/ Meiny Suzery

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H. Hadiyanto

^1,2,3

/ Marcelinus Christwardana

⁴

/ Meiny Suzery

^3,5

/ Heri Sutanto

^3,6

/ Ayu Munti Nilamsari

¹

/ Argino Yunanda

¹

Effects of Carrageenan and Chitosan as

Coating Materials on the Thermal Degradation of Microencapsulated Phycocyanin from

Spirulina sp.

1Diponegoro University, Department of Chemical Engineering, Jl Prof. Soedarto, SH, Tembalang, 50275 Semarang, Indonesia, E-mail: [email protected]

2Master Program of Environmental Science, Diponegoro University, Jl Imam Bardjo, SH, Semarang 50275, Indonesia, E-mail:

[email protected]

3Diponegoro University, Center of Biomass and Renewable Energy (C-BIORE), Jl Prof. Soedarto, SH, Tembalang, 50275 Se- marang, Indonesia, E-mail: [email protected], [email protected], [email protected]

4Institut Teknologi Indonesia, Department of Chemical Engineering, Jl. Raya Puspitek, Serpong, 15314 South Tangerang, In- donesia, E-mail: [email protected]. https://orcid.org/0000-0003-4084-1763.

5Faculty of Science and Mathematics, Diponegoro University, Department of Chemistry, Jl. Prof. Soedarto, SH-Tembalang Semarang, Indonesia, E-mail: [email protected], [email protected]

6Faculty of Science and Mathematics, Diponegoro University, Department of Physics, Jl. Prof. Soedarto, SH-Tembalang Se- marang, Indonesia

Abstract:

Phycocyanin is a natural substance that can be used as an antioxidant and food colorant. The quality of phycocyanin deteriorates when it is exposed to heat, and such deterioration is evidenced by decreases in its antioxidant activity and color. Encapsulation, which introduces a coating material over a substance of interest, has been applied to prevent changes in substance quality. The objective of the present research is to evaluate the kinetics of thermal degradation of phycocyanin coated with carrageenan or chitosan. Encapsulated phycocyanin samples were exposed to temperatures of 40, 50, or 60 °C for 90 min, and kinetics of the resulting degradation was evaluated to determine changes in sample quality. The results showed that the thermal degradation of encapsulated phycocyanin at 40–60 °C follows first-order reaction kinetics with reaction rate constants (k) of 4.67–

9.17 × 10^–5 s^-1and 3.83–7.67 × 10^–5 s^-1for carrageenan and chitosan, respectively, and that thekof encapsulated phycocyanin is slower than that obtained from samples without the coating materials (control). Encapsulation efficiencies (EE) of 68.66 % and 76.45 %, as well as loading capacities of 45.28 % and 49.16 %, were, respectively, obtained for carrageenan and chitosan.

Keywords:carrageenan, chitosan, phycocyanin, heating process, thermal degradation DOI:10.1515/ijfe-2018-0290

Received:September 15, 2018;Revised:December 23, 2018;Accepted:March 4, 2019

1 Introduction

Phycocyanin is the main carotenoid responsible for the color of blue-green algae or cyanobacteria; it can also be found in other microalgae, such as rhodophytes and cryptophytes [1, 2]. Phycocyanin is a pigment–protein complex from the family of phycobiliproteins, which are accessory pigments to chlorophyll [3]. The molec- ular weight and maximum absorption intensity of phycocyanin depend on the state of aggregation, such as temperature, protein concentration, pH solution, and the source of alga [4]. Two types of phycocyanins have been identified, namely, chloro-phycocyanin (C-PC) and allo-phycocyanin (A-PC); of these, the former makes up most of the phycobiliprotein structure [5]. In the industrial sector, phycocyanin is usually used as a natural colorant and often applied to food products [6]. Its bright color is believed to be more appealing than that of synthetic colorants. According to some researchers, phycocyanin can be utilized as an antioxidant to reduce free

H. Hadiyanto, Marcelinus Christwardanaare the corresponding authors.

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radicals. The carotenoid contains an open tetrapyrrole chain that can bind with a peroxy radical by donating hydrogen atoms and is bonded to the 10th C atom of a tetrapyrrole molecule [7, 8]. Phycocyanin has been used as a cosmetic additive and nutritional supplement for both humans and animals [9]. As phycocyanin is highly sensitive to fluorescent reagents, it can also be used in immunolabelling experiments as a label for antibodies and receptors [10].

Although phycocyanin presents many benefits, it is easily degraded due to the disposition of its protein aggregation state. This disposition may be attributed to several factors, such as light, temperature, pH, and protein concentration [11–13]. Sensitivity to heat and light, which manifests as precipitation and disappear- ance of color, is a limitation of phycocyanin when used as a food coloring agent or bioactive compound in the industrial sector [14].

