Glucoamylase as binding agent for non-animal protein food products

Abstract

The invention relates to the field of food. Disclosed herein is a method of making a non-animal protein food product or cultured meat food product, comprising: adding a heat labile glucoamylase protein formulation to an ingredient for making the food product, and processing the obtained glucoamylase protein formulation and ingredient composition into a non-animal protein food product or a cultured meat food product.

Description

Technical Field

[0001] This invention relates to the field of food. Background Technology

[0002] Food products containing non-animal proteins (such as plant proteins) as alternatives to animal-derived proteins are gaining attention due to consumer concerns about the environmental impact of animal-based products and the beneficial nutritional properties of non-animal protein-based foods. In particular, plant-based protein beverages are becoming increasingly popular as alternatives to dairy products such as milk, yogurt, or ice cream.

[0003] The many drawbacks associated with using animal-derived protein for human consumption—from the acceptability of raising animals for consumption to the fact that meat production is inefficient in terms of feed input to food output and carbon footprint—make the ongoing search for improved meat alternatives one of the most active food development activities in society today.

[0004] Historically, meat substitutes have used fermented vegetable sources, such as soybeans (e.g., tofu, fermented black beans) or gluten / wheat (e.g., seitan), to achieve a certain protein content. Today, modern technologies such as extrusion and 3D printing are used to create meat substitutes with a texture, flavor, and appearance more like meat. Soybeans and gluten remain favorable sources of such meat substitutes because they are widely available, affordable, relatively high in protein, and easy to process. However, intolerances or allergies to genetically modified (GM) soybeans and gluten are driving consumer demand for alternatives. Producers of meat substitutes are turning to other proteins, such as those from legumes (e.g., peas). However, the use of these alternative protein sources comes with new problems. Protein blends are often not as easy to process as traditional soybeans or gluten or combinations thereof, and in many cases, result in a lack of textured food proteins that mimic the nutrition, texture, appearance, and / or taste of animal-derived meat products. Consequently, consumers often find these meat substitutes unappealing and unpalatable. Therefore, there is a need in the field for attractive and palatable meat substitutes.

[0005] A common binder or binder used in dairy products or meat substitutes is methylcellulose. Methylcellulose is undesirable given food labeling requirements that require mention of such chemicals. Consumers increasingly expect food products with clean labels. Other common binders are wheat gluten or egg whites. The disadvantage of wheat gluten is that it is a potential allergen. Furthermore, the disadvantage of egg whites is that the resulting food is not vegan and is a potential allergen.

[0006] Typically, alternative adhesives or gelling agents are needed. Summary of the Invention

[0007] This invention provides: - A method for preparing non-animal protein food products or cultured meat food products, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product. - Food products that can be obtained through one of the methods described in this article; - Meat or fish alternative food products containing non-animal protein or cultured meat and heat-sensitive glucosyl amylase protein preparations; -A meat or fish substitute comprising textured plant protein, non-animal protein, water, flavorings, and a binder system, wherein the binder system comprises a heat-sensitive glucosyl amylase protein preparation; and - Heat-sensitive glucosylamylase protein preparations are used for obtaining binding in food products or for at least partially replacing adhesives in food products. Detailed Implementation

[0008] Food binders are food additives added to food products to improve texture by thickening or binding ingredients together. Food binders play a significant role in food production by, for example, improving texture, juiciness, and / or increasing volume.

[0009] Examples of widely used food binders include eggs, wheat flour, oatmeal, rice, milk, gelatin, guar gum, xanthan gum, (potato) starch, or methylcellulose.

[0010] There is a need in the art for alternative adhesives or alternative gelling agents. This application addresses this need.

[0011] As disclosed in the Experimental Section of this document, heat-sensitive glucosylamylase protein formulations can surprisingly be used as adhesives or gelling agents, preferably as gelling agents.

[0012] Throughout the specification and appended claims, the words “comprising,” “including,” and “having,” as well as variations such as “containing,” should be interpreted as inclusive. That is, where the context permits, these words are intended to express the possibility of including other elements or integers not specifically stated.

[0013] When used in this text, the articles “a” and “a kind” refer to one or more grammatical objects of the article (i.e., one or at least one). For example, “element” can mean one element or more elements.

[0014] In one aspect, the present invention provides a method for preparing non-animal protein food products or cultured meat food products, comprising: A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product.

[0015] Alternatively, this aspect of the invention can be described as follows: - A method for preparing non-animal protein food products, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product, or - A method for preparing cultured meat food products, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into cultured meat food products.

[0016] When used in this article, the term "non-animal protein food product" refers to a food product that does not contain animal protein.

[0017] As used in this article, the term "cultured meat food product" refers to a food product prepared from cultured meat. Cultured meat is meat produced through in vitro cell culture of animal cells. This is a form of cell agriculture. Cultured meat is produced using tissue engineering techniques traditionally used in regenerative medicine.

[0018] As used herein, the term "ingredients for the preparation of..." encompasses any ingredient used in the preparation of non-animal protein food products or cultured meat food products. Suitable ingredients depend on the final non-animal protein food product or cultured meat food product. As a non-limiting example, meat substitute products such as hamburger patties, sausages, or nuggets are described in more detail herein. Preferably, the meat substitute product is a hamburger. Typical ingredients of such meat substitutes are textured plant protein, (non-animal) protein, oil and / or fat, water, and a flavoring system. A heat-sensitive glucosylamylase protein preparation may be added to any of these ingredients, for example, to textured plant protein, to protein, to oil and / or fat, to water, or to a flavoring system. In one embodiment, all (or some) of the ingredients of the non-animal protein food product or cultured meat food product are added to each other (and optionally mixed), and a glucosylamylase protein preparation is added in a next step. In yet another embodiment, all the ingredients of the non-animal protein food product or cultured meat food product and the heat-sensitive glucosylamylase protein preparation are added to each other at approximately the same time. In one embodiment, a heat-sensitive glucosylamylase protein preparation may be added during the preparation of textured plant protein. Preferably, the term "ingredients for the preparation of..." does not include starch.

