Enhancement of Phycocyanin Extraction from Dry Spirulina platensis Powder by Freezing-Thawing Pre-treatment

Phycocyanin (PC) is a bioactive compound that can function as an antioxidant, anti-inflammatory, immunomodulatory, and anti-cancer agent. It can act as a potential material in preventing COVID-19 and curing those suffering from it. Spirulina platensis (SP) is one of the microalgae rich in proteins and PC. This study aimed to determine the optimum PC extraction from SP, using distilled water as solvent through freezing-thawing pre-treatment. The variables set in the investigation were water content in SP before freezing (24.7-84.9 % wet basis), soaking time (0.25, 1, 2, and 6 hours), raw materials’ storage period (1-13 months), freezing time (1-141 days), and the (solvent/biomass) ratio (20-440 mL/g). Spirulina platensis powder was soaked, frozen, thawed, and extracted in batch operation. The residue was extracted with the same solvent. The PC concentration in the filtrate was determined by measuring its absorbance using a spectrophotometer at wavelengths 615 and 652 nm. The experiment gave the optimum yield at a water content of 81.9% (wet basis), soaking time of 6 hours, freezing time of 1 day, and a solvent-to-biomass ratio of 100 mL/g. The optimum storage period of the raw material was one month. The phycocyanin IC₅₀ value of 1.485 mg/L.

Freezing-thawing pre-treatment; Phycocyanin; Spirulina platensis

      Microalgae are photosynthetic microorganisms that convert solar energy into chemical energy through photosynthesis in their chlorophyll. Microalgae can grow in fresh water and seawater. Microalgae have diverse nutritional content, especially protein, carbohydrates, and fats (Rosmahadi et al., 2021). Various microalgae that can function as a food source or energy include Botryococcus braunii, Chlorella vulgaris, Dunaliella tertiolecta, Spirulina platensis, and Tetraselmis suecica (Rosmahadi et al., 2021; Rosli et al., 2020). Spirulina platensis is one of the microalgae that can be a source of protein (Sela, Budhijanto, and Budiman, 2021; Vernes et al., 2019; Soni, Sudhakar, and Rana, 2017). It is preferable due to its easiness of being cultivated in fresh water. The content of PC in SP varies from 5 to 20% (Garcia and Mejia, 2021). Consuming Spirulina or phycocyanobilin-enriched Spirulina extracts may potentially boost type 1 interferon response in the circumstances of RNA viral infection (McCarty and DiNicolantonio, 2020). Phycocyanin isolation begins with the cell wall breaking. The bioactive substances inside the cell can get out more quickly through the broken cell wall so that the extraction of PC becomes fast. If the cell wall remains intact, the extraction will be prolonged because the molecules have to diffuse through it. In general, microalgae cell walls are pretty strong, thus requiring an extraordinary method to break them down. The success of PC extraction significantly hinges on this initial step (Chia et al., 2019).

       Various ways of cell wall breaking have been carried out, including sonication (Dianursanti et al., 2020; Pratiwi, Utama, and Arbianti, 2020; Pan-utai and Iamtham, 2019; Ilter et al., 2018; Rodrigues et al., 2018; Tavanandi et al., 2018), microwave (Wang, Zhang, and Fang, 2019; Ilter et al., 2018), homogenization with a stirrer (Rodrigues et al., 2019; Ilter et al., 2018; Tavanandi et al., 2018; Silveira et al., 2007), freeze-thawing (Chia et al., 2019; Ilter et al., 2018; Tavanandi et al., 2018), pulsed electric field (Jaeschke et al., 2019; Martínez et al., 2017), and high-pressure homogenization of up to 350 bars (Deniz, Ozen, and Yesil-Celiktas, 2016). Phycocyanin is very sensitive to temperatures above 60°C (Su et al., 2014; Chaiklahan, Chirasuwan, and Bunnag, 2012; Antelo, Costa, and Kalia, 2008), so a proper method is needed to extract it from SP. The disadvantages of conventional methods include the relatively long stirring time (Rodrigues et al., 2018; Silveira et al., 2007). The agitation process usually comes into contact with the ambient air, so the PC’s quality is not good if not immediately stored at low temperatures.

      This study used the freezing method to break the cell wall. This method is considerable because it can maintain the quality of the product gained, given that PC is easily damaged if left at room temperature (or higher) or exposed to ambient air. However, freezing also causes ice expansion which can break the cell wall due to volume changes (Dombrovsky et al., 2015). Therefore, the water amount in Spirulina must be precise to ensure a successful extraction. A small amount of water in the cell makes the ice expansion insufficient, thus preventing the cell wall from breaking. Several researchers have extracted phycocyanin from Spirulina platensis by the freezing-thawing method at a freezing temperature of -20°C (Prabakaran et al., 2020; Chentir et al., 2018) or -40°C (Tavanandi et al., 2018). Phycocyanin yielded 52.82%-62.76% with 4-6 freezing-thawing cycles (Prabakaran et al., 2020; Tavanandi et al., 2018). However, repeating freezing-thawing cycles are time and energy-consuming and only suitable for laboratory scales (Jaeschke et al., 2021). Therefore, it is necessary to study the freezing-thawing method with only one cycle using a freezing temperature slightly below 0°C to save energy and obtain satisfactory extraction results.
     In this research, distilled water used as a solvent and the appropriate water content used in Spirulina platensis will determine the success of the freezing process. If the cell lacks water, the expansion of ice inside the cell is not enough to break down the cell wall. Conversely, excess water will cause it to be outside the cell. It will freeze both inside the cell and outside the cell. The ice outside the cell will prevent the cell wall from breaking, thus decreasing the number of cells broken. As a result, the phycocyanin content will also decrease. This phenomenon indicates that an appropriate water content allowing the cell wall to break during freezing is necessary. In this case, the freezing-thawing method is superior, as it can damage the cell walls and obtain a better quality of PC produced. Therefore, this research aimed to determine the optimum water content in freezing SP to get a good extract. The variables studied were soaking time, the storage period of raw materials, freezing time, and the solvent-to-biomass ratio.