Several approaches, such as addition of stabilizers, structural modification, immobilization, and microencapsulation, have been attempted to slow down the degradation rate of phycocyanin [15]. Sodium azide and dithiothreitol, which are common stabilizers, can be used to preserve phycocyanin for analytical purposes, but these reagents are toxic and unsuitable for application to food-grade products [16]. Sugars and polyhydric al- cohols have also been used to stabilize the proteins in phycocyanin [17, 18]. Basic research on phycocyanin encapsulation has been conducted by our group for several years now and advanced research is well under- way [19–23]. Yan et al. prepared phycocyanin microcapsules but only focused on the properties of the resulting product, not its degradation kinetics [24].

Chitosan, a natural polysaccharide, is the most important derivative of chitin, which is derived from natural sources, such as insect exoskeletons; arthropods such as crustacean shells, shrimp, crab, and cephalopod beak;

and fungal cell walls [25]. The polysaccharide is a highly compatible and effective biomaterial that can be used in many applications, such as water treatment [26], food [27], cosmetics [28], agriculture [29], and biomedicine [30]. Chitosan can be dissolved in acidic solution and confer positive charges through amino protonation; it also presents gelation and membrane-forming properties [31]. Carrageenan is another natural polysaccharide derived from certain species of red seaweeds or Rhodophyta. Carrageenan has progressively been used as a biomaterial for pharmaceutical purposes to improve drug formulations and sustained drug release, and its benefits beyond the pharmaceutical industry are currently being investigated [32]. Liu et al. determined three types of carrageenan: kappa (κ)-carrageenan, lambda (λ)-carrageenan, and iota (ι)-carrageenan [33]. Of these types,λ-carrageenan exhibits the best solubility, whileκ-carrageenan shows the poorest; in fact,κ-carrageenan is only soluble in hot water [34]. The presence of anhydrous bridges in carrageenan molecules is a key factor influencing their gelation process, especially when usingκ- andλ-carrageenan.

The kinetic parameters, especially thermal degradation, are important to predict the changes in quality of bioproduct that occurs during heat processing in industry. Thermal degradation kinetic usually use first-order kinetic reaction in the approach [35]. It is also used to determine the proper heating and temperature time, and the energy needed in processing bioproducts that involve heating, so it does not lose its value.

Although preliminary investigations on phycocyanin encapsulation have been carried out by our group, our general characteristics are usually limited to the encapsulation efficiency (EE), load capacity, and in vitro release of phycocyanin. In the present study, carrageenan and chitosan were used as coating materials to fabricate microencapsulated phycocyanin. We focus on the kinetics of thermal degradation of the encapsulated products as we aim to apply phycocyanin as a food colorant in the industry. Deeper characterizations, including determination of microstructures, chemical bonding, antioxidant activity (AA), kinetic rates (k), activation energies (E_a), and color degradation of microencapsulated phycocyanin, are performed.

2 Experimental

2.1 Materials and method

Powdered phycocyanin extract fromSpirulinasp. (4.2 of purity based on the absorbance ratio A₆₂₀/A₂₈₀) was purchased from CV Neoalgae (Sukoharjo, Indonesia). Commercialκ-carrageenan and chitosan were purchased from a local chemical store in Semarang for use as coating materials. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium chloride, sodium chloride, acetic acid, and sodium tripolyphosphate (STPP) were obtained from Sigma–Aldrich (St Louis, MO, USA)

2.2 Fabrication of microencapsulated phycocyanin

A phycocyanin solution was prepared for encapsulation by carrageenan or chitosan. To obtain the phycocyanin solution, 5 mg of phycocyanin extract was mixed with 50 ml of citrate buffer (pH 6) at a ratio of 1:10; this ratio

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was selected because other ratios, e.g. 1:1 or 1:5, yielded a phycocyanin solution that appeared concentrated and may be difficult to process.

To fabricate phycocyanin microencapsulated by carrageenan, 2 % (w/v) carrageenan solution was prepared by mixing 2 g of carrageenan powder with 100 ml of 1 % (w/v) NaCl and stirring at 50 °C for 4 h to form a gel. This mixture was then left at room temperature to rest. The phycocyanin and carrageenan solutions were mixed at a volume ratio of 1:1 for 1 h to obtain a homogenized mixture, which was then taken up into a syringe, injected into a container with 0.5 M KCl solution, and then left for 15 min until a perfect matrix had formed.

Granules of the encapsulated phycocyanin were filtered and washed using 1 % (w/v) NaCl. Finally, phycocyanin encapsulated by carrageenan was dried at 10 °C in a freeze dryer for 24 h.

To fabricate phycocyanin microencapsulated by chitosan, 2 % (w/v) chitosan and 100 ml of 1 % (v/v) acetic acid were mixed for 4 h at room temperature until a chitosan gel solution was obtained. The phycocyanin and chitosan solutions were then mixed at a volume ratio 1:1 for 1 h, taken up into a syringe, and injected into a container with 0.5 M STTP solution. After 15 min, a chitosan matrix formed over the phycocyanin, and the granules were filtered and washed with 1 % (w/v) NaCl. The chitosan-encapsulated phycocyanin was dried at 10 °C in a freeze dryer for 24 h.

