CELL CULTURE EDIBLE Fish PRODUCT

Abstract

Provided herein are in vitro cultured fish compositions, methods and systems for culturing cells derived from fish sources.

Description

Background Art

[0001] Cell-cultured foods are part of the food alternatives that have become the focus of development by many companies worldwide as a means to address public health, environmental, and animal welfare issues related to animal farming and agriculture. Chemically contaminated and spoiled foods have serious impacts on personal health. Foodborne diseases and food poisonings have different origins - bacteria, viruses, parasites, molds, 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 Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) is the biogenic amine histamine. Biogenic amines (BAs) in foods pose a potential public health problem due to their physiological and toxicological effects. Consumption of foods containing high concentrations of biogenic amines is associated with health hazards. The range of adverse effects extends from mild stomach problems to major health fatalities. Chemical contaminants are closely related to serious consequences, lack of personal control, and long-term effects (Kher et al., 2011). According to the WHO, more than 200 diseases are transmitted through food, and the vast majority of people will contract a foodborne disease at some point in their lives. For example, in the United States, 48 million people (one-sixth) contract a foodborne disease every year. Among them, 128,000 are hospitalized, and 3,000 die from such diseases (Claudia Ruiz-Capillas et al., 2019). Food intake is the most likely source of human exposure to metals. Metals such as cadmium and lead easily enter the food chain. Heavy metals severely deplete specific nutrients in the body, thereby reducing immune defenses, damaging psychosocial abilities, and causing intrauterine growth retardation. Heavy metal intake is also associated with malnutrition and increases the incidence of gastrointestinal diseases (Khan et al., 2008). Food contaminants are also a major cause of cancer (Abnet, 2007). Exposure to polychlorinated biphenyls (PCBs) due to food contamination has an adverse impact on the neurodevelopment and immune response of children (Schantz et al., 2004). Pesticides in foods as contaminants also show serious health effects. Excessive amounts of these chemicals in foods can cause nerve and kidney damage, congenital disabilities, reproductive problems, and can be proven to be carcinogenic (Bassil et al., 2007). The accumulation of pesticides in body tissues can also lead to metabolic degradation (Androutsopoulos et al., 2013). There is also a risk of neurodevelopmental disorders such as attention deficit disorder, autism, cerebral palsy, and intellectual disabilities caused by industrial chemicals such as arsenic, PCBs, and lead in foods and water. The present application provides an edible fish product that is substantially free of contaminants and has an extended shelf life compared to traditional products. Summary of the Invention

[0003] The present invention relates to cell-cultured foods derived from fish sources and related cells. Compositions are also provided. In at least one embodiment, the fish source is bluefin tuna, and the edible product formed therefrom is substantially free of contaminants.

[0004] 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.

[0005] According to aspects of the present invention, provided herein are edible compositions that are substantially free of contaminants and contain a homogeneous mixture of in vitro cultured fish cells. The diameter of each cell can be less than about 20 μm to about 200 μm. In one embodiment, the diameter of each cell is less than 20 μm. In another embodiment, the diameter of each cell is less than 200 μm.

[0006] The edible compositions provided herein contain one, two, or three cell types. The cell types can include myoblasts, myotubes, fibroblasts, endothelial cells, neurons, red blood cells, preadipocytes, induced pluripotent stem cells, adipocytes, or combinations thereof.

[0007] According to aspects of the present invention, the edible composition can be in the form of a viscous slurry. The edible composition can be frozen or lyophilized.

[0008] The edible composition can be a liquid, semi-liquid, semi-solid, solid, or foam.

[0009] According to another aspect, an edible composition is described that contains an aggregate of non-fibrous in vitro cultured fish cells, wherein the cells are in a layered structure and each layer contains a homogeneous mixture of cells. The edible composition can contain a single cell type or can contain multiple cell types (e.g., 1 - 3 cell types, 4 or more cell types). In another embodiment, the edible composition contains an aggregate of non-fibrous in vitro cultured tuna cells, and the non-fibrous in vitro cultured tuna cells are arranged in a non-layered homogeneous structure. In another embodiment, the non-fibrous in vitro cultured cells are bluefin tuna cells.

[0010] According to another aspect, a method for preparing a bluefin tuna slurry substantially free of contaminants is described. The method includes harvesting myoblasts and fibroblasts from the muscle tissue of wild-caught Pacific bluefin tuna, harvesting preadipocytes from the subcutaneous fat of wild-caught Pacific bluefin tuna, in vitro amplifying the myoblasts, fibroblasts, and preadipocytes for at least 50 population doublings to obtain a stable cell line, amplifying the myoblasts and / or preadipocytes under suitable growth conditions, converting 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 diluted differentiation fluid, and concentrating the cells by centrifugation, sedimentation, or other separation methods to separate the cells from the liquid, thereby forming a slurry having a cell concentration of at least 10 6 cells / ml. The slurry can contain from about 10 6 cells / ml to about 10 9 cells / ml. The method can further include freezing or drying the slurry to form a solid product. The wild-caught Pacific bluefin tuna used in the methods described herein can weigh about 12 - 200 pounds.

[0011] The slurry used in the methods described herein can contain one or more cell types, e.g., myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells, embryo-derived cells, or induced pluripotent stem cells.

[0012] The amplification step can include inoculating 0.1 - 1 gram of tissue / well.

[0013] Treating the preadipocytes can cause the cells to transform from small proliferating cells to round and non-proliferating lipid-loaded cells. The growth conditions can include a pH ranging from 7.3 to 7.5, and the temperature can vary from 15°C to 30°C.

[0014] The media and systems described herein, as well as the related compositions, cells, cell biomass, and cell culture foods described herein, can be used in a variety of applications where cell viability, controlled proliferation, and contamination levels in the cells and related cell culture foods are desired. For example, the compositions described herein and the related cells, cell biomass, and cell culture foods described herein can be used to generate cell culture foods, such as foods substantially free of contaminants. Thus, exemplary fields of application include food manufacturing, food processing, and commercialization. Additional exemplary applications include the use of the media, compositions, methods, and systems described herein, as well as the related cells and cell biomass cell culture foods, in multiple fields, including basic biological research, applied biology, bioengineering, bioenergy, medical research, therapeutics, and other fields that can be determined by those skilled in the art upon reading the present invention.

[0015] Details of one or more embodiments of the present 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 DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A-1G shows a schematic representation of the marbling seen in the cell-cultured bluefin tuna product of the present invention.

[0017] Figure 2A and 2B shows the extended shelf life of the cell-cultured bluefin tuna product at 4°C compared to traditional bluefin tuna. The degree of color change in the cell-cultured product is significantly slower.

[0018] Figure 3 shows the shelf life stability of the cell-cultured bluefin tuna product at -80°C. The figure shows the color difference of the cell-cultured bluefin tuna after 11 days of storage at -80°C. The CIELAB values, a*, and b* of the cell-cultured bluefin tuna (BN BFT) are measured. Figure 3 shows that there is no difference in color (a* / b*) between day 0 and day 11, indicating the extended shelf life stability of the cell-cultured bluefin tuna product under -80°C storage conditions.

[0019] Figure 4 is a figure showing the color difference of the cell-cultured bluefin tuna after three months of storage at -20°C compared to traditional bluefin tuna. The color difference between the traditional bluefin tuna (traditional BFT) and the cell-cultured bluefin tuna sample (BN BFT) is quantified as ΔE (total color difference) calculated in the CIELAB color space system, which measures three values: the achromatic component L* (light and dark) and two color descriptors, a* (red and green) and b* (yellow and blue) values. Statistical analysis is performed using two-way ANOVA. The figure shows a significant reduction in color change (p<0.05) in the cell-cultured bluefin tuna compared to traditional bluefin tuna, indicating the extended shelf life of the cell-cultured bluefin tuna product under frozen storage conditions.

[0020] Figure 5 shows the degree of lipid oxidation of the cell-cultured bluefin tuna after two weeks of storage at 4°C compared to traditional bluefin tuna. Oxidation is measured by determining the amount of malondialdehyde (MDA) in each sample of traditional bluefin tuna (traditional BFT) and cell-cultured bluefin tuna (BN BFT) stored at 4°C for 14 days (n = 3). The figure shows a significant decrease (p<0.05) in the amount of MDA produced in the cell-cultured bluefin tuna compared to traditional bluefin tuna by two-way ANOVA, indicating reduced oxidation of the cell-cultured bluefin tuna product under refrigerated storage conditions. DETAILED DESCRIPTION OF THE INVENTION

[0022] Provided herein are cell culture edible foods, compositions, methods, and systems derived from fish sources, as well as 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., wild-caught bluefin tuna).

