WO2024028439A1 - Asthaxanthine complexée subissant un déplacement hypsochrome lors du chauffage et application dans des produits alimentaires - Google Patents

Asthaxanthine complexée subissant un déplacement hypsochrome lors du chauffage et application dans des produits alimentaires Download PDF

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WO2024028439A1
WO2024028439A1 PCT/EP2023/071558 EP2023071558W WO2024028439A1 WO 2024028439 A1 WO2024028439 A1 WO 2024028439A1 EP 2023071558 W EP2023071558 W EP 2023071558W WO 2024028439 A1 WO2024028439 A1 WO 2024028439A1
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astaxanthin
food product
exogenous
bound
complexing agents
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PCT/EP2023/071558
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English (en)
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Anton Pluschke
Fabian MACHENS
Marilena SCHMICH
Stuti Singh
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Novelty For Them Gmbh
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Publication of WO2024028439A1 publication Critical patent/WO2024028439A1/fr

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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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
    • A23L5/44—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives using carotenoids or xanthophylls
    • 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/75—Coating with a layer, stuffing, laminating, binding or compressing of original fish pieces
    • 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
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/70—Fixation, conservation, or encapsulation of flavouring agents
    • A23L27/72—Encapsulation
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • 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
    • A23L33/15—Vitamins
    • A23L33/155—Vitamins A or D
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
    • A23P20/10—Coating with edible coatings, e.g. with oils or fats
    • A23P20/105—Coating with compositions containing vegetable or microbial fermentation gums, e.g. cellulose or derivatives; Coating with edible polymers, e.g. polyvinyalcohol
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/211—Carotene, carotenoids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

Definitions

  • the present invention provides food products that include bound astaxanthin.
  • the food products are capable of changing colour by altering the binding state of the astaxanthin.
  • Also provided are methods of altering food product properties using bound astaxanthin and methods of making such food products.
  • Crustaceans such as lobster or prawn
  • Crustaceans naturally occur in various colours, often dark blue/purple.
  • a colour change from blue/purple to red can be observed.
  • This colour change is caused by the release of the red carotenoid astaxanthin.
  • astaxanthin In raw lobster, astaxanthin is bound in a protein complex called a-crustacyanin.
  • the astaxanthin conformation is slightly modified through its interaction with the protein, leading to a bathochromic shift of its colour towards blue/purple.
  • Thermal treatment degenerates the proteins, leading to the release of astaxanthin in its native conformation and intense red colour, causing the described colour change of crustaceans during cooking.
  • Food products such as seafood products
  • the resulting seafood analogues lack the characteristic change of colour from blue/purple to red during cooking, which is commonly observed for crustaceans.
  • a-crustacyanin is present in the shell and epidermis of many crustaceans, e.g. prawn and lobster.
  • This large molecular weight complex is composed of an octamer of dimeric p- crustacyanin (P-CRCN) subunits, with p-CRCN formed by two types of crustacyanin (CRCN) subunits (A and C) in association with two astaxanthin molecules.
  • P-CRCN dimeric p- crustacyanin
  • CRCN crustacyanin subunits
  • a and C crustacyanin
  • the characteristic slate-blue colouration of lobster derives from crustacyanin carotenoproteins present in the lobster carapace. These proteins bind the carotenoid astaxanthin (3,3'-dihydroxy-p, '-carotene-4, 4'-dione, AXT) and occur either as p- crustacyanins (dimers of 1 : 1 apoprotein-AXT complexes) or as a -crustacyanin (octamer of a- crustacyanins).
  • the X-ray structure of p-CR was resolved at a 3.2 A resolution, revealing that the two AXT molecules bind noncovalently at a distance of 7 A from each other at the heterodimeric subunit interface.
  • the CRT rings are coplanar with the polyene chain, which was suggested to extend the conjugation of the molecule and to perturb the electronic ground state.
  • the AXTs form hydrogen bonds with histidine residues, thus suggesting that a dipole moment is induced in the chromophore.
  • the structural studies also showed that the AXTs are bent in the binding pocket, which also could contribute to the large colour shift through a planarization and polarization mechanism.
  • the bathochromic shift of AXT arises from approximately 50 % (0.15-0.23 eV) from electrostatic effects, 50 % (0.15 eV) from steric contributions, and less than 1 % (0.004-0.02 eV) from exciton coupling between the two chromophores.
  • the invention is based on the surprising finding that complexed astaxanthin can be used in food products to produce a colour change upon heating.
  • the invention provides a way to recreate the cooking experience (colour change) of crustaceans, such as lobster or prawn in alternative, animal-free seafood.
  • Alternative seafood products can be coloured by using astaxanthin, thereby, for example recreating the colour of salmon or cooked prawn, lobster or crab.
  • the resulting seafood analogues lack the characteristic change of colour from blue/purple to red during cooking, which is commonly observed for crustaceans (Prawn, crab, lobster).
  • the present invention solves the problem by incorporation of complexed or bound astaxanthin (for example, as a carotenoid-protein complex) into food preparations such as plant-based seafood analogues.
  • a food product comprising exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin is bathochromicly shifted in comparison to unbound astaxanthin.
  • the complexing agent comprises at least one: a. crustacyanin (CRCN) subunit A protein or a homologue thereof; and/or b. CRCN subunit C protein or a homologue thereof.
  • CRCN crustacyanin
  • the complexing agent comprises at least one beta-CRCN.
  • the complexing agent comprises at least one alpha-CRCN.
  • the exogenous astaxanthin bound to one or more complexing agents is: on an outer surface of the food product; and/or in an internal volume of the food product.
  • the exogenous astaxanthin bound to one or more complexing agents is: crosslinked to the outer surface of the food product; and/or comprised in a surface film.
  • the surface film comprises sodium alginate.
  • the exogenous astaxanthin bound to one or more complexing agents is encapsulated.
  • the exogenous astaxanthin is unbound from the one or more complexing agents and is hypsochromicly shifted in comparison to bound astaxanthin.
  • the astaxanthin has been obtained by recombinant techniques.
  • the one or more complexing agents have been obtained by recombinant techniques.
  • the food product is vegetarian. In certain embodiments, the food product is vegan.
  • the food product is a seafood analogue.
  • the seafood analogue is a plant-based seafood analogue.
  • a composition for altering one or more properties of a food product comprising: exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin is bathochromicly shifted in comparison to unbound astaxanthin; and an encapsulation agent.
  • the encapsulation agent comprises sodium alginate.
  • the composition is a film.
  • a method of altering one or more properties of a food product comprising: incorporating exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin undergoes a bathochromic shift in comparison to unbound astaxanthin into the food product or on a surface thereof; heating the food product, thereby unbinding the exogenous astaxanthin from the complexing agent, wherein the unbound exogenous astaxanthin undergoes a hypsochromic shift in comparison to bound astaxanthin.
