Composition, preparation and use

Abstract

There is described a composition for use as a foodstuff colouring agent. The composition comprises a glycoprotein, wherein the glycoprotein comprises a phycocyanin and a pectin; wherein the phycocyanin is glycated by the pectin. Also described is a method for preparing the composition for use as a colouring agent. There is also described a foodstuff or a foodstuff colouring agent comprising the composition, or prepared by the method for preparing the composition; and the use of the composition, the foodstuff colouring agent, or composition prepared by the method for preparing the composition, as a foodstuff colouring agent.

Description

[0001] COMPOSITION, PREPARATION AND USE

[0002] Field of Invention

[0003] The present invention relates to a composition for use as a colouring agent and a method for preparing a composition for use as a colouring agent.

[0004] Background

[0005] Spirulina (Arthrospira platensis and A. maxima), and other microalga are rich sources of pigments, proteins and other valuable and bioactive components, such as phycocyanins. Phycocyanins are pigmented biliproteins, protein-pigment complex chromophores produced in prokaryotic cyanobacteria as well as certain eukaryotes. C-phycocyanin, also referred to as phycocyanin C and C-PC, is part of the light-harvesting phycobiliprotein family, found in cyanobacteria as an accessory pigment to chlorophyll. It is a bright blue protein-pigment complex that is used as a blue colouring agent in foods and / or beverages, and the like, acting as a natural replacement for artificial colourants such as Brilliant Blue FCF (that has been associated with health problems).

[0006] Cyanobacteria are used in aquaculture to produce phycocyanins (including C-phycocyanin), most commonly using Spirulina (Arthrospira platensis and A. maxima), a non-toxic, edible, filamentous cyanobacteria with spiral-shaped filaments or trichomes (although other genera such as Aphanizomenon and Anabaena are used). Like other phycobiliproteins, C-phycocyanin is highly water soluble, making it relatively easy to extract from cyanobactenal cell. Also, around 7 to 20% dry weight phycocyanin can accumulate in Spirulina, making it ideal for commercial production of phycocyanin as a food and / or beverage ingredient. Whilst they are currently used as a natural, non-toxic, colouring agents, there are limitations to the use of phycocyanins. For example, C- phycocyanin tends to degrade on exposure to heat, light and / or a certain pH, leading to a loss of blue colour. In particular, it is reported that there is a 50% loss of tertiary structure in C-phycocyanin after one minute of exposure to 65 °C, resulting in significant colour loss / change, and / or aggregation (and subsequent precipitation). This means the C- phycocyanin performs poorly with thermal processes, such as pasteurisation, baking or sugar cooking in confectionery products. Therefore, C-phycocyanin cannot be used as a blue colouring agent in some applications that require the application of heat, light and / or a certain pH during processing. As the production of many foods and / or beverages, and the like requires the application of heat, the uses of C-phycocyanin in these industries are limited. Improving the heat stability of C-phycocyanin would enable use of the protein as an ingredient in a broader range of products, helping food and beverage manufacturers transition away from artificial colourants to natural alternatives.

[0007] Therefore, there is a need for a blue colouring agent that can be safely used (i.e., is non-toxic to and / or has no negative health implications for humans) in foods and / or beverages, and the like. In particular, there is a need for naturally occurring, non-toxic blue colouring agents, that on heating can retain their blue colour without notable degradation. Specifically, it is observed that there is a lack of C-phycocyanin colouring agents that are heat stable (i.e., that can retain their blue colouration without notable degradation on heating).

[0008] Therefore, it is an object of the present invention to obviate or mitigate at least some of the disadvantages of the prior art. A further object of the invention is to provide a composition that comprises a phycocyanin colouring agent, that is stable, and that retains its colour with little to no degradation at elevated temperatures, for example, those temperatures used in the preparation of some foods and / or beverages, and the like. A still further object of the invention is to provide a composition that comprises a C-phycocyanin colouring agent, that is stable, and that retains its blue colour with little to no degradation at elevated temperatures, for example, those temperatures used in the preparation of some foods and / or beverages, and the like.

[0009] Definitions

[0010] By the term “foodstuff(s)” it is meant substances that are used as foods or beverages and / or to make foods or beverages, intended for human consumption.

[0011] By the term “colouring agent(s)” it is meant a colour or colouring additive, or a colour or colouring foodstuff(s), safe for use in foodstuff(s). For example, but not limited to, the Spirulina extract as referred to in the Code of Federal Regulations, Title 21 , Chapter I, Subchapter A, Part 73, Subpart A, Section 73.530.

[0012] By the term “pectin” it is meant a polysaccharide comprising a-1 ,4-linked D-galacturonic acid (GalA), and variety of neutral sugars like arabinose, galactose, rhamnose and lesser amounts of other sugars. The a-1 ,4- linked D-galacturonic acid can be acetylated and methyl esterified.

[0013] Pectins have a linear anionic backbone with regions having no side chains known as “smooth regions” and regions with non-ionic side chains known as “hairy regions”. Structural classes of pectin include the following: • Homogalacturonan (HG): This is the major type of pectin in cell walls of plants having 65% of pectin. It is partially methyl esterified at C-6 and O-acetylated at 0-2 or 0-3.

[0014] • Rhamnogalacturonan I (RG-I): This accounts for about 20 to 35% of pectin and has a more complex structure than HG. It has up to 100 repeating units of (1 ,2)-a-L-rhamnose-(1 ,4)-a-D-galacturonic acid.

[0015] A large amount of the rhamnose sugar is substituted at 0-4 by neutral side chains including arabinogalactan I and II, arabinan and galactan, among which galactan and arabinan are more abundant.

[0016] • Rhamnogalacturonan II (RG-II): This contains about 10% of pectin and is a more structurally complex. Highly branched component.

[0017] The main types of pectin are described in Noreen et al. Pectins functionalized biomaterials; a new viable approach for biomedical applications: A review. Int. J. Biol. Macromol. 2017;101 :254-272.

[0018] The degree of esterification (DE) of pectin, also sometimes referred to as degree of methylation (DM), is expressed as a ratio of the number of methyl-esterified carboxyl groups to the total amount of galacturonic acid units, and is often used to classify the different types of pectin.

[0019] By “high-methoxy” (HM), also referred to as “high-methoxyl” and “high- ester” pectin, it is meant pectin having a DE (or DM) greater than approx. 50%.

[0020] By the term “amidated pectin” it is meant pectin produced industrially synthesized through the reaction of ammonia with carboxymethyl groups (-COOCH3) on the pectin molecule. The degree of amidation (DA) is defined as the percentage of carboxylic acid groups of pectin present in amide form. By the term “glycation” it is meant the non-enzymatic reaction of a carbonyl group of a carbohydrate (including sugars and / or polysaccharides) with an amino group of a protein; and by the term “glycated” it is meant a compound that has undergone glycation (typically, “glycated” is used to refer to a protein that has undergone glycation with a carbohydrate).

[0021] By the term “glycoprotein” it is meant a compound containing a protein that is covalently bonded to a carbohydrate (including sugars and / or polysaccharides), typically where the carbonyl group of the carbohydrate is covalently bonded to the protein at an amino group.

[0022] Statements of Invention

[0023] According to a first aspect of the invention there is provided a composition for use as a foodstuff colouring agent, the composition comprising a glycoprotein, wherein the glycoprotein comprises: a phycocyanin; and a pectin; wherein the phycocyanin is glycated by the pectin.

[0024] At least part of the phycocyanin is glycated by the pectin.

[0025] Optionally, the glycation of the phycocyanin by the pectin is effected by applying heat, optionally wherein the heat applied is from approximately 95 to approximately 105 °C, optionally from approximately 98 to approximately 102 °C, optionally at approximately 100 °C. The heat applied is typically at approximately 100 °C. The phycocyanin may be present in an amount of from approximately 17.7 to approximately 28.6 %w / w, optionally in an amount of from approximately 24 to approximately 27 %w / w. The phycocyanin may be present in an amount of from approximately 24 to approximately 28 %w / w, optionally in an amount of from approximately 24 to approximately 27.1 %w / w. The phycocyanin may typically be present in an amount from approximately 27 to approximately 28 %w / w.

[0026] The pectin may be present in an amount of from approximately 34.3 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

[0027] The pectin may be present in an amount of from approximately 34.2 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

[0028] The pectin may be present in an amount of from approximately 34.2 to approximately 46.8 %w / w, optionally in an amount of from approximately

[0029] 34.2 to approximately 45.6 %w / w.

