Food comprising fish myoglobin

Abstract

The present invention relates to a fish or meat substitute or a pet food, comprising a myoglobin from or derived from eastern Pacific bonito, sword fish or Atlantic blue marlin.

Description

[0001] FOOD COMPRISING FISH MYOGLOBIN

[0002] Field

[0003] The present invention relates to a fish or meat substitute or a pet food, comprising a myoglobin from or derived from eastern Pacific bonito, sword fish or Atlantic blue marlin.

[0004] Background

[0005] The consumption of meat and fish products raises several sustainability and animal welfare concerns. Hence, there is an ongoing need for alternatives that address these issues, while maintaining the nutritional benefits of meat and fish.

[0006] Description

[0007] Fish or meat substitute

[0008] In an aspect there is provided a fish or meat substitute comprising a myoglobin from or derived from salmon, preferably Atlantic salmon (Salmo salar), eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans), more preferably eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans). Such a fish or meat substitute may be called a fish or meat substitute of or according to the invention. Unless explicitly mentioned otherwise, a fish or meat substitute refers to a fish or meat substitute of the invention herein. In the context of this application, the common names eastern Pacific bonito and blue bonito are used interchangeably.

[0009] A fish substitute is per definition not fish, not natural fish, not genuine fish. A fish substitute may be considered as a non-naturally occurring food or edible product. It means that within the context of this invention, a fish substitute is not fish from or derived from a fish, particularly from salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, preferably from eastern Pacific bonito, sword fish or Atlantic blue marlin. A fish substitute is synonymous of a fish replica or a fish analogue product or a fish-like product.

[0010] A meat substitute is per definition not meat, not natural meat, not genuine meat. A meat substitute may be considered as a non-naturally occurring food or edible product. It means that within the context of this invention, a meat substitute is not meat from or derived from an animal. A meat substitute is synonymous of a meat replica or a meat analogue product or a meat-like product.

[0011] A fish or meat substitute may have the same aspect (such as pattern, lettering), form, structure, composition (such as similar fat, protein and / or heme iron content), texture, color, flavor, palatability, aroma and / or appearance as fish or meat, respectively. Alternatively, it might have an aspect, form, structure, texture, color, flavor, palatability, aroma and / or appearance which is distinct from the one of fish or meat, respectively. In this application, any aspect such as form, structure, composition (such as similar fat, protein and / or heme iron content), texture, color, flavor, palatability, aroma, appearance and / or nutritional value preferably relates to the perception by a human or a pet.

[0012] Fish or meat substitutes may be formulated as a liquid, solid, semi-solid food product. Suitable examples of liquid food product include soup. Suitable examples of solid food products include a cell-based fish or meat or cultured fish or meat or plant-based fish or meat. Such plant-based fish or meat may comprise plant proteins such as soy proteins. In addition they comprise a myoglobin as defined herein. In an embodiment, this myoglobin is the sole source of heme-containing protein. Suitable examples of snacks include a protein bar or a protein shake.

[0013] In an embodiment, a fish or meat substitute of the invention is expected to mimic the aspect (such as pattern, lettering), form, structure, composition (such as similar fat, protein and / or heme iron content), flavor, palatability, texture, color, aroma, appearance and / or nutritional value of fish or meat. This is typically the case when the fish or meat substitute is a cell-based fish or meat or cultured fish or meat or plant-based fish or meat. This is mostly due to the presence of the myoglobin as described herein. The fish or meat substitute may not have all drawbacks of fish or meat. For example, the fish or meat substitute is healthier than fish or meat. Moreover, the impact of the fish or meat substitute on the environment (long term direct / indirect effect on climate change) is expected to be less detrimental than the known impact of fish or meat.

[0014] In an embodiment, a fish or meat substitute of the invention is expected to mimic the aspect (such as pattern, lettering), form, structure, composition (such as similar fat, protein and / or heme iron content), flavor, palatability, texture, color, aroma and / or appearance of fish or meat. This is typically the case when the fish or meat substitute is a cell-based fish or meat or cultured fish or meat or plant-based fish or meat. This is mostly due to the presence of the myoglobin as described herein. The fish or meat substitute may not have all drawbacks of fish or meat. For example, the fish or meat substitute is healthier than fish or meat. Moreover, the impact of the fish or meat substitute on the environment (long term direct / indirect effect on climate change) is expected to be less detrimental than the known impact of fish or meat.

[0015] In an embodiment, a fish or meat substitute of the invention is expected to mimic the composition (such as similar fat, protein and / or heme iron content), flavor, color and / or aroma of fish or meat without having all its drawbacks. This is mostly due to the presence of the myoglobin as described herein.

[0016] In an embodiment, a fish or meat substitute of the invention is expected to mimic the flavor and / or aroma of fish or meat without having all its drawbacks. This is mostly due to the presence of the myoglobin as described herein.

[0017] In an embodiment, a fish or meat substitute of the invention is expected to mimic the flavor of fish or meat without having all its drawbacks. This is mostly due to the presence of the myoglobin as described herein. In an embodiment, a fish or meat substitute of the invention is expected to mimics the aroma of meat without having all its drawbacks. This is mostly due to the presence of the myoglobin as described herein.

[0018] Within the context of the invention, the fish or meat experience may be mimicked when the fish or meat substitute generates an aroma and / or a flavor compound recognizable by humans and characteristic of some odorants and / or flavor compound released when fish or meat is cooked or grilled (“cooked or grilled fish or meat substitutes”). For example, the formation of some of these odorants may be catalyzed by the iron of heme present in myoglobin. Without being bound to this theory, the formation may be due to lipid oxidation and / or Maillard reactions. This situation may be mimicked with the myoglobin of the fish or meat substitute of the invention.

[0019] In the context of this invention, an “other meat substitute” or “a meat substitute not according to the invention” is preferably a plant-based burger comprising a recombinant soy leghemoglobin (LegH), more preferably the commercially available Impossible Burger.

[0020] In an embodiment, a fish or meat substitute of the invention, preferably a grilled fish or meat substitute of the invention, has an aroma characterized by a higher concentration of oxidized lipids, lipid oxidation products, pyrazines, pyrroles, alcohols, aldehydes and / or ketones in the volatile compounds obtained from the (grilled) fish or meat substitute relative to other fish or meat substitutes (i.e. not according to the invention). Higher preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300%. Preferred pyrazines are methylpyrazine, 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, and 2-ethyl-6- methylpyrazine. A preferred pyrrole is pyrrole. Preferred lipid oxidation products are 2- methylbutanal, and 3-methylbutanal. Preferred aldehydes are methional, nonanal, octanal, decanal and dodecanal. A preferred ketone is 2-acetylthiazole and 1-hydroxy-2-butanone. A preferred alcohol is 3-penten-2-ol. It is understood that the categories of compounds mentioned in this paragraph are not mutually exclusive, e.g. lipid oxidation products may be aldehydes or ketones. Aromas from fish or meat substitutes containing different concentrations of myoglobin may be analyzed using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME).

[0021] Within the context of the invention, the fish or meat experience may be mimicked when the color of the fish meat substitute is similar with the color of fish or meat. In this context, fish is preferably red fish. The color of fish or meat is determined by the concentration of heme-containing protein and / or by its oxidation state in the fish or meat. Therefore, the myoglobin as defined herein will define the color of the fish or meat substitute.

[0022] In an embodiment, a fish or meat substitute of the invention, preferably a raw meat substitute of the invention, is expected to mimic the color of a fish or meat, preferably of raw meat. Without being bound to this theory, the color of the fish or meat substitute is mostly due to the presence of the myoglobin. Preferably, the fish or meat substitute of the invention is able to mimic the color of fish or meat to a higher degree than other fish or meat substitutes (i.e. not according to the invention). In a preferred embodiment, the color of a fish or meat substitute of the invention, preferably of a raw meat substitute of the invention, is essentially stable for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 ,

[0023] 12, 13, 14, 15, 16, 17, 18, 19 or 20 days, preferably upon storage under constant light at 4°C. Preferably, the color of the fish or meat substitute of the invention is stable for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 days longer than the time period wherein the color of meat or other fish or meat substitutes (i.e. not according to the invention) is stable, more preferably wherein a AE (color change) from 0 up to 10 is defined as stable (see further). Essentially stable for at least X days preferably means a AE (color change) of the fish or meat substitute between 0 and 0.5, 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 12.5,

[0024] 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 between day 0 and day X. Most preferably, AE of the fish or meat substitute is not more than 10 upon storage for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. The color may be assessed via absorbance measurements using spectrometry.

[0025] Within the context of the invention, the fish or meat experience may be mimicked when the composition of the fish or meat substitute is similar with fish or meat composition. The composition of the fish or meat substitute may be seen to be similar to the fish or meat composition when the same or similar components are present and optionally when the quantities in which they are present is the same or similar with the quantities present in fish or meat. For example a similar fat, protein and / or heem iron content may be present in a fish or meat substitute as in fish or meat.

[0026] These animal myoglobins when formulated into a fish or meat substitute are also expected to provide roughly the same amount of total available iron or heme iron as fish or meat. Within this context, “roughly” means at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more. Usually the amount of total bioavailable iron or heme iron in the fish or meat substitute of the invention is not more than 120% of the total bioavailable iron or heme iron in genuine fish or meat. It means these myoglobins may be considered as a unique alternative fortificant to improve the iron status of the population.

[0027] These myoglobins are also expected to provide roughly a higher amount of total bioavailable iron or heme iron than the amount of total bioavailable iron or heme iron that is provided in a plant-based fish or meat analogue product. Within this context, “higher” means at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more.

[0028] In a preferred definition, nutritional value means the amount of total available iron or heme iron. According to this preferred definition, “mimicking the nutritional value of fish or meat” should be interpreted as “provide roughly the same amount of total available iron or heme iron as fish or meat”, as outlined above. Within the context of the invention, total iron is the total amount of iron which is bioavailable in a fish or meat substitute. Heme iron is the part of iron which is transported by a protein comprising a heme domain. Heme iron is available for absorption by the human body. Such a protein is myoglobin as defined herein. Availability of iron may be assessed as described in Proulx A. K., et al (2006)., J. Agric. Food. Chem., (54), 1518-1522). Briefly the ferritin concentration of a preparation is first normalized to cell protein concentration. Subsequently, the percentages of normalized ferritin is compared to the percentage of FeSC and expressed as relative biological values (RBV).

