RU2772991C2 - Food products copying meat - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to the food industry, namely to plant-based meat substitutes. A food product copying meat includes: from 0.01% to 5% by weight of protein containing heme iron; 10% by weight or more of plant protein; sugar, and amino acid or a sulfur-containing compound. The food product copying meat does not contain animal products. The preparation of the food product copying meat leads to obtaining at least two volatile compounds that have a small associated with meat compared to the reference food product copying meat that does not contain protein containing heme iron.

EFFECT: invention allows for obtaining a meat-substituting composition that precisely reproduces the taste, texture, aroma or color of a meat animal product.

13 cl, 17 dwg, 1 ex

Description

This application claims priority to US Patent Application Serial No. 61/572205, filed July 12, 2011, which is incorporated herein in its entirety by reference.

BACKGROUND TO THE INVENTION

Animal husbandry has a strong negative impact on the environment. It is now established that 30% of the earth's surface is devoted to animal husbandry, and that livestock makes up 20% of the biomass of all terrestrial animals. Because of this large scale, livestock contributes more than 18% of the resulting greenhouse gas emissions. Animal husbandry may be the biggest source of human water pollution, and animal husbandry is by far the biggest threat to biodiversity on earth. It has been found that if the world's population could switch from meat-containing foods to foods that do not contain materials derived from animal bodies, 26% of the earth's surface would be freed up for other uses. In addition, switching to a vegetarian diet would greatly reduce water and energy intake.

Meat consumption has a strong negative impact on human health. The health benefits of vegetarian food are widely recognized. If the population switched to vegetarian food, the reduction in health care costs would be significant.

Hunger is a global problem, with the world's 4 major cash crops (soybeans, corn, wheat and rice) already providing more than 100% of the population's calorie and protein needs, including every essential amino acid.

Plant-based meat substitutes have hardly led to a switch to a vegetarian diet. The current state of the art in meat replacement compositions includes the extrusion of a soy/grain mixture resulting in products that almost do not reproduce the sensation of cooking and eating meat. Typical disadvantages of these products, which are more homogeneous than equivalent meat products, are texture and mouthfeel. In addition, since these products must generally be sold pre-cooked, with added artificial flavors and flavors, they do not reproduce the flavors, odors, and other key features associated with cooking meat. As a result, these products are mainly of interest to a limited number of consumers who are already vegan/vegan, but have not been of interest to the larger segment of consumers who are accustomed to eating meat.

Described here are improved methods and compositions that more accurately reproduce the characteristics that consumers value in the preparation and consumption of meat, and that overcome the shortcomings and shortcomings of current meat substitutes.

SUMMARY OF THE INVENTION

In some aspects, the present invention relates to a meat replacement composition comprising a protein content wherein one or more isolated and purified proteins represent 10% or more by weight of said protein content, said meat replacement composition accurately reproducing taste, texture, aroma, or the color of a meat product derived from animal-based sources.

In one embodiment, the meat replacement composition accurately reproduces the color of said meat product in its raw and post-cooked state.

In another embodiment, one or more isolated and purified proteins represent 25% or more by weight of said protein content.

In another embodiment, one or more isolated and purified proteins represent 50% or more by weight of said protein content.

In another embodiment, one or more isolated and purified proteins represent 75% or more by weight of said protein content.

In another embodiment, one or more isolated and purified proteins represent 90% or more by weight of said protein content.

In another embodiment, gluten (gluten) does not make up 10% or more by weight of said protein content.

In another embodiment, each of said isolated, purified proteins is isolated and purified separately.

In another embodiment, the meat replacement composition comprises 1-7 isolated and purified proteins.

In another embodiment, each of said 1-7 isolated and purified proteins is from a different plant species.

In some embodiments, the meat substitute comprises less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 individual proteins isolated from one or more plant species.

In another embodiment, said protein content includes no more than a micro amount of any other proteins derived from one or more plant species.

In another embodiment, said one or more isolated and purified proteins are selected from the group consisting of leghemoglobin, non-symbiotic hemoglobin, hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, actinase, translation elongation factors, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumins, glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin, prolamine, phaseolin (protein), proteinopl asta, secalin, extensins, Triticeae gluten, zein, any seed storage protein, oleosins, caloleosins, steroleosins or other oil body proteins, plant storage protein A, plant storage protein B, mung bean seed 8S storage globulin.

In another embodiment, said one or more isolated and purified proteins are not isolated from an animal.

In another embodiment, said one or more isolated and purified proteins are from a single plant source.

In another embodiment, said one or more isolated and purified proteins are from a variety of plant-based sources.

In another embodiment, said one or more isolated, purified proteins are from a genetically modified organism.

In some embodiments, said genetically modified organism is a genetically modified bacterial or yeast organism.

In some embodiments, said isolated, purified protein is shaped into fibers.

In specific embodiments, these fibers resemble musculoskeletal fibers.

However, in more specific embodiments, said fibers are asymmetric fibers.

In some embodiments, the meat replacement composition further comprises one or more isolated and purified iron proteins.

In some embodiments, said one or more isolated and purified iron-containing proteins are selected from the group consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.

In a particular embodiment, said iron-containing protein comprises an amino acid sequence at least 70% homologous with SEQ ID NO: 1. SEQ ID NO: 1:

mvaftekqdalvsssfeafkanipqysvvfytsilekapaakdlfsflangvdptnpkltghaeklfalvrdsagqlkasgtvvadaalgsvhaqkavtdpqfvvvkeallktikaavgdkwsdelsrawevaydelaaaikka.

In a particular embodiment, said iron protein comprises an amino acid sequence at least 70% homologous with SEQ ID NO: 2. SEQ ID NO: 2: midqkekeli keswkriepn kneigllfya nlfkeeptvs vlfqnpissq srklmqvlgi lvqgidnleg liptlqdlgr rhkqygvvds ftlveeeakacll ksiqeylgta. In some embodiments, said iron protein comprises an amino acid sequence at least 80% homologous with SEQ ID NO: 2. In some embodiments, said iron protein comprises an amino acid sequence at least 90% homologous with SEQ ID NO: 2. In some embodiments, implementation of the specified iron-containing protein includes an amino acid sequence homologous to at least 98% of SEQ ID NO: 2.

In a particular embodiment, said iron protein comprises an amino acid sequence at least 70% homologous with SEQ ID NO: 3. SEQ ID NO: 3: MRKQPTVFEK LGGQAAMHAA VPLFYKKVLA DDRVKHYFKN TNMEHQAKQQ EDFLTMLLGG PNHYKGKNMA EAHKGMNLQN SHFDAIIENL AATLKELGVS DQIIGEAAKV the protein includes an amino acid sequence at least 80% homologous to SEQ ID NO: 3. In some embodiments, said iron protein includes an amino acid sequence 90% homologous to SEQ ID NO: 3. In some embodiments, said iron protein includes an amino acid sequence, at least 98% homologous to SEQ ID NO: 3.

In some embodiments, isolated and purified proteins are combined in one or more gels.

In some embodiments, the meat replacement composition further comprises one or more fats.

In specific embodiments, said one or more fats are derived from a plant-based source.

In another aspect, the present invention provides a meat replacement product that includes an indicator that shows the progress of cooking from raw to cooked, said meat replacement product from non-animal sources.

In some embodiments, the implementation of the specified indicator is a visual indicator that accurately reproduces the color change of the meat product in the specified course of cooking.

In one embodiment, said color change is from red to brown.

In one embodiment, said color change is from pink to white or tan.

In one embodiment, said visual indicator changes from transparent to opaque during said cooking progress.

In some embodiments, the indicator is an odor-related indicator that indicates the progress of cooking.

In one embodiment, said odor indicator is one or more volatile odorants released during cooking.

In some embodiments, said indicator comprises one or more isolated, purified iron-containing proteins.

In specific embodiments, said one or more isolated, purified iron-containing proteins are in a reduced state prior to preparation.

In one embodiment, said one or more isolated and purified iron-containing proteins are selected from the group consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.

In a particular embodiment, said iron-containing protein comprises an amino acid sequence at least 70% homologous with SEQ ID NO: 1. SEQ ID NO: 1:

mvaftekqdalvsssfeafkanipqysvvfytsilekapaakdlfsflangvdptnpkltghaeklfalvrdsagqlkasgtvvadaalgsvhaqkavtdpqfvvvkeallktikaavgdkwsdelsrawevaydelaaaikka.

In some embodiments, said one or more isolated and purified iron-containing proteins are not isolated from an animal. In some embodiments, the compositions of the present invention do not contain any proteins from an animal.

In specific embodiments, said one or more isolated and purified iron-containing proteins are from one or more plant-based sources.

However, in more specific embodiments, said one or more isolated, purified proteins are isolated from root nodules, roots, seeds, leaves, or stems of said one or more plant-based sources.

In other specific embodiments, said one or more plant-based sources are soybean or pea plants.

In one embodiment, said one or more plant-based sources comprise one or more plants of the legume family.

In some embodiments, said one or more isolated and purified iron-containing proteins in a reduced or oxidized state have a UV/Visible spectral line profile similar to that of an animal-derived myoglobin protein when in an equivalent reduced or oxidized state.

In a specific embodiment, the difference between the wavelength at which the maximum light absorption of said one or more isolated and purified iron-containing proteins is observed and the wavelength at which the maximum absorption of light by myoglobin derived from an animal-based source is observed is less than 5%.

In some embodiments, said one or more isolated, purified proteins are from a genetically modified organism.

In one embodiment, said genetically modified organism is a genetically modified bacterial or yeast organism.

In some embodiments, the meat replacement product does not contain methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the meat replacement product further comprises less than 1% wheat gluten.

In a more specific embodiment, said meat replacement product does not contain wheat gluten.

In other specific embodiments, said meat replacement product does not contain soy protein isolate.

In other specific embodiments, said meat replacement product does not contain soy protein concentrate.

In other specific embodiments, said meat replacement product does not contain soy protein.

In a more specific embodiment, said meat replacement product contains less than 5% carbohydrate.

In other specific embodiments, said meat replacement product does not contain tofu.

In some embodiments, said meat replacement product does not contain tofu and does not contain wheat gluten.

In some embodiments, said meat replacement product does not contain soy protein and does not contain wheat gluten.

In some embodiments, said meat replacement product does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, said meat replacement product contains less than 1% cellulose.

In some embodiments, said meat replacement product contains less than 5% insoluble carbohydrates.

In some embodiments, said meat replacement product does not contain soy protein and contains less than 1% cellulose.

In some embodiments, said meat replacement product does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, said meat replacement product does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, said meat replacement product does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In another aspect, the present invention provides a replica of muscle tissue comprising a protein content that includes one or more isolated and purified proteins, said replica of muscle tissue nearly matching in taste, texture, or color to an equivalent muscle tissue derived from an animal-based source.

In some embodiments, said one or more isolated and purified proteins comprise at least 50% by weight of said protein content. In some embodiments, said one or more isolated and purified proteins represent at least 40% by weight of said protein content. In some embodiments, said one or more isolated and purified proteins represent at least 30% by weight of said protein content. In some embodiments, said one or more isolated and purified proteins represent at least 20% by weight of said protein content. In some embodiments, said one or more isolated and purified proteins represent at least 10% by weight of said protein content.

In some embodiments, said one or more isolated and purified proteins comprise at least 50% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 40% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 30% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 20% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 10% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 5% by weight of the contents of said composition. In some embodiments, said one or more isolated and purified proteins make up at least 1% by weight of the contents of said composition.

In some embodiments, said protein content is from one or more non-animal sources.

In specific embodiments, said one or more non-animal sources is a plant-based source.

In other specific embodiments, said one or more non-animal sources are genetically modified yeast or bacteria.

In some embodiments, each of said one or more isolated proteins is isolated and purified separately.

In some embodiments, said one or more isolated proteins are selected from the group consisting of hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins , ciliate myoglobins, flavohemoglobins, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actins, translation factors 1 ,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumin, glycinin, conglycinin, globulin, vicilin, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin , prolamine, phaseolin (protein), proteinoplast, secalin, extensins, Triticeae gluten, zein, any seed storage protein, oleosins, caloleosins, steroleosins or other oil body proteins, vegetable storage protein A, vegetable storage protein B, mung bean seed 8S storage globulin.

In one embodiment, said seed storage protein is mung bean 8S protein.

In some embodiments, said protein content is suspended in a gel.

In some embodiments, said protein content is in the form of a gel.

In one embodiment, said gel comprises an isolated, purified cross-linking enzyme.

In some embodiments, said isolated, purified crosslinking enzyme is selected from the group consisting of transglutaminase, lysyl oxidases, and amine oxidases.

In a particular embodiment, said isolated, purified crosslinking enzyme is transglutaminase.

In some embodiments, said protein content is assembled into fibers.

In specific embodiments, these fibers are located isotropically.

In one embodiment, said fibers are asymmetric fibers.

In some embodiments, the muscle tissue replica further includes one or more isolated and purified iron-containing proteins.

In some embodiments, said one or more isolated and purified iron-containing proteins are selected from the group consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.

In a particular embodiment, said one or more isolated and purified iron-containing proteins comprise an amino acid sequence at least 70% homologous to SEQ ID NO: 1. SEQ ID NO: 1: In a particular embodiment, said one or more isolated and purified iron proteins comprise an amino acid sequence that is at least 80% homologous to SEQ ID NO: 1. In a specific embodiment, said one or more isolated and purified iron proteins comprise an amino acid sequence that is homologous to at least 90% of SEQ ID NO: 1. In a specific embodiment, said one or more isolated and purified iron-containing proteins comprise an amino acid sequence homologous to at least 95% of SEQ ID NO: 1. In a specific embodiment, said one or more isolated and purified iron-containing proteins include an amino acid sequence at least 98% homologous to SEQ ID NO: 1.

In specific embodiments, the muscle tissue copy comprises a protein content, wherein (i) one isolated and purified protein that is not an iron protein is 40-95% of said protein content, (ii) one or more isolated and purified iron protein is 1 -20% of the specified protein content, and (iii) one or more cross-linking reagents constitute 0.1-35% of the specified protein content.

In one embodiment, said protein content is 5-50% w/w or w/v of said copy.

In one embodiment, said single isolated and purified protein is mung bean 8S protein.

In one embodiment, said one or more isolated and purified iron proteins is leghemoglobin.

In one embodiment, said one or more crosslinking reagents is transglutaminase.

In some embodiments, the muscle tissue copy is free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the implementation of the copy of the muscle tissue, in addition, contains less than 1% wheat gluten. In specific embodiments, the implementation of the copy of the muscle tissue, in addition, contains less than 5% wheat gluten. In specific embodiments, the implementation of the copy of the muscle tissue, in addition, contains less than 10% wheat gluten. In specific embodiments, the implementation of the copy of the muscle tissue, in addition, contains less than 0.1% wheat gluten.

In a more specific embodiment, the specified copy of the muscle tissue does not contain wheat gluten.

In other specific embodiments, the implementation of the specified copy of the muscle tissue does not contain protein isolate from soy.

In other specific embodiments, the implementation of the specified copy of the muscle tissue does not contain soy protein concentrate.

In other specific embodiments, the implementation of the specified copy of the muscle tissue does not contain soy protein.

In a more specific embodiment, the specified copy of the muscle tissue contains less than 5% carbohydrates.

In other specific embodiments, the implementation of the specified copy of the muscle tissue does not contain tofu.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain tofu and does not contain wheat gluten.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain soy protein and does not contain wheat gluten.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, the implementation of the specified copy of the muscle tissue contains less than 1% cellulose.

In some embodiments, the implementation of the specified copy of the muscle tissue contains less than 5% insoluble carbohydrates.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain soy protein and contains less than 1% cellulose.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, the implementation of the specified copy of the muscle tissue does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In some embodiments, the muscle tissue copy is free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the implementation of the copy of the meat contains less than 1% wheat gluten. In specific embodiments, the implementation of the copy of the meat contains less than 5% wheat gluten. In specific embodiments, the implementation of the copy of the meat contains less than 10% wheat gluten. In specific embodiments, the implementation of the copy of the meat contains less than 0.1% wheat gluten.

