WO2024100067A1 - Dairy product or replica thereof supplemented with heme-comprising protein - Google Patents

Dairy product or replica thereof supplemented with heme-comprising protein Download PDF

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Publication number
WO2024100067A1
WO2024100067A1 PCT/EP2023/081041 EP2023081041W WO2024100067A1 WO 2024100067 A1 WO2024100067 A1 WO 2024100067A1 EP 2023081041 W EP2023081041 W EP 2023081041W WO 2024100067 A1 WO2024100067 A1 WO 2024100067A1
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Prior art keywords
replica
dairy product
myoglobin
seq
hemeprotein
Prior art date
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PCT/EP2023/081041
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French (fr)
Inventor
Hermes SANCTORUM
Andy DE JONG
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Paleo B.V.
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Publication date
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Publication of WO2024100067A1 publication Critical patent/WO2024100067A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/015—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1315—Non-milk proteins or fats; Seeds, pulses, cereals or soja; Fatty acids, phospholipids, mono- or diglycerides or derivatives therefrom; Egg products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/18—Vegetable proteins from wheat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A23L33/165—Complexes or chelates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a dairy product, or a replica thereof, supplemented with a hemeprotein.
  • the invention also relates to a method for preparing a dairy product, or a replica thereof wherein the method comprises adding a hemeprotein to a base dairy product, or a replica thereof.
  • Dairy products including milk have become one of the most important and commonly consumed food products in daily life. Dairy products, such as milk, are great sources of nutrients that are vital for the growth, health and maintenance of the body. These nutrients include protein, vitamin A, vitamin D, vitamin B12, potassium, phosphorus, riboflavin, zinc, choline, magnesium, selenium and calcium.
  • the amount of dairy products that one needs varies. This is mainly due to the different needs for different types of nutrients during different physical situations between consumers. For females, the amount can also depend on the maternity situation, for example, whether pregnant or breastfeeding. In early life, newborn babies are prone to iron deficiency due to the rapid growth and low iron in milk.
  • the present invention provides dairy products, and replicas thereof, comprising a significant amount of bioavailable iron.
  • a dairy product, or a replica thereof, supplemented with a hemeprotein is said to be supplemented with a hemeprotein if a hemeprotein has been added during any step of its preparation.
  • supplementation with a hemeprotein results in an increase of the hemeprotein concentration during the preparation, which is preferably reflected in an increased hemeprotein concentration in the (final) dairy product, or the replica thereof, as defined below.
  • a method for preparing a dairy product, or a replica thereof comprising adding a hemeprotein to a base dairy product, or a replica thereof.
  • a dairy product, or a replica thereof obtainable by a method comprising the addition of a hemeprotein to a base dairy product, or a replica thereof.
  • a dairy product or a replica thereof, comprising a hemeprotein.
  • a hemeprotein any preference relating to the hemeprotein listed herein may be applied to the hemeprotein in the fourth aspect of the invention.
  • the hemeprotein may be added or applied by manufacturer of a dairy product, the consumer of such dairy product, or any other producer of a dairy product or a replica thereof.
  • the products of the first, third and fourth aspect of the invention, and those obtained via the second aspect of the invention, comprise iron in the form of heme-iron.
  • This organic heme-iron is more easily taken up by the body, compared to the non-heme iron (e.g. present in transferrin), and provides a significant part of the bioavailable iron in non-vegetarian diets. Hence, it solves the unmet need in the art.
  • any specification of the sequence of the hemeprotein applies to the hemeprotein in the first, second and third aspect of this invention, and to the hemeprotein in the fourth aspect of this invention.
  • Dairy products are food products made from or containing a mammalian milk.
  • a mammalian milk is a white-colored liquid produced by the mammary glands of mammals. It is an emulsion or colloid of butterfat globules within a water-based fluid that contains dissolved carbohydrates and protein aggregates with minerals.
  • Mammalian milk is the primary source of nutrition for young mammals before they can digest solid food.
  • the nutrients comprised in dairy products can be broadly classified as macronutrients (protein, carbohydrate and fat) and micronutrients (vitamins and minerals).
  • Cow’s milk usually contains about 3.5 % protein, and 4.5-5.0 % carbohydrate (lactose) and depending on whether it is skimmed (fat- free milk), low-fat or whole milk (full-fat milk), the fat content ranges from 0.3-3.6%.
  • the vitamins and minerals in dairy products contribute to the essential functions of physiological processes, for example as summarized in table 1 below.
  • a milk may be the unprocessed mammalian milk or may be produced after optional homogenization or pasteurization of the mammalian milk, in several grades after standardization of the fat level, and the possible addition of the bacteria Streptococcus lactis and/or Leuconostoc citrovorum.
  • a cream may be obtained by skimming the higher-fat layer skimmed from the top of the mammalian milk before homogenization.
  • a butter may be made from the fat and protein components of churned cream.
  • a yoghurt may be obtained from the mammalian milk after fermentation by lactic acid bacteria, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus, which produce lactic acid that decreases pH and causes milk protein to coagulate. Lactic acid produced from fermentation acts on milk protein to give yoghurt its texture and characteristic tart flavor.
  • a cheese may be produced in a wide range of flavors, textures, and forms by coagulation of the milk protein casein.
  • a custard is a variety of culinary preparations that may be based on sweetened milk, cheese, or cream cooked with egg or egg yolk to thicken it, and sometimes also flour, corn starch, or gelatin.
  • An ice cream is a sweetened frozen food that may be made from milk or cream and eaten as a snack or dessert.
  • a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream relate to the general appearance of the food product.
  • dairy product which are made from or contain a mammalian milk, and replicas thereof, which are not of mammalian origin as described below, may be categorized in one of these categories.
  • a replica of a dairy product is a food product that resembles a dairy product but is neither made from nor contains milk produced by a mammary gland of a mammal. In other words, a replica of a dairy product is not of mammalian origin.
  • a dairy replica product is by definition, not a dairy product, not a natural dairy product, not a genuine dairy product.
  • the expression “a replica of a dairy product may be replaced by “a dairy food substitute”, “a dairy product substitute”, or “a non-natural dairy product”. It means that within the context of this invention, a replica of a dairy product is not a dairy product from or derived from a dairy animal such as a cow, a buffalo, a goat, a sheep or a horse.
  • cell-based, cultured, or plant-based dairy product substitutes may be considered replicas of dairy products.
  • To resemble in this context means to approach, to have essentially or substantially the same, to have the same, or to mimic one or more physical, chemical or sensory characteristics of a corresponding control or reference dairy product.
  • physical characteristics are color, viscosity, particle size distribution.
  • chemical characteristics are nutrient concentrations, pH, ionic strength.
  • sensory characteristics are form, structure, texture, flavor, color, aroma, appearance.
  • the dairy product is a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream.
  • the replica of a dairy product is a replica of a milk, a cream, a butter, a yoghurt, a cheese, a custard or of an ice cream.
  • the dairy product is a milk, wherein the milk is a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula.
  • the replica of a dairy product is a replica of a milk
  • the replica of a milk is a replica of a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula.
  • Whole milk means milk is taken as is from the mammal origin, without any sort of nutritional alteration, and processed for food safety, or a replica of such a dairy product. This means no nutrient is removed from nor supplemented to the milk. Whole milk is not considered raw milk and is safe to consume.
  • Whole milk is sometimes also referred to as fresh milk or regular milk.
  • Reduced-fat milk has at least 25% less fat than regular milk.
  • Low milk has less than 1 .5% fat of the total weight of the milk.
  • Fat- free (also known as skim) milk has less than 0.2% fat of the total weight of the milk.
  • Lactose-free milk is a type of milk where natural sugar lactose has been broken down.
  • Flavored milk is a sweetened dairy drink made with milk, sugar, flavorings, and sometimes food colorings.
  • Raw milk is unpasteurized milk directly from animal origin.
  • Breast milk or mother's milk is milk produced by the mammary glands of a human female.
  • Infant formula or baby formula also called infant milk or infant growth milk, is a manufactured dairy product or replica thereof designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottle-feeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water).
  • the U.S. Federal Food, Drug, and Cosmetic Act defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants because of its simulation of human milk or its suitability as a complete or partial substitute for human milk".
  • the starter formula is suitable for babies up to 6 months of age.
  • the follow-on formula is suitable for older children.
  • a comparison between human breast milk, infant formula and full-fat milk from cow shows the difference in nutrient composition.
  • a pregnant woman needs about 30 to 45 mg of iron per day (compared to 18 mg for adult women between 19 to 50 years old) to meet the new demands of extra blood volume, the developing placenta, and growing fetus.
  • Sufficient iron supply for the developing fetus, neonate and infant is critical to produce new red blood cells, muscle cells and brain development.
  • the iron content of human breast milk is low: 0.3 to 0.9 mg/L compared with 4 to 7 mg/L in supplemented infant formula based on cow milk.
  • the absorption rate however, is considerably higher.
  • Breastfed infants absorb up to 50% of consumed iron, compared with an average 10% absorption rate for formula-fed infants (Fomon et al., 1993).
  • a dairy product specifically an infant formula
  • Milk is usually produced from a dairy animal, such as a cow, a buffalo, a goat, a sheep, a horse, a llama or other less common animals such as a yak, a horse, a reindeer, a zebu, a giraffe and a donkey.
  • cow milk means milk (a type of dairy product) produced from a cow.
  • human (breast) milk means a (breast) milk from a human.
  • the dairy product supplemented with a hemeprotein is derived from a milk of a dairy animal such as a cow, a buffalo, a goat, a sheep, a horse, a yak, a horse, a llama, a reindeer, a zebu, a giraffe or a donkey, preferably from a milk of a cow.
  • a cow refers to an animal of the subspecies Bos primigenius taurus and not to a female specimen of other species such as yak or buffalo.
  • the replica of the dairy product is a vegan product, preferably a vegan milk.
  • vegan milk may be plant-based juice that resembles the texture, taste and qualities of conventional animal milk. It can also be used to make many products, such as replicas of dairy products.
  • vegan milk may be soya milk (soy milk), almond milk, coconut milk, rice milk, cashew milk, macadamia milk, flax milk, pea protein milk, banana milk, sunflower milk, peanut milk, oat milk, hazelnut milk and sunflower milk.
  • a hemeprotein as defined can refer to all proteins or protein subunits that are capable of covalently or noncovalently binding a heme moiety.
  • a hemeprotein as defined herein is preferably found to elicit oxygen binding activity or to carry an atom of iron bound to a heme.
  • the oxygen binding activity may be assessed by determining the ratio of oxygen-bound versus non-oxygen-bound myoglobin using spectrophotometry, such as the measurement and calculation method according to Tang et al. (Krzywicki revisited: equations for spectrophotometric determination of myoglobin redox forms in aqueous meat extract. J Food Sci. 2004).
  • the activity of carrying an atom of iron bound to a heme may be assessed by detecting the absorbance peak near 400 nm (the so called Soret band) caused by the presence of heme iron using spectrophotometry, or by using mass spectrometry.
  • Soret band the absorbance peak near 400 nm
  • the hemeprotein comprises a heme group.
  • Heme-containing polypeptides may transport or store oxygen.
  • Some examples of known heme-containing protein or polypeptide include hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin, androglobin, a globin E, a globin X, a globin Y, a flavohemoglobin, Hell's gate globin I, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.
  • HbN or HbO
  • the hemeprotein is a metalloprotein. In an embodiment, the hemeprotein is a globin, preferably hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin, more preferably myoglobin or hemoglobin, most preferably myoglobin.
  • Myoglobin is a relatively small globular protein of about 17 kD, found in heart and skeletal muscles. It carries a single heme group with an atom of iron capable of reversible oxygen binding (Fig. 1), allowing myoglobin to transport oxygen from the cell surface to mitochondria.
  • the hemeprotein is not a protein selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin and transferrin.
  • the replica dairy product does not comprise one or more proteins selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin and transferrin.
  • the hemeprotein may be represented by an amino acid sequence having at least 90% identity with any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NQ:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum) , SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:27 (Paramecium caudatum), SEQ ID NO:28 (Aspergillus niger), SEQ ID NO:29 (Zea mays), SEQ ID NQ:30 (Oryza
  • SEQ ID NO:31 Alkaopsis thaliana
  • SEQ ID NO:32 Pisum sativum
  • SEQ ID NO:33 Vigna unguiculata
  • SEQ ID NO:34 Sus scrofa
  • SEQ ID NO:35 Equus caballus
  • SEQ ID NO:36 Nicotiana benthamiana
  • SEQ ID NO:37 Bacillus subtilis
  • SEQ ID NO:38 Corynebacterium glutamicum
  • SEQ ID NO:39 Synechocystis PCC6803
  • SEQ ID NQ:40 Synechococcus sp.
  • the hemeprotein may be represented by an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NO:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum), SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), S
  • SEQ ID NO:31 Alkaopsis thaliana
  • SEQ ID NO:32 Pisum sativum
  • SEQ ID NO:33 Vigna unguiculata
  • SEQ ID NO:34 Sus scrofa
  • SEQ ID NO:35 Equus caballus
  • SEQ ID NO:36 Nicotiana benthamiana
  • SEQ ID NO:37 Bacillus subtilis
  • SEQ ID NO:38 Corynebacterium glutamicum
  • SEQ ID NO:39 Synechocystis PCC6803
  • SEQ ID NQ:40 Synechococcus sp. PCC 7335
  • SEQ ID NO:41 Nostoc commune
  • SEQ ID NO:42 Bacillus megaterium
  • the hemeprotein may be represented by an amino acid sequence of any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NQ:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum), SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:27 (Paramecium caudatum), SEQ ID NO:28 (Aspergillus niger), SEQ ID NO:29 (Zea mays), SEQ ID NQ:30 (Oryza sativa
  • SEQ ID NO:31 Alkaopsis thaliana
  • SEQ ID NO:32 Pisum sativum
  • SEQ ID NO:33 Vigna unguiculata
  • SEQ ID NO:34 Sus scrofa
  • SEQ ID NO:35 Equus caballus
  • SEQ ID NO:36 Nicotiana benthamiana
  • SEQ ID NO:37 Bacillus subtilis
  • SEQ ID NO:38 Corynebacterium glutamicum
  • SEQ ID NO:39 Synechocystis PCC6803
  • SEQ ID NQ:40 Synechococcus sp. PCC 7335
  • SEQ ID NO:41 Nostoc commune
  • SEQ ID NO:42 Bacillus megaterium
  • a dairy product, or a replica thereof, supplemented with a hemeprotein wherein the hemeprotein is an isolated hemeprotein.
  • An isolated hemeprotein is preferably defined as a hemeprotein which is not comprised in a cell or a tissue right before and/or during the addition.
  • the isolated hemeprotein is added (during the supplementation step) as a “separate” (or isolated) protein which is not comprised in a cell or a tissue.
  • isolated in this context does not refer to the origin of the hemeprotein (i.e. isolated from a source such as cell or a tissue), but to the fact that a separate protein is added. It is of course understood that the isolated myoglobin may become part of a cell or a tissue once it has been added to the dairy product, or the replica thereof.
  • the hemeprotein is a myoglobin.
  • Myoglobin contains 153 amino acids and as with other globins, consists of eight alpha helices connected by loops.
  • Myoglobin contains a porphyrin ring with an iron at its center.
  • a proximal histidine group (His-93) is attached directly to iron, and a distal histidine group (His-64) hovers near the opposite face.
  • Myoglobin is an iron- and oxygen-binding protein found in the cardiac and skeletal muscle tissue of vertebrates in general and in almost all mammals. Muscle cells use myoglobin to accelerate oxygen diffusion and act as localized oxygen reserves for times of intense respiration.
  • Hemoglobin is the iron-containing oxygen-transport metalloprotein in erythrocytes in vertebrates. Hemoglobin in the blood carries oxygen from the respiratory organs to the rest of the body. Myoglobin is a single subunit protein, while hemoglobin has four subunits. Both contain the heme group, which is responsible for binding oxygen. The heme group consists of an organic component protoporphyrin as well as an inorganic component that consists of an iron atom. There is not a single ubiquitous regulatory mechanism for heme biosynthesis. In mammalian cells, heme is predominantly synthesized in bone marrow (i.e., in erythroblasts and reticulocytes that still contain mitochondria) and liver cells.
  • heme biosynthesis begins with L-glutamate and proceeds through 5-amino-levulinate, a universal tetrapyrrole precursor, to uroporphyrinogen III. Uroporphyrinogen III is converted to protoheme IX which further produce heme, by incorporating iron, in the subsequent reactions.
  • Genetically engineered E.coli strains may be used for enhanced heme synthesis. For example, overexpression of gene constructs coaA gene and/or hemA induces increased expression of the intermediates ALA synthase or pantothenate kinase of heme biosynthesis pathway in E.coli.
  • the myoglobin may be derived from steppe mammoth (JVIammuthustrogontherii), woolly mammoth (Mammuthus primigenius), sheep, cow, pig, chicken, rabbit, mouse, rat or tuna.
  • the myoglobin is preferably from steppe mammoth or woolly mammoth.
  • the amino acid sequence is derived from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna, by addition, deletion and/or substitution of at least one amino acid. Addition, deletion and/or substitutions of two, three, four, five, six, seven, eight, nine or ten amino acids is also contemplated by the invention.
  • myoglobin amino acid sequence from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna have been disclosed later on by a given SEQ ID NO:1-8.
  • Such myoglobin derived from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna may also exert at least a detectable level of an activity of a myoglobin as explained later herein.
  • the myoglobin is a steppe mammoth myoglobin, woolly mammoth myoglobin, sheep myoglobin, cow myoglobin, pig myoglobin, chicken myoglobin, rabbit myoglobin or tuna myoglobin or a myoglobin derived from any of these myoglobins.
  • the myoglobin is steppe mammoth myoglobin (SEQ ID NO: 1) or derived therefrom.
  • the myoglobin is woolly mammoth myoglobin (SEQ ID NO: 2) or derived therefrom.
  • the myoglobin is sheep myoglobin (SEQ ID NO: 3) or derived therefrom.
  • the myoglobin is cow myoglobin (SEQ ID NO: 4) or derived therefrom. In an embodiment, the myoglobin is pig myoglobin (SEQ ID NO: 5) or derived therefrom. In an embodiment, the myoglobin is chicken myoglobin (SEQ ID NO: 6) or derived therefrom. In an embodiment, the myoglobin is rabbit myoglobin (SEQ ID NO: 7) or derived therefrom. In an embodiment, the myoglobin is tuna myoglobin (SEQ ID NO: 8) or derived therefrom.
  • the myoglobin may be represented by any one of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 or 8, preferably by SEQ ID NO: 1 or 2.
  • the myoglobin is from a steppe mammoth as defined earlier herein, 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 in combination with 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, 1 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
  • the myoglobin is from a woolly mammoth as defined earlier herein, 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 in combination with 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, 1 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,
  • the myoglobin is from sheep as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is from cow as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is from pig as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, 1 at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is from chicken as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 1 11 , I at position 116, A at position 1 17, E at position 118, A at position 121 , S at position 128, K at position 133, S at position 145
  • the myoglobin is from a rabbit as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, in combination with 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, 1 at position 31 , G at position 32, H at position 35, T at position 36, E at position
  • the myoglobin is from a tuna as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8 in combination with 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 ,
  • the myoglobin may be represented by one of the following amino acid sequences: a) having at least 90% identity with SEQ ID NO: 1 or 2, and having F at position 30 and/or Q at position 65; b) having at least 90% identity with SEQ ID NO: 3, 4 or 5, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, 4 or 5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150; c) having at least 90% identity with SEQ ID NO: 6, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 7: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49
  • the myoglobin is represented by a sequence having at least 90% identity with SEQ ID NO:1 or 2, and having F at position 30 and/or Q at position 65.
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from steppe mammoth (Mammuthus trogontherif), woolly mammoth (Mammuthus primigenius), sheep, cow, pig, chicken, rabbit or tuna, preferably from steppe mammoth or woolly mammoth.
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a steppe mammoth as defined earlier herein, 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 in combination with 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,
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a woolly mammoth as defined earlier herein, 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 in combination with 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
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a sheep as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a cow as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a pig as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a chicken as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111 , I at position 116, A at position 117
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a rabbit as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, in combination with 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 a
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a tuna as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8 in combination with 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
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin may be represented by one of the following amino acid sequences: a) having at least 90% identity with SEQ ID NO: 1 or 2, and having F at position 30 and/or Q at position 65; b) having at least 90% identity with SEQ ID NO: 3, 4 or 5, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, 4 or 5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150; c) having at least 90% identity with SEQ ID NO: 6, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 7: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31;
  • the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin may be represented by a sequence having at least 90% identity with SEQ ID NO:1 or 2, and having F at position 30 and/or Q at position 65.
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • SEQ ID NO: 3 100% identity or similarity with the SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • SEQ ID NO: 4 100% identity or similarity with the SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • SEQ ID NO: 5 100% identity or similarity with the SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, 1 at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • SEQ ID NO: 6 100% identity or similarity with the SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, 1 at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 11 1 , I at position 1 16, A at position 1 17, E at position 1 18, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150.
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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
  • the hemeprotein is a recombinant protein obtained from a microbial fermentation, preferably wherein the microbial fermentation comprises the extracellular secretion of the recombinant protein.
  • a microorganism may be a prokaryote, a eukaryote or a filamentous fungus.
  • a prokaryote may be a bacterium.
  • the bacterium may be a Gram positive/Gram negative bacterium 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, Ralst
  • Preferred bacteria include Aeribacillus pallidus, Aneurinibacillus terranovensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus halodurans, Bacillus pumilus, Brevibacillus thermoruber, Brevibacillus panacihumi, Cupriavidus basilensis, G.
  • Iraustophilus Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, Escherichia coll, Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti, Sphingobium sp., Novosphingobium sp., Sphingomonas henshuiensis, and Rhizobium radiobacter.