[0019] In the case of meat or fish substitutes based on high-moisture extrudate (HME), a heat-sensitive glucosylamylase protein preparation may be added during the preparation of the HME, or alternatively added to any other commonly used ingredients of the HME or meat or fish substitute (e.g., oils, fats, proteins, vitamins, minerals). Preferably, the meat or fish substitute does not contain starch.

[0020] In the case of meat or fish substitutes based on shear cell technology, a heat-sensitive glucosylamylase protein preparation can be added during the preparation of the protein plate, or alternatively added to any other commonly used ingredients (e.g., oils, fats, proteins, vitamins, minerals) of the protein plate or meat or fish substitute. Preferably, the meat or fish substitute does not contain starch.

[0021] In the case of non-animal protein food products that are vegan cheese (also known as plant-based dairy cheese alternatives), the term "ingredients used to prepare..." typically includes gums and / or non-animal protein sources as binders, vegetable fats or oils, and other ingredients (salts, calcium, acids, preservatives, flavorings, colorings). Preferably, the vegan cheese does not contain starch.

[0022] In the case of non-animal protein foods that are plant-based milk or yogurt (also known as plant-based dairy alternatives), the term "ingredients used to prepare..." typically includes plant-based milk (such as soy, oats, coconut, rice, almonds, and lactic acid bacteria (in yogurt)) and other ingredients (gum, vitamins, flavorings, or sugars, etc.). Preferably, the plant-based milk or yogurt does not contain starch.

[0023] In the case of non-animal protein foods, such as vegan omelets or other egg substitutes, the term "ingredients used to prepare..." typically includes plant-based proteins, binders (such as methylcellulose), and other ingredients (flavorings, colorings). Preferably, methylcellulose is replaced by glucosylamylase protein.

[0024] The steps of adding heat-sensitive glucosyl amylase protein preparations to ingredients used in the preparation of non-animal protein food products or cultured meat food products typically include: - Combining heat-sensitive glucosyl amylase protein with plant proteins, microbial proteins, or algal proteins. - Combining heat-sensitive glucosyl amylase protein with plant protein - Combining heat-sensitive glucosyl amylase protein with microbial protein - Combining heat-sensitive glucosyl amylase protein with fungal protein - Combining heat-sensitive glucosyl amylase protein with bacterial protein - Combining heat-sensitive glucosyl amylase protein with algal protein - Combining heat-sensitive glucosyl amylase protein preparations with textured plant proteins - Combining heat-sensitive glucosyl amylase protein preparations with plant-based milk -Preparation of a binder phase, wherein the heat-sensitive glucosylamylase protein preparation is combined with oil or fat and water. -Preparing a binder phase, wherein the heat-sensitive glucosyl amylase protein preparation is combined with oil or fat, water, and an aqueous colloid. - Combining heat-sensitive glucosyl amylase protein preparations with other dried ingredients - Combine heat-sensitive glucosyl amylase protein preparations with other drying ingredients, oils, and water. - Combine heat-sensitive glucosyl amylase protein preparations with seasonings (such as, but not limited to, salt and / or pepper), or - Combining heat-sensitive glucosyl amylase protein preparations with water and yeast extracts Preferably, the term "ingredients for the preparation of..." as used herein does not include starch as an ingredient.

[0025] The phrase “processing the obtained glucosylamylase and ingredient composition into a non-animal protein food product or a cultured meat food product” includes well-known steps such as mixing, kneading, fermentation, cooking, frying, baking, freezing, extrusion, shearing unit technology and many other steps.

[0026] The term "obtained glucosylamylase protein preparation and ingredient composition" refers to a composition obtained when a heat-sensitive glucosylamylase protein preparation is added to an ingredient used in the preparation of any of the said food products.

[0027] Glucoamylase is an enzyme produced by many microorganisms but used for industrial purposes, typically derived from the genus Aspergillus (…). Aspergillus ) or Rhizopus ( Rhizopus ) species, such as Aspergillus niger ( Aspergillus niger ), Aspergillus awamori ( Aspergillus awamori ) and Rhizopus oryzae ( Rhizopus oryzae Most preferably, glucosidase is produced by *Aspergillus niger*. Glucoamylase, also commonly known as amyloglucosidase or EC 3.2.1.3-glucan-1,4-α-glucosidase, is currently used for the saccharification of starch. Its main applications are in brewing, bioethanol production, baking, and the starch industry for the production of glucose syrup. The methods described herein are independent of the enzymatic activity of glucosidase. As disclosed in the experimental section of this document, glucosidase protein is well-suited as a binder or gelling agent; therefore, a method for preparing non-animal protein food products or cultured meat food products is disclosed herein, comprising... A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product, wherein the glucosylamylase protein preparation provides binding or gelling properties to the food product when the glucosylamylase protein preparation and ingredient composition or the food product is heated to at least 65 degrees Celsius.

[0028] Preferably, this document discloses a method for preparing non-animal protein food products or cultured meat food products, including... A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product, wherein the glucosylamylase protein preparation provides gelling properties to the food product when the glucosylamylase protein preparation and ingredient composition or the food product is heated to at least 65 degrees Celsius.

[0029] The binding properties are acquired through the gelation of heat-sensitive glucosyl amylase protein. Preferably, the gelation leads to the formation of an irreversible gel.