  • heating comprises increasing a temperature of the food product to around 65°C.
  • a method of producing a food product comprising exogenous astaxanthin bound to one or more complexing agents, the method comprising; obtaining exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin undergoes a bathochromic shift in comparison to unbound astaxanthin; i) applying the exogenous astaxanthin bound to one or more complexing agents to an outer surface of the food product; and/or ii) mixing the exogenous astaxanthin bound to one or more complexing agents with a food product formulation and forming the food product.
  • obtaining comprises: isolating the exogenous astaxanthin bound to one or more complexing agents from an animal; producing exogenous astaxanthin bound to one or more complexing agents by in vitro translation and transcription; recombinantly producing the one or more complexing agents and reconstituting the one or more complexing agents with the exogenous astaxanthin to form exogenous astaxanthin bound to one or more complexing agents; recombinantly producing the exogenous astaxanthin bound to one or more complexing agents in a host organism.
  • the exogenous astaxanthin bound to one or more complexing agents is applied to the outer surface of the food product, wherein the method further comprises crosslinking the exogenous astaxanthin to the outer surface of the food product.
  • the exogenous astaxanthin bound to one or more complexing agents is applied to the outer surface of the food product, wherein prior to applying the exogenous astaxanthin, the exogenous astaxanthin is formed into a film or applied as a film.
  • the film comprises at least one biopolymer.
  • the complexing agent is as described herein.
  • the exogenous astaxanthin bound to one or more complexing agents is as described herein.
  • the food product is as described herein.
  • Figure 1 shows absorption spectra of free astaxanthin and a and p-CRCN.
  • Figure 2 shows a-CRCN or p-CRCN isolated from lobster. The colour change can be observed from blue to red when heated.
  • Figure 3 shows a Plant-based prawn piece with isolated protein uncooked (left-hand side) and cooked (right-hand side) in all of A to D.
  • Figure 4 shows Plant-based prawns, raw without isolated protein (left-hand side), raw with isolated and encapsulated isolated protein (right-hand side)
  • Figure 5 shows plant-based prawns raw without isolated protein (left-hand side) cooked with isolated and encapsulated isolated protein (right-hand side).
  • the invention is partly based on the bathochromic shift that occurs to the compound astaxanthin when bound.
  • Astaxanthin is a carotenone that consists of beta, beta-carotene-4,4'- dione bearing two hydroxy substituents at positions 3 and 3' (the 3S,3'S diastereomer).
  • a carotenoid pigment found mainly in animals (crustaceans, echinoderms) but also occurring in plants. It can occur free (as a red pigment), as an ester, or as a blue, brown or green chromoprotein. It has a role as an anticoagulant, an antioxidant, a food colouring, a plant metabolite and an animal metabolite.
  • Astaxanthin is a member of the xanthophylls, because it contains not only carbon and hydrogen but also oxygen atoms. Astaxanthin consists of two terminal rings joined by a polyene chain. This molecule has two asymmetric carbons located at the 3, 3' positions of the p-ionone ring with a hydroxyl group (-OH) on either end of the molecule. In case one, the hydroxyl group reacts with a fatty acid, then it forms mono-ester, whereas when both hydroxyl groups are reacted with fatty acids, the result is termed a di-ester.
  • Astaxanthin exists in stereoisomers, geometric isomers, free and esterified forms. All of these forms are found in natural sources.
  • the stereoisomers (3S, 3'S) and (3R 37?) are the most abundant in nature.
  • Haematococcus biosynthesizes the (3S, 3'S)-isomer, whereas yeast Xanthophyllomyces dendrorhous produces (3/?, 3 ?)-isomer.
  • Synthetic astaxanthin comprises isomers of (3S, 3'S) (3/?, 3'S) and (3/?, 3'R).
  • Astaxanthin has the molecular formula C40H52O4. Its molar mass is 596.84 g/mol.
  • the natural sources of astaxanthin are algae, yeast, salmon, trout, krill, shrimp and crayfish.
  • Microorganism sources of astaxanthin include Chlorophyceae (e.g. Haematococcus pluvialis, Chlorococcum, Chlorella zofingiensis, and Neochloris wimmeri), Ulvophyceae (e.g. Enteromorpha intestinalis and Ulva lactuca), Florideophyceae (e.g. Catenella repens), Alphaproteobacteria (e.g. Agrobacterium aurantiacum and Paracoccus carotinifaciens), Tremellomycetes (e.g.
  • Xanthophyllomyces dendrorhous and Xanthophyllomyces dendrorhous Labyrinthulomycetes (e.g. Thraustochytriu sp. CHN-3), and Malacostraca (e.g. Pandal us borealis and Pandal us clarkia).
  • Astaxanthin is a lipophilic compound and can be dissolved in solvents and oils. Solvents, acids, edible oils, microwave assisted and enzymatic methods are used for astaxanthin extraction. Astaxanthin is accumulated in encysted cells of Haematococcus. Astaxanthin in Haematococcus has been extracted with different acid treatments, hydrochloric acid giving up to 80% recovery of the pigment. When encysted cells were treated with 40% acetone at 80 °C for 2 min followed by kitalase, cellulose, abalone and acetone powder, 70% recovery of astaxanthin was obtained. High astaxanthin yield has been observed with treatment of hydrochloric acid at various temperatures for 15 and 30 min using sonication.
  • Vegetable oils (soybean, corn, olive and grape seed) have been used to extract astaxanthin from Haematococcus.
  • the culture is mixed with oils, and the astaxanthin inside the cell is extracted into the oils, with the highest recovery of 93% with olive oil.
  • Astaxanthin (1.3 mg/g) has been extracted from Phaffia rhodozyma under acid conditions. Microwave-assisted extraction at 75 °C for 5 min resulted in 75% of astaxanthin; however, astaxanthin content is high in acetone extract. Astaxanthin yield from Haematococcus was 80%-90% using supercritical fluid extraction with ethanol and sunflower oil as co-solvent.
  • Astaxanthin has been extracted repeatedly with solvents, pooled and evaporated by rotary evaporator, then redissolved in solvent and absorbance of extract was measured at 476-480 nm to estimate the astaxanthin content. Further, the extract can be analysed for quantification of astaxanthin using high-pressure liquid chromatography and identified by mass spectra.
  • Astaxanthin may also be extracted from shellfish such as shrimp, prawns, crabs and lobster using the extraction methods described herein. However, in some examples wherein the food product is vegetarian or vegan, astaxanthin is not derived from an animal source.
  • Suitable host cells for recombinant production of astaxanthin include prokaryotic hosts and eukaryotic host cells such as bacterial cells, yeast cells, algae and fungi.