  • a preferred bacterium is Escherichia coli.
  • Preferred Escherichia coli strains include: 58, 679, WG1 , DH5a, TG1 , TOP10, K12, BL21 , BL21 DE3, XL1-Blue, XL10-Gold, TB1 , REG-12, W945, HB101 , DH1 , DP50, AB284, JC9387, AG1 , C600, Cavalli Hfr, Y10.
  • a eukaryote may be a yeast or a filamentous fungus.
  • 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.
  • yeasts the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha (also known as Ogataea henricii), Yarrowia lipolytica, Candida tropicalis and Pichia pastoris (also known as Komagataella phaffii) are preferred.
  • Preferred Pichia strains are selected from the following list: Bg09, Bg10, Bg11 , Bg12 (exemplified), Bg20, Bg21 , Bg22, Bg23, Bg24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430, X-33, GS115, KM71 , SMD1168, SMD1165, MC100-3, most preferred Bg10 and derivatives.
  • Preferred Saccharomyces strains are selected from the following list: S288C, CEN.PK family, CBS 2354, ATCC 2360, ATCC 4098, ATCC 4124, ATCC 4126, ATCC 4127, ATCC 4921 , ATCC 7754, ATCC 9763, ATCC 20598, ATCC 24855, ATCC 24858, ATCC 24860, ATCC 26422, ATCC 46523, ATCC 56069, ATCC 60222, ATCC 60223, ATCC 60493, ATCC 66348, ATCC 66349, ATCC 96581.
  • a preferred yeast is a Pichia strain, more preferably Pichia pastoris.
  • a filamentous fungus may be selected from the following list including: Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallinastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Ustilago and Trichoderma.
  • Preferred filamentous fungus are selected from the following list: Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus vadensis, Penicillium chrysogenum, Penicillium citrinum, Penicillium rubens, Penicillium oxalicum, Penicillium subrubescens, Rasamsonia emersonii, Talaromyces emersonii, Acremonium chrysogenum, Trichoderma reesei, Aspergillus sojae, and Chrysosporium lucknowense.
  • Preferred strains of filamentous fungus are selected from the following list: Aspergillus niger CBS 513.88, N593, CBS 120.49, N402, ATCC 1015 Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , ATCC 9576, ATCC 14488-14491 , ATCC 11601 , ATCC12892, Aspergillus vadensis CBS 113365, CBS 102787, IMI 142717, IBT 24658, CBS 113226, Penicillium chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Wisconsin 54-1255, Penicillium subrubescens CBS 132785, FBCC 1632, Talaromyces emersonii CBS 393.64, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC567
  • the microorganism used in the fermentation may be a bacterium, a yeast, a filamentous fungus or a cultured mammalian cell line, preferably Escherichia coli or Saccharomyces cerevisiae.
  • single, isolated, cultured mammalian cells may be considered as microorganisms.
  • the microorganism may be a bacterium, a yeast or a filamentous fungus.
  • the microorganisms in the context of this invention are useful for the production of a hemeprotein such as myoglobin.
  • the invention provides a method for the production of a myoglobin as defined herein, comprising culturing the microorganisms in a suitable medium and optionally recovering the microorganism and/or myoglobin.
  • the produced myoglobin does not comprise a signal peptide as defined elsewhere herein.
  • the microorganisms used may be a genetically engineered strains comprising gene constructs that expressing enzymes or intermediates of heme biosynthesis pathway.
  • such gene constructs may comprise hemA, hemL, hemB, hemD, hemF, hemG or hemH described in Junli Zhang et al,. Scientific Reports 2015.
  • Cell culturing may be performed for a duration of 14, 13.5, 13, 12.5, 12, 11 .5, 1 1 , 10.5, 10, 9.5, 9,
  • cell culturing is performed for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11 ,
  • the cell culturing will typically result in a production of at least 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 200 g/L or 300 g/L of a hemeprotein, preferably a my
  • the cell culturing will typically result in at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8, 9%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, preferably at least 1 %, more preferably at least 5%, most preferably at least 10% of the carbon source in the growth medium being converted to a hemeprotein, preferably a myoglobin.
  • Cell culturing may also be performed by implementation of a multiple step, preferably a two-step, culture method.
  • a production step of a hemeprotein such as a myoglobin may be preceded by a cellular biomass growth step, wherein only limited production or no production is taking place.
  • the different steps may be carried out using different culture modes and/or different growth media and/or different culture process parameter values, depending on the goal of each step and/or the cultured cell.
  • the biomass during the production step may or may not be actively growing.
  • the host cells and/or hemeprotein may optionally be recovered from the culture medium.
  • the hemeprotein may optionally be recovered from the recovered cellular biomass.
  • the recovered hemeprotein is purified.
  • purification of the hemeprotein will result in a purity of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, most preferably in a hemeprotein that is substantially pure.
  • the microorganism produces the hemeprotein, preferably the myoglobin, extracellularly.
  • the hemeprotein is transported out of the host cell after it is synthesized in the host cell.
  • both secretory and extracellular fermentations are considered to be extracellular productions.
  • an extracellular production process has the advantage that the downstream processing to recover the produced hemeprotein is more convenient, efficient and/or effective.
  • an extracellular production process may result in a composition comprising the hemeprotein with a high purity such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with minimal downstream processing compared to intracellular methods wherein the hemeprotein is not transported out of the host cell after its synthesis.
  • Purity may be measured as the weight percentage of the total protein fraction in the cell-free supernatant obtained at the end of a process or extracellular process according to the invention.
  • an extracellular production process has the advantage that the optional step of recovering hemeprotein does not comprise lysing the host cell. As a result, the extracellular process may result in a composition having a low concentration of nucleic acids originating from the host cell.
  • the relevant downstream processing technology that may be suitable for recovery and/or purification will depend on whether the hemeprotein is accumulated within the cultured cells or excreted. Said processing technology and the associated choice will be known to the skilled person and is discussed, for example, in Wesselingh, J.A and Krijgsman, J., 1st edition, Downstream Processing in Biotechnology, Delft Academic Press, NL, 2013.
  • the biomass may be recovered from the culture medium using e.g. centrifugation or filtration. If the produced hemeprotein is accumulated within the cells, it can then be recovered and/or purified from the biomass.
  • Recovery and/or purification may be performed according to any conventional recovery or purification methodology known in the art. Methods for recovery and/or purification of proteins are known to the skilled person and are discussed in standard handbooks, such as Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY, 2001 or Ausubel F. et al, eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, NY, 2003.
  • recovery and/or purification methods include chromatographic methods such as ultrafiltration, microfiltration, gel-filtration chromatography, ion-exchange chromatography, immunoaffinity chromatography, metal affinity chromatography, fractionation with precipitants such as ammonium sulfate and polyethylene glycol, gel electrophoresis and salting out and dialysis.
  • metal affinity chromatography or size-exclusion chromatography is used.
  • Recovery and/or purification may optionally be enhanced by linking the enzyme polypeptide to a sequence that facilitates purification, such as with a GST domain, using well-known molecular toolbox techniques.
  • the sequence that facilitates purification and/or signal peptide that facilitates excretion of the hemeprotein is removed from the final product using techniques known in the art, for example proteolysis by endopeptidases targeting a linker between the sequence that facilitates purification and/or the signal peptide and the hemeprotein.
  • the enzyme polypeptide is linked (fused) to a hexa-histidine peptide, such as the tag provided in a pET23a(+) vector (Genescript Biotech, Leiden, The Netherlands), among others, many of which are commercially available.
  • hexa-histidine peptide provides for convenient purification of the fusion protein.
  • the hemeprotein is obtained via recovering and/or purifying from the culture medium. This may be realized continuously with the production process or subsequently to it. In a preferred embodiment, the hemeprotein is obtained via recovering and/or purifying from the cultured cells. A filter may be used for the purification of the recovered hemeprotein. This may be realized continuously with the production process, by harvesting fractions of growing cells, or subsequently to it.
  • the hemeprotein is sterilized, shredded, spray dried, spray dried, freeze dried, blended, shaped, cubed, dosed or packed.
  • Sterilization refers to any process that removes, kills, or deactivates all forms of life (particularly microorganisms such as fungi, bacteria, spores, and unicellular eukaryotic organisms) and other biological agents such as prions present in or on a specific surface, object, or fluid. Sterilization can be achieved through various means, including heat, chemicals, irradiation, high pressure, and filtration.
  • Freeze drying also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure, then removing the ice by sublimation.
  • Packing aims to provide a protection for the product, to tamper resistance and to provide physical, chemical or biological needs. Packing may also contain nutrition facts, characteristics of the products, and an instruction/guide for use of the product.
  • the cultured host cells used in fermentation are immobilized. Immobilization of cells may be achieved by any means known to the skilled person as discussed in standard handbooks such as Guisan, J.M., Bolivar, J.M., Lopez-Gallego, F., Rocha-Martin, J. (Eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020.
  • the host cells can be immobilized to a semi-solid or solid support by three different methods. The first method involves polymerizing or solidifying a spore- or cell-containing solution.
  • polymerizable or solidifiable solutions examples include alginate, A-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinyle benzene, polyvinyl benzene, polyvinyl alcohol, epoxy carrier, cellulose, cellulose acetate, photocrosslinkable resin, prepolymers, urethane, and gelatin.
  • the second method involves cell adsorption onto a support. Examples of such supports include bone char, cork, clay, resin, sand porous alumina beads, porous brick, porous silica, celite, orwood chips.
  • the host cells can colonize the support and form a biofilm.
  • the third method involves the covalent coupling of the host cells to a support using chemical agents like glutaraldehyde, o-dianisidine (U.S. Pat. No. 3,983,000), polymeric isocyanates (U.S. Pat. No. 4,071 ,409), silanes (U.S. Pat. Nos. 3,519,538 and 3,652,761), hydroxyethyl acrylate, transition metal-activated supports, cyanuric chloride, sodium periodate, toluene, and the like.
  • Cultured host cells can be immobilized in any phase of their growth, for example after a desired cell density in the culture has been reached.
  • Suitable culture modes and/or different culture process parameter values will be known to the skilled person and are discussed in standard handbooks, such as Colin R. Phillips C.R., Poon Y. C., Immobilization of Cells: In Biotechnology Monographs book series (Biotechnology, volume 5), Springer, Berlin, Germany, 1988; Tampion J., Tampion M. D., Immobilized Cells: Principles and Applications, Cambridge University Press, UK, 1987.
  • immobilized cells are cultured in packed bed bioreactors, also known as plug-flow bioreactors, or expanded (fluidized) bed bioreactors. Suitable growth media and recovery and/or purification methods are further discussed elsewhere herein.
  • Myoglobins which may be represented by SEQ ID NO: 1-8 may be encoded by nucleic acids which may be represented by SEQ ID NO: 9-18, respectively, although it is understood that these nucleic acids sequence may need to be codon-optimized in order to be expressed by the microorganism.
  • the numbering of the myoglobin amino acid sequences originating from steppe mammoth (SEQ ID NO: 1), woolly mammoth (SEQ ID NO: 2), sheep (SEQ ID NO: 3), cow (SEQ ID NO: 4), pig (SEQ ID NO: 5), chicken (SEQ ID NO: 6), rabbit (SEQ ID NO: 7) used herein begins with methionine or Met or M at position 1 . All these amino acid sequences SEQ ID NO: 1 -7 count 154 amino acid residues.
  • SEQ ID NO: 8 The numbering of the myoglobin amino acid sequence originating from tuna (SEQ ID NO:8) used herein is slightly different from the consecutive numbering indicated in the sequence listing, as this sequence only counts 147 amino acid residues.
  • SEQ ID NO: 8 is also attributed 154 positions, wherein eight positions are considered to be gaps (1 , 2, 3, 6, 7, 51 , 120, 121).
  • the dairy product, or the replica thereof does not comprise a symbiotic hemoglobin produced by a plant in its root nodules. In an embodiment, the dairy product, or the replica thereof, does not comprise a leghemoglobin. In an embodiment, the myoglobin disclosed herein may be the sole source of heme-containing protein present in the dairy product of the replica thereof. It means that the dairy product, or the replica thereof, may comprise other proteins than the myoglobin as disclosed herein.
  • the weight fraction of hemeproteins in the total protein content in the dairy product, or the replica thereof is at least 0.001 %, and wherein the weight fraction of myoglobins in the heme-proteins in the dairy product, or the replica thereof, is at least 50%.
  • the concentration of the total iron content in the dairy product, or the replica thereof is from 0.1 up to 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, or 2.0 mg per 100 mL.
  • the dairy product which is preferably a milk, comprises a higher amount of bioavailable iron or heme iron than a corresponding control or reference diary product, which has not been supplemented with a hemeprotein.
  • the replica dairy product which is preferably a replica of a milk, comprises a higher amount of bioavailable iron or heme iron than a corresponding control or reference replica diary product, which has not been supplemented with a hemeprotein.
  • Iron bioavailability may be assessed or measured by change in hemoglobin and/or serum ferritin level or concentration in iron-deficient individuals, whole-body retention of radioiron, iron incorporation into erythrocytes or a reticulocyte-rich erythrocyte fraction, plasma iron response test, in vitro simulator model such as SHIME assay (Simulator of the Human Intestinal Microbial Ecosystem), compartmental modeling of iron absorption and/or dual isotopic tracer.
  • SHIME assay Simulator of the Human Intestinal Microbial Ecosystem
  • iron bioavailability may be assessed using any one of these assays or a combination thereof.
  • Example 2.2 illustrates that dairy products supplemented with a hemeprotein comprise a higher amount of bioavailable iron.
  • the iron bioavailability of the dairy product is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference dairy product which has not been supplemented with a hemeprotein.
  • the iron bioavailability of the replica of a dairy product is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference replica dairy product which has not been supplemented with a hemeprotein.
  • An advantage of the current invention is that the dairy products or replicas thereof have a higher iron bioavailability than similar dairy products or replicas that have been supplemented with other, non-heme forms of iron.
  • the iron bioavailability of the dairy product is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference dairy product which has been supplemented with a non-heme salt or complex of iron and has not been supplemented with a hemeprotein.
  • the iron bioavailability of the replica of a dairy product is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference replica dairy product which has been supplemented with a non-heme salt or complex of iron and has not been supplemented with a hemeprotein.
  • control or reference replicas are supplemented with iron(lll)pyrophosphate, iron(ll)lactate or iron(lll)pyrophosphate as shown in Table 4.
  • An additional advantage of the current invention is that the supplementation with a hemeprotein is compatible with most or all nutrients natively present in the dairy product or replica thereof.
  • the other forms of iron outlined above are typically not compatible with at least some of the nutrients.
  • the supplementation with these types of iron does not result in an increase of iron bioavailability and/or results in the decrease of the bioavailability of the nutrients in question.
  • the amount in the dairy product, or the replica thereof is ranged from 0.1 to 3 mg bioavailable iron or heme iron per 100 mL of dairy product, or replica thereof. In an embodiment such amount is ranged from 0.1 mg up to 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 .0 mg, 1.1 mg, 1 .2 mg, 1 .3 mg, 1 .4 mg, 1 .5 mg, 2.0 mg, 2.5 mg, 3.0 mg bioavailable iron or heme iron per 100 mL dairy product, or replica thereof.
  • such amount is 0.1 , 0.2, 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 mg bioavailable iron or heme iron per 100 mL dairy product, or replica thereof.
  • Myoglobin is a richly pigmented protein, which is the main reason forthe red color of animal muscle.
  • White meat such as chicken muscle, has lower amount of myoglobin compared to red meat, such as beef.
  • red meat such as beef.
  • most mammal’s blood color is due to the presence of hemoglobin in the blood.
  • the blood color is blue, which is from the copper-rich hemeprotein called hemocyanin.
  • the color of the dairy product, or the replica thereof is measured to compare with the color of a corresponding base diary product, or a replica thereof, which is not supplemented with the myoglobin.
  • Color measurements for milk are frequently reported based on different color units, as there is still currently a lack of international standards for milk color measurement (B. Milovanovic et al,. Foods, 2020). Color changes occur during the production phases which contains material that reflects specific wavelengths of light and therefore different colors of products.
  • the color of the dairy product may be measured using different instruments such as colorimeter or spectrophotometer, and different settings such as calibrations, illuminants, aperture sizes, observation angles, or number of readings taken per sample.
  • a colorimeter or spectrophotometer assesses the color in various sample solutions (pigments or colorants in food) by absorbing a particular wavelength of light and denotes the assessment in the form of some values using the Beer-Lambert law.
  • Minolta colorimeters or Hunter Associates Laboratory equipment may be used as an instrument.
  • a calibration of the instrument is performed each time before the measurement.
  • the light source of illu minant A 2848 K; tungsten-filament lighting
  • B 4900 K; direct sunlight
  • C (6800 K; average daylight from the total sky
  • D65 (6500 K; spectral distribution of mid-day sun
  • aperture size ranges from 8mm to 3.18cm may be used.
  • an observation angle of 10° may be used.
  • a technical replication of 4 to 6 may be used.
  • Data information from colorimeter or spectrophotometer may be analyzed and represented using the CIE L*a*b* color space or system to calculate lightness, redness, and yellowness.
  • CIE L*a*b* color system In food color measurement, researchers frequently use CIE L*a*b* color system to report color data (Sylwia Chudy et al,. 2020), which calculates lightness, redness and yellowness, if they are looking at the “true” human eye perception of color.
  • L* coordinate represents the brightness of the dairy product, or the replica thereof
  • a* coordinate represents the redness of the dairy product, or the replica thereof
  • b* coordinates represents the yellowness of the dairy product, or the replica thereof.
  • the dairy product or the replica thereof may have a L* coordinate ranges between 42.5 to 95.5, an a* coordinate ranges between 0 to 11 .8, and a b* coordinate ranges between 0 to 33.
  • the color of the dairy product, or the replica thereof may also be labeled according to the International Numbering System for Food Additives.
  • the milk product of this invention may be E100, E101 , E102, E103, E104, E107 or E110, or INS 100, INS 101 , INS 102, INS 103, INS 104, INS 107 or INS 110.
  • the color of the dairy product, or the replica thereof is white, beige, pearl, ivory, cream, or light yellow, preferably wherein the color is the same as a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein.
  • the color of the dairy product, or the replica thereof is not identical or similar to red, pink or orange.
  • the form, structure, texture, flavor, color, aroma and/or appearance of the dairy product, or the replica thereof are similar to those of a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein.
  • the dairy product or the replica thereof is expected to mimic the form, structure, texture, flavor, color, aroma and/or appearance of a corresponding base dairy product without having all its drawbacks. Alternatively, it might have an aspect in color, flavor and/or aroma which is distinct from the one of a milk.
  • Example 2.3 illustrates that the supplementation with a hemeprotein as disclosed herein has a minimal impact on the color of the chocolate plant-based dairy analogue.
  • Aromas from a dairy product, or a replica thereof, containing different concentrations of hemeproteins such as myoglobin may be analyzed using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME).
  • GC-MS gas chromatography-mass spectrometry
  • HS-SPME headspace solid-phase microextraction
  • the flavor of the dairy product or the replica thereof are similar to those of a corresponding base dairy product, which has not been supplemented with a hemeprotein.
  • the dairy product or the replica thereof does not taste acid, astringent, barny, bitter, cooked, cowy, feed, flat, foreign, garlic, malty, non-fresh, oxidized, metallic-oxidized, or soapy.
  • the sensory judging of the dairy product or the replica thereof for the purpose of quality control and shelf-life evaluation may be performed by the established guidelines, for example the American Dairy Science Association (ADSA) guidelines. Under this guideline, the dairy product or the replica thereof may be scored on a scale of 1-10 based on the defects.
  • ADSA American Dairy Science Association
  • the dairy product or the replica thereof may be supplemented with additional substances for an alternatively flavored dairy product, or replica thereof.
  • the additional substances may be from a fruit (e.g., strawberry, cherry, banana etc.), a vanilla, a chocolate, a nut (pecan, pistachio), coffee, caramel, peanut butter, tea, rum, or any food flavor aroma.
  • the flavor of the dairy product or the replica thereof has a similar level of sensory acceptability of a corresponding control or reference dairy product or the replica thereof.
  • the degree of liking of a product based on its sensory appeal may be assessed by a sensory evaluation or sensory acceptability test.
  • sensory acceptability may be tested though hedonic scales where the participants or consumers indicate how much they like or dislike the sample in terms of a specific sensory property, such as appearance, flavor, test, texture and/or the overall combination liking/acceptance.
  • a commonly used scale is the 9-point hedonic scale that ranges from “like extremely” to “dislike extremely”.
  • a “similar level” means a difference of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% difference in the average sensory property test results between the dairy product or the replica thereof and the corresponding dairy product or the replica thereof.
  • Example 2.4 illustrates that a replica of a dairy product according to the invention has similar sensory characteristics as a reference replica of a dairy product.
  • the acidity of a dairy product orthe replica thereof is represented by pH level measured by a pH meter.
  • the pH value of condensed milk may be about 6.33
  • the pH value of evaporated milk may be from 5.9 to 6.3
  • the pH value of buttermilk may be from 4.62 to 4.83
  • the pH value of whole milk may be from 6.4 to 6.9
  • the pH value of yoghurt may be from 4.4 to 4.8
  • the pH value of 40% cream may be from 6.44 to 6.80
  • the pH value of 20% cream may be from 6.50 to 6.68
  • the pH value of cheese may be from 4.1 to 7.44.
