Food Chemistry 297 (2019) 124993

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Optimization model for ultrasonic-assisted and scale-up extraction of T

anthocyanins from Pyrus communis ‘Starkrimson’ fruit peel
⁎
Tarun Belwala, Hao Huanga, Li Lia, Zhenhua Duanb, , Xuebing Zhangc, Halah Aalima,
⁎
Zisheng Luoa,
a
Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural
Affairs, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, People’s Republic of China
b
Institute of Food Science and Engineering, Hezhou University, Hezhou, People’s Republic of China
c
Hangzhou Wanxiang Polytechnic, Huawu Road 3, Hangzhou 310023, People’s Republic of China

A R T I C LE I N FO A B S T R A C T

Keywords: Ultrasonic effect on extraction of cyanidin-3-galactoside (Cy3-gal) from pear fruit peel was investigated and
Ultrasonic-assisted extraction compared with conventional extraction (CE) method. Different process factors were tested to determine the
Cyanidin-3-O-galactoside optimum conditions for ultrasonic-assisted extraction (UAE). Results revealed that under optimized UAE con-
Anthocyanins ditions (ultrasonic power = 162 W, temperature = 71 °C, trifluoroacetic acid = 3%, ethanol = 57%, ultra-
Viscosity
sonication time = 11 min, and sample to solvent ratio = 1:30 g/ml), Cy3-gal yield was significantly higher
Extraction mechanism
(0.34 3 ± 0.005 mg/g) than from CE (0.266 ± 0.004 mg/g), whereas the extract viscosity during UAE showed
Scale-up
a negative impact. UPLC-Triple-TOF/MS analysis detected a total number of 13 anthocyanin compounds, out of
which 8 were identified and that mainly consisted of cyanidin, delphinidin and petunidin compounds. Higher
yield of Cy3-gal under UAE compared to CE was also justified by higher deformations in the cell structure. The
possible mechanism of ultrasonication effect during the extraction process is also proposed in the present study.
During scale-up UAE process, the extraction yield of Cy3-gal was recorded higher under batch scale-up compared
to continuous operation. The present study is an attempt to optimize UAE of valuable anthocyanins from Pyrus
communis ‘Starkrimson’ fruit peel and further scaled-up for higher volume extraction which can be utilized for
industrial applications.

1. Introduction
The need and applications of plant-derived bioactive compounds
and extracts are increasing over the past several years mainly as nu-
traceuticals for promoting human health. Among several plant-derived
bioactive compounds, anthocyanins are one of the special classes of
flavonoid compounds which has been recognized for its multi-func-
tional and multi-industrial applications (Khoo, Azlan, Tang, & Lim,
2017). From transparency market research report (TMR, 2019), the
anthocyanin market in the year 2018 was over US$ 500 million, which
was expected to increase with a compound annual growth rate (CAGR)
of 4.6% from 2019 to 2026. Among other anthocyanin compounds,
cyanidin-3-O-galactoside (Cy3-gal) contain 3-O-beta-D-galactoside
group esterified to the 3rd position of the C ring of cyanidin. The natural
occurrence of Cy3-gal is limited to some fruit and berry species such as
Aronia melanocarpa (Chocke berries), Vaccinium macrocarpon (Cran-
berries), Vaccinium vitis-idaea (Lingon berries), Amelan chieralnifolia
(Saskatoon berries), Empetrum nigrum (Crow berries), Pyrus pyrifolia and
Pyrus communis (Lin & Harnly, 2008; Zhang et al., 2012; Fang, 2015).
Anthocyanins were well tested for their health effects and found ef-
fective against a variety of disease conditions (Li, Wang, Luo, Zhao, &
Chen, 2017; Belwal, Nabavi, Nabavi, & Habtemariam, 2017). Specifi-
cally, Cy3-gal was found to improve spatial memory (Tan et al., 2014),
and showed strong antioxidant and cytoprotective activity (Bellocco
et al., 2016). Moreover, anthocyanins are also gaining pace as a natural
colorant in food, bevrages, pharmaceutical and cosmetic products
(Khoo et al., 2017). Also, it has been reported that among different
anthocyanin compounds, cyanidin market share was found to be the
highest (~30%) compared to other common anthocyanins (TMR,
2019).
Extraction is an important and crucial step in plant and food pro-
cessing in terms of extracting desired bioactive compounds in their
optimum yield without affecting their functional qualities (Chemat,
Rombaut, Meullemiestre et al., 2017; Chemat, Rombaut, Sicaire et al.,

⁎
Corresponding authors.
E-mail addresses: duanzhenhua@hzu.gx.cn (Z. Duan), luozisheng@zju.edu.cn (Z. Luo).

https://doi.org/10.1016/j.foodchem.2019.124993
Received 4 March 2019; Received in revised form 10 June 2019; Accepted 10 June 2019
Available online 11 June 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
T. Belwal, et al.
2017; Belwal et al., 2018). Ultrasonic-assisted extraction (UAE) is one
of the advanced forms of classical extraction techniques and uses ul-
trasonic waves to generate cavitation bubbles which on collision gen-
erate a hot spot/micro-jet with higher temperature and pressure, thus
resulting in faster and efficient extraction of components from the plant
matrix (Chemat, Rombaut, Sicaire et al., 2017). The extract quality and
yield are significantly affected by various UAE factors, such as ultra-
sonic power/intensity, reaction vessel type, extraction temperature and
time, solvent pH, concentration and volume (Pandey, Belwal, Sekar,
Bhatt, & Rawal, 2018; Huang et al., 2019). More specifically, UAE has
been widely used and optimized for the extraction of anthocyanins from
plant samples (Zou, Wang, Gan, & Ling, 2011; Celli, Ghanem, & Brooks,
2015) and also in our previous work (Huang et al., 2019). Thus, de-
termination of significant process factors and optimization of their le-
vels for getting superior quality of extract are prerequisite.
Pyrus communis fruits are cultivated and consumed in larger quan-
tities especially in China (Silva, Souza, Barbieri, & De Oliveira, 2014).