In a more specific embodiment, the meat replica does not contain wheat gluten.

In other specific embodiments, the meat copy does not contain soy protein isolate.

In other specific embodiments, the meat replica does not contain soy protein concentrate.

In other specific embodiments, the implementation of the copy of the meat does not contain soy protein.

In a more specific embodiment, the copy of the meat contains less than 5% carbohydrates.

In other specific embodiments, the meat copy does not contain tofu.

In some embodiments, the meat replica does not contain tofu and does not contain wheat gluten.

In some embodiments, the meat replica does not contain soy protein and does not contain wheat gluten.

In some embodiments, the meat copy does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, the implementation of the copy of the meat contains less than 1% cellulose. In some embodiments, the meat copy contains less than 0.1% cellulose. In some embodiments, the implementation of the copy of the meat contains less than 10% cellulose. In some embodiments, the implementation of the copy of the meat contains less than 5% cellulose.

In some embodiments, the meat copy contains less than 5% insoluble carbohydrates.

In some embodiments, the meat replica does not contain soy protein and contains less than 1% cellulose.

In some embodiments, the meat replica does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, the meat replica does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, the meat replica does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In another aspect, the present invention provides an adipose tissue replica comprising a thickened emulsion, said thickened emulsion comprising a protein solution with fat droplets suspended therein.

In some embodiments, said fat blobs are from a non-animal source.

In some embodiments, said fat droplets are comprised of one or more vegetable oils.

In some embodiments, said one or more vegetable oils are selected from the group consisting of corn oil, olive oil, soybean oil, peanut oil, nut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oils, flaxseed oils, algae oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oils, rice bran oils, oils produced by bacteria, algae, archaea or fungi or genetically engineered bacteria, algae, archaea or fungi, triglycerides, monoglycerides, diglycerides, sphingosides, glycolipids, lecithin, lysolecithin, phosphatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid , caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, conjugated oleic acid or esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid or conjugated oleic acid, or glyceric esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid s, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid or conjugated oleic acid, or derivatives of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, which are triglycerides, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid.

In one embodiment, said one or more vegetable oils is rice bran oil or canola oil.

In some embodiments, said protein solution includes one or more isolated, purified proteins.

In some embodiments, said one or more isolated, purified proteins are 75% or more of the protein in said protein solution.

In some embodiments, said one or more isolated, purified proteins are from a non-animal source.

In some embodiments, said non-animal source is a plant-based source.

In some embodiments, said non-animal source is genetically modified yeast or bacteria.

In some embodiments, each of said one or more isolated proteins is isolated and purified separately.

In some embodiments, said one or more isolated proteins are selected from the group consisting of hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins , ciliate myoglobins, flavohemoglobins, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actins, translation factors 1 ,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumin, glycinin, conglycinin, globulin, vicilin, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin , prolamine, phaseolin (protein), proteinoplast, secalin, extensins, Triticeae gluten, zein, any seed storage protein, oleosins, caloleosins, steroleosins or other oil body proteins, vegetable storage protein A, vegetable storage protein B, mung bean seed 8S storage globulin.

In some embodiments, said one or more isolated, purified proteins is albumin protein, seed storage protein, or pea globulin protein.

In specific embodiments, said albumin protein is isolated pea albumin protein.

In some embodiments, said seed storage protein is mung bean 8S protein.

In some embodiments, said thickened emulsion comprises a protein solution comprising 1-3 isolated and purified proteins, said solution being 30-70% by volume of said emulsion; vegetable oil, and the specified vegetable oil is 30-70% of the volume of the specified emulsion; and an isolated, purified crosslinking enzyme, wherein said crosslinking enzyme is 0.5-5% w/v of said emulsion; wherein said vegetable oil is emulsified in said protein solution, wherein said emulsion is gelled with said crosslinking enzyme.

In other embodiments, said thickened emulsion comprises a protein solution comprising 1-3 isolated and purified proteins, said solution being 1-30% by volume of said emulsion; vegetable oil, and the specified vegetable oil is 70-99% of the volume of the specified emulsion; and an isolated, purified crosslinking enzyme, wherein said crosslinking enzyme is 0.5-5% w/v of said emulsion; wherein said vegetable oil is emulsified in said protein solution, wherein said emulsion is gelled with said crosslinking enzyme.

In some embodiments, the adipose tissue copy further includes a cross-linking enzyme.

In some embodiments, said crosslinking enzyme is transglutaminase.

In some embodiments, one of said 1-3 isolated and purified proteins is mung bean 8S protein, pea albumin protein, or pea globulin protein.

In particular embodiments, said vegetable oil is rice bran oil or canola oil.

In some embodiments, the adipose tissue copy is free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the adipose tissue replica also contains less than 1% wheat gluten.

In a more specific embodiment, the specified copy of the adipose tissue does not contain wheat gluten.

In other specific embodiments, said adipose tissue copy does not contain soy protein isolate.

In other specific embodiments, said adipose tissue copy does not contain soy protein concentrate.

In other specific embodiments, said adipose tissue copy does not contain soy protein.

In a more specific embodiment, the specified copy of adipose tissue contains less than 5% carbohydrates.

In other specific embodiments, said adipose tissue copy does not contain tofu.

In some embodiments, said adipose tissue replica does not contain tofu and does not contain wheat gluten.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain soy protein and does not contain wheat gluten.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, said adipose tissue copy contains less than 1% cellulose.

In some embodiments, said copy of adipose tissue contains less than 5% insoluble carbohydrates.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain soy protein and contains less than 1% cellulose.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, the implementation of the specified copy of adipose tissue does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In another aspect, the present invention provides a replica of connective tissue comprising a protein content comprising one or more isolated, purified proteins, said protein content being assembled into structures that nearly match the structure and appearance of connective tissue or skin.

In some embodiments, said protein content is from a non-animal source.

In some embodiments, said non-animal source is a plant-based source.

In some embodiments, said non-animal source is genetically modified yeast or bacteria.

In some embodiments, said one or more isolated proteins represent 50% or more by weight of said protein content.

In some embodiments, said one or more isolated proteins represent 90% or more by weight of said protein content.

In some embodiments, each of said one or more isolated proteins is isolated and purified separately.

In some embodiments, said one or more isolated proteins are selected from the group consisting of hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins , ciliate myoglobins, flavohemoglobins, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actins, translation factors 1 ,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumin, glycinin, conglycinin, globulin, vicilin, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin , prolamine, phaseolin (protein), proteinoplast, secalin, extensins, Triticeae gluten, zein, any seed storage protein, oleosins, caloleosins, steroleosins or other oil body proteins, vegetable storage protein A, vegetable storage protein B, mung bean seed 8S storage globulin.

In some embodiments, said one or more isolated and purified proteins is a prolamin family protein.

In some embodiments, said one or more isolated and purified proteins is zein.

In some embodiments, said protein content is suspended in a gel.

In some embodiments, said gel comprises an isolated, purified cross-linking enzyme.

In some embodiments, said isolated, purified crosslinking enzyme is selected from the group consisting of transglutaminase, lysyl oxidases, and amine oxidases.

In some embodiments, said isolated, purified crosslinking enzyme is transglutaminase.

In some embodiments, said protein content is shaped into a fiber.

In some embodiments, said fiber is produced by an extrusion process.

In some embodiments, said fiber is stabilized with protein crosslinks.

In some embodiments, the implementation of the fiber contains an isolated, purified cross-linking enzyme.

In some embodiments, said isolated, purified crosslinking enzyme is selected from the group consisting of transglutaminase, lysyl oxidases, and amine oxidases.

In some embodiments, said isolated, purified crosslinking enzyme is transglutaminase.

In another aspect, the present invention provides a meat replacement product comprising a muscle tissue copy, an adipose tissue copy, and a connective tissue copy; said muscle tissue copy, adipose tissue copy and/or connective tissue copy being assembled into a structure that nearly matches the physical structure of the meat.

In some embodiments, the meat replacement product includes two or more of said muscle tissue copies, adipose tissue copies, and connective tissue copies.

In some embodiments of the meat replacement product, said muscle tissue copy is 40-90% by weight of said product, said adipose tissue copy is 1-60% by weight of said product, and said connective tissue copy is 1-30%. in the weight relation from the specified product.

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

In some embodiments, said protein content is from a non-animal source.

In some embodiments, the non-animal based source is a plant based source.

In some embodiments, said non-animal source is genetically modified yeast or bacteria.

In some embodiments, 50% or more, by weight, of said protein content is one or more isolated, purified proteins.

In some embodiments, each of said one or more isolated proteins is isolated and purified separately from a different plant species.

In some embodiments, one or more of said isolated proteins is selected from the group consisting of hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinases, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, translation factors -1,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumin, glycinin, conglycinin, globulin, vicilin, conalbumin, gliadin, glutelin, gluten, glutenin , hordenin, prolamine, phaseolin (protein), proteinoplast, secalin, extensins, Triticeae gluten, zei on, any seed storage protein, oleosins, caloleosins, steroleosins or other oil body proteins, vegetable storage protein A, vegetable storage protein B, mung bean seed 8S storage globulin.

In some embodiments, the meat replacement product additionally includes one or more isolated and purified iron-containing proteins.

In some embodiments, said one or more isolated and purified iron-containing proteins are selected from the group consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins. In some embodiments, said iron-containing protein comprises an amino acid sequence at least 70% homologous to SEQ ID NO: 1:

mvaftekqdalvsssfeafkanipqysvvfytsilekapaakdlfsflangvdptnpkltghaeklfalvrdsagqlkasgtvvadaalgsvhaqkavtdpqfvvvkeallktikaavgdkwsdelsrawevaydelaaaikka.

In some embodiments, the meat replacement product does not contain methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the meat replacement product also contains less than 1% wheat gluten.

In a more specific embodiment, the meat replacement product does not contain wheat gluten.

In other specific embodiments, said meat replacement product does not contain soy protein isolate.

In other specific embodiments, said meat replacement product does not contain soy protein concentrate.

In other specific embodiments, said meat replacement product does not contain soy protein.

In a more specific embodiment, said meat replacement product contains less than 5% carbohydrate.

In other specific embodiments, said meat replacement product does not contain tofu.

In some embodiments, said meat replacement product does not contain tofu and does not contain wheat gluten.

In some embodiments, said meat replacement product does not contain soy protein and does not contain wheat gluten.

In some embodiments, said meat replacement product does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, said meat replacement product contains less than 1% cellulose.

In some embodiments, said meat replacement product contains less than 5% insoluble carbohydrates.

In some embodiments, said meat replacement product does not contain soy protein and contains less than 1% cellulose.

In some embodiments, said meat replacement product does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, said meat replacement product does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, said meat replacement product does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In another aspect, the present invention provides a food product comprising one or more isolated, purified iron-containing proteins, said food product being shaped for consumption by an animal.

In some embodiments, said one or more isolated, purified iron-containing proteins are from a non-animal source.

In some embodiments, said non-animal source is a plant-based source.

In some embodiments, said plant-based source includes one or more plants of the legume family.

In some embodiments, said one or more legume plants is a soybean or pea plant.

In some embodiments, said non-animal source is genetically modified yeast or bacteria.

In some embodiments, said iron-containing protein is selected from the group consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3 and cytochromes, HGbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.

In one embodiment, said iron-containing protein comprises an amino acid sequence at least 70% homologous to SEQ ID NO: 1:

mvaftekqdalvsssfeafkanipqysvvfytsilekapaakdlfsflangvdptnpkltghaeklfalvrdsagqlkasgtvvadaalgsvhaqkavtdpqfvvvkeallktikaavgdkwsdelsrawevaydelaaaikka.

In some embodiments, the food product does not contain methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In specific embodiments, the implementation of the food product, in addition, contains less than 1% wheat gluten.

In a more specific embodiment, said food product does not contain wheat gluten.

In other specific embodiments, said food product does not contain soy protein isolate.

In other specific embodiments, said food product does not contain soy protein concentrate.

In other specific embodiments, said food product does not contain soy protein.

In a more specific embodiment, said food product contains less than 5% carbohydrates.

In other specific embodiments, said food product does not contain tofu.

In some embodiments, said food product does not contain tofu and does not contain wheat gluten.

In some embodiments, said food product does not contain soy protein and does not contain wheat gluten.

In some embodiments, the implementation of the specified food product does not contain products of animal origin and contains less than 5% carbohydrates.

In some embodiments, said food product contains less than 1% cellulose.

In some embodiments, said food product contains less than 5% insoluble carbohydrates.

In some embodiments, said food product does not contain soy protein and contains less than 1% cellulose.

In some embodiments, the implementation of the specified food product does not contain soy protein and contains less than 5% insoluble carbohydrates.

In some embodiments, said food product does not contain wheat gluten and contains less than 1% cellulose.

In some embodiments, the implementation of the specified food product does not contain wheat gluten and contains less than 5% insoluble carbohydrates.

In another aspect, the present invention provides a method of making a meat replacement composition, comprising isolating and purifying one or more proteins and assembling said one or more proteins into a physical structure that nearly matches the physical structure of meat.

In another aspect, the present invention provides a method of replicating muscle tissue, comprising isolating and purifying one or more proteins and assembling said one or more proteins into a physical structure that nearly matches the physical structure of skeletal muscle.

In another aspect, the present invention provides a method for creating a copy of adipose tissue, comprising isolating and purifying one or more proteins, preparing a solution comprising one or more proteins, emulsifying one or more fats in said solution, and stabilizing said solution to form a thickened emulsion with one or more crosslinking reagents.

In another aspect, the present invention provides a method of creating a replica of connective tissue, comprising isolating and purifying one or more proteins and precipitating said one or more proteins, said precipitation resulting in one or more proteins forming physical structures that nearly correspond to the physical structure of connective tissue.

In some embodiments, said precipitation comprises dissolving said one or more proteins in a first solution and extruding the first solution into a second solution, said one or more proteins being insoluble in said second solution, said extruding causing said one or more proteins to precipitate.

In another aspect, the present invention provides a food product comprising one or more isolated, purified iron-containing proteins, said food product being shaped for consumption by an animal.

In another aspect, the present invention provides a food product comprising one or more isolated, purified iron-containing proteins, said food product being shaped for human consumption.

In another aspect, the present invention provides a food product comprising one or more isolated, purified iron-containing proteins, said food product being shaped for consumption by an animal. In another aspect, the present invention provides a food product comprising one or more isolated, purified iron-containing proteins, said food product being shaped for human consumption.

All publications and patent applications referred to in this specification are incorporated herein by reference to the same extent as if it were specifically and separately indicated that each individual publication or patent application is incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A patent or application data file contains at least one color drawing. Copies of this patent publication or patent application with color drawing(s) will be made available by the Office upon request and upon payment of the required fee.

New features of the present invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present invention will be achieved in view of the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are used, and the accompanying drawings, in which:

In FIG. 1 shows a comparison of superimposed GCMS profiles (in mirror image orientations) of otherwise identical muscle tissue copies prepared in the presence of leghemoglobin (LHb, profile on the right) or iron ion (profile on the left). In this figure, the vertical axis is the retention time in the gas chromatography separation step and the horizontal axis is the m/z ratios for the ions generated from the fragmentation of the respective separated volatile compounds. Selected compounds differentially represented in the two samples are labeled on the right side of the figure.

In FIG. 2 shows a portion of the root of a pea plant (Pisum sativum) with the root nodules cut to show the red color imparted by the leghemoglobin it contains. The cut root nodule turns red.

In FIG. 3 depicts leghemoglobin isolated from 1 ounce of pea roots. The red color usually attributed to the meat is unmistakable in a color photograph.

In FIG. 4 shows that leghemoglobins from different species are homologues and have similar color characteristics (FIG. 4). In FIG. 4, panel A, shows SDS-PAGE after electrophoresis of lysed root nodules of three species of plants in the legume family: (1) horse bean, (2) rugose pea, (3) soybean. The arrows indicate the corresponding leghemoglobins. Panel B shows the similarity of the UV/Vis line profile for leghemoglobins from two different plant species (horse bean and soybean).

