What Is Phycocyanin?

Spirulina is a genus of the cyanobacteria phylum, cyanophyceae class, segmental organisms order, and trematophyceae family. It is a filamentous, multicellular, spiral prokaryotic algae with high protein content and fast reproduction [1, 2]. Spirulina includes various strains such as Arthrospira platensis, Arthrospira maxima, and Arthrospira salina. Spirulina is rich in protein, fat, vitamins, minerals, chlorophyll, β-carotene, and polysaccharides, and is an ideal food and medicine resource for humans [3].

Spirulina contains phycocyanin, an important light-harvesting pigment protein, which is mainly composed of phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE). Phycobiliproteins are a safe and non-toxic protein resource. Not only are they a valuable edible and feed protein resource in nature, but they also have advantages in the research of the original theory of photosynthesis. Spirulina polysaccharides and phycocyanin are important active substances in spirulina [4]. In recent years, research conducted both domestically and abroad has shown that spirulina has a variety of functions, including anti-fatigue, anti-radiation, anti-viral, anti-tumor, anti-allergy and immunity-enhancing properties, which means that spirulina and its active ingredients have broad application prospects in the research and development of functional foods [5].

Phycocyanin is a type of photosynthetic auxiliary pigment commonly found in cyanobacteria cells. It is a special pigment protein composed of an open-chain tetrapyrrole compound and a dehydrogenase protein bound together by a sulfur chain bond [6]. Its theoretical research and applications have received widespread attention in recent years. The content of phycocyanin in spirulina is as high as 10% to 20%, and it is an important natural pigment for photosynthesis in spirulina cells. It can preferentially transfer light energy to photosystem II with almost 100% efficiency during photosynthesis [7, 8]. Phycocyanin can be widely used as a natural pigment in industries such as food, cosmetics, and dyes. Phycocyanin also has strong fluorescence and can be used to make fluorescent reagents, fluorescent probes, fluorescent tracers, etc., which are used in clinical medicine, immunochemistry, and biological engineering research fields [9, 10]. As an important physiologically active ingredient, it can also be made into medicines for healthcare. Phycocyanin is also an ideal photosensitizer with no toxic side effects [8].

Due to the good development prospects of phycobiliproteins and their high content in spirulina, research on phycobiliproteins in spirulina has become a hotspot in algae protein research. This paper introduces the research progress of spirulina phycobiliproteins in recent years in terms of extraction, purification, physicochemical properties, physiological activity, etc.

1 Extraction

The extraction of spirulina phycocyanin is often divided into two processes: protein dissolution and protein precipitation. Phycocyanin is an intracellular protein. To dissolve it, the cell wall and cell membrane must first be broken so that it dissolves in the extraction solution in a dissolved state. Current research indicates that the extraction of spirulina protein is still in the experimental research stage, and the extraction methods are not the same. Zheng Jiang [11] summarized the methods of cell disruption as: repeated freezing and thawing, chemical reagent treatment, swelling, ultrasonic and tissue crushing methods. There are 5 methods, and the protein precipitation methods are: salting out, crystallization, isoelectric point precipitation and ultrafiltration methods.

1.1 Among the cell disruption methods, the swelling method has a long extraction cycle, and the ultrasonic method has a poor extraction rate. Therefore, the more commonly used methods are the repeated freezing and thawing method and the chemical reagent treatment method, but there are also certain differences between the two. Lin Hongwei [12] et al. used sodium dodecyl sulfate (SDS) to destroy the cell membrane of Arthrospira platensis to extract phycobiliproteins, with an extraction rate of up to 98%, which was significantly better than the control group extracted by the freeze-thaw method. Zhang Yifang [13] and others used a combination of KCl and lysozyme to extract phycobiliproteins from the cell walls of spirulina, achieving a wall-breaking rate of over 95%. Compared with the freeze-thaw method, they concluded that the freeze-thaw method is only suitable for the preparation of small amounts of samples, and it is difficult to quickly freeze and thaw large amounts of samples. The chemical reagent method makes it more difficult to purify the protein later because of the addition of chemical reagents, and improper operation can easily cause protein denaturation. The freeze-thaw method is simple and convenient to operate. Therefore, the freeze-thaw method is more commonly used in experiments to extract small amounts of phycobiliproteins.