2.1. Materials

   Spirulina platensis powder was purchased from Nogotirto Algae Park, Yogyakarta, Indonesia. The content of water, protein, fat, ash, and carbohydrates was determined based on the proximate analysis (AOAC, 2010). The solvent used was distilled water.

2.2. Freezing-thawing pre-treatment and extraction
      The researchers prepared several specimens, each of which contained one gram of SP powder added with various amounts of distilled water to get different water contents. Each of them was soaked for 15 minutes, 1, 2, and 6 hours, and then let to freeze. After 24 hours of freezing, they were thawed and then added with distilled water for extraction using a vacuum filter. The absorbance was measured using a spectrophotometer. Figure 1 depicts the experimental procedures (where t₁ was soaking time, and t₂ was freezing time).

Figure 1 Experimental procedure of PC extraction from SP powder by the freezing-thawing method

2.3. The equilibrium of solid-liquid extraction

       Experiments on solid-liquid extraction equilibrium were carried out in batches, in which the residue from the first extraction was added with pure solvent (distilled water). After filtering, the second residue was added with distilled water and then filtered. The extraction was complete when no phycocyanin was found in the extract, as indicated by the absorbance at a wavelength of 652 nm near zero. 

2.4. Phycocyanin determination

       The concentration of phycocyanin in the filtrate was determined using a spectrophotometer at 615 nm and 652 nm, with the following equations (1) to (4) (Rodrigues et al., 2019; Pan-utai and Iamtham, 2019; Rodrigues et al., 2018; Deniz Ozen, and Yesil-Celiktas, 2016; Silveira et al., 2007).

where CPC was the concentration of chloro-phycocyanin (g/L), APC was the concentration of allophycocyanin (g/L), PCt was total phycocyanin (g/L), OD₆₁₅ was the filtrate’s optical density at 615 nm, and OD₆₅₂ was the filtrate’s optical density at 652 nm from a spectrophotometer.

The yield of phycocyanin (mg/g) was:

where V was the solvent volume (mL), and DB (dry basis) was the mass of SP powder (g).

2.5. Antioxidant activity

     Antioxidant activity was investigated by DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity. Spirulina platensis (water content 80%) 100 mg dissolved in 5 mL of ethanol p.a. The sample was extracted using sonication for 15 minutes, then filtered. Put the filtrate in a 10 mL volumetric flask, add ethanol to 10 mL, and mix homogeneously. We prepared various solutions concentrations of ethanolic extract. One mL of the sample was mixed with 1 mL of 0.15 mM DPPH in absolute ethanol. The mixtures were then incubated at room temperature for 30 mins in the dark. A spectrophotometer measured the absorbance at 517 nm to monitor the DPPH radical decrease. The IC₅₀ was de?ned as the concentration of ethanolic extract of phycocyanin to scavenge 50% initial DPPH radical, and it was re?ected by a 50% reduction of absorbance (Abdullah et al., 2020; Pan-utai and Iamtham, 2019).

        Based on the proximate analysis, Spirulina platensis contained 9.57% water, 39.77% protein, 0.8% fat, 7.12% ash, and 42.76% carbohydrates (by difference). Phycocyanin can function as an immunomodulator, reduce inflammatory level, and strengthens immunity. It does not trigger acute diseases and sub-chronic toxicity (Grover et al., 2021). According to D’Alessandro and Filho (2016), the structure of phycocyanin is shown in Figure 2.

Figure 2 The structure of phycocyanin

3.1. Effect of water content and soaking time

      Figure 3 shows the effects of water content and soaking time on the extraction yield. Having the same water contents, the longer the soaking time, the higher the PC content.

Figure 3 Effects of water content and soaking time on the extraction yield

       The longer the soaking time, the more sufficient the duration taken by the distilled water added to SP powder to diffuse into the biomass pores. If the water is already inside the cell before freezing, when it freezes, its phase inside the cell will change, its volume will expand, and the formed ice will break down the cell wall. Consequently, PC will quickly come out during the thawing and extraction stages. The more the cell walls are broken, the higher the PC content. When the soaking time was 15 minutes, the yield would be below 10%. For 15 minutes, not all of the water added to the biomass could enter the cell. At a water content of 81.9 %, the yield obtained was below those when the soaking times were 1, 2, and 6 hours. Of all the experiments, the highest PC content of 81.65% was obtained when after soaked for 6 hours before being frozen. Microscopic visualization of the cells after freezing (with soaking times of 1 and 6 hours before freezing) is present in Figure 4.