In FIG. Figure 5 compares reduced (2+ heme iron) and oxidized (3+ heme iron) soy leghemoglobin (Figure 5, panel A) and equine myoglobin (Figure 5, panel B) demonstrating similar absorption profiles in UV/visible range of two proteins. The present inventors purified leghemoglobin from soybean root nodules using the protocol described here. Purified equine myoglobin was purchased from SigmaAldrich. Soy leghemoglobin (FIG. 5, panel A) and equine myoglobin (FIG. 5, panel B) were reduced with 1 mM sodium hydrosulfite. The UV/Visible light absorption spectra of heme Fe3+ (blue line - higher peak in Figs. 5 and 6) and heme Fe2+ (red line) of soy leghemoglobin (Fig. 5, panel A) and equine myoglobin (Fig. 5, panel A) are shown. B). Inserts show the increase in the spectra in the UV/visible range in the region from 450 nm to 700 nm. (Fig. 5, panel C) Images of a 10-µl liquid drop of a 40 mg/mL soybean leghemoglobin solution in the heme Fe3+ oxidation state (left droplet), showing the characteristic rust color, and a 40 mg/mL soy leghemoglobin solution in the heme Fe2+ oxidation state (right drop) showing the characteristic red color and (right image) corresponding equine myoglobin samples.

In FIG. 6 shows examples of successful reduction of heme iron in leghemoglobin with sodium hydrosulfite and titanium citrate. In FIG. 6, the UV/Visible spectrogram of purified soy leghemoglobin, in which the heme iron is in the oxidized (+3) state, is represented by blue curves in each panel (blue curves have higher peaks in the main plots). The red curves in each panel are the UV/Vis spectra of the same species of leghemoglobin after reduction to grade (+2) (red lines) by the addition of (panel A) 1 mM sodium hydrosulfite or (panel B) 0.24% (in w/v ratio) of titanium citrate in a buffer containing 20 mM potassium phosphate pH 7.3, 100 mM sodium chloride. Inserts show the increase in the spectra in the UV/visible range in the region from 450 nm to 700 nm. For this example, leghemoglobin was purified from soybean root nodules using fractionation by saturation to 60/90% ammonium sulfate and buffer exchange with a buffer containing 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride. A stock solution of sodium hydrosulfite was prepared by dissolving 100 mM sodium hydrogen sulfite in 1 mM sodium hydroxide in water. The titanium citrate stock solution was prepared from 20% (w/v) titanium chloride in hydrochloric acid by mixing it with 0.2 M sodium citrate (1:10 v/v). The pH was adjusted using sodium carbonate to pH 7.0.

In FIG. 7 shows an example of flow-through purification of leghemoglobin from soybean root nodules. The figure shows SDS-PAGE fractionation of samples taken at various stages of purification of soy leghemoglobin: (lane 1) soy root nodule lysate; (lane 2) soybean root nodule lysate purified by fractionation using 60/90% (w/v) ammonium sulfate. Shown is the protein content of a protein precipitate obtained after fractionation with 90% ammonium sulfate, resuspended in a buffer containing 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTA; proteins from the 90% ammonium sulfate precipitated precipitate were further purified by anion exchange chromatography (FFQ GE Healthcare) in 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride. The leghemoglobin produced in the flow material is shown in lane 3. The flow material from the anion exchange chromatography column was fractionated using size exclusion chromatography (Sephacryl S-100 GE Healthcare) and the resulting leghemoglobin-containing fraction is shown in lane 4. Leghemoglobin content at various stages purifications were determined by determining the proportion of the corresponding leghemoglobin band on SDS-PAGE after electrophoresis in the corresponding sample using ImageDoc analysis software (BioRad). The purity (relative content) of leghemoglobin in the respective purification steps was: lysate: 32.7% (lane 1), fractionation using 60/90% (w/v) ammonium sulfate: 78% (lane 2), anion exchange chromatography : ~ 83% (lane 3) and gel chromatography: up to ~ 95% (lane 4).

In FIG. 8 shows an electrophoresis-stained SDS-PAGE analysis of (A) soy leghemoglobin expressed and purified using recombinant protein technology and (B) soy leghemoglobin purified from soybean root nodules. (A) Recombinant soy leghemoglobin A containing a His tag and a site for His tag removal by TEV protease was expressed in E. coli strain BL21 and purified using His tag affinity chromatography (Talon resin, CloneTech) . The left lane contains molecular weight standards, the right lane contains purified recombinant soy leghemoglobin A (arrow). The estimated molecular weight of recombinant soy leghemoglobin A is 17.1 kDa. (B) SDS-PAGE after electrophoresis of purified soy leghemoglobin from root nodules. The left lane contains molecular weight standards, the right lane contains purified soy leghemoglobin A (arrow). Mass spectrometric analysis of the purified material indicated that all four isoforms of soy leghemoglobin were present and were full length (data not shown). Estimated molecular weights (M.m.) of soy leghemoglobin isoforms range from M.m. 15.4 to 15.8 kDa.

In FIG. Figure 9 shows an example of 6 pieces of commercial meat analog (Quorn chicken analog), approximately 1 cm on the lateral surface, 4 of which (left and lower right) were soaked with a solution of approximately 10 mg/ml soy leghemoglobin in 20 mM potassium phosphate pH 7, 4 and 100 mM NaCl; the other two (upper right) were soaked in the same buffer without leghemoglobin. The rich pink color of the leghemoglobin injected pieces is evident in the color photographs in contrast to the pale tan color of the untreated pieces.

In FIG. 10 shows 4 pieces of leghemoglobin-infused Quorn chicken analogue during pan cooking at 350°C. The bottom two pieces have been flipped over to show the fried surface has turned brown. In the top two pieces, the heated part has turned grey-brown, while the cooler upper surface remains pink. In some embodiments, a solution containing heme-containing proteins, such as a leghemoglobin solution, is administered to the consumer until the consumer is the color of uncooked meat.

In FIG. 11 shows that 43 ml of mung bean protein solution (150 mg/ml in dialysis buffer) was mixed with 37 ml of leghemoglobin solution (46.5 mg/ml of leghemoglobin and 20 mg/ml of another soy root nodule protein) in 20 mM phosphate potassium, 100 mM NaCl, pH 7.3. 20 ml of transglutaminase solution (20% by weight) was added, the solutions were thoroughly mixed, dispensed into two 50 ml Falcon tubes and incubated overnight at room temperature. Final protein concentrations were 65 mg/ml for mung bean protein, 18 mg/ml leghemoglobin, 91 mg/ml total protein.

In FIG. 12 shows a "white" muscle tissue replica prepared by mixing 43 ml of mung bean protein solution (150 mg/ml) with 45 ml of 11.7 mg/ml leghemoglobin solution and 0.8% (w/v) transglutaminase solution. Final protein concentrations were 63 mg/ml for mung bean protein, 5.2 mg/ml leghemoglobin, 68 mg/ml total protein.

In FIG. 13 shows that mung bean-based adipose tissue replica prepared in Eppendorf tubes formed an opaque, off-white gel with a uniform texture, with no visible liquid that was not included in the gel. The gel was free-standing, elastic and resilient. The gel has a weak, pleasant aroma and a weak and pleasant smell. The taste is moderately salty.

In FIG. 14 shows a pea globulin based adipose tissue replica prepared in Eppendorf tubes that is very similar to the mung bean based adipose tissue replica except that it released some oil on compression.

In FIG. 15 shows connective tissue analog cords that were created using a 1:3 ratio in 70% ethanol loaded into a syringe with a 23G needle (ID 0.337 mm). The solution was slowly extruded from the bottom of a 5 inch high tank into an excess of 5 M NaCl solution. A solution of zein in ethanol, with a lower density than a solution of NaCl, floated up, pulling out the fibrous strands of solidifying zein. The NaCl was constantly stirred as strands began to form to help elongate the strands. The cords gathered together and became a hard, dense mass.

In FIG. 16 shows a prototype ground beef patty that was prepared by combining 62% (w/w) muscle tissue replica (62% (w/w) "dark muscle tissue replica" and 38% (w/w) "white muscle tissue replica"). ”), 29% (w/w) adipose tissue replica (from pea globulin and canola oil), 5% (w/w) connective tissue replica (FIG. 16, panel A). Prototype ground beef patty was prepared by combining 62% muscle replica (62% "dark muscle replica" and 38% "white muscle replica"), 29% adipose tissue replica (from mung bean seed 8S protein and rice oil). bran), 5% connective tissue analogue (Fig. 16, panel B). Prototype ground beef patty was prepared by combining 71% (w/w) muscle tissue replica (consisting of 60% "white muscle tissue analog", 40% "dark muscle tissue analog"), 23% adipose tissue (from pea globulin proteins and canola oil) (Fig. 16, panel C). Prototype ground beef patty was prepared by combining 67% white muscle replica with 28% adipose tissue replica (from pea globulins and rice bran oil) (FIG. 16, panel D).

In FIG. 17 shows a ground beef patty replica that was prepared by combining 62% (w/w) muscle tissue replica (62% (w/w) "dark muscle tissue replica" and 38% (w/w) "white muscle tissue replica"). ”), 29% (w/w) adipose tissue replica (from pea globulin and canola oil), 5% (w/w) connective tissue replica. The panel on the left shows the patty before cooking, and the panel on the right shows the same patty after cooking for about 2 minutes. Experts have identified the flavor from making a copy of the ground beef as distinctly "meaty".

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Here are described methods and compositions for creating commodities. The commodities may be intended for consumption by animals. For example, the subject of consumption may be food suitable for human consumption. Consumables may be authorized by the relevant regulatory authorities. May be sold in grocery stores or prepared in restaurants, schools, hospitals, military installations, prisons, shelters, chronic care facilities, commodities similar to existing human foods. The commodities could also be pet food. For example, dog food could be made in accordance with the present invention. The commodities may also be wildlife food. For example, commodities for non-domesticated carnivores could be provided.

The consumables of the present invention can compete with, complement or replace animal-based products. For example, commodities may be replicas of meat created entirely from plant-based sources. Commodities can be created that replicate the cut or appearance of the meat as it is currently sold. For example, a consumer item may be visually similar to, or indistinguishable from, ground beef or a specific cut of beef. Alternatively, commodities with a unique look or appearance may be created. For example, the consumable might contain a drawing or inscription that is based on the structure of the consumable. In some cases, commodities are similar to regular meat products after they are cooked. For example, it is possible to create a consumable that is larger than a regular cut of beef, but which, after cutting and cooking the consumable, looks just like regular cooked meat. In some embodiments, the consumable may bear some resemblance to a conventional form of meat in two directions, but not in a third direction. For example, a consumable may bear some resemblance to a cut of meat in two directions (for example, when viewed from above), but may be much longer (or thicker) than a regular cut. Thus, in some embodiments, a composition is provided that can be repeatedly cut into typically meat-shaped products.

A consumable can be created entirely from plant-based sources. In some cases, the commodity may be created from organic sources. Commodities can also be created from a combination of plant-based sources and animal-based sources. For example, the subject of consumption may be a product in the form of ground beef supplemented with plant-based products of the present invention.

Commodities can be created from local products. For example, commodities can be created from plants growing within a certain radius of a possible consumer. The radius could be 1, 10, 100 or 1000 miles, for example. Thus, in some embodiments, the present invention provides a method for creating a meat replica that does not contain products that have been transported 1, 10, 100, or 1000 miles prior to the meat replica being created.

The present invention provides methods for obtaining consistent properties from commodities when they are created from different sources. Thus, for example, a plant-based replica meat made from native plants in Iowa, USA will taste, aroma, and texture substantially similar to a plant-based replica made from native plants in Lorraine, France. This constancy makes it possible to advertise locally grown products with consistent properties. Permanence may result from concentration or purification of similar components in different locations. These components can be combined in predetermined ratios to ensure consistency. In some embodiments, a high degree of property consistency is possible using components (eg, isolated or concentrated proteins and fats) that originate from the same plant species. In some embodiments, a high degree of property consistency is possible using components (eg, isolated or concentrated proteins and fats) that originate from different plant species. In some embodiments, the same proteins can be isolated from different plant species. In some embodiments, the present invention provides a method comprising isolating similar plant constituents from plant-based sources at different locations, assembling the compositions provided herein at both locations, and selling the compositions, wherein the compositions assembled and sold at different geographic locations have unchanged physical and chemical properties. In some embodiments, the isolated constituents are from different plant populations in different locations. In some embodiments, one or more of the dedicated constituents are transported to separate geographic locations.

Consumables may require fewer resources to craft than domesticated animal commodities. Accordingly, the present invention provides meat replicas that require less water and energy to create than meat production. For example, a consumable may require less than about 10, 50, 100, 200, 300, 500, or 1000 gallons of water per pound of consumable to create. By comparison, beef can require upwards of 2,000 gallons of water per pound of meat.

A consumer product may require less land to create than a meat product with a similar protein content. For example, a consumable may require 30% or less of the land area required to produce a meat product with a similar protein content.

The commodity may provide health benefits compared to the animal product it replaces in the diet. For example, it may have less cholesterol and lower levels of saturated fat than comparable meat products.

The commodity may be beneficial to animal welfare when compared to the animal product it replaces in the diet. For example, it can be created without the need for stalling, force-feeding, premature weaning, disruption of mother-offspring relationships, or slaughtering animals for their meat.

A consumer product may have a lower carbon footprint than the meat products it replaces. For example, a commodity may result in net greenhouse gas emissions of 1%, 5%, 10%, 25%, 50%, or 75% of the greenhouse gas emissions of the livestock product it replaces.

The commodity may give rise to alternatives to animal products or combinations of animal products whose consumption is prohibited by religious beliefs. For example, the consumable may be a kosher pork chop.

The consumable can also be transported in multiples and built or assembled in a different location. If available, local ingredients can be used to create a consumable. They can be supplemented with components that are not available in a particular location. This makes it possible to create commodities, such as replicas of meat, using less energy in transportation than is required for meat. For example, local water can be used in combination with a kit that provides other components of the commodity. Using local water will reduce shipping weight, thus reducing costs and environmental impact.

Commodities may be created or assembled in whole or in part in areas where animal husbandry is impractical or impossible. The consumer item may be created or assembled in an urban environment. For example, a kit may be provided to a consumer to enable him to create a consumable. The consumer can use local water or use plants from a rooftop garden, such as in Shanghai. In another example, commodities could be created aboard a spacecraft, an interplanetary station, or a moon base. Accordingly, the present invention provides methods and systems for creating meat replicas for use in or training for space flight. For example, the present invention could be used to train at a ground base for space flight. Commodities could be created on an island or on a man-made platform in the sea, where keeping livestock is difficult or prohibited.

In some embodiments, commodities are designed that mimic the feeling of eating meat. The appearance, texture and taste of the commodities may be similar or indistinguishable from meat. Therefore, in some embodiments, the present invention provides methods for determining whether an animal or human can distinguish a consumable from meat.

One way to determine if a commodity is comparable to meat is to a) determine the properties of the meat and b) determine if the commodity has similar properties. Meat properties that can be tested include mechanical properties such as hardness, cohesiveness, brittleness, chewability, stickiness, viscosity, elasticity, or stickiness. Meat properties that can be tested also include geometric properties such as particle size and shape, and particle shape and orientation. Additional qualities may include moisture content and fat content. These properties can be described using terms such as "soft", "hard", or "hard" to describe hardness; "crunchy", "crunchy", "brittle", "chewing", "soft", "resilient", "brittle", "friable", "viscous" or "glutinous" to describe cohesiveness; "low viscosity" or "viscous" to describe viscosity; "plastic" or "elastic" to describe elasticity; "glutinous", "sticky", or "viscous" to describe stickiness; "grainy", "grainy" or "rough" to describe the shape and size of the particles; "fibrous", "cellular" or "crystalline" to describe the shape and orientation of the particles, "dry", "wet", "wet" or "watery" to describe the moisture content; or "oily" or "fatty" to describe the fat content. Thus, in one embodiment, a group of people may be asked to rate a particular meat, such as ground beef, according to properties that describe the meat. These ratings can be used as an indicator of meat properties. The commodities of the present invention can then be compared to meat to determine how similar the commodities are to meat. In some cases, the properties of the commodities are then altered to make the commodities more meat-like. Thus, in some embodiments, the commodities are judged to be meat-like according to human judgment. In some embodiments, the consumable is indistinguishable by humans from real meat.