1.2 In actual operation, several methods are often used in combination to dissolve phycobiliproteins as much as possible. For example, Gao Tianrong [14] and Wang Yong [15] used repeated freezing and thawing and ultrasound to break the cell walls of spirulina. Lin Hongwei [12] and others used a washing and cyclic freezing and thawing method to extract spirulina phycobiliproteins. The results showed that the extraction yield using Tween 20 as the extraction reagent was 65.1%, which was higher than that using the buffer solution cyclic freezing and thawing method.

After cell disruption, the phycobiliprotein dissolves in the extraction solution. At this time, it is quite important to choose an appropriate method for precipitation. The isoelectric point precipitation method uses the property of proteins having the lowest solubility at their isoelectric point. By adjusting the pH of the solution to the isoelectric point of the phycobiliprotein, the solubility of the phycobiliprotein is reduced and it precipitates. Zhang Yifang [13] and Tang Zhaohui [16] have used this method to precipitate phycocyanin. However, it is generally believed that phycocyanin is sensitive to pH, and poor pH control during precipitation can easily cause protein denaturation. The literature reports more on the use of salting-out to precipitate phycocyanin, and its precipitation effect is also generally recognized.

Ammonium sulfate solution is a commonly used salt solution. Zhang Yifang [13] and others have also used salt solutions such as magnesium sulfate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate to compare with ammonium sulfate solution for salting out. The results showed that ammonium sulfate salting out was effective, while the other salting out methods were less effective. However, there are various opinions on the salting out concentration of ammonium sulfate. Mostly, a 50% saturated ammonium sulfate solution is used for precipitation [4, 12, 17], but some use a 30% to 60% saturation [15, 18], and Lin Hongwei [19, 20] even uses 70% or 80% ammonium sulfate solutions. Hu Yibing [21] and others used ammonium sulfate solutions of different concentrations to establish a stepwise gradient salting-out method to separate and purify the phycobiliproteins of the dinoflagellate, with good results. H.  W. Siegleman [22] believes that salting out with ammonium sulfate solutions of different concentrations can also separate phycobiliproteins from other phycobiliproteins, while Peng Weimin [23] believes that it is impossible to separate phycobiliproteins from other phycobiliproteins by ammonium sulfate salting out. Nevertheless, the extraction of phycobiliproteins from spirulina always involves ammonium sulfate treatment to obtain a crude phycobiliprotein extract.

2 Purification methods

The extract of spirulina protein has a high content of impurity proteins, and the purity ratio (A620/A280) of phycocyanin must be above 4.0 to be of practical value [11]. Therefore, the crude extract must be further separated and purified to remove impurity proteins and increase the purity of phycocyanin. The purification methods currently reported in the literature include hydroxyapatite column chromatography, gel chromatography, ion exchange, and the less commonly used diatomaceous earth column chromatography. In practical applications, it is often necessary to use two or more methods at the same time to achieve better results.

Wei Ping [18] et al. passed the crude extract of phycocyanin through DEAE-Sephadex A-25 and hydroxyapatite (HA) adsorption columns, respectively, and then eluted the phycocyanin fraction once again through a HA column and passed it through a G-150 column to extract phycocyanin from Arthrospira maxima. The results show that a reagent-grade phycocyanin with a purity ratio of up to 4.18 can be obtained by using a homemade secondary hydroxyapatite column, and a phycocyanin with a single component can be obtained by further G-150 column chromatography.

Hu Yibing [21] and others used hydroxyapatite chromatography and Sephadex G-100 gel chromatography to obtain phycocyanin with a purity ratio greater than 5.0. Yin Gang [24] and others used Sephacryl S-200 gel chromatography and hydroxyapatite column chromatography to isolate and purify phycocyanin from artificially cultivated Spirulina platensis, obtaining pure phycocyanin. Zhang Chengwu [4] and others purified the phycobiliprotein twice by HA column chromatography and then purified it once more by Sephadex G-100 column chromatography and filtration to obtain electrophoretically pure phycobiliprotein. Yin Gang [25] and others also studied the use of DEAE Sepharose F F ion exchange and hydroxyapatite adsorption to isolate and purify phycobiliproteins from Spirulina platensis. The phycobiliproteins were identified as electrophoretically pure by isoelectric focusing. Yin Gang [26] and others used hydroxyapatite and Sephadex G-100 for column chromatography to isolate and purify phycobiliprotein with a purity ratio of 4.71.