  • the pH value of the dairy product, or the replica thereof is from 6 to 7, from 6 to 6.9, from 6 to 6.8, from 6 to 6.7, from 6 to 6.6, from 6 to 6.5, from 6.1 to 7, from 6.1 to 6.9, from 6.1 to 6.8, from 6.1 to 6.7, from 6.1 to 6.6, from 6.1 to 6.5, from 6.2 to 7, from 6.2 to 6.9, from 6.2 to 6.8, from 6.2 to 6.7, from 6.2 to 6.6, from 6.2 to 6.5, from 6.3 to 7, from 6.3 to 6.9, from 6.3 to 6.8, from 6.3 to 6.7, from 6.3 to 6.6, from 6.3 to 6.5, preferably wherein the dairy product, or the replica thereof, is a milk.
  • the appearance of the dairy product or the replica thereof are similar to those of a corresponding control or reference dairy product or of a corresponding control or reference replica dairy product, which has not been supplemented with a hemeprotein.
  • the appearance of a dairy product orthe replica thereof may be evaluated by the viscosity which is expressed by the unit poise. Viscosity may be measured both in absolute or relative terms. Absolute viscosity is the viscosity in poise or centipoise. Relative viscosity is the rate of flow of liquid. The viscosity of milk ranges between 1 .5 to 2.0 centipoise at 20°C. Due to the fat emulsion and colloidal particles, milk is more viscous than water.
  • the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% higher than the average concentration of heme-proteins in a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein.
  • the average concentration of hemeproteins in the milk of a specific species of an animal is determined over several corresponding animals, wherein no exogeneous or myoglobin has been added to the milk of the animals after harvesting.
  • the resulting dairy product, or replica thereof, of this invention is edible for humans or animals, and the addition of the hemeprotein using the method of this invention does not introduce toxicity or affect the food safety of said dairy product.
  • the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, preferably lower than 0.1 %, more preferably lower than 0.02%. It is understood that the hemeproteins comprised in the dairy product may originate from the addition of the hemeprotein, or from another source.
  • the (total) weight concentration of myoglobins in the dairy product, or the replica thereof is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1 .0%preferably lower than 0.1 %, more preferably lower than 0.02%. It is understood that the myoglobins comprised in the dairy product may originate from the addition of the myoglobin, or from another source.
  • the weight fraction of myoglobins in the heme-proteins in the dairy product, or the replica thereof is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 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%, or 75%.
  • the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, and the weight fraction of myoglobins in the hemeproteins in the dairy product, or the replica thereof, is at least 5%.
  • the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof is at least 0.001 %
  • the weight fraction of myoglobins in the hemeproteins in the dairy product, or the replica thereof is at least 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %
  • At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of hemeproteins in the dairy product, orthe replica thereof originate from the addition of the myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof, originate from the addition of the myoglobin (per weight).
  • At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof originate from the addition of the myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof, originate from the addition of the myoglobin (per weight).
  • the hemeprotein is added as part of a composition, wherein the weight concentration of the hemeprotein in the composition 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 %.
  • weight concentration may be expressed as a mass of myoglobin per total volume or mass of the composition.
  • the hemeprotein is added as part of a composition, wherein the weight fraction of the hemeprotein in the protein fraction comprised 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%.
  • weight fraction is a dimension less number which may be interpreted as a mass of hemeprotein per mass of protein.
  • the hemeprotein is added to the base dairy product, or the replica thereof, as part of a composition, wherein the hemeprotein protein purity of the composition is at least 80%.
  • the hemeprotein protein purity may be calculated from the mass of the hemeprotein divided by the mass of the total composition.
  • the hemeprotein is supplemented as part of an extract or composition.
  • the hemeprotein is supplemented as part of an extract or composition, said extract or composition may comprise the cell that produces it.
  • the weight concentration of the hemeprotein in the extract 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%.
  • weight concentration may be expressed as a mass of hemeprotein per total volume or mass of the extract.
  • the hemeprotein is supplemented as part of an extract or composition, said extract or composition may comprise the cell that produces it.
  • the weight fraction of the hemeprotein in the extract 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%.
  • weight fraction is a dimensionless number which may be interpreted as a mass of hemeprotein per mass of total weight of the extract.
  • the hemeprotein is supplemented to the base dairy product, or the replica thereof, as part of an extract or composition, said extract or composition may comprise the cell that produces it, wherein the weight concentration of the hemeprotein in the composition is at least 10%.
  • the hemeprotein is supplemented as part of an extract or composition, said extract or composition does not comprise the cell that produces it.
  • the hemeprotein is in the form of powder.
  • the amount of the hemeprotein needed to be added to the dairy product may be calculated or estimated based on the concentration of hemeprotein in the dairy product before the addition and the desired final concentration of hemeprotein in the dairy product.
  • the dairy product before addition of the hemeprotein preferably contains less than 0.0001 %, less than 0.0002 %, less than 0.0003 %, less than 0.0004 %, less than 0.0005%, less than 0.0006 %, less than 0.0007%, less than 0.0008 %, less than 0.0009 %, less than 0.001 % hemeproteins per weight basis.
  • a tool such as a container, a cup, a spoon, a blender, a mixer, may be used to add the hemeprotein.
  • the hemeprotein may be added directly to the base dairy product, or the replica thereof, or may be dissolved in an editable solvent, prior to be added to the base dairy product, or the replica thereof.
  • the hemeprotein is in the form of solid shapes, wherein the solid shaped hemeprotein has a pre-determined weight and/or volume.
  • the amount of the hemeprotein needed to be added to the dairy product may be calculated or estimated based on the concentration of the hemeprotein, and/or the volume of the solid-shaped hemeprotein, the total weight of the dairy product and the desired final amount of hemeprotein in the dairy product.
  • the dairy product before addition of the hemeprotein preferably contains less than 0.0001 %, less than 0.0002 %, less than 0.0003 %, less than 0.0004 %, less than 0.0005%, less than 0.0006 %, less than 0.0007%, less than 0.0008 %, less than 0.0009 %, less than 0.001 % hemeproteins per weight basis.
  • a tool such as a container, a cup, a spoon, a blender, a mixer, may be used to add the hemeprotein.
  • the myoglobin may be added directly to the base dairy product, or the replica thereof, or may be dissolved in a food grade solvent, prior to be added to the base dairy product, or the replica thereof.
  • the verb "to comprise” and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb “to consist” may be replaced by “to consist essentially of’ meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention.
  • to consist may be replaced by “to consist essentially of’ meaning that a dairy product, a replica dairy product, a myoglobin, a gene construct, a host cell (or methods) as described herein may comprise additional component(s) (or additional steps) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • references to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • at least a particular value means that particular value or more.
  • at least 2 is understood to be the same as “2 or more” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.
  • the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
  • the word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.
  • the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
  • Food safety measures such as good hygienic practice (GHP), good manufacturing practice (GMP), hazard analysis and critical control practice (HACCP), quality management and microbial risk assessment are implemented for the monitoring of food safety of this invention.
  • a nucleic acid molecule such as a nucleic acid molecule encoding a hemeprotein is represented by a nucleic acid or nucleotide sequence which encodes a protein fragment or a polypeptide or a peptide or a derived peptide. It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identity number (SEQ ID NO) is not limited to this specific sequence as disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.
  • nucleotide sequence that encodes an amino acid sequence that has at least 60% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.
  • 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%.
  • 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%.
  • Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
  • sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Identity and “similarity” can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference.
  • Sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).
  • a global alignment algorithms e.g. Needleman-Wunsch
  • sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical”
  • a global alignment is suitably used to determine sequence identity when the two sequences have similar 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), maximizing the number of matches and minimizing the number of gaps.
  • EMBOSS water uses the Smith-Waterman local alignment algorithm.
  • the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).
  • nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference.
  • BLASTx and BLASTn 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/.
  • conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.
  • protein or “polypeptide” or “amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.
  • amino acids or “residues” are denoted by three-letter symbols.
  • a residue may be any protein
  • gene means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
  • a gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) e.g. comprising a polyadenylation- and/or transcription termination site.
  • "Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e.
  • a “regulator” or “transcriptional regulator” is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.
  • promoter or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active under most physiological and developmental conditions.
  • An “inducible” and/or “repressible” promoter is a promoter that is physiologically or developmentally regulated to be induced and/or repressed, e.g. by the application of a chemical inducer or repressing signal.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
  • Gene constructs as described herein could be prepared using any cloning and/or recombinant DNA techniques, as known to a person of skill in the art, in which a nucleotide sequence encoding said hemeprotein is expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-lnterscience, New York (1987) and in Sambrook and Russell (2001 , supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).
  • expression vector generally refers to a tool in molecular biology used to obtain gene expression in a cell, for example by introducing a nucleotide sequence that is capable of effecting expression of a gene or a coding sequence in a host compatible with such sequences.
  • An expression vector carries a genome that is able to stabilize and remain episomal in a cell.
  • a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered.
  • a vector is capable of integrating into a cell's genome, for example through homologous recombination or otherwise.
  • a nucleic acid or DNA or nucleotide sequence encoding a hemeprotein is incorporated into a DNA construct capable of introduction into and expression in an in vitro cell culture.
  • a DNA construct is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.
  • a DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment.
  • the term “operably linked” has already been described herein.
  • a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide.
  • a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame.
  • enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
  • an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment.
  • suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001 , supra).
  • a transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognized by the host.
  • the selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001 , supra).
  • An expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment.
  • the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. Coli).
  • Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001 , supra) and in Metzger et al. (1988) Nature 334: 31-36.
  • suitable expression vectors can be expressed in, yeast, e.g. S.
  • a cell may thus be a prokaryotic or eukaryotic host cell.
  • a cell may be a cell that is suitable for culture in liquid or on solid media.
  • a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.
  • a promoter suitable to be used in a bacterium is lac, trp, tac, T7, phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL promoter from phage A.
  • a terminator suitable to be used in a bacterium is lac, trp, tac, T7 (used in example), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL terminator from phage A.
  • a preferred promoter used is a T7 promoter and/or a preferred terminator is the T7 terminator.
  • a preferred signal peptide for excretion is E. coli Sec-recognition peptide (SecA), E. coli Tet- recognition peptide, E. coli dsbA, E. coli phoA, E. coli pelB, E. coli MBP (maltose binding protein).
  • a marker suitable for E coli is ampicillin.
  • the proBA operon from E. coli strain K12 including its original transcription regulatory elements may be used to facilitate selection without antibiotics.
  • a promoter suitable to be used in yeast may be a constitutive promoter.
  • suitable constitutive promoters include: a glycolytic promoter selected from FBA1 , TPI1 , PGK1 , PYK1 , TDH3, ENO2, HXK2, PGI1 , PFK1 , PFK2, GPM1 gene or a non-glycolytic promoter of the TEF2 gene.
  • a suitable promoterto be used in yeast may be inducible. If the yeast is a Pichia, the methanol inducible promoter AOX1 is preferred.
  • GAL1 promoter galactose-inducible
  • the genes mentioned from which a promoter could be derived for a yeast as host cell could also be used to derive a terminator for the same yeast.
  • a preferred signal peptide for excretion for Pichia (and Saccharomyces) includes: the S. cerevisiae alpha mating factor pre- pro- secretion signal peptide, the S. cerevisiae Ost1 signal peptide, the S. cerevisiae Aga2 signal peptide and fusions thereof.
  • the following regulatory regions may be used.
  • the following promoters may be used: the Aspergillus niger glucoamylase promoter (g/aA), the Aspergillus nidulans alcohol dehydrogenase promoter (a/cA) or the Aspergillus oryzae taka-amylase A promoter (amyB), the Aspergillus niger alcohol dehydrogenase promoter (ac/hA), the Trichoderma reesei pyruvate kinase promoter (pki) or the Aspergillus nidulas glyceraldehyde- 3-phosphate dehydrogenase promoter (gpdA).
  • a preferred signal peptide for excretion for a filamentous fungus includes: the Aspergillus niger glucoamylase signal peptide (g/aA), the Aspergillus niger a-galactosidase signal peptide (AgIB) and the Trichoderma reesei cellobiohydrolase I (Cbhl).
  • a preferred promoter and terminator tor Aspergillus are the glucoamylase promoter and the glucoamylase terminator of Aspergillus niger.
  • Gene constructs described herein can be placed in expression vectors.
  • an expression vector comprising a gene construct as described in any of the preceding embodiments.
  • Expression may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of transgene expression in the transduced tissue on the level of the mRNA or the protein by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides or ELISA. Expression may be assessed at any time after administration of the gene construct, expression vector or composition as described herein.
  • expression may be assessed after 1 week, 2 weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9, weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, or more.
  • Suitable cell culturing methods for use in the process of producing recombinant protein are known to the skilled person, for example, in van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design, CRC Press, NY, 1991 . Such methods include, but are not limited to, submerged fermentation in liquid media, surface fermentation on liquid media and solid-state fermentations.
  • Cell culturing may, for example, be performed by cultivation in micro-titer plates, shake-flasks, small-scale benchtop bioreactors, medium-scale bioreactors and/or large-scale bioreactors in a laboratory and/or an industrial setting.
  • Suitable cell culturing modes include, but are not limited to, continuous, batch and/or fed-batch fermentation as well as their combinations. Cell culturing may be performed using continuous fermentation, batch fermentation, preferably fed batch fermentation.
  • culture medium hereinafter alternately referred to as “growth medium”, can be interpreted to encompass cases wherein the cultured cells are absent as well as cases wherein the cultured cells are present in the culture medium.
  • Culture broth refers to the culture medium wherein the cultured cells are present.
  • Culture supernatant refers to the culture medium wherein the cultured cells are absent.
  • Cell-free extract refers to a cell lysate not comprising the cellular debris. Cell culturing as part of the process of the invention can be performed under conditions conducive to the production of the introduced hemeproteins, which are known to the skilled person.
  • Such conditions depend not only on the chemical composition of the culture medium but also on other process parameters including culture duration, temperature, O2 levels in the culture broth and/or headspace, CO2 levels in the culture broth and/or headspace, pH, ionic strength, agitation speed, hydrostatic pressure and the like.
  • Cell culturing can take place using a culture medium comprising suitable nutrients, such as carbon and nitrogen sources and additional compounds such as inorganic salts and vitamins, using procedures known in the art (see, e. g. Bennett, W. and Lasure, L., 1 st edition, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • suitable growth media are available from commercial suppliers or may be prepared using published compositions that are suitable for the respective hosts (e.g. in catalogues of the Centraalbureau Voor Schimmelcultures collection (CBS) or of the American Type Culture Collection (ATCC)).
  • the exact composition of the growth medium and the values of culture process parameters are not critical features of the invention. Any growth medium composition may be contemplated, as long as it allows for growth of the host cell and production of the introduced hemeproteins.
  • the growth medium will typically comprise a carbon source to be used for the growth of the cultured cell.
  • suitable carbon sources may be added externally to the growth medium or may already be present in said medium. Carbon sources may be present or added individually or in mixtures of multiple carbon sources.
  • Second-generation feedstocks can be particularly attractive because of their lower carbon footprint.
  • Second-generation feedstocks will typically comprise lignocellulosic material.
  • Such material includes any lignocellulose and/or hemicellulose-based materials.
  • Such material may be sourced from agricultural, industrial or municipal, preferably agricultural, waste streams.
  • 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 examples include trees, shrubs and pastures, wheat, wheat straw, sugarcane bagasse, switchgrass, Japanese pampas grass, corn, corn stover, corn cob, canola stalk, soybean stalk, sweet corn, corn kernels, products and by-products from cereal milling (including wet milling and dry milling), such as corn, wheat, and barley, often referred to as “bran or fiber”, and municipal solids.
  • Biomass can also be grassy materials, agricultural residues, forestry residues, municipal solid waste, wastepaper, and pulp and paper mill residues.
  • Agricultural biomass includes branches, shrubs, tows, corn and corn straw, energy crops, forests, fruits, flowers, cereals, pastures, herbaceous crops, leaves, bark, needles, logs, roots, young trees, short term rotating woody crops, shrubs, switch herbs, trees, vegetables, fruits, vines, sugar beet pulp, wheat middlings, oat hulls, and hard and soft timber (not including toxic wood), and organic waste materials resulting from agricultural processes including agriculture and forestry activities, particularly forestry wood waste.
  • Agricultural biomass may be any of the foregoing alone, or any combination or mixture thereof.
  • Carbon sources such as organic acids, aldehydes, ketones, esters and alcohols may also be contemplated.
  • growth media comprising combinations of multiple different carbon sources may also be contemplated in the process of the invention.
  • Such media could, as a non-limiting example, combine more oxidized carbon sources such as organic acids with more reduced carbon sources such as alcohols.
  • 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 nitrate salts.
  • additional suitable compounds known in the art include phosphate, sulphate, metals such as magnesium, trace elements and vitamins.
  • the exact growth medium requirements will vary based on the host cell, e.g. between yeasts, bacteria and filamentous fungi, said requirements will be known to the skilled person. Accordingly, the growth medium may be a complete (rich) medium or a minimal medium, i.e. a medium comprising only the absolutely necessary components for growth depending on the cultured host cell.
  • process parameters can be assigned any value, as long as they allow for growth of the host cell and production of the introduced hemeproteins. Typically, said values will differ based on the host cell that is being cultured and will be known to the skilled person.
  • the process according to the invention is an oxygen-limited or aerobic process, meaning that cell culturing is performed under oxygen-limited or aerobic conditions, more preferably the process is oxygen-limited.
  • Oxygen-limited conditions also known as a micro-aerobic conditions, are culture conditions in which the oxygen consumption is limited by the availability of oxygen. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
  • the rate of oxygen consumption is at least about 5.5 mmol/L/h, more preferably at least about 6 mmol/L/h and even more preferably at least about 7 mmol/L/h.
  • Aerobic conditions are culture conditions in which the oxygen consumption is not limited by the availability of oxygen.
  • Cell culturing may be performed at a temperature value that is optimal for the cell, typically at a temperature range of 16-42 °C. In some embodiments, the temperature ranges between 20-40 °C, more preferably between 25-38 °C, most preferably between 28-36 °C. In some most preferred embodiments, a temperature value of about 30 or 36 °C is used.
  • Cell culturing may be performed at a pH value that is optimal for the cell.
  • 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.
  • the pH ranges from about pH 3.0 to about pH 9, more preferably from about pH 3.5 to pH 7.
  • a pH value of about 6 is used.
  • Cell culturing may be performed at an ionic strength value of the culture medium that is optimal for the cell, typically at a range between 50 mM - 2 M.
  • the ionic strength of the culture medium ranges between 75 mM - 1 M, more preferably between 100 mM - 750 mM. In some most preferred embodiments, an ionic strength value of about 100 mM is used.
  • Figure 1 Expression vector for animal myoglobin production in Pichia pastoris. A vector, carrying the codon-optimized sequence encoding cattle myoglobin has been generated. The expression cassette is produced by restriction using the two Bglll cut sites, allowing genome integration by recombination between chromosomal DNA and the AOX1 promoter on the one hand, and the AOX1 3’ fragment on the other hand.
  • Figure 2 Bioavailability of iron from different sources after passage through the in vitro GIT tract of different dairy (replica) samples. Samples were supplemented with different sources of iron, after passage through the in vitro SHIME system iron content was measured by ICP-MS to determine the amount of bioavailable iron. The data is expressed as relative values compared to the initial iron concentration of the same sample at the start of the assay.
  • Figure 3 Color effect of myoglobin in chocolate flavoured plant-based soy milk.
  • A Samples prepared with different concentrations of animal myoglobin obtained by precision fermentation in Pichia pastoris (0, 0.05, 0.1 % w/w) were imaged.
  • B Relative colorimetric L*a*b* values of chocolate flavoured plant-based soy milk containing various concentrations of animal myoglobin obtained by precision fermentation in Pichia pastoris (0, 0.05, 0.1 % w/w). Data are expressed as relative values compared to the samples without myoglobin.
  • C Relative color difference (AE) compared to the sample without animal myoglobin addition (0% w/w). All colorimetric assays were performed 3 times, error bars represent the standard deviation.
  • Example 1 Milk product based on rabbit myoglobin
  • Example 1.1 Production of purified rabbit myoglobin by Escherichia coll.
  • Full length rabbit myoglobin gene SEQ ID NO: 7 is synthesized and cloned in a modified pET-23a(+) vector, comprising the T7 promoter and terminator and a C-terminal hexa His-tag (Genscript Biotech, Leiden, the Netherlands).
  • the ampR marker gene originally present in said vector is replaced by the proBA operon from E. coll strain K12, including its original transcription regulatory elements, to facilitate selection without antibiotics.
  • the correctly assembled plasmids are confirmed by PCR and used to transform a proline auxotrophic E. coll protein production strain (E. coll K12 AproBA).
  • Transformed strains are incubated overnight in shake-flasks containing minimal medium (10.5 g/L K2HPO4, 4.5 g/L KH2PO4, 1.0 g/L (NH 4 )2SO4, 0.12 g/L MgSO4, 0.5 g/l Nacitrate, 2 g/L glucose, and 5.0 mg/L thiamine HClat 37°C and 150 rpm (pH 6).
  • 500 pl of the overnight culture is transferred to a 1 L shake-flask containing 500 mL minimal medium and incubated at 37°C and 150 rpm, until an GD600 of 0.4-1 is reached.
  • IPTG isopropyl-p-D-thiogalactoside
  • IPTG isopropyl-p-D-thiogalactoside
  • the culture is harvested and centrifuged at 3500 x g (4°C) for 15 min. The supernatant is discarded and the pellet is dissolved in 50 mL BugBuster Protein Extraction Reagent (Novagen), containing 1 KU Lysozyme/ml (Sigma-Aldrich), 25 U Benzonase® Nuclease and completeTM, EDTA-free Protease Inhibitor Cocktail (Roche). The dissolved pellet is incubated for 30 min at 4°C in a shaker.
  • the centrifugation step is repeated and the cell-free extract (supernatant) is collected and assayed by SDS-PAGE to confirm the production of myoglobins.
  • the production of rabbit myoglobins by the respective transformed strains is confirmed by the presence of protein bands of the correct size.
