Bioresource Technology 102 (2011) 38553860

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Environmentally friendly preparation of pectins from agricultural byproducts

and their structural/rheological characterization
Bockki Min a, Jongbin Lim a, Sanghoon Ko a, Kwang-Geun Lee b, Sung Ho Lee c, Suyong Lee a,
a
Department of Food Science and Technology, Carbohydrate Bioproduct Research Center, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, Republic of Korea
b
Department of Food Science and Biotechnology, Dongguk University-Seoul, 3Ga 26 Pil-dong, Jung-gu, Seoul 100-715, Republic of Korea
c
Genencor, A Danisco Division 2600 Kennedy Dr. Beloit, WI 53511, USA

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

Article history: Apple pomace which is the main waste of fruit juice industry was utilized to extract pectins in an envi-
Received 28 September 2010 ronmentally friendly way, which was then compared with chemically-extracted pectins. The water-based
Received in revised form 2 December 2010 extraction with combined physical and enzymatic treatments produced pectins with 693.2 mg g1
Accepted 2 December 2010
galacturonic acid and 4.6% yield, which were less than those of chemically-extracted pectins. Chemi-
Available online 7 December 2010
cally-extracted pectins exhibited lower degree of esterication (58%) than the pectin samples obtained
by physical/enzymatic treatments (69%), which were also conrmed by FT-IR analysis. When subjected
Keywords:
to steady-shear rheological conditions, both pectin solutions were shown to have shear-thinning proper-
Pectin
Environmentally friendly
ties. However, decreased viscosity was observed in the pectins extracted by combined physical/enzy-
Degree of esterication matic methods which could be mainly attributed to the presence of more methyl esters, thus limiting
Agricultural by products polymer chain interactions. Moreover, the pectins which were extracted by combined physical/enzy-
Rheology matic treatments, showed less elastic properties under high shear rate conditions, compared to the
chemically-extracted pectins.
2010 Elsevier Ltd. All rights reserved.

1. Introduction well-being trend, serious consumer concerns about chemical addi-

Fruit processing in the food industry gives rise to large amounts
of by-products. Specially, since the by-products of apple and citrus
fruits amount to up to 50% and 2535% of the processed fruits,
their annual wastes are estimated to be 34.2  106 and
15.6  106 Mton, respectively (Oreopoulou and Russ, 2007). There-
fore, there have been great interests in searching for proper dis-
posal methods of apple pomace and citrus peels which are the
residue left after juice extraction. Specically, a main focus has
been placed in the utilization of their certain components pectin
as a soluble dietary ber.
A number of methods have been tried to extract pectins from
various sources so far. Pectins are industrially obtained from apple
pomace and citrus peels in a chemical way with strong acids such
as oxalic (Koubala et al., 2008), hydrochloric (Choi, 1996; Hwang
et al., 1998), nitric (Constenla et al., 2002), and sulphuric acids
(Garna et al., 2007) which are regarded as conventional acid
extraction (Yapo, 2009). Even though these chemical procedures
have advantages from an efcient and economical point of view,
they may cause environmental problems by producing hazardous
contaminants that must be treated. Moreover, with the recent
Corresponding author. Tel.: +82 2 3408 3227; fax: +82 2 3408 4319.
E-mail address: suyonglee@sejong.ac.kr (S. Lee).
tives become so dominant that the growth rate of natural products
in the food industry has begun to rise (Sloan, 2010). Therefore,
new green efforts have been made in order to minimize the use
of harmful chemicals in the food industry and the processing for
pectin isolations is no exception, thereby introducing environ-
ment- and human-friendly technology. For examples, enzymatic
extraction has been conducted with polygalacturonase (Contreras-
Esquivel et al., 2006), (hemi)cellulase (Shkodina et al., 1998;
Zykwinska et al., 2008), protease (Zykwinska et al., 2008), and
microbial mixed enzymes (Ptichkina et al., 2008). Also, ultra-
sonic-(Panchev et al., 1988), autoclave-(Oosterveld et al., 1996,
2000), microwave-(Liu et al., 2006), and extrusion-assisted (Shin
et al., 2005) treatments have applied to extract pectins from agri-
cultural byproducts such as apple pomace, sugar beet pulp, and or-
ange peels. However, their main research focuses were placed on
the yield and/or chemical compositions of pectins extracted by dif-
ferent methods. Even, the combined effects of physical and enzy-
matic treatments for pectin extraction have not been reported
yet to our best knowledge.
In this study, pectin extraction from apple pomace has been
studied in an environmentally friendly way with combined physi-
cal and enzymatic treatments. Then, their chemical, structural, and
rheological properties were characterized and compared with
those of chemically-extracted pectins.

