Food Chemistry 178 (2015) 106–114

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

Ultrasound-assisted heating extraction of pectin from grapefruit peel:

Optimization and comparison with the conventional method
Wenjun Wang a,c,d, Xiaobin Ma a,c,d, Yuting Xu a,c,d, Yongqiang Cao e, Zhumao Jiang e, Tian Ding a,c,d,
Xingqian Ye a,b,c,d, Donghong Liu a,b,c,d,⇑
a
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, Zhejiang 310058, PR China
b
Fuli Institute of Food Science, Zhejiang University, Hangzhou, Zhejiang 310058, PR China
c
Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou, Zhejiang 310058, PR China
d
Zhejiang R & D Center for Food Technology and Equipment, Hangzhou, Zhejiang 310058, PR China
e
School of Life Sciences, Yantai University, Yantai, Shandong 264000, PR China

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

Article history: The extraction of pectin from grapefruit peel by ultrasound-assisted heating extraction (UAHE) was
Received 18 July 2014 investigated using response surface methodology and compared with the conventional heating extrac-
Received in revised form 14 January 2015 tion (CHE). The optimized conditions were power intensity of 12.56 W/cm2, extraction temperature of
Accepted 17 January 2015
66.71 °C, and sonication time of 27.95 min. The experimental optimized yield was 27.34%, which was
Available online 22 January 2015
well matched with the predicted value (27.46%). Compared with CHE, UAHE provided higher yield
increased by 16.34% at the temperature lowered by 13.3 °C and the time shortened by 37.78%. Image
Keywords:
studies showed that pectin extracted by UAHE showed better color and more loosen microstructure com-
Ultrasound assisted heating extraction
Grapefruit peel
pared to that extracted by CHE, although Fourier Transform Infrared Analysis indicated insignificant dif-
Pectin ference in their chemical structures. Furthermore, UAHE pectin possessed lower viscosity, molecular
Response surface methodology weight and degree of esterification, but higher degree of branching and purity than CHE pectin, indicating
Comparison that the former was preliminarily modified during the extraction process.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction innovative technologies, especially ultrasound, have been com-

Pectin is a family of complex heteropolysaccharides consisting
of a backbone of a-(1 ? 4) galacturonic acid residues which are
partially esterified with methyl alcohol or acetic acid at the carbox-
ylic acid. Pectin is widely used as a gelling, stabilizing and thicken-
ing agent in food systems such as jams and jellies, confectionery,
and fruit juice (Thakur, Singh, & Handa, 1997; Zouambia, Moulai-
Mostefa, & Krea, 2009). Citrus peels were usually discarded as
by-products of juice and can production, causing severe environ-
mental issues and economic wastes. However, the pectin content
of dry citrus peel is as high as 25–30%, making it one of the primary
pectin sources for commercial applications (Arslan & Togrul, 1996;
Srivastava & Malviya, 2011).
In the commercial extraction process, pectin is conventionally
extracted using hot water (60–100 °C) under the pH range from
1.5 to 3.0 for hours (Koubala, Mbome et al., 2008). The process is
time consuming and the yield of pectin is limited. Therefore, many
⇑ Corresponding author at: College of Biosystems Engineering and Food Science,
Zhejiang University, 866 Yuhangtang Rd., Hangzhou, Zhejiang 310058, PR China.
Tel.: +86 057188982169; fax: +86 057188982144.
E-mail address: dhliu@zju.edu.cn (D. Liu).
bined with heating extraction to improve extraction efficiency
and pectin yield. However, the application of ultrasound-assisted
heating extraction (UAHE) in pectin extraction is still at the stage
of laboratory research, and only few studies have been carried
out in the last few decades (Bagherian, Ashtiani, Fouladitajar, &
Mohtashamy, 2011; Panchev, Kirchev, & Kratchanov, 1988;
Panchev, Kirtchev, & Kratchanov, 1994; Xu et al., 2014), because
there exists a big issue that ultrasound could break down polymers
including pectin, leading to the modification of structural and
physico-chemical properties, which is unfavorable to its gelling
properties (Zhang, Ye, Ding et al., 2013; Zhang, Ye, Xue et al., 2013).
In recent years, pectin is suggested to possess various pharma-
ceutical activities including wound healing (Hokputsa et al., 2004),
lipase inhibition (Edashige, Murakami, & Tsujita, 2008; Kumar &
Chauhan, 2010), apoptosis induction of human cancer cell
(Jackson et al., 2007), as well as immunostimulating, anti-metasta-
sis, anti-ulcer, cholesterol decreasing effects, etc. (Yamada, 1996).
Grapefruit peel pectin which has short side chains is rich in
rhamnogalacturonan I backbones in particular compared with
orange, lime and lemon, possessing potential values in bioactivity
(Kaya, Sousa, Crepeau, Sorensen, & Ralet, 2014). However, unmod-
ified pectin is thought to be too large to be absorbed in the body

http://dx.doi.org/10.1016/j.foodchem.2015.01.080
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 W. Wang et al. / Food Chemistry 178 (2015) 106–114 107

