Chemical composition of the algae Arthrospira platensis .... ABSTRACT Based on the results of this study on the extraction of C-phycocyanin from Arthrospira platensis, the mechanical ex
INTRODUCTION
Preface
Since ancient times, people have utilized nutritious foods and natural remedies to promote health and combat illness With modern advancements in science and technology, extensive research has been conducted to discover rare natural substances with significant medicinal applications One such substance is C-phycocyanin (C-PC), a key component of phycobiliproteins found in various species of blue-green algae, especially Spirulina.
Arthrospira platensis is a pigment known for its high concentrations and extensive applicability across multiple sectors Numerous studies have demonstrated its effectiveness in pharmaceuticals, food products, and cosmetics, particularly in whitening creams and antioxidants Additionally, it has shown promise in medical applications, including cancer treatment.
Phycocyanin extraction methods have been extensively researched, yet many remain complex and suitable only for laboratory-scale experiments, making them impractical for industrial production As a result, Vietnam currently relies on costly imports of phycocyanin for commercial use, lacking the ability to source it domestically.
This study aims to present a simple, safe, and effective method for extracting phycocyanin and exploring its purification into powder form Key aspects include selecting optimal extraction conditions, preparation, refining processes, and product preservation The anticipated outcomes are expected to significantly advance the extraction of natural pigments for use in food, medicine, and large-scale industrial production.
Objectives
Finding a method to obtain C-phycocyanin simple, safe, effective and can be applied in industrial production
Find out a method to preserve C-phycocyanin into a solid form to preserve the preparation.
Requirements
Conducted various experiments to find a simple, efficient extraction method to obtain the optimal amount of C-phycocyanin Ensure technical when conducting experiments, experimental results must be accurate and truthful.
LITERATURE REVIEW
An overview of Arthrospira platensis
Spirulina (A platensis) has been revered since ancient times as a superfood, rich in high-quality protein, lipids, essential minerals, and bioactive compounds like phycocyanin, beta-carotene, chlorophyll, and gamma-linoleic acid It is also a source of valuable pigments, including vitamins B and E The phycocyanins in Spirulina are widely used as a natural edible colorant, celebrated for their vibrant blue hue and remarkable antioxidant, anti-cancer, and anti-inflammatory properties.
Spirulina, as highlighted by Lee et al (2013), is extensively researched and utilized across various sectors, including food production, nutritional supplements, and cosmetics It is celebrated as an ideal functional food, known for its essential health benefits.
WHO as the best food for mankind in the 21 st century (N Seyidoglu et al.,
Spirulina, comprising the species A platensis and A maxima, is a spiral-shaped cyanobacterium found in various water bodies globally It thrives in both freshwater and saltwater environments, particularly in alkaline conditions with a pH range of 8.5 to 11 A platensis has been utilized for food since ancient times, dating back approximately 3.5 billion years The species was first described in 1940 by French phycologist Dangeard, who obtained a sample from a colleague in equatorial Africa near Lake Chad In 1964, botanist Jean Léonard discovered edible greenish cakes made from A platensis being sold in local markets in Fort-Lamy, now known as N'Djamena, Chad.
In Vietnam, A platensis has been studied since the 1970s After the
1970s, there were many studies on Spirulina Recently, at the Institute for
In a study conducted by scientist Nguyen Duc Bach and colleagues in 2020, the application of red LEDs was found to effectively extend the cultivation time of Spirulina in Northern Vietnam As a result, several farms dedicated to the cultivation of A platensis have emerged in the country, including notable examples such as Vinh Hao in Binh Thuan province and Vnua Pharma in Hanoi.
Table 2.1 Scientific classification of Arthrospira platensis
The genus Spirulina comprises over 35 species, with the two most studied being S geitleri (S maxima), native to Africa, and S platensis, native to South America Other notable species include S prpvilca found in Peru, S jeejibai in Germany, S subsalsa in Ukraine, S laxissima in Kenya, and S pacifica in the United States.
The cell structure of Spirulina platensis was described in figure (2.1), (R.A
In 1953, Lewin noted that S platensis cells are cylindrical and organized in filaments, featuring a prominent central vacuole The thin and flexible cell wall of S platensis enables the cells to bend and twist, adapting to water currents.
Figure 2.1 Structure of Spirulina (Arthrospira platensis)
A platensis typically exhibits a spring-like, cyan-colored twist with uniform spirals, though its shape can vary slightly based on environmental conditions and developmental stages The algae fibers may elongate or twist, resulting in different forms such as a "C" or "S," but they consistently remain unbranched and heterocellular (S Boussiba et al., 1979).
In terms of size, adult algae are usually long from 250 1000 m, width
A platensis exhibits a spiral diameter ranging from 35 to 50 µm and a twisting step of 60 µm, with variations depending on the degree of torsion or straightness Structurally, it consists of nearly 100 cells per algae fiber, and its cells lack a typical nucleus with clearly defined boundaries (D Kumar, et al., 2014).
A platensis does not have chloroplasts but instead thylakoid bodies arranged in rings containing the pigments chlorophyl, phycocyanin, carotenoids Cell membranes also do not have a plant-like cellulose wall, but rather layers of peptidoglycan that are easily assimilated by digestive enzymes Because of these characteristics, A platensis is classified as a bacteriology, not algae as before (M.A.B Habib, et al., 2008)
A platensis exhibits two primary motor characteristics: free torsion, which involves a shape change from torsion to straight, and translational movement in aquatic environments facilitated by cylindrical air cells (D Kumar et al., 2014) This organism can achieve a movement speed of up to 5 microns per second, utilizing fimbria—fibers measuring 5-7 nm in diameter and 1-2 microns in length—located along its body These bristles function as paddles, enabling the cyanobacteria to navigate effectively (C D'souza et al., 2018).
