WO2023004459A1

WO2023004459A1 - Ingredients for meat mimetic products

Info

Publication number: WO2023004459A1
Application number: PCT/AU2022/050791
Authority: WO
Inventors: Mary Ann Augustin; Danyang Ying
Original assignee: v2food Pty Ltd
Priority date: 2021-07-27
Filing date: 2022-07-27
Publication date: 2023-02-02

Abstract

The present disclosure relates generally to texturized protein products for use as ingredients in food products, such as in meat mimetic food products. In particular, the disclosure relates to heat-treated protein sources and their use in the manufacture of texturized ingredients obtained by a thermo-mechanical process, methods for preparing said ingredients, and meat mimetic products comprising said ingredients.

Description

INGREDIENTS FOR MEAT MIMETIC PRODUCTS

FIELD

BACKGROUND

With a need to feed a growing world-wide population, estimated to reach 9.7 billion people by 2050, there is a need to rebalance the animal derived component of the world's food sources to achieve sustainable food systems and increased food and nutrition security. Plant- derived alternatives to meat products represent a growing market due to the shifting dietary patterns of consumers. Increasingly, consumers are concerned about the impact the food production system on the environment, climate change and animal ethics, and this influences the choices they make about food purchases. Vegans, vegetarians, and even animal meat eaters are driving demand for meat alternatives made entirely or substantially from non animal products.

However, many consumers are not willing to eliminate or reduce meat consumption on the basis of ecological and ethical reasons alone. Since the 1960s, textured protein products, prepared from soy flour or concentrate, or other plant-based protein sources ( e.g . wheat, pea, faba and canola), using extrusion technology have become a popular replacement for minced or ground meat. However, animal meat comprises a complex matrix of protein structures and fibres, within which are trapped fats, carbohydrates and water, and which contribute to the sensory, textural and structural characteristics (e.g. flavour, chewiness, juiciness) of the meat-containing food product, and recreating the complex fibrous texture of animal meat from non-meat protein sources, such as soy and pea proteins, has proven challenging. Beyond committed vegetarians and vegans, ready consumer acceptance of these traditional meat mimetic products is compromised due to the non- meat like textures - typically spongy, mushy or rubbery - and there is a need for texturized food ingredients for use in meat mimetic food products that can deliver various structures for desired textures, such as soft chewy, fibrous or cartilaginous, as found in animal meat.

The most common method used to prepare texturized ingredients for use in meat mimetic products is the extrusion process (see for example, Riaz, M.N (2011)., Textured Vegetable Proteins, Handbook of Food Proteins , (Phillips, GO; Williams, PA, Eds), Woodhead Publishing in Food Science Technology and Nutrition, 222, 395-418; and Day L., Swanson B.G. (2013) Functionality of Protein-Fortified Extrudates. Comprehensive Reviews in Food Science and Food Safety, 12, 546-564; and the references cited therein). Protein-rich material, such as protein concentrates or isolates, are combined with other components in an extruder barrel and subjected to heat and shear to produce differentiated textures useful in meat mimetic applications.

Traditionally, soy protein was used but today other sources of plant-based protein sources (e.g. wheat, pea, faba, canola) or proteins from algal sources have been included for producing meat analogues by extrusion. (Liu K, Hsieh F-H (2008) Protein protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems. Journal of Agricultural and Food Chemistry, 56, 2681-2687; Osen R, Toelstede S, Wild F, Eisner P, Schweiggert-Weisz U (2014) High moisture extrusion cooking of pea protein isolates: Raw material characteristics, extruder responses, and texture properties, Journal of Food Engineering, 127, 67-74; Pietsch VL, Emin MA, Schuchmann HP (2017) Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat analogue products. Journal of Food Engineering, 198, 28-35; Rhu G-H (2020) Extrusion cooking of high- moisture meat analogues. Chapter 7, IN Extrusion Cooking, Cereals & Grains Association, Elsevier; do Carmo CS, Knutsen SH, Malizia G , Dessev T, Geny A, Zobel H, Myhrer KS, Varela P, Sahlstrpm S (2021) Meat analogues from a faba bean concentrate can be generated by high moisture extrusion. Future Foods 3, 100014). Combinations of plant proteins and carbohydrate ingredients have also been combined and extruded to produce plant-based meat analogues by extrusion. (Palanisamy M, Topfl S, Aganovic K, Berger RG (2018) Influence of iota carrageenan addition on the properties of soya protein meat analogues. LWT - Food Science and Technology, 87, 546-552).

High moisture extrusion of plant-obtained proteins has been used as a means of introducing layered or fibrous structures, thereby producing a food product, or ingredient therefor, that may more closely mimic the fibrous texture of animal meat proteins. High moisture extrusion of plant proteins involves plasticizing the protein molecules by applying heat and shear to a protei water mixture ( greater than 40%, and typically about 45% to 55%, or even up to 70%, by weight of water) in a heated extruder barrel, followed by forcing the plasticized protein melt through a cooling die positioned at the end of the extruder. Forcing the melt through the cooling die results in molecular alignment of the peptide and protein molecules, and affords a fibrous anisotropic structure.

WO2015/153666 teaches that plant proteins can be combined to produce ground meat replicas that mimic ground animal meat, including fibrousness and heterogeneity in texture. A nutrient-dense meat structured protein product is disclosed in US9526267. where a meat structured protein product comprising protein fibres that are substantially aligned, and where a non-animal protein material and water are combined to form dough and sheared and heated. W02019191807 discloses an extrusion process for the manufacture of a fibrous -textured high-moisture protein foodstuff using a blend of dry proteinaceous materials and/or a stream of wet protein material. US 10477882 discloses a vegan meat-like product using extrusion and texturization of a pea protein in combination with konjac and fenugreek. US 10375981 discloses a meat substitute for use in manufacture of chicken dices, pulled meat, vegetarian sausage made by high moisture texturization of a peanut protein. EP3578053 describes the use of dehulled fava bean flour and vegetable protein flour for producing meat analogues by high moisture extrusion. US20180360088A1 claims a method for improving the quality of high moisture texturized peanut protein by using a cross-linking enzyme. KR102106067B 1 claims manufacture of artificial meat by extrusion of vegetable protein with green tea mixture comprising soy protein isolate, wheat gluten, com starch and green tea.

Differentiated structures can also be prepared using low moisture (<40% by weight of water) extrusion, however, the addition of other ingredients may be required. For example, PCT/AU2019/050883 describes the use of low moisture extrusion of mixtures of plant- derived proteins and carbohydrates to obtain protein-carbohydrate conjugate structures (Maillard reaction products) to imitate one or more textures of animal meat.

Most protein sources for use in these processes are protein-rich, such as protein concentrates containing about 65-80% protein content on a dry weight basis, and protein isolates, typically containing about >80-95% protein on a dry weight basis. These protein-rich sources are typically produced using water and energy intensive wet fractionation processes. The increasing awareness of the importance of sourcing sustainable ingredients has led to the growing interest in the use of dry fractionation techniques, alone or in combination with aqueous processing (hybrid fractionation), as these alternatives use less resources than processes that rely on aqueous fraction alone for concentration of protein. However, dry fractionation techniques can result in an extracted protein material having a lower protein content, typically about 50-70% on a dry weight basis. Due to their lower protein content, protein concentrates obtained from dry fractionation, or protein concentrates obtained by wet or hybrid fractionation and having similar protein contents, may be less desirable or suitable for use in the manufacture of texturized ingredients for meat mimetic s, as they require longer residency times, heating and/or the addition of other components during extrusion, in order to achieve the desired texture, such as fibrosity or chewiness.

There remains a continued need for methods and processes for producing texturized ingredients from plant protein sources having lower protein contents, than for example protein isolates, and that may mimic one or more textures found in animal meat, such as fibrous, chewy or cartilaginous textures. SUMMARY

The present disclosure is predicated on the finding that heat pre-treatment of lower protein content ( e.g . about 45-75% protein) plant-derived protein sources prior to undergoing further heat and shear (thermo-mechanical processing), such as in an extruder or shear cell process, may advantageously afford products with one or more improved textural or structural properties, compared to the corresponding products prepared from the same protein source that has not been pre-treated. Without limiting the disclosure by theory, this may be achieved, at least in part, by subjecting the protein source to heat pre-treatment under conditions sufficient to promote Maillard reactions and/or protein-protein interactions.

