Method for preparing pet food

Abstract

Described herein is a method for preparing pet food wherein the method comprises the addition of isolated myoglobin wherein the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, vigorous myoglobin, or tuna myoglobin.

Description

Technical Field

[0001] The present invention relates to a method for preparing pet food, wherein the method comprises adding isolated myoglobin, and the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, mammoth myoglobin or tuna myoglobin. Background Art

[0002] The increasing knowledge of pet nutrition and food technology has enabled the industry to develop and diversify in terms of the types, flavors, compositions, and shapes of pet food. However, commercial pet food is highly processed, which means that during industrial production, nutritional value may be lost along with flavor and texture.

[0003] In this context, efforts are being made to develop meat alternatives as pet food to reduce its ecological footprint. For these types of pet food, it is difficult to obtain flavors and textures close to those of unprocessed meat, and it is disadvantageous for achieving a threshold level of animal food acceptance.

[0004] Therefore, there is a continuing need in the art for pet food that mimics certain aspects of meat, such as flavor and texture, while being environmentally friendly.

[0005] Specification

[0006] Method for preparing pet food

[0007] In a first aspect of the present invention, there is provided a method for preparing pet food, wherein the method comprises adding isolated myoglobin, and the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, mammoth myoglobin or tuna myoglobin. Example 3 provides an example of such a method.

[0008] In an embodiment, the pet is a vertebrate. In an embodiment, the pet is a mammal, bird, fish, amphibian or reptile. In an embodiment, the pet is a mammal. Mammals are a group of vertebrates that make up the class Mammalia, characterized by the presence in females of mammary glands to produce milk for feeding (nursing) their young, a neocortex (region of the brain), soft fur or hair, and three middle ear bones. In an embodiment, the pet is a carnivore or omnivore, preferably a mammal. In a preferred embodiment, the pet can be a dog, cat, ferret, fox, bear, wolf, jaguar, lion, tiger, snake, piranha or alligator. In a preferred embodiment, the pet can be a dog or a cat.

[0009] In an embodiment, the rat is any species in the genus Rattus. In a more preferred embodiment, the rat is a black rat (Rattus rattus) or a brown rat (Rattus norvegicus). Different species within the genus Rattus may share the same myoglobin sequence.

[0010] In an embodiment, the pet food is dry pet food, semi-moist pet food, wet pet food, or any combination of these types of pet food. The pet food can be formulated as a liquid, solid, or semi-solid pet food. "Dry pet food" means pet food having a moisture content of less than 15% by weight. "Semi-moist pet food" means pet food having a moisture content between 55% and 65% by weight. "Wet-type food" means food having a moisture content between 65% and 85% by weight. Hereinafter, unless otherwise specified, all percentages mentioned should be understood to be by weight and based on the weight of the final pet food. Suitable examples of solid pet food include kibble, treats, cell-based meat and meat alternatives, cultured meat and meat, and plant-based meat and meat alternatives. Suitable examples of treats include protein bars, dental treats, meat chunks, or chew sticks. Such plant-based meat may contain plant proteins such as soy protein. Suitable examples of semi-moist and wet-type pet food include pate or loaf, gravy blocks or jelly blocks or broth blocks. In an embodiment, the pet food is pate or loaf, or gravy blocks.

[0011] In an embodiment, the pet food is a meat alternative. By definition, a meat alternative is not meat, not natural meat, nor real meat. A meat alternative can be considered a non-natural food or an edible product. This means that in the context of the present invention, a meat alternative is not meat derived from or obtained from rats, rabbits, mice, or tuna (or from or obtained from mammoths (if such an animal still existed, but it does not)). A meat alternative is a synonym for a meat replica or meat analogue product or meat-like product. Thus, it should be understood that cell-based meat, cultured meat, and plant-based meat can be considered meat alternatives. Cell-based meat is typically cultivated directly from animal stem cells. Cultured meat is typically produced through in vitro cell culture of animal cells as a form of cell agriculture. Plant-based meat is typically produced using plants and other non-animal products.

[0012] In an embodiment, the pet food is a meat substitute, wherein the meat substitute is a cell-based meat substitute, a cultured meat substitute, or a plant-based meat substitute. The meat substitute can be dry pet food, semi-moist pet food, wet pet food, or any combination of these types of pet food. The inventors have found that the "meat experience" of pet food containing meat can be mimicked in the meat substitute by adding isolated myoglobin as defined herein. Depending on the envisioned meat substitute, a person skilled in the art will know which formulation is most suitable.

[0013] In an embodiment, the pet food is a meat substitute, wherein the meat substitute mimics the appearance (such as patterns, printing), form, structure, composition (such as similar fat, protein, and / or heme iron content), palatability, flavor, texture, color, aroma, appearance, and / or nutritional value of meat. The acceptance, overall preference, or preference relative to another of a pet food, or the palatability of a pet food, can be evaluated by preference / acceptability testing. The palatability of pet food is typically measured using a single-bowl or double-bowl test or a lever-press test. For example, in a preference test, a control meal and a test meal are provided to the pet in a controlled environment. The experimenter records the ratio of the animal's selection of the test meal compared to the control meal. Larger tests can be conducted by pet owners in a home environment, requiring the pet owner to record "food preference" behaviors such as the time spent sniffing the food, licking the lips or nose, or ear or tail wagging. This is mainly due to the addition of isolated myoglobin as described herein. The meat substitute may not have all the drawbacks of meat. For example, the meat substitute can be healthier than meat. In addition, it is expected that the impact of the meat substitute on the environment (long-term direct / indirect impact on climate change) is less harmful than the known impact of meat.

[0014] Regarding nutritional value, Example 4 shows that the iron bioavailability of the pet food is higher than that of the corresponding pet food supplemented with non-heme iron. Example 5 details the color and color stability of the pet food. It is shown that adding myoglobin to the meat substitute can stabilize and / or improve the desired color characteristics of the meat substitute over time during refrigeration, regardless of whether the product is in a raw or cooked state. Example 6 reports the changes in the aroma characteristics of the meat substitute after adding myoglobin, indicating a significant increase in the levels of volatile compounds related to meat flavor in the heat-processed product containing myoglobin.

[0015] In an embodiment, the pet food is a meat substitute, where it is expected that the meat substitute mimics the composition of meat (such as similar fat, protein, and / or heme iron content), palatability, flavor, color, and / or aroma, but without all the drawbacks of meat. In an embodiment, it is expected that the meat substitute mimics the flavor and / or aroma of meat, but without all the drawbacks of meat. In an embodiment, it is expected that the meat substitute mimics the palatability of meat, but without all the drawbacks of meat. In an embodiment, it is expected that the meat substitute mimics the flavor of meat, but without all the drawbacks of meat. In an embodiment, it is expected that the meat substitute mimics the aroma of meat, but without all the drawbacks of meat. In embodiments of this context, this is mainly due to the presence of isolated myoglobin as described herein. Alternatively, it can have an appearance, form, structure, texture, color, palatability, flavor, aroma, and / or look different from that of meat.

[0016] In the context of the present invention, when the meat substitute produces an aroma and / or flavor compound characteristic of some odorants and / or flavor compounds that can be recognized by the pet and are released during meat cooking, the meat experience can be mimicked. For example, the heme iron present in the isolated myoglobin may catalyze the formation of some of these odorants. Without being bound by this theory, this formation may be due to lipid oxidation and / or the Maillard reaction ("Maillard products"). This situation can be mimicked with the isolated myoglobin within the meat substitute of the present invention.

[0017] In an embodiment of the present invention, where the pet food is a meat substitute, preferably a raw meat substitute, it is expected that the raw meat substitute mimics the bloody aroma and / or metallic aroma of meat. Preferably, the meat substitute is capable of mimicking the bloody aroma and / or metallic aroma of meat to a higher degree than other meat substitutes (i.e., meat substitutes not according to the present invention). In the context of the present invention, "raw" means not subjected to heat treatment, preferably uncooked and ungrilled.

[0018] In an embodiment of the present invention, where the pet food is a meat substitute, preferably a cooked meat substitute, it is expected that the cooked meat substitute mimics the roasted aroma of meat, especially roasted meat. Preferably, the meat substitute is capable of mimicking the roasted aroma of meat to a higher degree than other meat substitutes (i.e., meat substitutes not according to the present invention). In the context of the present invention, "cooked" means subjected to heat treatment, preferably sterilized, grilled, or roasted.

[0019] In the context of the present invention, especially in embodiments of this section, "other meat substitutes" or "meat substitutes not according to the present invention" are preferably plant-based meat substitutes containing recombinant soybean leghemoglobin (LegH).

[0020] In an embodiment, the aroma of the pet food is characterized by a higher concentration of Maillard compounds (i.e., compounds formed as a result of the Maillard reaction) during cooking compared to the corresponding pet food without added isolated myoglobin.

[0021] In an embodiment, the aroma of the pet food (preferably suitable for dogs) is characterized by a higher concentration of 3-methylbutanal, 2-methyl-butanal, pyrazine, pyrrole, methylpyrazine, 2-ethyl-6-methyl-pyrazine, methional, 2,6-dimethylpyrazine, 2-acetylthiazole, 2,5-dimethylpyrazine, octanal, and / or nonanal during cooking compared to the corresponding pet food without added isolated myoglobin.

[0022] In an embodiment, the aroma of the meat substitute of the present invention (preferably a grilled meat substitute) is characterized by a higher concentration of oxidized lipids, lipid oxidation products, pyrazine, pyrrole, aldehyde, and / or ketone in the volatile compounds obtained from the (grilled) meat substitute compared to other meat substitutes (i.e., not according to the present 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. Preferred pyrrole is pyrrole. Preferred lipid oxidation products are 2-methylbutanal and 3-methylbutanal. Preferred aldehydes are methional, nonanal, and octanal. Preferred ketone is 2-acetylthiazole. It should be understood that the categories of compounds mentioned in this paragraph are not mutually exclusive, for example, lipid oxidation products can be aldehydes or ketones.

[0023] The aroma of meat substitutes containing different concentrations of recombinant myoglobin can be analyzed using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME).

[0024] In the context of the present invention, when the color of the meat substitute is similar to the color of meat, the meat experience can be mimicked. The color of meat is determined by the concentration of heme proteins and / or by the oxidation state of the heme proteins in the meat. Therefore, the total amount of isolated myoglobin added to the meat substitute will determine the color of the meat substitute.

[0025] In an embodiment, it is expected that a meat substitute (preferably a raw meat substitute) mimics the color of meat (preferably raw meat). Without being bound by this theory, the color of the meat substitute is mainly due to the addition of isolated myoglobin. Preferably, the meat substitute of the present invention is capable of mimicking the color of meat to a higher degree than other meat substitutes (i.e., meat substitutes not according to the present invention).

[0026] In a preferred embodiment, preferably when stored at 4 °C under constant light, the color of the meat substitute (preferably a raw meat substitute) of the present invention is substantially stable 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. Preferably, the color of the meat substitute of the present invention is stable for a period of 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 color of meat or other meat substitutes. More preferably, a ΔE change of 0% to a maximum of 10% is defined as stable. Substantially stable for at least X days preferably means that between day 0 and day X, the ΔE change of the meat substitute is 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%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20%, where ΔE is. Most preferably, after 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 ΔE change of the meat substitute does not exceed 10%. The color can be evaluated via absorbance measurement results using spectrometry.

[0027] In the context of the present invention, when the composition of the meat substitute is similar to the composition of meat, the meat experience can be mimicked. The composition of the meat substitute can be considered similar to the meat composition when the same or similar components are present and optionally when the amounts of these components present are the same or similar to those present in meat. For example, similar fat, protein and / or heme iron contents may be present in the meat substitute as in meat.

[0028] Addition of isolated myoglobin

[0029] In a first aspect of the present invention, a method for preparing pet food includes adding isolated myoglobin. The isolated myoglobin can be added or applied by the manufacturer of the pet food, the consumer of the pet food, or any other producer of the pet food or related products.

[0030] In embodiments, the (total) weight concentration of heme protein 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 protein in pet foods produced by conventional or common methods that do not explicitly include the step of adding isolated myoglobin. In this context, the average concentration of heme protein in the meat of a particular species of animal is determined in several corresponding animals, where no exogenous or isolated myoglobin is added to the animal meat after harvest. In this context, the resulting pet food of the present invention is edible by the pets as defined herein, and adding myoglobin using the method of the present invention does not introduce toxicity or affect the food safety of the pet food.

[0031] In embodiments, the (total) weight concentration of heme protein 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 less than 10%, more preferably less than 5%.

[0032] In an embodiment, the (total) weight concentration of myoglobin 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 less than 10%, more preferably less than 5%. It should be understood that the myoglobin contained in the pet food can be derived from the addition of isolated myoglobin or from another source.

[0033] In an embodiment, the weight fraction of myoglobin in the heme protein 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%.

[0034] In an embodiment, the (total) weight concentration of heme protein 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 myoglobin in the heme protein in the pet food is at least 90%.

[0035] In an embodiment, the (total) weight concentration of heme protein in the pet food is at least 0.05%, and the weight fraction of myoglobin in the heme protein 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%.

[0036] In an embodiment, the (total) weight concentration of heme protein in the pet food is at least 0.1%, and the weight fraction of myoglobin in the heme protein 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%.

[0037] In an embodiment, the (total) weight concentration of heme protein in the pet food is at least 0.5%, and the weight fraction of myoglobin in the heme protein 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%.

[0038] In an embodiment, the (total) weight concentration of heme protein in the pet food is at least 1.0%, and the weight fraction of myoglobin in the heme protein 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%.

[0039] In an embodiment, the (total) weight concentration of heme protein in the pet food is at least 3.0%, and the weight fraction of myoglobin in the heme protein 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%.

[0040] In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the heme protein in the pet food is derived from the addition (by weight) of isolated myoglobin. In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the myoglobin in the pet food is derived from the addition (by weight) of isolated myoglobin.

[0041] In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the myoglobin in the pet food is derived from the addition (by weight) of isolated myoglobin. In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the myoglobin in the pet food is derived from the addition (by weight) of isolated myoglobin.

[0042] In an embodiment, isolated myoglobin is added as part of a composition, wherein the weight concentration of the isolated myoglobin in the composition is at least 10%, at least 15%, 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, the weight concentration can be expressed as the mass of myoglobin per total volume or total mass of the composition.

[0043] In an embodiment, isolated myoglobin is added as part of a composition, wherein the weight fraction of isolated myoglobin in the protein fraction contained in the composition is at least 10%, at least 15%, 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, the weight fraction is a dimensionless number, which can be interpreted as the mass of myoglobin per mass of protein.

[0044] In an embodiment, the isolated myoglobin is in the form of a liquid formulation (i.e., the composition can be a liquid). The amount of isolated myoglobin to be added to pet food can be calculated or estimated based on the concentration of myoglobin in the pet food before addition and the desired final concentration of myoglobin in the pet food. In this context, before adding the isolated myoglobin, the pet food 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 protein by weight.

[0045] In an embodiment, the isolated myoglobin is in the form of a powder (i.e., the composition can be a powder). The amount of isolated myoglobin to be added to pet food can be calculated or estimated based on the concentration of myoglobin in the pet food before addition and the desired final concentration of myoglobin in the pet food. In this context, before adding the isolated myoglobin, the pet food 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 protein by weight. Tools (such as containers, cups, spoons, blenders, mixers) can be used to add the isolated myoglobin. The isolated myoglobin can be added directly to the pet food, or it can be dissolved in an editable solvent and then added to the pet food.

[0046] In an embodiment, the isolated myoglobin is in the form of a solid shape, wherein the solid-shaped myoglobin has a predetermined weight and / or volume. The amount of isolated myoglobin to be added to pet food can 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 pet food, and the desired final amount of myoglobin in the pet food. In this context, prior to adding the isolated myoglobin, the pet food 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% by weight of heme protein. Tools (such as containers, cups, spoons, blenders, mixers) can be used to add the isolated myoglobin. The isolated myoglobin can be added directly to the pet food, or it can be dissolved in a food-grade solvent and then added to the pet food.

[0047] In an embodiment, the pet food is a meat substitute, wherein the isolated myoglobin is added to crosslink with another substance (such as legumin).

[0048] Isolated myoglobin

[0049] In an embodiment, the method of the present invention includes adding isolated myoglobin, wherein the isolated myoglobin is recombinant myoglobin produced by microbial fermentation, and the microorganism has been genetically modified to express the recombinant myoglobin. The myoglobin that can be represented by SEQ ID NO: 1-6 can be encoded by nucleic acids that can be represented by SEQ ID NO: 7-12, respectively, but it should be understood that these nucleic acid sequences may need to be codon-optimized for expression by the microorganism. Example 1 gives more details about the myoglobin used, while Example 2 provides the fermentation of the microorganism (i.e., Pichia pastoris strain).

[0050] In the present application, the numbering of the myoglobin amino acid sequences derived from rat (SEQ ID NO: 1), rabbit (SEQ ID NO: 2), mouse (SEQ ID NO: 3), steppe mammoth (SEQ ID NO: 4), and woolly mammoth (SEQ ID NO: 5) starts from methionine or Met or M at position 1. All of these amino acid sequences SEQ ID NO: 1-5 count 154 amino acid residues.

[0051] The numbering of the myoglobin amino acid sequence (SEQ ID NO:6) derived from tuna used in this text is slightly different from the consecutive numbering indicated in the sequence listing because the sequence only counts 147 amino acid residues. To directly compare SEQ ID NO:6 with SEQ ID NOs: 1-5, SEQ ID NO:6 is also given 154 positions, eight of which are considered gaps (2, 3, 6, 7, 51, 120, 121).