while modified pectin that has been broken down into smaller
fragments can be absorbed more easily. It is noteworthy that high
intensity (>10 W/cm2) power ultrasound is proved to be an effec-
tive tool to improve the bioactivities via modifying the molecular
properties of polysaccharides (Zhang, 2013; Zhou, Yu, Zhang, He,
& Ma, 2012). Therefore, it could be envisaged that high intensity
UAHE had the potential to simultaneously achieve the high-effi-
cient extraction and modification/preliminary modification of pec-
tin. From this point of view, it is of great importance to promote
further studies and applications in pectin extraction using this
innovative method.
To reduce the degradation effect of ultrasound, output power
intensity of ultrasound was commonly kept at a low level
(1–8 W/cm2) when it was used in the extraction of polysaccharides
(reviewed by (Ebringerova & Hromadkova, 2010)). Therefore, the
effect of ultrasound intensity on the yield of pectin is scarcely stud-
ied. In a previous study (Xu et al., 2014), we reported that the
mechanism of high intensity (>10 W/cm2) ultrasound enhanced
pectin yield was improving pectin extractability through disrupt-
ing vegetal tissue. It could be seen that ultrasound intensity had
significant effects on pectin extraction. Besides, extraction temper-
ature and sonication time were also essential variables of UAHE
(Bagherian et al., 2011; Xu et al., 2014). However, few works has
been performed in the optimization of UAHE conditions and explo-
ration of the combined effects of these variables in the process of
pectin extraction.
In the current study, response surface methodology (RSM) was
employed to study the extraction of grapefruit peel pectin using
UAHE, aiming at optimizing the conditions for the extraction of
pectin from grapefruit peel, as well as understanding the combined
effects of key operating variables (output power intensity of ultra-
sound, extraction temperature and sonication time) on the yield of
pectin. In addition, comparative analysis of extraction yield, molec-
ular structure, and physico-chemical properties of pectin extracted
by UAHE and CHE were also investigated.
2. Materials and methods
2.1. Material and chemical reagents
immersed 15 mm into solution contained in a jacketed cylinder
reactor (200 mL volume). During the experiments, the temperature
of mixture was measured with a digital thermometer located in the
center of the reactor and was held constantly at a desired value by
a thermostatic bath driving cooling water through jacket. Periodi-
cally agitation (10 s on: 50 s off) was performed to keep the mix-
ture temperature even and the material getting equal irradiation.
The reactor was sealed to prevent water leaks during extraction.
The sketch diagram of experimental set-up was shown in Supple-
mentary Fig. S1.
A calorimetric procedure was used to determine the effective
ultrasound power (P, W) transferred into the medium for each
condition tested (Raso, Manas, Pagan, & Sala, 1999). Power intensity
(I, W/cm2) dissipated from a probe tip with radius r is given by
I = P/pr2. For output power levels of 50, 60 and 70 W emitted by a
25 mm probe, the calculated power intensities were 10.18, 12.22
and 14.26 W/cm2.
2.3. Extraction and purification of pectin
The extracting and purifying of pectin was carried out according
to our previous method (Xu et al., 2014). Deionized water adjusted
by 0.5 M HCl to reach pH of 1.5 was used as the extraction solvent.
Three grams of peel particles was mixed with 150 mL extraction
solvent in the reactor. Extraction conditions for UAHE were shown
in Table 1; while for CHE, the extraction mixture was kept at 80 °C
for 90 min without ultrasound irradiation. After extraction, the
extraction mixture was quickly transferred into centrifugal tubes,
and placed in a cold-water bath for cooling. Then the mixture
was centrifuged at 9000 rpm for 30 min under 4 °C using a low-
temperature centrifuge (HITACHI CF16RXII, Japan). The volume of
the first filtrate was measured and recorded as Vfiltrate (Vf, mL). In
the next step, the filtrate was coagulated using equal volume of
95% ethanol and left for 2 h under 4 °C. The coagulated pectin
was separated by centrifuge and washed three times with 95% eth-
anol. It was dried at 40 °C in a laboratory drier until its weight was
constant; the dried pectin was labeled as Pectin I, and its weight (M,
g) was weighed with an analytical balance (Mettler Toledo GB204,
Switzerland). The yield (Y, %) of pectin was calculated according to
Panchev et al. (1988) as follows:

