[0001] 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.

[0002] Background

[0003] Crustaceans, such as lobster or prawn, naturally occur in various colours, often dark blue/purple. During the cooking process, a colour change from blue/purple to red can be observed. This colour change is caused by the release of the red carotenoid 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 (cooking) 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.

[0004] Food products, such as seafood products, can be coloured by using the natural carotenoid astaxanthin, thereby for example recreating the colour of salmon or cooked prawn, lobster or crab. However, the resulting seafood analogues lack the characteristic change of colour from blue/purple to red during cooking, which is commonly observed for crustaceans.

[0005] 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. These CRCN complexes are known to cause a bathochromic shift in the emission spectrum of astaxanthin from red to purple as seen in p-CRCN (Amax = 580-590 nm) or blue in the case of a-CRCN. ¹ The characteristic slate-blue colouration of lobster (Homarus gammarus) 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).

[0006] While the slate-blue colouration is the result of AXT having an absorption maximum (Amax) at ~630 nm in a-crustacyanin (580-590 nm in the p -crustacyanins), unbound AXT in solution absorbs at 475-500 nm, yielding an orange-red colouration. The absorption by AXT in a-crustacyanin is thus bathochromicly shifted by >0.50 eV, which is among the largest protein-induced spectral shifts known. In 2002, 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. Since then, the chemical origin of this shift has been the subject of numerous experimental studies that have identified a number of possible mechanisms. According to recent theories, 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.

[0007] Methods for isolation and crystallization of a-CRCN from animals (lobster and prawn shell) have been previously described. ²³ Methods for the recombinant expression (e.g. in E. coli) and subsequent reconstitution of mainly p-CRCN have also been described. ^4-

[0008] There is a need for improved food products that can undergo a colour change upon heating.

[0009] There is a need for improved seafood analogues.

[0010] There is a need for seafood analogues that more closely resemble natural seafood.

Brief summary of the disclosure

[0011] 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. However, 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.

[0012] Secondly, incorporating astaxanthin into plant-based seafood analogues also increases the nutritional value of the food (compared to other plant-based options), which is important for consumers not wanting to compromise on their personal health when looking for more sustainable options. [0013] In a first aspect of the invention, there is provided 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.

[0014] In certain embodiments, 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.

[0015] In certain embodiments, the complexing agent comprises at least one beta-CRCN.

[0016] In certain embodiments, the complexing agent comprises at least one alpha-CRCN.

[0017] In certain embodiments, 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.

[0018] In certain embodiments, 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.

[0019] In certain embodiments, the surface film comprises sodium alginate.

[0020] In certain embodiments, the exogenous astaxanthin bound to one or more complexing agents is encapsulated.

[0021] In certain embodiments, the exogenous astaxanthin is unbound from the one or more complexing agents and is hypsochromicly shifted in comparison to bound astaxanthin.

[0022] In certain embodiments, the astaxanthin has been obtained by recombinant techniques.

[0023] In certain embodiments, the one or more complexing agents have been obtained by recombinant techniques.

[0024] In certain embodiments, the food product is vegetarian. In certain embodiments, the food product is vegan.

[0025] In certain embodiments, the food product is a seafood analogue.

[0026] In certain embodiments, the seafood analogue is a plant-based seafood analogue. [0027] In another aspect of the invention there is provided 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.

[0028] In certain embodiments, the encapsulation agent comprises sodium alginate.

[0029] In certain embodiments, the composition is a film.

[0030] In another aspect of the invention there is provided a method of altering one or more properties of a food product, the method 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.

[0031] In certain embodiments, heating comprises increasing a temperature of the food product to around 65°C.

[0032] In another aspect of the invention there is provided 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.

[0033] In certain embodiments, 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.

[0034] In certain embodiments, 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.

[0035] In certain embodiments, 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.

[0036] In certain embodiments, the film comprises at least one biopolymer.

[0037] In certain embodiments, the complexing agent is as described herein.

[0038] In certain embodiments, the exogenous astaxanthin bound to one or more complexing agents is as described herein.

[0039] In certain embodiments, the food product is as described herein.

[0040] In another aspect of the invention there is provided a food product obtained by a method as described herein.

[0041] In another aspect of the invention there is provided use of exogenous astaxanthin bound to one or more complexing agents to form a food product that undergoes a hypsochromic shift when heated.

