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Food Hydrocolloids 38 (2014) 129e137

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Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Pectin extracted from apple pomace and citrus peel by subcriticalwater

Xin Wang, Quanru Chen, Xin Lü*

College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi Province 712100, China

a r t i c l e i n f o

Article history:Received 9 October 2013Accepted 2 December 2013

Keywords:PectinSubcritical waterApple pomaceCitrus peel

* Corresponding author. Tel./fax: þ86 29 8709 2195E-mail address: xinlu@nwsuaf.edu.cn (X. Lü).

0268-005X/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2013.12.003

a b s t r a c t

Subcritical water was used to extract pectin from citrus peel and apple pomace, in which the effect ofextraction temperature on properties of the pectins was investigated. The maximum yield of citrus peelpectin (CPP) and apple pomace pectin (APP) were 21.95% and 16.68% respectively. No significant dif-ferences were found in FTIR spectra of CPP and APP. According to DSC analysis, the endothermic propertyof pectin was affected by extraction temperature while the exothermic property of pectin was onlyaffected by its constituents and raw material. The pectin solutions exhibited shear-thinning propertiesand tended to be more elastic (G0 > G00) with frequency increase according to rheological analysis, whichwas also reflected in hydrogel analysis. Moreover, both CPP and APP scavenged more than 60% DPPHradical and 80% ABTS radical in vitro and the highest proliferation inhibition rates of colon cancer cell HT-29 by CPP and APP were 76.45% and 45.23% respectively.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Pectin is a natural high molecular compound widely-existing incell wall andmiddle lamella structure of all higher plants (Qiu, Tian,Qiao, & Deng, 2009). Pectin is usually considered as a complexpolysaccharide which consists of a-1,4-linked D-galacuronic acid,which is partly methyl esterified, and the side chain containsvarious neutral sugars, such as L-rhamnose, L-arabinose, and D-galactose (Mohnen, 2008; Xie, Li, & Guo, 2008). Pectin propertiesinclude gelatification, thickening and stabilization, giving it wide-spread use in food, medical, chemical, textile and other industrialfields (Sato et al., 2011). It is also reported that pectin had severalbiological and physiological functions, such as reduction of serumcholesterol (Brown, Rosner, Willett, & Sacks, 1999), delay of gastricemptying (Schwartz et al., 1988), immune-modulation(Inngjerdingen et al., 2007) and inducing apoptosis of colon can-cer cells (Olano-Martin, Rimbach, Gibson, & Rastall, 2003).

Generally themain feedstocks for commercial pectin productionare apple pomace and citrus peel (Willats, Knox, &Mikkelsen, 2006).China, now is the largest apple and condensed apple juice producerin the world (Qiu et al., 2009) as well as increasing yields of orangeand orange juice (Xie et al., 2008). Thus large amounts of applepomace and citrus peel, as the primary waste product of the juice

.

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manufacturing, are produced annually, which lead to wasting ofresources and create environmental problems. Numerous attemptshave been made to utilize them as a source of dietary fiber (Sudha,Baskaran, & Leelavathi, 2007), animal feed (Joshi & Sandhu, 1996),polyphenol (Li, Smith, & Hossain, 2006), and biofuel (Edwards &Doran-Peterson, 2012). Among them, pectin extraction is thoughtto be the most reasonable way for the apple pomace and citrus peelto be utilized (Shalini & Gupta, 2010). Pectin is industrially producedat acidic conditions with elevated temperature (Koubala et al.,2008). Acidic wastewater and environmental concerns make alter-native extractionmethods including ultrasound (Zhang et al., 2013),enzymatic (Ptichkina, Markina, & Rumyantseva, 2008), microwave(Fishman & Cooke, 2009) and subcritical water (Ueno, Tanaka,Hosino, Sasaki, & Goto, 2008) attractive.

Subcritical water is the water under subcritical temperatures andpressures with dielectric constant and the ion product greatlychanged (Marshall & Franck,1981; Teo, Tan, Yong, Hew, &Ong, 2010),which has proved to be effective for hydrolysis of lignocellulosicmaterial (Heitz et al., 1986) and pectin extraction from citrus peel(Carr, Mammucari, & Foster, 2011; Ueno et al., 2008). However,detailed reporting on the characteristics of pectin extracted bysubcriticalwaterhas not been found,whichwas focused in this study.

In this study, the pectin of apple pomace and citrus peel wasextracted by subcritical water without acid or alkaline addition,after which the physicochemical properties, rheological properties,gel properties and bioactive activities of the pectins wereinvestigated.

