Ultrasonics Sonochemistry 38 (2017) 92103

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Ultrasound assisted methods for enhanced extraction of

phycobiliproteins from marine macro-algae, Gelidium pusillum
(Rhodophyta)
Rochak Mittal a,c, Hrishikesh A. Tavanandi a,c, Vaibhav A. Mantri b, K.S.M.S. Raghavarao a,c,
a
Academy of Scientific and Innovative Research (AcSIR), India
b
CSIR Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), Bhavnagar, India
c
CSIR Central Food Technological Research Institute (CSIR-CFTRI), Mysuru, India

a r t i c l e i n f o a b s t r a c t

Article history: Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is
Received 14 July 2016 difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which
Received in revised form 12 February 2017 offer major hindrance for cell disruption. The present study is aimed at developing most suitable method-
Accepted 22 February 2017
ology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum
Available online 27 February 2017
(Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional
methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing
Keywords:
and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication
Seaweed
R-phycoerythrin
for different parameters such as ultrasonication amplitude (60, 90 and 120 mm) and ultrasonication time
R-phycocyanin (1, 2, 4, 6, 8 and 10 mins) at different temperatures (30, 35 and 40 C) was carried out. Kinetic parameters
Kinetics were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass
Energy transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power
Ultrasonication density were estimated to be 41.97 W, 14.81 W/cm2 and 0.419 W/cm3, respectively. Synergistic effect
Extraction of ultrasonication was observed when employed in combination with other conventional primary extrac-
tion methods. Homogenization in combination with ultrasonication resulted in an enhancement in effi-
ciency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication
resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods
employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency
of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrason-
ication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE
was present in the extract and remained intact even after processing. Microscopic studies indicated a
clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a
given primary extraction method. These combination methods were found to be effective for extraction
of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for down-
stream processing of biomolecules also from other macro-algae.
2017 Elsevier B.V. All rights reserved.

1. Introduction inflammatory, neuro-protective and hepato-protective properties

Phycobiliproteins from red marine macro-algae namely,
R-Phycoerythrin (R-PE, M.W. 240 kDa) and R-Phycocyanin (R-PC,
M.W. 136 kDa) are the aqueous soluble red and blue fluorescent
pigments, respectively. These bioactive molecules are reported
to have antioxidant [1,2], anti-diabetic, anti-tumor, anti-
Corresponding author at: CSIR Central Food Technological Research Institute
(CSIR-CFTRI), Mysuru, India.
E-mail address: raghavarao@cftri.res.in (K.S.M.S. Raghavarao).
[1,3]. Depending upon their source, phycoerythrin is classified into
B, R and C types and phycocyanin into C and R types, wherein B
stands for Bangiales, R for Rhodophyta and C for Cyanobacteria.
These are essentially low volume high value compounds. Depend-
ing on purity level, R-PE costs about (180 USD to 250 USD/mg) [4].
R-PE and R-PC can be used as an immuno-fluorescent probe due to
their fluorescent properties and are gaining importance in thera-
pies, cosmetics [3], for analytical purposes in biotechnology (cell
labeling) and as natural colorants in food processing [3]. These
biomolecules can be extracted from selected group of micro- as

http://dx.doi.org/10.1016/j.ultsonch.2017.02.030
1350-4177/ 2017 Elsevier B.V. All rights reserved.
 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103 93

well as macro-algae. Extraction of phycobiliproteins from micro- from the coast of Valinokkam, Tamil Nadu, India. The biomass
algae has been extensively reported in the literature [57]. was washed with filtered sea water for the removal of sand parti-
The marine macro-algae, for instance genus Gelidium, have been cles, epiphytes and other undesirable foreign materials before
an industrially important source of agar [8]. In addition to agar, transporting under cold conditions to CSIR-CFTRI, Mysore. Biomass
algal biomass can be used more effectively for the extraction of was stored at
40 2 C and desired quantity of biomass was
other bio-chemicals such as macro-elements (Na, K, Ca, Mg) [8], taken out as and when required for the experimentation.
proteins, lipids and cellulose [9,10], which also have health bene-
fits [8,11]. Thousands of tons of seaweed biomass is used to extract 2.1.2. Chemicals
these products [8]. Unlike micro-algae (which is cultivated in fresh The phosphate salt of analytical grade was procured from Mer-
water), macro-algae can be harvested from their natural habitat or ck, Bangalore. Standard of R-PE was procured from Sigma Aldrich
cultivated in the shallow waters of sea [8]. In addition, these Pvt. Ltd., Bangalore. All other chemicals and HPLC column used
macro-algae have natural pigments such as Phycoerythrin (PE) were procured from Merck, Bangalore.
and Phycocyanin (PC) as accessory photosynthetic pigments [8].
As the phycobiliproteins are intracellular, cell disruption is 2.2. Methods
required for their efficient release during extraction. There are sev-
eral methods available for extraction of phycobilins such as osmotic 2.2.1. Preparation of phosphate buffer
shock [12], maceration in presence of liquid nitrogen [13], freeze Double distilled water was used to prepare phosphate buffer of
grinding [4,14], freezing and thawing [2], ultrasonication [15] and 0.1 M and pH 6.8, [9] and used for primary extraction of phyco-
homogenization [16]. However, these methods are reported for biliproteins. In our preliminary studies also, the extraction buffer
characterization and physicochemical analysis of phycobiliproteins pH of 6.8 was found to be the most suitable.
and not for the purpose of downstream processing. The large
polysaccharides (for instance, agar and cellulose) present in the cell
2.2.2. Primary extraction methods
wall of macro-algae, offer major hindrance for cell disruption during
Different primary extraction methods such as ultrasonication,
the primary extraction of the metabolites. Hence, there is a need for a
maceration using mortar and pestle, maceration in presence of liq-
suitable method of cell disruption for their extraction.
uid nitrogen, homogenization, freezing and thawing, maceration in
Being a green technology, ultrasonication has attracted the
combination with freezing and thawing, homogenization in combi-
attention of researchers for its application in food and allied indus-
nation with ultrasonication, and maceration in combination with
tries [17]. Ultrasonication acts by creating compression and
ultrasonication were carried out to develop the most suitable
decompression through sound waves at frequency >20 kHz. Sev-
methodology for the primary extraction of R-PE and R-PC from
eral mechanisms for the action of ultrasound have been identified,
marine macro-algae. Overall work plan is presented in Fig. 1 and
which include fragmentation, erosion, sonocapillary effect, sono-
detailed procedures of all the methods attempted are explained
poration, local shear stress and destruction-detexturation of plant
in following sections.
cell wall matrix [18]. An overall effect of these mechanisms, results
in disruption of cell-wall. However, the relative extent of contribu-
2.2.2.1. Serial extraction. Serial extraction (repeated extraction) was
tion of a given mechanism may vary with the type of biomass and
carried out in order to estimate the maximum extractable content
process parameters. By employing ultrasonication, higher extrac-
of phycobiliproteins (R-PE and R-PC) present in the biomass.
tion yields can be achieved at lower process time [19] and often
Extraction buffer of pH 6.8 and solid-liquid ratio of 1:10 were
at lower temperatures and hence more suitable for thermolabile
employed. At lower solid-liquid ratio (1:6), the quantity of buffer
compounds [20].
was not sufficient to conduct the maceration experiments and at
In ultrasound assisted extraction processes, higher yields were
higher ratio (1:14), the concentration of desired biomolecules
observed for biomolecules such as carotenoids from pomegranate
was low, leading to absorbance lower than detectable limit in spec-
waste [21] and anthocyanins from blue berry [22]. Further, ultra-
troscopic analysis due to dilution. Hence, solid-liquid ratio of 1:10
sonication enjoys the advantage of direct scalability due to its abil-
was employed in all the primary extraction experiments.
ity of generation of progressively high intensity cavitation zones
5 g fresh biomass was macerated into fine paste using mortar
and therefore suitable for scale-up of the process [23].
and pestle and was added to 50 ml of 0.1 M phosphate buffer of
Gelidium pusillum (Stackhouse) Le Jolis, a common marine macro-
pH 6.8. The suspension was incubated for 1 hour at 4 C (with
alga of tropical cost, was selected as source for the phycobiliproteins
intermittent stirring) and centrifuged (REMI, PR 24) at 4 C and
in the present study. To the best of our knowledge, reports are not
15,000g for 12 mins. The pellet was re-suspended in fresh extrac-
available in the scientific literature on standardization of different
tion buffer and incubated for 1 hour at 4 C. This procedure was
methods for the extraction of phycobiliproteins from Gelidium pusil-
repeated until no detectable phycobiliproteins were extracted in
lum. Further, combination of different extraction methods for cell
the buffer and supernatant of all these primary extraction steps
disruption of marine macro-algae for the primary extraction of phy-
was pooled together for spectrophotometric analysis. This forms
cobiliproteins, and their comparison in terms of extraction yield and
the basis (100%) for the estimation of extraction efficiency of all
efficiency are scarce. Thus, the aim of present study was to standard-
the primary extraction methods attempted with respect to R-PE
ize different ultrasound assisted methods for the primary extraction
and R-PC as per the following equation.
of R-PE and R-PC from Gelidium pusillum and their comparison to
identify the most suitable method. Extraction efficiency
 