Free-form phycocyanin was also prepared as a control. During the preparation process, the intensity of light in the room was minimized, and the samples were covered with aluminum foil to prevent photodegradation.

The complete pictures of microencapsulated phycocyanin preparation can be shown in Figure S1 in Supple- mentary Material.

2.3 Encapsulation efﬁciency and phycocyanin load determination

Encapsulation efficiency (EE) and phycocyanin load are important indices used to describe the characteristics of microencapsulation. The EE was determined based on the phycocyanin mass before and after encapsulation.

The EE is calculated by using Eq 1 [36, 37]:

EE= m_initial−munencapsulated

m_initial ×100% (1)

wherem_initialis total weight of phycocyanin added andmunencapsulatedis total weight of free-non-encapsulated phycocyanin. In this case, value ofm_initial is 5 mg as shown in Section 2.2. Moreover, phycocyanin load was obtained by dissolving 25 mg of dried microencapsulated phycocyanin in 25 mL of Na-buffer pH 7.4 for 20 min.

Then allowed to stand for 30 min in order to precipitate chitosan or carrageenan. The filtrate was separated from the solution, dried and then measured the weight. The phycocyanin load was calculated as follows:

phycocyanin load= mencapsulated

m_microbeads ×100% (2)

where themencapsulatedis mass of phycocyanin in supernatant, obtained from weight of dried microencapsulated minus by weight of chitosan or carrageenan filtrate. On the other hand,m_microbeadsis weight of dissolved dried phycocyanin microcapsule which the value was 25 mg.

2.4 Heating process and antioxidant activity measurement

Samples were heated to simulate actual conditions in the industry. Here, 10 mg of microencapsulated phycocyanin was diluted with 10 ml of de-ionized (DI) water and then heated to temperatures of 40, 50, and 60 °C for 90 min. Thereafter, 1 ml of each sample was obtained every 15 min and subjected to DPPH radical scavenging capacity assay to determine the AA of the encapsulated phycocyanin during heating. The DPPH method was selected because it is simple, fast, easy, accurate, reliable, and practical.

The AA assay was carried out by mixing 1 ml of 0.1 mM DPPH solution (in methanol) with 1 ml of encapsulated phycocyanin sample and then incubating the mixture for 1 h. The incubated mixture was diluted using methanol to 5 ml and its absorbance was measured using a UV–Vis spectrophotometer (Optima SP-3000;

Optima Inc., Tokyo, Japan) at 515 nm [38]. AA was determined by

Antioxidant activity= (1− A_sample

A_blank ) ×100% (3)

A mixture consisting of 1 ml of 0.1 mM DPPH, 1 ml of DI water, and 3 ml of methanol was used as the blank solution.

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2.5 Phycocyanin content measurement

Quantification of encapsulated phycocyanin was performed on the basis of the phycocyanin concentration determined after heating fortminutes. Total phycocyanin content (TPC) was obtained by measuring and adding C-PC and A-PC contents using a UV–Vis spectrophotometer. The relative phycocyanin concentration was determined by dividing the obtained TPC at timetwith the initial TPC and then multiplying by 100 %. C-PC and A-PC were calculated as follows[39]:

C−PC= OD₆₂₀−0.475×OD₆₅₂

5.34 (4)

A−PC= OD₆₅₂−0.280×OD₆₂₀

5.09 (5)

where OD₆₅₂and OD₆₂₀are optical densities of phycocyanin at wavelengths of 652 and 620 nm, respectively.

2.6 Kinetic model analysis

The kinetics of degradation of encapsulated phycocyanin, which was determined in terms of AA, were investigated to verify whether the data could be fitted to a zero-order, first-order, or second-order kinetic model as

C_t− C₀= − 𝑘t (6)

lnC_t

C₀ = − 𝑘t (7)

1 C₀ − 1

C_t = − 𝑘t (8)

whereC_tis the AA of the microencapsulated phycocyanin at timet, C₀is the initial AA of the microencapsulated phycocyanin,tis the heating time, andkis the kinetic rate constant. Here, AA directly represented the concentration of phycocyanin, both in initial and at timet. All of the data were fitted to the models using linear regression, and the coefficient of determination (R²) was used as the criterion to confirm adequacy of fit. The half-life (t_1/2) of phycocyanin was obtained by dividing 0.693 withk.

2.7 Activation energy measurement

TheE_aof microencapsulated phycocyanin was measured by relating the appropriate kinetic model, as shown in eqs. (6), (7), or (8), and the Arrhenius equation:

𝑘 = C^−E^a^/(RT) (9)

ln𝑘 = −E_A R (1

T) −ln C (10)

where C is the apparent phycocyanin concentration, which can be represented by AA, kis the kinetic rate constant,E_ais the activation energy,Ris the ideal gas constant (8.314 J·mol^-1·K⁻¹), andTis temperature. The slope determined by fitting isE_a/R, soE_awas determined by multiplying the slope withR.