From the available report, in the year 2017–18 the global production of
Pears is recorded as 24.17 million metric tons, led by China with ap-
proximately 80% of the total production (Report on ‘Major pear pro-
ducing countries worldwide 2017/2018). The Starkrimson cultivar of
Pyrus communis, also known as ‘Hongpi’ in Chinese, is known for its
attractive crimson red color. Pyrus is consumed as raw fruit and also
used in the preparation of various food products such as pies, cakes,
cheese, carpaccio, risotto, jams, and ice creams, due to its pleasant taste
and low calorie value (Silva et al., 2014). The food processing of Pyrus
fruits mainly requires removal of its peel and core, which are con-
sidered as a waste (Featherstone, 2016). However, the peel contains
valuable bioactive compounds and has studied for its chemical profiling
and bioactivity. For instance, among ten different pear varieties, the
peel contained a higher concentration of bioactive compounds espe-
cially polyphenols as compared to its flesh (Li et al., 2014). The peel of
red cultivars of Pyrus fruit is also a rich source of anthocyanins, among
which concentration of Cy3-gal was reported the highest (∼93%)
(Huang, Zhang, Qin, Wenquan, & Wu, 2012). Moreover, studies (Wu
et al., 2019) revealed that Starkrimson has highest anthocyanin content
as compared to other red Asian and European pear cultivars (i.e., 5 Hao,
Red Zaosu, Red Sichou, Palacer, and Red Bartlett).
A number of studies have been conducted on UAE optimization of
anthocyanins from various plant sources. Nevertheless, very few studies
were conducted on utilizing food waste for bioactive compound re-
covery under optimized advanced extraction conditions. No such stu-
dies have been proposed for UAE optimization of anthocyanins (espe-
cially Cy3-gal) from fruit peel waste and further tested for its higher
volume extraction. Thus, keeping in mind the high anthocyanin con-
tents in fruit peel of Pyrus communis ‘Starkrimson’, the present study
was designed to test the ultrasonic effect on extraction of Cy3-gal and
further testing for its batch and continuous scale-up extraction process.
The UAE was also compared with the conventional extraction (CE)
method.
2. Materials and methods
2.1. Chemicals and reagents
Cyanidin-3-O-galactoside (Idaein chloride, ≥90%) and cyanidin-3-
O-glucoside (Kuromanin chloride, ≥95%) standards were procured
from Sigma Chemicals (Sigma-Aldrich, USA). 2,2-diphenyl-1-picrylhy-
drazyl (DPPH), 2,4,6-tri(2-pryridyl)1,3,5-triazine (TPTZ), ascorbic acid,
analytical grade ethanol, methanol, acetone, trifluoroacetic acid, hy-
drochloric acid, formic acid and LC-MS grade acetonitrile were pur-
chased from Aladdin Industrial Corporation (Shanghai, China). For
solvent dilution and mobile phase preparation, Millipore ultra-pure
water was used. All other chemicals are of analytical and HPLC grade.
2
Food Chemistry 297 (2019) 124993
2.2. Fruit collection and sample preparation
Pyrus communis ‘Starkrimson’ fruits were collected from Shanxi
Province, People Republic of China during its full ripening period. The
fruits were then directly stored in a refrigerator at 4 °C and processed
within a week. Fruits were peeled out (around 10% of the fruit weight)
with the help of a mechanical peeler and cut into smaller pieces with
minimal pressure and distortion. Peels were then put into the mortar
and pestle and ground using liquefied nitrogen. To make homogenized
powder sample, all the powdered samples were mixed together and
packed in an aluminum foil covered with polyethylene bags and stored
in a refrigerator at −80 °C.
2.3. Ultrasonic-assisted extraction: Instrumentation
2.3.1. Lower volume extraction
For lower volume UAE experiments, ultrasonic probe instrument
(Scientz-IID, Ningbo Scientz Biotechnology Co., Ltd., China) operated at
20 KHz frequency with variable power was used. A double wall ex-
traction vessel (total volume 50 mL, 4 cm diameter) having inlet and
outlet openings was used (Supplementary Fig. 1B). For maintaining the
temperature of the extraction vessel, the extraction vessel was con-
nected to a thermostated water bath (SDC-6, Ningbo Scientz Bio-
technology Co., Ltd., China) through its inlet and outlet openings. The
ultrasonic probe (6 mm) was connected to the master unit which con-
trols the ultrasonic power, pulse time, ultrasonication time, and also
displays the process temperature. The probe and the extraction vessel
were fixed inside a soundproof chamber.
2.3.2. Scaling-up UAE
The lower extraction volume (30 mL) UAE was tested to higher
volume i.e., 5X (150 mL), 15X (450 mL) and 100X times (3000 mL)
using the ultrasonic probe instrument (Scientz-IID, Ningbo Scientz
Biotechnology Co., Ltd., China), with some modifications. For instance,
for 5X times scale-up experiment, the extraction vessel was changed to
200 mL (total volume) having 6 cm of internal diameter and a 6 mm
diameter ultrasonic probe was used (Supplementary Fig. 1C), while for
15X, the vessel changed to 500 mL (total volume) with 8 cm of internal
diameter and a 15 mm diameter ultrasonic probe was used
(Supplementary Fig. 1D). Both 5X and 15X scale-up experiments were
conducted in batch mode, while 100X was operated in a continuous
mode.
For 100 X times scale-up experiment, a 600 mL (total volume)
double wall extraction vessel having 8.5 cm of internal diameter was
modified to a continuous flow solvent system, by introducing additional
openings in the internal wall (Supplementary Fig. 1E). During the ex-
traction process, ultrasonic probe of 15 mm diameter was used, and the
continuous flow of the solvent was maintained using peristaltic pump
connected to a 5 L solvent container. The pump was operated at a rate
of 80 mL/min for maintaining continuous and circulating flow of the
solvent from the solvent container into the reaction vessel and vice-
versa.
To determine the actual ultrasonic power P (W) entering into the
system, calorimetric measurement was conducted (Sicaire et al., 2016)
to access the actual power and calculated by Eq. (1):
dT
P = mCp
dt (1)
where, Cp is the heat capacity of the solvent at constant pressure (J
g−1 °C−1), m is the mass of solvent (g) and dT/dt is temperature rise per
second (°Cs−1).
To maintain the desired ultrasonic intensity (UI) with changing
extraction vessel volume and diameter during scale-up process, UI was
calculated by Eq. (2) (Sicaire et al., 2016):
T. Belwal, et al.
P 2.5. Conventional extraction (CE)
UI = 4 ×
π × D2 (2)
where, UI is the ultrasonic intensity (W/cm2), P is the actual ultrasonic
power (W) entering into the system, D is the vessel diameter (cm), π is
the constant (3.14).