Phycobiliprotein content from a given cell disruption method
 100
Phycobiliprotein content from serial extraction
2. Materials and methods 1

2.1. Materials
2.2.2.2. Ultrasonication. Different parameters affecting the extrac-
2.1.1. Algal biomass tion of R-PE and R-PC such as ultrasonication amplitude (60, 90
Biomass of marine macro-algae, Gelidium pusillum, was col- and 120 mm) and ultrasonication time (1, 2, 4, 6, 8 and 10 mins)
lected in morning hours during low tide period of April month were standardized. The experiments were carried out at 30, 35
94 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103

Fig. 1. Overall work plan for extraction and analysis of phycobiliproteins from fresh biomass of marine macro-algae Gelidium pusillum.

and 40 C (employing a water bath) at an ultrasonication pulse on recorded at adiabatic condition using thermocouple [26]. The
and off cycle of 1/1 second. 10 g of fresh biomass was washed with energy imparted by ultrasonication to the medium was estimated,
distilled water and excess water was removed using tissue paper from this data.
before adding to 100 ml of phosphate buffer (0.1 M, pH 6.8). The Ultrasonic power (P) imparted to the liquid system was calcu-
suspension was then subjected to ultrasonication (Q700 Qsonica lated using the following equation
Sonicator with 3/400 diameter high gain horn) followed by centrifu-  
dT
gation. The supernatant was collected for spectroscopic analysis P mC p 5
and the pellet for microscopy. dt
Extraction kinetics. The extraction of phycobiliproteins (R-PE or R- where m is the mass of the solvent (g), Cp the specific heat of the
PC) contained in the solid biomass of macro-algae can be described solvent, (J/g/C) and dT/dt is change in temperature with respect to
by the following equation [24] time (C/min).
dC L Power estimated was expressed in terms of Ultrasonic intensity
kC S
C L 2 2 (UI, W/cm2) [25] and Acoustic energy density (AED, W/cm3) [26]
dt
using following equations.
where CL is the concentration of R-PE or R-PC in the extraction
 