[0030] The pectin may be present in an amount of from approximately 34.3 to approximately 46.8 %w / w, optionally in an amount of from approximately

[0031] 34.3 to approximately 45.6 %w / w.

[0032] The pectin may be present in an amount of from approximately 43.8 to approximately 58.4 %w / w.

[0033] The phycocyanin may be C-phycocyanin. The pectin may be high-methoxy pectin, wherein the high-methoxy pectin has a degree of methylation of greater than approximately 50%, optionally at least approximately 69%, optionally at least approximately 70%, optionally from approximately 62 to approximately 75%, optionally from approximately 65 to approximately 75%, optionally from approximately 69 to approximately 75%, optionally from approximately 70 to approximately 75%.

[0034] The high-methoxy pectin may have a degree of methylation of at least approximately 70%. The high methoxy-pectin may have a degree of methylation of from approximately 62 to approximately 75%. The high methoxy-pectin may have a degree of methylation of from approximately 69 to approximately 75%.

[0035] The pectin may comprise at least 65 %w / w galacturonic acid.

[0036] The pectin may comprise a molecular weight of from approximately 60,000 to approximately 130,000 Da, optionally approximately 90,000 Da.

[0037] The pH of the composition on reconstitution may be from approximately 4 to approximately 5, optionally from approximately 4 to approximately 4.5, optionally from approximately 4.2 to approximately 4.4.

[0038] The composition may further comprises a non-polysaccharide sugar, optionally wherein the phycocyanin is glycated by the non-polysaccharide sugar, optionally wherein the non-polysaccharide sugar is a reducing monosaccharide, optionally dextrose, optionally wherein the non- polysaccharide sugar is present in an amount of from approximately 11.4 to approximately 29.2 %w / w, optionally from approximately 11 .5 to approximately 25.5 %w / w. The non-polysaccharide sugar may be present in an amount of from approximately 11 .4 to approximately 23.4 %w / w, optionally from approximately 11 .4 to approximately 22.8 %w / w.

[0039] The non-polysaccharide sugar may be present in an amount of from approximately 14.6 to approximately 29.2 %w / w.

[0040] The non-polysaccharide sugar may typically be dextrose.

[0041] Non-polysaccharide sugars are monosaccharides, reducing monosaccharides, disaccharides and / or oligosaccharides.

[0042] The weight ratio of phycocyanin to the pectin and dextrose may be from approximately 1 :2 to approximately 1 :4.

[0043] The composition may further comprise at least one of a buffer and a base, optionally a phosphate salt, optionally tripotassium phosphate, optionally wherein the at least one of a buffer and a base is present in an amount of from greater than 0 to approximately 1 %w / w. The at least one of a buffer and a base may typically be present in an amount of approximately 1 %w / w.

[0044] The base may typically be tripotassium phosphate, optionally wherein the tripotassium phosphate is present in an amount of approximately 1 %w / w.

[0045] The composition may further comprise other algal constituents, optionally wherein the other algal constituents are present in an amount of from approximately 8.8 to approximately 14.3 %w / w, optionally from approximately 12 to approximately 13.5 %w / w. The other algal constituents may be present in an amount of from approximately 13.3 to approximately 14 %w / w, optionally from approximately 13 to approximately 14 %w / w. The other algal constituents may be non-C-phycocyanin proteins. These are non-phycocyanin proteins extracted from Spirulina. That is, Spirulina proteins that are not phycocyanin and that are not C- phycocyanin.

[0046] The composition may comprise a glycoprotein, wherein the glycoprotein comprises: a phycocyanin; a pectin; and a non-polysaccharide sugar, wherein the phycocyanin is glycated by the pectin and / or the non- polysaccharide sugar.

[0047] The composition may comprise a glycoprotein, wherein the glycoprotein comprises: a phycocyanin; a pectin; and a non-polysaccharide sugar, wherein the phycocyanin is glycated by the pectin and / or the non- polysaccharide sugar, and wherein the pectin comprises at least 65 %w / w galacturonic acid.

[0048] The composition may comprise a glycoprotein, wherein the glycoprotein comprises: approximately 17.7 to approximately 28.6 %w / w C- phycocyanin; approximately 34.2 to approximately 58.4 %w / w pectin; and approximately 11 .4 to approximately 29.2 %w / w dextrose, wherein the C-phycocyanin is glycated by the pectin and / or the dextrose, and wherein the pectin comprises at least 65 %w / w galacturonic acid.

[0049] According to a second aspect of the invention there is provided a method for preparing a composition for use as a foodstuff colouring agent, the method comprising the steps of: adding a phycocyanin to a mixing vessel; adding a pectin to the mixing vessel; forming a mixture comprising the phycocyanin and the pectin; glycating the mixture comprising the phycocyanin and the pectin to form a glycoprotein comprising the phycocyanin glycated by the pectin.

[0050] The glycation may be carried out at a temperature of from approximately 95 to approximately 105 °C, optionally from approximately 98 to approximately 102 °C, optionally at approximately 100 °C. The glycation may typically be carried out at a temperature of approximately 100 °C.

[0051] The glycation may be carried out for at least approximately 60 minutes, optionally from approximately 50 to approximately 90 minutes, optionally from approximately 50 to approximately 80 minutes, optionally from approximately 50 to approximately 70 minutes, optionally from approximately 55 to approximately 90 minutes, optionally from approximately 55 to approximately 80 minutes, optionally from approximately 55 to approximately 70 minutes, optionally from approximately 60 to approximately 90 minutes, optionally from approximately 60 to approximately 80 minutes, optionally from approximately 60 to approximately 70 minutes. The glycation may typically be carried out for at least approximately 60 minutes. The glycation may be carried out at a relative humidity of from greater than 0 to approximately 20%, optionally at a relative humidity of from greater than 0 to approximately 10%.

[0052] The glycation may typically be carried out at a relative humidity of from greater than 0 to approximately 10%.

[0053] The method may comprise the further step of adjusting the pH of the phycocyanin in the mixing vessel to approximately 6 before the addition of the pectin, optionally wherein the pH adjustment is by the addition of at least one of a buffer and a base, optionally a phosphate salt, optionally tripotassium phosphate, optionally wherein the at least one of a buffer and a base is present in an amount of from greater than 0 to approximately 1 %w / w. The at least one of a buffer and a base may typically be present in an amount of approximately 1 %w / w.

[0054] The base may typically be tripotassium phosphate, optionally wherein the tripotassium phosphate is present in an amount of approximately 1 %w / w.

[0055] The method may comprise the further step of homogenising the mixture comprising the phycocyanin and the pectin before glycation, optionally wherein the homogenisation is by high shear mixing.

[0056] The method may comprise the further step of drying the mixture comprising the phycocyanin and the pectin before glycation, and optionally after homogenisation, optionally wherein the drying is by way of at least one of spray drying and lyophilisation. The phycocyanin may be present in an amount of from approximately 17.7 to approximately 28.6 %w / w, optionally in an amount of from approximately 24 to approximately 27 %w / w. The phycocyanin may be present in an amount of from approximately 24 to approximately 28 %w / w, optionally in an amount of from approximately 24 to approximately 27.1 %w / w. The phycocyanin may typically be present in an amount from approximately 27 to approximately 28 %w / w.

[0057] The pectin may be present in an amount of from approximately 34.3 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

[0058] The pectin may be present in an amount of from approximately 34.2 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

[0059] The pectin may be present in an amount of from approximately 34.2 to approximately 46.8 %w / w, optionally in an amount of from approximately

[0060] 34.2 to approximately 45.6 %w / w.

[0061] The pectin may be present in an amount of from approximately 34.3 to approximately 46.8 %w / w, optionally in an amount of from approximately

[0062] 34.3 to approximately 45.6 %w / w.

[0063] The pectin may be present in an amount of from approximately 43.8 to approximately 58.4 %w / w.

[0064] The phycocyanin may be C-phycocyanin. The pectin may be high-methoxy pectin, wherein the high-methoxy pectin has a degree of methylation of greater than approximately 50%, optionally at least approximately 69%, optionally at least approximately 70%, optionally from approximately 62 to approximately 75%, optionally from approximately 65 to approximately 75%, optionally from approximately 69 to approximately 75%, optionally from approximately 70 to approximately 75%.

[0065] The pectin may comprise at least 65 %w / w galacturonic acid.

[0066] The pH of the mixture comprising the phycocyanin and the pectin after addition of the pectin may be from approximately 4 to approximately 5, optionally from approximately 4 to approximately 4.5, optionally from approximately 4.2 to approximately 4.4.

