Microwave-accelerated heating in preparation of pectin nanoparticles cross-linked with Mg²⁺: process optimization and lyophilized product characterization

Abstract

Pectin nanoparticles (PNPs) having both pectin and nanomaterials properties, and have been used in food, pharmaceutical and cosmetics formulations for developing products with unique properties. In the present study, PNPs were prepared using ionotropic gelation method and Mg²⁺, as cross-linked ion, under microwave-accelerated heating technique. PNPs in the colloidal form were then lyophilized to prepare PNPs in the powder form. Results indicated that using obtained optimum amount of Mg²⁺ to pectin volume ratio (0.1) and microwave heating time (155 s) using response surface methodology, PNPs with minimum particle size (390 ± 10 nm) and polydispersity index (0.312 ± 0.010) and maximum zeta potential (-23.17 ± 1.5 mV) were produced in the colloidal form. The colloidal form of the PNPs was subjected to lyophilization conditions (pressure and temperature of 100 Pa and − 60 °C, for 48 h) and physico-chemical properties of the prepared PNPs in the powder, were assessed by Fourier transform-infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy and dynamic light scattering methods. Results indicated that the lyophilized PNPs were fabricated in rod shape with amorphous state and smooth surface. Results indicated that by decreasing the concentration of PNPs in the solutions from 1 to 0.01%, viscosity and density also decreased from 1.514 to 1.208 cp. and 1.0045 to 1.0017 g/mL, respectively. Preparation of PNPs as a promising bridge between natural polysaccharides and advanced nanotechnology applications, can explore PNPs role in various formulations and new products.

Introduction

Pectin, as a natural polymer, is composed of galacturonic acid units which found in various fruits and vegetables, especially citrus peel (e.g. orange, lemon, grapefruit and tangerine fruits)¹. This polysaccharide has been widely used in food and medicine areas. In food, pectin has been used as thickening, gelling, emulsifying and stabilizing agents for preparation of traditional and novel products (e.g. beverage, jam, juice sauce, and jelly)². In medicine, pectin utilizes as an effective nasal and oral drug delivery carrier for extending drug half-life and controlled release³. As compared to the pectin in its bulk, pectin nanoparticles (PNPs) have unique properties with numerous potential applications because of those higher surface to volume ratio⁴. In fact pectin is a molecule and PNPs compose various pectin chains and have high surface, which that increases attachment and reactivity of the PNPs into other molecules and drastically enhance PNPs properties and activities, such as excellent film forming properties, water solubility, and high biodegradability, bioactivity, bioavailability, bio-accessibility and biocompatibility⁵.

Generally, ionotropic gelation (IG) technique, which is known as a controlled diffusion method has been utilized in formation of the PNPs⁶. In IG process negatively charged of the main functional groups of pectin, including carboxyl and hydroxyl, react with positively charged divalent or multivalent ions such as, Mg²⁺, Ca²⁺ and Zn²⁺⁷. In fact, in preparation of PNPs, cross-linked ions interact with two carboxyl groups in the different pectin chins and the polymeric chins are combined together and formed an egg-box model⁸. Schematic of the preparation of PNPs using IG method shows in Fig. 1. Among the utilized cations, Magnesium has the lowest atomic mass (24.31 u) compared to calcium (40.1 u) and zinc (65.38 u). Additionally, the electron configurations of Mg (3s²), Ca (4s²), and Zn (3d¹⁰4s²) suggest that Mg has the fewest electron shells, potentially contributing to more efficient cross-linked agent⁹.

Fig. 1.

Schatic of preparation of PNPs using ionotropic gelation method.