[0042] In another aspect of the invention there is provided use of exogenous astaxanthin bound to one or more complexing agents as a food additive for a food product.

[0043] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0044] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0045] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0046] Various aspects of the invention are described in further detail below.

Brief description of the Figures

[0047] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0048] Figure 1 shows absorption spectra of free astaxanthin and a and p-CRCN. Taken from Chayen, N. E., Cianci, M., Grossmann, J. G., Habash, J., Helliwell, J. R., Nneji, G. A., Raftery, J., Rizkallah, P. J. & Zagalsky, P. F. (2003). Acta Cryst. D59, 2072-2082.

[0049] Figure 2 shows a-CRCN or p-CRCN isolated from lobster. The colour change can be observed from blue to red when heated.

[0050] 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.

[0051] Figure 4 shows Plant-based prawns, raw without isolated protein (left-hand side), raw with isolated and encapsulated isolated protein (right-hand side)

[0052] Figure 5 shows plant-based prawns raw without isolated protein (left-hand side) cooked with isolated and encapsulated isolated protein (right-hand side).

[0053] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0054] Various aspects of the invention are described in further detail below.

Detailed Description

ASTAXANTHIN AND COMPLEXING AGENTS

[0055] 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. It is a carotenone and a carotenol. It derives from a hydride of a beta-carotene. 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). The primary stereoisomer of astaxanthin found in the Antarctic krill Euphausia superba is 3R, 3'R, which contains mainly esterified form, whereas in wild Atlantic salmon it is 3S, 3'S which occurs as the free form. Astaxanthin has the molecular formula C40H52O4. Its molar mass is 596.84 g/mol.

[0056] 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).

[0057] 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.

[0058] Many strategies have been developed for this synthesis of astaxanthin with the oldest and still most widely used involves a Wittig reaction of two C15 phosphonium salts with a C10- dialdehyde. Other methods include the hydroxylation of canthaxanthin, a C10 +C20 +C10 synthesis via dienolether condensation, and the isomerization of a lutein extracted from marigold to zeaxanthin and then oxidation to astaxanthin. Synthetically produced astaxanthin may include (3S, 3’S), (3R, 3’S), (3S, 3’R), (3R, 3’R), in a 1 :2:2: 1 ratio, respectively

[0059] 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.

[0060] Suitable host cells for recombinant production of astaxanthin include prokaryotic hosts and eukaryotic host cells such as bacterial cells, yeast cells, algae and fungi. For example, the host may be Escherichia coli, Lactococcus lactis, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, Chlorophyceae, Ulvophyceae, Florideophyceae, Alphaproteobacteria, Tremellomycetes, Labyrinthulomycetes, and Malacostraca.

[0061] The invention provides food products as described herein that comprise bound astaxanthin. 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. Thus in some examples, when referring to unbound astaxanthin, astaxanthin with an electronic absorption spectra peak of from about around 470 nm to 495 nm is intended. In some examples, unbound or free astaxanthin refers to astaxanthin having an orange, red or orangey-red colour.

[0062] 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.

[0063] The chemical basis for the bathochromic shift is not fully known. However, a number of theories exist. For example, V. R. Salares, N. M. Young, H. J. Bernstein and P. R. Carey, Biochim. Biophys. Acta, 1979, 576, 176 put forward a polarization mechanism in which proximal charged groups and hydrogen-bonding to astaxanthin cause a charge redistribution and increased electron delocalization in the ground state of the carotenoid. Studies of reconstituted crustacyanin complexes containing modified carotenoids have furthermore established that the presence of C4 keto groups in conjugation with the polyene chain is an essential chemical feature of a carotenoid for its absorption to exhibit a large bathochromic shift. From x-ray crystallographic data of bound astaxanthin, a number of structural features with possible implications for the bathochromic shift were identified. For example, in the a- crustacyanin protein, the C4 keto groups and the C5=C6 bonds are almost fully conjugated with the polyene chain as the p-ionone rings, and the polyene chain are essentially coplanar. Since the p-ionone rings of unbound astaxanthin most likely adopt an out-of-plane configuration (inferred from C5=C6-C7=C8 dihedral angles of around 43° the protein may increase the conjugation and electronic delocalization. As for explicit protein-carotenoid interactions involving the C4 keto groups, it has also been proposed that a hydrogen-bonded histidine (His) and a hydrogen-bonded water molecule could contribute to the bathochromic shift by exerting a polarizing effect. Other research has also suggested that protonation of histidine residue of the a-crustacyanin protein substantially contributes to the bathochromic shift.