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137130

2. Materials and methods

2.1. Materials and reagents

Apple pomace was provided by Shaanxi Haisheng fresh fruitjuice Co. Ltd., China and oven-dried at 105 �C for 24 h. Citrusreticulata was purchased from a local supermarket and the peelwas stripped manually and oven-dried at 105 �C for 24 h. Where-after apple pomace and citrus peel were ground to pass through a100-mesh sieve and stored in desiccators at room temperature forlatter extraction.

Galacturonic acid, arabinose, rhamnase, xylose, galactose,glucose, mannose, DPPH and ABTS�þ were purchased from Sigmachemical Co. (St. Louis, MO, USA). All other chemicals used in thisstudy were analytical grade.

2.2. Extraction of pectin by subcritical water

An autoclave with 500 mL working volume was used for pectinextraction by subcritical water, in which a thermocouple and apressure gage were used to assay the temperature and pressureinside the reactor. The raw material and distilled water were addedinto the reactor with the solid to liquid ratio of 1:30. The extractiontemperature was set at 130 �C, 150 �C, 170 �C for apple pomacepectin extraction and 100 �C, 120 �C, 140 �C for citrus peel pectinextraction according to preliminary experiments. The extractiontime was set for 5 min. All the experiments were performed intriplicate, with the average value reported.

After subcritical water extraction, the water soluble portion wasretrieved by filtration and the filtrate was collected for alcoholprecipitation. The precipitate was washed by 5% (v/v) HCl in 60%isopropyl alcohol and anhydrous ethanol in turn for three times,whereafter it was dried at 105 �C for 24 h. The pectin yield wascalculated according to equation (1).

Pectin Yieldðwt%Þ ¼ PectinðgÞRaw MaterialsðgÞ � 100% (1)

2.3. Chemical composition and molecular weight analysis

The galacturonic acid was determined by modified carbazolemethod (Bitter & Muir, 1962). The degree of methyl esterificationwas determined by the titration of free carboxyl groups before andafter basic hydrolysis (Liu, Cao, Huang, Cai, & Yao, 2010). The proteincontent was determined via the Bradford method (Carlsson, Borde,Wölfel, Åkerman, & Larsson, 2011). Ash content was determined byincinerating dried samples at 575 �C for 8 h in a muffle furnace.

Neutral monosaccharides were released from pectin by acidhydrolysis with trifluoroacetic acid (2 M) at 120 �C for 1.5 h,whereafter trifluoroacetic acid was removed by rotary evaporationat 60 �C. The sodium borohydride was added into the solution atroom temperature for 1.5 h, then acetic anhydride and pyridinewere added to catalyze esterification reaction in boiling water for1.5 h, after which gas chromatography was used to determinealditol-acetate derivatization products of the monosaccharides(Blakeney, Harris, Henry, & Stone, 1983; Masmoudi et al., 2010). Gaschromatography (Shimadzu 2014C) with a high performancecapillary column, DB-17 (30 m � 0.25 mm ID, 0.25 mm film thick-ness, Agilent) was used to determine the neutral monosaccharidesderivatization products.

The molecular weight (Mw) of pectin samples were determinedby gel-permeation chromatography (GPC) as described by the ref-erences (Jia, Zhang, Lan, Yang, & Sun, 2013; Ying, Han, & Li, 2011).Waters HPLC apparatus (Waters Co. Ltd., USA) equipped with three

Ultrahydrogel linear columns (7.8 � 300 mm) in a series and amodel 2414 refractive index detector was used, by which the Mwwas investigated and calculated according to the calibration curve(LgMw ¼ �0.1316x þ 10.94, x means retention time, R2 ¼ 0.9938)obtained by using various standard dextrans.

2.4. Fourier transform infrared spectroscopy

An FT-IR spectrometer (Bruker Vetex70 FTIR instrument, Ger-many) was employed to investigate the characteristic spectra of theextracted pectins. Dried sample (1 mg) and potassium bromide(100 mg) was mixed, ground and pressed into tablets, thereafter itwas scanned within the range of 4000e400 cm�1 (Park, Khan, &Jung, 2006).

2.5. Thermal analysis

Differential scanning calorimetry (DSC Q2000 TA system, USA)was used to investigate the thermal properties of the pectins ac-cording to described method (Sharma & Ahuja, 2011). 5 mg driedand finely ground pectin sample was added into a standardaluminum crucible and immediately sealed. The crucible washeated from 40 �C to 300 �C at a heating rate of 10 �C/min in dy-namic inert nitrogen atmosphere (50 mL/min). Simultaneously, anempty standard aluminum crucible was used as reference.