  • the host may be Escherichia coli, Lactococcus lactis, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, Chlorophyceae, Ulvophyceae, Florideophyceae, Alphaproteobacteria, Tremellomycetes, Labyrinthulomycetes, and Malacostraca.
  • Bound astaxanthin refers to astaxanthin that is bound to one or more agents that a capable of causing a bathochromic shift of the astaxanthin.
  • Unbound or free astaxanthin has a peak in the electronic absorption spectra of around 470 nm to 495 nm depending on the solvent used (e.g. 472 nm in hexane or 492 nm in pyridine). This means that unbound or free astaxanthin has a colour that is red or orange.
  • unbound astaxanthin when referring to unbound astaxanthin, astaxanthin with an electronic absorption spectra peak of from about around 470 nm to 495 nm is intended.
  • unbound or free astaxanthin refers to astaxanthin having an orange, red or orangey-red colour.
  • bound astaxanthin When bound, for example, by a protein such as p-crustacyanin in a protein complex such as a-crustacyanin, the bound astaxanthin undergoes a bathochromic shift to have an electronic adsorption spectra peak at around 550 nm to 750 nm. This means that bound astaxanthin appears blue or purple in colour. Sometimes referred to as “slate-blue”. Thus, the term bound astaxanthin refers to astaxanthin that has undergone and/or is undergoing a bathochromic shift. In some examples, bound astaxanthin refers to astaxanthin that is blue, purple or slate-blue in colour.
  • the food products described herein include astaxanthin in its bound conformation and, as such, have a blue, purple or slate-blue colour.
  • the astaxanthin may be bound by any suitable complexing agent that is capable of binding to the astaxanthin and maintaining it in a bathochromicly shifted state.
  • the astaxanthin may be bound to a chemical moiety or protein moiety that induces a bathochromic shift.
  • the bound astaxanthin has an absorption maximum from 550 nm to 780 nm. In some examples, the bound astaxanthin has an absorption maximum of from 560 nm to 750 nm. In some examples, the bound astaxanthin has an absorption maximum of from 570 nm to 650 nm. In some examples, the bound astaxanthin has an absorption maximum of from 580 nm to 630 nm. [0066] In some examples, unbound astaxanthin has an absorption maximum of around 440 nm to 495 nm. In some examples, unbound astaxanthin has an absorption maximum of around 440 nm to 495 nm. In some examples, unbound astaxanthin has an absorption maximum of around 480 nm to 490 nm.
  • the bathochromic shift of unbound to bound astaxanthin is around 4000 cm -1 . In some examples, the hypsochromic shift of bound to unbound astaxanthin is around 4000 cm -1 .
  • exogenous refers to any substance derived from an external source.
  • the astaxanthin either in bound (complexed) form or the complexing agent, may each be obtained from a source that is separate and independent from the components of the food product itself.
  • a food product such as a plant oil may include astaxanthin produced by the plant the oil is obtained from. This would be considered to be endogenous astaxanthin.
  • Another example would be astaxanthin and complexed astaxanthin that naturally occurs in wild-type seafood (such as lobster, crab or prawns).
  • the complexed astaxanthin in such animals would be considered endogenous.
  • the term exogenous also excludes animals or cells that a meat product has been formed from that naturally or recombinantly produced complexed astaxanthin within the cells that form a cell based food product.
  • the complexing agent may be a synthetic protein molecule or nucleic acid that is capable of binding astaxanthin so as to induce a bathochromic shift of the astaxanthin.
  • the complexing agent is a naturally occurring or synthetic protein derived from one or more crustacyanin proteins (CRCN), crustacyanin-like proteins or homologues thereof.
  • CRCN crustacyanin proteins
  • Crustacyanins are members of the lipocalin family of hydrophobic ligandbinding proteins.
  • a-crustacyanin is a multimeric protein complex comprising 16 crustacyanin subunits.
  • the subunits that make up a-crustacyanin comprise 8 p-crustacyanin subunits, p-crustacyanin comprises a heterodimer formed by type I apocrustacyanins subunits and type II apocrustacyanins subunits. Each subunit binds a single astaxanthin molecule. These subunits may be referred to as apocrustacyanins.
  • Type I apocrustacyanin subunits include apocrustacyanin Ci, C2, and A1.
  • Type I subunits may be referred to as crustacyanin C subunits (CRTC).
  • Type II apocrustacyanin subunits include apocrustacyanin A2 and A3.
  • Type II subunits may be referred to as crustacyanin A subunits (CRTA). Examples of crustacyanin subunits are shown in Table 1.
  • the complexing agent comprises at least one protein selected from Table 1.
  • the food products described herein include at least one Type I apocrustacyanin subunit
  • at least one apocrustacyanin Ci, C2, and/or A1 examples include Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C1 subunit identified by UniProtKB number P80029.
  • the C1 subunit consists of 181 amino-acid residues, of which six are cysteine and none are methionine.
  • the complexing agent comprises at least one Type I crustacyanin subunit.
  • the complexing agent comprises at least one Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C1 subunit.
  • the complexing agent comprises a homologue of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C1 subunit. Examples of homologues of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C1 subunit are shown in Table 2 below.
  • the complexing agent comprises at least one protein comprising an amino acid sequence having at least 50% sequence identity to any one of the amino acid sequences in Table 2.
  • homologues of Homarus gammarus European lobster
  • Homarus vulgaris Crustacyanin-C1 subunit may include crustacyanins from other organisms classed as Type I and/or Type II crustacyanins.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 1. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1 In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1. In some examples, the complexing agent comprises a protein comprising an amino acid sequence according to SEQ ID NO: 1.
  • the complexing agent comprises a Homarus americanus (American lobster) H1 apocrustacyanin protein. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some examples, the complexing agent comprises a protein comprising an amino acid sequence according to SEQ ID NO: 10.
  • the complexing agent comprises a Macrobrachium rosenbergii (Giant fresh water prawn) Crustacyanin-like lipocalin.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 9.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9. In some examples, the complexing agent comprises a protein comprising an amino acid sequence according to SEQ ID NO: 9.
  • the food products described herein include at least one Type II apocrustacyanin subunit
  • Type II apocrustacyanin subunits include Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit identified by UniProtKB number P80007.
  • the A2 subunit consists of 174 residues and is similar to proteins of the retinol-binding protein superfamily.
  • the complexing agent comprises at least one Type II crustacyanin subunit.
  • the complexing agent comprises at least one Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit.
  • the complexing agent comprises a homologue of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit.
  • homologues of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit are shown in Table 3 below.
  • the complexing agent comprises at least one protein comprising an amino acid sequence having at least 50% sequence identity to any one of the amino acid sequences in Table 3. It will be understood by those skilled in the art that homologues of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit may include crustacyanins from other organisms classed as Type I and/or Type II crustacyanins.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 11. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 11 In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11 . In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 11 . In some examples, the complexing agent comprises a protein comprising an amino acid sequence according to SEQ ID NO: 11 .