[0064] 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. For example, the astaxanthin may be bound to a chemical moiety or protein moiety that induces a bathochromic shift.

[0065] In some examples, 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.

[0067] In some examples, 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 .

[0068] The astaxanthin referred to herein comprised in a food product may be referred to as exogenous. The term "exogenous" refers to any substance derived from an external source. For example, 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. For example, 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. In some examples, 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.

[0069] In some examples, 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.

[0070] In some examples, the complexing agent is a naturally occurring or synthetic protein derived from one or more crustacyanin proteins (CRCN), crustacyanin-like proteins or homologues thereof. Crustacyanins are members of the lipocalin family of hydrophobic ligandbinding proteins.

[0071] 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.

[0072] 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. In some examples, the complexing agent comprises at least one protein selected from Table 1.

Table 1 : Crustacyanin subunits

[0073] In some examples, the food products described herein include at least one Type I apocrustacyanin subunit For example, at least one apocrustacyanin Ci, C2, and/or A1. Examples of Type I apocrustacyanin subunits 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. In some examples, the complexing agent comprises at least one Type I crustacyanin subunit. In some examples, the complexing agent comprises at least one Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-C1 subunit. In some examples, 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. In some examples, 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. It will be understood by those skilled in the art that 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.

Table 2: Type I crustacyanin subunits and homologues

[0074] In some examples, 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.

[0075] In some examples, 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.

[0076] In some examples, the complexing agent comprises a Macrobrachium rosenbergii (Giant fresh water prawn) Crustacyanin-like lipocalin. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 9. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9. In some examples, the complexing agent comprises a protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9. In some examples, 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.

[0077] In some examples, the food products described herein include at least one Type II apocrustacyanin subunit For example, at least one apocrustacyanin A2 and/or A3. Examples of 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. Some regions of the sequence are most similar to the retinol-binding protein, P- lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. In some examples, the complexing agent comprises at least one Type II crustacyanin subunit. In some examples, the complexing agent comprises at least one Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit. In some examples, the complexing agent comprises a homologue of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit. Examples of homologues of Homarus gammarus (European lobster) (Homarus vulgaris) Crustacyanin-A2 subunit are shown in Table 3 below. In some examples, 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.

Table 3: Type II crustacyanin subunits and homologues

[0078] In some examples, 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 .

[0079] In some examples, 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. In some examples, 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.

[0080] “Identity” or “percent identity” refers to the degree of sequence variation between two given nucleic acid or amino acid sequences. For sequence comparison, typically, one sequence acts as a reference sequence to which test sequences are compared. When using a 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. Math.2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the world wide web at ncbi.nlm.nih.gov/). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., J. Mol. Biol.215: 403-410 (1990)). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. 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). For amino acid sequences, 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. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the 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)). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a 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.

[0081] As used herein, "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.

[0082] 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)).

[0083] In some examples, 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. For example, the dimeric protein may comprise at least amino acid sequences comprising any one of SEQ ID NOs: 1 to 39. In some examples, the dimeric protein comprises at least one Type I crustacyanin subunit and at least one Type II crustacyanin subunit.

[0084] In some examples, the dimeric protein may comprise at least one Crustacyanin-C1 subunit or homologue thereof and at least one Crustacyanin-A2 subunit or homologue thereof. For example, 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. 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: 35. In some examples, 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. In some examples, 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.

[0085] In some examples, the dimeric protein may comprise two Type I crustacyanin subunits. In some examples, the dimeric protein may comprise two Type II crustacyanin subunits. For example, 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.

[0086] In some examples, the dimeric protein may be formed by associating two monomers after production. In other examples, the dimeric protein may be produced as a fusion protein. For example, 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.

