Thermal Degradation Kinetics of Phycocyanin

Encapsulation as an Antioxidant Agent

A M Nilamsari2 , A Yunanda2, and  H Hadiyanto1 2,
1
Center of Biomass and Renewable Energy (C-BIORE), Chemical Engineering
Department, Diponegoro University
2
Department of Chemical Engineering Diponegoro University
Jl. Prof. Soedarto SH, Tembalang, Semarang, 50275
Email: hadiyanto@live.undip.ac.id

Email: hady.hadiyanto@gmail.com
Abstract. Phycocyanin is a blue-light pigment that found in Cyanobacteria and
two Eucaryotics algae such as Rhodophyta and Crytophyta. Phycocyanin is
soluble in water and has a strong fluorescent properties as an antioxidant and
normally used in food industry, cosmetic, biotechnology, and drug. However,
Phycocyanin is easily damaged by a heating process. The aim of this study is to
obtain the optimal condition of Phycocyanin encapsulation with different
coating materials, Chitosan and Carrageenan, by the calculation of heat
resistance of antioxidant activity (D), range of temperature that increase the rate
of degradation (Z), rate constant of degradation (k), and activation energy (Ea).
The ratio of Phycocyanin and the coating material are 2% (w/v) and 2 % (w/v).
Keyword: Phycocyanin, Antioxidant, Carrageenan, Chitosan, Degradation

1. Introduction

Antioxidants are compounds that can be used to protect food by slowing down the
damage, rancidity or change of colour caused by oxidation (Windono et al., 2001). Antioxidants
are able to act as hydrogen radical contributors or free radical acceptors to delay the initiation
phase of free radical formation. The presence of natural (such as Phycocyanin compounds) and
synthetic antioxidants can inhibit lipid oxidation, prevent damage, it change inorganic
components in foodstuffs to extend shelf life (Rohdiana, 2001). T-butyl hydroxy anisol (BHA)
and di-t-butyl hydroxytoluene (BHT) compounds are used as food antioxidants, but the
possibility of adverse side effects is not used for therapeutic agents. The development of natural
antioxidants has received considerable attention in recent years. The purpose is to preventive
medicine and for food industry. DPPH test is an effective and rapid method for estimating
antioxidant activity. The principle of this method is to measure the power of extracting an
ingredient against the free radical of DPPH (Damayanti, 2004).
Development of antioxidant components is widely used in the production of functional
foods. The functional properties of functional foods are to determined by the bioactive
components contained in them, example: dietary fiber, inulin, FOS and antioxidants (Marsono,
2007). Based on the source, antioxidants are divided into endogenous antioxidants and
antioxidant enzymes, such as: Superoxide Dismutase (SOD), Catalase (Cat), and Glutathione
Peroxidase (Gpx); as well as exogenous antioxidants, which are obtained from outside the
body / food. Various natural ingredients native to Indonesia contains many antioxidants with
various active ingredients, including vitamin C, E, pro vitamin A, organosulfure, α-tocopherol,
flavonoid, thymoquinone, statin, niacin, Phycocyanin, and others (Asri, 2014).
One type of antioxidant that comes from outside the human body is Phycocyanin.
Phycocyanin is the most important pigment in Spirulina sp. (Henrikson, 2009). Phycocyanin has
a significant content as an antioxidant, protector of liver function, and radical compounds
remover (Weil, 2000). Therefore, Phycocyanin is widely used in the field of food colouring and
cosmetics. Phycocyanin content in 10 grams of dried Spirulina is about 1400 mg or 14%, so it
has been recommended to consume 0.25-2.5 grams of Phycocyanin per day to boost the
immune system and inhibit cancer growth (Henrikson, 2009). There are several methods to
protect Phycocyanin from degdradation, microencapsulation is one of them.
Microencapsulation is a process where solid materials, liquids, and even gases can be
capsulated (encapsulated) with microscopic particle size, by forming thin wall covering around
the material to be capsulated (Ansel, 2007). The techniques used for microencapsulation are
spray drying, spray cooling / chilling, fluidization of bed drying, melt extrusion, melt injection,
centrifugal extrusion, coacervation, liposome capture, co-crystallization, emulsification,
rotational suspension rotation, and molecular inclusion (Nicolaas and Eyal, 2010). The active
ingredient in the Phycocyanin microencapsulation is sensitive to light, heat and oxygen to have
a limited shelf life. Microencapsulation process is to minimize the occurrence of quality
degradation of Phycocyanin. Therefore, thermal degradation kinetics model of Phycocyanin
microencapsulation needs to be further investigated.