2.1.3 Chemical composition of the algae Arthrospira platensis
A platensis is recognized as the most nutritionally complete food globally, boasting over 50 micronutrients, surpassing all other foods, including green vegetables, nuts, and herbs (J.C Dillon, et al., 1995).
A.platensis contains about 60% protein (protein) which is a higher source of texture than beef (18%), poultry (19%), fresh milk (3.7%) and eggs (14%) In particular, the protein in the algae A platensis is a synthesis of more than 18 amino acids, of which 8 are essential and all are easily digestible (up to 95%) due to the nature of vegetable protein (A Vonshak, 1997)
Table 2.2 Amino acid composition in Arthrospira platensis
Source: The role of Parry Organic Spirulina(A platensis) in Health
A platensis is a nutrient-dense superfood, rich in essential vitamins including vitamin A, vitamin E, and the B complex vitamins (B1, B2, B6, B12), boasting twice the B12 content of beef liver and 20 times the beta-carotene found in carrots Additionally, it contains double the vitamin E of wheat germ and is packed with vital minerals such as potassium, calcium, magnesium, and zinc This algae is also a significant source of essential fatty acids like GLA and dietary fiber (S.M Hosseini et al., 2013).
Table 2.3 Vitamin composition in Arthrospira platensis Vitamin composition Content (mg/100g)
Source: The role of Parry Organic Spirulina (A.platensis) in Health
A platensis is rich in essential anti-aging compounds, including phycocyanin, chlorophyll, and carotenoids, which play a crucial role in cell protection, setting it apart from other natural substances (J.C Dillon, et al., 1995).
Table 2.4 Pigmentation composition in Arthrospira platensis
Source: The role of Parry Organic Spirulina (Arthrospira platensis) in Health
2.1.4 The applications of Arthrospira platensis
Unique phytonutrients from the algae A platensis enhance the immune system and promote overall health by providing mild detoxification benefits for conditions such as kidney failure and hepatitis, while also lowering cholesterol and alleviating diffuse polyarthritis This algae aids in the prevention of atherosclerosis and supports weight management by satisfying the body's nutritional needs, thereby reducing appetite Additionally, A platensis is effective in managing diabetes, anemia, gastric and duodenal ulcers, pancreatitis, cataracts, and visual impairment, while also addressing hair loss It lowers cancer risk and serves as supportive nutrition for cancer patients undergoing treatments, enhancing the kidneys' ability to eliminate toxins Furthermore, it improves metabolism and combats malnutrition, making it beneficial for athletes by increasing physical strength and endurance.
An overview of C-phycocyanin
C-phycocyanin is a blue pigment found in cyanobacteria, rhydophyta, and cryptophyta (M Kuddus, et al., 2013) This pigment gives cyanobacteria their bluish color and is also known as cyanobacteria C-PC is formed by sulfur binding of the Cistein component in the polypeptide sequence with the vinyl group-bearing carbon of the tetrapyrol phycocyanobilin (C Romay, et al.,
2003) The structure of C-PC is shown in figure (2.2)
C-Phycocyanin (C-PC), a water-soluble pigment found in Spirulina algae, aggregates into groups and binds to cell membranes to form phycobilisomes This compound exhibits fluorescent and antioxidant properties, making it valuable in various industries C-PC and other phycobiliproteins are widely utilized in food, cosmetics, biotechnology, diagnostics, and pharmaceuticals.
The blue molecule is formed by three or more amino acids connected by peptide bridges, leading to various groups of phycobiliproteins The three primary types are phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), each associated with specific pigments Notably, phycocyanin, which contains the pigment phycocyanobilin, is referred to as C-phycocyanin (C-PC) and has a molecular weight of 232,000 Dalton C-PC exhibits a maximum visible absorbance between 615 nm and 620 nm, with peak fluorescence emission around 652 nm.
Phycocyanin is a pigment with a flattened elliptical structure, consisting of α subunits (12,000-18,500 Da) and β subunits (14,000-20,000 Da), typically found in a hexameric form denoted as (αβ)₆ It can also exist in trimeric (αβ)₃ and dimeric (αβ)₂ forms The molar mass of the monomer is approximately 44 kDa.
The phycobiliprotein has a molecular weight of 132 kDa, while its hexamer form weighs 260 kDa, with an isoelectric point of 5.8 At this pH, the protein tends to clump, impairing its water interaction capabilities It exhibits strong absorption in the wavelength range of 615-620 nm and luminescence at 647-652 nm, remaining stable within a pH range of 5.0-7.5 and a temperature of 25 ± 2 ℃ Deviations from these conditions can lead to color loss or denaturation of phycocyanin (N.T Eriksen, et al., 2008).