0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.12.019
3856 B. Min et al. / Bioresource Technology 102 (2011) 38553860
2. Methods
2.1. Materials
Apple pomace obtained from the First Fruits Co. (Seoul, Korea)
was used as a source of pectin. After the apple pomace was
oven-dried at 80 C, it was ground and screened with a 50 mesh
sieve. The apple pomace powder (7.19% moisture, 2.03% ash,
3.95% protein, 4.30% fat and 82.54% carbohydrate (by difference))
was stored in a plastic bag prior to pectin extractions. All the chem-
icals and reagents were of analytical grade.
2.2. Pectin extraction
Pectins were extracted from the apple pomace powder in two
different ways (chemical and combined physical/enzymatic meth-
ods). In the conventional chemical method (Koubala et al., 2008;
Rha et al., 2011), apple pomace powder was treated four times
with ethanol (85%) for 70 C for 20 min and ltered with miracloth
(Merck KGaA, Darmstadt, Germany). The residue (10 g) was then
mixed with oxalic acid/ammonium oxalate (0.25%, pH 4.6,
400 mL), which was maintained at 85 C for 1 h. The mixture was
ltered with miracloth and the ltrate was mixed with three vol-
umes of ethanol (96%). After centrifugation at 14,500  g for
10 min, the precipitates were washed with 70% and then 96% eth-
anol, followed by oven-drying (50 C).
On the other hand, another way to extract apple pomace pectins
was based on combined physical and enzymatic treatments with
distilled water (Min et al., 2010, 2009) (Fig. 1). The apple pomace
Fig. 1. Schematic of pectin extraction from apple pomace in (a) chemical and (b) environmentally friendly ways.
powder was suspended in distilled water at a concentration of
10% (w/v) and mixed with agitation for 1 h at room temperature.
After the suspension was ltered with miracloth, the residue was
mixed with distilled water (10%, w/v), homogenized for 3 min,
and then autoclaved at 121 C for 10 min. After cooling at room
temperature, it was followed by an enzymatic treatment with
Viscozyme L (Novozymes, Bagsvaerd, Denmark). The viscozyme
with 1.2  104 fungal b-glucanase unit was added to the suspen-
sion, which was maintained at 40 C and boiled to stop the enzy-
matic reaction after 1 h. Then, the reaction mixture was dialyzed
(8000 Molecular weight cutoff, Spectrum Laboratories Inc., CA,
USA) against distilled water for 24 h. After freeze-drying, the sam-
ples were stored in a plastic bag before further experimental
analysis.
2.3. Analysis of degree of esterication
The degree of esterication (%) was calculated from the molar
ratio of methanol to galacturonic acid (Klavons and Bennett,
1986; Smout et al., 2005). For methanol analysis, pectins were dis-
solved in distilled water (0.1%, w/v) and the total volume was ad-
justed to 50 mL by adding potassium hydroxide (25 mL, 1.0 N)
before they were incubated at ambient temperature for 30 min.
After the pH was adjusted to 7.5 with phosphoric acid, the solution
was diluted with distilled water. Alcohol oxidase (1 unit, Sigma
Aldrich Inc., MO, USA) was added to 1 mL of the diluted solution
and incubated at 25 C for 15 min. Then, 2,4-pentanedione in
2.0 M ammonium acetate and 0.05 M acetic acid (2 mL) was added,
 B. Min et al. / Bioresource Technology 102 (2011) 38553860 3857