[0030] Preferably, a heat-sensitive glucosyl amylase protein preparation is used to replace conventional binders (e.g., methylcellulose). Therefore, in other words, the present invention provides a method for reducing (preferably completely reducing) methylcellulose in the preparation of non-animal protein food products or cultured meat food products, the method comprising... A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product.

[0031] The term "thermostrophic glucosylamylase" refers to a glucosylamylase that exhibits enzymatic activity at relatively low temperatures (as opposed to heat-stable glucosylamylase). The enzymatic activity of glucosylamylase is determined, for example, by incubating the enzyme on a suitable substrate, such as maltose. Whether a glucosylamylase is heat-stable or heat-sensitive is determined by testing the enzyme activity on the substrate at different temperatures. Heat-sensitive glucosylamylase is defined herein as a glucosylamylase that exhibits enzymatic activity at temperatures below 70 degrees Celsius (i.e., heat-sensitive glucosylamylase has no activity or almost no activity at temperatures above 80 degrees Celsius).

[0032] In the aspects of the invention outlined above (i.e., methods for preparing non-animal protein food products or cultured meat food products, or methods for reducing (preferably completely reducing) methylcellulose in the preparation of non-animal protein food products or cultured meat food products), the methods may include a heating step, i.e., the methods described above further include a heating step to at least 65 degrees Celsius. As disclosed herein in the Experimental section, heating to at least 65 degrees Celsius results in the gelation of heat-sensitive glucosylamylase proteins. A suitable upper limit for heating depends on the final product and the heating method used (e.g., with or without pressure), and can be readily determined by those skilled in the art. That is, gelation as described herein refers to gelation after heating, preferably gelation after heating to at least 65 degrees Celsius.

[0033] The term "glucosylamylase" is explained above. The term "heat-sensitive glucosylamylase" is also explained above. Glucoamylase is typically sold as a glucosylamylase protein preparation, which contains not only glucosylamylase protein but also other components (such as salts and / or preservatives). Glucoamylase preparations have been described for use in and isolated from a variety of organisms, including microorganisms. Glucoamylase is typically a bacterium or fungus (e.g., Bacillus spp.). Bacillus Extracellular components of Aspergillus species (especially Aspergillus niger and Aspergillus oomori). Aspergillus awamori Glucoamylases from fungi, and their protein-engineered or evolved variants, are frequently used in industrial practice. Other fungal glucosylases, such as those from the genus Rhizopus, can also be used. Rhizopus), Penicillium ( Penicillium ) or Tara yeast ( Talaromyces The glucosidase is a protein derived from microorganisms. Typically, glucosidase protein preparations also contain other components, such as other proteins derived from or expressed by microorganisms. Therefore, heat-sensitive glucosidase protein preparations may contain other components. For example, the preparation of intracellular glucosidase requires cell disruption to release the glucosidase protein. Simultaneously, other cytoplasmic proteins / enzymes are released. For the purposes of this invention, a crude glucosidase protein preparation can be used.

[0034] Examples of suitable heat-sensitive glucosylamylase protein preparations include those containing the following glucosylamylase protein preparations. -Natural glucosidase protein -Fungal glucosyl amylase protein - Aspergillus glucoamylase protein -Aspergillus niger glucosyl amylase protein -Classic (non-GMO) glucosyl amylase protein, - Extracellularly produced glucosylamylase protein, and / or - Any combination thereof.

[0035] Preferably, the heat-sensitive glucosylamylase protein formulation comprises a glucosylamylase protein with an optimal pH range of pH 4 to 5, or the formulation comprises an Aspergillus glucosylamylase protein, more preferably an Aspergillus glucosylamylase endogenously expressed in Aspergillus. The expression of the glucosylamylase protein in Aspergillus is preferably enhanced by classical strain modification. Most preferably, the glucosylamylase protein formulation in which the glucosylamylase is endogenously expressed in Aspergillus and its expression is enhanced by classical strain modification.

[0036] Suitable examples of heat-sensitive glucosylamylase protein formulations are Amigase Mega L, Bakezyme AG800, Hazyme DCL products (DSM Food & Beverage), Sunson GA130L (Sunson), glucosylamylase GA-300S (Hunan NHY), Sunbake AMG NG (Suntaq), Distillase, Diazyme X4 (IFF), or AMG (Novozymes).

[0037] As described above and as shown in the Experimental Section of this document, other heat-sensitive glucosylamylase protein formulations may also be used. Even more preferably, the heat-sensitive glucosylamylase protein formulations used do not contain glycerol or any compounds typically used for the spray drying of enzymes (e.g., maltodextrin).

[0038] As described above, the method described herein does not depend on the enzymatic activity of heat-sensitive glucosylamylase, therefore the enzymatic activity of glucosylamylase can be inhibited by a suitable inhibitor without losing its function as a binder.

[0039] The phrase "natural glucosylamylase protein" refers to non-denatured glucosylamylase protein with enzymatic activity.

[0040] The heat-sensitive glucoamylase is used in this invention not because of its enzymatic activity, but as a (further) component in non-animal protein food products or cultured meat food products, more specifically, as a component that forms a gel upon heating.