2.4. UAE optimization experiments
2.4.1. Single factor experiments
Prior to single factor experiments, preliminary experiments were
conducted to select best solvent and acid combination. Among different
solvents (ethanol, methanol and acetone) and acids (hydrochloric acid,
formic acid and trifluoroacetic acid) tested during preliminary experi-
ments, ethanol and trifluoroacetic acid (TFA) combination were found
best for Cy3-gal extraction (data not shown). Single factor experiments
were carried out to determine the optimum level of each individual
factor (ultrasonic power, temperature, time, trifluoroacetic acid con-
centration, ethanol concentration and sample to solvent ratio) w.r.t.
Cy3-gal extraction yield. Each factors were tested at five levels, and a
total of 25 experiments were conducted by changing one factor levels at
a time while keeping other factors terms at a constant level. For each
experiment, 1 g peel powder sample was extracted with desired volume
and concentration of ethanol containing trifluoroacetic acid as per the
experimental design. The ultrasonic power and time were fixed based
on the experiment, and the process temperature of the extraction vessel
was controlled with a thermostated water bath. The pulse time for each
experiment was fixed at 2 s ON and 2 s OFF. After extraction, the extract
was cooled down to room temperature and centrifuged at 6730×g for
15 min at 15 °C using BIORIDGE Tabletop centrifuge (TGL-16 M,
Shanghai Lu Xiangyi centrifuge instrument Co., Ltd., China). The su-
pernatant was collected and stored in polypropylene tubes at −20 °C
for further experiments.
2.4.2. Central composite design (CCD)
Considering the results of preliminary experiments, CCD was ap-
plied to perform optimization experiments. A total of 44 experimental
runs (Supplementary Table 1) were conducted, including 5 factors
(ultrasonic power, temperature, time, TFA concentration and ethanol
concentration) varying at 3 levels i.e., lower (−1), middle (0) and
higher (+1). Cy3-gal was considered as the response factor and the
second order polynomial equation (Eq. (3)) was applied as-
k k k k
YCy3 − gal = β0 + ∑ βi Xi + ∑ βii Xi2 + ∑ ∑ βij Xi Xj
i=1 i=1 i=1 j=i+1 (3)
where, YCy3-gal is the response term for Cy3-gal yield, β0 is the inter-
ception coefficient, βi, βii and βij are the linear, quadratic and inter-
active terms, respectively. Independent variables were coded as Xi and
Xj. The significant linear, quadratic and interactive effect of factors on
the response was determined using Fisher’s test for analysis of variance
(ANOVA) at p < 0.05. Three-dimensional response surface graphs
were plotted using Design Expert version 11 software (Stat-Ease Inc.,
USA).
2.4.3. Determination of optimized condition and its validation
Keeping all factor levels within the range and Cy3-gal extraction
yield as maximum, the optimized conditions and predicted response
value were determined using a numerical optimization method (Design
Expert version 11 software). Based on the desirability index, the opti-
mized condition having maximum desirability was selected, and the
experiment was performed in triplicates. To validate the optimization
model the experimental value was compared with the model predicted
value.
3
Food Chemistry 297 (2019) 124993
Conventional extraction was carried out under the optimized UAE
condition without providing the sonication (Supplementary Fig. 1A).
2.6. In vitro antioxidant assays
2.6.1. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging
activity
DPPH free radical scavenging activity of the extract under optimum
UAE, scale-up and CE conditions were tested and compared, according
to Brand-Williams, Cuvelier, and Berset (1995), with minor modifica-
tions. The extract was diluted and then 2 mL of each test solution was
added to 4 mL of DPPH solution (0.10 mM). The mixture was shaken
vigorously and kept in dark for 30 min. The absorbance was measured
at 517 nm under UV–Vis spectrophotometer and the results were ex-
pressed in micromole ascorbic acid equivalent per gram sample (μmol
AAE/g). The experiments were carried out in triplicates.
2.6.2. Ferric-reducing antioxidant power (FRAP) assay
The FRAP assay was measured according to Benzie and Strain
(1996), with some modifications. Briefly, the FRAP reagent was pre-
pared just before starting the experiment by mixing sodium acetate
(0.1 M in acetic acid, pH = 3.6), TPTZ solution (10 mM in 40 mM HCl)
and 20 mM FeCl3 solution at a ratio of 10:1:1 (v:v:v). The diluted ex-
tract (0.1 mL) was added to 4.9 mL of FRAP reagent in a vial and then
incubated in water bath at 37 °C for 10 min. The absorbance was
measured at 593 nm under UV–Vis spectrophotometer and results were
expressed as micromole ascorbic acid equivalent per gram sample
(μmol AAE/g). The experiments were carried out in triplicates.
2.7. Viscosity analysis
To determine the viscosity of the extract under different extraction
conditions, viscosity analysis was performed using Rheometer MCR-302
(Anton Paar, Germany) according to Marták and Schlosser (Marták &
Schlosser, 2017) protocol. Briefly, a cone and plate system were applied
at 25 ℃ with a shear rate of 0.1 s−1. The graph between Cy3-gal con-
centration and viscosity was plotted using SPSS version 16.0 software
(IBM, USA).
2.8. High performance liquid chromatography (HPLC) analysis
HPLC analysis of Cy3-gal was conducted using Waters e2695-HPLC
system (Waters Corporation, MA, USA) and PDA detector (2998). The
samples for analysis were prepared by evaporating the solvent using
rotatory evaporator and then dried under reduced pressure in freeze
dryer. The dried sample then mix with 10 mL of ethanol and filtered
through 0.22 µm syringe filter (Millipore Co., Shanghai, China). For the
seperation and identification of Cy3-gal, a ZORBAX-SB C18 column
(100 mm × 4.6 mm, 1.8 µm column, Agilent Technologies Co., Santa
Clara, USA) was used with water (0.1% formic acid) and acetonitrile
(0.1% formic acid) as solvent A and B, respectively (Cai et al., 2016).
The injection volume was set to 10 μL and the gradient elution system
was used as: 0–2 min, 5% B; 2–25 min, 5% to 50% B; 25–35 min, 50% to
95% B; 35–37 min, 95% B, 37–40 min 95% to 5% B, with a flow rate of
0.8 mL min−1. The column temperature was maintained at 30 ± 1 °C.
The anothocyanins were monitored at 520 nm. Cy3-gal (Idaein
chloride) was used as standard anthocyanin compound from 2 to
10 ppm concentration to determine the limit of detection
(LOD = 1.22 mg/L), limit of quantification (LOQ = 3.72 mg/L), reten-
tion time (RT) and linear equation for the quantification of Cy3-gal
concentration in the sample. To differentiate between cyanidin-3-O-
galactoside and cyanidin-3-O-glucoside compounds having similar
molecular mass, a standard of cyanidin-3-O-glucoside was also run.
