Abstract
The aim of this work is to investigate the structural features, physicochemical, rheological, and emulsifying properties of pectin extracted from pumpkin “Cucurbita maxima” pulp and peel. After the peel and pulp of fresh pumpkin were solubilized in water at high temperature (80°C) and clarification, the obtained filtrate was treated with aluminum sulphate, which precipitates a fibrous coagulum. The results found that the major functional groups of pectin isolates identified by Fourier transform infrared are: –OH, CH2, C═C, –C–O–C–, and CH3. The results obtained using nuclear magnetic resonance methods suggest that these polysaccharides consist of two domains: the first one is homogalacturonan and the second is formed by rhamnogalacturonan. Mineral and metal ions on pectin surface detected using scanning electron microscopy coupled to energy dispersive X-ray analysis were different pulp and peel pectin. Rheological behavior of these biopolymers is suitably represented by the Herschel–Bulkley and power law models. The extracted pectin has a viscosity spectrum expressed by the following properties: G′, G′′, G*, and
Graphical abstract
1 Introduction
Pectin is one of the most complex polysaccharides, largely found in nature. It is extensively localized in the primary cell wall and middle lamella of higher plants. It provides consistent and mechanical resistance to vegetable tissues. The novel model of the chemical structure of pectin consists of three domains. The first one is composed of long chains of linear units of d-galacturonic acid (GalA) linked by (α-1,4), which represents the majority of total pectin (with 65%) [1]. In this domain, some of the carboxyl groups of GalA are partially methylated at the C-6 position or acetyl-esterified at the O-2 and O-3 positions. This domain is known as homogalacturonan (HG). The second domain consists of side chains inserted via rhamnose residues. This segment forms a rhamnogalacturonan (RG-I and RG-II) domain. It is rich in neutral sugars, including arabinose (Ara) and galactose (Gal). RG-I may also be methylated and acetylated [2]. Other minor sugars can exist in RG-II domain such as fucose, glucuronic acid, methyl-esterified glucuronic acid, apiose, 2-O-methylxylose, and 2-O-methylfucose. This domain is considered the most conserved domains among pectin molecules which represents 2–10% of pectin [3]. The final domain consisting of HG links to the GalA and xylogalacturonan (XG), the branched regions including RG-I, RG-II, and XG, are referred to as belonging to the hairy regions [4]. Other compounds of non-glycosidic nature may exist in the pectin substance such as phenolic groups, amide groups, and proteins, which can be attached to the side chains of RG-I regions. Recent research reported that HG and RG-I are varied between 5.7–95% and 1.4–82.5%, respectively [5]. Pectin can be classified according to its degree of methylation (DM) into two groups: high methyl-esterified (DM > 50%) and low methyl-esterified (DM < 50).
Pectin is usually extracted from food processing wastes such as apple pomace (10–15% dry basis), citrus peel (25–35% dry basis), and sugar beet (10–20% dry basis) [6,7]. This hydrocolloid polymer is a very important food additive because of its gelling, emulsifying, stabilizing, and thickening properties. It is designated as E440 in the list of food additives. Several studies have reported that the extraction conditions can affect the yield of pectin and its molecular weight, degree of esterification (DE), and branching contents. Consequently, the techno-functional properties of pectin are directly influenced. Currently, pectin research is focusing on other plant sources, such as pumpkin that has been subjected to only a limited number of studies. Pumpkin is largely consumed as a vegetable. Its high consumption is related to its high nutritional value and therapeutic properties such as anti-diabetic, anti-obesity, and lipid-lowering effects in various animals and cellular models [8]. Pumpkin is a promising alternative source of pectin and much work is clearly needed to fully characterize this pectin. The functional properties of pectin are based on their behavior in aqueous solutions, particularly on their shape and hydrodynamic volume. The intrinsic viscosity measurement is used to characterize pectin solutions as a hydrocolloid substance. When present in water, pectin usually forms colloidal solutions possessing gelling, thickening, and stabilizing functions. Food industries explore properties of pectin for new technological functionality in emulsions. According to Tamnak et al. [9], most polysaccharides are not recommended for the application in emulsion products because of their reduced hydrophobicity and high molecular weights. However, recent research works have reported that pectins extracted from pumpkin [10], apple and citrus [11], and sugar beet [7] exhibit high emulsifying capacity. They are able to form and stabilize emulsions. The emulsifying properties of these pectins can be related to high acetyl group content, by the presence of covalent bond of ferulic acids and protein or the low molecular weight.
Very few studies have been done on the pectin extracted using aluminum salts as solvent of precipitation; especially, when the chemical structure is not detailed. The present work has been deepened in a way that makes it possible to propose a description of the structural features of aluminum pectinates using sophisticated methods, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), and scanning electron microscopy coupled to energy dispersive X-ray (SEM-EDX) analysis, and compared to apple pectin and citrus pectin. Rheological properties, viscoelastic behavior, and emulsifying capacity of aluminum pectinates have been described.
In the previous work of Baississe and Fahloul [12], pectin was isolated from pumpkin pulp and peel by acid extraction using HCl of pH 1.8 and at high temperature treatment (80°C). The chemical composition, molecular weight, rheological behavior, and ζ-potential were determined. Chemical analysis of the extracted pectin showed an abundance of GalA which represents the totality of the uronic acid (52.91 ± 0.02–55.14 ± 0.10 g/100 g of dried samples). This biopolymer has values of 5.13 ± 0.04–5.65 ± 0.08 dL/g, 234.64–267.40 kDa, and 0.07 ± 0.00 for intrinsic viscosity [η], molecular weight, and flexibility, respectively. The hydrodynamic parameter of this pectin has values of the radius (R h) ranging from 32.39 ± 0.19 to 34.93 ± 0.40 nm, relaxation time (t r) from 2.77 ± 0.02 × 10−7 to 11.26 ± 0.00 × 10−7 ms, and diffusivity coefficient (D) from 2.17 to 8.73 ± 0.00 × 10−5 cm2/s. The rheological behavior was suitably represented by the Bingham and Herschel–Bulkley models for all concentrations. ζ-potential of the system constituted by pumpkin pectin and citric acid/sodium citrate buffer ranged from −28.10 ± 0.20 to + 0.35 ± 0.65 mV.
