Methods and compositions for consumer products

Abstract

The present application relates to methods and compositions for consumer products. Described herein are methods and compositions for producing non-meat consumer products. A meat substitute constructed from muscle analogs, fat analogs, and connective tissue analogs is described.

Description

[0001] This application is a divisional application of the invention patent application filed on January 13, 2014, with application number "201480013805.8" and titled "Method and Composition for Consumer Products". Related reference

[0002] This application claims priority to U.S. Application No. 13 / 941,211, filed July 12, 2013; U.S. Application No. 61 / 908,634, filed November 25, 2013; and U.S. Application No. 61 / 751,816, filed January 11, 2013; and relates to the following co-pending patent applications: PCT / US12 / 46560, PCT / US12 / 46552, 61,876,676, filed September 11, 2013; 61 / 751,818, filed January 11, 2013; and 61 / 611,999, filed March 16, 2012, all of which are incorporated herein by reference. Technical Field

[0003] This invention relates to consumer products, and more particularly to non-animal imitations of animal-based food products, which in some embodiments can be produced by breaking down non-animal materials into their constituent parts and reassembling those parts into consumer products. Background Technology

[0004] Animal farming has profound negative environmental impacts. Currently, it is estimated that 30% of the Earth's landmass is dedicated to animal farming, and livestock account for 20% of the total biomass of terrestrial animals. As a result, large-scale animal farming accounts for over 18% of net greenhouse gas emissions. Animal farming is likely the largest anthropogenic source of water pollution and is currently the greatest threat to biodiversity worldwide. It is estimated that if the world's human population could shift from a meat-based diet to one free of animal products, 26% of the Earth's landmass would be freed up for other uses. Furthermore, a shift to a vegetarian diet would significantly reduce water and energy consumption.

[0005] Meat consumption has profound negative effects on human health. The health benefits of a vegetarian diet are well-established. If the human population were to shift towards a more vegetarian diet, healthcare costs would decrease.

[0006] Hunger is a global problem, and the world’s four major commodity crops (soybeans, corn, wheat and rice) already supply more than 100% of the human population’s needs for calories and protein (including every essential amino acid).

[0007] Most plant-based meat substitutes fail to induce a shift towards vegetarianism. Current levels of meat substitute composition technology involve extruding soy / grain blends, producing products that largely fail to mimic the experience of cooking and consuming meat. A common limitation of these products is their homogeneous texture and mouthfeel compared to equivalent meat products. Furthermore, because these products must be sold pre-cooked with artificial flavors and aromas, they cannot replicate the smells, flavors, and other key characteristics associated with freshly cooked meat. Therefore, these products primarily appeal to a limited consumer base that has already transitioned to vegetarianism / veganism, but fail to attract the larger segment of consumers accustomed to eating meat.

[0008] Food is any substance that any animal (including humans) consumes or drinks to obtain nutrition or pleasure. It typically has a plant or animal origin and contains essential nutrients such as carbohydrates, fats, proteins, vitamins, or minerals. This substance is ingested by organisms and absorbed by the cells of organisms dedicated to producing energy, sustaining life, or stimulating growth.

[0009] Food typically has its origin in photosynthetic organisms, and is typically derived from plants. Some foods are obtained directly from plants; however, even animals used as food sources are fed plant-based foods. Edible fungi and bacteria are used to transform material from plants or animals into other food products, such as mushrooms, bread, and yogurt.

[0010] In most cases, depending on the purpose of the food, plants or animals are graded and separated into several different parts. Generally, certain parts of a plant (such as seeds or fruits) are more valued by humans than others and are selected for human consumption, while other less desirable parts (such as grass stalks) are typically used to feed animals.

[0011] Animals are typically slaughtered into smaller pieces of meat before being consumed, giving them a specific flavor and handling characteristics.

[0012] While many foods can be eaten raw, many undergo some form of preparation for reasons of safety, palatability, texture, or flavor. At its simplest level, this can involve washing, cutting, trimming, or adding other foods or ingredients. It can also involve mixing, heating or cooling, or fermentation, and individual foods can be combined with other food products to achieve desired blending properties.

[0013] In recent years, attempts have been made to introduce scientific rigor into the food preparation process in the fields of food science and molecular gastronomy. Food science broadly studies the safety, microbiology, preservation, chemistry, engineering, and physics of food preparation, while molecular gastronomy focuses on using scientific tools (such as liquid nitrogen), emulsifiers (such as soy lecithin), and gelling agents (such as calcium alginate) to transform food products into unexpected forms.

[0014] However, the raw materials are typically whole organisms (plants or animals); or isolated tissues, such as meat chops, fruiting bodies of fungi, or seeds of plants. In some cases, the isolated tissues are modified before food preparation, for example, into powder or by separating oil and most of the protein from the seeds.

[0015] Although all these items contain a mixture of proteins, carbohydrates, fats, vitamins, and minerals, the physical arrangement of these materials in the plant or animal of origin determines the intended use of the plant or animal tissue. This document discloses improved methods and compositions for the production of consumer products. Summary of the Invention

[0016] This document provides consumer products and methods of making them. Consumer products can be non-animal-based, such as those containing predominantly plant-based or entirely plant-based proteins and / or fats, and can be in the form of beverages (e.g., alcoholic beverages, such as liqueurs, or protein drinks), protein supplements, baked goods (e.g., bread or biscuits), condiments (e.g., mayonnaise, mustard), meat products, or meat substitute products (e.g., ground beef products). For example, the protein beverage can be a meal replacement drink, beer supplemented with the protein, or a distilled alcoholic beverage supplemented with the protein (e.g., vodka or rum). The condiment can be mayonnaise. The meat product can be minced meat, sausage, or meat substitutes, which may include muscle imitations, plant-based fats, and / or connective tissue. Aggregates comprising one or more proteins can be used to help the components in the consumer product (e.g., ground beef products) bind together.

[0017] Therefore, this document provides a consumer product comprising isolated and purified plant protein, wherein the isolated and purified plant protein has (i) a solubility of at least 25 g / L in solution at a temperature between about 2°C and about 32°C, wherein the pH of the solution is between 3 and 8, and the sodium chloride content is 0 to 300 mM; or (ii) a solubility of at least 1 mg / ml in solution at a temperature between 90°C and 110°C, wherein the pH of the solution is between 5 and 8, and the sodium chloride content is 0 to 300 mM. In some embodiments, the consumer product is a beverage, a protein supplement, a baked product, a condiment, a meat product, or a meat substitute product. In some embodiments, the beverage is an alcoholic beverage or a protein drink. In some embodiments, the alcoholic beverage is a milk wine. The milk wine may further comprise a non-dairy lipid emulsion, wherein the milk wine does not contain animal products. In some embodiments, the protein drink is a dietary substitute beverage, a beer supplemented with the protein, or a distilled alcoholic beverage supplemented with the protein. The condiment may be a mayonnaise imitation. In some embodiments, the meat product may be meat paste, a sausage imitation, or a meat substitute. In some embodiments, the isolated and purified plant protein has a size of at least 10 kDa. In some embodiments, the isolated and purified plant protein is not completely denatured. In some cases, the isolated and purified plant protein is not derived from soybean. In some embodiments, the isolated and purified plant protein comprises one or more of the following: RuBisCo, mung bean 8S globulin, pea globulin, pea albumin, lentil protein, corn protein, or oil body protein.

[0018] In some embodiments, the isolated and purified plant protein comprises dehydrated protein, hydrophilic protein, intrinsically disordered protein, or protein identified based on its ability to remain soluble after boiling at pH and salt concentrations comparable to those of food. In some embodiments, the consumer product further comprises plant-derived lipids or microbial-derived lipids. In some embodiments, the consumer product further comprises a second isolated and purified protein, and / or flavoring agents, taste enhancers, emulsifiers, gelling agents, sugars, or fiber.

[0019] The present invention also provides a consumer product comprising a aggregate of one or more isolated and purified proteins. In some embodiments, the consumer product is a meat imitation. In some embodiments, the consumer product further comprises plant-derived lipids or microbial-derived lipids. The plant-derived lipids or microbial-derived lipids may comprise lecithin and / or oil. The product may comprise up to about 1% by weight of lecithin. The product may comprise lecithin and the oil. In some embodiments, the oil is canola oil, palm oil, or cocoa butter. The product may comprise about 1% to about 9% of the oil. The one or more isolated and purified proteins may comprise plant proteins. The one or more plant proteins may comprise one or more pea proteins, chickpea proteins, lentil proteins, lupin proteins, other legume proteins, or mixtures thereof. In some embodiments, the one or more pea proteins are pea globulin, pea globulin, conjugated pea globulin, or mixtures thereof.

[0020] The present invention also provides a meat imitation comprising muscle imitation, connective tissue imitation, adipose tissue imitation, and a aggregate containing one or more isolated and purified proteins. The aggregate may further comprise plant-derived lipids or microbial-derived lipids. The plant-derived or microbial-derived lipids may be lecithin and / or oil. The meat imitation may be ground beef.

[0021] A consumer product is also provided comprising a cold-set gel containing one or more isolated and purified proteins from non-animal sources, along with salts. In some embodiments, the isolated and purified plant proteins comprise one or more of the following: RuBisCo, mung bean 8S globulin, pea globulin, pea albumin, lentil protein, corn protein, or oleosome protein. In some embodiments, the isolated and purified plant proteins comprise dehydrated proteins, hydrophilic proteins, or inherently disordered proteins. In some embodiments, the cold-set gel further comprises plant-derived lipids or microbial-derived lipids. In some embodiments, the plant-derived lipids or microbial-derived lipids are lecithin and / or oils.

[0022] The present invention further provides an adipose tissue imitation comprising one or more isolated plant proteins, one or more oils derived from plants or algae, and optionally phospholipids. In some embodiments, the phospholipids are lecithin. In some embodiments, the plant oils are selected from the group consisting of: 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 butter, cocoa butter, wheat germ oil, rice bran oil, and combinations thereof. In some embodiments, the fat release temperature of the adipose tissue replica is in the range of 23°C to 33°C, 34°C to 44°C, 45°C to 55°C, 56°C to 66°C, 67°C to 77°C, 78°C to 88°C, 89°C to 99°C, 100°C to 110°C, 111°C to 121°C, 122°C to 132°C, 133°C to 143°C, 144°C to 154°C, 155°C to 165°C, 1 The temperatures range from 66°C to 167°C, 168°C to 169°C, 170°C to 180°C, 181°C to 191°C, 192°C to 202°C, 203°C to 213°C, 214°C to 224°C, 225°C to 235°C, 236°C to 246°C, 247°C to 257°C, 258°C to 268°C, 269°C to 279°C, 280°C to 290°C, or 291°C to 301°C. In some embodiments, the percentage of fat released by the adipose tissue imitation during cooking is 0% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. In some embodiments, the isolated and purified plant protein comprises one or more of the following: RuBisCo, mung bean 8S globulin, pea globulin, pea albumin, lentil protein, corn protein, or oil body protein.

[0023] In some embodiments, the adipose tissue imitation comprises about 40% to about 90% of the oil. In some embodiments, the adipose tissue imitation comprises about 1% to about 6% of the isolated and purified plant protein. In some embodiments, the adipose tissue imitation comprises about 0.05% to about 2% of the phospholipids. In some embodiments, the firmness of the adipose tissue imitation is similar to that of beef adipose tissue.

[0024] A consumer product is also provided, comprising a heme-containing protein and (i) carbon monoxide and / or (ii) nitrite, wherein the consumer product does not contain meat. In some embodiments, the heme-containing protein comprises at least 0.01% of the composition. In some embodiments, the consumer product further comprises one or more ammonium, sodium, potassium, or calcium salts. In some embodiments, the isolated and purified protein is cross-linked.

[0025] Further, a consumer product is provided that comprises a gel emulsion, wherein the gel emulsion comprises:

[0026] a) Proteins that have been separated and purified;

[0027] b) A first lipid, which is solid at a selected temperature range when not in the consumer product; and

[0028] c) A second lipid, which is liquid at the selected temperature range when not in the consumer product; wherein the melting temperature of the mixture of the first and second lipids is similar to the melting temperature of lipids found in meat, and wherein the first and second lipids are plant-derived lipids or microbial-derived lipids.

[0029] The present invention also provides a method for manufacturing a consumer product, comprising:

[0030] a) Prepare a solution comprising isolated and purified plant protein, wherein the isolated and purified plant protein has (i) a solubility of at least 25 g / L in the solution at a temperature between about 2°C and 32°C, wherein the pH of the solution is between 3 and 8, and the sodium chloride content is 0 to 300 mM; or (ii) a solubility of at least 1 mg / ml in the solution at a temperature between 90°C and 110°C, wherein the pH of the solution is between 5 and 8, and the sodium chloride content is 0 to 300 mM; and

[0031] b) Add the solution to the beverage.

[0032] In some embodiments, the solution comprises two or more isolated and purified plant proteins. In some embodiments, the beverage is clarified. In some embodiments, the concentration of the isolated and purified plant proteins in the solution is at least 1% by weight. In some embodiments, the isolated and purified plant proteins are selected from the group consisting of: RuBisCo, lentil globulin, soybean globulin, pea globulin, pea albumin, gliadin, lentil protein, dehydrated protein, hydrophilic protein, and intrinsically disordered protein. In some embodiments, the isolated and purified plant proteins are freeze-dried prior to the preparation of the solution. In some embodiments, the beverage has an improved taste compared to a corresponding beverage without the isolated and purified proteins.

[0033] A method for extending the shelf life of a meat-free consumer product is also provided, the method comprising adding a heme-containing protein to the consumer product, wherein the heme-containing protein oxidizes more slowly than myoglobin under equivalent storage conditions. In some embodiments, the heme-containing protein comprises an amino acid sequence having at least 70% homology to the amino acid sequence set forth in any of SEQ ID NO: 1-27.

[0034] A further method is provided for producing a meat imitation comprising a cold-set gel, wherein the method includes:

[0035] a) Denature a solution containing at least one isolated and purified protein from a non-animal source under conditions in which the isolated and purified protein does not precipitate from the solution;

[0036] b) Optionally add any heat-labile component to the denatured protein solution;

[0037] c) To gel the denatured protein solution at about 4°C to about 25°C by increasing the ionic strength of the solution to form a cold-set gel; and

[0038] d) Incorporate the cold-setting gel into the meat imitation.

[0039] In some embodiments, the gel is induced using 5 to 100 mM sodium chloride or calcium chloride. In some embodiments, the heat-labile component is a protein or lipid or a mixture thereof. In some embodiments, the protein is a heme-containing protein. In some embodiments, the cold-set gel is formed in a matrix comprising frozen-arranged plant proteins.

[0040] In some embodiments, the isolated and purified protein from a non-animal source is a plant protein. In some embodiments, the plant protein is selected from the group consisting of: RuBisCo, lentil globulin, soybean globulin, pea globulin, pea albumin, prolysin, lentil protein, dehydrated protein, hydrophilic protein, and intrinsically disordered protein.

[0041] Further, an adipose tissue replica is provided, comprising

[0042] a) Separated and purified non-animal proteins;

[0043] b) Non-animal lipids; and

[0044] c) A three-dimensional matrix comprising fibers derived from non-animal sources, wherein the lipids and proteins are dispersed within the three-dimensional matrix, and wherein the three-dimensional matrix stabilizes the structure of the adipose tissue replica.

[0045] A connective tissue replica is also provided, comprising one or more isolated and purified proteins assembled into a fibrous structure by a solution spinning process. In some embodiments, the fibrous structure is stabilized by a cross-linking agent.

[0046] This article provides a method for imparting a beef-like flavor to a consumer product, comprising adding a heme-containing protein to the consumer composition, wherein the beef-like flavor is imparted to the consumer composition after cooking.

[0047] A method is also provided for making a poultry or fish composition taste like beef, the method comprising adding heme protein to the poultry or fish composition respectively.

[0048] In some embodiments, the heme-containing protein has an amino acid sequence that is at least 70% homologous to any of the amino acid sequences set forth in SEQ ID NO: 1-27.

[0049] A further method for producing a coagulant is provided, the method comprising:

[0050] a) Acidifying a solution of one or more plant proteins to a pH between 3.5 and 5.5, wherein the solution contains 100 mM or less sodium chloride; and

[0051] b) Separating the aggregate from the solution. In some embodiments, the pH is between 4 and 5. In some embodiments, the plant protein comprises one or more pea proteins, chickpea proteins, lentil proteins, lupin proteins, other legume proteins, or mixtures thereof. In some embodiments, the pea protein comprises isolated and purified pea globulin, isolated and purified pea globulin, isolated and purified conjugated pea globulin, or combinations thereof. In some embodiments, the isolated and purified pea protein comprises isolated and purified pea globulin and isolated and purified conjugated pea globulin. In some embodiments, the acidification step is carried out in the presence of plant-derived lipids or microbial-derived lipids. In some embodiments, the plant-derived lipids or microbial-derived lipids comprise oils and / or phospholipids.

[0052] This article provides a method for producing adipose tissue replicas, the method comprising forming an emulsion comprising one or more isolated plant proteins, one or more oils derived from plants or algae, and optionally phospholipids. In some embodiments, when the phospholipids are included, they are lecithin. In some embodiments, the plant oils are selected from the group consisting of: 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 butter, cocoa butter, wheat germ oil, rice bran oil, and combinations thereof. In some embodiments, the fat release temperature of the adipose tissue replica is in the range of 23°C to 33°C, 34°C to 44°C, 45°C to 55°C, 56°C to 66°C, 67°C to 77°C, 78°C to 88°C, 89°C to 99°C, 100°C to 110°C, 111°C to 121°C, 122°C to 132°C, 133°C to 143°C, 144°C to 154°C, 155°C to 165°C, 1 The temperatures range from 66°C to 167°C, 168°C to 169°C, 170°C to 180°C, 181°C to 191°C, 192°C to 202°C, 203°C to 213°C, 214°C to 224°C, 225°C to 235°C, 236°C to 246°C, 247°C to 257°C, 258°C to 268°C, 269°C to 279°C, 280°C to 290°C, or 291°C to 301°C. In some embodiments, the percentage of fat released by the adipose tissue imitation during cooking is 0% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. In some embodiments, the isolated and purified plant protein comprises one or more of the following: RuBisCo, mung bean 8S globulin, pea globulin, pea albumin, lentil protein, corn protein, or oil body protein. In some embodiments, the emulsion comprises about 40% to about 90% of the oil. In some embodiments, the emulsion comprises about 1% to about 4% of the isolated and purified plant protein. In some embodiments, the adipose tissue replica comprises about 0.05% to about 1% of the phospholipid. In some embodiments, the emulsion is formed by high-pressure homogenization, ultrasonic treatment, or manual homogenization.

[0053] A further method is provided for minimizing undesired odors or flavors in a composition containing plant proteins, the method comprising contacting the composition with a ligand having affinity for one or more lipid oxygenases.

[0054] A method for minimizing undesirable odors or flavors in a composition containing plant proteins is also provided, the method comprising contacting the composition with activated carbon and then removing the activated carbon from the composition.

[0055] A method for minimizing undesirable odors or flavors in a composition containing plant proteins is also provided, the method comprising adding a lipoxygenase inhibitor and / or an antioxidant to the composition.

[0056] The present invention further provides a chocolate-flavored spread, comprising:

[0057] a) Sugar

[0058] b) Chocolate seasoning and

[0059] c) Cream derived from plant-based milk.

[0060] This document provides a method for altering the texture of a consumer product during or after cooking, comprising incorporating one or more plant proteins having a low denaturation temperature into the consumer product. In some embodiments, at least one of the one or more plant proteins is isolated and purified. In some embodiments, the one or more plant proteins are selected from the group consisting of rubisco, pea protein, lentil protein, or other legume proteins. In some embodiments, the pea protein comprises pea albumin protein. In some embodiments, the consumer product becomes firmer during or after cooking.

[0061] A tissue replica is also provided, comprising frozen-arranged non-animal proteins. In some embodiments, the non-animal proteins are plant proteins. In some embodiments, the non-animal proteins are isolated and purified. In some embodiments, the tissue replica is a muscle tissue replica.

[0062] The present invention also provides a meat imitation comprising a tissue imitation containing frozen-arranged non-animal proteins.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials can be used to practice this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0064] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings and the claims. The word "comprising" in the claims may be replaced by "consistently consisting of" or "component of" according to standard patent law practice. Attached Figure Description

[0065] Figure 1 The amino acid sequence of an exemplary heme-containing protein.

[0066] Figure 2A It is a bar graph depicting the percentage of fat release based on the amount of lecithin.

[0067] Figure 2B It is a bar graph depicting the temperature of fat release based on the amount of lecithin.

[0068] Figure 2C It is a bar graph depicting the firmness of fat imitations based on the amount of lecithin.

[0069] Figure 3 It is a bar graph depicting the percentage of fat release from fat imitations containing different oils (canola oil, cocoa butter, coconut oil, or rice bran oil).

[0070] Figure 4 It is a bar graph depicting the fat release temperature of fat imitations containing different oils (canola oil, cocoa butter, coconut oil, or rice bran oil). Detailed Implementation

[0071] I. Consumer Goods

[0072] This document describes methods and compositions for producing consumer products. In some cases, the consumer product is a non-animal imitation of an animal-based food product, which can be produced by breaking down non-animal materials into their components and recombining those components into the consumer product. In other cases, the consumer product is not intended to imitate animal-based food and actually possesses its own unique characteristics suitable for food. Additionally, in some cases, the consumer product may serve as a carrier for nutritional preparations or pharmaceutical compositions rather than primarily as food.

[0073] The advantages of the consumer products described herein may include, for example, using less energy or water in their production compared to similar food products, not using animals in their production, creating healthier products, using raw materials that would otherwise be discarded, or allowing the exclusion (or non-inclusion) of certain components (e.g., allergens) from the consumer products. Consumer products may also have a higher degree of production consistency, leading to improved product quality control. Another advantage is that consumer products can be intentionally designed to have characteristics suitable for food preparation, making them superior to traditional food products.

[0074] Consumer products can be used for animal consumption, including human consumption. Consumer products can be food for livestock (e.g., dog food can be produced according to the present invention) or wild animals (e.g., food for non-domesticated carnivores).

[0075] Similar to existing human food, consumer goods can be sold in grocery stores, convenience stores, supermarkets and club shops, or prepared in restaurants (including fast food restaurants), schools, event venues, hospitals, military facilities, prisons, shelters or long-term care facilities.

[0076] Consumer products can be approved by the appropriate regulatory authority. For example, a consumer product can be formulated to meet the requirements of the U.S. Food and Drug Administration. The method of the present invention may include steps necessary to satisfy the regulatory authority.

[0077] The consumer products of this invention can mimic, compete with, complement, or replace conventional food products (referred to herein as "food products"). Food products can be any currently existing food. Consumer products of this invention can be made to mimic food products, such as equivalent meat products. Equivalent meat products can be white or dark meat. Equivalent meat products can be derived from any animal. Non-limiting examples of animals used to obtain equivalent meat products include farmed animals such as cattle, sheep, pigs, chickens, turkeys, geese, ducks, horses, and dogs; or hunted animals (whether wild or farmed) such as rabbits, deer, bison, buffalo, boars, snakes, pheasants, quails, bears, elk, antelopes, pigeons, wild pigeons, grouse, foxes, wild boars, goats, kangaroos, emus, alligators, crocodiles, turtles, groundhogs, marmots, opossums, partridges, squirrels, raccoons, whales, seals, ostriches, capybaras, coypus, guinea pigs, rats, mice, and wild rats; any kind of insects or other arthropods; or marine food such as fish, crabs, lobsters, oysters, musculoskeletons, scallops, abalone, squid, octopuses, sea urchins, tunicates, etc.

[0078] Although many meat products are typically derived from the skeletal muscle of an animal, it should be understood that meat can also be derived from other muscles or organs of an animal. In some embodiments, the equivalent meat product is a cut of meat derived from skeletal muscle. In other embodiments, the equivalent meat product is an organ, such as kidney, heart, liver, gallbladder, intestine, stomach, bone marrow, brain, thymus, lung, or tongue. Therefore, in some embodiments, the compositions of the present invention are consumer products that resemble skeletal muscle or organs.

[0079] Consumer products (e.g., meat substitutes) may comprise one or more of the following: a first composition comprising a muscle tissue replica, a second composition comprising a fat tissue replica, and / or a third composition comprising a connective tissue replica, wherein one or more compositions are combined in a manner that mimics the physical structure of meat. The present invention also provides unique compositions of muscle tissue replicas (referred to herein as “muscle replicas”), fat tissue replicas (referred to herein as “fat replicas”), and connective tissue replicas (referred herein as “connective tissue replicas”). In some embodiments, these compositions consist primarily or entirely of ingredients derived from non-animal sources (e.g., 10% or less of the ingredients are derived from animal sources). In alternative embodiments, meat substitute products comprising muscle, fat, and / or connective tissue replicas, or comprising one or more of said replicas, are partially derived from animal sources but supplemented with ingredients derived from non-animal sources. In some embodiments, up to 90% of the food product is derived from animal sources. In some embodiments, approximately 75% of the food product is derived from animal sources. In some embodiments, approximately 50% of the food product is derived from animal sources. In some embodiments, approximately 10% of the food product is derived from animal sources. In other alternative embodiments, the present invention provides meat products (e.g., beef, chicken, turkey, or pork products) substantially derived from animal sources, supplemented with one or more of muscle tissue imitations, fat imitations, and / or connective tissue imitations, wherein the imitations are substantially or entirely derived from non-animal sources. A non-limiting example of such meat products is an ultra-lean ground beef product supplemented with a non-animal-derived fat imitation, which improves texture and mouthfeel while retaining the health benefits of lower-fat animal products. Such alternative embodiments can produce products that more closely mimic the key characteristics associated with the preparation and consumption of meat, but at a lower cost and with less environmental impact, less animal welfare impact, or improved health benefits for consumers.

[0080] Examples of other food products that a consumer product can mimic or substitute include: beverages (such as milk wine or milk), protein drinks (e.g., RuBisCo can be used as a protein supplement in beer, distilled alcoholic beverages (such as vodka), juice, meal replacement drinks, or water), and pastes (such as Nutella). TM (Imitations of cream, corn cheese or mayonnaise), minced meat, blood sausage, meat enhancers, eggs, fish, sausage, tenderizer, spam, or frozen foods (such as ice cream, yogurt, kefir, sour cream or butter).

[0081] Consumer products can be meat imitations. Consumer products can be manufactured to mimic cuts or appearances of meat. For example, a consumer product may visually resemble or be indistinguishable from ground beef or specific cuts of beef. In one exemplary embodiment, the imitation is assembled in a manner that approximates the physical texture of natural ground meat (e.g., ground beef, ground chicken, or ground turkey). In other embodiments, the imitation is assembled in a manner that approximates different cuts of beef (e.g., sirloin, tenderloin, London steak, etc.). Alternatively, a consumer product can be manufactured to have a unique appearance or shape. For example, a consumer product may contain patterns (e.g., printed words or images) formed by the structure of the consumer product. In some cases, a consumer product may resemble a traditional food product after it is prepared. For example, a consumer product may be produced larger than a traditional cut of beef, but after being sliced ​​and cooked, it may look identical to traditionally cooked meat. In some embodiments, a consumer product may resemble a traditional food product in shape in two dimensions, but not in a third dimension. For example, a consumer product can resemble a cut of meat in two dimensions (e.g., when viewed from above), but can be much longer (or much thicker) than a conventional cut. In this example, the composition can be repeatedly cut into products traditionally shaped like meat.

[0082] Consumer products can be made from locally sourced products. For example, a consumer product can be made from plants that grow within a radius of the end consumer. For example, the radius could be 1, 10, 100, or 1000 miles. Therefore, in some embodiments, the present invention provides a method of producing consumer products that do not contain products that have already been shipped 1, 10, 100, or 1000 miles away.

[0083] This invention provides a method for producing consistent consumer product properties when the product is produced from various sources. For example, a plant-based meat substitute produced from local plants in Iowa, USA, will have a taste, aroma, and texture substantially similar to a plant-based meat substitute produced from local plants in Lorraine, France. This consistency allows for methods of advertising locally grown foods with consistent properties. This consistency can be achieved by the concentration or purification of similar components from different locations. These components can be combined in predetermined ratios to ensure consistency. In some embodiments, using components from the same plant species (e.g., isolated or concentrated proteins and fats) can enable a high degree of characteristic consistency. In some embodiments, using components from different plant species (e.g., isolated or concentrated proteins and fats) can enable a high degree of characteristic consistency. In some embodiments, the same protein can be isolated from different plant species (i.e., homologous proteins). In some embodiments, the invention provides a method comprising: isolating similar plant components from plant sources in different locations, assembling compositions provided therein in two locations, and marketing compositions wherein compositions assembled and marketed in different geographical locations have consistent physical and chemical properties. In some embodiments, the isolated components are derived from different plant populations in different locations. In some embodiments, one or more of the isolated components are shipped to independent geographical locations.

[0084] Consumer products may require fewer resources to produce than those produced from domesticated animals. Therefore, this invention provides meat imitations that require less water or energy to produce than meat. For example, the consumer products described herein may require less than about 10, 50, 100, 200, 300, 500, or 1000 gallons of water per pound of product. In comparison, producing beef may require more than 2000 gallons of water per pound of meat.

[0085] Consumer products may require less land to produce than meat products with similar protein contents. For example, the consumer products described herein may require 30% or less of the land area needed to produce meat products with similar protein contents.

[0086] Consumer products may offer health benefits compared to the animal products they replace in your diet. For example, they may have less cholesterol or lower saturated fat compared to equivalent meat products. The American Heart Association and the National Cholesterol Education Program recommend limiting dietary cholesterol intake to 300 mg per day, equivalent to 12 ounces of beef or two egg yolks. Consumer products described herein that are indistinguishable from animal products (such as ground beef) and have reduced or no cholesterol can help maintain a low-cholesterol diet. In another instance, the consumer products described herein may be cholesterol-free or contain higher levels of polyunsaturated fatty acids compared to the animal products they replace.

[0087] Consumer products can offer animal welfare benefits compared to the animal products they replace in the diet. For example, they can be produced without the need for restriction, force-feeding, premature weaning, disruption of the mother-offspring interaction, or slaughter of animals for their meat.

[0088] Consumer products can have a smaller "carbon footprint" than the meat products they replace. For example, a consumer product can generate 1%, 5%, 10%, 25%, 50%, or 75% of the net greenhouse gas emissions caused by the animal products it replaces. For instance, according to the Environmental Working Group's (2011) "meat eaters guide to climate change and health," beef production results in 27 kg of CO2 emissions per kilogram of beef consumed, while lamb production results in 39 kg of CO2 emissions per kilogram of beef consumed.

[0089] The consumer products described herein can provide alternatives to animal products or combinations of animal products that are forbidden by religious beliefs. For example, a consumer product could be a kosher pork chop.

[0090] Consumer products can also be shipped in component form and manufactured or assembled in different locations. Local components can be used to produce consumer products when available. Local components can be supplemented with those not available locally. This allows for methods of producing consumer products (e.g., meat imitations) using less shipping energy than meat would require. For example, local water can be used in combination with other components supplied to a consumer product. Using local water reduces shipping weight, thereby reducing costs and environmental impact.

[0091] The consumer products described herein can be produced or assembled, wholly or partially, in areas where animal husbandry is impractical or prohibited. The consumer products can also be produced or assembled in urban environments. For example, kits can be provided to users to enable them to produce the consumer products. Users can use local water or plants from rooftop gardens (e.g., in Shanghai). In another instance, the consumer products can be produced on spacecraft, space stations, or lunar bases. Therefore, the present invention provides methods and systems for producing meat replicas for space travel or for training in space travel. For example, the invention can be used for Earth-based training in space travel. The consumer products can also be produced on artificial platforms on islands or in the sea, where raising livestock is difficult or prohibited.

[0092] II. The nature of consumer goods

[0093] The consumer products described in this article are typically designed to mimic the experience of consuming a food product (such as meat). The appearance, texture, and taste of a consumer product can make it similar to or indistinguishable from a food product (such as meat). Consumer products can also be manufactured to possess the desired characteristics of a food product without incorporating other undesired characteristics. For example, a consumer product could be a replica steak that lacks cartilage or other components typically not consumed in a given food product.