2.8 Decimal reduction time measurement

The decimal reduction time (D-value) is the time required at a specific temperature and under specific conditions to reduce a concentration or activity by one decimal; in other words, it is the time required to achieve

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reduction of the initial population by 90 %. To determine theD-value, a thermal destruction rate curve was created as follows:

𝐷 = 𝑡

log𝑁_𝑡 (11)

log𝑁_𝑡= 1

𝐷𝑡 (12)

whereDis the decimal reduction time,tis time, andN_tis the AA of microencapsulated phycocyanin at timet.

The slope of the thermal destruction rate curve is 1/D.

2.9 Color degradation measurement

The color of phycocyanin before and after heating was analyzed using a colorimeter (Konica Minolta CR-400 Chroma Meter; Tokyo, Japan). The colorimeter was calibrated using a white reference tile before sample measurement. Approximately 10 ml of each sample was filled into a transparent glass surface (10 cm × 15 cm) and then analyzed. The L*a*b* coordinates (L*, lightness; a*, red-green; b*, for yellow-blue) were determined using the CIE scale, and color change (E*) was measured via:

ΔE^*= √ΔL^*2+Δa^*2+Δb^*2 (13)

whereΔE* is the color change,ΔL* is the difference inLbefore and after heating,Δa* is the difference in a before and after heating, andΔb* is the difference in b before and after heating.

2.10 Scanning electron microscopic analysis

The structure and particle size of the microencapsulated phycocyanin obtained after freeze-drying were evaluated with a Phenom ProX scanning electron microscope (Eindhoven, The Netherlands). The sample was at- tached to 1 inch-diameter SEM stubs using two-sided adhesive tape. Gold-palladium was used to coat the spec- imens via the plasma deposition method, and SEM was operated at a 15 kV accelerating voltage. Images were analyzed and recorded at 10,000–20,000 × magnification.

2.11 Fourier-transform infrared spectroscopic analysis

Chemical bonds between phycocyanin, carrageenan, and chitosan were evaluated by Fourier-transform infrared (FTIR) spectroscopy (Perkin Elmer Frontier; Waltham, MA, USA) within the wavenumber range of 450–

4000 cm⁻¹. The samples were not heated prior to analysis because they were already dry; thus, FTIR spectroscopy was performed at room temperature.

2.12 Statistical

All experiments had been done in triplicate and the results are expressed as mean values ± SD (standard deviation). The coefficient of determination (R²) was used as criteria for adequacy of fit.

3 Results and discussion

3.1 Physical and microstructural analysis of microencapsulated phycocyanin

Scanning Electron Microscope (SEM) was employed to describe the morphology and microscopic structure of the microencapsulated phycocyanin, and the results are shown in Figure 1(a–b). Two notable items could be

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observed in these figures: (i) The phycocyanin sample microencapsulated by carrageenan clearly presented a cubic form. Carrageenan-encapsulated phycocyanin revealed a particle size of around 2 µm and few pores on its surface, likely because, during freeze-drying, water on the carrageenan surface, but not in phycocyanin, is elim- inated from the granules. (ii) A large number of nanopores formed on the surface of the chitosan-encapsulated phycocyanin. These nanopores allowed the water in chitosan and phycocyanin to evaporate during freeze- drying. Thus, the chitosan-encapsulated phycocyanin showed irregular particle sizes of 2–4 μm.

Figure 1:Scanning Electron Microscope (SEM) micrographs of phycocyanin encapsulated with (a) carrageenan and (b) chitosan as coating materials. Photographs of phycocyanin encapsulated with (c) carrageenan and (d) chitosan.

The morphologies of phycocyanin encapsulated with carrageenan and chitosan are shown in Figure 1(c–d), respectively. Carrageenan-encapsulated phycocyanin showed a round and uniform shape. Compared with this sample, chitosan-encapsulated phycocyanin showed a flat non-uniform round shape. Freeze-drying is usually done under vacuum conditions. Because of the nanopores on the surface of chitosan, water on the surface of chitosan and phycocyanin was evaporated from the chitosan-coated sample. When the sample was completely dried, the air taken up by the vacuum pump in the drying chamber may further affect the structure of phycocyanin and chitosan. As such, the shape of this sample was shrunken and flat.

The EE and load capacities of the encapsulated phycocyanin samples are shown in Table 1. The EE and phycocyanin load of the chitosan-encapsulated phycocyanin were 76.45 % and 49.16 %, respectively, while the corresponding EE and phycocyanin load of the carrageenan-encapsulated sample were 68.66 % and 45.28 %, respectively. Adoption of chitosan as an encapsulation material for phycocyanin appears a very effective approach to protect the carotenoid since the EE obtained from this method is higher than that obtained in a pre- vious experiment that used alginate as an encapsulant (71.76 %) [23]. The polymer matrix of chitosan enabled good bonding with other molecules to trap phycocyanin within the microspheres despite the matrix featuring a large number of nanopores.

