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Characterization and swelling properties of composite gel

microparticles based on the pectin and κ-carrageenan

Elena A. Günter, Vladislav V. Martynov, Vladislav S. Belozerov,

Ekaterina A. Martinson, Sergey G. Litvinets

PII: S0141-8130(20)34111-8
DOI: https://doi.org/10.1016/j.ijbiomac.2020.08.024
Reference: BIOMAC 16399

To appear in: International Journal of Biological Macromolecules

Received date: 4 March 2020

Revised date: 7 May 2020
Accepted date: 3 August 2020

Please cite this article as: E.A. Günter, V.V. Martynov, V.S. Belozerov, et al.,
Characterization and swelling properties of composite gel microparticles based on the
pectin and κ-carrageenan, International Journal of Biological Macromolecules (2018),
https://doi.org/10.1016/j.ijbiomac.2020.08.024

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© 2018 Published by Elsevier.

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Characterization and swelling properties of composite gel microparticles based on the

pectin and -carrageenan

Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov a,b, Ekaterina A. Martinson
b
, Sergey G. Litvinets b

a
Institute of Physiology, Komi Science Centre, The Urals Branch of the Russian Academy of

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Sciences, 50, Pervomaiskaya str., Syktyvkar, 167982, Russia

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b
Vyatka State University, 36, Moskovskaya str., Kirov, 610000, Russia
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Abstract
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The aim of this research was to produce composite gel microparticles based on the pectin

(campion callus culture (SVC) and commercial apple (AU) pectins) and -carrageenan and
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investigate the relationship between the characteristics and swelling properties of the composite
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microparticles. The microparticles were obtained using emulsion dehydration techniques with
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successive incubation in calcium chloride solution. A significant positive correlation between the

Ca2+ content and the SVC concentration in gel formulations was shown. Decreasing degree of

methylesterification (DM) of the SVC pectin promoted Ca2+ binding in comparison with the AU

pectin. Increasing concentration of the pectin promoted increasing gel strength of the composite

microparticles. The higher gel strength of the composite microparticles based on the SVC pectin

was probably due to the lower DM and a higher linearity in comparison with the AU pectin. The

microparticle gel formulations with a higher pectin concentration had a lower swelling degree in

the simulated digestive fluids. The addition of the carrageenan to the gel formulations led to an

increase in the swelling degree in comparison with that without carrageenan. The correlation

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analysis indicated that increasing initial Ca2+ content and gel strength of the microparticles

promoted decreasing swelling degree of the composite microparticles.

* Corresponding author. Tel.: +7 8212 241001; fax: +7 8212 241001.

E-mail address: gunter-ea@mail.ru (E.A. Günter).

Keywords: composite gel microparticles; pectin; -carrageenan.

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1. Introduction

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Pectin is non-toxic, biodegradable and biocompatible polymer. This polysaccharide
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presents in the cell wall of all higher plants [1]. Pectins are used in the pharmaceutical industry
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as a carrier in colon-targeted drug delivery systems [2] and in food industry for binding,
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thickening, emulsifying and gelling [1]. Moreover, pectins possess cation-binding capacity and

they are used as biosorbents to remove toxic metal ions from humans or heavy metals from
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wastewater [3]. The pectic polysaccharides include homogalacturonan (HG),

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rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), xylogalacturonan (XGA) and

apiogalacturonan (AGA) [1, 4]. Until recently, it was accepted that HG, RG-I and RG-II form a
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continuous backbone. However, an alternative macromolecular structure has been postulated in

which HG is a side chain of RG-I, similar as arabinan and galactan [5]. HG is a linear,

unbranched molecule composed of 1,4-linked -D-galacturonic acid (GalA) residues. The GalA

residues can be methyl esterified at C-6 [1, 4, 5]. RG-I is a highly branched polysaccharide. The

backbone of RG-I consists of repeating disaccharide [→-D-GalpA-1,2--L-Rhap-1,4→]n. Part

of the rhamnose residues in RG-I is substituted at O-4 with side chains consisting of a single

sugar unit, mainly arabinose and galactose, or of complex polymers such as arabinogalactan,

arabinan and galactan [1, 4, 5]. RG-II has a highly conserved chemical structure. The backbone

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of RG-II is composed of 1,4-linked -D-GalA residues. The side chains contain eleven different

monosaccharides, among them several rather uncommon sugars, such as apiose, aceric acid and

2-keto-3-deoxy-D-manno-octulosonic acid [1, 4, 5]. Low methoxylated pectin can form a gel in

the presence of calcium ions. Ionic linkages are formed via calcium bridges between dissociated

carboxyl groups. The “egg-box” model describes the close packing of HG that occurs upon Ca2+-

induced gelling [4].

The pectin, an anionic polysaccharide, was combined with different compounds and

polymers to improve its properties [6]. Other polysaccharide that was used for the preparation of

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hydrogels is κ-carrageenan since it has strong gel formation ability, high swelling property and

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high water-holding capacity. Carrageenans are natural, water-soluble, sulfated galactans that are

isolated from marine red algae. These biopolymers are used in food and pharmaceutical as
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gelling, stabilizing and thickening agents and also as fat substitutes in the food industry. [7].
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Carrageenans formed by alternate units of D-galactose and 3,6-anhydro-galactose joined by -
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1,3- and -1,4-glycosidic linkage [8]. There are three most important types of commercially

available carrageenan: monosulfated -carrageenan, bisulfated ι-carrageenan and trisulfated -

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carrageenan, however only the first two can form gels [9]. The differences in the structure of κ-
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and ι-car result in different gelling properties. -Carrageenan can form a strong gel in the
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presence of specific monovalent cations, whereas ι-carrageenan is particularly sensitive to

divalent ions [10]. The gelation of -carrageenan involves coil-to-helix transition upon cooling

and subsequent cationdependent aggregation between helices [11-13]. The gelation of -

carrageenan occurs in the presence of monovalent or divalent cations and their mixtures [9].

