How About the Stability of Natural Pigment Phycocyanin Powder?

Phycocyanin is a photosynthetic pigment protein complex obtained by isolating and purifying it from microalgae cells [1]. It is a secondary pigment of chlorophyll [2]. Phycocyanin powder has been widely used in many fields. It can be used as a natural coloring agent in food [3], as an additive in the cosmetics industry, and as a nutritional supplement in the health care industry [4]. Due to its high sensitivity to fluorescent reagents, it can also be used in immunoassay experiments as a marker for antibodies and receptors [5]. In addition, phycobiliproteins have biological activities such as anti-tumor, anti-inflammatory, antioxidant, and immune regulation [6], and can also be used in the pharmaceutical industry as anti-inflammatory agents and antioxidants.

However, due to the different aggregation states of the protein in phycocyanin, it is prone to degradation. This property may be related to many factors, such as light, temperature, pH and protein concentration [7–8]. The sensitivity of phycobiliproteins to heat and light is manifested in the loss of color and the appearance of precipitation. Moreover, with the increase of light intensity and the increase of temperature, the activity of phycobiliproteins also decreases or even inactivates, which greatly restricts the application of phycobiliproteins [9]. Some studies have shown that the degradation rate of phycobiliproteins can be slowed down by adding stabilizers, structural modification, immobilization and microencapsulation, etc. [10], thereby improving the stability of phycobiliproteins. This paper reviews the structural characteristics of phycobiliproteins, the factors affecting stability and the methods to improve stability, with a view to providing theoretical reference for improving the stability of phycobiliproteins and expanding their scope of application.

1 Structural characteristics of Phycocyanin

Phycobilisomes (PBS) are light-harvesting complexes with an absorption range of 500–660 nm [11]. Each phycobilisome is composed of colored proteins called phycobiliproteins (PBP). These molecules are arranged in an antenna-like manner; the absorbed energy is transported to the reaction center of photosystem II with an efficiency of over 95%. Therefore, cyanobacteria can use red, yellow, green and, to a lesser extent, blue light [12]. It contains the colored proteins phycocyanin (PC, λmax=610–620 nm), phcoerythrin (PE,   PE, λmax=540~570 nm) and allophycocyanin (APC, λmax=650~655 nm) (Fig. 1 [13]). As shown in Figure 1, different phycobiliproteins are assembled in a specific order, so that energy can be efficiently transferred to the reaction center in one direction. The order is phycocyanin, then phycobilin, and finally allophycocyanin, which ultimately transfers to the reaction center photosystem I (photosystem I, PS I) and II (photosystem II, PS II) [14].

Phycobiliproteins are composed of a chain-opening tetrapyrrole chromophore, phycobilin, covalently attached to the protein molecule. The type and number of phycobilin chromophores causes phycobiliproteins to appear in different colors [15]. Changes in the structure of the chromophore cause phycobiliproteins to lose their color and antioxidant activity. Phycobiliproteins are located at the ends of peripheral rods and are adjacent to a core cylinder composed of allophycocyanin. Its basic structure is composed of monomers with two homologous subunits, α and β [11]. These monomers aggregate into (αβ)3 trimers with C3 facing each other to form a symmetry, and every two (αβ)3 form a (αβ)6 hexamer with triple symmetry [16‒17].  The hexamer further assembles into a rod or core cylinder, and the non-pigment linking protein is isolated in the large pore at the center of the trimer, hexamer, rod or core cylinder.

The protein structure is highly correlated with the stability of phycobiliproteins and promotes the protection of chromophores. Therefore, any factor that affects the stability and structure of the protein may prevent or accelerate the degradation of phycobiliproteins. Compared to trimers and monomers, the hexameric structure is more stable and provides higher protection for phycobiliproteins [18]. The maintenance of the linear conformation of phycobiliproteins is also important for preventing their degradation [19]; when the protein is denatured, the reduction in the hydrogen bond network causes the phycobiliprotein molecule to rearrange from the linear conformation to the cyclic conformation, resulting in color fading [20].

2 Stability studies of Phycocyanin

Phycocyanins are water-soluble protein-pigment complexes. The rate of phycobiliprotein degradation depends on the aggregation state of the protein, which is affected by light, pH, temperature and other factors.