In this work, pectin isolates were obtained from the pulp and peel of pumpkin “Cucurbita maxima” by hot water treatment and compared for their structural features, physicochemical, rheological properties, and electrophoretic charge. In this study, FT-IR and NMR spectroscopy methods were applied for the identification of monomer and glycosyl residue chains and functional groups such as OH, methyl, and acetyl groups. Mineral and metal compositions on the surface of pectin was determined using EDX coupled to the SEM. This pectin has been used in the preparation of the soybean oil emulsion in order to evaluate its emulsifying properties such as the emulsifying activity index, specific surface area, interfacial concentration, and the average droplet size characterized by equivalent surface mean diameter (d [3,2]).
2 Materials and methods
2.1 Pectin extraction
Peel and pulp of fresh pumpkin were separately treated with hot water in which the cell wall-bound pectin is released by high temperature (80°C) in a water bath (pH 5.94) (IKA-HEIZBAD HB-250 type) with continuous stirring by IKA provided with an overhead stirrer RW 28 basic for 60 min.
After clarification, the obtained filtrate was treated with aluminum sulphate which precipitates a fibrous coagulum; aluminum pectinate. The pH value was adjusted between 4 and 4.2 by ammonia. The fibrous pectin precipitate was washed, pressed, and freeze dried in a PHYWE Christ freeze drier type and stored in glass bottles.
2.2 Determination of the physicochemical composition
GalA content was determined by automated m-hydroxybiphenyl assay difference in response of glucuronic acid and galacturonic acid (GalpA) in the presence and absence of tetraborate was used to differentiate them [13]. The absorbance measured at 520 nm increased linearly with GalA concentration from 0–100 g/mL [14]. The methyl group’s dosage was realized according to the method recommended by Barros et al. [15]. DM was determined by titrimetric method [16]. The total polyphenolic content was determined by the colorimetric method using Folin–Ciocalteu reagent and the gallic acid standard as given by Sanchez-Aldana et al. [17]. Protein content determination was carried out by Kjeldahl method, and the coefficient conversion for pectin is 6.25 [18]. Ash content was determined to quantify the degree of purity of pectin extracted using AOAC method [18].
2.3 Morphology of pumpkin pectin by SEM-EDX
SEM involves scanning the surface of a sample by a focused beam of electrons with an accelerating voltage of 2 kV. These electrons interact with the surface of the sample. The signal emitted at each point on the surface of the object is synchronized with that of a video screen, allowing the formation of a composite image modulated by the detected signal intensity. The mineral elements are also detected using EDX analysis.
2.4 FT-IR spectroscopy analysis
FT-IR spectra of pumpkin pectin extracted from the pulp and peel were obtained in attenuated total reflection (ATR) mode which is applied to the solid sample using Alpha-P-Brucker type. In this mode, using a spatula, a small amount of the sample was placed on the plate so that it could cover the crystal. Spectrum is obtained after being scanned. The ATR-IR measurement was performed on an ATR crystal at a 45° incident angle, in the frequency range of 4,000–500 cm−1. The spectral data obtained were processed by Opus Touch IR spectroscopy software.
2.5 NMR spectroscopy of pumpkin pectin
NMR spectra were acquired on variants 400 MHz for 1H, 101 MHz for 13C, 41 MHz for 15N, and 400 MHz for HMBC of lyophilized data samples after being solubilized in DMSO at 70°C using a Bruker 400 MHz spectrometer (Bruker, Germany). Experiments were realized in room temperature. Chemical shifts were expressed as
3 Physicochemical and rheology properties
3.1 Physicochemical properties
3.1.1 Solubility
Pectin solutions were prepared by dissolving the sample into three solvents: citric acid/sodium citrate buffer, NaCl, and CaCl2. These solvents were used at 1 M for ionic strength while stirring rapidly, heating at 40°C for 40 min, and filtering using filter paper (125 mm of diameter). Quantity of the solubilized pectin was estimated by drying the filter paper for 1 h at 102°C. The pectin quantity g/100 mL(% w/v) were solubilized relative to the initial quantity using the following equation given by Li et al. [19] equation (1):
where m 1 is the mass (g) of filter paper after heating for 1 h at 105°C, m 2 and m 3 are the masses of the sample (g) dissolved in the solvent and filter paper after drying at 150°C for 1 h followed by cooling in a desiccator, respectively.
3.2 Dynamic viscosity
Dynamic viscosity of pectin solutions was determined using a rheometer analyzer (Anton Paar MCR-301 model). This test is realized with double gap geometry (rod: DG26, 716282 and the sample cylinder: DG26, 7 SS/20720) at room temperature. In this test, the flow behavior of pectin solutions at different concentrations (0.5–4.0 w/v) was characterized by a rotational programmable viscometer. The samples were poured into the cylinder of the rotational viscometer and shear rate was applied between 10 and 1,000 s−1.
3.3 Intrinsic viscosity and molecular weight
Intrinsic viscosity [η] is defined as the limit of
where c is the concentration of pectin solution,
The
where k m is the constant Martin equation
Molecular weight is one of the most important characterizations of the biopolymers. It can influence some important functional parameters of these macromolecules. The relationship between intrinsic viscosity and molecular weight (M w) is given by Mark–Houwink–Sakurada (M–H–S) equation (4) [21]:
where “k” and “a” are M–H–S constants.
Flexibility (ξ) of the chain of pectin is directly related to its molecular weight and its intrinsic viscosity. The limits b = 0.5 and b = 0.8 indicate the model situations of solvent and of good solvent, respectively (equation (5)).
3.4 Hydrodynamic parameters
Diffusion coefficient (D) of pectin in water can be evaluated by the Einstein relation [19]. This parameter is very important in terms of physicochemical characteristics and molecular biology. It is given by the following formula (6):
where T is the temperature (K), and η is the apparent viscosity.
Relaxation time (t r), which is associated with an individual relaxation of the polymer, is calculated on the basis of the diffusion coefficient (D) by the following formula (7) [19]:
where q is the scattering wave vector.
