Cell culture edible fish products

Abstract

Provided herein are in vitro cultured fish compositions, methods, and systems for culturing cells derived from a fish source. In at least one embodiment, the fish source is Bluefin tuna, and edible products formed therefrom are substantially free of contaminants. Provided herein are edible compositions comprising a homogenous mixture of in vitro cultured fish cells. The cells may each have a diameter of less than about 20 μm to about 200 μm.

Description

[Background Technology]

[0001] (background) Cell-cultured foods are part of a growing body of food alternatives that have been the focus of development by numerous companies worldwide as a means to address public health, environmental, and animal welfare issues associated with livestock and agriculture. Chemically contaminated and spoiled food has serious health implications. Foodborne illnesses and food poisoning have diverse origins (bacteria, viruses, parasites, mold, contaminants, etc.). In fact, some cases of food poisoning can be traced back to chemical and natural toxins. One of the toxins targeted by the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) is the biogenic amine histamine. Biogenic amines (BAs) in food constitute a potential public health problem due to their physiological and toxicological effects. Consumption of foods containing high concentrations of biogenic amines has been associated with health risks. The harmful effects range from minor stomach problems to major health disasters. Chemical contaminants are strongly associated with serious consequences, a lack of personal control, and long-term effects (Kher et al., 2011). According to the WHO, over 200 diseases are transmitted through food, and a large portion of the population will encounter a foodborne illness at some point in their lives. For example, in the United States, 48 ​​million people (one in six) suffer from a foodborne illness each year. Of these, 128,000 are hospitalized and 3,000 die from the illness (Claudia Ruiz-Capillas et al., 2019). Dietary intake is the most likely source of human exposure to metals. Metals (e.g., cadmium and lead) can easily enter the food chain. Heavy metals can severely deplete certain nutrients in the body, which can reduce immune defenses, impair psychosocial abilities, and cause intrauterine growth retardation. Heavy metal intake is also associated with malnutrition and increased rates of digestive disorders (Khan et al., 2008). Food contaminants are also a major cause of cancer (Abnet, 2007). Exposure to polychlorinated biphenyls (PCBs) resulting from food contamination can have adverse effects on children's neurological development and immune responses ( Schantz et al., 2004 ).Pesticides in food as contaminants also pose serious health risks. Excessive levels of these chemicals in food can cause nerve and kidney damage, birth defects, and reproductive problems, and excessive levels of these chemicals in food can be carcinogenic (Bassil et al., 2007). Accumulation of pesticides in body tissues can also result in metabolic degradation (Androutsopoulos et al., 2013). There is also a risk of neurodevelopmental disorders (such as attention deficit disorder, autism, cerebral palsy, and mental retardation) caused by industrial chemicals (such as arsenic, PCBs, and lead) in both food and water. The present application provides edible fish products that are substantially free of contaminants and have an extended shelf life compared to conventional products. Summary of the Invention [Means for solving the problem]

[0002] (Abstract) The present invention relates to cell-cultured foods and related cells derived from a fish source. Compositions are also provided. In at least one embodiment, the fish source is Bluefin tuna, and edible products formed therefrom are substantially free of contaminants.

[0003] The edible compositions provided herein can contain less than 0.1 parts per million (ppm) of contaminants. The contaminants can be environmental contaminants (e.g., mercury), food processing contaminants, unapproved adulterants, and food additives. Environmental contaminants include mercury, lead, cadmium, zinc, copper, nickel, chromium, arsenic, aluminum, fluoride, radon, and pesticides.

[0004] In accordance with one aspect of the present invention, provided herein is an edible composition substantially free of contaminants, comprising a homogenous mixture of in vitro-cultured fish cells. The cells may each be less than about 20 μm to less than about 200 μm in diameter. In one embodiment, the cells are each 20 μm in diameter. In another embodiment, the cells are each less than 200 μm in diameter.

[0005] The edible compositions provided herein include one, two, or three cell types, which may be myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, adipocytes, or a combination thereof.

[0006] The edible composition may be in the form of a viscous slurry, according to certain aspects of the present invention. The edible composition may be frozen or freeze-dried.

[0007] The edible composition may be liquid, semi-liquid, semi-solid, solid, or foam.

[0008] According to another aspect, the edible composition is described as comprising a population of non-filamentous in vitro cultured fish cells, the cells being in a layered morphology, with each layer comprising a homogenous mixture of cells. The edible composition may comprise a single cell type or may comprise multiple cell types (e.g., one to three cell types, four or more cell types). In another embodiment, the edible composition comprises a population of non-filamentous in vitro cultured tuna cells arranged in a homogenous morphology without layering. In yet another embodiment, the non-filamentous in vitro cultured cells are Bluefin tuna cells.

[0009] According to another embodiment, a method for producing substantially contaminant-free Bluefin tuna slurry is described, comprising the steps of harvesting myoblasts and fibroblasts from muscle tissue of wild-caught Pacific Bluefin tuna, harvesting preadipocytes from subcutaneous fat of wild-caught Pacific Bluefin tuna, expanding the myoblasts, fibroblasts, and preadipocytes in vitro for at least 50 population doublings to achieve stable cell lines, expanding the myoblasts and / or preadipocytes under suitable growth conditions, transferring the myoblasts to a differentiation medium to form myocytes or myotubes, treating the preadipocytes with a lipid-containing medium to form adipocytes, differentiating the myoblasts and / or preadipocytes in a dilute differentiation medium, and concentrating the cells by centrifugation, sedimentation, or other separation method to separate the cells from the liquid, thereby achieving at least 10 6 forming a slurry containing cells at a concentration of about 10 6 cells / ml~about 10 9 The slurry may contain 1000 cells / ml. The method may further include freezing or drying the slurry to form a solid product. The wild-caught Pacific Bluefin tuna used in the methods described herein may weigh from about 12 pounds to about 200 pounds.

[0010] The slurries used in the methods described herein may contain one or more cell types (e.g., myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells, embryo-derived cells, or induced pluripotent stem cells).

[0011] The growing step may include seeding 0.1 gram to 1 gram of tissue per well.

[0012] The preadipocyte treatment can cause the cells to transition from small, proliferating cells to round, lipid-laden, non-proliferating cells. The growth conditions can include a pH range of 7.3-7.5 and a temperature change from 15°C to 30°C.

[0013] The culture media and systems described herein, as well as the related compositions, cells, cell biomass, and cell culture foods described herein, can be used in connection with a variety of applications in which cell viability, controlled growth levels, and contamination levels in the cells and related cell culture foods are desired. For example, the compositions described herein, as well as the related cells, cell biomass, and cell culture foods described herein, can be used to produce cell culture foods (e.g., foods that are substantially free of contaminants). Thus, exemplary fields of application include food production, food processing, and commercialization. Further exemplary applications include the use of the culture media, compositions, methods, and systems described herein, as well as the related cells, cell biomass, and cell culture foods, in several fields, including basic biology research, applied biology, bioengineering, bioenergy, medical research, therapeutics, and additional fields that can be identified by one of ordinary skill in the art upon reading the present invention.

[0014] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0015] [Figure 1] 1A-1G show schematic diagrams of the marbling found in cell-cultured Bluefin tuna products of the present invention.

[0016] [Figure 2]Figures 2A and 2B show the extended shelf life of the cell cultured Bluefin tuna product compared to conventional Bluefin tuna at 4°C. The rate of color change is substantially slower in the cell culture product.

[0017] [Figure 3] Figure 3 shows the shelf-life stability of cell-cultured Bluefin tuna product at -80°C at 80°C. The graph shows the color difference of cell-cultured Bluefin tuna after 11 days of storage at -80°C. CIELAB values ​​(a* and b*) were measured for cell-cultured Bluefin tuna (BN BFT). Figure 3 shows that there is no color difference (a* / b*) between days 0 and 11, indicating extended shelf-life stability of the cell-cultured Bluefin tuna product at -80°C storage conditions.

[0018] [Figure 4] Figure 4 is a graph showing the color difference in cell-cultured Bluefin tuna compared to conventional Bluefin tuna after three months of storage at -20°C. The color difference between conventional Bluefin tuna (Conventional BFT) and cell-cultured Bluefin tuna samples (BN BFT) was quantified as ΔE (total color difference) calculated in the CIELAB color space system. The CIELAB color space system measures three values: an achromatic component, L* (light vs. dark), and two color descriptors, a* (red vs. green) and b* (yellow vs. blue). Statistical analysis was performed using two-way ANOVA. The graph shows a significant decrease (p<0.05) in color change between cell-cultured Bluefin tuna compared to conventional Bluefin tuna, indicating an extended shelf life of the cell-cultured Bluefin tuna product under frozen storage conditions.

