Mikroenkapsulasi Fikosianin dalam Maltodekstrin-Alginat: Formulasi dan Karakterisasi

Fikosianin merupakan sumber pewarna biru alami yang dapat diekstrak dari Spirulina sp. Karakteristik dari fikosianin adalah tidak stabil oleh cahaya, suhu, dan pH selama proses pengolahan dan penyimpanan. Metode mikroenkapsulasi dapat digunakan untuk melindungi fikosianin dari pengaruh eksternal di mana jenis dan konsentrasi enkapsulan yang digunakan dapat mempengaruhi karakteristik mikrokapsul fikosianin yang dihasilkan. Tujuan dari penelitian ini adalah untuk mengetahui formulasi terbaik dan karakterisasi mikrokapsul fikosianin dari Spirulina sp. dengan maltodekstrin dan alginat sebagai enkapsulan. Mikrokapsul diproduksi dengan lima perbedaan konsentrasi alginat dalam maltodekstrin, yaitu 0 %; 0,2 %; 0,4 %; 0,6 %; dan 0,8 % (b/b). Total enkapsulan yang digunakan adalah 10 % dari larutan mikropartikel fikosianin. Hasil penelitian menunjukkan bahwa peningkatan konsentrasi alginat dapat meningkatkan kadar fikosianin, kadar air, efisiensi enkapsulasi, bulk density, intensitas warna biru, dan ukuran partikel serta dapat memperbaiki morfologi mikrokapsul yang dihasilkan. Mikrokapsul fikosianin dengan konsentrasi alginat 0,6 % dan maltodekstrin 9,4 % memiliki kadar fikosianin, efisiensi enkapsulasi, dan intensitas warna biru paling tinggi.


INTRODUCTION
Phycocyanin is blue-colored phycobiliprotein contained in Spirulina sp. which can be used as alternative natural colorant of food, drink and cosmetics. Several reports have shown that phycocyanin has advantages as compared with blue synthetic colourant are that phycocyanin functions as antioxidant (Zheng et al., 2012), anti-coagulant activity (Jensen et al., 2016) and anti-cancer (Ravi et al., 2015). Phycocyanin will be denaturized at a temperature above 60°C (Martelli et al., 2014) and at a pH<5.0 (Duangsee et al., 2009), causing it to fade. Microencapsulation is one of the methods that can be used to protect phycocyanin from external influences and to facilitate distribution process. According to Ozkan and Bilek (2014), microencapsulation of natural food colourants is an active compound coating technology for the protection, stabilization, and the slow release of core material. The final size of the product is related with the term microencapsulation, that is of a micro scale (<1 mm), and nano-encapsulation, that is of a nano scale (< 1 µm). The active compound being coated is called core and the material coating is called encapsulant or wall (Carvajal et al., 2010).
Spray drying is a method most commonly used in food industry because it is easy to handle, fast process, lower costs, and produces small-sized products. The process of spray drying is used for microencapsulation of various natural colourants like carotenes (Kha et al., 2010), anthocyanin (Fang and Bhandari, 2011;Bakowska-Barczak and Kolodziejczyk, 2011), and betalains (Pitalua et al., 2010). However, this method can damage to the active compound being encapsulated due to the use of high temperature during spray drying process. To protect the active compound during spray drying process, a precise type and ratio of the encapsulant are needed.
Encapsulant can be selected from a variety of polymers based on the characteristics of the microcapsules. The type of encapsulant commonly used is maltodextrin, it is starch derivative compounds which has low solubility in water, high viscosity, and relatively low price, thus widely used as an encapsulant (Cakrawati and Handayani, 2017). Due to maltodextrin as an encapsulant has low emulsion stability, made the skin layer weak, so the active compound as core material to be less protected during spray drying (Hermanto et al., 2016). Therefore, it needs another biopolymer to combine encapsulants, such as alginate.
According to Chavarri et al. (2010), alginate is one of the linear anionic polysaccharides, which enables it to be used as the encapsulant. The benefit of using alginate as an encapsulant is that it is non-toxic, can develop a strong matrix, is water-soluble, is of low price, and is safe to be consumed. Hadiyanto et al. (2017), the phycocyanin microcapsule is produced by using an extrusion method with alginate as the encapsulant.
Based on the above description, research on phycocyanin microencapsulation using maltodextrin and alginate as an encapsulant by spray drying method needs to be conducted. The objective of this research is to determine the best formulation and characterization of phycocyanin microcapsules with maltodextrin and alginate as an encapsulants. The use of encapsulant with a precise formulation is expected to protect phycocyanin during the spray drying process, thus increasing the functional characteristics of phycocyanin microcapsules.