In some embodiments, subjects are asked to identify the commodities as a form of meat. In some embodiments, one property of the compositions of the present invention is that an animal, such as a human, will identify the composition as meat. In some embodiments, a human identifies a composition of the present invention as having properties equivalent to meat. In some embodiments, one or more properties of the meat are equivalent according to the human perception. Such properties include properties that can be checked. In some embodiments, a person identifies the commodities of the present invention as being more meat-like than meat substitutes found in the art.

In embodiments, the experiment may indicate that the consumer item is acceptable to consumers. A product quality assessment team can be used to test a variety of commodities described here. A number of members of the product quality assessment panel may try many samples of commodities, namely natural meat, in comparison with the consumable compositions described here. Parameters such as fat content can be standardized to, for example, 20% fat using mixtures of lean and fatty meats. Fat content can be determined using the Babcock method for meat (S. S. Nielson, Introduction to the Chemical Analysis of Foods (Jones & Bartlett Publishers, Boston, 1994)). You can prepare a mixture of ground beef and consumables of the present invention, prepared in accordance with the procedure described here.

Members of the product quality assessment team may be provided with samples in booths, under red light or white light, in an open user group. Samples may be assigned random three-digit numbers and may be rotated in the voting to avoid bias. Food quality panel members may be asked to rate samples for softness, juiciness, texture, taste, and overall acceptability using a hedonic scale from 1 = very dislike to 9 = very like, with a median of 5 = like or dislike. Members of the product quality assessment panel may be encouraged to rinse their mouths with water between samples and may be given the opportunity to comment on each sample.

The results of this experiment may indicate significant differences (p<0.05) or similarities between conventional meat and the compositions of the present invention.

These results will indicate that the compositions of the present invention are judged to be satisfactorily equivalent to real meat products. In addition, these results may indicate that the compositions of the present invention are preferred over other commercially available meat substitutes for members of the product quality assessment panel. Thus, in some embodiments, the present invention provides for commodities that are substantially similar to conventional meat.

The commodities of the present invention may also have physical characteristics similar to those of conventional meat. In one embodiment, the force required to pierce a 1" thick structure (e.g., a patty) made from the commodity of the present invention with a steel rod of constant diameter does not differ significantly from the force required to pierce a similar 1" thick meat structure (e.g., , ground beef cutlets) with a steel rod with a similar constant diameter. Accordingly, the present invention contemplates commodities with physical strength characteristics similar to those of meat.

In some embodiments, the composition of the present invention has meat-like reduction characteristics after cooking. In one embodiment, a consumable of the present invention with a similar fat and protein content to ground beef has the same reduction in size after cooking as real ground beef. Similar similarities in size reduction profiles can be achieved for the various compositions of commodities described here that are tailored to different meats.

In some embodiments, the consumable is compared to real meat based on olfactometer readings. In various embodiments, the olfactometer can be used to evaluate odor intensity and odor thresholds, odor over thresholds when compared to a control gas, determine hedonic scores to determine degree of discrimination, or relative intensity of odors. In some embodiments, the implementation of the olfactometer allows you to train and automatically evaluate groups of experts. Thus, in some embodiments, the consumable is a product that results in similar or identical olfactometer readings. In some embodiments, the implementation of the similarity is sufficient to be below the threshold of detection - human perception.

Gas chromatography-mass spectrometry (GCMS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to separate and identify various substances in a test sample. The GCMS may, in some embodiments, be used to evaluate the properties of a commodity. For example, volatile chemicals can be isolated from the headspace around meat. These chemicals can be identified using GCMS. This creates a profile of volatile chemicals in the free space around the meat. In some cases, it is also possible to determine each GCMS peak. For example, one could evaluate the odor sensation of the chemical compound responsible for each peak. This information could be used to further refine the profile. The GCMS could then be used to evaluate the properties of the commodity. The GCMS profile could be used to improve the quality of the commodity.

The characteristic flavoring and aromatic components are mainly formed during the cooking process as a result of chemical reactions of molecules, including amino acids, fats and sugars, which are found in plants as well as in meat. Therefore, in some embodiments, the consumable is tested for similarity to meat during or after cooking. In some embodiments, human scoring, human analysis, olfactometer readings, or GCMS measurements, or a combination thereof, are used to create a cooked meat odor map. It is also possible to create a map of the smells of a consumer item, for example, a copy of meat. These cards can be compared to determine how similar a cooked commodity really is to meat. In some embodiments, the odor map of the consumable during or after cooking is similar to or indistinguishable from such cooked or cooked meat. In some embodiments, the implementation of the similarity is sufficient to be below the threshold of detection - human perception.

In one aspect, the present invention provides a meat replacement product (alternatively referred to herein as a "commodity") that consists essentially or entirely of ingredients derived from non-animal sources, yet replicates the essential features associated with the preparation and consumption of an equivalent meat product of animal origin. The equivalent meat product may be white meat or dark meat. An equivalent meat product may be obtained from any animal. Non-limiting examples of animals used to produce an equivalent meat product include farmed animals such as, for example, cattle, sheep, pig, chicken, turkey, goose, duck, horse, dog, or game animals (either wild or farmed), such as, for example, rabbit, deer, bison, buffalo, wild pig, snake, pheasant, quail, bear, elk, antelope, pigeon, dove, grouse, fox, wild pig, goat, kangaroo, emu, alligator , crocodile, turtle, woodchuck, marmot, possum, partridge, squirrel, raccoon, whale, seal, ostrich, capybara, coypu, guinea pig, rat, mouse, vole, any kind of insect or other arthropod, seafood such as, for example, fish, crabs, lobsters, oysters, clams, scallops, abalone, squid, octopus, sea urchins, tunicates and others. Many meat products are usually derived from the skeletal muscle of an animal, but it is understood that meat may 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, for example, kidney, heart, liver, gallbladder, intestines, stomach, bone marrow, brain, thymus, lung, tongue. Accordingly, in some embodiments, the compositions of the present invention are commodities similar to skeletal muscle or organs.

In some aspects, the present invention provides meat replacement products comprising one or more of a first composition comprising a copy of muscle tissue, a second composition comprising a copy of adipose tissue, and/or a third composition comprising a copy of connective tissue, wherein one or more of the compositions are combined into a structure which mimics the physical structure of the meat. In other aspects, the present invention provides compositions for muscle tissue copy (here referred to as "muscle copy"), adipose tissue copy (here referred to as "fat copy"), and connective tissue copy (here referred to as "connective tissue copy"). In some embodiments, the compositions and meat replacement products consist primarily or entirely of ingredients derived from non-animal sources. In alternative embodiments, the muscle, fat, and/or connective tissue replica or meat replacement products comprising one or more of said replicas are partially derived from animal-based sources, but supplemented with ingredients derived from non-animal-based sources. However, in other alternative embodiments, the present invention provides meat products essentially derived from animal-based sources, but which are supplemented with one or more of a muscle tissue copy, a fat copy, and/or a connective tissue copy, said copies being essentially or entirely from non-animal sources. A non-limiting example of such a meat product is ultra-fat ground beef supplemented with a non-animal-derived copy of fat that improves texture and taste while maintaining the health benefits of the commodities that are low in animal fat. Such alternative embodiments result in products with properties that more closely mimic key features associated with meat preparation and consumption, but which are less expensive and have less environmental impact, less impact on animal welfare, or more health benefits for the consumer.

The physical structure of the meat replacement product can be manipulated by controlling the location, organization, arrangement, or orientation of the muscle, fat, and/or connective tissue copies described herein. In some embodiments, the implementation of the product is designed so that the copies described here are associated with each other as in meat. In some embodiments, the consumable is designed such that, after cooking, the copies described herein are bonded to each other as in cooked meat. In some embodiments, one or more copies of muscle, fat, and/or connective tissue are combined into a structure that mimics the physical structure of different cuts or cuts of meat. In a typical embodiment, the copies are combined into a structure that nearly matches the physical structure of natural ground beef. In other embodiments, the copies are combined into a pattern that nearly matches various cuts of beef, such as, for example, ribeye, filet mignon, London kebab, among others.

PROTEINS AND PROTEIN SOURCES

In some embodiments, any of the meat replacement products, muscle copy, fat copy, or connective tissue copy includes one or more isolated, purified proteins. In some embodiments, the meat replacement products consist of one or more of a muscle copy, a fat copy, and/or a connective tissue copy that includes one or more isolated, purified proteins. In other embodiments, the muscle copy, the fat copy, and/or the connective tissue copy includes one or more isolated, purified proteins. In some embodiments, approximately 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 of the protein content consists of one or more isolated, purified proteins. For the purposes of this document, "purified protein" will refer to a formulation in which the total relative weight content of protein components other than the target protein, which may be a single monomeric or multimeric protein molecule, is reduced by 2 or more times, 3 or more times, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or 1000 times or more compared to the starting material from which the target protein was isolated.

In some embodiments, one or more of the isolated, purified proteins are from non-animal sources. Non-limiting examples of non-animal sources include plants, fungi, bacteria, archaea, genetically modified organisms such as genetically modified bacteria or yeast, a chemical, or in vitro synthesis. In specific embodiments, one or more isolated, purified proteins are derived from plant-based sources. Non-limiting examples of plant-based sources include cereals such as, for example, corn, maize, rice, wheat, barley, rye, triticale, teff, oilseeds, including cottonseed, sunflower seed, safflower seed, rapeseed, green leafy vegetables such as, for example, lettuce, spinach, cabbage, collard greens, field cabbage, collard greens, mustard greens, young dandelion leaves, broccoli, cabbage, green material not normally consumed by humans, including crop biomass, including including switchgrass, Chinese cane, sorghum, other grasses, alfalfa, corn stubble, green material generally excluded from harvested plants, sugarcane leaves, tree leaves, root vegetables such as cassava, sweet potatoes, potatoes, carrots, beets, turnips, plants of the legume family, such as, for example, clover, peas, such as cowpeas, wrinkled peas, thermopsis, garden peas, beans, etc. e.g. soybeans, horse beans, lima beans, common beans, mutton peas, golden beans, pinto beans, lentils, wolf bean, prosopis, carob, soybeans and peanuts, vetch (Vicia), plants of the genus Stylosanthes, peanuts, indigo, acacia, plants of the genus Leucaena, cyamopsis and sesbania. One skilled in the art will appreciate that proteins that can be isolated from any organism in the plant kingdom can be used in the present invention.

Proteins abundant in plants can be isolated in high quantities from one or more source plants and are therefore an economical choice for use in any of the muscle, fat, connective tissue or meat replacement products. Accordingly, in some embodiments, the one or more isolated proteins comprise an abundant protein that is found at high levels in the plant and that can be isolated and purified in large quantities. In some embodiments, the abundant protein is 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% of the total protein content of the source plant. In some embodiments, the abundant protein is 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. In some embodiments, the abundant protein is about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20 %, 25%, 30%, 35%, 40%, 45%, 50% of the total dry weight of the source plant. In some embodiments, the abundant protein is about 0.5-5%, about 1-10%, about 5-20%, about 10-30%, about 15-40%, about 20-50% of the total dry matter. the mass of the source plant.

In specific embodiments, the one or more isolated proteins comprise an abundant protein that is found at high levels in plant leaves. In some embodiments, the abundant protein is 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% of the total protein content in the leaves of the source plant. In some embodiments, abundant protein is about 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about 30-70% of the total leaf protein. which is the source of the plant. In specific embodiments, one or more isolated proteins comprise ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Rubisco is a protein particularly useful for meat copies due to its high solubility and amino acid composition with near-optimal proportions of essential amino acids for human nutrition. In specific embodiments, the one or more isolated proteins comprise a ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase). In specific embodiments, one or more isolated proteins comprise a plant storage protein (VSP).

In some embodiments, the one or more isolated proteins comprise an abundant protein that is found at high levels in plant seeds. In some embodiments, the abundant protein is 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 in seeds of the source plant. In some embodiments, the abundant protein is about 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about 30-70% or >70% of the total protein content in the seeds of the source plant. Non-limiting examples of proteins found at high levels in plant seeds are seed storage proteins such as albumins, glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin, prolamine, phaseolin (protein), secalin, Triticeae gluten, zein, any seed storage protein, oleosins, caloleosins, steroleosins, or other oil body proteins.

In some embodiments, the one or more isolated proteins include proteins that interact with lipids and help stabilize the lipids in the structure. Without wishing to be bound by a particular theory, such proteins can improve the integration of lipids and/or fat copies with other components of the meat replacement product, resulting in improved flavor and texture in the final product. A non-limiting example of a plant lipid interacting protein is the oleosin family of proteins. Oleosins are lipid-interacting proteins found in plant oil bodies. Other non-limiting examples of plant proteins that can stabilize emulsions include lima bean seed storage proteins, pea albumins, pea globulins, mung bean 8S globulins, common bean 8S globulins.

COPIES OF MUSCLE TISSUE

A wide range of meat products includes a large proportion of skeletal muscle. Accordingly, the present invention provides a composition derived from non-animal sources that mimics or nearly matches key features of animal skeletal muscle. In another aspect, the present invention provides a meat replacement product that includes a composition derived from non-animal sources that mimics or nearly matches skeletal muscle. Such a composition will be referred to here as a "muscle copy". In some embodiments, the muscle copy and/or the meat replacement product comprising the muscle copy is derived in part from animal-based sources. In some embodiments, the muscle copy and/or the meat replacement product comprising the muscle copy is entirely from non-animal sources.

Many meat products include a large proportion of striated muscle, in which the individual muscle fibers are organized in a mostly isotropic manner. Accordingly, in some embodiments, the muscle replica includes fibers that are organized isotropically to some extent. In some embodiments, the fibers include a protein component. In some embodiments, the fibers include about 1%, 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% or more of the protein component.

In some embodiments, the protein component includes one or more isolated, purified proteins. For example, one or more isolated, purified proteins may include 8S globulin from mung bean seeds or an albumin or globulin fraction from pea seeds. These proteins are examples of proteins with suitable properties for making copies of meat, due to their ability to form gels with structures similar to those of animal muscle or adipose tissue. Described here are examples and embodiments of one or more isolated, purified proteins. The list of possible candidates here is essentially open and may include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses or genetically engineered microorganisms, or synthesized in vitro. It is possible to artificially create proteins that mimic the physical properties of animal muscle tissue. In some embodiments, one or more isolated, purified proteins are approximately 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.

The skeletal muscle of animals, such as beef cattle, typically contains significant amounts of glycogen, which can be somewhere around 1% of the mass of muscle tissue at the time of slaughter. After slaughter, some of this glycogen continues to be metabolized to produce products that include lactic acid, which contributes to lowering the pH of the muscle tissue, the desired meat quality. Glycogen is a branched polymer of glucose linked together via alpha (1→4) glycosidic bonds in straight chains, with branching points including alpha (1→6) glycosidic bonds. Plant starches, in particular amylopectins, are also branched polymers of glucose linked together via alpha (1→4) glycosidic bonds in straight chains, with branching points involving alpha (1→6) glycosidic bonds, and can therefore , be used as an analog of glycogen when creating copies of meat. Thus, in some embodiments, the muscle or meat replica includes starch or pectin.

Additional animal muscle components include sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides, and sulfur compounds. Thus, in some embodiments, the muscle copy may include sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides, and sulfur compounds. In some embodiments, the concentration 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 commodities is within 10% of the concentrations detectable in the muscle or meat that is being reproduced.