Table 1:Encapsulation efficiency and phycocyanin load of carrageenan- and chitosan-microencapsulated phycocyanin.

Coating material EE (%) Phycocyanin load (%)

Carrageenan 68.66 ± 2.45 45.28 ± 3.75

Chitosan 76.45 ± 3.11 49.16 ± 3.32

3.2 Chemical bonding characterization of microencapsulated phycocyanin

Figure 2 (line a) is the pure chitosan FTIR characteristic peaks. The presence of type III amide bond found at 1121.38 cm^-1, while the wavelength at 1215.85 cm^-1showed CH₃group of chitosan [40]. Wavelength at 1310.41 and 1660.12 cm^-1showed type II and I amide bond, respectively [41, 42]. Moreover, unique C=N bond could be found at 2400.87 cm^-1, and N-H stretch found at 3688.71, overlapped O-H bond [43].

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Figure 2:FTIR spectra of (a) pure chitosan, (b) pure carrageenan, (c) pure phycocyanin, (d) carrageenan-encapsulated phycocyanin, and (e) chitosan-encapsulated phycocyanin.

Figure 2 (line b) illustrates peaks characteristic of pure carrageenan. The peaks at 846.42, 932.25, and 1308.66 cm^-1 presented the presence of C–O–SO₄group on C4 of galactose, C–O bond of 3,6-anhydrogalactose, and S=O bond od sulfate esters, respectively, which exactly are the constituent components of carrageenan [44, 45]. The other components are type II and I amides bond, C=N bond, and N-H stretching which could be found at 1496.45, 1653.12, 2422.3, and 3390.34 cm^-1,respectively.

Figure 2 (line c) shows five characteristic peaks of phycocyanin as determined by FTIR. N–H stretching vibrations, which indicates the presence of lipids and proteins in phycocyanin, were observed at 3396.56 cm^-1 [46], while aliphatic C–H stretching vibrations were found at 2936.44 cm^-1 [47]. Phycocyanin also contained type-I amide (C=O) bonds, which appeared at 1652.39 cm^-1, and type-II amide bonds at 1546.7 cm^-1[48]. Because the phycocyanin used in this work was extracted from microalgae, phosphate and sulfate residues also appeared at 1080.31 cm^-1[49].

The main bonds formed by carrageenan-encapsulated phycocyanin are shown in Figure 2 (line d). The S=O bond of sulfate esters was observed at 1254.9 cm^-1, while the C–O bond of 3,6-anhydrogalactose appeared at wavenumbers of 1068.99 and 932.15 cm^-1[44]. The peak of galactose, as the carrageenan monomer, appeared to overlap with that of C–O peak, while the C–O–SO₄group on C₄of galactose was evidenced by the peak at 846.6 cm^-1[45]. All of the bonds found confirmed the presence of carrageenan on the surface of phycocyanin as a coating. The peaks of sulfate and phosphate residues from phycocyanin overlapped with the C–O bond of carrageenan. Amides of types I and II were observed at 1649.54 and 1542.6 cm^-1, respectively, while aliphatic C–

H stretching vibrations were found at 2930.7 cm^-1. N–H stretching vibrations of lipids and proteins appeared at 3439.44 cm^-1, and the O–H bond of carrageenan, which usually appears in the range of 3400–3500 cm^-1, overlapped with the peak of the N–H bond. A unique peak at 2370.8 cm^-1that was not found in the spectra of pure or carrageenan-encapsulated phycocyanin indicated the presence of the C=N bond. This result revealed that carrageenan and phycocyanin form the C=N bond upon bonding with each other.

Figure 2 (line e) shows the main FTIR peaks of chitosan-encapsulated phycocyanin. The peak at 1215.38 cm^-1 was attributed to the symmetrical deformation of the CH₃group, while the peak at 1157.01 cm^-1represented CO–NH deformation and type-III amides (CH₂group) [46]. The peak at 1468.2 cm^-1indicated N–H stretching or type-II amide deformation, while the peak at 1650.79 cm^-1revealed type-I amide bonds (C–N vibration of superimposed C = O linked to OH by H bonds) [47, 48]. The presence of the O–H group, unfortunately, could not

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be identified due to overlapping with the N–H stretching vibrations of lipid and proteins from phycocyanin, but can be observed in the range of 3400–3500 cm^-1 [49]. Moreover, all bonds found in phycocyanin, such as amide bonds of types I and II, phosphate and sulfate residues, and aliphatic C–H stretching bonds could also be clearly observed in the spectrum of the sample, which means chitosan bonded well with phycocyanin. The unique peak of C=N was observed at 2374.02 cm^-1, which means C=N bonds were formed in the chitosan- encapsulated phycocyanin