3.5 Rheological properties
Flow properties were characterized by rheometer equipment (model: Anton Paar rheometer MCR-301) with double gap geometry. Flow curves were plotted in the range of 0.01–1,000 s−1 at 20°C. The following models have been used to evaluate the rheological behavior of pectin:
Herschel–Bulkley model (equation (9)):
where τ is the shear stress (Pa), γ is the shear rate (s−1),
Viscoelastic measurements were made in dynamic conditions (oscillatory shear) using rheometer analyzer type Anton Paar rheometer MCR-301 in the frequency range 1–102 rad/s for the dynamic oscillatory movement of pectin solutions at different concentrations (0.5–4.0 w/v). The amplitude of shear deformation (30%) was verified to be within the limits of linear viscoelasticity. This analysis permitted to characterize the viscoelastic behavior by measuring the constraint modulus: G′ (Pa) represents the elastic modules and G″ (Pa) represents the viscous modulus.
3.6 ζ-potential
Measurement of the electrophoretic particle mobility (Doppler laser velocimetry) was determined by Zetasizer analyzer (model Malvern Zetasizer Nano ZS instrument). This instrument can be used for the determination of the ζ-potential. The surface charge of 1.00 wt% of pectin solutions was measured as a function of pH (4–9) and concentration (1–4%). The result was reported as zeta-potential (ζ) (expressed in mV), which represents the sum of the individual surface charge of different functional groups in the mixed biopolymer or individual biopolymer solutions.
3.7 EAI of extracted pectin
The emulsions were prepared by mixing 5 mL of soybean oil with 15 mL of pectin solution of 1% (v/v). The preparation was homogenized at 16 rpm for 90 s using Ultra Turrax high shear mixer, followed by high pressure homogenization performed with the homogenizer system (varied at 0.1–16 bar) (Model IK, Albertslund, Denmark). The volume of 50 μL of each emulsion was transferred into 5 mL of 0.1% sodium dodecyl sulfate. Once the emulsion formation was completed, the absorbance (A) was measured at 500 nm. According to Mu et al. [23], the EAI was calculated by using the following formula (10):
where T = 2.303, D is the dilution factor (1,000), C is the weight of pectin per unit volume (g/mL), L is the width of the optical path (0.01 m), and Φ is the oil volume fraction of the dispersed phase.
The diameter (μm) of the oil droplets was obtained after image analysis of the photographs taken by a digital camera (FINEPIX JV200, JAPAN) and observed under an optical microscope (ZEISS, 10 × 2.5 objective) [9]. The image analysis was carried out using Motic Images Plus software, version 2.0. The average droplet size of the emulsions prepared in the presence of pumpkin pectin was determined as d [3,2], mean diameter, given by the formula (11):
The specific surface area (
where Φ is the oil volume fraction of the dispersed phase and d [3,2] is the surface-volume mean diameter (µm), and C is the adsorbed pectin concentration (mg/mL).
3.8 Statistical analysis
The data reported in all tables are averages of triplicate observations. The averages, one- and two-way ANOVA, and Least significant difference were computed to measure variations in the observations with the help of sigma plot. Modde software version 6.0 was used to study the effect of 3 levels of pH (4, 7, and 9), 3 levels of ionic strength (0.001, 0.010, and 0.100 M), and 2 levels of concentration of pectin (0.5 and 1%) on ζ-potential.
4 Results and discussion
4.1 Chemical composition of pectin polysaccharide
The functionality of pectin is closely related to its physicochemical characteristics, including GalpA content, DE, molecular weight, etc. Consequently, it is necessary to determine their physicochemical properties for their potential application.
Physicochemical characteristics of the investigated pumpkin pectin extracted from the pulp (PuP) and the peel (PeP) are illustrated in Table 1. The total sugar content and GalpA were 55.54 ± 0.001 g/100 g, 51.06 ± 0.01 g/100 g, and 53.72 ± 0.20 g/100 g, 51.17 ± 0.03 g/100 g in PuP and PeP (dry basis), respectively. Similar values of pumpkin PuP were found by Bai et al. [27] ranging from 32.10 ± 3.90 to 77.00 ± 3.40 g/100 g.
Chemical composition and DM of PuP and PeP extracted by hot water treatment
| Composition | PuP | PeP |
|---|---|---|
| Total sugar (g/100 g) | 55.54 ± 0.001 | 53.72 ± 0.22 |
| GalA (g/100 g) | 51.06 ± 0.01ns | 51.17 ± 0.03ns |
| Total polyphenol (g/100 g) | 0.64 ± 0.01 | 0.71 ± 0.00 |
| Protein (g/100 g) | 0.41 ± 0.11ns | 0.45 ± 0.18ns |
| Ash (g/100 g) | 3.84 ± 0.44ns | 3.90 ± 0.47ns |
| DM (%) | 54.21 ± 1.09*** | 36.36 ± 0.49*** |
Values are means of triplicate measurements; (***) there is a statistically significant difference (P < 0.001), (ns) not a statistically significant difference between two mean values of pulp and peel pectin.
GalA is the major compound, as is common in pectic polysaccharides. Torkova et al. [26] found that different neutral sugars were present in all pectin samples such as rhamnose, arabinose, and galactose. Other types are not dominant in all pectin, which reflects the effect of plant origin, such as xylose is present in apple pectin and fructose in pumpkin pectin.
PuP has high methyl-esterification (DM > 50%); its degree of methyl esterification is 54.21 ± 1.09%, while the PeP was little methylated with a DM value of only 36.36 ± 0.49%. Total polyphenolic contents were 0.648 ± 0.010 g/100 g and 0.715 ± 0.00 g/100 g (dry sample) for PuP and PeP, respectively (Table 1). In a similar study, Bai et al. [27] extracted very low methylated pectin from pumpkin pulp using acid, with the DE ranging from 14.6 ± 2.8 to 36.7 ± 2.7%.