followed by incubation at 60 C for 15 min. After cooling down to
room temperature, the absorbance was measured at 412 nm.
The content of galacturonic acid was spectrophotometically
determined by the sulfamate/m-hydroxydiphenyl method (Filisetti-
Cozzi and Carpita, 1991; Yoo et al., 2003). 0.4 mL of pectin solutions
in distilled water (0.1%, w/v) was mixed with 4 M sulfamic acid
potassium sulfamate (pH 1.6, 40 lL) and agitated with a vortex
mixer. After H2SO4 containing 75 mM sodium tetraborate
(2.5 mL) was added, the mixture was agitated, cooled in an ice
bath, and brought to a boil for 15 min. After cooling in an ice bath,
meta-hydroxydiphenol reagent (80 lL) was added and after 5 min,
the absorbance was read at 520 nm with D-galacturonic acid as
standard.
2.4. Analysis of monosaccharide composition
The compositions of monosaccharides in extracted pectin sam-
ples were analyzed, according to the previous methods (Rascn-
Chu et al., 2009; Rha et al., 2011). Pectins (2 mg) were hydrolyzed
with 2 M triuoroacetic acid (0.4 mL) at 120 C for 4 h and the mix-
ture was evaporated at 40 C. Then, the hydrolysates were dis-
solved in distilled water (0.05%), ltered through 0.2 lm lter
(Toyo Roshi Kaisha, Ltd., Tokyo, JAPAN) and loaded into the
HPAEC-PAD system (BioLC, Dionex Corporation, CA, USA) with a
CarboPac PA1 column (4  250 mm, Dionex Corporation, CA,
USA). The eluent and ow rate were 18 mM NaOH and 1 mL min1,
respectively.
2.5. FT-IR analysis
Fourier transform infrared (FT-IR) spectra were obtained by a
FT-IR spectrometer (Nicolet Instrument Co., Madison, WI) in order
to investigate the structure of pectins extracted by two different
methods. Samples were incorporated with KBr and pressed into a
thin pellet which was used for FT-IR analysis.
2.6. Molecular weight measurement
The molecular weights of extracted pectins were determined by
using high performance size exclusion chromatography connected
with a refractive index detector (Agilent 1100 series, CA, USA). Pec-
tins (1 mg) was dissolved in 50 mM sodium nitrate (1 mL) and l-
tered through 5 lm lter. Then, the samples (0.2 lL) were injected
into columns in series (BioSep-SEC-S2000 and S4000, Phenomenex
Inc., CA, USA), which have exclusion limits of 3  105 and
1.5  106 Da, respectively. Sodium nitrate (50 mM) was eluted at
50 C with a ow rate of 1 mL min1.
SAS system (SAS Institute Inc., Cary, NC, USA), followed by Dun-
cans multiple range test for mean comparison.
3. Results and discussion
Fig 1 presents the experimental procedures for extracting pec-
tins from apple pomace by chemical and combined physical/enzy-
matic treatments. In the conventional chemical method (Fig. 1(a)),
pectins were extracted by using chemicals such as oxalic acid/
ammonium oxalate and ethanol, which needs to be removed. How-
ever, as shown in Fig. 1(b), apple pomace was subjected to thermo-
mechanical conditions, followed by cellulolytic enzyme treatment
during which distilled water was used as extraction solvent with-
out any assistance of chemical acids. Thus, the lack of chemicals
during pectin extraction may allow the food industry to move to-
ward environmentally friendly technology and sustainable
production.
The contents of galacturonic acid and neutral sugars of the pec-
tin samples extracted from apple pomace are presented in Table 1.
The chemically-extracted pectins showed a galacturonic acid con-
tent of 853.5 (mg g1 dry weight) while 693.2 mg g1 was included
in the samples extracted by combined physical/enzymatic treat-
ments. Thus, compared to the chemical extraction method, the
physical/enzymatic treatments produced pectins with lower
amount of galaturonic acid. It seemed that the thermo-mechanical
treatment and enzymatic hydrolysis allowed non-pectinuous poly-
saccharides to be solubilized, giving lower values of galacturonic
acid contents. As also can be seen in Table 1, arabinose was the
main neutral sugar and residues of rhamnose, galactose, glucose,
and xylose were found in both samples. Thus, the extracted sam-
ples contained pectic components with typical side chain sugar
residues (Fennema, 1996). It was interesting to note that less sugar
content was included in chemically-extracted pectins. It would be
possibly due to degradation of neutral sugar linkages which are
vulnerable to hydrolysis under acidic conditions (Garna et al.,
2007).
The degree of esterication was estimated to be 58% and 69% for
pectins extracted by chemical and physical/enzymatic treatments,
respectively (Table 2). Thus, it suggested that they belong to high
methoxyl pectins regardless of extraction methods. However,
lower degree of esterication was observed in the chemically-
extracted pectins. The use of the same chemical extraction to this
Table 1
Neutral sugar composition and galacturonic acid content of apple pomace pectins
which were extracted by chemical and physical/enzymatic treatments.