  • the cell-free extracts containing the soluble fraction of proteins is loaded to a HisTrap FF 1 mL column (Cytiva, MA, USA), coupled with an AKTA start system.
  • the column is equilibrated with 20 mM HEPES, 0.4 M NaCI, and 20 mM imidazole, pH 7.5, 1 mL/min flow rate.
  • the protein is eluted with 20 mM HEPES, 0.4 M NaCI, and 400 mM imidazole, pH 7.5.
  • the fractions containing the rabbit myoglobins are pooled, concentrated and confirmed by SDS-PAGE and Western Blotting using an anti-histidine tag antibody (Bio-Rad), which confirm the successful purification of all myoglobins.
  • the purified rabbit myoglobins are stored at -20°C for later use.
  • Example 1.2 Steps for production of milk product
  • the raw milk is then stored at temperature 4°C;
  • Emulsion stabilizer, folic acid, vitamin, mineral mix and myoglobins produced from previous step of myoglobin fermentation are added to the milk in a mixing tank.
  • the mixture is gently being stirred for 10 to 20 mins at 38 to 45°C in the mixing tank to allow the supplements to be dissolved.
  • the mixture is being continuously stirred to be homogeneous at a temperature of 65 to 80 °C.
  • the mixture is pasteurized at a temperature of 62° to 65°C for 30 mins, then the mixture is cooled at 15 to 20°C.
  • compositions according to the invention and reference examples are reported herein below:
  • Example 2 Milk product based on cattle myoglobin
  • the sequence coding for myoglobin from cattle was cloned downstream of the methanol-inducible A0X1 promoter of Pichia pastoris, and upstream of a histidine prototrophy selection marker and the A0X1 terminator.
  • Myoglobin is naturally found in the cytoplasm of muscle cells.
  • histidine-auxotroph his4 Pichia pastoris cells, and transformants were selected fortheir ability to grow in the absence of histidine.
  • histidine-auxotroph his4 Pichia pastoris cells
  • the presence of the myoglobin expression cassette in the genome of the histidine prototroph clones was then verified by PCR. Verified transformants were then tested for their ability to produce extracellular cattle myoglobin after methanol induction in 96 wells microplates. The presence of a methanol-inducible protein of the expected molecular weight was confirmed after protein electrophoresis and staining with Coomassie blue.
  • Example 2.2 Nutritional analysis of iron supplementation in dairy replicas
  • the plant-based soy infant growth milk resulted in 19.4% absorption of the total iron content by the end of the in vitro simulation.
  • the overall iron bioaccesibility was thus low (i.e., ⁇ 50% present in the bioaccessible and bioavailable fractions).
  • Example 2.3 Color and color stability of dairy replicas supplemented with myoglobin
  • color appearance of a material represents the response of retina rods and cones to the reflected radiation in the so-called ‘visible region’ of the electromagnetic spectrum, in the range between 400 and 700 nm.
  • visible region of the electromagnetic spectrum
  • color is the result of an interaction between a light source and pigments, by which energy is absorbed and emitted as complementary not-absorbed radiations in the visible region.
  • a commercial chocolate plant-based soy milk was supplemented with 0%, 0.05% or 0.1 % w/w of animal myoglobin obtained by precision fermentation in Pichia pastoris, as described in Example 2.1 , (corresponding to 0.16 mg/100mL and 0.32 mg/100mL of heme iron, respectively), to test whether the supplementation with the myoglobin influenced the colour of the final product. This is important for consumer acceptance, as a colour shift could make the product undesirable and create rejection despite its increased nutritional value. As shown in Fig. 3A, no differences visible by eye were observed in the three products with different myoglobin concentrations. The samples were examined by colorimetry and the relative value (compared to the sample without myoglobin) is shown in Fig.
  • Example 2.4 Sensory analysis of dairy replicas supplemented with precision fermentation derived myoglobin
  • the sequence coding for myoglobin from the steppe mammoth was obtained after DNA extraction from a molar sample from the so-called Adycha specimen, Illumina DNA sequencing, merging the reads and mapping them against the African savannah elephant (Loxodonta africana) genome [4], Multiple sequence alignments were performed with Clustal Omega [5] and visualized using Jalview 2.11 .1 .4 [6].
  • the myoglobin coding sequence was codon optimized for expression in Pichia pastoris (Komagataella phaffii) [7], The optimized sequence encoding the myoglobin from Bos taurus, preceded by the coding sequence of the Saccharomyces cerevisiae mating factor alpha were chemically synthetized by GenScript. Gene fragments were cloned into the pBDIPp5 vector, downstream of the AOX1 promoter and upstream of the AOX1 terminator, the HIS4 selection marker and the AOX1 3’ fragment. The vector was amplified in E. coll (DH10B), purified with the SmartPure Plasmid Kit (Eurogenetec) and verified by Sanger sequencing (Eurofins Genomics). An expression cassette was generated from the resulting vector by restriction with Bglll (New England Biolabs), which cuts upstream from the AOX1 promoter and downstream from the AOX1 3’ fragment.
  • the Pichia pastoris (Komagataella phaffii) strain GS115 (his4) was obtained from Life Technologies. Cell transformation was performed using the electroporation method essentially as previously described [8], Briefly, cells of the GS115 strain were grown in YPD (1 % yeast extract, 2% peptone and 2% D-glucose) medium. Cells in the exponentially growing phase were incubated for 30 min in YPD medium with 200 mM HEPES buffer (pH 8.0) and 25 mM dithiothreitol. Competent cells were then washed with ice-cold 1 M sorbitol, and transferred to a sterile electroporation cuvette (Bio-Rad).
  • Cells were then electroporated with 1-5 pg of linear expression cassette using a Gene-Pulser (BioRad) electroporator, and resuspended in 1 mL of YPD medium containing 1 M sorbitol before transfer to a sterile 1 ,5-mL eppendorf tube.
  • Cells were incubated at 28°C without agitation for 3 h, before plating onto agar plates containing solid MGY medium (Minimal Glycerol Medium: 1.34% Yeast Nitrogen Base with ammonium sulfate without amino acids, 2% D-glucose, 4 10 -5 % biotin with 2% agar). Plates were incubated for up to 4 days at 28°C.
  • Transformants able to grow in the absence of histidine were verified by PCR.
  • a DNA fragment specific for the myoglobin expression cassette was amplified using a pair of primers that anneal with the coding sequence of the Saccharomyces cerevisiae mating factor alpha.
  • PCR reactions were conducted using the OneTaq® Quick-Load® 2X Master Mix with Standard Buffer (New England Biolabs) according to the manufacturer’s instructions. PCR products were visualized after migration at 60V for 60 min in a 2% agarose TAE gel with ethidium bromide. The Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolabs) was used to control the size of the amplified fragment.
  • YPD agar (1 % yeast extract, 2% soy peptone and 3% D-glucose, 2% agar
  • a seed culture was then prepared in a 2L flask containing 200 mL of YPG (1 % yeast extract, 2 % soy peptone and 3% glycerol) medium, and incubated for 24h at 28°C in an orbital shaker incubator.
  • This seed culture was then used to inoculate a glass-vessel fermenter (Biostat B, Sartorius) containing 900 ml of modified BSM medium [9], A batch fermentation phase was conducted for about 14-16h , until all the glycerol was consumed. A glycerol fed batch phase was then conducted to further increase the biomass. The temperature was then lowered to 26°C, and a mixed glycerol:methanol was applied for 72h to induce myoglobin expression. Dissolved oxygen was maintained above 25% during the whole fermentation, while the pH was kept at 6 during the growth phase and 5 during the induction phase. After 96h of methanol induction, cells were removed by centrifugation at 4,000 rpm at 4°C.
  • Remaining cells were removed by microfiltration using cellulose filters with a pore diameter of 0.45 pm (Sartorius Hydrosart) using a tangential flow filtration device (Sartoflow, Sartorius). After ultrafiltration using cellulose filters with a molecular weight cut-off of 10 kD using the same device, the product was stored frozen at -20°C until use. When necessary for the assay, the ultrafiltration retentate was further concentrated using disposable ultrafiltration centrifuge devices, with a polyethersulfone membrane having a molecular-weight cutoff of 10 kD (Pierce Protein Concentrators, Thermo Scientific).
  • Alpro soy drink Alpro
  • Alpro Alpro soy drink
  • i 316.5 mg/100 mL of cattle myoglobin produced internally by precision fermentation in Pichia pastoris, which corresponds to approximately 1 mg/100 mL of heme iron or ii) 1 mg/100 mL of iron(lll)pyrophosphate (Fe4(P2O?)3) from Sigma Aldrich, after which the product was mixed thoroughly to ensure full solubility of the supplemented ingredient.
  • Alpro soya chocolate which contains 1 .5% of lean cocoa, which was supplemented with 0%, 0.05% or 0.1 % w/w of cattle myoglobin produced internally by precision fermentation in Pichia pastoris.
  • Assays were performed by ProDigest (Gent, Belgium). The experiment conducted to evaluate bioaccessibility/bioavailability of test compounds, makes use of an adapted SHIME® system representing the physiological conditions of the stomach and small intestine within the same reactor over time.
  • Four dairy milk or dairy milk replicate products were tested using an intake dose of 100 mL/reactor, each corresponding with a specific iron content (see section 2.3 above).
  • a specific gastric suspension is added to the reactor. After this, a standardized enzyme and bile liquid is added to simulate the small intestinal condition.
  • Incubation conditions i.e., pH profiles and incubation times
  • the protocol contains dynamic pH profiles as this mimics the in vivo condition more closely.
  • ICP-MS inductively coupled plasma - mass spectrometer
  • Binomial testing was performed on the results. For a triangle test binomial testing determines that to reject the null hypothesis (HO: all samples are equal) at 0.05 probability level, at least 7 correct answers are required for a panel size of 11. Similarly, from 8 or higher correct answers the null hypothesis can be rejected at the 0.01 probability level [10],

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Abstract

Described herein is a dairy product, or a replica thereof, supplemented with a hemeprotein, and a method for preparing such a dairy product, or a replica thereof.

Description

DAIRY PRODUCT OR REPLICA THEREOF SUPPLEMENTED WITH HEME-COMPRISING PROTEIN
Field
The present invention relates to a dairy product, or a replica thereof, supplemented with a hemeprotein. The invention also relates to a method for preparing a dairy product, or a replica thereof wherein the method comprises adding a hemeprotein to a base dairy product, or a replica thereof.
Background
Dairy products including milk have become one of the most important and commonly consumed food products in daily life. Dairy products, such as milk, are great sources of nutrients that are vital for the growth, health and maintenance of the body. These nutrients include protein, vitamin A, vitamin D, vitamin B12, potassium, phosphorus, riboflavin, zinc, choline, magnesium, selenium and calcium.
Depending on the age, gender, or level of activity, the amount of dairy products that one needs varies. This is mainly due to the different needs for different types of nutrients during different physical situations between consumers. For females, the amount can also depend on the maternity situation, for example, whether pregnant or breastfeeding. In early life, newborn babies are prone to iron deficiency due to the rapid growth and low iron in milk.
However, it is known that dairy products are a poor source of iron. Iron plays a pivotal role in several metabolic processes and is essential for health and physiological efficiency. Moreover, studies have shown that milk plays adverse effects on the iron storage of the body, via interference with the absorbance of iron from food and supplements.
There have been efforts in supplementing milk with iron, but the absorption of the added iron is low. Specifically, the effort of adding lactoferrin, does not affect iron status indicators in an infant fed with infant formula based on supplemented milk.
Hence, there is need in the art for dairy products, or replicas thereof, that are good sources of bioavailable iron.
Description
The present invention provides dairy products, and replicas thereof, comprising a significant amount of bioavailable iron.
In a first aspect of this invention, there is provided a dairy product, or a replica thereof, supplemented with a hemeprotein. A dairy product, or a replica thereof, is said to be supplemented with a hemeprotein if a hemeprotein has been added during any step of its preparation. Hence, supplementation with a hemeprotein results in an increase of the hemeprotein concentration during the preparation, which is preferably reflected in an increased hemeprotein concentration in the (final) dairy product, or the replica thereof, as defined below.
In a second aspect of this invention, there is provided a method for preparing a dairy product, or a replica thereof, wherein the method comprises adding a hemeprotein to a base dairy product, or a replica thereof.
In a third aspect of this invention, there is provided a dairy product, or a replica thereof, obtainable by a method comprising the addition of a hemeprotein to a base dairy product, or a replica thereof.
In a fourth aspect of the invention, there is provided a dairy product, or a replica thereof, comprising a hemeprotein. Wherever applicable, any preference relating to the hemeprotein listed herein may be applied to the hemeprotein in the fourth aspect of the invention.
In the method for preparing a dairy product, or a replica thereof, supplemented with a hemeprotein, wherein the method comprises adding the hemeprotein to a base dairy product, or a replica thereof, the hemeprotein may be added or applied by manufacturer of a dairy product, the consumer of such dairy product, or any other producer of a dairy product or a replica thereof.
The products of the first, third and fourth aspect of the invention, and those obtained via the second aspect of the invention, comprise iron in the form of heme-iron.
This organic heme-iron is more easily taken up by the body, compared to the non-heme iron (e.g. present in transferrin), and provides a significant part of the bioavailable iron in non-vegetarian diets. Hence, it solves the unmet need in the art.
All preferences and embodiments listed below relating to the dairy product, the replica thereof, the hemeprotein, the base dairy product, the method of for preparing, the supplementation/addition, etc. may be applied mutatis mutandis to each of these aspects of this invention. For example, and without being limiting, any specification of the sequence of the hemeprotein, applies to the hemeprotein in the first, second and third aspect of this invention, and to the hemeprotein in the fourth aspect of this invention.
Dairy product or replica thereof
Dairy products are food products made from or containing a mammalian milk. A mammalian milk is a white-colored liquid produced by the mammary glands of mammals. It is an emulsion or colloid of butterfat globules within a water-based fluid that contains dissolved carbohydrates and protein aggregates with minerals.
Mammalian milk is the primary source of nutrition for young mammals before they can digest solid food. The nutrients comprised in dairy products can be broadly classified as macronutrients (protein, carbohydrate and fat) and micronutrients (vitamins and minerals). Cow’s milk usually contains about 3.5 % protein, and 4.5-5.0 % carbohydrate (lactose) and depending on whether it is skimmed (fat- free milk), low-fat or whole milk (full-fat milk), the fat content ranges from 0.3-3.6%. The vitamins and minerals in dairy products contribute to the essential functions of physiological processes, for example as summarized in table 1 below.
Table 1 . nutrients in mammalian milk and their function
Figure imgf000004_0001
The nutrient concentration of dairy products varies, depending on what component of the mammalian milk they are made from. Several types of dairy products can be discerned based on their general appearance. A milk may be the unprocessed mammalian milk or may be produced after optional homogenization or pasteurization of the mammalian milk, in several grades after standardization of the fat level, and the possible addition of the bacteria Streptococcus lactis and/or Leuconostoc citrovorum. A cream may be obtained by skimming the higher-fat layer skimmed from the top of the mammalian milk before homogenization. A butter may be made from the fat and protein components of churned cream. A yoghurt may be obtained from the mammalian milk after fermentation by lactic acid bacteria, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus, which produce lactic acid that decreases pH and causes milk protein to coagulate. Lactic acid produced from fermentation acts on milk protein to give yoghurt its texture and characteristic tart flavor. A cheese may be produced in a wide range of flavors, textures, and forms by coagulation of the milk protein casein. A custard is a variety of culinary preparations that may be based on sweetened milk, cheese, or cream cooked with egg or egg yolk to thicken it, and sometimes also flour, corn starch, or gelatin. An ice cream is a sweetened frozen food that may be made from milk or cream and eaten as a snack or dessert. Note that the terms a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream relate to the general appearance of the food product. As such, both dairy product, which are made from or contain a mammalian milk, and replicas thereof, which are not of mammalian origin as described below, may be categorized in one of these categories.
A replica of a dairy product is a food product that resembles a dairy product but is neither made from nor contains milk produced by a mammary gland of a mammal. In other words, a replica of a dairy product is not of mammalian origin. A dairy replica product is by definition, not a dairy product, not a natural dairy product, not a genuine dairy product. The expression “a replica of a dairy product may be replaced by “a dairy food substitute”, “a dairy product substitute”, or “a non-natural dairy product”. It means that within the context of this invention, a replica of a dairy product is not a dairy product from or derived from a dairy animal such as a cow, a buffalo, a goat, a sheep or a horse. It is thus understood that cell-based, cultured, or plant-based dairy product substitutes may be considered replicas of dairy products. To resemble in this context means to approach, to have essentially or substantially the same, to have the same, or to mimic one or more physical, chemical or sensory characteristics of a corresponding control or reference dairy product. Examples of physical characteristics are color, viscosity, particle size distribution. Examples of chemical characteristics are nutrient concentrations, pH, ionic strength. Examples of sensory characteristics are form, structure, texture, flavor, color, aroma, appearance.
In an embodiment, the dairy product is a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream. In an embodiment, the replica of a dairy product is a replica of a milk, a cream, a butter, a yoghurt, a cheese, a custard or of an ice cream.
In an embodiment, the dairy product is a milk, wherein the milk is a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula. In an embodiment, the replica of a dairy product is a replica of a milk, wherein the replica of a milk is a replica of a whole milk, a reduced milk, a low-fat milk, a fat-free milk, an organic milk, a lactose-free milk, a flavored milk, a raw milk, a breast milk, or an infant milk formula, preferably wherein the infant milk formula is a starter formula or a follow-on formula. Whole milk means milk is taken as is from the mammal origin, without any sort of nutritional alteration, and processed for food safety, or a replica of such a dairy product. This means no nutrient is removed from nor supplemented to the milk. Whole milk is not considered raw milk and is safe to consume. Whole milk is sometimes also referred to as fresh milk or regular milk. Reduced-fat milk has at least 25% less fat than regular milk. Low milk has less than 1 .5% fat of the total weight of the milk. Fat- free (also known as skim) milk has less than 0.2% fat of the total weight of the milk. Lactose-free milk is a type of milk where natural sugar lactose has been broken down. Flavored milk is a sweetened dairy drink made with milk, sugar, flavorings, and sometimes food colorings. Raw milk is unpasteurized milk directly from animal origin.
Breast milk or mother's milk is milk produced by the mammary glands of a human female. Infant formula or baby formula, also called infant milk or infant growth milk, is a manufactured dairy product or replica thereof designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottle-feeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water). The U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants because of its simulation of human milk or its suitability as a complete or partial substitute for human milk". The starter formula is suitable for babies up to 6 months of age. The follow-on formula is suitable for older children. A comparison between human breast milk, infant formula and full-fat milk from cow shows the difference in nutrient composition.
Table 2. Content of nutrients in breast milk, infant formula and cow’s milk (per 100ml)
Figure imgf000006_0001
In average, a pregnant woman needs about 30 to 45 mg of iron per day (compared to 18 mg for adult women between 19 to 50 years old) to meet the new demands of extra blood volume, the developing placenta, and growing fetus. Sufficient iron supply for the developing fetus, neonate and infant is critical to produce new red blood cells, muscle cells and brain development. The iron content of human breast milk is low: 0.3 to 0.9 mg/L compared with 4 to 7 mg/L in supplemented infant formula based on cow milk. The absorption rate, however, is considerably higher. Breastfed infants absorb up to 50% of consumed iron, compared with an average 10% absorption rate for formula-fed infants (Fomon et al., 1993). Full-term infants are usually born with adequate iron stores to support hemoglobin synthesis through the first few months, but the risk of iron deficiency increases after about 4 months. This risk can be mitigated by providing a dairy product, specifically an infant formula, according to the present invention. Milk is usually produced from a dairy animal, such as a cow, a buffalo, a goat, a sheep, a horse, a llama or other less common animals such as a yak, a horse, a reindeer, a zebu, a giraffe and a donkey. The average nutrient composition in milk from different mammalian origins varies as shown in table 3. Herein, the term cow milk means milk (a type of dairy product) produced from a cow. Likewise, the term human (breast) milk, means a (breast) milk from a human. These and similar terms thus exclude replicas of dairy products, which are of non-mammalian origin.
In an embodiment, the dairy product supplemented with a hemeprotein, is derived from a milk of a dairy animal such as a cow, a buffalo, a goat, a sheep, a horse, a yak, a horse, a llama, a reindeer, a zebu, a giraffe or a donkey, preferably from a milk of a cow. Herein, a cow refers to an animal of the subspecies Bos primigenius taurus and not to a female specimen of other species such as yak or buffalo.
Table 3. Average composition of goat, sheep, cow, and human milk (per 100ml)
Figure imgf000007_0001
In an embodiment, the replica of the dairy product is a vegan product, preferably a vegan milk. Vegan milk may be plant-based juice that resembles the texture, taste and qualities of conventional animal milk. It can also be used to make many products, such as replicas of dairy products. Vegan milk may be soya milk (soy milk), almond milk, coconut milk, rice milk, cashew milk, macadamia milk, flax milk, pea protein milk, banana milk, sunflower milk, peanut milk, oat milk, hazelnut milk and sunflower milk.
Hemeprotein
Within the context of the invention, a hemeprotein as defined can refer to all proteins or protein subunits that are capable of covalently or noncovalently binding a heme moiety. A hemeprotein as defined herein is preferably found to elicit oxygen binding activity or to carry an atom of iron bound to a heme. For example, the oxygen binding activity may be assessed by determining the ratio of oxygen-bound versus non-oxygen-bound myoglobin using spectrophotometry, such as the measurement and calculation method according to Tang et al. (Krzywicki revisited: equations for spectrophotometric determination of myoglobin redox forms in aqueous meat extract. J Food Sci. 2004). For example, the activity of carrying an atom of iron bound to a heme may be assessed by detecting the absorbance peak near 400 nm (the so called Soret band) caused by the presence of heme iron using spectrophotometry, or by using mass spectrometry.