[0029] In an embodiment, the fish or meat substitute does not comprise a leghemoglobin which has been produced by a bacterium living in symbiosis in the root nodules of a soy plant and / or comprises as sole heme-containing protein the animal myoglobin as earlier defined herein.

[0030] In an embodiment, the myoglobin disclosed herein may be the sole source of heme-containing protein present in the fish or meat substitute of the invention. It means the fish or meat substitute of the invention may comprise other proteins than the myoglobin as disclosed herein. Examples of proteins that may be present include soy proteins.

[0031] Within the context of the invention, a heme-containing protein as defined can refer to all proteins or protein subunits that are capable of covalently or noncovalently binding a heme moiety. Hemecontaining polypeptides can transport or store oxygen. Some examples of heme-containing protein include globin, hemoglobin, leghemoglobin.

[0032] In another embodiment, the fish or meat substitute does not comprise a leghemoglobin which has been produced by a bacterium living in symbiosis in the root nodules of a soy plant. In an embodiment, the fish or meat substitute does not comprise a symbiotic hemoglobin. In an embodiment, the fish or meat substitute does not comprise a leghemoglobin. In an embodiment, the fish or meat substitute does not comprise a protein which has been produced by a bacterium living in symbiosis in the root nodules of a soy plant.

[0033] In an embodiment, the fish or meat substitute does not comprise a leghemoglobin which has been produced by a bacterium living in symbiosis in the root nodules of a soy plant and comprises as sole heme-containing protein the animal myoglobin as earlier defined herein.

[0034] In an embodiment, the fish or meat substitute comprises a similar amount of animal myoglobin as the counterpart genuine fish or meat. In an embodiment such amount is ranged from 0.1 to 5 weight % animal myoglobin. In an embodiment, the amount is at least 0.1 , 0.2, 0.3, 0.4, 0.5. 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0 or more weight % animal myoglobin. It is also encompassed by the invention to have amounts of at least 10%, 20%, 30%, 40%, 50%, of the final weight of the product. The skilled person knows that depending on the fish or meat substitute the amount may vary.

[0035] It is understood that a concentration such as a weight percentage of myoglobin in the fish or meat substitute according to the invention refers to the concentration of all myoglobin comprised in the fish or meat substitute, regardless of its source. This applies to all concentrations of myoglobin mentioned herein.

[0036] In the context of a method according to the invention, it is clear the concentrations below relate to concentrations “after the addition” of the isolated myoglobin.

[0037] In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.1% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1%, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.45%, or 0.4%.

[0038] In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.15% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1%, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.45%, or 0.4%.

[0039] In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.2% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1%, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.45%, or 0.4%.

[0040] In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.3% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1%, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.45%, or 0.4%.

[0041] In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.4% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1%, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, or 0.45%. In an embodiment, the weight percentage of the myoglobin in the fish or meat substitute according to the invention is from 0.5% up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1 %, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1 %, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25%, 1.2%, 1.15%, 1.1 %, 1.05%, 1 %, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, or 0.6%.

[0042] In an embodiment, the food ingredient comprises an amount of myoglobin that may be ranged from 0.1 to 100 weight % myoglobin. In an embodiment, the amount is at least 0.1 %, 1 .0%, 1 .5%, 2.0%, 2.5%, 3.0%, 4.0%, 4.5%, 5.0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the final weight of the product. The skilled person knows that depending on the food ingredient the amount may vary.

[0043] In an embodiment, the fish or meat substitute comprises or is prepared by mixing from 10% up to 40% of a protein source, from 5% up to 30% of a lipid source, from 0.1 % up to 5% of NaCI, from 0.1 % up to 5% of a fiber source, and from 0.1 % up to 5% of a myoglobin according to the invention. In a more preferred embodiment, the fish or meat substitute comprises or is prepared by mixing from 20% up to 30% of a protein source, from 10% up to 20% of a lipid source, from 0.5% up to 2% of NaCI, from 0.5% up to 1 .5% of a fiber source, and from 0.1 % up to 3% of a myoglobin according to the invention.

[0044] Preferably, the protein source is texturized soy protein, pea protein isolate, mung bean protein, textured wheat protein, rice protein or potato protein. In this context, texturized soy protein is a defatted soy flour product, also known as texturized vegetable protein (TVP) or soy fish or meat, as is clear to the skilled person.

[0045] Preferably, the lipid source is sunflower oil, coconut oil, canola oil or cocoa butter.

[0046] Preferably, the fiber source is methylcellulose or potato starch.

[0047] In a more preferred embodiment, the fish or meat substitute comprises or is prepared by mixing from 20% up to 30% of texturized soy protein, from 10% up to 20% of sunflower oil, from 0.5% up to 2% of NaCI, from 0.5% up to 1.5% of methylcellulose, and from 0.1 % up to 10% of a myoglobin according to the invention. Even more preferably, the weight percentage of the myoglobin in the fish or meat substitute according to the more preferred embodiment is from 0.1 % up to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1 %, 2.05%, 2%, 1.95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1 .65%, 1.6%, 1.55%, 1.5%, 1.45%, 1 .4%, 1.35%, 1.3%, 1.25%, 1.2%, 1 .15%, 1.1 %, 1.05%, 1 %, 0.95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, or 0.5%. In an embodiment, the fish or meat substitute comprises or is prepared by mixing from 20% up to 30% texturized soy protein, from 10% up to 20% sunflower oil, from 1 % up to 2% NaCI, from 0.5% up to 1 .5% methylcellulose, from 50% up to 70% water and from 0.2% up to 0.8% myoglobin.

[0048] In a preferred embodiment, the fish or meat substitute comprises or is prepared by mixing from 23% up to 27% texturized soy protein, from 13% up to 17% sunflower oil, from 1 .25% up to 1 .75% NaCI, from 0.75% up to 1 .25% methylcellulose, from 55% up to 60% water and from 0.45% up to 0.55% myoglobin.

[0049] In an embodiment, the fish or meat substitute comprises or is prepared by mixing 25% texturized soy protein, 15% sunflower oil, 1 .5% NaCI, 1 .0% methylcellulose, 57% water and 0.5% myoglobin, wherein these weight percentages are rounded to two significant digits.

[0050] In an embodiment, the fish or meat substitute comprises or is prepared by mixing from 15% up to 35% texturized soy protein, from 10% up to 20% sunflower oil, from 0.5% up to 2.5% NaCI, from 0.5% up to 1 .5% methylcellulose, from 50% up to 70% water and from 0.5% up to 1 .5% myoglobin. In a preferred embodiment, the fish or meat substitute comprises or is prepared by mixing from 22% up to 27% texturized soy protein, from 13% up to 17% sunflower oil, from 1 .25% up to 1 .75% NaCI, from 0.75% up to 1 .25% methylcellulose, from 50% up to 60% water and from 0.75% up to 1 .25% myoglobin.

[0051] In an embodiment, the fish or meat substitute comprises or is prepared by mixing 25% texturized soy protein, 15% sunflower oil, 1.5% NaCI, 1.0% methylcellulose, 57% water and 1.0% myoglobin, wherein these weight percentages are rounded to two significant digits.

[0052] In the fish or meat substitutes above, % refers to a weight percentage. Insofar as the weight percentages describing the composition of a fish or meat substitute do not add up to 100%, the addition of water to a corresponding percentage is assumed.

[0053] In an embodiment, the fish or meat substitute comprises a similar amount of bioavailable iron or heme iron as the counterpart genuine fish or meat. In an embodiment such amount is ranged from 0.3 to 20 mg bioavailable iron or heme iron per 100 gram fish or meat substitute. In an embodiment such amount is ranged from 0.1 mg up to 16.5 mg, 16.17 mg, 15.84 mg, 15.51 mg, 15.18 mg, 14.85 mg, 14.52 mg, 14.19 mg, 13.86 mg, 13.53 mg, 13.2 mg, 12.87 mg, 12.54 mg, 12.21 mg, 11.88 mg, 11 .55 mg, 1 1.22 mg, 10.89 mg, 10.56 mg, 10.23 mg, 9.9 mg, 9.735 mg, 9.57 mg, 9.405 mg, 9.24 mg, 9.075 mg, 8.91 mg, 8.745 mg, 8.58 mg, 8.415 mg, 8.25 mg, 8.085 mg, 7.92 mg, 7.755 mg, 7.59 mg, 7.425 mg, 7.26 mg, 7.095 mg, 6.93 mg, 6.765 mg, 6.6 mg, 6.435 mg, 6.27 mg, 6.105 mg, 5.94 mg, 5.775 mg, 5.61 mg, 5.445 mg, 5.28 mg, 5.115 mg, 4.95 mg, 4.785 mg, 4.62 mg, 4.455 mg, 4.29 mg, 4.125 mg, 3.96 mg, 3.795 mg, 3.63 mg, 3.465 mg, 3.3 mg, 3.135 mg, 2.97 mg, 2.805 mg, 2.64 mg, 2.475 mg, 2.31 mg, 2.145 mg, 1.98 mg, 1.815 mg or 1.65 mg bioavailable iron or heme iron per 100 gram fish or meat substitute. In another embodiment such amount is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1 .6, 1 .7, 1 .8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 ,

[0054] 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,

[0055] 9.8, 9.9, 10, 10.1 , 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11 , 11 .1 , 11.2, 11 .3, 11 .4, 11.5, 11 .6, 11 .7, 11 .8, 11 .9, 12, 12.1 , 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1 , 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1 , 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1 ,

[0056] 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1 , 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1 , 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1 , 18.2, 18.3, 18.4, 18.5, 18.6, 18.7,

[0057] 18.8, 18.9, 19, 19.1 , 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, or 20 mg bioavailable iron or heme iron per 100 gram fish or meat substitute.

[0058] In an embodiment, the fish or meat substitute does not comprise one or more milk proteins selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p- lactoglobulin, lactoferrin and transferrin, particularly wherein said milk protein is from a woolly mammoth. In another embodiment, the fish or meat substitute does not comprise one or more milk proteins selected from the group consisting of p-casein, K-casein, a-S1 -casein, a-S2-casein, a- lactalbumin, p-lactoglobulin, lactoferrin and transferrin, particularly wherein said milk protein is from a mammoth.