[0067] The pH of the composition after glycation may be from approximately 4 to approximately 5, optionally from approximately 4 to approximately 4.5, optionally from approximately 4.2 to approximately 4.4.

[0068] The method may further comprise the step of addition of a nonpolysaccharide sugar, optionally in a mixture with the pectin, optionally wherein the phycocyanin is glycated by the non-polysaccharide sugar, optionally wherein the non-polysaccharide sugar is a monosaccharide, optionally dextrose, optionally wherein the non-polysaccharide sugar is present in an amount of from approximately 11.4 to approximately 29.2 %w / w, optionally from approximately 11 .5 to approximately 25.5 %w / w.

[0069] The non-polysaccharide sugar may be present in an amount of from approximately 11 .4 to approximately 23.4 %w / w, optionally from approximately 11 .4 to approximately 22.8 %w / w. The non-polysaccharide sugar may be present in an amount of from approximately 14.6 to approximately 29.2 %w / w.

[0070] The non-polysaccharide sugar may typically be dextrose.

[0071] Non-polysaccharide sugars are monosaccharides, reducing monosaccharides, disaccharides and / or oligosaccharides.

[0072] The method may comprise the steps of: adding a phycocyanin to a mixing vessel; adding a pectin and a non-polysaccharide sugar to the mixing vessel; forming a mixture comprising the phycocyanin, the pectin and the non-polysaccharide sugar; glycating the mixture comprising the phycocyanin, the pectin and the non-polysaccharide sugar to form a glycoprotein comprising the phycocyanin glycated by the pectin and / or the non-polysaccharide sugar.

[0073] The method may comprise the steps of: adding a phycocyanin to a mixing vessel; adding a pectin and a non-polysaccharide sugar to the mixing vessel; forming a mixture comprising the phycocyanin, the pectin and the non-polysaccharide sugar; glycating the mixture comprising the phycocyanin, the pectin and the non-polysaccharide sugar to form a glycoprotein comprising the phycocyanin glycated by the pectin and / or the non-polysaccharide sugar, wherein the pectin comprises at least 65 %w / w galacturonic acid, wherein the non-polysaccharide sugar is dextrose, and wherein the glycation is carried out for at least approximately 60 minutes.

[0074] The method may comprise the composition for use as a foodstuff colouring agent is as described in the first aspect.

[0075] According to a third aspect of the invention, there is provided a composition prepared from the method of the second aspect.

[0076] The composition may be for use as a foodstuff colouring agent.

[0077] According to a fourth aspect of the invention there is provided a foodstuff or a foodstuff colouring agent comprising the composition of the first aspect, or the composition of the third aspect, or prepared by the method of the second aspect.

[0078] According to a fifth aspect of the invention there is provided the use of the composition of the first aspect, the composition of the third aspect, the foodstuff colouring agent of the fourth aspect, or a composition prepared by the method of the second aspect, as a foodstuff colouring agent.

[0079] According to a sixth aspect of the invention, there is provided a composition for use as a foodstuff colouring agent, the composition comprising a glycated phycocyanin, wherein the phycocyanin is glycated by pectin.

[0080] The composition of the sixth aspect contains all the features of the preceding aspects including, but not limited to, the first aspect. According to a seventh aspect of the invention, there is provided a compound for use as a foodstuff colouring agent, the compound comprising a glycated phycocyanin, wherein the phycocyanin is glycated by pectin.

[0081] The alternative features and different embodiments as described apply to each and every aspect and each and every embodiment thereof mutatis mutandis. For example, (and without exclusion of the other aspects) the alternative features and different embodiments as described for the first aspect apply to the second aspect (and vice versa), and the alternative features and different embodiments as described for the first and second aspects apply to the sixth and seventh aspects, mutatis mutandis.

[0082] Brief Description of the Figures

[0083] Embodiments of the invention will now be described, by way of example, with reference to the drawings, in which:

[0084] Fig.1 is an SDS PAGE of the C-phycocyanin glycoprotein (glycated by pectin) or the present invention against non-glycated C-phycocyanin.

[0085] Fig. 2 is a series of test tubes containing different C-phycocyanin- pectin compositions before glycation (top rows) and after glycation (bottom rows) and the differences in heat stability 24 hours after (image A) and 2 weeks after (image B) boiling for 30 seconds.

[0086] Detailed Description

[0087] Cultivation of Spirulina

[0088] Spirulina was cultured as described in WO2015110844, the method disclosed in which is further outlined below.

[0089] F / 2 sterile medium (CCAP [Culture Collection of Algae and Protozoa] recipe) supplemented with 2.5 g / 1 NaNOs (pH 8) was inoculated under aseptic conditions at 20 % (v / v) with Arthrospira platensis (CCMP [Culture Collection of Marine Phytoplankton] 1295 / Bigelow Laboratory US) (OD 0.11-0.12) in logarithmic growth phase. A stirred tank photobioreactor (STPBR) (Infers Labfors 4 benchtop modified bioreactor) with 680 nm red LEDs was operated with 2.75 litres of culture at 30 °C and 45 pmo m-2light intensity with 18:6 light: dark cycle and impeller speed 200 rpm with natural compressed air (approx. 0.03 % CO2) supplied at 0.08 LPM (WM (volume of air per volume of culture per minute) approx. 0.03 litres air per litres medium per minute, LPM) through a gauzed ring sparger. pH and dissolved oxygen were recorded online in 10 minute periods (Mettler Toledo probes). This process was continued until culture density reached approx. 6 to 8 g dry algal biomass (DAB) per litre, while ensuring the C- phycocyanin composition of the biomass was > 7% (dry weight). The culture was then diverted for harvesting.

[0090] Harvesting of Spirulina Biomass

[0091] Spirulina culture was fed from the STPBR to a 10,000 L AISI™ 316 stainless steel vented harvest vessel. The culture was gently agitated in the vessel prior to transfer via a pump to either a centrifugal separator (Alpha Laval™ LAPX high speed separator) or vibratory sieve (Cuccolini™ VLB 800), for separation of algal biomass. Depending on the method used, a wet algal biomass (WAB) paste of between approx. 10 to approx. 25% dry weight was generated for further processing. This can be used directly for extraction of C-phycocyanin, or it can be frozen in food grade tubs or bags for future use. The spent culture media can be returned to the STPBR system for reuse (> 50% reuse), to improve circularity and minimise nutrient wastage.

[0092] Downstream Processing of Spirulina Biomass for C-phycocyanin Extract Wet algal biomass (153 kg) was fed / pumped into a 900 L AISI™ 316 stainless steel, vented extraction tank along with 3.43 g / L potassium chloride (KCI) buffer to achieve a final dry weight content of between approx. 1 and approx. 2% (typically 1 .7%). The WAB - KCI buffer mixture was well agitated to ensure a homogenous solution. This mixture was then transferred to a high-pressure homogeniser (GEA Niro Soavi™ NS3024P), whereby cells were lysed at 400 bar.

[0093] The lysate was transferred at 1 ,200 L / H to a 1 ,600 L to a jacketed, AISI™ 316 stainless steel, vented mixing vessel (agitated at 400 rpm), and agitated for 15 minutes. Separation of C-phycocyanin extract from other proteins / cellular debris (“spent biomass”) can be aided using a sedimentation tank or a separator. In the present process, separation was expedited using a disc stack centrifuge separator, the residence time being optimised to improve the clarity of the extract, both in terms of maximising C-phycocyanin content (measured spectrophotometrically (i.e. Yoshikawa & Belay, 2008)), reducing spent biomass content (determined spectrophotometrically by the greenness of the extract (higher a* values in the CIELAB colour space), and minimisation of the allophycocyanin content. The purity of the C-phycocyanin extract, based upon the ratio of absorbance 620 nm / 280 nm should be greater than 0.8.