2.6. Rheological properties

The rheological properties of pectin were determined byrheometer (AR1000, TA instruments, USA) with a 20 mm parallelplate. The solution for rheological tests was prepared by mixingpectin with distilled water (2%, w/w). The sample solutions weresubjected to steady-shearing at 25 �C with the shear rates rangedfrom 0.02-100 s�1. Oscillatory measurements were used to deter-mine the storage modulus (G0) and loss modulus (G00) of pectinsolutions. Strain sweep (0.01e100% at 1 Hz) was applied to test thelinear viscoelastic region of the samples. And the frequencydependence of G0 and G00 was determined by a frequency sweep(0.1e10 Hz at 1% strain) (Zhang et al., 2013).

2.7. Gel properties

Gel preparation and gel properties assay were according todescribed method (Angioloni & Collar, 2009; Piermaria, de la Canal,& Abraham, 2008). Briefly, 0.5 g pectin sample was dissolved in17 mL distilled water, whereafter 35 g sucrose was added into thesolution and the pH was adjusted to 3 by 12.5% citric acid solution.The mixture solution was placed at 4 �C for 24 h. Before texturalanalysis, the prepared pectin gels were placed at room temperaturefor 0.5 h. A Texture Analyser TA.XT Plus (Stable Micro Systems, UK)was used to determine the textural properties of the pectin gel. Thepuncture test was performed using a cylindrical probe (5-mmradius, PC-0.5R). A standard one-cycle program was used tocompress the gels at 0.1 mm/s test speed, 1.0 mm/s pre-test speed,1.0 mm/s post-test speed, and 1 g originating force. The test wasstopped when a 10-mm depth of penetration had been reached.The whole procedure was repeated three times at 20 �C.

2.8. DPPH radical scavenging activity

The antioxidant activity of the pectins were determined basedon scavenging activity of the stable DPPH free radical (Rha et al.,2011). 2 mL pectin solution (0.5, 1.5, 2.5, 3.5, 4.5 mg/mL) wasadded into 100 mM DPPH in ethanol (4 mL). Then the mixture wasplaced in the dark at room temperature for 30 min, whereafter the

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137 131

absorbance of the resulting solution was determined at 517 nm.The DPPH scavenging activity was calculated according to equa-tion (2).

Scavenging activity %ð Þ ¼ B� S� Scð ÞB

� 100% (2)

where B, S, Sc were the absorbance of blank, sample, and samplecontrol respectively.

Determinations were repeated three times for each pectin so-lution. IC50 values was defined as the concentration of pectinsamples that scavenged 50% DPPH free radicals, which was used toevaluate the DPPH radical scavenging activity.

2.9. ABTS�þ radical scavenging activity

The ABTS�þ radical scavenging activity of sample was assayedaccording to described method (Braca et al., 2001). The ABTScation radical was produced by the reaction between 7 mM ABTSin H2O and 2.45 mM potassium persulfate, stored in the absence oflight at room temperature for 12 h. The ABTS solution was thendiluted by ethanol to an absorbance of 0.70 at 734 nm and equil-ibrated at room temperature. 2 mL pectin solutions at differentconcentration (0.5, 1.5, 2.5, 3.5, and 4.5 mg/mL) were added into4 mL ABTS. Then the mixture was placed in the dark at roomtemperature for 6 min, the absorbance of the mixture solution wasdetermined at 734 nm. The ABTS scavenging activity was calcu-lated using the same formula as DPPH radical scavenging activitycalculation. Determinations were repeated three times for eachpectin solution.

2.10. Cell proliferation inhibitory activity measurement of HT-29

The activity of cell proliferation inhibitory was determined byMTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazoliumbromide assay) (Wang et al., 2012). HT-29 (colon cancer cell) cellswere seeded at a density of 1 � 105/well into 96-well cell plateswith a 200 mL culture medium and allowed to adhere for 24 h(37 �C, 5% CO2, which were used throughout the assay), because theretention time of food in human colon will not exceed 24 h. Sub-sequently, the medium was removed and the cells were treatedwith 180 mL of medium containing extracted pectins at variousconcentrations (0.04, 0.2, 1, 5, 10 mg/mL) for 24 h. Each concen-tration of the pectin was repeated in five wells and control sampleswere treated likewise without pectin addition. 20 mL of MTT (5 mg/mL in PBS) was added into each well and the supernatant wascarefully removed after 4 h incubation. The resulting formazanwasdissolved in 200 mL DMSO and measured by absorbance at 490 nmusing a microplate reader (Bio-Rad). Proliferation inhibitory rate bythe pectin was calculated as percentage of cell viability, in whichcontrol sample was considered as 100%. The cell proliferationinhibitory activity was calculated as equation (3).