[0023] The term "about", when referring to a measurable value such as an amount, duration, and the like, means to encompass variations of ±20% or less, or in some cases ±15% or less, or in some cases ±10% or less, or in some cases ±5% or less, or in some cases ±1% or less, or in some cases ±0.1% or less from the specified value, as such variations are appropriate.

[0024] The term "cell line" is a term in the art that refers to a defined population of cells that can be maintained in culture for an extended period of time. A "stable cell line" is a cell line that exhibits genomic and phenotypic stability after 24 hours of seeding, has a doubling time of less than 96 hours, and a viability of more than 70%.

[0025] The term "myoblast" is a term in the art that refers to the precursor of a muscle cell (which is also called a muscle fiber). As will be understood by those skilled in the art, myoblasts differentiate into muscle cells through myogenesis. Depending on the type of muscle cell that the myoblast will differentiate into, myoblasts can be classified as skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts. Exemplary myoblasts of aquatic animals include skeletal muscle myoblasts and smooth muscle myoblasts.

[0026] The term "fibroblast" is a term in the art that refers to a cell type in animal connective tissue and synthesizes extracellular matrix components such as collagen. Fibroblasts produce the structural framework of animal tissues and play a key role in wound healing. Fibroblasts are the most common cells in animal connective tissue. Fibroblasts have a branched cytoplasm that wraps around an oval, speckled 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 a reduced amount of rough endoplasmic reticulum. Although fibroblasts are messy and dispersed when they have to cover a large space, in an aggregated state, they are usually locally arranged in parallel clusters. Exemplary fibroblasts include fibroblasts from muscle and other tissues such as brain, heart, or skin.

[0027] The term "adipocyte" is a term in the art that refers to a fat cell, which is also known as a lipocyte. Adipocytes are the cells that mainly make up adipose tissue and are specialized in storing energy as fat. Adipocytes can be derived from mesenchymal stem cells, which produce 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 referred to as white and brown fat, respectively, and contain two types of adipocytes. Adipocytes can be derived from preadipocytes resident in adipose tissue or from bone marrow-derived progenitor cells that migrate into adipose tissue. The cells used herein typically comprise adipocytes from white adipose tissue.

[0028] The term "preadipocyte" is a term in the art that denotes the progenitor cell of a mature, differentiated adipocyte, which can be stimulated to form adipocytes. Preadipocytes can be isolated from the subcutaneous or visceral adipose tissue of an animal.

[0029] Preadipocytes can be grown in a preadipocyte growth medium, which contains all the growth factors and supplements necessary for the optimal growth of undifferentiated preadipocytes. For example, preadipocytes can be grown in a preadipocyte growth medium containing endothelial cell growth supplement, epidermal growth factor, hydrocortisone, and / or heparin.

[0030] The formation of adipocytes from preadipocytes involves a tightly regulated process of cell differentiation called adipogenesis, in which mesenchymal stem cells differentiate into preadipocytes, and preadipocytes differentiate into adipocytes. The term "differentiation" refers to a process of change in the expression pattern in which pluripotent gene expression is altered to cell type-specific gene expression. Transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT / enhancer-binding protein (C / EBP) are the main regulators of adipogenesis. The characteristics of differentiated adipocytes include, but are not limited to, growth arrest, morphological changes, high expression of adipogenic genes, and the production of adipokines such as adiponectin, leptin, resistin (in mice, but not in humans), and TNF-α, as will be understood by those skilled in the art.

[0031] As used herein, the term "homogeneous product" is a product that is formed as a single article and is substantially uniform throughout with respect to its sensory and functional properties.

[0032] As used herein, the term "culture medium" refers to a composition in liquid, solid, or gel state that contains organic, inorganic, and / or biogenic components in which cells are capable of surviving, maintaining viability, or proliferating. Culture media typically include a basal medium.

[0033] As will be understood by those skilled in the art, the term "basal medium" as used herein refers to a medium that contains components necessary for cell survival and growth, such as amino acids, glucose, and ions such as calcium, magnesium, potassium, sodium, and phosphate.

[0034] An example of a basal medium is the basal medium formulation (www.sigmaaldrich.com / life-science / cell-culture / learning-center / media-formulations / basal.html). Those skilled in the art can identify other examples.

[0035] Exemplary biogenic components include serum. The medium can be chemically defined. For example, Lipid Mixture 1 available from SigmaAldrich (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 tocopheryl acetate, and 100 mg / ml Pluronic F-68 dissolved in cell culture water.

[0036] The medium in the sense of the present invention can have biogenic components, including fetal bovine serum (FBS) or fish liver oil fatty acids. For example, Lipid Mixture (1000×) available from Sigma Aldrich (www.sigmaaldrich.com / catalog / product / sigma / l5146?lang=en®ion=US) contains 4.5 g / L cholesterol, 10 g / L fish liver oil fatty acids (methyl ester), 25 g / L polyoxyethylene sorbitan monooleate, and 2.0 g / L D-α-tocopheryl acetate.

[0037] The International Organization for Standardization (ISO) defines food "texture" in its standard vocabulary for sensory analysis as "all rheological and structural (geometric and surface) properties of a food that are perceptible by mechanical, tactile, and, where appropriate, visual and auditory receptors" (ISO, 2008). Texture is a key quality parameter used in the fresh processed food industry to evaluate consumer acceptability. Among texture properties, hardness (firmness) is one of the most important parameters and is commonly used to determine the freshness of food. Elasticity, cohesiveness, adhesiveness, and chewiness are also important properties for evaluating the texture of meat products.

[0038] The "color" of food is a physical property that is commonly associated with food quality. The surface color of food can be easily measured by instruments that are used as quantitative quality tools. During the life of a product, the color may change, indicating a decline in product quality. To analyze the color change of a product, the International Commission on Illumination (CIE) proposed a general method for analyzing color in 1931. This method differentiates color into three different tristimulus values. More recently, the Munsell system further simplifies color quantification through a multi-dimensional approach. L*, a*, b*, h, and C readings can be quantified and compared. L represents the overall brightness of the sample. The a* value represents red or green in the sample, while the b* value represents yellow and blue. The hue (h) and chroma (C) values are derived from the a* and b* values, where the hue is expressed in radians or degrees within the color space, and the chroma is expressed as a measure of intensity, i.e., the distance from the achromatic center of the color space. Color analysis can indicate surface degradation of a product, and the color difference (ΔE) can help distinguish differences between storage treatments.

[0039] The Institute of Food Science and Technology (IFST) in the UK defines "shelf life" as "the period during which a food product remains safe while stored under recommended conditions; ensures the retention of desired sensory, chemical, physical, microbial, and functional properties; and complies with any nutritional data label declarations" (Shelf Life of Foods: Guidelines for Its Determination and Prediction, 1993).

[0040] "Substantially free" of heavy metal contaminants (e.g., mercury) means that the edible composition contains less than 0.1 parts per million [ppm] of one or more heavy metal contaminants (e.g., less than 0.1 ppm, less than 0.01 ppm, less than 0.001 ppm, or less than 0.0001 ppm).

[0041] Cultured fish cells and compositions

[0042] In certain embodiments of the present invention, media, methods, and systems for culturing fish cells (e.g., bluefin tuna cells) are described, as well as fish cells such as bluefin tuna cells that can be obtained and / or obtained therefrom. In embodiments of the present invention, compositions, methods, and systems are described, as well as related fish cells, fish cell biomass, and fish cell culture foods, which have a controllable cell lipid content and lipid uptake, and / or have improved cell differentiation and / or cell viability related to a set lipid content and lipid uptake. Fish cell culture foods can be in various forms, including, for example, slurries, powders, homogeneous single cells, homogeneous multi-cells, non-fibrous stratified single cells, or non-fibrous stratified multi-cell products.

[0043] In one embodiment, the source of the fish cells is any fish cells from a variety of species.