[0059] In an embodiment, the fish or meat substitute does not comprise one or more milk proteins selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p- lactoglobulin, lactoferrin and transferrin, particularly wherein said milk protein has not been produced by a mammal or a mammalian cell.

[0060] In a more preferred embodiment, the fish or meat substitute does not comprise a milk protein such as p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin or transferrin, particularly wherein said milk protein is from a woolly mammoth. In another more preferred embodiment, the fish or meat substitute does not comprise a milk protein such as p- casein, K-casein, a-S1 -casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin or transferrin, particularly wherein said milk protein is from a mammoth.

[0061] In another more preferred embodiment, the fish or meat substitute does not comprise a milk protein such as p-casein, K-casein, a-S1 -casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin or transferrin, particularly wherein said milk protein has not been produced by a mammal or a mammalian cell.

[0062] In an embodiment, the fish or meat substitute does not comprise a K-casein from a woolly mammoth. In an embodiment, the fish or meat substitute does not comprise a p-casein from a woolly mammoth. Pet food

[0063] In an aspect there is provided a pet food comprising a myoglobin from or derived from salmon, preferably Atlantic salmon (Salmo salar), eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans), more preferably eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans). Such a pet food may be called a pet food of or according to the invention. Unless explicitly mentioned otherwise, a pet food refers to a fish or meat substitute of the invention herein.

[0064] In an embodiment, the pet is a vertebrate. In an embodiment, the pet is a mammal, a bird, a fish, an amphibian, or a reptile. In an embodiment, the pet is a mammal. Mammals are a group of vertebrates constituting the class mammalia, characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or hair, and three middle ear bones. In an embodiment, the pet is a carnivore or an omnivore, preferably a mammal. In a preferred embodiment, the pet may be a dog, a cat, a ferret, a fox, a bear, a wolf, a jaguar, a lion, a tiger, a snake, a piranha, or an alligator. In a preferred embodiment, the pet may be a dog or a cat.

[0065] In an embodiment, the pet food is a dry pet food, a semi-moist pet food, a wet pet food, or any combination of these types of pet food. A pet food may be formulated as a liquid, solid, or semisolid pet food. By "dry pet food" is meant one that has a moisture content less than 15 percent by weight. By "semi-moist pet food" is meant one that has a moisture content between 55 to 65 percent by weight. By " wet type food" is meant one that has a moisture content between 65 to 85 percent by weight. Hereinafter, all percentages referred to are understood to be by weight unless specified otherwise and are based upon the weight of the final pet food product. Suitable examples of solid pet foods include kibbles, snacks, cell-based fish and meat and fish or meat substitutes, cultured fish and meat and plant-based fish and meat substitutes. Suitable examples of snacks include a protein bar, a dental snack, meat cubes or chew sticks. Such plant-based meats may comprise plant proteins such as soy proteins. Suitable examples of semi-moist and wet type pet foods include pate or loaf, chunks in gravy or jelly or soup. In an embodiment, the pet food is a pate or loaf, or chunks in gravy.

[0066] In an embodiment, the pet food is a fish or meat substitute (according to the invention). Characteristics of fish or meat substitutes according to the invention are described elsewhere herein and can be applied mutatis mutandis to the pet food according to this embodiment. Preferably, the myoglobin in the meat substitute is cross-linked with another substance, for example a pea vicilin protein.

[0067] In the context of pet food, particularly pet foods being fish or meat substitutes, it is understood that aspects such as form, structure, composition (such as similar fat, protein and / or heme iron content), texture, color, flavor, palatability, aroma, appearance and / or nutritional value preferably relate to the perception by a pet.

[0068] In an embodiment, the pet food is more palatable than a corresponding pet food not comprising a myoglobin from or derived from salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, preferably from eastern Pacific bonito, sword fish or Atlantic blue marlin. The degree of acceptance, the general liking or preference of one pet food over another, or the palatability of a pet food may be assessed by a preference / acceptance test. Palatability of pet foods is typically measured using a single-bowl or a two-bowl test or an operant lever-press test. For example, in a preference test, pets are offered a control meal and the test meal in a controlled environment. Experimenters record the rate at which the animals choose the test meal over the control meal. Larger tests can be conducted in a home setting by pet owners who are asked to record "food preference" behaviors such as time spent sniffing the food, licking lips or nose, ear or tail flicking, etc.

[0069] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% higher than the average concentration of heme-proteins in a corresponding pet food product not comprising a myoglobin from or derived from salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, preferably eastern Pacific bonito, sword fish or Atlantic blue marlin.

[0070] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 0.005%, 0.01 %, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1 %, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 6%, 7%, 8%, 9%, 10%, preferably lower than 10%, more preferably lower than 5%.

[0071] In an embodiment, the (total) weight concentration of myoglobins in the pet food is at least 0.005%, 0.01 %, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1 %, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 6%, 7%, 8%, 9%, 10%, preferably lower than 10%, more preferably lower than 5%.

[0072] In an embodiment, the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0073] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 0.005%, 0.01 %, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1 %, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 6%, 7%, 8%, 9%, 10%, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 90%.

[0074] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 0.05%, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0075] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 0.1 %, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0076] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 0.5%, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0077] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 1 .0%, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0078] In an embodiment, the (total) weight concentration of heme-proteins in the pet food is at least 3.0%, and the weight fraction of myoglobins in the heme-proteins in the pet food is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0079] Method

[0080] In an aspect there is provided a method of preparing a fish or meat substitute according to the invention, or a pet food according to the invention, comprising adding an isolated myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin, to an initial fish or meat substitute or to an initial pet food, respectively. Such a method may be called a method of or according to the invention. Wherever a fish or meat substitute or a pet food is mentioned herein, it is understood that a corresponding method of the invention is disclosed as well.

[0081] In an embodiment, the invention provides a method of preparing a fish substitute comprising adding an isolated myoglobin from eastern Pacific bonito, sword fish or Atlantic blue marlin to an initial fish substitute.

[0082] In an embodiment, an isolated myoglobin is a myoglobin which is not comprised in a cell or a tissue before the addition. In other words, the isolated myoglobin is added as a “separate” (or isolated) protein which is not comprised in a cell or a tissue. Thus, isolated in this context does not refer to the origin of the myoglobin (i.e. isolated from a source such as cell or a tissue), but to the fact that a separate protein is added. It is of course understood that the isolated myoglobin may become part of a cell or a tissue once it has been added to the fish or meat substitute or the pet food.

[0083] In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of hemeproteins in the fish or meat substitute or the pet food originate from the addition of the isolated myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the fish or meat substitute or the pet food originate from the addition of the isolated myoglobin (per weight).

[0084] In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the fish or meat substitute or the pet food originate from the addition of the isolated myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the fish or meat substitute or the pet food originate from the addition of the isolated myoglobin (per weight).

[0085] In an embodiment, the isolated myoglobin is added as part of a composition, wherein the weight concentration of the isolated myoglobin in the composition at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %. In this context, weight concentration may be expressed as a mass of myoglobin per total volume or mass of the composition.

[0086] In an embodiment, the isolated myoglobin is added as part of a composition, wherein the weight fraction of the isolated myoglobin in the protein fraction comprised in the composition is at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 1 1 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 %, at least 65%, at least 70%, 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. In this context, weight fraction is a dimensionless number which may be interpreted as a mass of myoglobin per mass of protein.

[0087] In an embodiment, the concentration of myoglobin in the initial fish or meat substitute or the initial pet food is less than 0.10 wt%, 0.09 wt%, 0.08 wt%, 0.07 wt%, 0.06 wt%, 0.05 wt%, 0.04 wt%, 0.03 wt%, 0.02 wt%, 0.01 wt%, 0.009 wt%, 0.008 wt%, 0.007 wt%, 0.006 wt%, 0.005 wt%, 0.004 wt%, 0.003 wt%, 0.002 wt%, 0.001 wt%. In an embodiment, it is preferable that the initial fish or meat substitute or the initial pet food substitute does not comprise myoglobin.

[0088] In an embodiment, the concentration of the myoglobin in the fish or meat substitute or the initial pet food substitute after the addition is in the range 0.05-0.5 wt.%, and the concentration of myoglobin in the initial fish or meat substitute or the initial pet food substitute is less than 0.01 wt.%.

[0089] In an embodiment, the isolated myoglobin is in the form of a liquid formulation (i.e. the composition may be a liquid). The amount of the isolated myoglobin needed to be added to the fish or meat substitute or the pet food may be calculated or estimated based on the concentration of myoglobin in the fish or meat substitute or the pet food before the addition and the desired final concentration of myoglobins in the fish or meat substitute or the pet food. In this context, the fish or meat substitute or the pet food before addition of the isolated myoglobin preferably contains less than 0.1 %, less than 0.2 %, less than 0.3 %, less than 0.4 %, less than 0.5%, less than 0.6 %, less than 0.7%, less than 0.8 %, less than 0.9 %, less than 1.0 % heme-proteins per weight basis.

[0090] In an embodiment, the isolated myoglobin is in the form of powder (i.e. the composition may be a powder). The amount of the isolated myoglobin needed to be added to the fish or meat substitute or the pet food may be calculated or estimated based on the concentration of myoglobin in the fish or meat substitute or the pet food before the addition and the desired final concentration of myoglobins in the fish or meat substitute or the pet food. In this context, the fish or meat substitute or the pet food before addition of the isolated myoglobin preferably contains less than 0.1 %, less than 0.2 %, less than 0.3 %, less than 0.4 %, less than 0.5%, less than 0.6 %, less than 0.7%, less than 0.8 %, less than 0.9 %, less than 1.0 % heme-proteins per weight basis. A tool, such as a container, a cup, a spoon, a blender, a mixer, may be used to add the isolated myoglobin. The isolated myoglobin may be added directly to the fish or meat substitute or the pet food, or may be dissolved in an editable solvent, prior to be added to the fish or meat substitute or the pet food.