Cell proliferation inhibitory activityð%Þ ¼ ODc� ODsODc

� 100%

(3)

where ODs and ODc were the absorbance of sample and controlrespectively.

2.11. Statistical analysis

All experiments were carried out in triplicate. Results werestatistically analyzed by ANOVA and followed by Duncan’s multiplerange test. Statistical significancewas set at confidence level of 95%.

3. Results and discussion

3.1. Yield, molecular weight and chemical composition of CPP andAPP

The yield, molecular weight and chemical composition of pectinextracted from citrus peel and apple pomace are shown in Table 1.The highest yield of CPP with 68.88% galacturonic acid was ob-tained at 120 �C, which was close to the glacturonic acid content ofpectin extracted by traditional methods (Lim, Yoo, Ko, & Lee, 2012).The statistical variance analysis revealed that the temperaturesignificantly affected the yield of the pectin as well as the gal-acturonic acid content. For APP, the highest yield of pectin with40.13% galacturonic acid was gained at 150 �C, however, the highestgalacturonic acid of 44.37% was obtained at 130 �C correspondingto 13.33% pectin yield, which possibly because pectinase was usedin concentrated apple juice production and parts of hemicellulosespolysaccharides reacted with pectin thus precipitated together athigher extraction temperature.

The neutral sugars are important for pectin’s physicochemicaland functional properties (Shin & Hwang, 2002). The main neutralsugars of CPP were rhamnose, arabinose and galactose, by which itcould be deduced that the structure of most extracted CPP possiblyconsisted of a rhamnogalacturonan backbone with arabinan and/orarabinogalactan-rich side chain (Oosterveld, Beldman, Schols, &Voragen, 2000). Small amounts of glucose, xylose and mannosewere also detected, which possibly came from hemicelluloses hy-drolysis and reacted or mixed with pectin, thus the total neutralsugar content increased from 5.98% to 10.91% with temperatureincrease. When temperature increased from 100 �C to 140 �C, thevalue of Rha/Gal A increased from 0.8 to 1.2 and the same trend hadbeen found in the value of (Gal þ Ara)/Rha (8.60e14.56), whichindicated that extracted pectin probably contained more side sugarchains as temperature increased (Sengkhamparn, Verhoef, Schols,Sajjaanantakul, & Voragen, 2009).

While for APP, the neutral sugar content was also significantlyaffected by the extraction temperature and the highest neutralsugar content was obtained at 170 �C, at which the content ofxylose, mannose and glucose were significantly higher than otherAPP. Higher glucose content compared with previous study(Masmoudi et al., 2010) was from fragments of the hemicellulosesand amorphous cellulose which were possibly reacted with pectinin subcritical water. On the contrary, the content of glucose in CPPwas similar as the pectin extracted by acidic methods (Lim et al.,2012), which might be caused by the high organic acid content incitrus peel and made the extraction process approached acid-likeprocess. Meanwhile, the galactose of APP was higher than CPP’swhile the arabinose was lower, which indicated that arabinoga-lactan side chains of APP and CPP were different. Since the value ofRha/Gal A (1.5e2.0) and (Gal þ Ara)/Rha (8.75e16.56) of APP wereslightly higher than CPP’s, APP possibly had more proportion ofhairy regions and side chains than CPP.

The DE of the pectins was affected by the extraction tempera-ture and raw materials. When the temperature increased from100 �C to 140 �C, the DE of CPP increased firstly then decreased, inwhich the highest DE of 74.74% was obtained at 120 �C. While forAPP, the DE of APP increased with temperature increase, in whichthe highest DE of 89.69% was obtained at 170 �C. Because the DE ofCPP and APP were more than 50%, they were classified into highester-methyl pectin and thus affected their properties, for example,higher sugar concentration and lower acidic conditionwere neededfor their gel formation (Liu et al., 2010) and theywere possible goodcholesterol-lowering agent (Davidson et al., 1998). The DE of CPPand APP was relatively higher than the commercial pectin (Ismail &Ramli, 2012), especially DE of APP, which possibly because the un-

Table

1Yield,m

olecularweigh

tan

dco

mpositionof

citruspee

lpectinan

dap

ple

pom

acepectinex

tractedby

subc

riticalwater.