[0094] The C-phycocyanin extract (700 - 850 L) was transferred to a jacketed, 900 L AISI™ 316 stainless steel, vented mixing vessel for basification to between pH approx. 5.8 and approx. 6.2 using concentrated tripotassium phosphate (approx. 10 to approx. 40 % (w / v)) and was agitated well for 30 minutes. The solution was basified prior to depth filtration to prevent retention (and subsequent clogging) of C-phycocyanin on the depth filtration filters. A pH of above 5.8 promotes C-phycocyanin solubility and aids its passage through the depth filters. After basification, the C- phycocyanin was passed through a plate heat exchanger to reduce temperature of the extract to approx. 2 to approx. 5 °C to minimise proliferation of food spoilage organisms. The chilled extract was clarified using depth filtration with sanitised filter sheets or lenticular filters (pore size < 1 pm) such as Seitz™ K-series filters made from cellulose fibres, diatomaceous earth and kieselguhr. The purpose of the depth filtration step is twofold; firstly, it significantly reduces the bioburden of extract, including removal of indicator food pathogens and spoilages organisms (i.e. yeasts, moulds, E. coli, Coliforms, staphylococcus). Secondly, it further clarifies the extract by removing small cell fragments and solids suspended in extract. Chilled, microfiltered extract is then stored at approx. 2 to approx. 5 °C for up to 18 hours prior to concentration and diafiltration.

[0095] The microfiltered extract was then concentrated using crossflow (tangential) ultrafiltration. The microfiltered extract (700 - 850 L) was circulated (1 ,500 - 1 ,800 L / H) through a crossflow system fitted with polyethersulfone membranes with 100 kDa molecular weight cut-off (MWCO) pores. This pore sizes allows the retention of C-phycocyanin in the retentate, while smaller molecules and compounds (e.g., smaller proteins, peptides, salts etc.) pass through the membranes into the permeate effluent. The retentate was circulated until the desired C- phycocyanin concentration was achieved, typically 25 - 35 g / L of C- phycocyanin, while maintaining transmembrane pressure between approx. 3.5 and approx. 4.5 bar, and the temperature of the retentate at approx. 2 to approx. 5 °C. The purity of the concentrate should be greater than 3 at this stage. Once the desired C-phycocyanin concentration was achieved, diafiltration of the C-phycocyanin concentrate commenced. A buffer (i.e. 0.1 M citrate buffer, pH 4.5), or sterile process water, was fed into a 1 ,000 L AISI™ 316 stainless steel crossflow buffer tank at a rate equal to the flow of permeate, hence retaining the total volume of the extract (comprising the C-phycocyanin, and also referred to as the retentate) at a steady state. Remaining salts were washed out of the C-phycocyanin concentrate, resulting in a purified liquid consisting of about 25 - 35 g / L C-phycocyanin and 10 - 20 g / L of non-C-phycocyanin Spirulina compounds (typically other proteins). Diafiltration was continued until approximately 5 volumes of buffer / water (5 times the remaining volume of C-phycocyanin concentrate) has been added, or the total dissolved solids of the permeate (ppm) reached equilibrium. The purified, concentrated C-phycocyanin extract had a purity of greater than 4.2, and was suitable for use in preparing a C-phycocyanin composition.

[0096] Preparation of C-phycocyanin-Pectin Concentrate

[0097] A mixture comprising C-phycocyanin and pectin was prepared using a high-ester (also referred to as high-methoxy or high-methoxyl) pectin (E440(i)) (which can be extracted from citrus peels) and standardised (i.e. , mixed) with dextrose (pectin and dextrose mixture obtained from Healan Ingredients™: Product Range HIP10 and Product Code PEC010). HIP10 is from approximately 60% to approximately 80% w / w pectin and from approximately 20% to approximately 40% by weight dextrose. The amount of pectin and dextrose mixture added was based upon the final C- phycocyanin content of the concentrate. Typically, the C-phycocyanin content of the concentrate is approx. 20 to approx. 35 g C-phycocyanin per litre, and the total (dry weight) of pectin and dextrose mixture added is 2.2 times the total C-phycocyanin content (calculated as C-phycocyanin content in grams multiplied by the volume (in litres) of the concentrate). For example, if the C-phycocyanin content of the concentrate is 20 grams per litre and there is one litre present, then 44 grams of pectin and dextrose mixture (in this case, high-ester pectin (E440(i)) supplied as per above) is added. Note that high-ester pectin in accordance with E440(i) comprises at least 65 % by weight galacturonic acid on the ash-free and anhydrous basis after washing with acid and alcohol.

[0098] The pectin and dextrose mixture was added to the C-phycocyanin concentrate in one of two ways. In a first method, the pectin and dextrose mixture was made into a 20% (w / v) paste in 1 % tripotassium phosphate solution, using vigorous mixing with a high shear rotor-stator homogeniser, ensuring that the pectin and dextrose mixture was fully dispersed and that no clumps remained. The paste was introduced to the C-phycocyanin concentrate, and the paste and the so-formed mixture was well mixed using a mixer or high shear homogeniser. In a second method, the pectin and dextrose mixture as a powder was added directly to the C- phycocyanin concentrate, which was first basified with 1 % tripotassium phosphate, ensuring the starting pH of the C-phycocyanin concentrate was 6 before addition of the pectin and dextrose mixture. This helps prevent the pectin setting when introduced, as pectin is acidic and, when the pH falls below 4, the pectin is harder to dissolve, and the mixture becomes very viscous and hard to pump. When introducing the powder directly to the concentrate, high shear homogenisation is used to effectively disperse the powder and prevent clumping. Regardless of the method used, the so-formed mixture of C-phycocyanin, pectin and dextrose is ready to be further treated by drying. Note that mixture concentrate can also be frozen at this point prior to drying.

[0099] The C-phycocyanin:pectin and dextrose mixture ratios used above were between 1 :2 and 1 :4. Therefore, the so-formed mixture, on a dry weight basis, (i.e., not in solution or not including the weight of water when in solution) is approximately 17.7 to approximately 28.6 % w / w C- phycocyanin, approximately 8.8 to approximately 14.3 % w / w other algal constituents (typically non-C-phycocyanin proteins), approximately 57.1 to approximately 72.7 % w / w high-ester pectin and dextrose (from approximately 34.3 to approximately 58.4 % w / w pectin and from approximately 11 .4 to approximately 29.2 % w / w dextrose) and up to approximately 1 % (of total solids) tripotassium phosphate. A composition comprising approximately 24 to approximately 27 % w / w C-phycocyanin is found to be comparable to E180 food colourant, and may further comprise approximately 12 to approximately 13.5 % w / w other algal constituents (typically non-C-phycocyanin proteins), approximately 58.5 to approximately 63 % w / w high-ester pectin and dextrose (from approximately 35 to approximately 50.5 % w / w pectin and from approximately 11 .5 to approximately 25.5 % w / w dextrose) and up to approximately 1 % (of total solids) tripotassium phosphate.

[0100] In the above process, a high-ester pectin (E440(i)) standardised with dextrose (pectin obtained from Healan Ingredients™: Product Range HIP10 and Product Code PEC010) was used. Other sugars, polysaccharides, and pectins were also used as further described in the Examples.

[0101] Drying of C-phycocyanin-Pectin Concentrate

[0102] The C-phycocyanin and pectin mixture was dried into a fine powder by spray drying using a Bucchi™ B-290 spray dryer. The mixture was spray dried using the following parameters. The outlet temperature was kept under approx. 85 °C to prevent thermal degradation of the C-phycocyanin. The inlet temperature was typically maintained at approx. 170 to approx. 180 °C, with an air flow of approx. 550 to approx. 800 litres / hour, with a feed flow of approx. 15 to approx. 20 ml / min. The final moisture content of the dried powder was < approx. 5 % (w / w). A scrubber is typically attached to the dryer to improve product yield. The target colour of the powder was E180 or above, calculated spectrophotometrically by measuring the absorption of a 0.25% (w / v) solution of the powder at 618 nm in 0.1 M citrate buffer. The Evalue was calculated using the following formula:

[0103] Absoption 618 nm Evalue = - - - x 100

[0104] 0.25

[0105] As an alternative to spray drying, the C-phycocyanin and pectin mixture can be dried by lyophilisation (Harvest Right™ Large Pharmaceutical Dryer) and milling (using a coffee grinder purchased from Amazon™). In this method, the C- phycocyanin - pectin concentrate was loaded onto sanitised food trays and loaded into the freeze dryer. In the dryer, the product is first frozen at - 50 °C until completely frozen, before a vacuum was applied until the pressure in the dryer was < 200 millibar. The product was then held at this pressure until all the readily removable water was removed, leaving a freeze-dried cake. The freeze-dried cake was recovered, then milled to produce a fine powder using the coffee grinder.