3.3 Effect of different coating materials on the thermal degradation of antioxidant activity and phycocyanin content

Figure 3(a) shows the decrease in AA of carrageenan-encapsulated phycocyanin during heating to 40, 50, and 60 °C. The AA of this sample decreased from 58.52 % to 44.66 % at 40 °C, from 51.43 % to 34.74 % at 50 °C, and from 41.69 % to 17.48 % at 60 °C. Thus, increased in temperature generally resulted in a higher decrease in AA. This finding might be expected since the coating material can be assumed to melt during heating and release the phycocyanin into the solution, which, in turn, causes the phycocyanin components (e.g. biliprotein) to degrade or called denature, resulting in a decrease in AA [50]. Since no stabilizer (e.g. sugar) was added to the phycocyanin solution, no glycosidic bonds are formed in the carotenoid, which, by itself, is relatively unstable [18, 51].

Figure 3:Degradation of the antioxidant activity of (a) carrageenan-encapsulated, (b) chitosan-encapsulated, and (c) free- form phycocyanin during heating to 40, 50, and 60 °C. Relative phycocyanin contents of (d) carrageenan-encapsulated, (e) chitosan-encapsulated, and (f) free-form phycocyanin at the same temperatures. Error bars show Standard Deviation.

The same phenomena were observed in the chitosan-encapsulated phycocyanin. The AA of this sample decreased from 61.48 % to 50.31 % at 40 °C, from 54.82 % to 39.91 % at 50 °C, and from 44.44 % to 27.23 % at 60 °C.

This decrease rate was slightly lower than that of carrageenan-encapsulated phycocyanin due to bonding dif- ferences between the coating material and phycocyanin especially the presence of CO–NH deformation and type-III amides (CH₂group) between phycocyanin and chitosan. The bonding structures of CO–NH deformation and type-III amides can be shown in Figure S2 in Supplementary Material. In the chitosan-encapsulated sample, water on the surface of the microcapsule surface and in the phycocyanin solution was evaporated

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during freeze-drying due to the presence of small pores on the chitosan coating. This absence of water pro- motes strong bonding between chitosan and phycocyanin. As such, the phycocyanin components in chitosan microcapsule difficult to degrade by increasing the temperature, for example, to 40 °C. At this temperature, the microencapsulated phycocyanin using chitosan was not completely melted and mixed in the solution, in contrast to the carrageenan-encapsulated sample, which completely melts at 40 °C. This finding confirms that bonding between chitosan and phycocyanin is stronger than that between carrageenan and phycocyanin on account of the presence of CO–NH deformation and type-III amides (CH₂group) in the former that are not dominant in the latter. The AA of free-form phycocyanin decreased from 94.5 % to 53.65 % at 40 °C, from 94 % to 39.13 % at 50 °C, and from 93.7 % to 18.47 % at 60 °C, as shown in Figure 3(c).

Quantification of the encapsulated phycocyanin was performed to investigate the relationship between AA and phycocyanin concentration. Thus, the relative concentration of phycocyanin after heating was determined, as shown in Figure 3(d–f). The relative concentrations of carrageenan-encapsulated phycocyanin decreased to 76.31 % at 40 °C, 67.54 % at 50 °C, and 41.93 % at 60 °C. By comparison, the relative concentrations of chitosan- encapsulated phycocyanin decreased to 81.83 % at 40 °C, 72.81 % at 50 °C, and 61.27 % at 60 °C. Moreover, the concentration of phycocyanin without any coating decreased dramatically to 56.77 % at 40 °C, 41.41 % at 50 °C, and 19.54 % at 60 °C. These results prove that both coating materials, chitosan and carrageenan, can slow down the thermal degradation of phycocyanin. In addition, the effects of chitosan in preserving AA and the phycocyanin concentration are better than those of carrageenan. Decreases in phycocyanin content clearly decreased AA.

3.4 Kinetic analysis of the thermal degradation of microencapsulated phycocyanin

Data of the degradation of AA of the samples were fitted to zero-order (eq. (2)), first-order (eq. (3)), and second- order (eq. (4)) kinetic models, and theR²of the resulting equation were obtained using Excel software. Fig- ure 4(a–c), respectively, show the zero-, first-, and second-order kinetic models of the thermal degradation of carrageenan-encapsulated phycocyanin. The range ofR²of the zero-order kinetic model was 0.857–0.987 with an average of 0.969, that of the first-order kinetic model was 0.991–0.995 with an average of 0.993, and that of second-order kinetic model was = 0.988–0.993 with an average of 0.990.

Figure 4:(a, d, g) Zero-order, (b, e, h) first-order, and (c, f, i) second-order kinetic models of the thermal degradation of (a–c) carrageenan-encapsulated, (d–f) chitosan-encapsulated, and (g–i) free-form phycocyanin.