Fresh grapefruit (Citrus paradisi Macf.) were picked from Taiz- Y ¼ M=3 ðgÞ  150 ðmLÞ=V f  100% ð1Þ
hou, China. The collected peel was first soaked in a water bath at
90 °C for 5 min to inactivate enzymes, then drying was carried
out in an oven with air circulation at 50 °C. The dried peels were
Table 1
grounded by an electric grinder (Qijian Q-250A3, Shanghai Bingdu Factors and levels for response surface methodology (RSM), and Box–Behnken
Electrical Appliance Co., Ltd., China) and passed through an 80 experimental design with independent variables.
mesh sieve to obtain particles (<180 lm). Finally, the vacuum-
Run Coded and uncoded variable levels Yield of
packed samples were stored inside desiccators until use. numbers pectin (%)
X1/power X2/extraction X3/sonication
Ethanol, HCl, NaOH, phenol, H2SO4, foline phenol, gallic acid,
intensity temperature (°C) time (min)
NaNO2 and Al(NO3)3 (Sinopharm Chemical Reagent Co., Ltd., (W/cm2)
Shanghai, China), trifluoroacetic acid (Aladdin Reagents, Shanghai,
1 1 (14.26) 0 (70) 1 (40) 26.01
China) and 1-phenyl-3-methyl-5-pyrazolone (PMP) (Acros Organ- 2 0 (12.22) 1 (60) 1 (20) 26.05
ics, USA) were analytical grade (purity P 99%). Chemicals used 3 0 (12.22) 0 (70) 0 (30) 27.23
for high-performance liquid chromatography (HPLC), including 4 1 (14.26) 1 (80) 0 (30) 23.51
methyl alcohol and acetonitrile, were HPLC-grade (Tedia, USA). 5 1 (14.26) 1 (60) 0 (30) 25.95
6 1 (14.26) 0 (70) 1 (20) 26.51
Rutin standard was purchased from Aladdin Reagents (Shanghai,
7 1 (10.18) 1 (80) 0 (30) 23.10
China), and monosaccharides standards were purchased from 8 1 (10.18) 0 (70) 1 (20) 25.14
Sigma–Aldrich (St. Louis, MO, USA). 9 0 (12.22) 0 (70) 0 (30) 27.14
10 0 (12.22) 0 (70) 0 (30) 27.15
11 1 (10.18) 1 (60) 0 (30) 25.53
2.2. Experimental set-up and characterization of ultrasonic field 12 0 (12.22) 1 (80) 1 (20) 24.14
13 0 (12.22) 1 (60) 1 (40) 25.82
Ultrasound was applied using a probe (25 mm diameter) sys- 14 0 (12.22) 0 (70) 0 (30) 27.51
15 0 (12.22) 1 (80) 1 (40) 24.68
tem (Sonics, VCX800, USA), which had a maximum power of 16 1 (10.18) 0 (70) 1 (40) 25.83
800 W, and operated at 20 kHz. Duty cycle of ultrasound pulse 17 0 (12.22) 0 (70) 0 (30) 27.32
was set at 50% (2 s on: 2 s off). The ultrasonic emitter was
108 W. Wang et al. / Food Chemistry 178 (2015) 106–114
The second extraction process was employed in CHE and three
selected UAHE conditions (10.18, 12.22 and 14.26 W/cm2 at 70 °C
for sonication 30 min) according to Panchev et al. (1988) with
minor modifications. After the first centrifugation, hot distilled
water (150 mL) of the same temperature with the first extraction
was mixed with the residue, and held without sonication for
30 min. Then the extraction mixture was cooled and centrifuged
(9000 rpm, 30 min), the filtrate was coagulated and dried. The
dried pectin was labeled as Pectin II. The flow chart of the extrac-
tion and purification of pectin from grapefruit peel was shown in
Supplementary Fig. S2.
2.4. Experimental design for RSM and data analysis
RSM with Box–Behnken design (BBD) was used to determine
the optimal conditions for pectin extraction from grapefruit peel.
A three-level-three-factor BBD was employed to explore the best
combination of three independent factors affecting extraction
process, including output power intensity of ultrasound (X1:
10.18–14.26 W/cm2), extraction temperature (X2: 60–80 °C), and
sonication time (X3: 20–40 min). The experimental ranges for the
factors were determined according to the results of our previous
study (Xu et al., 2014). The whole design consisted of 17 experi-
mental points, including 12 factorial points and 5 centre points.
Five replicates at the centre of the design were used to estimate
a pure error sum of squares. All the experiments were randomly
carried out in order to minimize the effect of unexplained variabil-
ity due to systematic errors. The experimental design and data
analysis were performed using Design Expert software (Version
7.0). The response variables were fitted to a quadratic polynomial
model, the general form of which was as follows:
X
3 X
3 X
2 X
3
Y ¼ b0 þ bi X i þ bii X 2i þ bij X i X j
i¼1 i¼1 i¼1 j¼iþ1
where Y is the response variable; b0, bi, bii and bij are the regression