[0019] [Figure 5]Figure 5 shows the extent of lipid oxidation in cell-cultured Bluefin tuna product compared to conventional Bluefin tuna after two weeks of storage at 4°C. Oxidation was measured by assaying the amount of malondialdehyde (MDA) per sample for conventional Bluefin tuna (Conventional BFT) and cell-cultured Bluefin tuna (BN BFT) for 14 days at 4°C (n=3). The graph shows a significant decrease (p<0.05) in the amount of MDA production in cell-cultured Bluefin tuna compared to conventional Bluefin tuna by two-way ANOVA, indicating reduced oxidation under refrigerated storage conditions for cell-cultured Bluefin tuna product. DETAILED DESCRIPTION OF THE INVENTION

[0020] (Detailed explanation) Provided herein are cell-cultured edibles, compositions, methods and systems derived from fish sources, and related cells and cell biomass. In one embodiment, the cells are derived from one or more primary cell lines isolated from wild-caught fish (e.g., from wild-caught Bluefin tuna).

[0021] The term "about," when referring to a measurable value (e.g., amount, time period, etc.), is intended to include a variation of no more than ±20%, or in some cases no more than ±15%, or in some cases no more than ±10%, or in some cases no more than ±5%, or in some cases no more than ±1%, or in some cases no more than ±0.1% from the specified value, where such a variation is appropriate.

[0022] The term "cell line" is a term of art that refers to a defined population of cells that can be maintained in culture for extended periods of time. A "stabilized cell line" is a cell line that exhibits genomic and phenotypic stability, a doubling time of less than 96 hours, and a viability of greater than 70% 24 hours after seeding.

[0023] The term "myoblast" is a term of art that refers to the precursor of muscle cells (also called muscle cells). Myoblasts differentiate into muscle cells through myogenesis, as understood by those skilled in the art. Depending on the type of muscle cell they differentiate into, myoblasts can be classified as skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts. Exemplary myoblasts in aquatic animals include skeletal muscle myoblasts and smooth muscle myoblasts.

[0024] The term "fibroblast" is a term of art referring to a type of cell in animal connective tissue that synthesizes components of the extracellular matrix (e.g., collagen). Fibroblasts generate the structural framework for animal tissues and play an important role in wound healing. Fibroblasts are the most common cell of connective tissue in animals. Fibroblasts have a branched cytoplasm surrounding a speckled, oval nucleus with two or more nucleoli. Active fibroblasts can be recognized by their abundant rough endoplasmic reticulum. Inactive fibroblasts (also called fibrocytes) are smaller, spindle-shaped, and have reduced amounts of rough endoplasmic reticulum. While loose and scattered when they must cover large spaces, fibroblasts often align locally in parallel clusters when crowded. Exemplary fibroblasts include those derived from muscle and other tissues (e.g., brain, heart, or skin).

[0025] The term "adipocyte" is a term used in the art to refer to fat cells (also known as lipocytes). Adipocytes are cells specialized for storing energy as fat and are the primary constituents of adipose tissue. Adipocytes can be derived from mesenchymal stem cells, which give rise to adipocytes through adipogenesis. In cell culture, adipocytes can also form osteoblasts, myocytes, and other cell types. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), which are also known as white fat and brown fat, respectively, and contain two types of adipocytes. Adipocytes can arise from either preadipocytes present in adipose tissue or bone marrow-derived progenitor cells that migrate into adipose tissue. As used herein, cells typically include adipocytes derived from white adipose tissue.

[0026] The term "preadipocyte" is a term of art that refers to a precursor of a mature differentiated adipocyte that can be stimulated to form an adipocyte. Preadipocytes can be isolated from subcutaneous or visceral adipose tissue of an animal.

[0027] The preadipocytes can be grown in a preadipocyte growth medium containing all the growth factors and supplements necessary for optimal growth of undifferentiated preadipocytes, for example, endothelial cell growth supplements, epidermal growth factor, hydrocortisone, and / or heparin.

[0028] The formation of adipocytes from preadipocytes involves a tightly regulated cell differentiation process (called adipogenesis), in which mesenchymal stem cells are committed to preadipocytes and preadipocytes differentiate into adipocytes. The term "differentiate" or "differentiation" refers to the process of expression pattern change in which pluripotent gene expression is converted to cell-type-specific gene expression. Transcription factors (e.g., peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer-binding protein (C / EBP)) are key regulators of adipogenesis. Characteristic properties of differentiated adipocytes, as understood by those skilled in the art, include, but are not limited to, growth arrest, morphological changes, high expression of lipogenic genes, and production of adipokines (e.g., adiponectin, leptin, resistin (in mice, but not in humans), and TNF-α).

[0029] As used herein, the term "homogeneous product" is a product that is formed as a unitary piece and is substantially uniform throughout the piece in terms of its organoleptic and functional properties.

[0030] As used herein, the term "culture medium" refers to a liquid, solid, or gel composition containing organic, inorganic, and / or biological components, in which cells can live, maintain viability, or grow. Culture media typically include a basal medium.

[0031] As used herein, the term "basal medium" refers to a culture medium that contains essential components for cell survival and growth, such as amino acids, glucose, and ions (e.g., calcium, magnesium, potassium, sodium, and phosphate), as understood by those skilled in the art.

[0032] An example of a basal medium is Basal Media Formulation (www.sigmaaldrich.com / life-science / cell-culture / learning-center / media-formulations / basal.html). Further examples may be identifiable by one of skill in the art.

[0033] Exemplary biological components include serum. The medium can be chemically defined. For example, Lipid Mixture 1, available from Sigma Aldrich (www.sigmaaldrich.com / catalog / product / sigma / l0288?lang=en®ion=US), contains non-animal-derived fatty acids (2 μg / ml arachidonic acid and 10 μg / ml each of linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid), 0.22 mg / ml cholesterol from New Zealand wool, 2.2 mg / ml Tween®-80, 70 μg / ml tocopherol acetate, and 100 mg / ml Pluronic® F-68, solubilized in cell culture water.

[0034] The medium in the sense of the present invention may contain biological components, such as fetal bovine serum (FBS) or cod liver oil fatty acids. For example, the Lipid Mixture (1000x) available from Sigma Aldrich (www.sigmaaldrich.com / catalog / product / sigma / l5146?lang=en®ion=US) contains cholesterol (4.5 g / L), cod liver oil fatty acid (methyl ester) (10 g / L), polyoxyethylene sorbitan monooleate (25 g / L), and D-α-tocopherol acetate (2.0 g / L).

[0035] Food "texture" is defined by the International Organization for Standardization (ISO) in its standard terminology for sensory analysis as "all the rheological and structural (geometric and surface) properties of a food that can be perceived by mechanoreceptors, tactile receptors, and, where appropriate, visual and auditory receptors" (ISO, 2008). Texture is an important quality parameter used in the fresh food and processed food industries to assess consumer acceptability. Among textural characteristics, firmness (hardness) is one of the most important parameters and is often used to determine the freshness of food. Elasticity, cohesiveness, adhesiveness, and chewability are also important properties for texture evaluation of meat-based products.

[0036] The "color" of a food is a physical characteristic commonly associated with its quality. The color of a food's surface can be easily measured using instruments used as a quantitative quality tool. Over the life of a product, color changes can indicate deterioration in the product's quality. To analyze product color changes, the International Commission on Illumination (CIE) proposed a universal method to be used in analyzing color in 1931. This method distinguishes color into three different tristimulus values. More recently, the Munsell system has made color quantification even easier through multidimensional methods. L*, a*, b*, h, and C readings can be quantified and compared. L indicates the overall lightness of a sample. The a* value indicates red or green in the sample, while the b* value indicates yellow or blue. Hue (h) and chroma (C) values ​​are derived from the a* and b* values; hue is expressed in radians or degrees in the color space, and chroma is expressed as a measure of intensity as a distance from the achromatic center of the color space. Color analysis can indicate surface alteration of the product, and color difference values ​​(ΔE) can be useful in distinguishing between preservation treatments.

[0037] The Institute of Food Science and Technology (IFST) in the UK has defined "shelf life" as "the period during which it is certain that a food will remain safe and will retain the desired organoleptic, chemical, physical, microbiological and functional characteristics; and will comply with any label declaration of nutritional data when stored under recommended conditions" (Shelf Life of Foods: Guidelines for Its Determination and Prediction, 1993).

[0038] "Substantially free" of heavy metal contaminants (e.g., mercury) means that the edible composition contains less than 0.1 parts per million (ppm) (e.g., less than 0.1 ppm, less than 0.01 ppm, less than 0.001 ppm, or less than 0.0001 ppm) of one or more heavy metal contaminants.