SEQ ID NO: 41 - Homarus qammarus CRCN A2-C1 translational fusion:

MDKIPDFWPGKCASVDRNKLWAEQTPNRNSYAGVWYQFALTNNPYQLIEKCVRNEYS FD GKQFVIKSTGIAYDGNLLKRNGKLYPNPFGEPHLSIDYENSFAAPLVILETDYSNYACLY SCID YNFGYHSDFSFIFSRSANLADQYVKKCEAAFKNINVDTTRFVKTVQGSSCPYDTQKTLGG SS GDGIPSFVTAGKCASVANQDNFDLRRYAGRWYQTHIIENAYQPVTRCIHSNYEYSTNDYG FK VTTAGFNPNDEYLKIDFKVYPTKEFPAAHMLIDAPSVFAAPYEVIETDYETYSCVYSCIT TDNY KSEFAFVFSRTPQTSGPAVEKTAAVFNKNGVEFSKFVPVSHTAECVYRA

[0087] The dimeric protein may be referred to as a p-crustacyanin. Thus in some examples, the complexing agent comprises a p-crustacyanin comprising at least two crustacyanin subunits as described herein.

[0088] In some examples, the complexing agent comprises a plurality of crustacyanin subunits. In some examples, the complexing agent comprises a plurality of dimeric proteins as described herein. For example, a plurality of p-crustacyanins.

[0089] In some examples, the plurality of crustacyanin subunits comprises at least one a- crustacyanin. For example, 16 crustacyanin subunits or 8 p-crustacyanins. [0090] 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. The term "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. The enzyme to perform the actual synthesis must also be present. After completion of in vitro transcription, the DNA template becomes a contaminant that must be removed, as do the enzymes that were added to promote synthesis. Any contaminants that were present in the DNA plasmid preparation or in the enzyme preparations must also be removed. 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. Such synthetic reaction systems are well-known in the art and have been described in the literature. The cell-free synthesis reaction may be performed as batch, continuous flow, or semi-continuous flow, as known in the art.

[0091] Single subunits may be produced individually and then combined together to form dimeric or multimeric crustacyanins as described herein. For example, 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.

[0092] In some examples, subunits may be recombinantly expressed in a single host organism and associated with each other within the host organism. For example, 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.

[0093] In some examples, multiple subunits may be expressed from a single expression vector. For example, the subunits may be expressed as fusion proteins as described herein.

[0094] In some examples, 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. In certain embodiments, 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. In some examples, 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. In a certain embodiment, 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.

[0095] In some examples, fusion proteins described herein may include at least one linker sequence. Suitable linkers for fusion proteins are known in the art. For example, 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.

[0096] The complexing agents described herein are provided in a food product bound to at least one astaxanthin molecule. For example, the complexing agent may include at least one crustacyanin subunit bound to an astaxanthin molecule.

[0097] The complexing agents may be produced and then isolated prior to being bound to astaxanthin that has been produced separately from the complexing agent. Alternatively, 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.

[0098] For example, 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.

[0099] In another example, 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.

[00100] In other examples, a host cell may recombinantly express a crustacyanin subunit, dimeric protein or multimer and also endogenously or recombinantly produce astaxanthin. For example, 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).

[00101] 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. For example, the host may be Escherichia coli, Lactococcus lactis, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, Chlorophyceae, Ulvophyceae, Florideophyceae, Alphaproteobacteria, Tremellomycetes, Labyrinthulomycetes, and Malacostraca.

[00102] The bathochromic shift of the bound astaxanthin may depend on the complexing agent to which it is bound. For example, astaxanthin bound to a single crustacyanin subunit may have a peak adsorption from about 550 nm to about 580 nm. For example, 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.

[00103] 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. In some examples, the complexed astaxanthin is applied to a surface of a formed food product. For example, sprayed, brushed or applied by dipping. In some examples, 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. In some examples, the complexed astaxanthin is mixed into a part of the food product. For example, mixed into a composition used to form the food product prior to or during the formation of the food product, thus distributing the complexed astaxanthin within an internal volume of the food product. For example, complexed astaxanthin may be mixed with a seafood analogue composition prior to forming the seafood analogue composition into a shaped food product. In some examples, the complexed astaxanthin may be applied to an outer surface of a food product and incorporated into an internal volume of the food product.

[00104] 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.

[00105] 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.