IG is a time-consuming process which, by its conjugation into other heating- accelerated techniques such as, ultrasonication, microwave, UV radiation and hydrothermal, it is possible to overcome that¹⁰. Due to microwave positive influences including rapid heating, uniform temperature distribution, more thermal effectiveness, nucleation promotion and formation of mono-dispersed particle size distribution, microwave-accelerated heating technique in combination to IG process has been efficiently utilized in organic NPs fabrication¹¹. Microwaves are electromagnetic waves with frequency of 200 MHz to 300 GHz. Frequency of 915 MHz is mostly uses in industrial applications, and the frequency of 2.45 GHz is common for microwave laboratory setups¹². Microwave penetrates into the polar and ionic materials and releases energy, therefore heat generates throughout inside of the materials¹³. For materials with low thermal conductivities such as polymers and ceramics, microwaves rapidly penetrate inside of the materials and significantly decrease processing time¹⁴.

Several technological and non-processing parameters have key effects on the characteristics of the formed organic NPs such as PNPs Those are including pH, temperature and pressure of the mixture solution, type and amount of biopolymer and cross-linked ions, biopolymer to cross-linked salt ratio, type of the heating-accelerated methods and those processing conditions including frequency, power and heating time¹⁵. In order to prepare PNPs with desirable characteristics such as small particle size and polydispersity index (PDI), and high stability (zeta potential value), optimization of the mentioned parameters is necessary¹⁶. Response surface methodology (RSM) as a statistical and mathematical tool has been used to product formulation and process optimization with numerous independent variables and responses. RSM has numerous advantages such as reduced runs of experiment, visual interpretation, handles interactions and curvature, predictive modeling, efficient optimization, versatility across fields, and cost and waste reduction¹⁷. In the present study an optimized novel combination of IG and microwave-accelerated heating methods, coupled with lyophilization was developed to prepare PNPs, which aligns with the principles of green chemistry.

Present study focused on, (i) optimization of processing parameters namely, microwave heating time (s) and volume ratio of cross-linked salt (Mg²⁺) solution to the pectin solution for preparation of PNPs with smallest particle size and highest stability (zeta potential), (ii) preparation of lyophilized PNPs using obtained optimum processing conditions for the fabricated PNPs in the colloidal form, and (iii) assessment of physico-chemical characteristics of the lyophilized PNPs.

Materials and methods

Materials

According to our previous study, extracted pectin from orange peel (with esterification degree, DE of 87), was used to prepare PNPs⁹. Potassium bromide (KBr), Magnesium chloride (MgCl₂) and distilled water (DW) were purchased from Merck Company (Darmstadt, Germany) Dr. Mojallali Company (Tehran, Iran), respectively.

PNPs preparation

According to the literature studies and based on IG method, MgCl₂ (0.03% W/V) and pectin (0.07% W/V) solutions were prepared and filtered by 0.2 μm nylon syringe filter and Whatman filter paper No. 1, respectively^9,18. For preparation of PNPs, a domestic microwave oven (MH8265DIS, LG Co., Seoul, South Korea) with 42 L capacity and constant power, voltage and frequency of 1200 W, 230 V and 50 Hz, respectively, was used and based on central composite design (CCD) defined volume ratios of the prepared Magnesium solution to the pectin solution were provided and exposed to the microwave irradiation for the defined times. The prepared PNPs in the colloidal form were then stored at closed tubes for additional analyses and process. For preparation of PNPs in the powder form, the provided PNPs in the colloidal form was placed into a laboratory freezer at − 20 °C for 48 h, and the frozen samples were then placed laboratory freeze dryer (Sahand Bioprocess Equipment Co., Tabriz, Iran) adjusted at pressure and temperature of 100 Pa and − 60 °C, for 48 h¹⁹. The lyophilized PNPs were used for further analyses.

Analyses

PNPs in the colloidal form

Polydispersity index (PDI), particle size and zeta potential values of the produced PNPs in the colloidal solution were assessed using a dynamic light scattering (DLS) particle size analyzer (Nanotrac Wave, Microtrac, USA)²⁰. pH values of the prepared solutions containing pectin, MgCl₂ and PNPs were measured using a laboratory digital pH meter (211, Hanna, Italy).