2.1 Effect of pH on the stability of phycobiliproteins

pH is the main factor affecting the aggregation and decomposition of phycobiliproteins in solution, such as monomers, trimers, hexamers and other oligomers. When pH changes, the charge and dissociation of phycobiliproteins also change, which affects stability. When the pH is close to 7.0, hexamers predominate, which is the most stable structure and prevents the denaturation of phycobiliproteins. However, at higher or lower pH, this structure is prone to dissociation, and its stability is reduced [21]. When the pH is acidic or alkaline, the conformation of the phycobiliprotein chromophore is changed, which changes its color and affects its stability [15, 22]. Ren Shuncheng et al. [23] found that the conformation of the phycobiliprotein chromophore remained stable and displayed bright blue at pH 4.0–7.0, while at pH<4.0 or pH>7.0 the blue color changed to green and precipitated at pH 2.5–3.0.

2.2 Effect of light on the stability of phycobiliproteins

Light can damage the structure of phycobiliproteins, which destabilizes the conformation of the chromophores attached to them and reduces the stability of the phycobiliproteins. The stability of phycobiliproteins gradually stabilizes as the light intensity decreases. Wu et al. [24] found that the degradation of phycobiliproteins under a light intensity of 100 μmol m−2 s−1 is higher than that under 50 μmol m−2 s−1 . When phycobiliproteins are exposed to light for a long time, they often lose their chromophores, thereby losing their color and stability [25]. Liang Xiao et al. [26] selected a light condition of 1500 Lx to irradiate phycobiliproteins. As the light time and intensity increased, the color of phycobiliproteins gradually lightened, and their retention rate decreased, indicating that not only the light intensity but also the light time will affect the stability of phycobiliproteins.

2.3 Effect of temperature on the stability of phycobiliproteins

An increase in temperature can lead to an extension reaction of the phycobiliprotein-phycocyanin structure, causing a conformational change from a linear to a cyclic form, thereby affecting the three-dimensional structure of phycobiliproteins. The determinants of the thermal stability of phycobiliproteins mainly include: the number of hydrogen bonds, the fraction of polar surface, the content of secondary structure and the difference in the ratio of surface area to volume [27]. MUNAWARO et al. [28] found that phycobiliproteins have the potential to maintain spectral intensity at 60 °C, but begin to decrease at 70 °C and higher temperatures, indicating that the pigment protein is thermally unstable during heating. Studies have found that the structure of phycocyanin is destroyed above 40 °C [29‒30]. Bcker et al. [31] determined the intermediate point transition temperatures of phycocyanin trimers and hexamers to be 58.4 °C and 60.9 °C. At 40 °C, there is no change in the absorbance or fluorescence spectrum of phycocyanin,  When the temperature is greater than 50 °C, the spectrum changes with the increase of temperature. Not only is phycobiliprotein sensitive to high temperatures, but its stability is also not high under low temperature conditions. CHOI et al. [32] stored phycobiliprotein at 4 °C, and the content of phycobiliprotein decreased by 10.39%, and its stability decreased.

2.4 Other factors

In addition to the above factors that affect the stability of phycobiliproteins, metal ions, additives, etc. can also affect the conformation of phycobiliprotein chromophores. The addition of metal ions affects the stability of phycobiliproteins, while the addition of emulsifiers or foaming agents can form bubbles around phycobiliproteins to protect them, thereby maintaining the stability of phycobiliproteins [22]. Zhang Yanyan et al. [33] found that the stability of phycobiliproteins was better in solutions with low concentrations of Mn2+, Al3+, Zn2+, and Cu2+. The stability of phycobiliproteins was not significantly affected by changes in the concentration of Na+ and Mg2+, while the higher the concentration of Fe3+, the more beneficial it was for the stability of phycobiliproteins. Some organic reagents can reduce the stability of phycobiliproteins. Zhao Bingbing et al. [34] added different concentrations of additives to phycobiliproteins and found that their stability decreased with the increase of ethanol, sodium benzoate, and citric acid. Among them, citric acid had a greater effect.

3 Methods for improving the stability of Phycocyanin

3.1 Adding stabilizers

Adding stabilizers is the simplest way to improve the stability of phycobiliproteins. This method is easy to apply, does not require complicated or expensive equipment, but does require the stabilizer to be highly safe, low in toxicity and harmless, and the amount of additive to be large. At present, the main stabilizers commonly used are sugar, sorbitol, benzoic acid, sodium azide and dithiothreitol. CHENTIR et al. [35] added polyethylene glycol-4000, sucrose and sorbitol to a 0.5 mg/mL phycobiliprotein solution. Polyethylene glycol-4000 had the best thermal stabilizing effect on phycobiliprotein, followed by sorbitol,  and the protective effect on the algin protein increases with the concentration of the stabilizer. FAIETA et al. [36] studied the effects of thermal effects and equivalent thermal effects on the discoloration of algin protein in water and solutions of different concentrations of sucrose and trehalose. At a constant temperature, the loss of color increases with time, while the concentration of the solute increases. This shows that the concentration of the stabilizer is positively correlated with the stability of phycobiliproteins, but prolonged heating will lead to a decrease in the stability of phycobiliproteins.