[0052] The microorganism can be a prokaryote, a eukaryote or a filamentous fungus. The prokaryote can be a bacterium. The bacterium can be a Gram-positive / Gram-negative bacterium selected 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,XyIeIIa, 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. lraustophilus, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, Escherichia coli, Corynebacterium glutamicum,Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti, Sphingobium sp., Novosphingobium sp., Sphingomonas henshuiensis, and Rhizobium radiobacter. Preferred bacteria are Escherichia coli. Preferred Escherichia coli strains include: 58, 679, WG1, DH5α, TG1, TOP10, K12, BL21, BL21 DE3, XL1-Blue, XL10-Gold, TB1, REG-12, W945, HB101, DH1, DP50, AB284, JC9387, AG1, C600, Cavalli Hfr, Y10.

[0053] The eukaryote can be yeast or 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. Among the yeasts, the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha (also known as Ogataea henricii), Yarrowia lipolytica, Candida tropicalis, and Pichia (also known as Komagataella phaffii) are preferred. Preferred Pichia strains are selected from the following list: Bg09, Bg10, Bg11, Bg12 (exemplary), Bg20, Bg21, Bg22, Bg23, Bg24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430, X-33, GS115, KM71, SMD1168, SMD1165, MC100-3, with Bg10 and derivatives being most preferred.Preferred yeast strains are selected from the following list: S288C, CEN.PK family, CBS2354, ATCC 2360, ATCC4098, 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. Preferred yeasts are Pichia strains, more preferably Pichia pastoris.

[0054] The filamentous fungus can be selected from the following list, which includes: 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 fungi 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 filamentous fungal strains 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, ATCC 12892, 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; Cephalosporium chrysogenum ATCC 36225 or ATCC 48272; Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921; Aspergillus sojae ATCC 11906; Chrysosporium lucknowense ATCC 44006. In a preferred embodiment, a genus of Aspergillus is used as the filamentous fungus. More preferably, an Aspergillus niger strain is used.

[0055] In an embodiment, the microorganism used in the fermentation can be a bacterium, yeast, filamentous fungus, or a cultured mammalian cell line, preferably Escherichia coli or Saccharomyces cerevisiae. In this context, a single, isolated, cultured mammalian cell can be considered a microorganism. In a further embodiment, the microorganism can be a bacterium, yeast, or filamentous fungus. In the context of the present invention, the microorganism can be used to produce myoglobin. Thus, in a further aspect, the present invention provides a method for producing myoglobin as defined herein, the method comprising culturing a microorganism in a suitable medium and optionally recovering the microorganism and / or myoglobin. Optionally, the produced myoglobin does not contain a signal peptide as defined elsewhere herein.

[0056] Cell culture can be carried out for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11, 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 day, where the duration may have a 20% deviation. Preferably, cell culture is carried out for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11, 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 day, where the duration may have a 10% deviation. More preferably, cell culture is carried out for 5 days, where the duration may have a 20%, most preferably 10% deviation.

[0057] Cell culture will typically result in the 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 myoglobin.

[0058] Cell culture 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 into myoglobin.

[0059] Cell culture can also be carried out by implementing a multi-step, preferably two-step, culture method. For example, a cell biomass growth step can be carried out before the myoglobin production step, where only limited production or no production occurs. Different steps can be carried out using different culture modes and / or different growth media and / or different culture process parameter values, depending on the objective of each step and / or the cells being cultured. The biomass during the production step may or may not grow actively.

[0060] The host cells and / or myoglobin can optionally be recovered from the culture medium. When present intracellularly, myoglobin can optionally be recovered from the recovered cell biomass. Optionally, the recovered myoglobin is purified. Preferably, the purification of myoglobin will result in a purity of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and most preferably substantially pure myoglobin.

[0061] In the examples, the microorganism produces myoglobin extracellularly. Myoglobin is transported out of the host cell after its synthesis in the host cell. In this context, both secretion and extracellular fermentation are considered extracellular production. Without being bound by this theory, the extracellular production method has the advantages that the downstream processing for recovering the produced myoglobin is more convenient, more efficient, and / or effective. In addition, compared to the intracellular method where myoglobin is not transported out of the host cell after its synthesis, the extracellular production method can produce a composition containing high-purity (such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) myoglobin with minimal downstream processing. The purity can be measured as the weight percentage of the total protein fraction in the cell-free supernatant obtained at the end of the method according to the invention or the extracellular method. Without being bound by this theory, the extracellular production method has the advantage that the optional step of recovering myoglobin does not include lysing the host cells. Thus, the extracellular method can produce a composition with a low concentration of nucleic acids derived from the host cell.

[0062] The relevant downstream processing techniques that may be applicable for recovery and / or purification will depend on whether myoglobin accumulates within the cultured cells or is secreted. Such processing techniques and the associated options will be known to the person skilled in the art and are discussed, for example, in the following literature: Wesselingh, J.A and Krijgsman, J., 1st edition, Downstream Processing in Biotechnology, Delft Academic Press, NL, 2013. During the recovery process, biomass can be recovered from the culture medium using, for example, centrifugation or filtration. If the produced myoglobin accumulates within the cells, it can be recovered and / or purified from the biomass. If myoglobin is secreted, 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 can be carried out according to any conventional recovery or purification method known in the art. Methods for the recovery and / or purification of proteins are known to the person skilled in the art and are discussed in standard manuals such as Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001; or Ausubel F. et al. eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 2003. Examples of widely used recovery and / or purification methods include chromatography, such as gel filtration chromatography, ion exchange chromatography, immunoaffinity chromatography, metal affinity chromatography, gel filtration chromatography, fractionation using 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 can optionally be enhanced by linking the enzyme polypeptide to a sequence that facilitates purification, such as a GST domain, using well-known molecular toolbox techniques. Optionally, the sequence that facilitates purification and / or the signal peptide that facilitates myoglobin secretion are removed from the final product using techniques known in the art, such as proteolysis by an endopeptidase that targets the linker between the sequence that facilitates purification and / or the signal peptide and myoglobin.In some embodiments, the enzyme polypeptide is linked (fused) to a hexa-histidine peptide, such as the tag provided in the pET23a(+) vector (Genescript Biotech, Leiden, the Netherlands), many of which are commercially available. As described, for example, in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), the hexa-histidine peptide provides convenient purification of the fusion protein.

[0063] In preferred embodiments, isolated myoglobin is obtained via recovery and / or purification from the culture medium. This can be achieved continuously during the production process or after the production process. In preferred embodiments, isolated myoglobin is obtained via recovery and / or purification from cultured cells. The recovered myoglobin can be purified using a filter. This can be achieved continuously during the production process by harvesting fractions of the growing cells or after the production process.

[0064] In embodiments, the isolated myoglobin is sterilized, minced, spray dried, freeze dried, blended, shaped, cubed, dosed or packaged. Sterilization refers to any process that removes, kills or inactivates all forms of life (especially microorganisms such as fungi, bacteria, spores and single-celled eukaryotes) and other biological agents such as prions present in or on a particular surface, object or fluid. Sterilization can be achieved in various ways, including heating, chemicals, irradiation, high pressure and filtration. Freeze drying, also known as lyophilization or cryodesiccation, is a low-temperature dehydration method that involves freezing the product, reducing the pressure and then removing the ice by sublimation. Packaging is intended to provide protection for the product, prevent tampering, and provide for physical, chemical or biological requirements. The packaging can also contain nutritional labels, product characteristics and instructions / guidelines for using the product.

[0065] In a preferred embodiment, the cultured host cells used in the fermentation are immobilized. Immobilization of the cells can be achieved by any means known to those skilled in the art, as discussed in standard manuals, such as Guisan, J.M., Bolivar, J.M., López-Gallego, F., Rocha-Martín, J. (eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020. Generally, host cells can be immobilized onto a semi-solid or solid support by three different methods. The first method involves polymerizing or solidifying a solution containing spores or cells. Examples of polymerizable or curable solutions include alginate, λ-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinylbenzene, polyvinylbenzene, polyvinyl alcohol, epoxy supports, cellulose, cellulose acetate, photocrosslinkable resins, prepolymers, polyurethanes, and gelatin. The second method involves cell adsorption onto a support. Examples of such supports include bone char, cork, clay, resins, sand, porous alumina beads, porous bricks, porous silica, diatomaceous earth, or wood chips. The host cells can colonize on the support and form a biofilm. The third method involves covalently coupling the host cells to the support using chemical reagents such as glutaraldehyde, o-dianisidine (U.S. Patent No. 3,983,000), polymeric isocyanates (U.S. Patent No. 4,071,409), silanes (U.S. Patent Nos. 3,519,538 and 3,652,761), 2-hydroxyethyl acrylate, transition metal-activated supports, cyanuric chloride, sodium periodate, toluene, etc. The cultured host cells can be immobilized at any stage of their growth, for example, after reaching the desired cell density in the culture. Suitable culture modes and / or different values of culture process parameters will be known to those skilled in the art and are discussed in standard manuals, 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, the immobilized cells are cultured in a packed bed bioreactor, also known as a plug flow bioreactor or an expanded (fluidized) bed bioreactor. Suitable growth media and recovery and / or purification methods are further discussed elsewhere herein.

[0066] In an embodiment, the isolated myoglobin is a myoglobin that was not included in a cell or tissue prior to addition. In other words, the isolated myoglobin is added as a "separate" (or isolated) protein not included in a cell or tissue. Thus, in this context, "isolated" does not refer to the source of the myoglobin (i.e., isolated from a source such as a cell or tissue), but rather to the fact that a separate protein is added. It should of course be understood that once the isolated myoglobin has been added to the pet food, the isolated myoglobin can become part of a cell or tissue.

[0067] The isolated myoglobin can be derived from a rat, rabbit, mouse, mammoth, or tuna. Preferably, the amino acid sequence is derived from a rat, rabbit, mouse, mammoth, or tuna by the addition, deletion, and / or substitution of at least one amino acid. The present invention also contemplates the addition, deletion, and / or substitution of two, three, four, five, six, seven, eight, nine, or ten amino acids. Examples of myoglobin amino acid sequences from rats, rabbits, mice, mammoths, or tuna are later disclosed by given SEQ ID NO: 1-6. Such isolated myoglobin derived from a rat, rabbit, mouse, mammoth, or tuna can also exhibit the activity of isolated myoglobin at least at a detectable level, as explained later herein. In the context of this application, unless otherwise expressly stated, a mammoth can be any species in the genus Mammuthus, such as the woolly mammoth (Mammuthus primigenius) or the steppe mammoth (Mammuthus trogontherii).

[0068] In an embodiment, the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, mammoth myoglobin, or tuna myoglobin, or a myoglobin derived from any one of these myoglobins. In an embodiment, the isolated myoglobin is rat myoglobin or a myoglobin derived from rat myoglobin. In an embodiment, the isolated myoglobin is rabbit myoglobin or a myoglobin derived from rabbit myoglobin. In an embodiment, the isolated myoglobin is mouse myoglobin or a myoglobin derived from mouse myoglobin. In an embodiment, the isolated myoglobin is steppe mammoth myoglobin or a myoglobin derived from steppe mammoth myoglobin. In an embodiment, the isolated myoglobin is woolly mammoth myoglobin or a myoglobin derived from woolly mammoth myoglobin. In an embodiment, the isolated myoglobin is tuna myoglobin or a myoglobin derived from tuna myoglobin.

[0069] In an embodiment, the isolated myoglobin can be represented by any one of SEQ ID NO: 1, 2, 3, 4, 5 or 6, preferably represented by SEQ ID NO: 1 or 2.

[0070] In an embodiment, the isolated myoglobin is from a rat as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 1, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 1 and combined with the following: G at position 6, Q at position 9, M at position 10, N at position 13, I at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, K at position 35, A at position 36, E at position 42, K at position 43, N at position 49, S at position 52, E at position 53, E at position 54, E at position 55, S at position 58, H at position 65, C at position 67, A at position 72, T at position 75, K at position 79, Q at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, V at position 111, Q at position 114, L at position 116, K at position 117, K at position 118, R at position 119, S at position 121, G at position 122, D at position 123, A at position 128, G at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0071] In an embodiment, the isolated myoglobin is from a rabbit as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:2, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:2 and combined with the following: A at position 6, Q at position 9, L at position 10, N at position 13, V at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, H at position 35, T at position 36, E at position 42, K at position 43, H at position 49, S at position 52, E at position 53, D at position 54, E at position 55, A at position 58, H at position 65, N at position 67, A at position 72, A at position 75, K at position 79, H at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, A at position 111, H at position 114, L at position 116, H at position 117, S at position 118, R at position 119, P at position 121, G at position 122, D at position 123, A at position 128, A at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0072] In an embodiment, the isolated myoglobin is from a mouse as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:3, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:3 and combined with the following: G at position 6, Q at position 9, L at position 10, N at position 13, V at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, K at position 35, T at position 36, D at position 42, K at position 43, N at position 49, S at position 52, E at position 53, E at position 54, D at position 55, G at position 58, H at position 65, C at position 67, A at position 72, T at position 75, K at position 79, Q at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, I at position 111, E at position 114, L at position 116, K at position 117, K at position 118, R at position 119, S at position 121, G at position 122, D at position 123, A at position 128, G at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0073] In an embodiment, the isolated myoglobin is from a steppe mammoth as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:4, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:4 and combined with the following: G at position 6, E at position 9, L at position 10, K at position 13, T at position 14, I at position 22, P at position 23, L at position 27, F at position 30, V at position 31, T at position 35, G at position 36, E at position 42, K at position 43, H at position 49, T at position 52, E at position 53, G at position 54, E at position 55, A at position 58, Q at position 65, V at position 67, A at position 72, G at position 75, K at position 79, H at position 82, Q at position 84, A at position 85, I at position 87, Q at position 88, P at position 89, H at position 92, S at position 93, T at position 96, I at position 102, D at position 110, A at position 111, H at position 114, L at position 116, Q at position 117, S at position 118, P at position 121, A at position 122, E at position 123, A at position 128, G at position 130, K at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0074] In an embodiment, the isolated myoglobin is from a woolly mammoth as defined above, preferably a myoglobin having at least 90% sequence identity to SEQ ID NO:5, more preferably a myoglobin having at least 90% sequence identity to SEQ ID NO:5 and combined with the following: G at position 6, E at position 9, L at position 10, K at position 13, T at position 14, I at position 22, P at position 23, L at position 27, F at position 30, V at position 31, T at position 35, G at position 36, E at position 42, K at position 43, H at position 49, T at position 52, E at position 53, G at position 54, E at position 55, A at position 58, Q at position 65, V at position 67, A at position 72, G at position 75, K at position 79, H at position 82, Q at position 84, A at position 85, I at position 87, Q at position 88, P at position 89, Q at position 92, S at position 93, T at position 96, I at position 102, D at position 110, A at position 111, H at position 114, L at position 116, Q at position 117, S at position 118, P at position 121, A at position 122, E at position 123, A at position 128, G at position 130, K at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0075] In an embodiment, the isolated myoglobin is from tuna as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:6, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:6 and combined with the following: D at position 9, A at position 10, K at position 13, C at position 14, Y at position 22, T at position 23, G at position 27, L at position 30, T at position 31, K at position 35, E at position 36, K at position 42, L at position 43, G at position 49, A at position 52, Q at position 53, A at position 54, D at position 55, G at position 58, H at position 65, A at position 67, K at position 72, E at position 75, A at position 79, S at position 82, A at position 84, A at position 85, L at position 87, K at position 88, P at position 89, N at position 92, S at position 93, T at position 96, I at position 102, E at position 110, V at position 111, K at position 114, M at position 116, H at position 117, E at position 118, A at position 122, G at position 123, Q at position 128, T at position 130, R at position 133, L at position 143, A at position 145, N at position 146, E at position 149, L at position 150 or S at position 153.

[0076] In an embodiment, the isolated myoglobin can be represented by one of the following amino acid sequences:

[0077] a) having at least 90% sequence identity with SEQ ID NO:1 and having at least one of the following amino acid combinations:

[0078] - M at position 10, and / or

[0079] - A at position 36, and / or

[0080] - S at position 58, and / or

[0081] - M at position 10 and A at position 36, and / or

[0082] - M at position 10 and S at position 58, and / or

[0083] - A at position 36 and S at position 58, and / or

[0084] - M at position 10 and A at position 36 and S at position 58;

[0085] b) having at least 90% sequence identity with SEQ ID NO:2 and having amino acid A at position 6;

[0086] c) having at least 90% sequence identity with SEQ ID NO:3 and having at least one of the following amino acid combinations:

[0087] - D at position 55, and / or

[0088] - I at position 111, and / or

[0089] - E at position 114, and / or

[0090] - D at position 55 and I at position 111, and / or

[0091] - D at position 55 and E at position 114, and / or

[0092] - I at position 111 and E at position 114, and / or

[0093] - D at position 55, I at position 111 and E at position 114;

[0094] d) having at least 90% identity with SEQ ID NO:4 or 5 and having F at position 30 and / or Q at position 65;

[0095] e) having at least 90% identity with SEQ ID NO:6 and having H at position 65 and / or position 94;

[0096] f) having at least 90% identity with SEQ ID NO:1, 2 or 3 and having at least one of the following amino acid combinations:

[0097] - L at position 22, and / or

[0098] - A or T at position 36, and / or

[0099] - G at position 122;

[0100] - L at position 22 and A or T at position 36, and / or

[0101] - L at position 22 and G at position 122; and / or

[0102] - A or T at position 36 and G at position 122, and / or

[0103] - L at position 22, A or T at position 36 and G at position 122;

[0104] g) having at least 90% identity to any one of SEQ ID NO: 1 to 5 and having I at position 87;

[0105] h) having at least 90% identity to SEQ ID NO: 1 or 3 and having at least one or more of the following amino acids or any combination thereof:

[0106] - N at position 49, and / or

[0107] - E at position 54, and / or

[0108] - C at position 67, and / or

[0109] - T at position 75, and / or

[0110] - Q at position 82, and / or

[0111] - K at position 117, and / or

[0112] - K at position 118, and / or

[0113] - S at position 121;

[0114] i) having at least 90% identity to SEQ ID NO: 4 and having at least one of the following amino acid combinations:

[0115] - H at position 92, and / or

[0116] - H at position 92 and F at position 30, and / or

[0117] - H at position 92 and Q at position 65, and / or

[0118] - H at position 92, F at position 30 and Q at position 65.