Neutral sugar/galacturonic acid Chemical Physical/enzymatic

2.7. Rheological measurements content (mg g1) extraction extraction
Galacturonic acid 853.5 693.2
A controlled-stress rheometer (AR1500ex, TA Instruments, USA) Rhamnose 9.6 15.47
Arabinose 37.3 78.04
with a 40 mm parallel plate was used to characterize the rheolog-
Galactose 23.3 26.2
ical properties of pectin solutions at different concentrations Glucose 16.9 14.8
(5, 6, 7 and 8%, w/w). For steady-shear measurements, shear rates Xylose 7.3 13.2
ranging from 1 to 500 s1 were used and the resulting stress was
recorded. Also, the samples were subjected to dynamic oscillatory
deformation at a frequency range from 0.01 to 10 Hz at a strain of Table 2
0.1% which was within the linear viscoelastic limit. All rheological Yield, degree of esterication, and molecular weight of apple pomace pectins which
measurements were carried out at 25 C and the reported curves were extracted by chemical and physical/enzymatic treatments.

were mean values of three measurements. Characteristics Chemical Physical/enzymatic

extraction extraction

2.8. Statistical analysis Degree of esterication 58 69

(%)
Yield (%) 7.7 4.6
All experiments were carried out at least in triplicate. Results
Molecular weight (kDa) 233.4 231.7
were statistically analyzed by analysis of variance (ANOVA) with
3858 B. Min et al. / Bioresource Technology 102 (2011) 38553860