Yield

(wt%)

Mw

(Da)

Galacturonic

acid

(wt%)

Deg

reeof

esterification

(%)

Protein(w

t%)

Ash

(wt%)

Neu

tral

suga

rco

mposition(w

t%)

Unkn

own

(wt%)

Rham

nose

Arabinose

Xylose

Man

nose

Gluco

seGalactose

C10

019

.78�

0.34

a63

,355

.00

60.77�

0.93

a71

.88�

0.79

a1.01

�0.08

a3.49

�0.15

a0.50

2.38

0.14

0.24

0.80

1.92

28.66

C12

021

.95�

0.05

b69

,489

.50

68.88�

1.05

b74

.74�

1.57

b2.19

�0.11

b4.30

�0.01

b0.48

3.10

0.12

0.21

0.59

2.52

17.61

C14

019

.21�

0.25

c56

,429

.99

52.33�

0.57

c68

.88�

1.35

c0.36

�0.05

c3.27

�0.03

a0.62

4.44

0.18

0.30

0.78

4.59

33.13

A13

013

.33�

0.30

a65

,721

.10

44.37�

0.23

a83

.41�

1.22

a0.37

�0.01

a2.07

�0.08

a0.67

2.99

1.49

0.62

14.95

4.23

28.24

A15

016

.68�

0.20

b53

,402

.16

40.13�

0.66

b85

.99�

1.37

a0.27

�0.02

b3.12

�0.06

b0.79

2.33

2.64

1.03

25.34

4.58

19.77

A17

010

.05�

0.23

c28

,253

.17

20.67�

0.38

c89

.69�

0.31

b0.24

�0.02

c1.84

�0.08

a0.41

1.39

4.45

1.90

40.94

5.40

22.76

a,b,cValues

within

aco

lumnfollo

wed

bydifferentlettersaresign

ificantlydifferentat

P<

0.05

usingDuncan’smultiple

range

test.

C10

0,C12

0an

dC14

0referto

thecitruspee

lpectinex

tractedat

100

� C,1

20� C

and14

0� C

resp

ective

ly,A

130,

A15

0an

dA17

0referto

thepectinex

tractedat

130

� C,1

50� C

and17

0� C

resp

ective

ly.D

ataareex

pressed

asmea

ns�

stan

darddev

iation

.

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137132

esterified and/or low esterified pectin had been hydrolyzed duringextraction by subcritical water. High methyl-esterified APP and CPPmade them more stable when they were subjected to endo-PG orhigh temperature (Liu et al., 2010), which was useful for food gradeedible film stability.

The protein of CPP increased firstly and then decreased withextraction temperature increase, while the protein of APPdecreased with temperature increase. The increase of proteincontent indicated that the protein was firstly separated and hy-drolyzed from raw material while the degradation of protein wasnot severe at relative lower temperature. The protein content ofCPP and APP were significantly lower than pectin extracted bytraditional method (Masmoudi et al., 2010), which was probablydue to protein degradation caused by subcritical water (Brunner,2009). Although the previous studies found that proteins couldbe linked to pectin or existed in free form (Garna et al., 2007;Kravtchenko, Berth, Voragen, & Pilnik, 1992), the decrease of pro-tein with temperature increase indicated that pectin and proteinspossibly had less interaction between each other in subcriticalwater.

Ash content of CPP and APP increased firstly and decreased thenwith extraction temperature increase, which were lower than thecommercial pectin (Miyamoto & Chang, 1992). Generally inorganicsalt bound to pectin will be separated together with pectin, forinstance calcium, magnesium and potassium (Ueno et al., 2008).Thus the ash content variation correlated with the pectin yield.

The molecular weight (Mw) of CPP ranged from 56 to 69 KDaand from 28 to 65 KDa for APP, inwhich highestMw of CPP and APPwas obtained at 120 �C and 130 �C respectively. The Mw of CPPincreased firstly then decreased then with temperature increasewhile theMw of APP decreased with temperature increase. TheMwof APP and CPP were significantly lower than the pectin extractedby conventional methods but in the general range of 8e1000 KDafor pectins from various fruit sources (Corredig, Kerr, & Wicker,2000), which was possibly due to their hydrolysis in subcriticalwater. Since Mw of pectin significantly affected the pectin proper-ties, such as gelling and rheological properties (Lim et al., 2012), thelower Mw would definitely affect its functional characteristics andwould discussed in later parts.