In an embodiment, the hemeprotein comprises a heme group. Heme-containing polypeptides may transport or store oxygen. Some examples of known heme-containing protein or polypeptide include hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin, androglobin, a globin E, a globin X, a globin Y, a flavohemoglobin, Hell's gate globin I, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.
In an embodiment, the hemeprotein is a metalloprotein. In an embodiment, the hemeprotein is a globin, preferably hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin, more preferably myoglobin or hemoglobin, most preferably myoglobin.
Myoglobin is a relatively small globular protein of about 17 kD, found in heart and skeletal muscles. It carries a single heme group with an atom of iron capable of reversible oxygen binding (Fig. 1), allowing myoglobin to transport oxygen from the cell surface to mitochondria.
Within the context of this invention, the hemeprotein is not a protein selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin and transferrin. In an embodiment, the replica dairy product does not comprise one or more proteins selected from the group consisting of p-casein, K-casein, a-S1-casein, a-S2-casein, a-lactalbumin, p-lactoglobulin, lactoferrin and transferrin.
In an embodiment, the hemeprotein may be represented by an amino acid sequence having at least 90% identity with any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NQ:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum) , SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:27 (Paramecium caudatum), SEQ ID NO:28 (Aspergillus niger), SEQ ID NO:29 (Zea mays), SEQ ID NQ:30 (Oryza sativa subsp. Japonica), SEQ ID NO:31 (Arabidopsis thaliana), SEQ ID NO:32 (Pisum sativum), SEQ ID NO:33 (Vigna unguiculata), SEQ ID NO:34 (Sus scrofa), SEQ ID NO:35 (Equus caballus), SEQ ID NO:36 (Nicotiana benthamiana), SEQ ID NO:37 (Bacillus subtilis), SEQ ID NO:38 (Corynebacterium glutamicum), SEQ ID NO:39 (Synechocystis PCC6803), SEQ ID NQ:40 (Synechococcus sp. PCC 7335), SEQ ID NO:41 (Nostoc commune), SEQ ID NO:42 (Bacillus megaterium). In a preferred embodiment, the hemeprotein may be represented by an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NO:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum), SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:27 (Paramecium caudatum), SEQ ID NO:28 (Aspergillus niger), SEQ ID NO:29 (Zea mays), SEQ ID NQ:30 (Oryza sativa subsp. Japonica), SEQ ID NO:31 (Arabidopsis thaliana), SEQ ID NO:32 (Pisum sativum), SEQ ID NO:33 (Vigna unguiculata), SEQ ID NO:34 (Sus scrofa), SEQ ID NO:35 (Equus caballus), SEQ ID NO:36 (Nicotiana benthamiana), SEQ ID NO:37 (Bacillus subtilis), SEQ ID NO:38 (Corynebacterium glutamicum), SEQ ID NO:39 (Synechocystis PCC6803), SEQ ID NQ:40 (Synechococcus sp. PCC 7335), SEQ ID NO:41 (Nostoc commune), SEQ ID NO:42 (Bacillus megaterium).
In a more preferred embodiment, the hemeprotein may be represented by an amino acid sequence of any one of SEQ ID NO:17 (Vigna radiata), SEQ ID NO:18 (Methylacidiphilum infernorum), SEQ ID NO:19 (Aquifex aeolicus), SEQ ID NQ:20 (Glycine max), SEQ ID NO:21 (Hordeum vulgare), SEQ ID NO:22 (Magnaporthe oryzae), SEQ ID NO:23 (Fusarium oxysporum), SEQ ID NO:24 (Fusarium graminearum), SEQ ID NO:25 (Chlamydomonas eugametos), SEQ ID NO:26 (Tetrahymena pyriformis), SEQ ID NO:27 (Paramecium caudatum), SEQ ID NO:28 (Aspergillus niger), SEQ ID NO:29 (Zea mays), SEQ ID NQ:30 (Oryza sativa subsp. Japonica), SEQ ID NO:31 (Arabidopsis thaliana), SEQ ID NO:32 (Pisum sativum), SEQ ID NO:33 (Vigna unguiculata), SEQ ID NO:34 (Sus scrofa), SEQ ID NO:35 (Equus caballus), SEQ ID NO:36 (Nicotiana benthamiana), SEQ ID NO:37 (Bacillus subtilis), SEQ ID NO:38 (Corynebacterium glutamicum), SEQ ID NO:39 (Synechocystis PCC6803), SEQ ID NQ:40 (Synechococcus sp. PCC 7335), SEQ ID NO:41 (Nostoc commune), SEQ ID NO:42 (Bacillus megaterium).
In an embodiment, there is provided a dairy product, or a replica thereof, supplemented with a hemeprotein, wherein the hemeprotein is an isolated hemeprotein. An isolated hemeprotein is preferably defined as a hemeprotein which is not comprised in a cell or a tissue right before and/or during the addition. In other words, the isolated hemeprotein is added (during the supplementation step) as a “separate” (or isolated) protein which is not comprised in a cell or a tissue. Thus, isolated in this context does not refer to the origin of the hemeprotein (i.e. isolated from a source such as cell or a tissue), but to the fact that a separate protein is added. It is of course understood that the isolated myoglobin may become part of a cell or a tissue once it has been added to the dairy product, or the replica thereof.
In an embodiment, the hemeprotein is a myoglobin. Myoglobin contains 153 amino acids and as with other globins, consists of eight alpha helices connected by loops. Myoglobin contains a porphyrin ring with an iron at its center. A proximal histidine group (His-93) is attached directly to iron, and a distal histidine group (His-64) hovers near the opposite face. Myoglobin is an iron- and oxygen-binding protein found in the cardiac and skeletal muscle tissue of vertebrates in general and in almost all mammals. Muscle cells use myoglobin to accelerate oxygen diffusion and act as localized oxygen reserves for times of intense respiration. Hemoglobin is the iron-containing oxygen-transport metalloprotein in erythrocytes in vertebrates. Hemoglobin in the blood carries oxygen from the respiratory organs to the rest of the body. Myoglobin is a single subunit protein, while hemoglobin has four subunits. Both contain the heme group, which is responsible for binding oxygen. The heme group consists of an organic component protoporphyrin as well as an inorganic component that consists of an iron atom. There is not a single ubiquitous regulatory mechanism for heme biosynthesis. In mammalian cells, heme is predominantly synthesized in bone marrow (i.e., in erythroblasts and reticulocytes that still contain mitochondria) and liver cells. In Escherichia coli, heme biosynthesis begins with L-glutamate and proceeds through 5-amino-levulinate, a universal tetrapyrrole precursor, to uroporphyrinogen III. Uroporphyrinogen III is converted to protoheme IX which further produce heme, by incorporating iron, in the subsequent reactions. Genetically engineered E.coli strains may be used for enhanced heme synthesis. For example, overexpression of gene constructs coaA gene and/or hemA induces increased expression of the intermediates ALA synthase or pantothenate kinase of heme biosynthesis pathway in E.coli.
In an embodiment, the myoglobin may be derived from steppe mammoth (JVIammuthustrogontherii), woolly mammoth (Mammuthus primigenius), sheep, cow, pig, chicken, rabbit, mouse, rat or tuna. In a preferred embodiment, the myoglobin is preferably from steppe mammoth or woolly mammoth. Preferably, the amino acid sequence is derived from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna, by addition, deletion and/or substitution of at least one amino acid. Addition, deletion and/or substitutions of two, three, four, five, six, seven, eight, nine or ten amino acids is also contemplated by the invention. Examples of myoglobin amino acid sequence from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna have been disclosed later on by a given SEQ ID NO:1-8. Such myoglobin derived from steppe mammoth, woolly mammoth, sheep, cow, pig, chicken, rabbit or tuna may also exert at least a detectable level of an activity of a myoglobin as explained later herein.
In an embodiment, the myoglobin is a steppe mammoth myoglobin, woolly mammoth myoglobin, sheep myoglobin, cow myoglobin, pig myoglobin, chicken myoglobin, rabbit myoglobin or tuna myoglobin or a myoglobin derived from any of these myoglobins. In an embodiment, the myoglobin is steppe mammoth myoglobin (SEQ ID NO: 1) or derived therefrom. In an embodiment, the myoglobin is woolly mammoth myoglobin (SEQ ID NO: 2) or derived therefrom. In an embodiment, the myoglobin is sheep myoglobin (SEQ ID NO: 3) or derived therefrom. In an embodiment, the myoglobin is cow myoglobin (SEQ ID NO: 4) or derived therefrom. In an embodiment, the myoglobin is pig myoglobin (SEQ ID NO: 5) or derived therefrom. In an embodiment, the myoglobin is chicken myoglobin (SEQ ID NO: 6) or derived therefrom. In an embodiment, the myoglobin is rabbit myoglobin (SEQ ID NO: 7) or derived therefrom. In an embodiment, the myoglobin is tuna myoglobin (SEQ ID NO: 8) or derived therefrom.
In an embodiment, the myoglobin may be represented by any one of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 or 8, preferably by SEQ ID NO: 1 or 2.
In an embodiment, the myoglobin is from a steppe mammoth as defined earlier herein, 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 in combination with 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, 1 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.
In an embodiment, the myoglobin is from a woolly mammoth as defined earlier herein, 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 in combination with 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, 1 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.
In an embodiment, the myoglobin is from sheep as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150. In an embodiment, the myoglobin is from cow as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the myoglobin is from pig as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, 1 at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the myoglobin is from chicken as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 1 11 , I at position 116, A at position 1 17, E at position 118, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150.
In an embodiment, the myoglobin is from a rabbit as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, in combination with 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, 1 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, 1 at position 143, A at position 145, K at position 146, E at position 149, L at position 150, or Q at position 153. In an embodiment, the myoglobin is from a tuna as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8 in combination with 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.
In an embodiment, the myoglobin may be represented by one of the following amino acid sequences: a) having at least 90% identity with SEQ ID NO: 1 or 2, and having F at position 30 and/or Q at position 65; b) having at least 90% identity with SEQ ID NO: 3, 4 or 5, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, 4 or 5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150; c) having at least 90% identity with SEQ ID NO: 6, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 7: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111 , I at position 116, A at position 117, E at position 118, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150; or d) having at least 90% sequence identity with SEQ ID NO: 7, and having an amino acid A at position 6; e) having at least 90% identity with SEQ ID NO: 8, and having H at position 65 and/or position 94.
In an embodiment, the myoglobin is represented by a sequence having at least 90% identity with SEQ ID NO:1 or 2, and having F at position 30 and/or Q at position 65. In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from steppe mammoth (Mammuthus trogontherif), woolly mammoth (Mammuthus primigenius), sheep, cow, pig, chicken, rabbit or tuna, preferably from steppe mammoth or woolly mammoth.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a steppe mammoth as defined earlier herein, 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 in combination with 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 11 1 , 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, 1 at position 143, A at position 145, K at position 146, E at position 149, L at position 150, or Q at position 153.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a woolly mammoth as defined earlier herein, 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 in combination with 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 11 1 , 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, 1 at position 143, A at position 145, K at position 146, E at position 149, L at position 150, or Q at position 153.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a sheep as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150. In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a cow as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a pig as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a chicken as defined earlier herein, preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6, more preferably a myoglobin comprising at least 90% sequence identity with SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111 , I at position 116, A at position 117, E at position 118, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a rabbit as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 7, in combination with 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 1 14, 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, 1 at position 143, A at position 145, K at position 146, E at position 149, L at position 150, or Q at position 153.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin is derived from a tuna as defined earlier herein, preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8, more preferably a myoglobin having at least 90% sequence identity with SEQ ID NO: 8 in combination with 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.
In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin may be represented by one of the following amino acid sequences: a) having at least 90% identity with SEQ ID NO: 1 or 2, and having F at position 30 and/or Q at position 65; b) having at least 90% identity with SEQ ID NO: 3, 4 or 5, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, 4 or 5: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150; c) having at least 90% identity with SEQ ID NO: 6, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 7: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111 , I at position 116, A at position 117, E at position 118, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150; or d) having at least 90% sequence identity with SEQ ID NO: 7, and having an amino acid A at position 6; e) having at least 90% identity with SEQ ID NO: 8, and having H at position 65 and/or position 94. In an embodiment, the myoglobin is a recombinant myoglobin, wherein the recombinant myoglobin may be represented by a sequence having at least 90% identity with SEQ ID NO:1 or 2, and having F at position 30 and/or Q at position 65.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 1 in combination with 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.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 2 in combination with 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 In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 3 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 3, N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 4 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO:5: N at position 13, Q at position 27, 1 at position 31 , N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 6: Q at position 6, Q at position 10, T at position 13, 1 at position 14, H at position 27, M at position 31 , H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 11 1 , I at position 1 16, A at position 1 17, E at position 1 18, A at position 121 , S at position 128, K at position 133, S at position 145 and/or F at position 150.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 7 in combination with 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, 1 at position 143, A at position 145, K at position 146, E at position 149, L at position 150, or Q at position 153.
In an embodiment, the amino acid sequence of the myoglobin or preferably the recombinant myoglobin identified earlier herein comprises a sequence that has 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 with the SEQ ID NO: 8 in combination with 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.
In an embodiment, the hemeprotein is a recombinant protein obtained from a microbial fermentation, preferably wherein the microbial fermentation comprises the extracellular secretion of the recombinant protein.
The microbial fermentation is performed in a microorganism. A microorganism may be a prokaryote, a eukaryote or a filamentous fungus. A prokaryote may be a bacterium. The bacterium may be a Gram positive/Gram negative bacterium 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, Xylella, Yersinia, Treponema, Vibrio, Streptococcus, Lactococcus, Zymomonas, Staphylococcus, Salmonella, Sphingomonas, Sphingobium, Novosphingobium, Brucella and Microscilla. Preferred bacteria include Aeribacillus pallidus, Aneurinibacillus terranovensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus halodurans, Bacillus pumilus, Brevibacillus thermoruber, Brevibacillus panacihumi, Cupriavidus basilensis, G. Iraustophilus, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, Escherichia coll, Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti, Sphingobium sp., Novosphingobium sp., Sphingomonas henshuiensis, and Rhizobium radiobacter. A preferred bacterium is Escherichia coli. Preferred Escherichia coli strains include: 58, 679, WG1 , DH5a, TG1 , TOP10, K12, BL21 , BL21 DE3, XL1-Blue, XL10-Gold, TB1 , REG-12, W945, HB101 , DH1 , DP50, AB284, JC9387, AG1 , C600, Cavalli Hfr, Y10.
A eukaryote may be a yeast or a filamentous fungus. Preferred yeasts include Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Cryptococcus, Debaromyces, Saccharomycecopsis, Saccharomycodes, Wickerhamia, Debayomyces, Hanseniaspora, Ogataea, Kuraishia, Komagataella, Metschnikowia, Williopsis, Nakazawaea, Torulaspora, Bullera, Rhodotorula, Sporobolomyces. Within yeasts, the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha (also known as Ogataea henricii), Yarrowia lipolytica, Candida tropicalis and Pichia pastoris (also known as Komagataella phaffii) are preferred. Preferred Pichia strains are selected from the following list: Bg09, Bg10, Bg11 , Bg12 (exemplified), Bg20, Bg21 , Bg22, Bg23, Bg24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430, X-33, GS115, KM71 , SMD1168, SMD1165, MC100-3, most preferred Bg10 and derivatives. Preferred Saccharomyces strains are selected from the following list: S288C, CEN.PK family, CBS 2354, ATCC 2360, ATCC 4098, ATCC 4124, ATCC 4126, ATCC 4127, ATCC 4921 , ATCC 7754, ATCC 9763, ATCC 20598, ATCC 24855, ATCC 24858, ATCC 24860, ATCC 26422, ATCC 46523, ATCC 56069, ATCC 60222, ATCC 60223, ATCC 60493, ATCC 66348, ATCC 66349, ATCC 96581. A preferred yeast is a Pichia strain, more preferably Pichia pastoris.
A filamentous fungus may be selected from the following list including: Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallinastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Ustilago and Trichoderma. Preferred filamentous fungus are selected from the following list: Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus vadensis, Penicillium chrysogenum, Penicillium citrinum, Penicillium rubens, Penicillium oxalicum, Penicillium subrubescens, Rasamsonia emersonii, Talaromyces emersonii, Acremonium chrysogenum, Trichoderma reesei, Aspergillus sojae, and Chrysosporium lucknowense. Preferred strains of filamentous fungus are selected from the following list: Aspergillus niger CBS 513.88, N593, CBS 120.49, N402, ATCC 1015 Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , ATCC 9576, ATCC 14488-14491 , ATCC 11601 , ATCC12892, Aspergillus vadensis CBS 113365, CBS 102787, IMI 142717, IBT 24658, CBS 113226, Penicillium chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Wisconsin 54-1255, Penicillium subrubescens CBS 132785, FBCC 1632, Talaromyces emersonii CBS 393.64, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC56765 or ATCC 26921 , Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006. In a preferred embodiment, Aspergillus is used as a filamentous fungus. More preferably, an Aspergillus niger strain is used.
In an embodiment, the microorganism used in the fermentation may be a bacterium, a yeast, a filamentous fungus or a cultured mammalian cell line, preferably Escherichia coli or Saccharomyces cerevisiae. In this context, single, isolated, cultured mammalian cells may be considered as microorganisms. In a further embodiment, the microorganism may be a bacterium, a yeast or a filamentous fungus. The microorganisms in the context of this invention are useful for the production of a hemeprotein such as myoglobin. Accordingly, in a further aspect, the invention provides a method for the production of a myoglobin as defined herein, comprising culturing the microorganisms in a suitable medium and optionally recovering the microorganism and/or myoglobin. Optionally, the produced myoglobin does not comprise a signal peptide as defined elsewhere herein. The microorganisms used may be a genetically engineered strains comprising gene constructs that expressing enzymes or intermediates of heme biosynthesis pathway. For example, such gene constructs may comprise hemA, hemL, hemB, hemD, hemF, hemG or hemH described in Junli Zhang et al,. Scientific Reports 2015.
Cell culturing may be performed for a duration of 14, 13.5, 13, 12.5, 12, 11 .5, 1 1 , 10.5, 10, 9.5, 9,
8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5 or 1 days, wherein said duration may deviate by 20%. Preferably, cell culturing is performed for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11 ,
10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5 or 1 days, wherein said duration may deviate by 10%. More preferably, the cell culturing is performed for 5 days, wherein said duration may deviate by 20%, most preferably by 10%.
The cell culturing will typically result in a production of at least 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 200 g/L or 300 g/L of a hemeprotein, preferably a myoglobin.
The cell culturing will typically result in at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8, 9%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, preferably at least 1 %, more preferably at least 5%, most preferably at least 10% of the carbon source in the growth medium being converted to a hemeprotein, preferably a myoglobin.
Cell culturing may also be performed by implementation of a multiple step, preferably a two-step, culture method. For example, a production step of a hemeprotein such as a myoglobin may be preceded by a cellular biomass growth step, wherein only limited production or no production is taking place. The different steps may be carried out using different culture modes and/or different growth media and/or different culture process parameter values, depending on the goal of each step and/or the cultured cell. The biomass during the production step may or may not be actively growing.
The host cells and/or hemeprotein may optionally be recovered from the culture medium. When present intracellularly, the hemeprotein may optionally be recovered from the recovered cellular biomass. Optionally, the recovered hemeprotein is purified. Preferably, purification of the hemeprotein will result in a purity of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, most preferably in a hemeprotein that is substantially pure.
In an embodiment, the microorganism produces the hemeprotein, preferably the myoglobin, extracellularly. The hemeprotein is transported out of the host cell after it is synthesized in the host cell. In this context, both secretory and extracellular fermentations are considered to be extracellular productions. Without being bound to this theory, an extracellular production process has the advantage that the downstream processing to recover the produced hemeprotein is more convenient, efficient and/or effective. Furthermore, an extracellular production process may result in a composition comprising the hemeprotein with a high purity such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with minimal downstream processing compared to intracellular methods wherein the hemeprotein is not transported out of the host cell after its synthesis. Purity may be measured as the weight percentage of the total protein fraction in the cell-free supernatant obtained at the end of a process or extracellular process according to the invention. Without being bound to this theory, an extracellular production process has the advantage that the optional step of recovering hemeprotein does not comprise lysing the host cell. As a result, the extracellular process may result in a composition having a low concentration of nucleic acids originating from the host cell.
The relevant downstream processing technology that may be suitable for recovery and/or purification will depend on whether the hemeprotein is accumulated within the cultured cells or excreted. Said processing technology and the associated choice will be known to the skilled person and is discussed, for example, in Wesselingh, J.A and Krijgsman, J., 1st edition, Downstream Processing in Biotechnology, Delft Academic Press, NL, 2013. In a recovery process, the biomass may be recovered from the culture medium using e.g. centrifugation or filtration. If the produced hemeprotein is accumulated within the cells, it can then be recovered and/or purified from the biomass. If it is excreted, it can be recovered from the cell-free medium or, if the biomass separation step is skipped, directly from the culture broth. Recovery and/or purification may be performed according to any conventional recovery or purification methodology known in the art. Methods for recovery and/or purification of proteins are known to the skilled person and are discussed in standard handbooks, such as Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY, 2001 or Ausubel F. et al, eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, NY, 2003. Examples of widely used recovery and/or purification methods include chromatographic methods such as ultrafiltration, microfiltration, gel-filtration chromatography, ion-exchange chromatography, immunoaffinity chromatography, metal affinity chromatography, fractionation with precipitants such as ammonium sulfate and polyethylene glycol, gel electrophoresis and salting out and dialysis. Preferably, metal affinity chromatography or size-exclusion chromatography is used. Recovery and/or purification may optionally be enhanced by linking the enzyme polypeptide to a sequence that facilitates purification, such as with a GST domain, using well-known molecular toolbox techniques. Optionally, the sequence that facilitates purification and/or signal peptide that facilitates excretion of the hemeprotein is removed from the final product using techniques known in the art, for example proteolysis by endopeptidases targeting a linker between the sequence that facilitates purification and/or the signal peptide and the hemeprotein. In some embodiments, the enzyme polypeptide is linked (fused) to a hexa-histidine peptide, such as the tag provided in a pET23a(+) vector (Genescript Biotech, Leiden, The Netherlands), among others, many of which are commercially available. As, for example, described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), hexa-histidine peptide provides for convenient purification of the fusion protein.