PNPs in the lyophilized form

Fourier transform-infrared spectroscopy (FT-IR)

Main functional groups presented in the fabricated PNPs were monitored by FT-IR spectroscopy (Tensor 27, Germany) in wavenumber range of 4000 to 400 cm^− 1 and using KBr pellets.

X-ray diffraction (XRD)

Microstructure of the resulted PNPs including amorphous and crystalline states of the formed PNPs was assessed by XRD (D5000, Siemens Co., Germany) using Cu Ka radiation.

Scanning electron microscopy (SEM)

Shape, size and porosity of the lyophilized PNPs were evaluated by scanning electron microscopy (SEM, mv 2300, Com scan, USA) and atomic force microscopy (AFM; NanoMagnetics EzAFM, Oxford, UK) was used to detect surface of the formed samples.

DLS

DLS also was used for determination of the particle size, PDI and zeta potential values of the lyophilized PNPs.

Rheological assessment

Viscosity and density of the prepared samples were measured using CAVIS-Capillary viscometer (Nanterre, France) using an Ostwald 150 capillary tube. the viscosity and density of the samples were expressed based on cp. and g/mL, respectively²¹.

Experimental design and data statistical analysis

CCD was selected to design of experiments according to two selected independent parameters including, volume ratio of Magnesium solution to pectin solution (X₁, 0.1–0.9) and microwave heating time (X₂, 60–180 s). According to our preliminary tests, the ranges for the Mg to pectin ratio in the solutions and microwave heating time were selected to the preparation of PNPs. According to Tables 1 and 13 experiments in randomized form, were generated by CCD, having 1 block and 5 repeated center points. RSM was also chosen to predict the values of particle size (Y₁, nm) and zeta potential (Y₂, mV) of the resulted PNPs as function of the independent parameters. In order to model of the PNPs preparation, a second-order polynomial equation was used (Eq. 1).

1

Table 1.

CCD and experimental and predicted values for particle size and zeta potential values of the colloidal solutions containing prepared PNPs.

Runs	Volume ratio of Mg to pectin (solutions)	Microwave heating time (s)	Particle size (nm)	Zeta potential (mV)
1	0.8	80	783	-16.85
2	0.8	165	882	-13.33
3	0.5	120	1372	-18.11
4	0.5	120	1478	-18.15
5	0.1	120	821	-22.19
6	0.5	180	*	*
7	0.2	80	1400	-20.83
8	0.5	60	1269	-15.45
9	0.5	120	1478	-18.15
10	0.2	165	685	-19.85
11	0.5	120	1588	-19.47
12	0.9	120	*	-17.46
13	0.5	120	1474	-16.88

* These data were out of range due to human /instrument errors and excluded in the model generation.

Where, the linear (X₁, X₂), quadratic (X₁², X₂²), and interaction (X₁.X₂) effects of the fabricated factors had coefficients of βi and βj, βii and βjj, and βij, respectively. Accuracy and suitability of the generated models were assessed through the coefficient of determination (R²) and the lack-of-fit P-value. Analysis of variance (ANOVA) was also used to study importance of the terms in the generated models based on small P-value (p < 0.05)^20,22. To illustrate the effectiveness of independent parameters on the responses, both three and two-dimensional surface and contour plots were plotted based on the generated models. Optimization procedures, including both graphical and numerical approaches, were employed to identify the optimal region and exact values of the fabricated variables for producing PNPs with small particle size and high stability, as manifested in zeta potential value. Three more additional tests were done using obtained optimum values for the independent variables to prepare PNPs and compared experimental values for the responses with those were predicted by the fitted models by comparison Tukey test with p < 0.05, as confirmation procedure²². All CCD, RSM, ANOVA, model generation and process optimization and validation were accomplished using Minitab software (V. 16, PA, USA).