[0091] In an embodiment, the isolated myoglobin is in the form of solid shapes, wherein the solid shaped myoglobin has a pre-determined weight and / or volume. The amount of the isolated myoglobin needed to be added to the fish or meat substitute or the pet food may be calculated or estimated based on the concentration of the isolated myoglobin, and / or the volume of the solid-shaped isolated myoglobin, the total weight of the fish or meat substitute or the pet food and the desired final amount of myoglobins in the fish or meat substitute or the pet food. In this context, the fish or meat substitute or the pet food before addition of the isolated myoglobin preferably contains less than 0.1 %, less than 0.2 %, less than 0.3 %, less than 0.4 %, less than 0.5%, less than 0.6 %, less than 0.7%, less than 0.8 %, less than 0.9 %, less than 1.0 % heme-proteins per weight basis. A tool, such as a container, a cup, a spoon, a blender, a mixer, may be used to add the isolated myoglobin. The isolated myoglobin may be added directly to the fish or meat substitute or the pet food, or may be dissolved in a food grade solvent, prior to be added to the fish or meat substitute or the pet food.

[0092] In an embodiment, the isolated myoglobin is a recombinant myoglobin produced from fermentation by a microorganism which has been genetically modified to express said recombinant myoglobin. The microorganism may be referred to as the host cell herein.

[0093] A microorganism may be a prokaryote, a eukaryote or a filamentous fungus. A prokaryote may be a bacterium. The bacterium may be a Gram positive / Gram negative bacterium sleeted from the following list: Absidia, Achromobacter, Acinetobacter, Aeribacillus, Aneurinibacillus, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter, Arzoarcus, Azomonas, Azospirillum, Azotobacter, Bacillus, Beijerinckia, Bradyrhizobium, Brevibacills, Burkholderia, Byssochlamys, Citrobacter, Clostridium, Comamonas, Cupriavidus, Corynebacterium, Deinococcus, Escherichia, Enterobacter, Flavobacterium, Fusobacterium, Gossypium, Klebsiella, Lactobacillus, Listeria, Megasphaera, Micrococcus, Mycobacterium, Norcadia, Porphyromonas, Propionibacterium, Pseudomonas, Ralstonia, Rhizobium, Rhodopseudomonas, Rhodospirillum, Rodococcus, Roseburia, Shewanella, Streptomycetes, Xanthomonas, Xylella, Yersinia, Treponema, Vibrio, Streptococcus, Lactococcus, Zymomonas, Staphylococcus, Salmonella, Sphingomonas, Sphingobium, Novosphingobium, Brucella and Microscilla. Preferred bacteria include Aeribacillus pallidus, Aneurinibacillus terranovensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus halodurans, Bacillus pumilus, Brevibacillus thermoruber, Brevibacillus panacihumi, Cupriavidus basilensis, G. Iraustophilus, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, Escherichia coll, Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti, Sphingobium sp., Novosphingobium sp., Sphingomonas henshuiensis, and Rhizobium radiobacter. A preferred bacterium is Escherichia coli. Preferred Escherichia coli strains include: 58, 679, WG1 , DH5a, TG1 , TOP10, K12, BL21 , BL21 DE3, XL1-Blue, XL10-Gold, TB1 , REG-12, W945, HB101 , DH1 , DP50, AB284, JC9387, AG1 , C600, Cavalli Hfr, Y10.

[0094] A eukaryote may be a yeast or a filamentous fungus. Preferred yeasts include Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Cryptococcus, Debaromyces, Saccharomycecopsis, Saccharomycodes, Wickerhamia, Debayomyces, Hanseniaspora, Ogataea, Kuraishia, Komagataella, Metschnikowia, Williopsis, Nakazawaea, Torulaspora, Bullera, Rhodotorula, Sporobolomyces. Within yeasts, the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha (also known as Ogataea henricii), Yarrowia lipolytica, Candida tropicalis and Pichia pastoris (also known as Komagataella phaffii) are preferred. Preferred Pichia strains are selected from the following list: Bg09, Bg10, Bg11 , Bg12 (exemplified), Bg20, Bg21 , Bg22, Bg23, Bg24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430, X-33, GS115, KM71 , SMD1168, SMD1165, MC100-3, most preferred Bg10 and derivatives. Preferred Saccharomyces strains are selected from the following list: S288C, CEN.PK family, CBS 2354, ATCC 2360, ATCC 4098, ATCC 4124, ATCC 4126, ATCC 4127, ATCC 4921 , ATCC 7754, ATCC 9763, ATCC 20598, ATCC 24855, ATCC 24858, ATCC 24860, ATCC 26422, ATCC 46523, ATCC 56069, ATCC 60222, ATCC 60223, ATCC 60493, ATCC 66348, ATCC 66349, ATCC 96581. A preferred yeast is a Pichia strain, more preferably Pichia pastoris.

[0095] A filamentous fungus may be selected from the following list including: Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallinastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Ustilago and Trichoderma. Preferred filamentous fungus are selected from the following list: Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus vadensis, Penicillium chrysogenum, Penicillium citrinum, Penicillium rubens, Penicillium oxalicum, Penicillium subrubescens, Rasamsonia emersonii, Talaromyces emersonii, Acremonium chrysogenum, Trichoderma reesei, Aspergillus sojae, and Chrysosporium lucknowense. Preferred strains of filamentous fungus are selected from the following list: Aspergillus niger CBS 513.88, N593, CBS 120.49, N402, ATCC 1015 Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , ATCC 9576, ATCC 14488-14491 , ATCC 11601 , ATCC12892, Aspergillus vadensis CBS 113365, CBS 102787, IMI 142717, IBT 24658, CBS 113226, Penicillium chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Wisconsin 54-1255, Penicillium subrubescens CBS 132785, FBCC 1632, Talaromyces emersonii CBS 393.64, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC56765 or ATCC 26921 , Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006. In a preferred embodiment, Aspergillus is used as a filamentous fungus. More preferably, an Aspergillus niger strain is used. In an embodiment, the microorganism used in the fermentation may be a bacterium, a yeast, a filamentous fungus or a cultured mammalian cell line, preferably Escherichia coli or Saccharomyces cerevisiae. In this context, single, isolated, cultured mammalian cells may be considered as microorganisms. In a further embodiment, the microorganism may be a bacterium, a yeast or a filamentous fungus. The microorganisms in the context of this invention are useful for the production of a myoglobin. Accordingly, in a further aspect, the invention provides a method for the production of a myoglobin as defined herein, comprising culturing the microorganisms in a suitable medium and optionally recovering the microorganism and / or myoglobin. Optionally, the produced myoglobin does not comprise a signal peptide as defined elsewhere herein.

[0096] Suitable cell culturing methods for use in a method according to the invention are known to the skilled person and are discussed, for example, in van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design, CRC Press, NY, 1991 . Such methods include, but are not limited to, submerged fermentation in liquid media, surface fermentation on liquid media and solid-state fermentations. Cell culturing may, for example, be performed by cultivation in micro-titer plates, shake-flasks, small-scale benchtop bioreactors, medium-scale bioreactors and / or large-scale bioreactors in a laboratory and / or an industrial setting. Suitable cell culturing modes include, but are not limited to, continuous, batch and / or fed-batch fermentation as well as their combinations. In an embodiment, cell culturing is performed using continuous fermentation. In a preferred embodiment, cell culturing is performed using batch fermentation. In a more preferred embodiment, cell culturing is performed using fed batch fermentation.

[0097] In the context of the invention, "culture medium”, hereinafter alternately referred to as "growth medium”, can be interpreted to encompass cases wherein the cultured cells are absent as well as cases wherein the cultured cells are present in the culture medium. "Culture broth” refers to the culture medium wherein the cultured cells are present. "Culture supernatant” refers to the culture medium wherein the cultured cells are absent. "Cell-free extract” refers to a cell lysate not comprising the cellular debris. Cell culturing as part of the process of the invention can be performed under conditions conducive to the production of the introduced myoglobins, which are known to the skilled person. Such conditions depend not only on the chemical composition of the culture medium but also on other process parameters including culture duration, temperature, O2 levels in the culture broth and / or headspace, CO2 levels in the culture broth and / or headspace, pH, ionic strength, agitation speed, hydrostatic pressure and the like. Cell culturing can take place using a culture medium comprising suitable nutrients, such as carbon and nitrogen sources and additional compounds such as inorganic salts and vitamins, using procedures known in the art (see, e. g. Bennett, W. and Lasure, L., 1stedition, More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable growth media are available from commercial suppliers or may be prepared using published compositions that are suitable for the respective hosts (e.g. in catalogues of the Centraalbureau Voor Schimmelcultures collection (CBS) or of the American Type Culture Collection (ATCC)). The exact composition of the growth medium and the values of culture process parameters are not critical features of the invention. Any growth medium composition may be contemplated, as long as it allows for growth of the host cell and production of the introduced myoglobins. The growth medium will typically comprise a carbon source to be used for the growth of the cultured cell. The skilled person understands that suitable carbon sources may be added externally to the growth medium or may already be present in said medium. Carbon sources may be present or added individually or in mixtures of multiple carbon sources. Examples of suitable carbon sources known in the art include simple sugars such as glucose, maltose, sucrose, xylose, arabinose, complex sugars such as maltodextrins, hydrolysed starch, starch, molasses, and second-generation feedstocks. Second- generation feedstocks can be particularly attractive because of their lower carbon footprint. Second- generation feedstocks will typically comprise lignocellulosic material. Such material includes any lignocellulose and / or hemicellulose-based materials. Such material may be sourced from agricultural, industrial or municipal, preferably agricultural, waste streams. Examples of suitable materials include (agricultural) biomass, commercial organic matter, municipal solid waste, virgin biomass such as waste paper and garden waste, or non-virgin biomass. General forms of biomass include trees, shrubs and pastures, wheat, wheat straw, sugarcane bagasse, switchgrass, Japanese pampas grass, corn, corn stover, corn cob, canola stalk, soybean stalk, sweet corn, corn kernels, products and by-products from cereal milling (including wet milling and dry milling), such as corn, wheat, and barley, often referred to as “bran or fiber”, and municipal solids. Biomass can also be grassy materials, agricultural residues, forestry residues, municipal solid waste, waste paper, and pulp and paper mill residues. Agricultural biomass includes branches, shrubs, tows, corn and corn straw, energy crops, forests, fruits, flowers, cereals, pastures, herbaceous crops, leaves, bark, needles, logs, roots, young trees, shortterm rotating woody crops, shrubs, switch herbs, trees, vegetables, fruits, vines, sugar beet pulp, wheat middlings, oat hulls, and hard and soft timber (not including toxic wood), and organic waste materials resulting from agricultural processes including agriculture and forestry activities, particularly forestry wood waste. Agricultural biomass may be any of the foregoing alone, or any combination or mixture thereof. Carbon sources such as organic acids, aldehydes, ketones, esters and alcohols may also be contemplated. The use of growth media comprising combinations of multiple different carbon sources may also be contemplated in the process of the invention. Such media could, as a non-limiting example, combine more oxidized carbon sources such as organic acids with more reduced carbon sources such as alcohols. Examples of suitable nitrogen sources known in the art include soy bean meal, corn steep liquor, yeast extract, whey protein, egg protein, casein hydrolysate, urea, ammonia, ammonium salts and nitrate salts. Examples of additional suitable compounds known in the art include phosphate, sulphate, metals such as magnesium, trace elements and vitamins. The exact growth medium requirements will vary based on the host cell, e.g. between yeasts, bacteria and filamentous fungi, said requirements will be known to the skilled person. Accordingly, the growth medium may be a complete (rich) medium or a minimal medium, i.e. a medium comprising only the absolutely necessary components for growth depending on the cultured host cell. Similar to the composition of the growth medium, process parameters can be assigned any value, as long as they allow for growth of the host cell and production of the introduced myoglobins. Typically, said values will differ based on the host cell that is being cultured and will be known to the skilled person. Preferably, the process according to the invention is an oxygen-limited or aerobic process, meaning that cell culturing is performed under oxygen-limited or aerobic conditions, more preferably the process is oxygen-limited. Oxygen-limited conditions, also known as a micro-aerobic conditions, are culture conditions in which the oxygen consumption is limited by the availability of oxygen. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing / mass transfer properties of the fermentation equipment used. Preferably, under oxygen-limited conditions in a liquid culture, the rate of oxygen consumption is at least about 5.5 mmol / L / h, more preferably at least about 6 mmol / L / h and even more preferably at least about 7 mmol / L / h. Aerobic conditions are culture conditions in which the oxygen consumption is not limited by the availability of oxygen.