[0119] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin is from a rat as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:1, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:1 and combined with the following: G at position 6, Q at position 9, M at position 10, N at position 13, I at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, K at position 35, A at position 36, E at position 42, K at position 43, N at position 49, S at position 52, E at position 53, E at position 54, E at position 55, S at position 58, H at position 65, C at position 67, A at position 72, T at position 75, K at position 79, Q at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, V at position 111, Q at position 114, L at position 116, K at position 117, K at position 118, R at position 119, S at position 121, G at position 122, D at position 123, A at position 128, G at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0120] In an embodiment, the isolated myoglobin is recombinant myoglobin, where the recombinant myoglobin is from a rabbit as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:2, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:2 and combined with the following: A at position 6, Q at position 9, L at position 10, N at position 13, V at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, H at position 35, T at position 36, E at position 42, K at position 43, H at position 49, S at position 52, E at position 53, D at position 54, E at position 55, A at position 58, H at position 65, N at position 67, A at position 72, A at position 75, K at position 79, H at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, A at position 111, H at position 114, L at position 116, H at position 117, S at position 118, R at position 119, P at position 121, G at position 122, D at position 123, A at position 128, A at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0121] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin is from a mouse as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:3, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:3 and combined with the following: G at position 6, Q at position 9, L at position 10, N at position 13, V at position 14, L at position 22, A at position 23, Q at position 27, L at position 30, I at position 31, G at position 32, K at position 35, T at position 36, D at position 42, K at position 43, N at position 49, S at position 52, E at position 53, E at position 54, D at position 55, G at position 58, H at position 65, C at position 67, A at position 72, T at position 75, K at position 79, Q at position 82, Q at position 92, S at position 93, T at position 96, V at position 102, E at position 110, I at position 111, E at position 114, L at position 116, K at position 117, K at position 118, R at position 119, S at position 121, G at position 122, D at position 123, A at position 128, G at position 130, S at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0122] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin is from a woolly mammoth as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 4, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 4 and combined with the following: G at position 6, E at position 9, L at position 10, K at position 13, T at position 14, I at position 22, P at position 23, L at position 27, F at position 30, V at position 31, T at position 35, G at position 36, E at position 42, K at position 43, H at position 49, T at position 52, E at position 53, G at position 54, E at position 55, A at position 58, Q at position 65, V at position 67, A at position 72, G at position 75, K at position 79, H at position 82, Q at position 84, A at position 85, I at position 87, Q at position 88, P at position 89, H at position 92, S at position 93, T at position 96, I at position 102, D at position 110, A at position 111, H at position 114, L at position 116, Q at position 117, S at position 118, P at position 121, A at position 122, E at position 123, A at position 128, G at position 130, K at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0123] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin is from a woolly mammoth as defined above, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:5, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO:5 and combined with the following: G at position 6, E at position 9, L at position 10, K at position 13, T at position 14, I at position 22, P at position 23, L at position 27, F at position 30, V at position 31, T at position 35, G at position 36, E at position 42, K at position 43, H at position 49, T at position 52, E at position 53, G at position 54, E at position 55, A at position 58, Q at position 65, V at position 67, A at position 72, G at position 75, K at position 79, H at position 82, Q at position 84, A at position 85, I at position 87, Q at position 88, P at position 89, Q at position 92, S at position 93, T at position 96, I at position 102, D at position 110, A at position 111, H at position 114, L at position 116, Q at position 117, S at position 118, P at position 121, A at position 122, E at position 123, A at position 128, G at position 130, K at position 133, I at position 143, A at position 145, K at position 146, E at position 149, L at position 150 or Q at position 153.

[0124] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin is from tuna as defined above, preferably myoglobin having at least 90% sequence identity with SEQ ID NO:6, more preferably myoglobin having at least 90% sequence identity with SEQ ID NO:6 and combined with the following: D at position 9, A at position 10, K at position 13, C at position 14, Y at position 22, T at position 23, G at position 27, L at position 30, T at position 31, K at position 35, E at position 36, K at position 42, L at position 43, G at position 49, A at position 52, Q at position 53, A at position 54, D at position 55, G at position 58, H at position 65, A at position 67, K at position 72, E at position 75, A at position 79, S at position 82, A at position 84, A at position 85, L at position 87, K at position 88, P at position 89, N at position 92, S at position 93, T at position 96, I at position 102, E at position 110, V at position 111, K at position 114, M at position 116, H at position 117, E at position 118, A at position 122, G at position 123, Q at position 128, T at position 130, R at position 133, L at position 143, A at position 145, N at position 146, E at position 149, L at position 150 or S at position 153.

[0125] In an embodiment, the isolated myoglobin is recombinant myoglobin, wherein the recombinant myoglobin can be represented by one of the following amino acid sequences:

[0126] a) having at least 90% sequence identity with SEQ ID NO:1 and having at least one of the following amino acid combinations:

[0127] - M at position 10, and / or

[0128] - A at position 36, and / or

[0129] - S at position 58, and / or

[0130] - M at position 10 and A at position 36, and / or

[0131] - M at position 10 and S at position 58, and / or

[0132] - A at position 36 and S at position 58, and / or

[0133] - M at position 10 and A at position 36 and S at position 58;

[0134] b) having at least 90% sequence identity with SEQ ID NO:2 and having amino acid A at position 6;

[0135] c) having at least 90% sequence identity with SEQ ID NO:3 and having at least one of the following amino acid combinations:

[0136] - D at position 55, and / or

[0137] - I at position 111, and / or

[0138] - E at position 114, and / or

[0139] - D at position 55 and I at position 111, and / or

[0140] - D at position 55 and E at position 114, and / or

[0141] - I at position 111 and E at position 114, and / or

[0142] - D at position 55, I at position 111 and E at position 114;

[0143] d) having at least 90% identity with SEQ ID NO:4 or 5 and having F at position 30 and / or Q at position 65;

[0144] e) having at least 90% identity with SEQ ID NO:6 and having H at position 65 and / or position 94;

[0145] f) having at least 90% identity with SEQ ID NO:1, 2 or 3 and having at least one of the following amino acid combinations:

[0146] - L at position 22, and / or

[0147] - A or T at position 36, and / or

[0148] - G at position 122;

[0149] - L at position 22 and A or T at position 36, and / or

[0150] - L at position 22 and G at position 122; and / or

[0151] - A or T at position 36 and G at position 122, and / or

[0152] - L at position 22, A or T at position 36 and G at position 122;

[0153] g) having at least 90% identity to any one of SEQ ID NOs: 1 to 5 and having an I at position 87;

[0154] h) having at least 90% identity to SEQ ID NO: 1 or 3 and having at least one or more of the following amino acids or any combination thereof:

[0155] - N at position 49, and / or

[0156] - E at position 54, and / or

[0157] - C at position 67, and / or

[0158] - T at position 75, and / or

[0159] - Q at position 82, and / or

[0160] - K at position 117, and / or

[0161] - K at position 118, and / or

[0162] - S at position 121.

[0163] In embodiments, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO: 1 and at least one combination of the following amino acid combinations:

[0164] - M at position 10, and / or

[0165] - A at position 36, and / or

[0166] - S at position 58, and / or

[0167] - M at position 10 and A at position 36, and / or

[0168] - M at position 10 and S at position 58, and / or

[0169] - A at position 36 and S at position 58, and / or

[0170] - M at position 10, A at position 36, and S at position 58.

[0171] In an embodiment, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO:2 and having amino acid A at position 6.

[0172] In an embodiment, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO:3 and in combination with at least one of the following amino acid combinations:

[0173] - D at position 55, and / or

[0174] - I at position 111, and / or

[0175] - E at position 114, and / or

[0176] - D at position 55 and I at position 111, and / or

[0177] - D at position 55 and E at position 114, and / or

[0178] - I at position 111 and E at position 114, and / or

[0179] - D at position 55, I at position 111, and E at position 114.

[0180] In an embodiment, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO:4 or 5 and having an F at position 30 and / or a Q at position 65.

[0181] In an embodiment, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO:6 and having an H at position 65 and / or at position 94.

[0182] In an embodiment, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO:1, 2 or 3 and in combination with at least one of the following amino acid combinations:

[0183] - an L at position 22, and / or

[0184] - an A or T at position 36, and / or

[0185] - a G at position 122;

[0186] - L at position 22 and A or T at position 36, and / or

[0187] - L at position 22 and G at position 122; and / or

[0188] - A or T at position 36 and G at position 122, and / or

[0189] - L at position 22, A or T at position 36, and G at position 122.

[0190] In embodiments, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO: 1 to 5 and having I at position 87.

[0191] In embodiments, the amino acid sequence of the isolated myoglobin or preferably recombinant myoglobin identified above comprises a sequence having 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% identity or similarity to SEQ ID NO: 1 or 3 and having at least one or more of the following amino acids or any combination thereof:

[0192] - N at position 49, and / or

[0193] - E at position 54, and / or

[0194] - C at position 67, and / or

[0195] - T at position 75, and / or

[0196] - Q at position 82, and / or

[0197] - K at position 117, and / or

[0198] - K at position 118, and / or

[0199] - S at position 121.

[0200] Pet food

[0201] In a second aspect of the present invention, there is provided a pet food obtainable by the methods described herein. The pet food according to this aspect contains myoglobin, wherein the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, mammoth myoglobin, tuna myoglobin or a combination of any two or three or four or five of said myoglobins.

[0202] All preferences and embodiments listed for the method for preparing pet food according to the first aspect of the present invention can be applied, with necessary modifications, to the pet food of the second aspect of the present invention. For example, any specification of the sequence of the isolated myoglobin added during the method can be applied to the sequence of the myoglobin contained in the pet food of the second aspect. This is obvious because the pet food of the second aspect can be obtained by the method of the first aspect.

[0203] In an embodiment, the pet food, which is preferably a meat substitute, does not contain one or more milk proteins selected from the group consisting of: β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and transferrin, wherein said milk proteins are from woolly mammoth. In another embodiment, the meat substitute does not contain one or more milk proteins selected from the group consisting of: β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and transferrin, wherein said milk proteins are from mammoth.

[0204] In an embodiment, the pet food, which is preferably a meat substitute, does not contain one or more milk proteins selected from the group consisting of: β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and transferrin, wherein said milk proteins are not produced by mammals or mammalian cells.

[0205] In a more preferred embodiment, the pet food, which is preferably a meat substitute, does not contain milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin, wherein the milk proteins are from woolly mammoths. In another more preferred embodiment, the meat substitute does not contain milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin, wherein the milk proteins are from mammoths.

[0206] In another more preferred embodiment, the pet food, which is preferably a meat substitute, does not contain milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin, wherein the milk proteins are not produced by mammals or mammalian cells.

[0207] In an embodiment, the pet food, which is preferably a meat substitute, does not contain κ-casein from woolly mammoths. In a further embodiment, the meat substitute does not contain a protein represented by a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:13. In a further embodiment, the pet food, which is preferably a meat substitute, does not contain the protein represented by SEQ ID NO:13.

[0208] In an embodiment, the pet food, which is preferably a meat substitute, does not contain β-casein from woolly mammoths. In a further embodiment, the meat substitute does not contain a protein represented by a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:14. In a further embodiment, the pet food, which is preferably a meat substitute, does not contain the protein represented by SEQ ID NO:14.

[0209] In an embodiment, the pet food, which is preferably a meat substitute, does not contain symbiotic hemoglobin produced by bacteria living symbiotically in soybean plant nodules. In an embodiment, the pet food, which is preferably a meat substitute, does not contain leghemoglobin. In an embodiment, the isolated myoglobin disclosed herein can be the sole source of the heme-containing protein present in the meat substitute. This means that the meat substitute can contain other proteins in addition to the isolated myoglobin as disclosed herein. Examples of proteins that may be present include soy proteins.

[0210] In the context of the present invention, a heme-containing protein as defined can refer to all proteins or protein subunits capable of covalently or non-covalently binding a heme moiety. Heme-containing polypeptides can transport or store oxygen. Some examples of heme-containing proteins include globin, hemoglobin, leghemoglobin.

[0211] In an embodiment, a pet food, preferably a meat substitute, is prepared by mixing (i.e., adding) the following: 10% to up to 40% of a protein source, 5% to up to 30% of a lipid source, and 0.05% to up to 5%, preferably 0.1% to up to 5% of isolated myoglobin. In a more preferred embodiment, a pet food, preferably a meat substitute, is prepared by mixing the following: 20% to up to 30% of a protein source, 10% to up to 20% of a lipid source, and 0.05% to up to 3%, preferably 0.1% to up to 3% of isolated myoglobin.

[0212] In an embodiment, a pet food, preferably a meat substitute, is prepared by mixing (i.e., adding) the following: 10% to up to 40% of a protein source, 5% to up to 30% of a lipid source, 0.1% to up to 5% of NaCl, 0.1% to up to 5% of a fiber source, and 0.05% to up to 5%, preferably 0.1% to up to 5% of isolated myoglobin. In a more preferred embodiment, a pet food, preferably a meat substitute, is prepared by mixing the following: 20% to up to 30% of a protein source, 10% to up to 20% of a lipid source, 0.5% to up to 2% of NaCl, 0.5% to up to 1.5% of a fiber source, and 0.05 to up to 3%, preferably 0.1% to up to 3% of isolated myoglobin.

[0213] Preferably, the protein source is textured soy protein, soy protein powder, concentrate or isolate, pea protein powder, concentrate or isolate, broad bean protein, mung bean protein, textured wheat protein, rice protein or potato protein. In this context, textured soy protein is a defatted soy flour product, also known as textured vegetable protein (TVP) or soy meat, which is clear to the person skilled in the art. Preferably, the lipid source is sunflower oil, coconut oil, rapeseed oil or cocoa butter, or any vegetable oil. Preferably, the fiber source is methylcellulose or potato starch.

[0214] In a more preferred embodiment, the pet food, which is preferably a meat substitute, is prepared by mixing: 20% to a maximum of 30% of textured soy protein, 10% to a maximum of 20% of sunflower vegetable oil, 0.5% to a maximum of 2% of NaCl, 0.5% to a maximum of 1.5% of methylcellulose, and 0.05% to a maximum of 5%, preferably 0.1% to a maximum of 5%, of isolated myoglobin. Even more preferably, the weight percentage of isolated myoglobin in the meat substitute according to the more preferred embodiment is 0.05% to a maximum of 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%. Most preferably, the weight percentage of isolated myoglobin in the meat substitute according to the more preferred embodiment is 0.1% to a maximum of 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%.

[0215] In an embodiment, pet food that is preferably a meat substitute is prepared by mixing: 20% to up to 30% of textured soy protein, 10% to up to 20% of sunflower oil, 1% to up to 2% of NaCl, 0.5% to up to 1.5% of methylcellulose, 50% to up to 70% of water, and 0.05% to up to 1%, preferably 0.2% to up to 0.8%, of isolated myoglobin. In a preferred embodiment, pet food that is preferably a meat substitute is prepared by mixing: 23% to up to 27% of textured soy protein, 13% to up to 17% of sunflower oil, 1.25% to up to 1.75% of NaCl, 0.75% to up to 1.25% of methylcellulose, 55% to up to 60% of water, and 0.45% to up to 0.55% of isolated myoglobin.

[0216] In an embodiment, pet food that is preferably a meat substitute is prepared by mixing: 25% of textured soy protein, 15% of sunflower oil, 1.5% of NaCl, 1.0% of methylcellulose, 57% of water, and 0.5% of isolated myoglobin, where these weight percentages are rounded to two significant figures.

[0217] In an embodiment, pet food that is preferably a meat substitute is prepared by mixing: 15% to up to 35% of textured soy protein, 10% to up to 20% of sunflower oil, 0.5% to up to 2.5% of NaCl, 0.5% to up to 1.5% of methylcellulose, 50% to up to 70% of water, and 0.5% to up to 1.5% of isolated myoglobin. In a preferred embodiment, pet food that is preferably a meat substitute is prepared by mixing: 22% to up to 27% of textured soy protein, 13% to up to 17% of sunflower oil, 1.25% to up to 1.75% of NaCl, 0.75% to up to 1.25% of methylcellulose, 50% to up to 60% of water, and 0.75% to up to 1.25% of isolated myoglobin.

[0218] In an embodiment, pet food that is preferably a meat substitute is prepared by mixing: 25% of textured soy protein, 15% of sunflower oil, 1.5% of NaCl, 1.0% of methylcellulose, 57% of water, and 1.0% of isolated myoglobin, where these weight percentages are rounded to two significant figures.

[0219] In the above meat substitute, % refers to weight percentage. If the sum of the weight percentages describing the composition of the meat substitute is less than 100%, it is assumed that water is added to the corresponding percentage.

[0220] In an embodiment, a pet food that is preferably a meat substitute contains a bioavailable iron or heme iron in an amount similar to that of the corresponding real meat. In an embodiment, this amount ranges from 0.3 mg to 20 mg of bioavailable iron or heme iron per 100 g of pet food. In an embodiment, this amount ranges from 0.1 mg to a maximum of 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, 11.22 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 of bioavailable iron or heme iron per 100 g of pet food.In another embodiment, this 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, 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, 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, 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, 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 of bioavailable iron or heme iron / 100 g of pet food.