In another aspect, the present invention provides methods for creating a copy of a muscle. In some embodiments, the compositions are shaped into asymmetrical fibers prior to incorporation into an article of consumption. In some embodiments, these fibers mimic muscle fibers. In some embodiments, the fibers are twisted fibers. In other embodiments, the fibers are extruded fibers. Accordingly, the present invention provides methods for producing asymmetric or twisted protein fibers. In some embodiments, fibers are formed by extruding the protein component through an extruder. Extrusion methods are well known in the art and are described in US6379738, US3693533, US20120093994, which are incorporated here by reference.

In some embodiments, extrusion can be performed using an MPF19 twin screw extruder (APV Baker, Grand Rapids, Mich.) with a chilled head. The chill head can cool the extrudate until the extrudate returns to atmospheric pressure, thereby greatly suppressing the expansion or swelling of the final product. In the MPF19, dry feed and liquid can be added separately and mixed in the housing. The extrusion parameters can be, for example: screw speed = 200 rpm, product temperature at die = 150° C., feed rate = 23 g/min and water flow rate = 11 g/min. The temperature of the product can be measured during extrusion using a thermoelectric element at the end of the extrusion body. Color, opacity, structure, and texture can be observed for each sample obtained. Samples obtained can optionally be dried at room temperature overnight, then ground to a fine powder (<60 mesh) using a Braun food grinder. The pH of the samples can be measured in duplicate using a 10% (w/v) suspension of the sample powder in distilled water.

COPY FAT

Animal fat is important for the feeling of eating cooked meat. Accordingly, the present invention provides a composition derived from non-animal sources that mimics the key features of animal fat. In another aspect, the present invention provides a meat replacement product that includes a composition derived from non-animal sources that mimics animal fat. Such a composition will be referred to as "fat copy". In some embodiments, the fat copy and/or the meat replacement product comprising the fat copy is derived in part from animal-based sources.

In some embodiments, the meat replacement product contains a fat component. In some embodiments, the fat content of the consumable is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% fat. In some embodiments, the fat copy includes a gel with fat droplets suspended therein. In some embodiments, the implementation of the gel is a soft, elastic gel, including proteins and optional carbohydrates. In specific embodiments, the proteins used in the gel are plant or microorganism proteins. In some embodiments, the proteins used in the fat copy may include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses, or genetically engineered microorganisms, or synthesized in vitro. It is possible to artificially create proteins that mimic the physical properties of animal fat.

The fat droplets used in some embodiments of the present invention may be from a number of sources. In some embodiments, the sources are non-animal sources. In specific embodiments, the sources are plant-based sources. Non-limiting examples of 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, linseed oil, algae oil, palm oil , palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, oils produced by bacteria, algae, archaea or fungi or genetically engineered bacteria, algae, archaea or fungi, triglycerides, monoglycerides, diglycerides, sphingosides, glycolipids, lecithin, lysolecithin, phosphatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eiko zanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, conjugated oleic acid or esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid or conjugated oleic acid, or glycerol esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated l inoleic acid or conjugated oleic acid, or triglyceride derivatives of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid.

In some embodiments, the fat droplets are derived from the oil of the pulp of the fruit or seed. In other embodiments, the source may be a yeast or mold. For example, in one embodiment, the fat droplets include triglycerides derived from Mortierella isabellina.

In some embodiments, vegetable oils are modified to resemble animal fats. Vegetable oils can be modified with flavorings or other substances to replicate the taste and smell of meat during and after cooking. Accordingly, certain aspects of the present invention include methods for testing the qualitative similarity between cooking-related properties of animal fat and cooking-related properties of vegetable oils in a consumable.

In some embodiments, the fat copy includes a protein component comprising one or more isolated, purified proteins. Purified proteins contribute to the flavor and texture of the meat replica. In some embodiments, the purified proteins can stabilize emulsified fats. In some embodiments, the purified proteins can form gels after denaturation and enzymatic crosslinking that mimic the appearance and texture of animal fat. Here are examples and embodiments of one or more isolated, purified proteins. In specific embodiments, the one or more isolated proteins include a protein isolated from plants of the legume family. Non-limiting examples of plants in the legume family are presented here, although variations with other legumes are possible. In some embodiments, the legume family plant is a pea plant. In some embodiments, the isolated, purified proteins stabilize the emulsions. In some embodiments, the isolated, purified proteins form gels after crosslinking or enzymatic crosslinking. In some embodiments, the isolated, purified proteins include seed storage proteins. In some embodiments, the isolated, purified proteins include albumin. In some embodiments, the isolated, purified proteins include globulin. In a specific embodiment, the isolated, purified protein is purified pea albumin protein. In another specific embodiment, the isolated, purified protein is purified pea globulin protein. In another specific embodiment, the isolated, purified protein is mung bean 8S globulin. In another specific embodiment, the isolated, purified protein is oleosin. In another specific embodiment, the isolated, purified protein is caloleosin. In another specific embodiment, the isolated, purified protein is Rubisco. In some embodiments, the protein component is approximately 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of copy fat, by dry weight or total weight. In some embodiments, the protein component is about 0.1-5%, about 0.5-10%, about 1-20%, about 5-30%, about 10-50%, about 20-70%, or about 30- 90% or more of a copy of fat, by dry weight or total weight. In some embodiments, the protein component includes a solution containing one or more isolated, purified proteins.

In some embodiments, the fat copy includes crosslinking enzymes that catalyze reactions leading to covalent crosslinks between proteins. Cross-linking enzymes can be used to create or stabilize the desired structure and texture of the adipose tissue copy, to mimic the desired texture of the desired animal fat equivalent. Non-limiting examples of crosslinking enzymes include, for example, transglutaminase, lysyl oxidase, or other amine oxidases (eg, Pichia pastoris lysyl oxidase). In some embodiments, crosslinking enzymes are isolated and purified from a non-animal source, examples and embodiments of which are provided herein. In some embodiments, the fat copy comprises at least 0.0001%, or at least 0.001%, or at least 0.01%, or at least 0.1%, or at least 1% (in terms of weight by volume) of the crosslinking enzyme. In specific embodiments, the cross-linking enzyme is transglutaminase.

In another aspect, the present invention provides methods for creating a copy of fat. In some embodiments, the fat droplets are suspended in a gel. In some embodiments, the present invention provides methods for producing fat droplets suspended in a gel. Fat can be isolated and homogenized. For example, a mixture of organic solvents may be used to aid mixing of the lipid. The solvent can then be removed. At this point in time, the lipid can be frozen, lyophilized, or stored. Thus, in some aspects, the present invention provides a method for isolating and storing a lipid that has been selected for characteristics similar to those of animal fat. The lipid layer or lump can then be hydrated. In the case of hydration, agitation or temperature changes may be used. Hydration may take place in a solution of the precursor in the gel. Once hydrated, the lipid suspension can be sonicated or extruded to further modify the properties of the lipid in solution.

In some embodiments, a copy of the fat is harvested that nearly matches the pattern of adipose tissue in the meat. In some embodiments, some or all of the fat replica components are suspended in a gel. In various embodiments, the gel may be a protein gel, a hydrogel or organogel, or a xerogel. In some embodiments, the gel may be thickened to the desired consistency using a polysaccharide or protein based agent. For example, starch obtained by washing the crushed parts of plants such as potatoes, cassava starch, corn starch, katakuriko starch, potato starch, sago starch, tapioca starch, alginine, guar gum, locust bean gum, xanthan gum, collagen, egg white, furcellaran, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, gum senegal, konjac, starch, pectin, amylopectin, or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, or fungi may used singly or in combination to thicken the gel to form an architecture or structure for the consumable.

In specific embodiments, the fat copy is an emulsion comprising a solution of one or more proteins and one or more fats suspended therein as droplets. In some embodiments, the emulsion is stabilized with one or more crosslinking enzymes to form a gel. In more specific embodiments, the one or more proteins in solution are isolated, purified proteins. However, in more specific embodiments, the isolated, purified proteins include a fraction enriched in purified pea albumin. In other more specific embodiments, the isolated, purified proteins include a fraction enriched in purified pea globulin. In other more specific embodiments, the isolated, purified proteins comprise a fraction enriched with purified mung bean 8S globulin. However, in more specific embodiments, the isolated, purified proteins include a Rubisco enriched fraction. In other specific embodiments, one or more fats are derived from plant-based oils. In more specific embodiments, one or more fats are derived from one or more of corn oil, olive oil, soybean oil, peanut oil, nut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil. flax seed oils, algae oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, oils produced by bacteria, algae, archaea or fungi, or genetically engineered bacteria, algae, archaea or fungi, triglycerides, monoglycerides, diglycerides, sphingosides, glycolipids, lecithin, lysolecithin, phosphatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid , caproic acid, capric acid, caprylic acid slots, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, conjugated oleic acid or oleic acid esters, palmitoleic acid, palmitic acid, myristic acid , lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid or conjugated oleic acid , or glycerol esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid or conjugated oleic acid, or triglyceride derivatives of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid. However, in more specific embodiments, one or more of the fats is rice bran oil. In another specific embodiment, one or more fats is canola oil. In other specific embodiments, the cross-linking enzyme is transglutaminase, lysyl oxidase, or another amine oxidase. However, in more specific embodiments, the cross-linking enzyme is transglutaminase. In specific embodiments, the copy fat is a high fat emulsion comprising a solution of purified pea albumin emulsified with 40-80% rice bran oil stabilized with 0.5-5% (w/v) transglutaminase with turning into a gel. In specific embodiments, the fat copy is a high fat emulsion comprising a solution of partially purified mung bean 8S globulin emulsified with 40-80% rice bran oil stabilized with 0.5-5% (w/v) ) transglutaminase with gelation. In specific embodiments, the fat copy is a high fat emulsion comprising a solution of partially purified mung bean 8S globulin emulsified with 40-80% canola oil stabilized with 0.5-5% (w/v) transglutaminase with gelation. In specific embodiments, the copy fat is a high fat emulsion comprising a solution of purified pea albumin emulsified with 40-80% rice bran oil stabilized with 0.0001-1% (w/v) transglutaminase with turning into a gel. In specific embodiments, the copy fat is a high fat emulsion comprising a solution of partially purified mung bean 8S globulin emulsified with 40-80% rice bran oil stabilized with 0.0001-1% (w/v ) transglutaminase with gelation. In specific embodiments, the fat copy is a high fat emulsion comprising a solution of partially purified mung bean 8S globulin emulsified with 40-80% canola oil stabilized with 0.0001-1% (w/v) transglutaminase with gelation.

COPY OF CONNECTIVE TISSUE

Animal connective tissue provides key structural features that are an important component of the meat-eating experience. Accordingly, the present invention provides a composition derived from non-animal sources that mimics key features of animal connective tissue. In another aspect, the present invention provides a meat replacement product that includes a composition derived from non-animal sources that mimics important structural and visual features of animal connective tissue. Such a composition will be referred to herein as a "copy of connective tissue". In some embodiments, the connective tissue copy and/or the meat replacement product comprising the connective tissue copy is derived in part from animal-based sources.

Animal connective tissue can generally be classified into fascial type and cartilaginous type. The fascial type is highly fibrous, resistant to stretch (high modulus of elasticity), and is characterized by a high protein content, a moderate water content (approximately 50%), and a low or no fat and polysaccharide content. Accordingly, the present invention provides a replica of connective tissue that replicates the key features of a fascial-type tissue. In some embodiments, the connective tissue copy comprises 50% protein by total weight, approximately 50% liquid by weight, and has low fat and polysaccharide component values.

The protein content of most of the connective tissue of the fascial type consists mainly of collagen. Collagen is characterized by a high proportion of proline and alanine, and is also assembled into characteristic elongated fibrils or rod-shaped, flexible structures. Prolamins are the only family of proteins found in non-animal based sources such as plant based sources. Prolamins are found in extremely high amounts in plants and are similar in amino acid composition to collagen. Among the proteins tested by the present inventors for this purpose, prolamins are particularly suitable because of their low cost and their ability to easily form fibers or layers when twisted or extruded. Non-limiting examples of prolamin family proteins include, for example, zein (found in corn), these include hordein from barley, gliadin from wheat, secalin, extensins from rye, kafirin from sorghum, avenin from oats. In fascial connective tissue, the prolamin family of proteins, alone or in combination, demonstrate suitability for the protein component because they are present in extremely high amounts, are similar in general amino acid composition to collagen (with a high proportion of proline and alanine), and are capable of being converted into thin layers and fibers. In addition to zein (found in corn), they include hordein from barley, gliadin from wheat, secalin, extensins from rye, kaffirin from sorghum, avenin from oats. Other proteins may need to be added to the prolamins to achieve the targeted lists of physicochemical and nutritional properties. The list of possible candidates here is essentially open-ended and may include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses or genetically engineered microorganisms or synthesized in vitro, animal-derived or recombinant collagen, extensins (glycoproteins with a high content of hydroxyproline, which are abundant in the cell wall, for example, Arabidopsis thaliana, the monomers of which are "collagen-like" rod-shaped flexible molecules). It is possible to artificially create proteins that mimic the physical properties of animal connective tissue.

The methods for forming fascial-type connective tissue will be those practiced in the art, with a shift towards methods for producing fibrous or fibrous-like structures by biological, chemical or physical means, singly or in combination, sequentially or in parallel, until the final formation . These methods may include extrusion or twisting.

Cartilage-type tissue is macroscopically homogeneous, resistant to compression, characterized by a higher water content (up to 80%), a lower protein (collagen) content, and a higher polysaccharide (proteoglycan) content (approximately 10% each).

Compositionally, cartilage-type connective tissue will be very similar to fascial-type tissue, with the relative ratios of each set to more accurately reproduce "meat" connective tissue.

The methods for forming cartilage-type connective tissue will be similar to those for fascial-type connective tissue, but with a shift towards methods for obtaining isotropically homogeneous structures.

The fat may be suspended in a gel. In some embodiments, the present invention provides methods for producing fat droplets suspended in a protein gel. Fat can be isolated from plant tissues and emulsified. High speed mixing, homogenization, agitation, or temperature changes can be used for emulsification. The lipid suspension can be sonicated or extruded to further modify the properties of the lipid in solution. At this point in time, in some embodiments, the other components of the consumable are added to the solution, followed by the thickener. In some embodiments, cross-linking agents (eg, transglutaminase or lysyl oxidase) are added to link the components of the consumable. In other embodiments, a thickener is added and the lipid/gel slurry is later combined with additional consumable components. In fascial connective tissue, the prolamin family of proteins, alone or in combination, demonstrate suitability for the protein component because they are present in extremely high amounts, are similar in general amino acid composition to collagen (with a high proportion of proline and alanine), and are capable of being converted into thin layers. In addition to zein (found in corn), they include hordein from barley, gliadin from wheat, secalin, extensins from rye, kaffirin from sorghum, avenin from oats. Other proteins may need to be added to the prolamins to achieve the targeted lists of physicochemical and nutritional properties. The list of possible candidates here is essentially open-ended and could include any major seed storage proteins, animal-derived or recombinant collagen, extensins (hydroxyproline-rich glycoproteins abundant in the cell wall, e.g., Arabidopsis thaliana, whose monomers are "collagen-like » rod-shaped flexible molecules).

In some embodiments, some or all of the components of the consumable are suspended in a gel. In various embodiments, the gel may be a hydrogel, an organogel, or a xerogel. The gel can be thickened using a polysaccharide or protein based agent. For example, starch obtained by washing the crushed parts of plants such as potatoes, cassava starch, corn starch, katakuriko starch, potato starch, sago starch, tapioca starch, alginine, guar gum, locust bean gum, xanthan gum, collagen, egg white, furcellaran, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, gum senegal, konjac, starch, pectin, amylopectin, or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, or fungi may used alone or in combination to thicken the gel to form the architecture or structure of the consumable. Enzymes that catalyze reactions leading to covalent crosslinks between proteins can also be used alone or in combination to form the architecture or structure of the commodities. For example, transglutaminase, lysyl oxidase, or other amine oxidases (eg, Pichia pastoris lysyl oxidase (PPLO)) can be used alone or in combination to shape the architecture or structure of the consumer product. In some embodiments, a plurality of gels with different components are combined to form a consumable. For example, a plant-based protein-containing gel may be combined with a plant-based fat-containing gel. In some embodiments, fibers or strands of proteins are oriented parallel to each other and then held in place using a gel containing plant-based fats.