Peng Weimin [23] and others used hydroxyapatite column chromatography to purify phycobiliprotein from spirulina to obtain phycobiliprotein with high purity. Lin Hongwei [19, 20] et al. first used a diatomite 545 column to fractionally elute and then used DEAE-cellulose ion exchange to purify spirulina to obtain phycocyanin with a purity ratio of 4.1. Zhang Jianping [27] and others first used hydroxyapatite column chromatography and then Sephadex G-150 dextran gel chromatography to obtain a purer phycocyanin. Wang Yong [15] and others studied and established a separation and purification procedure for Sephadex G-200, DEAE-Sephadex A-25, HA, and Sephadex G-200. The results of this method were ideal, with polyacrylamide gel electrophoresis (PAGE) showing a single electrophoretic band, and a purity ratio of up to 14, breaking through the previous maximum value of 10 reported both domestically and abroad. This is also a typical example of the combined use of multiple purification methods.

3. Physicochemical property research

Due to the broad application prospects of phycobiliproteins, the study of their physicochemical properties has become an important topic in the development of spirulina. In recent years, research on phycobiliproteins has delved into molecular composition, and significant progress has been made in the study of other physicochemical properties.

3.1 Spectral property research

Spectroscopy is one of the important characteristics of phycobiliproteins, and the study of spectroscopic properties provides an important basis for the identification of spirulina phycobiliproteins. At the same time, the maximum absorption can also be used for protein content determination, providing a simple and effective method for quality control of phycobiliprotein products. However, due to the differences in phycobiliprotein between different strains of spirulina and the different purity of the phycobiliprotein samples used by researchers, there are also differences in the reported spectroscopic properties.

Yin Gang [26] and others have shown that the ultraviolet-visible spectrum of spirulina platensis phycocyanin has characteristic absorption peaks at wavelengths of 278 nm, 360 nm, and 620 nm. Wei Ping [18] and others found that after purification, the phycocyanin of Spirulina maxima has characteristic absorption peaks at 620 nm and 348 nm after scanning with UV-Vis. Zhang Chengwu [4] and others measured the maximum absorption wavelength of purified phycocyanin of Spirulina platensis at 620 nm by scanning with UV-Vis. Peng Weimin [17] et al. measured the maximum visible absorption peak of the phycocyanin of Spirulina platensis to be 620 nm using a UV-visible spectrophotometer, and measured its fluorescence emission peak at room temperature to be 645 nm using a fluorescence spectrophotometer.

Wang Yong [15] and others found that the absorption wavelength of phycocyanin in salt-tolerant spirulina at pH 7.0 is 615 nm, and that as the pH decreases, the maximum visible absorption peak of phycocyanin shifts to a blue wavelength, and a red shift when the pH increases; the fluorescence excitation peak of phycocyanin under neutral conditions has two peaks at 590 nm and 635 nm, and the fluorescence emission peak has only one peak at 650 nm. Zhang Erxian [28] and others determined that the maximum absorption peak of phycocyanin is at 625 nm, and its fluorescence emission peak is at 648 nm. Yin Gang [24, 26] and others also performed infrared spectroscopy on phycobiliproteins and found that phycobiliproteins have absorption peaks at 3200, 1650, 1550, 1100, 1050 and 650 cm-1, which provides a richer basis for the identification of phycobiliproteins.

3.2 Amino acid composition of phycobiliproteins

Studying the amino acid composition of the protein is conducive to further exploring the internal structure and active groups of phycobiliproteins, and also provides a theoretical basis for other properties of phycobiliproteins. Yin Gang [24, 26], Liu Qifang [29], Li Jianhong [9], and others have all studied the amino acid composition of phycobiliproteins in spirulina. The results show that the amino acid composition of phycobiliproteins in spirulina of different strains is basically the same.