Food fillers and functional foods
C-PC extracted from A platensis is considered a food and cosmetic colorant in Japan but is still not recognized in many places, such as Europe Moreover, C-PC is gradually replacing synthetic color This pigment is also used in many fermented dairy products in many countries around the world Several studies have shown the function of C-PC in foods as a colorfast and rheological agent The C-PC is also used as a nutritional ingredient, especially in functional foods, dried biomass of A platensis is used as a functional ingredient in food In addition to nutritional value, C-PC also has the effect of increasing the body's immunity, anti-oxidant, anti-inflammatory, anti-viral, anti- cancer and lowering blood cholesterol ( Z Khan, et al., 2005)
C-PC is an essential nutrient and pharmaceutical ingredient with antioxidant and free radical scavenging capabilities These properties are attributed mainly to the phycobiliprotein group: C-PC is bleached during free radical damage, the antioxidant activity of free phycocyanobilin is comparable to that of phycocyanobilin bound in C-PC and the activity The antioxidant activity is increased by denaturation or trypsin hydrolysis of C-PC The free radical scavenging capacity of C-PC is found in Se-rich C-PCs, also known as Se-PCs Se-PCs obtained from A.platensis were grown in Se-enriched media
Both organic and intangible Se are present in pure Se-PC but not Se-amino acids Se-PC's free radical scavenging ability is due to its covalent attachment to
Se and its compounds (C Romay, et al., 2005)
2.2.4 Some studies on the use of C-phycocyanin
The study on C-PC's effects on mucosa, immune response, and allergic inflammation in C3H/HeN and BALB/cA mice revealed that C-PC enhances the secretion of IgA antibodies while decreasing IgE antibodies This indicates that C-PC boosts immune system activity against inflammatory diseases and mitigates allergic inflammation by lowering IgE levels (S Ayehunie, 1998).
Anti-oxidant and anti-inflammatory
The study of C-phycocyanin (C-PC) derived from A platensis highlights its significant antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective properties C-PC effectively neutralizes alkoxyl, hydroxyl, and peroxyl radicals, while also inhibiting lipid oxidation in mice Through 12 experiments, the anti-inflammatory effects of C-PC were clearly established, attributed to its capacity to eliminate oxidants and inhibit the activity of the COX-2 enzyme (S Ayehunie, 1998).
Protects heart and blood sugar
A study compared the effects of casein, A platensis, acetone extract from A platensis, and C-PC from A platensis on cholesterol solubility, absorption, and metabolism Results showed that A platensis significantly reduced cholesterol solubility in vitro and absorption in Caco-2 cells compared to casein Additionally, mice fed A platensis exhibited lower serum total cholesterol and atherogenic index, while beneficial cholesterol levels were higher than those in casein-fed mice (R González, et al., 2005).
C-PC is more effective than Spirulina in lowering blood cholesterol levels The authors concluded that lower cholesterol solubility, lower cholesterol absorption, and increased fecal steroid excretion were observed in the presence of A.platensis or C-PC There are studies that have demonstrated that, with the provision of chromium-rich A.platensis, it is beneficial to the blood sugar, total cholesterol and triglycerides in the blood of patients
Anti-cancer and anti-virus
In April 1996, researchers from the Microbiology Laboratory at Dana-Farber Institute of Oncology and Harvard Medical School, along with Earthrise Aquaculture, announced their investigation into the water extract from Spirulina (A platensis) They found that this extract can prevent HIV-1 replication in transformed T cell lines and peripheral blood mononuclear cells, effectively inhibiting viral replication at concentrations of 5-10 µg/ml This significant finding indicates that even small amounts of A platensis extract can reduce and completely halt the reproduction of the AIDS virus (HIV-1) (S Ayehunie et al., 1998).
The A platensis extract has been shown to be non-toxic to human cells while effectively preventing viral replication Research has highlighted an aqueous extract known as calcium-spirulan, which inhibits the replication of various viruses, including HIV-1, herpes simplex virus, cytomegalovirus, influenza A, mumps, and measles, in vitro Additionally, this extract has demonstrated protective effects against viral infections in both human and monkey cell cultures.
Scientific journals indicate that calcium-spirulan, a unique polymerized sugar molecule derived from A platensis containing sulfur and calcium, is an effective treatment for HIV-1 and HSV-1 infections in severely ill AIDS patients Research shows that field mice infected with the fatal herpes virus 9 exhibit rapid recovery when treated with this soluble essence (N.T Eriksen, et al., 2008) In Vietnam, the Research Institute for Technology Application is actively involved in this research.
(Ministry of Environmental Science & Technology) has succeeded in extracting phycocyanin from A platensis to treat cancer of the jaw and nasopharyngeal areas
Resistant to heavy metals, radioactive substances and toxins contained in drugs
The study was performed on arsenic poisoning patients with a combination of A platensis extract (250 mg) and zinc (2 mg), twice daily, for
The treatment's effectiveness was evaluated over 16 weeks by monitoring changes in skin manifestations, including tanning and keratosis, as well as the increase in arsenic levels excreted, with up to 47% of arsenic discharged from the scalp The findings indicated significant improvements in skin pigmentation and keratosis (A Herrera, et al., 1989).
Studies on C-phycocyanin extract in the world and Vietnam
2.3.1 Studies on C-phycocyanin extract in the world
Numerous global studies have focused on optimizing the extraction of C-phycocyanin, particularly through innovative methods A notable study by Y.K Kim et al (2015) demonstrated that combining ultrasonic and heat treatments effectively extracted high yields of C-phycocyanin from Spirulina, achieving yields between 10-15%.
Another study by M Qiu et al., (2016) compared the extraction methods of C-phycocyanin from Spirulina using ultrasonic and microwave treatments
Microwave treatment proved to be more effective in extracting C-phycocyanin, achieving yields of up to 25%, surpassing ultrasonic treatment In contrast, a study by L Chen et al (2017) demonstrated that a combination of high-speed centrifugation and ultrasonic extraction from Spirulina resulted in yields of up to 20%.
2.3.2 Studies on C-phycocyanin extract in Vietnam
In Vietnam, research on C-phycocyanin extract has highlighted its potential uses in the food and cosmetic sectors A study by Nguyen et al (2018) focused on the extraction and purification of C-phycocyanin from Spirulina, revealing its significant antioxidant properties The findings indicated that C-phycocyanin could serve as an effective natural antioxidant in the food industry.
