Article pubs.acs.org/JAFC

Characteristics of Two Calcium Pectinates Prepared from Citrus Pectin Using Either Calcium Chloride or Calcium Hydroxide Xiujun Guo,† Hanying Duan,† Chao Wang, and Xuesong Huang* Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China ABSTRACT: Calcium pectinate (CaP) was prepared from citrus pectin using either calcium chloride (C-CaP) or calcium hydroxide (HO-CaP) as the source of calcium for the reaction. The production yields and the rates of decalcification for the two calcium pectinates were compared and both found to be lower for C-CaP than for HO-CaP. In an attempt to explain these differences, certain chemical and structural characteristics of the two products, including functional groups (−CH3, CO, COO−), rheological properties, morphology, and egg-box junction zones, were investigated by Fourier transformation infrared (FTIR) spectroscopy, rheology, scanning electron microscopy (SEM), and X-ray diffraction (XRD). The results from FTIR showed that, with an increase in calcium content, the wavenumber values and peak areas of FTIR for −CH3, CO, and COO− groups all changed dramatically for C-CaP, while they were virtually unchanged for HO-CaP. Rheological analysis of the CaP gel showed that C-CaP had a stronger cross-linked network structure and a greater range of elastic behavior as compared to HOCaP. SEM images of two CaP gels showed irregular membranes. C-CaP maintained a tight structure and a smooth surface, whereas HO-CaP was loose and rough. The results from XRD revealed a higher degree of crystallinity within C-CaP than within HO-CaP, which indicated that C-CaP possessed compact, ordered, and stable egg-box junction zones while the junction zones in HO-CaP were metastable and loose. KEYWORDS: calcium pectinate, pectin, structure, egg-box junction zone



hydroxide (HO-CaP) as the source of calcium.4 Our earlier work found that HO-CaP had a higher yield and decalcification rate than did C-CaP; however, possible chemical characteristics to explain these findings were not investigated. Accordingly, the purpose of the work reported here was to evaluate the differences between HO-CaP and C-CaP in terms of functional group structure, rheological properties, morphological structure, and egg-box junction zones, with the analytical techniques utilized being Fourier transformation infrared (FTIR) spectroscopy, rheology, scanning electron microscopy (SEM), and X-ray diffractometry (XRD). These results ultimately may lead to a better understanding of the theoretical basis for the production and future application of CaP in related fields.

INTRODUCTION Pectin is a structural heteropolysaccharide consisting of Dgalacturonic acid units joined together by α-1,4-glycosidic linkages, with a few neutral sugars.1 The polygalacturonic acid is partly esterified with methyl groups, and the free acid groups may be partly or fully neutralized with cations, such as Ca2+, Al3+, Na+, and K+.2 Neutralization with Ca2+ leads to the formation of calcium pectinate (CaP). Previous research indicates that ionic bridges between Ca2+ and pectin carboxyl groups lead to the arrangement of chains and an ordered meshlike formation known as stable egg-box structures.3−5 CaP is widely used in the food and pharmaceutical industries due to its good biocompatibility and stable chemical properties. In 1977, pectin and pectate were listed as “generally recognized as safe” (GRAS) substances by the Food and Drug Administration.6 CaP has been widely investigated as a carrier of colon-targeted drugs due to its unique properties.7−9 CaP is also used in encapsulation of bioactive compounds. Early studies found that the stability of anthocyanin was greatly improved in the acidic gastric environment upon entrapment within CaP.10 CaP can as well form a barrier to control the diffusion of moisture in fried food.11 In addition, CaP is an intermediate product that is obtained during pectin extraction from citrus peel and apple pomace using the “salting out” method. This may provide a means for the production of pectin as a food additive after a further decalcification process.12,13 At present CaP is usually prepared by combining calcium chloride with pectin.7−10,13,14 This type of CaP is designed as C-CaP in the present study. The preparation conditions, functional groups, rheology, and morphology of C-CaP have been studied extensively.14−17 However, few studies have focused on the preparation of calcium pectinate using calcium © 2014 American Chemical Society



MATERIALS AND METHODS

Pectin Preparation. Crude pectin was extracted by an acid hydrolysis−ethanol precipitation process from citrus peel.18 Pectin was subsequently purified by precipitation and washed with ethanol three times. The purified pectin (DE = 68%, by the titrimetric method19) was subsequently lyophilized to dryness, and the dried samples were ground into a fine powder. Preparation of CaP. Pectin powder was suspended in deionized water (1%, w/v) and the suspension stirred for 3 h until the pectin powder was completely dissolved.16 Before addition of calcium reagents, the pH of the pectin solution was adjusted to 9.75 for the preparation of C-CaP,14 but not for the preparation of HO-CaP. Calcium reagents were subsequently added to the pectin solution to reach final concentrations of 10, 12, 14, 16, and 18 g of CaCl2/100 g of Received: Revised: Accepted: Published: 6354

