Characterization of partial acid hydrolysates of citrus pectin for their pasting, rheological and thermal properties

Abstract

Pectin was subjected to acid hydrolysis with hydrochloric acid for 30 and 60 min to prepare partial hydrolysates (PH30 and PH 60). The influence of acid hydrolysis on the physico-chemical and functional properties were assessed for their potential applications in foods. Acid hydrolysis significantly reduced the molecular weight and viscosity of pectin in a time dependent manner. Steady shear properties revealed a shear-thinning behavior for NP and PH 30 while Newtonian behavior was observed for PH 60. Oscillatory measurements revealed a viscoelastic behavior for NP while a viscous liquid like behavior was observed for PH30. DSC measurements also revealed reduced thermal stability of pectin hydrolysates in comparison to native pectin. The results of the present study suggested that pectin hydrolysates with improved solubility can be used in various food products as a source of dietary fiber without modifying the texture and palatability of food products.

Introduction

Pectin is a natural complex polysaccharide found in the cell wall and middle lamella of plants, mainly obtained as a byproduct from apple and citrus fruit wastes and is considered as an important source of dietary fiber (Min et al. 2011). Pectin is mainly used as thickening, gelling, emulsifying and stabilizing agent in food systems such as jams, jellies and marmalades (Mesbahi et al. 2005). It is an ionic polysaccharide composed of partially methylated poly-(1 → 4)-α-D-galacturonic acid residues. On the basis of degree of methylation, pectin is further categorized into low methoxyl pectin (25–50%) and high methoxyl pectin (50–80%). The functionality of pectin in food systems greatly depends on its degree of methylation and polymerization (Seshadri et al. 2003). Low methoxyl pectin requires the presence of divalent ions to form gels while high methoxyl pectin forms gel under acidic pH in the presence of high concentration of sugar. The hydrogen bonding and electrostatic interactions contribute to the thickening and gelling mechanism of pectin and its effectiveness in imparting thickening to the solution. The gelling ability of pectin depends upon degree of esterification, molecular weight, temperature and concentration used (Li et al. 2013). In dilute solutions, pectin molecules move freely apart but come in contact with each other as the concentration increases resulting in the aggregation of pectin molecules to form a structured thick network through hydrogen bonding (Hua et al. 2015).

Pectin is generally soluble in water but serves some technological drawbacks, requiring high temperature and high-speed mixing to achieve complete hydration resulting in the formation of lumps and thus increasing energy and production cost (Einhorn-Stoll et al. 2015; Kurita et al. 2012). Also, the usage of hydrocolloids is generally limited to low concentrations due to their adverse effect on food palatability and thus restricts their applications in food products (Dangi et al. 2019). Moreover, the physiological benefits are mainly dependent on the effective concentration of pectin to be used as a dietary component which in turn depends on its molecular weight and viscosity. The low solubility, thickening and gelling ability have also restricted the applications of pectin to be used as a promising additive in food products, particularly liquid foods to get effective physiological responses (Yamaguchi et al. 1994). In order to overcome these limitations, partial hydrolysis offers an approach to degrade pectin to achieve low viscosity and improved solubility making it suitable for providing effective functions in the human body (Ma et al. 2018).

In recent years, partial depolymerization of polysaccharides is gaining interest due to the associated positive health benefits of their hydrolysates. Acid hydrolysis is a cost-effective method to yield low molecular weight pectin and specific functional properties can be achieved with the aid of controlled hydrolysis. Partial hydrolysis led to the formation of pectin oligosaccharides and monosaccharides which are best known for their prebiotic effects and thus conferring advantages to the host (Gomez et al. 2014). Particularly, pectin hydrolysates have received attention due to its beneficial physiological functions such as immune modulation, anti-tumor and anti-cancerous properties (Combo et al. 2013). Prebiotic effect of pectin hydrolysates has been reported in infant formulas (Fanaro et al. 2005) but still pectin hydrolysates have not been much used in the commercial food products. To best of our knowledge the characterization of pectin hydrolysates for their pasting, thermal and rheological behavior still remained unexplored. Therefore, the objective of this study was to study the pasting and rheological behavior of citrus pectin acid hydrolysates to assess their suitability for incorporation into food systems in a concentration-based manner.