[0041] A sufficient amount of heat-sensitive glucosylamylase is added as a gelling agent, causing the glucosylamylase protein to form a gel upon heating. The amount of glucosylamylase protein preparation added to achieve the desired binding effect can be easily determined by a person skilled in the art. Guidance can be found in the experimental section of this document. Based on the total protein (present in the glucosylamylase protein preparation used), a dose of 0.05 to 20% or 0.05 to 10% is sufficient to cause binding or gelation. More preferably, at least 0.1% of the total protein (present in the glucosylamylase protein preparation used) is used, and a suitable range is at least 0.1% to 20%. Most preferably, at least 0.5% of the total protein (present in the glucosylamylase protein preparation used) is used, and therefore the most preferred range is at least 0.5% to 20%. The amount of glucosylamylase protein preparation provided is relative to the mass of the food product. Not all proteins in a glucosamine protein preparation are glucosamine proteins, but because the glucosamine-producing strains are engineered to produce as much glucosamine protein as possible, a 50% glucosamine protein content (based on the total protein in the glucosamine protein preparation) is typical. A dose of 0.025 to 10% or 0.025 to 5% is sufficient to induce binding, based on the glucosamine protein (present in the glucosamine protein preparation used). More preferably, at least 0.05% glucosamine protein (present in the glucosamine protein preparation used) is used, and a suitable range is at least 0.05% to 10%. Most preferably, at least 0.25% glucosamine protein (present in the glucosamine protein preparation used) is used, and a suitable range is at least 0.25% to 10%. The amount of glucosamine protein provided is relative to the mass of the food product. Those skilled in the art can determine the amount of glucosamine protein in a glucosamine protein preparation using well-known methods. For example, HP-SEC or SDS-PAGE analysis can be used to estimate the relative amount of glucosylamylase protein compared to other proteins in a glucosylamylase protein formulation.

[0042] As used herein, the term "non-animal protein food product" refers to a food product that does not contain any animal protein. Non-animal protein food products contain protein from other sources. Non-animal proteins are, for example, plant proteins, microbial proteins, or algal proteins. Suitable examples of microbial proteins are fungal or bacterial proteins. In other words, non-animal protein food products are, for example, plant protein food products, microbial protein food products, algal protein food products, fungal protein food products, or bacterial protein food products. As used herein, the term "plant protein food product" means "food product containing plant protein" (or "food product containing plant protein"; these phrases are used interchangeably herein), which refers to a food product containing at least 10% (based on all proteins present in the food product) of plant protein. Preferably, the plant-based food product contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (based on all proteins present in the food product) of plant protein. Most preferably, the plant protein food product contains only or exclusively (i.e., 100% based on all proteins present in the food product) plant protein and contains no animal-derived protein at all. As used herein, the term "plant protein" refers to any protein derived from a plant source. Preferably, plant protein is protein derived from cereals, false cereals, legumes, nuts, seeds, or other sources (e.g., coconut, potato, rapeseed, or tiger nut).

[0043] Examples of suitable grains are barley, fonio, corn, millet, oats, rye, sorghum, teff, rye, spelt, rice, or wheat.

[0044] Suitable examples of false grains are amaranth, buckwheat, or quinoa.

[0045] Examples of suitable legumes are lupins, peas, chickpeas, beans (preferably broad beans), duckweed, potatoes, peanuts, or soybeans.

[0046] Examples of suitable nuts are almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pistachios, or walnuts.

[0047] Examples of suitable seeds are rapeseed, chia seeds, flax seeds, pumpkin seeds, sesame seeds, or sunflower seeds.

[0048] As used herein, the term "microbial protein food product" means "food product containing microbial protein" (or "food product containing microbial protein"; these phrases are used interchangeably herein), which refers to a food product containing at least 10% (based on all proteins present in the food product) microbial protein. Preferably, the microbial protein food product contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (based on all proteins present in the food product) microbial protein. Most preferably, the microbial protein food product contains only or exclusively (i.e., 100% based on all proteins present in the food product) microbial protein and contains no animal-derived protein at all. Preferably, the microbial protein is fungal or bacterial protein, thus obtaining a fungal protein food product or a bacterial protein food product.

[0049] As used herein, the term "fungal protein food product" means "food product containing fungal protein" (or "food product containing fungal protein"; these phrases are used interchangeably herein), which refers to a food product containing at least 10% (based on all proteins present in the food product) of fungal protein. Preferably, the fungal protein food product contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (based on all proteins present in the food product) of fungal protein. Most preferably, the fungal protein food product contains only or exclusively (i.e., 100% based on all proteins present in the food product) fungal protein and contains no animal-derived protein at all. An alternative term for fungal protein is mycoprotein. Suitable examples of mycoprotein are derived from, for example, Fusarium moniliforme (… Fusarium venenatum Proteins from bacteria. Such products are marketed under the brand name Quorn. Other suitable sources of bacterial protein are... Neurospora crassa , Thermomucor indicae-seudaticae , Lentinula edodes (Shiitake) Pleurotus ostreatus (Oyster mushroom) Rhizopus , Fusarium oxysporum , Fusarium novum-yellowstonensis or Aspergillus oryzae (Koji).

[0050] As used herein, the term "bacterial protein food product" means "food product containing bacterial protein" (or "food product containing bacterial protein"; these phrases are used interchangeably herein), which refers to a food product containing at least 10% (based on all proteins present in the food product) of bacterial protein. Preferably, the bacterial protein food product contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (based on all proteins present in the food product) of bacterial protein. Most preferably, the bacterial protein food product contains only or exclusively (i.e., 100% based on all proteins present in the food product) bacterial protein and contains no animal-derived bacterial protein at all. Suitable examples of bacterial proteins can be obtained from Xanthobacter tagetidis or Cupriavidus necator .