Phycocyanin Microencapsulation
Phycocyanin was extracted using fresh water 1:100 (wPhycocyanin was extracted using aquadest 1:100 (w/v) with a homogenization speed of 300 rpm at a room temperature for 4 h. Next, the solution is centrifuged at 4800 g for 15 min to separate phycocyanin solution from Spirulina sp. Phycocyanin and encapsulant were homogenized at the speed of 10,000 rpm using a homogenizer. Than, phycocyanin microparticle solution was dried using spray dryer with an inlet temperature of 130 °C. Further information about phycocyanin microparticle solution formulation is shown in Table 1.

Moisture Content
Phycocyanin microcapsules (1 g) were weighed after it was dried in the oven at 105 °C until constant weight. The moisture content (%) was calculated based on the loss of weight before and after drying (Li et al., 2017).

Encapsulation Efficiency (EE)
The percentage EE was calculated as the ratio of active compound (phycocyanin content) in the microcapsule with active compound before microencapsulation (Mirhojati et al., 2017).

Colour
The colour of phycocyanin microcapsules were measured using chroma meter. L* is brightness, a* and b* show color in which -a* is greenish and +a* is reddish, -b* is bluish and +b* is yellowish (Ravichandran et al., 2014).

Particle Size Distribution
Phycocyanin microcapsules were dispersed in hexane using ultrasonic waves for 2 min and then particle size distribution was directly determined using Dynamic Light Scattering (Parrarud and Pranee, 2010).

Morphological Observation
Phycocyanin microcapsule's morphology was observed using modified method of Venil et al. (2016), in which the sample was coated with gold and then observed using an SEM at 5,000 magnification and 20 kV voltage.

Data Analysis
Results were analyzed with SPSS version 17 (International Business Machines Corporation, USA) using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test comparisons among means. Significance was defined at p< 0.05.

Phycocyanin Content
The result shows that the phycocyanin content of FM is lower than FMA (Table 2). There showed that more phycocyanin is trapped using maltodextrin-alginate than using maltodextrin alone. Novianty et al. (2015) stated that the use of alginate up to 1% concentration combined with maltodextrin as an encapsulant can protect phenol compound on liquid smoke during spray drying with an inlet temperature of 130 °C. According to Hadiyanto et al. (2017), alginate has capabilities of cross-linking formation with polymer bonds, thus increasing its ability to trap a compound.
Phycocyanin microcapsule with maltodextrin-alginate as an encapsulant produced by this research has phycocyanin content between 2.05 ± 0.03 (%) to 2.42 ± 0.10 (%). Earlier research (Dewi et al., 2016) showed that the use of 2 types of encapsulant (maltodextrincarrageenan) is more effective in trapping and protecting phycocyanin than the use of maltodextrin alone as an encapsulant.
The increase of alginate concentration of up to 0.6% and combination with maltodextrin cause increase phycocyanin content in microcapsule (Table 2), that is, 2.37 ± 0.05 (%). This is suspected as due to the increase of emulsion stability, correlated with the increase of alginate concentration. The characteristics of microcapsule can be influenced by emulsion stability, amount of solids, viscosity, particle size, encapsulant type, and microencapsulation method (Martins et al., 2014;Wilkowska et al., 2016).
At 0.8% alginate concentration, more of the phycocyanin produced sticks to the chamber and cyclone walls during the spray drying process.This is probably due to the fact that an increase of encapsulant concentration causes an increase of glass transition temperature, thus causing an unsuitable use of the inlet temperature. Sormoli et al. (2012) reported that glass transition temperature increases along with the increase of encapsulant concentration, which relates with the increase of polymer bond between the types of encapsulant used in lactose drying using spray dryer. Marcela et al. (2016) explained that the increase of alginate ratio as an encapsulant and the use of low inlet temperature cause more particles sticky to the walls of the spray dryer's chamber and the particles cannot be removed.