coefficients for intercept, linearity, square and interaction, respec-
tively; Xi and Xj represent the coded independent variables, which
were coded according to the equation:
X i ¼ ðX i  X 0 Þ=DX
where xi is the coded value of the variable Xi, X0 is the value of xi at
the centre point, and DX is the step change. The actual and coded
levels of the independent variables used in the experimental design
are shown in Table 1.
2.5. Pectin color
The CIELab coordinates (L⁄, a⁄, b⁄) of the pectin samples were
directly read with a Tristimulus Colorimeter (WSD-III, Kang Guang
Instrument Co., Beijing, China), with a white tile as the reference.
The L⁄ value represented the lightness, ranging from 0 (black) to
+100 (white); a⁄ value ranges from 100 (green) to +100 (red)
and b⁄ value ranges from 100 (blue) to +100 (yellow). The hue
angle (Hab ) and chroma (C⁄) were calculated according to the fol-
lowing equations:
 (GAE)/mg pectin; the content of total flavonoids was expressed
Hab ¼ tan1 ðb =a Þ
 2
2
C  ¼ a2 þ b
2.6. Scanning electron microscopy (SEM) analysis
Dried pectin particles were fixed onto a SEM specimen stub
with a double-sided adhesive tape prior to coating. Specimens
were coated with gold using a HITACHI E-1020 ion sputter (JEOL,
Japan) and subsequently photographed using Scanning Electron
Microscope Cambridge Stereoscan 260 coupled with energy dis-
persive X-ray microanalysis system (Leica, UK).
2.7. Fourier Transform Infrared Spectroscopy (FTIR) analysis
Pectin particles were mixed with KBr (1:100) and pressed into
KBr pellets before FTIR analysis. FTIR spectra Nicolet 5700 (Thermo
Fisher Scientific, USA) was applied at the absorbance mode in the
frequency range of 4000–400 cm1 at a resolution of 4 cm1. The
absorption spectra were obtained after denoising and baseline
correction.
2.8. Determination of physico-chemical properties of pectin
2.8.1. Intrinsic viscosity and viscosity-average molecular weight (MV)
Intrinsic viscosity was measured with an Ubbelohde capillary
viscometer with a capillary diameter of 0.58 mm (Jiaojiang Glass
Instrument Co., Taizhou, China) at 25 ± 0.1 °C. 0.1 M phosphate
buffer (pH = 7.0) was used as the solvent. 3.0 mg of dried sample
was dissolved in 10 mL solvent. According to the efflux time of
the pectin solution (t) and the solvent (t0), the relative viscosity
(gr = t/t0) was obtained. Specific viscosity was calculated from the
formula gsp = (t  t0)/t0. Intrinsic viscosity could be obtained by
one point method according to the following equation (Abdel-
Azim, Atta, Farahat, & Boutros, 1998).
g ¼ ½2 ðgsp  ln gr Þ0:5 =C ð6Þ
where C represented the concentration of pectin solution (g/L); g
was the intrinsic viscosity (L/g). The viscosity-average molecular
weight (MV) was calculated from the Mark–Houwink–Sakurada
ð2Þ
equation:
g ¼ k  MaV ð7Þ
where g was the intrinsic viscosity (L/g); k and a were constants
depending on the solution and temperature, which were
2.34  105 and 0.8224, respectively (Kar & Arslan, 1999).
ð3Þ
2.8.2. Degree of esterification (DE), total sugar content and
monosaccharide composition
DE of pectin was determined by the titrimetric method as
described by previous study (Liu, Cao, Huang, Cai, & Yao, 2010)
with minor modification. Total sugar content was determined by
phenol–sulfuric acid method in microplate format using glucose
as the standard (Masuko et al., 2005). Monosaccharide composition
was determined by HPLC method of pre-column derivatization
with 1-phenyl-3-methyl-5-pyrazoIone (PMP) as described by
Zhang, Ye, Ding et al. (2013).
2.8.3. Contents of total phenolics and flavonoids
Total phenolics and flavonoids were extracted using 80% (v/v)
ethanol at 4 °C for 24 h and determined according to the methods
of Toor and Savage (2005), with minor modifications. The content
of total phenolics was expressed in lg gallic acid equivalents
ð4Þ
in lg rutin equivalents (RE)/mg pectin.
ð5Þ 2.9. Statistical analysis and figure plotting
All experiments were performed in at least two replicates. The
statistical analysis of model was used SPSS software (Version
16.0). Other data was analyzed using analysis of variance (ANOVA)
by SPSS software (Version 16.0), and expressed as mean
value ± standard deviation. The confidence level for statistical
 W. Wang et al. / Food Chemistry 178 (2015) 106–114 109