In some embodiments, the use of a heat pre-treated protein source containing pre-conjugated biopolymers, such as protein-carbohydrate conjugates and/or protein-protein aggregates, may advantageously increase the development of further protein-carbohydrate conjugates and/or protein-protein aggregates formed during a heat and shear process (using, for example, extrusion or shear cell (e.g. conical or Couette cell) technology), and subsequent cooling process, to afford one or more textural ingredients, that contributes to a desired textured component in a texturized ingredient, such as a fibrous, a soft or hard chewy, or a cartilaginous texture. The contribution or improvement in desired structural or textural characteristic of the ingredient may be measured subjectively, for example by a sensory panel, or objectively, as measured by one or more parameters of Texture Profile Analysis, such as hardness, cohesiveness, springiness or chewiness, compared to the corresponding ingredient made from a protein source that has not been heat-treated. In a first aspect, the disclosure provides a process for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of heating a protein source having a protein content in the range of about 45-75% on a dry weight basis, at a temperature in the range of about 50-110°C for a period of 10 minutes-72 hours; and subjecting the heat-treated protein source to a thermo-mechanical process to produce a texturized ingredient. In some embodiments of the first aspect, the protein source is heated at a temperature of about 50°C, or 55°C or 60°C, or 65°C or 70°C, or 75°C or 80°C, or 85°C or 90°C, or 95°C, or 100°C or 105°C or 110°C. In further embodiments, the protein source is heated for a period of about 30 minutes -48 hours, such as from about 30-24 hours, such as about 1-12 hours, or 30-180 minutes, e.g. about 30, or 45, or 60, or 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes. In some embodiments, the protein source is heated for about 15, 20, 25, 30, 45 or 60 minutes at about >90-110°C; or about 30, or 45, or 60, or 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes at about 50, 55, 60, 65, 70, 75, 80, 85 or 90°C.

In some embodiments of the first aspect, including any embodiments above, the protein source is heated in an oven, such as a humidity oven, or heated in a fluid bed dryer.

In some embodiments of the first aspect, including any embodiments above, the protein source is heated in the presence of moisture, such as a relative humidity in the range of about 20-90%RH, such as about 50-90%RH, or 50-80%RH.,

In some embodiments of the first aspect, including any embodiments above, the protein source consists of or comprises a dry fractionated protein, for example, soy, faba bean, chickpea,, pea or lentil protein.

In some embodiments of the first aspect, including any embodiments above, the thermo mechanical process is extrusion. In further embodiments, the extrusion is conducted under low moisture conditions.

In some embodiments of the first aspect, including any embodiments above, the texturized ingredient (optionally hydrated) demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre-treatment,

In another aspect, there is provided a process for heat pre-treating a protein source having a protein content in the range of about 45-75% on a dry weight basis, for use in a thermo mechanical process to produce a texturized ingredient for a meat mimetic food product, wherein the texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre treatment, said process comprising the step of heating the protein source at a temperature in the range of 50-200°C.

In some embodiments, the protein source is heated at a temperature in the range of about 50- 110°C, such as about 50°C, or 60°C, or 70°C, or 80°C or 90°C or 100°C or 105°C or 110°C. In further embodiments, the protein source heated at a temperature of about 50-110°C for a period of about 10-180 minutes, such as about 30, or 45, 60, 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes. In some embodiments, the protein source is heated for about 15, 30, 45 or 60 minutes at about >90-110°C, or about 30, 45, 60, 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes at about 50, 55, 60, 65, 70, 75, 80, 85 or 90°C.

In some embodiments, the protein source is heated at a temperature in the range of about 100-200°C, such as about 100°C, 110°C, 120°C, 130°C, 140°C, 150°C In further embodiments, the protein source is heated at a temperature for a period of about 10-60 minutes, such as about 10, or 20, or 30, or 40, or 50 minutes.

In any one or more of the aspects or embodiments above, the protein source has a protein content in the range of about 50-70% on a dry weight basis, such as about 55-65%.

In any one or more of the aspects or embodiments above, the protein source is a dry fractionated protein obtained from a dry fractionation process. In any one or more of the embodiments above, the protein source is a protein concentrate obtained from a wet fractionation process. In any one or more of the embodiments above, the protein source is obtained from a hybrid fractionation process. In any one or more of the aspects or embodiments above, the protein is selected from one or more of chickpea, pea, cowpea, soy, lupin, canola, mung bean, lentil, quinoa, hemp, algal and faba bean protein. In further embodiments, the protein is selected from soy protein, faba bean protein, pea protein, chickpea protein or lentil protein.

In any one or more of the aspects or embodiments above, the protein source is obtained by a dry fractionation process and has protein content of about 50-70% protein on a dry weight basis. In further embodiments, the protein content is about 55-65% protein on a dry weight basis. In further embodiments, the protein source is obtained from soy, faba bean, pea, lupin or lentil

In any one or more of the aspects or embodiments above, the protein source is obtained by a wet fractionation process and has a protein content of about and has protein content of about 50-70% protein on a dry weight basis. In further embodiments, the protein content is about 55-65% protein on a dry weight basis. In further embodiments, the protein source is obtained from soy, chickpea, lentil, pea or faba bean.

In any one or more of the aspects or embodiments above, the protein source is heat pre treated in the absence of one or more additional or extrinsic carbohydrate sources.

In any one or more of the aspects or embodiments above, the protein source is heat pre treated in the presence of one or more additional carbohydrate sources, i.e. a mixture of protein source and additional carbohydrate source is subjected to heat pre-treatment. In some embodiments, including any of the above, the one or more additional carbohydrate sources may be selected from one or more of sugars (including reducing sugars), oligosaccharides, maltodextrin, pectin, dried glucose syrup, carrageenan (iota, kappa or lambda), gums, fibre, starch (including low amylose starches (0-25% amylose content) and high amylose starches (>40% amylose content)), resistant starch or modified starches, b- glucan or b-glucan rich sources or potato flour or other flour from plant sources. In further embodiments thereof, the one or more additional carbohydrate source(s) are added in a total amount of about 10%, or less, such as about 5% or less, on a dry weight basis. In any one or more of the aspects or embodiments above, the protein source is subjected to heat pre-treatment in the presence of added moisture, In further embodiments, the protein source is subjected to heat pre-treatment at a humidity in the range of about 50-90% R.H.

In any one or more of the aspects or embodiments above, the protein source is subjected to heat pre-treatment in an oven or fluid bed dryer.

Another aspect of the disclosure provides the use of a heat pre-treated protein source according to the disclosure in the manufacture of a texturized ingredient for a meat mimetic food product, wherein the heat pre-treated protein source has a protein content in the range of about 45- 75% on a dry weight basis, and wherein the resulting texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre treatment.

In another aspect, there is provided a method for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of subjecting a heat pre-treated protein source according to the disclosure having a protein content in the range of about 45-75% on a dry weight basis, to a thermo-mechanical process to produce a texturized ingredient, wherein the resulting texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre treatment.

In another aspect, there is provided a texturized ingredient, for use in a meat mimetic food product, prepared by subjecting a heat pre-treated protein source according to the disclosure to a thermo-mechanical process, wherein the heat pre-treated protein source has a protein content in the range of about 45- 75% on a dry weight basis, and wherein the texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre- treatment.

In any one or more aspects or embodiments above, the thermo-mechanical process is conducted in an extruder, preferably under low moisture conditions (barrel moisture of 40% (w/w) or less).

In any one or more aspects or embodiments above, the thermo-mechanical process is conducted in a shear cell, such as a conical or Couette cell preferably under low moisture conditions (moisture of 40% (w/w) or less). In any one or more aspects or embodiments above, the heat pre-treated protein source is optionally extruded or subjected to shear together with one or more further added carbohydrate source(s).

In another aspect, there is provided a meat mimetic food product comprising a texturized ingredient prepared according to the disclosure.

BREIF DESCRIPTION OF FIGURES

Figure 1A sets out a generalized exemplary scheme for the use of a heat pre-treated protein source to produce a texturized ingredient for use in a meat mimetic food product.