[0094] In some embodiments, the present invention provides a method for determining the suitability of a consumer product as a substitute for a food product, for example by determining whether an animal or human can distinguish the consumer product from a determined food product (e.g., a specific type of meat). One method for determining whether a consumer product is equivalent to a food product (e.g., meat) is a) defining the properties of the meat and b) determining whether the consumer product has similar properties.

[0095] Properties that can be tested or used to compare or describe food products or consumer goods include mechanical properties such as hardness, cohesiveness, brittleness, chewiness, adhesiveness, viscosity, elasticity, and tackiness. Testable food product properties also include geometric properties such as particle size and shape, as well as particle shape and orientation. The three-dimensional structure of the particles can also be tested. Other properties may include moisture content and fat content. These properties can be described using terms such as: "soft," "firm," or "hard" to describe hardness; "crumbly," "crunchy," "brittle," "chewy," "tender," "tough," "short," "mealy," "pasty," or "gummy" to describe cohesion; "thin" or "viscous" to describe viscosity; and "plastic" or "elastic" to describe elasticity. The terms "tic" describes elasticity; "sticky," "tacky," or "gooey" describes adhesiveness; "gritty," "grainy," or "coarse" describes particle shape and size; "fibrous," "cellular," or "crystalline" describes particle shape and orientation; "dry," "moist," "wet," or "watery" describes moisture content; or "oily" or "greasy" describes fat content. Therefore, in one embodiment, a group of people can rate a food product (e.g., ground beef) based on the properties described. Consumer products described herein can be rated by the same people to determine equivalence.

[0096] It can also evaluate the flavor of food products. Flavors can be scored based on their similarity to food products, such as "egg flavor," "fish flavor," "butter flavor," "chocolate flavor," "fruit flavor," "pepper flavor," "bacon flavor," "cream flavor," "milky flavor," or "beef flavor." Flavors can also be scored based on seven basic tastes: sweet, sour, bitter, salty, umami (minty), pungent / piquant, and metallic. Flavors can be described based on their similarity to experiences induced by chemical substances, such as dimethylglyoxal (butter flavor), 3-hydroxy-2-butanone (butter flavor), nonan-2E-enal (polyester), 1-octen-3-ol (mushroom flavor), hexanoic acid (sweat flavor), 4-hydroxy-5-methylfuranone (HMF, meat flavor), pyrazine (nut flavor), bis(2-methyl-3-furanyl)disulfide (barbecue flavor), decanone (mold / fruit flavor), isoamyl acetate (banana flavor), benzaldehyde (bitter almond flavor), cinnamaldehyde (cinnamon flavor), ethyl propionate (fruit flavor), methyl anthranilate (grape flavor), limonene (orange flavor), ethyl decanedioate (pear flavor), allyl hexanoate (pineapple flavor), ethyl maltitol (sugar, marshmallow), ethyl vanillin (vanilla flavor), butyric acid (rotten flavor), 12-methyltridecaldehyde (beef flavor), or methyl salicylate (wintergreen flavor). These ratings can be used as indicators of the nature of food products. The consumer product of the present invention can then be compared with food products to determine the degree of similarity between the consumer product and the food product. In some cases, the nature of the consumer product is then altered to make it more similar to a food product. Therefore, in some embodiments, the consumer product receives a rating similar to that of a food product based on human evaluation. In some embodiments, the consumer product is indistinguishable from real meat to humans.

[0097] It is possible to eliminate properties related to the source of a consumer product's components. For example, a consumer product may be made from components derived from beans, but it can become devoid of a "bean-like" flavor or texture. One way this can be achieved is by breaking down the component source material into isolated and purified components without using components that do not possess the desired characteristic properties of the source. Additionally, as described herein, undesirable flavors or odors (e.g., unwanted flavors or odors) in isolated and / or purified components can be minimized by deodorizing with activated charcoal or by removing enzymes (e.g., lipoxygenases (LOXs)) that may be present in trace amounts and can convert unsaturated triglycerides (e.g., linoleic acid or linolenic acid) into smaller and more volatile molecules. LOXes are naturally found in legumes (e.g., peas, soybeans, and peanuts) as well as rice, potatoes, and olives. When legume flour is graded and separated into individual protein fractions, LOXes can act as unwanted "time bombs," potentially developing unwanted flavors or odors during aging or storage. As shown in Example 34, compositions containing plant proteins (e.g., from ground cover plant seeds) can undergo purification to remove LOX using, for example, an affinity resin that binds to and removes LOX from the protein sample. The affinity resin can be linoleic acid, linolenic acid, stearic acid, oleic acid, propyl gallate, or epigallocatechin gallate attached to a solid carrier (e.g., beads or resin). See, for example, WO2013138793. Additionally, depending on the protein composition, certain combinations of antioxidants and / or LOX inhibitors can be used as effective agents to minimize the formation of undesirable flavors or odors in protein solutions, especially in the presence of fats and oils. The compounds may include, for example, one or more of the following: β-carotene, α-tocopherol, caffeic acid, propyl gallate, or epigallocatechin gallate. These may be included during protein purification or in subsequent food processing steps to mitigate the formation of undesirable flavors or odors in protein-rich foods.

[0098] In some compositions, individuals asked to identify the consumer product will identify it as a form of food product or as a specific food product, for example, an individual will identify the consumer product as meat. For instance, in some compositions, humans will identify the consumer product as having properties equivalent to meat. In some embodiments, based on human perception, one or more properties of the consumer product are equivalent to corresponding properties of meat. These properties include those that can be tested. In some embodiments, humans will identify the consumer product of the present invention as being more like meat than any meat substitute found in the art.

[0099] Experiments can demonstrate that the consumer product is acceptable to consumers. A panel can be used to screen multiple consumer products described herein. Multiple human panel members can test multiple consumer product samples, i.e., natural meat compared to the consumer compositions described herein, or meat substitutes compared to the consumer compositions described herein. A mixture of lean and fatty meat can be used to standardize variables (e.g., fat content) to, for example, 20% fat. Fat content can be determined using the Babcock method for meat (SS Nielson, Introduction to the Chemical Analysis of Foods (Jones & Bartlett Publishers, Boston, 1994). A mixture of ground beef prepared according to the procedures described herein and the consumer product of the present invention can be formulated.

[0100] Samples can be presented to panel members in an open consumer group (e.g., in a small room) under red or white light. Samples can be assigned random three-digit numbers and rotated in anonymous voting positions to prevent bias. Panel members can rate the samples on softness, juiciness, texture, flavor, and overall acceptability using a pleasure scale of 1 = very averse, to 9 = very fond, with a median of 5 = neither dislike nor averse. Panel members can rinse their mouths between samples and be given the opportunity to comment on each sample.

[0101] The results of this experiment can demonstrate significant differences or similarities between conventional meat and the composition of this invention.

[0102] These results demonstrate that the compositions described herein are deemed acceptablely equivalent to real meat products. Furthermore, these results demonstrate that the compositions described herein are more preferred by the panel members compared to other commercially available meat substitutes. Therefore, in some embodiments, the present invention provides consumer products that resemble conventional meat and are more meat-like than previously known meat substitutes.

[0103] The consumer product of the present invention can also have physical characteristics similar to food products (e.g., traditional meat). In one embodiment, the force required to pierce a 1-inch-thick structure (e.g., a meat patty) made of the consumer product of the present invention with a fixed-diameter steel bar is not significantly different from the force required to pierce a 1-inch-thick structure of a similar food product (e.g., ground beef patty) with a similar fixed-diameter steel bar. Therefore, the present invention provides a consumer product having physical strength characteristics similar to meat. In another embodiment, the tear cross-sectional area is 100 mm². 2 The force required for the sample of the present invention is the same as the tear cross-sectional area of ​​100 mm² measured in the same manner.2 The required force is not significantly different for animal tissue (muscle, fat, or connective tissue) samples. The force can be measured using, for example, a TA.XTPlus Texture analyzer (Texture Technology). Therefore, this invention provides consumer products with physical strength characteristics similar to those of meat.

[0104] The consumer products described herein can have cooking loss characteristics similar to food products (e.g., meat). For example, a consumer product can have fat and protein contents similar to ground beef and exhibit the same size reduction during cooking as real ground beef. Various compositions of consumer products described herein that match various meats can achieve similarity in size loss characteristics. The cooking loss characteristics of consumer products can also be engineered to be superior to those of food products. For example, consumer products can be produced that exhibit less loss during cooking but achieve similar taste and texture qualities to cooked products. One way to achieve this is by altering the lipid ratio based on the melting temperature of the consumer composition. Another way to achieve this is by altering the protein composition of the consumer product by controlling the protein concentration or by means of mechanisms that form tissue imitations.

[0105] In some embodiments, consumer products are compared to animal-based food products (e.g., meat) based on olfactory meter readings. In various embodiments, the olfactory meter can be used to assess odor concentration and odor threshold, or odor threshold compared to a reference gas, to determine a hedonic rating score for the degree of enjoyment, or the relative intensity of the odor. In some embodiments, the olfactory meter allows for the training and automated evaluation of expert panels. Therefore, in some embodiments, the consumer product is one that yields similar or identical olfactory meter readings. In some embodiments, the difference is small enough to be below the detection threshold perceived by humans.

[0106] Gas chromatography-mass spectrometry (GCMS) is a method that combines the characteristics of gas chromatography-liquid chromatography and mass spectrometry to separate and identify different substances within a test sample. In some embodiments, GCMS can be used to evaluate the properties of consumer products. For example, volatile chemicals can be separated from the headspace surrounding meat. These chemicals can be identified using GCMS. This yields a characteristic of the volatile chemicals in the headspace surrounding meat. In some cases, each peak of the GCMS can be further evaluated. For example, humans can rate the olfactory experience of the chemical that causes a particular peak. This information can be used to further refine the characteristic. GCMS can then be used to evaluate the properties of consumer products. GCMS characteristics can be used to optimize consumer products.

[0107] Characteristic flavor and aroma components are largely generated during the cooking process through chemical reactions of molecules, including amino acids, fats, and sugars found in plants and meats. Therefore, in some embodiments, consumer products are tested for similarity to meat during or after cooking. In some embodiments, olfactory maps of cooked meat are created using human ratings, human assessments, olfactory meter readings, or GCMS measurements, or combinations thereof. Similarly, olfactory maps of consumer products (e.g., meat imitations) can be created. These maps can be compared to assess the similarity between cooked consumer products and meat. In some embodiments, the olfactory map of a consumer product during or after cooking is similar to or indistinguishable from the olfactory map of cooked or being cooked meat. In some embodiments, the similarity is sufficient to exceed a detection threshold perceived by humans. Consumer products can be produced such that their characteristics resemble those of cooked food products, but uncooked consumer products may have properties different from those of pre-cooked, pre-cooked food products.

[0108] Shelf life is the length of time a consumer product is given before it is deemed unsuitable for sale, use, or consumption. Generally, it is important to keep meat products at around 2°C, as shelf life decreases with exposure to higher temperatures.

[0109] The shelf life of meat is determined by studying the sensory cues of meat products over time (odor, visual appearance of packaging, color, taste, and texture) and by conducting laboratory analyses under controlled conditions to determine how long the product remains safe, healthy, and enjoyable. Ground beef is used as an example, but similar conditions would apply to patties, ribs, and roasts from other meat types. Beef in its natural state is a deep blue-purple color. However, oxygen can penetrate the meat and react chemically with the myoglobin in it, producing a red color. Continued exposure to oxygen causes myoglobin oxidation, resulting in the red meat turning brown and developing an "unpleasant" flavor. To control this oxidation, various methods for storing and displaying meat products to increase their shelf life have been extensively studied. These methods include the use of vacuum packaging, modified atmosphere packaging (high oxygen), modified atmosphere packaging (low oxygen, with carbon monoxide), and / or high-pressure pasteurization (HPP).

[0110] The primary determinant of meat color is the concentration of iron-carrying proteins in the meat. In the skeletal muscle component of meat products, myoglobin is one of the main iron-carrying proteins. It is estimated that white chicken contains less than 0.05% myoglobin; pork and veal contain 0.1-0.3%; veal contains 0.4-1.0%; and older beef contains 1.5-2.0%. Typically, myoglobin in meat exists in three states: oxymyoglobin (Fe... 2+ (Oxygenation = bright red); Myoglobin (Fe)2+ (Non-oxygenated = slightly purple / magenta); and metmyoglobin (Fe 3+ (Oxidation = Brown). The conversion of oxymyoglobin to metmyoglobin in the presence of oxygen is considered the reason for the color change of minced meat from red to brown. Meat shelf-extending agents have been developed to prolong the red life of meat products, including but not limited to carbon monoxide, nitrite, sodium metabisulfite, Bombal, vitamin E, rosemary extract, green tea extract, catechins, and other antioxidants.

[0111] However, there are inherently more stable heme proteins, such as those from wind-producing bacteria ( Aeolian water (SEQ ID NO:3) or extreme acidophilic methanogens ( Methylacidiphilum infernorum The hemoglobin isolated from (SEQ ID NO: 2) will oxidize more slowly than thermophilic hemoglobin (e.g., myoglobin). The heme proteins described herein (see, for example...) Figure 1 It can also reduce heme-Fe through meat shelf-extending agents (such as carbon monoxide and sodium nitrite). 2+ The lifespan of the state is extended. Heme proteins can be selected to obtain desired color-preserving properties. For example, for sous-vide cooking, relatively unstable heme proteins (such as those from barley) are more suitable. Barley The heme protein can provide a brown product in which myoglobin presents as cooked while retaining its red, uncooked appearance. In some embodiments, the heme protein can be selected to have increased stability, in which, for example, meat imitations can retain an appealing rare appearance even when thoroughly cooked for food safety.

[0112] The primary determinant of rancidity and the development of undesirable flavors or odors is the oxidation of components of consumer products, including but not limited to fats. For example, the oxidation of unsaturated fatty acids is a known cause of putrid odors. In some embodiments, meat imitations have extended shelf lives because the chemical composition of the meat imitations is controlled so that the taste, texture, odor, and chemical properties do not react with oxygen to form undesirable flavors or odors. In some embodiments, meat imitations are less sensitive to oxidation due to the presence of higher levels of unsaturated fatty acids than found in beef. In some embodiments, meat imitations do not contain unsaturated fatty acids. In other embodiments, meat imitations contain higher levels of antioxidants, such as glutathione, vitamin C, vitamin A, and vitamin E, than found in meat; and enzymes, such as catalase, superoxide dismutase, and various peroxidases. In other embodiments, components that generate undesirable flavors or odors, such as lipoxygenases, are absent.

[0113] In some embodiments, the consumer products described herein exhibit increased stability under commercial packaging conditions. In some embodiments, the improved shelf life is achieved by using components with increased oxidative stability (e.g., lipids with reduced unsaturated fatty acid content) and / or by using more stable heme proteins (e.g., hemoglobin isolated from *Aeromonas hydrophila* (SEQ ID NO: 3) or *Extreme acidophilus methanogens* (SEQ ID NO: 2). In some embodiments, the improved shelf life is attributed to a combination of components used in the consumer product. In some embodiments, the consumer product is specifically designed for a particular packaging method.

[0114] III. Composition of Consumer Goods

[0115] The consumer products described herein include one or more isolated and purified proteins. "Isolated and purified protein" means a formulation in which, relative to the source material from which the specified protein was isolated, the cumulative abundance of the protein component by mass is reduced by a factor of 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, or 1000 or more. For clarity, isolated and purified protein is described as being isolated and purified relative to its starting material (e.g., plant or other non-animal source). In some embodiments, the term "isolated and purified" may indicate that the protein formulation is at least 60% pure, for example, more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. The fact that consumer products may contain materials other than isolated and purified protein does not alter the isolated and purified nature of the protein, as this definition typically applies to proteins added prior to their composition.

[0116] In some embodiments, one or more isolated and purified proteins constitute at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% by weight of the protein contents of the consumer product. In some embodiments, each of the one or more isolated proteins is isolated and purified separately.

[0117] The consumer products described herein may consist substantially or entirely of ingredients derived from non-animal sources (e.g., plant, fungal, or microbial sources). Plant sources may be organically grown. Proteins may be extracted from source materials (e.g., from animal tissues or plant, fungal, algal, or bacterial biomass, or from the supernatant of cultures that secrete proteins) or from combinations of source materials (e.g., multiple plant species). Consumer products may also be made from a combination of plant and animal sources. For example, a consumer product may be a ground beef product supplemented with the plant-based products of this invention.

[0118] A. The source of the product

[0119] As described above, isolated and purified proteins can be derived from non-animal sources, such as plants, algae, fungi (e.g., yeast or filamentous fungi), bacteria, or archaea. In some embodiments, isolated and purified proteins can be obtained from genetically modified organisms, such as genetically modified bacteria or yeast. In some embodiments, isolated and purified proteins are obtained through chemical synthesis or in vitro synthesis.

[0120] In some embodiments, one or more isolated and purified proteins are derived from plant sources. The isolated and purified proteins can be isolated from a single plant source, or multiple plant sources can serve as starting materials for protein isolation and purification. As described herein, the isolated and purified plant proteins are soluble in solution. The solution may contain EDTA (0-0.1 M), NaCl (0-1 M), KCl (0-1 M), NaSO4 (0-0.2 M), potassium phosphate (0-1 M), sodium citrate (0-1 M), sodium carbonate (0-1 M), sucrose (0-50%), urea (0-2 M), or any combination thereof. The solution may have a pH of 3 to 11. In some embodiments, the plant protein may have a solubility in solution of >25 g / L (e.g., at least 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or 225 g / L) at a temperature between about 2°C and about 32°C (e.g., between 3°C and 8, between 4°C and 8, between 5°C and 8, between 6°C and 8, between 7°C and 8, between 7°C and 8, between 6°C and 8, between 7°C and 8, between 7°C and 8, between 8 ...

[0121] Those skilled in the art will understand that proteins isolated from any organism in the plant kingdom can be used to produce the consumer products described herein. Non-limiting examples of plant sources include: cereal crops such as maize, oats, rice, wheat, barley, rye, millet, sorghum, buckwheat, amaranth, quinoa, black wheat (wheat-rye hybrids), and teff (sphagnum moschata). Eragrostis tef Oilseed crops, including cottonseed, sunflower seed, safflower seed, and Capsella asiatica ( ). Crambe ), genus *Hylocereus* Camelina ), mustard, rapeseed (Brassica napus type rapeseed ( Brassica napus )); Gum arabic ( Acacia ); or from plants of the legume family, such as clover, pen-shaped beans ( Stylosanthes ), Sesbania ( Sesbania ), wild pea (Vigna genus ( Vetches )), Genus Peanut ( Peanut ), Indigofera ( Indigofera ), Leucaea ( Leucaena ), genus *Guaranta* ( Cyamopsis ), peas (such as cowpeas, English peas, yellow peas, or green peas) or beans (such as soybeans, broad beans, lima beans, kidney beans, chickpeas, mung beans, black and white peas, lentils, lupins, peas, carob, soybeans, and peanuts (groundnuts) Peanut Leafy greens, such as lettuce, spinach, kale, green cabbage, radish, chard, mustard greens, dandelion greens, broccoli, or cabbage; or green substances that are not generally edible for humans, including biomass crops, such as switchgrass. Panicum virgatum )), genus *Ammonium* Miscanthus ), Luzhu ( Reed gift ), energy sugarcane, sorghum ( Sorghum Other grasses, alfalfa, corn stalks, kelp or other seaweed; green matter generally discarded from harvested plants, sugarcane leaves, tree leaves; root crops, such as cassava, sweet potato, potato, carrot, beet or bulrush; or coconut.

[0122] Proteins can be isolated from any part of a plant, including roots, stems, leaves, flowers, or seeds. For example, ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) can be isolated from, for example, alfalfa, carrot tips, corn stalks, sugarcane leaves, soybean leaves, switchgrass, Miscanthus, energy sugarcane, Reed, seaweed, kelp, algae, or mustard.

[0123] Plant-rich proteins can be extensively isolated from one or more plant sources, and are therefore an economical option suitable for any of the compositions provided herein (e.g., muscle, fat, or connective tissue imitations, meat substitute products, or others). Thus, in some embodiments, one or more isolated and purified proteins comprise abundant proteins found in high amounts in plants and capable of extensive isolation and purification. In some embodiments, the abundant protein constitutes about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total protein content of the source plant material. In some embodiments, the abundant protein constitutes about 0.5-10%, about 5-40%, about 10-50%, about 20-60%, or about 30-70% of the total protein content of the source plant material. In some embodiments, the protein-rich composition of the plant material comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the total dry matter weight. In some embodiments, the protein-rich composition of the plant material comprises about 0.5-5%, about 1-10%, about 5-20%, about 10-30%, about 15-40%, or about 20-50% of the total dry matter weight.

[0124] One or more isolated and purified proteins may comprise abundant proteins found in high amounts in plant leaves. In some embodiments, the abundant protein constitutes about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the total protein content of the leaves of the source plant. In some embodiments, the abundant protein constitutes about 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about 30-70% of the total protein content of the leaves of the source plant. In some embodiments, one or more isolated proteins comprise RuBisCo, which is particularly suitable for meat imitations due to its high solubility and amino acid composition close to the optimal ratio of essential amino acids for human nutrition. In a particular embodiment, one or more isolated proteins comprise ribulose-1,5-bisphosphate carboxylase oxygenase activator (RuBisCo activator). In some embodiments, one or more isolated and purified proteins comprise nutrient storage proteins (VSPs).

[0125] One or more isolated proteins may comprise abundant proteins found in high amounts in plant seeds. In some embodiments, the abundant protein constitutes about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% or more of the total protein content of the seed of the source plant. In some embodiments, the abundant protein constitutes about 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about 30-70% or >70% of the total protein content of the seed of the source plant. Non-limiting examples of proteins found in high amounts in plant seeds include seed storage proteins, such as albumin, glycinin, conglycinin, lentinin, globulin, pea globulin, conalbumin, gliadin, glutenin, gluten, barley gliadin, gliadin, bean globulin (protein), protein body, rye gliadin, wheat gluten or corn protein, or oil body proteins, such as oil body protein, oil body calcium protein or oil body sterol protein.

[0126] One or more isolated and purified proteins may include highly soluble proteins, such as dehydrated proteins, hydrophilic proteins, native unfolded proteins (also known as intrinsically disordered proteins), or other late embryo-enriched (LEA) family proteins. LEA proteins have been found in animals, plants, and microorganisms and are thought to act as osmotic protectants and stress-response proteins. See, for example, Battaglia et al. , Plant Physiol. , 148:6-24 (2008). The protein is also thermally stable. The solubility of the LEA protein in solution at a temperature between 90°C and 110°C (e.g., between 95°C and 105°C, 95°C or 100°C) may be at least 1 g / L (e.g., 2, 4, 6, 8, 10, 15, 20, 25, 50, 100, 150, 200 or 250 g / L), wherein the pH of the solution is between 5 and 8 (e.g., pH is 5, 5.5, 6, 6.5, 7, 7.5 or 8), and the sodium chloride content is 0 to 300 mM (e.g., 50, 100, 150, 200, 250 or 300 mM). In some cases, LEA proteins can be separated by heating the protein extract to 90°C to 110°C (e.g., 95°C or 100°C) and concentrating the LEA protein fraction by, for example, ultrafiltration after centrifugation or filtration of insoluble material. In some cases, plasma pH precipitation, trichloroacetic acid precipitation, and / or ammonium sulfate precipitation steps can be performed before or after the heating step to further remove non-LEA proteins. Heating the solution to 90°C–110°C denatures most proteins, thus removing most of the protein from the solution.

[0127] B. protein

[0128] Unbound by theory, it is believed that by isolating and purifying non-animal proteins (e.g., plant proteins), consumer products can achieve greater consistency and control over their properties. In some embodiments, the protein component of the consumer product comprises about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99% of one or more isolated and purified proteins. The isolated and purified proteins may be greater than 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% pure.

[0129] Proteins, after separation and purification, can be isolated from one or more other components not found in animal sources. For example, protein fractions can be isolated from plant isolates. In some cases, the isolated proteins can be purified, in which a particular type of protein is separated from other components found in non-animal sources. Proteins can be separated based on their molecular weight, for example by size exclusion chromatography, ultrafiltration, membrane chromatography, or density centrifugation. In some embodiments, proteins can be separated based on their surface charge, for example by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Proteins can also be separated based on their solubility, for example by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, or solvent extraction. Proteins can also be separated by their affinity for another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography may also include the use of antibodies with a specific binding affinity for the protein of interest, nickel NTA for His-tagged recombinant proteins, lectins bound to the sugar moieties of glycoproteins, or other molecules that specifically bind to the protein of interest.

[0130] Protein isolation allows for the elimination of unwanted material. In some embodiments, the isolated and purified protein is a protein that has been substantially separated from unwanted material (e.g., nucleic acids (e.g., RNA and DNA), lipid membranes, phospholipids, fats, oils, carbohydrates (e.g., starch, cellulose, and dextran), phenolic compounds, polyphenolic compounds, aromatic compounds, or pigments) from the seeds, leaves, stems, or other parts of a plant.

[0131] Separated and purified proteins can also be recombinantly produced using peptide expression techniques (e.g., heterologous expression using bacterial, insect, fungal (e.g., yeast), plant, or mammalian cells). In some cases, standard peptide synthesis techniques (e.g., liquid-phase or solid-phase peptide synthesis) can be used to synthesize proteins. In other cases, cell-free translation techniques can be used to synthesize proteins.

[0132] Proteins incorporated into consumer products can provide nutritional functions. In some cases, proteins are also used to alter the properties of consumer products, such as their flavor, color, odor, and / or texture. For example, meat substitute products may contain protein indicators that indicate the cooking progression from raw to cooked, wherein the meat substitute product is derived from a non-animal source.

[0133] Examples of proteins that can be isolated and purified and used in the consumer products described herein include ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose-1,5-bisphosphate aldolase, phosphofructokinase, triose phosphophosphoryl isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, protease, lipase, amylase, glycoprotein, lectin, mucin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actin, translation elongation factor, histone, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo), and ribulose-1,5-bisphosphate carboxylase oxygenase activator (RuBisCo activator). Albumin, soybean globulin, conglyzed soybean globulin, globulin, pea globulin, conalbumin, gliadin, gluten, gluten, barley gliadin, gliadin, bean globulin (protein), protein body, rye gliadin, extensin, wheat gluten, collagen, corn protein, sorghum gliadin, oat protein, dehydrated protein, hydrophilic protein, late embryo-enriched protein, natural unfolded protein, any seed storage protein, oil body protein, oil body calcium protein, oil body sterol protein or other oil body protein, nutrient storage protein A, nutrient storage protein B, mung bean seed storage 8S globulin, globulin, pea globulin and pea albumin.

[0134] In some embodiments, the isolated and purified proteins may be proteins that interact with lipids and contribute to the stabilization of lipids in a structure, proteins that bind lipids and contribute to the cross-linking of lipid structures, or proteins that bind lipids and contribute to the cross-linking of lipid structures and non-lipid-interacting proteins. Without wishing to be bound by any particular theory, the use of said proteins in the consumer products described herein can improve the integration of lipid and / or fat imitations with other components of meat substitute products, resulting in improved taste and texture of the final product. Non-limiting examples of lipid-interacting plant proteins include proteins in the oil body protein family. Oil body proteins are lipid-interacting proteins found in the oil bodies of plants. Other non-limiting examples of plant proteins that can interact with lipids and stabilize emulsions include seed storage proteins from the Great Northern bean, albumin from peas, globulin from peas, 8S globulin from mung beans, 8S globulin from common beans, prolyl glutenin, and lipid transfer proteins.

[0135] In some embodiments, one or more of the isolated and purified proteins may be iron-carrying proteins, such as heme-containing proteins. As used herein, the term "heme-containing protein" may be used interchangeably with "heme-containing polypeptide" or "heme protein" or "heme polypeptide" and includes any polypeptide that can covalently or non-covalently bind the heme moiety. In some embodiments, the heme-containing polypeptide is a globulin and may include a globulin fold comprising a series of seven to nine α helices. Globulin-type proteins may be of any class (e.g., class I, II, or III) and, in some embodiments, may transport or store oxygen. For example, the heme-containing protein may be a non-symbiotic hemoglobin or leghemoglobin. The heme-containing polypeptide may be a monomer, i.e., a single polypeptide chain, or may be a dimer, trimer, tetramer, and / or higher-order oligomer. Under conditions in which a consumer product containing heme protein is manufactured, stored, disposed of, or prepared for consumption, the oxygenated Fe of the heme protein... 2+ The lifespan of this state can be similar to that of myoglobin or can exceed it by 10%, 20%, 30%, 50%, 100%, or more. Under conditions in which heme-containing consumer products are manufactured, stored, disposed of, or prepared for consumption, the non-oxygenated Fe of the heme-containing protein... 2+ The lifespan of the state can be similar to that of myoglobin or can exceed it by 10%, 20%, 30%, 50%, 100%, or more.

[0136] Non-limiting examples of heme-containing polypeptides may include androglobin, cytoglobin, globin E, globin X, globin Y, hemoglobin, lentiglobin, flavin hemoglobin, Hell's gate globin I, myoglobin, invertebrate hemoglobin, β-hemoglobin, α-hemoglobin, protoglobin, cyanobacterial globin, cytoglobin, histone, neuroglobin, hemochloroglobin, truncated hemoglobin (e.g., HbN or HbO), truncated 2 / 2 globin, hemoglobin 3 (e.g., Glb3), cytochrome, or peroxidase.

[0137] Heme-containing proteins that can be used in the consumer products described herein can be derived from mammals (e.g., farmed animals such as cows, goats, sheep, horses, pigs, bulls, or rabbits), poultry, plants, algae, fungi (e.g., yeast or filamentous fungi), ciliates, or bacteria. For example, heme-containing proteins can be derived from mammals such as farmed animals (e.g., cows, goats, sheep, pigs, bulls, or rabbits); or poultry such as turkeys or chickens. Heme-containing proteins can also be derived from plants such as tobacco (…). Nicotiana tabacum / Nicotiana sylvestris / tobacco); corn ( Corn / corn), Arabidopsis thaliana ( Arabidopsis thaliana ), legumes, such as soybeans ( Glycine max / soybean), chickpeas ( Chickpea / garbanzo / chick pea), peas ( Pea / pea) variants, such as garden peas or sweet peas, common beans (... Common bean Varieties, such as green beans, black beans, navy beans, northern beans or black and white bean, purple and red cowpeas ( Unguiculata vine Varieties (cowpeas), mung beans ( Radiant vine / Mung beans), white lupins ( White lupine (Lupins) or alfalfa ( Medicago sativa (alfalfa); Brassica napus (canola); Wheat (Triticum) Wheat species. (Wheat, including wheat kernels and spelt wheat); upland cotton ( Gossypium hirsutum (Cotton); Rice ( White rice / rice); genus *Zizania* ( Zizania sps (Wild rice); sunflower ( Sunflower (Sunflower); beet ( Beetroot / sugarbeet); pearl millet ( Pennisetum gray / pearl millet); genus *Chenopodium* ( Chenopodium sp. (quinoa); Sesame (genus) Sesamum sp.(Sesame); Flax ( Common flax / flax); or barley ( Barley / barley). Heme-containing proteins can be isolated from fungi, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Pichia pastoris () Shepherd's pie ), rice blast fungus ( Magnaporthe rice Fusarium graminearum ( ) Fusarium grasses ) or Fusarium oxysporum ( Fusarium oxysporum Heme-containing proteins can be isolated from bacteria, such as *Escherichia coli*. Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Bacillus megaterium ( Bacillus megaterium ), Synechocystis ( Synechocystis sp. ), wind-producing bacteria, extreme acidophilic methanogenic bacteria, or thermophilic bacteria (e.g., bacteria that grow at temperatures above 45°C), such as thermophilic bacteria ( ). Thermophilus Heme-containing proteins can be isolated from algae, such as *Chlamydomonas oogatus*. Chlamydomonas eugametos Heme-containing proteins can be isolated from protozoa, such as Paramecium caudatum (…). Paramecium caudate ) or Tetrahymena piriformis ( Tetrahymena pyriformis In some embodiments, bacterial hemoglobin is selected from the group consisting of: *Heterotrophic bacilli*, *Schizophyllum commune*, and *Schizophyllum tansii*. Thermobifida brown Extreme acidophilic methanogenic bacteria (Hellgate), Synechocystis, or Bacillus subtilis. The sequences and structures of many heme-containing proteins are known. See, for example, Reedy et al. Nucleic Acids Research) , 2008, Volume 36, Database Special Issue D307-D313 and on the World Wide Web http: / / hemeprotein.info / heme.php Available heme protein database.