T. Belwal, et al.
2.9. UPLC-Triple-TOF/MS analysis
The ultra-high pressure liquid chromatography (UPLC) (Waters Co.,
Milford, USA) coupled with triple-TOF mass spectrophotometry (UPLC-
Triple-TOF/MS) 5600+ System (AB SCIEX Co., Framingham, USA) with
an electrospray ionization (ESI) source was used for the detection and
identification of anthocyanin compounds in the sample using Cai’s
protocol (Cai et al., 2016), with minor modifications. The sample pre-
paration, mobile phase and other conditions were similar as mentioned
above. Spectral information over the wavelength range of 200–800 nm
was collected. And the detection wavelength was set at 520 nm. The MS
condition was set at the positive ion mode with scan ranges from m/z
200–2000, capillary voltage at 3.0 kV, sampling cone at 35 V, collision
energy at 4 eV, source temperature at 100 ℃, desolvation temperature
at 300 ℃, and desolvation gas rate at 500 L/h. The data were analyzed
using the Peak View software, version 1.2 (Sciex, USA) and identified
based on the molecular ion and fragment ion peak and confirmed with
literature references. Both cyanidin-3-O-galactoside and cyanidin-3-O-
glucoside standard were used to confirm the identification of these two
compounds.
2.10. Scanning electron microscopy (SEM)
SEM of the fruit peel powder sample before and after extraction
(UAE and CE) was carried out using SEM analyzer (Model SU 8010,
Hitachi, Japan). Briefly, 1 g of the powder sample was mixed with 2 mL
of glutaraldehyde solution (2.5% v/v) in phosphate buffer (0.1 M, pH
7.0) and kept overnight at 4 °C. The glutaraldehyde was decanted and
the samples were washed three times in phosphate buffer (0.1 M, pH
7.0) for 15 min at each step and postfix with 1% osmium tetraoxide in
phosphate buffer for about 90 min. After decantation of osmium tetra-
oxide, the samples were washed three times with phosphate buffer
(0.1 M, pH 7.0). The samples now dehydrated with ascending order of
ethanol concentrations (30%, 50%, 70%, 80%, 90%, and 95%) for
about 15 min at each step and with absolute ethanol for two times
(20 min for each step). Finally, the samples were dehydrated under
critical point dryer (Model HCP-2, Hitachi, Japan) and coated with
gold–palladium in E-1010 ion sputter (Hitachi, Japan) for about
4–5 min and observed under SEM analyzer.
2.11. Fourier transform infrared spectroscopy (FT-IR)
FT-IR was carried out using FT-IR spectrophotometer (Thermo
Nicolet AVATAR 370FT-IR, Thermo Electron Corporation, USA). The
extract was first concentrated using rotary evaporator and then dried
using freeze dryer. A small amount (∼0.2 g) of freeze-dried extract was
mixed completely with KBr (∼1 g) and pellets were formed. The sample
pellets were then analyzed under FT-IR spectrophotometer and the
spectra were recorded in the region between 4000 and 600 cm−1. FT-IR
analysis was done with the help of OMNIC version 6.2 software
(Thermo Scientific, USA).
2.12. Statistical analysis
Statistical analysis were performed using SPSS software version
16.0 (IBM, USA). The 3D response surface graphs and regression coef-
ficient analysis were performed using design expert software version 11
(Stat-Ease Inc., USA). All experiments were performed in triplicates and
the significance was determined using Fisher’s test at p < 0.05.
3. Results and discussion
3.1. Selection of factors and their levels
With increasing ultrasonic power from 60 to 200 W, a significant
higher extraction yield (0.29 mg/g) of Cy3-gal was recorded (Fig. 1A).
4
Food Chemistry 297 (2019) 124993
However, it was interesting to note that a significant (p < 0.05) de-
creased in Cy3-gal extraction yield was recorded when applied lower
(60 W) and higher (400 and 600 W) ultrasonic power (Fig. 1A). Ultra-
sonic power played an important role in the extraction of compounds
due to cavitation effect. As seen from the single factor study, moderate
ultrasonication power of 200 W produce highest effect compared to
lower and higher power. The possible reason of lower extraction yield
at higher power could be the generation of high pressure and tem-
perature by intense collapse of cavitation bubbles during high power
ultrasonication (Nipornram, Tochampa, Rattanatraiwong, &
Singanusong, 2018; Arruda et al., 2019), which results in lower down
the anthocyanin concentration.
With increase in ultrasonic temperature from 15 to 70 °C, a sig-
nificant linear increase in Cy3-gal extraction yield was recorded, with
the maximum extraction yield of 0.30 mg/g (Fig. 1B) at 70 °C. Tem-
perature during ultrasonication provided additional support as to
loosen the sample cell structure and thus helps in penetration of sol-
vent. Moreover, a higher temperature of the medium results in produ-
cing stronger cavitation effect (Zhang, Hu, Yang, & Xu, 2012), subject
to the other extraction conditions. In the present study the rise in
temperature up-to maximum of 70 °C support well the Cy3-gal extrac-
tion, which was well supported by Thirunavukkarasu and Nithya
(2011) study where a temperature of 76.5 °C was found best for the
extraction of anthocyanins from eggplant peels. Also, studies reported
that higher temperature resulted in degradation of anthocyanins during
UAE (Zou et al., 2011, Gao, 2017).
Similarly, ethanol concentration of 35 and 65% showed higher Cy3-
gal extraction yield, while further increasing ethanol concentration
resulted in a significant decrease in Cy3-gal extraction yield (Fig. 1C).
The polarity of the solvent played an important role during extraction
of compounds. The polarity of ethanol and water in a mixture can fa-
cilitate the extraction of compounds that were soluble in the mixture of
solvents at a suitable ratio (Yilmaz & Toledo, 2006; Spigno, Tramelli, &
De Faveri, 2007). Similar effect was seen during the extraction of
polyphenolic compounds from fruits of eggplant (Ferarsa et al., 2018).
Meanwhile, TFA concentration of 3.5% support well for the extraction
of Cy3-gal (Fig. 1D). Acidified solvent was found to be effective in ex-
traction of anthocyanin compounds (Boulekbache-Makhlouf, Medouni,
Medouni-Adrar, Arkoub, & Madani, 2013).