2. Methods

2.1. Materials
In this study, phycocyanin is used as raw material. In addition, side materials are also
used such as: chitosan, carrageenan, DPPH, methanol, acetic acid, STTP, KCl, NaCl and
aquadest. Chitosan and carrageenan were used as a coating material. Methanol, acetic acid,
NaCl, and aquadest were used as solvent. STTP and KCl were used as a microencapsulation
media, and DPPH as a material test. Phycocyanin is powder form of Spirulina platensis. While
other materials obtained from laboratory in Diponegoro University.
2.2. Variables
Variabel yang dikendalikan dalam penelitian ini antara lain daging sapi, tepung tapioka
15% dari berat daging, garam 2%, bawang putih 2,5%, merica halus 0,8%, es batu 20%, serta
suhu penyimpanan bakso yaitu pada suhu kamar 25 0 C.The controlled variables in this research
were heating time during 90 minutes with observation time for every 30 minutes. 3.2.2.Variabel
respon dalam penelitian ini adalah tekstur bakso daging sapi yang dihasilkan, total bakteri, MPN
bakteri Coliform serta tingkat kesukaan konsumen terhadap bakso daging sapi.The response
variable in this study was the activity of antioxidant. Variabel bebas dalam penelitian ini antara
lain konsentrasi asap cair (0,5% - 1%), konsentrasi karagenan ( 0,5% - 1,5%).Independent
variables in this study were temperature (40°C, 50°C, 60°C) and coating materials (chitosan 2%
and carrageenan 2%).
2.3. Making Microencapsulation Phycocyanin
To make microencapsulation Phycocyanin is devided into two methods, they are with
chitosan coating agent and carrageenan coating agent. Once, coating of chitosan was done by
dissolving chitosan into 1% of acetic acid solvent. One percent of acetic acid solution was
prepared by dissolving 1 ml glacial acetic acid into 100 ml of aquadest. Then, 2 grams of
chitosan was dissolved in 100 ml of 1% acetic acid solution, stirred with magnetic stirrer for ±4
hours until homogenous to obtain chitosan 2% w/v 100 ml. After that, dissolve Phycocyanin in
aquadest with ratio of Phycocyanin and aquadest of 1:10. Then, Phycocyanin solution mixed
with chitosan solution with ratio of volume of 1:1. After that, stir the solution by using magnetic
stirrer until homogeneous. Then, homogeneous solution is inserted into syringe and injected into
STTP (Sodium Tripolyphosphate) solution 0.5 M. Wait for about 15 minutes until the matrix
perfectly formed. Then, filtered and washed the granules with 1% of NaCl. After that, dried it
by freeze drying method (10°C) and wait for about 24 hours. Finally, microencapsulation with
chitosan coating is formed. In the other hand, carrageenan coating was done by dissolved 2
grams of carrageenan in 100 ml of 1% NaCl solution and heated with stirring by using magnetic
stirrer at temperature about 40-50 °C until gel of carrageenan are formed, and carrageenan 2%
w/v 100 ml was obtained. After that, dissolved Phycocyanin in aquadest with ratio of
Phycocyanin and aquadest of 1:10. Then Phycocyanin solution mixed with carrageenan
solution with ratio of volume of 1:1. After that, stirred the mixture by using magnetic stirrer
until homogeneous. The homogeneous solution is inserted into syringe and injected into KCl 0.5
M. Wait for about 15 minutes until the matrix perfectly formed. Then filtered and washed the
granules with 1% NaCl. After that, dried by freeze drying method (10°C) and wait for about 24
hours. Finally, microencapsulation by carrageenan coating is formed.
2.4. Antioxidant Activity Test
The DPPH test is an effective and rapid method for estimating antioxidant activity. The
principle of this method is to measure the power of extracting an ingredient against the free
radical of DPPH (Damayanti, 2004). The various variables are temperature (40°C, 50°C, and
60°C) and heating time (0, 30, 60, 90 minutes). Antioxidant activity of phycocyain
microencapsulation analysis was conducted by dissolving 10 mg of microencapsulation into 10
ml of aquadest, then heated according to various temperature (40°C, 50°C, and 60°C). Then,
took 1 ml sample for various times (0, 30, 60, 90 minutes). To make a solution of 0.1 mM
DPPH by dissolving 2 mg DPPH into 50 ml of methanol. Then 1 ml of heated
microencapsulated Phycocyanin, 1 ml DPPH 0.1 mM, and 5 ml of methanol are incubated for
about 30 minutes. Then, take the solution that has been incubated to have an antioxidant activity
test. And also, prepared blank solution in the same way without using of Phycocyanin
microencapsulation. Perform absorbance measurement at optimum λ in wavelength of 515 nm.
Antioxidant capacity is to inhibit free radicals. Andayani et al. (2008) determined the equation
of antioxidant activity, it is:
Antioxidant Activity (100%) = {1- (A sample / A blank)} x 100%.
2.5 Color Stability Test with Digital Colorimeter
The color of the material can be measured by using a calorimeter tool. First, take 5 mm
samples on each variable. Then, put the sample in place just below the camera lens connected to
the Digital Colorimeter device. Point the camera at the part you want to know the color. View
and note the values of L, a and b on the device display.