[0138] For example, non-symbiotic hemoglobin can be derived from plants selected from the following groups: soybean, sprouted soybean, alfalfa, golden flax, black bean, black-eyed bean, northern bean, chickpea, mung bean, cowpea, black and white bean, pea pod, dried pea, quinoa, sesame, sunflower, wheat flour, spelt wheat, barley, wild rice, or rice.

[0139] Any of the heme-containing proteins described herein that can be used in the production of consumer products may have at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity with the corresponding wild-type heme-containing protein or fragment thereof containing a heme-binding motif. For example, a heme-containing protein may be compatible with... Figure 1 The amino acid sequences described in the text have at least 70% sequence identity, including non-symbiotic hemoglobins, such as those from mung bean (SEQ ID NO:1), barley (SEQ ID NO:5), maize (SEQ ID NO:13), and japonica rice subspecies (…). Rice sativa subsp. japonica (rice) (SEQ ID NO:14) or Arabidopsis thaliana (SEQ ID NO:15); Hellgate globulin I, for example from extreme acidophilic methanogens (SEQ ID NO:2); yellow heme protein, for example from *Aeromonas hydrophila* (SEQ ID NO:3); legume heme, for example from soybean (SEQ ID NO:4), pea (SEQ ID NO:16), or cowpea (SEQ ID NO:17); heme-dependent peroxidase, for example from *Magnaporum oryzae* (SEQ ID NO:6) or *Fusarium oxysporum* (SEQ ID NO:7); cytochrome c peroxidase from *Fusarium graminearum* (SEQ ID NO:8); from *Chlamydomonas mouseum* (SEQ ID NO:15); Chlamydomonas moewusii ) (SEQ ID NO:9), Tetrahymena piriformis (SEQ ID NO:10, truncated Group I), Paramecium cercis (SEQ ID NO:11, truncated Group I); truncated hemoglobin from Aspergillus niger ( Aspergillus niger Hemoglobin of SEQ ID NO:12; or mammalian myoglobin protein, such as that of European cattle ( Bos taurus (SEQ ID NO:18) Myoglobin, wild boar ( Sus scrofa (SEQ ID NO:19) Myoglobin or horse ( Equus caballus (SEQ ID NO:20) Myoglobin; derived from Nicotiana benthamiana ( Nicotiana benthamiana ) (SEQ ID NO:21), Bacillus subtilis (SEQ ID NO:22), Corynebacterium glutamicum ( Corynebacterium glutamicum ) (SEQ ID NO:23), Synechococcus sp. PCC6803 (SEQ ID NO:24), Synechococcus sp. PCC 7335 (SEQ ID NO:25), Nostoc commune ( Nostoc commune Heme protein of Bacillus megaterium (SEQ ID NO:26) or Bacillus megaterium (SEQ ID NO:27). See also Figure 1 .

[0140] The percentage of similarity between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program, a standalone version of BLASTZ containing BLASTP version 2.0.14. This standalone version of BLASTZ is available from Fish & Richardson's website (e.g., www.fr.com / blast / ) or the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). Instructions on how to use the Bl2seq program can be found in the readme accompanying BLASTZ. Bl2seq uses the BLASTP algorithm to perform the comparison between two amino acid sequences. To compare two amino acid sequences, the Bl2seq options are set as follows: -i is set to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired filename (e.g., C:\output.txt); and all other options remain at their default settings. For example, the following command can be used to generate an output file containing the comparison between two amino acid sequences: C:\Bl2seq -ic:\seq1.txt -jc:\seq2.txt -p blastp -oc:\output.txt. If the two sequences being compared share homology, the specified output file will present those homologous regions as the aligned sequences. If the two sequences being compared do not share homology, the specified output file will not present the aligned sequences. A similar procedure can be followed for nucleic acid sequences, except that blastn is used.

[0141] After alignment, the number of matches is determined by counting the positions of identical amino acid residues in both sequences. The percentage of agreement is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence and then multiplying the result by 100. Note that the percentage of agreement is rounded to one decimal place. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It should also be noted that the length value will always be an integer.

[0142] It should be understood that many nucleic acids can encode polypeptides with specific amino acid sequences. The degeneracy of the genetic code is well known in the art; that is, for many amino acids, there are more than one nucleotide triplet that can act as a codon for the amino acid. For example, using an appropriate codon bias table specific to a particular species, the codons in the coding sequence of a given enzyme can be modified to obtain optimal expression in that species (e.g., bacteria or fungi).

[0143] Heme-containing proteins can be extracted from source materials (e.g., from animal tissues or plant, fungal, algal, or bacterial biomass, or from the supernatant of cultures that secrete proteins) or from combinations of source materials (e.g., multiple plant species). Leghemoglobin can be readily obtained as an unused byproduct of commercial leguminous crops (e.g., soybeans, alfalfa, or peas). In the United States, the amount of leghemoglobin in the roots of these crops exceeds the myoglobin content of all red meat consumed in the US.

[0144] In some embodiments, the extract of heme-containing proteins comprises one or more heme-free proteins derived from source materials (e.g., proteins from other animals, plants, fungi, algae, or bacteria) or from combinations of source materials (e.g., different animals, plants, fungi, algae, or bacteria).

[0145] In some embodiments, the heme-containing protein is isolated and purified from other components of the source material (e.g., other animal, plant, fungal, algal, or bacterial proteins) using the techniques described above. As used herein, the term "isolated and purified" indicates that the heme-containing protein formulation is at least 60% pure, for example, more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure.

[0146] Heme-containing proteins can also be recombinantly produced using peptide expression technologies, such as heterologous expression using bacterial, insect, algal, fungal (e.g., yeast), plant, or mammalian cells. For example, heme-containing proteins can be expressed in *E. coli* cells. Heme-containing proteins can be tagged with heterologous amino acid sequences, such as FLAG, polyhistidines (e.g., hexahistidine, HIS tag), hemagglutinin (HA), glutathione S-transferase (GST), or maltose-binding protein (MBP), to aid in protein purification. In some embodiments, recombinant heme-containing proteins including an HIS tag and a protease (e.g., TEV) site to allow HIS tag cleavage can be expressed in *E. coli* and purified using His-tag affinity chromatography (Talon resin, CloneTech). In some cases, standard peptide synthesis techniques (e.g., liquid-phase or solid-phase peptide synthesis) can be used to synthetically produce heme-containing proteins. In some cases, cell-free translation techniques can be used to synthetically produce heme-containing proteins.

[0147] In some embodiments, the isolated and purified protein is substantially in its native fold and is water-soluble. In some embodiments, more than 50, 60, 70, 80, or 90% of the isolated and purified protein is in its native fold. In some embodiments, more than 50, 60, 70, 80, or 90% of the isolated and purified protein is water-soluble.

[0148] Proteins used in consumer products can be altered (e.g., hydrolyzed, cleaved, cross-linked, denatured, polymerized, extruded, electrospun, spray-dried, lyophilized, derivatized, or chemically modified). For example, proteins can be modified by covalently linking sugars, lipids, cofactors, peptides, or other chemical groups (including phosphate esters, acetates, methyl groups, and other natural or non-natural molecules). For example, the peptide backbone of a protein can be cleaved by exposure to acids or proteases or other means. For example, proteins can be denatured by exposure to heat or cold, changes in pH, exposure to denaturing agents (e.g., detergents, urea, or other dispersants), or mechanical stress (including shearing), i.e., their secondary, tertiary, or quaternary structures can be altered. The arrangement of proteins in solutions, colloids, or solid aggregates can be controlled to affect mechanical properties, including tensile strength, elasticity, deformability, stiffness, or hydrophobicity.

[0149] Proteins can also assemble into fibers, which can form a matrix for the structure of the composition. The three-dimensional matrix of the protein fibers can, for example, contain chemicals that promote the formation of intermolecular disulfide crosslinks (a mixture of glutathione, dithiothreitol (DTT), and β-mercaptoethanol (BME)). In some embodiments, the chemicals are proteins (thioredoxin, glutoredoxin). In some embodiments, the protein is an enzyme (disulfide isomerase). In some embodiments, the fibers are crosslinked by a chemical crosslinking agent with two reactive groups selected from the group consisting of: N-hydroxysuccinimide (NHS) ester, imino ester, aryl fluorine, aldehyde, maleimide, pyridyl dithiol, haloacetyl, aryl azide, diazacyclopropene, carbodiimide, acylhydrazine, and isocyanate.

[0150] In some embodiments, aggregates comprising one or more plant proteins can be formed and used, for example, as binders in meat or other imitations. Aggregation is a process in which a homogeneous solution of a polymer fed into the mixture undergoes phase separation to produce a polymer-rich, dense phase ('aggregate') and a solvent-rich phase (supernatant). Protein-polysaccharide aggregates have been used in the development of biomaterials. See, for example, Boral and Bohidar (2010). Journal of Physical Chemistry, Series B (Journal of Physical Chemistry B . ) Volume 114 (37): 12027-35; and Liu et al., (2010) Journal of Agricultural and Food ChemistryVolume 58: 552-556. The formation of the agglomerates is driven by associational interactions between relatively fed polymers. However, as described herein, agglomerates can be formed using proteins (e.g., plant proteins comprising one or more pea proteins, chickpea proteins, lentil proteins, lupin proteins, other legume proteins, or mixtures thereof). Generally, agglomerates can be formed by acidifying a low ionic strength solution (e.g., a buffer solution of 100 mM or less sodium chloride) to a pH of 3.5 to 5.5 (e.g., pH 4 to 5), said low ionic strength solution comprising one or more isolated and purified plant proteins, such as pea globulin or pea globulin (e.g., comprising a pea globulin moiety conjugated with pea globulin), a combination of both pea globulin and lentinan, or unfractionated pea proteins. Under these conditions, the proteins separate from the solution and the mixture can be centrifuged to cleanly separate the agglomerates. Unlike precipitates, such agglomerates are viscous materials that can be stretched by traction and melted upon heating. The process can be carried out in the presence of oil (up to 70%, such as palm or other oils) to form a creamy material. The binding properties of the coagulant can be adjusted as needed by changing the composition of the solution (the ratio of pea globulin to lentinan, the type and amount of oil used). In some embodiments, one or more gums (such as acacia gum or xanthan gum) can be used to form the coagulant. The coagulant can be used as a binder in beef patty imitations to bind and hold together the imitation fat, muscle, and connective tissue.

[0151] Bound materials with different adhesive and cooking characteristics can be prepared by combining wheat gluten (0-20%) and pea protein fractions (0-50%) in the presence of a plasticizer, such as glycerol (0-30%) or polyethylene glycol. If necessary, leghemoglobin or other heme-containing proteins can be added to the mixture. The material can be incorporated into the beef patty imitation during mixing to remove any clumps.

[0152] In some embodiments, proteins can undergo freeze-alignment to texturize them without compression. The method involves slowly freezing the protein-containing material to allow ice crystals to form. When cooled from one side, the ice crystals preferentially form in a direction perpendicular to the cooling side. After freezing, the ice can be removed from the material in a freeze dryer, leaving a material with several layers. The structure can then be stabilized by heating under pressurized, humid conditions to produce a material suitable for use in meat simulants. The freezing alignment of soy protein has been described by Lugay and Kim (1981) (see Freeze alignment: A novel method for protein texturization. pp. 177–187, Chapter 8 in DW Stanley, ED Murray, and DH Lees, eds., 1981. Utilization of Protein Resources. Westport, CT: Food & Nutrition Press, Inc.). Freezing-aligned proteins can undergo further processing (by immersion in a solution containing beef flavor and / or soy hemoglobin) and be combined with imitations of fat and connective tissue to form beef imitations. The imitation can also be used as a structure in which a cold-set gel (containing, for example, pea protein and myoglobin) or a cross-linked gel (containing, for example, pea protein and leghemoglobin) can be formed around the structure before it is combined with fat and connective tissue.

[0153] C. Lipids

[0154] The consumer products described herein may include lipid components. Lipids may be isolated and / or purified, and may be in the form of triglycerides, monoglycerides, diglycerides, free fatty acids, glycosphingolipids, glycolipids, phospholipids, or oils, or collections of said lipids (e.g., membranes, lecithin, lysophosphatidylcholine, or fat droplets containing a small amount of lipids in a predominantly aqueous phase). In some embodiments, the lipid source is derived from non-animal sources, including oils from genetically engineered bacteria, algae, archaea, or fungi (e.g., oils derived from plants, algae, fungi (e.g., yeast or filamentous fungi), seaweed, bacteria, or archaea). Non-limiting examples of vegetable oils include 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 butter, cocoa butter, wheat germ oil, or rice bran oil; or margarine. Oils can be hydrogenated (e.g., hydrogenated vegetable oils) or non-hydrogenated.

[0155] In some embodiments, lipids may be triglycerides, monoglycerides, diglycerides, free fatty acids, glycosphingolipids, glycolipids, lecithin, lysophosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, or phosphatidylserine; sphingolipids, such as sphingomyelin or ceramides; sterols, such as stigmasterol, sitosterol, campesterol, rapeseed sterol, stigmasterol, ergosterol, yeast sterol, coccosterol, dinoflagellate sterol, lanosterol, cholesterol, or episterol; lipid amides, such as N-palmitoylproline, N-stearoylglycine, N-palmitoylglycine, N-arachidoylglycine, N-palmitoyltaurine, N-arachidoylhistidine, or cannabinoids; Fatty acids, such as palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristone acid, caprylic acid, caprylic acid, caprylic acid, geranilic acid, undecanoic acid, linoleic acid (C18:2), eicosanoic acid (C22:0), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5), docosapentaenoic acid (C22:5), docosahexaenoic acid (C22:6), erucic acid (C22:1), conjugated linoleic acid, linolenic acid (C18:3), oleic acid (C18:1), trans-oleic acid (trans isomer of oleic acid), trans-isooleic acid (C18:1 trans11), or conjugated oleic acid; or esters of said fatty acids, including monoacylglycerol esters, diacylglycerol esters, and triacylglycerol esters of said fatty acids.

[0156] Lipids may comprise phospholipids, lipid amides, sterols, or neutral lipids. Phospholipids may comprise multiple amphiphilic molecules containing fatty acids (e.g., see above), glycerol, and a polar group. In some embodiments, the polar group is, for example, choline, ethanolamine, serine, phosphate esters, glycerol-3-phosphate esters, inositol, or inositol phosphate esters. In some embodiments, lipids are, for example, sphingolipids, ceramides, sphingomyelin, cerebrosides, gangliosides, ether lipids, phospholipid acetals, or PEGylated lipids.

[0157] In some embodiments, the lipids used in consumer products are cream portions derived from seeds, nuts, and legumes, including but not limited to sunflower seeds, safflower seeds, sesame seeds, rapeseed, almonds, macadamia nuts, grapefruit, lemon, orange, watermelon, pumpkin, cocoa, coconut, mango, winter squash, cashews, Brazil nuts, chestnuts, hazelnuts, peanuts, pecans, walnuts, and pistachios. As used herein, the term "cream portion" can refer to a separated emulsion comprising lipids, proteins, and water.

[0158] To obtain the cream portion from seeds, nuts, or legumes, one or more of the following steps may be performed. The seeds, nuts, or legumes may be blended for 1 to 30 minutes. For example, the seeds, nuts, or legumes may be blended by gradually increasing the speed to the maximum speed over 4 minutes, and then blending at the maximum speed for 1 minute. The seeds, nuts, or legumes may be blended in water or a solution containing all or some of the following: EDTA (0-0.1 M), NaCl (0-1 M), KCl (0-1 M), NaSO4 (0-0.2 M), potassium phosphate (0-1 M), sodium citrate (0-1 M), sodium carbonate (0-1 M), and / or sucrose (0-50%), at a pH of 3 to 11, to obtain a slurry. The slurry may be heated to 20°C to 50°C and centrifuged to obtain the cream portion (the top layer, also known as "cream"). Further purification of the cream fraction can be achieved by washing the cream fraction with a 0.1M to 2M urea solution, followed by centrifugation to separate the cream fraction. Alternatively, the residual liquid from a solution containing proteins in water (referred to as the "defatted" layer) can be used.

[0159] The “cream” can be used as is or undergo further purification steps. For example, washing and heating can remove color and flavor molecules (e.g., unwanted molecules) or unwanted particulate matter to improve texture and creaminess. Specifically, washing with a high pH buffer (pH>9) can remove bitter compounds and improve texture, washing with urea can remove storage proteins, washing at a pH below 9 followed by washing at a pH above 9 can remove unwanted color molecules, and / or washing with salt can reduce taste compounds. Heating can increase the removal of particulate matter, color, and flavor compounds. For example, the cream portion can be heated for 0-24 hours at a temperature ranging from 25°C to 80°C. In some embodiments, the resulting cream portion contains seed storage proteins. In some embodiments, the seed storage proteins are substantially removed from the resulting cream portion.

[0160] D. Fiber

[0161] Fibers can be separated and / or purified for inclusion in the consumer products described herein. Fibers can refer to non-starch polysaccharides, such as arabinoyl xylan, cellulose, and other plant components, such as resistant starch, resistant dextrin, inulin, lignin, waxes, chitin, pectin, beta-glucan, and oligosaccharides from any plant source.

[0162] Fibers can refer to proteins that have undergone extrusion and solution spinning, as described herein.

[0163] E. Sugar

[0164] In some embodiments, the consumer product may also contain sugars. For example, the consumer product may contain: monosaccharides, including but not limited to glucose (dextrose), fructose (levose), galactose, mannose, arabinose, xylose (D- or L-xylose), and ribose; disaccharides, including but not limited to sucrose, lactose, melibiose, trehalose, cellobiose, or maltose; sugar alcohols, such as arabinitol, mannitol, galactitol, or sorbitol; sugar acids, such as galacturonate, glucuronate, or glucuronide; oligosaccharides and polysaccharides, such as dextran; starch, such as corn starch or potato starch; pectin, such as apple pectin or orange pectin; raffinose, stachyose, or dextran; plant cell wall degradation products, such as salicin; and / or sugar derivatives, such as N-acetylglucosamine.

[0165] F. Gel formation

[0166] The components of the composition can be formed as a gel. In some embodiments, the gel comprises a protein derived from a non-animal source (e.g., plant or other non-animal sources, such as genetically modified yeast or bacteria). The gel can be formed using a variety of methods. Protein concentration, enzyme concentration, pH, and / or process temperature will affect the rate of gel formation and the quality of the final tissue replica.

[0167] Gels can be stabilized entirely through physical cross-linking between components. In some embodiments, gels can be produced by thermal / cold cycling, in which case the gel is stabilized by physical interactions (entanglement, hydrophobic interactions) between protein molecules. For example, a gel can be formed by heating a protein solution to a temperature of at least 40°C, 45°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C, and then cooling it to room temperature or to a temperature below 40°C.

[0168] In some embodiments, the gel can be formed by subjecting a composition containing proteins and any other components (e.g., lipids) to high-pressure processing.

[0169] In some embodiments, the gel can be produced by adjusting the pH of the solution. For example, the pH of a concentrated protein solution can be adjusted to a near-equipotential pH close to that of the major protein components by adding hydrochloric acid or other acids, or sodium hydroxide or other bases.

[0170] In some embodiments, the gel can be produced by immersing protein powder in a solution. For example, the protein powder can be immersed in a concentrated sodium hydroxide solution of at least 1%, 5%, 10%, 20% (wt / v), or more than 20%. In other examples, the protein powder can be immersed in a mixed water / ethanol solution.

[0171] In some embodiments, a cold-set gel is formed to avoid denaturation or decomposition of any heat-labile components (e.g., iron oxidation in the heme moiety or the generation of undesirable flavors). For general methods of forming cold-set gels, see Ju and Kilara A. (1998). Journal of Food Science , Vol. 63(2): 288-292; and Maltais et al., (2005) Journal of Food Science(Volume 70 (1): C67-C73). Generally, cold-set gels are formed by first thermally denaturing a protein solution below its minimum gelling concentration (depending on pH and the type of protein, typically <8% (w / v) for globular plant proteins (e.g., pea protein) at pH 6-9). The protein solution can be heated to a temperature above the denaturation temperature of the protein under conditions in which the protein does not precipitate from the solution (e.g., 0-500 mM sodium chloride, pH 6-9). The solution can be cooled back to room temperature or below, and any thermally unstable components (e.g., heme-containing proteins and / or oils) can be mixed in when the solution is sufficiently cold but before gelling. Gelation can be induced by adding sodium chloride or calcium chloride (e.g., 5 to 100 mM), and the solution can be incubated at room temperature or below to allow gel formation (typically several minutes to several hours). The resulting gel can be used as is in meat imitations or further processed (e.g., stabilized) before being incorporated into meat imitations.

[0172] In some embodiments, the gel may contain a cross-linking enzyme or be at least partially produced by (e.g., stabilized by) a cross-linking enzyme. The cross-linking enzyme may be, for example, transglutaminase, tyrosinase, lipoxygenase, protein disulfide reductase, protein disulfide isomerase, thiol oxidase, peroxidase, hexose oxidase, lysine oxidase, or amine oxidase.

[0173] In some cases, the gel may contain chemicals that promote the formation of intermolecular disulfide bonds between proteins. In some embodiments, the chemicals are proteins (e.g., thioredoxin, glutoredoxin). In some embodiments, the proteins are enzymes (disulfide isomerases).

[0174] The gel can be stabilized by chemically crosslinking two reactive groups selected from the group consisting of N-hydroxysuccinimide (NHS) ester, imino ester, aryl fluorine, aldehyde, maleimide, pyridyl dithiol, haloacetyl, aryl azide, diazacyclopropene, carbodiimide, acyl hydrazine and isocyanate.

[0175] In some embodiments, the gel can be stabilized by adding starch and gum.

[0176] In some embodiments, one or more of these methods are used in combination. For example, transglutaminase cross-linked gels can be further stabilized by heat / cold treatment.

[0177] G. Muscle Imitation

[0178] Many meat products contain a high proportion of skeletal muscle. Therefore, this invention provides a composition that can be derived from a non-animal source, mimicking or approximating key characteristics of animal skeletal muscle. Compositions derived from a non-animal source that mimic or approximate animal skeletal muscle can be used as components of consumer products (e.g., meat imitations). Such compositions will be referred to herein as "muscle imitations." In some embodiments, muscle imitations and / or meat substitute products containing muscle imitations are partially derived from animal sources. In some embodiments, muscle imitations and / or meat substitute products containing muscle imitations are entirely derived from non-animal sources.

[0179] Muscle tissue imitations may contain protein contents, which include one or more isolated and purified proteins, wherein the muscle tissue imitation approximates the taste, texture, or color of equivalent muscle tissue derived from animal sources.

[0180] Many meat products contain a high proportion of striped skeletal muscle, where individual muscle fibers are organized primarily in an anisotropic manner. Therefore, in some embodiments, muscle replicas contain fibers that are organized to some extent in anisotropic ways. The fibers may contain a protein component. In some embodiments, the fibers contain about 1% (wt / wt), about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% (wt / wt), or more than 99% of a protein component.

[0181] The connective tissue component of skeletal muscle substantially contributes to the texture, mouthfeel, and cooking properties of meat products. Connective tissue is composed of protein (collagen, elastin) fibers ranging from 0.1 to 20 micrometers. In some embodiments, a mixture of fibers with diameters of <1-10 micrometers and 10-300 micrometers is produced to mimic the fibrous composition of animal connective tissue. In some embodiments, the 3D matrix of the fibers is stabilized by protein cross-linking to mimic the tensile strength of animal connective tissue. In some embodiments, the 3D matrix of the fibers contains isolated and purified cross-linking enzymes. Cross-linking enzymes may be, for example, transglutaminase, tyrosinase, lipoxygenase, protein disulfide reductase, protein disulfide isomerase, thiol oxidase, peroxidase, hexose oxidase, lysine oxidase, or amine oxidase.

[0182] Some proteins (such as 8S globulin from mung bean seeds or albumin or globulin fractions from pea seeds) have advantageous properties for constructing meat imitations due to their ability to form gels with a texture similar to animal muscle or adipose tissue. See also the proteins identified in Sections III A and B. Proteins can be artificially engineered to mimic the physical properties of animal muscle tissue.

[0183] In some embodiments, one or more isolated and purified proteins constitute about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more by weight of the protein component of the meat imitation. In some embodiments, one or more isolated and purified proteins constitute about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more by weight of the protein content of the consumer product.

[0184] The skeletal muscle of animals (such as beef cattle) typically contains a considerable amount of glycogen, which can constitute about 1% of the muscle tissue mass at slaughter. After slaughter, a portion of this glycogen continues to be metabolized, yielding byproducts including lactic acid, which contributes to a decrease in the pH of the muscle tissue—a desirable quality in meat. Glycogen is a branched polymer of glucose linked together by α (1->4) glycosidic bonds in a straight chain, with the branching points containing α (1->6) glycosidic bonds. Starch from plants, specifically amylopectin, is also a branched polymer of glucose linked together by α (1->4) glycosidic bonds in a straight chain, with the branching points containing α (1->6) glycosidic bonds, and can therefore be used as an analogue of glycogen in the construction of meat imitations. Thus, in some embodiments, muscle or meat imitations comprise starch or pectin.

[0185] Other components of animal muscle tissue include sodium, potassium, calcium, magnesium and other metal ions, lactic acid and other organic acids, free amino acids, peptides, nucleotides, and sulfur-containing compounds. Therefore, in some embodiments, the muscle replica may include sodium, potassium, calcium, magnesium, other metal ions (e.g., iron, zinc, copper, nickel, lithium) or selenium, lactic acid and other organic acids (e.g., fatty acids), free amino acids, peptides, nucleotides, and sulfur-containing compounds (glutathione, β-mercaptoethanol, or dithiothreitol). In some embodiments, the concentrations of sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides, and / or sulfur-containing compounds in the muscle replica or consumer product are within 10% of the concentrations found in the imitated muscle or meat.

[0186] This invention also provides a method for producing muscle replicas. In some embodiments, the method includes shaping a composition into asymmetric fibers, which are then incorporated into a consumer product. In some embodiments, these fibers mimic muscle fibers. In some embodiments, the fibers are spun fibers. In other embodiments, the fibers are extruded fibers. Thus, this invention provides a method for producing asymmetric or spun protein fibers. In some embodiments, the fibers are formed by extruding protein components through an extruder. Extrusion methods are well known in the art and described, for example, in U.S. Patent Nos. 6,379,738, 3,693,533, and 20120093994, which are incorporated herein by reference. These methods can be applied to the production of the compositions provided herein.

[0187] Extrusion can be performed using, for example, a Leistritz Nano-16 twin-screw co-rotation extruder (American Leistritz Extruder Corp., USA, Sommerville, New Jersey). Effective cooling of the bobbin section helps limit protein denaturation. Effective cooling of the die section helps limit product expansion and excessive moisture loss. Protein feed and liquid are added separately: protein is fed through a volumetric piston feeder or a continuous auger feeder, and liquid can be added to the bobbin via a high-pressure liquid injection system. Die nozzles with various inner diameters and channel lengths allow for precise control of extrudate pressure, cooling rate, and product expansion. In some examples, extrusion parameters are: screw speed 100-200 rpm, die diameter 3 mm, die length 15 cm, product temperature at die end 50°C, feed rate 2 g / min, and water flow rate 3 g / min. The product temperature at the die is measured during extrusion using thermocouples.

[0188] Fibers can be produced by preparing a high-viscosity protein "dope" by adding sodium hydroxide to a concentrated protein solution or precipitated protein, and by forcing the solution through a small steel capillary (in some examples, a 27-bore hypodermic needle) into a coagulation bath using a piston-type device (in some examples, a syringe with a pump). In some examples, the bath is filled with a concentrated acid solution (e.g., 3 M hydrochloric acid). In some examples, the bath is filled with a buffer solution with a pH approximately equal to the plasma point of the protein. The coagulated protein solution jet forms fibers, which are collected at the bottom of the bath.

[0189] Spinning into fiber bundles can be produced by forcing a protein “spinning solution” through a spinneret with many small holes. In some instances, the spinneret is 1 cm². 2A stainless steel plate with approximately 25,000 holes, each hole being approximately 200 micrometers in diameter. In some embodiments, the muscle tissue replica is produced by immersing a 3D matrix of fibers (connective tissue replica) in a protein solution, resulting in a protein gel containing the 3D matrix of fibers.

[0190] H. Fat imitation

[0191] Animal fat is essential to the experience of consuming cooked meat and to some of its nutritional value. Therefore, this invention provides compositions derived from non-animal sources that mimic key characteristics of animal fat, including texture and / or flavor, by using components that mimic the chemical composition and physical properties of, for example, ground beef. In another aspect, this invention provides a meat substitute product comprising a composition derived from a non-animal source that mimics animal fat. Such compositions will be labeled herein as “adipose replicas”. In some embodiments, the fat replica and / or meat substitute product comprising the fat replica is partially derived from an animal source. Consumer products may also include fat replicas that mimic key characteristics of non-animal fat, including texture, flavor, firmness, fat release percentage, and / or fat release temperature. The fat content of the consumer product may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or 95% fat.

[0192] Ground beef is typically prepared by mixing lean beef with fat (adipose / fat) cut from rib cuts, with a fat tissue addition of 16-30% (Cox 1993). Without fat, meat processed by a grinder is tough, crisp, and dries quickly. Adding fat to lean beef allows the released fat during cooking to provide a liquid surface to aid in cooking, and it is primarily a product of fatty acids, contributing to the key beef flavor. Engineered fat tissue imitations, which play an equally crucial role in the texture and flavor of plant-based ground beef, significantly enhance both.

[0193] The fat imitations described here offer significant health benefits superior to beef fat tissue because their fatty acid composition can be controlled to reduce the amount of saturated fat. Furthermore, plant-based fat imitations are cholesterol-free. Plant-based fat imitations can contain a lower percentage of total fat while still releasing or retaining the same amount of fat to achieve the desired culinary properties, flavor, and texture.

[0194] As described herein, fat imitations can be produced comprising emulsions of plant-derived lipids and one or more isolated and purified proteins, wherein the composition (e.g., fatty acid composition), culinary characteristics (e.g., fat release temperature or fat release percentage), and physical properties (e.g., firmness) can be controlled to make the plant-based composition mimic animal fats. Fat tissue imitations include (1) vegetable oils containing triglycerides of fatty acids; (2) one or more isolated and purified proteins (e.g., plant proteins) from non-animal sources; and (3) phospholipids, such as lecithin. Proteins can be plant or microbial proteins as described above (e.g., RuBisCo, oil body proteins, albumins, globulins, or other seed storage proteins). See also the proteins described in Sections III A and B. Vegetable oils can be any of the oils described herein. See, for example, Section III C.

[0195] Fat imitations can be gel emulsions. In some embodiments, the gel is a soft, elastic gel containing proteins and optionally carbohydrates. The gel emulsion may contain a protein solution comprising a variety of proteins (e.g., 1-5 or 1-3 isolated and purified proteins), wherein the protein solution comprises 1-30% of the emulsion volume. The gel emulsion may contain fat droplets, wherein the fat droplets comprise 70-99% of the emulsion volume. The gel emulsion may contain isolated and purified cross-linking enzymes, wherein the cross-linking enzyme comprises 0.0005% to 0.5% of the emulsion volume by weight, 0.5-2.5% of the emulsion volume by weight, or less than 0.001% of the emulsion volume by weight. Emulsions of fat droplets in protein solutions can be stabilized by forming a gel using cross-linking enzymes (e.g., transglutaminase), by gelling proteins through heating and cooling the protein solution, by forming a cold-solid gel, by forming aggregates, or by a combination of these techniques as described in Section C on aggregates and Section F on gel formation.

[0196] In some embodiments, the fat imitation comprises a cross-linking enzyme that catalyzes a reaction that produces covalent cross-links between proteins. The cross-linking enzyme can be used to produce or stabilize the desired structure and texture of the fat tissue imitation to mimic the desired texture of an equivalent animal fat. In some embodiments, the cross-linking enzyme is isolated and purified from a non-animal source, examples and embodiments of which are described herein. In some embodiments, the fat imitation comprises at least 0.0001%, at least 0.001%, at least 0.01%, at least 0.1%, or at least 1% (wt / vol) of the cross-linking enzyme. The cross-linking enzyme may be selected from, for example, transglutaminase, tyrosinase, lipoxygenase, protein disulfide reductase, protein disulfide isomerase, thiol oxidase, peroxidase, hexose oxidase, lysine oxidase, and amine oxidase. In some embodiments, the cross-linking enzyme is transglutaminase, lysine oxidase (e.g., Pichia pastoris lysine oxidase), or other amine oxidase.