In a preferred embodiment, the hemeprotein is obtained via recovering and/or purifying from the culture medium. This may be realized continuously with the production process or subsequently to it. In a preferred embodiment, the hemeprotein is obtained via recovering and/or purifying from the cultured cells. A filter may be used for the purification of the recovered hemeprotein. This may be realized continuously with the production process, by harvesting fractions of growing cells, or subsequently to it.
In an embodiment, the hemeprotein is sterilized, shredded, spray dried, spray dried, freeze dried, blended, shaped, cubed, dosed or packed. Sterilization refers to any process that removes, kills, or deactivates all forms of life (particularly microorganisms such as fungi, bacteria, spores, and unicellular eukaryotic organisms) and other biological agents such as prions present in or on a specific surface, object, or fluid. Sterilization can be achieved through various means, including heat, chemicals, irradiation, high pressure, and filtration. Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure, then removing the ice by sublimation. Packing aims to provide a protection for the product, to tamper resistance and to provide physical, chemical or biological needs. Packing may also contain nutrition facts, characteristics of the products, and an instruction/guide for use of the product.
In a preferred embodiment, the cultured host cells used in fermentation are immobilized. Immobilization of cells may be achieved by any means known to the skilled person as discussed in standard handbooks such as Guisan, J.M., Bolivar, J.M., Lopez-Gallego, F., Rocha-Martin, J. (Eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020. Typically, the host cells can be immobilized to a semi-solid or solid support by three different methods. The first method involves polymerizing or solidifying a spore- or cell-containing solution. Examples of polymerizable or solidifiable solutions include alginate, A-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinyle benzene, polyvinyl benzene, polyvinyl alcohol, epoxy carrier, cellulose, cellulose acetate, photocrosslinkable resin, prepolymers, urethane, and gelatin. The second method involves cell adsorption onto a support. Examples of such supports include bone char, cork, clay, resin, sand porous alumina beads, porous brick, porous silica, celite, orwood chips. The host cells can colonize the support and form a biofilm. The third method involves the covalent coupling of the host cells to a support using chemical agents like glutaraldehyde, o-dianisidine (U.S. Pat. No. 3,983,000), polymeric isocyanates (U.S. Pat. No. 4,071 ,409), silanes (U.S. Pat. Nos. 3,519,538 and 3,652,761), hydroxyethyl acrylate, transition metal-activated supports, cyanuric chloride, sodium periodate, toluene, and the like. Cultured host cells can be immobilized in any phase of their growth, for example after a desired cell density in the culture has been reached. Suitable culture modes and/or different culture process parameter values will be known to the skilled person and are discussed in standard handbooks, such as Colin R. Phillips C.R., Poon Y. C., Immobilization of Cells: In Biotechnology Monographs book series (Biotechnology, volume 5), Springer, Berlin, Germany, 1988; Tampion J., Tampion M. D., Immobilized Cells: Principles and Applications, Cambridge University Press, UK, 1987. Preferably, immobilized cells are cultured in packed bed bioreactors, also known as plug-flow bioreactors, or expanded (fluidized) bed bioreactors. Suitable growth media and recovery and/or purification methods are further discussed elsewhere herein.
Myoglobins which may be represented by SEQ ID NO: 1-8 may be encoded by nucleic acids which may be represented by SEQ ID NO: 9-18, respectively, although it is understood that these nucleic acids sequence may need to be codon-optimized in order to be expressed by the microorganism.
In this application, the numbering of the myoglobin amino acid sequences originating from steppe mammoth (SEQ ID NO: 1), woolly mammoth (SEQ ID NO: 2), sheep (SEQ ID NO: 3), cow (SEQ ID NO: 4), pig (SEQ ID NO: 5), chicken (SEQ ID NO: 6), rabbit (SEQ ID NO: 7) used herein begins with methionine or Met or M at position 1 . All these amino acid sequences SEQ ID NO: 1 -7 count 154 amino acid residues.
The numbering of the myoglobin amino acid sequence originating from tuna (SEQ ID NO:8) used herein is slightly different from the consecutive numbering indicated in the sequence listing, as this sequence only counts 147 amino acid residues. In order to compare SEQ ID NO: 8 directly with SEQ ID NO: 1-7, SEQ ID NO: 8 is also attributed 154 positions, wherein eight positions are considered to be gaps (1 , 2, 3, 6, 7, 51 , 120, 121).
Further characteristics of the dairy product or replica thereof
In an embodiment, the dairy product, or the replica thereof, does not comprise a symbiotic hemoglobin produced by a plant in its root nodules. In an embodiment, the dairy product, or the replica thereof, does not comprise a leghemoglobin. In an embodiment, the myoglobin disclosed herein may be the sole source of heme-containing protein present in the dairy product of the replica thereof. It means that the dairy product, or the replica thereof, may comprise other proteins than the myoglobin as disclosed herein.
In an embodiment, the weight fraction of hemeproteins in the total protein content in the dairy product, or the replica thereof, is at least 0.001 %, and wherein the weight fraction of myoglobins in the heme-proteins in the dairy product, or the replica thereof, is at least 50%.
Preferably, the concentration of the total iron content in the dairy product, or the replica thereof, is from 0.1 up to 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, or 2.0 mg per 100 mL. In an embodiment, the dairy product, which is preferably a milk, comprises a higher amount of bioavailable iron or heme iron than a corresponding control or reference diary product, which has not been supplemented with a hemeprotein. In an embodiment, the replica dairy product, which is preferably a replica of a milk, comprises a higher amount of bioavailable iron or heme iron than a corresponding control or reference replica diary product, which has not been supplemented with a hemeprotein. Iron bioavailability may be assessed or measured by change in hemoglobin and/or serum ferritin level or concentration in iron-deficient individuals, whole-body retention of radioiron, iron incorporation into erythrocytes or a reticulocyte-rich erythrocyte fraction, plasma iron response test, in vitro simulator model such as SHIME assay (Simulator of the Human Intestinal Microbial Ecosystem), compartmental modeling of iron absorption and/or dual isotopic tracer. In the context of this invention, iron bioavailability may be assessed using any one of these assays or a combination thereof.
Example 2.2 illustrates that dairy products supplemented with a hemeprotein comprise a higher amount of bioavailable iron.
In an embodiment, the iron bioavailability of the dairy product, which is preferably a milk, is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference dairy product which has not been supplemented with a hemeprotein. In an embodiment, the iron bioavailability of the replica of a dairy product, which is preferably a replica of a milk, is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference replica dairy product which has not been supplemented with a hemeprotein.
An advantage of the current invention is that the dairy products or replicas thereof have a higher iron bioavailability than similar dairy products or replicas that have been supplemented with other, non-heme forms of iron.
In an embodiment, the iron bioavailability of the dairy product, which is preferably a milk, is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference dairy product which has been supplemented with a non-heme salt or complex of iron and has not been supplemented with a hemeprotein. In an embodiment, the iron bioavailability of the replica of a dairy product, which is preferably a replica of a milk, is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% higher than the iron bioavailability of a corresponding control or reference replica dairy product which has been supplemented with a non-heme salt or complex of iron and has not been supplemented with a hemeprotein. Examples of such control or reference replicas are supplemented with iron(lll)pyrophosphate, iron(ll)lactate or iron(lll)pyrophosphate as shown in Table 4. An additional advantage of the current invention is that the supplementation with a hemeprotein is compatible with most or all nutrients natively present in the dairy product or replica thereof. In contrast, the other forms of iron outlined above are typically not compatible with at least some of the nutrients. Hence, the supplementation with these types of iron does not result in an increase of iron bioavailability and/or results in the decrease of the bioavailability of the nutrients in question.
In an embodiment the amount in the dairy product, or the replica thereof, is ranged from 0.1 to 3 mg bioavailable iron or heme iron per 100 mL of dairy product, or replica thereof. In an embodiment such amount is ranged from 0.1 mg up to 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 .0 mg, 1.1 mg, 1 .2 mg, 1 .3 mg, 1 .4 mg, 1 .5 mg, 2.0 mg, 2.5 mg, 3.0 mg bioavailable iron or heme iron per 100 mL dairy product, or replica thereof. In another embodiment such amount is 0.1 , 0.2, 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 mg bioavailable iron or heme iron per 100 mL dairy product, or replica thereof.
Myoglobin is a richly pigmented protein, which is the main reason forthe red color of animal muscle. White meat, such as chicken muscle, has lower amount of myoglobin compared to red meat, such as beef. Similarly, most mammal’s blood color is due to the presence of hemoglobin in the blood. Especially, in vertebrate animals like whale and octopus, the blood color is blue, which is from the copper-rich hemeprotein called hemocyanin.
In the context of this invention, the color of the dairy product, or the replica thereof, is measured to compare with the color of a corresponding base diary product, or a replica thereof, which is not supplemented with the myoglobin. Color measurements for milk are frequently reported based on different color units, as there is still currently a lack of international standards for milk color measurement (B. Milovanovic et al,. Foods, 2020). Color changes occur during the production phases which contains material that reflects specific wavelengths of light and therefore different colors of products. The color of the dairy product may be measured using different instruments such as colorimeter or spectrophotometer, and different settings such as calibrations, illuminants, aperture sizes, observation angles, or number of readings taken per sample. A colorimeter or spectrophotometer assesses the color in various sample solutions (pigments or colorants in food) by absorbing a particular wavelength of light and denotes the assessment in the form of some values using the Beer-Lambert law. For example, Minolta colorimeters or Hunter Associates Laboratory equipment may be used as an instrument. For example, a calibration of the instrument is performed each time before the measurement. For example, the light source of illu minant A (2848 K; tungsten-filament lighting), B (4900 K; direct sunlight), C (6800 K; average daylight from the total sky), or D65 (6500 K; spectral distribution of mid-day sun) may be used. For example, aperture size ranges from 8mm to 3.18cm may be used. For example, an observation angle of 10° may be used. For example, a technical replication of 4 to 6 may be used. Data information from colorimeter or spectrophotometer may be analyzed and represented using the CIE L*a*b* color space or system to calculate lightness, redness, and yellowness. In food color measurement, researchers frequently use CIE L*a*b* color system to report color data (Sylwia Chudy et al,. 2020), which calculates lightness, redness and yellowness, if they are looking at the “true” human eye perception of color. In this context, L* coordinate represents the brightness of the dairy product, or the replica thereof, a* coordinate represents the redness of the dairy product, or the replica thereof, and b* coordinates represents the yellowness of the dairy product, or the replica thereof. For example, in the context of this invention, the dairy product or the replica thereof may have a L* coordinate ranges between 42.5 to 95.5, an a* coordinate ranges between 0 to 11 .8, and a b* coordinate ranges between 0 to 33. Alternatively, the color of the dairy product, or the replica thereof, may also be labeled according to the International Numbering System for Food Additives. For example, the milk product of this invention may be E100, E101 , E102, E103, E104, E107 or E110, or INS 100, INS 101 , INS 102, INS 103, INS 104, INS 107 or INS 110. In an embodiment, the color of the dairy product, or the replica thereof, is white, beige, pearl, ivory, cream, or light yellow, preferably wherein the color is the same as a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein. In a preferred embodiment, the color of the dairy product, or the replica thereof, is not identical or similar to red, pink or orange.
In an embodiment, the form, structure, texture, flavor, color, aroma and/or appearance of the dairy product, or the replica thereof, are similar to those of a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein. In an embodiment, the dairy product or the replica thereof is expected to mimic the form, structure, texture, flavor, color, aroma and/or appearance of a corresponding base dairy product without having all its drawbacks. Alternatively, it might have an aspect in color, flavor and/or aroma which is distinct from the one of a milk.
Example 2.3 illustrates that the supplementation with a hemeprotein as disclosed herein has a minimal impact on the color of the chocolate plant-based dairy analogue.
Aromas from a dairy product, or a replica thereof, containing different concentrations of hemeproteins such as myoglobin may be analyzed using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME).
In an embodiment, the flavor of the dairy product or the replica thereof are similar to those of a corresponding base dairy product, which has not been supplemented with a hemeprotein. In an embodiment, the dairy product or the replica thereof does not taste acid, astringent, barny, bitter, cooked, cowy, feed, flat, foreign, garlic, malty, non-fresh, oxidized, metallic-oxidized, or soapy. The sensory judging of the dairy product or the replica thereof for the purpose of quality control and shelf-life evaluation may be performed by the established guidelines, for example the American Dairy Science Association (ADSA) guidelines. Under this guideline, the dairy product or the replica thereof may be scored on a scale of 1-10 based on the defects. The dairy product or the replica thereof may be supplemented with additional substances for an alternatively flavored dairy product, or replica thereof. The additional substances may be from a fruit (e.g., strawberry, cherry, banana etc.), a vanilla, a chocolate, a nut (pecan, pistachio), coffee, caramel, peanut butter, tea, rum, or any food flavor aroma.
In a preferred embodiment, the flavor of the dairy product or the replica thereof has a similar level of sensory acceptability of a corresponding control or reference dairy product or the replica thereof. The degree of liking of a product based on its sensory appeal may be assessed by a sensory evaluation or sensory acceptability test. For example, sensory acceptability may be tested though hedonic scales where the participants or consumers indicate how much they like or dislike the sample in terms of a specific sensory property, such as appearance, flavor, test, texture and/or the overall combination liking/acceptance. A commonly used scale is the 9-point hedonic scale that ranges from “like extremely” to “dislike extremely”. In this context, a “similar level” means a difference of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% difference in the average sensory property test results between the dairy product or the replica thereof and the corresponding dairy product or the replica thereof.
Example 2.4 illustrates that a replica of a dairy product according to the invention has similar sensory characteristics as a reference replica of a dairy product.
Within the context of this invention, the acidity of a dairy product orthe replica thereof is represented by pH level measured by a pH meter. For example, the pH value of condensed milk may be about 6.33, the pH value of evaporated milk may be from 5.9 to 6.3, the pH value of buttermilk may be from 4.62 to 4.83, the pH value of whole milk may be from 6.4 to 6.9, the pH value of yoghurt may be from 4.4 to 4.8, the pH value of 40% cream may be from 6.44 to 6.80, the pH value of 20% cream may be from 6.50 to 6.68, the pH value of cheese may be from 4.1 to 7.44.
In embodiments, the pH value of the dairy product, or the replica thereof, is from 6 to 7, from 6 to 6.9, from 6 to 6.8, from 6 to 6.7, from 6 to 6.6, from 6 to 6.5, from 6.1 to 7, from 6.1 to 6.9, from 6.1 to 6.8, from 6.1 to 6.7, from 6.1 to 6.6, from 6.1 to 6.5, from 6.2 to 7, from 6.2 to 6.9, from 6.2 to 6.8, from 6.2 to 6.7, from 6.2 to 6.6, from 6.2 to 6.5, from 6.3 to 7, from 6.3 to 6.9, from 6.3 to 6.8, from 6.3 to 6.7, from 6.3 to 6.6, from 6.3 to 6.5, preferably wherein the dairy product, or the replica thereof, is a milk.
In an embodiment, the appearance of the dairy product or the replica thereof are similar to those of a corresponding control or reference dairy product or of a corresponding control or reference replica dairy product, which has not been supplemented with a hemeprotein. Within the context of this invention, the appearance of a dairy product orthe replica thereof may be evaluated by the viscosity which is expressed by the unit poise. Viscosity may be measured both in absolute or relative terms. Absolute viscosity is the viscosity in poise or centipoise. Relative viscosity is the rate of flow of liquid. The viscosity of milk ranges between 1 .5 to 2.0 centipoise at 20°C. Due to the fat emulsion and colloidal particles, milk is more viscous than water. Alternations in the physical nature of fat or protein hydrolysis, cooling or heating of milk affect the viscosity. For example clustering of fat globules affects viscosity, exemplified by the increased viscosity of cream compared to milk. Homogenization of milk results in the state of sub-division of dispersed constituents therefore also increasing the viscosity. Heating and concentration increase the viscosity due to increased total solids and changes in milk constituents. The viscosity may be measured using an Ostwald pipette, MacMichael Viscometer and a Falling Ball Viscometer.
In an embodiment, the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof, is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% higher than the average concentration of heme-proteins in a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein. In this context, the average concentration of hemeproteins in the milk of a specific species of an animal is determined over several corresponding animals, wherein no exogeneous or myoglobin has been added to the milk of the animals after harvesting. In this context, the resulting dairy product, or replica thereof, of this invention is edible for humans or animals, and the addition of the hemeprotein using the method of this invention does not introduce toxicity or affect the food safety of said dairy product.
In an embodiment, the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof, is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, preferably lower than 0.1 %, more preferably lower than 0.02%. It is understood that the hemeproteins comprised in the dairy product may originate from the addition of the hemeprotein, or from another source.
In an embodiment, the (total) weight concentration of myoglobins in the dairy product, or the replica thereof, is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1 .0%preferably lower than 0.1 %, more preferably lower than 0.02%. It is understood that the myoglobins comprised in the dairy product may originate from the addition of the myoglobin, or from another source.
In an embodiment, the weight fraction of myoglobins in the heme-proteins in the dairy product, or the replica thereof, is at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 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%, or 75%.
In an embodiment, the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof, is at least 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, and the weight fraction of myoglobins in the hemeproteins in the dairy product, or the replica thereof, is at least 5%.
In an embodiment, the (total) weight concentration of hemeproteins in the dairy product, or the replica thereof, is at least 0.001 %, and the weight fraction of myoglobins in the hemeproteins in the dairy product, or the replica thereof, is at least 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 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%, or 75%.
In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of hemeproteins in the dairy product, orthe replica thereof, originate from the addition of the myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof, originate from the addition of the myoglobin (per weight).
In an embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof, originate from the addition of the myoglobin (per weight). In a more preferred embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of myoglobins in the dairy product, or the replica thereof, originate from the addition of the myoglobin (per weight).
In an embodiment, the hemeprotein is added as part of a composition, wherein the weight concentration of the hemeprotein in the composition 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, weight concentration may be expressed as a mass of myoglobin per total volume or mass of the composition.
In an embodiment, the hemeprotein is added as part of a composition, wherein the weight fraction of the hemeprotein in the protein fraction comprised 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, weight fraction is a dimension less number which may be interpreted as a mass of hemeprotein per mass of protein. In an embodiment, the hemeprotein is added to the base dairy product, or the replica thereof, as part of a composition, wherein the hemeprotein protein purity of the composition is at least 80%. In this context, the hemeprotein protein purity may be calculated from the mass of the hemeprotein divided by the mass of the total composition.
In an embodiment, the hemeprotein is supplemented as part of an extract or composition.. In an embodiment, the hemeprotein is supplemented as part of an extract or composition, said extract or composition may comprise the cell that produces it. In an embodiment, the weight concentration of the hemeprotein in the extract 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%. In this context, weight concentration may be expressed as a mass of hemeprotein per total volume or mass of the extract.
In an embodiment, the hemeprotein is supplemented as part of an extract or composition, said extract or composition may comprise the cell that produces it. Wherein the weight fraction of the hemeprotein in the extract 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%. In this context, weight fraction is a dimensionless number which may be interpreted as a mass of hemeprotein per mass of total weight of the extract. In an embodiment, the hemeprotein is supplemented to the base dairy product, or the replica thereof, as part of an extract or composition, said extract or composition may comprise the cell that produces it, wherein the weight concentration of the hemeprotein in the composition is at least 10%.
In an embodiment, the hemeprotein is supplemented as part of an extract or composition, said extract or composition does not comprise the cell that produces it.
In an embodiment, the hemeprotein is in the form of powder. The amount of the hemeprotein needed to be added to the dairy product may be calculated or estimated based on the concentration of hemeprotein in the dairy product before the addition and the desired final concentration of hemeprotein in the dairy product. In this context, the dairy product before addition of the hemeprotein preferably contains less than 0.0001 %, less than 0.0002 %, less than 0.0003 %, less than 0.0004 %, less than 0.0005%, less than 0.0006 %, less than 0.0007%, less than 0.0008 %, less than 0.0009 %, less than 0.001 % hemeproteins per weight basis. A tool, such as a container, a cup, a spoon, a blender, a mixer, may be used to add the hemeprotein. The hemeprotein may be added directly to the base dairy product, or the replica thereof, or may be dissolved in an editable solvent, prior to be added to the base dairy product, or the replica thereof.
In an embodiment, the hemeprotein is in the form of solid shapes, wherein the solid shaped hemeprotein has a pre-determined weight and/or volume. The amount of the hemeprotein needed to be added to the dairy product may be calculated or estimated based on the concentration of the hemeprotein, and/or the volume of the solid-shaped hemeprotein, the total weight of the dairy product and the desired final amount of hemeprotein in the dairy product. In this context, the dairy product before addition of the hemeprotein preferably contains less than 0.0001 %, less than 0.0002 %, less than 0.0003 %, less than 0.0004 %, less than 0.0005%, less than 0.0006 %, less than 0.0007%, less than 0.0008 %, less than 0.0009 %, less than 0.001 % hemeproteins per weight basis. A tool, such as a container, a cup, a spoon, a blender, a mixer, may be used to add the hemeprotein. The myoglobin may be added directly to the base dairy product, or the replica thereof, or may be dissolved in a food grade solvent, prior to be added to the base dairy product, or the replica thereof.