Zhang Chengwu [4] and others analyzed the amino acid composition and content of the spirulina platensis phycobiliprotein and concluded that, except for tryptophan, which was not measured, phycobiliprotein contains 14 amino acids, only trace amounts of histidine and proline, and lacks methionine. Peng Weimin [17] used high performance liquid chromatography to analyze the amino acid composition of phycocyanin in Spirulina platensis. The results showed that the amino acid composition of phycocyanin and phycocyanin was similar, with phenylalanine being the most abundant, followed by aspartic acid, glutamic acid and tyrosine, while proline and histidine were less abundant. At the same time, the ratio of acidic amino acids to basic amino acids in phycocyanin is 2.14, which is higher than the 1.92 in other phycocyanins. Therefore, phycocyanin is considered to be an acidic protein, which also explains why the isoelectric point of phycocyanin is lower than that of other phycocyanins, as reported in the literature [29].

3.3 Biochemical characterization

3.3.1 Isoelectric point

The isoelectric point is one of the most prominent properties of proteins. The reported isoelectric points of spirulina phycocyanin vary, but all are between 3.4 and 4.8 [4, 13, 24, 26, 29]. This may be due to the differences in the properties of phycocyanin in different strains of spirulina, and another reason is that phycocyanin of different purities affects the consistency of the measurement results. The research results also found that the isoelectric point of phycocyanin is generally lower than that of other phycocyanin, which may be related to the composition of amino acids in the protein [17].

3.3.2 Study of phycocyanin subunits

Current research suggests that phycobiliproteins are composed of two subunits with different molecular weights, α and β, and are usually hexamers of two subunits (αβ)6 [15]. However, there are currently different opinions on the molecular weight of the subunits. Zhang Chengwu [4] et al. used 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze purified phycobiliproteins. They found that the phycobiliprotein of Spirulina platensis consists of two subunits, α and β, and that their molecular weights are 14,500 μ and 15,000 μ, respectively. Zhang Erxian [28] measured the molecular weights of the two phycobiliprotein subunits to be 1 4900μ and 17200μ. Peng Weimin [17] performed a regression analysis using the relative migration rate (X) of a standard protein and the logarithm of its corresponding molecular weight (Y) as parameters. The resulting regression equation is: Y = 1.0228X + 5.1255 (R2 = 0.9889). The calculated molecular weight of the α subunit of the phycobiliprotein in the blunt-cap spirulina is approximately 16.3 K D, and the molecular weight of the β subunit is approximately 18.9 K D, which is similar to the reports in the literature [30, 31].

3.4 Stability of phycobiliprotein

Zhang Yifang [13] believes that phycocyanin is stable below 40°C. At 45°C, its pigment begins to decompose, the optical density of the solution gradually decreases, and at 50°C, the optical density decreases rapidly. At 70°C, the optical density of the solution is 75% lower than the original value. A sugar solution can improve the thermal stability of phycocyanin. Light has a relatively small effect on phycocyanin. After 60 hours of exposure to light at 5000 lux, the optical density of the pH 5 solution remains unchanged. Studies have also shown that phycobiliproteins are stable between pH 4.0 and 8.5, with no change in optical density. The color of the solution lightens when the pH is greater than 8.5 or less than 4.0. The above research results show that phycobiliproteins are sensitive to temperature and pH but not light. This finding is of great significance for controlling the conditions during the extraction, purification and preservation of phycobiliproteins.

4 Physiological activity research

Phycocyanin is one of the important active ingredients in spirulina. Clinical research has shown that phycocyanin in spirulina can improve the body's immune system, promote animal cell regeneration, and inhibit the growth of cancer cells [32]. Therefore, it is of great significance to further study the physiological activity of phycocyanin. At present, the focus of research is mainly on the study of anticancer activity, and some progress has been made in the study of other activities.

4.1 Research on anticancer activity

Dong Qiang [33] and others studied the anticancer activity of phycocyanin (PC) on HeLa cells using two methods. The experiment proved that PC has a significant inhibitory effect on the growth of HeLa cells, and when the PC concentration is 80 mg·L-1, the inhibition rate of cancer cells reaches 31.0%. Shen Haiyan [34] and others used a semi-solid agar culture method and MT T assay to determine the effect of spirulina phycocyanin on the growth of human leukemia cell lines HL-60, K-562 and μ-937. The 3 tumor cell types were treated with different concentrations of spirulina phycocyanin under in vitro culture conditions. The results showed that spirulina phycocyanin had a varying degree of inhibitory effect on the 3 tumor cell types, and there was a concentration-dose effect, with a strong inhibitory effect at high concentrations. Guo Baojiang [35] and others studied the inhibitory effect of selenized phycocyanin extracted from selenium-enriched cultivated Spirulina platensis on liver cancer cells. Guo Baojiang [36] and others also studied the inhibitory effect of photo-immobilized phycocyanin on the in vitro liver cancer cell line 7402. The experiment showed that when the initial immobilized phycocyanin concentration was 20μg/w ell, the inhibition rate of 7402 cells reached 55%. As the concentration continued to increase, the inhibition rate of cancer cells decreased. When the concentration of phycocyanin reached 0.5mg/w ell and 1mg/w ell, the inhibition rate rebounded to 55% and 66%.