With increasing ultrasonic time from 7 to 21 min, the extraction
yield was significantly increased, with a maximum obtained as
0.29 mg/g (Fig. 1E). However, further increase in ultrasonic time sig-
nificantly decrease the extraction yield. Ultrasonic time played an im-
portant role during the extraction process. Generally, a longer extrac-
tion time results in increased extraction yield. However, in case of
ultrasonication as the time increased the chances of compound de-
gradation increases due to generation of excess heat and pressure. Si-
milar results were obtained elsewhere (Xu, Yuan, Xiao, & Sun, 2018)
The sample to solvent ratio showed non-significant effect between
some of the tested levels (Fig. 1F). For instance, ratio 1:10 and 1:30
showed non-significant differences. Similarly, 1:20 and 1:40, 1:40 and
1:50 showed no significant differences. Among all the tested ratio, 1:10
and 1:30 showed significantly higher Cy3-gal extraction yield as com-
pared to others.
While determining the effect of individual factors over its varying
levels on Cy3-gal extraction yield, the following order was recorded,
ultrasonic time > ultrasonic power > ethanol concentration >
ultrasonic temperature ≥ TFA concentration > sample to solvent
ratio. As compared to the other factors the effect of sample to solvent
ratio was found lowest, thus kept at a constant ratio of 1:30 (g/mL)
during multi-factorial experiments.
3.2. Multi-factorial experiments
3.2.1. Model fitness
Central composite design (CCD) was performed using five
T. Belwal, et al.
Fig. 1. Effect of (A) ultrasonic power, (B) ultrasonic temperature, (C) trifluoroacetic acid concentration, (D) ethanol concentration, (E) ultrasonication time, and (F)
sample to solvent ratio on Cy3-gal extraction yield.
independent factors (ultrasonic power, ultrasonic temperature, TFA
concentration, ethanol concentration, and ultrasonic time) which
varied over three levels (Supplementary Table 1). The linear, quadratic
and interactive effect of these factors over Cy3-gal extraction yield was
determined. The polynomial equation was estimated for YCy3-gal, and
the same has shown in Eq. (4) as:
YCy3 − gal = 0.2618 − 0.0184 X1 − 0.0852 X32 − 0.0235X1X2 − 0.0179X1
X3 − 0.0155X2X3 + 0.0144 X2X 4
where, YCy3-gal is the response term for Cy3-gal yield, X1, X2, X3, X4 are
the independent factors as ultrasonic power, ultrasonic temperature,
trifluoroacetic acid concentration, ethanol concentration, and ultra-
sonication time, respectively. The regression coefficient values are
given for each model term in numericals. Overall the CCD model
showed good significant (p < 0.05) model F-value (2.65) with only
1.3% chances of error (Table 1). The lack of fit value was also found to
be larger (4.17) and non-significant, which resemble model fitness.
Other statistical terms such as R2, predicted R2 and Adeq-Precision were
found satisfactory (Table 1). Overall the model showed significant
linear, quadratic and interactive effect between the factors over Cy3-gal
extraction yield.
3.2.2. Effect of UAE factors on Cy3-gal extraction
3.2.2.1. Linear effect. Among all the tested factors, ultrasonic power
(X1) showed significant (p < 0.01) linear effect over Cy3-gal extraction
yield (Table 1). With increasing ultrasonic power from 150 to 450 W, a
significant (p < 0.01) decrease in Cy3-gal extraction yield was
recorded (Fig. 2A). This was also justified by the negative regression
Food Chemistry 297 (2019) 124993
coefficient value (Table 1). UAE, which is generally considered to be
superior over CE techniques, also negatively affected the extraction
yield when operated over and below its optimum power/intensity. For
instance, ascorbic acid degradation was found to increase in
watermelon juice under high ultrasonic amplitude and higher
processing time (Rawson et al., 2011). Also, during higher ultrasonic
power/intensity, trans-lycopene degradation increased (Xu & Pan,
2013). Moreover, anthocyanin degradation was reported to increase
(4) with increasing ultrasonic amplitude and time in grape juice (Tiwari,
Patras, Brunton, Cullen, & O’Donnell, 2010) and strawberry juice
(Tiwari, O’Donnell, Patras, Brunton, & Cullen, 2009). The possible
mechanism of degradation of bioactive compounds under UAE is
mainly due to the formation of free radicals (water hydrolysis),
isomerization and heat effect (Fig. 3A), which was also explained by
Pingret, Fabiano-Tixier, and Chemat (2013). Likewise, it was also
postulated that an increase in ultrasonic power/intensity resulted in
more numbers of larger size bubble formation which collapse gently
and prevent the transmission of ultrasonic waves into the medium
(Rawson et al., 2011; Pingret et al., 2013).
3.2.2.2. Quadratic effect. Among all quadratic terms, only TFA
concentration was found to produce a significant (p < 0.01)
quadratic negative effect over Cy3-gal extraction yield (Table 1). As
TFA concentration increased from 1.5% to 2.5%, an increase in the Cy3-
gal extraction yield was recorded, which significantly decreased as TFA
concentration further increased to 3.5% (Fig. 2B). From the response
surface graph, it can be clearly estimated that the highest Cy3-gal
extraction yield was recorded when the TFA concentration was in
5
T. Belwal, et al. Food Chemistry 297 (2019) 124993

Table 1 (i.e., solubilization, polarity, mass transfer) of the solvent, while ul-
Regression coefficient analysis and analysis of variance (ANOVA) trasonication provided the cavitation effect, thus produce a synergistic
showing linear, quadratic and interactive effect of independent vari- effect on extraction.
ables. Similarly, a significant (p < 0.05) interaction between ultrasonic
Factor Regression coefficient (β) power and TFA concentration (X1X3) was recorded, which negatively
affected the Cy3-gal extraction yield (Table 1). With the increasing
Intercept, X0 0.2618*
ultrasonic power from 150 to 300 W along with increasing TFA con-
Linear centration from 1.5 to 2.5%, the model showed an increase in Cy3-gal
X1 −0.0184** extraction yield (Fig. 2B). However, further concurrently increase or
X2 0.0008
X3 0.0072
decrease in the ultrasonic power and TFA concentration resulted in
X4 −0.0055 lower down the extraction yield. The response surface graph (Fig. 2B)
X5 −0.0015 clearly depicted that the highest extraction yield could be obtained
Interactive when the ultrasonic power was kept at a lower level, while TFA con-
X1X2 −0.0235** centration at near middle level. The acidic environment was supported
X1X3 −0.0179* well the better extraction of anthocyanins from the plant matrix (Ma
X1X4 −0.0056
et al., 2012), which provided better stability and solubilization to the
X1X5 0.0010
X2X3 −0.0155*
compounds. Anthocyanins are present in plants as an equilibrium of
X2X4 0.0144* four molecular species, i.e., flavylium cation, quinoidal base, carinol
X2X5 0.0029 pseuodobase, and chalcone pseudobase. The appropriate acidic condi-
X3X4 −0.0041 tion is necessary for extraction of anthocyanins. With the change in pH,
X3X5 0.0019
flavylium cation reactivity towards nucleophilic reagents changes,
X4X5 −0.0009
which resulted in structural changes and thus produce differences in
Quadratic
color (Raso et al., 1999; Ma et al., 2012).