A study by T.M Hoang et al (2020) highlighted the benefits of C-phycocyanin extract in cosmetics, revealing its protective effects on skin cells against UV-B radiation This positions C-phycocyanin as a promising natural ingredient for sunscreens and skincare products, showcasing its potential in the cosmetic industry.
MATERIALS AND EXPERIMENTAL METHODS
Materials
The dried biomass of A platensis was obtained from the Institute for
Microalgae and Pharmacosmetics (IMPC), Vietnam National University of Agriculture Dried algae biomass is packed, vacuum sealed and stored in a cool room
3.1.2 Place and time of experiment
Location: Institute for Microalgae and Pharmacosmetics
Chemicals and equipments
Chemicals: Distilled water; NaH 2 PO 4 ; Na 2 HPO 4 ; NaCl; MgCl 2 - all chemicals are made in China; C-phycocyanin
Table 3.1 List of equipments used in the experiment
7 Sublimation dryer HT-FD6 Vietnam
Research methods
The extraction of C-phycocyanin involves several key steps: cell disruption, protein precipitation, purification, and concentration (D.P Jaeschke, et al., 2021) Initially, the cells of blue-green algae or cyanobacteria must be disrupted, which can be achieved through mechanical, chemical, or enzymatic methods The selection of the appropriate method is influenced by the specific type of algae or cyanobacteria used and the target yield of C-phycocyanin.
After disrupting the cells, the subsequent step involves precipitating the phycocyanin protein by adjusting the pH or salt concentration to render the protein insoluble The resulting precipitated C-PC is then isolated from the solution using techniques like centrifugation, filtration, or sedimentation (D.P Jaeschke, et al., 2021).
Some research methods proposed by many scientists are:
The centrifugation method is an effective technique for separating C-phycocyanin from algae, as noted by R Sathasivam et al (2018) This method utilizes centrifugal force to differentiate components of the algae mixture based on their density C-phycocyanin's higher density compared to other algae components facilitates its separation Consequently, this method achieves a high concentration of C-phycocyanin, making it ideal for large-scale production.
S Aishwarya, et al., (2017) suggested that the drying method is a simple and cost-effective approach to extract C-phycocyanin from algae This method involves drying the algae at a low temperature to preserve the quality of the pigment The dried algae are then ground to a fine powder, and the C-phycocyanin is extracted using a suitable solvent The advantages of this method include its simplicity, low cost, and the use of basic equipment However, the yield of C- phycocyanin using this method may be lower compared to other methods
N Mallick, el al., (2007) proposed that the mechanical grinding method using a grinder or blender is a straightforward approach to extract C-phycocyanin from algae This method involves grinding the algae in a blender or grinder to break down the cell walls and release the pigment The ground algae are then extracted using a suitable solvent, and the C-phycocyanin is separated from other pigments and impurities This method is easy to perform, and does not require expensive equipment However, the yield of C-phycocyanin using this method may be lower compared to other methods such as the centrifugation method
The extraction procedure of C-PC proposed in this study is based on the experiment of N Mallick, et al., (2007), and the implementation process is depicted in figure (3.1)
Figure 3.1 Schematic diagram of C-PC extraction process in
The process of extracting C-PC according to the diagram in figure (3.1) is basically as follows:
Dried algal biomass was immersed in various extraction solvents, including distilled water, phosphate buffer, NaCl, and MgCl2, followed by centrifugation to eliminate residue This process yielded C-phycocyanin (C-PC), which formed distinct layers on the surface The extract's optical density (OD) was measured at wavelengths of A620nm, A650nm, and A280nm to assess concentration and purity These results were utilized to optimize the extraction conditions for maximum C-PC efficiency.
3.3.2 Factors affecting the extraction of C-PC
Dried algal biomass was dissolved in various solvents, including distilled water, 0.1M phosphate buffer, 0.9% NaCl, and 1.2% MgCl2, and then incubated in a ring extract at 4°C for 4, 8, and 12 hours The C-PC extract underwent centrifugation at 1900 rpm for 30 minutes, after which the extract was collected for analysis of C-PC concentration and purity coefficient.
The experimental process is described by the following steps:
Step 1: Test tubes 1, 2, 3, 4 are numbered corresponding to the following solvent conditions: Distilled water, phosphate buffer 0.1M, NaCl solution
Step 2: Take 2 (g) dried algae into test tubes, then proceed to put 40 ml of different solvents into test tubes, shake well
Step 3: Wrap the test tubes in sealed paper, store in the refrigerator for 4 hours
Step 4: After extracting within 4 hours, the test tubes were obtained, then centrifuged by YSCF-TD4 machine for 20 minutes at 1900 (rpm)
Step 5: After centrifugation, collect the supernatant C-PC extract and measure the OD values of the samples at different values: A620nm, A650nm and A280nm
Step 6: Repeat the experiment with different conditions
Choose the optimal salt concentration
Investigation conditions: The fixation condition has been optimized in the above experiment, examining the solvent at different salt concentrations: 0.6; 0.9; 1.2 and 1.5 (%)
Choose the optimal extraction time
Extraction conditions were optimized based on prior experiments, with algae biomass soaked for varying durations of 4, 8, 12, 16, and 24 hours Following the soaking process, the extract was centrifuged, and the resulting solution was collected for analysis of C-PC concentration and purity.
Choose the optimal biomass-solvent volume ratio
Varying the biomass mass allows for the determination of different ratios of biomass to solvent volume In the conducted experiments, fixed conditions were optimized while surveying various weights of algae, specifically 1.50 g, 2.00 g, 2.50 g, 3.00 g, and 3.50 g.