[00106] In some examples, the complexed astaxanthin is encapsulated in at least one biopolymer, such as an edible biopolymer. For example, the biopolymer may comprise starch, pectin, chitin, chitosan, alginate, silk, elastin, collagen, gelatin, hemicellulose, lignin, cellulose, carrageenan, or a mixture thereof. In some examples, the biopolymer may be an aliginate or collagen. In some examples, the complexed astaxanthin is encapsulated in an alginate. For 1 example, sodium alginate. As used herein, the term “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.

[00107] In some examples, the complexed astaxanthin is mixed with an encapsulation agent to form a composition comprising encapsulated complexed astaxanthin. Such 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.

[00108] Once the composition is applied to the outer surface of the product, 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.

[00109] In some examples, 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. In some examples, the film forming agent is at least one biopolymer as described herein, such as an edible biopolymer. For example, the film forming agent may comprise starch, pectin, chitin, chitosan, alginate, silk, elastin, collagen, gelatin, hemicellulose, lignin, cellulose, carrageenan, or a mixture thereof. In some examples, the film forming agent may be an aliginate or collagen. In some examples, the film forming agent may be an alginate. Such as sodium alginate. Subsequently, the film forming agent may be polymerized or crosslinked as described above in respect of encapsulating agents.

[00110] 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.

[00111] 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. For example, the complexed astaxanthin may be included in the food product in an amount of at least 0.001 mg. For example, at least 0.001 , at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.006, at least 0.007, at least 0.008, at least 0.009, at least 0.01 , at least 0.02, at least 0.03, at least 0.04, at least 0.05, at least 0.06, at least 0.07, at least 0.08, at least 0.09, at least 0.1 , at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 1 , at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10 mg.

[00112] In some examples, 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. For example, 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 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2 mg, 2.05 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3 mg, 3.05 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, or 4 mg of complexed astaxanthin.

[00113] 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). As such, 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.

[00114] Thus, provided herein is the use of exogenous astaxanthin to form a food product that undergoes a hypsochromic shift when heated. For example, the food product may change from a blue, purple, or slate-blue colour to orange, red or orangey-red colour when heated.

[00115] Therefore, there is also provided a method of altering one or more properties of a food product that includes complexed astaxanthin as described herein. The food product may be a seafood analogue as described herein. In some examples, the one or more properties includes an appearance of the food product and/or a nutritional property of the food product. For example, 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.

[00116] In some examples, 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. For example, 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.

[00117] Also provided herein are methods of producing a food product that includes the complexed astaxanthin 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.

[00118] For example, obtaining may include isolating complexed astaxanthin from an animal. For example, from lobster shell. For example, by using methods as described in Zagalsky, P. F. Invertebrate carotenoproteins. Methods Enzymol. 111 , 216-247 (1985) and Chen, H. et al. Purification and characterisation of two novel pigment proteins from the carapace of red swamp crayfish (Procambarus clarkii). Foods 11 , (2022).

[00119] Obtaining may include producing exogenous astaxanthin bound to one or more complexing agents by in vitro translation and transcription as described herein.

[00120] In some examples, 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. et al. In vivo production of a recombinant carotenoid-protein complex. (2016) and EP2977462A1. The complexing agents may then be combined with synthetically produced astaxanthin or recombinantly produced astaxanthin. Methods of producing astaxanthin, either synthetically or recombinantly, are described herein. Examples of such methods include those described in Zhao, X., Shi, F. & Zhan, W. Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae. Lett. Appl. Microbiol. 61 , 354-360 (2015), Ma, Y., Li, J., Huang, S. & Stephanopoulos, G. Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica. Metab. Eng. 68, 152-161 (2021), Jiang, G. et al. Enhanced astaxanthin production in yeast via combined mutagenesis and evolution. Biochem. Eng. J. 156, 107519 (2020), Zhou, P. et al. Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae. Enzyme Microb. Technol. 100, 28-36 (2017) and Park, S. Y., Binkley, R. M., Kim, W. J., Lee, M. H. & Lee, S. Y. Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metab. Eng. 49, 105-115 (2018).

[00121] The complexing agents and astaxanthin may then be reconstituted in vitro. For example, by missing the astaxanthin and complexing agent to form complexed astaxanthin.

[00122] In some examples, obtaining complexed astaxanthin may include producing the exogenous astaxanthin bound to one or more complexing agents in a host organism. For example, 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. For example, as described in Jiang, G. et al. Enhanced astaxanthin production in yeast via combined mutagenesis and evolution. Biochem. Eng. J. 156, 107519 (2020).

