Fabrication of ultrafine Himalayan walnut oil Pickering emulsions by ultrasonic emulsification: Techno-functional properties of emulsions and microcapsules

Abstract

In present scenario, much of the attention has been put on the production and utilization of Pickering emulsions deciphering enhanced stability and applicability over wide environmental conditions. In this context the present study was carried out to elaborate effect of different wall materials and pH systems on the physicochemical, structural and morphological properties of Himalayan walnut oil Pickering emulsions by ultrasonic emulsification. In this study, concentrated Pickering emulsion of Himalayan walnut oil (HWO) was prepared utilizing soy protein isolate (SPI), maltodextrin (MD) stabilized by pectin at varying concentrations and pH systems (4.0, 7.0). With increase in pectin and SPI concentration and lowering MD, stable emulsions were obtained as deciphered by an Emulsion stability index (ESI) of 100 for 7 days at ambient storage. HWO Pickering emulsions were analysed for particle size measurements (2.13–13.64 µm) and depicted negative zeta potential values (−3.70 to −18.58). Lyophilized HWO microcapsules depicted moderate encapsulation efficiency (44.69–57.63 %) whereas the hygroscopicity values of the microcapsule ranged from (0.21–12.10 %). Thermogravimetric analysis (TGA) of the samples depicted the temperature of maximum degradation rate up to 550 °C whereas XRD spectra depicted amorphous nature of oil microcapsules. FTIR spectra revealed a close association between the SPI-MD-Pectin matrix. SEM analysis revealed stable oil globules entrapped in protein-polysaccharide matrix with no visible cracks and fissures.

1. Introduction

Persian walnut (Juglans regia L.) comprises one of the most important and principal cultivated nut species in the valley of Kashmir situated in north-western Himalayas which produces highest and best quality walnuts [1]. Himalayan walnuts are considered to be most desirable nuts in the international market due to its widespread consumer acceptance and organic nature. Walnut oil (WO), the principal component of walnut kernels is an important source of various essential fatty acids mostly polyunsaturated fatty acids (PUFA) (Linoleic and linolenic acid), monounsaturated fatty acids (MUFA) in addition to tocopherols, and phenolic compounds [2]. Major fatty acids present in WO include oleic (18:1), linoleic (18:2), and linolenic (18:3) fatty acids. Despite immense health and nutritional benefits, WO find minimal use in food industry due to its low stability and miscibility under different processing conditions [3].

In recent times much of the focus on emulsion production has shifted to the production of Pickering emulsions (PE) which refers to emulsions stabilized by solid particles of biopolymers such as proteins, polysaccharides or protein-polysaccharide complex [4]. Unlike conventional emulsification required for oil–water stabilization, in PE solid particles adsorbed at the oil–water interface inhibit interface interaction by forming a physical barrier through volume exclusion and particle adsorption characteristics [5]. This inclusive phenomenon endows the emulsion with distinct stabilization enabling its longevity regarding coalescence, physical stabilization and higher oxidative stability during storage [6], [7].

Soy protein isolate (SPI) has been seen as the material of great choice due to its availability, lower cost, biodegradable nature, oil binding and emulsifying properties [8] where as maltodextrin (MD), a hydrolyzed polysaccharide has multidimensional attributes like higher bulk density, low viscosity, medium solubility, higher binding affinity for hydrophobic materials, and lower oxygen permeability and its use as a cryoprotectant for increasing emulsion stability [9]. Pectin, an assorted polysaccharide has a range of properties like gelling nature which tends to form a dense matrix can be utilized as an active coating material for sensitive compounds [10].

Ultrasonic emulsification (UE) is a well-known sustainable green technology for fabrication of stable emulsion which involves the principle of acoustic cavitation leading to emulsion formation by shearing effect generated by ultrasound wave [11]. Various studies have utilized UE for effective emulsion stabilization for e.g. implication of invasive ultrasonics for development of functional dairy products [12], coconut oil emulsion with Gum acacia and maltodextrin [13] preparation of flaxseed oil and skimmed milk emulsion [14] and stabilization of walnut oil emulsion [15].

Various researchers have used protein-polysaccharide complex for successful encapsulation of oils like SPI-chitosan for algal oil encapsulation [16], [17], complexation of SPI- chitosan loaded with fish oil [18], flaxseed oil [19], tiger-nut, linseed and chia oils stabilized by sodium caseinate-lactose [20], [21], olive oil Pickering emulsions [22].

Prior research has invariably theorized the utilization of various protein-polysaccharide wall material combinations for hydrophobic material encapsulation, however encapsulation of Himalayan walnut oil (WO) in protein polysaccharide matrix by UE has mostly remained untested and undocumented till date. From the above context this study was designed to prepare Pickering emulsions of Himalayan WO using SPI-MD-pectin complex under pH system of 4.0 & 7.0. The overall goal of this study is to compare the complexation of SPI, MD and pectin as a function of pH and to explore their potential for stabilizing WO emulsions. It is hypothesized that complexation of SPI- MD-pectin will act as better emulsifiers with greater ability to migrate at the oil–water interface. Himalayan walnut oil Pickering emulsions were analyzed by investigating the structural morphological and thermal analysis with assessment of the stability of omega fatty acids by Gas chromatography. This approach enables for better understanding of the storage stability, complexation and re-dispersibility of these emulsions at industrial scale for futuristic food applications.