The compositions of the present invention may be puffed up or expanded upon heating, such as frying, baking, microwave heating, forced air heating, air tunnel heating, and the like, in accordance with methods well known in the art. technology.

In some embodiments, a plurality of gels with different components are combined to form a consumable. For example, a plant-based protein-containing gel may be combined with a plant-based fat-containing gel. In some embodiments, fibers or strands of proteins are oriented parallel to each other and then held in place using a gel containing plant-based fats.

In some embodiments, the copy of the meat does not contain animal products, contains less than 1% wheat gluten, does not contain methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the meat replica is free of animal products, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the copy of the meat is free of animal products, free of soy protein isolate, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the copy of the meat is free of animal products, free of soy protein concentrate, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the copy of the meat is free of animal products, free of soy protein, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the meat replica is free of animal products, free of tofu, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the meat replica does not contain materials derived from animal bodies, does not contain tofu, and does not contain wheat gluten.

In some embodiments, the meat replica does not contain animal products, does not contain soy protein, and does not contain wheat gluten.

In some embodiments, the meat copy is free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal.

In some embodiments, the meat replica does not contain animal products and contains less than 5% carbohydrates.

In some embodiments, the meat copy is free of animal products, free of soy protein, free of wheat gluten, free of methylcellulose, carrageenan, burnt sugar, konjac flour, gum arabic, and gum senegal, and contains less than 5% carbohydrate.

In some embodiments, the meat replica does not contain animal products and contains less than 1% cellulose.

In some embodiments, the meat replica does not contain animal products and contains less than 5% insoluble carbohydrates.

In some embodiments, the meat replica does not contain animal products, does not contain soy protein, and contains less than 1% cellulose.

In some embodiments, the meat replica does not contain animal products, does not contain soy protein, and contains less than 5% insoluble carbohydrates.

In some embodiments, the meat replica does not contain animal products, does not contain wheat gluten, and contains less than 1% cellulose.

In some embodiments, the meat replica does not contain animal products, does not contain wheat gluten, and contains less than 5% insoluble carbohydrates.

You can also adjust the percentage of different components. For example, non-animal substitutes for muscle tissue, adipose tissue, connective tissue, and blood components can be combined in varying ratios and into physical structures that most closely match the look and feel of meat. The various components may be arranged to provide consistency between the pieces of the consumable. The components may be arranged to ensure that no waste is generated from the consumable. For example, while a regular cut of meat may have parts that are not normally eaten, a copy of the meat may improve the meat by not including those non-edible parts. This improvement makes it possible to consume the entire created or transported product, which reduces waste and transportation costs. Alternatively, the meat replica may include non-edible portions to mimic the sensation of eating meat. Such parts may include bone, cartilage, connective tissue, or other materials commonly referred to as cartilage, or included materials that mimic these components. In some embodiments, the consumable may contain simulated non-edible portions of meat products that are intended to perform additional functions. For example, the simulated bone may be designed to distribute heat during cooking, making the consumable cook faster or more uniform than meat. In other embodiments, the implementation of the simulated bone may also serve to keep the product at a constant temperature during transportation. In other embodiments, the simulated non-edible portions may be biodegradable.

In some embodiments, meat replacement compositions comprising 10-30% protein, 5-80% water, 5-70% fat, comprising one or more isolated, purified proteins, do not contain animal protein. In specific embodiments, the meat replacement compositions include transglutaminase.

In some embodiments, the implementation of the article of consumption contains components that replicate the components of meat. The main component of meat is typically skeletal muscle. Skeletal muscle is typically composed of approximately 75 percent water, 19 percent protein, 2.5 percent intramuscular fat, 1.2 percent carbohydrates, and 2.3 percent other soluble non-protein substances. They include organic acids, sulfur compounds, nitrogen compounds such as amino acids and nucleotides, and inorganic substances such as minerals. Accordingly, in some embodiments, the implementation of the present invention provides for the copying of the approximate values in this composition for the product of consumption. For example, in some embodiments, the subject of consumption is a plant-based replica of meat, which may include approximately 75% water, 19% protein, 2.5% fat, 1.2% carbohydrates, and 2.3 percent other soluble non-protein substances. In some embodiments, the subject of consumption is a plant-based replica of meat comprising 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 subject of consumption is a plant-based replica of meat comprising 60-90% water, 5-10% protein, 1-20% fat, 0.1-5% carbohydrates, and 1-10 percent other soluble non-protein substances. In some embodiments, the subject of consumption is a plant-based replica of meat comprising 0-50% water, 5-30% protein, 20-80% fat, 0.1-5% carbohydrates, and 1-10 percent other soluble non-protein substances. In some embodiments, the copy contains between 0.01% and 5% by weight of heme containing protein. In some embodiments, the copy contains between 0.01% and 5% by weight of leghemoglobin. Some meats also contain myoglobin, a heme-containing protein that provides most of the red color and iron content in some meats. In some embodiments, the copy contains between 0.01% and 5% by weight of heme containing protein. In some embodiments, the copy contains between 0.01% and 5% by weight of leghemoglobin. It is understood that these percentages may vary in the meat, and it is possible to create replicas of the meat that nearly match the natural variations in the meat. In addition, in some cases, the present invention provides for improved copies of the meat, which include these components in typically unnatural percentages. For example, you can create a copy of meat with a higher fat content than the typical average fat content. The percentages of these components can also be changed to enhance other desired properties.

In some cases, the copy of the meat is created so that after cooking, the percentages of the components are similar to those in the cooked meat. Thus, in some embodiments, the uncooked commodity has different percentages of ingredients than uncooked meat, but after cooking, the commodity is similar to cooked meat. For example, meat can be replicated with a higher water content than the typical water content of raw meat, but after microwave cooking, the resulting product has similar component percentages to grilled meat.

In some embodiments, the object of consumption is a copy of the meat with a lower water content than the typical water content of the meat. In some embodiments, the present invention provides methods for hydrating the meat copy to achieve a meat-like water content in the meat copy. For example, a copy of meat with a water content that would be low for meat, such as 1%, 10%, 20%, 30%, 40%, or more than 50% water, is hydrated to about 75% water. After hydration, in some embodiments, the copy of the meat is then prepared for human consumption.

The consumable may have a protein component. In some embodiments, the protein component of the consumable is 10%, 20%, 30%, or 40%. In some embodiments, the protein content of the consumable is similar to that of meat. In some embodiments, the protein content of the commodity is greater than that of the meat. In some embodiments, the implementation of the subject of consumption contains less protein than meat.

The protein in a consumer product may come from a variety or combination of sources. Non-animal sources may provide some or all of the protein in a consumer product. Non-animal sources may include vegetables, fruits, nuts, grains, algae, bacteria, or fungi. The protein may be isolated or concentrated from one or more of these sources. In some embodiments, the subject of consumption is a copy of the meat, including protein derived only from non-animal sources.

In some embodiments, the protein is shaped into asymmetric fibers for inclusion in an article of consumption. In some embodiments, these fibers replicate muscle fibers. In some embodiments, the protein is twisted fibers. Accordingly, the present invention provides methods for producing asymmetric or twisted protein fibers. In some embodiments, the consumable contains a protein or proteins that contain all of the amino acids found in proteins that are essential for human nutrition. In some embodiments, the proteins added to the consumable are supplemented with amino acids.

MEAT COOKING INDICATORS

The release of odorants during cooking is an important aspect of meat consumption. In some embodiments, the consumer is an entirely non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable by humans as being typical of beef cooking. In some embodiments, the consumable, when cooked, produces a pleasant odor easily recognizable by humans as being typical of pork cooking. In some embodiments, the consumable is an entirely non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable by humans as typical of bacon preparation. In some embodiments, the consumable is an all-non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable by humans as being typical of chicken cooking. In some embodiments, the consumable is an entirely non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable to humans as typical of lamb cooking. In some embodiments, the consumable is an entirely non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable by humans as typical of fish cooking. In some embodiments, the consumable is an entirely non-animal replica of meat that, when cooked, produces a pleasant odor easily recognizable to humans as typical of turkey cooking. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, with an odor released during cooking. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, with an odorous substance formed as a result of chemical reactions that occur during cooking. In some embodiments, the subject of consumption is a replica of meat composed primarily or entirely of ingredients derived from non-animal sources, containing mixtures of proteins, peptides, amino acids, nucleotides, sugars, and polysaccharides, and fats in combination and spatial arrangements that allow these compounds undergo chemical reactions during cooking to form odorous substances and odor-producing compounds. In some embodiments, the consumable is a replica of meat composed primarily or entirely of ingredients derived from non-animal sources, with a volatile or non-persistent odor released during cooking. In some embodiments, the consumable is a method of preparing a meat replica, wherein the meat replicas consisting primarily or entirely of ingredients derived from non-animal sources are heated to release a volatile or non-persistent odorant.

Odorants released during the preparation of meat are formed as a result of reactions in which fats, protein, amino acids, peptides, nucleotides, organic acids, sulfur-containing compounds, sugars and other carbohydrates may be involved as reagents. In some embodiments, the odorants that come together during meat cooking are identified and located in close proximity to each other in the commodity such that the odorants are mixed when the commodity is cooked. Thus, in some embodiments, characteristic odor-producing and odoriferous substances are formed during the cooking process as a result of chemical reactions involving the amino acids, fats and sugars found in plants as well as in meat. Thus, in some embodiments, characteristic odor-producing and odoriferous substances are primarily formed during the cooking process as a result of chemical reactions involving one or more amino acids, fats, peptides, nucleotides, organic acids, sulfur compounds, sugars, and other carbohydrates detectable in plants and also in meat.

Some of the reactions that produce odorous substances released during meat cooking can be catalyzed by iron, specifically the heme iron in myoglobin. Thus, in some embodiments, some of the characteristic odor-generating and aromatic substances are formed during the cooking process as a result of chemical reactions catalyzed by iron. In some embodiments, some of the characteristic odor-generating and aromatic substances are formed during the cooking process as a result of chemical reactions catalyzed by heme. In some embodiments, some of the characteristic odor-generating and odoriferous substances are formed during the cooking process as a result of chemical reactions catalyzed by the heme iron in leghemoglobin. In some embodiments, some of the characteristic odor-generating and odoriferous substances are formed during the cooking process as a result of chemical reactions catalyzed by heme iron in the heme-containing protein.

Confirmation that the presence of leghemoglobin contributes positively to the pleasant odor of meat copies: A muscle tissue copy including pea meal, sunflower oil and glucose was heated for 10 minutes at 140ºC in the presence of either reduced leghemoglobin (LHb) or a mixture of iron (Fe3+) , sodium, and EDTA (EFS) in hermetically sealed containers containing fibers for microsolids recovery (SPME). These fibers contain polydimethylsiloxane (PDMS) which adsorbs volatile compounds for analysis by GC-MS. Analysis of multicopy GC-MS data shows consistent differences between LHb and EFS samples. Non-limiting examples of compounds found exclusively or in greater amounts in LHb samples are: 2-octanone, 2-methylfuran, which are often associated with the pleasant smell of cooked meat, and many other unidentified compounds. For example, in FIG. 1 shows a comparison of superimposed GCMS profiles (in mirror image orientations) of otherwise identical muscle copies prepared in the presence of leghemoglobin (LHb, profile on the right) or iron ion (profile on the left). In this figure, the vertical axis is the retention time in the gas chromatography separation step and the horizontal axis is the m/z ratios for the ions generated from the fragmentation of the respective separated volatile compounds. Selected compounds differentially represented in the two samples are labeled on the right side of the figure.

COLOR INDICATORS

The color of meat is an important part of the feeling of cooking and eating meat. For example, cuts of beef have a characteristic red color when raw, which gradually changes to brown as it cooks. In another example, white meats such as chicken or pork have a characteristic pink color in their raw state, which gradually changes to a white or brownish color during cooking. The degree of color measurement is used to show the progress of the beef cooking and determine the cooking time and temperature to obtain the desired doneness. In some aspects, the present invention provides a non-meat-based meat replacement 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 specific embodiments, the color indicator follows the color change of the cut of meat as the meat transitions from a raw state to a cooked state. In more specific embodiments, the color indicator colors the meat replacement product red prior to cooking, indicating a raw state, and causes the meat replacement product to change color to brown during cooking. In other specific embodiments, the color indicator colors the meat replacement product pink prior to cooking, indicating a raw state, and causes the meat replacement product to change color to white or brown during cooking.

The main nutritional factor that determines the color of meat is the concentration of iron-containing proteins in the meat. In the skeletal muscle meat component, one of the main iron-containing proteins is myoglobin. It is established that the white meat of chicken contains below 0.05%; pork and veal - 0.1-0.3%; meat of a young cow - 0.4-1.0%; and the meat of an old cow - 1.5-2.0%. Thus, in some embodiments, the object of consumption is a copy of the meat, which includes an iron-containing protein. In some embodiments, the meat copy comprises about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 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% , about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, or more than about 2% iron-containing protein by dry weight or total weight. In some cases, the iron-containing protein has been isolated or purified from a source. In other cases, the iron-containing protein has not been isolated and purified. In some instances, the source of the iron-containing protein is an animal-based source or a non-animal-based source, such as a plant, fungus, or genetically modified organisms such as, for example, bacteria or yeast. In some cases, the iron-containing protein is myoglobin. In some embodiments, the subject of consumption is a plant-based replica of meat that contains added animal myoglobulin. Thus, for example, a copy of the meat of a young cow may contain approximately 0.4-1% myoglobin. In some cases, the iron-containing protein is leghemoglobin. In some embodiments, the subject of consumption is a plant-based replica of meat that contains added leghemoglobin. Thus, for example, a copy of the meat of a young cow may contain approximately 0.4-1% leghemoglobin. In some cases, the iron-containing protein is cytochrome. In some embodiments, the consumption item is a plant-based replica of meat that contains added cytochrome. Thus, for example, a copy of the meat of a young cow may contain 0.4-1% cytochrome.

Another example of iron-containing proteins is hemoglobin, the iron-containing oxygen-binding protein in vertebrate erythrocytes. Hemoglobin is similar in color to myoglobin. In some embodiments, the present invention provides methods to conserve and recycle blood from livestock to replenish the color of a commodity. For example, blood is saved from the slaughterhouse, blood hemoglobin is used to enhance the color of the commodities. In some aspects, the subject of consumption is a copy of the plant-based meat containing hemoglobin.

Additional iron-containing proteins exist in nature. In some embodiments, the consumable includes an iron-containing protein that is not myoglobin. In some embodiments, the implementation of the subject of consumption does not contain myoglobin. In some embodiments, the implementation of the subject of consumption does not contain hemoglobin. In some embodiments, the object of consumption is a copy of the meat, which includes an iron-containing protein other than myoglobin or hemoglobin.

Examples of iron-containing proteins include hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, HgbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins. In various embodiments, these iron-containing proteins are added to the commodities to alter the commodities' apparent characteristics or iron content. In some embodiments, the subject of consumption includes a hemoprotein (eg, hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, HgbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins).

Leghemoglobin, similar in structure and physical properties to myoglobin, is readily available as a waste product from commercial legumes (eg soybeans, peas). The leghemoglobin in the roots of these crops in the US exceeds the myoglobin content in all red meat consumed in the US. In some embodiments, the subject of consumption is a meat replica composed primarily or entirely of ingredients derived from non-animal sources, including muscle replica, adipose tissue replica, connective tissue replica, and leghemoglobin. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, containing heme-containing protein. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, containing leghemoglobin. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, containing a member of the globin protein family. In some embodiments, the subject of consumption is a copy of the meat, consisting mainly or entirely of ingredients derived from non-animal sources, with a high content of iron from heme-containing protein. In some embodiments, the iron content is similar to that of meat. In some embodiments, the commodities have a characteristic red meat color, which color is produced by leghemoglobin.