  • the complexing agent comprises a Homarus americanus (American lobster) H2 apocrustacyanin protein. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 35. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 35. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 35. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 35.
  • the complexing agent comprises a protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 35. In some examples, the complexing agent comprises a protein comprising an amino acid sequence according to SEQ ID NO: 35.
  • Identity refers to the degree of sequence variation between two given nucleic acid or amino acid sequences.
  • sequence comparison typically, one sequence acts as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.
  • HSPs high-scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad.
  • test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1.
  • homologue refers to a protein that is functionally equivalent to the referenced protein, but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. In order to maintain the function of the protein, the substitutions may be conservative substitutions, replacing an amino acid with one having similar properties.
  • a homologue may refer to a protein which has an identity of at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% with the amino acid sequence of referred to. Algorithms for determining sequence identity are publicly available and include, e.g. BLAST, available through the National Center for Biotechnology Information (NCBI). One skilled in the art can determine if the sequences are similar to a degree that indicates homology and thus similar or identical function.
  • NCBI National Center for Biotechnology Information
  • a person skilled in the art can obtain a polynucleotide encoding a homologue of each protein by appropriately introducing substitution, deletion, insertion, and/or addition to the DNA encoding the protein, using methods such as site-specific mutagenesis (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp. 200 (1991)).
  • the complexing agent comprises at least one dimeric protein comprising at least two amino acid sequences as set forth in any one of Tables 1 , 2 and/or 3.
  • the dimeric protein may comprise at least amino acid sequences comprising any one of SEQ ID NOs: 1 to 39.
  • the dimeric protein comprises at least one Type I crustacyanin subunit and at least one Type II crustacyanin subunit.
  • the dimeric protein may comprise at least one Crustacyanin-C1 subunit or homologue thereof and at least one Crustacyanin-A2 subunit or homologue thereof.
  • the complexing agent may comprise a dimeric protein comprising an amino acid sequence according to SEQ ID NO: 1 and comprise an amino acid sequence according to SEQ ID NO: 11.
  • the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 1 and comprise an amino acid sequence according to SEQ ID NO: 35.
  • the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 10 and comprise an amino acid sequence according to SEQ ID NO: 11.
  • the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 10 and comprise an amino acid sequence according to SEQ ID NO: 35.
  • the dimeric protein may comprise two Type I crustacyanin subunits. In some examples, the dimeric protein may comprise two Type II crustacyanin subunits.
  • the complexing agent may comprise a dimeric protein comprising an amino acid sequence according to SEQ ID NO: 1 and comprise a second amino acid sequence according to SEQ ID NO: 1. In some examples, the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 1 and comprise an amino acid sequence according to SEQ ID NO: 10. In some examples, the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 11 and comprise a second amino acid sequence according to SEQ ID NO: 11. In some examples, the dimeric protein may comprise an amino acid sequence according to SEQ ID NO: 11 and comprise an amino acid sequence according to SEQ ID NO: 35.
  • the dimeric protein may be formed by associating two monomers after production.
  • the dimeric protein may be produced as a fusion protein.
  • a translational fusion protein comprising an amino acid sequence encoding a crustacyanin type II subunit (i.e. Type A) and an amino acid sequence encoding a crustacyanin type I subunit (i.e. Type C) as shown in SEQ ID NO: 41 below.
  • the dimeric protein may be referred to as a p-crustacyanin.
  • the complexing agent comprises a p-crustacyanin comprising at least two crustacyanin subunits as described herein.
  • the complexing agent comprises a plurality of crustacyanin subunits.
  • the complexing agent comprises a plurality of dimeric proteins as described herein. For example, a plurality of p-crustacyanins.
  • the plurality of crustacyanin subunits comprises at least one a- crustacyanin.
  • a- crustacyanin For example, 16 crustacyanin subunits or 8 p-crustacyanins.
  • the crustacyanins described herein may be produced by recombinant expression technologies or in vitro methods. “Recombinant expression” refers to the production of a peptide or protein by recombinant techniques, wherein generally, a nucleic acid encoding peptide or protein is inserted into a suitable expression vector which is in turn used to transform/transfect a host cell to produce the protein.
  • Recombinant when made in reference to a protein or a polypeptide, refers to a peptide, polypeptide or protein molecule, which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
  • Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature or to which it is ligated at a different location in nature.
  • Recombinant nucleic acid constructs may, for example, be introduced into a host cell by transformation/transfection.
  • Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells or as the result of subsequent recombination and/or repair events.
  • In vitro methods include in vitro transcription and translation. "In vitro transcription" refers to the chemical process by which mRNA is synthesized artificially from a DNA template, often referred to as a DNA plasmid. In vitro transcription mixtures also require that the raw materials for mRNA synthesis be present in the form of nucleotide bases.
  • In vitro translation refers to the cell-free synthesis of proteins or peptides in a reaction mix comprising biological extracts and/or defined reagents.
  • the reaction mix will comprise at least ATP, an energy source; mRNA; amino acids; enzymes and other reagents that are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc.
  • ribosomes e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc.
  • the cell-free synthesis reaction may be performed as batch, continuous flow, or semi-continuous flow, as known in the art.
  • Single subunits may be produced individually and then combined together to form dimeric or multimeric crustacyanins as described herein.
  • individual subunits may be expressed from respective expression vectors in the same or different host cells and then extracted and purified. Once extracted and purified, the individual subunits may be combined (for example, mixed under suitable conditions) to allow the subunits to associate into dimeric proteins (i.e. p-crustacyanin) or multimeric proteins (i.e. a-crustacyanin) as described herein.
  • subunits may be recombinantly expressed in a single host organism and associated with each other within the host organism.
  • a host organism may express one Type I subunit from one recombinant expression vector and a second Type I or Type II subunit from a second expression vector.
  • the individually expressed subunits may then bind to each other to form a dimeric protein as described herein within the host organism prior to being extracted and purified.
  • multiple subunits may be expressed from a single expression vector.
  • the subunits may be expressed as fusion proteins as described herein.
  • the amino acid sequences of the crustacyanins may include a purification tag.
  • a “purification tag” refers to a ligand that aids protein purification with, for example, size exclusion chromatography, ion-exchange chromatography, and/or affinity chromatography.
  • Purification tags and their use are well known to the art and may be, for example, poly-histidine (HIS), glutathione S-transferase (GST), c-Myc (Myc), hemagglutinin (HA), FLAG, or maltose-binding protein (MBP), V5, Green Fluorescent Protein (GFP), GSK, b- galactosidase (b-GAL), luciferase, NusA or Red Fluorescence Protein (RFP) tag.
  • polypeptides are operably linked to one or more purification tags (including combinations of purification tags).