[0044] In another embodiment, the fish source is any one or more of the seven tunas in the genus Thunnus. These include the northern bluefin tuna (T. thynnus), the longfin tuna (T. alalunga), the yellowfin tuna (T. albacares), the southern bluefin tuna (T. thynnus maccoyii), the bigeye tuna (T. obesus), the blackfin tuna (T. atlanticus), and the longtail tuna (T. tonggol). See https: / / www.britannica.com / animal / tuna-fish, which is incorporated herein by reference.

[0045] In another embodiment, the fish cells are derived from the genus Thunnus orientalis, wherein the fish cells are derived from bluefin tuna.

[0046] The cell-cultured fish products of the present invention differ from traditional fish-derived products in several aspects.

[0047] Contrary to traditional fish-derived products in which the cells are oriented such that the 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 disorganized within each layer. The connectivity of the cells within the cell-cultured bluefin tuna product is limited in terms of the number of cell junctions and the small amount of extracellular matrix. In contrast, in traditional fish-derived products, there are many interconnected cells and extracellular matrix. The amino acid profile and fatty acid profile of cell-cultured fish can be controlled and modified. In addition, the cell-cultured products can contain sugars, fibers, texturizing agents, natural pigments, natural flavorings that are not found in traditional products. The color of the cell-cultured products can also be controlled by adding natural coloring agent compounds (e.g., carotenoids such as astaxanthin) to the cell culture medium or adding natural coloring agent compounds (e.g., anthocyanins, beet juice, β-carotene, curcumin, spirulina, insect-derived colorants including but not limited to carmine, carotenoids such as astaxanthin, heme, leghemoglobin, lycopene, monascus red, paprika powder, or other natural colorants) to the final product to achieve the desired color (e.g., red, pink, white).

[0048] In some embodiments, the present invention relates to an edible composition comprising in vitro cultured fish cells arranged in a homogeneous or layered form. In vitro cultured fish cells are generally smaller than conventional fish cells. When proliferating, the diameter of each cultured cell can be less than 20 μm, for example, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 μm in diameter. Differentiated cultured cells are generally of medium size compared to a potentially smaller single myocyte with one nucleus (i.e., 10 - 30 μm) or a much larger in vitro myotube (i.e., 50 - 200 μm).

[0049] In certain embodiments, the diameter of the cultured cells can be less than 20 μm to 200 μm, such as 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, less than 175 μm, 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.

[0050] The fish cells are also substantially free of contaminants (e.g., environmental contaminants). Exemplary environmental contaminants include microplastics; persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), organochlorines (OCs), and polybrominated diphenyl ethers (PBDEs); heavy metals such as mercury, lead, cadmium, zinc, copper, nickel, chromium, arsenic, aluminum; fluorides; radon; microplastics; parasites, bacteria, and pesticides.

[0051] 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] of one or more heavy metal contaminants (e.g., less than 0.1 ppm, less than 0.01 ppm, less than 0.001 ppm, or less than 0.0001 ppm).

[0052] In addition to cells derived from the genus Thunnus, particularly cells of Thunnus thynnus, cells from a variety of fish species can be used. These species include, but are not limited to, bass, flounder, hake, yellowtail snapper, smelt, rainbow trout, quahog, blue crab, peekytoe crab, spanner crab, squid, eastern oyster, Pacific oyster, anchovy, herring, lingcod, moi, orange roughy, Atlantic sea bass, Nile perch, yellow perch, European oyster, Dover sole, sturgeon, tilefish, wahoo, yellowtail, sea urchin, Atlantic mackerel, sardine, black sea bass, European sea bass, hybrid striped bass, bream, cod, drum, haddock, grenadier, Alaska pollock, rockfish, pink salmon, snapper, tilapia, turbot, European perch, cisco, wolffish, quahog, surf clam, sea scallop, Jonah crab, snow crab, crayfish, calico scallop, Chinese white shrimp, sablefish, Atlantic salmon, coho salmon, skate, lantern crab, king crab, blue mussel, green oyster, pink shrimp, escolar, chum salmon, fall salmon, American shad, Arctic char, carp, catfish, sea bream, grouper, halibut, angel shark, butterfish, abalone, conch, stone crab, American lobster, spiny lobster, octopus, tiger shrimp, blue shrimp, bay shrimp, Pacific white shrimp, squid, barramundi, saithe, dogfish, cusk-eel, dolphin, opah, mako shark, swordfish, longfin tuna, yellowfin tuna, geoduck, squat lobster, sea scallop, rock shrimp, barracuda, toothfish, cobia, white croaker, eel, blue marlin, suckerfish, sockeye salmon, bluefin tuna, shrimp, crab, lobster, and echinoderms (e.g., sea cucumber and sea urchin).

[0053] Preferred aquatic animals include yellowtail (e.g., Seriola lalandi), dolphin (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, butterfish, salmon, sea bass, Patagonian toothfish (Dissostichus eleginoides), squid, clam, lobster, crab, scallop, shrimp, eel, bass (e.g., Micropterus salmoides), bluegill (Lepomis macrochirus), and carp (e.g., Hypophthalmichthys molitrix).

[0054] The homogeneous or layered mixture of in vitro cultured fish cells disclosed herein contains only one, two, three, or four different cell types, while traditional fish-derived products contain many different types of cells, including myoblasts, myotubes, endothelial cells, neurons, red blood cells, adipocytes, and combinations thereof. The mixture of in vitro cultured fish cells can form different product types, such as slurries, powders, homogeneous or layered sheets, and homogeneous or layered blocks. For example, in some embodiments, the cell culture slurry or powder product contains one or two cell types. The different cell types included in the cell culture product include myoblasts, myotubes, fibroblasts, endothelial cells, neurons, red blood cells, preadipocytes, induced pluripotent stem cells, adipocytes, and combinations thereof.

[0055] The cell culture edible fish products provided herein can be frozen or refrigerated or lyophilized so that they can be used later.

[0056] The edible composition can be liquid, semi-liquid, semi-solid, solid, or foamy.

[0057] In various embodiments, the cultured edible fish products according to the present invention can contain functional agents in addition to the cultured fish cells, including one or more texturizing agents that provide hardness, mouthfeel, and other texture characteristics such as cohesiveness, elasticity, and chewiness, and optionally additional functional agents that prevent lipid component peroxidation, reduce microbial contamination, and / or otherwise extend the product shelf life, and optionally sensory agents such as flavoring agents and coloring agents; nutritional supplements.

[0058] Functional agent

[0059] Texturizing agent

[0060] The texturizing agent and the concentration of each texturizing agent can be selected to achieve the degree of the above texture attributes and characteristics suitable for the intended purpose of the product. The product formulation can include one or more gelling agents and / or one or more thickening agents.

[0061] In addition to the above texturizing agents, the product can contain protein isolates / concentrates / structured proteins, including but not limited to soy, wheat, peas, chickpeas, water lentils, lentils, oats, rice, and potatoes. In addition, it can include 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 an exemplary embodiment, the total concentration of the reagents contributing to the texture can be between 1-40% (w / w) of the final product weight.

[0062] Food safety gelling agents applicable to products include, but are not limited to, sodium alginate, carrageenan, agar, gellan gum, konjac gum, and curdlan, etc. Similarly, a variety of food safety thickeners suitable for use include, but are not limited to, locust bean gum, gum arabic, carob gum, methylcellulose, psyllium husk, pea fiber, citrus fiber, and xanthan gum, etc. The concentration of specific texturizing agents varies according to the intended use of the product and can range from 0.05% to 10%.

[0063] In addition to hardness, certain texturizing agents further provide additional functional properties, including but not limited to thermal stability and browning. Such agents allow the product to remain solid during heating and / or to brown during cooking to produce an appetizing appearance and / or flavor. The concentration of such agents is selected to provide a product with acceptable thermal stability and / or browning in a controlled manner without imparting an unpleasant hardness to the consumer. In some embodiments, the texturizing agent is selected to provide emulsion stabilization in addition to other sensory or functional properties.

[0064] Some texturizing agents, such as locust bean gum and gellan gum, can have a synergistic effect in terms of texture. The specific combination and the absolute and relative concentrations of the individual agents comprising the combination are selected to obtain a product with a hardness close to that of traditional fish meat without sacrificing other desired sensory or functional characteristics.

[0065] Colorants

[0066] In traditional bluefin tuna, sarcoplasmic proteins and myoglobin are responsible for the red color of the muscle. The amount of myoglobin and fat in the meat of different parts gives them different colors ranging from dark red (akami) to pink / pale pink (chutoro and otoro; commonly referred to as toro).