However, in spite of wide range of applications, carrageenans have some drawbacks and

adverse effects on the biological systems. Hence, its modifications with natural and synthetic

polymers are performed [8]. Moreover, some limitations of κ-carrageenan based hydrogels, such

as low mechanical properties, have been attempted to solve by forming composites with

functional nanomaterials [14]. New biodegradable materials, blends and composites of

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carrageenan with natural polymers having suitable properties for biomedical applications and

food are gaining interest. Chitosan, starch, cellulose, chitin, alginate-polymer/carrageenan

composites and blends have been used extensively in drug delivery, tissue engineering and in

many other applications [8]. However, a small number of studies have been carried out on the

development of biomaterials based on the pectin and -carrageenan [15-17]. The blending of

natural polymers allows developing new biomaterials that exhibit combinations of properties that

could not be obtained from individual polymers. The blending of pectin and carrageenan allows

obtaining composites with new physicochemical properties that will improve their functionality.

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These composite biopolymers may have potential applications in the pharmaceutical and food

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industry.

Moreover, the method of preparation of composites influences its properties. Usually,

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pectin-calcium gels are obtained using the ionotropic gelation method. However, there is the
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exchange of calcium with the sodium ions in the simulated gastrointestinal fluids, which results
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in the structural changes leading to matrix erosion [18, 19]. In the present study, composite gel

microparticles from pectin and -carrageenan were prepared using the emulsion dehydration
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method with successive incubation in calcium chloride solution that gives particles stable and
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swelling in the simulated gastrointestinal conditions.

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Previously, we demonstrated that the calcium pectinate gel beads obtained from different

callus cultures pectins have different swelling behavior and the release of a drug in vitro from

beads [20-22]. The ability of the pectic gel microparticles to decrease the food intake in mice

after a single administration was shown [23].

The aim of this work was to produce composite gel microparticles based on the pectin

and -carrageenan, to investigate the gel strength, morphology and swelling properties of

composite microparticles made from different pectins and to evaluate the relationship between

the physicochemical characteristics and swelling properties of these microparticles.

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2. Materials and methods

2.1. Materials

The SVC pectin with a low degree of methylesterification (DM 10%) was obtained from

the Silene vulgaris (Moench) Garcke (Oberna behen (L.) I.) callus culture. Commercial apple

pectin (AU) (Classic AU 701) with a DM of 36-44% was purchased from Herbstreith & Fox,

Germany. The chemical characteristics of the SVC and AU pectins previously described [21] are

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shown in Table 1. -Carrageenan was purchased from Sigma, USA. All other chemicals were of

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analytical grade.
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2.2. Isolation and characterization of pectins
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Isolation of the SVC pectin was performed as described earlier [21, 24]. The uronic acid
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content was estimated using a reaction with 3,5-dimethylphenol in the presence of concentrated
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sulfuric acid [25]. Total protein content was determined according to the Lowry method [26].

Spectrophotometric measurements were made with an Ultrospec 3000 instrument (UK). The
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degree of methylesterification (DM) was determined using the method described previously [27].
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The specific viscosity was measured using an Oswald viscosimeter at 25°C in the distilled water.

Pectin average molecular masses (Mw) were determined using HP-SEC on a chromatographic

system (Shimadzu, Japan) by a previously described method [21]. The neutral sugars (% of total

amount) in the form of corresponding alditol acetates were quantified using GC in a Varian 450-

GC (Netherlands) chromatograph using myo-inositol as the internal standard [21, 28]. Using

sugar composition data, three ‘sugar ratios’ were calculated, based on methods from a previous

study [29]. The first sugar ratio measures the linearity of pectin (LP), comparing the pectic

backbone sugar GalA to the neutral pectic sugars (Ara + Gal + Rha + Xyl) of the side chains.

The second ratio compares the proportion of Rha to GalA, indicating the amount of

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rhamnogalacturonan in pectin overall. The third ratio compares the number of

rhamnogalacturonan I side-chain sugars (Ara + Gal) to Rha, measuring the extent of branching

of RG-I (BP) [29].

2.3. Preparation of composite gel microparticles based on the pectin and -carrageenan

The composite microparticles were prepared using the emulsion dehydration method

reported by Esposito et al. (2001) and Vaidya et al. (2009) with modifications [30, 31].

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Formulations of composite gel microparticles are presented in Table 2. Briefly, the pectin (0.9,

1.2, 1.4 or 1.6 %) and -carrageenan (0.1%) were dissolved in distilled water (3 ml) at 70°C.

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This mixture was dispersed into an emulsion composed of 25 ml isooctane and Tween 80
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(1.0%). The dispersion was stirred for 30 min at 1000 rpm (MR, Heidolph, Germany) to obtain
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stable water/oil emulsion. The dispersion was rapidly cooled up to 10°C, and 50 ml of acetone
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was added, in order to dehydrate the pectic droplets. The formulation was continuously stirred at

1000 rpm for 30 min for complete solvent evaporation. The resulting microparticles were filtered
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and incubated in a cross-linking solution of CaCl2 (0.2 M, 25 ml, pH 3.3) for 30 min under
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continuous magnetic stirring at 1000 rpm and washed three times with distilled water to remove

the cations non-cross-linked with pectin. The prepared microparticles were freeze-dried at the ice
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condenser temperature of -55°C and the pressure of 0.021 mbar (Beta 2-8 LD plus, Martin

Christ, Germany). The pectic microparticles without -carrageenan (1.0SVC and 1.0AU) were

referred to as a control.