3.2. Fourier transform infrared spectroscopy

The FT-IR spectra of APP and CPP are shown in Fig. 1, in whichthe characteristic absorption peaks of pectin were found both inAPP spectrum and CPP spectrum though different in the intensity.Compared with the previous study on FT-IR spectra of pectin(Kacurakova, Capek, Sasinkova, Wellner, & Ebringerova, 2000; Liuet al., 2010; Qiao et al., 2009), a number of same absorption peakswere found, briefly, 3600e3000 cm�1 indicated the OeH stretch-ing, 1748 cm�1 indicated ester carbonyl (C]O) and 1636 cm�1

indicated carboxylate ion stretching which could be used for DEquantification of pectic polysaccharides (Gnanasambandam &Proctor, 2000), the intensity and band area of 1748 cm�1 of APPand CPP were coincidence with DE analysis (Table 1). The finger-print region (1300e800 cm�1) can reflect certain variations ofpectin monosaccharides’ composition (Kamnev, Colina, Rodriguez,Ptitchkina, & Ignatov, 1998; Monsoor, Kalapathy, & Proctor, 2001),however, some researchers think that it is difficult to interpretbecause of spectrum overlap (Gnanasambandam & Proctor, 2000;Mccann, Shi, Roberts, & Carpita, 1994), which was also found inour study that it was difficult to assign the peaks of fingerprintregion to corresponding chemical analysis results. However,some typical characteristic peaks of pectin, for example, 882 cm�1

(pyranose ring), 1273 cm�1 (CeO dilatation vibration), 1453 cm�1

(eCH3 antisymmetric deformation or the eCH2e symmetric

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500

C100

C120

C140

A130

A150

A170

Tra

nsm

itta

nce

Tra

nsm

itta

nce

Wavenumbers, cm-1 mc,srebmunevaW -1

Fig. 1. FT-IR spectra of CPP and APP extracted by subcritical water.

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137 133

deformation), were found. Moreover, the weak peaks of amidebands (amide I: 1670 cm�1; amide II: 1588 cm�1) in samplesindicated that small amount of protein existed, which was coinci-dence with chemical analysis (Table 1).

3.3. Thermal properties

The effects of extraction temperature and raw material on thethermodynamic properties of pectin were examined by DSC be-tween 40 �C and 300 �C. As shown in Fig. 2, an endothermic peakand an exothermic peak were observed in the DSC thermograms ofall pectin samples. The parameters of the two peaks were listed inTable 2, such as melting temperature (Tm), melting enthalpy (DHm),degradation temperature (Td) and degradation enthalpy (DHd). Tmvalues ranged from 105.13 �C to 137.18 �C of CPP and 86.46 �Ce136.36 �C of APP, which was coincidence with previous studiesabout pectin and other polysaccharides (Cooke, Gidley, & Hedges,1996; Sharma & Ahuja, 2011). For the CPP, the Tm value of C120was significantly higher than others, which indicated that higherDE, galacturonic acid content and Mw made the pectin moleculartightly adsorb the water (Iijima, Nakamura, Hatakeyama, &Hatakeyama, 2000). However, it was more complex for APP, thehighest Tm and DHmwere found for A150while the DE, galacturonicacid and Mw were not the highest. Because endothermic phe-nomenon is ascribed to water evaporation (Einhorn-Stoll, Kunzek,& Dongowski, 2007), higher melting temperature and meltingenthalpy meant more energy was needed to absolutely removewater, thus C120 and A150 had better capacity to sustain wateraccording to their higher Tm and DHm. The second peak was causedby the degradation of pectin in the heat processing (Godeck,

Fig. 2. DSC thermograms of CPP and APP extracted by subcritical water.

Kunzek, & Kabbert, 2001). As shown in Table 2, although CPP andAPP showed little differences for Td and were stable below 240 �C,the DHd of APP was significantly lower than CPP possibly becauseits lower galacturonic acid content. Accordingly, Td of the pectinswas mainly affected by their constituents while DHd of the pectinswas mainly affected by galacturonic acid content.