Under food processing conditions, the stability of phycobiliproteins can be improved by adding proteins. Proteins can wrap up phycobiliproteins, thereby improving their stability [37]. ZHANG et al. [38] treated whey protein and phycobiliproteins at pH 3.0 and 80 °C for 1–20 min. A 10% whey protein solution was found to prevent phycobiliprotein aggregation, and natural whey protein was more effective than denatured hydrolyzed whey protein.

3.2 Chemical modification

Chemical modification is a technique that uses bifunctional reagents to covalently bind two chemical groups on the surface of a protein to strengthen the folded structure of phycobiliproteins and improve their stability. Formaldehyde, methylglyoxal, and propionates can be used to cross-link phycobiliproteins and stabilize their tertiary and quaternary structures, thereby improving their stability [39–41]. The stability of the pigment can also be maintained by covalently linking the protein to polysaccharides to preserve the extended tetrapyrrole structure.  SELIG et al. [42] evaluated the stabilizing effect of beet pectin, guar gum and soluble soy polysaccharides on phycocyanin. The results showed that beet pectin can stabilize phycocyanin, maintain its color, and reduce the ability of enzymes (such as alkaline protease, papain and bromelain) to degrade the protein.

3.3 Encapsulation technology

3.3.1 Microcapsule encapsulation

Microencapsulation technology involves using a core material of solid, liquid or gas, and then using a natural or synthetic polymeric material as a wall material to form a semi-permeable or sealed microparticle [43]. The wall material used for encapsulation must be biocompatible, biodegradable, low-toxicity and low-cost. Microencapsulation technology can effectively improve the stability, solubility and bioavailability of the core material. Microcapsules of phycocyanin can be prepared using a variety of methods, such as freeze-drying, spray drying, and extrusion encapsulation. Microcapsules of phycocyanin prepared using these methods have good heat resistance and high antioxidant activity [44‒45]. Different coating materials also affect the stability of phycocyanin, with maltodextrin and carrageenan as the best coating materials [46]. Lv Xiaoling et al. [47] prepared microcapsules of phycocyanin using air suspension coating. It was measured that under the optimal conditions (inlet air temperature 80 °C, core wall material ratio 1:1.5, atomization pressure 0.15 MPa, gelatin content in the wall material 20%), the stability of phycocyanin was increased by 26.21%,   and the storage stability increased by 75.1%. SCHMATZD et al. [48] used polyvinyl alcohol to encapsulate phycocyanin by electro-spraying technology. The phycocyanin-polyvinyl alcohol ultrafine particles have high heat resistance, with a heat resistance temperature of up to 216 °C, and maintain the antioxidant activity of phycocyanin.

3.3.2 Liposome encapsulation

Liposomes are formed from phospholipid molecules that have a tendency to form stable lipid bilayers in aqueous phases. They can not only effectively improve the stability and solution dispersibility of the substances encapsulated in the inner layer, but also play a role in the delivery system, enhancing the functional role of the active substances. They have great potential as drug carriers for targeted drug delivery in the treatment of diseases.

Algin protein can be embedded in different materials to improve its stability. CHUNG et al. [49] prepared algin protein liposomes using chitosan. The hydrophilicity of chitosan was used to disperse the liposomes evenly in solution and improve the thermal stability of algin protein. NOGUEIRA et al. [50] hydrated a chloroform solution of soy lectin with trimethylglycine, magnesium chloride, and phycocyanin were hydrated to form phycocyanin liposomes, which improved the stability and specificity of the protein, as well as its antioxidant, anti-inflammatory, and neuroprotective activities. SEYED et al. [51] prepared phycocyanin liposomes using diethylene glycol at 70 °C and found that the resulting liposomes had high stability in terms of sedimentation and suspended particles.

3.4 Other methods

The stability of phycobiliproteins can also be improved by methods such as high pressure. High pressure causes phycobiliproteins to form a more compact protein structure and aggregate with changes in secondary structure. ZHANG et al. [52] studied the effect of high pressure treatment on the structure and color stability of phycobiliproteins, phycobiliprotein whey protein and phycobiliprotein-carrageenan mixtures. High-pressure treatment of phycocyanin-whey protein and phycocyanin-carrageenan formed beneficial aggregates at pH 5.0, which reduced the corresponding phycocyanin loss. This provides a new way to improve the storage stability of phycocyanin under light conditions.