Results and discussion

Model generation

Based on design of experiment (CCD) and achieved experimental values for the responses (Table 1), two models were fitted to predict the particle size and zeta potential values of the resulted PNPs as function of the chosen fabricated variables, namely, volume ratio of Mg solution to pectin solution and microwave heating time. Coefficients of the model’s terms indicate in Table 2. Furthermore, P-values of the model’s terms, R² and p-value of the lack fit for all the fitted models indicate in Table 3.

Table 2.

Coefficient of the model terms.

Responses	Particle size (nm)	Zeta potential (mV)
Model terms
A0 (constant)	405.06	10.76
A1 (main)	12.88	0.23
A2 (main)	2320.81	-13.52
A11 (quadratic)	-0.10	0.00
A22 (quadratic)	-4679.76	12.27
A12 (interaction)	16.96	-0.05

1: volume ratio of Mg solution to pectin solution.

2: Microwave heating time (s).

Table 3.

P-value, coefficient of determination and lack-of-fit of the model terms.

Responses	Particle size (nm)	Zeta potential (mV)
Model terms
A1 (main)	0.061	0.012
A2 (main)	0.012	0.111
A11 (quadratic)	0.006	0.014
A22 (quadratic)	0.000	0.046
A12 (interaction)	0.003	0.258
R2 (%)	97.45	90.07
Lack-of -fit (p-value)	> 0.05	> 0.05

1: volume ratio of Mg solution to pectin solution.

2: Microwave heating time (s).

Results revealed that only linear effect of the volume ratio of Mg solution to pectin solution, and interaction term of the selected variables had nonsignificant effect on particle size and zeta potential of the formed PNPs in the colloidal form, respectively. High values of R² for all fitted models (higher than 90%) revealed high adequacy of the attained models for predicting of the responses as function of the chosen independent parameters²³. Results also indicated that P-values of lack-of-fit for both provided models were higher than 0.05 (P > 0.05) which those indicated suitable fitness of the models²⁴.

Influences of fabricated parameters on particle size of the prepared PNPs

Particle size of the produced NPs in the colloidal solution has key effect on the physical, chemical and biological attributes of the prepared PNPs²⁵. Generally, by decreasing and increasing size of the formed NPs, their activities increase and decrease, respectively. According to Table 1, particle size of the produced PNPs changed from 685 to 1588 nm. Effectiveness of the independent parameters on the particle size of fabricated PNPs in the colloidal form show in Fig. 2 (A and B). Based on the Fig. 2A, at constant and low, and high ratio of Mg solution to pectin solution, by rising the microwave heating time, particle size of the fabricated PNPs decreased and increased, respectively. This result could be related to the fact that by increasing the microwave heating time, temperature of the colloidal solution increased which that enhanced reaction between pectin chains and Magnesium ions, followed by nucleation and growth of the PNPs. At higher microwave heating times, temperature of the colloidal solution containing formed PNPs significantly increased, which that caused rising in kinetic energy and impact rate of the produced PNPs. These could enhance pectin chain scission and decrease Mg to pectin bonding, and lead to reduction in the particle size of the resulted PNPs²⁶. On the other hand, at constant microwave heating times, by increasing the volume ratio of Mg solution to pectin solution, particle size of the made PNPs increased and decreased, respectively.

Fig. 2.

Surface (A) and contour (B) plots for the particle size of the prepared PNPs as function of volume ratio of Mg solution to pectin solution and microwave heating time (s).

The result could be related to the chemical structure of pectin which, that is composed of galacturonic acid units with carboxylic groups. The negative charge of this functional group increased repulsion of the pectin chains in the colloidal solution and by increasing the amount of Mg²⁺, pectin chains were combined to each other and compacted PNPs with small particle size were produced¹⁸. Presence of the curvature in the Fig. 2A, indicated that interaction term of the selected independent parameters had significant (p < 0.05) effect on the particle size of the produced PNPs and was in agreement with the presented result in Table 3. According to the Fig. 2B, PNPs with minimum particle size prepared using minimum volume ratios of Mg solution to pectin solution and maximum heating times, and maximum volume ratios of Mg solution to pectin solution.