The protein content was only 0.41 ± 0.11 and 0.45 ± 0.18 g/100 g in dry sample of PuP and PeP, respectively (Table 1). This low content might be due to the leaching fractions during extraction under solvent effect. Ash content of pectin isolates was high as the results were 3.84 ± 0.44 and 3.90 ± 0.47 g/100 g (dry sample) for PeP and PuP, respectively (Table 1).
Several studies have found that the extraction conditions, vegetable origin, plant variety, and geographic regions have effects on the chemical composition, and particularly on GalpA and neutral monosaccharide contents.
4.2 Morphology of pumpkin pectin
Figure 1 reveals the morphology of the pumpkin pectin powder obtained by SEM magnification 500×. In these pictures, the PuP is presented as irregular and lumpy-shaped particles with irregular rough and smooth surface regions with pores. Whereas PeP has two regions, the first region is monostratal lamellate with low porosity at the rough surface and the second region presents an irregular shape with relatively smooth surface. On the surface, the PeP and PuP have a certain number of the mineral and metal ions such as C (41.75%), O (49.65%) Al (3.3%), N (3.43%), S (1.94%), Po (0.10%), and Cl (0.08%) for PuP and C (28.65%), O (54.61%), Al (9.72%), N (3.80%), S (2.81%), P (0.11%), K (0.15%), Ne (0.19%), and Fe (0.33%) for PeP, respectively (Figure 1). These results indicate that these two samples are different in mineral and metallic composition, suggesting that these biopolymers can be used in different applications such as the domain of semiconductor, an inhibitor of oxidation phenomenon, catalytic performance in nanocomposite, encapsulation, and the pharmaceutical domain.
Morphology at magnification 500× and microanalysis by energy dispersive X-ray analysis of pumpkin pectin extracted from pulp (a) and peel (b).
4.3 Infrared spectrum of pumpkin pectin
Analysis of the FT-IR spectra was realized in order to identify the totality of functional groups of pectin extracted by hot water hot treatment from pumpkin pulp and peel. Figure 2 illustrates the FT-IR spectrum of pumpkin pectin isolates from the pulp and peel. This figure shows that the polysaccharide extracted from pumpkin pulp was linked with the following wavenumbers: 3661.0, 3184.32, 2940.28, 2819.50, 1723.00, 1610.31, 1435.40, 1333.93, 1075.88, 1017.82, 969.74, 817.00, and 592.68 cm−1; while the peel pectin was linked with the following wavenumbers: 3641.90, 3252.09, 2921.50, 1770.80, 1633.61, 1441.41, 1075.01, 973.87, 677.43, 595.96, and 541.44 cm−1. Comparing this spectrum with the pectin cited in the literature [28,29,30,31,32] further confirms that the polysaccharides obtained in this study from pumpkin pulp and peel were pectin. A broad band at a range of 3,200–3,600 cm−1, concentered at 3184.32 cm−1 for PuP and 3252.09 cm−1 for PeP, were attributed to the O–H stretching vibration of intra- and inter-molecular hydrogen bonds [28,29].
FT-IR spectra of pectin polysaccharide isolate from pulp (a) and peel (b).
An absorption band at 2940.28 cm−1 for PuP and 2921.50 cm−1 for PeP, was attributed to C–H stretching of the CH, CH2, and CH3 of the methyl groups in the sugar ring [28]. The two strong absorption detected at 1723.00 and 1610.31 cm−1 for PuP and 1770.0 and 1633.61 cm−1 for PeP separately indicated the presence of the C═O stretching vibration of methyl-esterified (COO–CH3) and ionic carboxyl (COO–) groups [29], respectively. Other important absorption peaks detected at 1435.40 and 1219.30 cm−1 for PuP and 1441.41 and 1241.60 cm−1 for PeP were assigned to the bending vibration of C–O–H groups and stretch tension of carboxylic acid (C–O), respectively.
The band observed at around 1554.0 for PuP and 1547.60 cm−1 were corresponding to the amide of the protein fraction [29]. In addition, peaks were found between 1333.93 and 1017.82 cm−1 for PuP and between 1075.01 and 1311.80 cm−1 for PeP, indicating the presence of hydroxyl and ether groups, respectively. Finally, signals at approximately 969.74–592.68 for PuP and 973.87–541.44 cm−1 for PeP indicated the existence of C–O–C and C–O–H CH3–CO bands, respectively [30]. These bands were characterized by stretching vibration of functional groups of neutral sugars with pyranose configuration present in pectin, which is typical of these polysaccharids [31,32].
4.4 NMR spectroscopy of pumpkin pectin
The 1H NMR and 13C NMR data of PuP and PeP are reported in Figures 3 and 4 and Table 2. The 1H NMR spectrum of data samples shows that the domain of the signals detected in all spectra are corredponding to following compounds: α-1-4-d-GalpA residues of the pectin in the free (HG) and methoxylated (HGM), addid the regions α-1-5-arabinan, β-1-4-galactan and α-1-2-L-rhamnose. The regions of
1D and 2D NMR spectrum of pumpkin pectin extracted from pulp (a) 1H NMR, (b) 13C NMR, (c) 15N NMR, and (d) HMBC NMR.
1D and 2D NMR spectrum of pumpkin pectin extracted from peel (a) 1H NMR, (b) 13C NMR, (c) HMBC NMR, and (d) 15N NMR.
Chemical shifts of glycosyl residues of PuP and PeP obtained by 1H and 13C NMR spectroscopy
| Glycosyl residues | Chemical shifts,
|
|
|---|---|---|
| 1H | 13C | |
| 4-α-d-GalpA (1–4) | 3.37–3.65–3.70–4.18–4.63–4.66–4.98–4.99–5.04–5.05–5.10–5.15–5.28 | 55.75–62.90–68.68–68.27–70.26–71.45–71.29–76.61–77.45–77.58–99.90–99.75–107.86**–102.93–170.16–170.23–176.07–175.46–177.36 |
| 4-α-d-Me-GalpA (1–4) Acetyl groups + | 3.36–3.37–3.72–4.99–5.00–2.5 | 52.75–55.76–56.68–56.32–71.29–71.40–71.45–81.69–102.93–175.46–177.35 |
| 4–5.5 (β-Galp (1–4) | 3.72–3.65–3.73 | 61.66**–62.90**–78.62–78.89–82.23–82.43 |
| 1,2-α-Rhap | 1.33–1.35–1.53–3.37–3.50–4.04–4.18–4.33–4.99–5.13–5.28–5.34 | 17.3–62.90**–69.60–70.15–70.26–70.36–77.58–77.45–78.89–78.62–83.58–99.90–99.79 |
| α-Araf (1–5) | 4.18–4.22–5.04–5.10–5.13 | 21.04–21.71–62.90**–68–96*–68.68*–82.23–82.43–83.65*–76.54–76.61–107.86**–85.47*–86.17**–113.06*–114.39** |
*pectin from pulp, **pectin from peel.