4 Applications

Phycocyanin powder is widely used as a natural water-soluble pigment in the food, cosmetics and pharmaceutical industries. Phycocyanin not only improves the color of food, but also increases its functional ingredients. It also has the effect of stimulating erythrocyte colony formation, replenishing blood and improving lymphatic activity.

4.1 Application in food

Since phycocyanin powder is unstable to light and heat, its application in baked goods is limited. At present, its application is mainly concentrated in dairy products, jelly candies and other foods. MG et al. [53] added phycocyanin to yogurt at pH 4.5. At 4 °C, as the concentration of phycocyanin increased, the viscosity of the yogurt increased accordingly,  resulting in a significant decrease in Streptococcus thermophilus and Lactobacillus bulgaricus at 14th and 21st day, respectively. Alginin can increase the dehydration rate of yogurt and enhance the texture of yogurt. DEWI et al. [54] prepared gel sugar using alginin microcapsules. Maltodextrin and sodium alginate were used as coating materials to prepare alginin microcapsules.  then added 0, 1%, 3% and 5% of the microcapsules to the gel sugar at a temperature of 40 °C. Adding 5% of the microcapsules produced a bright blue color, and the microcapsules had a certain degree of persistence during the processing of the gel sugar.

4.2 Applications in the medical field

Alginin powder is biologically sensitive, biocompatible, bioabsorbable, and has low toxicity to the human body. It can be used as an antioxidant, neuroprotective agent, pancreatic anticancer agent, and can also promote skin wound healing. MADHYASTHA et al. [55] used alginin to conjugate silver nanoparticles to significantly reduce the toxicity of silver nanoparticles,  enhanced the movement of red blood cells to the wound surface, and reduced cell stress at the wound edge. MADHYASTHA et al. [56] isolated cyanopeptide β2 from the β-chain of phycocyanin, which can scavenge free radicals in plasma, enhance iron reduction capacity, and inhibit damage to DNA by reactive oxygen species,  thereby maintaining the integrity of the DNA. FERNÁNDEZ-ROJAS et al. [57] studied the preventive effect of phycocyanin on cisplatin (CP)-induced mitochondrial dysfunction in male CD-1 mice. The study showed that  phycoerythrin can reduce abnormal mitochondrial reactions. LIAO et al. [58] studied the therapeutic potential of phycocyanin as an anti-pancreatic cancer drug in vitro and in vivo. The study showed that phycocyanin exerts anti-pancreatic cancer activity by inducing apoptosis and autophagy cell death, thus confirming that phycocyanin is a promising anti-pancreatic cancer drug.

4.3 Other applications

Phycoerythrin powder has the advantages of high fluorescence quantum yield, high molar extinction coefficient and large Stokes shift, which are superior to many synthetic dyes currently in use. Phycoerythrin is now combined with immunoglobulin, protein A and antibiotic proteins to form fluorescent probes. ZHENG et al. [59] developed a relatively sensitive new fluorescence detection method for detecting phycoerythrin by making the purified phycoerythrin fluorescent probe compatible with the light emitting diode (LED)-charge coupled device (CCD) fluorescence density bar qualitative detection system. This method solves the problem of traditional purification methods  LED)-charge coupled device (CCD) fluorescence density bar qualitative detection system, developed a relatively sensitive new fluorescence detection method for detecting phycobiliproteins. This method solves the disadvantages of the traditional purification method, which is complex, has a low collection rate and insufficient quantity. It can provide convenient quantitative information and shows great potential for rapid detection in environmental and food safety research.

5 Outlook

As a natural pigment, phycocyanin powder can be used as a coloring agent in the cosmetics industry, as an additive in food, and as an anti-inflammatory agent, antioxidant, anticancer agent, immunomodulator and fluorescent detection probe in medicine. As people's understanding of the properties and functions of phycocyanin continues to deepen, its application prospects are becoming more and more extensive. However, stability has become a bottleneck that limits its application, so solving the problem of stability will greatly promote the scope and scale of its application. At present, although the stability of phycobiliproteins can be improved in various ways, such as adjusting the pH, adding stabilizers, cross-linking agents or encapsulating phycobiliproteins, the chemical interactions between phycobiliproteins and stabilizers and food substrates need to be further explored. It remains to be seen whether the stabilizers of phycobiliproteins will affect the nutrition and sensory properties of foods, and whether they can be used under food processing and production conditions. In addition, during the application and promotion of phycocyanin, it is necessary to solve the problems of efficient extraction and purification technology, and to thoroughly explore its bioavailability and the mechanism of its biological activity and effect. It is believed that with the continuous deepening of research on phycocyanin, the market application of phycocyanin will become even more extensive.

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