Materials and methods

Commercial citrus pectin having 80% galactouronic acid and 7% methoxyl content was purchased from TM-Media, Titan Biotech. Ethanol, HPLC grade water, sodium nitrate and other chemicals were of analytical grade.

Acid hydrolysis of pectin

Pectin hydrolysates were prepared according to the method described by Murata et al. (2005) with slight alterations. Pectin (1% solution, 0.2 M HCl, 70° C) was subjected to acid hydrolysis for 30 min and 60 min separately, neutralized with 1.0 M NaOH followed by precipitation with ethanol and centrifuged at 3000 rpm for 5 min. The hydrolysates obtained were washed with ethanol three times, oven-dried and grinded to obtain powder.

Molecular weight determination of pectin and its hydrolysates

Viscometery

The samples were dispersed in distilled water at different concentrations (0.1–0.5 w/v) and intrinsic viscosity (η) was determined using Ostwald’s viscometer at 25º C. Relative viscosity was measured in order to determine specific viscosity using following equation:

$${\eta }_r=\frac{\mathrm{t}}{{\mathrm{t}}_{\mathrm{o}}}$$

$${\eta }_{\mathit{sp}}={\eta }_r-1$$

where t is the flow time of pectin solution and t_o is the flow time of distilled water through the same viscometer. Specific viscosity was used to measure reduced viscosity at different concentrations using following relation:

$${\eta }_{\mathit{red}}=\frac{{\eta }_{\mathit{sp}}}{C}$$

Intrinsic was then measured by extrapolating concentration to zero. Viscosity average molecular weight (M_v) was calculated using the Mark-Houwink’s equation,

$$\left[\eta \right]={\text{KM}}_{\text{v}}^{\text{a}},$$

where K = 1.4 × 10^–6 and α = 1.43 (Zhou et al. 2010).

Gel permeation chromatography

GPC was used to estimate the weight average molecular weight (M_w), number average molecular weight (M_n) and polydispersity index (PDI) (M_w/M_n) of pectin and its hydrolysates. The molecular weight was determined using gel permeation chromatography system with a Malvern instrument (GPC max) consisting of an aqueous column A6000 (300 × 8 mm) connected to a differential refractometer detector. The samples (1 mg/ml) were dissolved in 0.05 M NaNO₃, filtered through 0.22 µm nylon filter before injection and eluted at a flow rate of 0.7 ml/min at 35 °C. The GPC was calibrated with the known molecular weight series of pullulan standards (0.342 k, 1.3 k, 6.0 k, 10.0 k, 22.0 k, 50.0 k, 110.0 k, 200.0 k, 400 k and 800 k Da, Fluka, USA). Molecular weight of pectin was determined using linear regression equation obtained for the standards used using conventional calibration. The molecular weight values obtained are relative to the pullulan standards.

Degree of polymerization

Average degree of polymerization (DP) of pectin and its acid hydrolysates was calculated from the viscosity average molecular weight using the following equation:

$$\mathrm{Average}\mathrm{DP}=\frac{\mathrm{molecular}\mathrm{weight}\mathrm{of}\mathrm{polymer}\mathrm{molecular}}{\mathrm{weight}\mathrm{of}\mathrm{monomer}}$$

where the molecular weight of pectin monomer was 180 (Morris et al. 2008).

Physico-chemical properties

Water absorption capacity (WAC) of pectin and its hydrolysates was determined using the standard method (AACC 2010) by taking 0.5 g sample. The WAC was expressed as g/g as the grams of water retained by the sample. Oil absorption capacity (OAC) was determined according to the method described by Lin et al. (1974) by using 100 mg sample in 1 ml oil. OAC is expressed as the grams of oil absorbed per gram of the sample. Swelling power and solubility of native and hydrolyzed pectin were measured following the method of Bae et al. (2009) by taking 0.3 g sample. Swelling power was determined as the ratio of wet precipitate weight to dry weight while solubility was expressed as percent weight of the dry supernatant to dry sample weight.

Rheological behavior

The steady flow and viscoelastic behavior of aqueous pectin solutions were determined using a rheometer (MCR 102, Anton Paar, Germany) provided with a cone and plate geometry (40 mm diameter, 0.08 mm gap and 1° cone angle). The samples were dissolved in distilled water to achieve a final concentration of 4, 6 and 8%. The solutions were vigorously stirred for 2 h to achieve complete dissolution before performing the rheological measurements.