3.3.3 Investigate the method of processing C-PC solution into dry powder 3.3.3.1 Treatment of C-PC solution into dry powder by freeze-drying method
This study evaluates the quality and content of C-PC before and after treatment using a sublimation dryer (HT-FD6) The drying process involved a duration of 36 hours, with a negative drying temperature ranging from -30℃ to -40℃ and a positive drying temperature of 20℃ The standard solvent exhibited a C-PC content of 3.15 mg/mL and a purity of 0.63 The methodology for converting the C-PC solution into powder is detailed in the study.
To begin the process, centrifuge 40 mL of the extracted C-PC solution to eliminate any residue Next, spread the C-PC solution thinly on a petri dish and place it in a metal tray, then freeze it at temperatures between -30℃ and -40℃ until the solution solidifies completely.
For optimal water evaporation, it is essential to create a vacuum environment with pressure below 50 Pa and temperatures below 0℃ This vacuum drying process effectively removes 90% of the water content in C-PC.
Step 3: The temperature in the chamber will be increased, dried to 20℃, the residual water in C-PC will be removed and kept until the water content in C-PC is about 1-5%
After sublimation drying, the C-PC will crystallize into a powder form and be vacuum packed to safeguard against moisture and oxidation It is essential to conduct a quality assessment of the resulting C-PC preparation.
PC content in the solution or the purity
3.3.3.2 Treatment of C-PC solution into dry powder by natural evaporation method
This method involves maintaining C-PC levels by naturally evaporating water from the solution, which initially contains a C-PC concentration of 3.15 mg/mL and a purity of 0.63 The experimental procedure is carried out systematically to achieve the desired results.
Step 1: Take 40 mL of C-PC solution and spread a thin layer on a metal tray The metal tray is then placed in a cold room of 20℃ and protected from direct light
After approximately 24 hours, the C-PC solution will have evaporated and dried, leaving the C-PC residue on the surface At this point, scrape off the C-PC to obtain it in solid form Place the collected C-PC into a bubble bag, vacuum seal it, and store it at 4℃ while protecting it from light.
Step 3: Conduct an assessment of the quality of the obtained C-PC preparation such as the C-PC content in the solution or the purity
The concentration and purity factor of C-PC was determined by UV-Vis spectrophotometer as follows:
The content of PC (mg/mL) = (A 620nm - 0.7A 650nm )/7.38
The purity factor of PC is calculated according to the ratio of
In which, A620nm and A650nm are the maximum absorbance and maximum luminescence of PC, and A280nm is the absorbance of total protein, respectively (J Abaldeet al., 1998)
The purity coefficient of C-PC is calculated as the ratio of A 620nm /A 280nm , a ratio between 0.6 and 0.8 is considered pure (W Song et al., 2013)
The results of the experiments were processed and graphed using Microsoft Excel 2016.
RESULTS AND DISCUSSION
The results of selecting the optimal conditions for the extraction
C-phycocyanin release occurs when algal cells are mechanically broken down and is significantly affected by the choice of solvents The selected solvents are crucial for the precipitation of C-phycocyanin in the solution Additionally, it is essential to consider the pH value and salt concentration as key factors in this process.
• Phosphate buffer solution contains Na2HPO4 0.1M
Change the extraction time: 4 hours, 8 hours, 12 hours
Experiments conducted under various solvent conditions revealed a noticeable color difference in the tubes, indicating varying concentrations of C-PC Tubes 2 and 3 exhibited a distinct color of the C-PC pigment, while tubes 1 and 4 showed a less pronounced color The experimental results are illustrated in Figure 4.1.
Figure 4.1 Image of C-PC solution after being extracted in different solvents
Note: (1) Distilled water; (2) phosphate buffer solution 0.1M; (3) NaCl solution 0.9%; (4) MgCl 2 solution1.2%
The effectiveness of solvents in disrupting cyanobacteria cell structures is crucial for extracting desired compounds While some solvents can effectively break down cell walls, they may also dissolve unwanted components, whereas others are more selective The results indicate that using a 0.9% NaCl solution and a 0.1M phosphate buffer yields a color pigment solution closest to C-PC, compared to extractions with distilled water and 1.2% MgCl\(_2\).
The structural transformation of algae before and after extraction provides insight into the cell disruption capabilities of various solvents and their effectiveness in extracting C-PC The experimental results are illustrated in the accompanying figure.
Test tube Initial After 8 hours
Figure 4.2 Images of algae structure before and after soaking for 8 hours, observed under a microscope with 40x magnification
Test tubes (1), (2), (3), and (4) utilized different extraction solvents: distilled water, phosphate buffer solution, 0.9% NaCl solution, and 1.2% MgCl2 solution, respectively In tubes (1) and (2), the algae structure was disrupted almost immediately upon adding the solvent, whereas in tubes (3) and (4), the breakdown occurred more gradually After 8 hours of extraction, the differences in the algae structure before and after treatment became evident, with the damaged structure facilitating the release of internal compounds Overall, while there was minimal variation in the solvents' effectiveness in breaking down the algae structure, the extraction process demonstrated notable differences in the speed of disruption.
(2), it seems that the structure of the algae was broken the most
Extraction time significantly influences the yield of C-PC, with longer extraction durations leading to higher C-PC extraction rates, while shorter times result in lower yields However, there is a threshold beyond which increasing extraction time does not enhance C-PC levels Additionally, various solvents exhibit distinct extraction profiles over time, as demonstrated by the experimental results, which are detailed in Table 4.1.