[0197] Fat imitations may comprise a gel in which fat droplets are suspended. The fat droplets used in some embodiments of the invention may be derived from a variety of sources. In some embodiments, the source is non-animal (e.g., plant-based). See, for example, the examples provided in Section III C. In some embodiments, the fat droplets are derived from animal products (e.g., butter, cream, lard, and / or fat). In some embodiments, the fat droplets are derived from fruit pulp or seed oil. In other embodiments, the source may be algae, yeast, or oil-producing yeast (e.g., Yersinia lipolytica). Yarrowia lipolytica ()) or mold. For example, in one embodiment, a fungus derived from *Morchella asiatica* ( Mortierella isabellina The fat droplets contain triglycerides. In some embodiments, the fat droplets contain synthetic or partially synthetic lipids.

[0198] In some embodiments, the fat droplets are stabilized by adding surfactants (including but not limited to phospholipids, lecithin, and lipid membranes). The lipid membranes may be derived from algae, fungi, or plants. In some embodiments, the surfactant constitutes less than 5% of the fat imitation. In some instances, the diameter of the fat droplets can range from 100 nm to 150 µm. The diameter of these stabilized droplets can be obtained by homogenization, high-pressure homogenization, extrusion, or ultrasonic treatment.

[0199] In some embodiments, vegetable oils are modified to resemble animal fats. Vegetable oils can be modified with seasonings or other agents, such as heme proteins, amino acids, organic acids, lipids, alcohols, aldehydes, ketones, lactones, furans, sugars, or other flavor precursors, to mimic the taste and smell of meat during and after cooking. Therefore, some aspects of the present invention relate to methods for testing the qualitative similarity between the cooking properties of animal fats and the cooking properties of vegetable oils in consumer products.

[0200] In some embodiments, additional polysaccharides (including flaxseed polysaccharide and xanthan gum) may be added to the fat imitation.

[0201] The production of plant-based fat imitations requires the stabilization of oil-in-water emulsions. Typically, animal adipose tissue contains approximately 95% fat and is stabilized by a phospholipid bilayer and bound proteins. The fat imitations described herein can be produced in some cases with up to 95% fat, in many conditions with 80% fat, or with even lower amounts (e.g., 50% or less) of fat, while mimicking the properties of animal fat. The achievement of a high fat percentage is controlled through emulsion stabilization.

[0202] Composition (e.g., fatty acid composition), cooking characteristics (e.g., fat release temperature or fat release percentage), and physical properties (e.g., firmness) can be manipulated by controlling the type and amount of fat, the amount of protein, the type and amount of lecithin, the presence of additives, and the gelling method.

[0203] In some embodiments, the protein component constitutes about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or more of the fat imitation by dry weight or total weight. In some embodiments, the protein component constitutes about 0.1-5%, about 0.5-10%, or more of the fat imitation by dry weight or total weight. In some embodiments, the protein component is 0.5 to 3.5% or 1 to 3% of the fat imitation by dry weight or total weight. In some embodiments, the protein component constitutes a solution containing one or more isolated and purified proteins. The type of protein can affect the stability of the emulsion; RuBisCo and pea albumin allow the fat imitation to be made with more than 90% fat. The addition of polysaccharides (including flaxseed and xanthan gum) helps emulsify the mixture, thereby increasing the fat content.

[0204] The type and amount of fat can be controlled by selecting the source of the fat and its lipid composition. Generally, oils with a higher content of saturated fatty acids emulsify better at lower protein concentrations, while oils with more unsaturated fatty acids require higher protein concentrations for emulsification. Protein is needed to stabilize the emulsion, and increasing the protein content increases stability. If too little protein is added to emulsify a sufficient amount of fat, the mixture will separate into layers.

[0205] Lecithin is also a modifier of emulsions and can stabilize or disrupt them depending on the amount of protein present and the type of oil used. For example, lecithin may disrupt the protein / fat matrix, resulting in a less stable emulsion, but it can be added in low amounts to adjust other physical properties. Emulsions made from oils with a high unsaturated fat content can be stabilized with a high lecithin content (1%) to prevent coagulation. Emulsions made from oils with a high saturated fat content can coagulate at a high lecithin content (1%), but remain very soft.

[0206] As described in this article, fat imitations that can vary in hardness from very soft to very hard can be prepared. The composition and amount of fat control the hardness of the imitation. Harder oils containing more long-chain saturated fats produce harder gels. Oils that produce softer gels typically contain more unsaturated fatty acids or short-chain saturated fatty acids. Generally, as long as the emulsion is held and does not separate, the hardness of the gel increases with the percentage of total fat. The amount of protein also contributes to the hardness of the imitation. Generally, increasing the protein concentration increases the hardness of the imitation. The amount of lecithin is a modulator of the hardness of the imitation. When the gel is formed with a high percentage of protein (3%), a higher amount of lecithin (1%) is much softer than a lower amount of lecithin (0.05%). When the protein content is reduced (1.8%), all gels are softer, and if the emulsion is held, the difference in hardness between low (0.05%) and high (1%) lecithin contents is minimal.

[0207] Adding polysaccharides (including but not limited to xanthan gum and flaxseed paste) to imitations can increase the firmness of fat imitation gels.

[0208] When a fatty imitation is cooked, fat leaks from the structured imitation as it cooks. There is usually fat retained in the cooked product; it is important to achieve a balance between the fat released to aid cooking and the fat retained for texture and flavor. The percentage of fat released (per total fat) can be determined by measuring the amount of fat released during cooking to completion. The percentage of fat released is reported as weight of fat released / total fat in the imitation. For example, the percentage of fat released from the fatty tissue imitation described herein during cooking could be 0 to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. Fatty imitations typically release 0–90% fat under standard cooking conditions. In contrast, beef fatty tissue typically releases 40–55% fat under equivalent conditions.

[0209] Although vegetable oils have a fixed melting temperature, fat imitations can release fat over a wide temperature range. The fat release temperature is the temperature at which fat is visibly released from the imitation at the cooking surface. As described herein, the fat release temperature of fat imitations can be adjusted based on the type and amount of fat, the amount of protein, the type and amount of lecithin, the presence of additives, the emulsification method, and the gelling method. The resulting fat release temperatures of fat imitations can range from 23°C to 33°C, 34°C to 44°C, 45°C to 55°C, 56°C to 66°C, 67°C to 77°C, 78°C to 88°C, 89°C to 99°C, 100°C to 110°C, 111°C to 121°C, 122°C to 132°C, 133°C to 143°C, 144°C to 154°C, 155°C to 165°C, and 166°C to... The temperatures range from 167℃ to 169℃, 170℃ to 180℃, 181℃ to 191℃, 192℃ to 202℃, 203℃ to 213℃, 214℃ to 224℃, 225℃ to 235℃, 236℃ to 246℃, 247℃ to 257℃, 258℃ to 268℃, 269℃ to 279℃, 280℃ to 290℃, or 291℃ to 301℃. Beef fat releases fat at 100-150℃, as measured.

[0210] Emulsification is also a factor that controls the temperature at which fat is released: once fat is incorporated into the imitation along with protein or protein and lecithin, the temperature at which fat is released increases significantly to a level higher than the temperature at which fat melts alone.

[0211] Fatty acid composition is also a factor in fat release temperature and fat release percentage. Vegetable oils containing a higher proportion of unsaturated fatty acids have low melting temperatures and many are liquid at room temperature. Vegetable oils containing a higher proportion of saturated fatty acids have higher melting temperatures and are solid at room temperature. Imitations with a higher content of unsaturated fats have higher fat release temperatures than the same imitations made with more saturated fatty acids. A gel made from 75% oil with a high amount of unsaturated fatty acids, high protein content (3%), and minimal lecithin content (0.05%) (where the mixture is emulsified by a hand-held homogenizer and gelled using a heat-cooling method) can be heated to 200°C with very little or no fat release. Imitations containing oils with more long-chain saturated fats typically have more fat release at high protein content, but release less total fat percentage compared to imitations containing oils with more short-chain fats and a low protein percentage. A gel made from oils with a higher proportion of short-chain saturated fatty acids, high protein content (3%), and minimal lecithin content (0.05%) can be heated to 200°C with very little fat release.

[0212] The percentage of fat released during cooking of fat imitations varies with the amount of protein and lecithin. Typically, fat imitations contain 1-3% protein by weight. Increasing the protein content leads to a higher fat release temperature and a lower percentage of released fat. Increasing the lecithin content to 1% can lower the fat release temperature to 60-115°C and increase the percentage of released fat (e.g., 25-30%). The source or composition of the lecithin used can modulate the amount of fat released and the temperature threshold for fat release. Not bound by a specific mechanism, lecithin is believed to destabilize emulsions by disrupting protein-protein interactions. In one embodiment, at a high protein concentration of 3%, increasing the lecithin content to 1% can lower the fat release temperature to 55-60°C and increase the percentage of leaked fat to 60-65%.

[0213] The method of emulsion preparation is also a factor in determining the amount of fat released. Emulsification forms a homogeneous mixture of fat retained within a protein matrix and lecithin. Emulsification methods can include high-pressure homogenization, ultrasonic treatment, or manual homogenization. Alternative methods produce characteristic differences in the size of oil droplets in the emulsion, which affects the stability of the resulting emulsion and the maximum fat concentration that can form a stable emulsion.

[0214] The method of gelling the imitation is also a factor in determining the amount of fat released. While fat imitations can form without gelation, gelation produces a firmer and more stable emulsion. Gelation methods are described above and can include, for example, the addition of a cross-linking enzyme (e.g., transglutaminase (TG)) or subjecting the emulsion to a heating / cooling cycle. For instance, treatment with TG or the heating / cooling method can, as described above, convert the emulsion into a gel. Furthermore, gelled emulsions formed by cross-linking catalyzed by TG typically release fat at higher temperatures than emulsions gelled by heating / cooling techniques. Gems formed by cross-linking with TG also typically release less fat than gels formed by heating-cooling techniques.

[0215] In some embodiments, fat imitations can be prepared having a protein content of <1.5% and a minimum lecithin content (0.05%), and a fat release temperature of 45-65°C and a high fat release rate (e.g., 70-90%). These gels exhibit a high percentage of fat release.

[0216] In some embodiments, a fat imitation can be prepared having a low protein content (<1.5%) and a high lecithin content (>1%), a low fat release temperature (e.g., 30-50°C, e.g., 30 to 45°C), and a moderate fat leakage percentage (45-65%). Therefore, lecithin can play a role in stabilizing emulsions in gels formed from oils containing short-chain or long-chain fatty acids at low protein concentrations.

[0217] In some embodiments, >2% rubisco or pea albumin can be used to produce a fat imitation with more than 70% fat. In some embodiments, a gel formed with >3% isolated and purified protein can produce a fat imitation with more than 70% fat.

[0218] In some embodiments, fat imitations made from oils having a high proportion of long-chain saturated fatty acids, 3% protein content, and a minimum lecithin content (0.05%) can release fat at a temperature similar to that of beef fat (50-100°C) and can release low to moderate amounts of fat (15-45%).

[0219] In some embodiments, fat imitations having a higher protein concentration (>3%) and a lecithin content (>1%) can have a fat release temperature of 50-70°C and a higher fat release rate (50-80%). At high protein and low lecithin concentrations, gels with higher saturated fatty acid content typically leak about 10% more fat than corresponding gels formed with unsaturated fats.

[0220] In some embodiments, protease treatment of the protein components prior to gel formation can lead to increased release of the protein.

[0221] In some embodiments, adipose tissue simulants stabilized by cross-linking enzymes release more fat than adipose tissue simulants stabilized by heat / cooling protein denaturation. In one embodiment, adipose tissue matrix comprising a mixture of mung bean 8S protein and canola or coconut oil, cocoa butter, olive oil, and palm oil retains more bulk when formed after heat / cooling denaturation than when formed by enzymatic cross-linking. In one embodiment, adipose tissue matrix formed by heat / cooling denaturation of a pre-formed protein-oil emulsion containing Rubisco and cocoa butter has a higher melting temperature than a similarly composed adipose tissue simulant stabilized by cross-linking enzymes.

[0222] In some embodiments, an adipose tissue replica constructed from 1.4% wt / v mung bean 8S protein, 90% wt / v canola oil, and 0.45% wt / v soybean lecithin can be homogenized in the presence of variable concentrations of sunflower oil body protein. The concentration of the oil body protein can range from 1:10 to 1:10. 6 Molar ratio of oleoprotein to triglycerides. It was observed that bulk retention after cooking increased with increasing oleoprotein concentration in the adipose tissue imitation.

[0223] The firmness of adipose tissue replicas constructed in the form of stabilized protein-fat emulsions can be adjusted by varying the protein concentration within the adipose tissue replica matrix. For example, a series of adipose tissue replicas formed with varying concentrations of Rubisco and 70-80% v / v sunflower oil exhibit different firmnesses. Adipose tissue replicas with 0% and 0.18% (wt / vol) Rubisco are extremely soft, while those formed with 1.6% (wt / vol) Rubisco are soft, and those formed with 1.9% (wt / vol) Rubisco have moderate firmness.

[0224] In one embodiment, the firmness of the fat imitation formed by stabilizing a protein oil emulsion can be adjusted by changing the amount of protein in the fat imitation. In one embodiment, a fat tissue imitation made of Rubisco and 70% sunflower oil is softer at a lower concentration (e.g., 1%) of Rubisco than at a higher concentration (e.g., 3%) of Rubisco.

[0225] In another aspect, the present invention provides a method for producing fat imitations. The fat can be separated and homogenized. For example, an organic solvent mixture can be used to help dissolve the lipids in a gel and then remove them to provide the final gel. At this point, the lipids can be frozen, lyophilized, or stored. Therefore, in one aspect, the present invention provides a method for separating and storing selected lipids to have characteristics similar to animal fat. The lipid membrane or lipid cake can then be hydrated. Hydration can be achieved using stirring or temperature changes. Hydration can be carried out from a precursor solution to a gel. After hydration, the lipid suspension can be sonicated, homogenized, high-pressure homogenized, or extruded to further alter the properties of the lipids in solution.

[0226] In some embodiments, the fat imitation is assembled into a tissue that approximates the adipose tissue in meat. In some embodiments, some or all components of the fat imitation are suspended in a gel (e.g., a protein gel). In other embodiments, the gel may be a hydrogel, an organic gel, or a dry gel. In some embodiments, the gel may be thickened to a desired consistency using polysaccharide- or protein-based reagents. For example, fecula, arrowroot powder, corn starch, katakuri starch, potato starch, sago, tapioca starch, alginate, guar gum, locust bean gum, xanthan gum, collagen, protein, red algae gum, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, arcadia gum, konjac, starch, pectin, amylopectin, or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, or fungi may be used alone or in combination to thicken the gel to form the architecture or structure of a consumer product.

[0227] In some embodiments, the tensile strength of the fat imitation mimics that of adipose tissue. The tensile strength of the gel emulsion can be increased by incorporating fibers. The fibers can be derived from non-animal sources, including but not limited to watermelon, jackfruit, pumpkin, coconut, green algae, corn, and / or cotton. In some embodiments, the fibers are derived from the self-polymerization of proteins (e.g., oleoproteins and prolysin). In some embodiments, the fibers are derived from electrospun or extruded proteins. The fibers can form a three-dimensional network or chain, wherein the diameter of each fiber can be less than 1 mm.

[0228] Fat imitations can be emulsions comprising solutions of one or more proteins and one or more fats suspended in droplets. Slowly adding the oil phase to the aqueous phase can provide a more stable emulsion and prevent accidental emulsion failure. In some cases, the addition of lecithin can destabilize protein-stabilized emulsions, leading to increased fat leakage when the imitation is cooked. In some embodiments, the emulsion is stabilized into a gel by one or more cross-linking enzymes. In some embodiments, the emulsion is stabilized by a matrix formed by inducing proteins into a gel using a heat-cooling technique or a cold-solid gelation technique. Heating protein-stabilized emulsions can cause protein denaturation, resulting in increased firmness of the fat imitation. Heating to sufficient temperatures can also reduce the viability of the natural microbiome by at least 100-fold. In some embodiments, the emulsion is stabilized by a gelled protein matrix formed by a combination of one or more protein cross-linking enzymes and a heat / cooling technique or a cold-solid gelation technique. After the emulsion has cooled sufficiently, but before gelation is complete, one or more optional ingredients may be added, such as heme-containing proteins (e.g., up to about 0.4%, such as 0.15, 0.2, 0.25, 0.3, or 0.4%), to give the fat a more natural pink color; and / or one of a variety of flavoring compounds (e.g., amino acids, sugars, thiamine, or phospholipids) to provide an improved flavor to the final product.

[0229] The solution may contain one or more proteins that have been isolated and purified, such as purified fractions rich in pea albumin, purified fractions rich in pea globulin, purified fractions rich in mung bean 8S globulin, and / or fractions rich in Rubisco. In other embodiments, one or more fats are derived from plant-based oils (rice bran oil or canola oil). See, for example, section III C. In some cases, the composition contains cross-linking enzymes, such as transglutaminase, lysyl oxidase, or other amine oxidases. Thus, in some embodiments, adipose tissue replicas can be made by: isolating and purifying one or more proteins; preparing a solution containing one or more proteins; emulsifying one or more fats in the solution; and stabilizing the solution into a gel emulsion using one or more cross-linking agents.

[0230] In some embodiments, the fat imitation is a high-fat emulsion containing a protein solution of purified pea albumin, said protein solution being emulsified with 40-80% rice bran oil and stabilized into a gel with 0.5-5% (wt / vol) transglutaminase.

[0231] In some embodiments, the fat imitation is a high-fat emulsion containing a protein solution of isolated mung bean 8S globulin, the protein solution being emulsified with 40-80% rice bran oil or 40-80% rapeseed oil and stabilized into a gel with 0.5-5% (wt / vol) transglutaminase.

[0232] Fats can be isolated from plant tissues and emulsified. Emulsification can be achieved using high-speed blending, homogenization, high-pressure homogenization, sonication, shearing, stirring, or temperature changes. The lipid suspension can be sonicated or extruded to further alter the properties of the lipids in solution. In some embodiments, other components of the consumer product are added to the solution, followed by the addition of a gelling agent. In some embodiments, a cross-linking agent (e.g., transglutaminase or lysyl oxidase) is added to bond the components of the consumer product. In other embodiments, a gelling agent is added and the lipid / gel suspension is subsequently combined with other components of the consumer product.

[0233] Melting point is controlled by adjusting fat composition.

[0234] The process of cooking meat is integral to the experience of using and enjoying it. An important property of meat is that when it is heated, fat is released from it. This fat lubricates the cooking surface, increases heat transfer, and is an integral part of the visual, auditory, and olfactory experience of cooking meat. The amount of fat released rather than retained during cooking varies with cooking temperature and contributes to the visual, auditory, and olfactory experience of cooking meat.

[0235] The composition and ratio of fatty acids in triglycerides and phospholipids, as well as the ratio of phospholipid head groups, contribute to the unique flavor characteristics of cooked meat. For example, an increase in the content of phosphatidylcholine and phosphatidylethanolamine in fat provides a stronger beef flavor. As discussed above, the flavor of meat imitations can be adjusted by changing the ratios and types of different oils and phospholipids that constitute the meat imitation. For example, the flavor of cooked meat imitations can be controlled by changing the amounts of phospholipids, sterols, and lipids (e.g., 0.2-1% wt / wt). In one embodiment, the flavor of cooked meat imitations can be controlled by changing the ratio of different phospholipid head groups.

[0236] In some embodiments, phospholipids comprise a plurality of amphiphilic molecules containing fatty acids, glycerol, and polar groups. For examples of fatty acids, phospholipids, polar groups, and sterols associated with phospholipids, see, for example, section III C. For examples of applicable vegetable oils, also see section III C.

[0237] Fat exhibits different properties in different cuts of meat, ranging from the structurally important properties of fat in bacon to the varying softening characteristics of marbling fat in Wagyu beef.

[0238] By controlling the melting point of fat tissue imitations in consumer products, it is possible to mimic the cooking experience of different meat types. For example, fat tissue imitations produced from fat with a melting point of 23°C to 27°C can have a melting point similar to that of fat tissue from Kobe beef; fat tissue imitations produced from fat with a melting point of 35°C to 40°C can have a melting point similar to that of fat tissue from regular ground beef; and fat tissue imitations produced from fat with a melting point of 36°C to 45°C can have a melting point similar to that of fat tissue from bacon. Fat tissue imitations can be produced and incorporated into consumer products such that the ratio of fat released to fat retained by the fat tissue imitation during cooking is similar to the fat properties of meat (e.g., ground beef).

[0239] In some embodiments, the fat release temperature of the fat imitation can be controlled by mixing different ratios of vegetable oils containing triglycerides and phospholipids (e.g., lecithin). The melting point of fat is determined by the chemical composition of its fatty acids. Generally, fats containing saturated fatty acids (e.g., C10:0, C12:0, C14:0, C16:0, C18:0, C20:0, C22:0) are solid at refrigerated temperatures (e.g., about 1°C to about 5°C) and at room temperature (e.g., about 20°C to 25°C). By controlling the fat release temperature of the fat imitation during cooking, the firmness of the fat imitation during refrigeration (e.g., about 1.5°C to about 4°C) and at ambient temperatures (e.g., about 20°C to 25°C) can be controlled. Fats containing monounsaturated fatty acids (e.g., C16:1 or C18:1) are generally solid at refrigerated temperatures and liquid at room temperature. Fats containing polyunsaturated fatty acids (such as C18:2, C18:3, C20:5, or C22:6) are generally liquid at refrigerated temperatures and at room temperature. For example, virgin coconut oil melts at about 24°C, while hydrogenated coconut oil melts at 36-40°C.

[0240] For example, adipose tissue replicas containing triglycerides and phospholipids that are liquid at room temperature (approximately 20°C to 25°C) will be softer than adipose tissue replicas containing triglycerides and phospholipids that are solid at refrigerated temperatures.

[0241] Adipose tissue imitations may contain oils from one or more sources that are liquid at both refrigerated temperatures and ambient room temperature (e.g., canola oil, sunflower oil, and / or hazelnut oil). In one embodiment, the adipose tissue imitation contains oils from one or more sources that are solid at refrigerated temperatures but liquid at room temperature (e.g., olive oil, palm oil, and / or rice bran oil). In one embodiment, the adipose tissue imitation contains oils from one or more sources that are solid at room temperature but liquid at oral temperature (approximately 37°C) (e.g., palm kernel oil, coconut oil, and / or cocoa butter). In one embodiment, the adipose tissue imitation contains oils from one or more sources that are solid at oral temperature (approximately 37°C) (e.g., oil from mango butter).

[0242] In one embodiment, the adipose tissue imitation comprises triglycerides and phospholipids with a high ratio of saturated fatty acids and is harder than adipose tissue imitations containing a higher ratio of monounsaturated and polyunsaturated triglycerides and lipids. For example, adipose tissue imitations containing sunflower oil are softer than those containing cocoa butter. Adipose tissue imitations can be formed with 0%, 0.18%, 1.6%, or 2.4% wt / v Rubisco with 70%, 80%, or 90% v / v sunflower or cocoa butter. Each adipose tissue imitation containing cocoa butter is harder than an imitation formed with sunflower oil.

[0243] In one embodiment, adipose tissue imitations made from a stable emulsion of mung bean 8S protein and sunflower oil are softer than those made from a stable emulsion of mung bean 8S protein and cocoa butter. The adipose tissue imitations are formed with 2%, 1%, or 0.5% wt / v mung bean 8S protein and 70%, 80%, or 90% v / v sunflower or cocoa butter. Each adipose tissue imitation containing cocoa butter is harder than the imitation formed with sunflower oil.

[0244] In one embodiment, a fat tissue imitation made from a stable emulsion of mung bean 8S protein and canola oil is softer than one made from a stable emulsion of an equal mixture of mung bean 8S protein and coconut oil, cocoa butter, olive oil, and palm oil. The fat tissue imitation can be formed using 1.4% wt / v mung bean 8S protein with a 50%, 70%, or 90% v / v sunflower oil or other oil mixture. Each fat tissue imitation containing an oil mixture is harder than one formed with sunflower oil.

[0245] In one embodiment, adipose tissue imitations made from a stable emulsion of soy protein and sunflower oil are softer than those made from a stable emulsion of soy protein and cocoa butter. The adipose tissue imitations are formed using 0.6%, 1.6%, or 2.6% wt / v soy with a mixture of 50%, 70%, 80%, or 90% v / v sunflower oil or other oils. Each adipose tissue imitation containing an oil mixture is harder than the imitation formed with sunflower oil.

[0246] In some embodiments, a fat tissue imitation comprising 0%, 0.18%, 1.6%, and 2.4% wt / v Rubisco with 70%, 80%, and 90% v / v cocoa butter is solid at room temperature but melts at approximately oral temperature. In some embodiments, a fat tissue imitation comprising 0.6%, 1.6%, and 2.6% wt / v soy with 50%, 70%, 80%, and 90% v / v cocoa butter is solid at room temperature but melts at approximately oral temperature. In some embodiments, a fat tissue imitation comprising 1.4% wt / v mung bean 8S protein with an equal mixture of 50%, 70%, and 90% v / v coconut oil, cocoa butter, olive oil, and palm oil is solid at room temperature but melts at approximately oral temperature. In one embodiment, the melting temperature of the fat tissue imitation will be similar to that of beef fat. In some embodiments, the fat imitation comprises oils having a 1:1 ratio of saturated to unsaturated fatty acids. In some embodiments, the fat tissue imitation contains equal amounts of cocoa butter and mango butter. In some embodiments, the fat tissue imitation contains equal amounts of coconut oil, cocoa butter, olive oil, and palm oil.

[0247] In one embodiment, the adipose tissue imitation containing triglycerides and phospholipids will have a fatty acid ratio similar to that found in beef (C14: 0.05% wt / wt, C16: 0.025%, C18: 0.020%, C18:1: 0.060%, C18:2: 0.025%, C18:3: 0.05%, C20:4: 0.02%, and C20:6: 0.02%). For example, the adipose tissue imitation may contain equal proportions of olive oil, cocoa butter, coconut oil, and mango butter. In another example, the adipose tissue imitation may contain equal proportions of olive oil and rice bran oil.

[0248] In one embodiment, the melting temperature of the fat tissue imitation will be similar to that of Kobe beef fat. In some embodiments, the fat imitation comprises oils with a 1:2 ratio of saturated to unsaturated fatty acids (e.g., 1 part coconut oil to 2 parts sunflower oil). In some embodiments, the fat tissue imitation contains equal amounts of olive oil, rice bran oil, cocoa butter, and mango butter.

[0249] I. Connective tissue imitations

[0250] Animal connective tissue provides key textural features that are an important component of the meat-eating experience. Therefore, this invention provides a composition derived from a non-animal source that mimics key features of animal connective tissue. This invention also provides a meat substitute product comprising a composition derived from a non-animal source that mimics important textural and visual features of animal connective tissue. Such compositions will be labeled herein as "connective tissue imitations." In some embodiments, connective tissue imitations and / or meat substitute products containing connective tissue imitations are partially derived from animal sources.

[0251] Animal connective tissue can generally be divided into fascial and cartilaginous types. Fascial tissue is highly fibrous, resistant to elongation (with a high modulus of elasticity), and has a high protein content, moderate water content (about 50%), and low to zero fat and polysaccharide content. Therefore, the present invention provides a connective tissue imitation that mimics the key characteristics of fascial tissue. In some embodiments, the connective tissue imitation comprises about 50% protein by total weight and about 50% by liquid weight, and has low fat and polysaccharide components.

[0252] The fibrous nature of fascial connective tissue primarily consists of collagen fibers. These collagen fibers have been observed to be rope-like or ribbon-like in shape, ranging from 1 to 20 micrometers in width. These fibers are composed of densely packed fine collagen fibrils, ranging from 30 to 100 nanometers in diameter. These fibrils are also integrated into an elastic and reticular fiber network, with individual fibers reaching up to 200 nanometers in diameter.

[0253] In one embodiment, the fascia-type connective tissue imitation consists of fibers or fibrous structures that may be composed of proteins. In some embodiments, the protein contents are derived from non-animal sources (e.g., plant sources, algae, bacteria, or fungi, see, for example, sections IIIA and B). In some embodiments, the isolated proteins constitute 50%, 60%, 70%, 80%, 90%, or more by weight of the protein contents. In some embodiments, multiple isolated proteins are separately isolated and purified and constitute the total protein contents.

[0254] In fascial connective tissue, the gliadin family of proteins, individually or in combination, demonstrates the suitability of protein components due to their high abundance, similarity to collagen in terms of global amino acid composition (high proportions of proline and alanine), and their ability to undergo membrane processing. In some embodiments, gliadin family proteins are selected from the group consisting of: corn proteins (found in maize), barley gliadin from barley, wheat gliadin from wheat, rye gliadin, rye extensin, sorghum gliadin, or oat proteins from oats. In some embodiments, one or more isolated and purified proteins are corn proteins. In some embodiments, other proteins may be used to supplement gliadin to achieve target specifications for physicochemical and nutritional properties. See the list in Sections III A and B, which includes any major seed storage proteins, animal-derived collagen or recombinant collagen or extensin (hydroxyproline-rich glycoproteins abundant in the cell wall, such as in Arabidopsis, whose monomers are "collagen-like" rod-shaped flexible molecules).

[0255] Proteins can be freeze-dried and milled and combined with one or more other ingredients (such as wheat gluten, fiber (such as bamboo fiber) or isolated soy protein).

[0256] Fibers or fibrous structures can be formed by extrusion. In some embodiments, extrusion is performed using a Lesterretz Nano-16 twin-screw co-rotating extruder (Lesterretz Extrusion Presses, Inc., Somerville, New Jersey, USA). Effective heating and cooling of the bobbin section is used to optimize the mechanical properties, bulkiness, and water content of the fibers. For example, the water content can be adjusted to approximately 50% to produce a stiff connective tissue imitation. Protein feed and liquid are added separately: protein is fed through a volumetric piston feeder, and liquid is added to the bobbin through a high-pressure liquid injection system. In some instances, the extrusion parameters are: screw speed 200 rpm, product temperature at the die 120°C, feed rate 2.3 g / min, and water flow rate 0.7 g / min. The product temperature at the die during extrusion is measured using thermocouples.

[0257] Fibers or fibrous structures can be formed by extruding them through filaments and multi-filament dies to produce fibrous structures. In some embodiments, multiple dies with different aperture sizes in the range of 10-300 micrometers can be used to produce hybrid fibrous tissue replicas with precise control over the size and composition of the fibers. Fibers of different sizes can be incorporated into the composition to control the properties of the composition.

[0258] Electrospinning can be used to produce fibers in the <1-10 micrometer range. In some embodiments, electrospinning is used to produce fibers in the <1-10 micrometer diameter range. For example, a concentrated solution of mung bean globulin containing 400 mM sodium chloride (140 mg / ml) can be mixed with a solution of poly(vinyl alcohol) or poly(ethylene oxide) (9% w / v) to obtain a mixed solution with 22.5 mg / ml mung bean globulin and 6.75% w / v of the corresponding polymer. The resulting solution is slowly pumped (e.g., at 3 µl / min) from a 5 ml syringe through a Teflon tube and a blunt 21-gauge needle using a syringe pump. The needle is connected to the positive terminal of a high-voltage power supply (e.g., a Spellman CZE 30 kV) and fixed 20-30 cm from the collecting electrode. The collecting electrode is an aluminum cylinder (approximately 12 cm long and 5 cm in diameter) encased in aluminum foil. The cylinder is connected to a mandrel rotated at approximately 600 rpm by an IKA RW20 motor. The mandrel is connected to the ground terminal of the high-voltage power supply. Protein / polymer fibers accumulate on the foil and, after electrospinning is complete, are removed from the foil and added to the tissue imitation.

[0259] The size and composition of fibers produced by the method of this invention affect the taste, texture, and mechanical properties of tissue imitations. Tissues containing 1 to 50% fibers in the <1-10 micrometer range and 10 to 50% fibers in the 10-300 micrometer range most closely resemble animal connective tissue in terms of taste, mouthfeel, and mechanical properties.