[0039] (Cultured fish cells and compositions) In certain embodiments of the present invention, culture media, methods, and systems for culturing fish cells (e.g., Bluefin tuna), and fish cells (e.g., Bluefin tuna cells) obtainable and / or obtained thereby, are described. In embodiments of the present invention, compositions, methods, and systems, and related fish cells, fish cell biomass, and fish cell culture foods are described that have controllable cellular lipid content and lipid uptake, and / or improved cell differentiation and / or cell viability with defined lipid content and lipid uptake. The fish cell culture foods can be in multiple forms (e.g., slurry product, minced product, homogenous single-cell product, homogenous multi-cell product, non-fibrous layered single-cell product, or non-fibrous layered multi-cell product).

[0040] In one embodiment, the source of fish cells is any fish cell from a variety of species.

[0041] In another embodiment, the fish source is any one or more of the seven species of tuna in the genus Thunnus, including northern bluefin tuna (T. thynnus), albacore (T. alalunga), yellowfin (T. albacares), southern bluefin tuna (T. thynnus maccoyii), bigeye tuna (T. obesus), Atlantic bluefin tuna (T. atlanticus), and longfin tuna (T. tongol). See https: / / www.britannica.com / animal / tuna-fish, which is incorporated herein by reference.

[0042] In another embodiment, the fish cells are sourced from the genus thynnus orientalis, and the fish cells are sourced from Bluefin tuna.

[0043] The cell-cultured fish products of the present invention differ from conventional fish-derived products in several ways.

[0044] In contrast to conventional fish-derived products in which cells are oriented so that muscle fibers and blood vessels are aligned, the cell-cultured fish products disclosed herein contain cells that are not oriented in an organized manner. The cell-cultured layered products disclosed herein are similarly unorganized within each layer. The connectivity of cells in cell-cultured bluefin tuna products is limited in terms of the number of cell bonds and low amounts of extracellular matrix. In contrast, in conventional fish-derived products, there are many cells bound to each other and to the extracellular matrix. The amino acid and fatty acid profiles of the cell-cultured fish can be controlled and modified. Furthermore, the cell-cultured products can contain sugars, fiber, texture modifiers, natural colors, and natural flavor enhancers not found in conventional products. The color of the cell culture product can also be controlled by adding natural color compounds (e.g., carotenoids, such as astaxanthin) to the cell culture medium to achieve a desired color (e.g., red, pink, white) or by adding natural color compounds (e.g., anthocyanins, beet juice, beta-carotene, curcumin, spirulina, insect-derived colors (including but not limited to carmine), carotenoids (e.g., astaxanthin), heme, leghemoglobin, lycopene, Monascus pigment, paprika, or other natural colors) to the final product.

[0045] In certain embodiments, the present invention relates to an edible composition comprising in vitro cultured fish cells arranged in a homogeneous or layered morphology. The in vitro cultured fish cells are generally smaller than conventional fish cells. When proliferating, the cultured cells may each have a diameter of less than 20 μm, e.g., less than 20 μm, less than 19 μm, less than 18 μm, less than 17 μm, less than 16 μm, less than 15 μm, less than 14 μm, less than 13 μm, less than 12 μm, less than 11 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, or less than 5 μm. Differentiated cultured cells tend to be medium-sized compared to single muscle cells with one nucleus, which can be small (i.e., 10 μm to 30 μm), or the much larger in vitro myotubes (i.e., 50 μm to 200 μm).

[0046] In certain embodiments, the cultured cells have a diameter of less than 20 μm to less than 200 μm, e.g., less than 20 μm, less than 21 μm, less than 22 μm, less than 23 μm, less than 24 μm, less than 25 μm, less than 26 μm, less than 27 μm, less than 28 μm, less than 29 μm, less than 30 μm, less than 31 μm, less than 32 μm, less than 33 μm, less than 34 μm, less than 35 μm, less than 36 μm, less than 37 μm, less than 38 μm, less than 39 μm, less than 40 μm, less than 41 μm, less than 42 μm, less than 43 μm, less than 44 μm, less than 45 μm, less than 46 μm, less than 47 μm, less than 48 μm, less than 49 μm. Less than 50 μm, less than 51 μm, less than 52 μm, less than 53 μm, less than 54 μm, less than 55 μm, less than 56 μm, less than 57 μm, less than 58 μm, less than 59 μm, less than 60 μm, less than 61 μm, less than 62 μm, less than 63 μm, less than 64 μm, less than 65 μm, less than 66 μm, less than 67 μm, less than 68 μm, less than 69 μm, less than 70 μm, less than 71 μm, less than 72 μm, less than 73 μm, less than 74 μm, less than 75 μm, less than 76 μm, less than 77 μm, less than 78 μm, less than 79 μm, less than 80 μm, less than 81 μm, less than 82 μm, less than 83 μm, less than 84 μm, Less than 85 μm, Less than 86 μm, Less than 87 μm, Less than 88 μm, Less than 89 μm, Less than 90 μm, Less than 91 μm, Less than 92 μm, Less than 93 μm, Less than 94 μm, Less than 95 μm, Less than 96 μm, Less than 97 μm, Less than 98 μm, Less than 99 μm, Less than 100 μm, Less than 101 μm, Less than 102 μm, Less than 103 μm, Less than 104 μm, Less than 105 μm, Less than 106 μm, Less than 107 μm, Less than 108 μm, Less than 109 μm, Less than 110 μm, Less than 111 μm, Less than 112 μm, Less than 113 μm, Less than 114 μm, Less than 115 μm, Less than 116 μm, Less than 117 μm , less than 118 μm, less than 119 μm, less than 120 μm, less than 121 μm, less than 122 μm, less than 123 μm, less than 124 μm, less than 125 μm, less than 126 μm, less than 127 μm, less than 128 μm, less than 129 μm, less than 130 μm, less than 131 μm, less than 132 μm, less than 133 μm, less than 134 μm, less than 135 μm, less than 136 μm, less than 137 μm, less than 138 μm, less than 139 μm, less than 140 μm, less than 141 μm, less than 142 μm, less than 143 μm, less than 144 μm, less than 145 μm, less than 146 μm, less than 147 μm, less than 148 μm,Less than 149 μm, Less than 150 μm, Less than 151 μm, Less than 152 μm, Less than 153 μm, Less than 154 μm, Less than 155 μm, Less than 156 μm, Less than 157 μm, Less than 158 μm, Less than 159 μm, Less than 160 μm, Less than 161 μm, Less than 162 μm, Less than 163 μm, Less than 164 μm, Less than 165 μm, Less than 166 μm, Less than 167 μm, Less than 168 μm, Less than 169 μm, Less than 170 μm, Less than 171 μm, Less than 172 μm, Less than 173 μm, Less than 174 μm, 175 μm The diameter may be less than 176 μm, less than 177 μm, less than 178 μm, less than 179 μm, less than 180 μm, less than 181 μm, less than 182 μm, less than 183 μm, less than 184 μm, less than 185 μm, less than 186 μm, less than 187 μm, less than 188 μm, less than 189 μm, less than 190 μm, less than 191 μm, less than 192 μm, less than 193 μm, less than 194 μm, less than 195 μm, less than 196 μm, less than 197 μm, less than 198 μm, less than 199 μm, or less than 200 μm.

[0047] The fish cells are also substantially free of contaminants (e.g., environmental contaminants). Exemplary environmental contaminants include microplastics, persistent organic pollutants (POPs) (e.g., polychlorinated biphenyls (PCBs), organochlorines (OCs), and polybrominated biphenyl ethers (PBDEs)), heavy metals (e.g., mercury, lead, cadmium, zinc, copper, nickel, chromium, arsenic, aluminum), fluoride, radon, microplastics, parasites, bacteria, and pesticides.

[0048] In some embodiments, the edible composition is substantially free of mercury and arsenic. Substantially free of heavy metal contaminants (e.g., mercury) means that the edible composition contains less than 0.1 parts per million (ppm) (e.g., less than 0.1 ppm, less than 0.01 ppm, 0.001 ppm, or less than 0.0001 ppm) of one or more heavy metal contaminants.