(mg/ml) at any given time t (mins), Cs the equilibrium concentra- 4P
UI 6
tion of R-PE or R-PC in the extract (mg/ml) and k is the 2nd order pD2
extraction rate constant (ml/mg. min.].
Eq. (2) can be written, by separating the variables, as  
P
AED 7
dC L V
k:dt 3
C S
C L 2 where, D is the probe diameter (cm), and V is the volume of the
The above equation can be integrated over the limits of t (0 to solvent in (cm3).
t) and CL (0 to CL) and can be written in linearized form as
2.2.2.3. Maceration using mortar and pestle. 10 g of fresh biomass
t 1 t
4 was taken and washed with distilled water and excess water was
C L kC 2S C S
removed using tissue paper. The biomass was subjected to macer-
which is in the form of y c mx. The kinetic parameters namely, ation using mortar and pestle for different time durations of 10, 20,
the second order rate constant k, equilibrium/saturation concen- 30 and 45 mins on addition of 100 ml of phosphate buffer (0.1 M,
tration Cs can be obtained from the Intercept and the slope of 6.8pH). The suspension obtained was centrifuged in a refrigerated
the plot of t/CL versus t. The initial extraction rate, Ri (mg/ml. centrifuge at 4 C and 15,000g for 12 mins. Supernatant and pellet
min.) at the low values of CL (as t tends to 0), also can be were taken for spectrophotometric and microscopic analyses,
obtained as Ri = kC2s . respectively.
Energy calculations. On application of Ultrasound, sound waves
propagate through liquid medium and a portion of power gets con- 2.2.2.4. Maceration in presence of liquid nitrogen. 400 mg of fresh
verted into heat, leading to rise in the temperature of the medium. biomass was taken and washed with distilled water and excess
The heat dissipated in the medium during ultrasonication was esti- water was removed using tissue paper. The biomass was subjected
mated by calorimetric measurements [25]. Medium was exposed to maceration in presence of liquid nitrogen and incubated over-
to ultrasonication and mean temperature rise with time was night at 4 C [9]. The suspension thus obtained was centrifuged
 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103
at 4 C and 15,000g for 12 mins and supernatant was taken for
spectrophotometric analysis.
2.2.2.5. Homogenization. 10 g of fresh biomass was taken and
washed with distilled water and excess water was removed using
tissue paper. 100 ml of buffer was added to this pre-weighed bio-
mass and was subjected to homogenization (Ultra-Turrax IKA
25T disperser, Germany with 18G tool) at 15,000 RPM. Homoge-
nization was carried out using ice jacket to avoid rise in tempera-
ture of buffer (the temperature was not allowed to increase beyond
35 C). The biomass was homogenized for different time intervals
of 5, 10, 15, 20, 25, 30, 35 and 45 mins. The suspension thus
obtained was centrifuged at 4 C and 15,000g for 12 mins and
supernatant was collected for spectrophotometric analysis while
the pellet was analyzed microscopically.
2.2.2.6. Freezing and thawing. 500 mg of fresh biomass was washed
with distilled water before adding 5 ml of buffer and subjecting to
10 cycles of freezing and thawing. Each cycle involves 3 hours of
freezing at
40 2 C and 1 hour of thawing to room temperature
(27 2 C). The suspension was subjected to centrifugation and
supernatant was collected for spectroscopic analysis and pellet
for microscopy.
2.2.2.7. Maceration in combination with freezing and thawing. 15 g of
algal biomass was macerated for 45 mins using mortar and pestle.
500 mg of macerated biomass was subjected to freezing and thaw-
ing as mentioned earlier in Section 2.2.2.6 and centrifuged at 4 C
and 15,000g for 12 mins. The supernatant was collected for spec-
trophotometric analysis and pellet for microscopy
2.2.2.8. Homogenization in combination with ultrasonication. 10 g of
fresh biomass was subjected to homogenization (as mentioned in
Section 2.2.2.5), and was then subjected to ultrasonication (as
mentioned in Section 2.2.2.2). The suspension was centrifuged at
4 C and 15,000g for 12 mins to obtain supernatant and pellet for
spectrophotometric and microscopic analyses, respectively.
2.2.2.9. Maceration in combination with ultrasonication. 10 g of fresh
biomass samples were macerated using mortar and pestle (as
mentioned in Section 2.2.2.3) and processed by ultrasonication
(as mentioned in Section 2.2.2.2). The suspension was centrifuged
at 4 C and 15,000g for 12 mins to obtain the supernatant for spec-
trophotometric analysis and pellet for microscopy.
2.2.3. Analyses
2.2.3.1. Spectroscopy. R-PE and R-PC contents were estimated by
measuring absorbance at 564 nm, 618 nm and 730 nm using dual
beam UVVisible spectrophotometer (Schimadzu, UV 1800, Japan).
The following equations were used for the estimation of phyco-
biliprotein contents [27].
R
PE 0:1247A564
A730
0:4583A618
A730  8
R
PC 0:154A618
A730 9
All the experiments were carried out in triplicates. Mean, stan-
dard deviations (SD) and p-value were calculated using Microsoft
excel-2010.
2.2.3.2. Chromatography. The extract obtained from maceration
+ ultrasonication was subjected to HPLC (Shimadzu-LC10AS,
Japan, PDA detector) for the conformation of presence of phyco-
biliproteins and their intactness even after processing. The column
(reproSil 200 SEC, 5 mm, 300  4.6 mm) was equilibrated at flow
rate of 1 ml/min. with PBS buffer (0.05 M phosphate buffer
+ 0.15 M NaCl, pH 7.0). The R-PE standard (cat. no. 52412, Sigma
95
Aldrich Pvt. Ltd., Bangalore) of 0.5 mg/ml and sample obtained
were injected and eluted with mobile phase (PBS buffer) at a flow
rate of 1 ml/min, analyzing at 564 nm for the peak of R-PE. Prior to
injection, samples were filtered by 0.45 mm syringe filters to
remove any suspended particles.
2.2.3.3. Microscopy. Microscopic studies were conducted to visual-
ize the extent of extraction (color change and cell disruption)
occurring from the algal strands in different primary extraction
methods employed. After centrifugation, the algal strands (from
the pellet) were mounted on glass slides without staining and visu-
alized under light microscope at 200X magnification (Olympus/BX-
40, Japan).
3. Results and discussion
Different primary extraction methods and their combinations
were employed for enhancing the primary extraction of R-PE and
R-PC from the marine macro-algae, Gelidium pusillum. The results
are discussed in the following sections
3.1. Serial extraction
R-PE and R-PC contents in algal biomass vary with season and
geographical location [28]. Hence, it was thought desirable to
express the results in terms of efficiency of primary extraction
method instead of Phycobiliprotein content alone. For this purpose,
the maximum extractable R-PE and R-PC from the biomass needs
to be estimated. Maceration using mortar and pestle was repeated
(as mentioned in Section 2.2.2.3) until no practically detectable
phycobiliproteins are extracted in the buffer. This forms the basis
(100%) for finding the efficiency of any given primary extraction
method. On carrying out serial extraction, it was observed that
after 5th cycle, practically no increase in phycobiliprotein content
in buffer was observed. Total R-PE and R-PC contents in the pooled
extract were estimated to be 2.03 and 1.28 mg/g dry biomass,
respectively. This total content of R-PE and R-PC was used as basis
to estimate the extraction efficiency of any given primary extrac-
tion method. The contents of R-PE and R-PC have been reported
as 1.98 and 0.28 mg/g dry biomass [10] and 2.30 and 1.15 mg/g
dry biomass [9].
3.2. Ultrasonication
Application of ultrasonication for extraction of biomolecules
from natural sources has been reported and known as Ultrasound
Assisted Extraction (UAE). However, practically no studies on UAE
of R-PE from macro-algae are reported [15]. Ultrasonication alone
was explored as a primary extraction method for primary extrac-
tion of phycobiliproteins, from unground Gelidium pusillum. Differ-
ent process parameters such as ultrasonication amplitude,
ultrasonication time and system temperature were standardized.
Extraction of R-PE and R-PC was carried out at three tempera-
tures, (30, 35 and 40 C) for 4 mins at ultrasonication amplitude
of 60 mm (the middle value of amplitude feasible with ultrasonica-
tion unit employed in the present study). It may be noted that
ultrasonication time of 4 mins is the minimum period with which
detectable amounts of R-PE and R-PC could be extracted into the
buffer. It can be seen from Fig. 2a that the maximum yield of R-
PE (0.06 mg/g dry biomass) and R-PC (0.03 mg/g dry biomass)
could be extracted at 30 C. Lower yields at higher temperatures
can be attributed to the degradation of these biomolecules [13].
Accordingly, all further experiments were carried out at 30 C.
In order to study the effect of ultrasonication amplitude, extrac-
tion of R-PE and R-PC was carried out at different amplitudes (60,
96 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103