[0260] Cartilage-type connective tissue is macroscopically homogeneous and compressibility-resistant, with a high water content (up to 80%), a low protein (collagen) content, and a high polysaccharide (proteoglycan) content (approximately 10% each). In terms of composition, cartilage-type connective tissue imitations are similar to fascia-type tissue imitations, where the relative ratios of each are adjusted to more closely mimic 'meat' connective tissue. During extrusion, the water content can be adjusted to approximately 60% to create a soft connective tissue imitation.

[0261] The method for forming cartilaginous connective tissue is similar to that used for fascial connective tissue, but the method for producing isotropic non-fibrous gel is preferred.

[0262] Connective tissue replicas can be made by: isolating and purifying one or more proteins; and precipitating one or more proteins, wherein the precipitation results in the formation of a physical structure resembling that of connective tissue. Precipitation may involve dissolving one or more proteins in a first solution; and extruding the first solution into a second solution, wherein the one or more proteins are insoluble in the second solution, wherein extrusion induces precipitation of the one or more proteins.

[0263] In some embodiments, some or all components of the consumer product are suspended in a gel (e.g., a protein gel). In various embodiments, the gel can be a hydrogel, an organic gel, or a dry gel. The gel can be thickened using polysaccharide- or protein-based reagents. For example, residues, arrowroot powder, corn starch, katakuri starch, potato starch, sago, tapioca starch, alginate, guar gum, locust bean gum, xanthan gum, collagen, protein, red algae gum, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, arcadia gum, konjac, starch, pectin, amylopectin, or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, or fungi can be used alone or in combination to thicken the gel, forming the architecture or structure of the consumer product. Enzymes that catalyze reactions that produce covalent cross-links between proteins can also be used alone or in combination to form the architecture or structure of the consumer product. For example, transglutaminase, tyrosinase, lysyl oxidase, or other amine oxidases (e.g., Pichia pastoris lysyl oxidase (PPLO)) can be used alone or in combination to form the architecture or structure of a consumer product by cross-linking component proteins. In some embodiments, multiple gels with different components are combined to form a consumer product. For example, a gel containing plant-derived proteins can be combined with a gel containing plant-derived fats. In some embodiments, the fibers or filaments of the proteins are oriented parallel to each other and then fixed in situ by coating them with a gel containing plant-based fats.

[0264] The compositions of the present invention can be fluffed or expanded by heating according to methods well known in the art, such as frying, baking, microwave heating, heating in a forced ventilation system, heating in a wind tunnel, etc.

[0265] In some embodiments, multiple gels with different components are combined to form a consumer product. For example, a gel containing plant-derived proteins can be combined with a gel containing plant-derived fats. In some embodiments, the fibers or filaments of the proteins are oriented parallel to each other and then fixed in situ by coating them with a gel containing plant-derived fats.

[0266] J. Omitted by composition

[0267] Because consumer products can be made from defined ingredients that can be separated and purified, it is possible to produce consumer products that do not contain certain components. In some cases, this makes it possible to produce consumer products that do not contain ingredients that may not be desired by consumers (e.g., proteins that may cause allergies in humans, or additives that can be omitted). In some embodiments, the consumer product does not contain animal products. In some embodiments, the consumer product does not contain or contains less than 1% wheat gluten. In some embodiments, the consumer product does not contain methylcellulose. In some embodiments, the consumer product does not contain carrageenan. In some embodiments, the consumer product does not contain caramel color. In some embodiments, the consumer product does not contain konjac flour. In some embodiments, the consumer product does not contain gum arabic (also known as acacia gum). In some embodiments, the consumer product does not contain wheat gluten. In some embodiments, the consumer product does not contain isolated soy protein. In some embodiments, the consumer product does not contain tofu. In some embodiments, the consumer product contains less than 5% carbohydrates. In some embodiments, the consumer product contains less than 1% cellulose. In some embodiments, the consumer product contains less than 5% cellulose. In some embodiments, the consumer product contains less than 5% insoluble carbohydrates. In some embodiments, the consumer product contains less than 1% insoluble carbohydrates. In some embodiments, the consumer product does not contain artificial colors. In some embodiments, the consumer product does not contain artificial flavorings.

[0268] In some embodiments, the consumer product contains one or more of the following characteristics: free of animal products; free of methylcellulose; free of carrageenan; free of konjac flour; free of gum arabic; less than 1% wheat gluten; free of wheat gluten; free of tofu; about 5% carbohydrates; less than 5% cellulose; less than 5% insoluble carbohydrates; less than 1% insoluble carbohydrates; free of edible colorings such as caramel color, paprika, cinnamon, beetroot color, carrot oil, lycopene extract, raspberry powder, carmine, cochineal extract, annatto, turmeric, saffron, FD&C No. 3 Red, No. 5 Yellow, No. 6 Yellow, No. 3 Green, No. 2 Blue, No. 1 Blue, No. 1 Purple, FD&C No. 40 Red-Allura Red AC and / or E129 (red gradient); and / or free of artificial flavorings. In some embodiments, the consumer product does not contain isolated soy protein. In other embodiments, the consumer product does not contain soy protein or soy protein concentrate.

[0269] In some embodiments, the muscle tissue imitation contains less than 10%, less than 5%, less than 1%, or less than 0.1% wheat gluten. In some embodiments, the muscle tissue imitation does not contain wheat gluten.

[0270] IV. Combination of Components

[0271] A. Imitation meat

[0272] Meat substitute products (or meat imitations) may comprise the compositions described herein. For example, meat imitations may comprise muscle imitations; adipose tissue imitations; and connective tissue imitations (or combinations thereof). Muscle imitations, adipose tissue imitations, and / or connective tissue imitations may be assembled in a manner approximating the physical structure of meat. In some embodiments, a binder (e.g., a coagulant) is used to help the imitations bind to each other.

[0273] The percentages of different components can also be controlled. For example, non-animal substitutes for muscle, adipose tissue, connective tissue, and blood components can be combined in different ratios and physical structures to most closely resemble the appearance and feel of meat. The various components can also be arranged to ensure consistency across consumer products. The components can be arranged to ensure that the consumer product does not generate waste. For example, while traditional cuts of meat may have typically inedible parts, meat imitations can be modified to exclude these inedible parts (e.g., bone, cartilage, connective tissue, or other materials commonly referred to as gristle). Such modifications allow for the consumption of the entire product produced or shipped, reducing waste and shipping costs. Alternatively, meat imitations may include inedible parts to simulate the experience of eating meat. These parts may include bone, cartilage, connective tissue, or other materials commonly referred to as gristle, or materials that mimic these components. In some embodiments, the consumer product may contain simulated inedible parts of a meat product, which are designed to provide secondary functionality. For example, the simulated skeleton can be designed to distribute heat during cooking, allowing the consumer product to cook faster or more evenly than meat. In other embodiments, the simulated skeleton can also be used to keep the consumer product at a constant temperature during shipment. In other embodiments, the simulated inedible portion can be biodegradable (e.g., biodegradable plastic).

[0274] In some embodiments, the meat substitute composition comprises 10-30% protein, 5-80% water, and 5-70% fat, wherein the composition includes one or more isolated and purified proteins. Such meat substitutes may not include animal protein. In some embodiments, the meat substitute composition contains transglutaminase.

[0275] In some embodiments, meat substitute products include muscle imitations, adipose tissue imitations, and connective tissue imitations, wherein the muscle imitations comprise 40-90% of the product by weight, the adipose tissue imitations comprise 1-60% of the product by weight, and the connective tissue imitations comprise 1-30% of the product by weight.

[0276] In some embodiments, the meat substitute product comprises 60-90% water; 5-30% protein content; and 1-20% fat; wherein the protein content comprises one or more isolated and purified plant proteins.

[0277] In some embodiments, the consumer product contains components that mimic the composition of meat. The primary component of meat is typically skeletal muscle. Skeletal muscle typically consists of approximately 75% water, 19% protein, 2.5% intramuscular fat, 1.2% carbohydrates, and 2.3% other soluble non-protein substances. These soluble non-protein substances include organic acids, sulfur-containing compounds, nitrogen-containing compounds (e.g., amino acids and nucleotides), and inorganic substances (e.g., minerals). Therefore, some embodiments of the invention provide a consumer product with an approximate composition that mimics this composition. For example, in some embodiments, the consumer product is a plant-based meat imitation containing approximately 75% water, 19% protein, 2.5% fat, 1.2% carbohydrates, and 2.3% other soluble non-protein substances. In some embodiments, the consumer product is a plant-based meat imitation containing between 60-90% water, 10-30% protein, 1-20% fat, 0.1-5% carbohydrates, and 1-10% other soluble non-protein substances. In some embodiments, the consumer product is a plant-based meat imitation containing 60-90% water, 5-10% protein, 1-20% fat, 0.1-5% carbohydrates, and 1-10% other soluble non-protein substances. In some embodiments, the consumer product is a plant-based meat imitation containing 0-50% water, 5-30% protein, 20-80% fat, 0.1-5% carbohydrates, and 1-10% other soluble non-protein substances.

[0278] In some embodiments, the meat imitation contains between 0.01% and 5% by weight of heme-containing protein. In some embodiments, the imitation contains between 0.01% and 5% by weight of leghemoglobin. Some meats also contain myoglobin (a heme-containing protein), which constitutes a large portion of the red and iron content of some meats. It should be understood that these percentages are variable in meat, and meat imitations can be produced to approximate the natural variations of meat. In embodiments including heme-containing protein and optional flavoring, k-carrageenan can be used to absorb some of the liquid generated by the flavoring and heme solution so that the minced tissue is not overly wetted. During flavoring, the heme mixture and k-carrageenan powder are evenly distributed on the tissue mixture to ensure the homogeneity of the final milled product.

[0279] It should be understood that when proteins are supplied in solution form, water removal techniques (such as freeze-drying or spray drying) can optionally be used to concentrate the proteins. The proteins can then be restored to the required amount of liquid to prevent the fragmented tissue from becoming too wet.

[0280] Additionally, in some cases, the present invention provides modified meat imitations that contain these components in non-natural percentages. The concentration of heme-containing proteins is a significant determinant of meat flavor and aroma. Therefore, for example, meat imitations can have a higher heme protein content than typical beef. For example, a meat imitation can be produced with a higher average fat content than typical beef. The percentages of these components can also be altered to enhance other desired properties.

[0281] In some cases, a meat imitation is designed to have component percentages similar to cooked meat when cooked. Thus, in some embodiments, the uncooked consumer product has different component percentages than uncooked meat, but when cooked, the consumer product resembles cooked meat. For example, a meat imitation can be made with a higher water content than typical raw meat, but when cooked in a microwave, the resulting product has similar component percentages (non-starch polysaccharides such as arabinoyl xylan, cellulose, and many other plant components such as resistant starch, resistant dextrin, inulin, lignin, waxes, chitin, pectin, beta-glucan, and oligosaccharides) to cooked meat.

[0282] In some embodiments, the consumer product is a meat imitation with a lower water content than typical meat. In some embodiments, the present invention provides a method for hydrating the meat imitation to give it a water content similar to that of meat. For example, a meat imitation with a lower water content than meat, such as 1%, 10%, 20%, 30%, 40%, or 50% water, can be hydrated to approximately 75% water. In some embodiments, once hydrated, the meat imitation is then cooked for human consumption.

[0283] Consumer products may contain a protein component. In some embodiments, the protein content of the consumer product is 10%, 20%, 30%, or 40%. In some embodiments, the protein content of the consumer product is similar to that of meat. In some embodiments, the protein content of the consumer product is greater than that of meat. In some embodiments, the consumer product has less protein than meat.

[0284] Proteins in consumer products can be derived from multiple sources or a combination of sources. Non-animal sources can provide some or all of the proteins in a consumer product. Non-animal sources can include vegetables, non-food biomass (such as carrot tips and miscanthus), seaweed, fruits, nuts, grains, algae, bacteria, or fungi. See, for example, sections III A and B. Proteins can be isolated or concentrated from one or more of these sources. In some embodiments, the consumer product is a meat imitation containing proteins obtained solely from non-animal sources.

[0285] In some embodiments, the protein is shaped into asymmetric fibers for incorporation into a consumer product. In some embodiments, these fibers mimic muscle fibers. In some embodiments, the protein is spun into fibers. Therefore, the present invention provides a method for producing asymmetric or spun protein fibers. In some embodiments, the consumer product contains one or more proteins having all the amino acids found in proteins essential for human nutrition. In some embodiments, the protein added to the consumer product is supplemented with amino acids.

[0286] Physical texture can be a determining factor in the response of meat substitutes to cooking. For example, the flavor of meat is modulated by particle size. Minced meat, mashed into a paste during cooking, provides a different flavor profile than coarser ground beef. The ability to control the relative size and orientation of individual tissue imitations allows for the modification of the flavor and aroma characteristics of the product during cooking. For instance, muscle tissue imitations and fat tissue imitations offer different flavor profiles when cooked independently or when mixed. Further changes in flavor characteristics can be observed based on methods that blend different tissue imitations.

[0287] The physical composition of meat substitute products can be manipulated by controlling the localization, organization, assembly, or orientation of the muscle, fat, and / or connective tissue imitations described herein. In some embodiments, the product is designed such that the imitations described herein bind together as in meat. In some embodiments, the consumer product is designed such that, after cooking, the imitations described herein bind together as in cooked meat.

[0288] The characteristic flavor and aroma components of meat are largely produced through chemical reactions during the cooking process, acting on amino acids, fats, and sugars found in both plants and meat. Therefore, in some embodiments, consumer products are tested for similarity to meat during or after cooking. In some embodiments, olfactory maps of cooked meat are created using human ratings, human assessments, olfactory meter readings, or GCMS measurements, or combinations thereof. Similarly, olfactory maps of consumer products (e.g., meat imitations) can be created. These maps can be compared to assess the similarity between cooked consumer products and meat. In some embodiments, the olfactory map of a consumer product during or after cooking is similar to or indistinguishable from the olfactory map of cooked or being cooked meat. In some embodiments, the difference is small enough to be below a detection threshold for human perception.

[0289] In some embodiments, individual tissue replicas are assembled in layers, sheets, blocks, and filaments with defined locations and orientations.

[0290] In some embodiments, the imitation is assembled in the process of passing through a plate of a meat grinder with holes set to be less than 1 / 2 inch (e.g., 1 / 2 inch). The grinder provides multiple functions, such as reducing particle size, providing additional mixing or work, and shaping the material into cylindrical portions, similar to what is typically done for ground beef. During assembly, grinding, and shaping, it is important to keep the imitation tissue cold (e.g., 4–15°C) to control microbial growth, limit flavor reactions, and maintain the fat in a solid state so that discrete flakes of fat will be maintained through the grinding process.

[0291] Prior to grinding, imitation tissue is typically broken down into defined particle sizes in some way. For example, in some embodiments, individual tissue imitations may be shaped into small flakes with a diameter of less than 1 cm or less than 5 mm before being combined with other tissue imitations. Fatty tissue can be pulverized into particles of approximately 3–7 mm. This is important for both the appearance of the final material and the fat leakage characteristics during cooking. This size range allows the fat particles in the final product to have a natural appearance. If the fatty tissue is too small (e.g., less than 2 mm), then insufficient fat will leak from the product when cooking.

[0292] Soft connective tissue imitations can be broken down into pieces with irregular edges and a length of approximately 1-3 mm. If the pieces are too large (e.g., larger than approximately 4 mm), the final product may have too much foam.

[0293] Viscous or noodle-like imitations, composed of amorphous or long noodle-like fragments, as well as raw imitations, can be artificially broken down into fragments with a diameter of approximately 1-3 cm. Obtaining particles within this size range ensures thorough mixing and suitable homogeneity of the final grinding material.

[0294] In some embodiments, the hard connective tissue imitation can be shredded at three levels (e.g., coarse, medium, and fine). Shredding at three levels provides a greater degree of heterogeneity than a single-step shredding process and makes the final product taste more like ground beef.

[0295] In gluten-containing formulations, another function of food grinders is to process gluten and develop a gluten network of aligned gluten molecules. For gluten-containing formulations, it is important to minimize the interaction between fat and the gluten network. This is done by pre-freezing fat imitations and minced tissue imitations before assembly, and by minimizing the amount of manipulation after adding fat. Over-processing fat imitations into gluten will break down or “shorten” the gluten network.

[0296] Finally, for gluten-containing condiments, let the patties rest at room temperature for 30 minutes or overnight at 4°C before cooking. This gives the gluten network time to relax, resulting in a better overall texture.

[0297] In some embodiments, the connective tissue replica is incorporated into the protein solution before the formation of the muscle tissue replica.

[0298] In some embodiments, the connective tissue imitation is incorporated directly into the emulsion prior to the formation of the adipose tissue imitation.

[0299] In some embodiments, adipose tissue imitations are added to muscle tissue imitations in the form of chains and sheets to mimic the effect of "marbled" or bacon.

[0300] Mixed meat tissue imitations can enhance the sensation of flavor, which includes, but is not limited to, polyaromatic compounds associated with fruity / peaty / metallic, nutty / green, peanut butter / mold, raw potato / baked / earthy, sour, spicy / caramel / almond, creamy, sweet, fruity / aged beer, musty / nutty / coumarin / licorice / walnut / bread, coconut / woody / sweet, penetrating / nauseating, minty, or roasted caramel aromas.

[0301] In some embodiments, the blended meat imitations increase the presence of volatile odorants, such as 2-pentyl-furan; 4-methylthiazole; ethylpyrazine; 2,3-dimethylpyrazine; acetic acid; 5-methyl-2-furanaldehyde; butyrolactone; 2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2-cyclopenten-1-one, 2-hydroxy-3-methyl; 3-acetyl-1h-pyrrolidone; pantothenic acid lactone; 1-methyl-1(H)-pyrrole-2-2-carboxaldehyde; caprolactam; 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. In some embodiments, undesired flavors, including but not limited to those of gasoline, petroleum, acid / rotten / fishy, ​​mild / woody / yogurt, fatty / honey / orange, pungent / sweet / caramel, and nutty / burnt green, are formed only in the individual meat imitations but do not accumulate in the blended meat imitations. In some embodiments, individual tissue imitations increase the presence of volatile odorants, including but not limited to nonane, 2,6-dimethyl; 3-methyl-3-hexene; pyridine; acetoin; octanal; 1-hydroxy-2-propanone; and / or vinylpyrazine. In some embodiments, the level at which all of the above compounds accumulate during cooking depends on the size of the tissue imitation unit and how it is mixed (coarse, fine, or blended).

[0302] In some embodiments, the blending of meat tissue imitations enhances the flavor profile, which includes, but is not limited to, polyaromatic compounds associated with fruity / peaty / metallic, nutty / green, peanut butter / mold, raw potato / baked / earthy, sour, spicy / caramel / almond, creamy, sweet, fruity / aged beer, musty / nutty / coumarin / licorice / walnut / bread, coconut / woody / sweet, penetrating / nauseating, minty, or roasted caramel aromas. In some embodiments, the mixed meat imitation increases the presence of volatile odorants, including but not limited to phenylacetaldehyde; 1-octen-3-one; 2-n-heptylfuran; 2-thiophenecarboxaldehyde; 3-thiophenecarboxaldehyde; butyrolactone; 2-undecenal; methyl-pyrazine; furfural; 2-decanone; pyrrole; 1-octen-3-ol; 2-acetylthiazole; (E)-2-octenal; decanal; benzaldehyde; (E)-2-nonenal; pyrazine; 1-hexanol; 1-heptanol; dimethyl trisulfide; 2-nonanone; 2-pentanone; 2-heptanone; 2,3-butanedione; heptanal; nonanal; 2-octanone; 1-octanol; 3-ethylcyclohexane Pentanone; 3-Octen-2-one; (E,E)-2,4-Heptadienal; (Z)-2-Heptadienal; 2-Heptone, 6-Methyl-; (Z)-4-Heptadienal; (E,Z)-2,6-Nonadienal; 3-Methyl-2-Butenal; 2-Pentyl-furan; Thiazole; (E,E)-2,4-Decadienal; Hexanoic acid; 1-Ethyl-5-methylcyclopentene; (E,E)-2,4-Nonadienal; (Z)-2-Decadienal; Dihydro-5-pentyl-2(3H)-furanone; trans-3-Nonen-2-one; (E,E)-3,5-Octen-2-one; (Z)-2-Octen-1-ol; 5-Ethyldihydro-2(3H)-furanone; 2-Butenal; 1-Penten-3-ol; (E)-2-Hexenal; Formic acid; Heptanyl ester; 2-Pentylthiophene; (Z)-2-Nonenal; 2-Hexylthiophene; (E)-2-Decenal; 2-Ethyl-5-methyl-pyrazine; 3-Ethyl-2,5-dimethyl-pyrazine; 2-Ethyl-1-hexanol; Thiophene; 2-Methyl-furan; Pyridine; Butyraldehyde; 2-Ethyl-furan; 3-Methyl-butyraldehyde; Chloroform; 2-Methyl-butyraldehyde; Methacrolein; 2-Methyl-propionaldehyde; Propanal; Acetaldehyde; 2-Propyl-furan; Dihydro-5-propyl-2(3H)- Furanone; 1,3-hexadiene; 4-decyne; pentanal; 1-propanol; heptanoic acid; trimethyl-ethanethiol; 1-butanol; 1-penten-3-one; dimethyl sulfide; 2-ethylfuran; 2-pentyl-thiophene; 2-propenal; 2-tetren-1-ol; 4-octene; 2-methylthiazole; methyl-pyrazine; 2-butanone; 2-pentyl-furan; 2-methyl-propanal; butyrolactone; 3-methyl-butanal; methyl-cyclothioethane; 2-hexyl-furan; butanal; 2-methyl-butanal; 2-methyl-furan; furan; octanal; 2-heptenal; 1-octene; heptyl formate; 3-pentyl-furan; and 4-penten-2-one.In some embodiments, the level at which all of the above compounds accumulate during cooking depends on the size of the tissue units and how they are mixed (coarse, fine, or blended).

[0303] The production of volatile odorants may be enhanced when fat, muscle, and connective tissue replicas come into contact with each other. In some embodiments, the production of volatile odorants is enhanced when fat, muscle, and connective tissue are tightly mixed with individual tissue replicas of an average size of 5 mm. In some embodiments, the production of volatile odorants is enhanced when fat, muscle, and connective tissue are tightly mixed with individual tissue replicas of an average size of 2 mm. In some embodiments, the production of volatile odorants is enhanced when fat, muscle, and connective tissue replicas are tightly mixed with individual tissue replicas of an average size of 1 mm.

[0304] In some embodiments, the meat substitute is optimized for specific cooking methods (optimized for cooking in a microwave oven or for cooking in a slow cooker).

[0305] In some embodiments, meat substitutes are optimized for use in dehydration.

[0306] In some embodiments, the meat substitute is optimized for rapid rehydration when the dehydrated meat imitation is exposed to water.

[0307] In some embodiments, the meat substitute is optimized for use as emergency, camping, or space food.

[0308] The methods described in this article can be used to provide meat imitations with defined cooking characteristics, allowing for the production of meat imitations optimized for specific cooking techniques. For example, slow cooking in a stew pot requires slow cooking to allow the connective tissue in the meat to gel; however, meat imitations can be designed where the connective tissue gels more easily, thus allowing the stew pot to be prepared quickly.

[0309] B. Indicators for cooking meat

[0310] Consumer products may include compositions that indicate whether the consumer product is being cooked or has been cooked. The release of odorants during cooking is an important aspect of meat consumption. In some embodiments, the consumer product is a meat imitation composed entirely of non-animal products that, upon cooking, produces an odor that can be recognized by humans as the typical odor of cooked beef. In some embodiments, the consumer product, upon cooking, produces an odor that can be recognized by humans as the typical odor of cooked pork, bacon, chicken, lamb, fish, or turkey. In some embodiments, the consumer product is a meat imitation composed primarily or entirely of ingredients derived from non-animal sources, having odorants released during cooking or produced by chemical reactions occurring during cooking. In some embodiments, the consumer product is a meat imitation composed primarily or entirely of ingredients derived from non-animal sources, containing a mixture of proteins, peptides, amino acids, nucleotides, sugars, and polysaccharides and fats in a combination and spatial configuration that allows these compounds to undergo chemical reactions during cooking to produce odorants and flavor compounds.

[0311] In some embodiments, the consumer product is a meat imitation composed primarily or entirely of ingredients derived from non-animal sources, having volatile or unstable odorants released during cooking.

[0312] In some embodiments, the indicator is a visual indicator that accurately simulates the color change of the meat product during the cooking process. The color change may be, for example, from red to brown, from pink to white or tan, or from translucent to opaque during the cooking process.

[0313] In some embodiments, the indicator is an olfactory indicator that indicates the progress of cooking. In one embodiment, the olfactory indicator is one or more volatile odorants released during cooking.

[0314] In some embodiments, the indicator comprises one or more isolated and purified iron-containing proteins. In some embodiments, one or more isolated and purified iron-containing proteins (e.g., heme-containing proteins, see Section III B) are in a reduced state prior to cooking. In some embodiments, one or more isolated and purified iron-carrying proteins in a reduced or oxidized state have UV-Vis characteristics similar to those of animal-derived myoglobin proteins in an equivalent reduced or oxidized state. The peak absorption wavelength of *Aeromonas hydrophila* hemoglobin is 413 nm; the peak absorption wavelength of *Extreme Acidophilus methanogens* hemoglobin is 412 nm; the peak absorption wavelength of soybean hemoglobin is 415 nm; and the peak absorption wavelengths of barley and mung bean non-symbiotic hemoglobins are each 412 nm. The peak absorption wavelength of bovine myoglobin is 415 nm.

[0315] In some embodiments, the difference between the peak absorption wavelength of one or more isolated and purified iron-containing proteins and the peak absorption wavelength of myoglobin derived from animal sources is less than 5%.

[0316] Flavoring agents released during meat cooking are generated through reactions involving substances that may act as reactants, such as fats, proteins, amino acids, peptides, nucleotides, organic acids, sulfur-containing compounds, sugars, and other carbohydrates. In some embodiments, flavoring agents bound during meat cooking are identified and brought close together in the consumer product, such that the flavoring agents bind when the consumer product is cooked. Thus, in some embodiments, characteristic flavor and aroma components are generated during the cooking process through chemical reactions involving amino acids, fats, and sugars found in plants and meats. Thus, in some embodiments, characteristic flavor and aroma components are largely generated during the cooking process through chemical reactions involving one or more amino acids, fats, peptides, nucleotides, organic acids, sulfur-containing compounds, sugars, and other carbohydrates found in plants and meats.

[0317] Some reactions that generate odorants released during meat cooking can be catalyzed by iron, specifically heme iron from myoglobin. Therefore, in some embodiments, some of the characteristic flavor and aroma components are generated during the cooking process via iron-catalyzed chemical reactions. In some embodiments, some of the characteristic flavor and aroma components are generated during the cooking process via heme-catalyzed chemical reactions. In some embodiments, some of the characteristic flavor and aroma components are generated during the cooking process via heme iron-catalyzed chemical reactions from legume hemoglobin. In some embodiments, some of the characteristic flavor and aroma components are generated during the cooking process via heme iron-catalyzed chemical reactions from heme proteins. For example, when analyzed by GC-MS, heme proteins (e.g., from *Aeromonas hydrophila*, *Extreme acidophilus methanogens*, soybeans, barley, or mung beans) provide volatile odorant characteristics significantly different from any subset of the three components when heated in the presence of cysteine ​​and glucose. The volatile flavor components that increase under these conditions include, but are not limited to, furans, acetone, thiazoles, furfural, benzaldehyde, 2-pyridinecarboxaldehyde, 5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophenecarboxaldehyde, 3-thiophene methanol, and decanol. Under these conditions, cysteine ​​and glucose, alone or in the presence of iron salts (e.g., ferrous gluconate), produce a sulfurous odor, but the addition of heme protein reduces the sulfurous odor and replaces it with flavors including, but not limited to, chicken broth, roasted mushrooms, molasses, and bread.

[0318] Additionally, heme proteins (e.g., from *Aeromonas hydrophila*, *Methanophila extremophila*, soybeans, barley, or mung beans) increase specific volatile flavor compounds when heated in the presence of minced chicken, and these compounds are increased in beef compared to chicken when analyzed by GC-MS. The volatile flavor components increased under these conditions include, but are not limited to, propionaldehyde, butyraldehyde, 2-ethyl-furan, heptanaldehyde, octanaldehyde, trans-2-(2-pentenyl)furan, (Z)-2-heptenal, (E)-2-octenal pyrrole, 2,4-dodecadienal, 1-octanal, or (Z)-2-decenal-2-undecenal.

[0319] C. Color indicators

[0320] The color of meat is an important part of the experience of cooking and eating it. For example, cuts of beef are characteristically red when raw and gradually turn brown during cooking. As another example, white meat (such as chicken or pork) is characteristically pink when raw and gradually turns white or light brown during cooking. The amount of color change is used to indicate the cooking progress of beef and to quantify cooking time and temperature to produce the desired level of doneness. In some aspects, the present invention provides a non-meat-based meat substitute product that provides a visual indicator of cooking progress. In some embodiments, the visual indicator is a color indicator that undergoes a color change during cooking. In some embodiments, the color indicator mimics the color change of a cut of meat from raw to cooked. In more embodiments, the color indicator turns the meat substitute product red before cooking to indicate a raw state and turns the meat substitute product brown during cooking. In other embodiments, the color indicator turns the meat substitute product pink before cooking to indicate a raw state and turns the meat substitute product white or brown during cooking.

[0321] The primary determinant of the nutritional definition of meat color is the concentration of iron-carrying proteins in the meat. In the skeletal muscle component of meat products, one of the main iron-carrying proteins is myoglobin. As described above, myoglobin content varies from less than 0.05% in white meat of chicken to 1.5-2.0% in older beef. Therefore, in some embodiments, the consumer product is a meat imitation containing iron-carrying proteins (e.g., heme-containing proteins). In some embodiments, the meat imitation contains approximately 0.05%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2%, or more of iron-carrying proteins (e.g., heme-containing proteins) by dry weight or total weight. In some cases, ferritin has been isolated and purified from its source. In other cases, ferritin has not been isolated and purified. In some cases, the source of ferritin is animal-derived or non-animal-derived, such as plants, fungi, or genetically modified organisms, such as plants, algae, bacteria, or fungi. In some cases, ferritin is myoglobin. In some embodiments, the consumer product is a plant-based meat imitation fortified with animal myoglobin. Thus, for example, a calf meat imitation may contain about 0.4-1% myoglobin. In some embodiments, the consumer product is a plant-based meat imitation fortified with leghemoglobin or cytochrome. Thus, for example, a calf meat imitation may contain about 0.4-1% leghemoglobin or cytochrome.

[0322] Another example of a ferritin is hemoglobin, an iron-containing oxygen-binding protein found in the red blood cells of vertebrates. Hemoglobin is similar in color to myoglobin. In some embodiments, the present invention provides a method for preserving and recycling blood from animal husbandry to enhance the color of a consumer product. For example, blood is preserved from a slaughterhouse, and the hemoglobin derived from the blood is used to enhance the color of the consumer product. In some aspects, the consumer product is a plant-based meat imitation containing hemoglobin.

[0323] Other iron-containing proteins are naturally occurring. In some embodiments, the consumer product contains an iron-containing protein that is not myoglobin. In some embodiments, the consumer product does not contain myoglobin. In some embodiments, the consumer product does not contain hemoglobin. In some embodiments, the consumer product is a meat imitation containing an iron-containing protein other than myoglobin or hemoglobin. For examples of heme-containing proteins, see, for example, Section III B and Figure 3For example, in some embodiments, the consumer product contains heme proteins (e.g., hemoglobin, myoglobin, neuroglobin, cytoglobin, legume hemoglobin, non-symbiotic hemoglobin, hellgate globin I, bacterial hemoglobin, ciliate myoglobin, or flavohemoglobin).

[0324] Leghemoglobin, similar in structure and physical properties to myoglobin, is readily available as an unused byproduct of commercial leguminous crops such as soybeans or peas. In the United States, the leghemoglobin content in the roots of these crops exceeds the myoglobin content of all red meat consumed in the US.