[0221] Example 4 shows that the iron bioavailability of pet food is higher than that of the corresponding pet food supplemented with non-heme iron. Thus, the animal myoglobin produced herein can be used to supplement plant-based meat alternatives with the same level of bioavailable heme iron as the conventional meat products it is mimicking, regardless of the presence of inhibitory plant-derived polyphenols.

[0222] General term

[0223] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are read in light of this disclosure.

[0224] In this document and its claims, the verb “to comprise” and its conjugations are used in their non-limiting sense to mean including the item(s) after the word, but not excluding items not specifically mentioned. Further, the verb “consisting of” may be replaced by “consisting essentially of,” meaning that a method as described herein may include one or more additional steps in addition to the specifically specified steps, the one or more additional steps not changing the unique characteristics of the invention. Further, the verb “consisting of” may be replaced by “consisting essentially of,” meaning that a pet food, meat alternative, isolated myoglobin, gene construct, host cell (or method) as described herein may include one or more additional components (or additional steps) in addition to the specifically identified components, the one or more additional components not changing the unique characteristics of the invention.

[0225] The mention of an element by the indefinite article “a” or “an” does not exclude the possibility of there being more than one of the element, unless the context clearly requires that there be one and only one of the element. Thus, the indefinite article “a” or “an” generally means “at least one.” As used herein, a specific value with “at least” means that specific 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... and so on.

[0226] In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and not necessarily to describe an order or a temporal sequence. It should be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein. The word "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be the given value (10) plus or minus 0.1% of that value. As used herein, the term "and / or" indicates that one or more of the stated circumstances can occur alone or in combination with at least one of the stated circumstances up to and including all of the stated circumstances.

[0227] In the context of this application, all percentages in the context of concentration or composition refer to weight percentages unless otherwise defined. In the context of this application, an expression such as "a parameter having a value of at least X, Y, or Z" should be construed to mean the stated parameter having a value of at least X, at least Y, or at least Z.

[0228] Various embodiments are described herein. Unless otherwise stated, each embodiment described herein can be combined together. All patent applications, patents, and printed publications cited herein are incorporated herein by reference in their entirety, except for any definitions, subject matter disclaimers, or disavowals, and to the extent that the incorporated material is inconsistent with the content expressly disclosed herein, in which case the language of this disclosure shall govern. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the invention. Indeed, the invention is in no way limited to the methods and materials described. The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0229] Sequence identity

[0230] In the context of the present invention, a nucleic acid molecule such as a nucleic acid molecule encoding a myoglobin isolate is represented by a nucleic acid or nucleotide sequence encoding a protein fragment or polypeptide or peptide or derivative peptide. It should be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derivative peptide or construct identified herein by a given sequence identifier number (SEQ ID NO) is not limited to the specific sequence so disclosed. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derivative peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derivative peptide.

[0231] Throughout this application, each time a specific nucleotide sequence SEQ ID NO encoding a given protein fragment or polypeptide or peptide or derivative peptide is mentioned (by way of example, SEQ ID NO:X), it can be replaced with:

[0232] i. A nucleotide sequence that comprises a nucleotide sequence having at least 60% sequence identity with SEQ ID NO:X; or

[0233] ii. A nucleotide sequence whose sequence differs from the sequence of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or

[0234] iii. A nucleotide sequence that encodes an amino acid sequence having at least 60% amino acid identity or similarity to the amino acid sequence encoded by the nucleotide sequence SEQ ID NO:X.

[0235] 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%.

[0236] Throughout this application, each time a specific amino acid sequence SEQ ID NO (taking SEQ ID NO:Y as an example) is mentioned, it can be replaced with: a polypeptide represented by an amino acid sequence comprising a sequence having at least 60% sequence identity or similarity with the 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%.

[0237] In a further preferred embodiment, each nucleotide sequence or amino acid sequence described herein has 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% identity or similarity to a given nucleotide sequence or amino acid sequence, respectively, based on their percent identity or similarity to the given nucleotide or amino acid sequence.

[0238] The terms "homology", "sequence identity", etc. are used interchangeably herein. "Sequence identity" is described herein as the 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 or a portion of two given SEQ ID NOs. The portion is preferably at least 50%, 60%, 70%, 80%, 90% or 100% of the two SEQ ID NOs. 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 matches between strings of these sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in the following documents: 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 of which is incorporated herein by reference.

[0239] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch), which optimally aligns the sequences over their entire length, while sequences with a very large difference in length are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). When sequences (when optimally aligned using, for example, the programs EMBOSS needle or EMBOSS water with default parameters) share at least a certain minimum percentage of sequence identity (as described below), these sequences can be referred to as "substantially identical" or "substantially similar".

[0240] Global alignment is suitable for determining sequence identity when two sequences have similar lengths. When the sequences have significantly different total lengths, 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), thus maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water are used, where the gap opening penalty = 10 (nucleotide sequences) / 10 (proteins), and the gap extension penalty = 0.5 (nucleotide sequences) / 0.5 (proteins). For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0241] Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can be further used as "query sequences" to search public databases, for example, to 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. [Journal of Molecular Biology] 215: 403-10 (incorporated herein by reference). BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the redox enzyme nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTx program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized, as described in Altschul et al., (1997) Nucleic Acids Res. [Nucleic Acids Research] 25(17): 3389-3402 (incorporated herein by reference). When using the 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 / .

[0242] Optionally, when determining amino acid similarity, a person skilled in the art can also consider so-called conservative amino acid substitutions. As used herein, a "conservative" amino acid substitution refers to the interchangeability of residues having similar side chains. The following table gives examples of categories of amino acid residues for conservative substitutions.

[0243]

[0244] Alternative categories of conservative amino acid residue substitutions:

[0245] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0246] Alternative physical and functional classifications of amino acid residues:

[0247]

[0248]

[0249] For example, a group of amino acids having aliphatic side chains are: glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxy side chains are serine and threonine; a group of amino acids having amide-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains are lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu.

[0250] Proteins and Amino Acids

[0251] The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to a molecule consisting of a chain of amino acids, without regard to a particular mode of action, size, three-dimensional structure, or source. In the amino acid sequences described herein, the amino acids or "residues" are represented by three-letter symbols. These three-letter symbols, as well as the corresponding single-letter symbols, are well known to those of skill in the art and have the following meanings: 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 (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. A residue can be any protein amino acid, any non-protein amino acid, such as D-amino acids and modified amino acids formed by post-translational modification, and any non-natural amino acid, as described herein.

[0252] Gene or coding sequence

[0253] The term "gene" means a DNA fragment that contains a region (the transcribed region) that is operably linked to a suitable regulatory region (e.g., a promoter) and is transcribed in a cell into an RNA molecule (e.g., mRNA). A gene typically contains several operably linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end), which includes, for example, a polyadenylation site and / or a transcription termination site. "Expression of a gene" refers to the process in which a DNA region that is operably linked to an appropriate regulatory region (particularly a promoter) is transcribed into RNA that is biologically active, i.e., capable of being translated into a biologically active protein or peptide. As used herein, a "regulatory factor" or "transcriptional regulatory factor" is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA by binding to a specific DNA sequence.

[0254] Promoter

[0255] 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, is located upstream with respect to the transcription direction of the transcription start site of the coding sequence, and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to directly or indirectly play a role in regulating 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, for example, by the application of a chemical inducer or a repressive signal.

[0256] As used herein, the term "operably linked" refers to the joining of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" to another nucleic acid sequence when the nucleic acid is placed in a functional relationship with the other nucleic acid sequence. For example, if a transcription regulatory sequence affects the transcription of a coding sequence, it is operably linked to the coding sequence. Operably linked means that the DNA sequences being joined are typically contiguous and, where two protein-coding regions are to be joined, are contiguous and in frame. The joining can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in place thereof, or by gene synthesis.

[0257] Gene constructs and expression vectors

[0258] Any of the cloning and / or recombinant DNA techniques known to those skilled in the art can be used to prepare the gene constructs described herein, in which the nucleotide sequence encoding the isolated myoglobin is expressed in a suitable cell (e.g., a cultured cell or a cell of a multicellular organism), as described in the following references: Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, ibid.); both of these references are incorporated herein by reference in their entirety. See also 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).

[0259] The phrase "expression vector" or "vector" generally refers to a tool in molecular biology for achieving gene expression in cells, for example by introducing a nucleotide sequence capable of influencing the expression of a gene or coding sequence into a host compatible with such a sequence. An expression vector carries a genome that can be stable and remain free in a cell. In the context of the present invention, the cell may be intended to encompass the cell used to prepare the construct or the cell to which the construct will be administered. Alternatively, the vector can integrate into the genome of the cell, for example by homologous recombination or other means.

[0260] These expression vectors typically include at least a suitable promoter sequence and optionally a transcription termination signal. Additional factors necessary or helpful in achieving expression can also be used, as described herein. A nucleic acid or DNA or nucleotide sequence encoding myoglobin is incorporated into a DNA construct capable of being introduced into an in vitro cell culture and expressed therein. Specifically, the DNA construct is suitable for replication in a prokaryotic host (such as bacteria, for example Escherichia coli), or can be introduced into cultured mammalian, plant, insect (such as, for example, Sf9), yeast, fungal or other eukaryotic cell lines.

[0261] A DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA fragment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide coding fragment. The term "operably linked" has been described herein. For example, if a promoter or enhancer stimulates the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence. If the DNA of a signal sequence is expressed as a preprotein involved in polypeptide secretion, then the DNA of the signal sequence is operably linked to the DNA encoding the polypeptide. Generally, operably linked DNA sequences are contiguous, and in the case of a signal sequence, both contiguous and in the reading frame. However, enhancers do not need to be adjacent to the coding sequences whose transcription they control. The linkage is accomplished by ligation at convenient restriction sites or by insertion of linkers or adaptors in place thereof, or by gene synthesis.

[0262] The selection of an appropriate promoter sequence typically depends on the host cell chosen for expressing the 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). Transcription regulatory sequences typically include heterologous enhancers or promoters recognized by the host. The selection of an appropriate promoter depends on 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). Expression vectors include a replication system and transcription and translation regulatory sequences as well as an insertion site for the polypeptide coding segment. In most cases, the replication system functions only in the cell (bacterial cell, such as E. coli) used to generate the vector. Most plasmids and vectors do not replicate in cells infected with the vector. Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334 :31 - 36 describe examples of workable combinations of cell lines and expression vectors. For example, suitable expression vectors can be expressed in the following: yeast (e.g., Saccharomyces cerevisiae), e.g., insect cells, e.g., Sf9 cells, mammalian cells (e.g., CHO cells), and bacterial cells (e.g., E. coli). Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culturing in liquid or solid media. Alternatively, the host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.

[0263] For example, if bacteria (preferably E. coli) are used as the host cell, the following regulatory regions can be used. Promoters suitable for use in bacteria are lac, trp, tac, T7, phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from the rrnB ribosomal RNA operon, and the PL promoter from phage λ. Terminators suitable for use in bacteria are lac, trp, tac, T7 (used in the examples), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from the rrnB ribosomal RNA operon, and the PL terminator from phage λ. The preferred promoter used is the T7 promoter and / or the preferred terminator is the T7 terminator. The preferred signal peptides for secretion are the E. coli Sec recognition peptide (SecA), the E. coli Tet recognition peptide, E. coli dsbA, E. coli phoA, E. coli pelB, and E. coli MBP (maltose - binding protein). A marker applicable to E. coli is ampicillin. Alternatively, the proBA operon from E. coli strain K12 (including its native transcriptional regulatory elements) can be used to facilitate antibiotic - free selection.

[0264] In another example, if yeast is used as the host cell, the following regulatory regions can be used. The promoter suitable for yeast can be a constitutive promoter. Examples of suitable constitutive promoters include: glycolytic promoters selected from the FBA1, TPI1, PGK1, PYK1, TDH3, ENO2, HXK2, PGI1, PFK1, PFK2, GPM1 genes or non-glycolytic promoters of the TEF2 gene. The suitable promoter for yeast can be inducible. If the yeast is Pichia pastoris, the methanol-inducible promoter AOX1 is preferred. Otherwise, when the yeast is Saccharomyces cerevisiae, the GAL1 promoter (galactose-inducible) can be used. The genes from which the promoters mentioned above can be derived for use in yeast as a host cell can also be used to derive terminators for the same yeast. Preferred signal peptides for secretion in Pichia (and Saccharomyces) include: the pre-pro-secretory signal peptide of Saccharomyces cerevisiae α mating factor, the Saccharomyces cerevisiae Ost1 signal peptide, the Saccharomyces cerevisiae Aga2 signal peptide, and their fusions.

[0265] In another example, if filamentous fungi are used as the host cell, the following regulatory regions can be used. The following promoters can be used: the Aspergillus niger glucoamylase promoter (glaA), the Aspergillus nidulans alcohol dehydrogenase promoter (alcA), or the Aspergillus oryzae taka amylase A promoter (amyB), the Aspergillus niger alcohol dehydrogenase promoter (adhA), the Trichoderma reesei pyruvate kinase promoter (pki), or the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA). The genes from which the promoters mentioned above can be derived for use in filamentous fungi as a host cell can also be used to derive terminators for the same filamentous fungi. Preferred signal peptides for secretion in filamentous fungi (preferably Aspergillus) include: the Aspergillus niger glucoamylase signal peptide (glaA), the Aspergillus niger α-galactosidase signal peptide (AglB), and the Trichoderma reesei cellobiohydrolase I (CbhI). The preferred promoter and terminator for Aspergillus (more preferably Aspergillus niger) are the glucoamylase promoter and glucoamylase terminator of Aspergillus niger.

[0266] The gene constructs described herein can be placed in an expression vector. Thus, in another aspect, an expression vector is provided that contains the gene construct as described in any one of the foregoing examples.

[0267] Expression can be evaluated by any method known to those skilled in the art. For example, expression can be evaluated as follows: measuring the level of transgene expression in the transduced tissue at the mRNA or protein level using standard assays known to those skilled in the art, such as qPCR, RNA sequencing, RNA blot analysis, Western blot analysis, mass spectrometry of protein-derived peptides, or ELISA. Expression can be evaluated at any time after administration of the gene constructs, expression vectors, or compositions described herein. In some embodiments herein, expression can be evaluated 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 longer after administration.

[0268] Suitable cell culture methods for the method of producing recombinant proteins are known to those skilled in the art, such as van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design, CRC Press, New York, 1991. Such methods include, but are not limited to, submerged fermentation in liquid medium, surface fermentation in liquid medium, and solid-state fermentation. Cell culture can be carried out, for example, by culturing in microtiter plates, shake flasks, benchtop bioreactors, pilot-scale bioreactors, and / or large-scale bioreactors in a laboratory and / or industrial setting. Suitable cell culture modes include, but are not limited to, continuous, batch, and / or fed-batch fermentation and combinations thereof. Cell culture can be carried out using continuous fermentation, batch fermentation, preferably fed-batch fermentation.

[0269] In the context of the present invention, "medium", hereinafter alternatively referred to as "growth medium", can be interpreted to cover both the situation where cultured cells are absent and the situation where cultured cells are present in the medium. "Culture broth" refers to the medium in which cultured cells are present. "Culture supernatant" refers to the medium in which cultured cells are absent. "Cell-free extract" refers to a cell lysate that does not contain cell debris. Cell culture as part of the method of the present invention can be carried out under conditions that facilitate the production of the introduced myoglobin, and such conditions are known to those skilled in the art. These conditions depend not only on the chemical composition of the medium, but also on other process parameters, including culture duration, temperature, O 2 level in the culture broth and / or headspace, CO 2Horizontal, pH, ionic strength, stirring speed, hydrostatic pressure, etc. Cell culture can be carried out using a culture medium with procedures known in the art, which contains suitable nutrients (such as carbon sources and nitrogen sources) and additional compounds (such as inorganic salts and vitamins) (see, for example, Bennett, W. and Lasure, L., 1st Edition, More Gene Manipulations in Fungi, Academic Press, California, 1991). Suitable growth media are available from commercial suppliers or can be prepared using publicly available compositions suitable for the corresponding host (for example, within the catalogs of the Centraalbureau voor Schimmelcultures (CBS) in the Netherlands or the American Type Culture Collection (ATCC)).

[0270] The exact composition of the growth medium and the values of the culture process parameters are not critical features of the present invention. Any growth medium composition can be considered, as long as it permits the growth of the host cells and the production of the introduced myoglobin. The growth medium typically contains a carbon source for the growth of the cells being cultured. Those skilled in the art understand that a suitable carbon source can be added externally to the growth medium or may already be present in the medium. The carbon source can be present or added alone or in the form of a mixture of multiple carbon sources. Examples of suitable carbon sources known in the art include monosaccharides such as glucose, glycerol, maltose, sucrose, xylose, arabinose, complex sugars such as maltodextrin, hydrolyzed starch, starch, molasses, and second-generation feedstocks. Second-generation feedstocks may be particularly attractive due to their lower carbon footprint. Second-generation feedstocks typically contain lignocellulosic materials. Such materials include any lignocellulose- and / or hemicellulose-based materials. Such materials can be derived from agricultural waste streams, industrial waste streams, or municipal waste streams, 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. Biomass in general form includes trees, shrubs, and forages, wheat, wheat straw, bagasse, switchgrass, miscanthus, corn, corn stover, corn cobs, rape straw, soybean straw, sweet corn, corn kernels, products and by-products from cereal milling (including wet and dry milling), such as corn, wheat, and barley milling, commonly referred to as "bran or fiber", and municipal solid waste. Biomass can also be forage, agricultural residues, forestry residues, municipal solid waste, waste paper, and pulp and paper mill residues. Agricultural biomass includes branches, shrubs, tows, corn and corn stover, energy crops, forests, fruits, flowers, grains, forages, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short-rotation woody crops, shrubs, switch herb, trees, vegetables, fruits, grapevines, beet pulp, wheat middlings, oat hulls, and hardwoods and softwoods (excluding toxic woods), as well as organic wastes generated during agricultural processing, including agricultural and forestry activities, particularly forestry wood wastes. Agricultural biomass can be any of the foregoing individually or any combination or mixture thereof. Carbon sources such as organic acids, aldehydes, ketones, esters, and alcohols can also be considered. The use of a growth medium comprising a combination of multiple different carbon sources can also be considered in the method of the present invention. As a non-limiting example, such a medium can combine a more oxidized carbon source (such as an organic acid) with a more reduced carbon source (such as an alcohol). Examples of suitable nitrogen sources known in the art include soybean meal, corn steep liquor, yeast extract, whey protein, egg protein, casein hydrolysate, urea, ammonia, ammonium salts, and nitrates. Examples of other suitable compounds known in the art include phosphates, sulfates, metals such as magnesium, trace elements, and vitamins. The exact growth medium requirements will vary depending on the host cell, for example, between yeast, bacteria, and filamentous fungi, and such requirements will be known to those skilled in the art.Thus, the growth medium can be a complete (rich) medium or a minimal medium (i.e., a medium containing only the components necessary for growth), depending on the host cell being cultured.

