WO2016099261A1 - Natural blue photopigments, methods for producing them and to uses thereof as colorant. - Google Patents

Abstract

The invention relates to natural blue photopigments, methods for producing them and to uses thereof as colorant, in particular in food, confectionary, ice creams and beverages. Provided is a coloring material comprising a blue pigment obtained or obtainable from Cyanidioschyzon merolae. The blue pigment has a thermal stability until at least 70ºC, and a purity factor of 5 or higher, preferably 8 or higher, more preferably 10 or higher. Also provided is a method for providing a thermostable natural blue C. merolae pigment.

Description

Title: Natural blue photopigments, methods for producing them and to uses thereof as colorant.

The invention relates to natural blue photopigments, methods for producing them and to uses thereof as colorant, in particular in food, confectionary, ice creams and beverages.

Consumers have a strong preference for natural colorants, especially in food products. A range of red, yellow and red colorants from natural sources have been approved as additives in food products. Natural blue colorants still are a challenge because the color blue is not widespread in nature.

In the last few years, there has been growing interest in the possible usages of phycocyanin in food coloring, as a natural dye, as a fluorescent label, as a nutraceutical, and pharmaceutical. Stable

phycocyanin has been produced chemically and by protein engineering, and new purification procedures allow highly pure phycocyanin to be obtained at high yields. But the application of phycocyanin in food, as a dye, in cosmetics, as fluorescent marker, and other applications is limited as the currently commercially available natural phycocyanin dyes have a relatively low stability with respect to temperature, light, and/or air, resulting in the loss of its intense blue color. The stability can possibly be enhanced using certain chemicals or additives but this contradicts the natural origin of the phycocyanin and thus limits its use in many products.

Phycocyanins are accessory pigments of the light harvesting complex of a range of micro-algae and cyanobacteria. Phycocyanins have been associated with free-radical scavenging, inhibition of lipid oxidation, anti-inflammation, and improving weakened immune systems.

Phycocyanins are members of the phycobiliprotein family and are composed of a multimeric protein (a and b subunits; 70 tot 110 kDa) to which a chromophore is attached. The chemical structure of the chromophore is very similar to bilirubin, a degradation product of heme. The chromophore is the actual light-absorbing unit, with an absorption maximum of about 620 nm and an emission maximum at about 650 nm. The protein part confers the thermostability to the phycocyanin, with maxima of 70°C reported for certain thermophilic cyanobacteria.

The US food company Mars recently applied for permission of the FDA to use a blue pigment from the green algae Spirulina as colorants for candy, in particular M&Ms. This blue colorant is obtained by extracting a phycocyanin-rich fractions from a Spirulina culture. The maximum

temperature at which this colorant can be used is about 50 to 60°C.

Moon et al. (Korean J. Chem. Eng., 31(3), 490-495 (2014)) described the isolation and characterization of thermostable phycocyanin from the extremophilic red microalgal species Galdieria sulphuraria. When the phycocyanin solution was incubated for 30min at a temperature ranging from 0 to 95°C, the absorption of the spectra remained consistent until 60°C, with steady reduction in absorbance at 620 nm after that point. The heat induced denaturation on phycocyanin is an irreversible process. The denaturation of protein increased from 65 to 95°C, and the stability of G. sulphuraria phycocyanin decreased quickly as the temperature was elevated from 75 to 95°C.

Thus, whereas natural blue pigments are available, their use is still limited due to their susceptibility to increased temperatures. In particular, many food products need to be heated to properly homogenise the different ingredients. Additives such as coloring compounds need to be added at an elevated temperature before the mixture solidifies in order to achieve a homogeneous mixture. This posses restrictions on the thermal properties of the additives. An example are wine gums that are made from a mixture of gelatin and glucose syrup that are heated and thoroughly blended. As gelatin starts to solidify at temperatures of about 60 to 70°C, any color or flavour additive needs to be added above the gelation

temperature of the gelatin.

Thus, there is a clear need for natural blue which offer a wider application range e.g. which can be used in industrial processes involving exposure to high temperatures (e.g. pasteurization) and/or low pH values. To this end, the inventors set out to find natural blue photopigments which show an enhanced thermostability as compared to known natural blue pigments. In particular, they aimed at providing a blue photopigment that can be readily isolated from a natural source, and has a temperature tolerance of above 70°C, preferably above 75°C.