significance was set at a probability value of 0.05. The figures, of the determination coefficient (R2) calculated from the quadratic
including the three-dimensional response surface plots, two- regression model was 0.9855, which implied that 98.55% of the
dimensional contour plots, were plotted using Origin software variations could be explained by the fitted model. The value of
(Version 8.5). the adjusted determination coefficient (R2Adj = 0.9668) is close to
R2, indicating a high degree of correlation between the observed
3. Results and discussion and predicted values. Besides, a low value of the coefficient of vari-
ation (C.V. = 0.95%) indicated a higher degree of precision and reli-
3.1. Extraction of pectin from grapefruit peel ability of the experimental values.
The corresponding variables would be more significant if the
Supplementary Fig. S3 showed the dry weights of pectin after F-value becomes greater and the p-value becomes smaller, and
first and second extraction under different extraction conditions. values of p-value less than 0.05 showed model variables were
It could be seen that the weight of pectin obtained after the first significant (Atkinson & Donev, 1992). Therefore, the F-value
extraction using UAHE was lower than that of CHE. This was (52.85) and p-value (<0.01) implied that this model was very sig-
because ultrasound disintegrated the material, adversely affecting nificant. The lack-of-fit test measures the failure of the model to
the separation of liquid and solid phases (Xu et al., 2014). It was represent the data at points that are not included in the regression
noted that researchers had investigated the effects of temperature within the tested range. As shown in Table 2, F-value (4.68) and p-
and time of UAHE on pectin yield, reporting that the pectin yield of value (0.0850) of lack-of-fit implied that it was not significantly
UAHE was lower than that of CHE (Bagherian et al., 2011), which relative to the pure error. All these results indicated that the model
was contradictory with other studies (Panchev et al., 1988, 1994; was adequate for predicting the yield of pectin under any combina-
Xu et al., 2014). Overlooking the pectin absorbed in the residue tion of the variables within the employed range. The significance of
would probably lead to the improper conclusion. each coefficient was also determined using F-value and p-value:
Considering this disadvantage, the second extraction was nec- the linear coefficient (X1, X2), cross product coefficients (X1X3,
essary in order to obtain the dissolved pectin completely. Through X2X3) and quadratic term coefficients (X21, X22 and X23) significantly
the second extraction with hot distilled water, little pectin was affected the yield of pectin (p < 0.05).
recovered from the residue after CHE, while about 40–50% of Pectin
I (pectin obtained by the first extraction) was recovered from the 3.3. Analysis of response surfaces
residue after UAHE. The total weight of pectin extracted by UAHE
was significantly higher than that by CHE (p < 0.05). Response surfaces were plotted to study the effects of variables
and their interactions on the yield of pectin. Three-dimensional
response surface plots and two-dimensional contour plots, as pre-
3.2. Statistical analysis and model fitting using RSM
sented in Fig. 1, provided a method to visualize the relationship
between responses and experimental levels of each variable, and
The values of responses (yield of pectin) at different experimen-
the type of interactions between each two tested variables. In each
tal combinations were given in Table 1. By employing multiple
figure, the variable not shown was kept at level 0 (center value of
regression analysis on the experimental data, the predicted
the testing ranges).
response Y for the yield of pectin could be obtained by the follow-
As shown in Fig. 1A, when sonication time (X3) was set at 0
ing regression model:
level, both power intensity of ultrasound (X1) and extraction tem-
Y ¼ 27:27 þ 0:30X 1  1:04X 2 þ 0:012X 3  0:0025X 1 X 2 perature (X2) presented significantly quadratic effects on the yield
of pectin. The yield of pectin first increased rapidly with the
 0:30X 1 X 3 þ 0:29X 2 X 3  1:07X 21  1:67X 22  0:32X 23 ð8Þ increase of power intensity or extraction temperature, but
decreased after reaching a peak. The decline of the yield might
where Y is the yield of pectin (%) and X1, X2, X3 are the coded values
of the test variables, power intensity of ultrasound (W/cm2); extrac- be due to the degradation effects of pectin caused by high ultra-
sound intensity and/or extraction temperature (Bagherian et al.,
tion temperature (°C); and sonication time (min), respectively.
The statistical significance of regression equation was checked 2011; Panchev et al., 1988; Zhang, Ye, Ding et al., 2013). Besides,
it has been reported that temperature could significantly affect
by F-test, and the analysis of variance (ANOVA) for response sur-
face quadratic polynomial model was shown in Table 2. The value the power output of ultrasound. At ambient pressure, in the range
of 20–70 °C, the power output was hardly affected; however, at
temperatures higher than 70 °C it decreased drastically and at
100 °C no power output was detected (Raso et al., 1999). This
Table 2
Predicted regression model of relationship between response variable (yield of pectin)
was because the vapor pressure of the heated liquid was elevated
and independent variables (X1, X2, X3). when the temperature was high, which allowed cavitation to be
achieved at lower acoustic intensity, thus reducing the strength
Variables Sum of squares DF Mean square F-value p-Value
of shear forces in the vicinity of the bubble (Mason & Lorimer,
Modela 28.51 9 3.17 52.85 <0.0001 2002).
X1 0.71 1 0.71 11.81 0.0109
The circular contour plot on the right (Fig. 1A) showed that the
X2 8.65 1 8.65 144.33 <0.0001
X3 1.25  10–3 1 1.25  103 0.021 0.8893 mutual interactions between power intensity and extraction tem-
X1X2 2.50  103 1 2.50  103 4.17  104 0.9843 perature were insignificant (p > 0.05). However, our previous study
X1X3 0.35 1 0.35 5.91 0.0454 showed synergistic effects between ultrasound and heating on the
X2X3 0.34 1 0.34 5.71 0.0482
yield of pectin by comparing different extraction conditions with/
X21 4.85 1 4.85 80.98 <0.0001
X22 11.80 1 11.80 196.76 <0.0001
without ultrasound and/or heating (Xu et al., 2014). The disparity
X23 0.44 1 0.44 7.36 0.0301 could be explained that the synergistic effects between ultrasound
Residual 0.42 7 and heating were not significant in the tested ranges of variables.
Lack of fit 10.18 3 0.11 4.68 0.085 The quadratic effects of power intensity (X1) on the yield of pec-
Pure error 0.093 4 0.023
tin could be found in Fig. 1B, when extraction temperature (X2) was
Cor. total 28.93 16
fixed at 0 level. On the other hand, when power intensity was kept
a
R2 = 0.9855, R2Adj = 0.9668, C.V. = 0.95%. at a lower level (10.18 W/cm2), the yield of pectin increased evi-
110 W. Wang et al. / Food Chemistry 178 (2015) 106–114