Flow behavior of pectin solutions were determined by conducting steady shear measurements at 25 °C in the shear range from 0.1 to100 s⁻¹. The obtained flow curve data was analyzed using the following models:

$$\text{Power}\text{law}\text{model},\tau =K{\gamma }^n.$$

$$\text{Herschel}-\text{Bulkley}\text{model},\tau ={\sigma }_{\text{o}}+K{\gamma }^n.$$

where τ is the shear stress (Pa), σ_o is the yield stress (Pa), γ^. is the shear rate (s⁻¹), n is the flow behavior index (dimensionless) and K is the consistency coefficient (Pa sⁿ).

Viscoelastic behavior was determined using the frequency sweep measurements. Strain sweep test was performed to determine the maximum strain value within the linear viscoelastic region (LVE) followed by frequency sweep tests that were carried out in the frequency range from 0.1 to 100 rad/s at a constant strain (0.05%) within the LVE range. Dynamic viscoelastic moduli (G′, G″ and tan δ) were determined as a function of frequency (ω).

Pasting properties

Pasting profiles of pectin and its hydrolysates were studied to assess the influence of temperature on the viscosity of pectin solutions using rapid visco analyzer (Newport Scientific, Australia). The samples (4 and 6%) were taken into a canister, placed in the RVA and stirred at 960 rpm for 1 min and at 160 rpm for the remaining test. The temperature profile started from holding the samples at 80 °C for 2 min, followed by cooling to 30 °C at a rate of 4 °C/min.

FT-IR studies

FT-IR spectroscopy was used to assess the structural modification of pectin when subjected to acid hydrolysis. The powdered samples were directly placed on to sampling unit and spectra were recorded between 4000 and 400 cm⁻¹ using a FT-IR spectrophotometer (Bruker Alpha, Platinum ATR, Germany).

Thermal properties

Thermal behavior of pectin and its hydrolysates was determined using differential scanning colorimeter (DSC 25, TA instruments, USA). Powder samples (3.5 mg ± 0.1) were weighed in an aluminum pan and placed in the instrument using empty pan as a reference. Thermal scans were conducted in a temperature range of 50–450 °C with a heating rate of 10 °C/min. Thermal parameters including onset temperature (T_o), peak temperature (T_p), conclusion temperature (T_c) and enthalpy (∆H) were determined.

Statistical analysis

The data were analyzed using one-way Analysis of Variance (ANOVA) using SPSS version 19.0 at a significant difference (p < 0.05). All experiments were done in triplicates and the mean values ± SD were reported.

Results and discussion

Intrinsic viscosity and viscosity average molecular weight

Viscosity of aqueous pectin solutions depends on the intrinsic factors such as molecular mass, size, shape, volume and surface charge of the molecule and is used to determine the hydrodynamic parameters of the polymers. It has been reported that intrinsic viscosity is the volume per unit mass that the pectin occupied in the solution instead of the real viscosity (Hua et al. 2015). The η of NP (3.20 dL/g) was found to be higher than that of its acid hydrolysates, PH 30 (2.05 dL/g) and PH 60 (1.35 dL/g), as shown in Fig. 1a. The obtained values of intrinsic viscosity for NP are in accordance with those reported for commercial citrus pectin (Li et al. 2013). It was found that acid hydrolysis caused significant reduction in the intrinsic viscosity due to the cleavage of glycosidic bonds of the main polymer backbone. Intrinsic viscosity is used to determine the viscosity average molecular weight, which was observed to be lower for PH 30 (1.79 × 10⁴) and PH 60 (1.34 × 10⁴) in comparison to its intact counterpart (2.44 × 10⁴). It was found that molecular weight of pectin subjected to acid hydrolysis depends on the duration of the hydrolysis and increased hydrolysis time resulted into reduction in molecular weight to a greater extent. Also, the degree of polymerization was observed to be higher for NP as compared to its acid hydrolysates (Table 1) and confirmed the depolymerization of glycosidic bonds upon acid hydrolysis resulting into decrease in molecular weight and hence lower degree of polymerization. Molecular weight and concentration are the key factors that influence the functional properties of polymers and the above results suggested that pectin can be degraded to various M_w to attain desired characteristics.