[0325] In some embodiments, the consumer product is a meat imitation primarily or entirely composed of ingredients derived from non-animal sources and containing heme proteins (such as legumehemoglobin or members of the globulin protein family). For example, the meat imitation may be primarily or entirely composed of ingredients derived from non-animal sources (including muscle tissue imitations, adipose tissue imitations, connective tissue imitations) and heme proteins. In some embodiments, the consumer product is a meat imitation primarily or entirely composed of ingredients derived from non-animal sources, having a high iron content derived from heme proteins. In some embodiments, the iron content is similar to that of meat. In some embodiments, the consumer product has the distinctive red color of meat, provided by legumehemoglobin.

[0326] Heme proteins (such as the heme-containing proteins described in Section III B) can be used as indicators of the end of cooking for consumer products. Therefore, one embodiment of the invention is a method of cooking a consumer product comprising detecting leghemoglobin that migrates from the interior of the product to its surface as it is cooked. Another embodiment of the invention is a method of cooking a consumer product comprising detecting a color change from red to brown as the product is cooked.

[0327] In some embodiments, an increased shelf life is provided by extending the desired red lifespan of food products (e.g., based on non-meat meat substitutes).

[0328] In one embodiment, the present invention provides a heme protein that imparts a desired color to a non-meat meat substitute. In some embodiments, the heme protein is derived from a non-animal source, such as a plant, fungus, or a genetically modified organism, such as a plant, algae, bacteria, or fungus. See, for example, section III B. In some embodiments, the shelf life of the heme protein is extended by treatment with a meat shelf-extending agent.

[0329] In some embodiments, the meat shelf-extender is selected from the group consisting of: carbon monoxide, nitrite, sodium metabisulfite, Bombal, rosemary extract, green tea extract, catechins, and other antioxidants.

[0330] In one embodiment, the present invention provides a heme protein that imparts a desired flavor characteristic to a food product (e.g., a non-meat meat substitute). In some embodiments, the ability of the heme protein to generate the desired flavor characteristic is similar to that of myoglobin.

[0331] In some embodiments, the lifetime of heme protein’s ability to generate the desired flavor characteristics is 10%, 20%, 30%, 50%, or 100% or more greater than that of myoglobin.

[0332] D. Food products containing isolated and purified heme proteins

[0333] In some embodiments, the heme proteins described herein are added to meat or consumer products described herein to enhance the properties of the meat or consumer products. For example, a solution containing heme proteins may be injected into raw (e.g., raw white meat) or cooked meat to improve the sensory properties of the meat during cooking and to add a “beef-like” flavor (e.g., to white meat, such as chicken).

[0334] In another example, a solution of heme protein can be dropped onto meat or the consumer product of this invention to enhance its appearance. In one embodiment, heme protein can be used to enhance the advertising, photography, or visual text of food products (such as meat or meat substitutes).

[0335] In another embodiment, heme protein is added to consumer products as an iron supplement.

[0336] In one application of the invention, heme protein can be used as a food dye. In one embodiment, heme protein can be used in a variety of applications as a safe, digestible alternative to FD&C 40 Red-Allure Red AC, E129 (red gradient). The non-limiting list of possible uses will include drawing, particularly in the form of, for example, body painting or as artificial blood.

[0337] In some embodiments, the present invention provides a method for obtaining heme proteins (e.g., legume-based heme) from plants. Legume-based heme can be obtained from a variety of plants. Various legume species and varieties (e.g., soybean, broad bean, lima bean, cowpea, English pea, yellow pea, lupin, common bean, chickpea, peanut, alfalfa, pea hay, clover, lespedeza, or black-and-white pea) contain nitrogen-fixing root nodules (e.g., root nodules from pea plants) in which legume-based heme plays a crucial role in controlling oxygen concentration. In one embodiment, legume-based heme protein is purified from root nodules of legumes (e.g., soybean, broad bean, or pea) using ion-exchange chromatography. In one embodiment, legume-based heme is purified from root nodules of soybean, broad bean, or sweet pea.

[0338] Plants can be grown using standard agricultural methods, with the exception that in some cases, fertilizers are applied and the soil is rich in rhizobia (bacteria). Rhizobium Natural nitrogen-fixing bacteria. The entire root or nodule can be collected and dissolved using a grinder-blender in, for example, 20 mM potassium phosphate (pH 7.4), 100 mM potassium chloride, and 5 mM EDTA. During this process, leghemoglobin is released into the buffer. The nodule lysate containing leghemoglobin can be removed from cell debris by filtration through a 5 μm filter. In some embodiments, filtration is followed by centrifugation (7000 g, 20 min). The clarified leghemoglobin-containing lysate is then filtered through a 200 nm filter and applied to an anion exchange column (High Prep Q; High Prep DEAE, GE Healthcare) on a high-performance liquid chromatography system (GE Healthcare). The leghemoglobin in the eluent is collected and concentrated to the desired concentration via a 3 kDa filter membrane. The purity (partial abundance) of the purified soybean leghemoglobin was analyzed by SDS-PAGE gel electrophoresis: in the solution, soybean leghemoglobin was present at 20-40%, while after anion exchange purification, it was present at 70-80%. In another embodiment, the soybean leghemoglobin flow-through from anion exchange chromatography was applied to size exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). The soybean leghemoglobin eluted as two eluates corresponding to the dimer and monomeric substances. The purity (partial abundance) of the leghemoglobin was analyzed by SDS-PAGE and determined to be approximately 90-100%.

[0339] Proteins from leguminous root nodules can be transferred to a 10 mM sodium carbonate (pH 9.5), 50 mM sodium chloride buffer solution, filtered through a 200 nm filter, and applied to an anion exchange column on a rapid protein liquid chromatography instrument (GE Healthcare). Leghemoglobin binds to the anion exchange matrix and is eluted using a sodium chloride gradient. The purity (partial abundance) of leghemoglobin can be analyzed by SDS-PAGE, and determined to be approximately 60-80%.

[0340] Undesirable small molecules from leguminous roots can be removed from purified leguminous hemoglobin by passing it through an anion exchange resin. These small molecules permeate the root-nodule lysate with a gradually changing brown color, thus degrading the color quality of the leguminous hemoglobin solution. In one embodiment, the anion exchange resin is FFQ, DEAE, Amberlite IRA900, Dowex 22, or Dowex 1x4. Leguminous hemoglobin purified by ammonium sulfate fractionation (60% wt / v and 90% wt / v ammonium sulfate) or by anion exchange chromatography is buffer-exchanged in 20 mM potassium phosphate (pH 7.4) and 100 mM sodium chloride, and the solution is passed through one of the aforementioned anion exchange resins. The flow-through can be collected, and its color compared to the solution color before passing through the anion exchange resin. As evaluated by visual inspection, an improvement in the color of the purified soybean hemoglobin solution can be observed (from yellow / brown to a more pronounced red), however, the degree to which the yellow-brown hue is removed varies.

[0341] Alternatively, heme-containing proteins can be recombinantly produced as described in Chapter III B. For example, non-symbiotic hemoglobin from mung beans can be recombinantly expressed in *E. coli* and purified using anion-exchange chromatography or cation-exchange chromatography. Cell lysates can be loaded onto FF-Q resin in a rapid protein liquid chromatography instrument (GE Healthcare). Mung bean non-symbiotic hemoglobin is eluted in the eluent. The purity (partial abundance) of mung bean non-symbiotic hemoglobin is analyzed by SDS-PAGE and determined as a fraction of total protein: 12% in the *E. coli* lysate and 31% after purification on FFQ. UV-Vis analysis of the purified protein reveals the spectral characteristics of a heme-binding protein.

[0342] Alternatively, the cell lysate can be loaded onto FF-S resin in a rapid protein liquid chromatography instrument (GE Healthcare). Mung bean non-symbiotic hemoglobin can bind to the FF-S column and be eluted using a sodium chloride gradient (50 mM–1000 mM). The purity (partial abundance) of mung bean non-symbiotic hemoglobin can be analyzed by SDS-PAGE, showing 13% in the *E. coli* lysate and 35% after purification on FFQ. UV-Vis analysis of the purified protein reveals the spectral characteristics of the heme-binding protein.

[0343] In some embodiments, heme protein is used as an ingredient in food products where a blood-like flavor is desired. The heme-containing protein of the present invention was tasted by a group of volunteers and described in each case as tasting like blood.

[0344] Heme proteins, such as soy leghemoglobin, can be combined with other plant-based meat imitation components. In some embodiments, the heme protein is captured in a gel containing other components (e.g., lipids and other proteins). In some aspects, multiple gels are combined with non-gel heme proteins. In some embodiments, the heme protein is combined with other compounds of the consumer product to ensure that the heme protein can diffuse throughout the consumer product. In some embodiments, the consumer product is immersed in a solution containing heme proteins (e.g., soy leghemoglobin solution) for, for example, 1, 5, 10, 15, 30, or 45 minutes or 1, 5, 10, 15, 20, or 30 hours.

[0345] Given the use of heme proteins in coloring consumer products, they are suitable for detecting whether a product contains a specific heme protein. Therefore, in some embodiments of the present invention, methods for determining whether a product contains heme proteins are included. For example, ELISA, adjacent linkage analysis, luninex analysis, or Western blot analysis can be performed to determine the presence of leghemoglobin or other heme-containing proteins in food products (e.g., meat or meat imitations). In one embodiment, a detection method is used to determine whether meat has been altered by leghemoglobin or other heme-containing proteins.

[0346] E. Imitation mayonnaise spread.

[0347] Mayonnaise is a thick, creamy sauce. Traditional mayonnaise is a stabilized emulsion of oil and egg yolks. It is believed that lecithin and protein from the egg yolks stabilize the emulsion. Traditional commercially available mayonnaise typically contains 70-80% (wt / wt) fat and 5% (wt / wt) egg yolks. Lower-fat commercially available products may contain approximately 20% (wt / wt) fat. Consumer products may contain compositions with properties comparable to mayonnaise.

[0348] In one embodiment, purified plant protein can be used as a substitute for egg white to create a stable, creamy protein-fat emulsion with a visual and gustatory appearance similar to traditional mayonnaise. The fat (approximately 20-80% wt / wt) can be derived from a single or multiple sources as described herein. This non-traditional mayonnaise product can be used in all culinary applications where traditional mayonnaise is used. In one embodiment, vinegar and / or lemon and / or lime juice are added as flavor additives. In one embodiment, the purified plant protein is not soy protein. In one embodiment, the flavor can be adjusted by adding mustard, spices, herbs, and / or pickles.

[0349] Mayonnaise imitations may comprise a mixture of non-animal proteins. In one embodiment, the mayonnaise imitation is a mixture of 50% (wt / v) rice bran oil and 7% (wt / v) mung bean 8S protein. In another embodiment, the mayonnaise imitation is a mixture of 70% (wt / v) sunflower oil or cocoa butter, 2.4% (wt / v) RuBisCo, 0.29% (wt / wt) soy lecithin, and optionally 8 µM oil body protein.

[0350] The mixture can be emulsified, and the stability of the emulsion can be controlled by adjusting the size of the oil-water-protein particles using high-pressure homogenization or sonication. Oil can be added in liquid form. Protein can be added in solution form in a buffer solution. Soy lecithin can be resuspended in water and sonicated before being mixed with the oil and protein solution. The resulting oil, protein, and lecithin solution can be homogenized, for example, first at 5000 psi and then at 8000 psi, or sonicated for 2 minutes at 40% duty cycle at the maximum setting. The resulting product has a thickness, texture, creaminess, and visual appearance similar to one of the traditional mayonnaises. In some cases (e.g., using mung bean 8S protein and rice bran oil), the product color is pale grayish-white.

[0351] F. Counterfeit milk wine

[0352] Traditionally, lager contains dairy cream and alcohol as its base. Examples of alcohol include whiskey, Irish whiskey, Scotch whiskey, rum, vodka, grappa, or fermented fruit (such as cherry liqueur), plum brandy, tequila, or herbal bisque. Lager imitations can be produced by replacing the dairy cream in lager with a portion of non-dairy cream from a plant source. In one embodiment, the dairy cream in lager can be replaced by a stable emulsion with a consistency similar to dairy cream, consisting of vegetable fats and isolated or purified proteins. In one embodiment, the purified vegetable proteins and / or vegetable fats can be derived from one or more sources as described herein. For example, lager may include a portion of sunflower butter, RuBsiCo, and whiskey, along with one or more optional flavorings (such as vanilla, chocolate, and / or coffee).

[0353] G. Alcoholic beverages rich in protein

[0354] Traditionally, alcoholic beverages contain negligible to low amounts of protein. Adding plant protein to various alcoholic beverages will positively affect their flavor, mouthfeel, physical state, and increase their nutritional protein content. Additionally, the presence of protein in the various alcoholic beverages used in cocktails will positively affect their flavor, mouthfeel, physical state, and increase their nutritional protein content. Different categories of alcoholic beverages contain different amounts of alcohol. For example, winecoolers contain approximately 4-7% alcohol, beer contains approximately 3-10% alcohol, wine contains approximately 8-14% v / v alcohol, liqueurs contain approximately 17-20% alcohol, whiskey contains approximately 40% alcohol, and vodka contains approximately 35-50% alcohol. Furthermore, some traditional alcoholic beverages include sugar (e.g., Bacardi Razz at 10% wt / v).

[0355] Therefore, alcoholic beverages can be supplemented by adding, for example, 0.1-5% wt / v purified plant protein and optional sugar (1-15% wt / v). The sugar can be, for example, cane sugar, brown sugar, sucrose, or glucose. For instance, 180 mg / ml of purified Rubisco in 20 mM potassium phosphate (pH 7.0) and 150 mM NaCl can be added to whiskey. Adding 5% wt / v Rubisco to Jameson whiskey forms a soft gel with a consistency similar to traditional jello shots.

[0356] For example, alcoholic beverages rich in Rubisico, Mung Bean 8S, and Pea Globulin were created by adding purified Rubisico, Mung Bean 8S, and Pea Globulin proteins, respectively, to Corona beer, Pinot Grigio wine, and Jameson whiskey at final protein concentrations of 0.5%, 1%, and 5%, respectively. Corn protein was added to an aqueous solution of 60% ethanol and 5% sucrose, at concentrations of 0.5%, 1%, and 5%, respectively.

[0357] Pea protein is extracted from pea flour as follows: The flour is resuspended in a 5%, 20%, or 40% ethanol and 5% sucrose aqueous solution, followed by incubation at room temperature for 1 hour. Any undissolved solids are removed by centrifugation at 5000 g for 10 minutes. The resulting supernatant solution is clear. The 5% ethanol solution is particularly suitable.

[0358] A sensory panel evaluated all protein-rich alcoholic beverages as having distinct aromas and flavors compared to non-protein-rich beverages. In some cases, the resulting aromas and flavors were judged neutral compared to the control; in others, they were judged more appealing; and in still others, they were judged less appealing. In specific instances, the addition of both 0.5% wt / v and 1% wt / v concentrations of mung bean 8S protein to Jameson whiskey softened its aroma and mouthfeel. Adding 0.5% wt / v mung bean to Jameson whiskey imparted a slightly creamy flavor, with an aroma reminiscent of a traditional White Russian cocktail. Adding 5% wt / v corn protein to Jameson whiskey produced aromas and flavors characterized by moldy beans and raw potatoes.

[0359] In another example, Corona beer enriched with 0.5% wt / v pea globulin develops an aroma that is hoppy and similar to one of the Indian Pale Ales, with a flavor profile that carries pea characteristics. Adding 0.5% wt / v and 5% wt / v mung bean 8S protein transforms the Corona aroma into a sweet peony one, with enhanced hop notes. The flavor is neutral with 0.5% wt / v mung bean 8S and carries vegetal-nutty characteristics with 5% wt / v mung bean 8S.

[0360] In another example, a Pinot Grid wine enriched with 1% wt / v pea 8S protein was found to have additional aromas of sweetness and orange, and the flavor profile shifted to one with peanut butter characteristics. Adding 1% wt / v pea globulin adjusted the aromas to strongly musty oak and wet leaf notes, and the flavor profile shifted to one with earthy characteristics. Adding 5% wt / v Rubisco generated aromas and flavors of wet hay.

[0361] Compared to the corresponding solution without corn protein, the 60% ethanol, 5% sucrose solution rich in corn protein carried the characteristic aroma of roasted corn chips. There was no difference in flavor.

[0362] Compared to the protein-free control, the 5%, 20%, and 40% ethanol and 5% sucrose solutions rich in pea protein all exhibited earthy odors and flavors. Additionally, a pea flavor was detected, and bitterness increased at higher alcohol contents.

[0363] H. Chocolate Spread

[0364] Chocolate spread is a chocolate-flavored spread whose traditional main ingredients are cocoa powder, dairy milk, vegetable oil, and sugar. Traditional chocolate spread is a hard or soft solid at ambient room temperature and melts at temperatures below the melting point of cocoa butter. The product can be used as a spread on bread, crepes, and pancakes, as a icing for cakes and cookies, as a filling for chocolate candies, or as a filling for non-dairy chocolate cakes.

[0365] In one embodiment, dairy milk and dairy products (e.g., ice cream, whey, cream, yogurt, sour cream, or dairy fat) are partially replaced with non-dairy cream prepared as described herein. In one embodiment, the non-dairy cream portion is derived from a single or multiple sources described herein. In one embodiment, dairy milk and dairy products are replaced with any non-dairy milk as described herein. In one embodiment, dairy milk and dairy products are replaced with purified plant proteins as described herein. In one embodiment, dairy milk and dairy products are replaced with soft solids stabilized emulsions made from one or more vegetable oils and one or more purified plant proteins.

[0366] I. Other applications:

[0367] In one embodiment, the non-dairy vegetable shortening portion can be used as a substitute for dairy milk and dairy milk products to make non-dairy milk chocolate bars or non-dairy milk chocolate candies.

[0368] In one embodiment, the non-dairy vegetable cream portion and purified plant protein can be used as substitutes for dairy milk and dairy milk products to make non-dairy milk chocolate bars or non-dairy milk chocolate candies.

[0369] In one embodiment, a non-dairy vegetable shortening portion and plant protein can be used to make chocolate mousse. Traditional main chocolate mousse ingredients are bittersweet or semi-sweet chocolate, dairy butter, and eggs. In one embodiment, dairy butter can be replaced by a non-dairy vegetable shortening portion. In another embodiment, dairy butter and eggs can be replaced by a non-dairy vegetable shortening portion and a stabilizing plant seed storage protein (e.g., pea albumin).

[0370] In one embodiment, a vegetarian consumer product, such as a meat paste analogue, can be prepared. A vegetarian meat paste analogue can be prepared by finely chopping 10 g of fat imitation and heating it in a skillet with finely chopped scallions for 2–3 minutes. Muscle imitation (20 g), prepared without connective tissue imitation fibers, can be chopped into ½-inch cubes and browned in the fat and scallion mixture for another 3–5 minutes. The mixture can be forced through a sieve until homogenized. The still-warm skillet can be rinsed with a tablespoon of Madeira white wine without allowing it to completely evaporate. The liquid from the skillet is added to the homogenized mixture, spices (salt, pepper) are added for flavor, and the mixture is forced through a sieve again. After freezing in the refrigerator (e.g., for 15 minutes), the meat paste is ready to be served.

[0371] In some embodiments, other fats are used to produce leaner or fatter meat pastes than muscle imitations. For example, the meat paste may contain 0.5-10%, about 5%-40%, about 10%-60%, or about 30-70% or >70% fat tissue imitations.

[0372] In some embodiments, a muscle tissue imitation with a higher iron content can be used in meat paste to make it a closer imitation of pork or bird liver paste. For example, the muscle tissue imitation may contain about 1%, about 1.5%, about 2%, or >2% heme protein.

[0373] In some embodiments, muscle tissue imitations with lower iron content can be used in meat paste to make it a closer imitation of bird or fish paste. For example, muscle tissue imitations may contain about 1%, about 0.5%, about 0.2%, or <0.2% heme protein.

[0374] In one embodiment, a vegetarian consumer product, such as a blood sausage analogue, can be produced. Vegetarian blood sausage is made from a blood analogue produced by mixing a solution of heme protein and purified plant protein. For example, 35 ml of a mixture of soy heme protein (120 mg / ml) and pea albumin (100 mg / ml) (which closely resembles a blood composition) can be carefully mixed with a slurry of corn flour in brine (6:5 w / v flour to water ratio). A tablespoon of chopped onion can be sautéed with 10 g of chopped adipose tissue analogue, mixed with a few raisins, and cooled to room temperature before being mixed with the blood / powder mixture. The mixture can be seasoned (e.g., using salt, pepper, parsley, and / or cinnamon), loaded into herbivore sausage casings, and boiled in near-boiling water for approximately 45 minutes. After cooling, the sausage can be eaten as is or further cooked, such as smoked, crisped in an oven, or baked.

[0375] In some embodiments, a muscle imitation may be included in the formulation to mimic meat / blood sausage. In some embodiments, barley, buckwheat, oats, rice, rye, sorghum, wheat, or other grains may be used in blood sausage. In some embodiments, bread, chestnuts, potatoes, sweet potatoes, starch, or other fillers may be added to or substituted for the grains in blood sausage.

[0376] Example

[0377] Example 1: Protein isolation.

[0378] All steps were performed at 4°C or room temperature. Centrifugation was performed at 8000 g for 20 minutes at 4°C or room temperature. The powder was resuspended in a specific buffer, the suspension was centrifuged, and the supernatant was microfiltered through a 0.2 μm PES membrane and then concentrated by ultrafiltration on a 3 kDa, 5 kDa, or 10 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system.

[0379] Once fractionated, all fractions of ammonium sulfate precipitate of interest were stored at -20°C until further use. Before use in experiments, the precipitate was resuspended in 10 volumes of 50 mM potassium phosphate buffer (pH 7.4), +0.5 M NaCl. The suspension was centrifuged, and the supernatant was microfiltered through a 0.2 μm PES membrane and then concentrated by ultrafiltration on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system using 3 kDa, 5 kDa, or 10 kDa molecular weight cutoff PES membranes. Protein composition at individual fractionation steps was monitored by SDS-PAGE, and protein concentration was measured by standard UV-Vis methods.

[0380] (i) Pea albumin: Dried green or yellow pea flour is used as the source of pea albumin. The flour is suspended in 10 volumes of 50 mM sodium acetate buffer (pH 5) and stirred for 1 hour. The dissolved protein is separated from unextracted protein and pea seed debris by centrifugation (8000 g, 20 min) or filtration through a 5-micron filter. The supernatant or filtrate is collected separately. Solid ammonium sulfate is added to this crude protein extract to 50% wt / v saturation. The solution is stirred for 1 hour and then centrifuged. Ammonium sulfate is added to the supernatant from this step to 90% wt / v saturation. The solution is stirred for 1 hour and then centrifuged to collect pea albumin protein in spheroids. The spheroids are stored at -20°C until further use. The protein is recovered from the spheroids and prepared for use as described above, except that the final buffer may contain 0-500 mM sodium chloride.

[0381] In some embodiments, the powder was suspended in 10 volumes of 50 mM NaCl (pH 3.8) and stirred for 1 hour. The dissolved protein was separated from the unextracted protein and pea seed debris by centrifugation (8000 g, 20 min). The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 10 kDa cutoff PES membrane.

[0382] (ii) Pea globulin: Dried green pea flour was used to extract pea globulin protein. The flour was suspended in 10 volumes of 50 mM potassium phosphate buffer (pH 8) and 0.4 M sodium chloride and stirred for 1 hour. The dissolved protein was separated from the pea seed debris by centrifugation. The supernatant was subjected to ammonium sulfate fractionation at 50% and 80% saturation in two steps. 80% globules containing the globulin of interest were stored at -20°C until further use. The protein was recovered from the globules and prepared for use as described above.

[0383] iii) Soybean 7S and 11S globulins: Globulins from soybean flour were separated as follows: First, low-fat / defatted soybean flour was suspended in 4–15 volumes of 10 (or 20) mM potassium phosphate (pH 7.4). The slurry was centrifuged at 8000 rcf for 20 min, or clarified by filtration through a 5 μm filter, and the supernatant was collected. The crude protein extract contained both 7S and 11S globulins. The solution was then filtered through a 0.2 μm filter and concentrated using a 10 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system or by means of anion exchange resin, and then used in experiments. 11S globulins were separated from 7S proteins by isoelectric precipitation. The pH of the crude protein extract was adjusted to 6.4 with dilute HCl, stirred for 30 min–1 h, and then centrifuged to collect the 11S precipitate and the 7S protein in the supernatant. The 11S fraction was resuspended in 10 mM potassium phosphate (pH 7.4), and the protein fraction was microfiltered and concentrated before use.

[0384] Soy protein can also be extracted by suspending defatted soy flour in 4-15 volumes (e.g., 5 volumes) of 20 mM sodium carbonate (pH 9) (or water, pH adjusted to 9 after adding the flour) or 20 mM potassium phosphate buffer (pH 7.4) and 100 mM sodium chloride to reduce undesirable flavors in the purified protein. The slurry is stirred for one hour and centrifuged at 8000×g for 20 minutes. The extracted protein is ultrafiltered and then processed as described above; alternatively, the supernatant is collected and filtered through a 0.2-micron membrane and concentrated using a 10 kDa cutoff PES membrane.

[0385] (iv) Mung Bean 8S Globulin: Mung bean powder was used to extract 8S globulin as follows: First, the powder was suspended in 4 volumes of 50 mM potassium phosphate buffer (pH 7) (+0.5 M NaCl for laboratory-grade purification). After centrifugation, the protein in the supernatant was fractionated by adding ammonium sulfate at 50% and 90% saturation in two separate steps. The precipitate from the 90% fraction contained 8S globulin and was stored at -20°C until further use. The protein was recovered from the globules and prepared for use as described above.

[0386] Mung bean globulin can also be extracted by suspending the powder in 4 volumes of 20 mM sodium carbonate buffer (pH 9) (or water, adjusted to pH 9 after adding the mung bean powder) to reduce undesirable flavors in the purified protein fraction. The slurry is then centrifuged (or filtered) to remove solids, ultrafiltered, and then processed as described above.

[0387] (v) Late embryo enrichment of proteins: Powders (including but not limited to mung bean and soybean powder) were suspended in 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl, and stirred at room temperature for 1 hour, followed by centrifugation. Acid (HCl or acetic acid) was added to the supernatant to a concentration of 5% (v / v), stirred at room temperature, and then centrifuged. The supernatant was heated to 95°C for 15 minutes and then centrifuged. The supernatant was precipitated by adding trichloroacetic acid to a concentration of 25%, centrifuged, and then washed with acetone. The heating and acid washing steps can also be performed in reverse order.

[0388] (vi) Pea gluten precipitate: Dried green pea flour was suspended in 5 × (w / v) 60% ethanol and stirred at room temperature for one hour, then centrifuged (7000 g, 20 min), and the supernatant was collected. The ethanol in the supernatant was evaporated by heating the solution to 85°C and then cooling to room temperature. Ice-cold acetone (1:4 v / v) was added to precipitate the protein. The solution was then centrifuged (4000 g, 20 min), and the protein was recovered in the form of pale beige spheroids.

[0389] (vii) Corn gluten-glucoprotein: Suspend corn gluten concentrate or powder in 5 × (w / v) 60% ethanol, stir at room temperature for one hour, and then centrifuge. Evaporate the ethanol in the supernatant by heating, and then centrifuge the solution to recover the protein in spheroid form.

[0390] (viii) RuBisCO was fractionated from alfalfa greens as follows: First, the leaves were milled in a blender with 4 volumes of cold 50 mM potassium phosphate buffer (pH 7.4 buffer) (0.5 M NaCl + 2 mM DTT + 1 mM EDTA). The resulting slurry was centrifuged to remove debris, and the supernatant (crude solubility) was used for further purification. Proteins in the crude solubility were fractionated by adding ammonium sulfate to 30% (wt / v) saturation. The solution was stirred for 1 hour and then centrifuged. The pellets from this step were discarded, and additional ammonium sulfate was added to the supernatant to reach 50% (wt / v) ammonium sulfate saturation. The solution was centrifuged again after stirring for 1 hour. The pellets from this step contained RuBisCO and were kept at -20°C until use. The protein was recovered from the pellets and prepared for use as described above.

[0391] RuBisCO can also be purified by adjusting the crude solution to 0.1 M NaCl and applying it to an anion exchange resin. Weakly bound protein contaminants are washed with 50 mM potassium phosphate buffer (pH 7.4 buffer) + 0.1 M NaCl. RuBisCO is then eluted with a high ionic strength buffer (0.5 M NaCl).

[0392] The RuBisCO solution was decolorized (pH 7-9) by passing it through a column packed with activated carbon. The colorant bound to the column, while the Rubisco was separated in the filtrate.

[0393] Alternatively, the RuBisCO solution can be decolorized by incubating it with FPX66 (Dow Chemicals) resin packed in a column (or in batch mode). The slurry is incubated for 30 minutes, and then the liquid is separated from the resin. The colorant binds to the resin, and RuBisCO is collected in the column flow-through.

[0394] In some embodiments, RuBisCO is isolated from spinach leaves by first grinding the leaves together with 4 volumes of 20 mM potassium phosphate buffer (pH 7.4 buffer + 150 mM NaCl + 0.5 mM EDTA) in a blender. The resulting slurry is centrifuged to remove debris, and the supernatant (crude dissolved product) is filtered through a 0.2 micrometer membrane and concentrated using a 10 kDa cutoff PES membrane.

[0395] In some embodiments, RuBisCO is extracted from alfalfa or wheatgrass juice powder by mixing the powder with 4 volumes of 20 mM potassium phosphate buffer (pH 7.4 buffer + 150 mM NaCl + 0.5 mM EDTA) in a blender. The resulting slurry is centrifuged to remove debris, and the supernatant (crude dissolved product) is filtered through a 0.2 micrometer membrane and concentrated using a 10 kDa cutoff PES membrane.

[0396] (ix) Leghemoglobin. Soybean root nodules were suspended and dissolved in 20 mM potassium phosphate (pH 7.4), 100 mM potassium chloride, and 5 mM EDTA using a grinder-blender. During this process, leghemoglobin was released into the buffer. The root nodule lysate containing leghemoglobin was removed from cell debris by filtration through a 5-micron filter. In some embodiments, centrifugation (7000 g, 20 min) followed by filtration. The clarified leghemoglobin-containing lysate was then filtered through a 0.2-micron filter and applied to an anion exchange column (High Prep Q; High Prep DEAE, GE Healthcare) on a rapid protein liquid chromatography instrument (GE Healthcare). Leghemoglobin in the eluent was collected and concentrated to the desired concentration on a 3 kDa molecular weight cutoff PES membrane using a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system. The purity (partial abundance) of purified soybean leghemoglobin was analyzed by SDS-PAGE gel electrophoresis: in the solution, soybean leghemoglobin was present at 20-40%, while after anion exchange purification, it was present at 70-80%. In another embodiment, soybean leghemoglobin flocculent from anion exchange chromatography was applied to size exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). Soybean leghemoglobin eluted as two eluates corresponding to the dimer and monomeric substances. The purity (partial abundance) of soybean leghemoglobin was analyzed by SDS-PAGE and determined to be approximately 90-100%. UV-Vis spectral analysis (250-700 nm) showed spectral characteristics consistent with those of heme-loaded soybean leghemoglobin.

[0397] (x) Non-symbiotic hemoglobin from mung beans was cloned into the pJexpress401 vector (DNA2.0) and transformed into *E. coli* BL21. Cells were grown in LB medium containing soy peptone (in place of tryptone), kanamycin, 0.1 mM ferric chloride, and 10 µg / ml 5-aminolevulinic acid. Expression was induced by 0.2 mM IPTG, and cells were grown at 30°C for 20 h. *E. coli* cells expressing mung bean non-symbiotic hemoglobin were collected and resuspended in 20 mM MES buffer (pH 6.5), 50 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2. A small amount of DNase I and a protease inhibitor were added. Cells were lysed by sonication. The lysate was centrifuged at 16,000 g for 20 min and then filtered through a 200 nm filter to remove cell debris. The cell lysate was then loaded onto FF-S resin in a rapid protein liquid chromatography instrument (GE Healthcare). Non-symbiotic hemoglobin from mung beans was bound to an FF-S column and eluted using a sodium chloride gradient (50 mM–1000 mM). The purity (partial abundance) of the non-symbiotic hemoglobin was analyzed by SDS-PAGE, revealing 13% in the *E. coli* lysate and 35% after purification on an FFQ. UV-Vis analysis of the purified protein revealed the spectral characteristics of the heme-binding protein.