[0049] In addition to tuna cells from the genus Thunnus (particularly Bluefin tuna cells), cells from a wide variety of fish species can be used, including bass, flounder, hake, scup, smelt, rainbow trout, hard mussels, blue crab, peekytoe crab, Japanese crab, cuttlefish, Eastern oyster, Pacific oyster, anchovy, herring, golden mullet, moi, orange roughy, Atlantic Ocean perch, Nile perch (Lake Victoria perch), yellow perch, European oyster, and European sole. sole, sturgeon, tilefish, Wahoo, yellowtail, sea urchin, Atlantic mackerel, sardine, black sea bass, European sea bass, hybrid striped bass, bream, cod, drum, haddock, hoki, pollock, rockfish, pink salmon, snapper, tilapia, turbot, walleye, lake whitefish, wolffish, hard clams, surf clams, cockles, Jonah crab, snow crab, crayfish, bay scallops scallop, Korean shrimp, sablefish, Atlantic salmon, coho salmon, skate, Dungeness crab, king crab, mussel, green lipped mussel, Arctic shrimp, escolar, Chinook salmon, chum salmon, American shad, Arctic char, carp, catfish, John dory, grouper, halibut, monkfish, pompano, abalone, conch, stone crabcrab), American lobster, spiny lobster, octopus, black prawn, marsh shrimp, gulf shrimp, whitefish, squid, barramundi, cusk, dogfish, kingklip, dolphinfish, sunfish, mako shark, swordfish, albacore, yellowfin tuna, giant clams, squid lobster, sea scallop, rock shrimp, barracuda, Chilean sea bass, cobia, white croaker, eel, blue marlin, mullet, sockeye salmon, bluefin tuna, shrimp, crab, lobster, and echinoderms (e.g., sea cucumber and sea urchin).

[0050] Preferred aquatic animals include yellowtail (e.g., Seriola lalandi), dolphinfish (Coryphaena hippurus), red snapper (Lutjanus campechanus), bluefin tuna (e.g., Thunnus orientalis and Thunnus thynnus), yellowfin tuna (Thunnus albacares), cod (e.g., Gadus morhua, Gadus Macrocephalus, Gadus ogac), flounder, halibut, herring, mackerel, pompano, salmon, sea bass, toothfish (Dissostichus eleginoides, squid, clams, lobster, crab, scallop, shrimp, eel, bass (e.g., Micropterus salmoides), bluegill (Lepomis macrochirus), and carp (e.g., Hypophthalmichthys molitrix).

[0051] The homogenous or layered mixture of in vitro cultured fish cells disclosed herein contains only one cell type, two different cell types, three different cell types, or four different cell types, whereas conventional fish-derived products contain many different cell types, including myoblasts, myotubes, endothelial cells, neurons, erythrocytes, adipocytes, and combinations thereof. The mixture of in vitro cultured fish cells can be formed into various product types, such as slurries, minced meat, homogenous or layered sheets, and homogenous or layered blocks. For example, in some embodiments, the cell culture slurry product or minced meat product contains one or two cell types. The various cell types contained in the cell culture product include myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, adipocytes, and combinations thereof.

[0052] The cell cultured food fish products provided herein can be frozen or refrigerated or freeze-dried so that they can be used at a later time.

[0053] The edible composition may be liquid, semi-liquid, semi-solid, solid, or foam.

[0054] In various embodiments, cultured fish food products according to the present invention may contain functional agents in addition to the cultured fish cells, including one or more texture modifiers that provide firmness, mouthfeel, and other textural characteristics (such as cohesiveness, springiness, and chewiness), as well as optional additional functional agents that prevent peroxidation of lipid components, reduce microbial contamination, and / or otherwise extend the shelf life of the product, and optional sensory agents (e.g., flavorings and colors); and dietary supplements.

[0055] (functional agent) (Texture agent) The texture modifiers and the concentrations of each may be selected to achieve a degree of the above textural properties and characteristics appropriate for the intended purpose of the product. The product formulation may include one or more gelling agents and / or one or more thickening agents.

[0056] In addition to the texture improvers mentioned above, the product may contain protein isolates / protein concentrates / texturized proteins (including, but not limited to, soy, wheat, pea, chickpea, hemp, duckweed, lentil, oat, rice, and potato). It may also contain oils, fats, and / or hydrogenated vegetable oils and shortenings (including, but not limited to, sunflower, safflower, rapeseed, canola, soy, coconut, palm, and algae). These ingredients also contribute to the overall texture and mouthfeel of the product. In exemplary embodiments, the total concentration of texture-contributing agents may be between 1% (w / w) and 40% (w / w) of the final product mass.

[0057] Food-safe gelling agents suitable for use in the above products include, but are not limited to, sodium alginate, carrageenan, agar, gellan gum, konjac gum, and curdlan gum, among others. Similarly, various food-safe thickening agents suitable for use include, but are not limited to, guar gum, gum arabic, locust bean gum, methylcellulose, psyllium husk, pea fiber, citrus fiber, and xanthan gum, among others. The concentration of a particular texture modifier will vary according to the intended use of the product and may range from 0.05% to 10%.

[0058] In addition to firmness, certain texture modifiers further provide additional functional properties, including, but not limited to, heat stability and browning. Such agents allow the pieces to remain solid during heating and / or brown during cooking to develop an appetizing appearance and / or flavor. The concentration of such agents is selected to provide a product that is acceptably heat stable and / or browns in a controlled manner, without being too hard for the consumer. In some embodiments, the texture modifier is selected to provide emulsion stabilization in addition to other sensory or functional properties.

[0059] Some texture modifiers (e.g., locust bean gum and gellan gum) may have a synergistic effect on texture. The particular combination, as well as the absolute and relative concentrations of the individual agents that make up the combination, is selected to achieve a product with a firmness that approximates that of conventional fish meat without sacrificing other desirable organoleptic or functional properties.

[0060] (coloring agent) In traditional Bluefin tuna, the sarcoplasmic protein myoglobin is responsible for the red color of the muscle. The amount of myoglobin and fat in different fillets gives them distinct colors ranging from dark red (lean) to pinkish red / pink (chutoro and otoro; commonly called toro).

[0061] In the cell culture product, there is no native myoglobin, and therefore the natural colorants described in the above embodiments can be used in powder, liquid, or emulsion form to achieve various color shades of the product described in the above embodiments. The colorants can be used alone or in combination to achieve the desired result. In one example, the concentration of the colorants ranges from 33.0 to 62.8 L. * values, a range of 4.77 to 21.9 * values, and b in the range of 4.72 to 15.4 *The concentration can be varied from 0.001% to 5% to achieve the respective values.

[0062] The addition of natural coloring alone does not ensure the proper color; Bluefin tuna cells are responsible for adding some color characteristics as well as changing the overall color tone. In one embodiment, the L * value, a * value, b * The values ​​changed from 50.18 to 69.89, 18.46 to 17.24, and 14.30 to 14.26 with the addition of Bluefin tuna cells. Similarly, the texture of the Bluefin tuna cells can also be manipulated by changing the overall firmness of the edible product from 656g to 442g.

[0063] (sensory agent) In addition to the texture improver, the homogenous in vitro cultured fish cell product and the layered in vitro cultured fish cell product may optionally be formulated with one or more sensory agents (including, but not limited to, natural colors and natural flavoring agents). Suitable colors are those that provide the desired color at a pH typically found in meat obtained from live fish, i.e., pH 6-7. While colors derived from insects or other sources that are safe for human consumption may also be used, food-derived colors are preferred for reasons of safety and consumer appeal. Suitable colors include, but are not limited to, food-derived colors (e.g., anthocyanins, carotenoids, red radish powder, beet juice, leghemoglobin, lycopene, Monascus pigment, and paprika) and insect-derived colors (including, but not limited to, carmine).

[0064] The flavor of very fresh fish is characterized by a mild, delicate aroma, due in part to volatile 6-, 8-, and 9-carbon carbonyls and alcohols resulting from the action of lipoxygenase on long-chain polyunsaturated fatty acids. In various exemplary embodiments, one or more of these compounds are added to further enhance the flavor provided by cultured Bluefin tuna cells. The flavor enhancer may include vegetable oil, coconut oil, palm oil, algae oil, sunflower oil, safflower oil, soybean oil, olive oil, avocado oil, grapeseed oil, peanut oil, or canola oil.

[0065] Flavor can also be modified through the use of salts (including sodium chloride and potassium chloride); acidic ingredients (eg, vinegar, citric acid, tartaric acid, malic acid, folic acid, fumaric acid, and lactic acid), among others.

[0066] Texture improvers provided as sodium, potassium, calcium, or magnesium salts may affect flavor aspects (e.g., saltiness or bitterness), and in such cases, the type and concentration of other flavor enhancers (e.g., salt and alkaline components) are adjusted to provide the desired flavor profile.