Fig. 1. Response surface plots and contour plots showing the effect of power intensity of ultrasound (X1), extraction temperature (X2) and sonication time (X3) on the yield of
grapefruit peel pectin.

dently with increasing of sonication time (X3) from 20 to 30 min;
but beyond 30 min, the yield of pectin increased slowly as sonica-
tion time ascended. From Fig. 1C, when power intensity (X1) was
fixed at 0 level, extraction temperature (X2) showed significantly
quadratic effects on the yield of pectin. When extraction tempera-
ture was at a lower level (60 °C), the yield of pectin first increased
and decreased with the increasing of sonication time (X3). This
decrease could be due to the long extraction time leading to the
degradation of pectin macromolecules (Bagherian et al., 2011;
Panchev et al., 1988). The elliptical contour plots shown in Fig. 1B
and C indicated that the mutual interactions between power inten-
sity/extraction temperature and sonication time were significant.
These results demonstrated that the effect of power intensity or
extraction temperature on the yield of pectin was significantly
influenced by the changing of sonication time, and vice versa.
3.4. Optimization of UAHE parameters and validation of the model
As shown in Supplementary Table S1, the optimal extraction
conditions for achieving the maximal yield of pectin obtained by
differentiation of the quadratic polynomial model were as follows:
power intensity of 12.56 W/cm2, extraction temperature of
66.71 °C and sonication time of 27.95 min; the predicted maximal
yield corresponding to these conditions was 27.46%. However, con-
sidering the feasibility in experiment, the optimal extraction con-
ditions were slightly modified as follows: power intensity of
 W. Wang et al. / Food Chemistry 178 (2015) 106–114 111

Fig. 2. Comparison of pectin particles through ordinary and SEM images (CP: pectin extracted by conventional heating extraction; UP: pectin extracted by ultrasound-
assisted heating extraction).

12.56 W/cm2 (amplitude of 58%), extraction temperature of
67.7 °C, and sonication time of 28 min. The experimental yield of
pectin was about 27.34 ± 0.25% (n = 3), which was close to the pre-
dicted value. This result further confirmed that the model was ade-
quate for predicting the yield of pectin using UAHE.
3.5. Comparison between CHE and UAHE
(80 °C). The results were in accordance with many published stud-
ies on the extraction of polysaccharides, including pectin, hemicel-
luloses and other water-soluble polysaccharides, with the
assistance of ultrasound (Ebringerova & Hromadkova, 2010), defi-
nitely demonstrating that UAHE can be applied in the extraction
of pectin from various vegetal resources with higher efficiency
and less energy consumption.

3.5.1. Extraction yield of pectin 3.5.2. Image study

As shown in Supplementary Table S1, the yield of pectin
obtained by UAHE (27.34 ± 0.25%) was 16.34% higher than that
obtained by CHE (23.50 ± 0.57%), and the extraction time of the for-
mer (56 min, half of which is the sonication time) was 37.8%
shorter than the latter (90 min). Besides, the extraction tempera-
ture of UAHE (66.7 °C) was also reduced compared to CHE
Fig. 2 exhibited the ordinary and SEM images of pectins
extracted by different methods. Fig. 2A and B were the particles
(<80 lm) of CHE pectin (CP) and UAHE pectin (UP), of which the
color was different: CP was grey brown, whereas UP was light yel-
low. Difference in the color of different pectins was further
reflected by the CIELab coordinates as shown in Supplementary
112 W. Wang et al. / Food Chemistry 178 (2015) 106–114