[0271] Similar to the composition of the growth medium, any value can be assigned to the process parameters as long as they allow the growth of the host cell and the production of the introduced myoglobin. Generally, the values will vary depending on the host cell being cultured and will be known to the person skilled in the art. Preferably, the method according to the invention is an oxygen-limited or aerobic method, meaning that the cell culture is carried out under oxygen-limited or aerobic conditions, more preferably the method is oxygen-limited. Oxygen-limited conditions, also known as microaerophilic conditions, are culture conditions where the oxygen consumption rate is limited by the availability of oxygen. The degree of oxygen limitation depends on the amount and composition of the incoming gas stream and the actual mixing / mass transfer characteristics of the fermentation equipment used. Preferably, in liquid culture under oxygen-limited conditions, the oxygen consumption rate is at least about 5.5 mmol / L / h, more preferably at least about 6 mmol / L / h, even more preferably at least about 7 mmol / L / h. Aerobic conditions are culture conditions where the oxygen consumption rate is not limited by the availability of oxygen.

[0272] The cell culture can be carried out at a temperature value optimal for the cells, typically in the temperature range of 16°C - 42°C. In some embodiments, the temperature range is between 20°C - 40°C, more preferably between 25°C - 38°C, and most preferably between 28°C - 36°C. In some of the most preferred embodiments, a temperature value of about 30°C or 36°C is used.

[0273] The cell culture can be carried out at a pH value optimal for the cells. 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 8.5, about pH 9. In a preferred embodiment, the pH range is from about pH 3.0 to about pH 9, more preferably from about pH 3.5 to pH 7. In some of the most preferred embodiments, a pH value of about 6 is used.

[0274] The cell culture can be carried out at a medium ionic strength value optimal for the cells, typically in the range between 50 mM - 2 M. In some embodiments, the ionic strength range of the medium is between 75 mM - 1 M, more preferably between 100 mM - 750 mM. In some of the most preferred embodiments, an ionic strength value of about 100 mM is used.

[0275] Sequence Listing Overview

[0276]

[0277] Detailed sequence

[0278] - SEQ ID NO:1 Rat Myoglobin

[0279] MGLSDGEWQMVLNIWGKVEGDLAGHGQEVLISLFKAHPETLEKFDKFKNLKSEEEMKSSEDLKKHGCTVLTALGTILKKKGQHAAEIQPLAQSHATKHKIPVKYLEFISEVIIQVLKKRYSGDFGADAQGAMSKALELFRNDIAAKYKELGFQG

[0280] - SEQ ID NO:2 Rabbit Myoglobin

[0281] MGLSDAEWQLVLNVWGKVEADLAGHGQEVLIRLFHTHPETLEKFDKFKHLKSEDEMKASEDLKKHGNTVLTALGAILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISEAIIHVLHSKHPGDFGADAQAAMSKALELFRNDIAAQYKELGFQG

[0282] - SEQ ID NO:3 Mouse Myoglobin

[0283] MGLSDGEWQLVLNVWGKVEADLAGHGQEVLIGLFKTHPETLDKFDKFKNLKSEEDMKGSEDLKKHGCTVLTALGTILKKKGQHAAEIQPLAQSHATKHKIPVKYLEFISEIIIEVLKKRHSGDFGADAQGAMSKALELFRNDIAAKYKELGFQG

[0284] - SEQ ID NO:4 Steppe Mammoth Myoglobin

[0285] MGLSDGEWELVLKTWGKVEADIPGHGLEVFVRLFTGHPETLEKFDKFKHLKTEGEMKASEDLKKQGVTVLTALGGILKKKGHHQAEIQPLAHSHATKHKIPIKYLEFISDAIIHVLQSKHPAEFGADAQGAMKKALELFRNDIAAKYKELGFQG

[0286] - SEQ ID NO:5 Woolly Mammoth Myoglobin

[0287] MGLSDGEWELVLKTWGKVEADIPGHGLEVFVRLFTGHPETLEKFDKFKHLKTEGEMKASEDLKKQGVTVLTALGGILKKKGHHQAEIQPLAQSHATKHKIPIKYLEFISDAIIHVLQSKHPAEFGADAQGAMKKALELFRNDIAAKYKELGFQG

[0288] - SEQ ID NO:6 Tuna myoglobin

[0289] MADFDAVLKCWGPVEADYTTIGGLVLTRLFKEHPETQKLFPKFAGIAQADIAGNAAVSAHGATVLKKLGELLKAKGSHAAILKPLANSHATKHKIPINNFKLISEVLVKVMHEKAGLDAGGQTALRNVMGIIIADLEANYKELGFSG

[0290] - SEQ ID NO:7 Rat myoglobin nucleic acid

[0291] ATGGGCCTGAGCGATGGCGAATGGCAGATGGTGCTGAACATTTGGGGCAAAGTGGAAGGCGATCTGGCGGGCCATGGCCAGGAAGTGCTGATTAGCCTGTTTAAAGCGCATCCGGAAACCCTGGAAAAATTTGATAAATTTAAAAACCTGAAAAGCGAAGAAGAAATGAAAAGCAGCGAAGATCTGAAAAAACATGGCTGCACCGTGCTGACCGCGCTGGGCACCATTCTGAAAAAAAAAGGCCAGCATGCGGCGGAAATTCAGCCGCTGGCGCAGAGCCATGCGACCAAACATAAAATTCCGGTGAAATATCTGGAATTTATTAGCGAAGTGATTATTCAGGTGCTGAAAAAACGCTATAGCGGCGATTTTGGCGCGGATGCGCAGGGCGCGATGAGCAAAGCGCTGGAACTGTTTCGCAACGATATTGCGGCGAAATATAAAGAACTGGGCTTTCAGGGC

[0292] - SEQ ID NO:8 Rabbit myoglobin nucleic acid

[0293] ATGGGCCTGAGCGATGCGGAATGGCAGCTGGTGCTGAACGTGTGGGGCAAAGTGGAAGCGGATCTGGCGGGCCATGGCCAGGAAGTGCTGATTCGCCTGTTTCATACCCATCCGGAAACCCTGGAAAAATTTGATAAATTTAAACATCTGAAAAGCGAAGATGAAATGAAAGCGAGCGAAGATCTGAAAAAACATGGCAACACCGTGCTGACCGCGCTGGGCGCGATTCTGAAAAAAAAAGGCCATCATGAAGCGGAAATTAAACCGCTGGCGCAGAGCCATGCGACCAAACATAAAATTCCGGTGAAATATCTGGAATTTATTAGCGAAGCGATTATTCATGTGCTGCATAGCAAACATCCGGGCGATTTTGGCGCGGATGCGCAGGCGGCGATGAGCAAAGCGCTGGAACTGTTTCGCAACGATATTGCGGCGCAGTATAAAGAACTGGGCTTTCAGGGC

[0294] - SEQ ID NO:9 Mouse Myoglobin Nucleic Acid

[0295] ATGGGCCTGAGCGATGGCGAATGGCAGCTGGTGCTGAACGTGTGGGGCAAAGTGGAAGCGGATCTGGCGGGCCATGGCCAGGAAGTGCTGATTGGCCTGTTTAAAACCCATCCGGAAACCCTGGATAAATTTGATAAATTTAAAAACCTGAAAAGCGAAGAAGATATGAAAGGCAGCGAAGATCTGAAAAAACATGGCTGCACCGTGCTGACCGCGCTGGGCACCATTCTGAAAAAAAAAGGCCAGCATGCGGCGGAAATTCAGCCGCTGGCGCAGAGCCATGCGACCAAACATAAAATTCCGGTGAAATATCTGGAATTTATTAGCGAAATTATTATTGAAGTGCTGAAAAAACGCCATAGCGGCGATTTTGGCGCGGATGCGCAGGGCGCGATGAGCAAAGCGCTGGAACTGTTTCGCAACGATATTGCGGCGAAATATAAAGAACTGGGCTTTCAGGGC

[0296] - SEQ ID NO:10 Nucleic acid of Mammuthus trogontherii myoglobin

[0297] ATGGGACTCAGCGACGGGGAATGGGAGTTGGTGTTGAAAACCTGGGGGAAAGTGGAGGCTGACATCCCGGGCCATGGGCTGGAAGTCTTCGTCAGGCTCTTCACAGGTCATCCCGAGACCCTGGAGAAGTTCGACAAGTTTAAGCACCTGAAGACAGAGGGCGAGATGAAGGCCTCCGAGGACCTGAAGAAGCAAGGTGTTACTGTGCTCACTGCCCTGGGGGGCATCCTCAAGAAGAAAGGGCATCACCAGGCGGAGATTCAGCCCCTGGCCCATTCTCATGCCACCAAGCACAAGATCCCCATCAAGTATCTGGAGTTCATCTCGGACGCCATCATCCACGTTCTGCAAAGCAAGCATCCTGCGGAATTTGGCGCTGATGCCCAGGGAGCCATGAAAAAGGCCTTGGAGCTGTTCCGGAATGACATTGCGGCCAAGTATAAGGAGCTGGGGTTCCAGGGCTAG

[0298] - SEQ ID NO:11 Nucleic acid of Mammuthus primigenius myoglobin

[0299] ATGGGACTCAGCGACGGGGAATGGGAGTTGGTGTTGAAAACCTGGGGGAAAGTGGAGGCTGACATCCCGGGCCATGGGCTGGAAGTCTTCGTCAGGCTCTTCACAGGTCATCCCGAGACCCTGGAGAAGTTCGACAAGTTTAAGCACCTGAAGACAGAGGGCGAGATGAAGGCCTCCGAGGACCTGAAGAAGCAAGGTGTTACTGTGCTCACTGCCCTGGGGGGCATCCTCAAGAAGAAAGGGCATCACCAGGCGGAGATTCAGCCCCTGGCCCAGTCTCATGCCACCAAGCACAAGATCCCCATCAAGTATCTGGAGTTCATCTCGGACGCCATCATCCACGTTCTGCAAAGCAAGCATCCTGCGGAATTTGGCGCTGATGCCCAGGGAGCCATGAAAAAGGCCTTGGAGCTGTTCCGGAATGACATTGCGGCCAAGTATAAGGAGCTGGGGTTCCAGGGCTAG

[0300] -SEQ ID NO:12 Nucleic acid of tuna myoglobin

[0301] ATGGCTGACTTTGATGCAGTTCTGAAGTGTTGGGGTCCAGTGGAGGCGGACTACACCACCATTGGAGGCCTGGTTCTGACCCGTTTATTCAAAGAGCACCCTGAGACCCAGAAGCTGTTCCCCAAATTCGCTGGCATCGCCCAGGCTGACATAGCCGGTAACGCAGCTGTTTCTGCTCATGGTGCCACTGTGCTGAAGAAACTTGGAGAGCTGCTGAAGGCCAAAGGCAGTCACGCTGCCATCCTAAAACCACTGGCAAACAGCCATGCCACTAAGCACAAGATTCCCATTAATAACTTCAAGCTGATTTCTGAGGTCCTTGTGAAGGTCATGCATGAGAAGGCAGGACTCGATGCCGGTGGGCAGACAGCCCTGAGGAACGTGATGGGTATCATCATCGCCGACCTTGAGGCCAACTACAAAGAGCTGGGCTTCTCTGGCTGA

[0302] - SEQ ID NO:13 Woolly mammoth κ-casein

[0303] MKGFLLVVNILLLPLFLAAEVQNQEESRCLEKDERWFCQKAVKYIPNDYVLKSYYRYEPNYNQFRAAVPINNPYLIYLYPAKQVAVRPHTQIQWQVPSNIYPSPSVPHTYLKPPFIIPPKKTQDKPIIPPTGTVASIEATVEPKVNTVVNAEASSEFIATNTPEATTVPVISPQI

[0304] - SEQ ID NO:14 Woolly mammoth β-casein

[0305] MKVFILACLVAFALGRETVENLSSSEIRQFYSEQKPEGVKHEEQQREDEHQNKIQPLFQPQPLVYPFAEPIPYTVFPPNAIPLAQPIVVLPFPQPEVQLPEAKEITFPRQKLMSFLKSPVMPFFDPQPNLGTDLENLHLPLPLLQPLRHQLHQPLAQTPVLPLPLSLPKVLPVPQQVIPYPQRGRPIQNLLYEEPLLDPTRKIYPVAQPLAPVYNPVAYMIGIPCCSTLLTYLHQSSRSQYPIQNKLGYLIAMPKKVRPT BRIEF DESCRIPTION OF THE DRAWINGS

[0306] Figure 1 . Comparison of myoglobin sequences of six animal species. The sequence alignment shows sequences from the following species: tuna (Thunnus orientalis, SEQ ID NO:6), steppe mammoth (Mammuthus trogontherii, SEQ ID NO:4), rat (Rattus norvegicus, SEQ ID NO:1), mouse (Mus musculus, SEQ ID NO:3), rabbit (Oryctolagus cuniculus, SEQ ID NO:2), and cow (Bos taurus, SEQ ID NO:15). The amino acid conservation level is indicated by shading using BLOSUM62 scores.

[0307] Figure 2. Three-dimensional structures of myoglobins from 6 animal species. Predicted structures of wild-type myoglobins from the following species are shown as ribbon diagrams: rabbit (Oryctolagus cuniculus, SEQ ID NO:2), mouse (Mus musculus, SEQ ID NO:3), rat (Rattus norvegicus, SEQ ID NO:1), steppe mammoth (Mammuthus trogontherii, SEQ ID NO:4), tuna (Thunnus orientalis, SEQ ID NO:6), and cow (Bos taurus, SEQ ID NO:15). Heme is shown in black.

[0308] Figure 3 . Expression vectors for the production of animal myoglobins in Pichia pastoris. Six vectors have been generated that carry codon-optimized sequences encoding the 6 myoglobins listed above. Expression cassettes were generated by restriction using two BglII cleavage sites, allowing genomic integration by recombination between chromosomal DNA on the one hand and the AOX1 promoter and on the other hand the AOX1 3' fragment.

[0309] Figure 4 . Confirmation of the presence of myoglobin expression cassettes in Pichia pastoris. Agarose gel picture after electrophoresis of PCR products obtained after amplification of genomic DNA from different yeast colonies (up to 8 per myoglobin construct) using primers specific for the myoglobin expression cassette.

[0310] Figure 5 . Production of extracellular animal myoglobin (Mb) in Pichia pastoris. Cell-free supernatants were collected after methanol-induced fermentation in 96-well plates and analyzed by SDS-PAGE, revealing proteins with the expected molecular weight of myoglobin. Clones expressing myoglobin from cow, steppe mammoth, tuna, or rabbit are shown. Commercially available myoglobin purified from horse heart muscle was used as a standard.

[0311] Figure 6 . Matrix for plant-based wet pet food. A plant-based matrix for wet pet food was prepared without myoglobin (A) or containing (B) 0.1% w / w myoglobin.

[0312] Figure 7 . Color stability of myoglobin obtained by precision fermentation in Pichia pastoris. At 4 °C, during a 2-month storage period, colorimetric ΔE values were calculated for three independent myoglobin samples (labeled A, B, and C).

[0313] Figure 8. Color effects of different animal myoglobins (Mb) in plant-based wet pet food. Raw and heat-sterilized samples without myoglobin or containing 0.1% w / w rabbit or bovine myoglobin, which was obtained by precision fermentation in Pichia pastoris, were prepared. (A) Samples were imaged on day 0. (B) The color effects of myoglobin in plant-based pet food were quantified. The bar graphs show the colorimetric L*a*b* values for each sample.

[0314] Figure 9 . Color and color stability effects of myoglobin in plant-based wet pet food. (A) Raw and heat-sterilized samples prepared with different concentrations of myoglobin (obtained by precision fermentation in Pichia pastoris) (0, 0.2% w / w, 0.5% w / w, 1% w / w) were imaged on day 0 and (B) after storage at 4 °C for 20 days. (C) Colorimetric L*a*b* values for each sample on day 0. (D) Relative color difference (ΔE) after storage at 4 °C for 20 days. Data are represented as relative values compared to the corresponding raw or sterilized sample without myoglobin.

[0315] Figure 10 . Plant-based wet pet food containing bovine or rabbit myoglobin obtained by precision fermentation in Pichia pastoris. Plant-based wet pet food without myoglobin, containing 0.1% bovine myoglobin, or containing 0.1% rabbit myoglobin was prepared.