0.16 26
R-PE (mg/g) 24
0.14
22
Yield (mg/g dry biomass)

0.12 R-PC (mg/g)

20
0.1 18
16
0.08
14

t/CL
0.06 12
0.04 10
8 30 C
0.02 6
35 C
0 4
30 35 40 2 40 C
Temperature (oC) 0
2 4 6 8 10 12
Fig. 2a. Effect of temperature on ultrasound assisted extraction of Time (mins)
phycobiliproteins.
Fig. 2d. Second order extraction kinetics (R-PE).

0.16
R-PE 16
0.14 (mg/g)
Yield (mg/g dry biomass)

14
0.12

0.1 12

0.08 10
t/CL
0.06 8
0.04
6
0.02 30 C
4
0 35 C
60 90 120 2
Sonicaon amplitude (m) 40 C
0
Fig. 2b. Effect of ultrasonication amplitude on extraction of phycobiliproteins. 2 4 6 8 10 12
Time (mins)

Fig. 2e. Second order extraction kinetics (R-PC).

0.180
0.160
further increase in ultrasonication time resulted in an increase in
Yield (mg/g dry biomass)

0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000
2 4 6 8
Time (mins)
Fig. 2c. Effect of ultrasonication on extraction of phycobiliproteins.
90 and 120 mm) at system temperature of 30 C and ultrasonication
time of 4 mins. The results are shown in Fig. 2b. It can be seen from
figure that the maximum yield of R-PE (0.15 mg/g dry biomass)
and R-PC (0.1 mg/g dry biomass) could be obtained at 120 mm
(the highest amplitude feasible with ultrasonication unit).
Accordingly, further extraction experiments were carried out at
system temperature of 30 C and ultrasonication amplitude of
120 mm for different ultrasonication time intervals of 1, 2, 4, 6, 8
and 10 mins. The results are shown in Fig. 2c. It can be observed
from the figure that it resulted in the maximum yield of 0.16 mg/
g dry biomass for R-PE and 0.11 mg/g dry biomass for R-PC. These
values are very low compared to 2.03 mg/g dry biomass of R-PE
and 1.28 mg/g dry biomass of R-PC obtained in serial extraction.
Ultrasonication was not carried out for more than 10 mins because
temperature above 35 C (in spite of using cooling jacket) which
is not desirable for stability of R-PE [13]. Hence, operating temper-
ature of 30 C, ultrasonication amplitude of 120 mm and ultrasoni-
cation time of 10 mins were inferred to be the best possible
conditions for ultrasonication. In view of these low yields, ultra-
R-PE sonication alone does not appear to be a suitable primary extrac-
tion method for disintegration of cell wall and extraction of
R-PC
phycobiliproteins from macro-algae.
10
3.2.1. Extraction kinetics
The experimental data of the concentration of R-PE and R-PC as
a function of time has been plotted as per Eq. (4) in Figs. 2d and 2e.
The values of kinetic parameters such as extraction capacity (equi-
librium or saturation concentration, Cs), extraction rate constant
(k) and initial extraction rate (Ri) for each temperature are given
in Tables 1. It could be observed from the table that temperature
has a strong influence on the kinetic parameters.
The second-order kinetics of extraction of several biomolecules
such as antioxidants from pomegranate marc [29], and polyphe-
nols from Oak chips [30] and Picea abies bark [31] have been
reported in literature.
3.2.2. Energy calculations
During ultrasonication, a portion of power gets converted to
heat, leading into rise in the temperature of the medium. The heat
dissipated in the medium during ultrasonication was estimated by
calorimetric measurements. The power, ultrasound intensity and
 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103 97

Table 1
Kinetic parameters of phycobiliproteins extraction.
 
Temp (C) Slope C s Slope
1 Intercept Ri Intercept
1
k Ri R2
C 2s

R-PE (R-Phycoerythrin)
30 1.467 0.682 8.632 0.116 0.250 0.99
35 0.861 1.161 6.733 0.149 0.111 0.98
40 0.678 1.475 6.944 0.144 0.066 0.95
R-PC (R-Phycocyanin)
30 0.660 1.515 6.923 0.144 0.063 0.97
35 0.372 2.688 4.684 0.213 0.029 0.99
40 0.314 3.185 4.575 0.219 0.022 0.97

Table 2
Comparison of different pre-treatments for extraction of Phycobiliproteins.

Sl. Extraction Method R-PE Content (mg/g dry R-PC Content (mg/g dry R-PE extraction efficiency R-PC extraction efficiency
No. biomass) biomass) (%) (%)
1. Serial Extraction* 2.03 0.04 1.28 0.03
2. Maceration + Ultrasonicationa 1.56 0.01 1.19 0.01 76.80 93.13
3. Homogenization 1.41 0.01 0.95 0.01 69.61 74.14
+ Ultrasonication a
4. Homogenization aloneb 1.29 0.04 0.80 0.07 63.5 62.50
5. Maceration aloneb 1.19 0.03 0.81 0.03 58.77 63.20
6. Maceration + Freezing and 0.90 0.03 0.61 0.02 44.33 47.27
Thawingc
7. Maceration with Liquid nitrogen 0.54 0.05 0.34 0.03 26.65 26.64
8. Freezing and Thawing alonec 0.17 0.04 0.29 0.02 8.47 22.73
*
Forms the basis for finding the efficiency of all the pretreatment methods attempted.
a & c: for both R-PE and R-PC are significantly different (p < 0.05).
b: for R-PE is significantly different (p < 0.05) and for R-PC not significantly different (p>0.05).