  • a step of purifying, collecting, obtaining, or isolating a protein may therefore include size exclusion chromatography, ion-exchange chromatography, or affinity chromatography.
  • a step of purifying a crustacyanin protein (or a conjugate comprising it) utilizes affinity chromatography and, for example, an s28 affinity column or an affinity column comprising an antibody that binds the crustacyanin protein or the conjugate comprising it.
  • a step of purifying a fusion protein linked to a purification tag utilizes affinity chromatography and, for example, an affinity column that binds the purification tag.
  • fusion proteins described herein may include at least one linker sequence.
  • Suitable linkers for fusion proteins are known in the art.
  • peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n peptide linkers where "n" is generally an integer from 1 to 10.
  • the complexing agents described herein are provided in a food product bound to at least one astaxanthin molecule.
  • the complexing agent may include at least one crustacyanin subunit bound to an astaxanthin molecule.
  • the complexing agents may be produced and then isolated prior to being bound to astaxanthin that has been produced separately from the complexing agent.
  • the complexing agent may be produced in the presence of astaxanthin from a separate source or astaxanthin that is produced concurrently with the complexing agent and form complexes with astaxanthin in situ prior to isolation and purification.
  • a crustacyanin subunit, dimeric protein or multimer may be produced using recombinant or in vitro techniques as described herein.
  • the crustacyanin protein or proteins may then be mixed with astaxanthin produced by any of the methods described herein under conditions that lead to the crustacyanin binding to the astaxanthin and causing a bathochromic shift of the astaxanthin.
  • a crustacyanin subunit, dimeric protein or multimer may be produced by in vitro transcription and translation in the presence of astaxanthin and spontaneously bind to the astaxanthin forming bound astaxanthin complexes that cause the astaxanthin to undergo a bathochromic shift.
  • a host cell may recombinantly express a crustacyanin subunit, dimeric protein or multimer and also endogenously or recombinantly produce astaxanthin.
  • a host cell may be engineered to express enzymes required for astaxanthin production, such as crtB (phytoene synthase), crtY (lycopene cyclase), crtE (geranylgeranyl diphosphate synthase), crtl (phytoene dehydrogenase/phytoene desaturase), crtZ (P- carotene hydroxylase), and/or crtW (P-carotene ketolase).
  • the host cell may also be engineered to simultaneously express a crustacyanin subunit, dimeric protein or multimer as described herein. This leads to the formation of bound or complexed astaxanthin in the host cell (i.e. in vivo).
  • Suitable host cells for recombinant production of complexing agents such as the crustacyanin subunits, dimers or multimers described herein include prokaryotic hosts and eukaryotic host cells such as bacterial cells, yeast cells, algae and fungi.
  • the host may be Escherichia coli, Lactococcus lactis, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, Chlorophyceae, Ulvophyceae, Florideophyceae, Alphaproteobacteria, Tremellomycetes, Labyrinthulomycetes, and Malacostraca.
  • the bathochromic shift of the bound astaxanthin may depend on the complexing agent to which it is bound.
  • astaxanthin bound to a single crustacyanin subunit may have a peak adsorption from about 550 nm to about 580 nm.
  • astaxanthin bound to a single Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin- A2 subunit may have a peak adsorption of about 565 nm.
  • p-crustacyanin comprising a Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit and Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C2 subunit may have a peak adsorption of about 580 nm.
  • a p-crustacyanin comprising an H1 and H2 apocrustacyanin from Homarus americanus (American lobster) may have a peak adsorption of about 570 nm. In the case of a multimeric a-crustacyanin, the peak adsorption may be about 630 nm.
  • the complexed astaxanthin (i.e. astaxanthin bound to a complexing agent) may be incorporated into or onto a food product as described herein by any suitable method.
  • the complexed astaxanthin is applied to a surface of a formed food product. For example, sprayed, brushed or applied by dipping.
  • the complexed astaxanthin when the complexed astaxanthin is in a solid form, such as in the form of a powder, the complexed astaxanthin may be applied by sprinkling, dusting or scattering the complexed astaxanthin onto the outer surface of the food product.
  • the complexed astaxanthin is mixed into a part of the food product.
  • complexed astaxanthin may be mixed with a seafood analogue composition prior to forming the seafood analogue composition into a shaped food product.
  • the complexed astaxanthin may be applied to an outer surface of a food product and incorporated into an internal volume of the food product.
  • the complexed astaxanthin may be incarcerated into or onto the food product in a liquid or powdered form. Selection of the form of the complexed astaxanthin will depend on the food product and how the complexed astaxanthin is to be incorporated. For example, complexed astaxanthin may be in a buffer solution such as phosphate buffer.
  • the complexed astaxanthin may be encapsulated prior to incorporation into or onto a food product.
  • the complexed astaxanthin may be encapsulated by any suitable method, such as using spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation.
  • the encapsulating agent may be any suitable edible or food-safe agent such as fats, starches, dextrins, alginates, proteins and/or lipid materials. Suitable material and methods are described in Augustin, Mary Ann & Sanguansri, Luz & Margetts, C. & Young, B. (2001). Encapsulation of food ingredients. Food Australia. 53. 220- 223.
  • the complexed astaxanthin is encapsulated in at least one biopolymer, such as an edible biopolymer.
  • the biopolymer may comprise starch, pectin, chitin, chitosan, alginate, silk, elastin, collagen, gelatin, hemicellulose, lignin, cellulose, carrageenan, or a mixture thereof.
  • the biopolymer may be an aliginate or collagen.
  • the complexed astaxanthin is encapsulated in an alginate.
  • sodium alginate refers to a sodium salt of alginic acid and can be formed by a reaction of alginic acid with a sodium-containing base such as sodium hydroxide or sodium carbonate.
  • the complexed astaxanthin is mixed with an encapsulation agent to form a composition comprising encapsulated complexed astaxanthin.
  • a composition may then be applied to an outer surface of the food product by any suitable method. For example, by spraying, dipping or brushing.
  • the encapsulated complexed astaxanthin may be polymerized or crosslinked. Methods of polymerizing or crossing linking an encapsulation agent will depend on the encapsulation agent used. For example, for sodium alginate, the complexed astaxanthin may be polymerized or crosslinked by applying calcium chloride solution, for example, by spraying, dipping, or brushing to the complexed astaxanthin. As such, the encapsulated complexed astaxanthin may form a film on the outer surface of the food product.
  • the complexed astaxanthin is applied to an outer surface of the food product.
  • An edible film forming agent is then applied on top of the complexed astaxanthin.
  • Film forming agents may be similar agents to those used for encapsulation, for example, fats, starches, dextrins, alginates, proteins and/or lipid materials.
  • the film forming agent is at least one biopolymer as described herein, such as an edible biopolymer.