2.6 FTIR test and SEM test

FTIR test is used to determine the vibration of the functional groups of the compound.
From the FTIR spectrum will be known by the function of functional vibrations in the
Phycocyanin microencapsulation. While, SEM test is used to know the surface morphology
analysis of Phycocyanin microencapsulation.

3. Result and discussions

3.1 Determine Thermal Degradation Kinetic of Phycocyanin Encapsulation with Carrageenan
Thermal degradation kinetics of Phycocyanin encapsulation with carragenan are
detemined by various parameters, including parameters of k (1/minute), D (minutes), Z (°C) and
Ea (kJ/mol). By Arrhenius equation, the order of the reaction can be determined. After a suitable
order has been determined, the rate constant of reaction (k) is known by slope of the order
equation.
 (A) 70.000

Antioxidant Activity (%)

60.000
50.000
40.000
30.000
20.000
10.000 Temperature 40°C
0.000 Linear (Temperature
0 10 2040°C)
30 40 50 60 70 80 90 100
Time (minutes)

(B) 0
Ln Antioxidant Activity

40
4.
0
80
3.
0
20
3.
0
60
2. Temperature
0 40°C
00 0
2. 10 20 30 40 50 60 70 80 90 100
Time (minutes)

Figure 3.1 Thermal degradation kinetics of antioxidant activity (A) zero order & (B) 1st order

Based on Figure 3.1 maximum regression is 1st order. So, thermal degradation kinetics
of antioxidant activity of carrageenan are using 1st order equation with R2 equal to 0.9161-
0.9899. The results are according to Jaiswal and Abu-Ghannam (2007) that the kinetics model
of antioxidant capacity by DPPH method of 1st order equation with R2 is equal to 0.95-0.97.
3.000

f(x) = - 0.02x + 3.58

R² = 0.86
Log D

2.500

2.000
40 42 44 46 48 50 52 54 56 58 60
Temperature (°C)

Figure 3.2 Determination curve of Z

According to the data from Table 3.1, there is a degdradation process in antioxidant
during heating process. The value of D, are 769.231 minutes for D40°C, 400 minutes for D50°C,
and 333.333 minutes for D60°C. Thus, the differences in temperature and heating time also make
a different effect of degdradation kinetics of antioxidant activity in Phycocyanin encapsulation.
Based on Figure 3.2, it showed that regression equation is y=-0.0176x+3.5835 with R2=0.8563.