1.6 0.700
1.4
0.600
Yield (mg/g dry biomass)
Yield (mg/g dry biomass)

1.2
0.500
1
0.400
0.8

0.6
0.300

0.4 0.200
0.2 0.100
0
5 10 15 20 25 30 35 45
0.000
Time (mins) R-PE R-PC

R-PE G R-PC G R-PC H R-PE H Liquid Nitrogen Grinding 10 minutes Grinding 10 minutes

Fig. 3. Effect of maceration and homogenization on extraction of phycobiliproteins. Fig. 4. Effect of maceration in presence of liquid nitrogen on the extraction of
phycobiliproteins.

acoustic power density were estimated, based on these measure-

ments, to be 41.97 W, 14.81 W/cm2 and 0.419 W/cm3, respectively.
3.4. Maceration in presence of liquid nitrogen

400 mg of fresh biomass was macerated in presence of liquid

3.3. Maceration using mortar and pestle
The fresh biomass was subjected to maceration using mortar
and pestle for different time durations and the results are shown
in Fig. 3. It can be seen from the figure that the degree of extraction
(mg/g dry biomass) increased with an increase in time of pre-
treatment and the maximum degree of extraction was obtained
after 45 mins of maceration. However, at this juncture, the biomass
turned into slimy consistency, making further maceration by mor-
tar and pestle not feasible. The content of R-PE and R-PC were
found to be 1.19 and 0.81 mg/g dry biomass, respectively. Based
on the values of serial extraction, it translates into extraction effi-
ciency of 59 and 63% for R-PE and R-PC, respectively.
nitrogen for 10 mins. Low temperature achieved due to liquid
nitrogen turned the biomass brittle, facilitating the maceration
(by mortar and pestle) of biomass. The powder obtained was added
to 4 ml buffer. Overnight incubation resulted in yield of 0.54 and
0.34 mg/g dry biomass of R-PE and R-PC, respectively as shown
in Fig. 4. The corresponding values in maceration (for 10 mins)
without liquid nitrogen were observed to be 0.57 and 0.38 mg/g
dry biomass (Fig. 4). As per the conventional wisdom, low temper-
ature processing facilitates the processing of biomolecules. How-
ever, in the present case, a decrease in phycobiliprotein content
was observed in presence of liquid nitrogen. Similar results are
reported in literature, where decline in the Phycoerythrin content
was observed when maceration in presence of liquid nitrogen
98
1.2
1
Yield (mg/g dry biomass)
0.8
0.6
0.4
0.2
0 teins, if maceration of macro-algal biomass is carried out prior to
control 1 2 3 4 5 6
Cycles
R-PE with F&T aer maceraon
R-PE without maceraon
Fig. 5. Effect of freezing and thawing cycles on extraction of phycobiliproteins.
was carried out [4,32]. Further, it was advocated not to store phy-
coerythrin at freezing temperature [33]. Based on the values of
serial extraction, the extraction efficiency of the present method
was estimated to be about 26% for both R-PE and R-PC, which is
very low. Thus, maceration in presence of liquid nitrogen, the most
commonly used laboratory method, was not observed to be very
effective for extraction of phycobiliproteins from Gelidium pusillum.
However, relatively higher yields of R-PE (2.30 mg/g dry biomass)
and R-PC (1.15 mg/g dry biomass) from Gelidium pusillum were
reported by maceration in presence of liquid nitrogen [9].
3.5. Homogenization
Homogenization is a widely used method for primary extrac-
tion. Homogenization was carried out for different time periods
(545 mins) and the results are shown in Fig. 3. Significant increase
in the yield of R-PE (from 0.54 to 1.29 mg/g dry biomass) and R-PC
(from 0.32 to 0.80 mg/g dry biomass) after 45 mins of pre-
treatment can be observed from the figure. The maximum content
of phycobiliproteins observed at the end of homogenization trans-
lates into extraction efficiencies of 63.5 and 62.5% for R-PE and R-
PC, respectively. It can be noted that these values are higher than
that of maceration in presence of liquid nitrogen and maceration
alone. The increase in extraction yield in case of homogenization
alone was found to be statistically significant for R-PE (p < 0.05)
in comparison with maceration alone, whereas no significant dif-
ference was observed for R-PC (p > 0.05).
3.6. Freezing and thawing
Freezing and thawing is a method, where pressure on cell wall
exerted by freezing intracellular fluids as well as extracting buffer,
leads to development of cracks in the cell wall, facilitating leaching
out of the intracellular contents into the extraction buffer. The bio-
mass was subjected to 10 cycles of freezing and thawing and the
results are presented in Fig. 5. Degree of extraction, after an initial
decrease, increased upto 7th cycle above which it decreased again
resulting in maxima at 7th cycle. The maximum yields were
0.29 mg/g dry biomass for R-PE and 0.17 mg/g dry biomass for R-
PC, (that is, extraction efficiency of 14.28% and 13.2% for R-PE
and R-PC, respectively). It appears that at around 7th cycle of freez-
ing and thawing, the cell wall of the macro-algal biomass was bro-
ken resulting in maximum release of phycobiliproteins (hence
maxima at 7th cycle). These results (decrease in phycobiliproteins
content at higher cycles) match with our earlier observation made
on degradation of phycoerythrin during maceration in presence of
liquid nitrogen and also those reported in literature [4,32]. In the
absence of any pre-treatment, the phycobiliprotein content in the
R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103
cell is intact and getting released into the extract buffer on freezing
and thawing by virtue of increased hydrostatic pressure on the cell.
At the same time, due to temperature fluctuation, the phyco-
biliproteins tend to undergo degradation. As an overall result of
these two processes occurring during freezing and thawing, an ini-
tial decrease followed by an increase and a decrease was observed
both in case of R-PE and R-PC. It can be inferred from these results
that process time can be reduced significantly (without the need of
7 cycles) without compromising on the release of the phycobilipro-
7 8 9 10 freezing and thawing. Further, freezing and thawing alone was
not able to effectively extract R-PE and R-PC, possibly due to the
R-PC with F&T aer maceraon fact that this method alone was not able to break the polysaccha-
R-PC without maceraon ride complex matrix present in cell wall. Accordingly experiments
were carried out by subjecting the algal biomass to maceration for
45 mins in mortar and pestle prior to freezing and thawing and the
results are discussed in the following section.
3.7. Maceration in combination with freezing and thawing
Algal biomass was macerated for 45 mins and then subjected to
freezing and thawing, and the results are shown in Fig. 5. As
hypothesized in the previous section, a 3 fold improvement could
be achieved, and the maximum phycobiliprotein content could
be achieved at 1st cycle itself with 0.9 mg/g dry biomass for R-PE
and 0.6 mg/g dry biomass translating into extraction efficiency of
44% and 47% for R-PE and R-PC, respectively. Significant increase
in phycobiliprotein yield was not observed, with further increase
in freezing and thawing cycles and a sudden decrease was
observed after 6th cycle. Although, maceration prior to the freezing
and thawing significantly improved the performance, it is lower
when compared with homogenization and maceration. Statistically
significant difference was observed in the yields of both R-PE and
R-PC when freezing and thawing (with and without maceration)
were compared. (p < 0.05).
3.8. Ultrasound assisted methods
As mentioned earlier, ultrasonication alone when used as pri-
mary extraction method resulted in very low yield of phycobilipro-
teins when compared to serial extraction. On the other hand,
maceration for 10 mins has resulted in yield of 0.58 mg/g dry bio-
mass of R-PE and 0.38 mg/g dry biomass of R-PC. Similarly, homog-
enization for 10 mins has resulted in 0.90 mg/g dry biomass of R-
PE and 0.54 mg/g dry biomass of R-PC. Hence, it was thought desir-
able to exploit the advantages of both conventional methods
(homogenization and maceration) and ultrasonication in order to
explore the synergy.
3.8.1. Homogenization in combination with ultrasonication
Homogenization is the most widely used method for primary
extraction. Homogenization alone has resulted (as shown in
Fig. 3) in significant increase in yield of R-PE from 0.54 to
1.29 mg/g dry biomass (extraction efficiency from 26.6 to 63.5%)
and R-PC from 0.32 to 0.80 mg/g dry biomass (extraction efficiency
from 25.0 to 62.5%) in the extract with respect to homogenization
time. It may be noted that by homogenization, out of 2.03 mg/g dry
biomass of R-PE and 1.28 mg/g dry biomass of R-PC (maximum
possible values in serial extraction), only 1.29 mg/g dry biomass
of R-PE and 0.80 mg/g dry biomass of R-PC could be extracted. In
order to increase the extraction efficiency further by exploiting
the advantages of homogenization and ultrasonication, it was
thought prudent to explore the synergistic effect of ultrasonication
with homogenization of algal biomass. Accordingly, the homoge-
nized cell biomass was subjected to ultrasonication for different
time periods (1 to 10 mins) and the results are shown in Fig. 6.