  • the film forming agent may comprise starch, pectin, chitin, chitosan, alginate, silk, elastin, collagen, gelatin, hemicellulose, lignin, cellulose, carrageenan, or a mixture thereof.
  • the film forming agent may be an aliginate or collagen.
  • the film forming agent may be an alginate. Such as sodium alginate.
  • the film forming agent may be polymerized or crosslinked as described above in respect of encapsulating agents.
  • Encapsulating or applying a surface film over the complexed astaxanthin may control the release of the astaxanthin when the food product is heated. For example, when cooked. By encapsulating or applying a film to the complexed astaxanthin, leaching or seeping of the astaxanthin out of the food product upon heating may be avoided, thus improving the appearance of the food product when cooked.
  • the complexed astaxanthin may be included in the food product in a suitable amount to enable a change in one or more properties such as appearance and/or nutritional properties.
  • the complexed astaxanthin may be included in the food product in an amount of at least 0.001 mg.
  • the astaxanthin may be in the food product in an amount from 0.001 to 10 mg. In some examples, the complexed astaxanthin may be included in the food product in an amount of from 0.001 to 5 mg. In some examples, the complexed astaxanthin may be included in the food product in an amount from 0.001 to 4 mg.
  • the food product may comprise about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.015 mg, 0.02 mg, 0.025 mg, 0.03 mg, 0.035 mg, 0.04 mg, 0.045 mg, 0.05 mg, 0.055 mg, 0.06 mg, 0.065 mg, 0.07 mg, 0.075 mg, 0.08 mg, 0.085 mg, 0.09 mg, 0.095 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 1.05 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6
  • the complexed astaxanthin as described herein, is able to undergo a hypsochromic shift when heated due to the release of the astaxanthin from the complexing agent (i.e. unbinding).
  • food products described herein, including complexed astaxanthin are able to undergo a colour change by causing the release of the complexed astaxanthin from the complexing agent. For example, by the application of heat.
  • exogenous astaxanthin to form a food product that undergoes a hypsochromic shift when heated.
  • the food product may change from a blue, purple, or slate-blue colour to orange, red or orangey-red colour when heated.
  • the food product may be a seafood analogue as described herein.
  • the one or more properties includes an appearance of the food product and/or a nutritional property of the food product.
  • the complexed astaxanthin as described herein may be provided to improve the nutritional properties of a food product by acting as a source of astaxanthin when cooked and then ingested by a consumer.
  • the food product is heated to a temperature of at least about 65°C. In some examples, the food product is maintained at a temperature of at least about 65°C for a set period of time.
  • the set period of time may be a period of time sufficient to unbind the astaxanthin from the complexing agent. For example, a period of time sufficient to denature a crustacyanin subunit, dimer or multimer as described herein.
  • the method includes obtaining exogenous astaxanthin bound to a complexing agent.
  • the exogenous complexed astaxanthin may be obtained by any suitable method as described herein.
  • obtaining may include isolating complexed astaxanthin from an animal. For example, from lobster shell.
  • isolating complexed astaxanthin from an animal For example, from lobster shell.
  • Obtaining may include producing exogenous astaxanthin bound to one or more complexing agents by in vitro translation and transcription as described herein.
  • obtaining may include recombinantly producing the complexing agents as described herein.
  • Examples of such methods include those described in Ferrari, M. et al. Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 846-853 (2012), Hara, K. Y., Yagi, S., Hirono-Hara, Y. & Kikukawa, H. A Method of Solubilizing and Concentrating Astaxanthin and Other Carotenoids. Mar. Drugs 2021 , Vol. 19, Page 462 19, 462 (2021), Bourcier, C. C.
  • the complexing agents and astaxanthin may then be reconstituted in vitro. For example, by missing the astaxanthin and complexing agent to form complexed astaxanthin.
  • obtaining complexed astaxanthin may include producing the exogenous astaxanthin bound to one or more complexing agents in a host organism.
  • genetically engineering a host to recombinant express a complexing agent such as a crustacyanin subunit, dimer or multimer as described herein and to produce astaxanthin.
  • a complexing agent such as a crustacyanin subunit, dimer or multimer as described herein and to produce astaxanthin.
  • the obtained complexed astaxanthin may then be applied to the surface of a food product such as a seafood analogue as described herein.
  • the obtained complexed astaxanthin may be added to and mixed with a food product formulation.
  • a food product formulation for example, a seafood analogue formulation or composition prior to forming the composition into a food product.
  • food products that include bound exogenous astaxanthin.
  • the term food product refers to any substance, preparation, composition or object that is suitable for consumption, nutrition, oral hygiene or pleasure and which is intended to be introduced into the human or animal oral cavity, to remain there for a certain period of time and then to either be swallowed or to be removed from the oral cavity again (e.g., chewing gum).
  • Food products include all substances or products intended to be ingested by humans or animals in a processed (e.g., cereals) or a semi-processed (e.g., butchered meat) state. This also includes substances that are added to orally consumable products (particularly food and pharmaceutical products) during their production, treatment or processing and intended to be introduced into the human or animal oral cavity.
  • a processed e.g., cereals
  • a semi-processed e.g., butchered meat
  • Food products include processed and/or semi-processed products, such as, for example: baked goods (e.g., bread, biscuits, cake, cookies, and other pastries), sweets (e.g., chocolates, chocolate bar products, other bar products, fruit gum, coated tablets, hard candies, toffees and caramels, and chewing gum), non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black or green tea beverages enriched with extracts of green or black tea, Rooibos tea, other herbal teas, fruitcontaining lemonades, isotonic beverages, soft drinks, nectars, fruit and vegetable juices, and fruit or vegetable juice preparations), instant beverages (e.g., instant cocoa beverages, instant tea beverages, and instant coffee beverages), cereal products (e.g., breakfast cereals, muesli bars, and pre-cooked instant rice products), dairy products (e.g., whole fat or fat reduced or fat-free milk beverages, rice pudding, yoghurt, kefir, cream cheese, soft cheese,
  • the food product is a meat alternative or meat analogue.
  • meat analogue or “meat substitute”, “imitation meat”, and “meat alternative” are used interchangeably.
  • a meat analogue refers to a food product that is not produced by the slaughter of an animal but has structure, texture, aesthetic qualities, and/or other properties comparable or similar to those of slaughtered animal meat, including livestock (e.g., beef, pork), game (e.g., venison), poultry (e.g., chicken, turkey, duck), and/or fish or seafood.
  • livestock e.g., beef, pork
  • game e.g., venison
  • poultry e.g., chicken, turkey, duck
  • fish or seafood e.g., uncooked, cooking, and cooked meat-like food products.
  • Seafood refers to the marine and freshwater species in Phylum Arthropoda, Class Malacostraca, Orders Decapoda and Euphausiacea (e.g.