It was surprisingly found that the extremophilic red microalga Cyanidioschyzon merolae, growing optimally at 55°C, contains a very thermostable blue pigment (presumably a phycocyanin) which is very thermostable, with a maximum temperature tolerance of 82°C, i.e. far above the optimal growth temperature of the organism, while at 85°C it is stable for at least a few seconds. It was found to be stable in the pH range of 3 to 8. In addition, as C. merolae cells have almost no cell wall and are very susceptible to osmotic shock, the blue pigment can be obtained very easily by washing cells with pure water. This results in a clean blue pigment extract with almost no contamination (purity factor of 12 and higher).

Accordingly, in one embodiment the invention provides a thermostable blue pigment obtained or obtainable from Cyanidioschyzon merolae. Also provided is a coloring material comprising the C. merolae blue pigment. The blue pigment preferably has a thermal stability until at least 70°C, more preferably at least 75°C. For example, incubation of the pigment for up to 30 minutes at 70°C results in a remaining concentration (CR, %) of the pigment relative to the initial concentration of at least 95. It is noted that natural blue pigments from rhodophytes were known in the art. For example, Carra et al. (Phytochemistry 1966, Vol. 5 pg. 993-997) and Eriksen et al. (2008) Appl Microbiol Biotechnol 80: 1- 14), disclose

phycocyanins isolated from, respectively, Tolypothrix tenuis and Galdieria sulphuraria. Whereas it may be derivable from the art that C. merolae is a rhodophyte that can make phycocyanin, it was highly surprising and not predictable that the C. merolae phycocyanin is stable at temperatures far higher than the maximum growth temperature of this organisms (up to about 55/60°C). For example, Moriyama et al. (FEBS Journal 275, 2899- 2918) describe that the DNA polymerase enzyme from C. merolae is stable at 50°C but not at 60°C, which is clearly in line with the optimal

temperature at which C. merolae grows. Hence, nothing in the art could have predicted the unique and favourable properties of C. merolae blue pigment. The unexpected high thermostability makes it possible to use phycocyanin at higher temperatures than the currently available natural blue pigments, like phycocyanin from Spirulina. Therefore, the invention does not simply provide a further pigment but solves the technical problem that known pigments do not have the wide industrial applicability sought for, in particular processes which involve exposure to increased

temperatures.

As is shown herein below, the blue pigment can be easily purified to a purity index of 5 or higher, preferably 8 or higher, more preferably 10 or higher.

The purity index is expressed as the ratio of A618 to A280, wherein A618 is the maximum absorbance of the blue pigment (the C. merolae phycocyanin) and A280 is the absorbance of total proteins. Colorants with purity factors of

1 or higher are considered to be food grade.

Accordingly, the invention also provides a consumable comprising a coloring material comprising the thermostable blue pigment from C.

merolae cells. The consumable is for example a food or beverage product, preferably selected from the group consisting of neutraceuticals,

confectionary, ice creams and beverages. In one aspect, the C. merolae blue pigment is the sole colorant. Of course, the blue pigment can be

supplemented or mixed with one or more other (natural) pigments to obtain a desired color. Exemplary food, confectionary, desert or beverage products include a (blue) sport drink, a (blue) soda drink, (blue) ice cream, a (blue) wine gum, a (blue) button-shaped candy or a (blue) jelly bean. In a specific aspect, the C. merolae pigment is used for the manufacture of blue wine gums. However, the pigment can be used for any other coloring application or in any other type of product, such as paints, cosmetic products, clothing items, leather products, and the like.

In a further embodiment, the invention relates to a method for providing a thermostable natural blue pigment. The method comprises the steps of

(i) culturing Cyanidioschyzon merolae cells to provide a biomass;

(ii) suspending the biomass after harvesting in ultrapure water;

(iii) homogenizing the suspension followed by a period of maceration;

(iv) centrifugation of the suspension; and

(v) collecting the blue colored supernatant.