Fig. 1.

Intrinsic viscosity plots (a) and GPC chromatograms (b) of pectin and its hydrolysates

Table 1.

Molecular weight, intrinsic viscosity and physico-chemical properties of pectin and its hydrolysates

Samples	η (dL/g)	Mv (Da)	DP	Mw (Da)	Mn (Da)	PDI	WAC (g/g)	OAC (g/g)	SP (g/g)	Sol (%)
NP	3.20 ± 0.02c	2.44 × 104 ± 0.02c	135.55 ± 1.93c	8.58 × 105 ± 0.03c	9.01 × 104 ± 0.02c	9.51 ± 0.01c	19.84 ± 0.26c	7.39 ± 0.68b	7.35 ± 0.03c	57.21 ± 1.04a
PH 30	2.05 ± 0.03b	1.79 × 104 ± 0.07b	99.44 ± 1.81b	1.74 × 105 ± 0.02b	5.28 × 104 ± 0.01b	3.31 ± 0.02b	4.60 ± 0.25b	8.46 ± 0.70b	2.16 ± 0.01b	89.25 ± 1.42b
PH 60	1.35 ± 0.02a	1.34 × 104 ± 0.03a	74.44 ± 1.56a	5.40 × 104 ± 0.04a	2.50 × 104 ± 0.02a	2.12 ± 0.02a	1.07 ± 0.09a	12.85 ± 0.62a	1.58 ± 0.17a	137.70 ± 5.11c

Values are mean ± S.D of triplicates

Values in the same column with different letters are significantly different (p < 0.05)

NP—Native pectin, PH – pectin hydrolysate, η—intrinsic viscosity, M_v—viscosity average molecular weight, DP—degree of polymerization, M_w—weight average molecular weight, M_n—number average molecular weight, PDI—polydispersity index, WAC- water absorption capacity, OAC- oil absorption capacity, SP- swelling power, Sol- solubility

Gel permeation chromatography

The GPC chromatogram revealed the molecular weight distribution of native pectin and its hydrolysates (Fig. 1b). It was observed that hydrolysis caused a significant reduction in the weight average molecular weight (M_w), number average molecular weight (M_n) and polydispersity index (PDI) of the NP. Also, the GPC curves of hydrolysates were shifted towards the right side in retention time in comparison to native pectin which indicated the presence of low molecular weight fragments. The M_w and M_n of NP were observed to be 8.58 × 10⁵ and 9.01 × 10⁴, which reduced to 1.74 × 10⁵ and 5.28 × 10⁴ after 30 min hydrolysis followed by further reduction to 5.4 × 10⁴ and 2.5 × 10⁴ after 60 min hydrolysis (Table 1). PDI is the ratio of weight average molecular weight and number average molecular weight (M_w/M_n) and measures the molecular weight distribution of the polymers. PDI was found to be higher for NP (9.51) in comparison to its hydrolysates, PH 30 (3.31) and PH 60 (2.12). Such higher PDI for native pectin have also been reported in the literature for low methoxyl pectin (Li et al. 2013; Hua et al. 2015). Intense peaks were observed for acid hydrolysates depicting the homogeneity of pectin molecules in terms of molecular weight whereas broad peak was obtained for native pectin suggesting the complexity of the polymer molecular structure having wide distribution of molecular weights. Acid hydrolysis decreased molecular weight and intrinsic viscosity in a time-dependent manner due to reduction in the chain length of the polymer backbone resulting into the generation of oligosaccharides and monosaccharides having weakened intermolecular aggregations (Ho et al. 2017). Reduction in molecular weight of pectin have been reported earlier in literature with the aid of acidic, enzymatic and ultrasound depolymerization (Ma et al. 2016; Round et al. 2010; Zhang et al. 2013).

Water and oil absorption capacity

Water absorption capacity plays an important role in maintaining the texture, stability and overall quality of food systems. The acid hydrolysis significantly (p < 0.05) decreased the WAC of native pectin from 19.84 to 1.07 g/g as shown in Table 1. It was observed that the affinity of pectin towards water decreased which can be explained in terms of reduced molecular weight and intrinsic viscosity of the hydrolysates. The presence of more hydroxyl groups in the structure of native pectin allowed more water interaction through hydrogen bonding and hence higher WAC while acid depolymerization led to the reduction in the number of the hydrophilic groups resulting into lower WAC in case of hydrolysates.