The acetyl groups were corresponded to the region with signals at 2.08–2.13 ppm for the pectin extracted from the pulp and at 1.90–2.30 ppm for the pectin extracted from the peel. Similarly, Linares-Garcia et al. [33] and Roman et al. [30] found that the acetyl groups can be detected at 1.9, 2.05, 2.13, and 2.3 ppm. The low signal obtained in these samples corresponds to the regions with 1.37–0.30 ppm for PuP and 1.36–0.15 ppm for PeP, indicating the presence of methyl portion of 2 and 2,4 linked rhamnose residues [34]. Similarly, Zhao et al. [35] found that this group can be observed at the following proton peaks: 1.17 and 1.23 ppm at C6 position of rhamnose residue. These results demonstrated that data samples can be rich in rhamnose residue. Roman et al. [30] found that following chemical shifts 3.8, 4.0, 4.5, and 5.0 ppm corresponding to COOMe, H-3 (HG, HGM) and H-2 (HG, HGM), H-4 (HG, HGM) and H-5 (HG, HGM), and H-1 (G, E), respectively.
These proton peaks were detected in data sample studies (Table 2). In these samples, other regions were detected with the following signals (in ppm): 7.13–8.18, 10.89–12.45, and 15.14–14.53 and 7.27–8.24, 11.24–12.68, and 14.57–14.90 for the pectin obtained from pulp and peel, respectively. Typical signals of monomers or oligomeres β-1,4 d-galactan chain attached to arabinan units have the value at
In the 13C NMR spectrum (Table 2 and Figures 3 and 4), PuP and PeP present anomeric carbon signals from 90.38–100.23 and 91.75–107.86, respectively. Non-anomeric carbon signals are present in data samples, with chemical shifts ranging from 68.26 to 81.69 ppm for PuP and from 61.66 to 83.68 ppm for PeP. The carboxyl carbon of GalpA gave signals in the region from 170.16 to 177.36 ppm for PuP and from 170.23 to 175.46 ppm for PeP samples. Zhao et al. [35] found that the furan conformation of arabinose can be observed at region with low peak at 107.48 ppm and at 82.36 ppm. The peak at 107 ppm is detected in PeP but not in PuP, while the peak at 82 ppm is detected in PeP and PuP. Another peak at
In Figures 3 and 4, the combining 2D HMBC and 15N NMR spectroscopy are applied for identification of sugar ring carbon/hydrogen signals. In HMBC spectrum, the principal chemical shifts have the following values (in ppm) for PuP: 3.5/196; 3.43/170.50; 3.44/172.17; 3.28/176.57; 3.23/110.02; 3.46/85.65; 3.28/77.12; 2.72/179.53; 2.55/177.2; 2.73/104.72; 2.40/113.69; 2.33/116.18; 2.14/147.0 2.09/111.65, and 2.18/82.56. For PeP, the peaks were present with the following values (in ppm): 3.57/115.3; 3.57/57.72; 3.49/183.26; 3.75/72.49; 3.13/77.76; 3.02/76.00; 2.98/81.4; 2.60/110.06; 2.83/81.85; 2.4/103.14; 2.25/175.00; 1.67/173.65; 1.49/82.17, and 1.33/76.67. In 15N spectrum, the peaks detected have the following values (in ppm) ranging from 20.00/1.00–420.00/25.00 in PeP and PuP.
These different results suggested that the polysaccharides isolate from pumpkin pulp and peel are composed of two domains: the first one is HG which is formed by d-GalA which can be acetylated or methyl esterified and the second is rhamnogalacturonan (RG-I) with free and methyl-esterified GalpA residue regions and branched with β-1,4-d-Galactan and α-Araf-derived side chains. Other substations non-glycosidic present in these polysaccharides such as polyphenol groups and protein. Figure 5 found the proposition of structural features the polysaccharides isolate from pumpkin pulp and peel.
Structural features of pectin isolate from pumpkin pulp and peel: (a) HG, (b) HG esterified, and (c) RG-I.
5 Physicochemical and rheological properties
5.1 Physicochemical properties
5.1.1 Solubility
The solubility of all biopolymers differs from that of small molecular solids by a certain number of specific characters. In dissolution, the pectin undergoes a swelling phenomenon during which the solvent penetrates into the solid mass by removing aggregates formed by the macromolecules. The three-dimensional form appears due to the formation of a network that the solvent can only dilate and the swelling persists. The results reported in Table 3 show that these biopolymers have almost complete solubility in the critical acid/sodium citrate buffer (95.69% for pulp pectin and 89.26% for peel pectin). While they are poorly soluble in the other two solvents used in this study.