An illustrative, non-limiting example of a milk product according to the invention is shown in an Example of this specification.
General terms
Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs and read in view of this disclosure.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a method as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a dairy product, a replica dairy product, a myoglobin, a gene construct, a host cell (or methods) as described herein may comprise additional component(s) (or additional steps) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value. As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
In the context of this application, all percentages in the context of a concentration or composition referto weight percentages, unless defined otherwise. In the context ofthis application, expressions such as “a parameter having a value of at least X, Y or Z” should be interpreted as said parameter having a value of at least X, of at least Y, or of at least Z.
Various embodiments are described herein. Each embodiment as identified herein may be combined unless otherwise indicated. All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.
Food safety measures such as good hygienic practice (GHP), good manufacturing practice (GMP), hazard analysis and critical control practice (HACCP), quality management and microbial risk assessment are implemented for the monitoring of food safety of this invention.
Sequence identity
In the context of the invention, a nucleic acid molecule such as a nucleic acid molecule encoding a hemeprotein is represented by a nucleic acid or nucleotide sequence which encodes a protein fragment or a polypeptide or a peptide or a derived peptide. It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identity number (SEQ ID NO) is not limited to this specific sequence as disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.
Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given protein fragment or polypeptide or peptide or derived peptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: X; or ii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) due to the degeneracy of the genetic code; or
Hi. a nucleotide sequence that encodes an amino acid sequence that has at least 60% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.
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%.
Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.
Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively.
The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference.
“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).
A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) I 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) I 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).
Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov/.
Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.
Figure imgf000037_0002
Alternative conservative amino acid residue substitution classes:
Figure imgf000037_0001
For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.
Proteins and amino acids
The terms "protein" or "polypeptide" or “amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. In amino acid sequences as described herein, amino acids or “residues” are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to a person of skill in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non- proteinogenic amino acid such as D-amino acids and modified amino acids formed by post- translational modifications, and also any non-natural amino acid, as described herein.
Gene or coding sequence
The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) e.g. comprising a polyadenylation- and/or transcription termination site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide. As used herein, a “regulator” or “transcriptional regulator” is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.
Promoter
As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" and/or “repressible” promoter is a promoter that is physiologically or developmentally regulated to be induced and/or repressed, e.g. by the application of a chemical inducer or repressing signal.
As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
Gene constructs and expression vectors
Gene constructs as described herein could be prepared using any cloning and/or recombinant DNA techniques, as known to a person of skill in the art, in which a nucleotide sequence encoding said hemeprotein is expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-lnterscience, New York (1987) and in Sambrook and Russell (2001 , supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).
The phrase "expression vector" or "vector" generally refers to a tool in molecular biology used to obtain gene expression in a cell, for example by introducing a nucleotide sequence that is capable of effecting expression of a gene or a coding sequence in a host compatible with such sequences. An expression vector carries a genome that is able to stabilize and remain episomal in a cell. Within the context of the invention, a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered. Alternatively, a vector is capable of integrating into a cell's genome, for example through homologous recombination or otherwise.
These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. A nucleic acid or DNA or nucleotide sequence encoding a hemeprotein is incorporated into a DNA construct capable of introduction into and expression in an in vitro cell culture. Specifically, a DNA construct is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.
A DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term “operably linked” has already been described herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.
The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001 , supra). A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognized by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001 , supra). An expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment. In most cases, the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. Coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001 , supra) and in Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media. Alternatively, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.
For example if a bacterium (preferably E. coli) is used as host cell, the following regulatory regions may be used. A promoter suitable to be used in a bacterium is lac, trp, tac, T7, phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL promoter from phage A. A terminator suitable to be used in a bacterium is lac, trp, tac, T7 (used in example), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, PL terminator from phage A. A preferred promoter used is a T7 promoter and/or a preferred terminator is the T7 terminator. A preferred signal peptide for excretion is E. coli Sec-recognition peptide (SecA), E. coli Tet- recognition peptide, E. coli dsbA, E. coli phoA, E. coli pelB, E. coli MBP (maltose binding protein). A marker suitable for E coli is ampicillin. Alternatively, the proBA operon from E. coli strain K12, including its original transcription regulatory elements may be used to facilitate selection without antibiotics.
In another example, if a yeast is used as host cell, the following regulatory regions may be used. A promoter suitable to be used in yeast may be a constitutive promoter. Examples of suitable constitutive promoters include: a glycolytic promoter selected from FBA1 , TPI1 , PGK1 , PYK1 , TDH3, ENO2, HXK2, PGI1 , PFK1 , PFK2, GPM1 gene or a non-glycolytic promoter of the TEF2 gene. A suitable promoterto be used in yeast may be inducible. If the yeast is a Pichia, the methanol inducible promoter AOX1 is preferred. Otherwise the GAL1 promoter (galactose-inducible) may be used when the yeast is S. cerevisiae. The genes mentioned from which a promoter could be derived for a yeast as host cell could also be used to derive a terminator for the same yeast. A preferred signal peptide for excretion for Pichia (and Saccharomyces) includes: the S. cerevisiae alpha mating factor pre- pro- secretion signal peptide, the S. cerevisiae Ost1 signal peptide, the S. cerevisiae Aga2 signal peptide and fusions thereof.
In another example, if a filamentous fungus is used as host cell, the following regulatory regions may be used. The following promoters may be used: the Aspergillus niger glucoamylase promoter (g/aA), the Aspergillus nidulans alcohol dehydrogenase promoter (a/cA) or the Aspergillus oryzae taka-amylase A promoter (amyB), the Aspergillus niger alcohol dehydrogenase promoter (ac/hA), the Trichoderma reesei pyruvate kinase promoter (pki) or the Aspergillus nidulas glyceraldehyde- 3-phosphate dehydrogenase promoter (gpdA). The genes mentioned from which a promoter could be derived for a filamentous fungus as host cell could also be used to derive a terminator for the same filamentous fungus. A preferred signal peptide for excretion for a filamentous fungus, preferably an Aspergillus includes: the Aspergillus niger glucoamylase signal peptide (g/aA), the Aspergillus niger a-galactosidase signal peptide (AgIB) and the Trichoderma reesei cellobiohydrolase I (Cbhl). A preferred promoter and terminator tor Aspergillus (more preferably for Aspergillus niger) are the glucoamylase promoter and the glucoamylase terminator of Aspergillus niger. Gene constructs described herein can be placed in expression vectors. Thus, in another aspect there is provided an expression vector comprising a gene construct as described in any of the preceding embodiments.
Expression may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of transgene expression in the transduced tissue on the level of the mRNA or the protein by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides or ELISA. Expression may be assessed at any time after administration of the gene construct, expression vector or composition as described herein. In some embodiments herein, expression may be assessed after 1 week, 2 weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9, weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, or more.
Suitable cell culturing methods for use in the process of producing recombinant protein are known to the skilled person, for example, in van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design, CRC Press, NY, 1991 . Such methods include, but are not limited to, submerged fermentation in liquid media, surface fermentation on liquid media and solid-state fermentations. Cell culturing may, for example, be performed by cultivation in micro-titer plates, shake-flasks, small-scale benchtop bioreactors, medium-scale bioreactors and/or large-scale bioreactors in a laboratory and/or an industrial setting. Suitable cell culturing modes include, but are not limited to, continuous, batch and/or fed-batch fermentation as well as their combinations. Cell culturing may be performed using continuous fermentation, batch fermentation, preferably fed batch fermentation.
In the context of the invention, "culture medium”, hereinafter alternately referred to as "growth medium”, can be interpreted to encompass cases wherein the cultured cells are absent as well as cases wherein the cultured cells are present in the culture medium. "Culture broth” refers to the culture medium wherein the cultured cells are present. "Culture supernatant” refers to the culture medium wherein the cultured cells are absent. "Cell-free extract” refers to a cell lysate not comprising the cellular debris. Cell culturing as part of the process of the invention can be performed under conditions conducive to the production of the introduced hemeproteins, which are known to the skilled person. Such conditions depend not only on the chemical composition of the culture medium but also on other process parameters including culture duration, temperature, O2 levels in the culture broth and/or headspace, CO2 levels in the culture broth and/or headspace, pH, ionic strength, agitation speed, hydrostatic pressure and the like. Cell culturing can take place using a culture medium comprising suitable nutrients, such as carbon and nitrogen sources and additional compounds such as inorganic salts and vitamins, using procedures known in the art (see, e. g. Bennett, W. and Lasure, L., 1st edition, More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable growth media are available from commercial suppliers or may be prepared using published compositions that are suitable for the respective hosts (e.g. in catalogues of the Centraalbureau Voor Schimmelcultures collection (CBS) or of the American Type Culture Collection (ATCC)).
The exact composition of the growth medium and the values of culture process parameters are not critical features of the invention. Any growth medium composition may be contemplated, as long as it allows for growth of the host cell and production of the introduced hemeproteins. The growth medium will typically comprise a carbon source to be used for the growth of the cultured cell. The skilled person understands that suitable carbon sources may be added externally to the growth medium or may already be present in said medium. Carbon sources may be present or added individually or in mixtures of multiple carbon sources. Examples of suitable carbon sources known in the art include simple sugars such as glucose, glycerol, maltose, sucrose, xylose, arabinose, complex sugars such as maltodextrins, hydrolysed starch, starch, molasses, and second- generation feedstocks. Second-generation feedstocks can be particularly attractive because of their lower carbon footprint. Second-generation feedstocks will typically comprise lignocellulosic material. Such material includes any lignocellulose and/or hemicellulose-based materials. Such material may be sourced from agricultural, industrial or municipal, preferably agricultural, waste streams. Examples of suitable materials include (agricultural) biomass, commercial organic matter, municipal solid waste, virgin biomass such as waste paper and garden waste, or non-virgin biomass. General forms of biomass include trees, shrubs and pastures, wheat, wheat straw, sugarcane bagasse, switchgrass, Japanese pampas grass, corn, corn stover, corn cob, canola stalk, soybean stalk, sweet corn, corn kernels, products and by-products from cereal milling (including wet milling and dry milling), such as corn, wheat, and barley, often referred to as “bran or fiber”, and municipal solids. Biomass can also be grassy materials, agricultural residues, forestry residues, municipal solid waste, wastepaper, and pulp and paper mill residues. Agricultural biomass includes branches, shrubs, tows, corn and corn straw, energy crops, forests, fruits, flowers, cereals, pastures, herbaceous crops, leaves, bark, needles, logs, roots, young trees, short term rotating woody crops, shrubs, switch herbs, trees, vegetables, fruits, vines, sugar beet pulp, wheat middlings, oat hulls, and hard and soft timber (not including toxic wood), and organic waste materials resulting from agricultural processes including agriculture and forestry activities, particularly forestry wood waste. Agricultural biomass may be any of the foregoing alone, or any combination or mixture thereof. Carbon sources such as organic acids, aldehydes, ketones, esters and alcohols may also be contemplated. The use of growth media comprising combinations of multiple different carbon sources may also be contemplated in the process of the invention. Such media could, as a non-limiting example, combine more oxidized carbon sources such as organic acids with more reduced carbon sources such as alcohols. Examples of suitable nitrogen sources known in the art include soybean meal, corn steep liquor, yeast extract, whey protein, egg protein, casein hydrolysate, urea, ammonia, ammonium salts and nitrate salts. Examples of additional suitable compounds known in the art include phosphate, sulphate, metals such as magnesium, trace elements and vitamins. The exact growth medium requirements will vary based on the host cell, e.g. between yeasts, bacteria and filamentous fungi, said requirements will be known to the skilled person. Accordingly, the growth medium may be a complete (rich) medium or a minimal medium, i.e. a medium comprising only the absolutely necessary components for growth depending on the cultured host cell.
Similar to the composition of the growth medium, process parameters can be assigned any value, as long as they allow for growth of the host cell and production of the introduced hemeproteins. Typically, said values will differ based on the host cell that is being cultured and will be known to the skilled person. Preferably, the process according to the invention is an oxygen-limited or aerobic process, meaning that cell culturing is performed under oxygen-limited or aerobic conditions, more preferably the process is oxygen-limited. Oxygen-limited conditions, also known as a micro-aerobic conditions, are culture conditions in which the oxygen consumption is limited by the availability of oxygen. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, under oxygen-limited conditions in a liquid culture, the rate of oxygen consumption is at least about 5.5 mmol/L/h, more preferably at least about 6 mmol/L/h and even more preferably at least about 7 mmol/L/h. Aerobic conditions are culture conditions in which the oxygen consumption is not limited by the availability of oxygen.
Cell culturing may be performed at a temperature value that is optimal for the cell, typically at a temperature range of 16-42 °C. In some embodiments, the temperature ranges between 20-40 °C, more preferably between 25-38 °C, most preferably between 28-36 °C. In some most preferred embodiments, a temperature value of about 30 or 36 °C is used.
Cell culturing may be performed at a pH value that is optimal for the cell. In some embodiments, the culture pH value is about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9. In preferred embodiments, the pH ranges from about pH 3.0 to about pH 9, more preferably from about pH 3.5 to pH 7. In some most preferred embodiments, a pH value of about 6 is used.
Cell culturing may be performed at an ionic strength value of the culture medium that is optimal for the cell, typically at a range between 50 mM - 2 M. In some embodiments, the ionic strength of the culture medium ranges between 75 mM - 1 M, more preferably between 100 mM - 750 mM. In some most preferred embodiments, an ionic strength value of about 100 mM is used.
Overview of the sequence listing
Figure imgf000044_0001
Figure imgf000045_0001
Legends to the figures
Figure 1 — Expression vector for animal myoglobin production in Pichia pastoris. A vector, carrying the codon-optimized sequence encoding cattle myoglobin has been generated. The expression cassette is produced by restriction using the two Bglll cut sites, allowing genome integration by recombination between chromosomal DNA and the AOX1 promoter on the one hand, and the AOX1 3’ fragment on the other hand. Figure 2 — Bioavailability of iron from different sources after passage through the in vitro GIT tract of different dairy (replica) samples. Samples were supplemented with different sources of iron, after passage through the in vitro SHIME system iron content was measured by ICP-MS to determine the amount of bioavailable iron. The data is expressed as relative values compared to the initial iron concentration of the same sample at the start of the assay.
Figure 3 — Color effect of myoglobin in chocolate flavoured plant-based soy milk. (A) Samples prepared with different concentrations of animal myoglobin obtained by precision fermentation in Pichia pastoris (0, 0.05, 0.1 % w/w) were imaged. (B) Relative colorimetric L*a*b* values of chocolate flavoured plant-based soy milk containing various concentrations of animal myoglobin obtained by precision fermentation in Pichia pastoris (0, 0.05, 0.1 % w/w). Data are expressed as relative values compared to the samples without myoglobin. (C) Relative color difference (AE) compared to the sample without animal myoglobin addition (0% w/w). All colorimetric assays were performed 3 times, error bars represent the standard deviation.
Examples
By way of example and not limitation, the following non-limiting examples are illustrative of various embodiments provided by the present disclosure.
Example 1: Milk product based on rabbit myoglobin
Example 1.1: Production of purified rabbit myoglobin by Escherichia coll.
Full length rabbit myoglobin gene SEQ ID NO: 7 is synthesized and cloned in a modified pET-23a(+) vector, comprising the T7 promoter and terminator and a C-terminal hexa His-tag (Genscript Biotech, Leiden, the Netherlands). The ampR marker gene originally present in said vector is replaced by the proBA operon from E. coll strain K12, including its original transcription regulatory elements, to facilitate selection without antibiotics. The correctly assembled plasmids are confirmed by PCR and used to transform a proline auxotrophic E. coll protein production strain (E. coll K12 AproBA). Transformed strains are incubated overnight in shake-flasks containing minimal medium (10.5 g/L K2HPO4, 4.5 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 0.12 g/L MgSO4, 0.5 g/l Nacitrate, 2 g/L glucose, and 5.0 mg/L thiamine HClat 37°C and 150 rpm (pH 6). 500 pl of the overnight culture is transferred to a 1 L shake-flask containing 500 mL minimal medium and incubated at 37°C and 150 rpm, until an GD600 of 0.4-1 is reached. IPTG (isopropyl-p-D-thiogalactoside) at a concentration of 100 pM is added to the culture, after which the culture is incubated for 24 h at 16°C and 150 rpm. The culture is harvested and centrifuged at 3500 x g (4°C) for 15 min. The supernatant is discarded and the pellet is dissolved in 50 mL BugBuster Protein Extraction Reagent (Novagen), containing 1 KU Lysozyme/ml (Sigma-Aldrich), 25 U Benzonase® Nuclease and complete™, EDTA-free Protease Inhibitor Cocktail (Roche). The dissolved pellet is incubated for 30 min at 4°C in a shaker. The centrifugation step is repeated and the cell-free extract (supernatant) is collected and assayed by SDS-PAGE to confirm the production of myoglobins. The production of rabbit myoglobins by the respective transformed strains is confirmed by the presence of protein bands of the correct size.
For purification, the cell-free extracts containing the soluble fraction of proteins is loaded to a HisTrap FF 1 mL column (Cytiva, MA, USA), coupled with an AKTA start system. The column is equilibrated with 20 mM HEPES, 0.4 M NaCI, and 20 mM imidazole, pH 7.5, 1 mL/min flow rate. The protein is eluted with 20 mM HEPES, 0.4 M NaCI, and 400 mM imidazole, pH 7.5. The fractions containing the rabbit myoglobins are pooled, concentrated and confirmed by SDS-PAGE and Western Blotting using an anti-histidine tag antibody (Bio-Rad), which confirm the successful purification of all myoglobins. The purified rabbit myoglobins are stored at -20°C for later use.
Example 1.2: Steps for production of milk product
1 . Raw cow milk is obtained and examined for the freshness and quality.
2. The raw milk is then stored at temperature 4°C;
3. Emulsion stabilizer, folic acid, vitamin, mineral mix and myoglobins produced from previous step of myoglobin fermentation are added to the milk in a mixing tank.
4. The mixture is gently being stirred for 10 to 20 mins at 38 to 45°C in the mixing tank to allow the supplements to be dissolved.
5. The mixture is being continuously stirred to be homogeneous at a temperature of 65 to 80 °C.
6. The mixture is pasteurized at a temperature of 62° to 65°C for 30 mins, then the mixture is cooled at 15 to 20°C.
7. 20ml to 50ml of the mixture is sampled for color, aromatic, safety and nutritional analysis. The rest of the mixture is then transferred into aseptic tank, in order to be packed into products.
Illustrative, non-limiting examples of compositions according to the invention and reference examples are reported herein below:
Figure imgf000047_0001
Figure imgf000048_0001
Example 2: Milk product based on cattle myoglobin
Example 2.1: Extracellular production of myoglobin in Pichia pastoris
The sequence coding for myoglobin from cattle was cloned downstream of the methanol-inducible A0X1 promoter of Pichia pastoris, and upstream of a histidine prototrophy selection marker and the A0X1 terminator. Myoglobin is naturally found in the cytoplasm of muscle cells. To facilitate recombinant protein purification, we fused a sequence encoding a signal peptide in frame with the myoglobin coding sequence, in order to target nascent myoglobin proteins to the secretory pathway for excretion outside the cell.
This construct was used to transform histidine-auxotroph (his4) Pichia pastoris cells, and transformants were selected fortheir ability to grow in the absence of histidine. The presence of the myoglobin expression cassette in the genome of the histidine prototroph clones was then verified by PCR. Verified transformants were then tested for their ability to produce extracellular cattle myoglobin after methanol induction in 96 wells microplates. The presence of a methanol-inducible protein of the expected molecular weight was confirmed after protein electrophoresis and staining with Coomassie blue.
In order to obtain larger amount of material for the preparation of plant-based meat substitutes, fermentation runs were conducted in a 10 L fermenter. The cell-free supernatant was purified and concentrated by ultrafiltration, to obtain a concentrated myoglobin solution that also contains residual yeast proteins. The observed protein purity of myoglobin in the final product is typically in the range of 75-85%. Since the yeast that is used as a production host, Pichia pastoris, has an established history of safe use in food production, e.g. for multiple Generally Recognized As Safe (GRAS) products in the United States, and has been granted Qualified Presumption of Safety (QPS) in the European Union, consumption of Pichia proteins does not pose any safety issue. On the other hand, the presence of yeast proteins might even contribute to the palatability of the plant-based meat substitute containing the myoglobin ingredient.
Example 2.2: Nutritional analysis of iron supplementation in dairy replicas
So far, there have been efforts in supplementing milk with iron, but the absorption (bioavailability) of the added iron is low. Specifically, the effort of adding lactoferrin does not affect iron status indicators in an infant fed with infant formula based on supplemented milk [1], An additional challenge for the supplementation of plant-based milk analogues may be that the absorption of nonheme iron is inhibited by phytic acid (myo-inositol hexaphosphate), found in cereals, legumes nuts and seeds, and by polyphenol compounds, which are abundant in fruits and vegetables [2], Interestingly, the bioavailability of iron from heme iron sources is 2- to 7-fold higher than the bioavailability of non-heme iron [3], Therefore, the animal myoglobins (heme proteins) produced here can be used to supplement (plant-based) dairy products to improve the bioavailability of iron of the final product, regardless of the presence of inhibitory plant-derived polyphenols.
Various (plant-based) dairy products were assessed for iron (Fe) bioavailability during upper gastrointestinal passage under fed conditions. The corresponding physiological conditions of the stomach and small intestine were simulated making use of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) technology platform. This in vitro approach to study the gastrointestinal tract and intestinal microbial processes offers an excellent experimental setup to study the bioavailability of selected (food) ingredients such as minerals and vitamins. Here, the bioavailable iron content, i.e. the amount of iron absorbed by the body, of 4 different products (dairy and plant-based milk analogues) was assessed at the end of passage through the upper gastrointestinal tract (GIT). The bioaccessible mineral fraction (soluble fraction) was also determined. The products tested here are listed in Table 4.