[0067] In some cases, the single-cell Bluefin tuna products further include flavor enhancers that complement the flavor of the fish (e.g., soy sauce, ginger, sesame, herbs (e.g., parsley, dill, chervil, and / or coriander, among others); juices and / or extracts from citrus fruits (e.g., calamansi, lemon, lime, orange, grapefruit, kumquat, and / or yuzu, among others); and / or black pepper, chili pepper, and / or white pepper, among others).

[0068] The flavor enhancer may be added in its natural form and / or as a semi-dry or dry powder, encapsulation, extract or absolute, among others.

[0069] (nutritional supplement) Optionally, the nutritional content of the homogenous in vitro cultured fish cell products provided herein can be supplemented with, for example, amino acids, peptides, proteins, and / or lipids, the latter of which (including but not limited to, dietary supplements, including amino acids, vitamins, minerals, and / or carbohydrates) can be provided in the cell culture medium for subsequent uptake and incorporation by the cells. Exemplary lipids include one or more of polyunsaturated fatty acids, saturated fatty acids, and / or sterols, alone or in combination with an effective amount of nervonic acid. In some embodiments, each supplemented lipid (e.g., polyunsaturated fatty acids, saturated fatty acids, and / or sterols) is present at a concentration of about 10 μg / ml or greater.

[0070] In other alternative embodiments, nutritional supplements (including the lipids, amino acids, vitamins, minerals and / or carbohydrates described above) and dietary fiber may be added to the cell culture medium or post-harvest cell slurry, or may be added during product formulation.

[0071] (preservative) Optionally, the homogenous in vitro cultured fish cell product may be formulated with agents to reduce microbial load and / or peroxidation, increase product shelf life, and improve consumer safety. Examples of such agents include antioxidants suitable for use in producing homogenous single-cell products, such as 2,4,5-trihydroxybutyrophenone (THBP), algae extract, anoxomer, apigenin, ascorbic acid, baicalein, plant extracts (including, but not limited to, blueberry, ginseng, wolfberry, grape seed, and green tea), butylated hydroxytoluene (BHT), butylhydroxyanisole, butylhydroxyanisole (BHA), carnosol, carotenoids, catalase, catechin, creatine, dilauryl thiodipropionate (cilauryl thiodipropionate), and the like. thiodipropionate), epicatechin gallate, ethoxyquin, lipoic acid, mitoquinol, morin, myricetin, N-acetylcysteine, phenols, pinostrobin, proanthocyanidin dimer B2, propionyl-L-carnitine, propyl gallate, quercetin, resveratrol, rosemary extract, rutin, sauquinone, tert-butylhydroquinone (TBHQ), 2,4,5-trihydroxybutyrophenone (THBP), thiazolidinedione, thiodipropionic acid, thymol, and tocopherol.

[0072] In some embodiments, the antioxidants are plant-derived and include, but are not limited to, algae extract, apigenin, ascorbic acid, baicalein, plant extracts (including but not limited to blueberry, ginseng, wolfberry, grape seed, and green tea), carnosol, carotenoids, catalase, catechin, epicatechin gallate, lipoic acid, morin, myricetin, pinostrobin, proanthocyanidin dimer B2, propionyl-L-carnitine, quercetin, resveratrol, rosemary extract, rutin, sauquinone, thymol, and tocopherol.

[0073] (pollutants) Bluefin tuna (BFT) are apex predators and long-lived fish that accumulate mercury through dietary transfer. Tissue mercury concentrations in bluefin tuna (BFT) frequently exceed the human intake threshold guideline of 1 μg / g wet weight (ww) for large predatory fish established by the U.S. Food and Drug Administration and the World Health Organization. According to Tseng et al. (2021), total mercury levels in muscle tissue of Pacific bluefin tuna (BFT) ranged from 0.49 to 5.65 μg / g (ww) (mean = 2.00 ± 0.83 μg / g (ww), n = 261), with approximately 94% exceeding the safe intake guideline of 1 μg / g (ww). Additionally, Table 1 (n=19) summarizes the levels of environmental contaminants (including mercury and arsenic) in Pacific and Atlantic Bluefin tuna samples. See https: / / www.pnas.org / doi / 10.1073 / pnas.2111205118, which is incorporated herein by reference in its entirety. Cell-cultured Bluefin tuna according to the present invention are significantly low in all environmental contaminants. See Table 1 below, which shows naturally occurring levels in wild-caught and farmed Bluefin tuna. "Significantly low" levels refer to levels of any one or more environmental contaminants that are at least 60% below the levels allowed by state and / or federal guidelines. Table 2 shows the levels of heavy metals (arsenic, cadmium, lead, and mercury) in conventional (farmed) Pacific and Atlantic bluefin tuna and cell culture bluefin tuna (n=3). [Table 1] [Table 2]

[0074] (Storage stability study) The stability of fish and fish products during refrigerated and frozen storage is affected by various conditions, including packaging, temperature, relative humidity, temperature fluctuations, product composition, processing conditions, etc. Significant physical and chemical changes occur in fish and fish products during refrigerated and frozen storage, such as color deterioration / changes, oxidation / hydrolysis of lipids and proteins causing off-flavors, production of potentially harmful compounds (e.g., histamine), texture changes such as softening, loss of nutrients, flavor and taste deterioration, and weight loss due to moisture loss. A deterioration in at least one property can be considered a loss of shelf life.

[0075] Color change (browning) and lipid oxidation are concerns that limit the shelf life of traditional bluefin tuna (Tuna tuna) in both refrigerated and frozen storage. Under long-term cryogenic storage, tuna browns due to excessive metmyoglobin (MetMb) production, which negatively impacts its commercial value and results in food waste. MetMb, present in muscle fibers, is the most important color-producing substance in tuna (Singh, Benjakul, Zhou, Zhang, & Deng, 2021). Generally, the bright red color of tuna muscle is primarily associated with the presence of red pigments, particularly oxymyoglobin (OxyMb). However, OxyMb converts to brown MetMb during processing and storage (Hoa et al., 2020). MetMb is primarily formed due to Mb oxidation, a free radical generated during lipid oxidation and trimethylamine oxide production (Grunwald, Tatiyaborworntham, Faustman, & Richards, 2017). See also Ying Bu et al., Food Science and Technology (2022) and references cited therein. https: / / www.sciencedirect.com / science / article / pii / S0023643821018685.

[0076] In one example, a * / b * The ratio of α-tocopherol (α, β-tocopherol) significantly decreased by 60% in southern bluefin tuna during the first two days of refrigerated storage, causing the color to change from red to brown (Ying Bu et al., 2022). This study also suggests that its texture softened, with significant decreases in firmness (76%), chewability (76%), elasticity (11%), and resilience (45%) over a six-day storage period. This study also found significant increases in volatile nitrogen compounds and lipid oxidation products during storage, suggesting unacceptable quality deterioration. In another example, a similar trend was observed for the color of Pacific Bluefin tuna stored in a refrigerator, with a * / b * The ratio decreased by 88% over a 10-day storage period. Similar observations were reported in Bluefin tuna stored under frozen conditions at -18°C and -55°C. See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9368106 / .

[0077] In the case of cell culture Bluefin tuna (homogeneous Bluefin tuna prototype) packaged in parafilm and stored under refrigeration at 4°C, the overall color deterioration was at a much slower rate, with a 10-day refrigerated storage time. * / b * There was only a 16% decrease in value.

[0078] In another example, during frozen storage at -80°C, the product packaged in parafilm exhibited excellent freeze-thaw stability, with only <0.5% drip loss and color retention. It is noted that "mass loss" during frozen storage is important from an economic and quality standpoint. The smaller "mass loss" associated with cell-cultured bluefin tuna according to the present invention is yet another benefit offered to consumers. This appears to be consistent with the observations of Jinfeng Wang et al. (2022), whose data suggest that there was little quality change during short-term frozen storage at 18°C. However, at a frozen storage temperature of -55°C, the bluefin tuna exhibited significantly improved quality compared to a frozen storage temperature of -18°C. The contents of this reference are incorporated herein in their entirety.

[0079] In various embodiments, edible compositions according to the present invention may optionally include functional agents (e.g., dietary supplements, preservatives, pH adjusters, emulsifiers, emulsion stabilizers, flow improvers, humectants, and moisture retention agents, among others). Such agents provide various benefits, including, but not limited to, facilitating manufacturing, product formation, moisture enhancement, moisture retention, and / or improved shelf life.

[0080] (Product form) The homogeneous and layered in vitro cultured fish cell products can be formed in a wide variety of shapes, including, but not limited to, saku blocks (i.e., uniform pieces suitable for preparing sashimi and other sliced ​​presentations, for searing, or for further processing into mince, steaks, fillets and loins, sashimi, dices, slices, and cubes).