[0051] As used herein, the term "algae protein food product" means "food product containing algae protein" (or "food product containing algae protein"; these phrases are used interchangeably herein), which refers to a food product containing at least 10% (based on all proteins present in the food product) of algae protein. Preferably, the algae protein food product contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (based on all proteins present in the food product) of algae protein. Most preferably, the algae protein food product contains only or exclusively (i.e., 100% based on all proteins present in the food product) algae protein and contains no animal-derived algae protein at all. Suitable examples of algae protein are Chlamydomonas, Spirulina (commercially produced by Damhert), Euglena gracilis, Odontella, Saccharina (commercially produced by Viva Maris), or Chlorella (commercially produced by Alver).

[0052] The non-animal protein food products described herein (e.g., microbial protein food products, fungal protein food products, bacterial protein food products, or algal protein food products) or the cultured meat food products described herein are typically sold as meat substitute food products or fish substitute food products. Therefore, a method for preparing meat substitute food products or fish substitute food products is also described herein, including... A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a meat substitute food product or a fish substitute food product.

[0053] Alternatively, this aspect of the invention can be described as follows: - A method for preparing meat substitute food products, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a meat substitute food product, or - A method for preparing a fish alternative food product, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a fish substitute food product.

[0054] When used in the context of this article, the terms meat or fish substitute, meat or fish analog product, or meat or fish substitute refer to a product that does not contain animal or fish protein and is therefore suitable as a vegetarian or vegan meat or fish substitute and has an appearance that mimics that of an animal meat or fish-based product. Meat or fish substitutes can be patties, small food pieces, sausages, cold cuts, spreads, sticks, or any other form.

[0055] Alternatively, any of the methods described above (i.e., methods for preparing non-animal protein food products or cultured meat food products or methods for reducing (preferably completely reducing) methylcellulose in the preparation of non-animal protein food products or cultured meat food products) can be used to produce non-animal protein food products, such as cheese, milk, eggs or yogurt products, such as plant-based cheese (or vegan cheese), plant-based yogurt or plant-based milk substitutes, plant-based ice cream, etc.

[0056] Therefore, this article also describes a method for preparing non-animal protein food products, including A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product, wherein the food product is a plant-based cheese (or vegan cheese), a plant-based yogurt, a plant-based milk substitute, or a plant-based ice cream.

[0057] The heat-sensitive glucose amylase protein formulation used in any of the methods described herein is added to obtain binding properties, rather than being added to process starch / dextrin or maltose into glucose.

[0058] In another aspect, the present invention further provides a food product obtainable by any of the methods described herein, and therefore such food product may be a meat substitute or fish substitute or a non-animal protein food product or a cultured meat food product or any of the above-mentioned food products. This food product differs from other food products in that it contains heat-sensitive glucosylamylase. Prior art uses of glucosylamylase are limited to its use in the baking industry, brewing, starch processing, high-fructose corn syrup production, bioethanol production, etc. In this document, glucosylamylase is used as a binder or gelling agent, preferably as a binder or gelling agent to replace methylcellulose. The amount of heat-sensitive glucosylamylase added is much higher than its conventional use in saccharification in various industries, where typically ppm amounts of glucosylamylase protein are added.

[0059] This invention also provides meat or fish alternative food products comprising non-animal protein or cultured meat and a heat-sensitive glucosyl amylase protein preparation. The explanations provided above regarding the different features of the method claims also apply to this part of the invention.

[0060] The additional component of this meat or fish substitute food product is non-animal fat.

[0061] The present invention also provides meat or fish substitutes comprising textured plant protein, non-animal protein, water, flavoring agents, and a binder system, wherein the binder system comprises a heat-sensitive glucosylamylase protein preparation. In one embodiment, the heat-sensitive glucosylamylase protein preparation may also be present in the textured plant protein.

[0062] Preferably, the meat or fish substitute of the present invention comprises textured plant protein (TVP). Preferably, the textured plant protein is an extruded plant protein product. This results in changes in the protein structure, producing a fibrous, sponge-like matrix with a texture similar to meat. The textured plant protein can be rehydrated or dehydrated. Preferably, the textured plant protein is selected from soy protein, pea protein, lentil protein, lupin protein, wheat gluten, rapeseed protein, fava bean protein, or combinations thereof. Given that soy is an allergen, the textured plant protein of the present invention is preferably soy-free. Preferably, the meat or fish substitute of the present invention is soy-free.

[0063] Preferably, the meat or fish substitute of the present invention comprises 5-30% (w / w), preferably 6-25% (w / w), preferably 8-20% (w / w), or preferably 10-15% (w / w) of the meat or fish substitute, of the amount of textured plant protein.

[0064] Preferably, the meat or fish substitute of the present invention comprises textured plant protein, wherein the protein content is 50-99% (w / w), preferably 55-90% (w / w), and preferably 60-85% (w / w).

[0065] Preferably, the texturized plant protein of the present invention is hydrated to an amount of water greater than 10% (w / w) of the texturized plant protein, preferably 20% to 80% (w / w) of the texturized plant protein, and preferably 30% to 70% (w / w) of the texturized plant protein.

[0066] As used herein, the term "binder" or "binding agent" refers to a substance used to hold particles and / or fibers together into cohesive clumps. It is an edible substance used in the final product to capture food components with a matrix, with the aim of forming a viscous product and / or thickening the product. The binder of the present invention can contribute to a smoother product texture, add texture to the product, help retain moisture and / or help maintain the shape of a viscous product; for example, by helping particles aggregate. The amount of glucosylamylase protein formulation required to achieve the desired binding effect can be readily determined by those skilled in the art. Guidance can be found in the experimental section of this document. Based on the total protein (present in the glucosylamylase protein formulation used), 0.05 to 10% is sufficient to induce binding.

[0067] Preferably, the meat or fish substitute of the present invention does not contain methylcellulose and / or wheat gluten.