2.4. Size and morphology of composite microparticles

The dry microparticles (n = 40) of each formulation were characterized according to

projected equivalent diameter (the diameter of a circle with the equivalent area) using an optical

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microscope (Altami, Russia) fitted with a camera and an image analysis system (ImageJ 1.46r

program, National Institutes of Health, USA). An image of a linear scale was used for calibration

under the same optical conditions. One pixel corresponds to 0.024 mm.

The surface morphology was studied using a scanning electron microscope (SEM)

(JEOL, JSM6510LV, USA) at 10 kV. Previously, the dry microparticles were coated with

platinum (ion sputter, 30 sec) using a vacuum spraying machine (JEOL JFC-1600, Japan).

Digital images were obtained at magnifications of 100× (scale bar 100 m) and 1000× (scale bar

10 m).

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2.5. Energy-dispersive X-ray spectroscopy
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The content of Ca2+, carbon and oxygen (wt%) in the microparticles was determined
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using energy-dispersive X-ray spectroscopy (EDX) (Oxford Instruments INCA, United
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Kingdom). Previously, the dry microparticles were coated with platinum (ion sputter, 30 sec)

using a vacuum spraying machine (JEOL JFC-1600, Japan). The accelerating voltage was 20 kV,
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the distance was 12 mm and the probe size was 61-68. The chemical elements were determined
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based on the X-ray spectrum. All analyses were performed in five replicate samples.
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2.6. Texture analysis

A Texture Analyser (TA-XT Plus, Texture Technologies Corp., Stable Micro Systems,

UK) was used for a compression test of the wet gel microparticles. The gel microparticles were

compressed with a 12 mm diameter (P/0.5R) cylinder probe. The pre- and post-test speed was

10.0 mm/s and the test speed was 0.1 mm/s until a deformation of 50%. The experiments were

carried out at 25 °C. All analyses were performed in ten replicate samples. The calculations of

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maximum peaks were performed using Texture Exponent 6.1.4.0 software (Stable Micro

Systems, UK).

2.7. Swelling characterization of composite microparticles

The dry microparticles (5 mg) were immersed subsequently in the simulated gastric

(SGF, 10 ml), intestinal (SIF, 10 ml) and colonic (SCF, 10 ml) fluids for 2, 4 and 18 h,

respectively. The simulated fluids SGF (pH 1.25, KCl 1.12 g/l, NaCl 2.0 g/l, CaCl 2 0.11 g/l,

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KH2PO4 0.4 g/l, 1N HCl), SIF (prepared by adding 1N NaHCO3 to the SGF to obtain a final pH

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7.0) and SCF (pH 7.0, pectinase (EC 3.2.1.15.; 1.7 mg/ml; activity 1.18 U/mg; Sigma, USA))

were prepared according to [32, 33], with modifications. The microparticles were shaken (100
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rpm) in an orbital shaker incubator (Titramax 1000, Heidolph, Germany) at 37 °C. After a
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predetermined time interval, the projected equivalent diameter of microparticles was measured
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as described in Section 2.4. The swelling degree (SD) was calculated using the following

equation [34]: SD% = [(D1-D0) / D0] × 100, where D1 is the diameter of the particle after a
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determined contact time with the liquid and D0 is the initial diameter.
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2.8. Statistical analysis

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The values were given as the mean ± standard deviation (S.D.). The statistical

significance of the differences between two means was evaluated by the Student’s t-test. A value

of p < 0.05 was considered statistically significant. To reveal the relationship between the

studied parameters, the correlation coefficient was calculated, and its significance was assessed.

Statistical calculations were performed with the Excel Microsoft software (version Windows

2010).

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3. Results and discussion

3.1. Characterization of gel microparticles

Increasing concentration of the SVC pectin or apple pectin (AU) from 0.9 to 1.6 % into

gel formulations led to decreasing projected equivalent diameter of dried microparticles (Table

2). To reveal the relationship between the diameter of the microparticles and the pectin

concentration, correlation analysis was performed. A significant negative correlation between the

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diameter and the SVC concentration (R2 = -0.827, p < 0.05) was revealed. The diameter of

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microparticles correlated significantly with the AU pectin concentration (R2 = -0.810, p < 0.05).

This negative correlation indicated that increasing concentration of the pectin into gel
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formulations promoted decreasing microparticle diameter.
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The projected equivalent diameter of the 0.9SVC-0.1Car – 1.4SVC-0.1Car microparticles
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was 1.3–1.5-fold higher than that of the 1.0-SVC microparticles without carrageenan. The

diameter of dried 0.9AU-0.1Car microparticles was 1.4-fold higher than that of the 1.0-AU
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microparticles without carrageenan. The diameter of the 1.2AU-0.1Car – 1.6AU-0.1Car

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microparticles was 1.2–1.4-fold lower than that of the 1.0-AU microparticles.

EDX analysis showed that the content of carbon and oxygen was 50–55 wt% and 43–46
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wt%, respectively. The relationship between the Ca2+ content in the microparticles and the

concentration of the SVC pectin was revealed. A significant positive correlation between the

Ca2+ content and the SVC concentration (R2 = 0.840, p < 0.05) was shown that appeared to be

related to more COO- groups to bind the Ca2+ ions (Table 3). The Ca2+ content in the gel

formulations based on the AU pectin was similar (1.9-2.6 wt%) (Table 2). The Ca2+ content in

the gel formulations based on the SVC pectin (3.6-4.6 %) was higher than that in the

microparticles based on the AU pectin. This difference may be attributed to the DM of pectin

(Table 1). Decreasing DM of pectin promoted Ca2+ binding. The SVC pectin with a lower DM

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had more COO- groups to bind the Ca2+ ions. These findings are in agreement with our earlier

data, which demonstrated that the Ca2+ content was 1.8–1.9-fold higher in the microparticles

from the SVC pectin with the lower DM and higher linearity than that in the microparticles from

the LMC and AU pectins [23]. These data are in agreement with the data of [1] who

demonstrated that the calcium ion-binding capacity of pectin increased with decreasing DM,

when a higher amount of nonmethoxylated negatively charged galacturonic acid residues are

present. Moreover, the OSO3- groups of -carrageenan interact with the Ca2+ ions through ionic

bonds. The -carrageenans form a network of three-dimensional double helices, resulting from

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the “crosslinking” of the adjacent spiral chains that contain OSO3- groups oriented towards their

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external part [12]. Schematic representation of the interaction between Ca2+ and molecules of

pectin or -carrageenan is presented in Fig. 1.