[0081] (In vitro cultured fish cell slurry product) In another embodiment of the present invention, the in vitro cultured fish cell product is a slurry. The slurry can be a viscous slurry. Viscosity is a measure of a fluid's resistance to flow. A viscous slurry can be defined based on specific rheological studies to determine the key parameters preferred in a cell culture slurry according to its intended purpose. For example, a viscosity in the range between 25,000 cP and 50,000 cP. Viscosities outside this range are also possible depending on the specific product application.

[0082] The slurry can be used in the manufacture of products including, but not limited to, dietary supplements and food ingredients. In one embodiment, dietary supplements can be produced by filling freeze-dried cultured fish cells into capsules. Such capsules can be used in the same manner as fish oil capsules, but can offer the advantage of having a uniform and reproducible lipid profile while being free of environmental contaminants. The capsules themselves are made from animal-free materials such as cell culture gelatin, modified tapioca starch, and / or plant cellulose (e.g., hydroxypropyl methylcellulose (also known as hypromellose)), the latter of which offers the advantage of delaying the release of its contents until it reaches the small intestine, thereby optimizing lipid absorption.

[0083] In other embodiments, the slurry may be used as a food ingredient in fresh, frozen, or dried form, alone or in combination with other animal and / or plant materials, for the production of various food products (e.g., baby food, pet food, and other products).

[0084] The slurry can be dispensed by a variety of methods during the production of cultured food products, including, for example, Bluefin tuna cell products, with the methods selected according to factors that may include, but are not limited to, viscoelastic properties, shear rate, pressure, temperature, and, if mixing occurs simultaneously with dispensing, mixing requirements. Suitable devices for dispensing the slurry include, but are not limited to, positive displacement pumps (e.g., rotary, internal gear, screw, shuttle block, flexible or sliding vane, circumferential piston, helical twisted root (e.g., Wendelkolben pump) or water ring vacuum pumps); piston pumps; auger pumps; peristaltic pumps; progressive cavity pumps; and extruders.

[0085] Homogeneous single-cell and multi-cell preparations from in vitro cultured fish cells The homogeneous single-cell type product of the in vitro cultured fish cells of the present invention can be produced using a single-cell type slurry containing cells selected from, inter alia, myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, and adipocytes.

[0086] In yet another embodiment, a homogenous multi-cell product of in vitro cultured fish cells can be produced using a multi-cell type slurry derived from a culture containing one, two, three, four or more cell types, e.g., myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, adipocytes, and combinations thereof. Alternatively, a multi-cell type product can be prepared from a slurry of two, three, four or more single cell types that are then combined in ratios appropriate for the intended purpose of the final product. In an exemplary embodiment according to the invention, the cells are Bluefin tuna cells.

[0087] Multicellular products containing both muscle cells and adipocytes can be blended to produce products with fat contents that mimic the fat content of high-fat, medium-fat, or low-fat (lean) wild-caught or farmed fish fillets. In exemplary embodiments, Bluefin tuna cultured cell products are formulated to mimic Bluefin tuna fillets characterized as otoro (high fat), chutoro (medium fat), or akenomi (lean). In some embodiments, multicellular products each having various ratios of muscle cells and adipocytes can be combined to form layered products that mimic the layered, wild-caught Bluefin tuna fillets classified as toro.

[0088] In certain embodiments, the multi-cell product provides a more desirable impact profile than a product containing only muscle, for example, the multi-cell product may be formulated using lipid-packed fish fat cells such that it provides a lipid profile that provides optimal nutritional benefits.

[0089] The homogenous single-cell or homogenous multi-cell products can be formed into blocks as shown in schematic (g) below, and the blocks further processed into mince. The products can also be formed as sheets of uniform or variable thickness, as described below, and the sheets can then be combined into a layered product.

[0090] The pieces served to the consumer (e.g., entrée fillets, sashimi, and cubes (the last of which includes "poke" cubes served as either an appetizer or main course) may be formulated with one or more texture modifiers, colorants, dietary supplements, and other food ingredients to mimic the texture, color, and other sensory and nutritional aspects of traditional Bluefin tuna meat that are pleasing to the consumer.

[0091] Both single-cell and multi-cell fish culture cell products intended for use as soft meal products (e.g., infant and baby foods) and pet foods can be formulated with one or more texture modifiers of selected types and concentrations to produce semi-solid or semi-liquid products.

[0092] (minced meat) Mincing of in vitro cultured fish cells is best performed by processing large pieces formed from a homogenous product of either a single cell type or multiple cell types.

[0093] The single-cell or few-cell slurry may be combined with one or more texturizers or one or more binders to produce pieces with a texture optimized for processing by chopping into small pieces, the size of which varies according to the intended use of the mince. In some embodiments, the pieces are partially or completely frozen as a preliminary step to facilitate chopping.

[0094] Since chopping results in "breaking" the gel formed by the texture modifier, thereby releasing moisture, pieces intended for use in making mince may be formulated with small amounts of dry ingredients to promote moisture retention, including, but not limited to, textured proteins, flours, and gums, among others.

[0095] Two or more types of mince (each made from pieces with different cell type compositions) may be combined depending on the intended use of the final mince.

[0096] (Non-fibrous layered product of in vitro cultured fish cells) Embodiments of the present invention also include edible compositions comprising a population of non-fibrous in vitro cultured fish cells, the cells being in a layered morphology, as shown in the schematic diagram, and substantially free of contaminants. The compositions contain multiple layers of cultured cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers), which may or may not be equidistant. Individual layers or the overall layered morphology may contain a single cell type or multiple cell types. The overall layered morphology may contain a single cell type or multiple cell types (e.g., 2, 3, 2-3, 2-4 cell types). The cell types of the individual layers and / or the multilayered product may be any one or combination of myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, and adipocytes.

[0097] The slurry used to make the layered product includes at least one texture modifier that enables the slurry to achieve a semi-solid or solid form when set; the slurry may optionally further include one or more of the functional and / or sensory agents described above.

[0098] The non-fibrous layered product can be manufactured in many ways, but generally requires a step of forming the layers, which in turn requires a step of dispensing a corresponding slurry, followed by a step of hardening (solidifying), the slurry used to manufacture the layered product comprising at least one texture modifier that enables the slurry to achieve a semi-solid or solid form when hardened; the slurry may optionally further comprise one or more of the functional and / or sensory agents described above.

[0099] Layered products can be manufactured by separately forming and curing individual layers as individual pieces, and then assembling the pieces to create the layered product. In such cases, the layers can be allowed to passively adhere to one another, for example, through electrostatic interactions (e.g., hydrogen bonding). Alternatively, the layers can be bonded together using a bonding agent or an enzymatic method (e.g., using transglutaminase).

[0100] Layered products can also be produced as a single piece by dispersing and curing successive layers of slurry to create a vertical stack. Layered products can also be produced by forming the layers simultaneously, for example, as plates, sheets, or cylinders in which the layers are immediately adjacent and horizontally aligned. Layered products with a marbled appearance can be produced by mixing or swirling two or more layers before curing, or by distributing successive layers of slurry in a manner that creates heterogeneous and highly irregular layers. Combinations of these methods can also be used to produce products with various layering patterns.

[0101] Depending on the viscosity of the slurry used, the individual layers may be cured sequentially or simultaneously.

[0102] Individual layers or layer pieces can be formed by dispensing the slurry into a mold or onto a stationary or moving platform or conveyor using one or more stationary or moving dispensing devices to form separate or continuous pieces. The layers or layer pieces can have the same or larger dimensions as the finished product. If the pieces are larger than the dimensions of the finished product, they can be processed into pieces having the desired final dimensions. Other methods are also possible.

[0103] In yet another embodiment, the layered product is produced using a food manufacturing system that deposits slurries sequentially one on top of the other. In this method, the layers can be cured simultaneously if a high viscosity slurry is used, or sequentially after each layer is deposited before the next layer is deposited if a low viscosity slurry is used. The slurry can be dispensed by a number of methods, as described above.

[0104] In yet another embodiment, the individual layers are formed using pipe setting techniques known in the art.

[0105] (Manufacturing method) For homogenous products, the cell slurry is mixed with the above functional ingredients, except for the texture modifier, at 15°C to 45°C for 5 to 15 minutes, after which the texture modifier is added at 45°C to 75°C and mixed for an additional 5 to 15 minutes. After final mixing, the mixture is poured into molds and rapidly cooled to form a homogenous block of the product. Finally, the product is flash-frozen to -80°C and packaged in high-barrier packaging. For layered products with marbling as shown in the schematic diagram, the marbling layer is prepared, consisting of a high-fat emulsion and some texture modifiers that help form the product, and it is kept warm until the colored fraction is prepared. The colored mixture is prepared in a similar manner as for the homogenous products and then randomly layered with the white marbling emulsion to obtain the appearance as shown in the schematic diagram.