[0098] Cell culturing may be performed at a temperature value that is optimal for the cell, typically at a temperature range of 16-42 °C. In some embodiments, the temperature ranges between 20-40 °C, more preferably between 25-38 °C, most preferably between 28-36 °C. In some most preferred embodiments, a temperature value of about 30 or 36 °C is used.

[0099] Cell culturing may be performed at a pH value that is optimal for the cell. In some embodiments, the culture pH value is about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, about pH

[0100] 8.5, about pH 9. In preferred embodiments, the pH ranges from about pH 3.0 to about pH 9, more preferably from about pH 3.5 to pH 7. In some most preferred embodiments, a pH value of about 6 is used.

[0101] Cell culturing may be performed at an ionic strength value of the culture medium that is optimal for the cell, typically at a range between 50 mM - 2 M. In some embodiments, the ionic strength of the culture medium ranges between 75 mM - 1 M, more preferably between 100 mM - 750 mM. In some most preferred embodiments, an ionic strength value of about 100 mM is used.

[0102] Cell culturing may be performed for a duration of 14, 13.5, 13, 12.5, 12, 11 .5, 1 1 , 10.5, 10, 9.5, 9,

[0103] 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5 or 1 days, wherein said duration may deviate by 20%. Preferably, cell culturing is performed for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11 ,

[0104] 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5 or 1 days, wherein said duration may deviate by 10%. More preferably, the cell culturing is performed for 5 days, wherein said duration may deviate by 20%, most preferably by 10%. The cell culturing will typically result in a production of at least 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 200 g / L or 300 g / L of a myoglobin.

[0105] The cell culturing will typically result in at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, preferably at least 20%, more preferably at least 40%, most preferably at least 60% of the carbon source in the growth medium being converted to a myoglobin.

[0106] Cell culturing may also be performed by implementation of a multiple step, preferably a two-step, culture method. For example, a production step of a myoglobin may be preceded by a cellular biomass growth step, wherein only limited production or no production is taking place. The different steps may be carried out using different culture modes and / or different growth media and / or different culture process parameter values, depending on the goal of each step and / or the cultured cell. The biomass during the production step may or may not be actively growing.

[0107] The host cells and / or myoglobin may optionally be recovered from the culture medium. When present intracellularly, the myoglobin may optionally be recovered from the recovered cellular biomass. Optionally, the recovered myoglobin is purified. Preferably, purification of the myoglobin will result in a purity of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, most preferably in a myoglobin that is substantially pure.

[0108] In an embodiment, the microorganism produces the myoglobin extracellularly. The myoglobin is transported out of the host cell after it is synthesized in the host cell. In this context, both secretory and extracellular fermentations are considered to be extracellular productions. Without being bound to this theory, an extracellular production process has the advantage that the downstream processing to recover the produced myoglobin is more convenient, efficient and / or effective. Furthermore, an extracellular production process may result in a composition comprising the myoglobin with a high purity such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with minimal downstream processing compared to intracellular methods wherein the myoglobin is not transported out of the host cell after its synthesis. Purity may be measured as the weight percentage of the total protein fraction in the cell-free supernatant obtained at the end of a process or extracellular process according to the invention. Without being bound to this theory, an extracellular production process has the advantage that the optional step of recovering myoglobin does not comprise lysing the host cell. As a result, the extracellular process may result in a composition having a low concentration of nucleic acids originating from the host cell.

[0109] In an embodiment, an extracellular process results in a composition comprising less than or equal to 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1 %, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1 %, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1 %, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1 %, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 % of nucleic acids originating from the host cell.

[0110] The relevant downstream processing technology that may be suitable for recovery and / or purification will depend on whether the myoglobin is accumulated within the cultured cells or excreted. Said processing technology and the associated choice will be known to the skilled person and is discussed, for example, in Wesselingh, J.A and Krijgsman, J., 1st edition, Downstream Processing in Biotechnology, Delft Academic Press, NL, 2013. In a recovery process, the biomass may be recovered from the culture medium using e.g. centrifugation or filtration. If the produced myoglobin is accumulated within the cells, it can then be recovered and / or purified from the biomass. If it is excreted, it can be recovered from the cell-free medium or, if the biomass separation step is skipped, directly from the culture broth. Recovery and / or purification may be performed according to any conventional recovery or purification methodology known in the art. Methods for recovery and / or purification of proteins are known to the skilled person and are discussed in standard handbooks, such as Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY, 2001 or Ausubel F. et al, eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, NY, 2003. Examples of widely used recovery and / or purification methods include chromatographic methods such as gel-filtration chromatography, ion-exchange chromatography, immunoaffinity chromatography, metal affinity chromatography, gel-filtration chromatography, fractionation with precipitants such as ammonium sulfate and polyethylene glycol, gel electrophoresis and salting out and dialysis. Preferably, metal affinity chromatography or size-exclusion chromatography is used. Recovery and / or purification may optionally be enhanced by linking the enzyme polypeptide to a sequence that facilitates purification, such as with a GST domain, using well-known molecular toolbox techniques. Optionally, the sequence that facilitates purification and / or signal peptide that facilitates excretion of the myoglobin is removed from the final product using techniques known in the art, for example proteolysis by endopeptidases targeting a linker between the sequence that facilitates purification and / or the signal peptide and the myoglobin. In some embodiments, the enzyme polypeptide is linked (fused) to a hexa-histidine peptide, such as the tag provided in a pET23a(+) vector (Genescript Biotech, Leiden, The Netherlands), among others, many of which are commercially available. As, for example, described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), hexa-histidine peptide provides for convenient purification of the fusion protein.

[0111] In a preferred embodiment, the isolated myoglobin is obtained via recovering and / or purifying from the culture medium. This may be realized continuously with the production process or subsequently to it. In a preferred embodiment, the isolated myoglobin is obtained via recovering and / or purifying from the cultured cells. A filter may be used for the purification of the recovered myoglobin. This may be realized continuously with the production process, by harvesting fractions of growing cells, or subsequently to it. In an embodiment, the isolated myoglobin is sterilized, shredded, spray dried, freeze dried, blended, shaped, cubed, dosed or packed. Sterilization refers to any process that removes, kills, or deactivates all forms of life (particularly microorganisms such as fungi, bacteria, spores, and unicellular eukaryotic organisms) and other biological agents such as prions present in or on a specific surface, object, or fluid. Sterilization can be achieved through various means, including heat, chemicals, irradiation, high pressure, and filtration. Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure, then removing the ice by sublimation. Packing aims to provide a protection for the product, to tamper resistance and to provide physical, chemical or biological needs. Packing may also contain nutrition facts, characteristics of the products, and an instruction / guide for use of the product.

[0112] In a preferred embodiment, the cultured host cells used in fermentation are immobilized. Immobilization of cells may be achieved by any means known to the skilled person as discussed in standard handbooks such as Guisan, J.M., Bolivar, J.M., Lopez-Gallego, F., Rocha-Martin, J. (Eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020. Typically, the host cells can be immobilized to a semi-solid or solid support by three different methods. The first method involves polymerizing or solidifying a spore- or cell-containing solution. Examples of polymerizable or solidifiable solutions include alginate, A-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinyle benzene, polyvinyl benzene, polyvinyl alcohol, epoxy carrier, cellulose, cellulose acetate, photocrosslinkable resin, prepolymers, urethane, and gelatin. The second method involves cell adsorption onto a support. Examples of such supports include bone char, cork, clay, resin, sand porous alumina beads, porous brick, porous silica, celite, orwood chips. The host cells can colonize the support and form a biofilm. The third method involves the covalent coupling of the host cells to a support using chemical agents like glutaraldehyde, o-dianisidine (U.S. Pat. No. 3,983,000), polymeric isocyanates (U.S. Pat. No. 4,071 ,409), silanes (U.S. Pat. Nos. 3,519,538 and 3,652,761), hydroxyethyl acrylate, transition metal-activated supports, cyanuric chloride, sodium periodate, toluene, and the like. Cultured host cells can be immobilized in any phase of their growth, for example after a desired cell density in the culture has been reached. Suitable culture modes and / or different culture process parameter values will be known to the skilled person and are discussed in standard handbooks, such as Colin R. Phillips C.R., Poon Y. C., Immobilization of Cells: In Biotechnology Monographs book series (Biotechnology, volume 5), Springer, Berlin, Germany, 1988; Tampion J., Tampion M. D., Immobilized Cells: Principles and Applications, Cambridge University Press, UK, 1987. Preferably, immobilized cells are cultured in packed bed bioreactors, also known as plug-flow bioreactors, or expanded (fluidized) bed bioreactors. Suitable growth media and recovery and / or purification methods are further discussed elsewhere herein.