[0067] In cell culture products, there is no natural myoglobin. Therefore, the natural colorants mentioned in the previous embodiments can be used in the form of powder, liquid, or emulsion to obtain products with different hues as mentioned in the above embodiments. The colorants can be used alone or in combination to obtain the desired results. In one example, the concentration of the colorants ranges from 0.001% to 5% to achieve L*, a*, and b* values in the ranges of 33.0–62.8, 4.77–21.9, and 4.72–15.4, respectively.

[0068] Adding only natural colors does not ensure proper color, and bluefin tuna cells are responsible for adding some color features as well as altering the overall hue. In one embodiment, when bluefin tuna cells are added, the L*, a*, b* values of the product change from 50.18, 18.46, 14.30 to 69.89, 17.24, 14.26, respectively. Similarly, the texture of bluefin tuna cells can be manipulated by changing the overall hardness of the edible product from 656 g to 442 g.

[0069] Sensory enhancers

[0070] In addition to texturizing agents, homogenized and layered in vitro cultured fish cell products can optionally be formulated with one or more sensory agents, including but not limited to natural colorants, natural flavorings. Suitable colorants are those that provide the desired color at a pH prevalent in meat obtained from live fish, i.e., a pH of 6 to 7. For reasons of safety and consumer appeal, food-derived colorants are preferred, although colorants derived from insects or other sources safe for human consumption can also be used. Suitable colorants include but are not limited to food-derived colorants such as anthocyanins, carotenoids, carrot powder, beet juice, leghemoglobin, lycopene, monascus red, and paprika, and insect-derived colorants including but not limited to carmine.

[0071] The flavor of very fresh fish is characterized by a mild, delicate aroma, which is due in part to volatile 6-, 8-, and 9-carbonyl compounds 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 the cultured bluefin tuna cells. Flavorings can include vegetable oil, coconut oil, palm oil, algal oil, sunflower oil, safflower oil, soybean oil, olive oil, avocado oil, grapeseed oil, peanut oil, canola oil.

[0072] Flavor can also be adjusted by using salts including sodium chloride and potassium chloride; acidic components such as vinegar, citric acid, tartaric acid, malic acid, folic acid, fumaric acid, and lactic acid.

[0073] Texturizing agents provided as salts of sodium, potassium, calcium, or magnesium can affect flavor aspects such as saltiness or bitterness. In such cases, the type and concentration of other flavorings (such as salts and alkaline components) are adjusted in order to give the desired flavor profile.

[0074] In some cases, the single-cell bluefin tuna product further includes flavorings that complement the flavor of fish, such as soy sauce, ginger, sesame, herbs such as parsley, dill, capers, and / or cilantro; fruit juices and / or extracts from citrus fruits such as calamansi, lemon, lime, orange, grapefruit, kumquat, and / or pomelo; and / or black pepper, red pepper, and / or white pepper.

[0075] Flavoring agents can be added in their natural form and / or as semi-dry or dry powder, capsules, extracts or neat oils, etc.

[0076] Nutritional supplements

[0077] Optionally, the homogeneous nutritional components of the in vitro cultured fish cell products provided herein can be supplemented with, for example, amino acids, peptides, proteins, and / or lipids, the latter including but not limited to nutritional supplements, including amino acids, vitamins, minerals, and / or carbohydrates, which can be provided in the cell culture medium and then absorbed and incorporated 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 acid, saturated fatty acid, and / or sterol) is present at a concentration of about 10 μg / ml or higher.

[0078] In other alternative embodiments, nutritional supplements including lipids, amino acids, vitamins, minerals, and / or carbohydrates as described above, as well as nutritional fibers, can be added to the cell culture medium or cell slurry after harvest, or can be added during product formulation.

[0079] Preservatives

[0080] Optionally, the homogeneous in vitro cultured fish cell products can be formulated with reagents that reduce the microbial load and / or peroxidation, increase the product shelf life, and enhance consumer safety. Examples of such reagents include antioxidants suitable for the production of homogeneous single cell type products, including but not limited to, 2,4,5-trihydroxybutyrophenone (THBP), algae extracts, anoxomer, apigenin, ascorbic acid, baicalein, plant extracts (including but not limited to blueberries, ginseng, goji berries, grape seeds, and green tea), butylated hydroxytoluene (BHT), butylated hydroxyanisole, butylated hydroxyanisole (BHA), carnosol, carotenoids, catalase, catechins, creatine, dilauryl thiodipropionate, epigallocatechin gallate, ethoxyquin, lipoic acid, mitoquinol, morin, myricetin, acetylcysteine, phenols, pinosylvin, procyanidin dimer B2, propionyl-L-carnitine, propyl gallate, quercetin, resveratrol, rosemary extract, rutin, sauchinone, TBHQ (tert-butylhydroquinone), tert-butylhydroquinone (TBHQ), THBP (2,4,5-trihydroxybenzyl ketone), thiazolidine, thiodipropionic acid, thymol, and tocopherols.

[0081] In some embodiments, the antioxidants are plant-derived and include, but are not limited to, algae extracts, apigenin, ascorbic acid, baicalein, plant extracts (including, but not limited to, blueberries, ginseng, goji berries, grape seeds, and green tea), carnosol, carotenoids, catalase, catechins, epigallocatechin gallate, lipoic acid, morin, myricetin, pinocembrin, proanthocyanidin dimer B2, propionyl-L-carnitine, quercetin, resveratrol, rosemary extract, rutin, sauchinone, thymol, and tocopherols.

[0082] Pollutant

[0083] Bluefin tuna are top predator and long-lived fish and accumulate mercury through dietary transfer. The tissue mercury concentration in bluefin tuna (BFT) often exceeds the human consumption guideline threshold of 1 μg·g-1 wet weight (w.w.) for large predatory fish set by the US Food and Drug Administration and the World Health Organization. According to Tseng et al. (2021), the total mercury levels in the muscle tissues of Pacific bluefin tuna (Pacific BFT) ranged from 0.49 to 5.65 μg·g-1 w.w. (mean = 2.00 ± 0.83 μg·g-1 w.w., n = 261), where ~94% exceeded the 1 μg·g-1 w.w. of the safe consumption guideline. Meanwhile, Table 1 (n = 19) summarizes the levels of environmental pollutants 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. All environmental pollutants in the cell-cultured bluefin tuna according to the present invention are significantly lower. See Table 1 below, showing the naturally occurring levels in wild-caught and farmed bluefin tuna. The "significantly lower" levels refer to the levels of any one or more environmental pollutants being at least 60% lower than the levels allowed by state and / or federal guidelines. Table 2 shows the levels of heavy metals - arsenic, cadmium, lead, and mercury in traditional (farmed) Pacific and Atlantic bluefin tuna and cell-cultured bluefin tuna (n = 3).

[0084] Table 1 Metal contents of wild-caught and farmed bluefin tuna detected by ICP-MS (n = 19). All values are expressed in parts per billion (ppb). Heavy metals mercury and lead were detected in all tested samples.

[0085] Parts per billion Chu-toro O-toro Arsenic (As) 1490–4660 1960–9800 Cadmium (Cd) <10 <10 Mercury (Hg) 195–633 179–423 Lead (Pb) <10 <10 Iron (Fe) 3860–10200 3750–8860 Manganese (Mn) 143 Zinc (Zn) 3410–5460 2740–4660 Copper (Cu) 150–323 123–291 Tin (Sn) <10 <10 Antimony (Sb) <10 <10 Chromium (Cr) 11.0–26.1 10.1–59.2 Nickel (Ni) 15.8–33.0 10.9–29.5 Selenium (Se) 259–571 203–618 Molybdenum (Mo) <10 <10 Silver (Ag) <10 <10 Barium (Ba) <10 <10 Beryllium (Be) <10 <10 Cobalt (Co) <10 <10 Thallium (Tl) <10 <10 Vanadium (V) <10 <10 Strontium (Sr) 44.0–92.4 39.6–122.0

[0086] Table 2. Heavy metal contents of farmed Pacific and Atlantic traditional bluefin tuna and cell-cultured bluefin tuna detected by ICP-MS (n = 3). All values are expressed in parts per million (ppm).