[00123] The obtained complexed astaxanthin may then be applied to the surface of a food product such as a seafood analogue as described herein.

[00124] In some examples, the obtained complexed astaxanthin may be added to and mixed with a food product formulation. For example, a seafood analogue formulation or composition prior to forming the composition into a food product.

FOOD PRODUCTS

[00125] Provided herein are 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).

[00126] 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. 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, hard cheese, dried milk powder, whey, butter, buttermilk, partly or wholly hydrolyzed products containing milk proteins, ice creams, frozen yogurts, novelty ice cream bars and sandwiches), non-dairy milk beverages (e.g., soy milk, nut milks, oat milk, baby formulas), products from soy protein or other soy bean fractions (e.g., soy milk and products prepared thereof, beverages containing isolated or enzymatically treated soy protein, soy flour containing beverages, preparations containing soy lecithin, fermented products such as tofu or tempeh products prepared thereof and mixtures with fruit preparations and, optionally, flavoring substances), fruit preparations (e.g., jams, fruit ice cream, fruit sauces, and fruit fillings), vegetable preparations, sauces and/or dressings (e.g., mayonnaise, remoulade, Hollandaise sauce, barbeque sauce, steak sauce, hot chili sauce, ketchup, mustard, or horseradish sauce), snack articles (e.g., baked or fried potato chips (crisps) or potato dough products, and extrudates on the basis of maize or peanuts), bread products (e.g., sliced bread, rolls, tortillas and muffins), ready-made meals, smoothies and soups (e.g., weight-loss/meal-replacement smoothies, nutritional protein drinks, dry soups, instant soups, and pre-cooked soups), processed meats (e.g., sliced deli meats, sausages, pates, canned meats), and/or nut butters (e.g., almond butter, peanut butter, soy nut butter, cashew butter, hazelnut butter).

[00127] In some examples, the food product is a meat alternative or meat analogue. The term “meat analogue” or “meat substitute”, “imitation meat”, and “meat alternative” are used interchangeably.

[00128] 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. The term refers to 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. shrimp, crayfish, lobsters and crabs); 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).

[00129] In some examples, 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.

[00130] In some examples, the food product is a seafood analogue or seafood alternative. In some examples, the seafood analogue is an analogue of an animal in the order Decapoda or Euphausiacea. For example, the seafood analogue may be a prawn, shrimp, crab, crayfish or lobster analogue. As described herein, 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.

[00131] A number of methods for making seafood analogues are known in the art. In some examples, 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. For example, see “Ng, Ee Theng, et al. "Cultured meat-a patentometric analysis." Critical Reviews in Food Science and Nutrition (2021): 1-11.” and the references included therein. For example, 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.

[00132] In some examples, the seafood analogue is a plant-based seafood analogue. For example, 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. In other examples, 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). In examples, 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. In some examples, 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. In other examples, 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.

[00133] 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. For example, 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.

[00134] Examples of plant-based seafood alternatives and methods of making such seafood alternatives can be found in US11241024B1 and W02022043067A1.

[00135] In some examples, 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. For example, as described in WO2021178254A1

[00136] 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.

[00137] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context they are used by those of skill in the art. [00138] The addition of astaxanthin to food products, such as seafood analogues, either in an uncooked product (i.e. complexed astaxanthin) or cooked product (i.e. unbound astaxanthin) may also have improved nutritional properties.

[00139] 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.

[00140] Astaxanthin may also help to activate white blood cells (T-cells) and natural killer (NK) cells, thereby providing immune system support.

[00141] 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.

[00142] 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.

[00143] 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.

[00144] Astaxanthin may also help to reduce LDL or bad cholesterol and can raise HDL or good cholesterol.

CLAUSES

1. 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.