X12 0.0141
X22 0.0033 Another significant (p < 0.05) negative interactive effect between
X32 −0.0852** ultrasonic temperature and TFA concentration (X2X3) was recorded
X42 0.0010 (Table 1). With an increase in temperature from 40 to 60 °C along with
X52 0.0488 TFA concentration from 1.5% to 2.5%, an increase in Cy3-gal extraction
Model (F-value) 2.65*
yield was recorded (Fig. 2C). The highest extraction yield of Cy3-gal
Lack of fit (F-value) 4.17
R2 0.6978 was achieved when the temperature was set at a lower level, while the
Adjusted R2 0.4349 TFA concentration at an around the middle level (Fig. 2C). Further
Predicted R2 −0.3006 increase or decrease in temperature and TFA concentration resulted in a
Adeq Precision 5.3353
non-significant decrease in Cy3-gal extraction yield.
X1 = ultrasonic power, X2 = ultrasonic temperature,
The interaction between (X2X4) was found significantly (p < 0.05)
X3 = trifluoroacetic acid concentration, X4 = ethanol concentration, positive (Table 1). Concurrently decrease in ultrasonic temperature and
X5 = ultrasonication time; Level of significance ethanol concentration resulted in a significant increase in Cy3-gal ex-
* p < 0.05. traction yield (Fig. 2D). During ultrasonication the temperature and
** p < 0.01. solvent concentration have profound effect over solvent polarity, den-
sity, surface tension and viscosity (Chemat, Zill-E-Huma, & Khan, 2011;
between 2.0% and 3.0%. However, further changes in TFA Huang et al., 2019); which upon changes results in change in ultrasonic
concentration beyond this region was not found much effective in effect and subsequently the extraction yield (Fang et al., 2018). Also, in
extracting Cy3-gal from the fruit peel powder. With respect to change in some cases it has been noticed that higher temperature decreased the
TFA concentration, the pH and acidic condition of the solvent changed, cavitation effect by modulating solvent physical characteristics, thereby
which might affect the extraction efficiency and stability of the lower down the extraction yield (Rawson et al., 2011; Fang et al.,
anthocyanins as reported elsewhere (Raso, Mañas, Pagán, & Sala, 2018).
1999; Cortez, Luna-Vital, Margulis, & Gonzalez de Mejia, 2017).
3.2.3. Optimized condition and its validation
Based on the CCD model and the significant linear, quadratic and
3.2.2.3. Interactive effect. Based on the ANOVA, four significant interactive effects of the process factors, optimized UAE conditions for
interactive effects between the factors were recorded (Table 1). maximizing Cy3-gal extraction yield were determined. For maximum
A negative significant (p < 0.01) interactive effect between ultra- desirability, the optimized condition was determined as, ultrasonic
sonic power and temperature (X1X2) was recorded (Fig. 2A). Cy3-gal power of 162 W, ethanol concentration of 57% with 3% of TFA con-
extraction yield was found to increase significantly when ultrasonica- centration, sample to solvent ratio of 1:30 g/mL, ultrasonic temperature
tion operated at lower power and higher temperature (Fig. 2A). Ultra- of 71 °C and time of 11 min. Under optimized UAE condition, Cy3-gal
sonication operated at lower power was also found significantly better extraction yield was recorded as 0.343 ± 0.005 mg/g, which was
as observed during the linear effect, which revealed that with in- much closer to the model predicted value of 0.336 mg/g and thus va-
creasing ultrasonic power, the degradation or lower extraction of an- lidated the model.
thocyanins started and resulted in lower Cy3-gal extraction yield. The
degradation or lower extraction mechanism was explained previously 3.3. Viscosity and Cy3-gal extraction yield
(Pingret et al., 2013; Xu & Pan, 2013). Moreover, lower ultrasound
power operated at a lower temperature was found ineffective in ex- Extracts obtained under different extraction conditions were ex-
tracting Cy3-gal from fruit peel powder (Fig. 2A). However, as the amined for its viscosity. The Cy3-gal extraction yield were arranged in
temperature increased along with a decrease in ultrasonic power, a ascending order with respective viscosity of the extract and plotted on a
significant increase in Cy3-gal extraction yield was recorded (Fig. 2A), graph (Fig. 4). As seen from the graph, Cy3-gal extraction yield was
which revealed a significant (p < 0.01) negative interactive effect found to be mostly increased with the decrease in viscosity, except for
(X1X2). Higher temperature during ultrasonication process changes the few. In general, the graphs showed comparatively higher viscosity
surface properties (i.e., loosen the cell wall) and physical characteristics value (A) with corresponding lower Cy3-gal value (Fig. 4). Moreover, it

6
T. Belwal, et al.
Fig. 2. Response surface graphs showing the linear, quadratic, and interactive effect of different factors over Cy3-gal extraction yield.
is clearly observed that when the viscosity value was comparatively
lower (B) than others (A), a higher Cy3-gal extraction yield was re-
corded (Fig. 4). In general, higher viscosity of the extract during UAE
decreased the formation of cavitation bubbles and restrict their move-
ment to a limited area and thus negatively affected the sonication ef-
fect, mass transfer, and extraction of compounds from the plant sample
(Fig. 3B). The negative effect of viscosity on extraction yield during
ultrasonication was also reported during our previous research (Huang
et al., 2019). It has been reported that among other physical para-
meters, the viscosity is one of the main parameters controlling the ca-
vitation effect of ultrasound (Webb, Payne, & Coussios, 2011). Also, it
was found that lower viscosity of the solvent during ultrasonication
positively supports the cavitation and heating effect of ultrasound
(Hamidi et al., 2014). Moreover, higher viscosity might also supports
the agglomeration process of the powdered sample, which affected the
extraction yield. The viscosity of the solvent during extraction depends
on many factors, such as solvent concentration, sample to solvent ratio,
extraction temperature, ultrasonic power/intensity and time, which
when changed might affect the viscosity and extraction yield, as ob-
served during the present study.