[0113] Myoglobin The myoglobin comprised in the fish or meat substitute according to the invention, or in the pet food according to the invention, or added as an isolated myoglobin in a method according to the invention may have the following characteristics. Wherever a myoglobin is disclosed below, a corresponding fish or meat substitute, pet food or method according to the invention is disclosed as well. Wherever reference is made to the myoglobin, it is understood that this may also mean the isolated myoglobin added during a method according to the invention.

[0114] A myoglobin may be said to be from a species when the amino acid sequence of the myoglobin is the same as the sequence of a myoglobin found in the species. Examples of myoglobin amino acid sequences from salmon, eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans) have been disclosed later on by a given SEQ ID NO.

[0115] A myoglobin may be said to be derived from a species when the amino acid sequence of the myoglobin is derived from the sequence of a myoglobin found in the species, by addition, deletion and / or substitution of at least an amino acid. Addition, deletion and / or substitutions of two, three, four, five, six, seven, eight, nine or ten amino acids is also contemplated by the invention. A myoglobin derived from a species may also exert at least a detectable level of an activity of a myoglobin of the species as explained later herein.

[0116] In an embodiment, the salmon is an Atlantic salmon (Salmo salar).

[0117] In an embodiment, the myoglobin is from salmon, eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans).

[0118] In an embodiment, the myoglobin is from or derived from eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans).

[0119] In an embodiment, the myoglobin is not from or derived from salmon.

[0120] In an embodiment, the myoglobin is not from or derived from a species in the family Salmonidae.

[0121] In an embodiment, the myoglobin is not from or derived from a species in the subfamily Salmoninae. In an embodiment, the myoglobin is not from or derived from a species of the genera Salmo or Oncorhynchus.

[0122] In an embodiment, the myoglobin is not from or derived from Atlantic salmon (Salmo salar).

[0123] In an embodiment, the myoglobin is from Atlantic salmon, eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) or Atlantic blue marlin (Makaira nigricans).

[0124] In an embodiment, the myoglobin is from or derived from salmon, preferably from salmon, more preferably from Atlantic salmon.

[0125] In an embodiment, the myoglobin is from or derived from eastern Pacific bonito, preferably from eastern Pacific bonito.

[0126] In an embodiment, the meat substitute is from or derived from sword fish, preferably from sword fish.

[0127] In an embodiment, the myoglobin is from or derived from Atlantic blue marlin, preferably from Atlantic blue marlin.

[0128] SEQ ID NO: 1 represents the amino acid sequence of a salmon myoglobin, particularly of an Atlantic salmon myoglobin.

[0129] SEQ ID NO: 2 represents the amino acid sequence of a eastern Pacific bonito myoglobin. SEQ ID NO: 3 represents the amino acid sequence of a sword fish myoglobin.

[0130] SEQ ID NO: 4 represents the amino acid sequence of an Atlantic blue marlin myoglobin.

[0131] SEQ ID NO: 5 represents the amino acid sequence of tuna, specifically Pacific bluefin tuna (Thunnus orientalis).

[0132] SEQ ID NO: 1-5 relate to functional myoglobins, i.e. myoglobins from which the initial methionine has been cleaved off. The corresponding translated myoglobins, in which said methionine is present, are represented by SEQ ID NO: 6-10, respectively.

[0133] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0134] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0135] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 1 , 2, 3 or 4.

[0136] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0137] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0138] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 2, 3 or 4.

[0139] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0140] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0141] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 1 .

[0142] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0143] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0144] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 2.

[0145] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0146] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0147] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 3.

[0148] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0149] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0150] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence identity with any one of SEQ ID NO: 4.

[0151] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0152] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 1 , 2, 3 or 4.

[0153] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0154] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0155] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 2, 3 or 4.

[0156] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0157] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0158] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 1 .

[0159] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0160] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0161] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 2.

[0162] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0163] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0164] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 3.

[0165] In an embodiment, the myoglobin can be represented by an amino acid sequence having at least 70, 70.5, 71 , 71 .5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80,

[0166] 80.5, 81 , 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5,

[0167] 91 . 91 .5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, or 99.5 % sequence similarity with any one of SEQ ID NO: 4.

[0168] In the embodiments below, the numbering of positions in (amino acids sequences representing) myoglobin is defined as in Figure 1 .

[0169] In an embodiment, the myoglobin is represented by an amino acid sequence having at least one of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

[0170] In an embodiment, the myoglobin is represented by an amino acid sequence having one of the following combinations:

[0171] - histidine at position 20; or

[0172] - methionine at position 50; or

[0173] - isoleucine at position 56; or

[0174] - isoleucine at position 106; or

[0175] - histidine at position 20, and methionine at position 50; or - histidine at position 20, and isoleucine at position 56; or

[0176] - histidine at position 20, and isoleucine at position 106; or

[0177] - methionine at position 50, and isoleucine at position 56; or

[0178] - methionine at position 50, and isoleucine at position 106; or

[0179] - isoleucine at position 56, and isoleucine at position 106; or

[0180] - histidine at position 20, and methionine at position 50, and isoleucine at position 56; or

[0181] - histidine at position 20, and methionine at position 50, and isoleucine at position 106; or

[0182] - histidine at position 20, and isoleucine at position 56, and isoleucine at position 106; or

[0183] - methionine at position 50, and isoleucine at position 56, and isoleucine at position 106; or

[0184] - histidine at position 20, and methionine at position 50, and isoleucine at position 56, and isoleucine at position 106.

[0185] In an embodiment, the myoglobin is represented by an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% sequence identity with any one of SEQ ID NOs: 2, 3 or 4, and having at least one of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

[0186] In an embodiment, the myoglobin is represented by an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% sequence similarity with any one of SEQ ID NOs: 2, 3 or 4, and having at least one of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

[0187] In an embodiment, the myoglobin is represented by an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% sequence identity with any one of SEQ ID NOs: 2, 3 or 4, and having all of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

[0188] In an embodiment, the myoglobin is represented by an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% sequence similarity with any one of SEQ ID NOs: 2, 3 or 4, and having all of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

[0189] In an embodiment, the myoglobin comprises (i.e. is represented by an amino acid sequence comprising) a motif Xa-R-Xb, wherein:

[0190] • Xa can be any amino acid except for leucine or isoleucine, preferably Xa is methionine

[0191] • R comprises or consists of a sequence having at least 75% identity with AGNAA (SEQ ID NO: 11)

[0192] • Xb can be any amino acid except valine, preferably Xb is isoleucine

[0193] In embodiments, R comprises 1 , 2, 3, 4, and preferably 5 amino acids. In a particular embodiment, R comprises 5 amino acids. In embodiments, R has 75%, 85%, 95% and preferably 100% identity or similarity to AGNAA, wherein Xa is any amino acid except leucine or isoleucine, preferably Xa is methionine. In embodiments, R has 75%, 80, 85%, 90%, 95% and preferably 100% identity or similarity to AGNAA, wherein Xb is any amino acid except valine and, preferably Xb is isoleucine. In a particular embodiment, R has 75%, 80, 85%, 90%, 95% and preferably 100% identity or similarity to AGNAA, wherein Xa is methionine. In a particular embodiment, R has 75%, 80, 85%, 90%, 95% and preferably 100% identity or similarity to AGNAA, wherein Xb is isoleucine. In a particular embodiment, R has 75%, 80, 85%, 90%, 95% and preferably 100% identity or similarity to AGNAA, wherein Xa is methionine and Xb is isoleucine. In one embodiment, R has one, two or three mismatches with the AGNAA sequence, wherein Xa is any amino acid except leucine or isoleucine and, preferably Xa is methionine. In one embodiment, R has one, two or three mismatches in the AGNAA sequence, wherein Xb is any amino acid except valine and, preferably Xb is isoleucine. In a particular embodiment, R has one, two or three mismatches in the AGNAA sequence, wherein Xa is methionine and Xb is isoleucine.

[0194] In an embodiment, the myoglobin comprises (i.e. is represented by an amino acid sequence comprising) a motif R1-Xc-R2 represented, wherein:

[0195] • R1 comprises or consists of the amino acid triplet VLK or VLQ

[0196] • Xc is any amino acid except cysteine, preferably Xc is histidine or phenylalanine

[0197] • R2 comprises or consists of the amino acid triplet WGP or WEP

[0198] In embodiments, R1 comprises 1 , 2, and preferably 3 amino acids. In a particular embodiment, R1 comprises 3 amino acids. In embodiments, R2 comprises 1 , 2, and preferably 3 amino acids. In a particular embodiment, R2 comprises 3 amino acids. In embodiments, R1 comprises or consists of the amino acid triplet VLK or VLQ, and Xc is any amino acid except cysteine, preferably Xc is histidine or phenylalanine. In embodiments, R2 comprises or consists of the amino acid triplet WGP or WEP, and Xc is any amino acid except cysteine, preferably Xc is histidine or phenylalanine. In embodiments R1 comprises or consists of the amino acid triplet VLK or VLQ, R2 comprises or consists of the amino acid triplet WGP or WEP, and Xc is any amino acid except cysteine, preferably Xc is histidine or phenylalanine.

[0199] Without being bound to this theory, it is an advantage of myoglobins from eastern Pacific bonito, sword fish or Atlantic blue marlin that they are more thermostable and are more strongly bound to (i.e. have a higher affinity with) their heme cofactor. This results in a higher activity of the myoglobin in fish substitutes comprising these types of myoglobins, as the myoglobins will retain their native conformation for a longer time and / or under a broader range of temperatures. This may have one or more of several advantages:

[0200] - The myoglobins are less prone to aggregate, precipitate, lose heme, or deform, especially during productions processed characterized by a higher temperature, such as spray drying, but also during long-term storage or even during fermentation.

[0201] - The myoglobins are more stable in the corresponding fish substitutes, and thus keeping their red color for a longer time. - The myoglobins are more stable in non-fish and non-meat applications that are not stored cold, like iron fortification in beverages, nutrition bars, proteins an nutritional powders, cookies, etc.