Table 3.1 Calculation of D, k, Z, and Ea

Temperature D k Z Ea
 (°C) (minute) (minute-1) (°C) (kJ/mol)
40 769.231 0.0031
50 400 0.0058 54.945 0.80406
60 333.333 0.0068

From Table 3.1., it has been known that the reaction rate constant (k) at D 40°C, D50°C, and
D60°C are 0.0031 min -1, 0.0058 min -1, 0.0068 min -1.

-3.000
0.0160.0170.0180.0190.0200.0210.0220.0230.0240.0250.026
-3.500
-4.000
-4.500
Ln k

-5.000
f(x) = - 96.71x - 3.32
-5.500 R² = 0.95
-6.000
-6.500
-7.000
1/T

Figure 3.3 Energy activation degradation of antioxidant activity

Based on Figure 3.3, the activation energy (Ea) is determined by using linear regression
between 1/T (x-axis) and Ln k (y-axis), then the slope is multiplied by R constant (8.314 J/mol
K). With linear regression equation of y=-96.712x-3.3177, R2 = 0.9541, so we obtained
activation energy for 0.80406 kJ/mol. The figure shows the minimum energy is required to
initiate the degradation of antioxidant activity in Phycocyanin microencapsulation. The lower of
Ea value will be easier to be decreased.

3.2. Determine Thermal Degradation Kinetic of Phycocyanin Encapsulation with Chitosan

Thermal degradation kinetics of Phycocyanin encapsulation with carragenan are
detemined by various parameters, including parameters of k (1/minute), D (minutes), Z (°C) and
Ea (kJ/mol). By Arrhenius equation, the order of the reaction can be determined. After a suitable
order has been determined, the rate constant of reaction (k) is known by slope of the order
equation.
(A) 70.000
% Activity Antioxidant

60.000
50.000
40.000
30.000
20.000
Temperature
10.000 40°C
0 10 20 30 40 50 60 70 80 90 100
Time (minutes)

(B)
 4.500

Ln Activity Antioxidant
4.000
3.500
3.000
2.500 Temperature 40°C
Linear (Temperature
2.000 40°C)
0 10 20 30 40 50 60 70 80 90 100
Time (minutes)

Figure 3.4 Thermal degradation kinetics of antioxidant activity (A) zero order & (B) 1st order
Based on Figure 3.1 maximum regression is 1st order. So, thermal degradation kinetics
of antioxidant activity of carrageenan are using 1st order equation with R2 equal to 0.9161-
0.9899. The results are according to Jaiswal and Abu-Ghannam (2007) that the kinetics model
of antioxidant capacity by DPPH method of 1st order equation with R2 is equal to 0.95-0.97.
4.000

3.500
Log D

3.000

2.500

2.000
40 45 50 55 60 65 70
Temperature (°C)

Figure 3.5 Determination curve of Z

According to the results in Table 3.2, can be seen a decrease in antioxidant activity during
the heating process. D40°C is 833 minutes, D50°C is 417 minutes, D60°C 370 minutes. The value of D
(Decimal Reduction Time) is the time required to reduce levels of one cycle of the log at a
specific temperature. Benefits to determine the value D is to declare the heat treatment time
period that is required at a specific temperature to reduce 90% or one-tenth the initial values.
The larger the value of D indicates the higher heat resistance of the antioxidant activity in a
particular heating temperature. Thus, differences in temperature and heating time affect the
kinetics of decrease in antioxidant activity in the Phycocyanin microencapsulation (Sukasih,
2009).
Based on the determination of the value curve Z in Figure 3.5 regression equation
y=0,0176x + 3.5835 R2= 0.8563. The smaller value indicates that more sensitive Z value D by a
change in the heating temperature. By recognizing the value Z, allowing calculation of the
heating temperature needs that can destroy the antioxidant activity of the product (Sukasih,
2009).