Figure 2 graphically depicts a typical Texture Profile Analysis (TPA) profile for determining hardness, cohesiveness, springiness and chewiness of a texturized ingredient product produced by subjecting a heat pre-treated protein source to thermo-mechanical treatment. DESCRIPTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of’, and variations such as “consists essentially of’ will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the invention defined.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

The singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise.

The term “invention” includes all aspects, embodiments and examples as described herein.

As used herein, “about” refers to a quantity, value or parameter that may vary by as much as 10%, 5%, or 1-2% of the stated quantity, value or parameter, and includes at least tolerances accepted within the art. When a value is stated as a whole number, "about" may also refer to 1 or 2 whole numbers either side, as appropriate, for example, "about 50", may include values of 48, 49, 50, 51 and 52. When prefacing a recited range or list of values, it is intended to apply to both upper and lower limits of the range and each member of the list.

Unless the context indicates otherwise, features described below may apply independently to any aspect or embodiment of the invention.

As used herein, a texturized ingredient comprises a product obtained by subjecting a heat pre-treated protein source to thermo-mechanical processing, such as in an extruder or shear cell ( e.g . a conical or Couette cell), so as to afford a component or ingredient for use in a meat mimetic food product that mimics one or more textures found in animal meat. In some embodiments, the texturized ingredient provides a fibrous, chewy and/or or cartilaginous- like textured component intended to mimic one or more components found in animal meat.

As used herein, a meat mimetic food product refers to a food product that mimics, resembles or performs in a manner similar to an animal-derived meat product in any one or more physical or sensory factors, including pertaining to appearance, taste, texture, mouthfeel (moistness, chewiness, fattiness etc), aroma, or other physical properties, including structure, texture, storage, handling, and/or cooking.

Protein sources suitable for use according to the disclosure include non-animal (e.g. plant, fungal, or algal) -derived protein sources, i.e. proteins, polypeptides and/or amino acids derived from a non-animal source, such as a plant source, for example beans (e.g. soy beans, kidney beans, lima beans, black beans, faba beans, mung beans), chick peas (garbanzo), peas, lentils, lupin, cowpea, canola, quinoa, hemp and algae. The protein source used may obtained from a single protein source or a mixture of one or more protein sources.

The protein source may be obtained from a native or naturally occurring plant, fungus or algae, or a genetically modified or mutated plant, fungus, or algae, or mixtures thereof. In still other embodiments, the protein source may comprise synthetic or biosynthetically generated protein or polypeptide molecules. As used herein, unless otherwise indicated, "protein source" refers to protein in a dry form, for example as a powder and/or flakes, and may be obtained by a dry, wet or hybrid fractionation process. The protein source comprises about 45-75% protein on a dry weight basis, such as about 45-50%, or about 50-55%, or 55-60%, or about 60-65%, or about 65- 70%, or about 70-75%. In some embodiments, the protein source has a protein content of about 50-70%, such as about 55-65% on a dry weight basis. Flakes may be converted to smaller particle size (powder) form, for example by pounding or milling. The powder form has a higher surface area and may thereby potentially facilitate or expedite the formation of Maillard reaction products and/or protein-protein aggregates. Thus, in some embodiments, the protein source is in powder form, or comprises a powder form.

Although reference is made to a dry form of a protein source (as opposed to a protein source in the form of an aqueous mixture or slurry), it will be understood that the dry form can contain moisture. Accordingly, the protein source may comprise up to about 10% moisture by weight, such as about 1%, 2%, 3%, 4%, 5%, 6,%, 7%, 8%, 9% or 10%.

Dry fractionated protein concentrates, and wet or hybrid fractionated protein concentrates retain a greater proportion of native carbohydrates and water soluble saccharides from the original source compared to wet-fractionated protein isolates (protein contents of or greater than about 80% on a dry weight basis). Without intending to limit the disclosure by theory, the higher proportion of endogenous carbohydrates and saccharides in such a protein may facilitate the formation of protein-carbohydrate conjugates and Maillard reaction products during the heat pre-treatment process. In contrast, protein sources with higher protein contents, e.g. protein isolates having a protein content in the range of >80-90% or greater, have had a significant proportion of the endogenous water soluble carbohydrates and saccharides removed during processing, and may, on their own, be less amenable to the formation of desirable protein-carbohydrate conjugates and other Maillard reaction products. The method of fractionation may also determine the carbohydrate content of a protein source. In particular, wet fractionated protein concentrates generally have a lower carbohydrate content compared to a dry fractionated protein concentrate, since water-soluble carbohydrates are washed out during processing. Thus in some embodiments, the protein source comprises at least about 5% on a dry weight basis of endogenous carbohydrates (including dietary fibre and saccharides), such as at about 5-6%, or about 7-8%, or about 9- 10%, or about 11-14%, or about 15-20%, or more. In further embodiments, the endogenous carbohydrates advantageously include reducing groups to facilitate the formation of protein- carbohydrate conjugates and other Maillard reaction products.

In some embodiments, the protein source comprises or consists of a protein source obtained by a dry fractionation process. Dry fractionation involves dry milling of the non-animal- derived material (e.g. plant material) during which the starch and protein components are detached from the cellular matrix and subsequently separated, to afford a protein-enriched fraction. Methods therefor are known in the art, see for example: Schutyser, M.A.I., et al, Trends in Food Science & Technology, 45 (2015), 327-335, and the references cited therein; S. Thakur, et al, (2019) Pulse Flour Characteristics from a Wheat Flour Miller’s Perspective: A Comprehensive Review. Comprehensive Reviews in Food Science and Food Safety, 775-797; Tyler, R. T., et al, 1981. Air classification of legumes of legumes. I. Separation efficiency, yield and composition of the starch and protein fractions. Cereal Chem. 58, 144-148; Pelgrom, P. J. M., et al., 2014. Preparation of functional lupine protein fractions by dry separation. LWT Food Sci. Technol. 59, 680-688; Pelgrom, P. J. M., et al, 2015. Method development to increase protein enrichment during dry fractionation of starch-rich legumes. Food Bioproc Tech. 8, 1495-1502; Trends in Food Science & Technology, Volume 86, April 2019, Pages 340-351, “Dry fractionation methods for plant protein, starch and fiber enrichment: A review ”, A. Assatory et al, and W02006/006845 Al; the contents of which are incorporated herein by reference. Some examples of dry fractionated proteins include soy, faba bean, pea and lentil protein.

In some embodiments the protein source consists of or comprises a protein source prepared by a wet fractionation method. Wet fractionation may typically involve processing methods involving alkali or HCL solubilisation/dilution, followed by isoelectric precipitation to obtain protein-rich fractions. A further spray drying step affords dry powders. Methods for wet fractionation are known in the art, see for example Schutyser et al, supra·, Wu, S., Murphy, et al., 1999. Pilot-plant fractionation of soybean glycinin and b-conglycinin. J. Amer. Oil Chem. Soc. 76(3), 285-293; Arrutia, F., et al., 2020. Oilseeds beyond oil: Press cakes and meals supplying global protein requirements. Trends Food Sci. Technol. 100, 88- 102; Berghout, J.A.M., et al, 2014. The potential of aqueous fractionation of lupin seeds for high-protein foods. Food Chem. 159, 64-70; and Ntone, E., et al, 2020. Not sequentially but simultaneously: Facile extraction of proteins and oleosomes from oilseeds, Food Hydrocoll. 102, 105598; and the references cited therein.

In other embodiments, the protein source consists of or comprises a protein source obtained by a hybrid dry and wet fractionation process, see for example Schutyser et al, supra, and the references cited therein.

A form of protein source having a lower protein content, for example a protein flour, or protein concentrate, for example, having a protein content of about 45-60%, may be supplemented or combined with a form of protein having a higher protein content, provided that the total protein content is in the range of about 45-75% on a dry weight basis. Thus, in one example a protein source may be further supplemented by a "protein isolate" (the dry powder form obtained by drying the product of a wet extraction process), which contains greater than about 80% protein on a dry weight basis, for example about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94 or 95 % protein on a dry weight basis. Some exemplary protein isolates include soy, pea, lupin, chickpea and faba bean isolates.