Effectiveness of processing factors on zeta potential of the prepared PNPs

Zeta potential is defined as surface electrical charge density of the prepared NPs which, directly influences the stability of them. In fact, high zeta potential values indicate high repulsion between formed NPs in the colloidal systems and can prevent aggregation or coalescence of them. On the other hand, low zeta potential values show weak repulsion between the formed NPs and causes flocculation or phase separation in the colloidal solution²⁷. Zeta potential is highly pH-dependent and results indicated that pH values of the pectin and Mg solutions were 6.6 and 6.9, respectively. Furthermore, pH values of the prepared 13 colloidal solutions containing formed PNP (Table 1) were constant and nearby 6.7 ± 0.1. As clearly observed in Table 1, zeta potential of the produced PNPs varied from − 13.33 to -22.19 mV, which indicates moderate to high stability of the formed PNPs. Influences of the volume ratio of Mg solution to pectin solution and microwave heating time on the zeta potential of the resulted PNPs present in Fig. 3 (A and B). As can be seen in Fig. 3A, at constant volume ratio of Mg solution to pectin solution, rising in heating time caused increasing and decreasing in the zeta potential value of the formed PNPs, respectively. On the other hand, at constant microwave heating time, by increasing the volume ratio of Mg solution to pectin solution, zeta potential of the resulted PNPs decreased.

Fig. 3.

Surface (A) and contour (B) plots for the zeta potential of the prepared PNPs as function of volume ratio of Mg solution to pectin solution and microwave heating time (s).

Result could be related to the fact that, heating promotes dissociation of carboxyl groups (COOH), increasing the number of negatively charged sites (COO⁻) on the PNPs. More freely COO⁻ groups remain exposed, leading to a higher (more negative) zeta potential. Extended heating time, allows Mg²⁺ to bind more COO⁻, reducing the number of free charges²⁸. Absence of the curvature in this figure demonstrated that interaction term of the selected variables had nonsignificant effect on the zeta potential of the made PNPs and was in line with the presented result in the Table 3. As clearly observed in the Fig. 3B, PNPs with maximum zeta potential were prepared using minimum volume ratio of Mg solution to pectin solution.

Optimization of the PNPs preparation process

Graphical optimization for preparation of PNPs with small particle size and high zeta potential values shows in Fig. 4 (white zone).

Fig. 4.

Graphical optimization based on overlade contour plot for prediction area zone (white) for both selected independent variables.

Furthermore, numerical optimization based on generated models indicated that PNPs with small particle size (387 nm) and high zeta potential (-22.00 mV) were prepared using volume ratio of Mg solution to pectin solution and microwave heating time of 0.10 and 155 s, respectively. Three additional experimental runs were done to produce PNPs using attained optimal values for the processing parameters and statistical analysis indicated that there were nonsignificant differences between the experimental values of the particle size (390 ± 10 nm) and zeta potential (-23.17 ± 1.5 mV) and their predicted values, which validated the experimental design, modeling and optimization of the PNPs preparation procedure using CCD and RSM. Particle size and zeta potential distributions of the resulted PNPs using achieved optimum volume ratio of Mg solution to pectin solution and microwave heating time show in Figs. 5A and B, respectively. Based on Fig. 5A, highlighted relatively sharp and narrow peak indicated that the prepared PNPs had moderate uniformity in size and was in line with small value of the obtained PDI for the formed PNPs (0.312 ± 0.010)²⁰. According to Fig. 5B, narrow distribution around a high absolute zeta potential (-23.17 mV) indicated strong repulsion and good stability of the formed PNPs in the colloidal solution. Jacob et al. fabricated PNPs using high methoxylated pectin using Mg²⁺, as crosslinker with particle size and zeta potential values of 850 nm, -28.1 mV, respectively¹⁸. Jonassen et al.²⁹ also prepared PNPs using low and amidated low methoxylated pectin and Zn²⁺, as crosslinker, with particle size and zeta potential values ranging 360 to 620 nm and 17 to -24 mV, respectively. Ergin et al.³⁰ also produced PNPs using Ba²⁺, as crosslinker, with particle size and zeta potential values ranging 203.7 to 552.7 nm and − 10.9 to -25.5 mV, respectively. Generally, zeta potential value of the prepared PNPs has indirect relationship with pectin degree of esterification (DE). In low DE pectin, there is high numbers of free COO⁻ group in the pectin structure which some of them are interacted with cationic crosslinkers and most of them are remain in their free state and increase zeta potential value of the formed PNPs^21,28.