[0202] Due to this, and other mechanisms, myoglobins from eastern Pacific bonito, sword fish or Atlantic blue marlin lead to fish substitutes that are better at mimicking an aspect, form, structure, composition, flavor, texture, color, aroma, appearance and / or nutritional value of fish, compared to other fish myoglobins.

[0203] Examples 2 and 3 show the higher thermostability and heme affinity of myoglobin form eastern Pacific bonito, sword fish or Atlantic blue marlin, compared to myoglobin from Pacific bluefin tuna. The thermostability of a protein can be determined using the protocol outlined in Example 2. The affinity with heme can be determined using the protocol outlined in Example 3.

[0204] Fish or meat

[0205] In an aspect there is provided a fish or meat product comprising a myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin; wherein said myoglobin was not produced by the animal, or any cells comprised therein, from which said fish or meat product is derived. In other words, the fish or meat product is derived from an animal, but comprises a myoglobin not produced therein.

[0206] In an embodiment is provided a fish or meat product comprising an exogeneous myoglobin from or derived from salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, preferably eastern Pacific bonito, sword fish or Atlantic blue marlin. Herein, an exogeneous myoglobin is defined as a myoglobin which is not endogenously present in said fish or meat.

[0207] In an embodiment is provided a fish or meat product comprising myoglobin, wherein the concentration of myoglobin in said fish or meat product is higher than the average concentration of myoglobin in fish meat from which said fish or meat product is derived. Herein, the concentration of myoglobin should be interpreted as the total concentration of all myoglobin variants. The average concentration of myoglobin in fish or meat from which said fish or meat product is derived means the average concentration, determined over several corresponding animals, of myoglobin in said fish or meat, wherein no exogeneous myoglobin has been added after harvesting.

[0208] In an aspect, there is provided a method for preparing fish or meat, wherein the method comprises adding an isolated myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin, to a base fish or meat. All preferences for methods according to the invention can be applied mutatis mutandis to this method for preparing fish or meat.

[0209] Dairy products In an aspect, there is provided a dairy product, or a replica thereof, supplemented with a myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin. A dairy product, or a replica thereof, is said to be supplemented with a myoglobin if the myoglobin has been added as an isolated myoglobin during any step of its preparation. Hence, supplementation with a myoglobin results in an increase of the myoglobin concentration during the preparation, which is preferably reflected in an increased myoglobin concentration in the (final) dairy product, or the replica thereof. All preferences for methods according to the invention can be applied mutatis mutandis to this supplementation.

[0210] In an aspect, there is provided a method for preparing a dairy product, or a replica thereof, wherein the method comprises adding an isolated myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin, to a base dairy product, or a replica thereof. All preferences for methods according to the invention can be applied mutatis mutandis to this method for preparing a dairy product.

[0211] In an aspect, there is provided a dairy product, or a replica thereof, obtainable by a method comprising the addition of a myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin, to a base dairy product, or a replica thereof.

[0212] In an aspect, there is provided a dairy product, or a replica thereof, comprising a myoglobin from or derived from salmon, preferably Atlantic salmon, eastern Pacific bonito, sword fish or Atlantic blue marlin, more preferably eastern Pacific bonito, sword fish or Atlantic blue marlin.

[0213] A replica of a dairy product is a food product that resembles a dairy product but is neither made from nor contains milk produced by a mammary gland of a mammal. In other words, a replica of a dairy product is not of mammalian origin. A dairy replica product is by definition, not a dairy product, not a natural dairy product, not a genuine dairy product. The expression “a replica of a dairy product may be replaced by “a dairy food substitute”, “a dairy product substitute”, or “a non-natural dairy product”. It means that within the context of this invention, a replica of a dairy product is not a dairy product from or derived from a dairy animal such as a cow, a buffalo, a goat, a sheep or a horse. It is thus understood that cell-based, cultured, or plant-based dairy product substitutes may be considered replicas of dairy products. To resemble in this context means to approach, to have essentially or substantially the same, to have the same, or to mimic one or more physical, chemical or sensory characteristics of a corresponding control or reference dairy product. Examples of physical characteristics are color, viscosity, particle size distribution. Examples of chemical characteristics are nutrient concentrations, pH, ionic strength. Examples of sensory characteristics are form, structure, texture, flavor, color, aroma, appearance. In an embodiment, the dairy product is a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream. In an embodiment, the replica of a dairy product is a replica of a milk, a cream, a butter, a yoghurt, a cheese, a custard or of an ice cream.

[0214] In an embodiment, the dairy product is a milk, wherein the milk is a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula. In an embodiment, the replica of a dairy product is a replica of a milk, wherein the replica of a milk is a replica of a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula. Whole milk means milk is taken as is from the mammal origin, without any sort of nutritional alteration, and processed for food safety, or a replica of such a dairy product. This means no nutrient is removed from nor supplemented to the milk. Whole milk is not considered raw milk and is safe to consume. Whole milk is sometimes also referred to as fresh milk or regular milk. Reduced-fat milk has at least 25% less fat than regular milk. Low milk has less than 1 .5% fat of the total weight of the milk. Fat- free (also known as skim) milk has less than 0.2% fat of the total weight of the milk. Lactose-free milk is a type of milk where natural sugar lactose has been broken down. Flavored milk is a sweetened dairy drink made with milk, sugar, flavorings, and sometimes food colorings. Raw milk is unpasteurized milk directly from animal origin.

[0215] Breast milk or mother's milk is milk produced by the mammary glands of a human female. Infant formula or baby formula, also called infant milk or infant growth milk, is a manufactured dairy product or replica thereof designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottle-feeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water). The U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants because of its simulation of human milk or its suitability as a complete or partial substitute for human milk". The starter formula is suitable for babies up to 6 months of age. The follow-on formula is suitable for older children. A comparison between human breast milk, infant formula and full-fat milk from cow shows the difference in nutrient composition.

[0216] Milk is usually produced from a dairy animal, such as a cow, a buffalo, a goat, a sheep, a horse, a llama or other less common animals such as a yak, a horse, a reindeer, a zebu, a giraffe and a donkey. The average nutrient composition in milk from different mammalian origins varies as shown in table 3. Herein, the term cow milk means milk (a type of dairy product) produced from a cow. Likewise, the term human (breast) milk, means a (breast) milk from a human. These and similar terms thus exclude replicas of dairy products, which are of non-mammalian origin. In an embodiment, the dairy product supplemented with a myoglobin, is derived from a milk of a dairy animal such as a cow, a buffalo, a goat, a sheep, a horse, a yak, a horse, a llama, a reindeer, a zebu, a giraffe or a donkey, preferably from a milk of a cow. Herein, a cow refers to an animal of the subspecies Bos primigenius taurus and not to a female specimen of other species such as yak or buffalo.

[0217] In an embodiment, the replica of the dairy product is a vegan product, preferably a vegan milk. Vegan milk may be plant-based juice that resembles the texture, taste and qualities of conventional animal milk. It can also be used to make many products, such as replicas of dairy products. Vegan milk may be soya milk (soy milk), almond milk, coconut milk, rice milk, cashew milk, macadamia milk, flax milk, pea protein milk, banana milk, sunflower milk, peanut milk, oat milk, hazelnut milk and sunflower milk.

[0218] General terms

[0219] Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs and read in view of this disclosure.

[0220] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a dairy product, a replica dairy product, a myoglobin, a gene construct, a host cell (or methods) as described herein may comprise additional component(s) (or additional steps) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

[0221] Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.

[0222] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value. As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0223] In the context of this application, all percentages in the context of a concentration or composition referto weight percentages, unless defined otherwise. In the context ofthis application, expressions such as “a parameter having a value of at least X, Y or Z” should be interpreted as said parameter having a value of at least X, of at least Y, or of at least Z.

[0224] Various embodiments are described herein. Each embodiment as identified herein may be combined unless otherwise indicated. All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0225] Food safety measures such as good hygienic practice (GHP), good manufacturing practice (GMP), hazard analysis and critical control practice (HACCP), quality management and microbial risk assessment are implemented for the monitoring of food safety of this invention.

[0226] Sequence identity

[0227] In the context of the invention, a nucleic acid molecule such as a nucleic acid molecule encoding a myoglobin is represented by a nucleic acid or nucleotide sequence which encodes a protein fragment or a polypeptide or a peptide or a derived peptide. It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identity number (SEQ ID NO) is not limited to this specific sequence as disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.

[0228] Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given protein fragment or polypeptide or peptide or derived peptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: X; or ii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) due to the degeneracy of the genetic code; or

[0229] Hi. a nucleotide sequence that encodes an amino acid sequence that has at least 60% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

[0230] Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0231] Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0232] Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively.

[0233] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference.

[0234] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).

[0235] A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) I 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) 1 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0236] Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov / .

[0237] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0238] Alternative conservative amino acid residue substitution classes: For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

[0239] Proteins and amino acids

[0240] The terms "protein" or "polypeptide" or “amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. In amino acid sequences as described herein, amino acids or “residues” are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to a person of skill in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non- proteinogenic amino acid such as D-amino acids and modified amino acids formed by post- translational modifications, and also any non-natural amino acid, as described herein.

[0241] Gene or coding sequence

[0242] The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) e.g. comprising a polyadenylation- and / or transcription termination site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide. As used herein, a “regulator” or “transcriptional regulator” is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.

[0243] Promoter

[0244] As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" and / or “repressible” promoter is a promoter that is physiologically or developmentally regulated to be induced and / or repressed, e.g. by the application of a chemical inducer or repressing signal.

[0245] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0246] Gene constructs and expression vectors

[0247] Gene constructs as described herein could be prepared using any cloning and / or recombinant DNA techniques, as known to a person of skill in the art, in which a nucleotide sequence encoding said myoglobin is expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-lnterscience, New York (1987) and in Sambrook and Russell (2001 , supra) both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).

[0248] The phrase "expression vector" or "vector" generally refers to a tool in molecular biology used to obtain gene expression in a cell, for example by introducing a nucleotide sequence that is capable of effecting expression of a gene or a coding sequence in a host compatible with such sequences. An expression vector carries a genome that is able to stabilize and remain episomal in a cell. Within the context of the invention, a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered. Alternatively, a vector is capable of integrating into a cell's genome, for example through homologous recombination or otherwise.