1.8
1.6
Yield (mg/g dry biomass)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
control 1 2 4 6
Time (mins)
45R-PE M+U 45R-PE H+U
Fig. 6. Effect of ultrasonication on extraction of R-PE and R-PC from macerated and
homogenized biomass.
An increase in extraction yield can be observed from the figure for
R-PE from 1.29 to 1.41 mg/g dry biomass (extraction efficiency
from 63.5 to 69.6%) and for R-PC from 0.80 to 0.95 mg/g dry bio-
mass (extraction efficiency from 62.5 to 74%), over the ultrasonica-
tion period of 10 mins.
Homogenization + ultrasonication resulted in higher extrac-
tion efficiency when compared to homogenization alone or ultra-
sonication alone. This could be because, homogenization resulted
in more of cutting of biomass rather than shearing (in the present
design). As a result, the overall surface area could not increase to
the required extent. Similarly, ultrasonication (20 kHz frequency
and 120 mm amplitude) appeared to be not sufficient enough to
break the macro-algal strands. Thus it resulted in lower extraction
efficiency in both these methods. Whereas, homogenization
+ ultrasonication involves cavitation (compression and decom-
pression) of the biomass which are already homogenized thereby
facilitating the release of phycobiliproteins.
3.8.2. Maceration in combination with ultrasonication
As mentioned earlier, maceration of algal biomass for 45 mins
resulted in an yield of 1.19 mg/g dry biomass for R-PE and
0.81 mg/g dry biomass for R-PC (as shown in Fig. 3) out of 2.03
and 1.28 mg/g dry biomass, respectively, translating to an extrac-
tion efficiency of 58.77 and 63.2%, respectively. In order to increase
the extraction efficiency further, it was thought desirable to
explore the synergistic effect of ultrasonication with maceration.
Accordingly, the macerated algal biomass was subjected to ultra-
sonication for different time periods (1 to 10 mins) and the results
are shown in Fig. 6. Significant increase in the yield of R-PE from
1.19 to 1.56 mg/g dry biomass (an increase in extraction efficiency
from 58.77 to 76.80%) and R-PC from 0.81 to 1.19 mg/g dry bio-
mass (an increase in extraction efficiency from 63.2 to 93.13%)
was observed.
Maceration + ultrasonication resulted in higher extraction effi-
ciency when compared to maceration alone or ultrasonication
alone. This could be because, maceration involves only attrition
which could reduce the size of algal strands only to some extent
while ultrasonication (20 kHz frequency and 120 mm amplitude)
appeared to be not sufficient enough to break the macro-algal
strands, thereby resulting in lower extraction efficiency. Whereas,
maceration + ultrasonication involves cavitation (compression
and decompression) of the biomass which are already macerated
thereby facilitating the release of phycobiliproteins. The efficacy
of ultrasonication for enhanced extraction of different biomole-
cules such as anthocyanin and b-carotene has been reported in
the literature [3436].
Maceration + ultrasonication was observed to result in better
yield and efficiency when compared to homogenization +
ultrasonication. The increase in extraction yield (of both R-PE
and R-PC) was found to be statistically significant in case of
R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103 99
1.6
1.4
1.2
Yield (mg/g dry biomass)
1
0.8
0.6
0.4
0.2
8 10 0
control 1 2 4 6 8 10
Time (mins)
45R-PC M+U 45R-PC H+U
0+R-PE 10R-PE 20R-PE 30R-PE 45R-PE
0+R-PC 10R-PC 20R-PC 30R-PC 45R-PC
Fig. 7. Effect of duration of maceration and ultrasonication on extraction of
phycobiliproteins.
maceration + ultrasonication when compared with homogeniza-
tion + ultrasonication (p < 0.05).
In order to explore the possibility of reducing the time of mac-
eration prior to ultrasonication, experiments were carried out for
different time periods of maceration (045 mins) followed by
ultrasonication for extraction of phycobiliproteins from algal bio-
mass. The results are shown in Fig. 7. The yield of R-PE and R-PC
gradually increased (0.57 to 1.03 mg/g dry biomass for R-PE and
0.38 to 0.95 mg/g dry biomass for R-PC) with respect to time period
of maceration (1030 mins) prior to ultrasonication. This translates
into an increase in extraction efficiency from 40 to 61% in case of
R-PE and from 30 to 46% in case of R-PC. It can be seen from the
Fig. 7 that extraction yield increased with increase in maceration
time till 45 mins. This reconfirming that 45 mins of maceration
is required prior to ultrasonication for better extraction of
phycobiliproteins.
Maceration + ultrasonication has resulted in the highest yield
of phycobiliproteins, making it the best among all the primary
extraction methods attempted. In general, favorable effect of ultra-
sound on extraction can be attributed to its mechanical effect on
the process in terms of cell disruption, increasing the penetration
of the solvent into the solid matrix [37] and thus improving mass
transfer. During sonication of liquids at high intensities, the sound
waves that propagate into the liquid media results in altering low
and high pressure cycles with rates depending on the frequency
and amplitude. During low-pressure cycle, high intensity ultra-
sonic waves create small vacuum bubbles or voids in the liquid.
When the bubbles attain a volume at which they can no longer
absorb energy, they collapse violently during the high pressure
cycle [38]. This implosion of cavitation bubble [39], results in jets
that hit the cells at very high velocity (>400 km/h) in water [40]
resulting in shear force that breaks the cell envelops mechanically
improving the mass transfer. Ultrasound can also lead to perme-
abilization of the cell membrane leading to increased extraction
[41]. The profound effect of ultrasonication on extraction of R-PE
and R-PC from macro-algae can be explained as the following.
When solid-liquid system is exposed to ultrasonication, shear force
and turbulence are generated around the solids in the solvent due
to oscillation and collapse of cavitation bubbles, thus bringing the
local shear stress into effect. It can be seen from images of biomass
in Fig. 9f and c (subjected to maceration alone for 45 mins and
maceration in combination with ultrasonication, 45 + 10 mins)
that ultrasonication resulted in disruption of matrix of the cell
wall. The disruption observed can be attributed to detexturation
effect during ultrasonication on macerated biomass. This further
weakens the cell wall matrix and helps sonocapillary effect to play
its role. Sonocapillary effect increases the depth and velocity of
penetration of solvent into the disrupted matrix, leading to
100 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103