Table 3.2 Calculation of D, K, Z, and Ea

Temperatur value D k values of Ea
e (°C) (min) (min-1) Z (kJ/mol)
(° C)
40 833 0.0027 56.818
50 417 0 , 0055 857.3
 60 370 0.0062

In Table 3.2 known reaction rate constant (k) at a temperature of 40°C, 50°C and 60°C
respectively, are 0.0027 min-1; 0.0055 min-1; and 0.0062 min-1. While the reaction rate constant
(k) on research Francine, et al (2008) study of extract Phycocyanin get value k at 50°C and
60°C respectively at 0.0012 min-1 and 0.0108 min-1, This difference can occur because of
differences in the sample and the thermal treatment is given.

-5
-5.10.015 0.017 0.019 0.021 0.023 0.025 0.027
-5.2
-5.3
-5.4
Ln k

-5.5
-5.6
-5.7
-5.8
-5.9
-6
1/T

Figure 3.6 The curve determining the activation energy (Ea)

While the results of the activation energy (Ea) was determined using linear regression
between 1/T (axis x) with Ln k (y axis), then the slope is multiplied by the constant R (8.314
J/mol K). With linear regression equation y=-103,11x-3,2807, R2=0,9263 activation energy
generated is 857.3 kJ/mol. In the study Francine, et al (2008) studied about getting Ea of
Phycocyanin. Phycocyanin extract amounted to 466.788 kJ / mol. The figure shows the
minimum amount of energy required to initiate the reaction was decreased of antioxidant on
microencapsulated Phycocyanin. The lower the value, the more easily Ea decreased antioxidant
activity. Therefore, microencapsulation method is to reduce the effect of a decrease in
antioxidant activity by a heating process.

3.3. Effect of Coating Materials on Antioxidant Activity

A B

C
 Figure 3.7 Heating time relationship of antioxidant activity

Based on Figure 3.7 shows that the effect of heating time (minutes) of antioxidant
activity (%) at different temperatures (40°C, 50°C and 60°C) where the tested sample is
Phycocyanin microencapsulation with chitosan agent and carrageenan agent. Based on the
phenomenon, show that the longer of heating time, will show graph tends to fall. This
phenomenon occurs in both the microencapsulated Phycocyanin with chitosan coating material
or microencapsulated with a coating Phycocyanin carrageenan. This is because Phycocyanin is
biliprotein, as globular proteins in general may experience structural damage due to heat (Ó Ó
Carra & hEocha 1976; Lehninger 1982). Damage to these structures called denaturation, the
change in the structure of regular naturally become irregular in three-dimensional configuration
(Hawab et al., 1989). Then, the longer the heating time Phycocyanin defective number is also
growing. The less its number of active Phycocyanin cause decreased antioxidant activity. In
addition, based on test conducted that a coating agent of chitosan antioxidant activity greater
than the antioxidant activity by the carrageenan. This is because the microencapsulation process
Phycocyanin with carrageenan carried out at a temperature of 40-50°C, Phycocyanin very
susceptible to heat, it is causing there are some damaged due to Phycocyanin.

3.4. Comparison of Color Stability Test Carrageenan and Chitosan Coating Agent
Table 3.3 The color stability of the coating agent carrageenan and chitosan
No. Type ΔL Δa Δb ΔE
1. Carrageenan 40°C -2,453 -3,91 -3,601 5,854
2. Chitosan 40°C -0,76 -5,769 -1,75 6,075
3. Carrageenan 50°C -1,195 -1,032 -4,375 4,651
4. Chitosan 50°C -2,449 -4,848 -0,891 5,504
5. Carragenan 60°C -3,236 -1,987 -0,383 3,817
6. Chitosan 60°C -4,924 -2,055 -6,582 8,437

The results of the color measurement on the Phycocyanin microencapsulation with

colorimeter are presented in table 3.3, where in Phycocyanin microencapsulated either by using
carrageenan or chitosan coating material has a color of L(-), which indicates that the darker the
color of the solution Phycocyanin. Value of a(-) is ndicates that leads to the green color and the
value of b(-) is indicates that the color leads to blue, as well as AE shows how much the color
changes. Phycocyanin microencapsulation with chitosan has a blue color firmer than with
carrageenan. Blue indicates the rich content of Phycocyanin. Phycocyanin a natural blue
pigment contained in Spirulina sp. (Sedjati et al., 2012) which has the properties as an
antioxidant, however, is not resistant to temperature Phycocyanin (Chaiklahan et al, 2012; Yan
et al, 2014). If given a high temperature, then extract Phycocyanin will be damaged. This can be
evidenced by the increasing values of L and b along with rising temperatures.