4.2 Other activity research

Spirulina phycocyanin also has certain activity in other areas. Wang Yuanxun [37] and others found that feeding mice with extracted spirulina phycocyanin significantly improved exercise endurance. Zhang Chengwu [38] proved in an animal experiment that spirulina phycocyanin has anti-radiation effects, and the results also showed that phycocyanin may promote the recovery of hematopoietic function in irradiated animals. Tang Mei [39] and others found that phycocyanin can promote PHA-induced proliferation of normal mouse splenic lymphocytes, enhance the hemolytic ability of spot-forming cells and the content of hemolysin in the serum, and significantly resist the damage of hydrocortisone to the body's immune function.

Zhao Jingquan [40] and others used competitive reaction kinetics to study the scavenging effect of phycocyanin in spirulina against hydroxyl radicals. The results showed that phycocyanin has a strong scavenging effect on hydroxyl radicals, and the measured scavenging reaction rate constant was between (2.8–5.6) × 109L ·mol–1 ·S–1. Tang Mei [41] and others studied the effect of spirulina phycocyanin (PC) on the function of human peripheral lymphocytes. The results showed that PC can promote the effect of PHA on stimulating lymphocyte transformation, and there is a dose-dependent relationship. PC can restore the ability of T cells to form E rosettes after cyclophosphamide damage, especially the ability to form active E rosettes (Ea).

Experimental studies have shown that spirulina phycocyanin has physiological activities such as anti-tumor, anti-radiation, anti-fatigue, immunity enhancement and free radical scavenging, which provides an important basis for decision-making in the development of spirulina in the fields of functional foods and pharmaceuticals.

5 Research on phycocyanin products [42, 43]

Phycocyanin is an important active ingredient in spirulina, and its unique physical and chemical properties are valued in product development research. The School of Life Sciences at Peking University used spirulina sludge to prepare and purify phycocyanin monomers, which were coupled with purified DFI antibodies. The conjugates were then purified to obtain phycocyanin-labeled antibodies as fluorescent probes. The Institute of Chemical Metallurgy of the Chinese Academy of Sciences and the Institute of Oceanology of the Chinese Academy of Sciences have also carried out research on the development of fluorescent markers and diagnostic reagents for phycobiliproteins, and research on diagnostic reagents and diagnostic kits (screening, labeling, and detection techniques for fluorescent reagents). It is hoped that a technology and a popular phycobiliprotein-labeled hepatitis B virus surface antigen diagnostic kit that can replace other fluorescent markers and enzyme markers can be obtained. At the same time, spirulina protein has made great progress in food research, especially functional food research. There are already more than 10 kinds of spirulina tablets and capsules in China, which have been approved by the Ministry of Health as health products according to different functions. However, since the extraction of spirulina protein is still in the experimental research stage, there is no suitable process method for industrial production, making spirulina protein expensive and limiting its application to a certain extent.

6 Conclusion

Research on spirulina in China began in the 1970s. Although there have been significant developments over the past 30 years, most of the research is still at the laboratory stage. According to literature reports, it is difficult to extract and purify the active ingredient spirulina phycocyanin, and it will take some time to develop a mature production process. There are also not many functional foods based on spirulina phycocyanin, and research and development in the pharmaceutical field is still in its infancy. Therefore, in the next few years, research and development of phycocyanin will focus on the following areas: 1. Explore a method for the industrial production of large quantities of phycocyanin to reduce its cost and promote its widespread development and utilization. 2. Based on the results of research on the activity of phycocyanin, expand the development of phycocyanin from functional foods to pharmaceuticals and the development of medical diagnostic reagents to further develop its utilization value. Third, continue to conduct in-depth research on the physical and chemical properties of phycocyanin and establish a sound quality control method to provide quality assurance for the research and production of phycocyanin products, especially pharmaceuticals.