Table S2. For UP, the L⁄ value representing the lightness was (O–H), C–H of CH2 groups, and C–O, respectively. An absorption
higher; while C⁄ value representing the chroma was significantly at 1746 cm1 was caused by C@O stretching vibration of
(p < 0.05) lower than that of CP. Besides, the different Hab values methylesterified carboxyl groups, while the absorption at about
indicated that their color types were different. Non-enzymatic 1640–1645 cm1 was caused by C@O stretching vibration of free
browning, which negatively affects the color of pectin, is favored carboxyl groups. Some of carboxyl group signals might also origi-
by heating and includes a wide number of reactions such as Mail- nate from phenolic compounds as indicated by the presence of
lard reaction, caramelization, and chemical oxidation of phenolic peaks at 1523 cm1 for the aromatic ring vibrations. The average
compounds (Manzocco, Calligaris, Mastrocola, Nicoli, & Lerici, of the ratio of the peak area at 1745 cm1 (COO–R) over the sum of
2000). In the current study, the brown color of CP was probably the peak areas of 1745 cm1 (COO–R) and 1645 cm1 (COO–) was
attributed to the higher temperature and longer time during calculated as DE (Pappas et al., 2004). Therefore, it could be con-
extraction process (Supplementary Table S1). On the other hand, cluded that both CP and UP were belong to high-methoxyl (HM)
the higher content of total phenolic compounds as shown in Table pectin (DE > 50%) (Thakur et al., 1997). The three absorption peaks
3 also should be considered. between 1010 and 1100 cm1 indicated the samples contained
SEM images with different magnifications (Fig. 2C–F) providing pyranose and furanose; the peaks at 920 cm1 and 827 cm1
visual surface morphology features of dried pectin particles referred to the absorption of D-glucopyranosyl and a-D-mannopy-
showed that ultrasound affected the microstructures of pectin. As ranose, respectively (Zhang, 2007). The absorption at 827 cm1
shown in Fig. 2C, CP had a flat, but rough surface, while UP showed was significantly reduced in UP compared to that in CP, confirming
a smooth, but wrinkled surface (see Fig. 2D). Fig. 2E showed that CP that the former contained lower amount of Man. This result was
had a compact texture, but UP was much loosen (see Fig. 2F). The further testified by the monosaccharides component analysis as
different microstructures signified that ultrasound irradiation shown in Table 3. FTIR analysis showed that different extraction
might disrupt the crosslinks between pectin molecules and reorga- methods had little influence on the chemical structure of pectin.
nized the pectin matrix. Besides, the decreased DE (Table 3) made
UP possessed higher negative charges, which could interfere with 3.5.4. Physico-chemical properties of pectin
the interchain association of pectin chains (Thakur et al., 1997). As mentioned above, different extraction conditions could lead
to the variation in pectin structure and properties. Here intrinsic
viscosity, MV, DE and monosaccharides composition were investi-
3.5.3. Chemical structure analysis by FTIR
gated in order to compare the physico-chemical properties of CP
The infrared spectra of pectin obtained by two different
and UP. As shown in Table 3, the intrinsic viscosity of UP was sig-
extraction methods were illustrated in Fig. 3. For grapefruit peel
nificantly (p < 0.05) decreased than CP, demonstrating that the MV
pectin, the peaks at around 3434, 2930–2935, and 1145 cm1,
of the former (109.54 kDa) was lower than that of the latter
which were the characteristic absorption peaks of polysaccharides,
(132.01 kDa). This result was agreed with our previous conclusion
were attributed to the stretching vibrations of hydroxyl groups
that UAHE had more severe degrading effect than CHE (Xu et al.,
2014). Besides, DE of CP and UP were 67.59% and 65.52%, respec-
Table 3 tively, which were in accordance with the results of FTIR analysis
Physico-chemical properties of pectins extracted by conventional heating method (Fig. 3). Although the DE of UP was slightly lower than that of
(CHE) and ultrasound-assisted heating extraction (UAHE).
CP, the difference was insignificant (p > 0.05).
Physico-chemical properties CPA UPB Table 3 also showed the content of total sugar, GalA and neutral
Intrinsic viscosity (dL/g) 4.10 ± 0.26a 3.26 ± 0.20b sugars in the pectins extracted by CHE and UAHE. It could be seen
MVC (kDa) 132.01 ± 10.98a 109.54 ± 8.00b that the total sugar contents of both pectins were about 78%, of
DED (%) 67.59 ± 1.20a 65.52 ± 2.44a which GalA was the main type of monosaccharide, accounting for
Total sugar content (%, w/w) 77.85 ± 5.33a 78.85 ± 6.54a 55.20 and 50.03 mol% of the total monosaccharides of CP and UP
Total phenolics content (lg GAEE/mg) 7.06 ± 0.20a 4.21 ± 0.11b
Total flavonoids content (lg RE/mg) 2.47 ± 0.27a 1.76 ± 0.38a
respectively. GalA constituted the backbone of homogalacturonan
(HG) and rhamnogalacturonan (RG) regions of pectin. In the case
Monosaccharides composition (mol%)
Man 1.80 ± 0.14a 0.98 ± 0.27b
of neutral sugars, rhamnose (Rha), galactose (Gal) and arabinose
Rha 7.28 ± 1.66a 7.58 ± 1.03a (Ara) were the main components, comprising more than half of
GalA 55.20 ± 0.54a 50.03 ± 1.18b the total neutral sugars. Rha, together with GalA, was the main
Glc 13.99 ± 3.89a 10.82 ± 1.42a structural unit of RG region; while Ara and Gal constituted the side
Gal 13.84 ± 1.18a 14.25 ± 2.47a
chains of RG I. Moreover, xylose (Xyl), the main unit of xylogalac-
Xyl 3.28 ± 0.45a 2.73 ± 0.42a
Ara 4.43 ± 0.44b 12.72 ± 3.21a turonan (XG), and fructose (Fuc), which existed in the side chains
Fuc 0.18 ± 0.08b 0.89 ± 0.30a of RGII, occupied small percentages, indicating that the pectin of
Molar ratios of monosaccharidesF grapefruit peel also contained XG and RG II, with much less fre-
GalA/(Rha + Gal + Xyl + Ara + Fuc)G 1.92 ± 0.23a 1.33 ± 0.23b quency than RG I (Maxwell, Belshaw, Waldron, & Morris, 2012).
Rha/GalAH 0.13 ± 0.03a 0.15 ± 0.02a Besides, mannose (Man) and glucose (Glc) presented in the pec-
(Gal + Ara)/RhaI 2.55 ± 0.36a 3.54 ± 0.27a tins might originate from non-pectic polysaccharides such as the
Values represent means ± standard derivatives of three replicates; values with mannan family of hemicellulose (mannan, glucomannan, galacto-
different small case superscript letters in the same column indicate significant mannan, and galacoglucomannan) and cellulose that were bound
difference (p < 0.05), with the same letters indicate insignificant differences to pectin side chains (Handford et al., 2003; Zykwinska, Ralet,
(p > 0.05).
A Garnier, & Thibault, 2005). UP contained relatively lower amounts