Table 4. Dairy (replica) products and their corresponding iron source and concentration (https://ods.od.nih.gov/factsheets/lron-HealthProfessional/).
Figure imgf000049_0001
As shown in Fig. 2, for the soy-based milk analogue supplemented with animal myoglobin obtained by precision fermentation in Pichia pastoris, 41 .8% of the total Fe content was absorbed during the in vitro simulation. The overall iron bioaccessibility at SI end was high (i.e., >80% present in the bioaccessible and bioavailable fractions).
This stands in contrast to the same commercial soy milk analogue that was supplemented with iron(lll)pyrophosphate (Fe4(P2O?)3). The bioaccessible iron content was partially absorbed through the dialysis membrane, resulting in 33.5% of the total iron content being absorbed during the in vitro simulation. The overall Fe bioaccessibility at SI end was moderate (i.e., between 50 and 80% present in bioaccessible and bioavailable fractions). When comparing the two studied infant growth milks, the dairy infant growth milk showed limited bioavailability (25.7%) of the supplemented iron(ll)lactate (CeHioFeOe). While it was thus not possible to draw firm conclusions concerning the Fe bioaccessibility and bioavailability upon administration of the dairy-based infant formula, the results point towards a high overall bioaccessibility and a limited bioavailability.
The plant-based soy infant growth milk resulted in 19.4% absorption of the total iron content by the end of the in vitro simulation. The overall iron bioaccesibility was thus low (i.e., <50% present in the bioaccessible and bioavailable fractions).
Overall, it was concluded that the plant-based soy milk alternative supplemented with animal myoglobin obtained by precision fermentation in Pichia pastoris resulted in the highest overall bioavailable iron content by the end of the in vitro simulation. There was an increase of 20% compared to the same commercial soy milk supplemented with iron(lll)pyrophosphate, a 54% increase compared to the bioavailable iron in the soy-based infant growth milk and 39% compared to the dairy infant growth milk.
These results demonstrate that the addition of heme iron via the supplementation of the dairy replicate with myoglobin obtained by precision fermentation in Pichia pastoris leads to the highest bioavailability of all tested samples and would likely result in the highest uptake during the upper GIT passage, compared to non-heme iron supplementation. This not only demonstrates the great potential of supplementation with animal myoglobin produced by precision fermentation in plantbased (infant) dairy analogues, but also shows that it could improve the iron uptake in fortified dairy products.
Example 2.3: Color and color stability of dairy replicas supplemented with myoglobin
Consumers routinely use product color and appearance to select or reject product. From a physical point of view, the color appearance of a material represents the response of retina rods and cones to the reflected radiation in the so-called ‘visible region’ of the electromagnetic spectrum, in the range between 400 and 700 nm. From a chemico-physical point of view, color is the result of an interaction between a light source and pigments, by which energy is absorbed and emitted as complementary not-absorbed radiations in the visible region.
A commercial chocolate plant-based soy milk was supplemented with 0%, 0.05% or 0.1 % w/w of animal myoglobin obtained by precision fermentation in Pichia pastoris, as described in Example 2.1 , (corresponding to 0.16 mg/100mL and 0.32 mg/100mL of heme iron, respectively), to test whether the supplementation with the myoglobin influenced the colour of the final product. This is important for consumer acceptance, as a colour shift could make the product undesirable and create rejection despite its increased nutritional value. As shown in Fig. 3A, no differences visible by eye were observed in the three products with different myoglobin concentrations. The samples were examined by colorimetry and the relative value (compared to the sample without myoglobin) is shown in Fig. 3B: there was a small decrease for both samples in lightness (L*) and redness (a*), and no difference in yellowness (b*). No statistically significant difference was observed. These results suggest that adding myoglobin has a minimal impact on the color of the chocolate plant-based dairy analogue. In addition, the AE value, a measure for color difference between two samples, was calculated using the 0% myoglobin chocolate soy milk alternative as the reference. Both the 0.05% and 0.1 % myoglobin samples have low AE values (1 .44 and 2.12 respectively - Fig. 3C) and are below or close to the threshold of 2, indicating that little or no recognizable color change was observed.
Example 2.4: Sensory analysis of dairy replicas supplemented with precision fermentation derived myoglobin
To determine whether perceptible sensory differences between the chocolate flavoured plant-based milk analogue with 0% or 0.1 % w/w of animal myoglobin produced by precision fermentation in Pichia pastoris could be observed by consumers, a triangle test was carried out. This is a discrimination test designed primarily to determine whether a perceptible sensory difference exists or not between two products (such as additional ingredients, change of ingredients, packaging, processing, storage and so on).
An untrained panel of 11 participants analysed the smell and colour of the presented products. Out of 11 responses 5 were correct, and 6 incorrect. Binomial statistical testing showed the null hypothesis cannot be rejected, meaning that there is no statistically significant difference between the analysed samples (0% animal myoglobin and 0.1 % animal myoglobin). It can be concluded that the addition of myoglobin cannot be detected by smell or by colour of the product, and thus has good sensory acceptability. These results are in line with the results obtained by colorimetry in Example 2.3.
Example 2.5: Materials and methods
Sequence analysis
The sequence coding for myoglobin 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 coding for myoglobin from the steppe mammoth (Mammuthus trogontherii) was obtained after DNA extraction from a molar sample from the so-called Adycha specimen, Illumina DNA sequencing, merging the reads and mapping them against the African savannah elephant (Loxodonta africana) genome [4], Multiple sequence alignments were performed with Clustal Omega [5] and visualized using Jalview 2.11 .1 .4 [6].
Construction of expression plasmids
The myoglobin coding sequence was codon optimized for expression in Pichia pastoris (Komagataella phaffii) [7], The optimized sequence encoding the myoglobin from Bos taurus, preceded by the coding sequence of the Saccharomyces cerevisiae mating factor alpha were chemically synthetized by GenScript. Gene fragments were cloned into the pBDIPp5 vector, downstream of the AOX1 promoter and upstream of the AOX1 terminator, the HIS4 selection marker and the AOX1 3’ fragment. The vector was amplified in E. coll (DH10B), purified with the SmartPure Plasmid Kit (Eurogenetec) and verified by Sanger sequencing (Eurofins Genomics). An expression cassette was generated from the resulting vector by restriction with Bglll (New England Biolabs), which cuts upstream from the AOX1 promoter and downstream from the AOX1 3’ fragment.
Strain engineering
The Pichia pastoris (Komagataella phaffii) strain GS115 (his4) was obtained from Life Technologies. Cell transformation was performed using the electroporation method essentially as previously described [8], Briefly, cells of the GS115 strain were grown in YPD (1 % yeast extract, 2% peptone and 2% D-glucose) medium. Cells in the exponentially growing phase were incubated for 30 min in YPD medium with 200 mM HEPES buffer (pH 8.0) and 25 mM dithiothreitol. Competent cells were then washed with ice-cold 1 M sorbitol, and transferred to a sterile electroporation cuvette (Bio-Rad). Cells were then electroporated with 1-5 pg of linear expression cassette using a Gene-Pulser (BioRad) electroporator, and resuspended in 1 mL of YPD medium containing 1 M sorbitol before transfer to a sterile 1 ,5-mL eppendorf tube. Cells were incubated at 28°C without agitation for 3 h, before plating onto agar plates containing solid MGY medium (Minimal Glycerol Medium: 1.34% Yeast Nitrogen Base with ammonium sulfate without amino acids, 2% D-glucose, 4 10-5% biotin with 2% agar). Plates were incubated for up to 4 days at 28°C.
Transformants able to grow in the absence of histidine were verified by PCR. A DNA fragment specific for the myoglobin expression cassette was amplified using a pair of primers that anneal with the coding sequence of the Saccharomyces cerevisiae mating factor alpha. PCR reactions were conducted using the OneTaq® Quick-Load® 2X Master Mix with Standard Buffer (New England Biolabs) according to the manufacturer’s instructions. PCR products were visualized after migration at 60V for 60 min in a 2% agarose TAE gel with ethidium bromide. The Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolabs) was used to control the size of the amplified fragment.
Positive clones were then screened fortheir ability to express myoglobin. Cells were grown in deep, 96 wells microtiter plates in BMGY medium (1 % yeast extract, 2% peptone, 100 mm Potassium Phosphate buffer (pH6), 1 .34% Yeast Nitrogen Base with ammonium sulfate without amino acids, 4 10-5% biotin, 1 % glycerol). 1 % methanol was added to exponentially growing cells, to induce myoglobin expression. Samples were collected 72h after the start of the methanol induction, and analysed by SDS-PAGE to assess myoglobin levels (see below).
The best producing strains were selected for further experiments, and were cryopreserved as a master cell bank at -80 °C prior to use. Results shown here were obtained using the PAL004 (cattle myoglobin) strain.
Recombinant protein production and purification
Cells from the master cell bank were plated on a YPD agar (1 % yeast extract, 2% soy peptone and 3% D-glucose, 2% agar) plate and incubated for 2 days at 28°C. A seed culture was then prepared in a 2L flask containing 200 mL of YPG (1 % yeast extract, 2 % soy peptone and 3% glycerol) medium, and incubated for 24h at 28°C in an orbital shaker incubator. This seed culture was then used to inoculate a glass-vessel fermenter (Biostat B, Sartorius) containing 900 ml of modified BSM medium [9], A batch fermentation phase was conducted for about 14-16h , until all the glycerol was consumed. A glycerol fed batch phase was then conducted to further increase the biomass. The temperature was then lowered to 26°C, and a mixed glycerol:methanol was applied for 72h to induce myoglobin expression. Dissolved oxygen was maintained above 25% during the whole fermentation, while the pH was kept at 6 during the growth phase and 5 during the induction phase. After 96h of methanol induction, cells were removed by centrifugation at 4,000 rpm at 4°C. Remaining cells were removed by microfiltration using cellulose filters with a pore diameter of 0.45 pm (Sartorius Hydrosart) using a tangential flow filtration device (Sartoflow, Sartorius). After ultrafiltration using cellulose filters with a molecular weight cut-off of 10 kD using the same device, the product was stored frozen at -20°C until use. When necessary for the assay, the ultrafiltration retentate was further concentrated using disposable ultrafiltration centrifuge devices, with a polyethersulfone membrane having a molecular-weight cutoff of 10 kD (Pierce Protein Concentrators, Thermo Scientific).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
For protein electrophoresis, 20 pl of cell-free supernatant were mixed with an equal volume of 2X protein loading buffer (100 mM Tris (pH6,8); 4 mM EDTA, 4% SDS, 20% glycerol; 0.02% bromophenol blue, 4% p-mercaptoethanol). Samples were incubated at 95°C for 5 minutes, and the appropriate volume loaded in a 15% polyacrylamide gel on a vertical mini-PROTEAN gel apparatus (Bio-Rad) at 200 V for 40 minutes. Myoglobin from horse heart muscle (Sigma) was used as standard. The molecular weight marker (PageRuler™ Prestained Protein Ladder) was purchased from Thermofisher. After electrophoresis, proteins in the gel were stained with Coomassie Brilliant Blue G-250 (Bio-Rad).
Iron supplementation of dairy and plant-based milk alternatives
The commercial dairy and plant-based milk products used were the following: 1 . Alpro soy drink (Alpro) which was supplemented by either i) 316.5 mg/100 mL of cattle myoglobin produced internally by precision fermentation in Pichia pastoris, which corresponds to approximately 1 mg/100 mL of heme iron or ii) 1 mg/100 mL of iron(lll)pyrophosphate (Fe4(P2O?)3) from Sigma Aldrich, after which the product was mixed thoroughly to ensure full solubility of the supplemented ingredient.
2. Soya growing up drink 1-3+ (Alpro) supplemented by the manufacturer with 2.1 mg/100 mL as stated on the product packaging of iron(lll)pyrophosphate (Fe4(P2O?)3).
3. Infant growth milk (Nutrilon) supplemented by the manufacturer with 1.2 mg/100 mL of iron(ll)lactacte (CeHioFeOe) as stated on the product packaging.
4. Alpro soya chocolate (Alpro) which contains 1 .5% of lean cocoa, which was supplemented with 0%, 0.05% or 0.1 % w/w of cattle myoglobin produced internally by precision fermentation in Pichia pastoris.
Iron bioavailability
Assays were performed by ProDigest (Gent, Belgium). The experiment conducted to evaluate bioaccessibility/bioavailability of test compounds, makes use of an adapted SHIME® system representing the physiological conditions of the stomach and small intestine within the same reactor over time. Four dairy milk or dairy milk replicate products were tested using an intake dose of 100 mL/reactor, each corresponding with a specific iron content (see section 2.3 above). The test products were added to the reactor at the start of the gastric simulation. All experiments were performed in biological triplicate (n = 3) to account for biological variability. In order to mimic fed conditions, a specific gastric suspension is added to the reactor. After this, a standardized enzyme and bile liquid is added to simulate the small intestinal condition. Incubation conditions (i.e., pH profiles and incubation times) are optimized in order to resemble in vivo conditions in the different regions of the gastrointestinal tract (gastric phase and small intestinal phase) for fed conditions. The protocol contains dynamic pH profiles as this mimics the in vivo condition more closely.
In order to detect the concentration of iron in the different samples, inductively coupled plasma - mass spectrometer (ICP-MS) was used. Samples were collected at the end of the in vitro upper GIT tract passage. At the end of the small intestinal transit, the total mineral content of the pooled absorbed fraction (i.e., the bioavailable fraction) was determined. The dialysis solutions collected after 30 min, 105 min, and 180 min of small intestinal incubation were therefore pooled and analyzed as a single sample. For determination of the total mineral content samples were analyzed without prior centrifugation and filtration. For determination of the soluble mineral content, acidification with HNO3 was applied prior to analysis. For determination of the total mineral content, destruction of the samples using the nitric acid method was performed prior to analysis. This destruction was generally carried out at 120°C.
Colorimetry analyses
Color values were read in plant-based chocolate milk analogues supplemented with 0, 0.05 and
0.1 % w/w cattle myoglobin added. Prior to performing sample color readings, the colorimeter (HunterLab ColorFlex EZ Model 45/0, Hunter Associates Laboratory Inc., Rustin, VA, USA) was calibrated using standard black and white tiles. Color readings (where L* describes the lightness of the sample (0 = black, 100 = white), a* describes ranges from green (-) to red (+), and b* describes ranges from blue (-) to yellow (+)) were recorded with a D65 illuminant and a 10° standard observer using the Commission Internationale de I’Eclairage (CIE L*a*b*) color scale. The same volume of each sample was then placed into a glass cuvette and read for L*a*b* values. EasyMatch QC version 4.98 software (Hunter Associates Laboratory Inc., Rustin, VA, USA) was used for data capturing. Color difference (AE) values were calculated according to the following equation, where L, a, b are the values of the sample, and Lo, ao, and bo are the initial color values:
AE = J(L - L0)2 + (a - a(l)2 + (b - b0)2
The experiment was repeated with 3 biological replicates (n=3). Statistically significant differences between the different test products were calculated by performing Wilcoxon tests at a confidence interval of 95% (p < 0.05).
Sensory panel triangle test
In this test, three samples are displayed to the panellists at the same time. Two of these assessed samples are the same, and one of them is different. Samples are presented at random, making combinations such as AAB, ABA, BAA, BBA, BAB, and ABB. After coding random samples with three digits, assessors identify the odd one out, assessing samples from left to right. Statistically, assessors are likely to get it right 1 out of 3 times or 33.3%.
During this triangle test, 11 untrained panellists (7 male, 4 female, age range: 24-57 years old) were presented with two samples without cattle myoglobin (0% w/w) and one sample with 0.1 % w/w of myoglobin (randomized order). The panellists participated to this assay individually and were separated from each other while participating in the sensory test. The 11 panellists smelled the sample and looked at the colour to determine which sample was the one containing myoglobin.
Binomial testing was performed on the results. For a triangle test binomial testing determines that to reject the null hypothesis (HO: all samples are equal) at 0.05 probability level, at least 7 correct answers are required for a panel size of 11. Similarly, from 8 or higher correct answers the null hypothesis can be rejected at the 0.01 probability level [10],
References
1. Bjbrmsjb, M.; Hernell, O.; Lbnnerdal, B.; Berglund, S.K. Reducing Iron Content in Infant Formula from 8 to 2 Mg/I Does Not Increase the Risk of Iron Deficiency at 4 or 6 Months of Age: A Randomized Controlled Trial. Nutrients 2021 , 13, 1-11 , doi:10.3390/nu13010003.
2. Heath, A.-L.M.; Fairweather-Tait, S.J. Clinical Implications of Changes in the Modern Diet: Iron Intake, Absorption and Status. Best Pract. Res. Clin. Haematol. 2002, 15, 225-241 , doi:10.1053/beha.2002.0208. 3. Food, E.P. on F.A. and N.S. added to Scientific Opinion on the Safety of Heme Iron (Blood Peptonates) for the Proposed Uses as a Source of Iron Added for Nutritional Purposes to Foods for the General Population, Including Food Supplements. EFSA J. 2010, 8, 1-31 , doi : 10.2903/j.efsa.2010.1585.
4. van der Valk, T.; Pecnerova, P.; Diez-del-Molino, D.; Bergstrom, A.; Oppenheimer, J.; Hartmann, S.; Xenikoudakis, G.; Thomas, J.A.; Dehasque, M.; Saghcan, E.; et al. Million- Year-Old DNA Sheds Light on the Genomic History of Mammoths. Nature 2021 , 591, 265- 269, doi: 10.1038/s41586-021-03224-9.
5. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Sbding, J.; et al. Fast, Scalable Generation of High-Quality Protein Multiple Sequence Alignments Using Clustal Omega. Mol. Syst. Biol. 2011 , 7, doi:10.1038/msb.2011.75.
6. Waterhouse, A.M.; Procter, J.B.; Martin, D.M.A.; Clamp, M.; Barton, G.J. Jalview Version 2-A Multiple Sequence Alignment Editor and Analysis Workbench. Bioinformatics 2009, 25, 1189-1191 , doi:10.1093/bioinformatics/btp033.
7. Love, K.R.; Shah, K.A.; Whittaker, C.A.; Wu, J.; Bartlett, M.C.; Ma, D.; Leeson, R.L.; Priest, M.; Borowsky, J.; Young, S.K.; et al. Comparative Genomics and Transcriptomics of Pichia Pastoris. BMC Genomics 2016, 17, 1-17, doi:10.1186/s12864-016-2876-y.
8. Cregg, J.M. Pichia Protocols; 2007; ISBN 9781588294296.
9. Pais-Chanfrau, J.M.; Trujillo-Toledo, L.E. Optimization of Culture Medium for Large-Scale Production of Heterologous Proteins in Pichia Pastoris to Be Used in Nanoscience and Other Biotechnological Fields. Recent Adv. Biol. Nanotechnol. 2016, 8, doi:10.4172/0974- 8369.1000279.
10. Lawless, H.T.; Heymann, H. Discrimination Testing. In; 2010; pp. 79-100.

Claims

Claims
1 . A dairy product, or a replica thereof, supplemented with a hemeprotein.
2. The dairy product, according to claim 1 , which is a milk, a cream, a butter, a yoghurt, a cheese, a custard or an ice cream.
3. The replica of the dairy product according to claim 1 , which is derived from the dairy product of claim 2.
4. The replica according to claim 1 or 3, which is a vegan product, preferably a vegan milk.
5. The dairy product, or the replica thereof, according to any one of the preceding claims, wherein the hemeprotein is a myoglobin.
6. The dairy product, or the replica thereof, according to claim 5, wherein the myoglobin is an animal myoglobin derived from steppe mammoth, woolly mammoth, pig, sheep, cow, chicken or tuna.
7. The dairy product, or the replica thereof, according to claim 5 or 6, wherein the myoglobin represented by a sequence having at least 90% identity with SEQ ID NO:1 or 2, and having F at position 30 and/or Q at position 65.
8. The dairy product, or the replica thereof, according to any one of the preceding claims, wherein the hemeprotein is a recombinant protein obtained from a microbial fermentation, preferably wherein the microbial fermentation comprises the extracellular secretion of the recombinant protein.
9. The dairy product, or the replica thereof, according to any one of the preceding claims, wherein the weight fraction of hemeproteins in the total protein content in the dairy product, of the replica thereof, is at least 0.001 %, and wherein the weight fraction of myoglobins in the hemeproteins in the dairy product, or the replica thereof, is at least 50%.
10. The dairy product, or the replica thereof, according to any one of the preceding claims, wherein the iron bioavailability of the dairy product, or the replica thereof, is at least 50% higher than the iron bioavailability of a corresponding control or reference dairy product, or a corresponding control or reference replica thereof, which has not been supplemented with a hemeprotein.
11 . The dairy product or the replica thereof, according to any one of the preceding claims, wherein the color of the dairy product, or the replica thereof, is white, beige, pearl, ivory, cream, or light yellow, preferably wherein the color is the same as a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein. The dairy product, or the replica thereof, according to any one of the preceding claims, wherein the form, structure, texture, flavor, color, aroma and/or appearance of the dairy product, or the replica thereof, are similar to those of a corresponding base dairy product, or a replica thereof, which has not been supplemented with a hemeprotein. A method for preparing a dairy product, or a replica thereof, as defined in claims 1 to 12, wherein the method comprises adding the hemeprotein to a base dairy product, or a replica thereof. The method for preparing a dairy product, or a replica thereof, according to claim 13, wherein the hemeprotein is added to the base dairy product, or the replica thereof, as part of a composition, wherein the hemeprotein protein purity of the supplemented composition is at least 80%.
PCT/EP2023/081041 2022-11-10 2023-11-07 Dairy product or replica thereof supplemented with heme-comprising protein WO2024100067A1 (en)

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