The red alga Cyanidioschyzon merolae is a small (2 μιη in diameter), unicellular, photoautotrophic organism that inhabits sulphate-rich hot springs. The organization of a C. merolae cell is very simple: it contains one each of the nucleus, the mitochondrion and the chloroplast, but has no rigid cell wall. The cell also has a minimum set of membranous structures: simple architectures of the endoplasmic reticulum and the Golgi body, a single microbody (peroxisome), and a small number of lysosomes (vacuoles).

C. merolae strains are publicly available and can be obtained from culture collection, such as NIES (National Institute for Environmental Studies), Microbial Culture Collection, Biodiversity Resource Conservation Section, Center for Environmental Biology and Ecosystem Studies, Japan. In one embodiment of the present invention, the wild-type C. merolae strain 10D is used (Toda et al., Biochim Biophys Acta 1998, 1403: 72-84). In a preferred aspect, Cyanidioschyzon merolae strain NIES 1804 (De Luca, Taddei et Varano) is used.

Cyanidioschyzon merolae cells can be cultured using known procedures to provide a biomass. For example, they can be maintained by gyratory culture (130 rpm), in 2x Allen's medium at pH 2.3 and 42°C under continuous light.

Cultured cells can be harvested by centrifugation, after which the biomass is suspended in de-ionized (also referred to as "ultrapure") water. This treatment causes rupture of the cells by a hypo-osmotic shock, and release of their contents including the blue pigment. De-ionized (DI) water is water in which the ions have been removed. Ions can be removed in a number of ways, for instance by using anion and cation exchange resins in a variety of configurations. This can yield up to 17 megohm water, which is very pure. Distillation will also remove ions. The third and most commonly used technique is dual reverse osmosis (RO) which can also give ultra pure water.

Preferably, the aqueous suspension is homogenized by vigorous mixing, e.g. by vortexing. Following hypo-osmotic shock, the cells are left for some time to macerate for a time period that is sufficient to extract the blue pigment from the cells. Maceration is typically performed at room temperature for at least 10-30 minutes. Very good results were obtained upon prolonged maceration during one or more hours. Accordingly, in one embodiment a method of the invention comprises maceration for at least 2 hours, preferably at least 3 hours.

After maceration, the cell suspension can be centrifuged to remove debris and the blue colored supernatant can be collected in a fresh tube. The supernatant can be concentrated or partially or fully dried. Water can be removed by methods known in the art, such as by freeze-drying, mild vacuum evaporation, drying chamber. If desired, the crude extract can be subjected to a further treatment to purify the blue pigment. As shown herein below, fractional ammonium sulphate precipitation was suitably used to obtain blue pigment having a purity index of more than 15. In a specific embodiment, purification comprises subjecting the crude extract to ammonium sulphate precipitation at about 40% saturation via addition of solid ammonium sulphate, and recovery of the blue pigment in the precipitate.

The invention also provides a natural blue pigment obtainable by a method according to the invention. As mentioned herein above, the pigment is characterized among others by a thermal stability of up to at least 70°C.

Furthermore, it was found that the blue pigment can withstand exposure to acidic pH. Hence, the invention provides a natural thermostable acid resistant blue pigment. As will be appreciated by the skilled person, a coloring material or natural blue pigment as provided herein finds its use in any type of (industrial) coloring process. In view of its unique characteristics, it is however advantageously used in an (industrial) process involving exposure of the coloring material or pigment to a temperature of at least 70°C and/or a pH below 5.5. In one embodiment, said exposure has a duration of at least 15 minutes, preferably at least 30 minutes, more preferably at least 1 hour. In another embodiment, said exposure has a duration of up to 60 seconds, preferably up to 45 seconds, more preferably up to 15 seconds.

In one embodiment, the blue pigment of the invention is used in a process involving exposure to a temperature of at least 75°C, and / or to a pH below 5. Exemplary processes include pasteurization, incorporation in acidic beverages, and the preparation of wine gums.

LEGEND TO THE FIGURES

Figure 1 : Effect of different maceration times of C. merolae cells in pure water on phycocyanin yields.

Figure 2: UV-VIS absorption spectra of phycocyanin isolated from

Cyanidioschyzon merolae at each step of purification. Step 1. Crude extract; 2. Supernatant Fraction 0-20%; 3. Supernatant of fraction 20-40%; 4.