The results above were confirmed by determining the R²of the chitosan-encapsulated sample, as shown in Figure 4(d–f). Here, the range ofR²for the zero-order kinetic model was 0.977–0.994 with an average of 0.986, that for the first-order model was 0.991–0.996 with an average of 0.994, and that for the second-order kinetic model was 0.986–0.991 with an average of 0.989.

In Figure 4(g–i), the range ofR²for the zero-order kinetic model of free-form phycocyanin was 0.894–0.979 with an average of 0.946, that for the first-order kinetic model is 0.989–0.996 with an average of 0.994, and that for the second-order kinetic model is 0.981–0.995 with an average of 0.988.

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These above-mentioned values are more or less similar and can be used to describe the thermal degradation of microencapsulated phycocyanin, although first-order kinetic model has a little bit higher value. To support in selection of reaction kinetic order, some relevant references related to thermal degradation kinetic were studied and the result is first-order kinetic model is more suitable to be used for this purpose [52–54].

An important parameter in kinetic studies isk. Thekvalues obtained from the slope of linear regression based on the first-order kinetic thermal degradation model at different temperatures are presented in Table 2;

lnk, t_1/2, andR²are also shown in the same table. Three notable findings are obtained. First, as temperature increased,kalso increased, which means temperature is the main cause of phycocyanin degradation. Second, thekof carrageenan-encapsulated phycocyanin was higher than that of the chitosan-encapsulated sample. This finding confirms that chitosan was better able to slow down the degradation of phycocyanin compared with carrageenan or no encapsulation. Special bonds between phycocyanin and chitosan, i. e. CO–NH deformation and type-III amides (CH₂group), help prevented the degradation of biliprotein in the former. Finally, thet_1/2 of chitosan-encapsulated phycocyanin was higher than that of the carrageenan-encapsulated sample. Thus, to degrade the activity of phycocyanin by half, the time required by the former is longer than that required by the latter. This result confirms that the bonds between phycocyanin and chitosan, such as CO–NH deformation and type-III amides (CH₂group), are essential in preventing the destruction of the components of the carotenoid.

Table 2:Kinetic reaction rates and the corresponding determination coefficients of the thermal degradation of phycocyanin with and without encapsulation.

Coating material

Temperature (°C)

k(× 10^–5 s^-1) lnk t_1/2(h) R²

40 4.92 ± 0.20 −9.92 ± 0.06 3.92 ± 0.16 0.991

Carrageenan 50 6.92 ± 0.15 −9.58 ± 0.04 2.78 ± 0.06 0.992

60 16.08 ± 0.08 −8.73 ± 0.02 1.20 ± 0.06 0.995

40 3.73 ± 0.12 −10.20 ± 0.03 5.16 ± 0.17 0.991

Chitosan 50 5.55 ± 0.08 −9.80 ± 0.02 3.47 ± 0.05 0.996

60 8.75 ± 0.18 −9.34 ± 0.05 2.20 ± 0.05 0.994

40 10.38 ± 0.21 −9.17 ± 0.06 1.85 ± 0.14 0.996

Control 50 16.15 ± 0.27 −8.73 ± 0.08 1.19 ± 0.02 0.996

60 29.65 ± 0.29 −8.12 ± 0.1 0.65 ± 0.01 0.989

Determination of E_a can help support other kinetic findings, such as k, and predict the minimum energy required to initiate the degradation reaction of microencapsulated phycocyanin. The values of ln k in Ta- ble 2 were used to calculate E_a through eq. (6), and the results were plotted in the graph shown in Fig- ure 5, while other calculation results are displayed in Table 3. Chitosan-encapsulated phycocyanin (E_a = 51.11±1.43 kJ·mol^-1) clearly showed a higher E_a than carrageenan-encapsulated or free-form phycocyanin (45.37±2.12 and 36.87±3.29 kJ·mol^-1, respectively). This finding indicates that a higher minimum energy was required to degrade chitosan-encapsulated phycocyanin than other forms of phycocyanin. CO–NH deformation and type-III amides (CH₂group) may plan an important role in this result. A substance with a lowE_awas easily degraded [14]. The highR²(range, 0.957–0.999) obtained in this experiment indicate that our data are robust enough to be used forE_adetermination.

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Figure 5:Arrhenius plot of the thermal degradation of encapsulated phycocyanin for activation energy determination.

Error bars show standard deviation.

Table 3:Activation energy and determination coefficients of the thermal degradation on phycocyanin with and without encapsulation.

Coating material E_a/R E_a(kJ mol^-1) R²

Carrageenan 5457.50 ± 254.64 45.37 ± 2.12 0.957

Chitosan 6147.37 ± 172.32 51.11 ± 1.43 0.999

Control 4434.29 ± 395.44 36.87 ± 3.29 0.926

TheD-value is the time required to reduce levels of one log cycle at a specific temperature. A highD-value reflects the high resistance of a substance to a particular heating temperature. The log of AA was plotted, as shown in Figure 6, and the correspondingD-values and coefficients of determination are shown in Table 4.