2. The food product of clause 1 , wherein 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.

3. The food product of clauses 1 or 2, wherein the complexing agent comprises at least one beta-CRCN.

4. The food product of any of clauses 1 to 3, wherein the complexing agent comprises at least one alpha-CRCN.

5. The food product according to any preceding clause, wherein 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.

6. The food product according to clause 5a, 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 food product of clause 6, wherein the surface film comprises at least one biopolymer. The food product according to any preceding clause, wherein the exogenous astaxanthin bound to one or more complexing agents is encapsulated. The food product of any preceding clause, wherein upon heating the exogenous astaxanthin is unbound from the one or more complexing agents and is hypsochromicly shifted in comparison to bound astaxanthin. The food product according to any preceding clause, wherein the astaxanthin and/or the one or more complexing agents have been obtained by recombinant techniques. The food product according to any preceding clause, wherein the food product is vegetarian and/or vegan. The food product according to any preceding clause, wherein the food product is a seafood analogue. The food product according to clause 12, wherein the seafood analogue is a plantbased seafood analogue. 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. The composition of clause 14, wherein the encapsulation agent comprises at least one biopolymer. The composition of clause 14 or 15, wherein the composition is a film. A method of altering one or more properties of a food product, the method 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. The method of clause 17, wherein 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; 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. The method of clause 19, wherein obtaining comprises: a. isolating the exogenous astaxanthin bound to one or more complexing agents from an animal; b. producing exogenous astaxanthin bound to one or more complexing agents by in vitro translation and transcription; c. 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; d. recombinantly producing the exogenous astaxanthin bound to one or more complexing agents in a host organism. The method of clauses 19 or 20, wherein 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 method of any of clauses 19 to 21 , wherein 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 method of clause 22, wherein the film comprises at least one biopolymer. The composition of any of clauses 14 to 16 or the method according to any of clauses 17 to 23, wherein 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 composition of any of clauses 14 to 16 and 24 or the method according to any of clauses 17 to 24, wherein the complexing agent comprises at least one beta-CRCN. The composition of any of clauses 14 to 16, 24 and 25 or the method according to any of clauses 17 to 25, wherein the complexing agent comprises at least one alpha- CRCN. 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. on an outer surface of the food product; and/or b. in an internal volume of the food product. The method according to clause 27a, 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 method according to clause 28, wherein the surface film comprises at least one biopolymer. The composition of any of clauses 14 to 16 and 24 to 26 or the method according to any of clauses 17 to 29, the exogenous astaxanthin bound to one or more complexing agents is encapsulated. The composition of any of clauses 14 to 16, 24 to 26 and 30 or the method according to any of clauses 17 to 30, wherein upon heating the exogenous astaxanthin is unbound from the one or more complexing agents and is hypsochromicly shifted in comparison to bound astaxanthin. The composition of any of clauses 14 to 16, 24 to 26 and 30 to 31 or the method according to any of clauses 17 to 18 and 24 to 31 , wherein the astaxanthin and/or the one or more complexing agents have been obtained by recombinant techniques. The composition of any of clauses 14 to 16, 24 to 26 and 30 to 32 or the method according to any of clauses 17 to 18 and 24 to 32, wherein the food product is vegetarian and/or vegan. The composition of any of clauses 14 to 16, 24 to 26 and 30 to 33 or the method according to any of clauses 17 to 18 and 24 to 33, wherein the food product is a seafood analogue. The composition or method according to clause 34, wherein the seafood analogue is a plant-based seafood analogue. 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 use according to clause 37, wherein 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 use according to any of clauses 37 or 38, wherein the complexing agent comprises at least one beta-CRCN. The use according to any of clauses 37 to 39, wherein the complexing agent comprises at least one alpha-CRCN. The use according to any of clauses 37 to 40, wherein 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 use according to clause 42, wherein the surface film comprises at least one biopolymer. The use according to any of clauses 37 to 43, wherein the exogenous astaxanthin bound to one or more complexing agents is encapsulated. 45. The use according to any of clauses 37 to 44, wherein the astaxanthin and/or the one or more complexing agents have been obtained by recombinant techniques.

46. The use according to any of clauses 37 to 45, wherein the food product is vegetarian and/or vegan.

47. The use according to any of clauses 37 to 46, wherein the food product is a seafood analogue.

48. The use according to clause 47, wherein the seafood analogue is a plant-based seafood analogue.

[00145] Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

Example 1

[00146] a-CRCN or p-CRCN are isolated from animal sources, in this particular example from the lobster Homarus americanus using previously described techniques.

[00147] In short: 25 g lobster (Homarus americanus) shell was removed from a frozen, uncooked lobster. The shell was washed and dried and then frozen in liquid nitrogen. The frozen shell was crushed under liquid nitrogen using a motar and pestel. The ~ 1 cm shell pieces were subsequently ground to a fine powder using a Retsch Mill M200 with steel milling containers pre-cooled in liquid nitrogen(90 s milling at 30 Hz for each sample).