The oil absorption capacity of native pectin was much lower than water absorption capacity indicating its hydrophilic nature. However, similar trend was not observed for acid hydrolysates and oil absorption capacity was found higher when compared to water absorption capacity (Table 1). Moreover, hydrophobicity increased with increased hydrolysis times. Highest OAC was observed for PH60 (12.83 g/g) whereas lowest for NP (7.39 g/g). Acid hydrolysis caused disintegration of the high molecular weight fractions into low molecular weight fractions and might have exposed hydrophobic groups resulting into increased oil absorption capacity. These results implied that pectin hydrolysates could be beneficial in flavor retention, palatability and shelf life of foods. Increase in oil absorption capacity after depolymerization has also been reported for other plant seed gums (Hamdani et al. 2017, 2018).

Swelling power and solubility

Swelling power describes the ability of polymers to bind and hold water in its structure. Significant differences (p < 0.05) were found in swelling power of native pectin and its hydrolysates reflecting variations among the structure of NP and PH to associate with water owing to their differences in the molecular weight and intrinsic viscosity. Maximum swelling power was observed for NP (7.35 g/g) while minimum for PH60 (1.58 g/g) (Table 1). About 70% reduction in swelling power occurred after 30 min hydrolysis followed by further 26% reduction after 60 min hydrolysis. The obtained results of swelling were in accordance with the WAC results suggesting the breakdown and weakening of intermolecular associations of the polymer chain upon acid degradation.

An opposite trend from swelling power was obtained for solubility of the pectin and its hydrolysate. The results given in Table 1 showed that solubility of pectin significantly (p < 0.05) increased after acid hydrolysis and maximum solubility was observed for PH60 (137%). Solubility increased by 56% after hydrolysis of 30 min and by 140% after hydrolysis of 60 min, respectively. The increased solubility of pectin hydrolysates might be useful for preparation of hydrogels (Abd Alla et al. 2012). As observed from molecular weight estimation, the acid hydrolysis caused an increase in the proportion of oligosaccharides and monosaccharides having ability to hydrate rapidly as compared to NP being a high molecular weight polymer (Hamdani et al. 2017).

Rheological properties of pectin and its acid hydrolysates

Flow behavior

Rheology describes the flow characteristics of polymers and used to determine the potential applications of polymers in food systems. The flow behavior of aqueous solutions of pectin and its hydrolysates was assessed at various concentrations in the shear range from 0.1-100 s⁻¹. All solutions except PH 60 displayed a non-Newtonian shear thinning behavior as viscosity decreased with increasing shear rates (Fig. 2a–c). Reduction in viscosity with increasing shear rates could be attributed to the fact that increasing shear rates caused a reduction in the aggregation of molecules due to disruption of entanglements and molecules started aligning in the direction of flow (Hussain et al. 2015). The viscosity values of pectin and its hydrolysates increased with increasing concentrations and thus depicted the dependency of viscosity on concentration which could be due to the restriction in chain mobility that occurred at higher concentrations resulting into formation of entanglements to form a thick solution.

Fig. 2.

Viscosity curve (a, b, c) and Flow behavior curves (d, e, f) of NP (native pectin), PH 30 and PH 60 (pectin hydrolysate) at different concentrations

The flow measurement data was fitted to Herschel-Bulkley and Power law model and flow parameters shown in Table 2 and Fig. 2d–f revealed that Herschel-Bulkley model describes the flow behavior better with higher regression coefficients (R²). Yield stress was observed for all samples which indicated the presence to some cross-linked structure that must be broken down to initiate flow and an increase in yield stress was observed with increasing concentration. The n value reflects the flow behavior i.e. whether the sample is Newtonian or non-Newtonian. The n value closer to 1 shows the Newtonian fluid while n < 1 shows the non-Newtonian behavior. It was observed that NP and PH 30 showed pseudoplastic behavior, but the degree of pseudo-plasticity decreased after hydrolysis. The values for n decreased with increasing concentration of NP and PH 30 suggesting the formation of entanglements at higher concentration. However, no significant differences (p < 0.05) were observed as the concentration of PH 60 increased and it displayed a Newtonian like liquid behavior. This was further confirmed by consistency values (K) which were observed to be maximum for NP at 8% concentration while almost negligible values were obtained for PH 60 at all concentrations. There might be a possibility that the concentration of pectin hydrolysates was not sufficient to cause any entanglements in the solution, thus concentrations above 8% could initiate aggregations and thus their thickening effect can be explored. It can be suggested from flow results that controlled hydrolysis can be used to obtain desired molecular weight range having specific applications. Pectin hydrolysates can be used to increase the dietary fiber content particularly, in liquid food products such as beverages without modifying their rheological properties.