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Increasing concentration of the SVC or AU pectin from 0.9 to 1.6 % into gel formulations
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resulted in increasing initial gel strength of the microparticles (Table 2). A significant positive
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correlation between the initial gel strength and the concentration of SVC (R2 = 0.844, p < 0.05)

or AU (R2 = 0.956, p < 0.05) was shown (Table 3). This correlation indicated that increasing
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concentration of the pectin promoted increasing gel microparticle strength. The lower initial gel
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strength of the microparticles from the AU pectin was probably due to the higher DM and a
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lower linearity in comparison with the SVC pectin (Table 1).

The initial gel strength of the microparticles with carrageenan based on the 0.9–1.4%

concentration of the SVC or AU pectin was 1.3–1.7-fold and 1.5–10.0-fold lower than that of the

1.0-SVC and 1.0-AU microparticles without carrageenan, respectively. Increasing concentration

of the SVC or AU pectin up to 1.6 % into gel formulations with carrageenan resulted in

increasing gel strength in comparison with the microparticles without carrageenan. Pectin

contributed more to gel strength than carrageenan in these composite hydrogels. It was shown

that -carrageenan has high water sorption ability related to the highly polar sulfate groups [35].

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Decreasing gel strength of the composite microparticles is probably related to this property of -

carrageenan.

The surface morphology of dried gel microparticles is presented in Figs. 2 and 3. The

microparticles had a spherical or an elongated shape. The surface of all microparticles was

grooved and rough (Fig. 2). The surface microrelief of the microparticles based on the SVC

pectin and carrageenan was with an increased amount of small wrinkles (Fig. 3a-d), while the

surface of the microparticles without carrageenan was grooved (Fig. 3e). The surface microrelief

of the microparticles based on the AU pectin and carrageenan was with large wrinkles and ridges

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(Fig. 3f-i). The microparticles based on the AU pectin without carrageenan had a grooved

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microrelief (Fig. 3k). The formation of the wrinkles and ridges on the microparticle surface was

probably due to the presence of carrageenan in the gel formulations.

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3.2. Swelling behavior of the microparticles
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The swelling degree of dry microparticles in SGF (pH 1.25), SIF (pH 7.0) and SCF (pH
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7.0 + pectinase) is shown in Fig. 4. The study of swelling behavior of the microparticles revealed
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that the degree of swelling increased with an increase in pH of the medium. The swelling degree

of all microparticles based on the SVC pectin after 2 h of incubation in SGF was 22-30 % (Fig.
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4a). This may be due to the protonation of the carboxyl groups of pectin in acidic medium,

reducing the electrostatic repulsion among these groups, favoring shrinkage and reducing the

solubility of polymer chains [18]. At higher pH values, the carboxylic acid groups were

converted into negatively charged carboxylate ions, resulting in electrostatic repulsion between

the different polymer chains and network expansion [19]. The higher swelling in SIF (pH 7.0)

can be due to the exchange of calcium ions in the microparticles with the sodium ions of SIF,

resulting in water uptake, swelling and structural changes leading to matrix erosion [18, 19].

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The pectin concentration had an effect on the swelling rate. An increase in the

concentration of the SVC pectin from 0.9 to 1.6 % into gel formulations led to a decrease of the

swelling degree of the microparticles in the simulated digestive fluids. A significant negative

correlation between the SVC concentration and the swelling degree of the microparticles in SGF

(R2 = -0.995, p < 0.05), SIF (R2 = -0.997, p < 0.05) and SCF (R2 = -0.988, p < 0.05) was

revealed (Table 3). These results indicated that the microparticles with a higher pectin

concentration had a lower swelling degree in the simulated digestive fluids. This can be due to

the increased crosslinking density of pectic molecules. The addition of the carrageenan to the

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0.9SVC-0.1Car gel formulation led to an increase in the swelling degree in comparison with the

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1.0SVC gel formulation without carrageenan (Fig. 4a).

These data are in agreement with the data of [14] who demonstrated that the carrageenan-
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based hydrogel films exhibited excellent absorption ability with increasing immersion time in a
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simulated body fluid. The high swelling ratio of the hydrogel films is mainly caused by the
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excellent water holding capacity due to the hydrophilicity of carrageenan that is greatly

influenced by the degree of “crosslinking” that depends on the degree of substitution of

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carrageenan [8, 14]. The high swelling capacity of carrageenan-based hydrogel films may be due
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to the high hydrophilicity of carrageenan crosslinked with CuCl2 [14]. Hydrophilic character of

the hydrogel films based on the -carrageenan and pectin was shown to increase with increasing
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-carrageenan content in the polymer matrix [15]. It was shown that the swelling degree of the

alginate-carrageenan films in water increased gradually with increasing carrageenan content in

complex films [35]. The water sorption ability of carrageenan is much larger than the sorption

ability of alginate. The water sorption ability of carrageenan is probably related to highly polar

sulfate groups in the structure. Moreover, the entrapment of κ-carrageenan within synthetic

methacrylate-based hydrogels improved the swelling properties of composite hydrogels [6].