[0316] Figure 11 . Aroma characteristics of raw and heat-sterilized plant-based wet pet food with animal myoglobin obtained by precision fermentation in Pichia pastoris. (A) Distribution of different classes of volatile organic compounds in raw and heat-sterilized plant-based wet pet food without myoglobin or containing 0.1% w / w myoglobin (obtained by precision fermentation in Pichia pastoris) detected by SPME-GC-MS. Data are represented as relative values. The proportion of pyrazines is indicated at the top of each bar. (B) Principal component analysis (PCA) scores and load plots for PC1 and PC2 of volatile organic compounds emitted from raw and heat-sterilized plant-based wet pet food without myoglobin or containing 0.1% w / w myoglobin. Selected volatile organic compounds (loadings) are depicted by arrows, and the corresponding compound names are listed on the right side of the figure. (C) Correlation loadings for PC1 and PC2 of volatile organic compounds (n = 111) emitted from raw and heat-sterilized plant-based wet pet food without myoglobin and containing 0.1% w / w myoglobin.

[0317] Figure 12.Conventional meat-related volatile organic compounds and their relationship to the addition of myoglobin to plant-based wet pet food. (A) Levels (peak areas) of selected volatile organic compounds detected in raw and heat-sterilized plant-based wet pet food without myoglobin or containing 0.1% w / w myoglobin (obtained by precision fermentation in Pichia pastoris). Asterisks indicate statistically significant differences between sample pairs (t-test): (*) = p < 0.05, (***) = p < 0.001. (B) Aroma descriptions of selected volatile organic compounds also found in cooked conventional meat (The Good Scent Company information system) [1-4].

[0318] Example

[0319] By way of example and not limitation, the following non-limiting examples illustrate various embodiments provided by this disclosure.

[0320] Example 1: Selection of target protein

[0321] Myoglobin (Mb) is a relatively small globular protein, approximately 17 kD, present in cardiac and skeletal muscle. It carries a single heme group that is capable of reversible oxygen binding ( Figure 1 ), enabling myoglobin to transport oxygen from the cell surface to the mitochondria. Mb is also the main pigment responsible for the color of meat. In oxidized Mb (oxymyoglobin), the iron atom of the heme group coordinates with the four nitrogen atoms of the porphyrin ring, the so-called "proximal" histidine (His93) in the Mb chain, and an oxygen molecule. This can be further stabilized by hydrogen bonding between the oxygen molecule and another histidine (i.e., the "distal" histidine (His64)) in the heme binding pocket of Mb. In this form, Mb has a typical bright red color. In the absence of oxygen, Mb is a deeper red (deoxymyoglobin). When the heme iron is oxidized from ferrous (Fe(II)) to ferric (Fe(III)) state, it cannot bind oxygen, and Mb becomes brown (metmyoglobin), as seen in cooked meat. Heme iron oxidation also reduces the affinity of Mb for heme, leading to increased heme loss and subsequent protein unfolding [5].

[0322] Since myoglobin is present in the cardiac and skeletal muscle of vertebrates, it is also present in meat and fish products, and the concentration of myoglobin varies depending on the species, type of muscle tissue, and animal age. Reported concentrations of myoglobin in muscle tissues from various animal species are listed in Table 1 below.

[0323] Table 1. Myoglobin concentrations in muscle tissues from six animal species. The reported myoglobin concentrations are indicated as % (w / w of muscle wet mass). Data for the Asian elephant are used here as a proxy for the steppe mammoth (extinct species, from which muscle tissue samples for analysis are not available).

[0324] Species Myoglobin concentration Reference Cattle 0.243％ [6] 0.199% to 0.364% [7] 0.1% to 2% [8] 0.2% to 1.8% [9] Tuna 0.037% to 2.44%

[10] 0.0193％

[11] 0.965％

[12] 0.023% to 0.157%

[13] 0.13% to 2.37%

[14] Rabbit 0.02％

[15] 0.057% to 0.072%

[16] 0.08％

[17] 0.04％

[18] Mouse 0.000149% to 0.00023%

[19] Rat 0.07% to 0.12%

[20] Asian elephant 0.46％

[21]

[0325] The amino acid sequence of Mb is highly conserved during evolution and shows relatively little variation between species ( Figure 1 ). However, single amino acid changes that strongly affect the relative affinity of Mb for dioxygen and carbon dioxide, its autoxidation rate, or its aggregation propensity have been reported

[22] . Here, when screening candidate Mbs produced as ingredients for meat substitutes in pet food, we carefully examined the sequences of various vertebrate species. We included extant species whose muscle tissue is commonly used for meat consumption as well as the extinct family Elephantidae, namely the steppe mammoth (Mammuthus trogontherii)

[23] . We previously described relevant substitutions in the myoglobin sequence of this species: histidine (His92) at position 92 replaces glutamine ( Figure 1 ), which confers a higher positive surface charge (net surface charge (Z Mb ) = 2.67 at pH 6.5, compared to, for example, myoglobin from the Asian elephant with a Z Mb = 2.11 at pH 6.5)

[24] . This is reminiscent of the adaptation of Mb to deep diving in cetaceans, where the increased positive surface charge leads to a reduced attractive interaction between Mb molecules at contact distance, thus increasing Mb stability and preventing its aggregation [25, 26]. Overall, Mb from the steppe mammoth presents several features that make it particularly attractive as an ingredient in meat substitutes. Here, we evaluated the production and characteristics of recombinant Mb from the steppe mammoth and from cattle (Bos taurus), the latter being used as a reference because this extant species is widely used for consumption of its red meat. In addition, we reasoned that pets are unlikely to suffer from adverse food reactions (AFRs) due to ingesting myoglobin from animals that they are evolutionarily adapted to consume. For this reason, we also considered myoglobin from species that are known to be prey for the ancestors of modern pets, namely the rabbit (Oryctolagus cuniculus), which was prey for wolves and the first domesticated dogs

[27] , the house mouse (Mus musculus) or the rat (Rattus norvegicus), which were prey for the first domesticated cats

[28] . Finally, we also examined myoglobin from tuna (Thunnus orientalis), which, as a widely exploited fish, has a particularly high myoglobin concentration in its muscle (Table 1) and is very attractive to cats

[29] .

[0326] Example 2: Extracellular production of myoglobin in Pichia pastoris

[0327] Sequences encoding Mb from Mammuthus trogontherii, bovine, rat, rabbit, mouse, and tuna ( Figure 1 , Figure 2 ) were cloned downstream of the methanol-inducible AOX1 promoter and upstream of the histidine prototrophic selection marker and AOX1 terminator in Pichia pastoris ( Figure 3 ). Myoglobin is naturally present in the cytoplasm of muscle cells. To facilitate recombinant protein purification, we frame-fused the sequence encoding the signal peptide with the myoglobin coding sequence to target the nascent Mb protein to the secretory pathway for extracellular secretion ( Figure 3 ).

[0328] His4 auxotrophic Pichia pastoris cells were transformed with these constructs, and transformants were selected for their ability to grow in the absence of histidine. The presence of the myoglobin expression cassette in the genome of the histidine prototrophic clones was then verified by PCR ( Figure 4 ).

[0329] For each construct, the ability of the verified transformants to produce extracellular animal myoglobin after methanol induction was then tested in 96-well microtiter plates. The presence of the methanol-inducible protein with the expected molecular weight was confirmed after protein electrophoresis and staining with Coomassie blue ( Figure 5 ).

[0330] To obtain larger amounts of material for the preparation of plant-based meat alternatives, fermentation runs were carried out in a 10 L fermenter. The cell-free supernatant was purified and concentrated by ultrafiltration to obtain a concentrated myoglobin solution that also contained residual yeast proteins. The protein purity of myoglobin observed in the final product was typically in the range of 75% - 85%. Since the yeast Pichia pastoris used as the production host has an established history of safe use in food production, for example, in the United States for a variety of generally recognized as safe (GRAS) products and has been granted qualified presumption of safety (QPS) in the European Union

[30] , the consumption of Pichia proteins does not pose any safety concerns. On the other hand, the presence of yeast proteins may even contribute to the palatability of plant-based meat alternatives containing myoglobin components [31, 32].

[0331] Example 3: Preparation of plant-based pet food

[0332] Commercial pet foods have long been based on meat and meat-derived by-products because these products are generally highly palatable to pet mammals, including dogs and cats. The development towards more animal- and environmentally-friendly food production and the convenient delivery forms of commercial pet foods (e.g., grain-based kibbles) have influenced the increased popularity and availability of plant-based pet foods. The main proteins in such plant-based pet foods have historically been derived from soybeans, corn, and wheat (gluten). More recently, additional plant proteins have become available, including pea, potato, and rice proteins. Since palatability is one of the main criteria in pet diet decisions

[33] , producers are developing various strategies to impart the desired "meat-like" characteristics to their pet foods.

[0333] Here, we describe the development of a plant-based model pet food in which the properties of animal myoglobin produced in Pichia pastoris can be evaluated. Two main types of wet pet foods, "meat paste or meatloaf" and "gravy chunks", are distinguished. We selected the meat paste, which is typically prepared by processing a mixture of edible components under heat to produce a homogeneous semi-solid mass structured by heat-coagulated proteins. This homogeneous mass is usually packaged in single or multiple servings, which are then sealed and sterilized. At the time of packaging, the homogeneous mass assumes the shape of the container. It has been shown that incorporating Maillard components into sterilized wet pet foods containing recombinant meat significantly increases the palatability of the product

[34] .

[0334] To develop our plant-based model pet food (Table 2, Figure 6 ), we formulated a matrix similar to commercial wet pet foods in "meat paste" form and subjected it to similar processing conditions as described above, to the extent permitted by laboratory conditions.

[0335] Table 2. Composition of the plant-based model meat substitute compared to conventional pet foods. The nutritional composition of the plant-based model pet food developed herein was compared to the nutritional composition of commercial pet foods as described in

[35] and

[36] .

[0336]

[0337] We then added animal myoglobin obtained by precision fermentation to this pet food matrix and studied the effects of myoglobin addition on the aroma profile, color, and texture of the plant-based wet pet food.

[0338] Example 4: Nutritional value

[0339] Although pet food producers must pay special attention to ensure adequate intake of certain nutrients such as protein, amino acids (e.g., taurine, carnitine, methionine, lysine, and tryptophan), vitamins (e.g., vitamins A, B3, B9, and B12), minerals (e.g., calcium, iron, zinc, and copper), and fats

[37] , it is important to remember that all species require specific nutrients rather than specific ingredients. A study based on the analysis of blood samples showed that only two out of 20 dogs fed a vegetarian diet had any deficiencies. The parameters that showed significant differences between the groups fed a meat or vegetarian diet were iron, vitamin B12, and folic acid, but these elements were not outside the reference range

[38] .

[0340] Conventional meat products constitute an important source of dietary iron. Animal tissues are actually a source of heme iron (i.e., iron coordinated with porphyrin) (Table 3), while plants and legumes contain non-heme iron

[39] .

[0341] Table 3. Heme iron concentration bound to myoglobin in muscle tissues from six animal species. Based on the molar mass of heme iron of 616.5 Da and the molecular weight of the corresponding myoglobin, the concentration of heme iron bound to myoglobin in muscle tissues (indicated in mg / 100 g) was calculated according to the reported myoglobin concentration listed in Table 1.

[0342]

[0343] The bioavailability of iron from heme iron sources is 2 to 7 times that of non-heme iron

[40] . When formulating plant-based foods, another challenge is that the absorption of non-heme iron is inhibited by phytic acid (inositol hexaphosphate) found in grains, legumes, nuts, and seeds and by polyphenolic compounds abundant in fruits and vegetables

[39] .

[0344] We previously demonstrated that the heme iron present in animal myoglobin obtained by precision fermentation in Pichia pastoris yeast shows the same bioavailability as the heme iron present in conventional beef

[24] . Therefore, the animal myoglobin produced here can be used to supplement plant-based meat alternatives with the same level of bioavailable heme iron as the conventional meat products it is meant to mimic, regardless of the presence of inhibitory plant-derived polyphenols.

[0345] Example 5: Color and color stability of recombinant myoglobin

[0346] Consumers usually select or reject products based on their color and appearance

[41] . From a physical perspective, the color appearance of a material represents the response of the retinal rods and cones to the reflected radiation in the so-called "visible region" of the electromagnetic spectrum (ranging between 400 nm and 700 nm). From a chemo-physical perspective, color is the result of the interaction between a light source and a pigment, through which energy is absorbed and emitted as complementary unabsorbed radiation in the visible region

[42] .

[0347] Meat products are characterized by their red color, which is mainly imparted by myoglobin

[43] . To mimic the meat-like appearance, different food colorants are added to meat substitute products on the market, such as beet juice extract, pomegranate syrup, soy leghemoglobin, carrot juice extract, and lycopene

[44] .

[0348] The surface color of meat and meat analogs is routinely measured using a colorimeter equipped with a pulsed xenon arc lamp that irradiates the sample with a standardized light beam (with D65 as the standard illuminant). Then, a photocell is used to collect the light reflected from the sample surface and is used to calculate the coordinates in the color space

[45] .

[0349] Here, we tracked the colorimetric stability of independent myoglobin samples over a 2-month period, which were obtained by precision fermentation in Pichia pastoris and stored in the dark at 4 °C. In all three samples, the color difference (ΔE) observed at any time point did not reach or exceed 2 ( Figure 7 ), indicating that no recognizable color change was observed

[46] .

[0350] The color effects of different animal myoglobins in a plant-based matrix were tested. We first evaluated the addition of rabbit and bovine myoglobins (0.1% w / w), obtained by precision fermentation in Pichia pastoris, to a plant-based wet pet food matrix. Then we examined the color of the resulting products, either raw or freshly heat-sterilized ( Figure 8 A). In the raw products, for both rabbit and bovine myoglobins, the lightness (L*) decreased, while the redness (a*) increased ( Figure 8 B). The increase in redness was more pronounced for bovine myoglobin. Hernandez et al. (2016) previously measured the L*, a*, and b* values of beef (Longissimus lumyoglobinorum) samples

[47] . The L* value (42.9) of the raw sample containing 0.1% bovine myoglobin that we measured here was consistent with the reported L* value (37.61) of raw beef found in that previous study

[47] . et al. measured the color changes of rabbit meat during aging. The L* value of raw rabbit meat measured 45 minutes after death was 59.30

[48] . The L* value of the raw sample containing 0.1% rabbit myoglobin that we measured was 60.27, which is consistent with the findings of this author. In heat-sterilized products containing myoglobin, the brightness also decreased similarly ( Figure 8 B).

[0351] Regarding redness, the a* value of the raw sample was more than twice as high in the sample containing bovine myoglobin as in the sample containing rabbit myoglobin (7.56 and 3.01, respectively). After baking, the samples showed similar values (7.02 and 6.42 for bovine myoglobin and rabbit myoglobin, respectively). The a* value measured by Hernandez et al. on raw beef samples was 14.42

[47] , and this higher value may be explained by the lower concentration of bovine myoglobin in our samples compared to the concentration observed in bovine muscle (Table 1, also see below). For the rabbit samples, et al. measured an a* value of 3.15

[48] in samples 7 hours after death. This is comparable to the a* value of 3.01 for the sample containing 0.1% rabbit myoglobin that we measured. The naturally occurring concentration in rabbit meat (Table 1) is comparable to the concentration used in our study.

[0352] Finally, in both the raw and sterilized products, after adding both myoglobins, the yellowness (b*) decreased to a similar extent ( Figure 8 B). The difference in b* values between the raw samples with and without myoglobin was not significant (23.34 and 21.71, respectively). Baking caused a slight decrease in the b* value, and this effect increased in the presence of myoglobin. This indicates that adding myoglobin and baking the samples reduced the yellow hue contributed by the large amount of soybean matrix.

[0353] In a similar method, we examined the color of raw or freshly heat-sterilized plant-based wet pet food samples containing different final concentrations (0, 0.2% w / w, 0.5% w / w, 1% w / w) of myoglobin, which was obtained by precision fermentation in Pichia pastoris. In the raw product, as the myoglobin concentration increased, the brightness (L*) decreased while its redness (a*) increased ( Figure 9 A and Figure 9 C). In the heat-sterilized product, the brightness also decreased with the myoglobin concentration ( Figure 9 A and Figure 9 C). In both the raw and sterilized products, the yellowness (b*) decreased with increasing myoglobin concentration ( Figure 9 A and Figure 9 C). In summary, this indicates that adding myoglobin reduced the unwanted yellow hue caused by the large amount of plant-derived protein in products intended to mimic meat.

[0354] In addition to L*a*b* values, two other factors related to perceived color attributes are often used. The first factor is chroma (C*), which refers to the vividness or dullness of a color

[49] . In plant-based wet pet food samples, the C* value decreases as the myoglobin concentration increases (from 22.8 in the raw sample without myoglobin to 9.3 in the sterilized sample containing 1% myoglobin, respectively), indicating a more vivid color. Interestingly, the C* values measured in our samples containing bovine myoglobin (as Figure 8 shown) are slightly higher than the C* values of beef reported by Hernandez et al. (24.5 and 16.93, respectively)

[47] , and are four times higher in the samples containing rabbit myoglobin than those reported by et al. (20.1 and 5.28, respectively)

[48] . This indicates that myoglobin obtained by precision fermentation may contribute to a more vivid product color during manufacturing.

[0355] The second factor often used in colorimetry is hue angle (h ab ), which is the angle with the a* axis and is related to color descriptions in common language (red, yellow, green, blue, etc.)

[49] . Meat color is located in the first quadrant of the a*b* plane (a*, b* > 0, 0 ≤ h ab ≤ 90°), where the larger the h ab value indicates less redness. Here, the h ab values of the samples are in the positive range (1.45 - 0.96 for the raw sample without myoglobin and the raw sample with myoglobin on day 0, respectively), and decrease as the myoglobin concentration increases. Low and decreasing hue angle values indicate significant redness when plant-based pet food samples contain added myoglobin. It is worth noting that the h ab values measured in our samples containing rabbit myoglobin are consistent with the h values measured by ab et al.