In some advantageous embodiments, the protein source (which may be a mixture of different two or more different protein sources, for example, different originating sources, or different protein/carbohydrate contents) is subjected to a heat pre-treatment step prior to thermo mechanical processing (such as in an extruder or a concial or Couette cell) under conditions which promote the formation of protein-carbohydrate conjugates between proteins/peptides/amino acids and carbohydrates with a reducing sugar group (Maillard reaction products) and/or protein-protein aggregates. Thus, a "heat pre-treated" protein source may advantageously comprise protein-carbohydrate conjugates (Maillard reaction products) and/or protein-protein aggregates formed as a result of a heat pre-treatment step (preferably, under Maillard reaction conditions). Thus in some embodiments, a heat pre-treatment step comprises exposing the protein source to heat, and optionally moisture, for a suitable period of time and under conditions so as to form a heat pre-treated protein source comprising protein-carbohydrate conjugates and/or protein-protein aggregates.

In some embodiments, dependent on the extent of formation and/or sensitivity of the testing method, the presence of protein-carbohydrate conjugates and/or protein-protein aggregates may be detected or quantified by any suitable means known in the art, for example colour change, SDS Page or FTIR,

Suitable temperatures for the heat pre-treatment step may be in the range of from about 50- 200°C, such as in the range from about 60-200°C, or 80-190°C, or 90-180°C, or 100-170°C or 110-160°C or 120-150°C, and may be dependent upon the nature of the protein source, the amount and types of endogenous carbohydrates present, any additional carbohydrate added and the heating conditions ( e.g . length of time, moisture/humidity, heating equipment, etc). The protein source may be subjected to heat at a single temperature such as about 50°C, or about 60°C, or about 70°C, or about 80°C, or about 90°C, or about 100°C, or about 110°C, or about 120°C, or about 130°C, or about 140°C, or about 150°C, or about 160°C, or about

170°C, or about 180°C, or about 190°C or about 200°C; or may be subjected to a range of temperatures, for example a range of temperatures starting at a lower temperature, which is then increased to one or more higher temperature over the duration of the heat treatment. While heat -pre-treatment conditions may include the absence or very low levels of moisture

(e.g. up to 1, 2, 5, 10, 15 or 20% relative humidity), the presence of moisture may increase the rate or extent of formation of protein-carbohydrate conjugates and/or protein-protein aggregates. Suitable moisture conditions may include uncontrolled humidity, or controlled relative humidity, for example, in the range of about 20-90% RH, such as about 50-90% R.H., for example about 55%, or about 60%, or about 65%, or about 70% or about 75%, or about 80%, or about 85% or about 90%R.H.. In some embodiments, suitable heat pre-treatment intervals may range from about 5-60 minutes, such as about 10, 15, 20, 30, 35, 40, 45, 50, or 60 minutes, about 1-3 hours ( e.g ., about 1, 1.5, 2, 2.5 or 3 hours), about 7-12 hours, about 15-18 hours, or about 24 hours or about 48-72 hours. In some embodiments, at lower temperatures (e.g. less than 100°C, longer intervals (e.g. 1-72 hours) may be beneficial. In some embodiments, at higher temperatures (e.g. > 100°C, such as 110-160°C), shorter intervals (e.g. 1-90 minutes, such as 5-60, or 10-30 minutes) may be beneficial. In some embodiments there is provided a process for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of heating a protein source having a protein content in the range of about 45-75% on a dry weight basis, at a temperature in the range of about 50-110°C for a period of 10 minutes-72 hours The heat pre-treatment step may be performed with any suitable heating equipment, such as conventional ovens, and drying cabinets, temperature-humidity ovens and fluid bed dryers.

Conventional ovens and drying cabinets with controlled relative humidity (for example from 0-90%, such as 50-80%, e.g. about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%) can be used for the heat pre-treatment of the protein source (optionally together with a further carbohydrate ingredient, such as disclosed herein). Examples of conditions include heating at about 50-100°C, such as 60-90°C, for long intervals (for example between 30 minutes-48 hours, such as 1, 2, 3, 5, 7, 12, 18, 24 or 36 hours). In some further examples, conditions may include heating at about 60-90°C for 30, 35, 40, 45, 50, 55 or 60 minutes. Alternatively, the protein source can be heated at higher temperatures (for example, about <100-200°C, e.g., 105, 110, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 195°C), for a shorter period of time (for example, about 2 min-90 min, such as 5, 10, 15, 20, 25, 30, 45, 60, 75 or 90 min). Some exemplary conditions may include heating at about 130-<150°C for about 1-30 minutes, such as about 1-10 minutes, e.g. about 2-3, 4-5, 6-8, 9-10, 12-15, 17-20, 22-25 and 27-30 minutes.. The rate of reaction is dependent on the nature of the protein matrix and carbohydrates present, and reactions are promoted above the glass transition temperature.

Alternatively a fluid bed may be used to treat the protein source (optionally together with a further carbohydrate ingredient, such as disclosed herein) at various combinations of temperatures, humidity's, and processing times, including those as disclosed above, (e.g. about 50- 100°C, such as about 60-90°C, with or without humidity for a period of 5-60 min).

Alternatively continuous heat treatment equipment (for example, as supplied by Revtech Process Systems, France), using a combination of electrically generated heat and vibrational transport in a closed stainless steel spiral,) may be used for pre-treatment of the dry powdered protein- source (optionally together with a further carbohydrate ingredient, such as disclosed herein) using various combinations of temperature, relative humidity and times as disclosed above (e.g. 130 - 200°C, 1-100% steam, 2-15 min).

In some embodiments, the heat pre-treatment is carried out under reduced pressure, such as in a pressure cooker, for example about 1.7-2.0 bar (absolute).

In some embodiments, there is provided a process for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of heating a protein source having a protein content in the range of about 45-75% on a dry weight basis, at a temperature in the range of about 50-110°C for a period of about 10 minutes to-72 hours; and subjecting the heat-treated protein source to a thermo-mechanical process to produce a texturized ingredient, wherein the protein source comprises or consists of a dry fractionated protein such as soy, faba bean, chick pea, lupin, lentil or pea; and wherein the protein source is heated at a relative humidity in the range of about 20-90% R.R, such as about 50-80% RH. . In some embodiments of the disclosure, the heat pre-treatment step can form protein- carbohydrate conjugates between proteins/peptides/amino acids and intrinsic or native carbohydrates, particularly those having one or more reducing (carbonyl - aldehyde or ketone) groups, present in the extracted protein source, i.e. in the absence of added or extrinsic carbohydrates.

In some embodiments of the disclosure, a further extrinsic carbohydrate source may be added to the protein source to increase the development of protein-carbohydrate conjugates during the pre-treatment step. The additional extrinsic carbohydrate source may contain one or more of the same native or instrinsic carbohydrates, and/or a different carbohydrate. The additional carbohydrate source may advantageously include reducing groups. Thus, a heat pre-treated protein source may further contain protein-carbohydrate conjugates formed between the protein source and the additional extrinsic carbohydrate source. A carbohydrate source, which may include two or more different sources, can be mixed or blended with the powdered protein source or an aqueous suspension/dispersion of the protein source. Some examples of carbohydrate sources may include one or more of sugars (including reducing sugars), starches, gums, pectins and fibres, and may include monosaccharides, disaccharides, trisaccharides, polysaccharides and oligosaccharides and mixtures of two, three or four thereof, and be in any suitable form, such as milled, ground or powdered, or aqueous suspension, dispersion or solution. The carbohydrate source may be obtained from a single or multiple plant sources. Some suitable examples may include any one, or a mixture of two or more of starch (e.g. potato, rice, wheat, corn, oat, pea, cassava), resistant starch, for example, retrograded starch, high amylose starch (e.g. having at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% amylose content, such as Hylon V, Hylon VII, Hi-Maize 1043, Hi-Maize 240, Hi-Maize 260, Novelose 330, Novelose, 240, Novelose 260) pectin, fructans (e.g. inulins) b-glucan, carrageenan (iota, kappa, lambda), maltodextrin, pectin, methyl cellulose, alginate, guar gum, xanthan gum, gum carboxymethyl cellulose, locust bean gum, gellan gum, cellulose, hemicellulose, gums, flour (e.g. milled or ground from a grain, legume or tuber, such as wheat, rice, corn, oat, rye, barley, quinoa, amaranth, potato, carrot) and edible fibre such as oat bran, wheat bran rice bran, barley bran com bran, and carrot fibre. The amount of additional carbohydrate added to the protein source may depend on the nature and form of the protein source, for example a wet fractionated protein source which has had a proportion of soluble carbohydrates removed during processing, may require the addition of more carbohydrate than a dry fractionated protein source. The type of carbohydrate added may also influence the quantity added, for example, less may be required of a carbohydrate with more reducing groups than a carbohydrate with fewer reducing groups, or the water-holding capacity of an added carbohydrate may affect the available water for generating Maillard reaction products. In further embodiments thereof, the one or more additional carbohydrate source(s) are added to the protein source (having a protein content in the range of about 45-75%), in a total amount of about 10% or less on a dry weight basis, such as about 9%, or 8%, or7%, or 6%, or about 5.0% or less, on a dry weight basis, e.g., about 4.0%, or 3.0%, or 2.5%, or 2.0%, or 1.5%, or 1.0%, or 0.5%, or 0.1%