[0106] Glycation of C-phycocyanin-Pectin Composition

[0107] The dried powder was weighed into 5 kg batches, packaged in heat-proof polyethylene terephthalate (PET) roasting bags or heat-proof aluminium pail liners. The bags were placed in a preheated oven, ensuring that all the powder was heated to approximately 100 ± 5 °C for at least 1 hour, which ensured glycation and a heat stabilised composition. Experiments (see Examples) have shown that heating the dried C-phycocyanin and pectin mixture for at least of 1 hour ensures that the pectin molecules glycate with the C-phycocyanin molecules, without excessive loss of blue colour. After heating, the packages were immediately sealed and cooled to ambient temperature (approximately 20 °C). The heat stabilised glycated C-phycocyanin was then ready to be used in dry form, or to be eluted in a sterile citrate buffer up to 15% (w / v), to create a ready to use solution for food and beverage manufacturers (i.e., for use in foodstuffs). In the presence of water (naturally contained in the C-phycocyanin and pectin mixture) the reactive carbonyl groups of pectin (e.g., the polysaccharide galacturonic acid), and / or dextrose sugars when present, bind with the amino acids via a glycation reaction, which is part of the Maillard reaction, forming a glycoprotein which is more heat stable. If the C-phycocyanin and pectin mixture is not heated sufficiently, the heat stability of the resultant composition is compromised, and turbidity and / or aggregation is seen when heating the composition in solution. While the C-phycocyanin and pectin mixture can be heated for longer, discolouration of the composition can occur, as well as caramelisation and formation of other side products, creating off-colours and flavours. The heating process also destroys spores and vegetative cells of potential food pathogens and spoilage organisms.

[0108] Examples

[0109] The following example compositions were prepared as described above, with any deviations from the methods described apparent from the context of the examples as described below.

[0110] Sugar Screening

[0111] A range of sugar types were screened for their suitability for glycating to C- phycocyanin. This included monosaccharides, disaccharides and polysaccharides. Both reducing and non-reducing sugars were selected, with non-reducing sugars unable to glycate with proteins. A range of ‘dry’ glycation conditions were also explored throughout the investigation, from cooler, 45 °C glycations (for 1 to 5 days) through to hotter incubations for shorter periods (for example, 130 °C for 30 minutes). All glycations were performed on lyophilised C-phycocyanin: sugar mixes (initially at a 1 :2 weight (i.e. , grams) ratio), in an 80% humidity environment. Conventional spray drying (inlet temp 180 to 190 °C) was used as an ultra-high temp / short time comparison for some sugars. Performance was determined by observing changes in powder colour after glycation, or if no colour changes were observed then the performance of the powder solution when exposed to heat challenges was determined. Key outcomes, for analysed powders, are shown in Tables 1Aand 1 B.

[0112] Table 1 A: Screening of sugars to improve heat stability of C-phycocyanin through dry glycation: types, characteristics and spray drying results. Table 1B: Screening of sugars to improve heat stability of C-phycocyanin through dry glycation: glycation results at different temperatures and times.

[0113] Colour deterioration was noted for several glycated powders, particularly mono or disaccharides. Ribose and lactose powders rapidly turned green (and insoluble), most likely through the Maillard chemical reaction, which can form a variety of intermediate products as described in Hemmler, D., et al. Evolution of Complex Maillard Chemical Reactions, Resolved in Time. Sci Rep 7, 3227 (2017). The darkening of the sucrose - C- phycocyanin powder is likely due to caramelisation, whereas the lightening of the pectin - C-phycocyanin powder at higher temperatures could be due to C-phycocyanin degradation, exacerbated by the low acidity caused by the pectin itself.

[0114] Powders which showed improved heat stability (when eluted in buffers or syrups and heated) included trehalose, kappa-carrageenan and pectin powders. The limited heat stability shown by trehalose - C-phycocyanin powders is likely an effect of steric hinderance of the trehalose molecules. The protective effect of kappa-carrageenan on C-phycocyanin could be due to electrostatic interactions between sulphate groups on the kappa- carrageenan molecules and C-phycocyanin amino acids, helping prevent aggregation when exposed to heat, as reported elsewhere.

[0115] The most promising performance was seen with pectin and C-phycocyanin when spray dried or glycated at approximately 100 °C ± °5 C. The mechanism proposed is glycation between the reactive carbonyl group of the sugar and the nucleophilic amino group of exposed amino acid on C- phycocyanin proteins to form glycoproteins. Glycation Optimisation

[0116] Glycation optimisation was performed on a spray dried pectin: C- phycocyanin powder, formulated with C-phycocyanin:HM pectin and dextrose mixture ratio of 1 .2.2, and spray dried on a Bucchi™ 290. After glycation, powders were dissolved in citrate buffer to give solutions with 618 nm absorbance = 2. Solutions were then heated at 80 °C and absorbance changes and 618 nm monitored every 10 mins. Glycation conditions and the results of the absorbance studies are shown in Table 2 below.

[0117] Table 2: Remaining blue intensity (at 618 nm) after 80 °C incubation of C- phycocyanin-pectin (HIP 24-01 - See Table 3A) powders made with different glycation conditions.

[0118] Powders with no glycation, or glycation temperatures < 80 °C showed turbidity and pigment precipitation soon after heating, whereas powders with glycation at 100 °C stayed clear, with 20% of pigment intensity retained, even after 60 mins heating. A 1 -hour glycation was sufficient to achieve the improvement in heat performance, with slightly more colour degradation observed with longer glycation periods.

[0119] SDS PAGE Study An SDS PAGE study was performed on a C-phycocyanin glycated using HIP 24-01 (see Table 3A), where the glycation had been performed for 1 hour at 100 °C. 50 or 100 mg of the glycated C-phycocyanin powder were dissolved in 5 ml of deionised water and were fully mixed. Additionally, 50 or 100 mg of ScotBio Blue™ P-TRE, a conventional C-phycocyanin powder, were also dissolved in 5 ml of deionised water. One hundred microlitres of each solution were heated to 85 °C for 2 minutes with 100 pl of Tricine SDS Sample Buffer (2X). Fifteen pl of each mixture were loaded in triplicate into a Novex™ WedgeWell™ 8 to 16%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 15-well, along with 15 pl of peqGOLD Protein Marker IV™ into wells next to samples. The gel was run at 200 V in a X-Cell Surelock Mini-cell™ electrophoresis system, with Tris-Glycine Running buffer dosed with NuPage ™ antioxidant. After 40 minutes, the gel was removed, washed thoroughly, and stained overnight in Coomassie® brilliant blue G-250 solution electrophoresis gel stain. The gel is shown in Figure 1 , with conventional C-phycocyanin shown in wells 1 - 3 and 7 — 9, and C-phycocyanin glycated with pectin (HIP 24-01) shown in wells 4 - 6 and 10 - 12. Wells 1 - 6 correspond to approximately 5 mg of C- phycocyanin per well, and wells 7 - 12 approximately 2.5 mg of C- phycocyanin per well. The image clearly shows the loss of characteristic C-phycocyanin sub-units (shown in wells 1 - 3, 7 - 9) between 15 - 35 kDa with a distinctive band (approximately 110 kDa) in wells 4-6 and 10 - 12, indicative of a C-phycocyanin-pectin glycoprotein.

[0120] Screening of Different Pectin Types

[0121] To test if the functionality observed in the previous pectin-C-phycocyanin heated powders was common to other pectins, several other commercially available pectins were obtained, including high and low-methoxyl pectins (E440(i)) (standardised (i.e. , mixed) with either dextrose or sucrose), and amidated Pectin (E440(ii)). Chemical data was obtained from the suppliers, where possible (see Table 3A).

[0122] Table 3A: Selected chemical properties of pectin containing compositions screened in study. All pectins have a polygalacturonic acid content of at least 65 % w / w.

[0123] In this instance, powders were made my combining a 15% (w / v) pectin paste with a C-phycocyanin concentrate, homogenising the mixture at 5,000 rpm for 5 minutes with a high shear mixer, then spray drying the mixture. The mixtures were formulated to contain C-phycocyanin: pectin and dextrose mixture weight (i.e. , grams) ratios of 1 :4. The resulting powder was collected, then a small 1 g portion was baked in an oven at approximately 100 °C ± °5 C for 1 hour on aluminium trays. The colour of the powders before and after baking was assessed by determining Evalue 618nm and CIELAB values of 0.25% solutions in citrate buffer. The buffer solutions were then boiled in test tubes for 30 seconds to identify differences in heat stability. The results are summarised in Tables 3B and 3C.

[0124] Table 3B: Colour changes (colour and hue) of powders in screening study before and after glycation at approximately 100 °C for approximately 1 hour.