3.4. Rheological properties

It is shown in Fig. 3 that both CPP and APP solution show theshear-thinning fluid flow feature since the viscosities decreasewhen the shear rate increase, which is caused by the random coil ofpectin polysaccharides (Gan, Abdul Manaf, & Latiff, 2010; Zhanget al., 2013). For APP, A130 had the highest viscosity followed byA150 and A170 at the beginning of shearing. It is interesting that theviscosities of A130 and A150 rapidly decrease at approximately1 S�1 shear rate, but the viscosity of A170 decrease quickly at thebeginning of shearing. This is possibly because Mw of A130 andA150 were relatively 2-fold of A170 which mean longer chainlength and better coherent network structure, thus the inter- andintra- interaction among the pectin polysaccharides of A130 andA150 are stronger than A170. Therefore, the viscosity of A130 is thehighest followed by A150 and A170. Under the shear force, thelower the Mw is, the quicker the viscosity of pectin decreasebecause their shorter chain and weaker inter- or intra- interaction.Moreover, it is indicated that the pectin extracted at lower extrac-tion temperature had potential to form a solution with higherviscosity. For CPP, C120 had the highest viscosity followed by C100and C140. Similar trend can be found that the viscosity of CPP so-lution decrease with extraction temperature increase, in whichviscosity of C140 decrease quickly at the beginning same as A170.According the above results, it can be deduced that the viscosities ofboth APP and CPP solution are affected byMw and galacturonic acidcontent since highest viscosity corresponded to highest Mw and

Table 2Thermal properties of CPP and APP determined by DSC.

Tm (�C) DHm (J/g) Td (�C) DHd (J/g)

C100 105.13 115.7 243.39 78.79C120 137.18 115.6 240.1 81.79C140 106.94 91.93 237.27 63.81

A130 86.46 87.91 238.72 17.33A150 136.36 136.9 241.74 15.65A170 115.25 103.7 243.86 1.886

Tm, temperature of melting (�C); DHm, melting enthalpy (J/g); Td, temperature ofdegradation (�C); DHd, degradation enthalpy (J/g).

Fig. 3. The flow behavior of CPP and APP solutions.

Table 3Properties of CPP and APP gel.

Hardness (g) Gel strength (g$s) Viscous force (g) Stickiness (g$s)

C100 7.536 524.576 �4.454 �27.467C120 13.025 925.602 �6.456 �45.469C140 3.689 268.238 �2.486 �8.908

A130 7.567 100.34 �4.138 �46.85A150 3.441 59.177 �1.495 �9.056A170 3.137 53.943 �1.123 �4.099

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137134

galacturonic acid content (A130 and C120). Moreover, the anti-shearing capabilities of APP were higher than CPP, which indi-cated that the viscosity of pectin solution was not only affected byMw and galacturonic acid content but other factors, such as the DE,neutral sugars of pectin molecular (Gan, et al., 2010) and theirconformational structure.

Frequency sweeps were performed at 1% strain to determine thefrequency dependence of G0 and G00. It is shown in Fig. 4 that bothAPP and CPP solution exhibited typical behavior of polysaccharidesolution whose loss modulus (G00) was greater than the storagemodulus (G0) at low frequency which indicated that viscous prop-erties was predominant because the dynamic balance betweenpectin molecular entanglement and shearing. When the frequencyincreased, G00 and G0 crossed at certain frequency, which was anindication of viscoelastic behavior of the material and defined thebeginning of the elastic behavior or approaching gel state (Zhanget al., 2013). After the cross point, the G0 became higher than G00

because the inter chain entanglements of pectinmolecular orientedconsistently and might behave like a gel (Peressini, Bravin, Lapasin,Rizzotti, & Sensidoni, 2003; Piermaria et al., 2008). The frequency ofthe cross point decreased with extraction temperature increase forCPP and APP, which indicated that pectin molecules of low mo-lecular weight were easily oriented consistently and showed elasticproperties.

Fig. 4. Frequency sweeps of CPP (A) a

3.5. Gel properties

The properties of pectin hydrogel could be evaluated byhardness, gel strength, viscous force and stickiness (Hurler,Engesland, Poorahmary Kermany, & �Skalko-Basnet, 2012). Gelproperties of APP and CPP are shown in Table 3. For the CPP, thehighest gel hardness, gel strength, viscous force and stickinesswere obtained when the pectin was extracted at 120 �C, at whichthe galacturonic acid, DE and Mw were the highest leading tomore hydrogen bond formation and hydrophobic interactions toform a tight network in gel. For the APP, the highest gel hardness,gel strength, viscous force and stickiness were obtained whenthe pectin was extracted at 130 �C with the highest Mw andgalacturonic acid content. Compared the gel strength with stick-iness of CPP and APP gel, it was indicated that the elasticity ofpectin gel was significantly higher than viscosity, which might bemore suited to use as gels rather than thickeners in food industry(Vriesmann & Petkowicz, 2013). Generally higher DE had positiveeffect on gel properties since it was benefit to multiple hydro-phobic interactions and hydrogen bonds in the junction zones ofthe polymeric network formation (Rascón-Chu et al., 2009),however, it was found that APP gel properties were different (gelstrength for instance), which was a hint that gel network wasnot only affected by DE but also dominantly affected by Mwand galacturonic acid or conformational structure (Fraeye et al.,2010).