References

[1] Yao Baozhen. Nutritional evaluation and health-promoting function of spirulina [J]. Food Research and Development, 1998, 6(2): 2.

[2] He Jia, Zhao Qimei, et al. Research on the stability of spirulina phycocyanin [J]. Acta Biologica Sinica, 1998, 15(5): 17.

[3] Claudio S, Mass production of Spirulina [J], Experientia, 1982, 38: 40-43.

[4] Zhang Chengwu, Zeng Zhaqi, et al. Isolation, purification and physicochemical properties of spirulina platensis phycobiliproteins [J], Natural Product Research and Development, 1996, 8(2): 29-34.

[5] Hou Jianshe, Xue Fengzhao, et al. The main physiological regulatory functions of spirulina [J], Food Research and Development, 2001, 22 (1): 31-34.

[6] Yi Guoliang, Jiang Lijin. Model reaction of algal phycobiliprotein biosynthesis [J], Acta Chimica Sinica, 1991, (49): 94-97.

[7] Zeng Fanjie, Lin Qishan, et al. Isolation and characterization of R-phycoerythrin from the red alga Porphyra dentata [J], Acta Biochimica et Biophysica Sinica, 1992, 24 (6): 545-551.

[8] Glazer A N. A macromolecular complex optimized for light energy transfer [J]. Biochim Biophys Acta, 1984, 768: 29-51.

[9] Li Jianhong, Tai Zihou, et al. Properties of the great spirulina phycobiliprotein [J]. Journal of Nanjing University, 1996, 32(1): 59-63.

[10] Zhang Chengwu, Yin Zhimin, et al. Development and utilization of phycobiliproteins [J], Chinese Journal of Marine Drugs, 1998 (4): 26-29.

[11] Zheng Jiang. Research progress in the extraction and purification of phycobiliproteins [J], Food Science, 2002, 23 (11): 159-161.

[12] Lin Hongwei, Wu Zhengqing, et al. A new process for extracting spirulina blue protein [J], Guangxi Chemical Industry, 1997, 26(4): 5-7.

[13] Zhang Yifang, Liu Xuchuan, et al. Extraction and stability test of spirulina protein [J], Journal of Yunnan University (Natural Science Edition), 1999, 21(3): 230-232.

[14] Gao Tianrong, Wei Xiaokui, et al. Research on the comprehensive utilization process of spirulina [J], Journal of Yunnan Normal University, 2002, 22(2): 42-43.

[15] Wang Yong, Qian Kaixian, et al. Research on the separation and purification of high-purity phycocyanin and its spectral characteristics [J], Progress in Biochemistry and Biophysics, 1999, 26(5): 457-460.

[16] Tang Zhaohui, Jiang Jialun. Preliminary report on the extraction and characterization of spirulina platensis phycobiliproteins [J].

[17] Peng Weimin, Shang Shutian, et al. Study on the properties of spirulina platensis phycobiliproteins [J].

[18] Wei Ping, Li Huan, et al. Extraction and purification of phycocyanin from Spirulina maxima [J]. Journal of Nanjing University of Chemical Technology, 1999, 21(3): 62-64.

[19] Lin Hongwei, Qin Haicuo, et al. A new process for the extraction and purification of phycobiliproteins from Spirulina platensis [J], Fine Chemicals, 1998, 15(1): 18–20.

[20] Lin Hongwei, Liang Hong. Separation and purification of phycobiliproteins from Spirulina platensis [J], Guangxi Chemical Industry, 2002, 31(1): 30–31.

[21] Hu Yibing, Hu Hongjun, et al. Research on the large-scale extraction and purification of phycobiliproteins from a spirulina rich in phycobiliproteins [J], Wuhan Botanical Research, 2002, 20 (4): 299-302.

[22] Siegelman H W, Kycia J H. Algal biliproteins: handbook of phycological methods (Edited by Johan A H) [J]. Combridge U niversity press, 1978, 71-79.