Precipitation of fraction 20-40%; 5. Supernatant of fraction 40-60%; and 6. Precipitation of fraction 40-60% Figure 3: Effect of different temperature on CR (%) of phycocyanin solution The solution was incubated for 30 min. at the indicated temperature. CR, (%) refers to the remaining concentration of phycocyanin relative to the initial concentration. Figure 4: Solubility of C. merolae phycocyanin subjected to 80°C, pH 4.2 for up to 150 min.

Figure 5: Effect of incubating phycocyanin extract at 80°C and different pH for up to 150 min on the CR (%) value.

Figure 6: Effect of incubating phycocyanin extract at pH values between 2.5 and 4 at 80°C for up to 150 sec.

Figure 7: Effect of different pH and various incubation periods at room temperature (25°C) on crude phycocyanin concentration. EXPERIMENTAL SECTION

The section herein below exemplifies the isolation and characterization of the blue pigment (also referred to as phycocyanin) of Cyanidioschyzon merolae

Material and methods

Species and growth condition

The red unicellular microalgae Cyanidioschyzon merolae strain NIES 1804 was obtained from NIES (National Institiute for Environmental

Studies), Microbial Culture Collection, Biodiversity Resource Conservation Section, Center for Environmental Biology and Ecosystem Studies, Japan. The culture was cultivated in Allen medium (Nagasaka and Yoshimura, 2008), which was composed of (per liter) 1.32 g (NH₄)SO₄, 0.272 g KH₂PO . 0.247 g MgSO .7H₂O, 0.073 g CaCl₂.2H₂O, 0.011 g FeCl₃ and 1 mL trace element. C. merolae cells were grown in a 1L bioreactor at 40°C, sparged with regular air and continuous light. Efficiency of phycocyanin extraction

Cultures were harvested by centrifugation at 10.000 x g for 10 minutes at 4°C. To investigate the efficiency of phycocyanin extraction with ultrapure water at optimum maceration time, 0.2 g wet biomass were extracted by 2 mL ultrapure water and vortexing for 5 minutes and maceration at different incubation time (15, 30, 60, 90, 150, 90 ± 1 minutes). After maceration, the samples were centrifuged to remove debris and analysis with

spectrophotometer.

Preparation of phycocyanin extract Phycocyanin containing biomass was harvested by centrifugation at 10,000 g for 10 minutes at 4°C. Washed cell biomass was resuspended in ultrapure water. Resuspended cells were mixed well for 5 minutes and kept at 4 °C for 3 hours. The cell debris was removed by centrifugation at 15.000 x g for 10 minutes at 4 °C and blue colored supernatant was collected in fresh tubes.

Purification of phycocyanin

Phycocyanin crude extracts were further purified through ammonium sulfate precipitation. Two cycles of the process were performed, with initial precipitation at 20% saturation, 40% saturation and followed by third step at 60% saturation. Solid ammonium sulfate was dissolved by stirring at 400 rpm and the precipitate was recovered by centrifugation at 10,000 g at 4°C for 30 minutes. The pellet was dissolved in 0.05 M acetate buffer pH 4.5. Spectroscopic estimation of phycocyanin

The absorbance of phycocyanin were measured on a DR3900

spectrophotometer (Hach-Lange). The purity of phycocyanin was assessed by calculating the ratio of Αβιβ to A280, where Αβιβ is the maximum

absorption of Phycocyanin and A280 is the absorbance of total protein. The calculation of concentration of phycocyanin using the following equation:

(OD₆₂₀ -0.474X(OD_6S2) )

Phycocyanin (mg mL ) = 5.34

Properties of phycocyanin

To investigate the effect of different temperatures on the phycocyanin stability, 1 mL of phycocyanin solution was incubated for 30 minutes at different temperature (20 tol00°C).

To determine the thermostability of the extracted phycocyanin, the samples were incubated at 80°C, and the absorbance at 624 nm was measured at regular intervals (0-150 minutes). The remaining concentration of phycocyanin (CR, %) relative to the initial concentration was calculated using the following equation : CR (%) = C/C₀ x 100; which is CR (%) is the remaining concentration of phycocy anin as a percentage of the initial concentration.