Moisture Content
Differences in alginate concentration as an encapsulant cause significantly difference the moisture content of phycocyanin microcapsule ( Table 2). The moisture content of phycocyanin microcapsule produced in this research is between 2.97±0,07 (%) to 3.35±0.18 (%). The higher of the alginate concentration can increase the moisture content of phycocyanin microcapsule. The moisture content of microcapsule is also influenced by the type and composition of encapsulant which can hinder evaporation. The increase of encapsulant composition that is not accompanied with inlet temperature increase can cause low evaporation rate (Sormoli et al., 2012;Marcela et al., 2016). Different result had been reported by Fazaeli et al. (2012), the moisture content of microcapsules was decreased with addition of encapsulant concentration. These results could be explained that additional concentration of encapsulant caused an increase in total solids and a reduction in total moisture for evaporation.

Antioxidant Activity
The research results show that the higher the concentration of alginate used as an encapsulant along with maltodextrin can increase antioxidant activity of phycocyanin microcapsules ( Table 2). The increase of antioxidant activity is correlated with the amount of active compound that can be encapsulated (Bakowska-Barczak and Kolodziejczyk, 2011). Similar findings result had been reported by Dewi et al. (2016), that the high level of phycocyanin content causes an increase in antioxidant activity of phycocyanin microcapsules.
The increase of alginate concentration used as an encapsulant can increase antioxidant activity of phycocyanin microcapsules. The same result is also shown by Hadiyanto et al. (2017) that the use of alginate with a higher concentration can increase antioxidant activity and reduce IC50 value of phycocyanin microcapsules.

Encapsulation Efficiency (EE)
EE is defined the amount of phycocyanin that can be encapsulated. The lowest EE value is found in FM, that is, 29.74±0.30 (%) and the highest EE value is found in FMA6, that is, 40.74±0.86 (%) ( Table 2). This is closely related with phycocyanin content found in phycocyanin microcapsule, in which the higher content of phycocyanin is found in phycocyanin encapsulated using maltodextrin-alginate as contrasted to maltodextrin alone. The type of encapsulant play role in effectively protecting the active compound from the effect of high temperature during spray drying. The type and concentration of the encapsulant can affect the encapsulation efficiency of microcapsules (Murali et al., 2014;Mirhojati et al., 2017). Shinde and Nagarsenker (2011) explained that microencapsulation is considered successful if the powder produced contains the active compound with maximum retention.
Based on the research, the increase of alginate concentration of up to 0.6% can cause an increase of the EE of phycocyanin microcapsules. The increase of alginate concentration also causes the thickening of phycocyanin microparticle solution. According to Pitchaon et al. (2013), the high viscosity and thickness of the solution contribute to the reduced microparticle porosity. The high microparticle solution viscosity can also prevent active solution diffusion to microparticle surface. But in this research, the alginate concentration increase of 0.8% produces phycocyanin microcapsule with EE value that is not significantly different with an alginate concentration of 0.6%. However, a certain alginate concentration (maximum) can cause the formation of a more solid network with smaller pores, thus less active compound is trapped in the pores (Sevda and Rodrigues, 2011;Soliman et al., 2013). Table 3 shows that an increase of alginate concentration as an encapsulant can cause significantly increase the bulk density of phycocyanin microcapsules.

Bulk Density
There is between 0.56±0.01 kg • m -3 and 0.86±0.02 kg • m -3 . According to the observed by Janiszewska and Wlodarczyk (2013), the bulk density value is closely related with the moisture content of phycocyanin microcapsules. The type and composition of the encapsulant can influence evaporation rates, which influences the weight of each phycocyanin microcapsule particles (Fazaeli et al., 2012).
Bulk density is also influenced by the particle size of phycocyanin microcapsules. Caparino et al. (2012) explained that bulk density can increase due to the decrease of particle size. Small size particles provide wider surface contact area per volume unit. Based on this research, phycocyanin microcapsule encapsulated using maltodextrin-alginate has more nanometer-sized particles than phycocyanin microcapsule encapsulated using maltodextrin alone.