3.4. UPLC-Triple-TOF/MS analysis of anthocyanins
UPLC-Triple-TOF/MS analysis was conducted to find out the
number of possible anthocyanins present in the extract under optimized
condition. The precursor ion and the fragment ion peak (m/z value)
revealed the presence of a total number of 13 anthocyanin compounds
(Supplementary Table 2), out of which 8 were identified based on the
published literature, standards, and MS database. Among the identified
7
Food Chemistry 297 (2019) 124993
anthocyanins, cyanidin derivatives were mostly present in the extract.
The MS chromatograms of the identified compounds are presented in
Supplementary Fig. 2.
Cy3-gal showed its molecular ion peak at 7.1 min with an maximum
absorption peak at 517 nm. The [M] + showed the molecular weight of
449 Da, while the fragment ion showed as 287 Da, which resemble for
cyanidin. Similar MS data was revealed elsewhere in Pear fruit extracts
(Lin & Harnly, 2008; Zhang et al., 2012; Hamidi et al., 2014). However,
similar molecular ion peak and fragment ion peak was also seen at
6.7 min, which resembled to be cyanidin-3-O-glucoside (Huang et al.,
2012; Zhang et al., 2018), as confirmed by the standards retention time
(Supplementary Fig. 3). With molecular ion peak as 611 Da [M]+ and
fragmentation ion peak at 449 and 287 Da, the compound appeared to
be cyanidin-3-O (6-caffeoyl-glucoside) or Cyanidin 3-O-glucoside-5-O-
glucoside at retention time of 7.4 min (Lantzouraki et al., 2015;
Kapusta, Cebulak, & Oszmiański, 2017). Peaks at 8.2 and 8.5 min were
appeared to be cyanidin-3-rutinoside (595, 449, 287 Da) and cyanidin-
3-O-arabinoside (419, 317, 287 Da), respectively. These compounds
were also previously identified in Pear fruits as reported by Galvis
Sánchez, Gil-Izquierdo, & Gil, 2003; Huang et al., 2012. With molecular
ion peak of 465 Da and fragment ion peak of 303 Da at 11.1 min, the
compound was tentatively found to be delphinidin-3-O-glucoside or
delphinidin-3-O-galactoside (Kähkönen, Heinämäki, Ollilainen, &
Heinonen, 2003; Lantzouraki et al., 2015; Zhang et al., 2018). The peak
at 15 min tentatively appeared to be petunidin-3-O-(6″-O-coumaryl)-
glucoside, which was confirmed by the molecular ion of 625 Da and
fragment ion of 479, 317 Da (Kapusta et al., 2017). The peak at
15.2 min having molecular ion peak of 479 Da with fragment ion as
317 Da tentatively appeared to be petunidin-3-O-galactoside or
T. Belwal, et al. Food Chemistry 297 (2019) 124993

Fig. 3. Ultrasonication effects on extraction of compounds. (A) Possible mechanism of ultrasonication on the extraction and degradation of bioactive compounds. A
higher power/intensity of ultrasonic wave generated a higher number of cavitation bubbles along with micro-jet, which resulted in the formation of free radicals (due
to water hydrolysis), isomerization and increased heat effect. Also, larger size cavitation bubbles collapse gently which prevent extraction. (B) The behaviour of
cavitation bubbles under relative low viscosity and high viscosity extraction medium during UAE.

Fig. 4. Graph showing Cy3-gal extraction yield versus viscosity trends over different experimental runs. (A) higher viscosity and lower Cy3-gal value, (B) lower
viscosity and higher Cy3-gal value.

8
T. Belwal, et al.
Fig. 5. Modified UAE conditions under continuous scale-up mode. (A) pictorial representation of the process and (B) real processing instrument setup. Different
notations are, (1) ultrasonication controller, (2) closed ultrasonic chamber, (3) ultrasonic probe, (4) modified extraction vessel, (5) temperature probe, (6) ther-
mostatic water bath, (7) peristaltic pump, and (8) solvent tank.
petunidin-3-O-glucoside (Zhang et al., 2018). Based on the fragmented
ion peaks, the unknown compounds were tentatively identified as de-
rivatives of cyanidin, delphinidin, and petunidin (Supplementary
Table 2).
3.5. CE versus UAE
3.5.1. Cy3-gal extraction yield and in vitro antioxidant activity
CE was performed under the optimized condition, except providing
the ultrasonic effect (Supplementary Fig. 1A). Under CE, Cy3-gal ex-
traction yield was recorded as 0.266 ± 0.004 mg/g (Fig. 1A), which
was significantly lower than that produced during UAE
(0.343 ± 0.005 mg/g) (Supplementary Fig. 1B). This makes a clear
judgment that ultrasonication significantly increased the extraction
yield of anthocyanin when operated under optimized conditions. Si-
milar effect of improving extraction yield in case of UAE over CE was
9
Food Chemistry 297 (2019) 124993
reported elsewhere (Ferarsa et al., 2018; López et al., 2018). However,
some studies reported the degradation effect of ultrasonication on
bioactive compounds (Tiwari et al., 2010; Tiwari et al., 2009; Pingret
et al., 2013), and thus one must take equal concern on the degradation
effect of ultrasonication and subsequently select the process conditions.
The in vitro antioxidant activity of the extract under optimized UAE
condition was also tested and compared with CE to check the functional
activity of the extract under different extraction conditions. The results
revealed that both DPPH and FRAP in vitro antioxidant activity were
found significantly higher in the extract obtained under UAE (DPPH,
157.54 ± 1.34 μmol AAE/g; FRAP, 96.73 ± 1.12 μmol AAE/g) com-
pared to CE (DPPH, 114.18 ± 1.20 μmol AAE/g; FRAP,
74.26 ± 1.06 μmol AAE/g), which signify the better functional activity
of the extract under UAE. The higher antioxidant activity under UAE
might be due to higher recovery of anthocyanins. Similar effect of UAE
compared to CE on improvement of antioxidant activity was found
T. Belwal, et al.
elsewhere (Roidaki, Zoumpoulakis, & Proestos, 2015; Zhang, Birch, Xie,
Yang, & Bekhit, 2019).
3.5.2. FT-IR analysis
FT-IR analysis of the extract under both CE and UAE condition
showed similar band recognition (Supplementary Fig. 4). The band
from 600 to 1000 cm−1 region showed the presence of the aromatic
ring with a sharp absorbance at 1078/1079 cm−1 due to the stretching
vibration of CeOeC esters (Swer, Mukhim, Bashir, & Chauhan, 2018).