[0125] Table 3C: Colour changes (CIELAB) of powders in screening study before and after glycation at approximately 100 °C for approximately 1 hour. Considerable differences were observed in the performance and processing characteristics of each pectin, although a significant improvement in heat stability was seen for all pectins after glycation as is illustrated in Figure 2. In Figure 2, there are two photographs (A and B) of a series of test tubes containing different C-phycocyanin-pectin compositions before glycation (top rows) and after glycation (bottom rows) and the differences in heat stability 24 hours after (image A) and 2 weeks after (image B) boiling for 30 seconds. As labelled in Figure 2, the test tubes are in groups of three for each pectin and in the following order: HIP 1 , HIP 2C, HIP 5, HIP 10, and HIP 43.

[0126] The amidated pectins (HIP 5 and HIP 43) showed some degree of heat stability prior to glycation, indicating that some glycation has occurred during the initial spray drying, however no additional improvement was achieved after glycation (i.e. , after heating at approximately 100 °C ±°5 C for at least approximately 1 hour). All HM pectin samples, standardised with dextrose, showed significant improvement in heat stability after glycation, with no aggregation or turbidity observed. HIP 10 was the best performing product, showing the least colour degradation after boiling. Furthermore, samples HIP 2C and HIP 10 retained their characteristic blue hue for over 2 weeks, whereas other samples had turned green.

[0127] Similar results were seen when repeated with another HM pectin sample (HIP 24-01), and when repeated with C-phycocyanin: pectin and dextrose mixture weight (i.e., grams) ratios anywhere in the region from 1 :2 to 1 :4. It was also demonstrated that pectin can be incorporated into C- phycocyanin concentrate as either a 10 - to 20% paste or mixed directly in powder form into the concentrate using a high shear mixing tool. Powder dissolution was improved by firstly basifying the C-phycocyanin concentrate to around pH 6 using 1 % tripotassium phosphate solution, achieving a final C-phycocyanin - pectin mixture of pH 4 to pH 5, typically, of pH 4 to pH 4.5, and in most cases to pH 4.2 to 4.4. It is found that a pH of 4.3 can be of benefit. Table 4 illustrates the range of relative amounts of components in the glycated C-phycocyanin composition and for three different pectin compositions (HIP 10, HIP 2C and HIP 24-01 ). Table 4: %w / w in glycated C-phycocyanin composition.

[0128] Table 5 shows some further examples of C-phycocyanin glycated by pectin. Table 5: Examples of C-phycocyanin glycated by pectin with Evalue.

[0129] Based on analysis of the examples presented above, it was found that the following ranges of components formed C-phycocyanin - pectin glycoproteins that showed heat stability: • approximately 17.7 to approximately 28.6 % w / w C-phycocyanin; and

[0130] • approximately 34.3 to approximately 58.4 % w / w high-ester pectin. Other components that may be present can include:

[0131] • approximately 8.8 to approximately 14.3 % w / w other algal constituents (typically non-C-phycocyanin proteins);

[0132] • approximately 11 .4 to approximately 29.2 % w / w dextrose; and

[0133] • up to approximately 1 % w / w tripotassium phosphate (acting as a buffer and / or a base).

[0134] The following ranges of components also formed C-phycocyanin - pectin glycoproteins that showed heat stability and improved retention of an intense blue colour and were comparable to E180 blue food colourants:

[0135] • approximately 24 to approximately 27 % w / w C-phycocyanin; and

[0136] • approximately 35 to approximately 50.5 % w / w high-ester pectin.

[0137] Other components that may be present can include:

[0138] • approximately 12 to approximately 13.5 % w / w other algal constituents (typically non-C-phycocyanin proteins);

[0139] • approximately 11 .5 to approximately 25.5 % w / w dextrose; and

[0140] • up to approximately 1 % w / w tripotassium phosphate (acting as a buffer and / or a base).

[0141] Glycation Conditions - Relative Humidity

[0142] To test if relative humidity levels during glycation altered the heat stability and colour degradation of powders, glycation was performed at low relative humidity (i.e., less than 10%) and high relative humidity (i.e., more than 90%).

[0143] Glycation was performed on a spray dried pectin: C-phycocyanin powder, formulated with C-phycocyanin: HM pectin and dextrose mixture ratio of 1 :2.2, and spray dried on a Bucchi™ 290. The specific formulation comprised 27.1 % w / w C-phycocyanin, 58.5 % w / w high-ester pectin and dextrose (from approximately 35.1 to approximately 46.8 % w / w pectin and from approximately 11 .7 to approximately 23.4 % w / w dextrose), 13.3 % other algal constituents (typically non-C-phycocyanin proteins) and 1 % w / w (of total solids) tripotassium phosphate.

[0144] For glycation, a small 1 g portion of the spray-dried powder was baked in an oven at approximately 100 °C ± °5 C for 1 hour with a relative humidity level of either <10% relative humidity or >90% relative humidity. Relative humidity measurements were taken using an Elitech GSP-6 Temperature & Humidity Data Logger Humidity Probe. For each experiment (i.e. , low relative humidity and high relative humidity), relative humidity levels in the oven were measured before glycation (i.e., before the temperature was raised to 100 °C) and after glycation. Before glycation, relative humidity was measured continuously during pre-heating of the oven until the temperature reached 80 °C. After the glycation temperature treatment (i.e., heating at approximately 100 °C ± °5 C for 1 hour), the relative humidity was measured when the temperature dropped to 80 °C. Relative humidity levels were confirmed to be within the respective low and high humidity ranges before and after glycation. For the low relative humidity experiment, relative humidity levels were below 10% before and after glycation. For the high relative humidity experiment, relative humidity levels were above 90% before and after glycation. In the oven, humidity levels were maintained using a shallow water trough at the bottom of the oven.

[0145] The colour of the powders before and after baking was assessed by determining Evalue 618nm and CIELAB values of solutions in 0.1 M citrate buffer. 0.25 g of pre-glycated powder and each post-glycated powder was dissolved in 0.1 M trisodium citrate buffer (pH 6), separately. Each powder was agitated gently for 30 minutes until fully dissolved, then further diluted 10-fold in buffer, before reading on an Agilent 8453 DAD Spectrophotometer.

[0146] The Evalue was then calculated using the following formula:

[0147] Absoption 618 nm

[0148] Evalue = - — — — - x 100

[0149] CIELAB values were also calculated for each of the solutions on the Agilent 8453 DAD Spectrophotometer to determine the change in colour associated with the humidity (using The CIELAB 1976 colour space). Standard illuminant D65 with 10 observers were used. Delta E values were calculated between the glycated-powder solutions and the preglycated powder to determine colour differences using the formula: whereby L*i, a*i and b*i are the L* a* and b* values of the pre-glycated powder, and L*2, a*2 and b*2 are the L* a* and b* values of the glycated powder.

[0150] The results are summarised in Table 6.

[0151] Table 6: Co our changes (Colour Value, CIELAB and Delta E) of powders before and after glycation at low relative humidity (<10% relative humidity) and high relative humidity (>90% relative humidity).

[0152] The results show considerable colour loss and change is observed in a higher relative humidity glycation (i.e. , at more than 90% relative humidity), indicated by the decrease in colour value and the larger Delta E score. Low humidity glycation (i.e., glycation at below 10% relative humidity) results in minimal colour change from the pre-glycated powder, while also conferring added heat stability.

[0153] Stability Testing for Food and Beverage Applications

[0154] A composition according to the present invention (Composition A) was tested against a commercially available blue colourant, LINABLUE®. LINABLUE®does not comprise a pectin, such that the phycocyanin is nonglycated. Composition A and LINABLUE® were compared in model food and beverage systems.

[0155] Composition A was prepared as described above. In this example, the mixture comprising C-phycocyanin and pectin was prepared using a high- ester pectin (E440(i)) and standardised (i.e., mixed) with dextrose (pectin and dextrose mixture obtained from Healan Ingredients™: Product Range HIP10 and Product Code PEC010). The properties of HIP10 pectin were as defined in Table 3A. The mixture (C-phycocyanin: pectin and dextrose mixture) of Composition A comprised, on a dry weight basis, 28 % w / w C- phycocyanin, 57 % w / w high-ester pectin and dextrose (from approximately 34.2 to approximately 45.6 % w / w pectin and from approximately 11 .4 to approximately 22.8 % w / w dextrose), 14 % other algal constituents (typically non-C-phycocyanin proteins) and 1 % w / w (of total solids) tripotassium phosphate.

[0156] It was calculated that for a phycocyanin purity ratio of around 4 (determined by 620nm / 280nm), the ratio of phycocyanins to other algal biomass is approximately 0.5.