Leghemoglobin, in some embodiments, is used as an indicator that the preparation of the consumable is complete. Thus, one embodiment of the present invention is a method of preparing a consumer product, including the detection of leghemoglobin, which moved from the interior of the product to the surface during the preparation of the product. Another embodiment of the present invention is a method for preparing a consumer product, comprising detecting a color change from red to brown during the preparation of the product.

The heme protein, in some embodiments, is used as an indicator that the preparation of the commodities is complete. Thus, one embodiment of the present invention is a method of preparing a consumer product, comprising detecting a heme-containing protein that has moved from the interior of the consumer product to the surface during product preparation. Another embodiment of the present invention is a method for preparing a consumer product, comprising detecting a color change from red to brown during the preparation of the product.

A heme-containing protein from the group of hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, HgbI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins, in some embodiments, is used as an indicator that the preparation of the consumable is finished. Thus, one embodiment of the present invention is a method of preparing a consumer product, including the detection of leghemoglobin, which moved from the interior of the product to the surface during the preparation of the product. Another embodiment of the present invention is a method for preparing a consumer product, comprising detecting a color change from red to brown during the preparation of the product.

Foods containing isolated, purified leghemoglobin

In some embodiments, leghemoglobin is added to meat to improve the properties of the meat. For example, a solution containing leghemoglobin can be injected into raw or cooked meat. In another example, the leghemoglobin solution is dropped onto the meat or commodities of the present invention to improve appearance. In one embodiment, advertising, photography or videography of food products such as meat or meat substitute is enhanced with leghemoglobin.

Sources of leghemoglobin

In some embodiments, the present invention provides methods for producing leghemoglobin from plants. Leghemoglobin can be obtained from a variety of plants. Various types of legumes and their varieties, such as soybeans, horse beans, lima beans, cowpeas, wrinkled peas, thermopsis, wolf beans, common beans, mutton peas, peanuts, alfalfa, hay vetch, clover, Japanese clover and pinto beans , contain nitrogen-assimilating root nodules in which leghemoglobin plays a key role in controlling oxygen concentrations (eg root nodules of the pea plant, Figure 2). In FIG. 3 shows 100 ml of leghemoglobin solution isolated from 30 grams of pea root nodules. Leghemoglobins from different species are homologues and have similar color characteristics (FIG. 4). In FIG. 4, panel A, shows SDS-PAGE after electrophoresis of lysed root nodules of three species of plants in the legume family: (1) horse bean, (2) rugose pea, (3) soybean. The arrows indicate the corresponding leghemoglobins. Note that leghemoglobin is the highest protein in each lysate. Panel B shows the similarity of the UV/Vis line profile for leghemoglobins from two different plant species (horse bean and soybean). The present inventors purified leghemoglobin from fava bean (green) and soybean (red) root nodules using the protocol described elsewhere herein. The UV/Vis spectra of both proteins show that the heme iron is in the reduced (+2) state. Note that they are almost completely overlapped, in keeping with their visually identical red color. The heme iron in the respective leghemoglobins was reduced to +2 oxidation state by incubation of faba bean and soybean leghemoglobin with 10 mM sodium hydrosulfite in buffer containing 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride. Sodium hydrogen sulfite was then removed from the leghemoglobin solution using gel filtration. The inset shows the enhancement of the UV/Vis spectra from 450 nm to 700 nm. Some plant species express several isoforms of leghemoglobin (for example, soybeans contain four isoforms of leghemoglobin). Minor variations in a particular amino acid sequence can change the net charge of a protein at a particular pH and can change a particular structural conformation of the iron-containing heme group in leghemoglobin. Differences in the structural conformation of the heme group in different leghemoglobins can affect the rates of oxidation and reduction of heme iron. These differences may contribute to the color and odor-producing characteristics of different leghemoglobins.

Leghemoglobin has an absorption spectrum and appearance virtually identical to those of animal muscle myoglobin. In FIG. Figure 5 compares reduced (2+ heme iron) and oxidized (3+ heme iron) soy leghemoglobin (Figure 5, panel A) and equine myoglobin (Figure 5, panel B) demonstrating similar absorption profiles in UV/visible range of two proteins. The present inventors purified leghemoglobin from soybean root nodules using the protocol described here. Purified equine myoglobin was purchased from SigmaAldrich. Soy leghemoglobin (FIG. 5, panel A) and equine myoglobin (FIG. 5, panel B) were reduced with 1 mM sodium hydrogen sulfite. The UV/Visible light absorption spectra of heme Fe3+ (blue line) and heme Fe2+ (red line) of soybean leghemoglobin (FIG. 5, panel A) and equine myoglobin (FIG. 5, panel B) are shown. Inserts show the increase in the spectra in the UV/visible range in the region from 450 nm to 700 nm. (Fig. 5, panel C) Images of a 10-µl liquid drop of a 40 mg/mL soybean leghemoglobin solution in the heme Fe3+ oxidation state (left drop), showing the characteristic rust color, and a 40 mg/mL soy leghemoglobin solution in the heme Fe2+ oxidation state (right drop) showing the characteristic red color and (right image) corresponding equine myoglobin samples.

In other embodiments, leghemoglobin can be obtained from non-plant based sources, such as organisms such as bacteria or yeast that have been genetically modified to express high levels of leghemoglobin.

The degree of oxidation of the iron ion in leghemoglobin is important for its color. Leghemoglobin with heme iron in the +2 oxidation state is bright red, while leghemoglobin with heme iron in the +3 oxidation state turns red-brown. Thus, when using leghemoglobin as the source of the red color in the meat copy, it is desirable to reduce the heme iron from the +3 oxidation state to the +2 oxidation state. Heme iron in leghemoglobin can be converted from an oxidized (+3) state to a reduced (+2) state using reducing reagents. Examples of successful reduction of heme iron in leghemoglobin with sodium hydrosulfite and titanium citrate are shown in FIG. 6. In FIG. 6, the UV/Visible spectrogram of purified soy leghemoglobin, in which the heme iron is in the oxidized (+3) state, is represented by the blue curves in each panel. The red curves in each panel are the UV/Vis spectra of the same species of leghemoglobin after reduction to grade (+2) (red lines) by the addition of (panel A) 1 mM sodium hydrosulfite or (panel B) 0.24% (in w/v ratio) of titanium citrate in a buffer containing 20 mM potassium phosphate pH 7.3, 100 mM sodium chloride. Inserts show the increase in the spectra in the UV/visible range in the region from 450 nm to 700 nm. For this example, leghemoglobin was purified from soy root nodules using fractionation by saturation to 60/90% ammonium sulfate and buffer exchange with a buffer containing 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride. A stock solution of sodium hydrosulfite was prepared by dissolving 100 mM sodium hydrogen sulfite in 1 mM sodium hydroxide in water. The titanium citrate stock solution was prepared from 20% (w/v) titanium chloride in hydrochloric acid by mixing it with 0.2 M sodium citrate (1:10 v/v). The pH was adjusted using sodium carbonate to pH 7.0.

Leghemoglobin can be purified from legume root nodules, for example pea or soybean root nodules (Fig. 1 shows pea root nodules, Fig. 2 shows leghemoglobin isolated from them). Soybean root nodules were thoroughly cleaned to remove soil and extraneous root tissue prior to root nodule lysis in 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTA and 1 mM ascorbic acid. Root nodules were lysed by crushing root nodules using a Vitamix grinder. In the case of some samples, the polymer polyvinylpyrrolidone was added at a rate of 30% w/v to assist in the removal of small phenolic plant molecules that mediate the oxidation of heme iron in leghemoglobin. The root nodule lysate was fractionated using ammonium sulfate in two steps, first ammonium sulfate was added to 60% w/v. The precipitate was discarded and the supernatant was made up to 90% w/v ammonium sulfate. Leghemoglobin was obtained as a precipitate in a fraction containing 90% ammonium sulfate. Ammonium sulfate precipitated leghemoglobin was resuspended in 20 mM potassium phosphate, 1 mM EDTA, 50 mM sodium chloride, and the ammonium sulfate was removed using dialysis or size exclusion chromatography in the same buffer. In some cases this was the last purification step, while in other cases the leghemoglobin was further purified using anion exchange chromatography (GE Healthcare FFQ) sometimes followed by gel filtration (Sephacryl S-100, GE Healthcare). Soy leghemoglobin from the precipitate precipitated with 90% ammonium sulfate was loaded onto anion exchange chromatography columns (FFQ or DEAE, GE Healthcare) in various buffers (20 mM potassium phosphate pH 7.4 containing 0-100 mM sodium chloride, 20 mM Tris pH 8 containing 0-100 mM sodium chloride, 20 mM sodium pyroboric acid pH 9.8, 20 mM sodium chloride, 20 mM sodium carbonate pH 9, 20 mM sodium chloride) and purified either in flow material or using sodium chloride (0- 1 M salt gradient). An example of soybean root nodule leghemoglobin purification in flow material is shown in FIG. 7. The figure shows fractionation by SDS-PAGE of samples obtained at various stages of soybean leghemoglobin purification: (lane 1) soybean root nodule lysate; (lane 2) soybean root nodule lysate purified by fractionation using 60/90% (w/v) ammonium sulfate. Shown is the protein content of a protein precipitate obtained after fractionation with 90% ammonium sulfate, resuspended in a buffer containing 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTA; proteins from the 90% ammonium sulfate precipitated precipitate were further purified by anion exchange chromatography (FFQ GE Healthcare) in 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride. The leghemoglobin produced in the flow material is shown in lane 3. The flow material from the anion exchange chromatography column was fractionated using size exclusion chromatography (Sephacryl S-100 GE Healthcare) and the resulting leghemoglobin-containing fraction is shown in lane 4. Leghemoglobin content at various stages purification was determined by determining the proportion of the corresponding leghemoglobin band on SDS-PAGE after electrophoresis in the corresponding sample using ImageDoc analysis software (BioRad). The purity (relative content) of leghemoglobin in the respective purification steps was: lysate: 32.7% (lane 1), fractionation using 60/90% (w/v) ammonium sulfate: 78% (lane 2), anion exchange chromatography : ~83% (lane 3) and gel chromatography: up to ~95% (lane 4).

Leghemoglobin can also be produced by genetically engineering the bacterium or fungus that produces it. One illustrative example is shown in FIG. 8. In FIG. 8 shows an electrophoresis-stained SDS-PAGE analysis of (A) soy leghemoglobin expressed and purified using recombinant protein technology and (B) soy leghemoglobin purified from soybean root nodules. (A) Recombinant soy leghemoglobin A containing a His tag and a site for His tag removal by TEV protease was expressed in E. coli strain BL21 and purified using His tag affinity chromatography (Talon resin, CloneTech) . The left lane contains molecular weight standards, the right lane contains purified recombinant soy leghemoglobin A (arrow). The estimated molecular weight of recombinant soy leghemoglobin A is 17.1 kDa. (B) SDS-PAGE after electrophoresis of purified soy leghemoglobin from root nodules. The left lane contains molecular weight standards, the right lane contains purified soy leghemoglobin A (arrow). Mass spectrometric analysis of the purified material indicated that all four isoforms of soy leghemoglobin were present and were full length (data not shown). Estimated molecular weights (M.m.) of soy leghemoglobin isoforms range from M.m. 15.4 to 15.8 kDa.

Leghemoglobin, purified from soy root nodules and fava beans, was tasted respectively by a group of volunteers, and in each case it was identified as a blood-like piece.

Leghemoglobin can be isolated from the root nodules of legumes such as soybean, horse bean, cowpea, lima bean, mutton pea, pea, wolf bean, Japanese lotus, or other legumes. A root nodule (eg root nodules of a pea plant, Fig. 1) is obtained and homogenized in an aqueous solution; soluble proteins, including leghemoglobin, are obtained after removal of insoluble material by precipitation and filtration. Leghemoglobin can be purified by selective precipitation and/or chromatography and/or the use of molecules with specific affinity for leghemoglobin. (Fig. 2 showing 100 ml of leghemoglobin solution isolated from 30 grams of pea root nodules).

Heme-containing proteins, such as leghemoglobin, can be combined with other components of the plant-based meat copy. In some embodiments, heme-containing proteins are fixed in a gel that contains other components, such as lipids and/or proteins. In some aspects, a plurality of gels are combined with non-gel-based heme-containing proteins. In some embodiments, heme-containing proteins and other consumable compounds are combined, ensuring that the heme-containing proteins can diffuse through the consumable. In some embodiments, the consumable is in a solution containing heme proteins, such as a solution of leghemoglobin. In some embodiments, the consumer item is soaked in a solution containing heme proteins, such as leghemoglobin solution, for 1, 5, 10, 15, 20, or 30 hours. In some embodiments, the consumable is soaked in a solution containing heme proteins, such as leghemoglobin solution, for 1, 5, 10, 15, 30, or 45 minutes.

In FIG. Figure 9 shows an example of 6 pieces of commercial meat analog (Quorn chicken analog), approximately 1 cm on the lateral surface, 4 of which (left and lower right) were soaked with a solution of approximately 10 mg/ml soy leghemoglobin in 20 mM potassium phosphate pH 7.4 and 100 mM NaCl; the other two (upper right) were soaked in the same buffer without leghemoglobin. Note the intense pink color of the leghemoglobin injected pieces as opposed to the pale yellow-brown color of the untreated pieces.

In FIG. 10 shows 4 pieces of leghemoglobin-infused Quorn chicken analogue during pan cooking at 350°C. The bottom two pieces have been flipped over to show the fried surface has turned brown. Pay attention to the top two pieces, the heated part has turned gray-brown, while the colder upper surface remains pink. In some embodiments, a solution containing heme-containing proteins, such as a leghemoglobin solution, is administered to the consumer until the consumer is the color of uncooked meat.

Considering the suitability of heme-containing proteins for coloring commodities, it is useful to determine if a product contains a particular heme-containing protein. Accordingly, the present invention includes, in some embodiments, methods for determining whether a product contains a heme protein. Methods for detecting proteins are well known in the art. For example, an ELISA or close ligation assay or Luninex assay or Western blot assay can be performed to determine if leghemoglobin is present in a food product such as meat or meat replica. In one embodiment, detection methods are performed to determine if the meat has been altered with leghemoglobin.

Examples

Described here is an exemplary composition for a muscle tissue copy comprising one or more isolated, purified plant proteins.

Purification of proteins for copy components

Golden bean seeds, dried garden pea seeds were purchased in the form of ground flour and used to purify the respective seed storage proteins. Rubsico has been purified from the fresh alfalfa plant. Protein composition at individual fractionation steps was monitored by SDS-PAGE, and protein concentrations were determined using standard UV/Vis and Pierce analysis methods.

Mung bean 8S globulins: Mung bean meal was resuspended in 50 mM potassium phosphate buffer pH 7 and 0.5 M NaCl at a ratio of 1:4 (w/v) and the mixture was incubated for 1 h. centrifugation, and the proteins in the supernatant were fractionated by adding ammonium sulfate in 2 stages: 50% (w/v) and then 90% (w/v). The protein precipitated in the 90% fraction contained mung bean 8S globulins and was stored at -20°C until further use.

Pea albumins: Dried garden pea seed meal was resuspended at a ratio of 1:10 (w/v) in 50 mM sodium acetate pH 5 buffer and incubated for 1 h. fractionation by precipitation with ammonium sulfate in two stages: 50% (w/v) and then 90% (w/v). The ammonium sulfate solutions were stirred for 1 hour and the ammonium sulfate precipitated proteins were removed by centrifugation. Proteins of interest were precipitated at 90% (w/v) ammonium sulfate. The precipitate was stored at -20°C until further use.