Table 2.

Flow parameters of native and hydrolyzed pectin at different concentrations

Samples	Power n	Law K (Pa sn)	Model R2	Herschel σ (Pa)	Bulkley n	Model K (Pa sn)	R2
4% NP	0.84 ± 0.02c	0.73 ± 0.04b	0.91	0.06 ± 0.01a	0.80 ± 0.02ab	1.87 ± 0.76b	0.99
6% NP	0.74 ± 0.03c	4.05 ± 0.15c	0.91	0.31 ± 0.03a	0.66 ± 0.02ab	5.86 ± 0.10c	0.99
8% NP	0.71 ± 0.02c	11.41 ± 0.42d	0.93	3.70 ± 0.72c	0.52 ± 0.01a	29.21 ± 1.26d	0.99
4% PH 30	0.86 ± 0.02c	0.05 ± 0.01a	0.86	0.03 ± 0.01a	0.92 ± 0.01b	0.03 ± 0.00a	0.99
6% PH 30	0.59 ± 0.05b	2.22 ± 1.13c	0.84	1.35 ± 0.66b	0.77 ± 0.02ab	0.43 ± 0.17a	0.99
8% PH 30	0.56 ± 0.04ab	2.55 ± 1.15c	0.81	1.62 ± 1.10b	0.59 ± 0.03a	2.17 ± 0.97b	0.99
4% PH 60	0.38 ± 0.03a	0.16 ± 0.02a	0.56	0.34 ± 0.07a	1.02 ± 0.04bc	0.005 ± 0.00a	0.80
6% PH 60	0.44 ± 0.14ab	0.23 ± 0.14a	0.64	0.34 ± 0.05a	1.38 ± 0.33c	0.001 ± 0.00a	0.44
8% PH 60	0.39 ± 0.11a	0.53 ± 0.06a	0.62	0.57 ± 0.32a	1.21 ± 0.19c	0.189 ± 0.17a	0.82

Values are mean ± S.D of triplicates

Values with different letters in the same column are significantly different (p < 0.05)

Dynamic rheological behavior

Frequency sweep measurements were done to determine the viscoelastic behavior of pectin and its hydrolysates at different concentrations and frequency parameters i.e. storage modulus (G′) and loss modulus (G″) are used to differentiate polymer solutions and gels. Frequency sweep tests were only performed for NP and PH 30 because PH60 displayed almost Newtonian behavior with negligible consistency even at the highest concentration used and linear viscoelastic (LVE) range could not reach as the instrument was not able to determine the strain value. Thus, only flow properties were determined for PH 60. The rheograms of both NP and PH 30 showed that the magnitude of G′ and G″ increased with increase in frequency (ω). It was interesting to note that only NP showed elastic behavior and crossover of G′ and G″ occurred indicating the prevalence of entanglements in the solution and representing a gel –like behavior (Fig. 3a). At higher frequencies, the values of G″ were higher than G’ until a crossover frequency after which G’ dominated over G″ at low frequencies. Frequency is the inverse of time and higher frequency means shorter time and vice-versa. Therefore, it can be suggested that native pectin solutions require some time for entanglements to form an ordered gel-like structure. Unlike other polysaccharides like xanthan gum, native pectin displayed the characteristics of weak gel as strong gels have G′ > G″ at high frequencies and G′ < G″ at low frequencies. The possible reason for this behavior could be the absence of divalent ions in the aqueous solution and being a low methoxyl pectin, it requires divalent ions to form gels. The magnitude of G’ and G″ increased when concentration increased from 4 to 6% but decreased at 8% concentration. However, opposite trend was seen for PH 30 solution, which displayed a viscous behavior instead of elastic nature (Fig. 3b). The values for G″ were higher than G’ throughout the frequency range and the magnitude of G’ and G″ increased with increasing concentration indicating liquid like behavior of pectin hydrolysate. This behavior can be attributed to low molecular weight and viscosity of the PH 30 solutions which resulted from degradation of the polymer chain after acid hydrolysis. Molecular entanglements are the function of intrinsic factors and extrinsic factors and play an important role in determining the functionality of pectin as gelling and thickening agent (Li et al. 2013).