[0068] In a preferred embodiment, the meat or fish substitute of the present invention further comprises nutrients, preferably comprising both vitamins and minerals, preferably vitamins selected from the group consisting of B2, B3, B6, and B12, and preferably minerals selected from the group consisting of iron, selenium, and zinc. As used herein, the term "nutrient" refers to substances that provide nutritional value to the meat or fish substitute of the present invention, such as vitamins, minerals, trace elements, and antioxidants. The advantage of adding these nutrients is that the meat or fish substitute of the present invention more closely approximates the nutritional value of a genuine meat burger without introducing off-flavors into the meat or fish substitute.

[0069] In one embodiment, the meat or fish substitute of the present invention further comprises vegetable oils and / or vegetable fats. The vegetable oils and / or fats may be algal oil, fungal oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil, flaxseed oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango oil, cocoa butter, wheat germ oil, borage oil, blackcurrant oil, sea buckthorn oil, macadamia oil, saw palm oil, conjugated linoleic acid oil, arachidonic acid-enriched oil, docosahexaenoic acid (DHA)-enriched oil, eicosapentaenoic acid (EPA)-enriched oil, palm stearic acid, sea buckthorn berry oil, macadamia oil, saw palm oil, or rice bran oil; or margarine or other hydrogenated fats. In some embodiments, for example, the oil is algal oil. In a preferred embodiment, the vegetable oil of the present invention is sunflower oil and / or the vegetable fat of the present invention is coconut fat.

[0070] Preferably, the amount of vegetable oil is in the range of 2-20% (w / w) of the meat or fish substitute, for example, 5-15% (w / w) or 7-12% (w / w). Preferably, the amount of vegetable fat is in the range of 0.5% to 5% (w / w) of the meat or fish substitute, for example, 1% to 3% (w / w) of the meat or fish substitute.

[0071] Preferably, the meat substitute is a hamburger patty, food pieces, minced meat, meatballs, or sausage. Preferably, the meat substitute does not contain starch. Most preferably, it is a hamburger patty. Preferably, the hamburger does not contain starch.

[0072] Preferably, the fish substitute is a batter-based fish substitute, a fish burger substitute, a smoked fish substitute, a fish salad substitute, or a fish ball substitute. Preferably, the fish substitute does not contain starch.

[0073] Preferably, the meat or fish substitute comprises protein or protein isolate or protein concentrate. Examples of proteins may be barley, fonio, corn, millet, oats, rye, sorghum, tamarisk, rye, spelt wheat, rice, wheat, amaranth, buckwheat, quinoa, lupin, pea, chickpea, legumes (preferably broad beans), duckweed, potato, lentils, peanut, soybean, almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, walnut, canola, chia, flax, squash, sesame, sunflower, fungal protein (e.g., Quorn), mushrooms, or algae (e.g., Chlamydomonas, Spirulina, Euglena, Odontella, Saccharina, Chlorella). More preferably, the meat or fish substitute product of the present invention comprises 0.001 to 20% (w / w) of protein.

[0074] Preferably, the meat or fish substitute contains a flavoring agent, flavor reagent, or flavor precursor. Examples of flavoring agents may be yeast extracts or processed flavoring agents. More preferably, the meat or fish substitute product of the present invention contains 0.001 to 5% (w / w) of a flavoring agent.

[0075] In one embodiment, the meat or fish substitute of the present invention comprises a flavor modifier or a seasoning agent with modified properties. More preferably, the meat or fish substitute of the present invention comprises 0.001 to 1% (w / w) of a flavor modifier or a seasoning agent with modified properties.

[0076] Preferably, the meat or fish substitute of the present invention contains salt, preferably NaCl. The amount of salt is preferably in the range of 0.001 to 5% (w / w) of the meat or fish substitute of the present invention.

[0077] In a preferred embodiment, the meat or fish substitute of the present invention comprises a colorant, preferably in the range of 0.01 to 10% by weight, more preferably in the range of 0.1 to 5% by weight, and most preferably in the range of 0.2 to 2% by weight. In a preferred embodiment, the colorant of the present invention comprises or is beetroot or beetroot powder. The advantage of using beetroot is that it provides the product with a meat-like color without introducing an off-flavor. The colorant of the present invention may also be or comprise carotenoids. Preferably, the carotenoids are selected from the group consisting of α- or β-carotene, 8'-apo-β-carotene aldehyde, 8'-apo-β-carotene ester (e.g., ethyl ester), bis(oxalic acid), capsanthin, capsorubin, safflower xanthin, canthaxanthin, astaxanthin, astaxanthin ester, lycopene, lutein, zeaxanthin, or crocin and their derivatives.

[0078] In one embodiment, the meat or fish substitute of the present invention comprises heme, heme protein, heme-containing protein, or (large) molecules having chelated iron. More preferably, the meat or fish substitute of the present invention comprises 0.001 to 5% (w / w) of heme, heme protein, heme-containing protein, or (large) molecules having chelated iron.

[0079] In one embodiment, the meat or fish substitute of the present invention comprises an amount of water in the range of 50 to 80% (w / w), preferably 55 to 70% (w / w).