[0398] (xi) Heme protein with an N-terminal His6 epitope tag and TEV cleavage site was synthesized, cloned into the pJexpress401 vector (DNA2.0), and transformed into *E. coli* BL21. The transformed cells were grown in LB medium containing soy peptone (as a substitute for tryptone), kanamycin, 0.1 mM ferric chloride, and 10 µg / ml 5-aminolevulinic acid. Expression was induced by 0.2 mM IPTG, and cells were grown at 30°C for 20 h. *E. coli* cells expressing heme protein were collected and resuspended in 50 mM potassium phosphate (pH 8), 150 mM NaCl, 10 mM imidazole, 1 mM MgCl2, 1 mM CaCl2, DNase I, and a protease inhibitor. Cells were lysed by sonication and clarified by centrifugation at 9000 × g. The solution was incubated with NiNTA resin (MCLAB), washed with 5 column volumes (CV) of 50 mM potassium phosphate (pH 8), 150 mM NaCl, and 10 mM imidazole, and eluted with 50 mM potassium phosphate (pH 8), 150 mM NaCl, and 500 mM imidazole. SDS-PAGE and UV-Vis spectroscopy confirmed the expected molecular weight and complete heme loading, respectively.

[0399] In some embodiments, the transformed cells were grown in a seed culture medium containing: 10 g / L glucose monohydrate, 8 g / L potassium monophosphate, 2.5 g / L Sensient Amberferm 6400, 2.5 g / L Sensient Tastone 154, 2 g / L diammonium phosphate, 1 mL / L trace metal mixture (Teknova 1000× Trace Metal Mixture Catalog No. T1001), 1 g / L magnesium sulfate, 0.25 mL 0.1 M ferric chloride solution, 0.5 mL / L Sigma antifoaming agent 204, and 1 mL / L kanamycin sulfate 1000× solution. 250 mL of the culture medium was used in (4)-1 L baffled shake flasks, each inoculated with 0.25 mL of glycerol stock culture from a single vial. The shake flasks were incubated at 37°C for 5.5 h with stirring at 250 RPM. 40 L of seed culture was steam-sterilized in a 100 L bioreactor, cooled to 37°C, and the pH was adjusted to 7.0. After reaching a shake-flask OD of 2.5, 800 mL of shake-flask culture was inoculated. Aeration was supplied to the bioreactor at 40 L / m³, and agitation was maintained at 250 RPM. After 2.2 h of growth, reaching an OD of 2.20, 22 L of culture was transferred to a final 4 m³ culture tank. 3 In the bioreactor. The starting culture medium for the final bioreactor contained the following components, steamed in situ: 1775 L deionized water, 21.75 kg monopotassium phosphate, 2.175 kg diammonium phosphate, 4.35 kg ferric ammonium citrate, 8.7 kg ammonium sulfate, 10.875 kg Senxin Amberferm 6400, and 10.875 kg Senxin Tastone 154. After 30 minutes of steaming, the culture medium components were cooled to 37°C and sterilized before the following were added: 2.145 L of 0.1 M ferric chloride solution, 59.32 kg of 55% w / w glucose monohydrate, 3.9 L of trace metal mixture (Tianhuihua 1000× Trace Metal Mixture Catalog No. T1001), 10.88 L of 200 g / L diammonium phosphate, 36.14 L of 1 M magnesium sulfate, 2.175 L of Sigma-Aldrich defoamer 204, and 2.175 L of kanamycin sulfate 1000× solution. The pH was controlled at 7.0 by adding 30% ammonium hydroxide. (The last sentence appears to be incomplete and possibly refers to a different process.) 3Aeration was supplied at a rate of / min, and dissolved oxygen was maintained at 25% by varying agitation between 60-150 RPM. Additional nutrients were supplied at two time points (EFT=4 and EFT=8). Each addition consisted of 5.5 kg of Senxin Amberferm 6400, 5.5 kg of Senxin Tastone 154, and 4.4 kg of diammonium phosphate in a hyperbaric solution (100 g / L for Amberferm and Tastetone, and 200 g / L for diammonium phosphate). A sterile glucose solution of 55% w / w glucose monohydrate was fed into the bioreactor to maintain a residual glucose concentration of 2-5 g / L. After reaching an OD of 25°C, the temperature was lowered to 25°C, and the culture was induced with 0.648 L of 1 M isopropyl β-D-1-thiogalactopyranoside. The culture was allowed to grow for a total time of 25 hours. At this point, the culture was diluted 1:1 with deionized water, centrifuged, and the filtrate was concentrated to 50% v / v solids. The cell centrifuge filtrate was frozen at -20°C. The filtrate was thawed to 4°C and diluted in 20 mM potassium phosphate (pH 7.8), 100 mM NaCl, and 10 mM imidazole, and homogenized at 15,000 PSI. The homogenized cells were filtered through a tangential flow filter (TFF) at 0.2 μm, and the filtered solubility was directly loaded onto a zinc-fed IMAC column (GE). The binding proteins were washed with 10 column volumes (CV) of 20 mM potassium phosphate (pH 7.4), 100 mM NaCl, and 5 mM histidine, and eluted with 10 CV of 500 mM potassium dihydrogen phosphate and 100 mM NaCl. Eluted leghemoglobin was concentrated and permeated using a 3 kDa molecular weight cutoff PES membrane and TFF. The concentrated sample was reduced with 20 mM sodium dithionite and desalted using G-20 resin (GE). The desalted leghemoglobin sample was frozen in liquid nitrogen and stored at -20°C. The concentration and purity of leghemoglobin were determined by SDS-PAGE and UV-Vis analysis.

[0400] (xi) Oil body protein. Sunflower oil body purification from sunflower seeds. Sunflower seeds were mixed at a ratio of 1:3 wt / v in 100 mM sodium phosphate buffer (pH 7.4), 50 mM sodium chloride, and 1 mM EDTA. The oil body was collected by centrifugation (5000 g, 20 min) and resuspended at a ratio of 1:5 wt / v in 50 mM sodium chloride and 2 M urea, and stirred at 4°C for 30 min. The 2 M urea washing and centrifugation steps were repeated. The oil body collected by centrifugation was resuspended in 100 mM sodium phosphate buffer (pH 7.4) and 50 mM sodium chloride. The centrifugation and washing steps were repeated again, and the final washed oil body fraction was obtained from the last centrifugation step. The oil bodies were resuspended at 10% wt / w in 100 mM sodium phosphate buffer (pH 7.4), 50 mM sodium chloride, and 2% wt / v vegetable oil fatty acid salts, homogenized at 5000 psi, and incubated at 4°C for 12 h. The solution was centrifuged (8000 g, 30 min), the top layer was removed, and the dissolved fraction was collected. SDS-PAGE analysis showed that oil body proteins were the major proteins present in the dissolved fraction. The oil body protein concentration was 2.8 mg / ml.

[0401] (xii) Total Pea Protein: Dried green or yellow pea flour was used to extract total pea protein. The flour was suspended in 10 volumes of 20 mM potassium phosphate buffer (pH 8) and 100 mM sodium chloride and stirred for 1 hour. The dissolved protein was separated from the pea seed debris by centrifugation. The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 10 kDa cutoff PES membrane.

[0402] (xiii) Pea pea globulin and pea pea protein: Dried green or yellow pea flour was used to extract total pea protein as described above. The resulting crude pea mixture was fractionated into pea pea globulin and pea pea protein using ion-exchange chromatography. The material was loaded onto a Q agarose resin and the eluent was collected as the salt concentration changed from 100 mM to 500 mM NaCl. Pea pea globulin was collected at 350 mM NaCl, and pea pea protein was collected at 460 mM NaCl. The collected eluent was concentrated using a 10 kDa cutoff PES membrane.

[0403] (xv) Total protein from lentils: Air-separated lentil powder was used to extract a crude mixture of lentil proteins. The powder was suspended in 5 volumes of 20 mM potassium phosphate buffer (pH 7.4) and 0.5 M sodium chloride and stirred for 1 hour. The dissolved proteins were separated from the unextracted proteins and lentil seed debris by centrifugation (8000 g, 20 min). The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 10 kDa cutoff PES membrane.

[0404] (xvi) Lentil albumin: Air-separated lentil powder was suspended in 5 volumes of 50 mM sodium chloride (pH 3.8) and stirred for 1 hour. The dissolved protein was separated from the unextracted protein and lentil seed debris by centrifugation (8000 g, 20 min). The supernatant was collected, filtered through a 0.2 μm membrane, and concentrated using a 10 kDa cutoff PES membrane.

[0405] (xvii) Chickpea (Garbanzo bean) total protein: Chickpea flour was suspended in 5 volumes of 20 mM potassium phosphate buffer (pH 7.4) and 0.5 M sodium chloride and stirred for 1 hour. The dissolved protein was separated from the unextracted protein and chickpea seed debris by centrifugation (8000 g, 20 min). The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 10 kDa cutoff PES membrane.

[0406] (xviii) Chickpea albumin: Chickpea flour was suspended in 5 volumes of 50 mM sodium chloride (pH 3.8) and stirred for 1 hour. The dissolved protein was separated from the unextracted protein and lentil seed debris by centrifugation (8000 g, 20 min). The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 10 kDa cutoff PES membrane.

[0407] (xix) Amaranth powder dehydrated protein: Amaranth powder was suspended in 5 volumes of 0.5 M sodium chloride (pH 4.0) and stirred for 1 hour. The dissolved protein was separated from the unextracted protein and lentil seed debris by centrifugation (8000 g, 20 min). The supernatant was collected and filtered through a 0.2 μm membrane and concentrated using a 3 kDa cutoff PES membrane. Further concentration of the dehydrated protein from this fraction was obtained by boiling the concentrated protein material, swirling it at 8000 g for 10 min, and collecting the supernatant.

[0408] Example 2: Constructing muscle tissue analogs

[0409] To prepare the muscle tissue replica, 8 ml of mung bean protein solution (114 mg / ml in 20 mM phosphate buffer (pH 7.4) and 400 mM sodium chloride) was mixed with 16 ml of bean leghemoglobin solution (6 mg / ml bean leghemoglobin in 20 mM potassium phosphate, 400 mM NaCl, pH 7.3). The resulting mixture was concentrated using an Amicon rotary concentrator (10 kDa cutoff) to a final concentration of 61 mg / ml mung bean 8S globulin and 6.5 mg / ml bean leghemoglobin. Approximately 400 mg of transglutaminase powder was added to the solution, thoroughly mixed, and aliquoted into two 50 ml Falcon tubes, and incubated overnight at room temperature. The final total protein concentration was 67.5 mg / ml. The muscle tissue replica formed a slightly reddish-brown opaque gel with a small amount (<1 ml) of dark red, venous-blood-colored inclusions.

[0410] Example 3: Fat tissue imitations with increased tensile strength

[0411] Combine 40 ml aliquots of rice bran oil and 40 ml aliquots of mung bean protein (114 mg / ml) in a 250 ml Pyrex beaker. Place the beaker in a water bath and emulsify using a Branson Sonifer 450 sonicator (12 mm tip, 6 min, 60% duty cycle, power level 5).

[0412] In an 18 cm × 18 cm × 2.5 cm synthetic rubber Ikea plastic ice cube tray, 48 mg of electrospun fibers (from connective tissue example 14) were laid longitudinally and as evenly as possible on the bottom of a triangular mold measuring 13.97 cm × 1.27 cm × 1.5875 cm. Approximately 20 ml of rice bran oil / mung bean protein emulsion was then poured onto the top of the fibers. An additional 20 ml of emulsion was then poured into a similarly sized blank mold on the same tray, which served as a control.

[0413] Float the ice cube tray in boiling water for 15 minutes, then remove and let cool to room temperature.

[0414] Using a razor blade, cut each of the resulting gels into three segments, each 4.66 cm long and with a cross-sectional area of ​​1 cm². 2 A Stable Micro Systems TA XTExpress enhanced texture analyzer, connected to a TA-96B probe, was used to assess tensile strength. The tensile strength of the fatty acid imitation containing fibers was 23 kPa, while the tensile strength of the fatty acid imitation without fibers was 20 kPa.

[0415] Example 4: Fat imitations with a high fat percentage

[0416] A fat tissue imitation was cross-linked with 2% transglutaminase (Ajinomoto Activa® TI). The imitation contained a protein-oil emulsion formed with 3.3% wt / v pea globulin, 70% wt / v oil of an equal mixture of coconut oil, cocoa butter, olive oil, and palm oil, and 0.5% wt / v lecithin. After drainage and dehydration, the resulting gel was moderately soft and had a confirmed fat content of 75% (wt / wt).

[0417] The adipose tissue matrix containing a protein-oil emulsion was formed with 1.6 wt / v Rubisco and 80 wt / v cocoa butter. The resulting gel was soft.

[0418] Example 5: Method for preparing adipose tissue replicas

[0419] If necessary, melt the oil by heating to room temperature or by gentle heating. If the oil is solid at room temperature, keep it close to its melting point for the remainder of the procedure. Obtain the protein according to the prescribed protocol (see Example 1). Weigh the lecithin and resuspend it in water, then sonicate to produce a homogeneous solution. Combine the components in the prescribed ratio and, if necessary, bring them to volume with a buffer solution (20 mM sodium phosphate (pH 7.4) with 50 mM sodium chloride added), then subject them to homogenization or sonication to control the particle size within the emulsion. Then, gel the emulsion by either: (a) heating / cooling; (b) cross-linking with transglutaminase; or (c) heating / cooling followed by the addition of transglutaminase. Prepare control samples (neither heated / cooled nor cross-linked with transglutaminase) for comparison. Emulsions stabilized by heating / cooling are prepared by placing the emulsion in a 90–100°C water bath for five minutes, then allowing the sample to cool slowly to room temperature. Emulsions stabilized by transglutaminase crosslinking were prepared by adding transglutaminase to 2% wt / v and incubating at 37°C for 12–18 hours. Emulsions stabilized by heating / cooling followed by transglutaminase addition were prepared by first undergoing a heating / cooling process, and then adding the enzyme after the sample had cooled to room temperature. All emulsions were incubated at 37°C for 8–12 hours.

[0420] Example 6: Methods for analyzing adipose tissue imitations

[0421] After different gelling treatments, the gel emulsions were brought to room temperature for evaluation. The total volume of the gel emulsion and the volume of water and / or oil (if the gel emulsion was not in a single phase) were recorded. The firmness of the adipose tissue replica was evaluated by gently poking the gel emulsion. Cooking experiments were conducted by transferring the mass to a heated surface and measuring the liquid temperature immediately after cooking.

[0422] Example 7: Fat imitations of beef fat

[0423] The adipose tissue replica was prepared by gelling a purified mung bean 8S protein solution emulsified with equal volumes of cocoa butter, coconut butter, olive oil, and palm oil. The mung bean 8S protein was purified as described in Example 1 and had a concentration of 140 mg / ml in 20 mM potassium phosphate (pH 7.4) and 400 mM NaCl. The fat mixture was prepared by melting individual fats from a solid to a liquid state at 45°C for 30 minutes. The liquid fats (cocoa butter, coconut butter, olive oil, and palm oil) were then mixed at a 1:1:1:1 (v / v) ratio. The protein-fat emulsion was formed by mixing a 70% v / v liquid fat mixture with 4.2% wt / v mung bean 8S protein and 0.4% wt / v soy lecithin, and emulsifying by vortexing for 30 seconds followed by sonication for 1 minute. After homogenization, the fat-protein emulsion was observed to be in a single liquid phase, as determined by visual inspection.

[0424] One adipose tissue replica emulsion was stabilized by cross-linking with 0.2% wt / v transglutaminase at 37°C for 12 hours. Another adipose tissue replica was stabilized by protein gelation through heating to 100°C in a water bath followed by cooling to ambient room temperature. The resulting adipose tissue replicas were in a single phase. The adipose tissue replica matrix formed by transglutaminase was a softer solid than that formed by heat / cooling-induced gelation.

[0425] Example 8: Fat imitations of Kobe beef fat

[0426] The adipose tissue replica was prepared by gelling a purified pea globulin protein emulsion with equal amounts of cocoa butter, coconut butter, olive oil, and palm oil. The pea globulin protein was purified as described in Example 1 and its concentration in 20 mM potassium phosphate (pH 8) and 400 mM NaCl was 100 mg / ml. The fat mixture was prepared by melting individual fats from a solid to a liquid state at 45°C for 30 minutes. The liquid fats (cocoa butter, mango butter, olive oil) were then mixed at a 2:1:1 (olive oil:cocoa butter:mango butter) v / v ratio. The protein-fat emulsion was formed by quantitatively mixing the liquid fat mixture with a 5% wt / v solution of pea globulin protein at a 1:1 ratio and emulsifying for 30 seconds using a handheld homogenizer at maximum setting. After homogenization, the fat-protein emulsion was in a single liquid phase, as determined by visual observation. The emulsion was stabilized by cross-linking with 0.2% wt / v transglutaminase at 37°C for 12 hours. The resulting adipose tissue imitation was a soft solid in a single phase and had a salty flavor.

[0427] Example 9: Adipose tissue imitation with the fatty acid distribution of beef:

[0428] The adipose tissue replica was prepared by gelling a purified pea globulin protein emulsion with equal amounts of cocoa butter, mango butter, olive oil, and rice bran oil. The pea globulin protein was purified as described in Example 1 and its concentration in 20 mM potassium phosphate (pH 8) and 400 mM NaCl was 100 mg / ml. The fat mixture was prepared by melting individual fats from a solid to a liquid state at 45°C for 30 minutes. The liquid individual fats (cocoa butter, mango butter, olive oil, and rice bran oil) were then mixed at a 1:1:1:1 v / v ratio. The protein-fat emulsion was formed by mixing a 50% v / v liquid fat mixture with 5% wt / v pea globulin protein and emulsifying for 30 seconds using a handheld homogenizer at maximum setting. After homogenization, the fat-protein emulsion was in a single liquid phase, as determined by visual observation. The emulsion was stabilized by cross-linking with 0.2% wt / v transglutaminase at 37°C for 12 hours. The resulting adipose tissue imitation was a soft solid in a single phase and had a salty flavor.

[0429] Example 10: An adipose tissue imitation in which the hardness of the adipose tissue under refrigeration and ambient temperature is controlled by the melting temperature of the fat in the adipose tissue imitation.

[0430] Fat tissue imitations made from a stable emulsion of Rubisco and sunflower oil are softer than those made from a stable emulsion of Rubisco and cocoa butter. The fat tissue imitations were formed with 0.18%, 1.6%, and 2.4% wt / v Rubisco and 70%, 80%, and 90% v / v sunflower or cocoa butter, respectively. Each fat tissue imitation containing cocoa butter was harder than the corresponding imitation formed with sunflower oil. Fat tissue imitations containing 0.18%, 1.6%, and 2.4% wt / v Rubisco and 70%, 80%, and 90% v / v cocoa butter were solid at room temperature but melted near oral temperature. In fat tissue imitations formed with varying concentrations of Rubisco (0.18, 1.6, 1.9% wt / v) and 70-80% v / v sunflower oil, the imitations became harder with increasing protein content in the fat tissue imitation matrix. The adipose tissue simulant with 0.18% wt / v Rubisco is extremely soft; the adipose tissue simulant with 1.6% wt / v Rubisco is soft; and the adipose tissue simulant with 1.9% wt / v RuBisco is of medium firmness.

[0431] Fat tissue imitations made from a stable emulsion of mung bean 8S protein and sunflower oil are softer than those made from a stable emulsion of mung bean 8S protein and cocoa butter. The fat tissue imitations were formed using 2%, 1%, and 0.5% wt / v mung bean 8S protein with 70%, 80%, and 90% v / v sunflower or cocoa butter, respectively. Each fat tissue imitation containing cocoa butter was harder than the corresponding imitation formed with sunflower oil.

[0432] Fat tissue imitations made from a stable emulsion of mung bean 8S protein and canola oil are softer than their counterparts made from an equal mixture of mung bean 8S protein and coconut oil, cocoa butter, olive oil, and palm oil. The fat tissue imitations were formed using 1.4% wt / v mung bean 8S protein with 50%, 70%, and 90% v / v sunflower oil or other oil mixtures. Each fat tissue imitation containing an oil mixture was harder than the corresponding imitation formed with sunflower oil. Fat tissue imitations containing 1.4% wt / v mung bean 8S protein with equal mixtures of 50%, 70%, and 90% v / v coconut oil, cocoa butter, olive oil, and palm oil were solid at room temperature but melted near oral temperature.

[0433] Fat tissue imitations made from a stable emulsion of soy protein and sunflower oil are softer than those made from a stable emulsion of soy protein and cocoa butter. The fat tissue imitations are formed using 0.6%, 1.6%, and 2.6% wt / v soy with 50%, 70%, 80%, and 90% v / v sunflower or oil mixtures. Each fat tissue imitation containing an oil mixture is harder than the corresponding imitation formed with sunflower oil. Fat tissue imitations containing 0.6%, 1.6%, and 2.6% wt / v soy protein with 50%, 70%, 80%, and 90% v / v cocoa butter are solid at room temperature but melt near oral temperature.

[0434] Example 11: Cooking of adipose tissue imitations: The structure of the adipose tissue matrix controls the melting point during cooking.

[0435] The stabilized protein-oil emulsions constructed as described in Examples 5 and 6 above, and the adipose tissue formed with 2% w / v Rubisco and 50%, 70% and 90% v / v cocoa butter melt at higher temperatures when formed after heat / cool denaturation, and melt at lower temperatures when formed by cross-linking with transglutaminase.

[0436] Example 12: Cooking adipose tissue: The arrangement and structure of proteins and fats within the adipose tissue matrix control the amount of fat released and retained by the adipose tissue imitation during cooking.

[0437] During cooking of adipose tissue imitation matrices containing protein-oil emulsions, formed with 2% w / v Rubisco and 50%, 70%, or 90% v / v cocoa butter, adipose tissue imitations formed after heating / cooling denaturation retain more adipose tissue imitation blocks after cooking than those formed by cross-linking with transglutaminase. The released substance is liquid and oily.

[0438] During the cooking of adipose tissue imitation matrices containing protein-oil emulsions, and those formed with 2.6% and 0.6% w / v soy protein and 50%, 70%, or 90% v / v cocoa butter, more adipose tissue imitation blocks are retained when formed after heat / cool denaturation than when formed by cross-linking with transglutaminase. The released substance is liquid and exhibits an oily appearance.

[0439] Example 13: Cooked adipose tissue imitations: Substances in which the concentration of specific proteins within the adipose tissue matrix is ​​controlled and maintained after cooking.

[0440] A series of adipose tissue imitators, constructed from 1.4% wt / v mung bean 8S protein, 90% wt / v canola oil, and 0.45% wt / v soybean lecithin, were homogenized, and sunflower oil body protein was added to the emulsion at varying concentrations, ranging from 1:10 to 1:10. 6 Changes in the molar ratio of oil body protein to triglycerides. When the ratio of oil body protein to oil was higher in the adipose tissue imitation, an increase in bulk retention was observed after cooking.

[0441] A series of adipose tissue imitations prepared with varying concentrations of Rubisco and 70% v / v sunflower oil retained more bulk after cooking as the concentration of Rubisco increased. After cooking, the adipose tissue imitation containing 0% wt / v Rubisco completely melted, while the adipose tissue imitation containing 1.9% wt / v Rubisco retained 10% bulk, and the adipose tissue imitation containing 2.4% wt / v Rubisco retained 20% bulk.

[0442] Example 14: Connective tissue analogues

[0443] Connective tissue fiber imitations were manufactured by electrospinning a solution containing 22.5 mg / ml of lentinan (400 mM sodium chloride), 6.75% w / v poly(vinyl alcohol), and trace amounts of sodium azide (0.007% w / v). The resulting solution was pumped from a 5 ml syringe at 3 µl / min through a Teflon tube and a blunt 21-gauge needle using a syringe pump. The needle was connected to the positive terminal of a 17 kV Spelman CZE 30 kV high-voltage power supply and fixed 12 cm from an aluminum cylinder (approximately 12 cm long and 5 cm in diameter) encased in aluminum foil. A roller was connected to a mandrel rotated at approximately 220 rpm by an IKA RW20 motor. The mandrel was connected to the grounding terminal of the high-voltage power supply. The protein / polymer fibers accumulated on the foil were scraped off and used as connective tissue imitations.

[0444] Example 15: Extending the lifespan of reduced (heme-FE2+) soybean leghemoglobin

[0445] Horse myoglobin was purchased from Sigma. The myoglobin was resuspended at 10 mg / ml in 20 mM potassium phosphate (pH 8.0) and 100 mM NaCl. SDS-PAGE analysis showed that the protein purity was approximately 90%.

[0446] Soybean leghemoglobin was purified from soybean root nodules using ammonium sulfate precipitation (60% / 90% fractionation) as detailed in Example 1. The resuspended 90% ammonium sulfate leghemoglobin was further purified by anion exchange chromatography (HiTrap Q FF5 mL FPLC column) in 20 mM potassium phosphate (pH 8.0) and 100 mM NaCl. Leghemoglobin was eluted into the eluent. SDS-PAGE analysis showed a protein purity of approximately 70%. The leghemoglobin was buffer-exchanged in 20 mM potassium phosphate (pH 7.4) and 100 mM NaCl, and concentrated to 10 mg / mL using a 3.5 kDa membrane concentrator.

[0447] Carbon monoxide treatment: Myoglobin in 10 mg / ml of 20 mM potassium phosphate (pH 8.0) and 100 mM NaCl, and lentiglobin in 10 mg / ml of 20 mM potassium phosphate (pH 7.4) and 100 mM NaCl were first degassed under vacuum at 4°C for 1 hour, and then perfused with carbon monoxide gas for 2 minutes. Then, by adding 10 mM sodium dithionite and 0.1 mM sodium hydroxide for 2 minutes, the globulins were removed from the heme-Fe... 3+ Reduced to heme-Fe 2+ Sodium dithionite and sodium hydroxide were removed from the protein solution by size exclusion chromatography (PD-10 desalting column) in 20 mM potassium phosphate (pH 8.0), 100 mM NaCl, and 20 mM potassium phosphate (pH 7.4), 100 mM NaCl, respectively. Globulin eluates showing a peak red color as evaluated by visual assessment were collected. UV-Vis spectroscopy confirmed the presence of heme-Fe in both proteins. 2+ The presence of the gas was observed. After desalting, the solution was re-perfused with gas for 2 minutes. The color of the solution was evaluated by acquiring UV-Vis spectra (250 nm–700 nm) every 20 minutes using a nanodrop spectrophotometer. The control sample was not treated with carbon monoxide.

[0448] Sodium nitrite treatment: Myoglobin in 10 mg / ml of 20 mM potassium phosphate (pH 8.0) and 100 mM NaCl and stigmoglobin in 20 mM potassium phosphate (pH 7.4) and 100 mM NaCl were removed from heme-Fe by adding 10 mM sodium dithionite and 0.1 mM sodium hydroxide for 2 minutes. 3+ Reduced to heme-Fe 2+Sodium dithionite and sodium hydroxide were removed from the protein solution by size exclusion chromatography (PD-10 desalting column) in 20 mM potassium phosphate (pH 8.0), 100 mM NaCl, and 20 mM potassium phosphate (pH 7.4), 100 mM NaCl, respectively. Globulin eluates showing a peak red color, as evaluated by visual assessment, were collected. UV-Vis spectroscopy confirmed the presence of heme-Fe in both proteins. 2+ The presence of the state was observed. Sodium nitrite was then added from 100 mM nitrite in phosphate buffer (pH 7.4) to a final concentration of 1 mM. The heme-Fe group was tracked as a function of time using a spectrophotometer, recording the UV-Vis spectrum (250–700 nm). 2+ The lifetime of the state. The control sample was not treated with sodium nitrite.

[0449] In Microsoft Excel, the amplitude of the absorbance peak at 540 nm was plotted to analyze the heme-Fe content of myoglobin and beta-hemoglobin samples treated with carbon monoxide and sodium nitrite. 2+ Lifetime data analysis was performed. The baseline of the 540 nm absorbance was determined by the state of the UV-Vis spectrum of the globulin solution before the addition of any additives, dithionite reduction, or desalting. A built-in curve fitting function was used to generate an exponentially optimal line fit, the exponent of which is directly related to the half-life of the peak amplitude.

[0450] Heme-Fe in myoglobin and bean hemoglobin solutions 2+ The lifetimes of the heme-Fe state and the accompanying red color in the absence of carbon monoxide and sodium nitrite are approximately 6 hours and 4 hours, respectively. The addition of sodium nitrite reduces the heme-Fe... 2+ The lifespan of the heme-Fe complex is extended to over seven days. Adding carbon monoxide increases the heme-Fe complex's red color. 2+ The state and the accompanying redness extend the lifespan to more than two weeks.

[0451] Example 16: Preparation of meat imitations by varying the particle size of individual tissue imitation units to control odor generation during cooking.

[0452] Muscle tissue imitations and adipose tissue imitations were prepared separately and then combined into meat tissue imitations, allowing for size variations in individual tissue imitation units to control odor generation during cooking. Individual fat, muscle, and connective tissue imitations were constructed as follows.

[0453] The muscle tissue replica was prepared as in Example 2. The muscle tissue replica formed a slightly reddish-brown, opaque gel with a small amount (<1 ml) of dark red, venous-blood-colored inclusions. The connective tissue replica was prepared as in Example 14. The adipose tissue replica was prepared as in Example 7.

[0454] Meat imitations with a lean-to-fat ratio of 85 / 15 were prepared by combining individual muscle, connective, and adipose tissues to allow for particle size variation in the individual tissue imitations. (a) 2.1 g muscle imitation with 0.9 g of large fat imitations of 5–10 mm size (“coarse mixture”); (b) 2.1 g muscle imitation with 0.9 g of fat imitations chopped to 2–3 mm size (“fine mixture”); and (c) 2.1 g muscle imitation with 0.9 g of thoroughly blended fat imitations <1 mm size (“blended mixture”). A “muscle only” control sample contained 3 g of muscle imitation alone. A “fat only” control sample contained 3 g of fat imitations in the form of particles of 5–10 mm size. Meat, muscle, and adipose tissue samples were cooked in sealed glass vials at 150°C for 10 minutes. The odor characteristics of the samples were analyzed by a panel of testers and by GC-MS.

[0455] Sensory olfactory analysis of meat imitation samples by a group of testers showed that the size of individual tissue units and their degree of mixing within the meat tissue imitations were related to the generation of different odors. Individually cooked muscle tissue imitations generated odors associated with store-bought gravy, a slightly orange tang, and star anise. Individually cooked fat tissue imitations generated odors associated with musty, putrid, and sweet smells. Cooked meat tissue imitations (coarse-grained) generated odors associated with store-bought gravy, sweetness, a slightly musty smell, and star anise. Cooked meat tissue imitations (fine-grained) generated odors associated with soy sauce, musty smell, a slightly putrid smell, and beef broth. Cooked meat tissue imitations (very fine-grained) generated odors associated with sweetness, mustiness, and soy sauce. All samples except the fat tissue imitation generated odors associated with roasted meat, however, in varying intensities.

[0456] Analysis of GCMS data indicates that the size of individual tissue units and their degree of mixing within meat tissue imitations have a profound effect on the formation of aromatic compounds during cooking. Specifically, these compounds contribute to the formation of aromatic compounds associated with: fruity / green bean / metallic flavors (2-pentyl-furan); nutty / green flavors (4-methylthiazole); peanut butter / mold flavors (pyrazine, ethyl); raw potato / baked / earthy flavors (pyrazine, 2,3-dimethyl); sour flavors (acetic acid); spicy / caramel / almond flavors (5-methyl-2-furanaldehyde); creamy flavors (butyrolactone); sweet flavors (2,5-dimethyl-3-(3-methylbutyl)pyrazine); and fruity / aged beer flavors (2-cyclopenten-1-one, 2-hydroxyl). -3-methyl); musty / nutty / coumarin / licorice / walnut / bread (3-acetyl-1H-pyrrolidone); coconut / woody / sweet (pantolytic acid lactone); penetrating (1-H-pyrrole-2-2-carboxaldehyde, 1-methyl); minty (caprolactam); caramel (4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl). A variety of aromatic compounds associated with the odor are only present in mixed meat imitations, but not in individual tissue imitations. For example, some other aromatic compounds associated with the aromas of gasoline (nonane, 2,6-dimethyl), petroleum (3-hexene, 3-methyl); acidic / rotten / fishy (pyridine); mild / woody / yogurt (acetoin); fatty / honey / orange (octanal); pungent / sweet / caramel (2-acetone, 1-hydroxy); and nutty / burnt green (vinylpyrazine) are present only in individual meat imitations but do not accumulate in mixed meat imitations. Furthermore, the level at which all of the above compounds accumulate during cooking depends on the size of the tissue units and how they are mixed (coarse, fine, or very fine (blended)).