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The swelling degree of the 1.0-SVC microparticles without carrageenan was similar to

the 1.2SVC-0.1Car swelling degree in SGF and SIF. The microparticles without carrageenan

degraded partially in SCF due to the destruction of the pectin by pectinase.

The relationship between the initial Ca2+ content in the microparticles based on the SVC

pectin and its swelling degree was revealed. The initial Ca2+ content correlated significantly with

the swelling degree of the microparticles in SGF (R2 = -0.802, p < 0.05), SIF (R2 = -0.880, p <

0.05) and SCF (R2 = -0.827, p < 0.05) (Table 3). This negative correlation indicated that the

microparticles with a higher Ca2+ content had a lower swelling degree in the simulated digestive

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fluids.

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A significant negative correlation between the initial gel strength of the microparticles

based on the SVC pectin and its swelling degree in SGF (R2 = -0.892, p < 0.05), SIF (R2 = -
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0.818, p < 0.05) and SCF (R2 = -0.768, p < 0.05) was shown (Table 3). This correlation indicated
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that increasing initial gel strength of the microparticles promoted decreasing swelling degree.
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The gel strength of the microparticles based on carrageenan and/or the SVC pectin after

successive incubation in SIF and SCF was 1.2–2.5-fold and 150–380-fold lower than the initial
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gel strength of the microparticles, respectively (Fig. 5). A significant positive correlation
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between the microparticle gel strength after incubation in SIF and the concentration of SVC (R2

= 0.819, p < 0.05) indicated that increasing concentration of the pectin promoted increasing
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microparticle gel strength (Fig. 5a). The gel formulation with the highest concentration of SVC

(1.6SVC-0.1Car) had the highest gel strength after incubation in SIF. The gel strength of the 1.0-

SVC microparticles without carrageenan after incubation in SIF was 1.3–2.3-fold higher than

that of the microparticles with carrageenan and 0.9–1.4% of SVC (Fig. 5a). The gel strength of

the 1.0-SVC microparticles after incubation in SIF was 2.6-fold lower than that of the 1.6SVC-

0.1Car microparticles.

A significant negative correlation between the gel strength of the microparticles during

incubation in SIF and its swelling degree in SIF (R2 = -0.794, p < 0.05) was revealed. This

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correlation indicated that decreasing gel strength of the microparticles promoted increasing

swelling degree in SIF. The entrapment of κ-carrageenan within pectic hydrogels decreased gel

strength and increased the swelling properties of composite microparticles based on 0.9–1.4% of

SVC.

The gel strength of the microparticles based on the SVC pectin and carrageenan or on

SVC without carrageenan was similar after incubation in SCF (Fig. 5b). The values of the gel

strength of these microparticles were extremely low (0.0088-0.0094 N). This observation may be

due to a decrease in the crosslinking of pectic molecules under the action of pectinase.

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The microparticles based on the AU pectin swelled gradually in SGF (Fig. 4b). The

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microparticle swelling degree after 2 h of incubation in SGF was 20-41 %. All microparticles

based on the AU pectin destroyed in SIF within 0.5 h probably due to the lower gel strength of
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these microparticles and the initial Ca2+ content. Moreover, a high pH (7.0) and the presence of
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salts (sodium and phosphates) have been attributed to the destruction of the microparticles based
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on the AU pectin and κ-carrageenan. Increasing concentration of the AU pectin from 0.9 to 1.2-

1.6 % into gel formulations resulted in decreasing swelling degree of the microparticles in SGF.
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A correlation between the AU concentration and the swelling degree in SGF (R2 = -0.597, p <
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0.05) was not found. The initial Ca2+ content in these microparticles (R2 = -0.710, p < 0.05) and

the initial gel strength of the microparticles (R2 = -0.709, p < 0.05) correlated significantly with
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the microparticles swelling degree in SGF (Table 3). These correlations indicated that increasing

initial Ca2+ content and gel strength of the microparticles promoted decreasing swelling degree of

the microparticles based on the AU pectin. The swelling degree of the 1.0-AU microparticles

without carrageenan was similar to the 1.6AU-0.1Car swelling degree in SGF.

This behavior can be due to the structural differences of the pectins, in particular, the

lower DM and the higher linearity of the SVC pectin in comparison with the AU pectin (Table

1), and therefore a higher amount of nonmethoxylated negatively charged galacturonic acid

residues binding the Ca2+ ions. This results in an increase of the calcium ion-binding capacity of

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pectin, gel microparticles strength and a decrease in the swelling degree. Moreover, the higher

swelling of the microparticles based on the AU pectin can be due to large wrinkles on the surface

of the microparticles (Fig. 3).

4. Conclusions

Composite gel microparticles based on -carrageenan and campion callus culture pectin

(SVC) or apple pectin (AU) were produced using emulsion dehydration techniques with

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successive incubation in calcium chloride solution. Gel microparticles were formed by

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ionotropic gelation during incubation in calcium chloride solution. A significant positive

correlation between the Ca2+ content and the SVC concentration was shown that appeared to be
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related to more COO- groups to bind the Ca2+ ions. Decreasing DM of the SVC pectin (DM 10%)
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promoted Ca2+ binding in comparison with the AU pectin (DM 40%). Moreover, the OSO3-
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groups of -carrageenan interacted with the Ca2+ ions through ionic bonds. Increasing

concentration of the pectin promoted increasing microparticle gel strength. The higher gel
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strength of the microparticles from the SVC pectin was probably due to the lower DM and a
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higher linearity in comparison with the AU pectin.

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The relationship between the characteristics and swelling behavior of the composite

microparticles was revealed. The microparticle gel formulations with a higher pectin

concentration had a lower swelling degree in the simulated digestive fluids. The addition of the

carrageenan to the gel formulations led to an increase in the swelling degree in comparison with

the gel formulation without carrageenan. The correlation analysis indicated that increasing initial

Ca2+ content and gel strength of the microparticles promoted decreasing swelling degree of the

composite microparticles. The gel strength of the composite microparticles decreased during

incubation in the simulated digestive fluids. The entrapment of κ-carrageenan within pectic

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hydrogels decreased gel strength and increased the swelling properties of composite

microparticles based on 0.9–1.4% of SVC.