The extrinsically added carbohydrate source may be obtained from the native or naturally occurring plant, or a genetically modified or mutated plant, or mixtures thereof. In still other embodiments, the carbohydrate source may comprise, synthetic (e.g. chemically esterified) or biosynthetically (e.g. fermentation) generated monosaccharides, disaccharides, polysaccharides and oligosaccharides molecules, and may be the same or different to endogenous carbohydrates present.

In some embodiments, the carbohydrate source contains a reducing sugar group, i.e. having a free carbonyl group able to participate in reaction with amino groups in the protein source to form Maillard reaction products. The carbohydrate source may include a reducing sugar (e.g. more mono- di- or trisaccharides or oligosaccharides, such as glucose, galactose, fructose, glyceraldehyde, ribose, xylose, cellobiose, maltose, isomaltose, lactose and maltotriose), and/or other carbohydrate source, (e.g. pectin, starch, carrageenan (iota, kappa or lamba), maltodextrin).

In some embodiments, a carbohydrate source has been mechanically or chemically treated prior to mixing with the protein source, for example to increase the number of reducing carbonyl groups. Suitable treatment process may include microfluidization, ultrasound treatment or high pressure extrusion. For example, starch may be mechanically or chemically treated to increase the content of resistant starch, for example, from a low amylose starch, having an amylose content of less than about 25-30%, to an amylose content of at least about 50-80%. In some embodiments the carbohydrate source may be an esterified carbohydrate, such as high methoxy pectin, with about >50% degree of esterification, as well as low methoxy pectin (about <50% degree of esterification, or substituted fatty acids starch esters, e.g. acetylated, propionated or butylated starches with various degrees of substitution.

In one or more embodiments, optionally one or more other Maillard reaction promoting agents may be added to the protein source, alone or as a protein-carbohydrate mixture or blend. Examples of such agents may include, ascorbic acid, or agents that possess an amino group, for example one or more amino acids, such as alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, aspartic acid, glutamic acid, arginine, histidine, and lysine, peptides, polypeptides or hydrolysed protein.

In one or more embodiments, optionally one or more crosslinking agents to promote covalent coupling between carbohydrate-carbohydrate, carbohydrate -protein and/or protein-protein molecules may be mixed with the protein source (and optionally an additional carbohydrate source). Crosslinking agents may include chemical and/or enzymatic crosslinking agents. Some examples include transglutaminase (TG), and oxidative enzymes such as laccases, tryosinases and peroxidases, as well as phenolic compounds such as caffeic acid, catechin, ferullic acid and tannic acid. Enzymes that catalyse the breakdown of larger molecules, such as amyloglucosidases, ligninolytic enzymes, proteases and pectinases may also be added (see for example, Gatt, E., Industrial Crops and Products, 122, 329-339, 2018).

Formation of Maillard reaction products may be facilitated by higher pH conditions, and, thus, in some embodiments a pH adjusting agent may be added to increase the pH of the protein source or protein-carbohydrate mixture. Thus, in some embodiments, pre-treatment process is carried out at a pH of or greater than 7. In some more embodiments, the pre treatment process is carried out at a pH of about 7.5 or greater, or a pH of about 8.0 or greater, or a pH of about 8.5 or greater, or a pH of about 9. The desired pH may be achieved or adjusted, by a pH adjusting agent. Exemplary pH adjusting agents include any organic or inorganic agent capable of raising, lowering or maintaining the pH of the protein source or protein-carbohydrate mixture. Some examples of pH adjusting agents include citrates, citric acid, tartaric acid, tartrates, lactic acid, malic acid, bicarbonates, carbonates, phosphates and hydroxides, such as sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, calcium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate, potassium phosphate. In some embodiments, the pH adjusting agent may be added in an amount of up to about 10% by weight, for example, up to 0.5-1%, 1-2%, or 3-5%, or 7-8% by weight. In some embodiments, the pH adjusting agent is added to an aqueous suspension or dispersion of the protein source. In further embodiments thereof, the resulting aqueous suspension or dispersion with added pH adjusting agent may be directly subjected to heat pre-treatment conditions. In other embodiments thereof, the aqueous suspension or dispersion with added pH adjusting agent is gently dried ( e.g . evaporation, spray drying, fluid bed drying, freeze drying under conditions that do not promote Maillard reactions or protein-protein aggregation) before being subjected to heat pre-treatment conditions.

The addition of one or more carbohydrate sources, Maillard promoting agents, crosslinking agents and/or pH adjusters to the protein source may require intimate blending with the protein source prior to the heat pre-treatment step. In some embodiments, this may be achieved by ensuring all materials are of substantially similar size followed by thorough blending. In some embodiments, all components are dispersed in an aqueous mixture, followed by subsequent gentle drying (e.g. as described supra ) to powder form.

In some embodiments, in addition to improving desired textural attributes, the protein- carbohydrate conjugates formed in the protein source by the heat pre-treatment step may further afford useful techno-functional properties such as improved heat stability, emulsifying, foaming, gelling, water retention or absorption, and viscosity building properties, and accordingly in one or more embodiments use of a heat pre-treated protein source in a thermo-mechanical process to produce a meat mimetic food ingredient may provide further advantages related to one or more such properties of the protein-carbohydrate conjugates formed in the heat pre-treatment step. For example, depending on the extent of the Maillard reaction and the type of protein-carbohydrates treated, other desirable properties such as antioxidative and anti-microbial properties (Naik, Wang & Cordelia Selomulya (2021), Improvements of plant protein functionalities by Maillard conjugation and Maillard reaction products, Critical Reviews in Food Science and Nutrition,, Federica A. Higa & Michael T. Nickerson (2021), Plant Protein-Carbohydrate Conjugates: A Review of Their Production, Functionality and Nutritional Attributes, Food Reviews International,, Kutzli, Weiss, Gibis (2021). Glycation of Plant Proteins Via Maillard Reaction: Reaction Chemistry, Technofunctional Properties, and Potential Food Application. Foods 2021, 10, 376; Biihler J.M., et al, Foods, 2020, 9, 1077 D01:10.3390/foods9081077; and WO2018/183729 Al; the contents of which are incorporated herein by reference.

Extrusion and shear cell processes for the manufacture of texturized ingredients for meat mimetic food products are well known in the art (see, for example, the references cited above; Krintaris, G. A., et al, On the use of the Couette cell technology for large scale production of textured soy-based meat replacers., Journal of Food Engineering, 2016,169, 205-213; Cornet, S. H. V., et al, Thermo-mechanical processing of plant proteins using shear cell and high moisture extrusion cooking, Critical Reviews in Food Science and Nutrition (2021), Jan 6, 1-18, DOI: j_.(y_.08()/j_.()408398,2020J 864618, and the references cited therein; the contents of which are incorporated herein by reference).

As used herein "low moisture" refers to the total moisture content of the materials inside the extruder barrel or shear cell of 40% (w/w) or less. In calculating the initial moisture content, the respective moisture contents of the ingredients, such as the protein sources and any other ingredients, is taken into consideration. In some embodiments, low moisture refers to a moisture content in the range of 35-40% (w/w) (e.g., about 35%, 36%,. 37%, 38% or 39 %) or 30-35% (w/w) (e.g., about 30%, 31%, 32%,. 33%, or 34%) or 25-30% (w/w) (e.g., about 25%, 26%, 27%, 28%, or 29%) or about 20-25%, or about 15-20%, or about 10-15% or about 5-10%.