study yielded pectins with around 58% degree of esterication
from ambarella and lime peels (Koubala et al., 2008), which could
be favorably compared to our results.
The structures of pectins extracted by two different methods
were compared by using FT-IR analysis (data not shown). For both
samples, a broad and intense band at 3333 cm1 was observed,
which would be related to hydroxyl groups (OH). Also OCH3 bond
stretching from methyl esters of galaturonic acids produced
absorption bands at 2931 cm1. Two absorption peaks appeared
at 1620 cm1 and 1740 cm1, which corresponded to free carboxyl
and ester carbonyl groups, respectively (Gnanasambandam and
Proctor, 2000; Yang and Yen, 2002). It was interesting to note that
the peak intensity ratio of free carboxyl to ester carbonyl groups
was higher in the chemically-extracted pectins. Thereby, the FT-
IR results conrmed the difference in the degree of esterication
between two pectin samples as already mentioned in Table 2.
Table 2 also presented that the yield of chemically-extracted
pectins was 7.7% while 4.6% yield was observed in the pectins ob-
tained by physical/enzymatic treatments. Thus, the chemical
method seemed to be effective in extracting pectins from apple
pomace in terms of yield. Yield of pectins from apple pomace var-
ied depending on extraction conditions. When pectin extractions
from apple pomace were carried out under acidic conditions, the
yields were ranged from 2.9 to 16% (Garna et al., 2007; Rascn-
Chu et al., 2009). Also, the acidic extraction with assistance of
extrusion produced pectins of which yield was around 510%
(Hwang et al., 1998). Fissore et al. (2009) applied commercial cell
wall hydrolytic enzymes to obtain pectins, showing 11.7% yield.
A preceding study (Koubala et al., 2008) demonstrated that the
same chemical method with oxalic acid/ammonium oxalate, gave
rise to greater yields of pectin, compared to water-based extrac-
tion. Thus, hot acid extraction methods are reported to produce
high yield of pectins (Garna et al., 2007).
The molecular weight of physically/enzymatically-extracted
pectin was investigated and compared with that of chemically-
extracted pectin (Table 2). The pectins obtained by physical/enzy-
matic treatments exhibited a molecular weight of 231 kDa which
was slightly lower than that of chemically-treated pectins
(233 kDa). The chains of pectin molecules might be slightly de-
graded during thermo-physical treatments, consequently reducing
their molecular weight. Also, the hydrolytic activity of the cell wall
degrading enzyme used in this study might contribute partly to the
molecular weight decrease.
10
Apparent Viscosity (Pa.s)
Fig 2 exhibits the ow behaviors of both pectins extracted by
chemical and physical/enzymatic treatments at different concen-
trations (5, 6, 7 and 8%, w/w). As expected, an increase in their con-
centrations caused a signicant viscosity increase. It was also
interesting to note that they exhibited Newtonian plateau at low
shear rates while the viscosity decrease was observed with
increasing shear rates. Moreover, these shear-thinning features be-
came more pronounced with increasing concentrations. When the
ow behaviors at the same concentrations were compared, a dra-
matic difference in the apparent viscosity was observed between
two extraction methods. The solutions of chemically-extracted
pectins showed signicantly higher viscosity than those obtained
by physical/enzymatic methods. Even, the chemical extraction
method with oxalic acid/ammonium oxlate produced pectins with
higher viscosity by a half order of magnitude at a concentration of
8%.
As previously mentioned in Table 2, higher degree of esterica-
tion was observed in the pectins which were physically/enzymati-
cally extracted from apple pomace, indicating that there were more
carboxyl groups in the methyl ester form (COOCH3). The presence
of methyl esters does not allow pectin chains to be readily associ-
ated, consequently limiting the extent of chain interactions. Con-
comitantly, the formation of junction zones is retarded and less
resistance to shear ow exists. It would be why pectins obtained
by physical/enzymatic treatments exhibited lower viscosity than
chemically-extracted pectins at the same concentrations. These re-
sults were in good agreement with previous studies that reported a
decrease in the hardness of pectins gels with increasing degree of
esterication (Kim et al., 2008). In addition, since viscosity is a func-
tion of molecular weight, the viscosity variations between two pec-
tin samples could be partly attributed to their molecular weights.
Moreover, these ow behaviors were characterized by tting the
curves into the Williamson model given by:
g0
g
1 kc_ n
where g is apparent viscosity, g0 is zero-shear rate viscosity, k is a
time constant, and n is dimensionless power-law index for the
Williamson equation. The Williamson model parameters tted from
the data in Fig. 2 are listed in Table 3. For both samples, zero-shear
rate viscosity increased with increasing concentrations. Moreover,
increases in k which is equal to the reciprocal of shear rate at
g g20 , were observed at higher concentrations. It indicated that
the transition from Newtonian to shear-thinning behaviors was
shifted to lower shear rates due to less movement of pectin chains
with enhancing concentrations. The positive values of n which is
less than unity conrmed the shear-thinning properties of all pectin

solutions. In addition, high values of n suggested that more pro-

nounced shear-thinning behavior was observed in the chemically-
extracted pectins at the same concentration.
1
Table 3
Magnitude of the Willamson-model parameters of pectins from apple pomace which
were extracted by chemical and physical/enzymatic treatments (means with different
letters in the same column differ signicantly at p < 0.05).