Table 4.1 Survey results of C-PC extraction with different solvents
Content of PC (mg/mL)
Figure (4.3) and figure (4.4) show the comparative correlation between the content and purity of C-PC changes under different solvent conditions over a period of 4; 8 and 12 (hours)
Figure 4.3 The change of C-PC content according to the solvent
The maximum concentration of extracted C-PC reached 3.02 mg/mL using a 0.1M phosphate buffer over an 8-hour period, while the use of NaCl solvent yielded an extracted C-PC content of 2.96 mg/mL.
Figure 4.4 The change of purity of C-PC with solvent
Research by R Ktari et al (2020) demonstrated that a 0.5 M NaCl solution effectively extracted C-Phycocyanin (C-PC) from Spirulina, achieving an extraction efficiency of 2.87 mg/mL Similarly, H.T Phuc et al (2020) utilized a 1 M MgCl2 solution for C-PC extraction from Spirulina, resulting in an efficiency of 2.6 mg/mL.
Y Jiang, et al., (2019) used phosphate buffer to extract C-PC from Spirulina
The results showed that C-PC extraction efficiency reached 2.9 mg/mL
The purity results achieved using phosphate buffer and 0.9% NaCl were similar, with values of 0.68 and 0.64, respectively In contrast, the purity levels obtained with MgCl2 1.2% and distilled water were significantly lower, at 0.55 and 0.45 Purity values ranging from 0.6 to 0.8 are deemed clean, indicating that phosphate buffer and 0.9% NaCl are effective solvents for achieving acceptable purity levels.
In this study, 0.9% NaCl was selected as the extraction solvent for C-phycocyanin from A platensis, owing to its safety, affordability, and health benefits when utilized in food applications.
4.1.2 Choose the optimal salt concentration
The ideal salt concentration for C-PC extraction is influenced by factors such as the solvent type, extraction method, and target purity level Experiments were conducted under various environmental conditions, testing different salt concentration levels.
Carry out extraction experiments in different salt concentrations, then calculate the results and record them in table (4.2)
Table 4.2 Survey results of C-PC extraction at different salt concentrations
Content of PC (mg/mL)
The extraction of C-PC is significantly affected by salt concentration, as higher salt levels lead to solvation of salt molecules, which decreases the surrounding water molecules around protein surfaces This reduction allows hydrophobic surfaces to come closer together, resulting in precipitation Consequently, when comparing NaCl at varying concentrations, lower concentrations are more effective in dissolving protein, resulting in a greater yield of extracted C-PC.
Research by L Zhang et al (2018) demonstrated that a 1M NaCl solution can extract C-phycocyanin (C-PC) from Arthrospira platensis, resulting in a concentration of approximately 0.31 mg/mL after 8 hours Similarly, a study by D Singh et al (2018) utilized the same 1M NaCl solution to extract C-PC from Spirulina platensis, achieving a significantly higher concentration of about 2.5 mg/mL after the same duration.
Experimental results show that using NaCl solvent with a concentration of 0.9%, the amount of C-PC extract reached 2.88 (mg/mL) with a purity of 0.63 and the highest of the investigated concentrations
Figure 4.5 The change of C-PC content according to salt concentration
Figure 4.6 The change of purity of C-PC with salt concentration
4.1.3 Choose the optimal extraction time
The extraction time can have a significant impact on the extraction of C-
Longer extraction times typically lead to higher yields of C-PC, as more of the compound dissolves in the solvent However, extending the extraction duration may also heighten the risk of oxidation or degradation of C-PC.
Short extraction times can lead to lower yields and purity levels of C-PC, as insufficient time may prevent adequate dissolution in the solvent The optimal extraction duration varies based on factors such as the solvent type, extraction method, and target purity level A table detailing the optimal extraction time under various conditions is provided below.
After conducting the survey conditions, the results are calculated and recorded in the table (4.3)
Table 4.3 Survey results of C-PC extraction over time course
Content of PC (mg/mL)
Survey results on the method of processing C-PC solution into dry
After extraction, C-PC solution was treated by freeze-drying
(3) (4) Figure 4.11 Image of the experimental process of maintaining C-PC by freeze-drying method
Note: Images are for illustrative purposes only and cannot be the main result
1 C-PC solution is spread evenly on petri dishes
3 C-PC solution after being sublimated to remove water
After extraction, solvent C-PC was spread in a thin layer on a metal dish, then stored in cold conditions and protected from light
Figure 4.12 Image of the experimental process of maintaining C-PC by natural evaporation method
1 The C-PC solution is spread evenly and thinly on the metal tray
2 The tray is sealed to avoid direct light and is kept in a cold room condition
3 C-PC solution after evaporating all natural water
4.2.2 The quality comparison between freeze-drying method and natural drying
The C-PC solution, derived from two methods, was dissolved in distilled water and its optical density (OD) was measured at various wavelengths: A620 nm, A650 nm, and A280 nm, to assess concentration and purity.
The results after calculation are compared with a 40 (mL) solvent containing C-PC as a standard with a C-PC content of 3.15 (mg/mL) and purity of 0.63
Table 4.5 Investigate the quality of C-PC when using freeze-drying and natural drying methods
C-PC content in dried algae (mg/g)
Experimental results indicate that the freeze-drying method for preserving C-PC outperforms natural evaporation After sublimation drying, the C-PC content remained stable at 2.86 (mg/mL) with a purity of 0.61, while the natural evaporation method yielded only 1.92 (mg/mL) and a lower purity of 0.51, compared to the original solution levels of 3.15 (mg/mL) and 0.63.
Sublimation drying technology effectively removes nearly all the water from the C-PC solution while preserving its structure, ensuring that the content and purity of C-PC remain largely intact In contrast, the natural evaporation method exposes C-PC to air, resulting in oxidation that diminishes both its content and quality.