[48] in rabbit meat (1.42 and 1.07, respectively).

[0356] Finally, we measured the color difference in plant-based wet pet food samples after 20 days of refrigerated storage. The color differences observed in the raw and sterilized products decreased to a similar extent as the myoglobin concentration increased ( Figure 9 B and Figure 9 D).

[0357] Overall, these results show that the addition of myoglobin to plant-based meat alternatives can stabilize and / or improve the desired color characteristics of the meat alternative over time during refrigeration, regardless of whether the product is in the raw or cooked state. The addition of myoglobin obtained by precision fermentation increases the redness and decreases the yellowness of plant-based wet pet food, and thus its desired product appearance. Differences were observed between myoglobins of different species, with most of the measured parameters corresponding to findings reported in the literature for the respective conventional meat products. This suggests that when substituting conventional meat with plant-based alternatives, the use of myoglobins of different species produced by precision fermentation may contribute to a more realistic sensory experience.

[0358] Example 6: Aroma analysis

[0359] As with human food, palatability plays an important role in the success of pet food in the market. Palatability depends on the appearance, odor, taste, and texture of the product

[50] .

[0360] The aroma characteristics of many food products that can be used as raw materials for pet food have been widely studied [51 - 55]. Pet food is a complex object of aroma component research due to its diverse formulations. They can contain different grains (barley, oats, rice, wheat, etc.), meat sources (beef, chicken, pork, duck, turkey, venison, buffalo, etc.), animal and / or vegetable oils, and are supplemented with vitamins and minerals, antioxidants, and other additives

[56] .

[0361] Koppel et al. detected 54 volatile compounds in dry dog food, including aldehydes, esters, acids, alcohols, pyrazines, terpenes, furans, and ketones, with aldehydes and ketones being the main volatiles

[57] . Yin et al. reported a total of 55 volatile compounds in dog food sprayed with six palatability enhancers

[58] . Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography - mass spectrometry (GC-MS) was used to identify volatile compounds. Nine compounds that significantly contributed to the palatability of dog food were heptanal, nonanal, octanal, (E)-2-hexenal, (E,E)-2,4-decadienal, 2-pentylfuran, 4-methyl-5-thiazoleethanol, 2-furfurylthiol, and (E)-2-decenal. The contribution of the nine key aroma compounds was further analyzed through preference tests. (E)-2-decenal, 2-furfurylthiol, and 4-methyl-5-thiazoleethanol showed higher first-choice, consumption rates, and unit contribution rates (the relative amount of additional food consumed per unit amount of volatiles sprayed), and were crucial for the overall preferred aroma of dog food.

[0362] Myoglobin is important not only for the color of meat, but also for its sensory quality, and is related to the serum-like taste and metallic mouthfeel of meat

[59] . During meat cooking, aroma formation occurs mainly through lipid oxidation and the Maillard reaction. The latter occurs between the amino groups in proteins and the carbonyl groups from reducing sugars and / or lipid oxidation reaction products. Heme iron can affect these reactions and / or their kinetics

[60] .

[0363] In this study, animal myoglobin was obtained by precision fermentation in Pichia pastoris and added to plant-based model wet pet food formulations ( Figure 10 ). Then, headspace solid-phase microextraction (HS-SPME) was performed on raw and heat-sterilized samples, followed by gas chromatography-mass spectrometry (GC-MS) to determine their aroma profiles.

[0364] Depending on heat treatment and the presence of myoglobin, up to 1561 different aromatic compounds were detected in plant-based wet pet food samples. Among them, 111 were identified from the deconvoluted peaks and used for compound class and PCA analysis ( Figure 11 ). For the purposes of this study, aromatic compounds known to impart meaty aromas and / or produced during the Maillard reaction were selected and further analyzed ( Figure 11 , Figure 12 ).

[0365] The aroma profiles of raw samples with or without 0.1% myoglobin were mainly composed of aldehydes (29.5% and 40.7% respectively), followed by alcohols and ketones ( Figure 11 A). Heat sterilization of the samples led to a decrease in aldehydes (23.0% and 20.7% respectively), while the proportions of alcohols and ketones remained relatively stable. Compared to the unbaked counterparts (3.7% and 1.6% without and with myoglobin respectively), both sterilized samples with and without myoglobin showed a two-fold increase in the amount of furans (6.2% and 17.2% for sterilized samples without and with myoglobin respectively). Caramel, sweet, fruity, nutty, meaty, and burnt flavor impressions were associated with these compounds

[61] .

[0366] Principal component analysis (PCA) was performed to visualize the relationships among plant-based wet pet foods containing animal myoglobin (obtained by precision fermentation in Pichia pastoris) in terms of their volatile profiles (111 volatile compounds detected), with each sample replicated 3 times. The PCA biplot (combined score and loading plot) ( Figure 11 B) showed that the first two principal components (PCs) explained 80.8% of the total variance. The selection of volatile organic compounds (loadings, n = 12) was depicted by arrows, and their compound names were located on the right side of the figure ( Figure 11 B).

[0367] The first PC accounted for 57.4% of the variance and separated the raw plant-based wet pet food samples from the heat-sterilized samples. PC2 explained 23.4% of the variance, and its additional contribution separated the samples containing 0.1% w / w myoglobin from those without myoglobin. In the resulting score plot, the technical replicates (n = 3) clustered together, and the different samples were clearly separated. Twelve selected compounds (chosen based on their importance in meaty aroma, also see Figure 12 B) contributed significantly to the group of sterilized 0.1% w / w myoglobin samples ( Figure 11 B). The loading plot demonstrated how each of the 111 identified volatile organic compounds affected the two main principal components ( Figure 11 C). The volatile organic compounds mainly contributed to the quadrant of the samples containing 0.1% w / w myoglobin. The highest concentration of loadings was observed in the lower left quadrant, which corresponded to the sterilized plant-based wet pet food containing 0.1% animal myoglobin obtained by precision fermentation in Pichia pastoris.

[0368] Most interestingly, the presence of myoglobin in combination with heat treatment led to a four-fold increase in the amount of pyrazines (1.4% and 5.7% for the sterilized samples without and with myoglobin, respectively). Pyrazines are heterocyclic compounds that contribute to the characteristic odor and taste of food. These compounds are responsible for the "roasted-like" aroma of roasted meat or coffee beans. They are mainly formed during the thermal processing of food, such as coffee, cocoa, roasted nuts and seeds, grains and cereal products, meat products, and wine. Their formation is closely related to the Maillard reaction, which involves the reaction between α-dicarbonyls and amino acid groups to produce aminoketones

[62] .

[0369] In addition to pyrazines, several aromatic compounds were formed to a greater extent when the samples contained myoglobin obtained by precision fermentation in Pichia pastoris ( Figure 11 、 Figure 12 ). Some of these compounds have been reported to be found in conventional meat, are related to the Maillard reaction, and contribute to the "meaty" taste [1 - 4]. Compared with the sterilized pet food without myoglobin, when only 0.1% myoglobin was added, the peak areas of pyrazines (such as methylpyrazine, 2-ethyl-6-methylpyrazine, 2,6-dimethylpyrazine, and 2,5-dimethylpyrazine) increased significantly ( Figure 12 A). Compared with the sterilized samples without myoglobin, the levels of several aldehydes (methional, octanal, and nonanal), pyrrole, some lipid oxidation products (2-methylbutanal and 3-methylbutanal), and ketone (2-acetylthiophene) were also significantly increased in the heat-sterilized samples containing myoglobin ( Figure 12 A).

[0370] A two-way ANOVA was performed to evaluate the effect of the interaction between heat treatment and myoglobin addition on the volatiles formed. For all the selected pyrazine compounds, the interaction between heat treatment and myoglobin addition was significant (p < 0.0001). A Tukey pairwise comparison test was then performed, which confirmed that the combination of heat treatment and the presence of myoglobin led to a significant increase in the amount of pyrazines formed.

[0371] Example 7: Adverse food reactions

[0372] Adverse food reactions (AFR) in dogs and cats are common problems that can cause skin and / or gastrointestinal signs. Food allergy (classified as an immune-mediated reaction) is one type of AFR, and other reactions such as lactose intolerance are classified as non-immune reactions

[63] . Limited data are available on the prevalence of AFR in companion animals, but it is estimated that 1% of dogs and cats have AFR. Based on published studies, the prevalence of AFR in dogs with skin signs referred to dermatology centers is estimated to be between 7.6% and 12%

[64] . Verlinden et al. reported similar data: the authors estimated that 5% - 15% of dogs with skin or ear disorders are food allergic, and for cats it is 1% - 10%

[65] .

[0373] The most common food allergens and their prevalence in causing skin AFR in dogs (n = 297) and cats (n = 79) are listed in Table 4. In addition to this list, barley, rabbit meat, chocolate, kidney beans, and tomatoes have also been reported as food allergens in individual dogs (prevalence 0.3%). Eggs, barley, and rabbit meat have also been reported as allergens in individual cats (prevalence 1.2%)

[66] .

[0374] Table 4. Most commonly reported food allergens causing skin adverse food reactions in dogs (n = 297) and cats (n = 79). Source

[66] .

[0375] Food Dog Cat Beef 34％ 18％ Dairy products 17％ 4％ Fish - 17％ Chicken 15％ 5％ Wheat 13％ 4％ Lamb 5％ 3％ Corn - 4％

[0376] In addition, Dodds and colleagues studied the immunoreactivity of cat saliva (n = 1000), which showed that low-reactive foods were lamb, cow's milk, pork, turkey, wheat, and white fish, while high-reactive foods were millet, white potato, rice, and salmon

[67] .

[0377] Importantly, we conducted a literature search and found no reports of AFR caused by myoglobin in cats and dogs. These findings are also supported by a study that identified the allergens causing canine AFR to lamb, beef, and cow's milk

[68] . All dogs (n = 10) had IgE against bovine (bovine or cattle) IgG. Additionally, this was the only allergen detected in cow's milk. In lamb and beef, the major allergens were between 51 kDa and 58 kDa in size and were identified as phosphoglucomutase and IgG heavy chain. Other IgE-binding proteins with molecular masses of 27 kDa, 31 kDa, 33 kDa, 37 kDa, and 42 kDa were also detected in some serum samples. Since the size of myoglobin proposed in this patent is approximately 17 kDa, we can conclude that the authors did not detect any binding of IgE to myoglobin

[68] .

[0378] In addition to the literature search, we also performed a bioinformatics in silico analysis. Sequence homology searches were performed using the AllergenOnline database version 21 based on the amino acid sequences of different myoglobins (tuna (Thunnus orientalis), steppe mammoth (Mammuthus trogontherii), woolly mammoth (Mammuthus primigenius), rat (Rattus norvegicus), mouse (Mus musculus), rabbit (Oryctolagus cuniculus), and cow (Bos taurus)). A prophylactic search method (alignment of 80 amino acids, described in the methods section) was used. No significant homology with allergens present in the database was identified for the different myoglobin AA sequences. Significant homology was defined according to the Codex Alimentarius guidelines 2003

[69] , where an identity match of more than 35% was defined as the threshold to consider the possibility of cross-reactivity.

[0379] The AllergenOnline database consists of a peer-reviewed list of allergens (2290 protein sequence entries of unique, proven, or putative allergens from 412 species). The disadvantage of using this database is that most entries are human allergens since a large number of studies are on human allergenicity. However, canine allergies show a high similarity to human allergies (especially atopic dermatitis)

[70] , thus supporting the bioinformatics predictive search of the AllergenOnline database. Therefore,

[68] showed that the identified canine beef and lamb allergens were similar to those found for human meat allergens.

[0380] In summary, these results indicate that different myoglobins (tuna (Thunnus orientalis), steppe mammoth (Mammuthus trogontherii), woolly mammoth (Mammuthus primigenius), rat (Rattus norvegicus), mouse (Mus musculus), rabbit (Oryctolagus cuniculus), and cattle (Bos taurus)) are unlikely to cause AFR in pets consuming these functional ingredients.

[0381] Example 8: Materials and methods

[0382] The following materials and methods have been used in Examples 1 - 7.

[0383] Sequence analysis

[0384] Sequences encoding myoglobins from Rattus norvegicus, Oryctolagus cuniculus, Mus musculus, Thunnus orientalis, and Bos taurus were obtained from Uniprot (accession numbers: Q9QZ76, P02170, P04247, P68190, and P02192, respectively). The sequence encoding myoglobin from the steppe mammoth (Mammuthus trogontherii) was obtained after DNA extraction from a molar sample of the so - called Adycha specimen, Illumina DNA sequencing, read merging, and mapping of these reads against the African savannah elephant (Loxodonta africana) genome

[23] .

[0385] Using ClustalOmega

[71] for multiple sequence alignment and Jalview 2.11.1.4

[72] for visualization.

[0386] Protein modeling

[0387] Using the automated protein structure homology modeling server SWISS-MODEL (Waterhouse et al. 2018) to model the structures of the 6 myoglobins listed above (see 3.1). For 5 mammalian species, the high - resolution X - ray structure of deoxymyoglobin from Sus scrofa Protein Data Bank (PDB) accession number: 1myg.1.A) was used as a template. For myoglobin from Thunnus thynnus, the X - ray structure of myoglobin from Thunnus atlanticus (PDB accession number 2nx0.1) was used as a template. Using DeepView v4.1 To visualize all protein structures and generate images

[73] .

[0388] Construction of expression plasmids

[0389] Codon optimization of the myoglobin coding sequence for expression in Pichia pastoris (Komagataella phaffii)

[74] . The optimized sequences encoding myoglobin from Bos taurus and Mammuthus primigenius were chemically synthesized by GenScript after the coding sequence of the Saccharomyces cerevisiae mating factor α. The gene fragments were cloned into the pBDIPp5 vector (Bos taurus and Mammuthus primigenius), downstream of the AOX1 promoter and upstream of the AOX1 terminator, HIS4 selectable marker, and AOX1 3' fragment. For the other four myoglobins, the optimized sequences were chemically synthesized by IDT after the coding sequence of the Saccharomyces cerevisiae mating factor α and the AOX1 promoter, and before the AOX1 terminator and HIS4 selectable marker. The gene fragments were cloned into the pFL38 vector to replace the original URA3 selectable marker. The vectors were amplified in Escherichia coli (DH10B), purified using the SmartPure plasmid kit (Eurogenetec), and verified by Sanger sequencing (Eurofins Genomics). Expression cassettes were generated from the resulting vectors by restriction of the plasmids with BglII (New England Biolabs, which cuts upstream of the AOX1 promoter and downstream of the AOX1 3' fragment).

[0390] Strain engineering

[0391] The Pichia pastoris (Komagataella phaffii) strain GS115 (his4) was obtained from Life Technologies. Cell transformation was performed using electroporation essentially as previously described

[75] . Briefly, cells of the GS115 strain were grown in YPD (1% yeast extract, 2% peptone, and 2% D-glucose) medium. Cells in exponential growth phase were incubated in YPD medium containing 200 mM HEPES buffer (pH 8.0) and 25 mM dithiothreitol for 30 minutes. The competent cells were then washed with ice-cold 1 M sorbitol and transferred to a sterile electroporation cuvette (Bio-Rad). The cells were then electroporated with 1 - 5 μg of the linear expression cassette (see 3.3) using a Gene-Pulser (Bio-Rad) electroporator and resuspended in 1 mL of YPD medium containing 1 M sorbitol, and then transferred to a sterile 1.5 mL Eppendorf tube. The cells were incubated without agitation at 28 °C for 3 h and then plated onto agar plates containing solid MGY medium (minimal glycerol medium: 1.34% yeast nitrogen base (containing ammonium sulfate, without amino acids), 2% D-glucose, 4 × 10 -5 % biotin, and 2% agar). The plates were incubated at 28 °C for up to 4 days.

[0392] Transformants able to grow in the absence of histidine were verified by PCR. A DNA fragment specific to the myoglobin expression cassette was amplified using a primer pair that annealed to the coding sequence of the mating factor α of Saccharomyces cerevisiae. According to the manufacturer's instructions, a PCR reaction was performed using a 2X master mix with standard buffer (New England Biolabs). After migrating for 60 minutes at 60 V in a 2% agarose TAE gel containing ethidium bromide, the PCR products were visualized. The Purple 1kb Plus DNA ladder (New England Biolabs) was used to control the size of the amplified fragments.

[0393] Positive clones were then screened for the ability to express Mb. Cells were grown in BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH 6), 1.34% yeast nitrogen base (containing ammonium sulfate, without amino acids), 4 × 10 -5 % biotin, 1% glycerol) in 96-well deep-well microtiter plates. 1% methanol was added to exponentially growing cells to induce Mb expression. Samples were collected 72 h after the start of methanol induction and analyzed by SDS-PAGE to assess Mb levels (see below).

[0394] The best-producing strains were selected for further experiments and cryopreserved at -80 °C as a master cell bank prior to use. The results shown here were obtained using the PAL004 (bovine myoglobin) and PAL096 (rabbit myoglobin) strains.

[0395] Recombinant protein production and purification

[0396] Cells from the master cell bank were plated on YPD agar (1% yeast extract, 2% soy peptone, and 3% D-glucose, 2% agar) plates and incubated at 28 °C for 2 days. A seed culture was then prepared in a 2 L flask containing 200 mL of YPG (1% yeast extract, 2% soy peptone, and 3% glycerol) medium and incubated at 28 °C in an orbital shaker incubator for 24 h. This seed culture was then used to inoculate a glass vessel fermenter (Biostat B, Sartorius) containing 900 ml of modified BSM medium

[76] . The batch fermentation phase was carried out for approximately 14 - 16 h until all the glycerol was consumed. A glycerol fed-batch phase was then carried out to further increase the biomass. The temperature was then reduced to 26 °C, and a mixed glycerol:methanol was applied for 72 h to induce Mb expression. Dissolved oxygen was maintained above 25% throughout the fermentation, while the pH was kept at 6 during the growth phase and at 5 during the induction phase.