Stretching band at 1203 cm−1 showed the presence of pyran ring, a
typical absorption characteristic for flavonoid compounds (Pereira, de
Arruda, & Stefani, 2015). Sharp band at 1674 and 1434 cm−1 corre-
sponds to the stretching vibration of the C]C aromatic ring (Ahmed,
Amer, & Al-Bahate, 2014). The band at 3384 cm−1 appeared due to the
vibration and OH groups (Swer et al., 2018). The band between 2500
and 3400 cm−1 appeared due to the presence of phenol and aromatic
groups, which corresponds to the A and B rings of the anthocyanin
pigment (aglycon) (Saati, 2015).
Overall the FT-IR spectra under both the extraction conditions
showed the presence of anthocyanin functional groups, such as, sugar
vibrations and OeH (2500–3400 cm−1), C]C (1674 and 1434 cm−1),
CeOeC (600–1000 cm−1), and C]O (1674 cm−1), which revealed
UAE as effective means for extracting anthocyanins without breaking or
changing any characteristic functional group.
3.5.3. Cell structural analysis
SEM was performed to investigate the changes in cell structure
under different extraction conditions. The untreated Pear fruit peel
powder showed unbroken and smooth cell wall (Supplementary
Fig. 5A), while the sample underwent deformations during the extrac-
tion process (Supplementary Fig. 5B and C). The cells under CE were
rough, wrinkled and not much broken (Supplementary Fig. 5B) as
compared to UAE (Supplementary Fig. 5C). The higher deformation
during UAE opens the cells to extract out the cell components, thereby
increased the extraction yield (Ferarsa et al., 2018; Khadhraoui et al.,
2019).
The deformation under ultrasonication is due to the formation and
collision of cavitation bubbles, which produce micro-jet having higher
temperature and pressure (Chemat, Rombaut, Sicaire et al., 2017), re-
sulted in deformations in plant cells (Supplementary Fig. 5C). This
further breaks cells into smaller fragments and increased solubilization
and subsequent mass transfer and extraction of the compounds.
3.6. UAE scale-up: Model projection
Based on the low volume (30 mL) UAE optimization model, a scale-
up model was projected and tested under batch [5X (150 mL) and 15X
(450 mL)] and continuous [100X (3000 mL)] extraction process
(Fig. 5). For batch scale-up model, the extraction process was conducted
under similar conditions as for the optimized UAE conditions, while for
the continuous process the extraction conditions were modified (Fig. 5).
Under batch scale-up UAE process, Cy3-gal extraction yield was ob-
tained as 0.315 ± 0.004 mg/g and 0.302 ± 0.006 mg/g for 5X and
15X scale-up, respectively, which were closer to the lower volume
(30 mL) optimized extraction yield (Supplementary Fig. 1B). Under the
continuous extraction process (100X), a lower Cy3-gal extraction yield
of 0.285 ± 0.004 mg/g was recorded compared to the batch extraction
process. However, the continuous UAE extraction process extracted
Cy3-gal in a comparably good concentration w.r.t. CE, thus can be
further considered for larger volume extraction with further con-
siderations and optimization of instrumentation design and process
factors.
Scaling-up of extraction method from the laboratory to pilot-/full-
scale is an important prerequisite for utilizing larger amount of raw
materials for efficiently extracting larger amount of bioactive com-
pounds. Moreover, higher demand and wider applications of bioactive
10
Food Chemistry 297 (2019) 124993
compounds in food and health products make UAE a compelling can-
didate for developing scaling-up process. In recent years, very few
studies have been conducted on scaling-up of the extraction and re-
covery of bioactive compounds, which showed mixed effect on re-
covery. For instance, ultrasound-assisted maceration extraction
(UAME) of polyphenols from maritime pine sawdust was successfully
optimized and scaled-up from lower to higher extraction volume
(Meullemiestre, Petitcolas, Maache-Rezzoug, Chemat, & Rezzoug,
2016). Similarly, optimization of UAE of lipids from microalgae cells
was successfully performed and recommended for its larger volume
extraction (Adam, Abert-Vian, Peltier, & Chemat, 2012). However, in
one of the extraction scale-up model, UAE of proteins from soybean
slurry during scale-up model did not work well as compared to the
lower volume extraction (Preece, Hooshyar, Krijgsman, Fryer, &
Zuidam, 2017). These extraction scale-up models clearly showed the
posiibility of increasing the extraction volume with better recovey and
challenges. The present study is an attempt to this direction and the
outcomes clearly indicated that scaling-up of UAE process for valuable
anthocyanins could be possible, however more concern on the in-
strumentation and processing conditions need to be given.
4. Conclusions
The present study is an attempt to determine the ultrasonication
factors, mechanism and especially their relation with viscosity for op-
timizing cyanidin-3-O-galactoside extraction yield from Pyrus communis
‘Starkrimson’ fruit peel. And further project a scale-up model for higher
volume extraction. The results revealed a significant role of ultra-
sonication during extraction of anthocyanins, which was justified by
higher Cy3-gal extraction yield and deformations of the cell surface
compared to conventional extraction. Moreover, the lower viscosity of
the extracting solvent during UAE played a vital role and well sup-
ported the extraction process. The FT-IR results showed characteristic
anthocyanin functional groups after the UAE process, which revealed
that the degradation effect of ultrasonication could be prevented under
the optimized condition. Under optimized UAE condition, UPLC-Triple-
TOF/MS detected a total of 13 anthocyanin compounds out of which 8
were identified. The low volume UAE method was also scaled-up to
higher volume extraction and found that the batch scale-up process
showed promising results. However, the scale-up UAE process further
needs more detailed examination in term of instrumentation design and
method development.
Ultrasonication as such is considered to be a green extraction pro-
cess which ideally requires lower consumption of organic solvents,
energy and processing time (Chemat, Rombaut, Meullemiestre et al.,
2017a). In the present study the lower concentration of ethanol, ul-
trasonic power and extraction time was found to be optimum in ex-
tracting Cy3-gal, thus the process could be considered as green ex-
traction process. The present study could be further used to consider
Pyrus communis ‘Starkrimson’ fruit peel for extraction of valuable an-
thocyanins under optimized extraction conditions and utilized in var-
ious food, beverages, health and pharmaceutical products.
Declaration of Competing Interest
None.
Acknowledgments
The research was financially supported by the Key Research and
Development Program of Zhejiang Province (2018C02049), China;
Special Fund for Science and Technology Base and Talent of Guangxi
(GKAD17195088), China; Technology Innovation Programme of
Zhejiang Province (ZJWR0102001), China; Hangzhou Science and
Technology Development Program (20180432B31), China and China
Postdoctoral Science Foundation (2018M642443), China.
T. Belwal, et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.124993.
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