[0087] Parts per million, w / w Conventional bluefin tuna Cell-cultured bluefin tuna Arsenic (As) 1.57±0.26 <0.01 Cadmium (Cd) 0.003±0.00 <0.001 Mercury (Hg) 0.412±0.004 <0.005 Lead (Pb) <0.01 <0.01

[0088] Storage stability study

[0089] 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. The main physical and chemical changes occur during the refrigerated and frozen storage of fish and fish products, such as color degradation / change, off - flavors caused by lipid and protein oxidation / hydrolysis, generation of potentially hazardous compounds such as histamine, texture changes such as softening, nutritional loss, flavor and taste degradation, weight loss due to drip loss of moisture, etc. The deterioration of at least one property can be considered as the loss of shelf - life.

[0090] In traditional bluefin tuna, color change (browning) and lipid oxidation are problems that limit its refrigerated and frozen shelf - life. During long - term low - temperature frozen storage, due to the production of excessive metmyoglobin (MetMb), the color of tuna turns brown, which has a negative impact on its commercial value and also leads to food waste. Mb, which exists in muscle fiber cells, is the most important coloring substance in tuna (Singh, Benjakul, Zhou, Zhang, & Deng, 2021). Generally, the bright red color of tuna muscle is mainly related to the presence of red pigments, especially oxymyoglobin (OxyMb). However, OxyMb is converted to brown MetMb during processing and storage (Hoa et al., 2020). MetMb is mainly formed due to Mb oxidation, free radicals generated during lipid oxidation, and the formation of trimethylamine N - oxide. (Grunwald, Tatiyaborworntham, Faustman, & Richards, 2017). Also see Ying Bu et al, Food Science and Technology (2022) and the references cited therein. https: / / www.sciencedirect.com / science / article / pii / S0023643821018685

[0091] In one example, during the first two days of refrigerated storage when the color of southern bluefin tuna changed from red to brown, the ratio of a* / b* decreased significantly by 60% (Ying Bu et al. 2022). This study also showed that during a 6-day storage period, the texture softened, and the hardness (76%), chewiness (76%), springiness (11%), and resilience (45%) decreased significantly. This study also found that during storage, volatile nitrogenous compounds and lipid oxidation products increased significantly, indicating unacceptable quality deterioration. In another example, a similar trend was observed for the color of Pacific bluefin tuna stored in a refrigerator, where the ratio of a* / b* decreased by 88% during a 10-day storage period. Similar observations were also reported for bluefin tuna stored under frozen conditions at -18 and -55°C. https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9368106 /

[0092] In the case of cell-cultured bluefin tuna (homogenized bluefin tuna prototype) packaged in plastic wrap and stored under refrigeration at 4°C, the overall color degradation rate was much slower, and the a* / b* value decreased by only 16% after 10 days of refrigerated storage.

[0093] In another example, during frozen storage at -80°C, the product packaged in plastic wrap showed excellent freeze-thaw stability, with only <0.5% drip loss and retention of color attributes. It should be noted that "weight loss" during frozen storage is important from both an economic and quality perspective. The lower "weight loss" associated with cell-cultured bluefin tuna according to the present invention is another benefit provided to consumers. This appears to be consistent with the observations of Jinfeng Wang et al. (2022), where the data showed little quality change during short-term frozen storage at 18°C. However, at a frozen storage temperature of -55°C, bluefin tuna showed significantly improved quality compared to a frozen storage temperature of -18°C. The content of this reference is incorporated herein in its entirety.

[0094] In various embodiments, the edible composition according to the present invention may optionally include functional agents such as nutritional supplements, preservatives, pH regulators, emulsifiers, emulsion stabilizers, fluidity enhancers, wetting agents, and humectants, among others. Such agents provide various benefits, including but not limited to facilitating preparation, product formation, humidification, moisture retention, and / or improving shelf life.

[0095] Product form

[0096] Homogeneous and stratified in vitro cultured fish cell products can form various shapes, including but not limited to saku blocks (i.e., suitable for preparing sashimi and other sliced products for grilling or further processing into minced, fish steak, fish fillet and loin, sashimi, diced, flaked and cubed homogeneous blocks).

[0097] Plasma products of in vitro cultured fish cells

[0098] In another embodiment of the present invention, the in vitro cultured fish cell product is a plasma. The plasma can be a viscous plasma. Viscosity is a measure of the resistance to flow of a fluid. The viscous plasma can be defined based on certain rheological studies to determine the key parameters preferred in the cell culture plasma according to its intended purpose. For example, the viscosity ranges between 25,000 cP and 50,000 cP. It is also possible for the viscosity to exceed this range, depending on the specific product application.

[0099] The plasma can be used to prepare products including but not limited to nutritional supplements and food ingredients. In one embodiment, the nutritional supplement can be prepared by filling capsules with freeze-dried cultured fish cells. Such capsules can be used in the same way as fish oil capsules, but offer the advantages of having a uniform and reproducible lipid profile while being free of environmental contaminants. The capsules themselves can be made of animal-free materials, such as, for example, cell culture gelatin, modified tapioca starch and / or plant cellulose such as hydroxypropyl methylcellulose (also known as hypromellose), the latter offering the advantage of delaying the release of its contents until it reaches the small intestine, thus optimizing lipid absorption.

[0100] In other embodiments, the plasma can be used as a food ingredient in fresh, frozen or dried form, either alone or in combination with other animal and / or plant materials, for the production of various foods such as baby foods, pet foods and other products.

[0101] The plasma can be dispensed by a variety of methods during the preparation of edible products (including, for example, bluefin tuna cell products), which are selected based on factors including but not limited to: viscoelasticity, shear rate, pressure, temperature, and mixing requirements in cases where mixing and dispensing occur simultaneously. Devices suitable for dispensing the plasma include but are not limited to positive displacement pumps such as rotary, internal gear, screw, slider, flexible vane or sliding vane, circumferential piston, helical root (e.g., Wendelkolben pump) or liquid ring vacuum pumps; piston pumps; screw pumps; peristaltic pumps; screw pumps; and extruders.

[0102] Homogeneous single cell type products and multi-cell type products of in vitro cultured fish cells

[0103] The homogeneous single cell type product of in vitro cultured fish cells can be produced using a single cell type slurry that contains cells selected from myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, adipocytes, and the like.

[0104] In another embodiment, the homogeneous multiple cell type product of in vitro cultured fish cells can be produced using a multiple cell type slurry from a culture containing one, two, three, four or more cell types, such as myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, adipocytes, and combinations thereof. Alternatively, the multiple cell type product can be prepared from two, three, four or more single cell type slurries by combining the slurries in a ratio suitable for the intended purpose of the final product. In an exemplary embodiment according to the present invention, the cells are bluefin tuna cells.

[0105] The multiple cell type product containing both muscle cells and adipocytes can be formulated to produce a product having a fat content that mimics the fat content of wild-caught or farmed fish fillets with high fat, medium fat or low fat (lean). In an exemplary embodiment, the bluefin tuna cultured cell product is formulated to mimic bluefin tuna slices characterized as otoro (high fat), chutoro (medium fat) or akami (lean). In some embodiments, multiple cell type products each having different ratios of muscle cells and adipocytes can be combined to form a layered product that mimics the multi-layered natural bluefin tuna slices characterized by toro (abdomen).

[0106] In certain embodiments, the multiple cell type product provides a more desirable nutritional profile than a product containing only muscle. For example, the multiple cell type product can be formulated with lipid-loaded fish adipocytes such that they have a lipid profile that provides optimal nutritional benefits.

[0107] The homogeneous single cell or multiple cell type product can form a block as shown in schematic diagram (g), which is further processed into minced pieces as described below. The product can also form sheets of uniform or variable thickness, which can then be combined into a layered product as described below.

[0108] Articles provided to consumers, such as fish fillets, sashimi, and fish blocks (the latter including "poke" fish blocks provided as an appetizer or main course) in the center of a plate, can be formulated with one or more texturizing agents, colorants, nutritional supplements, and other food ingredients to mimic the texture, color, and other sensory and nutritional aspects of traditional bluefin tuna meat, and this pleases the consumer.

[0109] Single cell type and multi-cell type fish cultured cell products intended to be used as soft diet products, such as baby food, children's food, and pet food, can be formulated with one or more texturizing agents, and the type and concentration of the texturizing agent are selected to produce a semi-solid or semi-liquid product.