CP: pectin extracted by CHE.
B
UP: pectin extracted by UAHE. of associated Man (0.98 mol%) and Glc (10.82 mol%) than those of
C
MV: Viscosity-average molecular weight. CP (1.80 and 13.99 mol%), verifying that the covalent linkages
D
DE: degree of esterification. between pectin and non-pectic polysaccharides were cleaved by
E
GAE: gallic acid equivalent.
F
ultrasound irradiation. Similar results obtained by Sun, Sun, Sun,
Molar ratios of monosaccharides: exhibiting the primary structural properties
of pectin molecules.
and Su (2004) also showed that ultrasonication could cleave the
G
GalA/(Rha + Gal + Xyl + Ara + Fuc): the linearity of pectin. ether linkages between lignin and hemicelluloses. These results
H
Rha/GalA: the contribution of RG to pectin population. illustrated that ultrasound irradiation could increase the extract-
I
(Gal + Ara)/Rha: the length of side chains attached to RG-I. ability and purity of pectin.
 W. Wang et al. / Food Chemistry 178 (2015) 106–114
Fig. 3. The Fourier Transform Infrared Spectra (FTIR) of pectin extracted by different methods (CP: pectin extracted by conventional heating extraction; UP: pectin extracted
by ultrasound-assisted heating extraction).
3.5.5. Primary structure of pectin molecules
The types of monosaccharides of the pectins extracted by differ-
ent methods did not change, but the ratio among different mono-
saccharides presented some differences. Compared to CP, the mol%
of GalA of UP was significantly (p < 0.05) decreased, and the mol%
of Ara and Fuc increased, while no significant difference occurred
to other neutral sugars (Table 3). Furthermore, as for UP, the ratio
of GalA/(Rha + Gal + Xyl + Ara + Fuc), representing the linearity of
pectin molecules, was significantly (p < 0.05) lower than CP. This
demonstrated that the UP molecules possessed higher percentage
of side chains than CP, which could be explained by the following
two points. On the one hand, the contribution of RG to UP (Rha/
GalA) was higher than CP, indicating that the former had larger
quantities of side chains than the latter. This result indicated that
ultrasound irradiation had more severe degradation effects on
the HG region of the backbone than thermal acid alone. On the
other hand, the length of side chains ((Gal + Ara)/Rha) of UP was
higher than that of CP, which was attributed to that neutral sugar
linkage was more susceptible to hydrolysis at low pH (Garna et al.,
2007). The shorter extraction time and the lower temperature dur-
ing UAHE process protected side chains from degradation.
Many studies have proved that pectin properties could be sig-
nificantly affected by the extraction conditions. The decreased
molecular weight, GalA content, and DE might have negative
effects on the gelling power of pectin, thereby its gelling, stabiliz-
ing and thickening properties were negatively influenced (Belafi-
Bako, Cserjesi, Beszedes, Csanadi, & Hodur, 2012; Koubala, Kansci
et al., 2008; Koubala, Mbome et al., 2008; Masmoudi et al., 2012;
Yapo, Robert, Etienne, Wathelet, & Paquot, 2007). Fortunately,
these changes of properties (not including phenolics and flavo-
noids contents) were appropriately beneficial for the bioactivities
of pectin. Vayssade et al. (2010) showed that okra pectin which
has a structure of almost pure RGI with short galactan side chains
reduced contact and adhesion between cells, increased apoptosis
and decreased cell proliferation in a mouse melanoma cell line.
Maxwell et al. (2012) reviewed small molecular weight pectin frag-
ments that preferentially have RGI regions of arabinogalactan
113
chains and galactan chains can bind to the carbohydrate recogni-
tion domain (CRD) on the pro-metastatic protein galectin-3
(GAL3), in the potential role of preventing and reducing carcino-
genesis. There are two possible reasons to elucidate. On the one
hand, smaller size and lower viscosity made pectin easier to be
absorbed and function in the bloodstream and small intestine; on
the other hand, the better conserved side chains, e.g. arabans,
galactans, and arabinogalactans, could provide more bioactive
regions (Leclere, Cutsem, & Michiels, 2013; Maxwell et al., 2012).
These results indicated that pectin extracted by UAHE underwent
preliminary modification under thermal acid combined ultrasound
irradiation during the extraction process, and it could be further
processed to obtain modified pectin with higher bioactivities.
3.5.6. Prospect of UAHE of pectin in a larger scale
Using ultrasound, full extractions can now be completed in
shorter time with high productivity, giving higher purity of the
final product and consuming only a fraction of the energy normally
needed for CHE. This experiment showed that UAHE process of
pectin and the effects above have been achieved in small scale, in
the potential role of applying in a larger scale. To run out industrial
trials or to scale-up laboratory experiments, REUS (www.etsreus.
com, FRANCE) has developed reactors from 30 to 1000 L. For this
type of apparatus, the extraction solvent and the natural products
are mixed into a container and the ultrasounds are directly applied
to the mixture (Chemat, Zill e, & Khan, 2011). It is of great impor-
tance to promote further studies in reproducibility and bioactivi-
ties of UAHE pectin in order to meet the demand of production
and product quality in industrial application. All in all, these reveal
that there is an opportunity to develop UAHE pectin in a larger
scale even in an industrial reactor.
4. Conclusions
The extracting variables of UAHE were optimized through RSM
and the optimized conditions were power intensity of 12.56 W/
cm2, extraction temperature of 66.71 °C, and sonication time of
114 W. Wang et al. / Food Chemistry 178 (2015) 106–114
27.95 min. A comprehensive comparison carried out between
UAHE and CHE showed that UAHE provided higher pectin yield
with lower temperature and shorter extraction time. Besides,
although the chemical structure was not influenced by different
extraction methods, the color, microstructure, physico-chemical
properties and primary structure of UP were significantly different
from those of CP. The results demonstrated that UAHE was an effi-
cient and economic technique for extracting pectin from plant
resources. Pectin produced by this novel method was preliminarily
modified, and could be further processed to be modified pectin in
order to improve its bioactivities.
Acknowledgements
This work was financially supported by National Natural Sci-
ence Foundation of China (Project 31371872).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
01.080.
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