  • Phylum Mollusca Classes Bivalvia, Gastropoda and Cephalopoda (e.g. shellfish)
  • Phylum Echinodermata Classes Echinoidea and Holothuroidea (e.g. sea urchins and sea cucumbers)
  • Phylum Chordata Class Actinopterygii, Orders Pleuronectiformes, Perciformes, Scorpaeniformes, Gadiformes, Anguilliformes (e.g. pelagic fish, demersal fish and reef fish).
  • the food product is vegetarian. In some examples, the food product is vegan. In some examples, all of the components of the food products described herein may be vegetarian. That is to say, the components are not made from or with the aid of products or components derived from animals that have died, have been slaughtered, or animals that die as a result of being eaten. In some examples, the food products described herein may be vegan. That is to say, the components are not sourced from or derived from an animal or animal product. Food products that are "vegan" are free of any animal products or animal by-products.
  • the food product is a seafood analogue or seafood alternative.
  • the seafood analogue is an analogue of an animal in the order Decapoda or Euphausiacea.
  • the seafood analogue may be a prawn, shrimp, crab, crayfish or lobster analogue.
  • the invention provides food products that are capable of undergoing a colour change when cooked that is similar to or resembles the colour change seen in animals such as prawn, shrimp, crab, crayfish or lobster when cooked. Therefore, the invention may provide a seafood analogue that more closely mimics natural seafood (e.g. change in colour) when cooked.
  • the seafood analogue may be a cultured seafood analogue or cell based seafood analogue.
  • Cultured cell based seafood analogues may also be known as cultured seafood, in vitro seafood, cellular agriculture products, or artificial seafood.
  • Such products are formed by in vitro culturing of non-human animal cells (for example, non-human animal myocytes) to form a structure that resembles cuts of meat obtained from a farmed animal.
  • non-human animal cells for example, non-human animal myocytes
  • the seafood analogue may be a cultured seafood product made using a method as described in W02020149791 A1 and WO2021111263A1 , which are incorporated herein in their entirety.
  • the seafood analogue is a plant-based seafood analogue.
  • seafood analogues formed from non-animal derived protein such as a plant protein, for example, a vegetable protein, in particular soy protein or pea protein.
  • the non-animal derived protein may additionally or alternatively comprise a fungal protein, a protein extracted from a microorganism, or a recombinantly produced protein (such as a recombinantly produced animal protein).
  • the non-animal derived protein may comprise two or more different non-animal derived proteins.
  • the non-animal derived protein may be in pure form of protein isolate or a protein concentrate.
  • the non- animal derived protein may comprise an oil seed protein, a vegetable protein, a legume protein, a tubular protein and/or a pulse protein.
  • the protein may be a defatted meal with a high protein content, such as soybean meal, soy protein isolate, providing a protein content of greater than about 55%.
  • Plant-based meat alternatives are typically based on or comprise vegetable protein, such as pea protein, soy protein, wheat protein, or gluten.
  • Seafood analogues and the compositions or formulations thereof may also include additional agents such as fats, binders, or texturisers added.
  • Seafood analogues may also include additional agents in order to make the sensory properties, such as texture, taste, smell and visual properties, more similar to animal-derived seafood.
  • one or more fats, texturizers, bulking agents, thickeners, preservatives, flavour enhancers, antimicrobial agents, pH modulators, desiccants, vitamins, minerals, sweeteners, salts, metals, curing or pickling agents, colouring agents, or any combination thereof may be added.
  • the seafood analogue may be derived from a microorganism. That is to say that the protein and/or fat portion of the analogue may be produced by or extracted from a microorganism.
  • a microorganism For example, as described in WO2021178254A1
  • the formulations and compositions used for forming seafood analogues may be in a fluid form, such as a liquid, paste, emulsion, gel, or hydrogel state prior to being formed into a seafood analogue food product.
  • the fluid form may be processed, for example, heated, chilled, compressed or manipulated in order to form a solid or semi-solid seafood analogue food product that has a defined shape and texture similar to naturally occurring seafood.
  • Astaxanthin may have antioxidant properties that help protect cells from free radicals and oxidative stress. Astaxanthin may also neutralize reactive oxygen on the inner and outer layers of cell membranes.
  • Astaxanthin may also help to activate white blood cells (T-cells) and natural killer (NK) cells, thereby providing immune system support.
  • T-cells white blood cells
  • NK natural killer
  • Astaxanthin may also help to reduce inflammation. Astaxanthin may act on reactive oxygen species to reduce proteins that can cause inflammatory diseases like celiac disease, rheumatoid arthritis, heart disease, and diabetes.
  • Astaxanthin may also help to protect skin from ultraviolet (UV) damage. Astaxanthin accumulates in the epidermis and dermis layers of the skin, helping to block UV penetration and reducing existing damage.
  • UV ultraviolet
  • Astaxanthin is a smaller molecule, which means it can cross the blood-brain barrier and may help prevent cognitive disorders such as Alzheimer’s disease.
  • Astaxanthin may also help to reduce LDL or bad cholesterol and can raise HDL or good cholesterol.
  • a food product comprising exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin is bathochromicly shifted in comparison to unbound astaxanthin.
  • the complexing agent comprises at least one: a. crustacyanin (CRCN) subunit A protein or a homologue thereof; and/or b. CRCN subunit C protein or a homologue thereof.
  • CRCN crustacyanin
  • exogenous astaxanthin bound to one or more complexing agents is: a. on an outer surface of the food product; and/or b. in an internal volume of the food product.
  • exogenous astaxanthin bound to one or more complexing agents is: a. crosslinked to the outer surface of the food product; and/or b. comprised in a surface film.
  • the surface film comprises at least one biopolymer.
  • exogenous astaxanthin bound to one or more complexing agents is encapsulated.
  • upon heating the exogenous astaxanthin is unbound from the one or more complexing agents and is hypsochromicly shifted in comparison to bound astaxanthin.
  • a composition for altering one or more properties of a food product comprising: a. exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin is bathochromicly shifted in comparison to unbound astaxanthin; and b. an encapsulation agent.
  • composition of clause 14 wherein the encapsulation agent comprises at least one biopolymer.
  • composition of clause 14 or 15 wherein the composition is a film.
  • a method of altering one or more properties of a food product comprising: a. incorporating exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin undergoes a bathochromic shift in comparison to unbound astaxanthin into the food product or on a surface thereof; b. heating the food product, thereby unbinding the exogenous astaxanthin from the complexing agent, wherein the unbound exogenous astaxanthin undergoes a hypsochromic shift in comparison to bound astaxanthin.
  • heating comprises increasing a temperature of the food product to around 65°C.