4.2.3 Recovery efficiency of the refining process
This study focused on the efficient purification of C-phycocyanin (C-PC) by accurately weighing 2.5 g of pure C-PC and mixing it with 40 mL of 0.9% NaCl solution The extraction process was conducted for 12 hours at 4°C, followed by centrifugation at 1900 rpm for 20 minutes Finally, the solution was purified into a powder through freeze-drying and weighed.
Table 4.6 Recovery efficiency of the whole process of refining C-PC to powder form Theoretical mass (g) Actual weight (g) Recovery efficiency(%)
Various global studies have explored methods for extracting C-phycocyanin from algae For instance, H Dhakal et al (2018) employed an ultrasonic mixed extraction method using ethanol and water, achieving a maximum extraction efficiency of 17.3% from Spirulina In contrast, O.P Ruiz et al (2019) utilized high-pressure treatment on Arthrospira platensis, resulting in a significantly higher extraction efficiency of 85.4% Additionally, S.K Basha et al (2019) applied saline extraction (NaCl) on Spirulina, yielding an efficiency of 20.8% Notably, the current study demonstrates that the mechanical extraction method is the simplest and most cost-effective, with an extraction efficiency of 81.2%.
CONCLUSION AND SUGGESTION
Conclusion
Through the study of simple C-phycocyanin extraction method from the green- microalgae Arthrospira platensis, the following conclusions were drawn:
1 The mechanical extraction method, which involves breaking algae cells to release C-PC, is a straightforward and low-cost approach that is easy to implement Despite having a low concentration and purity, the resulting product still satisfies the safety requirements for use
2 In this study, C-phycocyanin extraction procedure was extracted with the following conditions: 0.9% NaCl buffer solution; the algal mass-volume solvent ratio is 0.0625; extraction time is 14 hours Centrifuge the solution at 1900 (rpm) for 20 minutes at 4°C
3 The product after extraction by the optimized process reached a concentration of 3.15 (mg/mL) with purity A 620nm /A 620nm = 0.63, the efficiency of the entire process including the extraction process and make powder products of C- phycocyanin is 81.2%
4 The superiority of freeze drying method in making dry solid products compared to natural evaporation method has been shown In particular, this method does not significantly reduce the quality and purity of C-PC.
Suggestion
Based on the results obtained from the experimental process and some limitations of the study, recommendations were made as follows:
1 Conducted surveys with various other methods to improve C-PC content as well as purity Some promising methods such as high-pressure extraction, ultrasonic extraction or lyzozyme treatment have great applications
2 Conduct a survey on methods to increase the purity of C-PC extract such as using ammonium sulphate salt, using activated carbon, chitosan In addition, some other C-PC purification methods such as sepandex gel filtration chromatography, high performance liquid chromatography (HPLC) or simpler can use multiple extraction methods
A Herrera, A Napoleone, A Hohlberg (1989), “Recovery of C- phycocyanin from the cyanobacterium Spirulina maxima”, Journal of Applied
A Vonshak, (1997), “Spirulina platensis (Athrospira): physiology, Cell Biology and Biotechnology”, Taylor and Francis,1, pp.230-233
C D‟souza, H.N Pradeep, C.A Nayak (2018), “Extraction of phycocyanin from Spirulina plantesis using sonication”, pp.27974-27978
C Romay, R González1, N Ledón1, D Remirez1, V Rimbau (2003),
“Phycocyanin: A Biliprotein with Antioxidant, Anti Inflammatory and Neuroprotective Effects Current Protein and Peptide Science”, 1, pp.207-216
C Sili, G Torzillo, A Vonshak, (2012), “Ecology of Cyanobacteria II: Their Diversity in Space and Time”, Springer Dordrecht Heidelberg New York London, (25), pp.677-705
D Kumar, D.W Dhar, S Pabbi, N Kumar, S Walia, (2014),
“Extraction and purification of C-phycocyanin from Spirulina platensis”, Indian
“Phycocyanin from Spirulina: A review of extraction methods and stability”,
Affiliations expand, PMID: 33992333 DOI: 10.1016/j.foodres.2021.110314
H Dhakal, B.R Pokharel, B Shrestha, P Poudel, A Wagle, N Wagle,
(2018), “Extraction of C-phycocyanin from Spirulina platensis using ultrasound-assisted extraction”, Journal of Applied Research on Medicinal and Aromatic Plants, 9, pp.55-61
H.T.H Tran, T.T.N Nguyen, T.M Hoang, T.T Nguyen, T.N Do,
(2020), “Extraction of C-phycocyanin from Spirulina platensis using high- speed centrifugation”, Journal of cosmetic dermatology, 19(2), pp.245-252
J Abalde, L Betancourt, E Torres, A Cid, C Barwell (1998),“ Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp” Plant Science, 136, 1, pp.109-120
J Léonard, P Compère, (1967), “Spirulina platensis blue alga of great nutritional value due to its high protein content”, (37), pp.1–23
J.C Dillon, A.P Phuc, J.P Dubacq, (1995), “Nutritional value of the algae Spirulina”, World Rev nutr diet, 77, pp.32-46