To determine the pH stability of phycocyanin, the samples were incubated at 80°C in different pH solutions (pH 2, 3, 4 or 5), and the absorbance at 624nm was measured at regular intervals (0-60 minutes).

Blue Gelatin Wine Gums

Add one part gelatin (Bloom 250) to two parts of warm water (approx. 55°C) and mix vigorously until the gelatin is dissolved and store overnight at room temperature. Heat the gelatin solution in a water bath at 60°C for minimal 2 hours and remove the foam. Add an appropriate amount of concentrated C. merolae blue pigment (obtained from pure water treated cell concentrate) to this solution.

Dissolve 33.0 g saccharose in 11.2 g water while heating to 60°C. Add sugar syrup (DE42) and heat till 100°C while continuous stirring. Add water to compensate the water loss due to evaporation.

Let the mixture cool down till about 75-80°C and add the appropriate amount of gelatin (e.g. 15.2 g of a 33% gelatin solution for wine gums with a final concentration of gelatin of 5%) while stirring vigorously. Add the appropriate amount of citric acid, coloring agent in the case of artificial colorants, flavor compound and keep stirring. Pour the mixture in a preheated funnel and pour small volumes in the prepared fatty maize starch moulds. Cover the wine gums with some starch and dry for at least 36 hours at 32 °C. Results

Efficiencies of phycocyanin extraction Fig. 1 shows the effect of different maceration times of C. merolae cells in pure water on phycocyanin yields. Maceration of up to 90 minutes gives relatively low amounts of phycocyanin. Maceration of more than 90 minutes gives considerably higher amounts of free phycocyanin. Purification of phycocyanin

Table 1. Determination of spectrophotometric purity of thermostable phycocyanin.

Supernatant Supernatant Pellet Supernatant Pellet crude Fraction of Fraction of Fraction of Fraction of Fraction of extract 20% 40% 40% 60% 60%

(NH₄)₂S0₄ (NH₄)₂S0₄ (NH )₂S0₄ (NH )₂S0₄ (NH )₂S0₄

Volume of

40 40 40 10 40 10 sample

dilution 1:5 1:5 1:5 1:20 1:5 1:20

652 0,098 0,096 0,032 0,059 0,019 0,031

624 0,496 0,469 0, 167 0,271 0,042 0, 147

562 0, 145 0, 135 0,049 0, 135 0,017 0,048

280 0,05 0,051 0,025 0,015 0,073 0,014 purity index 9,92 9,26 6,78 18,07 0,58 10,78

Total C-PC 16,8 15,8 5,6 9,2 1,2 5

Yield 100 94,31 33,81 54,06 7,38 31,32 Fig. 2 shows the UV-VIS absorption spectra of phycocyanin isolated from Cyanidioschyzon merolae at each step of purification. Step 1. Crude extracts; 2. Supernatant Fraction 0-20%; 3. Supernatant of fraction 20-40%; 4. Precipitation of fraction 20-40%; 5. Supernatant of fraction 40-60%; and 6. Precipitation of fraction 40-60%.

Properties of Phycocyanin

Figure 3 shows the effect of different temperature on CR (%) of phycocyanin solution (incubation time: 30 minutes). The phycocyanin solution was incubated for 30 min. at the indicated temperature, after which the solution was centrifuges and the absorption of the supernatant was measured at 624 nm. Up to 75°C, the phycocyanin is clearly soluble. Between 75°C and 85°C the blue colorant precipitates and the solution becomes more and more transparent. Above 85°C, the phycocyanin quickly precipitates. This result clearly demonstrates that the C. merolae phycocyanin is thermostable up to 80 to 82°C.

Figure 4 shows the solubility of C. merolae phycocyanin subjected to 80°C, pH 4.2 for up to 150 min.

Figure 5 shows the effect of incubating phycocyanin extract at 80°C and different pH for up to 150 min on the CR (%) value. Above pH 3 the extract is clearly more stable than at pH values lower than 3.