[0397] After 96 h of methanol induction, the cells were removed by centrifugation at 4,000 rpm at 4 °C. Using a tangential flow filtration device (Sartoflow, Sartorius), the remaining cells were removed by microfiltration using a cellulose filter with a pore size of 0.45 μm (Hydrosart, Sartorius). After ultrafiltration using a cellulose filter with a molecular weight cut-off of 10 kD using the same device, the product was stored frozen at -20 °C until use. When assay was required, the ultrafiltration retentate was further concentrated using a disposable ultrafiltration centrifugal device (Pierce Protein Concentrator, Thermo Scientific) with a polyethersulfone membrane having a molecular weight cut-off of 10 kD.

[0398] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0399] For protein electrophoresis, 20 μl of cell-free supernatant was mixed with an equal volume of 2X protein loading buffer (100 mM Tris (pH 6.8); 4 mM EDTA, 4% SDS, 20% glycerol; 0.02% bromophenol blue, 4% β-mercaptoethanol). The samples were incubated at 95 °C for 5 minutes and an appropriate volume was loaded into a 15% polyacrylamide gel in a vertical mini-PROTEAN gel apparatus (Bio-Rad) at 200 V for 40 minutes. Myoglobin from horse heart muscle (Sigma) was used as a standard. Molecular weight markers (PageRuler TM Pre-stained protein ladder) were purchased from Thermo Fisher. After electrophoresis, the proteins in the gel were stained with Coomassie Brilliant Blue G-250 (Bio-Rad).

[0400] Plant-based meat alternatives as pet food

[0401] The basic formulation of the plant-based wet pet food consisted of 20% w / w soy protein isolate (Bodyfit, NL), 10% w / w rapeseed oil and 70% w / w tap water. To achieve a myoglobin level of 0.1% w / w in the pet food, a certain volume of tap water was replaced with a myoglobin solution. This depended on the concentration of myoglobin in the solution. The matrix was mixed for 3 minutes using a hand-held mixer. 10 g of the matrix was transferred to a 250 mL Schott bottle using a plastic syringe. Baking was carried out in a pressure cooker at 121 °C for 30 minutes (for aroma analysis).

[0402] Colorimetric analysis

[0403] First, the color stability over time was measured in selected bovine myoglobin samples. These samples were first frozen at -20 °C immediately after production and kept frozen and in the dark until use. At the start of the experiment, the samples were wrapped in aluminum foil to protect from light and placed at 4 °C to thaw for 4 hours. The samples were kept under these conditions for 2 months and sampled at non-equidistant intervals.

[0404] Prior to colorimetric measurements, dilution was carried out with distilled water such that a total of 10 mg was present in 30 mL of the sample for colorimetry, allowing reproducible measurement of color values. Prior to taking the sample color readings, the colorimeter (HunterLab ColorFlex EZ Model 45 / 0, Hunter Associates Laboratory Inc., Reston, Virginia, USA) was calibrated using standard black and white color tiles. Using the Commission Internationale de l'Eclairage (CIE L*a*b*) colorimetric system, color readings were recorded with a D65 illuminant and a 10° standard observer (where L* describes the lightness of the sample (0 = black, 100 = white), a* describes the range from green (-) to red (+), and b* describes the range from blue (-) to yellow (+)). Then, equal volumes of each sample were placed in glass cuvettes and the L*a*b* values were read. Data capture was performed using EasyMatch QC version 4.98 software (Hunter Associates Laboratory Inc., Reston, Virginia, USA). The color difference (ΔE) value was calculated according to the following equation, where L, a, b are the values of the sample, and L 0 、a 0 and b 0 are the initial color values:

[0405]

[0406] Second, color values were read in plant-based wet pet foods supplemented with 0, 0.1% w / w, 0.2% w / w, 0.5% w / w, and 1% w / w bovine or rabbit myoglobin. The plant-based wet pet food consisted of 20% w / w soy protein isolate (BodyFit, NL), 10% w / w rapeseed oil, and 70% w / w H 2It consists of O. Different volumes of myoglobin solution are added to this mixture to replace that volume of water to obtain a matrix with a desired myoglobin concentration. A part of the sample is kept raw, and another part is placed in a 250 mL Schott bottle and heat-processed by autoclaving at 121 °C for 30 min. Then all the samples are transferred to 35 mm plastic Petri dishes. Readings are taken in these dishes as they can accommodate the measuring slot of the colorimeter. The measurement procedure is described above. In some cases, the samples are placed at 4 °C and left standing with the lid on for 20 days, after which colorimetric measurements are taken. The chroma (C*) and hue angle (h ab ) are calculated using the following formula

[49] :

[0407]

[0408] Aroma analysis

[0409] As described in paragraph 3.8, 70 g of a plant-based model wet pet food is prepared. 0.1% w / w of bovine myoglobin is added to this base matrix. The sample is heat-processed at 121 °C for 30 minutes and cooled to room temperature. Then 2 g of the pet food sample is transferred to a 20 mL glass vial for aroma profiling.

[0410] Solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) is used to analyze the aroma from plant-based meat alternatives with or without myoglobin. All samples are tested in the raw state (before baking) and after baking in a pressure cooker at 121 °C for 30 minutes. The samples are analyzed in triplicate and in batches to minimize the time spent at room temperature. For each sample, 2 g of the material is inserted into 20 mL for extraction using a PDMS / DVB / CAR (gray) SPME fiber, an incubation temperature of 45 °C for 20 min, and an extraction time of 40 minutes. The extracted compounds are injected into a DB-5MS (30 m x 0.25 mm x 1 μm) column using a splitless injector at 250 °C. TOF detection is performed in scan mode (35 - 350 amu, 10 scans / s). The SPME-GC-MS spectra are processed for peak deconvolution and compound identification.

[0411] Texture analysis

[0412] For texture characterization, as described in paragraph 3.8, prepare a base formulation of 500 g (containing 0.1% bovine myoglobin) or 550 g (without myoglobin) of model wet pet food. Fill a silicon tray with a cubic mold (3.3 x 3.3 x 3.3 cm) with 37 g (40 mL) of model wet pet food using a syringe and cook in an oven with a full tray of water (steam-generating oven) at 180 °C for 15 minutes. Prepare 12 replicates of cubes with and without myoglobin. Then wrap the samples in aluminum foil and store overnight in a container at 4 °C before analysis.

[0413] To obtain measurements of hardness (defined as peak force [g]) and adhesiveness (defined as negative peak force [g]), perform TPA. The TPA sequence involves contacting, compressing, and withdrawing from the product back to the original contact point, then repeating the whole cycle again. Use a Lloyd LF Plus texture analyzer and the accompanying computer software to measure the force-time curve. Compress the sample under a 5 cm diameter cylindrical probe at a test speed of 2 mm / s and a compression distance of 1 cm for 5 seconds.

[0414] Evaluate the differences in different texture parameters between samples with or without myoglobin by Student's t-test.

[0415] Adverse reaction prediction

[0416] By running a homology search of the myoglobin amino acid protein sequence listed above on a AllergenOnline database (i.e., a comprehensive list of putative allergenic proteins developed for the purpose of food safety assessment) to evaluate allergenicity. Perform an 80-mer sliding window homology search with default parameters. Any sliding window with a sequence showing more than 35% identity to any sequence stored in the database will be considered a match and thus indicate a possible cross-reactivity with known allergens.

[0417] In addition to in silico allergenicity prediction, perform a literature search on the Google Scholar and Pubmed databases using the following search queries: "Myoglobin allergy in cats", "Myoglobin allergy in dogs", "Myoglobin allergy in pets".

[0418] Example 9: Additional dog food product containing isolated rabbit myoglobin

[0419] Produce purified rabbit myoglobin by E. coli.

[0420] The full-length rabbit myoglobin gene SEQ ID NO:2 was synthesized and cloned into a modified pET-23a(+) vector that contains a T7 promoter and terminator as well as a C-terminal hexa-His tag (GenScript Biotech Corporation, Leiden, the Netherlands). The ampR marker gene originally present in the vector was replaced by the proBA operon from Escherichia coli strain K12 (including its native transcriptional regulatory elements) to facilitate antibiotic-free selection. The correctly assembled plasmid was confirmed by PCR and used to transform a proline auxotrophic E. coli protein production strain (E. coli K12ΔproBA). The transformed strain was incubated overnight at 37 °C and 150 rpm (pH 6) in a shake flask containing minimal medium (10.5 g / L K 2 HPO 4 、4.5 g / L KH 2 PO 4 、1.0 g / L (NH 4 ) 2 SO 4 、0.12 g / L MgSO4, 0.5 g / l sodium citrate, 2 g / L glucose, and 5.0 mg / L thiamine·HCl). 500 μl of the overnight culture was transferred to a 1 L shake flask containing 500 mL of minimal medium and incubated at 37 °C and 150 rpm until an OD600 of 0.4 - 1 was reached. IPTG (isopropyl-β-D-thiogalactopyranoside) at a concentration of 100 μM was added to the culture, and then the culture was incubated at 16 °C and 150 rpm for 24 h. The culture was harvested and centrifuged at 3500 x g (4 °C) for 15 min. The supernatant was discarded, and the pellet was resuspended in 50 mL of BugBuster protein extraction reagent (Novagen) that contains 1 KU lysozyme / ml (Sigma-Aldrich), 25 U nuclease, and cOmplete TM EDTA-free protease inhibitor mixture (Roche). The resuspended pellet was incubated in a shaker at 4 °C for 30 min. The centrifugation step was repeated, and the cell-free extract (supernatant) was collected and assayed by SDS-PAGE to confirm myoglobin production. Rabbit myoglobin production in the corresponding transformed strain was confirmed by the presence of a protein band of the correct size.

[0421] For purification, the cell-free extract containing the protein soluble fraction was loaded onto a HisTrap FF 1 mL column (Cytiva, Massachusetts, USA) coupled to an AKTA start system. The column was equilibrated with 20 mM HEPES, 0.4 M NaCl, and 20 mM imidazole (pH 7.5) at a flow rate of 1 mL / min. The protein was eluted with 20 mM HEPES, 0.4 M NaCl, and 400 mM imidazole (pH 7.5). The fractions containing rabbit myoglobin were pooled, concentrated, and confirmed by SDS-PAGE and Western blotting using an anti-histidine tag antibody (Bio-Rad) to confirm the successful purification of all myoglobin. The purified rabbit myoglobin was stored at -20 °C for later use.

[0422] Production of pet food

[0423] A 100 kg dry dog food control product was prepared with the ingredients in the proportions shown in Table 5. The ingredients of the dry base were weighed and placed in a mixer. These ingredients were blended and ground with a hammer mill and transferred to a storage bin.

[0424] Table 5 Dog food formula

[0425]

[0426]

[0427] The dry base mixture was fed into an extruder at 250 kg / h, and fish oil was fed into the extruder at 2.4 kg / h. Water (10 L / h) and steam (27 L / h) were added to the mixture of ingredients in the extruder barrel. The extruder screw speed was 900 rpm, and the conditions of the molten material were maintained at 20 bar and at least 104 °C. As the ropes of the expanded material left the extruder die, these ropes were cut into coarse grains by a cutter rotating at 900 rpm across the die surface. The coarse grains were fed into a dryer and dried at 125 °C for 21 minutes to a moisture content of 9.2%. After spraying all the liquids, the coated coarse grains were tumbled for another 5 minutes.

[0428] A small amount of the coarse grains was sampled for color stability, aroma, and nutritional analysis. Then the coated coarse grains were filled into polyethylene bags and sealed.

[0429] Example 10: Additional cat food product containing isolated rat myoglobin

[0430] Prepare 1 kg of a homemade cat food product with the ingredients in the proportions shown in Table 6. Weigh the ingredients and place them in a mixer. Blend these ingredients with a blender. After blending the mixture, add the isolated myoglobin to the mixture and blend for another 2 min. Measure the isolated myoglobin in powder form using a household scale. Alternatively, if the isolated myoglobin is in cube form, with each cube being 10 g, add 5 cubes to the mixture and then blend for another 2 min. Then place the mixture in a mold and bake it in an oven at 180 °C for 30 min. The mixture can also be further processed in a dryer to a moisture content of approximately 10%.

[0431] Table 6. Homemade Cat Food Recipe

[0432] Ingredient g Whole grain Approximately 700 Vegetable Approximately 250 Egg 50 Mineral premix 10 Vitamin A premix 5 Fish oil 5 Isolated rat myoglobin 50

[0433] The ingredients can be changed and alternative types of ingredients can be added. Vegetables can include spinach, lettuce, kale, lentils, peas, chickpeas, or beans. Grains can include rice, corn, barley, oats, wheat. Tubers can include potatoes, sweet potatoes. Sprouts can include soybeans, lentils, rye, sesame. Flowers can include artichokes, broccoli, cauliflower. Stems can include celery, asparagus. Seeds can include pumpkins, sunflowers.

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Claims

1. A method for preparing pet food, wherein the method comprises adding isolated myoglobin, and the isolated myoglobin is rat myoglobin, rabbit myoglobin, mouse myoglobin, mammoth myoglobin or tuna myoglobin.

2. The method according to claim 1, wherein the isolated myoglobin is not contained in cells or tissues before the addition.

3. The method for preparing pet food according to claim 1 or 2, wherein the isolated myoglobin is added as part of a composition, and the weight fraction of the isolated myoglobin in the protein fraction contained in the composition is at least 75%.

4. The method for preparing pet food according to any one of the preceding claims, wherein the weight concentration of heme protein in the pet food is at least 1%, and the weight fraction of myoglobin in the heme protein is at least 50%.

5. The method for preparing pet food according to any one of the preceding claims, wherein the pet is a carnivore.

6. The method for preparing pet food according to any one of the preceding claims, wherein the pet is a dog or a cat.

7. The method for preparing pet food according to any one of the preceding claims, wherein the pet food is dry pet food, semi-moist pet food, wet pet food or any combination of these types of pet food.

8. The method for preparing pet food according to any one of the preceding claims, wherein the pet food is a meat substitute.

9. The method for preparing pet food according to claim 8, wherein the meat substitute is a cell-based meat substitute, a cultured meat substitute or a plant-based meat substitute.

10. The method for preparing pet food according to claim 8 or 9, wherein the meat substitute mimics the appearance, form, structure, composition, palatability, flavor, texture, color, aroma, appearance and / or nutritional value of meat.

11. The method for preparing pet food according to claim 10, wherein compared with the corresponding pet food without added isolated myoglobin, the aroma of the meat substitute is characterized by a higher concentration of Maillard compounds during cooking.

12. The method for preparing pet food according to any one of the preceding claims, wherein the isolated myoglobin is recombinant myoglobin obtained from microbial fermentation, and the microorganism has been genetically modified to express the recombinant myoglobin.

13. The method for preparing pet food according to claim 12, wherein the microorganism is a bacterium, yeast, filamentous fungus or cultured mammalian cell line, preferably Escherichia coli or Saccharomyces cerevisiae.

14. The method for preparing pet food according to any one of the preceding claims, wherein the isolated myoglobin is represented by one of the following amino acid sequences: a) A sequence having at least 90% sequence identity with SEQ ID NO:1 and having at least one of the following amino acid combinations: - M at position 10, and / or - A at position 36, and / or - S at position 58, and / or - M at position 10 and A at position 36, and / or - M at position 10 and S at position 58, and / or - A at position 36 and S at position 58, and / or - M at position 10, A at position 36 and S at position 58; b) a sequence having at least 90% sequence identity with SEQ ID NO:2 and having amino acid A at position 6; c) a sequence having at least 90% sequence identity with SEQ ID NO:3 and having at least one of the following amino acid combinations: - D at position 55, and / or - I at position 111, and / or - E at position 114, and / or - D at position 55 and I at position 111, and / or - D at position 55 and E at position 114, and / or - I at position 111 and E at position 114, and / or - D at position 55, I at position 111 and E at position 114; d) a sequence having at least 90% identity with SEQ ID NO:4 or 5 and having F at position 30 and / or Q at position 65; e) a sequence having at least 90% identity with SEQ ID NO:6 and having H at position 65 and / or position 94; f) a sequence having at least 90% identity with SEQ ID NO:1, 2 or 3 and having at least one of the following amino acid combinations: - L at position 22, and / or - A or T at position 36, and / or - G at position 122; - L at position 22 and A or T at position 36, and / or - L at position 22 and G at position 122; and / or - A or T at position 36 and G at position 122, and / or - L at position 22, A or T at position 36 and G at position 122; g) a sequence having at least 90% identity with any one of SEQ ID NO:1 to 5 and having I at position 87; h) a sequence having at least 90% identity with SEQ ID NO:1 or 3, and having at least one or more of the following amino acids or any combination thereof: - N at position 49, and / or - E at position 54, and / or - C at position 67, and / or - T at position 75, and / or - Q at position 82, and / or - K at position 117, and / or - K at position 118, and / or - S at position 121; i) a sequence having at least 90% identity with SEQ ID NO:4, and having at least one of the following amino acid combinations: - H at position 92, and / or - H at position 92 and F at position 30, and / or - H at position 92 and Q at position 65, and / or - H at position 92, F at position 30 and Q at position 65.

15. A method for preparing pet food according to any one of the preceding claims, wherein the isolated myoglobin is represented by any one of SEQ ID NO:1 to 6, preferably by SEQ ID NO:1 or 2.

16. A pet food obtainable by the method according to any one of the preceding claims.

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