In some embodiments, higher moisture conditions may be used, for example, greater than 40% (w/w) moisture, such as about 45% (w/w), 50% (w/w), 55% (w/w), 60% (w/w), 65% (w/w), 70% (w/w) or 75% (w/w).

In some embodiments, the heat pre-treated protein source is subjected to thermo-mechanical processing (heat and shear) in an extruder, optionally in the presence of one or more additional carbohydrate sources ( e.g . as listed supra), which may be the same or different to any carbohydrate source subjected to the earlier heat-pre-treatment step along with the protein source. In some embodiments, the one or more additional carbohydrate source(s) added to the extruder may be added in a total amount of up to about 10% (w/w) of the total amount of heat-pre treated protein source, such as about 0.5, or 1.0%, or 1.5%, or 2.0%, or 2.5%, or 3.0%, or 4.0% or 5.0%, or 6.0%, or 7.0%, or 8.0% or 9.0% or 10.0%.

In some embodiments, the extrusion process is performed under low moisture conditions as defined supra. Thus, subjecting the heat pre -protein source (and optionally an additional carbohydrate source(s)) to extrusion comprises the step of mixing the heat-pre-treated protein source and any other components in an extruder. The heat-pre-treated protein source and any other ingredients are typically fed into a stationary smooth or grooved bore barrel, containing one or more rotating screw shafts within the barrel, which is terminated by a restriction (breaker plate) and, optionally, a die. In some embodiments, single or double screws mix and transport the ingredients down the shaft of the barrel, thereby subjecting the ingredients to a mechanical shear. Conditions, such as temperature and or pressure, within the barrel may be zoned to provide a varying temperature/pressure profile to which the mixture is subjected. At the end of the barrel, the mixture may be forced through a die. Traditionally such as in high moisture extrusion of protein, a holding die which is actively cooled, for example by a water jacket, is used as a cooling zone to “set” the plasticized mixture. In some embodiments, the holding die is cooled, for example with water at a temperature of 20, 25 or 30°C, however, in some embodiments of the present disclosure, the holding die is heated to maintain or increase the temperature of the melt exiting the extruder, or to slow the rate of cooling of the melt or maintain a specified temperature. Suitable heating temperatures for the die may be in the range of about 50-120°C, for example 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115 or 120°. In other embodiments, the die is neither actively heated nor cooled (also referred to herein as at ambient temperature). In some embodiments, the use of an ambient temperature (non-heated) die or heated die (as opposed an actively cooled die), may potentially generate further protein-carbohydrate conjugation and/or cross-linking of proteins (protein-protein aggregates).

Suitable extrusion equipment is known in the art, and may include single or twin screw extruders, or multiple screw extruders, such as in a planetary roller extruder. Heating of the mixture within the barrel may be effected by the heat generated by the physical shear process and/or by an external heat generating source, e.g. electrical heating.

One or more optional ingredients (i.e. may be present or absent) may be added to the extruder barrel together with the heat pre-treated protein source (optionally heat pre-treated with an additional carbohydrate source or other agent as described supra). One such ingredient may include a further carbohydrate source as described herein. Some other non-limiting examples of optional ingredients include pH adjusting agents, Maillard reaction promoting agents, crosslinking agents, enzymes, phenolic compounds, flavouring agents, colouring agents, fats, oils (e.g. plant derived oils, such as canola, sunflower, olive, coconut, vegetable, palm, peanut, flaxseed, cotton seed, corn, safflower, rice bran oil, optionally added in an amount of from about 0.5, 1, 2, 3 or up to about 5%(w/w)), lipids, fatty acids (e.g. omega-6 (e.g. linoleic acid) and omega-3 fatty acids (such as ALA, EPA, DHA) and their esters, and phospholipids containing such fatty acids, binding agents, emulsifiers (e.g. lecithin, polysorbates (20, 40, 60 80)), antioxidants, surfactants, salts, and nutritional agents e.g. essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), vitamins (e.g. A, B (1, 2, 3, 5, 6, 7, 9, and 12), C, D, E, K), minerals (calcium, phosphorus, magnesium, sodium, potassium, zinc, iodine, iron, copper.), and phytonutrients, such as carotenoids (e.g. a- and b- carotene, b-cryptoxanthin, lycopene, lutein), flavonoids (e.g. flavanols, flavonols flavones, flavonones, isoflavones, ), polyphenols (e.g. anthocyanins, quercetin, ellagic acid)). Where the added ingredient is potentially heat/shear sensitive and may potentially be degraded under extmsion/shear cell conditions, an ingredient may optionally be microencapsulated. While in some embodiments one or more optional ingredients may be included in the barrel/shear cell mixture, in some embodiments one or more such ingredients may be excluded. It will be understood that one or more optional agents may serve one or more functions.

In one or more embodiments, the texturized food ingredient may be dried, for example at room temperature, or at a temperature in the range of about 50-95°C, such as about 55 °C 60°C, or 65°C, or 70°C, or 75°C, or 80°C or 85°C or 90°C, for any suitable period, such as from about 1-24 hours, such as 2„ 3, 4, or 5 hours or 10-12 hours or 15-20 hours.

In one or more embodiments, the use of a pre-treated protein source according to the disclosure may improve the efficiency of the extrusion/shear process (compared to the use of the corresponding non-pre-treated protein source by any one or more of: reducing residency time in the extruder barrel or shear cell, reducing residency time in and/or length of the cooling die, reducing the temperature in one or more zones of the extruder barrel, reducing the number of temperature zones in the extruder barrel, and changing the design or configuration or the screw(s).

In one or more embodiments, the use of a pre-treated protein source according to the disclosure may afford a texturized food ingredient that has a more desirable texture (compared to the use of a non-pre-treated protein source), such as one or more of increased fibre length, or degree of fibrosity, or other objective texture measurements determined using a texture analyser, or by sensory evaluation of the product. In some embodiments, the use of a pre-treated protein source according to the disclosure may afford an extruded food ingredient that (optionally hydrated) has a more desirable texture with regard to one or more parameters of texture profile analysis (TPA), such as hardness, cohesiveness, springiness or chewiness. Thus in some embodiments, the resulting food ingredient prepared by subjecting the heat pre-treated protein source to thermo-mechanical treatment, such as extrusion or shear, and optionally hydrated has one or more of increased hardness, cohesiveness, springiness or chewiness compared to the corresponding food ingredient prepared under the same conditions from the same protein source, but that has not been subjected to heat pre treatment. In further embodiments, the resulting food ingredient demonstrates increased chewiness compared to the corresponding product prepared from the protein source which has not been subjected to heat pre-treatment. Methods for the determination of hardness, cohesiveness, springiness and chewiness are known in the art and further described herein.

In some advantageous embodiments of the disclosure, the heat pre-treatment conditions may also remove or mask undesirable flavours and/or odors from the protein source, and/or produce desirable flavours and/or odours, and thus provide an ingredient with more desirable flavor and/or odour attributes.