[0080] In another aspect, the present invention also provides the use of a heat-sensitive glucosylamylase protein formulation for obtaining binding in food products or for at least partially replacing adhesives or gelling agents in food products. The adhesive can be part of an adhesive system. For example, the adhesive methylcellulose can be part of an adhesive system that additionally includes oil, gum, and water. Preferably, at least 10% or at least 20, 30, or 40%, more preferably at least 50, 60, or 70%, even more preferably at least 80, 90, or 95% of the adhesive is replaced by the glucosylamylase, and most preferably 100% of the adhesive is replaced by the glucosylamylase. An example of an adhesive that can be at least partially replaced is methylcellulose. The use of the heat-sensitive glucosylamylase protein formulation for at least reducing the amount of methylcellulose in food products is described herein. Preferably, the food product is a meat substitute food product or a fish substitute food product or any of the above-mentioned food products. More preferably, methylcellulose is completely replaced as an adhesive. Preferably, the food product is a meat substitute, and most preferably a hamburger substitute. The explanations provided above regarding the different features of the method and product claims also apply to this part of the invention.

[0081] The present invention also provides the use of heat-sensitive glucosylamylase protein preparations as gelling agents. Preferably, the use of heat-sensitive glucosylamylase protein preparations as gelling agents in non-animal protein food products or cultured meat food products.

[0082] The invention will be explained in more detail in the following embodiments, which are not intended to limit the invention.

[0083] Example Rheological measurement Dynamic oscillatory rheology was performed using an Anton Paar Physica MCR302 rheometer with a cup and Bob geometry (CC27), and the procedure is shown in Table 1. Rheological data were obtained during the measurements, expressed as complex modulus G* [Pa] and phase angle [°]. Measurements were performed by filling cups with 17–20 mL of protein dispersion. To prevent the samples from drying out during the experiment, the samples in the cups were coated with a thin layer of sunflower oil. Detailed test procedure: Temperature scans were performed, the samples were heated, and then cooled in steps of 2 °C per minute within the temperature range of 25–95 °C, followed by holding at a final temperature of 95 °C for 10 minutes. During the heat setting and cooling phases, data were collected at a constant frequency of 1 Hz and a strain of 0.1%. Rheological data were collected at 30-second intervals. After the gel cooled, it was held at 25 °C for 10 minutes at a frequency of 0.1 Hz and a strain of 0.1%. During this step, rheological data were collected at 1-minute intervals. Subsequently, strain scanning was performed on the heat-set protein gel by increasing the strain from 0.1 to 100% at a constant frequency of 0.1 Hz and a constant temperature of 25 °C.

[0084]

[0085] Table 1: Rheometer Program Settings Texture Characterization (TPA) Texture characterization was performed on a texture analyzer equipped with the "Exponent" software (TA.TA.Xtplus, Stable Microsystems Ltd, Surrey, UK). A cylindrical probe with a diameter of 45 mm and a 5 kg weighing sensor were used. Double compression tests were performed at a test speed of 3 mm / sec up to 30% deformation, with a 2-second delay between the first and second compressions. All samples were measured in 5-fold increments, and the mean and standard deviation were calculated. Hardness, cohesion, elasticity, and resilience were calculated using the instrument software. From this, adhesiveness (hardness * cohesion) and chewiness (hardness * cohesion * elasticity) could be calculated.

[0086] Example 1: Heat setting of glucosylamylase protein preparation The thermosetting behavior of different glucosylamylase protein formulations was tested by heating 1 mL of sample in 2 mL Eppendorf tubes at 60, 70, or 80 °C for 5 minutes. Liquid enzymes were tested directly, while powdered enzymes were first resuspended at 15% (w / v) in cold 50 mM phosphate buffer (pH 7.0), and the solids were removed by centrifugation before testing. After heating, the development of the self-supporting gel was recorded by inverting the tubes. The glucosylamylase protein formulations tested were:

[0087] Table 2: Thermogelation behavior of commercial glucosylamylase. - indicates no gelation, ± indicates weak gel formation, + indicates formation of a self-sustaining gel. After testing the enzyme sets of globular proteins, many of them did not show gelation after incubation at 80°C for 10 minutes. Examples of non-thermal gelling enzymes are: • Bacterial amylase (Bacillus amyloliquefaciens) • Arabinofuranosinase (Aspergillus niger) • Xylanase (Aspergillus niger) • Xylanase (Trichoderma reesei) • Protease (Aspergillus oryzae) • Lipase (Rhizopus oryzae) • Protease (Rhizomucor miehei) • β-glucosidase (Aspergillus niger) • Succharomyces cerevisiae The test at lower temperatures (60 or 70 degrees Celsius) produced a wider variety of enzymes (globulin) that did not show gelation after incubation at 60 or 70 degrees Celsius for 10 minutes.

[0088] Example 2: Gel strength and elastic behavior Prepare an 11.1% (w / v) solution of egg white powder (Sanovo) in water by gently stirring until a homogeneous suspension is formed. The suspension contains approximately 10% (w / v) protein. Dilute Amigase Mega (30% protein solution) 3-fold with water to obtain an approximately 10% (w / v) protein solution. Suspend Sunbake AMG NG (60% protein particles) in cold water at 10% (w / v) and remove solids by centrifugation. Similarly, prepare a 2.5% (w / v) solution of egg white and Amigase Mega.

[0089] Thermal gelation was performed in a rheometer as described above. The thermal gelation initiation temperature of the glucoamylase sample was very similar to that of egg white, occurring at 65-70°C at high protein concentrations and at 70-75°C at low protein concentrations. Figure 1A and 2A The maximum modulus of the saccharifying enzyme after heating was comparable to that of low-concentration egg white, but slightly lower for a 10% solution. The gel strength increased further upon cooling to 25°C. The complex shear modulus of the cooled gel was almost unaffected by applied strain. Figure 1B and2B This indicates that the glucoamylase gel exhibits highly elastic behavior and does not break when strain is applied.