Declaration of interest: The authors report no conflicts of interest.

Funding: This research did not receive any specific grant from funding agencies in the public,

commercial, or not-for-profit sectors.

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References

ro
[1] I. Fraeye, T. Duvetter, E. Doungla, A.V. Loey, M. Hendrickx, Fine-tuning the properties

of pectin-calcium gels by control of pectin fine structure, gel composition and

-p
environmental conditions, Trends Food Sci. Technol. 21 (2010) 219–228.
re
[2] L.S. Liu, M.L. Fishman, K.B. Hicks, Pectin in controlled drug delivery – a review,
lP

Cellulose 14 (2007) 15–24.

[3] M. Celus, C. Kyomugasho, A.M. Van Loey, T. Grauwet, M.E. Hendrickx, Influence of
na

pectin structural properties on interactions with divalent cations and its associated
ur

functionalities, Compr. Rev. Food Sci. Food Saf. 17 (2018) 1576–1594.

[4] K.H. Caffall, D. Mohnen, The structure, function, and biosynthesis of plant cell wall
Jo

pectic polysaccharides, Carbohydr. Res. 344 (2009) 1879–1900.

[5] J.-P. Vincken, H.A. Schols, R.J.F.J. Oomen, M.C. McCann, P. Ulvskov, A.G. Voragen,

J., R.G.F. Visser, If homogalacturonan were a side chain of rhamnogalacturonan I.

Implications for cell wall architecture, Plant Physiol. 132 (2003) 1781–1789.

[6] N. Pettinelli, S. Rodriguez-Llamazares, V. Abella, L. Barral, R. Bouza, Y. Farrag, F.

Lago, Entrapment of chitosan, pectin or κ-carrageenan within methacrylate based

hydrogels: Effect on swelling and mechanical properties, Mater. Sci. Eng. C. 96 (2019)

583–590.

16
 Journal Pre-proof
[7] C.A. Running, R. Falshaw, S. Janaswamya, Trivalent iron induced gelation in lambda-

carrageenan, Carbohydr. Polym. 87 (2012) 2735–2739.

[8] K.M. Zia, S. Tabasum, M. Nasif, N. Sultan, N. Aslam, A. Noreen, M. Zuber, A review on

synthesis, properties and applications of natural polymer based carrageenan blends and

composites, Int. J. Biol. Macromol. 96 (2017) 282–301.

[9] I. Kwiecie´n, M. Kwiecie´n, Application of polysaccharide-based hydrogels as probiotic

delivery systems – A review, Gels. 4 (2018) 47–61.

[10] V.T.N.T. Bui, B.T. Nguyen, T. Nicolai, F. Renou, Mixed iota and kappa carrageenan gels

of
in the presence of both calcium and potassium ions, Carbohydr. Polym. 223 (2019)

ro
115107.

[11] Ö. Pekcan, Ö. Tari, Cation effect on gel–sol transition of kappa carrageenan, Polym.
-p
Bull. 60 (2008) 569–579.
re
[12] V.L. Campo, D.F. Kawano, D. Braz da Silva Jr., I. Carvalho, Carrageenans: Biological
lP

properties, chemical modifications and structural analysis – A review, Carbohydr. Polym.

77 (2009) 167–180.
na

[13] B.T. Nguyen, T. Nicolai, L. Benyahia, C. Chassenieux, Synergistic effects of mixed salt
ur

on the gelation of  -carrageenan, Carbohydr. Polym. 112 (2014) 10–15.

[14] L. Jaiswal, S. Shankar, J.-W. Rhim, Carrageenan-based functional hydrogel film

Jo

reinforced with sulfur nanoparticles and grapefruit seed extract for wound healing

application, Carbohydr. Polym. 224 (2019) 11519.

[15] V.D. Alves, N. Costa, I.M. Coelhoso, Barrier properties of biodegradable composite

films based on kappa-carrageenan/pectin blends and mica flakes, Carbohydr. Polym. 79

(2010) 269–276.

[16] C.L. Lopez-Pena, D.J. McClements, Optimizing delivery systems for cationic

biopolymers: Competitive interactions of cationic polylysine with anionic -carrageenan

and pectin, Food Chem. 153 (2014) 9–14.

17
 Journal Pre-proof
[17] K. Lefsih, C. Delattre, G. Pierre, P. Michaud, T.M. Aminabhavi, F. Dahmoune, K.

Madani, Extraction, characterization and gelling behavior enhancement of pectins from

the cladodes of Opuntia ficus indica, Int. J. Biol. Macromol. 82 (2016) 645–652.

[18] R. Awasthi, G.T. Kulkarni, M.V. Ramana, T. de Jesus Andreoli Pinto, I.S. Kikuchi, D.

Dal Molim Ghisleni, M. de Souza Braga, P. De Bank, K. Dua, Dual crosslinked pectin–

alginate network as sustained release hydrophilic matrix for repaglinide, Int. J. Biol.

Macromol. 97 (2017) 721–732.

[19] K. Benfattoum, N. Haddadine, N. Bouslah, A. Benaboura, P. Maincent, R. Barillé, A.

of
Sapin‐ Minet, M.S. El‐ Shall, Formulation characterization and in vitro evaluation of

ro
acacia gum–calcium alginate beads for oral drug delivery systems, Polym. Adv. Technol.