[0110] Flakes

[0111] Flakes of in vitro cultured fish cells are optimally formed by processing large chunks formed from single cell type or multi-cell type homogeneous products.

[0112] Single cell type or multi-cell type pastes can be combined with one or more texturizing agents or one or more binders to produce chunks having a texture optimized for processing by chopping into small pieces, the size of which varies according to the intended use of the flakes. In an embodiment, as a preliminary step, the chunks are partially or fully frozen to facilitate chopping.

[0113] Because chopping will cause the "destruction" of the gel formed by the texturizing agent, thereby releasing moisture, a small amount of dry ingredients can be used to formulate the chunks for producing flakes to promote moisture retention. Such dry ingredients include, but are not limited to, textured proteins, flours, gums, etc.

[0114] Depending on the intended use of the final flakes, two or more flakes can be combined, each produced from chunks having different cell type compositions.

[0115] Non-fibrous, layered products of in vitro cultured fish cells

[0116] Embodiments of the present invention also include edible compositions comprising a collection of non-fibrous in vitro cultured fish cells, wherein the cells are in a layered structure and are substantially free of contaminants, as shown in the schematic diagram. The composition comprises multiple layers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers) of cultured cells, which may or may not be equidistant. The individual layers or the layered structure as a whole can comprise a single cell type or multiple cell types. The layered structure as a whole can comprise a single cell type or multiple cell types (e.g., 2, 3, 2-3, 2-4 cell types). The cell types of the single layer and / or multiple layer products can be any one or combination of myoblasts, myotubes, fibroblasts, endothelial cells, neurons, erythrocytes, preadipocytes, induced pluripotent stem cells, and adipocytes.

[0117] The paste for producing the layered product contains at least one texturizing agent that causes the paste to reach a semi-solid or solid form upon solidification; they can optionally further contain one or more functional and / or sensory agents as described above.

[0118] Non-fibrous layered products can be produced in a variety of ways, but generally require a step of forming layers, which in turn requires dispensing and then solidifying (curing) the corresponding slurry. The slurry used to produce the layered product contains at least one texturizing agent that causes the slurry to reach a semi-solid or solid form upon solidification; they may optionally further contain one or more of the functional and / or sensory agents as described above.

[0119] The layered product can be produced by separately forming and solidifying individual layers into separate blocks and then assembling the blocks to produce the layered product. In this case, the layers can be allowed to passively adhere to each other, for example, by electrostatic interactions such as hydrogen bonding. In an alternative, adhesives or enzymatic methods (such as methods using transglutaminase) can be used to bind the layers together.

[0120] The layered product can also be produced in one piece by sequentially dispensing and setting slurry layers to produce a vertical stack. The layered product can also be produced by simultaneously forming the layers, for example, as a plate, sheet, or cylinder, where the layers are adjacent and horizontally aligned. A layered product with a marbled appearance can be produced by mixing or vortexing two or more layers before solidification, or by dispensing successive slurry layers in a way that produces non-uniform, highly irregular layers. Combinations of these methods can also be used to produce products with different layered patterns.

[0121] Depending on the viscosity of the slurry used, the individual layers can be solidified sequentially or simultaneously.

[0122] Individual layers or layered blocks can be formed by dispensing the slurry into a mold or onto a fixed or movable platform or conveyor belt using one or more fixed or movable dispensing devices to form discrete or continuous blocks. The layers or layered blocks can have the same or larger dimensions as the finished product. When the blocks are larger than the dimensions of the final product, they can be machined into blocks with the desired final dimensions. Other methods are also possible.

[0123] In another embodiment, a layered product is produced using a food preparation system that sequentially deposits the slurry layer by layer. In this method, if a high-viscosity slurry is used, the layers can be solidified simultaneously, or in the case of a low-viscosity slurry, the layers are sequentially solidified after each layer is deposited and before the next layer is deposited. As described above, the slurry can be dispensed by a variety of methods.

[0124] In another embodiment, individual layers are formed using tube-forming techniques known in the art.

[0125] Preparation method

[0126] For homogeneous products, the cytoplasmic body is mixed with the above-mentioned functional components except the texturizing agent at 15 - 45 °C for 5 - 15 minutes, then the texturizing agent is added at 45 - 75 °C and further mixed for 5 - 15 minutes. After the final mixing, the mixture is poured into a mold and rapidly cooled to form a homogeneous block of the product. Finally, the product is flash-frozen to -80 °C and packaged in a high-barrier package. For the marbled layered product as shown in the schematic diagram, a marbled layer consisting of a high-fat emulsion and some texturizing agent is prepared to assist in forming the product and kept warm until the coloring component is prepared. The coloring mixture is prepared in a similar manner to the homogeneous product and then randomly layered with the white marbled emulsion to obtain the appearance as shown in the schematic diagram.

[0127] In connection with the experimental section, which details the invention embodiments described below only by way of illustration, further details regarding the compositions, methods, and systems described herein will become more apparent. Examples

[0128] The cell-cultured edible foods and related cells, compositions, and systems described herein are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

[0129] Example 1: Cytoplasmic bodies of in vitro cultured fish cells

[0130] In this example, cells derived from bluefin tuna are concentrated into a viscous paste, which can be used as is, frozen, or dried and stored until use. The cytoplasmic body can contain one or more cell types, such as myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells, or induced pluripotent stem cells.

[0131] Origin of bluefin tuna cell line

[0132] Pacific bluefin tuna (12 - 100 pounds) are wild-caught and identified by visual inspection and genomic sequencing. Myoblasts are harvested from muscle tissue. Typically, 6 - 24 grams of tissue are processed via enzymatic and mechanical dissociation and seeded at 0.5 - 1.0 grams of tissue / well.

[0133] Expansion of bluefin tuna cells

[0134] The stable cell line of bluefin tuna cells is expanded under growth conditions. The cells proliferate under growth pH and temperature conditions in a dilution of approximately 10 3 to 10 6 cells / mL.

[0135] Formation of bluefin tuna cell paste

[0136] A concentrated cell paste is formed by concentrating cells from a dilute growth or differentiation solution. Concentration is performed by centrifugation, sedimentation, or other separation methods that separate the cells from the liquid. The paste contains more than 10 6 The cell concentration per mL and up to 10 9 , depending on the cell size. These slurries are viscous solutions similar to suspensions that can flow like liquids. Optionally, the product is then frozen or dried to form a solid product.

[0137] Cell Plasma Products

[0138] The cell paste is used to prepare products including but not limited to nutritional supplements and food ingredients. Nutritional supplements are prepared by filling capsules with freeze-dried cultured bluefin tuna cells. Such capsules are used in the same manner as fish oil capsules, but provide a uniform and reproducible lipid profile while not containing the advantages of environmental pollutants. The capsules are made of animal-free materials, such as cell culture gelatin, modified tapioca starch and / or plant cellulose such as hydroxypropyl methylcellulose (also referred to as hypromellose), wherein the latter provides the advantage of delaying the release of its contents until it reaches the small intestine, thereby optimizing lipid absorption.

[0139] Cell paste is used as a food ingredient in fresh, frozen or dried form, alone or in combination with other animal and / or plant materials, in the production of various foods such as baby food, pet food and other products.

[0140] Example 2: Homogeneous, single cell type product of in vitro cultured bluefin tuna cells

[0141] Homogenized bluefin tuna products are prepared by combining a bluefin tuna cell paste as described above in Example 1 with one or more of the aforementioned food ingredients. These homogenized products are formed as a single product that is substantially uniform throughout with respect to its organoleptic and functional attributes.

[0142] Single cell type products are prepared using the cell paste of single cell type, such as myoblasts, myotubes, preadipocytes, adipocytes, fibroblasts, endothelial cells or induced pluripotent stem cells. Both cell type and form depend on the desired purpose of the final product. In one embodiment, the homogenous single cell type product is formed into a block, which is further processed into a broken piece (see Example 4 below), or is formed into a sheet with uniform or variable thickness, which is combined into a layered product (see Example 3 below) with desired hardness, mouthfeel, nutrition, outward appearance or other product attributes.

[0143] Texture Agent

[0144] The texturing agents and the concentration of each texturing agent can be selected to achieve a degree of hardness, cohesiveness, springiness, and chewiness suitable for the intended purpose of the product.