Fig. 8. HPLC chromatogram for R-PE. (a) Sample, (b) R-PE Standard.

a. Control c. Maceration (45 mins) d. Homogenization (45 e. Homogenization (45

+ Sonication (10 mins) mins) + Sonication (10 mins)
mins)

b. Serial Extraction f. Maceration (45 mins) g. Maceration (45 mins) h. Freezing &Thawing(6
+ Freezing &Thawing(6 cycles)
cycles)

Fig. 9. Microscopic images of macro-algal strands after different primary extraction methods.

2.25
Overall Results
2
Yield (mg/g dry biomass)
1.75
1.5
1.25
1
0.75
0.5
0.25
0
Pre-treatment Methods
Fig. 10. Comparison of different primary extraction methods for the extraction of
phycobiliproteins from biomass of Gelidium pusillum.
enhanced extraction during ultrasonication assisted extraction.
[18]
In present study, it can be seen from Fig. 9c that the exposure to
ultrasonication, resulted in disruption of matrix of the cell wall
(possibly by local shear stress, detexturation and sonocapillary
effect) which in turn increases the surface area of biomass exposed
to the solvent thus explaining the enhanced extraction of
phycobiliproteins.
One of the major advantages of ultrasonication assisted method
is, ultrasonication itself being a mechanical method does not
involve any additional chemical solvent and in turn, does not gen-
erate any additional waste in the process. It is energy efficient
when compared to any known conventional method. [42] In addi-
tion, water/buffer, an eco-friendly solvent was used for the extrac-
tion of R-PE and R-PC. The additional power of 41.97 W spent
during ultrasonication + maceration is worth expending, consider-
ing the increase in yield (when compared to maceration alone) of
these high value low volume compounds (by 18.03% and 29.93%,
respectively). Thus, ultrasound assisted method for extraction of
phycobiliproteins would easily qualify to be a green technology.
The other advantages of this process is that, under mild conditions
(like the conditions used in the present process), the possibility of
degradation of other cell components such as agar, cellulose,
a. Control
b. Serial Extraction
Fig. 11. Microscopic images illustrating the effect of ultrasonication on cell disruption.
R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103 101
R-PE xylans [43] is vey less and can be extracted from the spent biomass
R-PC (left after extraction of Phycobiliproteins). However, degradation of
these polysaccharides was observed during extraction at relatively
higher temperatures.
3.9. HPLC
In order to confirm the presence of R-PE and its intactness even
after processing, the sample obtained by maceration in combina-
tion with ultrasonication was examined by chromatography. It
can be seen from the chromatogram obtained by HPLC
(Fig. 8a and b), that the peaks obtained at 564 nm, both for the
sample (Fig. 8a) and R-PE standard (Fig. 8b) are having almost
same retention time, that is, 2.87 and 2.90 mins, respectively.
The slight difference observed can be explained by the fact that
the standard of R-PE used and extract sample are from different
genus of macro-algae, that is, Porphyra and Gelidium. It can be
noted that the peak of R-PE in extract sample is at 564 nm, its char-
acteristic absorption wavelength, indicating the presence of R-PE
and its intactness after the processing. In ultrasonication assisted
extraction, there exists a possibility of degradation of natural prod-
ucts as evidenced by the chemical analysis. However, Li et al (2013)
[44] did not observe any evidence for caratinoid degradation while
Sun et al (2010) [45] reported that ultrasonic degradation of b-
carotene is typically slow in comparison with more volatile aro-
matics that diffuse more readily into the cavitation bubble in the
aqueous solvent. On the other hand, Jacotet-Navarro et al (2016)
[42] have observed degradation of cell wall polymers such as cel-
lulose during ultrasonication assisted extraction.
3.10. Microscopy
The optical microscopic studies were carried out (after each pri-
mary extraction method), as described in Section 2.2.3.3, to
observe the extent of extraction and cell disruption. The micro-
scopic images are shown in Fig. 9. It can be seen from the figure
that the effect of primary extraction methods to be prominent with
is significant difference in the extent to which cell is ruptured,
which in turn facilitated the release of phycobiliproteins into the
c. Homogenization d. Homogenization e. Homogenization
(10mins) (45mins) (45mins) +
Ultrasonication (10 mins)
f. Maceration (10 mins) g. Maceration (45 mins) h. Maceration (45 mins) +
Ultrasonication (10 mins)
102 R. Mittal et al. / Ultrasonics Sonochemistry 38 (2017) 92103
extraction buffer. A clear relation between the extraction efficiency
of the phycobiliproteins (R-PE and R-PC) and the degree of cell dis-
ruption due to a given primary extraction method could be
observed from the images in Fig. 11b to h. It can be noted that
the amount of color remained in the biomass after pre-treatment
is inversely related to degree of extraction, in other words cell
disruption.
Considerable difference in color and morphology of strands/fil-
aments of algal biomass can be observed from the images (Fig. 9)
with respect to different primary extraction methods. The image
of strand/filament that was not subjected to any primary extrac-
tion method (control) can be observed to be intact and rich in pig-
ment (Fig. 9a). On the other hand, microscopic image obtained
after serial extraction indicated a good level of cell disruption of
the algal strands which are almost colorless (Fig. 9b), in turn indi-
cating release of maximum extractable phycobiliproteins.
The image of the algal strands corresponding to maceration (45
mins) in combination with ultrasonication (10 mins) reached near
colorlessness (very close to that of serial extraction), conforming
that this being the most efficient primary extraction method. The
color intensity of the images of algal strands increased in the order