[0106] Further details regarding the compositions, methods and systems described herein will become apparent from the detailed invention of the following examples, which are provided for illustrative purposes only, in the experimental section of the specification below. [Example]

[0107] (Example) The cell culture edibles described herein, as well as related cells, compositions, and systems, are further described in the following examples, which are provided for illustrative purposes and are not intended to be limiting.

[0108] Example 1: Cell slurry of in vitro cultured fish cells In this example, cells from Bluefin tuna were concentrated into a viscous slurry. This viscous slurry can be used directly or frozen or dried and stored before use. The cell slurry can contain one or more cell types (e.g., myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells, or induced pluripotent stem cells).

[0109] (Bluefin tuna cell line derived) Pacific bluefin tuna (12 to 100 pounds) were wild-caught and identified visually and by genome sequencing. Myoblasts were harvested from muscle tissue. Typically, 6 to 24 grams of tissue were treated with enzymes and mechanical dissociation and seeded at 0.5 to 1.0 grams of tissue per well.

[0110] (Bluefin tuna cell proliferation) A stable cell line of Bluefin tuna cells was grown under growth conditions. The cells were cultured at approximately 10 3 cells / mL~10 6 The cells were grown at a dilution of 100 cells / mL under growth pH and temperature conditions.

[0111] (Bluefin tuna cell slurry formation) A concentrated cell slurry was formed by concentrating cells from the diluted proliferation or differentiation solution described above. Concentration was achieved through centrifugation, sedimentation, or other separation methods to separate the cells from the liquid. The slurry contained up to 10 cells per mL, depending on the size of the cells. 6 Beyond 10 9The slurries contained cell concentrations up to 1000 μg / ml. These slurries were viscous solutions similar to suspensions that could flow like liquids. Optionally, the products were then frozen or dried to form solid products.

[0112] (cell slurry product) The cell slurry is used in the manufacture of products including, but not limited to, dietary supplements and food ingredients. Dietary supplements are produced by filling capsules with freeze-dried cultured Bluefin tuna cells. Such capsules are used in the same manner as fish oil capsules, but offer the advantage of having a uniform and reproducible lipid profile while being free of environmental contaminants. The capsules are manufactured from animal-free materials (e.g., cell-cultured gelatin, modified tapioca starch, and / or plant cellulose (e.g., hydroxypropyl methylcellulose (also known as hypromellose)) which offers the advantage of delaying the release of its contents until it reaches the small intestine, thereby optimizing lipid absorption.

[0113] The cell slurry is used as a food ingredient in fresh, frozen, or dried form, alone or in combination with other animal and / or plant materials, for the production of a variety of food products, such as baby food, pet food, and other products.

[0114] Example 2: Homogeneous single-cell preparation of in vitro cultured Bluefin Tuna cells Homogeneous Bluefin tuna products are produced by combining the Bluefin tuna cell slurry described above in Example 1 with one or more of the food ingredients described above. These homogeneous products are formed as a unitary piece that is substantially uniform throughout the piece with respect to its organoleptic and functional properties.

[0115] Single-cell products are produced using a cell slurry of a single cell type (e.g., myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells, or induced pluripotent stem cells). Both the cell type and morphology depend on the intended purpose of the final product. In one example, the homogenous single-cell products are formed into blocks, which are further processed into mince (see Example 4 below), or processed into sheets of uniform or variable thickness, which are combined into layered products with desired firmness, texture, nutrition, appearance, or other product properties (see Example 3 below).

[0116] (Texture Agent) The texture modifiers and the levels of each are selected to achieve the appropriate degree of firmness, cohesiveness, elasticity and chewiness for the intended purpose of the product.

[0117] (Product form) Homogeneous in vitro cultured Bluefin tuna cell products can be formed into a wide variety of shapes, including, but not limited to, saku (blocks, i.e., uniform pieces suitable for preparing sashimi and other sliced ​​presentations, for searing, or for further processing into mince, steaks, filets and loins, sashimi, dices, fillets, and cubes).

[0118] (functional agent) In addition to firmness, certain texture modifiers further provide additional functional properties (including, but not limited to, heat stability and browning), which allow the pieces to remain solid during heating and / or brown during cooking to produce an appetizing appearance and / or flavor; in such cases, the concentration of such agents is selected to provide a product that is acceptably heat stable and / or browns in a controlled manner, without being objectionably firm. Furthermore, in some embodiments, the texture modifier is selected to provide emulsion stabilization in addition to other sensory or functional properties.

[0119] Example 3: Homogeneous multi-cell type preparation of in vitro cultured Bluefin Tuna cells Homogeneous multi-cell type products of in vitro cultured Bluefin tuna cells are produced using a multi-cell type slurry derived from a culture containing one, two, three, four or more cell types. In an alternative method, the multi-cell type products are produced from a slurry of two, three, four or more cell types combined in ratios appropriate for the intended purpose of the final product.

[0120] A multi-cell type product containing both muscle cells and adipocytes is formulated to provide a product that mimics the fat content of wild-caught or farmed Bluefin tuna fillets characterized as otoro (high fat), chutoro (medium fat), or akenmi (lean fat).

[0121] Example 4: Mincing of in vitro cultured Bluefin Tuna cells Mince of in vitro cultured Bluefin tuna cells is optimally formed by processing large pieces formed from either the single cell type or multi-cell type homogenous products described in Examples 2 and 3 above.

[0122] The single-cell or multi-cell slurry is combined with one or more texture modifiers or one or more binders to produce pieces with a texture optimized for processing by chopping into small pieces, the size of which varies depending on the intended use of the mince. In an alternative method, the pieces are partially or completely frozen as a preliminary step to facilitate chopping. Because chopping results in "breaking" the gel formed by the texture modifier, resulting in the release of moisture, pieces intended for use in producing mince are formulated with small amounts of dry ingredients (e.g., sodium alginate, fiber, psyllium husk, etc.) to promote moisture retention.

[0123] Example 5: Non-filamentous, layered, single-cell product of in vitro cultured Bluefin Tuna cells A non-filamentous layered single-cell product of in vitro cultured Bluefin tuna cells is produced by first creating two or more individual layers of the homogenous single-cell product described in Example 2 above, and then combining the layers. See Figures 1A-1G.

[0124] While the layered morphology can be achieved in many ways, it generally requires the steps of forming the layers, dispensing the corresponding slurries, then curing (solidifying) the layers, and then assembling the layers. Depending on the viscosity of the slurries used, the individual layers may be cured sequentially or simultaneously before or after assembling. In one example, each layer is made independently and cured (i.e., solidified); the layers are then assembled by stacking them vertically. In this example, the layers may be formed by dispensing the slurry in separate or continuous pieces into a mold or onto a platform. If the slurry is dispensed onto a platform as continuous pieces, the pieces may have the same length and width as the finished product, in which case they are dispensed, cured, and then assembled by stacking them vertically. If the pieces are larger than the dimensions of the final product, they may be cut into smaller pieces that are then cured and stacked, or in the alternative, the larger pieces may be cured and then stacked to form an intermediate form that is then cut into pieces having the desired length and width.

[0125] Example 6: Non-filamentous, stratified, multicellular product of in vitro cultured Bluefin Tuna cells A non-fibrous layered multi-cell type product of in vitro cultured Bluefin tuna cells is produced using individual layers that can each contain one or more cell types to produce a product with two or more cell types in the aggregate. In addition to the cell type composition, the layers contain one or more texture-enhancing agents described in Example 2 above (which may be the same or different for each individual layer), and one or more additional functional and / or sensory agents.

[0126] Marbling of the product may be achieved through the layering of the individual layers, which may be stabilized with food grade emulsifiers and have individual formulations including the texturizing, coloring, and organoleptic ingredients described above.

[0127] Example 7: Improved Refrigerated and Frozen Shelf Life of In Vitro Cultured Bluefin Tuna Cell Products The non-fibrous layered product produced according to one or more of the above examples was packaged in paraffin and stored under refrigeration at 4°C for 10 days. It was then removed and re-examined for color deterioration compared to conventional Bluefin tuna (which was used as a reference). As noted in Ying Bu et al., "Generally, the bright red color of tuna muscle is primarily associated with the presence of red pigments, particularly oxymyoglobin (OxyMb). However, OxyMb converts to brown MetMb during processing and storage...MetMb is primarily formed due to Mb oxidation, and free radicals are generated during lipid oxidation and trimethylamine oxide production." The overall color deterioration of the tuna product according to the present invention was at a significantly slower rate, and a * / b * There was only a 16% decrease in the ΔE value. Referring to Ying Bu, this suggests a slower rate of lipid oxidation. Figures 2A and 2B show the difference in color degradation under refrigerated conditions. Figure 2A shows shelf life as a function of ΔE value, while Figure 2B shows the same data as * / b * as a function of the value.