Fig. 3.

Dynamic rheological properties (a, b) mechanical spectra and (c, d) loss tangent (tan δ) of NP and PH 30 at different concentrations measured at 25 °C

Tan δ is the ratio of G’ and G″ and used to classify polymers on the basis of their viscoelasticity. Tan δ > 1 indicates the prevalence of viscous behavior while tan δ < 1 reveals the elastic character of the polymer. As can be seen from Fig. 3c the tan δ values of NP were lower than 1 indicating the dominance of elastic behavior due to its higher molecular weight and greater branching. The tan δ values decreased with increasing concentration suggesting the formation of aggregations through hydrogen bonding in the polymer solution. The tan δ values for PH 30 were higher than 1 indicating the liquid like behavior in a concentration dependent manner (Fig. 3d). These results showed the dependency of G’ and G″ on frequency and concentration. The concentration of pectin hydrolysate might not be sufficient to induce entanglements in the solutions as molecules moves freely in the dilute solution apart from each other. Therefore, these results suggested the potential of partial hydrolysates to be used at higher concentrations in the food systems without modifying its rheological properties.

Pasting properties

Rapid visco analyzer (RVA) was used to assess the influence of temperature on the viscosity of pectin and its acid hydrolysates. Pectin is a water-soluble polysaccharide and its viscosity depends on its molecular weight and dissolution temperature. It was observed that viscosity of the aqueous solutions of both pectin and its hydrolysates increased in a concentration dependent manner (Fig. 4a). It was evident from the RVA results that temperature had a prominent influence on the RVA viscosity and viscosity continued to decrease till the temperature remained at 80 °C. However, as the temperature began decreasing, the viscosity started rising reaching the final viscosity. This effect can be explained in terms of the intra and inter molecular associations that occurred in the aqueous solutions at low temperatures. Thermal transitions led to the increased mobility of molecules resulting into decreased viscosities, whereas low temperatures restricted the movement of molecules resulting into increased molecular interactions to form an entangled network through hydrogen bonding (Morris 1990). Acid hydrolysis caused a significant reduction in viscosities of pectin solutions. Maximum viscosity was found for 6% NP (1090cp) while minimum for 4% PH60 (7cp). There was a 90% reduction in the viscosity after 30 min hydrolysis followed by 98% reduction after 60 min hydrolysis at a same concentration of 6%. It was also observed that the lowering of temperature did not induced any changes in the viscosity of the PH 60 which was also confirmed by the flow behavior results revealing a Newtonian behavior. Reduction in the chain length of polysaccharide due to degradation of glycosidic bonds upon acid hydrolysis might be the cause of decrease in viscosity. Viscosity is directly linked to molecular weight and chain length; higher the molecular weight higher will be the viscosity. The obtained RVA results are also consistent with results obtained for other parameters of the study. Overall these results suggest the suitability of RVA to differentiate polysaccharides on the basis of viscosity and to assess their suitability for different food applications.

Fig. 4.

Viscometric RVA profiles (a), FT-IR spectra (b) and thermal scans (c) of pectin and its hydrolysates