Solubility, chemical composition, and molecular parameters of PuP and PeP extracted by water treatment at 80°C and dissolved in 0.2 M citric acid/sodium citrate buffer at 20°C
| Solubility and physicochemical and hydrodynamic parameters | PuP | PeP | |
|---|---|---|---|
| Solubility (%) | Citric acid/sodium citrate buffer at 1 M | 95.69 | 89.26 |
| Calcium chloride at 1 M | 73.75 | 74.66 | |
| Sodium chloride at 1 M | 81.05 | 83.68 | |
| Physicochemical and hydrodynamic parameters | [η] (dL/g) | 7.68 ± 0.18*** | 4.06 ± 0.97*** |
| K | 0.06 ± 0.005 | 0.11 ± 0.0.10 | |
| R 2 | 0.740 ± 0.16 | 0.963 ± 0.01 | |
| M w (kDa) | 407.48 ± 8.45 *** | 171.25 ± 39.26 *** | |
| ξ | 0.063 ± 0.000ns | 0.067 ± 0.0.001ns | |
| Diffusion coefficient (D) ×10−5 (cm2/s) | 1.90 ± 0.00ns | 2.96 ± 0.01ns | |
| Relaxation time (τs) × 10−9 (ms) | 2.45 ± 0.000ns | 3.82 ± 0.000ns | |
| Radius of gyration (R g) (nm) | 56.9 ± 0.017*** | 34.28 ± 0.00*** | |
Values are means of triplicate measurements; (***) there is a statistically significant difference (P < 0.001), (ns) not a statistically significant difference between two mean values of pulp and peel pectin.
5.2 Intrinsic viscosity and molecular weight determination
The values of intrinsic viscosity [η] are given in Table 3. These values varied between 4.06 ± 0.97 and 7.68 ± 0.18 dL/g. A statistically significant difference (P < 0.001) is obtained between PuP and PeP for [η] values. The [η] of pectin increases when dissociation of its macromolecules increases. It corresponds to the hydrodynamic specific volume occupied by the polymer unit mass in the reference solvent system.
Similar values were reported by Yoo et al. [36] and Torkova et al. [26] for pumpkin pectin and by Morris et al. [21] for pectin extracted from citrus.
The dependence of the specific viscosity ηsp as a function of the covering parameter C[η] of the pectin isolated from pumpkin shows that in solutions, the PuP has two critical concentrations: “C*”: 0.28 g/dL and “C**”: 0.30–0. 77 g/dL, whereas the PeP does not have the critical concentration in the range of concentrations studied in this work.
The PuP presents high value of M w, 407.48 ± 8.45 kDa, whereas the PeP presents low value 171.25 ± 39.26 kDa (Table 3). This difference can be explained by a stronger enzymatic degradation of the pectin in the peel than in the pulp. These polysaccharides have a similar flexibility (Table 3). Similar results for M w were cited in bibliography. Torkova et al. [26] found the values ranging from 72.2 ± 7.7 to 169.1 ± 16.0 kDa for the pectin extracted from pumpkin pulp (Cucurbita maxima). Whereas, Fissore et al. [37] extracted the pectin with a highly polymerized structure (136.00–1309.00 kDa) from some pumpkin.
5.3 Hydrodynamic parameters
The main hydrodynamic parameters are intrinsic viscosity and molecular weight which allow the determination of many very interesting parameters from a hydrodynamic point of view, such as the diffusion coefficient (D), radius of gyration (R
g), and the relaxation time (
6 Rheological properties
6.1 Modeling Rheological behavior
Figure 6 shows that a solution of pectin isolate could have three different rheological behaviors: thinning, thickening, and Newtonian. This property can be influenced by many properties such as M w, shape, and rigidity. The rheological behavior of pectin isolate was fitted using two models to describe its rheological behavior. Figure 7 and Table 4 reveals the data obtained by this modeling. The experimental data well fitted with both models for all concentrations of the PuP, whereas, for the PeP the experimental data were better adjusted with the Herschel–Bulkley model when compared to the power law model for all concentrations. In this case, pectin solutions exhibit a shear-thinning or pseudo-plastic behavior, in which apparent viscosity of these solutions decreased when the shear rate increased, whereas deformation stresses increased with the shear rate (Figure 6).
Modeling the rheological behavior of pectin extracted from pumpkin pulp and peel solubilized at various concentrations in 0.2 M citric acid/sodium citrate at 20°C. (a): pulp, (b): peel.
Storage modulus (G′) and loss modulus (G″) as a function of angular frequency of pumpkin pectin extracted from pulp and peel solubilized at various concentrations in 0.2 M citric acid/sodium citrate at 20°C.
Rheological properties of aqueous solutions of pumpkin pectin extracted from pulp and peel by water treatment at pH 1.8 after modeling of flow experimental data (20°C)
| Concentration of pectin (%) | |||||||
|---|---|---|---|---|---|---|---|
| 0.5 | 1 | 2 | 3 | 4 | |||
| Model parameters | |||||||
| Pulp | Power law model | n | 0.994 | 1.013 | 0.943 | 0.913 | 0.83 |
| K p (mPa s n ) | 1.30 | 1.57 | 4.46 | 5.24 | 12.97 | ||
| R 2 | 0.99 | 0.99 | 0.99 | 0.99 | 0.99 | ||
| Herschel–Bulkley | K C (mPa s n ) | 0.71 | 1.23 | 3.32 | 2.32 | 3.83 | |
| τ 0 (mPa) | 28.67 | 16.26 | 36.86 | 93.99 | 214.28 | ||
| n | 1.08 | 1.05 | 0.98 | 1.03 | 1.01 | ||
| R 2 | 0.99 | 0.99 | 0.99 | 0.99 | 0.99 | ||
| Peel | Power law model | n | 1.004 | 1.018 | 0.965 | 0.898 | 0.981 |
| K p (mPa sn) | 1.31 | 1.20 | 2.03 | 3.92 | 2.25 | ||
| R 2 | 0.99 | 0.99 | 0.98 | 0.99 | 0.99 | ||
| Herschel –Bulkley | K C (mPa s n ) | 0.89 | 0.95 | 1.46 | 2.32 | 1.14 | |
| τ 0 (mPa) | 19.79 | 11.89 | 20.87 | 43.646 | 50.75 | ||
| n | 1.06 | 1.05 | 1.01 | 0.974 | 1.08 | ||
| R 2 | 0.99 | 0.99 | 0.99 | 0.99 | 0.99 | ||
| Viscoelastic spectrum of pumpkin pectin | |||||||
| Pulp | G′ (Pa) | nd | 57.631 | 16.551 | 11.029 | 3.367 | |
| G″(Pa) | nd | 34.384 | 6.289 | 6.700 | 3.187 | ||
| G* (Pa) | nd | 67.120 | 17.718 | 12.919 | 4.700 | ||
|
|
831.08 | 206.353 | 144.595 | 83.036 | |||
| tan (δ) | nd | 0.597 | 0.381 | 0.612 | 1.006 | ||
| δ(°) | nd | 0.538 | 0.363 | 0.521 | 0.788 | ||
| Peel | G′ (Pa) | nd | 0.280 | 0.262 | 0.427 | 0.303 | |
| G″(Pa) | nd | 0.249 | 0.186 | 0.153 | 0.235 | ||
| G*(Pa) | nd | 0.41 | 0.39 | 0.48 | 0.42 | ||
|
|
18.34 | 24.25 | 26.44 | 18.24 | |||
| tan (δ) | nd | 1.520 | 4.60 | 1.02 | 1.20 | ||
| δ (°) | nd | 0.98 | 1.35 | 0.79 | 0.897 | ||
G*: complex modulus, G′: storage modulus (real part of G*), G″: loss modulus (imaginary part of G*),
nd: not determined.