Two important phenomena were noted in this experiment. First,D-value increased as the heating temperature increased. Second, theD-value of chitosan-encapsulated phycocyanin was higher than that of carrageenan- encapsulated or free-form phycocyanin. These phenomena similar to those observed during determination of t_1/2in Table 2, except that the t_1/2 values refer to a time when phycocyanin activity is reduced to 50% of its initial activity, whileD-values refer to a time when phycocyanin activity is 90% of the original activity. When t_1/2andD-values were compared, the decrease in rate of degradation slowed from 90 % to 50% of the initial phycocyanin activity.

Figure 6:Thermal degradation rate curves of (a) carrageenan-encapsulated, (b) chitosan-encapsulated, and (c) free-form phycocyanin to determine decimal reduction times.

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Table 4:Decimal reduction times and the corresponding determination coefficients of the thermal degradation of phycocyanin with and without encapsulation.

Coating material Temperature (°C) D (h) R²

Carrageenan

40 13.55 ± 1.21 0.988

50 6.08 ± 1.17 0.980

60 4.25 ± 1.12 0.991

Chitosan

40 14.01 ± 1.22 0.992

50 6.17 ± 1.06 0.987

60 4.46 ± 1.12 0.995

40 6.46 ± 2.02 0.996

Control 50 4.30 ± 1.45 0.996

60 3.34 ± 1.61 0.989

3.5 Effect of different coating materials on the color degradation of phycocyanin

Investigating the color degradation of microencapsulated phycocyanin is important because this degradation is related to degradation of AA. According to Hadiyanto et al. [18], the color of fresh phycocyanin solution is light blue, CIE coordinates of L* = 66.69, a* =−14.42, and b* =−15.35. Decreases inΔL*,Δa*, andΔb* shown in Figure 7(a–c) could be attributed to the effect of increasing temperature. TheΔL* value was decreased to negative value, which indicates that the phycocyanin color was lighter as increased in temperature, where the more negative value was control variable. It means, color of phycocyanin could change easily without formed as microcapsule. Microencapsulated phycocyanin using carrageenan and chitosan had more positiveΔa* value where the value around–2, which means these samples had a more blue-green color composition compared to the control. As the temperature increases,Δb* value of control variable also decreased, and the value was –7.7 which lower than microencapsulated phycocyanin using carrageenan or chitosan. It means that color of phycocyanin sample in control variable easier to change.ΔE* increased under the same conditions, as shown in Figure 7(d). To determine the effect of the coating material on color degradation, chitosan was used to mi- croencapsulate phycocyanin. The product demonstrated a bright light compared with another sample because of its lowΔE* value as discoloration becomes increasingly apparent with increasingΔE*. Adopting chitosan or carrageenan as the coating material of microcapsules only appears to prevent physical color changes. Sugar can be added in to the phycocyanin solution before encapsulation to stabilize the color change during heating via polymerization between phycocyanin and sugar [55]. According to Huang [56], polymerization of phycocyanin by sugar results in resistance to enzymatic reactions and protection from thermal degradation, including color degradation.

Figure 7:Correlations of (a)ΔL*, (b)Δa*, (c)Δb*, and (d)ΔE* with heating to 40, 50, and 60 °C. Error bars show standard deviation.

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4 Conclusion

This research reports the use of carrageenan and chitosan as phycocyanin coating materials to retard deterioration of the AA and color of the carotenoid due to high-temperature exposure. The effects of temperature on phycocyanin with and without encapsulation were thoroughly investigated, and findings indicated that carrageenan and chitosan could be used as effective coating materials to prevent the thermal degradation of phycocyanin. Phycocyanin degradation showed first-order reaction kinetics when heated to 40–60 °C and revealed kof 4.67–9.17 × 10^–5 s^-1and 3.83–7.67 × 10^–5 s^-1for carrageenan and chitosan, respectively; by comparison, the correspondingkobtained from the control (without coating) were 10.38–29.65 × 10^–5 s^-1. The EEs were 68.66 % for carrageenan and 76.45 % for chitosan, while the loading capacities were 45.28 % for carrageenan and 49.16 for chitosan, respectively. Encapsulation prevented color degradation, as evidenced by the lowerΔE observed in encapsulated systems compared with that in the control.

Taken together, the results indicate that encapsulated phycocyanin could be applied to the food industry, especially in food supplements containing antioxidant compounds with heat-sensitive properties.

Acknowledgements

This research was financially supported by the Indonesian Ministry of Research, Technology, and Higher Edu- cation through Research Grant PUSN 2017-2019. The authors thank to C-BIORE for their facilities and guidance.

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Supplementary Material: The online version of this article offers supplementary material (DOI:https://doi.org/10.1515/ijfe-2018-0290).