[0145] Product form

[0146] Homogenized in vitro cultured bluefin tuna cell products can form various shapes, including but not limited to saku (blocks, i.e., suitable for preparing sashimi and other sliced products for grilling or further processed into minced, fish steaks, fillets and loin, sashimi, diced, flaked and cubed homogeneous pieces).

[0147] Functional agents

[0148] In addition to hardness, certain texturizing agents further provide additional functional properties, including but not limited to heat stability and browning, and these agents allow the product 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 acceptable heat stability and / or brown in a controlled manner without an unpleasant hardness of the product. Additionally, in some cases, the texturizing agent is selected to provide emulsion stability in addition to other sensory or functional properties.

[0149] Example 3: Homogeneous, multiple cell type product of in vitro cultured bluefin tuna cells

[0150] A homogeneous multi - cell - type product of in vitro cultured bluefin tuna cells is produced using multi - cell - type slurries from a culture containing one, two, three, four or more cell types. Alternatively, the multi - cell - type product can be prepared from two, three, four or more single - cell - type slurries combined in a ratio suitable for the intended purpose of the final product.

[0151] A multi - cell - type product containing both myocytes and adipocytes can be formulated to produce a product with a fat content that mimics the fat content of wild - caught or farmed bluefin tuna slices characterized by otoro (high - fat), chutoro (medium - fat) or akami (lean).

[0152] Example 4: Granules of in vitro cultured bluefin tuna cells

[0153] The minced in vitro cultured bluefin tuna is optimally formed by processing large chunks formed from single - cell - type or multi - cell - type homogeneous products as described in Examples 2 and 3 above.

[0154] A slurry of a single cell type or multiple cell types is combined with one or more texturizing agents or one or more binders to produce a block having a texture optimized for processing by chopping into small pieces, the size of which varies according to the intended use of the crumbs. In an alternative embodiment, as a preliminary step, the block is partially or fully frozen to facilitate chopping. Since chopping will cause the "destruction" of the gel formed by the texturizing agent, thereby releasing moisture, a small amount of dry ingredients (such as sodium alginate, fiber, psyllium husk, etc.) can be used to formulate the block for producing the crumbs to promote moisture retention.

[0155] Example 5: Non-fibrous, layered, single cell type product of in vitro cultured bluefin tuna cells

[0156] A non-fibrous, stratified single cell type product of in vitro cultured bluefin tuna cells is produced by first producing separate layers of two or more homogeneous single cell type products (as described in Example 2), and then combining these layers. See Figure 1 A-1G.

[0157] The stratified structure can be achieved in various ways, but generally requires steps to form the layers, which in turn requires dispensing the corresponding slurries, then solidifying (curing) the layers, and then assembling multiple layers. Depending on the viscosity of the slurries used, the individual layers can be solidified sequentially or simultaneously, either before or after assembling them. In one embodiment, each layer is produced independently and allowed to solidify (i.e., cure); then the layers are assembled by stacking them vertically. In this embodiment, the layers can be formed by dispensing the slurry as discrete or continuous blocks into a mold or onto a platform. In the case where the slurry is dispensed as a continuous block onto the platform, these blocks can have the same length and width as the finished product, in which case they solidify after dispensing and then are assembled by vertical stacking. In the case where the blocks are larger than the size of the final product, they can be cut into smaller blocks, then solidified and stacked, or in an alternative, the larger blocks can be solidified and then stacked to produce an intermediate form, and this intermediate form is cut into multiple blocks having the desired length and width.

[0158] Example 6: Non-fibrous, layered, multiple cell type product of in vitro cultured bluefin tuna cells

[0159] A non-fibrous, stratified multiple cell type product of in vitro cultured bluefin tuna cells is produced using separate layers, each layer may comprise one or more cell types to produce a product having generally two or more cell types. In addition to the cell type composition, the layers contain one or more texturizing agents, which can be the same or different for each individual layer, and one or more additional functional and / or sensory agents, as described in Example 2 above.

[0160] The marbling of the product can be achieved by layering of individual layers as described above. The individual layers can be stabilized with a food-grade emulsifier and can have a separate formulation containing the aforementioned texture, coloring, and sensory components.

[0161] Example 7: Improved refrigerated and frozen shelf life of in vitro cultured bluefin tuna cell products

[0162] A non-fibrous layered product prepared according to one or more of the above embodiments is packaged in plastic wrap and stored at 4 °C refrigeration for 10 days. Thereafter, it is removed and the color degradation is examined compared to a traditional bluefin tuna used as a reference. As described by Ying Bu et al, "Generally, the bright red color of tuna muscle is mainly associated with the presence of red pigments, especially oxygenated myoglobin (OxyMb). However, OxyMb is converted to brown MetMb during processing and storage... MetMb is mainly formed due to Mb oxidation, free radicals generated during lipid oxidation, and trimethylamine oxide formation". The overall color degradation rate of the tuna product according to the present invention is much slower, with the a* / b* value only decreasing by 16%. Referring to Ying Bu, this indicates a lower lipid oxidation rate. Figure 2A and 2B shows the difference in color degradation under refrigeration conditions. Figure 2A shows the shelf life as a function of the ΔE value, while Figure 2B shows the same data as a function of the a* / b* value.

[0163] In another embodiment, the product used in the above embodiment is placed in a refrigerator at -80 °C for 11 days and then thawed for evaluation. The product packaged in plastic wrap shows excellent freeze-thaw stability, with only <0.5% drip loss and retains its color properties. The data shows that when the product is stored at -80 °C for 11 days, its color does not change significantly. Figure 3 shows the shelf life of the cell-cultured bluefin tuna product at -80 °C. This shows that the cell-cultured bluefin tuna has a longer shelf life compared to traditional bluefin tuna.

[0164] In another embodiment, the product used as above is placed in a refrigerator at -20 °C for three months and compared with wild-caught Pacific bluefin tuna (traditional bluefin tuna). Figure 4 shows the difference in color degradation under -20 °C conditions. The color degradation as a function of the ΔE value is significantly higher in traditional bluefin tuna than in the cell-cultured bluefin tuna samples, indicating an extended shelf life of the cell-cultured bluefin tuna product under frozen storage conditions.

[0165] In another example, traditional bluefin tuna and cell-cultured bluefin tuna were vacuum-packed and stored at 4 °C for 14 days, and lipid oxidation, measured as the amount of malondialdehyde (MDA) generated, was evaluated using the improved protocols described in Quevedo et al. (2013), Scheffler et al. (2010), and Sonar et al. (2020). Figure 5 It was shown that the amount of MDA generated in cell-cultured bluefin tuna was significantly lower compared to traditional bluefin tuna, indicating an enhanced shelf-life retention of the cell-cultured bluefin tuna product. The heavy metals in both samples, including arsenic, cadmium, lead, and mercury, were also evaluated by ICP-MS (Table 2). The cell-cultured bluefin tuna had significantly lower contaminant levels compared to wild-caught and farmed traditional bluefin tuna.

[0166] The above embodiments are provided to provide a complete invention and description to those of ordinary skill in the art: embodiments of how to prepare and use an edible composition comprising bluefin tuna cells substantially free of contaminants, and related compositions, methods, and systems of the present invention, and are not intended to limit the scope of what the inventors consider to be 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 other cells, compositions, methods, and systems according to the scope of various embodiments and claims. All patents and publications mentioned in the specification indicate the level of skill of those skilled in the art to which the present invention pertains.

[0167] The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other inventions) cited in the background, invention overview, detailed description of the invention, and examples is hereby incorporated by reference herein. All references cited in the present invention are incorporated by reference to the same extent as if each reference were incorporated by reference in its entirety individually. However, if any inconsistency occurs between the cited references and the present invention, the present invention prevails.

[0168] The terms and expressions that have been used herein are used as terms of description rather than limitation, and the use of these terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the invention claimed. Accordingly, it should be understood that although the present invention has been specifically disclosed by way of embodiments, exemplary embodiments, and optional features, those skilled in the art can make various modifications and variations to the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0169] It should also 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 context clearly dictates otherwise. Unless the context clearly dictates otherwise, the term "plurality" includes two or more referents. 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, unless otherwise defined.