The value of the flow behavior index is greater than or equal to zero and lower than or equal to one (0
6.2 Viscoelasticity behavior
For PuP, the values of G′, G″, and G* decrease with increase in the concentration of pectin in the solution. For complex viscosity (
6.3 ζ-potential of pectin
In order to determine the factors influencing the ζ-potential of pectin, the combined effects of pH/ionic strength, pH/pectin concentration, and ionic strength/pectin concentration on pectin ζ-potential were investigated by the surface response method. Figure 8 reveals the result of this investigation.
Surface plots showing the interaction effects of (a) concentrations of pectin (w%) and pH and (b) ionic strength and pH on ζ-potential of pectin extracted from pumpkin.
For PuP pectin, the regression model adjusted to ζ-potential data was obtained with R 2 = 0.899 for the combined effect of pH and concentration C (equation (14)):
For the combined effect of pH and ionic strength IS on ζ-potential, the regression model adjusted to ζ-potential data was obtained with R 2 = 0.955 (equation (15)):
For the combined effect of pectin concentration and ionic strength on ζ-potential, the regression model adjusted to ζ-potential data was obtained with R 2 = 0.825 (equation (16)):
For PeP pectin, for the combined effect of pH and concentration, the regression model adjusted to ζ-potential data was obtained with R 2 = 0. 897 (equation (17)):
For the combined effect of pH and ionic strength on ζ-potential, the regression model adjusted to ζ-potential data was obtained with R 2 = 0.949 (equation (18)):
The calculated optimum of ζ-potential (–46.35 mV) is obtained for the following conditions: pH = 9, concentration = 1%, and ionic strength = 0.01 M for PuP; however, for PeP the optimum value of ζ-potential is –22.17 mV, this value is obtained for the following conditions: pH = 8.99, concentration = 0.5%, and ionic strength = 0.85 M.
These results confirmed that ζ-potential is highly influenced by the three factors studied in this work. The pectin can present low stability and form an agglomerate/flocculate if it approaches its isoelectric point. Also, the polyelectrolyte behavior of this macromolecule increases with the decrease in ion valences [39]. The relationship of colloidal particle concentration and ζ-potential is very complex and usually determined by both surface adsorptions, the ζ-potential increased with the concentration of the particles. Lan et al. [40] found that the extraction conditions have a very important effect on the stability of pectin particles in solution, especially on the ζ-potential. This parameter has values ranging from +30 mV to –35 mV.
These results confirm that pectin carries ionic sites of the same sign close together and interdependent on each other which nominated polyelectrolytes. In solution, these kinds of macromolecules dissociate into multivalent ions and counter ions whose distribution in the solvent is very influenced by the electric field. This information is confirmed by the result given by SEM-EDX. This result found that the PeP has a metal-rich fraction compared to PuP, which can be explicated by a very large difference in ζ-potential values for both samples: –46.35 mV for PuP and –22.17 mV for PeP. This result is very important and can be offered to new applications of pectin, especially in biodegradable films, coating, encapsulation, and pharmaceutical domains.
6.4 EAI of extracted pectin
Table 5 illustrates the EAI, specific surface area, and interfacial concentration of pectin isolate from pulp and peel (1%) at pH 3.53. The emulsifying properties were assessed based on the emulsion droplet size distribution represented by d [3,2].
The values of the EAI, specific surface area, and interfacial concentration are 58.34 ± 2.35 m2/g, 1.97 m2/ml, and 3.37 ± 0.01 mg/m2 and 55.72 ± 0.40 m2/g, 1.28 m2/ml, and 5.19 ± 0.01 mg/m2 for the emulsion prepared in the presence of PuP and PeP, respectively. Nakauma et al. [24] reported results of (Γ) ranging from 1 to 4 mg/m2 for prepared emulsions based on sugar beet pectin (<5%) and soybean oil at pH 3. While Funami et al. [25] found the values of (Γ) and (S
EAI, specific surface area, interfacial concentration, and d [3,2] of PuP and PeP
| EAI (m2/g) | d [3,2] (µm) | S
|
Γ (mg/m2) | |
|---|---|---|---|---|
| Pectin extracted from pulp | 58.34 ± 2.35 | 0.76 | 1.97 | 3.37 ± 0.01 |
| Pectin extracted from peel | 55.72 ± 0.40 | 1.17 | 1.28 | 5.19 ± 0.01 |
The emulsion droplet size distribution found that the values of d [3,2] were 0.76 µm for the emulsion prepared with pulp pectin and 1.17 µm for the emulsion prepared with peel pectin. Pulp pectin is therefore a higher emulsifying agent. The values of d [3,2] ranging from 0.56 ± 0.04 to 3.00 ± 0.25 µm, from 0.55 to 0.82 µm, and from 1.3 to 3.7 µm for sugar beet pectin, soybean soluble polysaccharide, and okra pectin, respectively, are reported in the bibliography [41]. The emulsifying activity of pectin was most probably due to the non-carbohydrate residues such as protein fraction, acetyl groups, or total polyphenolic residues [42,43].