Figure 6 shows the effect of incubating phycocyanin extract at pH values between 2.5 and 4 at 80°C for up to 150 sec. The C. merolae phycocyanin is stable at low pH values for a few seconds. Figure 7 shows the effect of different pH and various time incubations at room temperature (+ 25°C) on crude phycocyanin concentration. This clearly demonstrates that even at low pH values the C. merolae phycocyanin is stable at room temperature, indicating that it can be used in beverages or food products with a low pH value that are stored at room temperature. Blue gelatin wine gums

The light blue wine gums had acceptable texture and appearance. The blue color was evenly distributed and no precipitation was visible. The wine gums kept their blue color for at least 6 weeks when stored at room temperature.

REFERENCES

Eisele LE. SH Bakhru, X Liu, R MacColl, MR Edwards (2000) Studies on C-phycocyanin from Cyanidium caldarium, s eukaryote at the extremes of habitat. Biochinica et Biophysica Acta 1456: 99-107

Eriksen NT (2008) Production of phycocyanin - a pigment with applications in biology, biotechnology, foods and medicine. Appl Microbiol Biotechnol 80: 1-14

Fukui, K., T. Saito, Y. Noguchi, Y. Kodera, A. Matsushima, H. Nashimura, Y. Inada (2004) Relationship between color development and protein conformation in the phycocyanin molecule. Dyes and pigments 63: 89-94

Graziani, G. A. Schiavo, M.A. Nicolai, S. Buono, V. Foglino, G. Pinto, A. Pollio. 2013. Microalgae as Human Food: Chemical and Nutrional Characteristics of the thermo-acidophilic microalga Galdieria sulphuraria. Food Function (2013) 4: 144-152

Gross, W. 2000. Ecophysiology of algae livig in highly acidic environments. Hydrobiologia 433: 31-37 Moon, M., S.K. Mishra, C.W. Kim, W.I. Suh, M.S. Park, J.W. Yang (2013) Isolation and characterization of thermostable phycocyanin from Galdieria sulphurarial. Korean J. Chem. Eng.

Sorensen, L., A. Hantke, N.T. Eriksen (2013) Purification of the photosynthethic pigment C -phycocyanin from heterotrophic Galdieria sulphuraria. J. Sci Food Agric 93: 2933-2938.

Claims

Claims

1. A coloring material comprising a blue pigment obtained or obtainable from Cyanidioschyzon merolae.

2. Coloring material according to claim 1, wherein the blue pigment has a thermal stability until at least 70°C, preferably at least 75°C.

3. Coloring material according to claim 1 or 2, having purity factor of 5 or higher, preferably 8 or higher, more preferably 10 or higher.

4. Coloring material according to any one of the preceding claims, wherein Cyanidioschyzon merolae is Cyanidioschyzon merolae strain NIES 1804.

5. A consumable comprising a coloring material according to any one of claims 1-4.

6. Consumable according to claim 5, being a food or beverage product, preferably selected from the group consisting of confectionary, ice creams and beverages.

7. Consumable according to claim 6, being a wine gum, a button- shaped candy or a jelly bean.

8. Method for providing a thermostable natural blue pigment, comprising the steps of

(i) culturing Cyanidioschyzon merolae cells to provide a biomass;

(ii) suspending the biomass after harvesting in ultrapure water; (iii) vortexing the suspension followed by a period of maceration; (iv) centrifugation of the suspension and

(v) collecting the blue colored supernatant.

9. A method according to claim 8, wherein maceration is performed for at least 2 hours, preferably at least 3 hours.

10. A natural blue pigment obtainable by a method according to claim 8 or 9.

11. Use of a coloring material according to any one of claims 1-4 or a natural blue pigment according to claim 10, in an industrial process involving exposure of the coloring material or pigment to a temperature of at least 70°C and/or a pH below 5.5.

12. Use according to claim 11, wherein said exposure has a duration of at least 15 minutes, preferably at least 30 minutes, more preferably at least 1 hour.

13. Use according to claim 11 or 12, involving exposure to a

temperature of at least 75°C, and / or to a pH below 5.

14. Use according to any one of claims 11-13, wherein the industrial process is selected from the group consisting of pasteurization, incorporation in acidic beverages, and the preparation of wine gums.