(2017) 1–12.
-p
[20] E.A. Günter, O.V. Popeyko, P.A. Markov, E.A. Martinson, S.G. Litvinets, E.A. Durnev,
re
S.V. Popov, Yu. S. Ovodov, Swelling and morphology of calcium pectinate gel beads
lP

obtained from Silene vulgaris callus modified pectins, Carbohydr. Polym. 103 (2014)

550–557.
na

[21] E.A. Günter, O.V. Popeyko, Calcium pectinate gel beads obtained from callus cultures
ur

pectins as promising systems for colon-targeted drug delivery, Carbohydr. Polym. 147

(2016) 490–499.
Jo

[22] E.A. Günter, P.A. Markov, A.K. Melekhin E.A., V.S. Belozerov, Martinson, S.G.

Litvinets, S.V. Popov, Preparation and release characteristics of mesalazine loaded

calcium pectin-silica gel beads based on callus cultures pectins for colon-targeted drug

delivery, Int. J. Biol. Macromol. 120 (2018) 2225–2233.

[23] E.A. Günter, D.S. Khramova, P.A. Markov, O.V. Popeyko, A.K. Melekhin, V.S.

Belozerov, E.A. Martinson, S.G. Litvinets, S.V. Popov, Swelling behavior and satiating

effect of the gel microparticles obtained from callus cultures pectins, Int. J. Biol.

Macromol. 123 (2019) 300–307.

18
 Journal Pre-proof
[24] E.A. Günter, Yu.S. Ovodov, Polysaccharides of cell cultures of Silene vulgaris, Appl.

Biochem. Microbiol. 43 (2007) 84–90.

[25] A.I. Usov, M.I. Bilan, N.G. Klochkova, Polysaccharides of algae, 48. Polysaccharide

composition of several calcareous red algae: isolation of alginate from Corallina

pilulifera P. et R. (Rhodophyta, Corallinaceae), Bot. Marina. 38 (1995) 43–51.

[26] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the

folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.

[27] P.J. Wood, I.R. Siddiqui, Determination of methanol and its application to

of
measurement of pectin ester content and pectin methyl esterase activity, Analytical

ro
Biochem. 39 (1971) 418–428.

[28] W.S. York, A.G. Darvill, M. McNeil, T.T. Stevenson, P. Albersheim, Isolation and
-p
characterization of plant cell wall and cell-wall components, Methods Enzymol. 118
re
(1985) 3–40.
[29] K. Houben, R.P. Jolie, I. Fraeye, A.M. Van Loey, M.E. Hendrickx, Comparative study of
lP

the cell wall composition of broccoli, carrot, and tomato: structural characterization of
na

the extractable pectins and hemicelluloses, Carbohydr. Res. 346 (2011) 1105–1111.

[30] E. Esposito, R. Cortesi, G. Luca, C. Nastruzzi, Pectin based microspheres: A

ur

preformulatory study, Ann. NY Acad. Sci. 944 (2001) 160–179.

Jo

[31] A. Vaidya, A. Jain, P. Khare, R.K. Agrawal, S.K. Jain, Metronidazole loaded pectin

microspheres for colon targeting, J. Pharm. Sci. 98 (2009) 4229–4236.

[32] K.L.B. Chang, J. Lin, Swelling behavior and the release of protein from chitosan-pectin

composite particles, Carbohydr. Polym. 43 (2000) 163–169.

[33] C. Gebara, K.S. Chaves, M.C.E. Ribeiro, F.N. Souza, C.R.F. Grosso, M.L. Gigante,

Viability of Lactobacillus acidophilus La5 in pectin–whey protein microparticles during

exposure to simulated gastrointestinal conditions, Food Res. Int. 51 (2013) 872–878.

19
 Journal Pre-proof
[34] G.F. Oliveira, P.C. Ferrari, L.Q. Carvalho, R.C. Evangelista, Chitosan-pectin

multiparticulate systems associated with enteric polymers for colonic drug delivery,

Carbohydr. Polym. 82 (2010) 1004–1009.

[35] Y.H. Roh, C.S. Shin, Preparation and characterization of alginate-carrageenan complex

films, J. Appl. Polym. Sci. 99 (2006) 3483–3490.

Table 1 reprinted from Carbohydrate Polymers, 147, Günter, E. A., & Popeyko, O. V.,

Calcium pectinate gel beads obtained from callus cultures pectins as promising systems for

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colon-targeted drug delivery, 490–499, Copyright (2016), with permission from Elsevier.

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Table 1

Characterization of callus culture pectins.

Pectin Contents (% of total amount) Mw (kDa) DM (%) Specific Sugar ratios

GalA Gal Ara Rha Glc Xyl Man Protein viscosity LP BP Amount of
(dl/g) RG
SVC 91.0±0.1 2.3±0.2 1.6±0.02* 1.3±0.1 0.4±0.1* 0.2±0.05* 0.1±0.01 3.1±0.2 410±24 9.6±1.2* 2.0±0.2 15.9±0.7* 3.0±0.4* 0.01±0.002
AU 91.4±0.2 2.4±0.4 0.3±0.04 1.4±0.1 1.6±0.1 2.9±0.1 0 0 401±5 40.0±1.0 2.1±0.1 10.6±1.5 2.0±0.3 0.01±0.002
DM - degree of methylesterification; Mw - molecular weight; LP - linearity of pectin; BP - branching of RG-I; RG-I –

rhamnogalacturonan I; Amount of RG – amount of rhamnogalacturonan in pectin overall. The data are presented as the mean ±

S.D. (n = 3). * The differences are significant (p < 0.05) compared to the AU.

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Table 2

Characterization of gel microparticles.

