hydroxypropyl starch, oxidised starch, and combinations thereof.

50. The foodstuff of claim 48 or 49, wherein the starch concentration in the composition is between 10 and 50% w / w.

51. The foodstuff of claim 48 or 49, wherein the mass ratio of myoglobin with respect to starch is between 1 : 1 to 1:3.

52. The foodstuff of any one of claims 44 to 51, wherein the buffer is a sodium phosphate buffer with a pH ranging from 5.8 to 6.2 with a concentration between 50 and 140 mM.

53. The foodstuff of claim 52, wherein the buffer additionally comprises one or more additives selected from ascorbic acid, cysteine, glucose, glutamic acid, thiamine, or sodium chloride.

54. The foodstuff of claim 53, wherein the ascorbic acid concentration is 0.25% w / w.

55. The foodstuff of any one of claims 52 to 54, wherein the buffer comprises sodium phosphate between 50 to 140 mM at pH 6.0, 10 mM cysteine, 10 mM glucose, 10 mM glutamic acid, and ImM thiamine.

56. The foodstuff of any one of claims 44 to 55, wherein the mean particle size distribution of the microencapsulated myoglobin is between 1 and 100 pm.

57. The foodstuff of any one of claims 44 to 56, wherein the composition comprises at least 20% of the total myoglobin microencapsulated.

58. The foodstuff of any one of claims 44 to 57, wherein the plant-based meat analogues are selected from soy protein, pea protein, textured soy protein, textured pea protein, chia fibre, methylcellulose, and mixtures thereof.

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