[0249] These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. A nucleic acid or DNA or nucleotide sequence encoding a myoglobin is incorporated into a DNA construct capable of introduction into and expression in an in vitro cell culture. Specifically, a DNA construct is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.

[0250] A DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term “operably linked” has already been described herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0251] The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001 , supra). A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognized by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001 , supra). An expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment. In most cases, the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. Coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001 , supra) and in Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media. Alternatively, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.

[0252] For example if a bacterium (preferably E. coli) is used as host cell, the following regulatory regions may be used. A promoter suitable to be used in a bacterium is lac, trp, tac, T7, phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL promoter from phage A. A terminator suitable to be used in a bacterium is lac, trp, tac, T7 (used in example), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL terminator from phage A. A preferred promoter used is a T7 promoter and / or a preferred terminator is the T7 terminator. A preferred signal peptide for excretion is E. coli Sec-recognition peptide (SecA), E. coli Tet- recognition peptide, E. coli dsbA, E. coli phoA, E. coli pelB, E. coli MBP (maltose binding protein). A marker suitable for E coli is ampicillin. Alternatively, the proBA operon from E. coli strain K12, including its original transcription regulatory elements may be used to facilitate selection without antibiotics.

[0253] In another example, if a yeast is used as host cell, the following regulatory regions may be used. A promoter suitable to be used in yeast may be a constitutive promoter. Examples of suitable constitutive promoters include: a glycolytic promoter selected from FBA1 , TPI1 , PGK1 , PYK1 , TDH3, ENO2, HXK2, PGI1 , PFK1 , PFK2, GPM1 gene or a non-glycolytic promoter of the TEF2 gene. A suitable promoterto be used in yeast may be inducible. If the yeast is a Pichia, the methanol inducible promoter AOX1 is preferred. Otherwise the GAL1 promoter (galactose-inducible) may be used when the yeast is S. cerevisiae. The genes mentioned from which a promoter could be derived for a yeast as host cell could also be used to derive a terminator for the same yeast. A preferred signal peptide for excretion for Pichia (and Saccharomyces) includes: the S. cerevisiae alpha mating factor pre- pro- secretion signal peptide, the S. cerevisiae Ost1 signal peptide, the S. cerevisiae Aga2 signal peptide and fusions thereof.

[0254] In another example, if a filamentous fungus is used as host cell, the following regulatory regions may be used. The following promoters may be used: the Aspergillus niger glucoamylase promoter (g / aA), the Aspergillus nidulans alcohol dehydrogenase promoter (a / cA) or the Aspergillus oryzae taka-amylase A promoter (amyB), the Aspergillus niger alcohol dehydrogenase promoter (ac / hA), the Trichoderma reesei pyruvate kinase promoter (pki) or the Aspergillus nidulas glyceraldehyde- 3-phosphate dehydrogenase promoter (gpdA). The genes mentioned from which a promoter could be derived for a filamentous fungus as host cell could also be used to derive a terminator for the same filamentous fungus. A preferred signal peptide for excretion for a filamentous fungus, preferably an Aspergillus includes: the Aspergillus niger glucoamylase signal peptide (g / aA), the Aspergillus niger a-galactosidase signal peptide (AgIB) and the Trichoderma reesei cellobiohydrolase I (Cbhl). A preferred promoter and terminator tor Aspergillus (more preferably for Aspergillus niger) are the glucoamylase promoter and the glucoamylase terminator of Aspergillus niger. Gene constructs described herein can be placed in expression vectors. Thus, in another aspect there is provided an expression vector comprising a gene construct as described in any of the preceding embodiments.

[0255] Expression may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of transgene expression in the transduced tissue on the level of the mRNA or the protein by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides or ELISA. Expression may be assessed at any time after administration of the gene construct, expression vector or composition as described herein. In some embodiments herein, expression may be assessed after 1 week, 2 weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9, weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, or more.

[0256] Legends to the figures

[0257] Figure 1 — Numbering of the amino acid sequence of myoglobin from Pacific bluefin tuna (Thunnus orientalis), eastern Pacific bonito (Sarda chiliensis), sword fish Xiphias gladius) and Atlantic blue marlin (Makaira nigricans).

[0258] Examples

[0259] Example 1: Myoglobin sequences and structures

[0260] For this analysis, the functional myoglobin sequences of Pacific bluefin tuna (Thunnus orientalis), eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) and Atlantic blue marlin (Makaira nigricans) were obtained.

[0261] Translated myoglobin generally start with an initial methionine. Functional myoglobin is the sequence found when myoglobin is extracted from animal cells, that excludes the initial methionine (as it is cleaved off to give myoglobin its function). The sequences are numbered in such a way that the initial methione is at -1 and the next amino acid residue (i.e. the first residue of the functional myoglobin) is at +1 (see Figure 1).

[0262] The 3D structures for these sequences were obtained in pdb format using AlphaFold v2 (Deepmind) monomer model in template mode using BFD database including relaxation, using default parameters, according to doi:10.1038 / s41586-021-03819-2.

[0263] Example 2: Thermostability of myoglobin from eastern Pacific bonito, sword fish and Atlantic blue marlin

[0264] The thermostability of the proteins of Example 1 was determined, namely of the functional myoglobin sequences of Pacific bluefin tuna (Thunnus orientalis), eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) and Atlantic blue marlin (Makaira nigricans).

[0265] The web-based SCooP V1 .0 was used to predict thermodynamic stability (= folding free energy AGfoid in Gibbs-Helmholtz equation) at various temperatures T, according to Fabrizio Pucci, Jean Marc Kwasigroch, Marianne Rooman, SCooP: an accurate and fast predictor of protein stability curves as a function of temperature, Bioinformatics, Volume 33, Issue 21 , November 012017, Pages 3415-3422, https: / / doi.Org / 10.1093 / bioinformatics / btx417. The following protocol was followed:

[0266] 1 . For every protein, upload the PDB protein structure file into SCooP.

[0267] 2. Select the "Unknown Host organism" option as the Organism.

[0268] 3. When the prediction has finished running, go to the text file of the result and copy the equation for AG (denoted Delta G(T)).

[0269] 4. Paste the equation in Geogebra Graphing Calculator for better visualisation.

[0270] 5. Make sure the natural logarithm is applied to the last term of the equation by manually adding the e to have loge.

[0271] 6. Name the equations from your different proteins A(T) = ... , B(T) = ..., etc.

[0272] 7. Obtain Gfoid (Gibbs-Helmholtz folding energy) at 20°C in kJ / mol.

[0273] 8. More negative values correspond with more thermostable proteins.

[0274] In conclusion, myoglobin from other fish (such as Thunnus orientalis) is less thermostable than a myoglobin from Sarda chiliensis, Xiphias gladius and Makaira nigricans.

[0275] Example 3: Heme affinity of myoglobin from eastern Pacific bonito, sword fish and Atlantic blue marlin

[0276] The heme affinity of the proteins of Example 1 was determined, namely of the functional myoglobin sequences of Pacific bluefin tuna (Thunnus orientalis), eastern Pacific bonito (Sarda chiliensis), sword fish (Xiphias gladius) and Atlantic blue marlin (Makaira nigricans).

[0277] A protein-ligand docking experiment is done using the AMDOCK tool (V1.5.2), which is a graphical interface to prepare ligand and protein files and uses Autodock vina for actual docking.

[0278] • As a ligand, the 3D structure of heme (PROTOPORPHYRIN IX CONTAINING FE) is obtained from structure file 1 MYT (in RCSB database).

[0279] • As a protein, the generated 3D structures of fish MB are used one by one.

[0280] • Constrained search space to box of 23 A with center on iron molecule of heme pose obtained by structurally aligning experimentally determined structure 1 MYT with proteins of interest.

[0281] • Docking parameters: exhaustiveness = 32, AMBER to prepare ligand file, pH 7.4 for protonation, max number of poses = 10.

[0282] • Best pose is analysed for each protein and verified to be similar to experimentally determined pose of heme in 1 MYT. • A higher dissociation constant Kd means a lower affinity with heme.

Claims

Claims1 . A method of preparing a fish substitute comprising adding an isolated myoglobin from eastern Pacific bonito, sword fish or Atlantic blue marlin to an initial fish substitute.

2. The method of preparing a fish substitute of claim 1 , wherein the isolated myoglobin is not comprised in a cell or a tissue before the addition.

3. The method of preparing a fish substitute of claim 1 or 2, wherein the isolated myoglobin is added as part of a composition, wherein the weight fraction of the isolated myoglobin in the protein fraction comprised in the composition is at least 5%.

4. The method of preparing a fish substitute of any one of claims 1 to 3, wherein the concentration of the myoglobin in the fish substitute after the addition is in the range 0.05-0.5 wt.%, and wherein the concentration of myoglobin in the initial fish substitute is less than 0.01 wt.%.

5. The method of preparing a fish substitute of any one of claims 1 to 4, wherein the isolated myoglobin is a recombinant myoglobin obtained from fermentation by a microorganism which has been genetically modified to express the recombinant myoglobin.

6. The method of preparing a fish substitute of claim 5, wherein the isolated myoglobin is obtained from a secretory or extracellular fermentation.

7. The method of preparing a fish substitute of claim 5 or 6, wherein the microorganism is a yeast, preferably Pichia pastoris.

8. The method of preparing a fish substitute of any one of claims 1 to 7, wherein the isolated myoglobin is represented by an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2, 3 or 4, and wherein the amino acid sequence has at least one of the following: histidine at position 20, methionine at position 50, isoleucine at position 56, and isoleucine at position 106.

9. The method of preparing a fish substitute of claim 8, wherein the amino acid sequence is identical to SEQ ID NOs: 2, 3 or 4.

10. A fish substitute obtainable by a method as defined in any one of claims 1 to 9.11 . The fish substitute of claim 10, which is a cell-based fish substitute, a cultured fish substitute, or a plant-based fish.

12. The fish substitute of claim 10 or 11 , which mimics an aspect, form, structure, composition, flavor, texture, color, aroma, appearance and / or nutritional value of fish.

13. The fish substitute of any one of claims 10 to 12, which is a pet food, preferably wherein the pet is a dog or a cat.

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