FT-IR studies

The FT-IR spectroscopy was used to study the possible structural changes in the pectin after being subjected to acid hydrolysis as acid degradation might have induced some chemical changes in the structure of pectin. The acid hydrolysates showed the characteristic peaks of pectin in the FT-IR spectrum with a slight difference in the peak intensities as shown in Fig. 4b. The dominant peaks in spectral region between 3000 and 3600 cm⁻¹ of NP (3565, 3383 and 3307), PH 30 (3274) and PH 60 (3275) indicates the stretching of O–H groups and water involved in the hydrogen bonding (Wang et al. 2014). The second peak near 2900 cm⁻¹ was common for all samples and represented the stretching of C-H bond of CH, CH₂ and CH₃ methyl esters of galacturonic acid (Liu et al. 2010). The peak around 1735 cm⁻¹ contributed to the stretching of ester carbonyl group (C=O). Acid hydrolysis caused an appearance of the stretching of COO group around 1600 cm⁻¹ and peaks at this region were only observed for hydrolysates which is an indicative of the reduction in the degree of methylation in hydrolysates as compared to NP. The peak around 1410 showed the presence of uronic acids (Lopez-Torrez et al. 2015). The modification of the peak shape and intensities reveals the different arrangement of molecules in the structure of intact and degraded pectin including decrease in the molecular weight and viscosity. Moreover, intense peaks were observed for acid hydrolysates which can be ascribed to the increased association of pectin with water which in turn partially explains the improved solubility of acid hydrolysates in comparison to native pectin. The peaks in the spectral region 1010–1150 cm⁻¹ was indicative of the presence of pyranose rings in the molecules (Wang et al. 2014). The peak around 915 cm⁻¹ and 860 cm⁻¹ revealed the presence of D-glucopyranosyl and b-D-mannose in the structure of native pectin as well as acid hydrolysates (Zhang et al. 2013). Overall, FT-IR results revealed no major structural changes occurred after acid hydrolysis as both hydrolysates had superimposable spectra to that of NP.

Thermal properties

DSC is an important tool used to study the transitions occurring in the polysaccharide during heating in an inert atmosphere. Also, the thermal stability of pectin as a promising food additive is of important consideration to determine their applicability in foods which are thermally processed and subjected to high temperatures such as baking and sterilization (Hussain et al. 2018). The DSC thermograms as shown in Fig. 4c showed endothermic peaks in the temperature from 50–150 °C for all samples. Single endothermic peak was observed for native pectin while two endothermic peaks were found for acid hydrolysates. The thermal events in the first peak around 100 °C contributed to the loss of water resulting from evaporation and dehydration. The second peak for acid hydrolysates occurring between 150–200 °C might be due to cleavage of the glycosidic bonds of the polymer backbone. Also, wide peak was observed in the case of NP as compared to acid hydrolysates and indicates the heterogeneity in the molecular weight distribution of NP. These results are also consistent with PDI results of the GPC study. In the endothermic events, the onset, peak and conclusion temperature moves towards higher temperature side whereas ∆H decreased suggesting the degradation of pectin molecular structure after acid hydrolysis.

Above 200 °C exothermic events occurred which corresponded to the thermal decomposition and elimination of the volatile products (Combo et al. 2013). A decrease in To, Tp, Tc and ∆H was observed for PH 30 and PH 60 indicating less thermal stability of acid hydrolysates in comparison to NP. A slight reduction of about only 2% was observed in the peak temperatures after 30 min hydrolysis followed by 7% reduction after 60 min of hydrolysis. Similarly, ∆H decreased by 30% followed by 50% reduction after hydrolysis of 30 and 60 min respectively. This clearly indicated the complex structure of native pectin in comparison to acid hydrolysates and much higher energy was needed to decompose the intact polymer. Partial hydrolysis of pectin led to a reduction in the thermal decomposition temperatures and provides useful information in the characterization of pectin hydrolysates for incorporating into food products as a dietary ingredient and can also be added to the products that are subjected to high temperatures such as breads, biscuits and cakes. Similar results have been reported for depolymerized sugar beet pectin (Combo et al. 2013).

Conclusion

The NP was found more viscous having high molecular weight and PDI in comparison to its acid hydrolysates. The decrease in molecular weight and intrinsic viscosity was observed to be a function of time and increased hydrolysis times caused reduction to the greater extent. Water absorption and swelling power decreased while oil absorption capacity and solubility increased after acid hydrolysis. Flow results showed that P and PH 30 exhibited a non-Newtonian pseudoplastic behavior in a concentration-based manner while PH60 displayed a Newtonian behavior. This was further confirmed by oscillatory experiments where PH 30 showed a liquid like behavior whereas NP showed an elastic behavior at low frequencies. Rheological and RVA behavior can provide useful information regarding the applicability of native pectin and its hydrolysates in food systems particularly liquid products such as beverages. It can be concluded from the present study that acid hydrolysis provides an approach to obtain hydrolysates varying in molecular weight with specific desired applications.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.