3.6. In vitro antioxidant activity

DPPH radical scavenging activity has been widely accepted as atool for estimating the antioxidant activity of substances in a modelsystem. The DPPH radical scavenging activities of CPP and APPincreased in a dose-dependent manner as shown in Fig. 5(A). Whenthe concentrationwas higher than 4.5mg/mL, the DPPH scavengingactivity of all pectin samples were more than 60% and the IC50 ofCPP and APP were significantly lower than the previous report

nd APP (B) solution at 1% strain.

Fig. 5. In vitro anti-radical activities of CPP and APP. (A) DPPH radical scavenging assay.(B) ABTS radical scavenging assay.

Fig. 6. Effects of CPP and APP on HT-29 cell proliferation. Columns refer to mean valueof five parallel experiments; bars refer to standard deviation.

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137 135

(Dalonso & Petkowicz, 2012), which indicated that CPP and APPextracted by subcritical water had higher in-vitro antioxidant ac-tivities. It was interesting that the IC50 of APP and CPP increasedslightly as the extraction temperature increase, which was possiblydue to hydrolysis of pectin reduced proton donation from hydroxyland uronyl group of the monosaccharide units, carboxyl group ofgalacturonic acid units and acetyl groups (Nara, Yamaguchi, Maeda,& Koga, 2009). Compared the result of CPP with the APP, it wasindicated that the DPPH scavenging activity of CPP was slightlystronger than APP.

In order to further verify the antioxidant activities of the APPand CPP, the ABTS radical scavenging activities of APP and CPPwere also investigated. The results of ABTS radical scavengingactivity were consistent with the DPPH test as shown in theFig. 5(B). Both CPP and APP showed higher antioxidant activitiesin a dose-dependent manner as it was in DPPH assay. The IC50

values of CPP and APP varied slightly and the highest radicalscavenging activity was 99.65% of A130 and 98.49% of C100respectively, which indicated that APP and CPP extracted bysubcritical water had higher activity to scavenge radical, such asDPPH and ABTS.

3.7. Inhibitory effect of pectin on HT-29 cell proliferation

It is shown in Fig. 6 that both APP and CPP significantly inhibitedHT-29 cells proliferation especially at high concentration. Generallythe proliferation inhibition rate of APP and CPP increased in a dose-dependent manner and inhibitory activities of CPP were higherthan APP at same concentration. For the CPP, the highest prolifer-ation inhibition rate of 76.45% appeared at 10 mg/mL of C100.Furthermore, the dose required for 50% growth inhibition (IC50values) of CPP increased with extraction temperature increase,which indicated that the antitumor activity against the HT-29 cellsof CPP decreased as the extraction temperature increase. For APP,the inhibition rate of APP was lower than CPP and the IC50 values ofAPP were higher than 10 mg/mL since the highest inhibition rate ofA150 at 10 mg/mL was 45.23% which was lower than 50%. It hasbeen reported that pectin inhibited cell proliferation and inducedapoptosis in several cancer cell lines (Olano-Martin et al., 2003; Ye,Wang, Zhou, Liu, & Zeng, 2008), inwhich the temperature-modifiedginseng pectin had dramatically increased anti-proliferative effectand induced apoptosis accompanied by the activation of caspase-3(Cheng et al., 2011). Similarly, during the extraction of pectin fromapple pomace and citrus peel by subcritical water, the reaction ormolecular rearrange of pectin and/or hemicellulose would occurdue to the hydrolysis and reaction in subcritical water, thus higheranti-proliferation activity was obtained.

4. Conclusions

Pectin of apple pomace and citrus peel was extracted bysubcritical water and the properties of APP and CPP were signifi-cantly affected by extraction temperature and raw material.Further, the results of MTT and radical scavenging assay showedthat these extracted pectin had anti-oxidative activities and anti-tumor activity against the HT-29. However, further investigationsare needed to explore conformational information of the extractedAPP and CPP as well as mechanism of the antioxidant and anti-tumor bioactivities.

Acknowledgments

The authors thank Science Foundation of Shaanxi Province,China (Grant No. 2010JQ3012) and Agricultural Scientific and

X. Wang et al. / Food Hydrocolloids 38 (2014) 129e137136

Technical Innovation Project of Shaanxi Province, China (Grant No.2010NKC-11), The Scientific Research Foundation for the ReturnedOverseas Chinese Scholars, State Education Ministry (Grant No. 41)for their financial support.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.foodhyd.2013.12.003.

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