of Maceration + Ultrasonication (Fig. 9c), Homogenization + Ultra-
sonication (Fig. 9d), Homogenization alone (Fig. 9e), Maceration
alone (Fig. 9f), Maceration + Freezing and thawing (Fig. 9g), and
Freezing and thawing alone (Fig. 9h). It may be noted that this
order corresponds to the decreasing order of efficiency of the dif-
ferent primary extraction methods as indicated in Table 2 and as
can be seen also from Fig. 10.
3.11. Synergistic effect of ultrasonication
It can be seen from Table 2 that out of all the primary extraction
methods attempted, ultrasonication did not result in similar
efficacy in case of Gelidium pusillum as observed in case of other
natural sources. The ultrasonication with 20 kHz frequency and
120 mm amplitude appeared to be not enough to break the
macro-algal strands. Whereas, ultrasonication when applied as a
combination with either maceration or homogenization resulted
in much higher extraction efficiency as can be seen in Fig. 11. This
indicates that ultrasonication of higher amplitude and frequency is
required for cell disruption of Gelidium pusillum.
When homogenization and maceration were carried out alone
for different time intervals, it can be seen from the Fig. 11c and d
and Fig. 11f and g, respectively that there is considerable difference
in the degree of extraction when these unit operations are carried
out for 10 mins and 45 mins. However, phycobiliprotein content
could still be seen in macro-algal strands even after 45 mins of
homogenization or maceration. However, when the samples
obtained after 45 mins of homogenization or maceration were sub-
jected to ultrasonication for 10 mins, higher extraction efficiency in
case of homogenization was observed as can be seen from Fig. 11e
and the highest extraction efficiency was observed in case of mac-
eration as can be seen from Fig. 11h. The algal strands obtained
after these combined primary extraction methods appeared much
lighter in color in case of homogenization in combination with
ultrasonication. The strands were almost colorless (closer to that
of obtained from serial extraction) in case of maceration in combi-
nation with ultrasonication and appeared more splintered (com-
pared to the samples obtained after 45 mins of homogenization
or maceration alone). These observations reiterate the synergistic
effect of ultrasonication. In other words, the increase in discol-
oration of the macro-algal strands that are subjected to different
primary extraction methods directly reflected in an increase in
extraction efficacy as reported in Table 2.
Ultrasonication being effective when used in combination may
be explained in the following way. Phycobiliproteins being
membrane bound pigments will not get detached from membrane
and leach into extraction buffer unless cell wall is ruptured. Pri-
mary extraction methods such as homogenization and maceration
alone are not adequate to achieve the desired extraction efficiency.
The primary extraction methods in combination with ultrasonica-
tion only could result in synergistic effect in turn in better extrac-
tion efficiency thereby facilitating the release of phycobiliproteins.
Thus, it can be inferred that maceration in combination with ultra-
sonication and homogenization in combination with ultrasonica-
tion are the suitable methods for extraction of phycobiliproteins
from Gelidium pusillum. To summarize, ultrasound assisted extrac-
tion resulted in a decrease in the process time, an increase in
extraction yield, effective utilization of equipment, reduction in
the layover time which in turn lead to reduction in the operation
cost [44]. By employing large scale UAE reactors available, there
is scope for scaling up of this process and developing an energy
efficient industrial process. [23,46]
4. Conclusions
Primary extraction of R-PE and R-PC from marine macro-algae,
Gelidium pusillum by using ultrasonication and conventional meth-
ods like maceration, maceration in presence of liquid nitrogen,
homogenization and, freezing and thawing (alone and in combina-
tions) is reported for the first time. Ultrasonication did not give
good results when employed alone despite optimization. Kinetic
parameters were estimated for ultrasonication based on second
order mass transfer kinetics. Based on calorimetric measurements,
power, ultrasound intensity and acoustic power density were esti-
mated to be 41.97 W, 14.81 W/cm2 and 0.419 W/cm3, respectively.
Ultrasonication when employed in combination with other
primary extraction methods (homogenization and maceration), it
resulted in higher extraction yields indicating synergistic effect.
Maceration in combination with ultrasonication demonstrated
higher ability to disrupt complex polysaccharide matrix of the cell
wall, resulting in the highest degree of extraction (77 and 93% of
R-PE and R-PC, respectively). Second best results were observed
in case of homogenization in combination with ultrasonication in
terms of extraction efficiency (69.6 and 74.1% for R-PE and R-PC,
respectively). Microscopic studies confirmed these observations
of primary extraction methods with a clear relation between
degree of extraction and colorlessness of microscopic images. HPLC
analysis was carried out and the chromatograms confirmed the
presence of R-PE and its intactness even after processing. These
ultrasonication assisted methods can be employed for the
enhanced extraction of biomolecules from other macro-algae.
Acknowledgement
Authors thank the Director, CSIR-CFTRI, Mysuru, for the infras-
tructural facilities at the institute and Dr. C. R. K. Reddy, CSIR-
CSMCRI, Bhavnagar, Gujarat for facilitating the collection of bio-
mass and Dr. V. Veeragurunathan, CSIR-CSMCRI, Marine Algal
Research Station, Mandapam, Tamil Nadu for providing algal bio-
mass. Rochak Mittal and Hrishikesh A Tavanandi gratefully
acknowledge CSIR, Government of India, for the fellowship.
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