[0170] When a Markush group or other group is used herein, all individual members of the group and all combinations and possible sub - combinations of the group are intended to be individually included in the invention. Unless otherwise stated, each combination of components or materials described or exemplified herein can be used to practice the invention. One of ordinary skill in the art will understand that methods, system elements and materials other than those specifically exemplified can be employed in the practice of the invention without undue experimentation. All known functional equivalents of any such methods, apparatus elements and materials are intended to be included in the invention. When ranges are given in the specification, for example, temperature ranges, frequency ranges, time ranges or composition ranges, all intermediate ranges and all sub - ranges, as well as all individual values included within the given range are intended to be included in the invention. Any one or more individual members of the ranges or groups disclosed herein can be excluded from the claims of the invention. The invention described herein by way of example can be practiced appropriately without the presence of any element or elements, limitation or limitations not specifically disclosed herein.

[0171] Numerous embodiments of the invention have been described. The specific embodiments provided herein are examples of useful embodiments of the invention, and it will be apparent to those skilled in the art that the invention can be practiced using a large number of variations of the genetic circuits, genetic molecular components and method steps set forth in this specification. As will be apparent to those skilled in the art, the methods and systems that can be used in this method and system can include a large number of optional compositions as well as processing elements and steps.

[0172] In particular, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the appended claims.

[0173] References

[0174] Abnet, 2007 Abnet C. (2007). Carcinogenic food contaminants. Cancer Invest. 25, 189–196. 10.1080 / 07357900701208733

[0175] Androutsopoulos et al., 2013. Androutsopoulos V., Hernandez A., Liesivuori J., Tsatsakis A. (2013). A mechanistic overview of health associated effects of low levels of organochlorine and organophosphorous pesticides. Toxicology 307, 89–94. 10.1016 / j.tox.2012.09.011

[0176] Bassil K., Vakil C., Sanborn M., Cole D., Kaur J., Kerr K. (2007). Cancer health effects of pesticides. Can. Fam. Phys. 53, 1704–1711

[0177] Chiesa LM, Labella GF, Panseri S, Pavlovic R, Bonacci S, Arioli F. Distribution of persistent organic pollutants (POPS) IN wild Bluefin tuna (Thunnus thynnus) from different FAO capture zones. Chemosphere. 2016 Jun;153:162 - 9. doi:10.1016 / j.chemosphere.2016.03.010. Epub 2016 Mar 24. PMID: 27016811.

[0178] Claudia Ruiz - Capillasnd and Ana M. Herrero, (2019). Impact of Biogenic Amines on Food Quality and Safety. Foods 8(2)62.

[0179] Grandjean P., Landrigan P. (2006). Developmental neurotoxicity of industrial chemicals. Lancet 368, 2167–2178. 10.1016 / S0140 - 6736(06)69665 - 7

[0180] Grunwald,N.Tatiyaborworntham,C.Faustman,M.P.Richards,“Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varyinghistidine content and hemin affinity”,Food Chemistry,227(2017),pp.289-297,10.1016 / j.foodchem.2017.01.035Wang,J.;Zhang,H.;Xie,J.;Yu,W.;Sun,Y.(2022).Effects of Frozen Storage Temperature on Water-Holding Capacity andPhysicochemical Properties of Muscles in Different Parts of BluefinTuna.Foods 2022,11,2315.https: / / doi.org / 10.3390 / foods11152315Kher S.,De JongeJ.,Wentholt M.,Deliza R.,de Andrade J.,Cnossen H.,et al.(2011).Consumerperceptions of risks of chemical and microbiological contaminants associatedwith food chains:a cross-national study.Int.J.Consum.Stud.37,73–83.10.1111 / j.1470-6431.2011.01054.xKhan et al.,2008;Khan S.,Cao Q.,Zheng Y.,Huang Y.,ZhuY.(2008).Health risks of heavy metals in contaminated soils and food cropsirrigated with wastewater in Beijing,China.Environ.Pollut.152,686–692.10.1016 / j.envpol.2007.06.056

[0181] Schantz et al.,2004 Schantz S.,Gardiner J.,Gasior D.,McCaffrey R.,Sweeney A.,HumphreyH.(2004).Much ado about something:the weight of evidencefor PCB effects onneuropsychological function.Psychol.Schools 41,669–679.10.1002 / pits.20008

[0182] Shelf Life of Foods:Guidelines for Its Determination and Prediction,1993;

[0183] Singh,Benjakul,Zhou,Zhang,&Deng,2021-Effect of squid penchitooligosaccharide andepigallocatechin gallate on discoloration and shelf-life of yellowfin tuna slices duringrefrigerated storage.Food Chemistry,351(10)(2021),p.129296,10.1016 / j.foodchem.2021.129296

[0184] Tseng et.al.,2021 Bluefin tuna reveal global patterns of mercurypollution and bioavailabilityin the world's oceans,Proceedings of theNational Academy of Sciences,September 2021DOI:10.1073 / pnas.2111205118;

[0185] Ying Bu et al.,2022; Changes in quality characteristics of southernbluefin tuna(Thunnusmaccoyii)during refrigerated storage and theircorrelation with color stability,LWT,Volume154,15 January 2022,112715.

Claims

1. An edible composition comprising a homogeneous mixture of in vitro cultured fish cells, wherein each of said cells has a diameter of less than 20 μm to 200 μm and is substantially free of one or more contaminants.

2. The edible composition according to claim 1, wherein each of said cells has a diameter of less than 20 μm and is substantially free of one or more contaminants.

3. The edible composition according to claim 1, wherein each of said cells has a diameter of less than 200 μm and is substantially free of one or more contaminants.

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

5. The edible composition according to 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. The edible composition according to any one of the preceding claims, wherein the composition comprises one, two, or three cell types.

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

8. The edible composition according to any one of the preceding claims, wherein the composition is in the form of a viscous paste or powder.

9. The edible composition according to any one of the preceding claims, wherein the composition is frozen or lyophilized.

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

11. The edible composition according to any one of the preceding claims, wherein the composition contains less than 0.1 parts per million [ppm] of the one or more contaminants.

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

13. An edible composition comprising an aggregate of non-fibrous in vitro cultured fish cells, wherein the cells are in a layered structure and are substantially free of contaminants, and each layer comprises a homogeneous mixture of cells.

14. The edible composition according to any one of claims 1, 2, and 3, wherein the composition comprises one cell type.

15. The edible composition according to any one of claims 1, 2, and 3, wherein the composition comprises 1 - 3 cell types.

16. An edible composition comprising a homogeneous mixture of in vitro cultured fish cells derived from the genus Thunnus, wherein each of said cells has a diameter of less than 20 μm to 200 μm and is substantially free of one or more contaminants.

17. An edible composition comprising a homogeneous mixture of in vitro cultured fish cells derived from Thunnus thynnus, wherein each of said cells has a diameter of less than about 20 μm to about 200 μm and is substantially free of one or more contaminants.

18. An edible composition comprising a homogeneous mixture of in vitro cultured fish cells derived from Thunnus thynnus, wherein each of said cells has a diameter of less than 20 μm and is substantially free of one or more contaminants.

19. The edible composition according to claim 16, wherein the tuna comprises belly (Toro) cuts.

20. The edible composition according to claim 16, wherein the tuna comprises loin (akami) cuts.

21. The edible composition according to claim 16, which comprises a color profile having L 46.56 - 70.59, a* 12.21 - 38.76 and b* 1.24 - 61.

68.

22. The edible composition according to claim 16, which comprises a colorant concentration of 0.001% to 5% to achieve ranges of L*, a* and b* values of 33.0 - 62.8, 4.77 - 21.9 and 4.72 - 15.4, respectively.

23. The edible composition according to claim 16, wherein the edible composition comprises a refrigerated shelf life of 4 - 14 days.

24. The edible composition according to claim 16, wherein the edible composition comprises a frozen shelf life of 90 - 120 days.

25. The edible composition according to claim 16, wherein the edible composition is a single cell consistency product.

26. The edible composition according to claim 24, wherein the single cell consistency product comprises a single cell type or multiple cell types.

27. The edible composition according to claim 24, wherein the product is marbled.

28. An edible composition, which comprises a collection of non - fibrous in vitro cultured fish cells derived from bluefin tuna, wherein the cells are in a layered structure and are substantially free of contaminants, and each layer comprises a homogeneous mixture of cells.

29. The edible composition according to claim 16, wherein the edible composition comprises a longer shelf life than traditional bluefin tuna.

30. The edible composition according to claim 23, wherein the edible composition comprises a longer shelf life than traditional bluefin tuna.

Images