[0157] The C-phycocyanin and pectin mixture of Composition A was dried as described above.

[0158] After spray drying (and before glycation), the dried powder was found to have a colour value comparable to E190-E200.

[0159] The dried powder was placed in a preheated oven and heated to 100 °C for 1 hour, which ensured glycation and a heat stabilised composition. After glycation, the colour value of the glycated Composition A dropped to a value of approximately E180 which was comparable to the colour value of LINABLUE®

[0160] LINABLUE® (DIC LIFETEC Co., Ltd., purchased from Pigment Tokyo) comprises 40% w / w Spirulina extract (comprising C-phycocyanin and other algal constituents), 55 % w / w trehalose and 5 % w / w trisodium citrate.

[0161] The ratio of C-phycocyanin to other algal constituents in LINABLUE®was determined using the method outlined in Yoshikawa et al. Singlelaboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arthrospira) supplements and raw materials by spectrophotometry. Journal ofAOAC Int. 2008 May- Jun;91 (3):524-9. PMID: 18567296. The total phycobiliprotein concentration in 100 mg of LINABLUE® was determined by addition of C-phycocyanin (cPC) and allophycocyanin (aPC). The other algal constituents were determined by the deduction of phycobiliproteins, trehalose and trisodium citrate from the total weight of powder.

[0162] Therefore, the relative amounts of components in LINABLUE® were determined to be as follows: 28 % w / w C-phycocyanin, 55 % w / w D- trehalose, 12 % other algal constituents and 5 % w / w trisodium citrate.

[0163] Trehalose is a non-reducing sugar. As outlined above, non-reducing sugars are unable to glycate with proteins.

[0164] LINABLUE® was found to have a blue colour intensity comparable to E180 food colourant.

[0165] Beverage Applications

[0166] The heat stability of glycated Composition A and LINABLUE® was compared in model beverage systems. Two separate experiments were used to determine the performance of the compositions in respect to pH, pasteurisation temperature, starting dosage and pasteurisation time.

[0167] Experiment 1 : Glycated Composition A and LINABLUE® were each dosed in citrate buffer (0.1 M) at pH 3, 4.5 or 6 to create a 0.1 % w / v solution.

[0168] The solutions were preheated to either 60, 75, 90 or 99 °C. Degradation of the colour of the solutions was monitored over 30 minutes incubation.

[0169] This experiment was designed to simulate hot beverage systems.

[0170] The results of Experiment 1 are provided in Tables 7-9.

[0171] Table 7: Pigment degradation of glycated Composition A and LINABLUE® over 30 minutes in pH 3 model beverage system. Table 8: Pigment degradation of glycated Composition A and LINABLUE® over 30 minutes in pH 4.5 model beverage system.

[0172] Table 9: Pigment degradation of glycated Composition A and LINABLUE® over 30 minutes in pH 6 model beverage system.

[0173] A summary of the results Experiment 1 are provided in Table 10.

[0174] Table 10: Ratio of glycated Composition A to LINABLUE® remaining after 30 minutes at different temperatures and in different buffer systems.

[0175] The results show that after 30 minutes incubation, the blue intensity (measured at 618 nm) was between 2.5 - 10.7 times higher with glycated Composition A than LINABLUE®, under all temperature treatments at 75°C and above.

[0176] The blue intensity (at 618 nm) was also 8.9 times higher with glycated Composition A than LINABLUE® at low pH (pH 3) under 60 °C temperature treatment.

[0177] The best comparative performance was observed in drink systems at pH 3 to pH 4.5 (i.e. , under acidic conditions). Typical low acidity beverages include carbonated soft drinks and fruit juices. Glycated Composition A showed improved heat and acid stability when compared to LINABLUE®. Experiment 2: Glycated Composition A and LINABLUE® were each dosed in citrate buffer (0.1 M) at pH 3, 4.5 or 6 to create either 0.025%, 0.05%, 0.1 % or 0.2% w / v solutions. The solutions were incubated at 95 °C.

[0178] Degradation of the colour of the solutions was monitored over 20 minutes incubation.

[0179] The results of Experiment 2 are provided in Tables 11 -13.

[0180] Table 11: Pigment degradation of glycated Composition A and LINABLUE® at different dosages over 20 minutes in pH 3 model beverage system. Table 12: Pigment degradation of glycated Composition A and LINABLUE® at different dosages over 20 minutes in pH 4.5 model beverage system.

[0181] Table 13: Pigment degradation of glycated Composition A and LINABLUE® at different dosages over 20 minutes in pH 6 model beverage system.

[0182] The results show that LINABLUE® degrades at a greater rate than glycated Composition A in model beverage systems at pH 3 to pH 6. In particular, the colour loss and rate of colour loss is significantly higher, with the majority of the colour of LINABLUE® lost within 5 minutes at 95 °C. With glycated Composition A, between 37 - 52% of blue colour remains even after 10 minutes heating at 95 °C.

[0183] When exposed to temperatures greater than 65°C phycocyanin proteins rapidly denature, causing them to precipitate out of solution. It was found that compositions of the present invention (i.e., comprising a C- phycocyanin-pectin glycoprotein) prevent this precipitation from occurring, allowing solutions to retain their blue colour for longer.

[0184] Industrial meaningful conclusions from Experiments 1 and 2 are provided in Table 14.

[0185] Table 14: Performance outcomes of glycated Composition A and LINABLUE® at common pasteurisation conditions.

[0186] The results of Experiments 1 and 2 show that glycated Composition A outperforms LINABLUE® and is the best choice of natural blue colourant for all beverage applications (including low acidity beverages) requiring pasteurisation or heating.

[0187] Food Applications

[0188] The heat stability of glycated Composition A and LINABLUE® was compared in model syrup systems. Two separate experiments were used to determine the performance of the compositions in respect to pH, sugar content, starting dosage and heating time at 95 °C.

[0189] Experiment 3: Glycated Composition A and LINABLUE® were each dosed into hot (95 °C) model syrup systems (at 50%, 65%, 80% and 95% glucose syrup) to create a 0.1 % w / w solution. Each concentration of syrup was amended to either pH 3, 4.5 or 6. Degradation of the colour of the solutions was monitored over 20 minutes incubation.

[0190] The results of Experiment 3 are provided in Tables 15 to 18. Table 15: Colour degradation of glycated Composition A and LINABLUE® in 50% glucose syrup solution at pH 3, 4.5 and 6.

[0191] Table 16: Colour degradation of glycated Composition A and LINABLUE® in 65% glucose syrup solution at pH 3, 4.5 and 6.

[0192] Table 17: Colour degradation of glycated Composition A and LINABLUE® in 80% glucose syrup solution at pH 3, 4.5 and 6. Table 18: Colour degradation of glycated Composition A and LINABLUE® in 95% glucose syrup solution at pH 3, 4.5 and 6. The results show that glycated Composition A has greater stability than LINABLUE® at pH 3 in 50% glucose syrups, with 20-25% less blue colour degradation at each time point.

[0193] As the glucose concentration increased, pigment degradation of both glycated Composition A and LINABLUE® decreased. Without wishing to be bound by theory, this may be due to the glucose having a protective effect on the colouring agents and preventing pigment degradation.

[0194] However, advantageously, the compositions of the present invention have been found to retain colour at low pH (pH 3) and low sugar concentration (50% glucose syrup) model systems. This is particularly advantageous in syrup and confectionary applications that require retention of colour at an acidic pH, such as postmix syrups and pectin gummies.

[0195] Experiment 4: Glycated Composition A and LINABLUE® were each dosed in 65% glucose syrup at pH 3, 4.5 or 6 to create either 0.025%, 0.05%, 0.1 % or 0.2% w / w solutions. The solutions were incubated at 95 °C.

[0196] Degradation of the colour of the solutions was monitored over 20 minutes incubation.

[0197] The results of Experiment 4 are summarised in Tables 19-21 . Table 19: Pigment degradation of glycated Composition A and LINABLUE® in 65% glucose syrup at different dosages over 20 minutes in pH 3 model beverage system. Table 20: Pigment degradation of glycated Composition A and LINABLUE® in 65% glucose syrup at different dosages over 20 minutes in pH 4.5 model beverage system.

[0198] Table 21: Pigment degradation of glycated Composition A and LINABLUE® in 65% glucose syrup at different dosages over 20 minutes in pH 6 model beverage system.

[0199] The results showed that, in pH 3 syrups, glycated Composition A showed (up to 14%) less degradation than LINABLUE® over 20 minutes. In pH 4.5 syrups, degradation is broadly similar for the first 5 mins for both pigments at all dosages.