[0128] In another example, a product as shown in the above example was placed in a -80°C freezer for 11 days and then thawed for evaluation. The product packaged in paraffin exhibited excellent freeze-thaw stability, with only <0.5% drip loss and retention of color characteristics. The data shows that there is no significant change in the color of the product when stored at -80°C for 11 days. Figure 3 shows the shelf life of cell-cultured Bluefin tuna product at -80°C, demonstrating that the cell-cultured Bluefin tuna has a longer shelf life compared to conventional Bluefin tuna.

[0129] In another example, the product used as described above was placed in a refrigerator at -20°C for three months and compared to wild-caught Pacific Bluefin tuna (traditional Bluefin tuna). Figure 4 shows the difference in color degradation under -20°C conditions. Color degradation as a function of ΔE values ​​was significantly greater for the traditional Bluefin tuna than for the cell-cultured Bluefin tuna samples, indicating an extended shelf life for the cell-cultured Bluefin tuna product under frozen storage conditions.

[0130] In another example, conventional and cell-cultured bluefin tuna were vacuum-packaged, stored under refrigeration at 4°C for 14 days, and evaluated for lipid oxidation, measured as a function of malondialdehyde (MDA) production, using modified protocols described in Quevedo et al. (2013), Scheffler et al. (2010), and Sonar et al. (2020). Figure 5 shows a significant decrease in the amount of MDA produced in the cell-cultured bluefin tuna compared to conventional bluefin tuna, indicating enhanced shelf-life retention for the cell-cultured bluefin tuna product. Both samples were also evaluated for heavy metals (including arsenic, cadmium, lead, and mercury) by ICP-MS (Table 2). The cell-cultured bluefin tuna had significantly lower contaminant levels than wild-caught conventional bluefin tuna and farmed conventional bluefin tuna.

[0131] The examples set forth above are provided to provide those of skill in the art with a complete invention description of the edible compositions comprising Bluefin tuna cells substantially free of contaminants and related compositions, methods, and systems of the present invention, as well as how to make and use embodiments thereof, and the examples set forth above are not intended to limit the scope of what the inventors regard as their invention. Those skilled in the art will recognize how to adapt the features of the exemplary cells, compositions, methods, and systems disclosed herein to additional cells, compositions, methods, and systems in various embodiments and within the scope of the appended claims. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains.

[0132] The entire disclosure of each document cited in the background, summary, detailed description and examples (including patents, patent applications, papers, abstracts, laboratory manuals, books or other inventions) is hereby incorporated by reference into this specification.All references cited in this invention are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.However, if any inconsistency occurs between the cited document and this invention, this invention shall prevail.

[0133] The terms and expressions that have been used in this specification are used as words of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present invention. Thus, while the present invention has been specifically disclosed by embodiments, it should be understood that exemplary embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0134] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0135] When a Markush or other group is used herein, all individual elements of the group, as well as all combinations and possible subcombinations of the group, are intended to be individually included in the present invention. Any combination of components or materials described or exemplified herein may be used to practice the present invention, unless otherwise specified. Those skilled in the art will recognize that methods, system elements, and materials other than those specifically exemplified may be used in the practice of the present invention without undue experimentation. All functional equivalents known in the art for any such method, device elements, and materials are intended to be included in the present invention. Whenever a range (e.g., a temperature range, frequency range, time range, or composition range) is provided herein, all intermediate ranges and subranges and individual values ​​contained within the provided range are intended to be included in the present invention. Any one or more individual elements of a range or group disclosed herein may be excluded from the claims of the present invention. The inventions suitably illustratively described herein may be practiced in the absence of any element or group of elements, limit, or group of limits not specifically disclosed herein.

[0136] Many embodiments of the present invention have been described. The specific embodiments presented herein are examples of useful embodiments of the present invention. It will be apparent to those skilled in the art that the present invention can be practiced using many variations of the genetic circuits, genetic molecular components, and method steps shown herein. As will be apparent to those skilled in the art, methods and systems useful for the present methods and systems may include numerous optional compositional and processing elements and steps.

[0137] In particular, it is understood that various modifications can be made without departing from the spirit and scope of the invention, and accordingly, other embodiments are within the scope of the appended claims. [ka]

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Claims

1. 1. An edible composition comprising a homogenous mixture of in vitro cultured fish cells, the cells each having a diameter of less than 20 μm to less than 200 μm, and the edible composition being substantially free of one or more contaminants.

2. 10. The edible composition of claim 1, wherein the cells are each less than 20 μm in diameter and are substantially free of one or more contaminants.

3. 10. The edible composition of claim 1, wherein the cells are each less than 200 μm in diameter and are substantially free of one or more contaminants.

4. 10. The edible composition of any one of the preceding claims, wherein the contaminant is an environmental contaminant.

5. 5. The edible composition of claim 4, wherein the environmental contaminant is selected from the group consisting of mercury, lead, cadmium, zinc, copper, nickel, chromium, arsenic, aluminum, fluoride, radon, persistent organic pollutants, and pesticides.

6. 10. An edible composition according to any one of the preceding claims, comprising one, two or three cell types.

7. 7. The edible composition of claim 6, wherein the cell type is selected from the group consisting of myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, and adipocytes.

8. 10. An edible composition according to any one of the preceding claims in the form of a viscous slurry or mince.

9. 10. An edible composition according to any one of the preceding claims which is frozen or freeze-dried.

10. 10. An edible composition according to any one of the preceding claims which is a liquid, semi-liquid, semi-solid, solid or foam.

11. 10. An edible composition according to any preceding claim, comprising less than 0.1 parts per million (ppm) of said one or more contaminants.

12. 12. The edible composition of claim 11, wherein the contaminant is mercury.

12. An edible composition comprising a population of non-fibrous in vitro cultured fish cells, the cells being in a layered form and substantially free of contaminants, each layer comprising a homogenous mixture of cells.

13. 4. The edible composition of claim 1, 2 or 3, comprising one cell type.

14. 4. The edible composition of any one of claims 1, 2 and 3, comprising between one and three cell types.

15. 1. An edible composition comprising a homogenous mixture of in vitro cultured fish cells derived from the genus Tuna, wherein the cells each have a diameter of less than 20 μm to less than 200 μm, and the edible composition is substantially free of one or more contaminants.

16. 1. An edible composition comprising a homogenous mixture of in vitro cultured fish cells derived from Bluefin tuna, wherein the cells each have a diameter of less than about 20 μm to less than about 200 μm, and the cells are substantially free of one or more contaminants.

17. 1. An edible composition comprising a homogenous mixture of in vitro cultured fish cells derived from Bluefin tuna, wherein the cells are each less than 20 μm in diameter and are substantially free of one or more contaminants.

18. 17. The edible composition of claim 16, wherein the tuna comprises a fillet of fatty tuna.

19. 17. The edible composition of claim 16, wherein the tuna comprises a lean fillet.

20. L 46.56-70.59, a ＊ 12.21-38.76 and b ＊ 17. The edible composition of claim 16, comprising a color profile of 1.24 to 61.

68.

21. L in the range of 33.0 to 62.8 ＊ Values, a range of 4.77 to 21.9 ＊ values, and b in the range of 4.72 to 15.4 ＊ 17. The edible composition of claim 16, comprising a color concentration of 0.001% to 5% to achieve each of the values.

22. 17. The edible composition of claim 16, comprising a refrigerated shelf life of 4 to 14 days.

23. 17. The edible composition of claim 16, comprising a frozen storage period of 90 to 120 days.

24. 17. The edible composition of claim 16, which is a single cell consistent product.

25. 25. The edible composition of claim 24, wherein the single cell consistent product comprises a single cell type or multiple cell types.

26. 25. The edible composition of claim 24, wherein the product is marbling.

27. An edible composition comprising a population of non-filamentous in vitro cultured fish cells derived from Bluefin tuna, the cells being in a layered form and substantially free of contaminants, each layer comprising a homogenous mixture of cells.

28. 17. The edible composition of claim 16, comprising a longer shelf life than conventional Bluefin tuna.

29. 24. The edible composition of claim 23, comprising a longer shelf life than conventional Bluefin tuna.

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