Fig. 5.

Particle size (A) and zeta potential (B) distributions for the prepared PNPs using obtained optimal conditions.

Characteristics of the produced lyophilized PNPs using achieved optimal conditions

FT-IR spectra of the lyophilized PNPs shows in Fig. 6. The main functional groups present in the prepared PNPs were hydroxyl (–OH), carboxylic acid (–COOH), and methyl ester (–COOCH₃) appear in the wavenumber ranges of 3745 –3300 cm^{− 1}, 1745 –1440 cm^{− 1}, and 1100 –1000 cm^{− 1}, respectively. The centered peaks at wavenumbers of 2927, 1260 –1100 cm⁻¹ were related to C–H ether bonds linking galacturonic acid units (R-O-R)²². The centered peak at 823.71 cm^{− 1} was related to the presence of Mg²⁺ in the prepared PNPs and reconfirmed by findings of Jacob et al.¹⁸.

Fig. 6.

FT-IR spectra of the prepared Mg cross-linked PNPs using attained optimal conditions.

Figure 7 (A, B, C and D) indicated SEM images of the prepared PNPs using obtained optimum conditions with different magnifications of 1, 10, 250 and 500. As can be seen in these figures, the prepared PNPs had rod-shape with high porous structure which that was related to the dual role of freeze drying in development of cross-linked pectin chains and enhancing their porosity as a result of the nucleation of ice, following by dehydration in a fast and single process³¹. This result with in agreement with findings of Jacob et al.¹⁸.

Fig. 7.

SEM images of the prepared Mg cross-linked PNPs using attained optimal conditions with different magnifications of 1 (A), 10 (B), 250 (C) and 500 X (D).

Figure 8 shows XRD pattern of the fabricated PNPs using attained optimum values for the independent variables. Result indicated that there was a broad peak centered at 2θ value of 20°. In our previous study, XRD result indicated that the extracted pectin from orange peel (with high DE) had amorphous state and two highlighted peaks were observed at 2θ values of 13 and 20°⁹. Dixit et al., also reported that extracted pectin from citrus peels by ethanol and under acidic conditions had mainly amorphous state and their XRD patterns have distinctive peaks at 2θ values of 13.56 and 22.56°³². Obtained result was in line with findings of Ergin et al.³⁰. They also found that the prepared PNPs had a peak around 2θ value of 20° and semi-crystalline structure.

Fig. 8.

XRD pattern of the prepared Mg cross-linked PNPs using attained optimal conditions.

Figure 9, indicates AFM image of the formed PNPs under microwave heating and attained optimum conditions. According to this figure, surface of the formed PNPs was flat. Furthermore, sample appears to be in the form of a polymer network rather than discrete particles. It seems that due to partial hydration of the formed PNPs during storage, the samples were agglomerated and formed the polymeric chains This result was inline with finding of Jonassen et al.²⁹.

Fig. 9.

AFM image of the prepared Mg cross-linked PNPs using attained optimal conditions.