[0457] Similar to meat tissue imitations, it was found that the structure and particle size of beef tissue modulate its response to cooking. For example, the flavor of meat is modulated by particle size. Beef samples were prepared as follows: samples of beef muscle and beef fat were separately cut with a knife, and: (a) “Grinding” was performed, in which the tissue blocks cut by the knife were passed through a standard meat grinder. An 80 / 20 (wt / wt) lean / fat ground beef sample was prepared by mixing the muscle and fat tissue blocks in an appropriate ratio prior to grinding. This sample preparation was referred to as a “fine-size particle mixture”. (b) The particle size of the ground tissue was further reduced by cryogenic grinding of the tissue in liquid nitrogen and crushing it into an extremely fine powder (particle size <1 mm) using a mortar and pestle. This sample preparation was referred to as an “extremely fine-size particle mixture”. All samples were cooked at 150°C for 10 minutes in sealed glass vials. The odor characteristics of the samples were analyzed by a group of testers and by GC-MS (as described in Example 1). The “muscle only” control sample contained 3 g of muscle tissue alone. The “fat only” control sample contained 3 g of fat tissue alone. The ground beef sample contained 3 g of an 80 / 20 (wt / wt) muscle / fat mixture.

[0458] Sensory olfactory analysis of beef samples by a group of testers showed that the size of individual tissue units and their degree of mixing within the sample were related to the generation of different odors. Cooked beef muscle alone generated typical odors associated with cooked ground beef. Cooked fat tissue imitations alone generated a slightly sweet odor and an odor associated with grilled mushrooms. Cooked ground beef with a “mixture of fine-sized particles” generated typical odors associated with cooked ground beef, with the characteristic slightly sweet odor of cooked fat present. Cooked ground beef with an “extremely fine-sized particle mixture” generated odors associated with cooked ground beef, but the characteristic slightly sweet odor of cooked fat was not detected.

[0459] Analysis of GCMS data indicates that the particle size of individual tissue units plays a role in the formation of aromatic compounds during cooking. Specifically, the formation and / or amount of various aromatic compounds by individual tissue samples or ground beef samples vary with tissue particle size. Some aromatic compounds that differ between fine and very fine-grained muscle tissues include: 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl; 3-acetyl-1H-pyrrololine; 1-(6-methyl-2-pyrazinyl)-1-ethylone; 2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2-furanaldehyde, 5-methyl; acetic acid; vinylpyrazine; pyrazine, 2,3-dimethyl; 2-propanone, 1-hydroxy; octanal; acetoin; 4-methylthiazole; pseudo-2-pentyl-furan; 2-pentyl-furan. Some aromatic compounds that differ between fine and very fine adipose tissue: triethylene glycol: 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl; caprolactam; 1-(6-methyl-2-pyrazinyl)-1-ethylone; 2-cyclopenten-1-one, 2-hydroxy-3-methyl; butyrolactone; 2-furanaldehyde, 5-methyl; ethylone, 1-(2-furanyl); acetic acid; 2-ethyl-5-methylpyrazine; pyrazine, 2,3-dimethyl; pyrazine, ethyl; octanal; acetoin; 4-methylthiazole; pseudo-2-pentyl-furan; pyridine; nonane, 2,6-dimethyl. Some aromatic compounds that differ between fine and very fine 80 / 20 muscle / fat samples: 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl; caprolactam; 1H-1-pyridine; 3-carboxynitrile, 4-ethyl-2-oxo-2,5; 1-H-pyrrole-2-2-carboxaldehyde, 1-methyl; 2-cyclopenten-1-one, 2-hydroxy-3-methyl; 2,5-dimethyl-3-(3-methylbutyl)pyrazine; butyrolactone; 2-furanaldehyde, 5-methyl; acetone, 1-(2-furanyl); acetic acid; vinylpyrazine; 2-ethyl-5-methylpyrazine; pyrazine, 2,3-dimethyl; 2-propanone, 1-hydroxy; octanal; acetoin; 2-pentyl-furan.

[0460] Example 17: The contribution of leghemoglobin to flavor

[0461] Beef flavor and aroma can be produced in non-beef consumer products by adding heme protein. Ground chicken (90% lean, 10% fat) was tightly bound with coarse cotton cloth and mixed with recombinant soybean leghemoglobin or recombinant bovine myoglobin to a final concentration of 0.5–1.0% wt / wt. The recombinant heme protein was expressed in *E. coli* and purified by nickel affinity purification as described in Example 1. The heme protein was reduced with 20 mM sodium dithionite before being mixed with the chicken. Sodium dithionite was removed from the sample using a Zeba desalting column (Thermo Scientific). The soybean leghemoglobin was desalted to 20 mM potassium phosphate (pH 7.4) and 100 mM NaCl. The myoglobin was desalted to either 20 mM potassium phosphate (pH 7.4) and 100 mM NaCl or 20 mM sodium citrate (pH 6.0) and 100 mM NaCl. The reduced heme protein sample was divided into two portions, and half of the sample was bubbled with carbon monoxide (CO) for 2 minutes. After mixing the heme protein sample with minced chicken, the mixture was poured into round molds and incubated overnight at 4°C. The rounds were baked in an oven or pan-fried at 165°C until each round reached an internal temperature of 165°C. A panel of judges tasted rounds containing chicken alone, chicken mixed with buffer, or chicken or beef (90% lean, 10% fat) mixed with bean heme or myoglobin + / - CO. Judges completed a questionnaire evaluating the aroma and flavor of each round. Judges rated the aroma and flavor of each round as follows: 1 = chicken, 2 = chicken + slight beef, 3 = 50 / 50 chicken + beef, 4 = beef + slight chicken, 5 = beef. Table 2 shows the average score obtained for each round. Percentages indicate the final concentration of heme protein wt / wt (abbreviations: KP = 20 mM potassium phosphate (pH 7.4), 100 mM NaCl buffer; NC = 20 mM sodium citrate (pH 6.0), 100 mM NaCl buffer; n / d = not determined). Adding recombinant lentinan or myoglobin to chicken results in increased beef odor and flavor. The perceived levels of beef flavor and odor increase with the content of myoglobin and lentinan. Lentinan and myoglobin provide the same benefits to flavor and odor.

[0462] Table 2

[0463]

[0464] Example 18: Preparation of non-dairy milk wine

[0465] The liqueur was made from sunflower butter fraction, RuBsiCo, and whiskey (Jameson). The sunflower butter fraction was prepared by mixing sunflower seeds with a 40 mM sodium phosphate (pH 8.0) and 400 mM sodium chloride buffer. The seed fragments were collected by centrifugation at 5000 g for 20 minutes to form pellets. The butter fraction was then resuspended in a 10 mM potassium phosphate (pH 7.4) buffer and collected by centrifugation at 5000 g for 20 minutes. Rubsico was purified as described in Example 1 and used as a 25 mg / ml stock solution in 20 mM potassium phosphate (pH 7.0) and 150 mM NaCl.

[0466] In one example, the liqueur was made as follows: 11.4% wt / v sunflower butter, 40% wt / v Jameson whiskey, 0.4–1.6% wt / v Rubsico, 0.5% wt / v vanilla extract, 0.5% wt / v espresso, and 1.5% wt / v chocolate powder. The resulting mixture was homogenized at 5000 psi.

[0467] In another example, the liqueur is made from sunflower butter and whiskey (Jameson) and sugar: 11.4% wt / v sunflower butter, 40% v / v Jameson whiskey, 0.5% v / v vanilla extract, 0.5% v / v espresso, 1.5% wt / v cocoa powder and 8% wt / v sugar.

[0468] The beverage is served at ambient room temperature or refrigerated. The resulting beverage is beige to light chocolate in color. Tasting results indicate that the beverage has a very creamy, alcoholic flavor similar to dairy liqueur. Refrigerated products are preferred. The emulsion is stable at room temperature for at least one week (maximum time tested).

[0469] Example 19 - Chocolate Spread

[0470] The chocolate spread is made from 34% (wt / wt) cane sugar, 22% (wt / wt) cocoa powder, 19% (wt / v) pistachio cream portion, and 12% (v / v) almond milk. The cane sugar and cocoa powder (Ghirardelli) were commercially available. The almond skim milk was made as follows: Almonds were blanched by immersing them in 100°C water for 30 seconds. The blanched nuts were collected and cooled by immersion in ice water. The almonds were air-dried. The almonds were then rehydrated by immersion in 2°C water for 16 hours. The rehydrated almonds were drained, mixed with water at a 1:2 wt / v ratio, and blended in a Vitamix blender for 5 minutes. The blended slurry was collected in a refrigerated container and cooled by stirring with a freeze-drying rod. After the slurry cooled to 10°C, it was placed at 2°C for up to 12 hours. Almond skimmed milk and cream were separated by centrifugation at 7480 g for 30 minutes at 4°C. The almond milk separated into three layers: dense spherical particles of insoluble solids; a transparent to translucent aqueous layer (referred to as "almond skimmed milk"); and a lighter, creamy, opaque layer (referred to as "almond cream"). The almond milk was then sterilized at 75°C for 16 seconds, refrigerated, and stored at 2°C.

[0471] The pistachio cream fraction was prepared as follows: Pistachios were mixed with a 100 mM sodium carbonate (pH 9.5) buffer containing 400 mM sodium chloride and 1 mM EDTA, and then centrifuged at 5000×g for 20 minutes. The cream fraction was collected and washed again in the same buffer. After centrifuging at 5000 g for 20 minutes, the cream fraction was collected and washed again in a 20 mM sodium phosphate (pH 7.4) buffer containing 50 mM sodium chloride and 1 mM EDTA. After centrifuging at 5000 g for 20 minutes, the cream fraction was collected, washed again in a neutral (pH 7.4) buffer, and centrifuged at 5000 g for 20 minutes. The pistachio cream fraction was collected and stored at 4°C.

[0472] The chocolate spread is made as follows: Melt the cane sugar in the almond milk, then add the cocoa powder to the sugar-milk mixture while stirring and let it melt. Next, add the sugar, milk, and cocoa to the pistachio cream portion and stir together. Pour the resulting mixture into molds and let it sit in the refrigerator or freezer for 24 hours.

[0473] In another example, the chocolate spread is made from 42% (wt / wt) cane sugar, 27% (wt / wt) cocoa powder, 31% (wt / v) sunflower butter portion, and 23% (v / v) almond skim milk. All ingredients and procedures except for the sunflower butter portion are as described above.

[0474] The sunflower butter fraction was produced as follows: Sunflower seeds were mixed with 5 times their weight volume of a 40 mM potassium phosphate solution (pH 8) containing 400 mM NaCl and 1 mM EDTA, then cooled to 20°C and centrifuged. The top butter layer was removed and mixed with the same buffer solution, then heated at 40°C for 1 hour. The slurry was cooled to 20°C and centrifuged; the butter layer was removed and mixed with 5 times its weight volume of a 100 mM sodium carbonate solution (pH 10) containing 400 mM NaCl, then centrifuged. The top layer was then mixed with 5 times its weight volume of water and centrifuged again. The resulting butter fraction was extremely buttery, white, and neutral in taste.

[0475] In another example, the chocolate spread is made from 37% (wt / wt) cane sugar, 23% (wt / wt) cocoa powder, 13% (wt / v) sunflower butter portion and 7% (wt / wt) cocoa butter and 20% v / v almond skim milk.

[0476] In another example, the chocolate spread is made from 37% (wt / wt) cane sugar, 23% (wt / wt) cocoa powder, 13% (wt / v) sunflower butter portion and 7% (wt / wt) coconut oil and 20% v / v almond skim milk.

[0477] In another example, the chocolate spread is made from 37% (wt / wt) cane sugar, 23% (wt / wt) cocoa powder, 13% (wt / v) sunflower butter portion, 7% (wt / wt) palm oil, and 20% v / v almond skim milk.

[0478] In another example, the chocolate spread is made by mixing equal amounts of the spread described above and pistachio oil with 1.8% (wt / wt) cane sugar, 1.13% (wt / wt) cocoa powder, 88% (wt / v) pistachio butter portion and 9% almond skim milk.

[0479] In another example, the chocolate spread is made from 8.5% (wt / wt) cane sugar, 5.4% (wt / wt) cocoa powder, 81% (wt / v) sunflower butter portion and 4.6% (v / v) almond skim milk by mixing the chocolate spread described above with sunflower butter in a 2:1 ratio.

[0480] Visual and textural examination of all products indicated that they formed a stable, solid, creamy spread at room temperature. All products remained firm and solid at refrigerated and refrigerator temperatures. Tasting results for all products indicated a highly desirable, rich, creamy texture that melted in the mouth, receiving positive feedback from tasters. Individual taster preferences varied regarding liking or disliking of pistachio or coconut flavors, and preferences for more or less sweetness and more or less cocoa flavor. One particular sample was described as resembling a milk chocolate spread, with sunflower butter contributing a neutral flavor.

[0481] Example 20 - Generating adipose tissue replicas

[0482] Adipose tissue replicas are produced using the ingredients listed in Table 3.

[0483] Table 3

[0484]

[0485] Lecithin (SOLEC) TM F-dehydrated soybean lecithin, The Solae Company, St. Louis, Missouri (MO) was prepared at a concentration of 50 mg / ml in 20 mM potassium phosphate, 100 mM NaCl (pH 8.0) buffer and sonicated (Sonifier simulated cell disruptor model 102C, BRANSON Ultrasonics Corporation, Danbury, Connecticut) for 30 seconds.

[0486] Pea pea globulin protein is supplied in liquid form containing approximately 140 mg / g pea globulin in a 20 mM potassium phosphate, 100 mM NaCl (pH 8.0) buffer.

[0487] Coconut oil (Shay and Company, Milwaukie, Oregon (OR)) and cocoa butter (Cocoa Family, Duarte, California (CA)) are melted by heating to 50-70°C, then combined and kept warm until needed.

[0488] Mix the buffered protein solution, additional buffer, and lecithin slurry in a 32-ounce metal beaker and equilibrate to room temperature. Form an emulsion using a handheld homogenizer (OMNI Model GLH, OMNI International, Kennesaw, Georgia, equipped with a G20-195ST 20 mm generator probe). Place the homogenizer probe into the protein-lecithin mixture and turn it on to speed 4. Then, slowly add warm oil over a process of approximately 2 minutes while continuously moving the probe back and forth in the mixture.

[0489] The emulsion was then heat-set by placing the metal beaker in a 95°C water bath. Using a clean spatula, the emulsion was stirred every 20 seconds for a total of 3 minutes. The beaker was then removed from the water bath and stored at 4°C for several hours until completely cooled.

[0490] Example 21 - Generating live tissue replicas

[0491] The raw tissue replicas are produced using the ingredients listed in Table 4.

[0492] Table 4:

[0493]

[0494] The buffer was 20 mM potassium phosphate, 100 mM NaCl (pH 7.4). Heme protein was prepared at a concentration of 55 mg / g in the 20 mM potassium phosphate, 100 mM NaCl (pH 7.4) buffer. The 17× flavor precursor mixture precursor is described in Example 27. Pea globulin was prepared in the 20 mM potassium phosphate, 500 mM NaCl (pH 8) buffer and then lyophilized before use. The final protein concentration of the dried material was 746 mg / g. Pea globulin was prepared in the 20 mM potassium phosphate, 200 mM NaCl (pH 8) buffer and then lyophilized before use. The final protein concentration of the dried material was 497 mg / g.

[0495] Mix the liquid components (buffer, heme, and flavor precursor mixture) in a plastic beaker. Then add the dried pea globulin and pea globulin, and gently stir at room temperature for 1 hour while fully rehydrating. Then add the dried transglutaminase preparation (ACTIVA® TI, Ajinomoto, Fort Lee, NJ) and stir for about 5 minutes until dissolved. Then stop stirring and allow the mixture to gel at room temperature until firm. After the gel has formed, refrigerate the raw tissue replica until use.

[0496] Example 22 - Indurated Connective Tissue Imitation

[0497] The simulant for connective tissue was made using isolated soy protein (Supro Ex38), wheat gluten (Cargill), and bamboo fiber (α-fiber B-200, The Ingredient House) as follows. The purified protein was freeze-dried and then ground using a standard coffee grinder. Commercially available isolated soy protein and wheat gluten powders were used as is.

[0498] The connective tissue imitation contains 49% isolated soy protein, 49% wheat gluten, and 2% bamboo fiber. The ingredients are thoroughly mixed and loaded into the loading tube of the extruder's batch feeder. A twin-screw extruder (Nano 16, Lesterretz Extruders), a high-pressure water injection pump (Eldex), and a custom die nozzle (stainless steel tubing, 3 mm ID, 15 cm length, pressure rating 3000+ PSI) are used for connection with Hy-Lok double-ferrule fittings, and a custom die with a threaded nozzle and a 10 mm ID, 20 mm long flow channel are employed.

[0499] The dry mixture is fed into the extruder at a rate of 1 g / min. Water is pumped into the second zone of the extruder barrel. The water feed rate is adjusted to match the dry mixture feed rate to provide 55% moisture content in the final extrudate. The temperature gradients are maintained along the extruder barrel as follows: feed zone -25°C, zone 1 -30°C, zone 2 -60°C, zone 3 -130°C, zone 4 -130°C. The die is neither actively heated nor cooled. The die nozzle is actively cooled (by applying a moistening agent) to maintain the extrudate temperature below 100°C.

[0500] The hard connective tissue replica obtained through this process is a dark grayish-white ("cappuccino") material, formed into 3mm thick filaments, with a tensile strength similar to that of animal connective tissue (3 MPa).

[0501] Example 23. Soft connective tissue imitation

[0502] The soft connective tissue imitation was made as in Example 22, except that the water feed rate was adjusted to the dry mixture feed rate to provide 60% moisture content in the final extrudate. The temperature gradients were maintained along the extruder barrel as follows: feed zone -25°C, zone 1 -30°C, zone 2 -60°C, zone 3 -115°C, zone 4 -115°C. The die was neither actively heated nor cooled. The die nozzle was actively cooled (by applying a wetted tissue) to maintain the extrudate temperature below 100°C.

[0503] The soft connective tissue imitation obtained through this process is a pale grayish-white material, formed into 3 mm thick filaments, which have low tensile strength (<0.1 MPa) and a significant tendency to longitudinally divide into strips and fine fibers.

[0504] Example 24. Process of producing a fine connective tissue imitation (corn protein fiber):

[0505] The fine connective tissue imitation is prepared using corn gluten protein powder, glycerol (FCC grade), polyethylene glycol (PEG 400 or PEG 3350), ethanol, sodium hydroxide (FCC grade), and water. Corn gluten powder and PEG 3350 at a 35% w / w corn gluten ratio are dissolved in an 85% ethanol aqueous solution to achieve a final corn gluten concentration of 57% w / w. The pH of the solution is adjusted to 7.0 using a 1M sodium hydroxide solution in ethanol. The assembly is performed using an injection pump equipped with a 1-12 ml syringe, a rotating nozzle (hypercutaneous needle, 18-27mm or plastic nozzle, 18-24mm), a heated silicone ribbon, and a heated fan. The current collector is rotated using a computer-controlled motor to rotate the Delrin rod, which acts as the current collector.

[0506] The solution is loaded into a syringe, which is then attached to an injection pump connected to an 18-bore plastic tip. A silicone heating band is wrapped around the tip to maintain it at a high temperature. After the solution is extruded from the tip and forms a droplet, it is picked up with a scraper and carefully transferred to a current collector rod to form a filament between the tip and the current collector. The extrusion rate is optimized to produce a uniform flow of material from the tip (5 ml / h for a 12 ml syringe and an 18-bore tip). The current collector rotation speed is 3 RPM. A heating fan is installed to blow hot air onto the wound fiber. After winding, the fiber is cured in a 120°C oven for 24 hours.

[0507] The fine connective tissue replicas obtained through this process are translucent yellow materials shaped into fibers 300 micrometers thick. They are semi-flexible in air and become extremely flexible and elastic in the presence of water, maintaining a high tensile strength (6 MPa) similar to that of animal connective tissue.

[0508] Example 25. Noodles

[0509] The noodles are prepared using purified pea pea globulin (freeze-dried) and isolated soy protein (SuproEX38, Solbar Q842 (CHS)) or soy protein concentrate (Hisolate, Harvest Innovations). To prepare the noodles, 67% concentrated or isolated soy protein and 33% crushed pea pea globulin powder are thoroughly mixed and loaded into the loading tube of the extruder's batch feeder. The dry mixture is fed into the extruder at a rate of 1–2 g / min. Water is pumped into the second zone of the extruder barrel at a rate of 3.6–5.3 ml / min to maintain the final moisture content of the extrudate at 72.5%. The temperature gradient is maintained along the extruder barrel as follows: feed zone -25°C, zone 1 -30°C, zone 2 -60°C, zone 3 -100°C, zone 4 -100°C. The temperature in zone 1 can vary within the range of 25–45°C. Zone 2 temperature can vary within the range of 45-65℃. Zones 3 and 4 temperatures can vary within the range of 95-100℃. The die is neither actively heated nor cooled. The die nozzle is passively cooled by ambient air to ensure the extrudate temperature remains below 100℃.

[0510] The noodles obtained through this process are a pale yellow material, formed into 1.5 mm thick filaments, with low tensile strength (<0.1 MPa) and a medium viscous texture.

[0511] Example 26. Preparation of Viscous Tissue Imitations

[0512] The viscous texture imitation was prepared using purified pea pea globulin (freeze-dried) and purified pea globulin (freeze-dried). To prepare the viscous texture imitation, 50% crushed pea globulin powder and 50% crushed pea globulin powder were thoroughly mixed and loaded into the loading tube of the extruder's batch feeder. The dry mixture was fed into the extruder at a rate ranging from 0.4 to 0.8 g / min. Water was pumped into the second zone of the extruder barrel at a rate ranging from 1.6 to 3.2 ml / min to maintain the final moisture content of the extrudate at 80%. As the total throughput increased from 2 g / min to 4 g / min, the screw speed increased from 100 to 200 RPM. Larger die diameters (4 mm and above) also helped prevent backflow at higher feed rates. The temperature gradient is maintained along the extruder barrel as follows: feed zone -25℃, zone 1 -30℃, zone 2 -60℃, zone 3 -90℃, zone 4 -90℃. The die is neither actively heated nor cooled. The die nozzle is passively cooled by ambient air. The die nozzle is kept unclogged by allowing the gel material to solidify.

[0513] The viscous tissue replicas obtained through this process are translucent, water-white materials shaped into irregular bulbs ranging from 1 to 5 cm in size, with a viscous, paste-like texture.

[0514] Example 27. Preparation of flavor precursor mixtures

[0515] The flavor precursor mixture was prepared by mixing concentrated stock solutions of each additive to make a 17× solution. Table 5 contains the chemical composition of the mixture and the mM concentration of each component in the final burger. The concentrated flavor precursor mixture was aseptically filtered and adjusted to pH 5.5–6.0 using NaOH, and then used in the burger at a 1× concentration.

[0516] Table 5.

[0517] Chemical composition of flavor precursor mixtures.

[0518]

[0519] Example 28 - Freezing and arranging to produce tissueed proteins for use as muscle replicas

[0520] This example describes a non-extrusion method for producing textured protein materials that can be used in meat imitations.

[0521] The muscle tissue replica was prepared as follows: First, a lentil protein gel was prepared by mixing a 4.5% (w / v) lentil protein solution in 20 mM potassium phosphate buffer (pH 7.4) + 100 mM sodium chloride with 20% (v / v) canola oil (from Jedwards International). The mixture was gelled by heating at 95°C for 15 minutes and then slowly cooling to room temperature (at a rate of 1°C / min). The gel was then poured into a container and frozen at -40°C until completely frozen by placing it in a liquid nitrogen bath. The frozen material was then dried in a freeze dryer. When the material was completely dry, it was stabilized by high-pressure treatment (121°C, 15 minutes). The resulting material was a tissue-formed muscle tissue replica from plant protein.

[0522] The arranged muscle imitations were then pre-soaked in water for 5 minutes, cut into 3-4 mm long pieces, and then combined with 10 g of fat imitations, 10 g of connective tissue imitations, and 5 g of cryogel to form 50 g beef patty imitations. The internal sensory panel concluded that the improved fibrous texture of the patties was a result of the cryo-arranged tissues.

[0523] The muscle tissue replica was also prepared by first forming a material with the frozen arrangement described above. After steam cooking the tissue replica at 121°C for 10 minutes, the material was immersed in a solution of heat-denatured pea globulin (6% w / v in 20 mM potassium phosphate buffer (pH 7.4) + 100 mM sodium chloride, heat-denatured by heating at 95°C for 30 minutes), 1% horse myoglobin (w / v) (Sigma), and 40% (v / v) canola oil (from Jedwalds International). Gelation was induced by adding 20 mM calcium chloride. The sample was allowed to stand at room temperature for 5 minutes to allow gel formation. The resulting muscle replica contained the arranged material in a cold-solid gel form reminiscent of beef muscle in a steak.

[0524] Example 29 - Cold gelation of proteins for meat applications

[0525] In one example, a cold-set gel containing myoglobin was prepared as follows: First, a 6% (w / v) pea globulin solution in a 20 mM potassium phosphate buffer (pH 7.4) containing 100 mM sodium chloride was thermally denatured for 30 minutes at 100°C. The solution was then cooled to room temperature. Canola oil (from Jedwalds International) and horse myoglobin (Sigma) were added to final concentrations of 20% (v / v) and 1% (w / v), respectively. Gel formation was induced by adding 20 mM calcium chloride. A 50 g beef patty replica was formed by combining 5 g of the cold gel with 10 g of adipose tissue replica, 10 g of connective tissue replica, and 25 g of muscle tissue replica. 5 ml of a 7% (w / w) coarse lentil protein solution was added to the mixture, and a patty was formed.

[0526] Example 30 - Binding materials in meat imitations

[0527] In one example, the beef imitation was prepared as follows: First, a coagulant was prepared from a solution of 3% (w / v) pea globulin and lentinan in 20 mM potassium phosphate (pH 7.4) + 100 mM sodium chloride (pea globulin:lentinan ratio of 3:1). Molten palm oil (from Jedwalds International) was added to the solution to a final concentration of 5%, and the mixture was vortexed. The emulsion was then acidified to pH 5 by adding hydrochloric acid while stirring. The slurry was then centrifuged at 5000×g for 10 minutes, and the liquid top layer was decanted from the coagulant.

[0528] A 50 g beef patty replica was formed by combining the coagulant at 10% with adipose tissue replicas (20%), connective tissue replicas (20%), and muscle tissue replicas (50%). 5 ml of a 7% crude lentil protein solution was added to the mixture, and patties were formed. Patties containing the coagulant as a binding material were observed to have greater cohesiveness than patties without the coagulant.

[0529] Example 31 - Assemble sticky and noodle-shaped fragmented imitations and hamburger imitations

[0530] The shredded tissue imitations and hamburger imitations were prepared using the ingredients listed in Table 6. The material was kept cold (4–15°C) throughout all pre-processing steps.

[0531] Table 6

[0532]

[0533] The adipose tissue replica from Example 6 was frozen into a solid block following a final heat-cooling step. Optionally, 0.2% by weight of heme protein in the form of a 20 mg / ml liquid solution could be added, and the mixture was artificially processed into fat. The fat was then artificially pulverized into small pieces with a diameter of 3-7 mm while cold.

[0534] The soft connective tissue replica from Example 23 was produced into long, thin, thread-like pieces via an extrusion process. The soft connective tissue was shredded in a single step using a Mini-Prep® Plus Processor (Model DLC-2L Cuisinart, Stamford, Connecticut). Approximately 200 g of soft connective tissue was placed in the Mini-Prep® Plus Processor and processed for 60 seconds at the shredding setting to produce pieces 1–3 mm long with irregular edges.

[0535] The viscous tissue imitations and noodle tissue imitations (see Examples 25 and 26) were produced as long noodle-shaped blocks or amorphous blocks, respectively, by an extrusion process. The raw tissue imitation from Example 21 was provided as a solid block by an enzymatic cross-linking process. All three of these tissue imitations were artificially broken down into blocks with a diameter of 1-3 cm.

[0536] The hard connective tissue imitation from Example 22 was produced into long, thin, linear flakes via an extrusion process. The hard connective tissue imitation was then shredded at three levels—coarse, medium, and fine—in a mini-shredder (Mini-Prep® Plus Processor Model DLC-2L Cuisinart, Stamford, Connecticut). 160–200 g of the hard connective tissue imitation was placed in the mini-shredder and processed for 90 seconds at the shredding setting. One-third of the material was removed as the coarse shred. The remaining material in the shredder was then processed for another 60 seconds, and one-third of the initial weight was removed as the medium shred. The remaining material in the shredder was then processed for another 30 seconds to produce the fine shred.

[0537] Soybean hemoglobin was freeze-dried and then reconstituted in a 17× flavor precursor mixture (see Example 27), and the pH was adjusted to 6.0 with 10 N NaOH to prepare flavor and heme protein solutions.

[0538] Following the pre-processing described above, manually mix the soft connective tissue, viscous, raw, pasta, hard connective tissue, and 2 / 3 of the fat in a bowl. Typical batch sizes are 100 g to 2000 g. Then, drip the flavor and heme solution onto the mixed tissue imitation and gently mix by hand. Next, sprinkle the k-carrageenan powder onto the mixture and mix by hand. Keep all materials cold (4–15°C) during assembly, grinding, and shaping. Grind the mixture using a vertical mixer equipped with a food grinder attachment (KitchenAid® Professional 600 Series 6 Quart Bowl-Lift Vertical Mixer Model KP26M1XER and KitchenAid® Food Grinder Model FGA, St. Joseph, Michigan (MI)) at speed setting 1. Feed the food grinder material via a screw conveyor through a rotating blade mounted in front of a fixed orifice plate.

[0539] Co...

Claims

1. A method for imparting a beef flavor and aroma to a consumer product, the method comprising adding a heme-containing protein from a plant, fungus, algae, bacteria, or ciliate to the consumer product, wherein, after cooking, a beef-like flavor is imparted to the consumer product; wherein the consumer product is white meat, and wherein the heme-containing protein is leghemoglobin or myoglobin.

2. The method according to claim 1, wherein the white meat is chicken or pork.

3. The method according to claim 2, wherein the chicken is minced chicken.

4. The method according to claim 1, wherein the heme-containing protein is lentiglobin.

5. The method of claim 1, wherein the heme-containing protein has a UV-Vis spectrum similar to that of myoglobin derived from animals.

6. The method of claim 1, wherein the consumer product does not contain beef animal products.

7. The method according to claim 1, wherein the heme-containing protein is Myoglobin.

8. The method of claim 1, further comprising adding 0.01%-5% by weight of a heme-containing protein to the consumer product.

9. The method of claim 1, further comprising adding 0.4%-1% by weight of a heme-containing protein to the consumer product.

10. The method of claim 1, wherein the consumer product further comprises one or more plant proteins selected from the group consisting of RuBisCo, pea protein, lentil protein, and other legume proteins.

11. The method of claim 10, wherein the pea protein comprises pea albumin protein.

12. The method of claim 10, wherein the one or more plant proteins are present in the consumer product in an amount between about 1% and about 30% by weight.

13. The method of claim 10, wherein the one or more plant proteins have a solubility of at least 25 g / L in an aqueous solution at a temperature between about 2°C and about 32°C, wherein the aqueous solution has a pH between 3 and 8 and has a sodium chloride content of 0 to 300 mM.

14. The method of claim 10, wherein the one or more plant proteins are isolated.

15. The method of claim 1, wherein volatile compounds are generated during cooking, wherein the volatile compounds are selected from the group consisting of: propionaldehyde, butyraldehyde, 2-ethylfuran, heptanaldehyde, octanaldehyde, trans-2-(2-pentenyl)furan, (Z)-2-heptenal, (E)-2-octenal pyrrole, 2,4-dodecadienal, 1-octanaldehyde, and (Z)-2-decenal-2-undecenal.

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