Gel microparticle Diameter (mm) Ca (wt%) Initial gel strength (N)

a
0.9SVC-0.1Car 0.395 ± 0.050 3.55 ± 0.37 1.379 ± 0.102a
a
1.2SVC-0.1Car 0.412 ± 0.110 3.68 ± 0.50 1.761 ± 0.125a
a
1.4SVC-0.1Car 0.356 ± 0.092 4.61 ± 0.02 1.828 ± 0.503a
1.6SVC-0.1Car 0.279 ± 0.078 4.37 ± 0.39 3.591 ± 0.678a
1.0SVC 0.281 ± 0.073 5.26 ± 1.05 2.329 ± 0.277
0.9AU-0.1Car 0.464 ± 0.106b 1.93 ± 0.61 0.006 ±0.001b
b
1.2AU-0.1Car 0.266 ± 0.052 2.63 ± 0.19 0.039 ± 0.015b
1.4AU-0.1Car 0.262 ± 0.043b 1.86 ± 0.29 0.040 ± 0.011b
b
1.6AU-0.1Car 0.277 ± 0.054 1.98 ± 0.27 0.076 ± 0.015b
1.0AU 0.334 ± 0.073b 1.65 ± 0.37 0.060 ± 0.015
The data are presented as the mean ± S.D. p < 0.05 vs. 1.0SVC; b p < 0.05 vs.
a

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1.0AU.

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Table 3

Correlation analysis of the characteristics of composite gels, concentration of the pectin and the

swelling degree of the microparticles.

Characterization of composite Concentration of Swelling degree (%)

gel the pectin (%) SGF, SIF, SCF,
2 h (n=8) 6 h (n=8) 24 h (n=8)
SVC
Concentration of the pectin (%) -0.995* -0.997* -0.988*
2+ * *
Ca content (wt%) 0.840 -0.802 -0.880* -0.827*
Initial gel strength (N) 0.844* -0.892* -0.818* -0.768*
AU
Concentration of the pectin (%) -0.597
2+
Ca content (wt%) -0.141 -0.710*

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Initial gel strength (N) 0.956* -0.709*
2
Correlation coefficients (R ) are presented. *Statistically significant (p < 0.05).

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Figure captions:

Fig. 1. Schematic representation of the interaction between Ca2+ and molecules of pectin or -

carrageenan.

Fig. 2. Scanning electron micrographs of the gel microparticles: (a) 0.9SVC-0.1Car, (b) 1.2SVC-

0.1Car, (c) 1.4SVC-0.1Car, (d) 1.6SVC-0.1Car, (e) 1.0SVC, (f) 0.9AU-0.1Car, (g) 1.2AU-

0.1Car, (h) 1.4AU-0.1Car, (i) 1.6AU-0.1Car, (k) 1.0AU; magnification 100×, scale bar 100 m.

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Fig. 3. SEM images of the gel microparticles: (a) 0.9SVC-0.1Car, (b) 1.2SVC-0.1Car, (c)
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1.4SVC-0.1Car, (d) 1.6SVC-0.1Car, (e) 1.0SVC, (f) 0.9AU-0.1Car, (g) 1.2AU-0.1Car, (h)

1.4AU-0.1Car, (i) 1.6AU-0.1Car, (k) 1.0AU; magnification 1000×, scale bar 10 m.
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Fig. 4. Swelling behavior of composite gel microparticles based on the SVC (A) and AU (B)

pectins in the simulated gastric (SGF), intestinal (SIF) and colonic (SCF) fluids.
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Fig. 5. The gel strength of various gel formulations after successive incubation in the simulated
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intestinal (A) and colonic (B) fluids. The data are presented as the mean ± S.D. * p < 0.05 vs.

1.0SVC.

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Fig. 1.
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International Journal of Biological Macromolecules.
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Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov b, Ekaterina A. Martinson b,
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Sergey G. Litvinets b
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Fig. 2.

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International Journal of Biological Macromolecules.

Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov b, Ekaterina A. Martinson b, Sergey G. Litvinets b

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Fig. 3.

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International Journal of Biological Macromolecules.

Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov b, Ekaterina A. Martinson b, Sergey G. Litvinets b

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A 80 SGF SIF SCF

70

Swelling degree (%)

60

50

40

30

20 0.9SVC-0.1Car
1.2SVC-0.1Car
10 1.4SVC-0.1Car
1.6SVC-0.1Car
1.0SVC

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0 1 2 3 4 5 6 7 24
Time (h)

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B 80 SGF SIF SCF

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60
Swelling degree (%)

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0.9AU-0.1Car
20
1.2AU-0.1Car
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1.4AU-0.1Car
10 1.6AU-0.1Car
1.0AU
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0 1 2 3 4 5 6 7 24
Time (h)

Fig. 4.
International Journal of Biological Macromolecules.

Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov b, Ekaterina A. Martinson b,

Sergey G. Litvinets b

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Fig. 5.
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International Journal of Biological Macromolecules.

Elena A. Günter a,*, Vladislav V. Martynov a, Vladislav S. Belozerov b, Ekaterina A. Martinson b,

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Sergey G. Litvinets b
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CRediT author statement

Elena A. Günter: Conceptualization; Data curation; Methodology; Project administration;

Supervision; Validation; Writing- Original draft preparation; Writing- Reviewing and Editing.
Vladislav V. Martynov: Investigation; Visualization.
Vladislav S. Belozerov: Investigation.
Ekaterina A. Martinson: Resources.
Sergey G. Litvinets: Resources; Software.

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Highlights

 Composite gel microparticles based on the pectin and -carrageenan were obtained.

 The emulsion dehydration method with successive incubation in calcium chloride

solution was used.

 The microparticles from the pectin with the lowest DM had the highest Ca2+ content.

 A higher pectin concentration in the microparticles promoted a lower swelling degree in

the digestive fluids.

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 The addition of the carrageenan to the gel formulations led to an increase in the swelling

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degree.


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Increasing Ca2+ content and gel strength of the microparticles promoted decreasing the

swelling degree.
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