[23] Peng Weimin, Shang Shutian, et al. Extraction of phycobiliproteins from the dinoflagellate Sp.~D (Spirulina platensis) [J], Food Science, 1999 (6): 48-49.

[24] Yin Gang, Liu Zheng, et al. Isolation, purification and characterization of phycobiliproteins from the dinoflagellate Spirulina platensis [J], Journal of Tsinghua University (Natural Sciences), 1999, 39 (6): 20-22.

[25] Yin Gang, Yin Jin, et al. Study on the purification of phycobiliproteins from spirulina by anion exchange and hydroxyapatite chromatography [J], Ion Exchange and Adsorption, 2000, 6 (2): 128-133.

[26] Yin Gang, Li Hui, et al. Separation and purification of phycobiliproteins and polysaccharides from spirulina and product characteristics [J], Fine Chemicals, 1999, 16 (2): 10-13.

[27] Zhang Jianping, Zhang Jingmin, et al. Isolation and structural characterization of R-phycoerythrin [J], Biophysical Journal, 1997 (6): 173-178.

[28] Zhang Erxian, Chen Yanli, et al. Purification of phycobiliproteins from Spirulina platensis and their scavenging of free radicals [J], Taiwan Strait, 1999, 18 (2): 172-176.

[29] Liu Qifang, Wang Houle, et al. Isolation and characterization of phycobiliproteins from Spirulina salina [J], Acta Hydrobiologica Sinica, 1988, 12 (2): 146-153.

[30] HIL DITCH C M, SMITH A J, et al. Phycocyanin from the cyanobacterium aphanothece halophytic [J], Phytochemistry, 1991, 30(11): 3515-3517.

[31] XIA A D, ZHOU J C, et al. Energy transfer kinetics in phyconyanin from cyanobacterium w estiellopsis prolific studied by pump-probe techniyues [J], Biochemical and Biophysical Research Communications, 1991, 179(1): 558-564.

[32] Schwarty J L, Shkiar G. Growth inhibition and destruction of oral cancer cells by extracts of Spirulina [J]. Proc Amer Acad Oral Pathol, 1986, 40: 23-27.

[33] Dong Qiang, Qian Kaixian, et al. Research on the anticancer activity of phycocyanin [J], Journal of Zhejiang University (Engineering Science), 2001, 35 (6): 672-675.

[34] Shen Haiyan, Wang Xixia, et al. Effect of spirulina phycocyanin on the growth of human leukemia cell lines HL-60, K-552 and μ-937 [J]. Marine Science, 2000, 24 (1): 45-48.

[35] Guo Baojiang, Wu Yanqing. Research on the anti-liver cancer effect of selenized phycocyanin as a biomaterial [J], Journal of South China Normal University (Natural Science Edition), 2001 (3): 40-44.

[36] Guo Baojiang, Xu Hede, et al. The inhibitory effect of photo-immobilized phycocyanin on the in vitro liver cancer 7402 [J], Ion Exchange and Adsorption, 2000, 16 (6): 547-552.

[37] Wang Yuanxun. Research on Spirulina to improve exercise tolerance [J], Journal of the Chinese Society of Sports Science, 1985, 4: 20–21.

[38] Zhang Chengwu, Zeng Zhaqi, et al. Protective effect of spirulina platensis polysaccharide and phycocyanin on acute radiation sickness in mice [J], Journal of Nutrition, 1996, 18(3): 327–330.

[39] Tang Mei, Jin Ying, et al. The effect of spirulina phycocyanin on the immune function of mice, Journal of Jinan University (Natural and Medical Sciences) [J], 1998, 19 (5): 93-97.

[40] Zhao Jingquan, Zhang Jianping. Study of the reaction kinetics of phycobiliproteins with hydroxyl radicals using pulsed photolysis [J]. Science Bulletin, 2000, 45(1): 32-36.

[41] Tang Mei, Jin Ying, et al. The effects of spirulina chlorophyllin and algin on the function of human peripheral blood lymphocytes [J], Journal of South China Normal University (Natural Science Edition), 1998 (4): 63-67.

[42] Li Dingmei. Overview and prospects of the development of China's microalgae industry (1) [J]. Grain and Feed Industry, 2001, (5): 26-27.

[43] Hou Jianshe. The development and current situation of spirulina food research and development in China [J]. Food Research and Development, 2000, 21(4): 23-26.