The presence of neutral sugar methyl and acetyl groups could positively influence the intrinsic viscosity, the molecular weight, and the diffusion coefficient. However, a strong significant correlation, especially for the relaxation time (p < 0.001) can be observed. These same components have a strong positive correlation with the flexibility of pectin and its emulsifying properties.
6.5 Correlation between composition, physicochemical, rheological, and emulsifying properties of pectin
The interfacial property of pectin is generally attributed to many factors including acetyl content, protein fraction, molecular weight, degree of methylation, internal charge distribution, etc.
Statistical analysis of the correlation between composition, physicochemical, rheological, and emulsifying properties of pulp and peel pectins are illustrated in Table 6.
Correlation between composition and physicochemical, rheological, and emulsifying properties of extracted pectin
| [η] | Ξ | M w | D | τ s | EAI | |
|---|---|---|---|---|---|---|
| CC | CC | CC | CC | CC | CC | |
| P | P | P | P | P | P | |
| Uronic acids | –0.98 | 0.99 | –0.98 | –0.98 | 0.99 | 0.93 |
| 0.015* | 0.008** | 0.017* | 0.016* | 0.006** | 0.06ns | |
| GalA | –0.67 | 0.70 | 0.68 | –0.68 | 0.69 | 0.69 |
| 0.318ns | 0.295ns | 0.319ns | 0.320ns | 0.309ns | 0.309ns | |
| Neutral oses | 0.97 | –0.98 | 0.97 | 0.97 | 0.99 | –0.90 |
| 0.025* | 0.018* | 0.028* | 0.026* | 0.006** | 0.09ns | |
| Methyl groups | 0.98 | –0.98 | 0.97 | 0.98 | 0.99 | –0.93 |
| 0.019* | 0.011* | 0.02* | 0.020* | 0.006** | 0.06ns | |
| Protein | 0.02 | –0.04 | 0.02 | 0.27 | –0.09 | 0.17 |
| 0.97ns | 0.955ns | 0.98ns | 0.729ns | 0.910ns | 0.82ns | |
| Polyphenol groups | 0.27 | –0.31 | 0.26 | 0.27 | –0.35 | –0.07 |
| 0.73ns | 0.682ns | 0.734ns | 0.729ns | 0.649ns | 0.92ns |
P: P-Value; CC: correlation coefficient, ns: not significant.
A strong positive correlation can be observed between uronic acid content, flexibility, and relaxation time of pectin, on the one hand, and between uronic acid content and emulsifying activity on the other hand. Furthermore, a negative correlation can be observed between uronic acid content and viscosity, and molecular weight and diffusion coefficient. However, a weak correlation is shown between GalA content and different parameters studied.
The presence of neutral oses and methyl groups could positively influence intrinsic viscosity, molecular weight, and diffusion coefficient. However, a strong significant correlation can be observed especially for the relaxation time (p < 0.001). These same components have a strong positive correlation on the flexibility of pectin and its emulsifying properties [43].
The presence of proteins and polyphenols do not have a significant correlation between different parameters studied in this work. This last result seems to be contacted by previous results which attribute protein fraction to an important role in the emulsifying properties of pectins. However, Cuevas-Bernardino et al. [42,43] have reported that the role of proteins is not critical in the emulsifying activity, indeed for two hawthorn accessions pectins having comparable amounts of protein, they were found to exhibit different emulsifying potentials. This can be explained by the fact that profile and interaction mode of protein and polyphenols fraction extracted from pectin differ according to botanical source.
According to Kaya et al. [44], 20 and 80% of the Rhamnose residues are substituted at C-4, depending on the botanical source of pectins and processing condition witch can significantly effect a confirmation of galacuronan backbone and its flexibility as a consequence. This situation is a consequence of the degree of methoxyl which is proportional to the M
w and [
7 Conclusion
This study was designed to evaluate the physicochemical, rheological, and emulsifying properties of pectin extracted by hot water treatment (80°C) from pumpkin “Cucurbita maxima” pulp and peel.
The extraction method used in this study allows isolating the pectin polysaccharide with homogalacturonan and rhamnogalacturonan regions substituted with β-(1, 4)-galactan and α-Araf-derived side chains. These regions are esterified by methyl and acetyl groups. The presence of polyphenol substances is detected. These biopolymers have on their surface a certain number of mineral (C, O, S, N, and Cl) and metal ions (Al), for PuP and C, O, Al, S, P, K, N, Ni, and Fe for PeP. This result is very important and can open new applications for these biopolymers in many domains.
These results may be economically important since acid extraction (industrial extraction) can be replaced by water extraction. In order to improve the yield, the sequential extraction with water can be proposed. These results motivate use to explore the interaction mechanisms of different parameters.
Pumpkin is a fairly important source of pectin with strong emulsifying activity; these substances can substitute other types of emulsifiers used at concentrations greater than 1% such as gum Arabic (15%), soybean soluble polysaccharides, etc. Pumpkin is a promising alternative source for pectin material and much work is clearly needed to fully characterize this pectin, as well as exploring the full potential of this source.
The rheological properties and emulsifying activity of food products are very important for the design, evaluation, and development of process equipment.
Acknowledgments
The authors would like to thank the food process engineering team of AgroParisTech-Center Massy, Technical platform for physico-chemical analysis of Laghouat and CRPAC Bou Ismail,Tipaza, Algeria for technical help. Baississe S. would like to thank Doctor M. GHELLAM (Food Engineering Department, Faculty of Engineering, Ondokuz Mayis University, Samsun, Turkey) for help in reviewing and editing and Mr Kahoul Mohamed Amine, Research Laboratory Engineer “Food Science,” Department of Food Technology, University of Batna Algeria, for help in experiments.
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Funding information: The authors state no funding involved.
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Author contributions: Baississe Salima: data curation, investigation, methodology, conceptualization, validation, and writing – original draft. Fahloul Djamel: investigation, validation, and supervision. Dridi Seloua: supervision, visualization, and review & editing. Mezdour Samir: investigation, methodology, conceptualization, validation, and review & editing.
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Conflict of interest: The authors declare that they have no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: All data generated or analyzed during this study are included in this published article.
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