J.C Lee, M.F Hou, H.W Huang, F.R Chang, C.C Yeh, Tang, J.Y Chang, H.W Marine, (2013), “Algal natural products with anti-oxidative, anti- inflammatory, and anti-cancer properties”, Cancer Cell Int, 13, pp.55-61
L Chen, M Qiu, Y Guo, J Chen (2017), “Extraction of C-phycocyanin from Spirulina platensis using high-speed centrifugation and ultrasonic extraction”, Marine drugs, pp.331-334
L.V Lăng, (1999), “Spirulina - nuôi trồng và sử dụng trong y dược và dinh dưỡng”, NXB Y học
M Kuddus, P Singh, G Thomas, A.A Hazimi (2013), “Recent
Developments in Production and Biotechnological Applications of C- phycocyanin”, pp.1-9
M Qiu, L Chen, Y Guo, J Chen, (2016), “Extraction of C- phycocyanin from Spirulina platensis using microwave and ultrasonic treatments”, Marine drugs, 14(2), pp.40-41
M.A.B Habib, M Parvin, T.C Huntington, M.R Hasan (2008), “A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals”, F.A.O Fisheries and Aquaculture Circular, 1, pp.33-34
M.R Palomares, L Nunez, D Amador, (2001), “Practical application of aqueous two‐ phase systems for the development of a prototype process for C‐phycocyanin recovery from Spirulina maxima”, Journal of Chemical
N Mallick, L.C Rai, (2007), “Inhibition of Fe (III)-EDTA- photodegradation of C-phycocyanin, the pigment-protein complex from
Spirulina platensis, by UV-B radiation”, Process Biochemistry, 42(2), pp.253-
N Seyidoglu, S Inan, C Aydin, (2017), “A prominent superfood:
Spirulina platensis, superfood and functional food - The development of superfoods and their roles as medicine”, IntechOpen, DOI: 10.5772/66118
N.T Eriksen, (2008), “Production of phycocyanin-a pigment with applications in biology, biotechnology, foods and medicine”, Appl Microbiol Biotechnol, 80, pp.1-14
Nguy n c B ch, Nguy n Phan Khu , Ph Th C m Mi n, Kim Anh
Nghiên cứu của Nguyễn Thị Hiền (2020) tập trung vào ảnh hưởng của ánh sáng LED đến sinh trưởng, hàm lượng sắc tố và khả năng thích ứng của tảo xoắn Arthrospira platensis trong môi trường nuôi cấy tại Việt Nam Kết quả nghiên cứu được công bố trong Tạp chí Khoa học.
N ng nghi p Vi t Nam, 18(8), pp.637-648
O.P Ruiz, V.M.M Juarez, M.R.R Burgueno, (2019), “Extraction of C- phycocyanin from Arthrospira platensis by high-pressure homogenization”,
Journal of Applied Phycology, 31(6), pp.3481-3491
P.D Karkos, S.C Leong, C.D Karkos, N Sivaji, D.A Assimakopoulos
(2011), „„Spirulina in Clinical Practice: Evidence-Based Human Applications”, Evidence-Based Complementary and Alternative Medicine, pp.1-4
R González, S Rodríguez, C Romay, O Ancheta, A González, J Armesto, D Remirez, N Merino, (1999), “Anti-inflammatory activity of phycocyanin extract in acetic acid-induced colitis in rats”, Pharmacol Res, 39(1), pp.45-59
“Green synthesis of C-phycocyanin from Spirulina platensis and its anticancer potential”, Journal of Photochemistry and Photobiology, 178, pp.251-258
R.A Lewin, (1953), “The cell structure and growth of Spirulina platensis in culture”, Annals of the New York Academy of Sciences”, 56(5), pp.867-880
R.G Fisher, N.E Woods, H.E Fuchs, R.M Sweet, (1980), “Three
Dimensional Structures of C-phycocyanin and B-Phycoerythrin at 5-A Resolution”, The Journal of Biological Chemistry, 255(11), pp.5082-5089
S Aishwarya, S Rajeshwari, P Ramasamy, (2017), “Comparative study on the extraction of C-phycocyanin from Arthrospira platensis using different methods”, Journal of Microbiology and Biotechnology Research, 7(4), pp.84-
S Ayehunie, A Belay, T.W Baba, R.M Ruprecht, (1998), “ Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis)”, 18(1), pp.7-12, DOI: 10.1097/00042560-199805010-00002
S Boussiba (1979) “Isolation and characterization of phycocyanins from the Blue-Green alga Spirulina platensis” Arch microbiol, 120, pp.155–159
(2013), “Spirulina paltensis: Food and Function”, Curr Nutr Food Sci, 9, pp.189–193
“Extraction and purification of C-phycocyanin from dry Spirulina powder and evaluating its antioxidant, anticoagulation and prevention of DNA damage activity”, Journal of Applied Pharmaceutical Science, 3(08), pp.149-153
“Protective effect of C-phycocyanin extracted from Spirulina platensis against
UV-B radiation in vitro”, Journal of cosmetic dermatology, 17(4), pp.514-520
W Song, C Zhao, S Wang, (2013) “A large-scale preparation method of high purity C-phycocyanin”, International Journal of Bioscience, Biochemistry Bioinformatics, 3(4), pp.293-297
W Song, C Zhao, S Wang, (2013), “A Large-Scale Preparation Method of High Purity C-phycocyanin”, International Journal of Bioscience, Biochemistry and Bioinformatics, 3(4), pp.294-297
W.S Park, H.J Kim, M Li, D.H Lim, J Kim, J.J Kwak, C.M Kang, M.G Ferrruzi, M.J Ahn (2018), “Two classes of pigments, carotenoids and C- phycocyanin, in Spirulina powder and their antioxidant activities”, Molecules,
Y.K Kim, H.K Kim, Y.C Kim, (2015), “Extraction of C-phycocyanin from Spirulina using ultrasonic and heat treatments”, Journal of food science,
Z Khan, P Bhadouria, P.S Bisen, (2005), “Nutritional and therapeutic potential of Spirulina”, Curr Pharm Biotechnol, 6, pp.373-379.