  • a method of producing a food product comprising exogenous astaxanthin bound to one or more complexing agents comprising; a. obtaining exogenous astaxanthin bound to one or more complexing agents, wherein when bound the exogenous astaxanthin undergoes a bathochromic shift in comparison to unbound astaxanthin; b. i) applying the exogenous astaxanthin bound to one or more complexing agents to an outer surface of the food product; and/or ii) mixing the exogenous astaxanthin with a food product formulation and forming the food product.
  • obtaining comprises: a.
  • the method according to any of clauses 17 to 18 and 24 to 26, wherein the exogenous astaxanthin bound to one or more complexing agents is: a.
  • a food product obtained by any one of clauses 19 to 35. Use of exogenous astaxanthin bound to one or more complexing agents to form a food product that undergoes a hypsochromic shift when heated.
  • the complexing agent comprises at least one: a. crustacyanin (CRCN) subunit A protein or homologue thereof; and/or b. CRCN subunit C protein or homologue thereof.
  • the complexing agent comprises at least one beta-CRCN.
  • the complexing agent comprises at least one alpha-CRCN.
  • the exogenous astaxanthin bound to one or more complexing agents is: a. on an outer surface of the food product; and/or b. in an internal volume of the food product.
  • the use according to clause 41a, wherein the exogenous astaxanthin bound to one or more complexing agents is: a. crosslinked to the outer surface of the food product; and/or b. comprised in a surface film.
  • the surface film comprises at least one biopolymer.
  • a-CRCN or p-CRCN are isolated from animal sources, in this particular example from the lobster Homarus americanus using previously described techniques.
  • Shell powder was mixed with 0.3 M boric acid (pH6.8, adjusted with TRIS). 1 L boric acid was used for per 25 g shell powder. The mixture was incubated at 4 °C overnight (with magnetic stirrer bar at 200 rpm). The mixture was then centrifuged at 10.000 xg for 30 min at 4 °C. The pellet was resuspended in the same volume of 10 % EDTA (adjusted to pH7). Keept stirring at 4 °C for 4 h. The slurry was centrifuged after 4 hrs (30 min, 10.000xg, 4 °C) to remove the shell powder. The blue supernatant contained the a-CRCN.
  • Proteins are precipitated with 50 % (w/v) of solid ammonium-sulfate and subsequent stirring at 4°C overnight.
  • the extract was centrifuged (30 min, 10.000 xg, 4 °C) to pellet the precipitated protein. Protein pellets were subsequently either stored at -20 °C or re-dissolved in 20 mM sodium phosphate buffer, pH7 prior to storage at -20°C.
  • the extract was subsequently applied on the surface of a plant-based prawn e.g. by spraying, painting or dipping ( Figures 2 - 5).
  • Example 2 [00151] The concentrated dark blue solution of a-CRCN in phosphate buffer as obtained in Example 1 , was applied on the surface of a piece of vegan prawn, by pipetting several drops of the solution directly on the prawn. After a short air drying period, the vegan prawn piece was fried in a pan, with the a-CRCN-treated side facing up. After a short while, the colour change documented in Figure 3 A-D was observed.
  • the prawn piece was covered with a solution of sodium alginate in water (up to 10 % w/V) and induced polymerization by dipping or spraying into/with ⁇ 10 % w/V calcium chloride solution.
  • Example 1 The a-CRCN solution as obtained in Example 1 was directly mixed with the sodium alginate solution as described in Example 3 in such a ratio that the solution was visibly blue, then applied to the prawn, followed by polymerization induced by calcium chloride application. The results are documented in Figures 4 and 5.
  • L* indicates lightness
  • a* is the red/green coordinate
  • b* is the yellow/blue coordinate.
  • An increase in a* from 0.1 to 3.5 confirmed the visual increase in the red colour.
  • CRCN subunits of type A and C are recombinantly produced in a suitable prokaryotic or eukaryotic protein expression host, such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • a suitable prokaryotic or eukaryotic protein expression host such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • the protein sequences used in this example are shown below.
  • the individual proteins of type A and C are then purified and combined with commercially available astaxanthin in order to reconstitute a-CRCN and or p-CRCN complexes.
  • the resulting complexes will be incorporated in alternative seafood as outlined in Examples 2 to 5.
  • proteins shown below may be used with a different protein tag for purification, e.g. a NusA tag or they may be used without any tag or a tag that can be removed after purification.
  • a translational fusion of two CRCN subunits of type A and C is recombinantly produced in a suitable prokaryotic or eukaryotic protein expression host, such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • a suitable prokaryotic or eukaryotic protein expression host such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • the protein sequences used in this example are shown below.
  • the translationally fused apo-proteins are then purified and combined with commercially available astaxanthin in order to reconstitute a-CRCN and/or P-CRCN complexes.
  • the resulting complexes will be incorporated in alternative seafood as outlined in Examples 2 to 5.
  • the protein shown below may be used with a different protein tag for purification, e.g. a NusA tag or may be used without any tag or a tag that can be removed after purification.
  • a single CRCN subunits of type A or C is recombinantly produced in a suitable prokaryotic or eukaryotic protein expression host, such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • a suitable prokaryotic or eukaryotic protein expression host such as Escherichia coli or Saccharomyces cerevisiae using methods known in the art.
  • the protein sequences used in this example are identical to the one shown in Example 6.
  • the proteins may be used with a different protein tag for purification, e.g. a NusA tag or they may be used without any tag or a tag that can be removed after purification.
  • CRCN subunits of type A or C or a translational fusion of two subunits are produced simultaneously produced by in vitro transcription-translation in the presence of astaxanthin, using methods known in the art. Upon purification, the resulting complexes will be incorporated in alternative seafood as outlined in Examples 2 to 5.
  • CRCN subunits or complete CRCN complexes may optionally be carried out in expression hosts, that have been genetically engineered to express the astaxanthin biosynthesis pathway, in order to produce astaxanthin in the cells during protein expression and thus allow for the in vivo assembly of crustacyanin subunits with astaxanthin to form a-CRCN and or p-CRCN complexes, rather than relying in in vitro assembly of these complexes.

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Abstract

La présente invention divulgue des produits alimentaires qui comprennent de l'astaxanthine liée. Les produits alimentaires sont capables de changer de couleur par modification de l'état de liaison de l'astaxanthine. L'invention concerne également un procédé de modification d'une ou de plusieurs propriétés d'un produit alimentaire, des compositions destinées à être utilisées dans de tels procédés, un procédé de formation des produits alimentaires comprenant de l'astaxanthine liée et l'utilisation d'astaxanthine liée exogène pour former un produit alimentaire qui subit un déplacement hypsochrome lorsqu'elle est chauffée.
PCT/EP2023/071558 2022-08-03 2023-08-03 Asthaxanthine complexée subissant un déplacement hypsochrome lors du chauffage et application dans des produits alimentaires WO2024028439A1 (fr)

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