Colour
The use of alginate with maltodextrin as an encapsulant can cause a decrease in L value and an increase of (-)b value (Table 3). Thus, the phycocyanin encapsulated using maltodextrin-alginate has more dark and blue colour. The blue colour produced from phycocyanin microcapsule is related with the phycocyanin content. The intensity of blue colour increased with increasing phycocyanin content (Sedjati et al, 2012). The color of phycocyanin microcapsule produced is also influenced by the type of encapsulant and the drying condition. Based on the previous research (Dewi et al., 2017), phycocyanin microcapsule with different encapsulant types (maltodextrin, maltodextrin-alginate, and maltodextrin-carrageenan) have different brightness (L) values. The (L) value from the highest to the lowest is as follows, phycocyanin microcapsule with maltodextrin-carrageenan, with maltodextrin-alginate, and with maltodextrin alone as an encapsulant. This is probably caused by the browning reaction of maltodextrin during the drying process, which influences the final color of the sproduct.

Particle Size Distribution
Based on particle size distribution analysis, the particle size of phycocyanin microcapsules with maltodextrin as an encapsulant has the following distribution: 82.8% sized 1,853 nm and 17.2% sized 181.4 nm. While phycocyanin microcapsules with  (Fig. 1). The different sizes of phycocyanin microcapsules are influenced by encapsulation method, type of encapsulant, and ratio between encapsulant and active compound (Mirhojati et al., 2017). Use of maltodextrin-alginate as an encapsulant can produce phycocyanin microcapsules with bigger size than phycocyanin microcapsules encapsulated using maltodextrin alone. Briones and Sato (2010) explained that the increase of microcapsule particle size is related to the number of active compounds that can be encapsulated. Carvalho et al. (2014) added that bigger microcapsule particle size is also caused by the use of more than one types of the encapsulant, thus creating two or more layers that cover the active compound. Figure 2 shows that the phycocyanin microcapsules with maltodextrin-alginate as an encapsulant have a spherical shape than phycocyanin microcapsules with maltodextrin as an encapsulant, which has a rather irregular shape, wrinkled and with the wavy surface ( Fig. 2). According to Harris et al. (2011), microcapsules with the wrinkled and wavy surface are caused by the rapid solvent evaporation during the spray drying process. Najafi et al. (2011) added that the differences in microcapsule morphology are influenced by the atomization and the drying condition. Phycocyanin encapsulated using maltodextrin alone produces microcapsules that are more fragile (cracked) (Fig. 2a). Meanwhile, no cracks are found on phycocyanin microcapsule with 0.6% maltodextrinalginate as an encapsulant (Fig. 2b). This is because of the mannuronic acid and guluronic acid content found in alginate which can influence the ability to form a gel. Fertah et al. (2017) stated that alginate with a higher ratio of mannuronic and guluronic acids can increase its ability to develop stable and strong gel. The same result was also shown by Dewi et al. (2016), phycocyanin encapsulated using maltodextrincarrageenan produce whole microcapsules without b) a a cracks as the result of interaction between maltodextrin and carrageenan which forms a stable complex polyelectrolyte. This is an advantage because microcapsules have low permeability against gas, and can increase protection and retention towards the active compound being encapsulated.

CONCULUSION
The higher concentration of alginate combined with maltodextrin as an encapsulant will increase moisture content, bulk density, and particle size of phycocyanin microcapsules. With a formulation of 9.4% maltodextrin, 0.6% alginate, and 90% phycocyanin extract, we can produce phycocyanin microcapsules with the best characteristics, namely phycocyanin content, the efficiency of encapsulation, the highest bluish colour, a rounder shape and solidity without cracks of particles. This shows that the use of maltodextrin combine with alginate can protect phycocyanin during spray drying process.

ACKNOWLEDGEMENT
The writer wishes to thank Diponegoro University that has funded this research, based on the Research Microencapsulation of L-ascorbic acid by spray drying using sodium alginate as wall material. Journal of Encapsulation and Adsorption Sciences, 6.
Thermal stability improvement of blue colorant Cphycocyanin from Spirulina platensis for food industry applications.