Pea globulins: Dried garden pea seed meal was resuspended at a ratio of 1:10 (w/v) in 20 mM potassium phosphate buffer pH 8, 0.4 M sodium chloride and stirred for 1 h. After centrifugation, the supernatant was fractionated using ammonium sulfate. First, the supernatant was adjusted to 50% (w/v) ammonium sulfate and the precipitated proteins were removed. The 50% (w/v) ammonium sulfate supernatant was then adjusted to saturation = 80% (w/v) ammonium sulfate. Protein precipitated with 80% (w/v) ammonium sulfate contained globulins of interest. The precipitate was stored at -20°C until further use.

RuBisCO: Fractionation of RuBisCO from green alfalfa leaves (or other green plants, e.g. soybeans, spinach, etc.) was performed by first grinding the leaves using 4 volumes of cold 50 mM K-phosphate buffer pH 7.4 (c (in laboratory) or without (in the field) 0.5 M NaCl + 2 mM DTT + 1 mM EDTA) in a chopper. The resulting suspension was centrifuged to remove debris and the supernatant (crude lysate) was used in further purification steps. The proteins in the crude lysate were fractionated by adding ammonium sulfate to saturation = 30% (w/v). The solution was stirred for 1 hour and then centrifuged. The precipitate from this step was discarded and additional ammonium sulfate was added to the supernatant until saturation = 50% (w/v) ammonium sulfate. After stirring for 1 h, the solution was centrifuged again. The precipitate from this step contained RuBisCO and was stored at -20° C. until used.

Obtaining Plant Proteins

Mung bean seed 8S protein was purified by ammonium sulfate fractionation as described. The pellet was resuspended in 20 mM potassium phosphate pH 7.4 and 0.5 M sodium chloride and the ammonium sulfate was removed by dialysis against the same buffer. Any precipitate was removed by centrifugation at 16,000×g, 10 min, and the protein was concentrated to the desired concentration. Pea globulins were purified by ammonium sulfate fractionation as described. The protein precipitate was resuspended in 20 mM potassium phosphate pH 7.4 and 0.4 M sodium chloride and the ammonium sulfate was removed by dialysis against the same buffer. Any precipitate was removed by centrifugation at 16,000×g, 10 min, and the protein was concentrated to the desired concentration. Pea albumin was purified by fractionation using ammonium sulfate as described. The protein precipitate was resuspended in 20 mM potassium phosphate pH 7.4 and 0.1 M sodium chloride and the ammonium sulfate was removed by dialysis against the same buffer. Any precipitate was removed by centrifugation at 16,000×g, 10 min, and the protein was concentrated to the desired concentration.

Creation of analogue of muscle tissue

Mung bean seed 8S protein was purified by ammonium sulfate fractionation as described. To prepare the gels, 200 g of the precipitate was dissolved in 400 ml of dialysis buffer (20 mM potassium phosphate, 400 mM NaCl, pH 7.3) and the resulting solution was dialyzed for 6 hours against 5 L of dialysis buffer replaced twice with fresh buffer. The protein solution was centrifuged at 12000×g for 15 min to remove debris. The protein was concentrated by dialysis for 36 hours against 5 L of a 30% w/w solution of PEG 8000 (polyethylene glycol, MW 8000) in dialysis buffer. The final protein concentration was 150 mg/ml.

Leghemoglobin was purified from soy root nodules. Legume root nodules were cleaned to remove soil and extraneous root tissue prior to root nodule lysis in 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTA and 1 mM ascorbic acid. Root nodules were lysed by crushing root nodules using a grinder in the form of a juicer. Insoluble material was removed by centrifugation. The root nodule lysate was fractionated using ammonium sulfate in two steps, first ammonium sulfate was added to 60% w/v and the solution was incubated for 1 h, 4°C. The precipitate was discarded and the supernatant was adjusted to 90% w/v ammonium sulfate and incubated for 12 h, 4°C. Leghemoglobin was obtained as a precipitate in the fraction containing 90% ammonium sulfate and resuspended in 20 mm potassium phosphate, 1 mm EDTA, 100 mm sodium chloride. SDS-PAGE analysis showed that the protein solution contained 70% leghemoglobin and 30% other root nodule proteins. Ammonium sulfate was removed using size exclusion chromatography in the same buffer. Leghemoglobin was concentrated by dialysis for 48 hours against 30% PEG 8000 (polyethylene glycol, MW 8000) in 20 mM potassium phosphate pH 7.3, 100 mM sodium chloride. The total protein concentration was 57 mg/ml. Spectra in the UV / visible range indicate that leghemoglobin is in the oxidized state of heme iron. Therefore, leghemoglobin was incubated with 5 mM sodium hydrogen sulfite for 5 minutes and the sodium hydrogen sulfite was removed using gel filtration in a buffer containing 20 mM potassium phosphate, 100 mM sodium chloride. Leghemoglobin was further concentrated to 35.4 mg/ml. UV/Visible line profile analysis confirmed that leghemoglobin is in the reduced state of heme iron.

Transglutaminase was purchased from Activa TI, Ajimoto. A stock solution (20% w/v) was prepared in a buffer containing 20 mM potassium phosphate, pH 7.3, 100 mM sodium chloride.

To prepare a dark muscle tissue analog (Figure 11), 43 ml of mung bean protein solution (150 mg/ml in dialysis buffer) was mixed with 37 ml of leghemoglobin solution (46.5 mg/ml of leghemoglobin and 20 mg/ml of another soy root nodule protein) in 20 mM potassium phosphate, 100 mM NaCl, pH 7.3. 20 ml transglutaminase solution (20% w/w) was added, the solutions were thoroughly mixed, dispensed into two 50 ml Falcon tubes and incubated overnight at room temperature. Final protein concentrations were 65 mg/ml for mung bean protein, 18 mg/ml leghemoglobin, 91 mg/ml total protein.

A "white" muscle tissue replica (Figure 12) was prepared by mixing 43 ml mung bean protein solution (150 mg/ml) with 45 ml 11.7 mg/ml leghemoglobin solution and 0.8% (w/v) transglutaminase solution. Final protein concentrations were 63 mg/ml for mung bean protein, 5.2 mg/ml leghemoglobin, 68 mg/ml total protein.

The “dark” muscle tissue analogue formed an opaque gel of dark chocolate color, with a homogeneous texture, with a shiny surface and a small amount (<1 ml) of dark red blood-colored liquid at the top. The gel was free-standing, elastic but brittle, similar in appearance to thin Jell-O. The gel has a moderate aroma with the characteristic features of beans and blood undoubtedly distinguishable. The scent is dominated by bean and iron/blood characteristics, with weaker herbal and medicinal/chemical odors. The taste is salty, with a taste of blood remaining in the mouth for a long time.

The "white" muscle analog was very similar, but much lighter, like a cappuccino, in color. It was also brittle, 2-3 times less resistant to compression.

Adipose tissue analog

Adipose tissue replica using mung bean 8S globulin fraction was prepared as follows: 15 ml of mung bean protein solution (150 mg/ml) in dialysis buffer was mixed with 15 ml of rice bran oil. 6 ml transglutaminase solution (20% w/w) was added, the solutions were thoroughly emulsified using a speed #2 homogenizer (VWR). The emulsion was aliquoted into 1.6 ml Eppendorf tubes and incubated overnight at room temperature. After that, the tubes were heated at 95°C for 5 min in a thermal block, and allowed to cool to room temperature on a laboratory bench. Final concentrations were 75 mg/ml mung bean protein, 50 wt % oil.

Adipose tissue analog using pea globulin (100 mg/ml) was prepared in the same manner. In addition, adipose tissue replica was prepared from pea globulin and either rice bran oil or canola oil, in large quantities in the same manner, but without aliquoting the emulsion into Eppendorf tubes. Instead, emulsions in 50 ml Falcon tubes were spun overnight on a shaker and subsequently incubated at 90°C for 30 minutes.

The adipose tissue replica based on mung bean (Fig. 13) and prepared in Eppendorf tubes formed an opaque off-white gel with a uniform texture, with no visible liquid that was not included in the gel. The gel was free-standing, elastic and resilient. The gel has a weak, pleasant aroma and a weak and pleasant smell. The taste is moderately salty.

The pea globulin based adipose tissue analog (FIG. 14) prepared in Eppendorf tubes was very similar to the mung bean based fat analog, except that it released some oil on compression. The adipose tissue replica prepared in 50 ml Falcon tubes was similar in appearance, texture, and flavors, but significantly softer (2 times softer for canola oil and 3 times softer for rice bran oil, according to definitions of compressibility).

Connective tissue analogue

Prototypes of the connective tissue analog have been developed using zein derived from 100% yellow corn gluten meal or from commercial sources such as Amazein (Prairie Gold, Bloomington, IL). Zein proteins were dissolved in 70-90% ethanol using the desired ratios of 1:3 to 1:5 (dry matter:solution). By precipitating zein proteins, for example by changing the pH in a controlled manner, large zein structures with physicochemical properties that can be freely manipulated are obtained. For example, in FIG. 15 shows connective tissue analog cords that were created using a 1:3 ratio in 70% ethanol loaded into a syringe with a 23G needle (ID 0.337 mm). The solution was slowly extruded from the bottom of a 5 inch high tank into an excess of 5 M NaCl solution. A solution of zein in ethanol, with a lower density than a solution of NaCl, floated up, pulling out the fibrous strands of solidifying zein. The NaCl was constantly stirred as strands began to form to help elongate the strands. The cords gathered together and became a hard, dense mass.

Prototypes of copies of ground beef, created from gels from plant proteins and vegetable oils.

Prototype ground beef patty was prepared by combining 62% (w/w) muscle tissue replica (62% (w/w) "dark muscle tissue replica" and 38% (w/w) "white muscle tissue replica"), 29 % (w/w) adipose tissue replica (from pea globulin and canola oil), 5% (w/w) connective tissue replica (FIG. 16, panel A). Prototype ground beef patty was prepared by combining 62% (w/w) muscle replica (62% "dark muscle replica" and 38% "white muscle replica"), 29% adipose tissue replica (from bean seed 8S protein golden and rice bran oil), 5% connective tissue analogue (Fig. 16, panel B). Prototype ground beef patty was prepared by combining 71% (w/w) muscle tissue replica (consisting of 60% "white muscle tissue analog", 40% "dark muscle tissue analog"), 23% adipose tissue (from pea globulin proteins and canola oil) (Fig. 16, panel C). Prototype ground beef patty was prepared by combining 67% white muscle replica with 28% adipose tissue replica (from pea globulins and rice bran oil) (FIG. 16, panel D).

In a further test, the effect of cooking spear ground beef patties by pan frying at 350°F was evaluated. A ground beef patty replica was prepared by combining 62% (w/w) muscle tissue replica (62% (w/w) "dark muscle tissue replica" and 38% (w/w) "white muscle tissue replica"), 29 % (w/w) adipose tissue replica (from pea globulin and canola oil), 5% (w/w) connective tissue replica (FIG. 17). The panel on the left shows the patty before cooking, and the panel on the right shows the same patty after cooking for about 2 minutes. Experts have identified the flavor from making a copy of the ground beef as distinctly "meaty".

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SEQUENCE LIST

<110> SAND HILL FOODS, INC.

<120> METHODS AND COMPOSITIONS FOR CONSUMER ITEMS

<130> 41751-701.601

<140> PCT/US2012/46560

<141> 2012-07-12

<150> 61/572.205

<151> 2011-07-12

<160> 3

<170> Patent in version 3.5

<210> 1

<211> 144

<212> Protein

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<220>

<223> Artificial sequence description: synthetic

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Met Val Ala Phe Thr Glu Lys Gln Asp Ala Leu Val Ser Ser Ser Phe

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Ser Ile Leu Glu Lys Ala Pro Ala Ala Lys Asp Leu Phe Ser Phe Leu

35 40 45

Ala Asn Gly Val Asp Pro Thr Asn Pro Lys Leu Thr Gly His Ala Glu

50 55 60

Lys Leu Phe Ala Leu Val Arg Asp Ser Ala Gly Gln Leu Lys Ala Ser

65 70 75 80

Gly Thr Val Val Ala Asp Ala Ala Leu Gly Ser Val His Ala Gln Lys

85 90 95

Ala Val Thr Asp Pro Gln Phe Val Val Val Lys Glu Ala Leu Leu Lys

100 105 110

Thr Ile Lys Ala Ala Val Gly Asp Lys Trp Ser Asp Glu Leu Ser Arg

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Ala Trp Glu Val Ala Tyr Asp Glu Leu Ala Ala Ala Ile Lys Lys Ala

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Phe Lys Glu Glu Pro Thr Val Ser Val Leu Phe Gln Asn Pro Ile Ser

35 40 45

Ser Gln Ser Arg Lys Leu Met Gln Val Leu Gly Ile Leu Val Gln Gly

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Ile Asp Asn Leu Glu Gly Leu Ile Pro Thr Leu Gln Asp Leu Gly Arg

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Arg His Lys Gln Tyr Gly Val Val Asp Ser His Tyr Pro Leu Val Gly

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Asp Cys Leu Leu Lys Ser Ile Gln Glu Tyr Leu Gly Gln Gly Phe Thr

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<---

Claims (18)

1. A meat copying food product, comprising: from 0.01% to 5% by weight of a protein containing heme iron; 10% or more by weight of vegetable protein; sugar; and an amino acid or sulfur-containing compound; wherein the meat-copying food product does not contain animal products, wherein the preparation of the meat-copying food product results in at least two volatile compounds that have a meat-related odor compared to a control meat-copying food product that does not contain a protein containing heme iron. 2. The meat-copying food product of claim 1, wherein the one or more heme iron containing proteins are selected from the group consisting of leghemoglobin, non-symbiotic hemoglobin, protoglobin, cytochrome, cyanoglobin, and flavohemoglobin. 3. The meat-copying food product of claim 1, wherein the heme iron-containing protein comprises an amino acid sequence at least 70% homologous to any of the amino acid sequences indicated by SEQ ID NOs: 1-3. 4. The meat-copying food product of claim 1, wherein the heme iron-containing protein comprises an amino acid sequence at least 80% homologous with any of the amino acid sequences indicated by SEQ ID NOs: 1-3. 5. The meat-copying food product of claim 1, wherein the heme iron-containing protein comprises an amino acid sequence at least 90% homologous to any of the amino acid sequences indicated by SEQ ID NOs: 1-3. 6. The meat copying food product according to claim 1, wherein the vegetable protein is selected from the group consisting of ribosomal protein, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, actin, translation elongation factor, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rubisco activase), albumin, glycinin, conglycinin, globulin, vicilin, conalbumin, gliadin, glutelin, gluten, glutenin, hordenin, prolamine, phaseolin, proteinoplast, secalin, extensin, Triticeae gluten, zein, any seed storage protein, oleosin, caloleosin, steroleosin, plant storage protein A, plant storage protein B, storage globulin 8S mung bean seeds. 7. The meat copying food product according to claim 1, wherein the sugar is glucose. 8. The meat-replicating food product of claim 1, wherein the meat-replicating food product further comprises fat from a non-animal source. 9. The meat copying food product according to claim 8, wherein the fat is selected from the group consisting of corn oil, olive oil, soybean oil, peanut oil, nut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil. oil, sunflower oil, linseed oil, algae oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil and rice bran oil. 10. The meat-copying food product of claim 1, wherein the meat-copying food product further comprises a nucleotide. 11. The meat-copying food product of claim 1, wherein the meat-copying food product is characterized by one or more of the following: no methylcellulose, no carrageenan, no burnt sugar, no konjac flour, no gum arabic, no gum senegal , contains less than 1% wheat gluten, does not contain soy protein isolate, does not contain soy protein concentrate, does not contain soy protein, contains less than 5% carbohydrates, does not contain tofu, contains less than 1% cellulose, contains less than 5% insoluble carbohydrates. 12. The meat copying food product of claim 1, wherein the heme iron containing protein is globin. 13. The meat copying food product according to claim 1, wherein the at least two volatile compounds comprise 2-octanone and 2-methylfuran.

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