3.5. FTIR Test of Phycocyanin Microencapsulation with Carrageenan and Chitosan

 Figure 3.8 FTIR test phycocyanin with carrageenan

Figure 3.9 FTIR test carrageenan (SK Bajpai, 2014)

Table 3.4 Identification of compounds FTIR test results and reference

No. X Y reference (λ)
1 3439,44 26,5 3309,96
2 2930,7 29,27 2943,47
3 2370,8 30,72 1722,49
4 1649,54 27,67 1427,37
5 1068,99 28,58 1149,61
6 610,28 30,88 620,89

Based on the FTIR test results Phycocyanin with carrageenan and carrageenan reference
FTIR showed no significant difference. Both have functional groups are ethers, esters, and
alcohols. This is in accordance with the references is that the algae biomass to absorb metal ions
via complex formation interaction between the metal ions with functional groups such as
-COOH main constituent polysaccharides and peptides groups (-CO, NH 2) as a constituent of
pectin and protein that acts as a donor pair of electrons to a metal ion (Gupta and Rastogi,
2008).
 Figure 3.10 FTIR test results phycocyanin with chitosan

Figure 3.11 FTIR Chitosan (ShahidaYasmeen, et al, 2016)

Table 3.5 Identification of compounds FTIR test results phycocyanin + chitosan

No. X Y Reference (λ)
1 3404,12 27,77 3478,68
2 2928,8 29,76 2924,1
3 1650,79 29,38 1656,87
4 1418,2 30,95 1571,05
5 1215,38 29,85 1422,52
6 1157,01 29,05 1378,16

Based on the results of FTIR test phycocyanin with chitosan and reference of FTIR test
chitosan showed that no significant difference. Both have functional groups are ethers, esters,
and alcohols. This is in accordance with the references is that the algae biomass to absorb metal
ions via complex formation interaction between the metal ions with functional groups such as
-COOH main constituent polysaccharides and peptides groups (-CO, NH 2) a constituent of
pectin and protein that acts as a donor pair of electrons to a metal ion (Gupta and Rastogi,
2008).

3.6 SEM test Phycocyanin Microencapsulation with Carrageenan and Chitosan

 Figure 3.12 SEM test results phycocyanin with carrageenan

Figure 3.13 SEM test results phycocyanin with chitosan

The results of the analysis of Phycocyanin microencapsulation by SEM showed that the
microencapsulated formed round with uneven surfaces caused by the particle coating material
that each carrageenan and chitosan. Microencapsulation surface pores using chitosan coating
material is denser than the surface of the microencapsulated using carrageenan coating material,
it is because the adhesion of the coating material chitosan deeper due to the heating in the
coating process the material.

Conclusions
Based on the value of D obtained show that the difference in temperature and the
heating time affect the kinetics decline in Phycocyanin microencapsulation antioxidant activity.
As for the value of Z, the smaller value indicates more sensitive Z value D by a change in the
heating temperature. Differences in reaction constant value (k) in each of the coating material
can occur because of differences in the sample and the thermal treatment is given. While, the
activation energy (Ea) showed that the lower the value of Ea is the easier decreased antioxidant
activity. Phycocyanin microencapsulation either by using the coating material carrageenan or
chitosan has a color of L(-), which indicates that the color of the solution Phycocyanin was
getting dark, the value of a(-) indicates that the color leads to green, and the value of b(-)
indicates that the color leads to blue, and AE shows how much the color changes. Phycocyanin
microencapsulation either by using carrageenan or chitosan coating material having functional
groups such as -COOH and cluster peptide (-CO, NH 2). Phycocyanin microencapsulation has an
uneven surface due to adhesion of the coating material on Phycocyanin extract.

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