The texturized food ingredients (fresh, or dried, and optionally further hydrated) may be used to prepare meat mimetic food products for human or animal consumption, such as ground or minced meat, by combining one or more of the thermo-mechanical processed food ingredients (for example having different textures to mimic one or more textures and/or sensory attributes of animal meat) with additional ingredients, including any such ingredients as previously described, to form meat mimetic food products. Some examples of additional ingredients may include: binding and thickening agents ( e.g ., gums, pectins, starches, egg, potato flakes, potato flour, flours made from milled or ground grains and legumes (e.g. wheat, rice, rye oats barley, buckwheat, corn, lupin, chickpea, lentil, bean etc), protein-carbohydrate Maillard Reaction Products,), flavouring agents (e.g. salt, meat flavour (such as pork, beef or chicken flavour), herbs, spices, vegetable flavour (e.g. celery, onion, garlic), yeast extract, sugars, (e.g. glucose, sucrose, dextrose), natural and artificial sweeteners, smoke flavour, monosodium glutamate), fats, oils and lipids (e.g. coconut oil, palm stearin, olive oil, vegetable oil, canola oil and other oils mentioned previously), colouring agents, nutritional agents (e.g. amino acids, vitamins, minerals, resistant starch and dietary fibre as mentioned previously) and preservatives. While in some embodiments that meat mimetic product does not contain any animal derived ingredients, optionally, the meat mimetic food product may contain a proportion (for example from up to about 1-2%, 5%, 10%, 20%, 30%, 40% or 50 % w/w) of an animal derived (e.g. fish, shellfish, poultry (e.g. chicken, duck, goose, turkey), bovine, porcine, ovine, caprine, equine) ingredient, such as muscle, blood, fat, cartilage and connective tissue, offal (organs), gelatin, casein and caseinates (e.g. calcium or sodium), whey, milk protein concentrate, milk protein isolate, and egg or components thereof (e.g. albumin, lecithin). In some embodiments, the food ingredients may be used in the preparation of a meat mimetic food product such as a ground or shredded meat product, for example, burger patties, kebabs, meat balls, meat loaves, sausages, meat sauces and fillings (e.g. chilli, bolognaise, taco fillings, pie fillings), and other formed or shaped meat products (optionally crumbed) such as nuggets, steaks, cutlets, schnitzels, fingers and strips. In some further embodiments the food ingredient may be used in the preparation of burger patties. In some embodiments the food product is free or substantially free of one or more agents that cause allergic or intolerant reactions, such as gluten, dairy, egg or nuts.

The disclosure is further described by reference to the following examples, which are intended to illustrate one or more embodiments, and are not to be construed as limiting the generality described above. EXAMPLES

Materials

Dry fractionated fababean protein (Vitessence Pulse CT 3602, supplier code: 37401G00) The dry fractionated fababean protein was purchased from Ingredion ANZ Pty Ltd (Riverview Business Park, 3 Richardson Place, North Ryde, NSW 2113, Australia). The manufacturer of the material was AGT Foods (625 42bd Street, NE, Minot, North Dakota, USA, 58703). The material contains 59.5% protein, 3.7% fat, 12.5% carbohydrate( including 1.7% sugar), 13.4% dietary fibre, 5.7% ash and approximately 5% moisture.

Soy protein concentrate (Wilcon F)

The soy protein concentrate was obtained from Qinhuangdao Goldensea Foodstuff (Qinhuangdao, Hebei, China). The product contains 70% protein, <6.5% ash, <1% fat, <4.5% crude fibre and <6% moisture. Dried glucose syrup (DGS)

DGS was Fieldose 30 from Itochu, Sydney, NSW, Australia.

Methodologies for the characteristation of powders and extrudates

Moisture content:

Samples (3.0-4.0 g) were dried in an oven at 105°C for 24 h. The dried weight of the extrudates were taken. % moisture and dry matter content were calculated as follows.

% moisture = 100 x (weight before drying- weight after drying/weight before drying) % dry matter = 100 - % moisture.

Aw:

Water activity was measured using a dew point water activity meter (AquaLab 4TE, USA). A sample was filled at around a half of a sample cup then placed in the sample chamber until the water activity value was stable.

Density:

Density of dried TVP conjugate was measure using a 500 mL plastic container. The accurate volume of that plastic container was determined before the density measurement by measuring the volume of water filled in the container using a volumetric cylinder. The TVP conjugate was filled into the plastic container with a gently tap then levelled at the top of container. Weight of the sample was measured. The average of sample weight in at least 3 measurements was used to determine the density of dried TVP conjugate expressed as kg/m³. Water sorption properties:

Extrudates containing protein, protein-carbohydrate or protein-carbohydrate-oil were covered with 25°C water (1 part extrudate: 3 parts water) and left for 30 min. The hydrated extrudates were drained and squeezed by hand to expel the water. The weights of the expelled water and hydrated extrudates were recorded. The water uptake by the extrudates was calculated. For comparison between the hydration properties of the extrudates with different moisture contents, the water uptake was also expressed as % water uptake on a dry ingredient basis.

Pre-treatment of powder ingredients

Pre-treatment using fluid bed

The pre-treatment was carried out in Glatt GPCG-2 fluid bed, which was equipped with top spray nozzle to add moisture as required. For each pre-treatment, 3 kg of powder protein raw material was loaded to the fluid bed which was operated with air flow of 50 M3/h (as determined with sufficient fluidization of the material was achieved) and at inlet air temperature of 120°C. for 2 h. Pre-treatment 1 was carried out without moisture; pre treatment 2 was carried out with the addition of moisture by continuous spraying of water to the powder during operation. During the fluid-bed treatment, the powders were heated up to 90°C for at least 1 hour for the dry treatment whilst at the moist treatment, the temperature of the powders only reached about 60°C due to the cooling and evaporation of sprayed water..

Pre-treatment using humidity oven

The pre-treatment was carried out in a Temperature + Humidity oven (Thermoline L + M, Thermoline Scientific Pty Ltd, Australia) operated at 110°C with or without 80%RH humidity for 1 hour (see Table 3 for detail of conditions). The temperature of 110°C was selected because a higher temperature would disable the humidifying function of the oven. The humidity was set at 80%RH and it took ~30 min to reach 75%RH and plateaued. The protein powders (FBPC and SPC) of 3 kg each were laid evenly on a perforated stainless- steel tray with aluminium foil. The thickness of the protein powders was around 15-20 mm. One thermocouple was inserted into the middle of each protein powder to record the powder temperature during treatment. The oven was pre-heated to the set temperature (and humidity). When the oven was opened to load the powders, the temperature dropped to 60°C and humidity to 40%RH. After loading of the powders and door closed, the air temperature inside the oven rose quickly to the set point within 5 min, however, it took about 25 min for the humidity to reach 75%RH and materials temperature reach 105°C and then plateaued. The powders were taken out after 1 hour treatment.

Caking and aggregation was observed for powders treated with heat and humidity. No caking observed after heat treatment without humidity. Slight darkening was observed for all the treated protein powders.

Sample identification, pre-treatment conditions, moisture content and water activities for control, fluidbed and oven treated powders are set out in Tables 1 and 2.

Table 1. Moisture content and water activity of FBPC and SPC powders - Fluidbed

Sample ID Pre-treatment Condition Moisture, % Aw

FBPC

T1A-FBPC Control - untreated 8.38% 0.4150

T1B-FBPC Fluidbed 90°C, 2hr 2.94% 0.0217

T1C-FBPC Fluidbed 60°C, 2hr+Moist 4.98% 0.0996

SPC

T2A-SPC Control-untreated 7.36% 0.2241

T2B-SPC Fluidbed 90°C, 2hr 2.77% 0.0211

T2C-SPC Fluidbed 60°C, 2hr+Moist 8.47% 0.2611

T2D-SPC+5%DGS Fluidbed 60°C, 2hr+Moist 5.63% 0.1266

Table 2. Moisture content and water activity of SCP powders-oven

Sample Pre-treatment Condition Moisture, % Aw

SPC

T4A-SPC Control - untreated 7.36% 0.2241

T4B-SPC Oven dry heat 110^eC, 1 hr 3.88% 0.0443

T4C-SPC Oven 110°C x 80%RH, 1 hr 7.91% 0.3302 Extrusion

The extrusion was carried out in a lab scale co-rotating twin screw extruder (KDT30-II, Jinan Kredit, China) with 30 mm screw diameter (D), 20:1 screw length/diameter ratio). The extruder was set at 30°C, 50°C, 100°C and 90°C for the barrel temperature from powder feed end to the die end. The die used was a rectangular die with opening of 1 x 4 mm² and land length of 5 mm. The extrusion was operated at a dry feed rate around 4 kg/h and barrel moisture at around 39-40%. Drying of the extrudates was carried out in a batch drying oven at 65°C overnight. Moisture content, water activity, density and water absorption of extrudates is set out in Table 3

able 3: Moisture content, water activity, density and water absorption of extrudates

Pre-treatment Condition

Sample ID Moisture, % Aw Density (g/l) WA (%dmb)