Table 4, indicates viscosity and density of the prepared colloidal solutions containing different concentrations of pectin and the prepared PNPs (1, 0.1 and 0.01%). Generally, extracted pectin from citrus peels, behaves like Newtonian liquids up to their 3% concentrations. More than this certain concentration, pectin solution transforms into pseudoplastic behavior³³. Dilute solutions of pectin contain homogenously dispersed pectin molecules that are too far apart to interact with one another and thus there is only slight increase in viscosity due to distortion in the velocity pattern of the liquid by an increased proportion of hydrated molecules³⁴. Results demonstrated that by increasing the concentrations of both pectin and PNPs in the solutions density also increased (Table 4). As can be seen in Table 4, viscosity of the solution containing of the prepared PNPs (1%) was lower than that of solution containing pectin (1%). It seems in the case of pectin, many pectin chains aggregated and increased the viscosity of the solution, rapidly. However, in the case of PNPs, due to small particle size, high surface to volume ratio and lower amounts of pectin chains, the viscosity of solution is lower than that of the solution containing pectin. Results also indicated that in both solutions containing pectin and PNPs, by increasing of their concentrations from 0.01 to 1%, density of the solutions also increased. According to Table 4, density of the solution containing of the prepared PNPs (1%) was higher than that of solution containing pectin (1%). Obtained results could be related to higher surface to volume ratio of the prepared PNPs, which that causing higher water absorption of the PNPs, as compared to that of the pectin³⁵.

Table 4.

Density and viscosity of the pectin and prepared PNPs solutions with three different concentrations.

Samples	Concentration (% W/V)	Density (g/mL)	Viscosity (cp.)
Pectin	1	1.0038 ± 0.0001b	2.128 ± 0.05a
	0. 1	1.0016 ± 0.0002cd	1.245 ± 0.08c
	0. 01	1.0014 ± 0.0001d	1.184 ± 0.02d
PNPs	1	1.0045 ± 0.0001a	1.514 ± 0.03b
	0. 1	1.0019 ± 0.0001c	1.222 ± 0.02c
	0. 01	1.0017 ± 0.0002cd	1.208 ± 0.05c

Values were expressed as the mean ± standard deviation (n = 3).

Different small letters in the same column indicate significant (p < 0.05) differences.

Conclusion

Results of the present study indicated that selected independent variables, namely microwave heating time and volume ratio of Mg solution to pectin solution had strong on physico-chemical properties of the formed PNPs. In fact, by increasing microwave heating time, temperature of the colloidal solution containing formed PNPs, its kinetic energy and impact rate of the produced PNPs enhance which those cause nucleation promotion and formation of uniform PNPs with small particle size PNPs. Furthermore, results indicated that by increasing the volume ratio of Mg solution to pectin solution in solution, zeta potential of the resulted PNPs decreased. Optimization of these two synthetic parameters resulted PNPs with small particle size and high stability in the colloid form. However, further optimizations for other processing parameters such as, pH value of the solutions, and concentrations of pectin and cross-linked ion, can improve the specification of the formed PNPs. Lyophilization also successfully resulted rod-shaped, porous and amorphous PNPs, which had different particle size, stability, viscosity, and density in the solutions, as compared to the pectin. Prepared PNPs using developed method in the present study for can be used easily in numerous food and pharmaceutical formulations and products to improve their physico-chemical and organoleptic attributes. Lack of reproducibility and stability of the prepared PNPs due to its source variability, degree of esterification and amidation, are the main limitations of PNPs preparation. Furthermore, Lab-scale methods including ionotropic gelation, microwave heating and lyophilization are not easily scalable to industrial production.

Author contributions

S. B.: Methodology, Software, Resources, Formal analyses, Data curation. H. J.-M.: Supervision, Project administration, Conceptualization, Investigation, Writing – review and editing. N. R.: Supervision, Validation.

Funding

This research was funded by the Sahand University of Technology (grant no. 1057).

Data availability

Data will be made available on request.

Competing interests

The authors declare no competing interests.