February 12, 2014 June 10, 2014 June 10, 2014 June 10, 2014 dx.doi.org/10.1021/jf5004545 | J. Agric. Food Chem. 2014, 62, 6354−6361

Journal of Agricultural and Food Chemistry

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Table 1. Effect of the Calcium Concentration and Source on the Gelation Characteristics and CaP Yield CaP type C-CaP

HO-CaP

concn (g/100 g of pectin) abbrev CaCl2 10 CaCl2 12 CaCl2 14 CaCl2 16 CaCl2 18 Ca(OH)2 10.8 Ca(OH)2 Ca(OH)2 Ca(OH)2 Ca(OH)2

11.6 12.5 13.3 14.1

CaP yield (mean ± SD) (%)

gelling behavior The mixture became viscous flowable and did not gel at all after 0.5 h of standing. The mixture became viscous and initiated a certain gel formation after 0.5 h of standing A gel formed immediately as CaCl2 solution was added; however, it was not strong enough for a real gelation. A compact gel was formed as CaCl2 solution was added. A compact gel was formed before all of the CaCl2 solution was added. The mixture became viscous flowable with addition of Ca(OH)2. Bits of broken gel could be found in the bottom of the solution. The mixture became viscous and initiated a certain gel formation. A gel formed when Ca(OH)2 suspension was added. A compact gel was formed when Ca(OH)2 suspension was added. A compact gel formed when Ca(OH)2 suspension was added. Undissolved Ca(OH)2 was observed in the gel.

pectin (abbreviated as CaCl2 10, CaCl2 12, CaCl2 14, CaCl2 16, and CaCl2 18) and 10.8, 11.6, 12.5, 13.3, and 14.1 g of Ca(OH)2/100 g of pectin [abbreviated as Ca(OH)2 10.8, Ca(OH)2 11.6, Ca(OH)2 12.5, Ca(OH)2 13.3, and Ca(OH)2 14.1]. A gel was formed after the mixture stood for 0.5 h. The end product (C-CaP or HO-CaP) was then separated through filtration, washed with deionized water, and lyophilized. The yield of CaP was calculated using the following equation: m X (%) = CaP × 100 mpectin (1)

68.2 ± 1.61 77.6 ± 1.14 79.1 ± 1.53

52.8 ± 1.08 81.6 ± 1.74 83.9 ± 0.29

Pro rheometer (Malven, Worcestershire, U.K.) equipped with a parallel plate geometry (diameter 20 mm, gap size 0.5 mm, temperature 20 ± 0.1 °C). To assess the viscoelastic properties, a frequency sweep test was performed over a range of 0.01−10 Hz at a strain of 1%. A strain sweep test was carried out over a range of 0.1− 100% at 1 Hz. The storage modulus (G′) and loss modulus (G″) were measured as a function of the frequency and strain. Each experiment was conducted in three replicates. SEM. To examine the internal microstructure of CaP, lyophilized CaP gels [CaCl2 14 or Ca(OH)2 12.5] were scanned with scanning electron microscopy (Philips XL-30 ESEM, Royal Philips, Holland). Before scanning, the lyophilized CaP gel sample was fixed on the conductive material of the copper vector and then sputter coated with gold−palladium under argon. The photographs were recorded using an accelerating voltage of 20 kV.15,21 XRD. The structure of the egg-box junction zone was investigated with a Bruker MSAL-XD2 Discovery X-ray diffractometer (Germany) equipped with a Cu Kα source and Ni filter. The X-ray diffractometer was operated under the following conditions: voltage 36 kV, current 20 mA, scanning regions of the diffraction angle 2θ = 10−30°, and scanning rate 0.05 deg (2θ) min−1.22 Standard sample holders were carefully filled with approximately 0.5 g of CaP powder [CaCl2 14 or Ca(OH)2 12.5]. The relative crystallinity was measured according to Nara and Komiya,23 and the separation methods of the crystalline and amorphous portions are shown in Figure 1. The diffractogram was smoothed using the fit Gaussian method with Origin 7.0 (OriginLab Corp., Northampton MA). The relative crystallinity was calculated using the following formula:

where X is the yield of CaP (%), mCaP is the mass of CaP (g), and mpectin is the mass of pectin (g). Decalcification Process. To obtain the decalcified pectin (DCpectin), C-CaP (CaCl2 14) and HO-CaP [Ca(OH)2 12.5] were suspended in a 60% acidified ethanol−water solution (the pH was adjusted to 0.5 with 37% HCl) in a ratio of 1:40 (w/v), and the resulting suspension was homogenized for 2 min. The suspension was filtered, washed with 95% ethanol, and then heated at 35 °C in an aircirculated oven for 4 h. To separate the soluble pectin and insoluble CaP, DC-pectin was again suspended in deionized water in a ratio of 1:100 (w/v) with stirring. Five replications were performed. The pectin recovery was calculated as mDC‐pectin − mI‐CaP Y (%) = × 100 mDC‐pectin (2) where Y refers to pectin recovery (%), mDC‑pectin is the mass of DCpectin (g), and mI‑CaP is the mass of insoluble CaP (g). FTIR. An FTIR analysis was carried out to investigate the CaP and DC-pectin functional groups. These functional groups included free carboxyl, an ionized carboxyl group, and a carbomethoxy group. Frequently, functional group changes will cause a measurable change in the infrared spectrum of the molecule. According to Table 1, CaP was generated only when the calcium concentration fell within the range of 12−16 g of CaCl2/100 g of pectin or 11.6−13.3 g of Ca(OH)2/100 g of pectin. Therefore, only the CaPs formed in the above concentration ranges were then used for FTIR analysis. Moreover, the DC-pectins prepared from C-CaP (CaCl2 14) and HO-CaP [Ca(OH)2 12.5] were also used for the FTIR analysis. For the spectroscopic measurements, a 2% (w/w) sample was mixed with KBr before compression into a disk20 and then ground into a fine powder using an agate mortar. Each disk was scanned at a resolution of 4 cm−1 over a spectral range of 400−4000 cm−1 using an Equinox 55 FTIR spectrometer (Bruker, Germany). FTIR spectral parameters, including band frequency and band area, were acquired by a software package (OMNIC FTIR software, version 6.0, Thermo Fisher Scientific, Madison WI). The data represent the mean of triplicate experiments. Rheology. The rheological properties of the C-CaP gel (CaCl2 14) and HO-CaP gel [Ca(OH)2 12.5] were characterized using a Kinexus

Figure 1. Calculation of the relative degree of crystallinity in the X-ray diffractogram. αc (upper area) and αa (lower area) indicate the crystalline and amorphous portions, respectively. 6355

dx.doi.org/10.1021/jf5004545 | J. Agric. Food Chem. 2014, 62, 6354−6361

Journal of Agricultural and Food Chemistry Xc =

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αc αa + αc

(3)

where Xc is the crystallinity, αa is the area of the amorphous region, and αc is the area of the crystalline region.



RESULTS Effect of the Calcium Concentration and Source on the CaP Yield. Table 1 provides a summary of the CaP gelling state and yield obtained under varied calcium concentrations and sources. A CaP gel formed when the calcium concentration was in the range of 12−16 g of CaCl2/100 g of pectin or 11.6− 13.3 g of Ca(OH)2/100 g of pectin. The rate of CaP gel coagulation accelerated with increased calcium concentration, regardless of the calcium source. At 14 g of CaCl2/100 g of pectin and 12.5 g of Ca(OH)2/100 g of pectin, a better gelling quality and greater yield were achieved. In addition, at all concentrations investigated, the yield of CaP from Ca(OH)2 was significantly higher than it was from CaCl2. As indicated from earlier studies, pectin can form a gel in the presence of calcium by the formation of the egg-box junction zones. Thus, the gel strength increases with increasing calcium concentration, since the number of junction zones formed is also increased.3,24 If the calcium concentration were too low, the egg-box junctions would be insufficient to form the gel, but if the calcium concentration were too high, inferior gels would form due to instant gel formation in some areas, which would not only lower the CaP yield, but also negatively affect the gel structure. Therefore, the calcium concentration was a critical factor in the formation of the egg-box structure. Calcium Binding Stability of CaP. The calcium binding stability of CaP was determined by the pectin recovery (eq 2). Higher pectin recovery indicates easier decalcification and less stability for CaP. During decalcification, H+ in acidified alcohol can displace Ca2+ and thus convert CaP into pectin.25 As shown in Table 2, the average pectin recovery from HO-CaP (90.9%)

Figure 2. FTIR spectra of C-CaP and HO-CaP. Two areas, A (3500− 1350 cm−1) and B (

Characteristics of two calcium pectinates prepared from citrus pectin using either calcium chloride or calcium hydroxide.

Calcium pectinate (CaP) was prepared from citrus pectin using either calcium chloride (C-CaP) or calcium hydroxide (HO-CaP) as the source of calcium f...
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