[00148] 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.

[00149] 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.

[00150] 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.

Example 3

[00152] a-CRCN was applied to a vegan prawn as described in Example 2.

[00153] After applying the solution to the surface, 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 4

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.

Example 5

All the described steps of Examples 2 to 4 can be carried out by using the precipitated protein in solid form.

Results - Examples 1 to 5

[00154] The colour change was observed during cooking and clearly visible once cooked. L*a*b* colour analytics were further carried using a handheld colorstriker instrument and confirmed the colour change.

TABLE 4 - Measured colour change

Table 4: colorstriker instrument measurements

[00155] Where L* indicates lightness, a* is the red/green coordinate, and b* is the yellow/blue coordinate. An increase in a* from 0.1 to 3.5 confirmed the visual increase in the red colour.

Example 6

[00156] Individual 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. 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.

[00157] Alternatively, the 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.

[00158] Examples of crustacyanin proteins are provided in Table 5

Table 5: Crustacyanin proteins

Example 7

[00159] 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. 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. Alternatively, 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.

Homarus qammarus CRCN A2-C1 translational fusion with 6x His tag

MDKIPDFVVPGKCASVDRNKLWAEQTPNRNSYAGVWYQFALTNNPYQLIEKCVRNEY SFD GKQFVIKSTGIAYDGNLLKRNGKLYPNPFGEPHLSIDYENSFAAPLVILETDYSNYACLY SCI DYNFGYHSDFSFIFSRSANLADQYVKKCEAAFKNINVDTTRFVKTVQGSSCPYDTQKTLG G SSGDGIPSFVTAGKCASVANQDNFDLRRYAGRWYQTHIIENAYQPVTRCIHSNYEYSTND Y GFKVTTAGFNPNDEYLKIDFKVYPTKEFPAAHMLIDAPSVFAAPYEVIETDYETYSCVYS CIT TDNYKSEFAFVFSRTPQTSGPAVEKTAAVFNKNGVEFSKFVPVSHTAECVYRAAAALEHH H HHH (SEQ ID NO: 47)

Example 8

[00160] 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. The protein sequences used in this example are identical to the one shown in Example 6. Alternatively, 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.

[00161] The proteins are then purified and combined with commercially available astaxanthin in order to reconstitute a-CRCN and or p-CRCN complexes containing only one type of CRCN subunits. The resulting complexes will be incorporated in alternative seafood as outlined in Examples 2 to 5.

Example 8

[00162] 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.

Example 9

[00163] The recombinant expression of 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.

[00164] For implementation of the astaxanthin biosynthesis, typically the following enzymes need to be overexpressed ⁷ :

- crtB (phytoene synthase)

- crtY (lycopene cyclase)

- crtE (geranylgeranyl diphosphate synthase)

- crtl (phytoene dehydrogenase/phytoene desaturase)

- crtZ (P-carotene hydroxylase)

- crtW (P-carotene ketolase)

[00165] For each of the enzymes above, multiple source organisms are available.

[00166] Additional modifications within the host genome may be required to enhance the astaxanthin yield.

[00167] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[00168] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[00169] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[00170] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Sequences

References

1. Van Wijk, A. A. C. et al. Spectroscopy and quantum chemical modeling reveal a predominant contribution of excitonic interactions to the bathochromic shift in a-crustacyanin, the blue carotenoprotein in the carapace of the lobster Homarus gammarus. J. Am. Chem. Soc. 127, 1438-1445 (2005).

2. Zagalsky, P. F. [9] Invertebrate carotenoproteins. Methods Enzymol. 111 , 216-247 (1985). 3. Helliwell, J. R. The structural chemistry and structural biology of colouration in marine Crustacea, in Crystallography Reviews vol. 16231-242 (Taylor & Francis Group, 2010).

4. 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).

5. 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).

6. Bourcier, C. C. et al. In vivo production of a recombinant carotenoid-protein complex. (2016).

7. Jiang, G. et al. Enhanced astaxanthin production in yeast via combined mutagenesis and evolution. Biochem. Eng. J. 156, 107519 (2020).