[0200] In pH 6 syrups, degradation is broadly similar for the first 2 mins for both pigments at all dosages.

[0201] Calculated performance outcomes after 20 minutes heating at 95°C are provided in Table 22. The results were obtained from Experiments 3 and 4.

[0202] Table 22: Performance outcomes of glycated Composition A and LINABLUE® after 20 minutes heating at 95°C.

[0203] Glycated Composition A shows better performance (increased colour retention) at low pH (pH 3) in 50% glucose syrups. A minimum of 35% of blue colour remains in all tested syrup systems with all doses (0.025-0.2% w / w).

[0204] The results of Experiments 3-4 show that glycated Composition A outperforms LINABLUE® and is the best choice of natural blue colourant for many syrup and confectionary applications (particularly low acidity syrups).

[0205] Based on the stability testing for food and beverage applications, the following ranges of components formed C-phycocyanin - pectin glycoproteins that showed heat stability, acid stability and improved retention of an intense blue colour: approximately 28 % w / w C-phycocyanin; and approximately 34.2 to approximately 45.6 % w / w high-ester pectin.

[0206] Other components that may be present can include:

[0207] • approximately 14 % w / w other algal constituents (typically non-C- phycocyanin proteins);

[0208] • approximately 11 .4 to approximately 22.8 % w / w dextrose; and

[0209] • approximately 1 % w / w tripotassium phosphate (acting as a buffer and / or a base).

[0210] The composition of the present invention provides a blue colouring agent that can be safely used (i.e. , is non-toxic to and / or has no negative health implications for humans) in foods and / or beverages, and the like. The composition of the present invention provides a non-toxic blue colouring agent, that on heating can retain its blue colour without notable degradation. Specifically, the present invention provides a C-phycocyanin based colouring agent that is more heat stable than conventional C- phycocyanin products, meaning that it can be used as an ingredient in a broader range of products, helping food and beverage manufacturers transition away from artificial colourants to natural alternatives. The present invention also provides a C-phycocyanin based colouring agent that is more acid stable than conventional C-phycocyanin products.

[0211] Various modifications and variations to the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

Claims1 . A composition for use as a foodstuff colouring agent, the composition comprising a glycoprotein, wherein the glycoprotein comprises: a phycocyanin; and a pectin; wherein the phycocyanin is glycated by the pectin.

2. The composition of claim 1 , wherein the phycocyanin is present in an amount of from approximately 17.7 to approximately 28.6 %w / w, optionally in an amount of from approximately 24 to approximately 27 %w / w.

3. The composition of any preceding claim, wherein the pectin is present in an amount of from approximately 34.2 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

4. The composition of any preceding claim, wherein the phycocyanin is C-phycocyanin.

5. The composition of any preceding claim, wherein the pectin is high- methoxy pectin, wherein the high-methoxy pectin has a degree of methylation of greater than approximately 50%, optionally at least approximately 69%, optionally at least approximately 70%, optionally from approximately 62 to approximately 75%, optionally from approximately 65 to approximately 75%, optionally from approximately 69 to approximately 75%, optionally from approximately 70 to approximately 75%.

6. The composition of any preceding claim, wherein the pectin comprises at least 65 %w / w galacturonic acid.

7. The composition of any preceding claim, wherein the pH of the composition on reconstitution is from approximately 4 to approximately 5, optionally from approximately 4 to approximately 4.5, optionally from approximately 4.2 to approximately 4.4.

8. The composition of any preceding claim, wherein the composition further comprises a non-polysaccharide sugar, optionally wherein the phycocyanin is glycated by the non-polysaccharide sugar, optionally wherein the non-polysaccharide sugar is a reducing monosaccharide, optionally dextrose, optionally wherein the non- polysaccharide sugar is present in an amount of from approximately11 .4 to approximately 29.2 %w / w, optionally from approximately11 .5 to approximately 25.5 %w / w.

9. The composition of any preceding claim, wherein the composition further comprises at least one of a buffer and a base, optionally a phosphate salt, optionally tripotassium phosphate, optionally wherein the at least one of a buffer and a base is present in an amount of from greater than 0 to approximately 1 %w / w.

10. A method for preparing a composition for use as a foodstuff colouring agent, the method comprising the steps of: adding a phycocyanin to a mixing vessel; adding a pectin to the mixing vessel; forming a mixture comprising the phycocyanin and the pectin;glycating the mixture comprising the phycocyanin and the pectin to form a glycoprotein comprising the phycocyanin glycated by the pectin.11 . The method of claim 10, wherein the glycation is carried out at a temperature of from approximately 95 to approximately 105 °C, optionally from approximately 98 to approximately 102 °C, optionally at approximately 100 °C.

12. The method of claim 10 or claim 11 , wherein the glycation is carried out for at least approximately 60 minutes, optionally from approximately 50 to approximately 90 minutes, optionally from approximately 50 to approximately 80 minutes, optionally from approximately 50 to approximately 70 minutes, optionally from approximately 55 to approximately 90 minutes, optionally from approximately 55 to approximately 80 minutes, optionally from approximately 55 to approximately 70 minutes, optionally from approximately 60 to approximately 90 minutes, optionally from approximately 60 to approximately 80 minutes, optionally from approximately 60 to approximately 70 minutes.

13. The method of claims 10 to 12, wherein the glycation is carried out at a relative humidity of from greater than 0 to approximately 20%, optionally at a relative humidity of from greater than 0 to approximately 10%.

14. The method of claims 10 to 13, wherein the method comprises the further step of adjusting the pH of the phycocyanin in the mixing vessel to approximately 6 before the addition of the pectin, optionally wherein the pH adjustment is by the addition of at leastone of a buffer and a base, optionally a phosphate salt, optionally tripotassium phosphate, optionally wherein the at least one of a buffer and a base is present in an amount of from greater than 0 to approximately 1 %w / w.

15. The method of claims 10 to 14, wherein the method comprises the further step of homogenising the mixture comprising the phycocyanin and the pectin before glycation, optionally wherein the homogenisation is by high shear mixing.

16. The method of claims 10 to 15, wherein the method comprises the further step of drying the mixture comprising the phycocyanin and the pectin before glycation, and optionally after homogenisation, optionally wherein the drying is by way of at least one of spray drying and lyophilisation.

17. The method of claims 10 to 16, wherein the phycocyanin is present in an amount of from approximately 17.7 to approximately 28.6 %w / w, optionally in an amount of from approximately 24 to approximately 27 %w / w.

18. The method of claims 10 to 17, wherein the pectin is present in an amount of from approximately 34.2 to approximately 58.4 %w / w, optionally in an amount of from approximately 35 to approximately 50.5 %w / w.

19. The method of claims 10 to 18, wherein the phycocyanin is C- phycocyanin.

20. The method of claims 10 to 19, wherein the pectin is high-methoxy pectin, wherein the high-methoxy pectin has a degree of methylation of greater than approximately 50%, optionally at least approximately 69%, optionally at least approximately 70%, optionally from approximately 62 to approximately 75%, optionally from approximately 65 to approximately 75%, optionally from approximately 69 to approximately 75%, optionally from approximately 70 to approximately 75%.21 . The method of claims 10 to 20, wherein the pectin comprises at least 65 %w / w galacturonic acid.

22. The method of claims 10 to 21 , wherein the pH of the mixture comprising the phycocyanin and the pectin after addition of the pectin is from approximately 4 to approximately 5, optionally from approximately 4 to approximately 4.5, optionally from approximately 4.2 to approximately 4.4.

23. The method of claims 10 to 22, wherein the method further comprises the step of addition of a non-polysaccharide sugar, optionally in a mixture with the pectin, optionally wherein the phycocyanin is glycated by the non-polysaccharide sugar, optionally wherein the non-polysaccharide sugar is a monosaccharide, optionally dextrose, optionally wherein the non-polysaccharide sugar is present in an amount of from approximately 11.4 to approximately 29.2 %w / w, optionally from approximately 11 .5 to approximately 25.5 %w / w.

24. A composition prepared from the method of claims 10 to 23.

25. A foodstuff or a foodstuff colouring agent comprising the composition of claims 1 to 9, or the composition of claim 24, or prepared by the method of claims 10 to 23.

26. The use of the composition of claims 1 to 9, the composition of claim 24, the foodstuff colouring agent of claim 25, or a composition prepared by the method of claims 10 to 23, as a foodstuff colouring agent.

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