FBPC - Post extrusion drying: 65°C overnight dry

EX-T1A Control - untreated 6.2 0.37 536 83.11

EX-TIB Fluidbed, 2hr, Dry 6.2 0.39 555 93.72

EX-TIC Fluidbed, 2hr, Moist 6.2 0.37 590 87.28

SPC - Post extrusion drying: 65°C overnight dry

EX-T2A Control-untreated 6.9 0.35 538 115.07

EX-T2B Fluidbed, 2hr, Dry 7.1 0.36 517 117.31

EX-T2C Fluidbed, 2hr, Moist 7.1 0.3936 504 120.02

EX-T2D (SPC+5%DGS) Fluidbed, 2hr , Moist 7.5 0.3910 477 133.51

SPC - Post extrusion drying: 65°C overnight dry

EX-T4A-R Control - untreated 6.04 0.3125 489 134.07

EX-T4B-R Oven dry heat 110°C, 1 hr 6.19 0.3034 475 137.4

EX-T4C-R Oven 110°C x 80%RH, 1 hr 6.51 0.3148 443 156.34

Texture Profile Analysis

Texture of hydrated extruded ingredients was evaluated with texture profile analysis (TPA) method using a tensile compression testing machine (Instron 4465 HI 840, UK) equipped with a 500 N load cell and a 35 mm cylinder probe

Sample preparation: Dried samples (12.8 g) were weighed into a sample container (60 mL specimen container with screw lid, Westlab, Victoria) to achieve a volume of the hydrated samples of 45 - 50 mL. The sample particles were arranged to minimise the gap between particles by gently and randomly moving the spatula across the samples (approximately 8 times), and to ensure the particles were packed and formed a flat surface. Water (27.2 g, 25°C) was gently poured evenly over the sample. The sample container was closed with the lid and the sample was left to hydrate for 15 min before the TPA measurement. Test performance: The TPA test was set to a compression mode for 2 compression cycles of 15 mm at 60 mm/min. Five replicates were measured for each sample. A typical TPA profile is shown in Figure 2. Parameters of TPA (hardness, cohesiveness, springiness and chewiness) were determined according to:

Hardness = the force at the 1st peak Cohesiveness = Area 2 / Area 1

Springiness = Distance 2 / Distance 1

Chewiness = Hardness x Cohesiveness x Springiness.

The results are set out in Table 4

Table 4: Texture analysis profile of re-hydrated extrudates

Pre-treatment Condition

Sample ID Hardness (N) Cohesiveness Springiness Chewiness (N)

FBPC - Post extrusion drying: 65°C overnight dry

EX-T1A Control - untreated 20.3 ±0.8 0.38 ±0.01 0.45 ± 0.03 3.5 ±0.1

EX-TIB Fluidbed, 2hr, Dry 24.5 ±0.8 0.38 ±0.01 0.45 ± 0.01 4.2 ±0.1

EX-TIC Fluidbed, 2hr, Moist 18.9 ± 1.8 0.43 ± 0.04 0.52 ±0.04 4.2 ± 0.8

SPC - Post treatment: 65°C overnight dry

EX-T2A Control-untreated 34.7 ±1.1 0.51 ±0.01 0.54 ±0.00 9.5 ±0.3

EX-T2B Fluidbed, 2hr, Dry 42.7 ±2.1 0.52 ±0.00 0.59 ±0.08 13.3 ± 1.7

EX-T2C Fluidbed, 2hr, Moist 43.5 ±2.4 0.52 ±0.00 0.64 ±0.03 14.7 ± 1.5 I

EX-T2D Fluidbed, 2hr, Moist

(SPC+5%DGS) 32.6 ±0.1 0.51 ±0.00 0.65 ±0.07 10.8 ±1.3

SPC - Post extrusion drying: 65°C overnight dry

EX-T4A-R Control - untreated 27.5 ±2.5 0.48 ± 0.00 0.52 ±0.02 6.9 ± 0.5

EX-T4B-R Oven 110°C, 1 hr 31.5 ±0.2 0.50 ±0.01 0.57 ±0.02 9.0 ± 0.5

EX-T4C-R Oven 110°C x 80%RH lhr 31.2 ±2.1 0.50 ±0.01 0.62 ±0.04 9.7 ±0.1

Claims

1. A process for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of heating a protein source having a protein content in the range of about 45-75% on a dry weight basis, at a temperature in the range of about 50- 110°C for a period of about 10 minutes to-72 hours; and subjecting the heat-treated protein source to a thermo-mechanical process to produce a texturized ingredient.

2. The process according to claim 1 wherein the protein source is heated at a temperature of about 50°C, or 55°C or 60°C, or 65°C or 70°C, or 75°C or 80°C, or 85°C or 90°C, or 95°C, or 100°C or 105°C or 110°C.

3. The process according to claim 1 or 2 wherein the protein source is heated for a period of 30 minutes to 24 hours, such as about 60-180 minutes

4. The process according to claim 3 wherein the protein source is heated for about 30, or 45, or 60, or 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes.

5. The method according to any one or claims 1-4, wherein the texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre-treatment,

6. A process for heat pre-treating a protein source having a protein content in the range of about 45-75% on a dry weight basis, for use in a thermo-mechanical process to produce a texturized ingredient for a meat mimetic food product, wherein the texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre treatment, said process comprising the step of heating the protein source at a temperature in the range of 50-200°C.

7. The process according to claim 6 wherein the protein source is heated at a temperature in the range of about 50-100°C, such as about 50°C, or 60°C, or 70°C, or 80°C or 90°C or 100°C.

8. The process according to claim 7, wherein the protein source is heated for aperiod of about 30-180 minutes, such as about 30, or 45, 60, 75, or 90, or 105, or 120, or 135 or 150, or 165, or 180 minutes.

9. The process according to claim 6, wherein the protein source is heated at a temperature in the range of about >100-200°C, such as about 105°C, 110°C, 120°C, 130°C, 140°C, 150°C

10. The process according to claim 9, wherein the protein source is heated for a period of about 10-60 minutes, such as about 10, or 15, or 20, or 30, or 40, or 45, or 50 minutes.

11. The process according to any one of claims 1-10 wherein the protein source has a protein content in the range of about 50-70% on a dry weight basis, such as about 55-65%, and optionally contains up to about 10% moisture by weight.

12. The process according to claim 11 wherein the protein source is a dry fractionated protein obtained from a dry fractionation process.

13. The process according to claim 11 wherein the protein source is a wet fractionated protein obtained from a wet fractionation process.

14. The process according to any one of claims 1-13, wherein the protein is selected from one or more of chickpea, pea, cowpea, soy, lupin, canola, mung bean, lentil, quinoa, hemp, algal and faba bean protein.

15. Thje process according to claim 14 wherein the protein is selected from soy, faba bean, pea, chickpea, lentil or lupin.

16. The process according to any one of claims 1-15, wherein the protein source is heat treated in the absence of one or more additional or extrinsic carbohydrate sources.

17. The process according to any one of claims 1-15 wherein the protein source is heat treated in the presence of one or more additional or extrinsic carbohydrate sources.

18. The process according to claim 17, wherein the one or more additional carbohydrate sources are added in a total amount of about 10%, or less, such as about 5% or less, on a dry weight basis.

19. The process according to any one of claims 1-18, wherein protein source is subjected to heat treatment in the presence of added moisture, such as at a humidity in the range of about 20-90% R.H, such as about 50-90%RH..

20. The process according to any one of claims 1-19, wherein, the protein source is subjected to heat treatment in an oven or fluid bed dryer.

21. A process for the manufacture of a texturized ingredient for a meat mimetic product comprising the step of subjecting a heat pre-treated protein source according to any one of claims 6-20, to a thermo-mechanical process to produce a texturized ingredient, wherein the texturized ingredient demonstrates one or more improved parameters of hardness, cohesiveness, springiness and chewiness, compared to the corresponding ingredient prepared from the same protein source which has not been subjected to heat pre-treatment.

22. The process according to any one of claims 1-5 and 21, wherein the thermo mechanical process is conducted in an extruder.

23. The process according to claim 22 conducted under low moisture conditions (barrel moisture of 40% (w/w) or less).

24. The process according to claim 23, wherein the heat pre-treated protein source is optionally extruded or subjected to shear together with one or more further added carbohydrate source(s).

25. The process according to any one of claims 1-5 and 21-23 wherein the texturized ingredient is dried at a temperature in the range of about for about 50-95°C for a period of about 1-24 hours.

26. A texturized ingredient produced by the process of any one of claims 1-5 -and 21-25.

27. A meat mimetic food product comprising a texturized ingredient according to claim 26.