Extraction method Concentration g0 k n R2

(%)
0.1
1 10 100 Chemical 5 0.638e 1.76 E03e 0.906a 0.99
extraction 6 1.147c 3.09 E03d 0.783b 1
Shear rate (1/s) 7 3.158b 7.27 E03b 0.761c 1
8 4.480a 1.16 E02a 0.692d 1
Phy/Enz-5% Phy/Enz-6% Phy/Enz-7% Phy/Enz-8%
Physical/enzymatic 5 0.135h 4.63 E04f 0.741c 0.98
Chemical-5% Chemical-6% Chemical-7% Chemical-8% extraction 6 0.242g 4.96 E04f 0.555e 0.99
7 0.450f 8.96 E04f 0.486f 1
Fig. 2. Flow behaviors of apple pomace pectins which were extracted by chemical 8 0.968d 3.68 E03c 0.414g 0.99
and physical/enzymatic treatments.
 B. Min et al. / Bioresource Technology 102 (2011) 38553860
The dynamic viscoelastic properties of pectins at different con-
centrations were characterized as a function of frequency (Fig. 3).
For all the samples, the storage (G0 ) and loss (G00 ) moduli increased
with increasing frequency with higher frequency dependence of
the G0 . Both values of G0 and G00 were also greater in the chemi-
cally-extracted pectin samples than those obtained by physical/
enzymatic treatments at the same concentration. Thus, more poly-
mer chain interactions in the chemically-extracted pectins with
low degree of esterication appeared to cause more increases in
their viscoelastic properties. Tand (G00 /G0 ) indicates the relative
contribution of elastic and viscous components to the rheological
a way. Since environmentally friendly processing without any use
b pectin. LWT-Food Sci. Technol. 35, 216221.
Fig. 3. Dynamic viscoelastic properties of pectins from apple pomace which were
extracted by chemical and physical/enzymatic treatments ((a) G0 and G00 , (b) tan).
3859
properties of a material. Thus, more contribution of increasing con-
centrations to elastic properties could be conrmed by the reduc-
tion of tand as seen in Fig. 3(b). Also, the values of tand for both
pectin samples showed close overlap at lower frequency while
they were not superimposed at high frequency. Even, the deviation
of the superposition took place at lower frequency with increasing
concentrations. Through a frequency sweep test, a rheological
properties of a material can be observed at short times (high fre-
quency) and long times (low frequency) which correspond to high
and low shear rates, respectively (Wrolstad et al., 2005). Therefore,
the pectins chemically obtained seemed to exhibit more elastic
properties with increasing frequency (that is, under high shear rate
conditions). It would be possibly explained by stronger network
formation of the pectins which were produced in a chemical
of chemicals can positively inuence the consumer preferences, re-
search should be further focused on fundamental interactions be-
tween environmentally friendly pectins and other food
ingredients (such as starch and proteins) for extending its practical
use in processed foods.
4. Conclusions
Combined physical and enzymatic approaches were applied to
extract pectins from apple pomace in an environmentally friendly
way. The physical/enzymatic treatments produced high methoxyl
pectins with 69% degree of esterication of which structural and
rheological properties were distinctly compared with those of
chemically-extracted pectins. Thereby, it appears that higher
amounts of green-labeled pectins are incorporated to food formu-
lations by controlling the rheology and texture of foods as much as
chemically-extracted pectins. It can consequently give more poten-
tial chances to provide deriving health benets from pectins with-
out any signicant quality changes of food products.
Acknowledgements
This study was supported by Technology Development Program
for Agriculture and Forestry, Ministry for Food, Agriculture,
Forestry and Fisheries (No. 20083061), Republic of Korea.
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