[0090] Example 3: Use of Amigase Mega in Vegan Hamburgers The ingredients shown in Table 3 are used in the following order for hamburger preparation. First, add the caramel and beetroot powder to water 1. This solution is used to hydrate TVP TU-crumble caramel 180 (ADM) and hydrate at room temperature for at least 45 minutes. During these 45 minutes, mix the product for 15 seconds every 15 minutes using a Hobart kitchen machine.

[0091] To prepare the binder phase, the solid components (texturant 1; MC, gellan gum, pectin) and oil were first mixed in a Magimix for 60 seconds. Then, Aglufiber (texturant 2) and the protein components were stirred into water 2. This dispersion was mixed in a Magimix for 5 minutes. The final dispersion or ice water (for the MC burger) was slowly added while mixing under high shear. This phase was then mixed for another 5 minutes. At this stage, a paste-like emulsion was prepared, which was then mixed with hydrated TVP until the dough appeared uniform. Finally, the dried portion of soy protein isolate, flavoring agents, and salt were incorporated into the dough, followed by frozen coconut fat. The added protein concentration in the test sample in the final burger was calculated to be 3% (w / w), replacing the 3% soy protein isolate (SPI) in the control burger.

[0092] Each 130-gram hamburger was then shaped using a mold. The hamburgers were flash-frozen for 90 minutes and then transferred to a regular freezer. The hamburgers were stored in the freezer for at least three nights before use. The day before cooking, the hamburgers were removed from the freezer and thawed in the refrigerator. The initial temperature of the hamburgers before cooking was 7°C. The cooking plate was set to 160°C, and the hamburgers were cooked alternately on each side for 3 minutes, until the core temperature was above 75°C.

[0093]

[0094] Table 3: Ingredients of vega-burgers using glucosyl amylase in the binder phase. MC: Methylcellulose; GPA: Gellan gum, pectin, Aglufiber; AM: Amigase Mega L; HU: Hunan HY01; EWP: Egg white protein.

[0095] As described above, the hardness of raw and cooked hamburger samples was determined using a 25 mm probe via TPA. Raw hamburgers were analyzed at 10°C, and cooked hamburgers at 50°C. The relative increase in hardness during cooking is an indicator of the effectiveness of the test components in thermal gelation. It is calculated by dividing the hardness (g) of the cooked hamburger by the hardness (g) of the raw hamburger. The results are shown in Table 4, indicating that different samples containing glucosylamylase protein resulted in a significant increase in hardness during cooking, even exceeding the relative increase in hardness when using methylcellulose or egg white. A good increase in the stability of the cooked samples was also detected by a professionally trained panel review.

[0096]

[0097] Table 4: The hardness of vegan hamburgers increases exponentially during cooking.

[0098] Legend of the drawings Figure 1: Rheological evaluation of 2.5% and 10% amylase (Amigase Mega L) protein aqueous solutions compared to egg white at the same dilution. A; Relationship between modulus (G*, Pa) and temperature. B; Relationship between modulus (G*, Pa) and strain (%) in heat-set gels at 25 °C.

[0099] Figure 2: Rheological evaluation of 10% glucosylamylase protein (Amigase Mega L and Sunbake AMG NG) and egg white with the same protein concentration in water. A; Relationship between modulus (G*, Pa) and temperature. B; Relationship between modulus (G*, Pa) and strain (%) in heat-set gels at 25 °C.

Claims

1. A method for preparing non-animal protein food products or cultured meat food products, comprising: A heat-sensitive glucosylamylase protein preparation is added to the ingredients used to prepare the food product, and the resulting glucosylamylase protein preparation and ingredient composition are processed into a non-animal protein food product or a cultured meat food product.

2. The method of claim 1, wherein the glucosylamylase protein preparation provides binding properties with the food product when the glucosylamylase protein preparation and ingredient composition or the food product is heated to at least 65 degrees Celsius.

3. The method according to claim 1 or 2, further comprising heating to at least 65 degrees Celsius.

4. The method according to any one of the preceding claims, wherein the glucosyl amylase protein preparation comprises -Natural glucosidase protein -Fungal glucosyl amylase protein - Aspergillus glucoamylase protein -Aspergillus niger glucosyl amylase protein -Classic (non-GMO) glucosyl amylase protein, - Extracellularly produced glucosylamylase protein, and / or - Any combination thereof.

5. The method according to any one of the preceding claims, wherein the non-animal protein is a plant protein, a microbial protein, or an algal protein.

6. The method according to claim 5, wherein the microbial protein is a fungal protein or a bacterial protein.

7. The method according to any one of the preceding claims, wherein the non-animal protein food product or the cultured meat food product is a meat substitute food product or a fish substitute food product.

8. The method according to any one of the preceding claims, wherein the amount of glucosylamylase protein is 0.025 to 10%, preferably 0.05 to 10%, more preferably 0.25 to 10% (relative to the mass of the food product).

9. A food product obtainable by the method of any one of claims 1 to 8.

10. Meat or fish alternative food products containing non-animal protein or cultured meat and heat-sensitive glucosyl amylase protein preparations.

11. The meat or fish substitute food product of claim 10, further comprising non-animal fat.

12. A meat or fish substitute comprising textured plant protein, non-animal protein, water, flavoring agent and binder system, wherein the binder system comprises a heat-sensitive glucosylamylase protein formulation.

13. The use of heat-sensitive glucosylamylase protein preparations for obtaining binding in food products or for at least partially replacing adhesives in food products.

14. The use according to claim 13, wherein all adhesives are replaced by the glucosylamylase protein formulation.

15. The use according to claim 13 or 14, wherein the adhesive is methylcellulose.

16. The use according to claim 13 or 14, wherein the food product is a meat substitute food product or a fish substitute food product.

17. Use of heat-sensitive glucosyl amylase protein preparations as gelling agents.

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