2. Materials and methods

2.1. Materials and chemicals

Walnuts were procured from the local markets of Kashmir, India. The walnuts were manually shelled and subsequently the kernels were stored in polyethylene pouches at −20 °C till further analysis. High esterified (DE 60–65 %) pectin was procured from Sigma Aldrich. Maltodextrin (DE 20), and defatted soyabean meal was procured from Himedia, Mumbai, India. All the other chemicals and solvents used in this research were purchased either from Himedia India or Sigma Aldrich and were of analytical grade.

2.2. Ultrasound-assisted extraction (UAE) of walnut oil

Walnut kernels (100gm) were macerated and finely homogenized by employing laboratory scale polytron homogenizer (Model PT-1200C, Switzerland) at 7000 rpm for 20 min. The finely ground walnut paste was mixed with n-Hexane (1:2) and the slurry was immersed in an ultrasonic bath with frequency of 40 kHz for a duration of 10 mins for extraction. The slurry was subjected to centrifugation at 12000 rpm for 30 min to extract walnut oil. The walnut oil was stored in amber bottles at −20 °C till further analysis.

2.3. Extraction of soy protein isolate

Protein was extracted from defatted soybean flour by isoelectric precipitation using the method of Papalamprou [23] with slight modifications. Briefly, defatted soybean flour (30 gm) was mixed with distilled water (1:10 w/v) and the pH was adjusted to 8.0 using 1 M NaOH. The solution was stirred intermittently for 45 min at 25 °C before centrifugation at 4500 rpm for 20 min to collect the supernatant. A second extraction step was performed on the precipitate using the same conditions and the supernatants were pooled. The pH of the solution was adjusted to 4.8 with 0.1 M HCl and centrifuged to recover the protein. The protein was washed with distilled water, re-dispersed in water and the pH of the solution was adjusted to 7.0 The protein solution was freeze-dried and the powder was packed in polyethylene pouches and stored at −20 °C.

2.4. Preparation of Pickering emulsions by UE

Oil-in-water emulsions were formulated according to the method of Chang [24] using different concentrations of soy protein isolate (SPI), maltodextrin (MD), and pectin (Pec) as shown in Table 1. The SPI was dispersed in Milli-Q water and adjusted to pH 4.0 and 7.0 using 1.0 M HCl or 1.0 M NaOH. To ensure maximum dispersion, these were stirred at 500 rpm overnight at 4 °C. MD was then dissolved in the SPI solution and stirred at 500 rpm for 3 h at room temperature (22–23 °C). Oil-in-water emulsions were prepared by homogenizing WO (10 %), MD and SPI solutions using a Polytron PT 2100 Homogenizer (Kinematica AG, Lucerne, Switzerland) at 15,000 rpm for 5 min at room temperature and the slurry was immersed in an ultrasonic bath with frequency of 40 kHz for 10 mins for maximum dispersion. Pectin was added to the initial emulsion at different concentrations and the pH of primary emulsions was readjusted to 4.0 and 7.0 before sample homogenization. The prepared WO emulsions were freeze-dried (Operon MPS-55, Hwanggeum Korea) and stored in polyethylene pouches at 4 °C.

Table 1.

Emulsion design using variable concentration of SPI, MD and pectin (g/100 g emulsion).

Strategy	Formulation	Oil	SPI	MD	Pectin	Core: Wall	Total Solids (° Bx)
F1	SPI-MD	10	2	18	−	1:2	30
F2	SPI-MD-Pec	do	2	17.9	0.1	do	do
F3	SPI-MD-Pec	do	4	15.8	0.2	do	do
F4	SPI-MD-Pec	do	6	13.7	0.3	do	do
F5	SPI-MD-Pec	do	8	11.6	0.4	do	do

F1-F5 are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0 and pH 4.0.

The above formulations (F1-F5) prepared at pH system 4.0 were designated as F4¹-F4⁵ and the formulations prepared at pH system 7.0 were designated as F7¹-F7⁵.

2.5. Fatty acid profile

Fatty acid methyl esters (FAME) from the HWO samples were obtained by alkaline treatment with 2 M KOH in methanol at room temperature (0.1 g of oil dissolved in 2.0 mL of hexane with 0.2 mL of KOH in methanol solution). Gas chromatography was carried out for FAME separation and quantification following the European commission regulation CEE 2568/91. The analysis was performed in an Agilent 7890A Gas Chromatograph equipped with a Flame Ionization Detector (FID). A standard fatty acid methyl ester mixture (C₄-C₂₄) (Sigma Chemical Co.) was used to identify the sample peaks. Commercial mixtures of fatty acid methyl esters were used as reference data for the relative peak area. Quantitative analyses of the fatty acids were performed using heptadecanoic acid methyl ester as internal standard.

2.6. Particle size analysis

Particle size of the freeze-dried emulsions was measured following the method of using a Mastersizer 2000 laser light scattering instrument (Malvern Instruments Ltd., Worcestershire, United Kingdom) equipped with a Hydro 2000S sample handling unit (containing water). The emulsion samples were taken, immediately after homogenization.

2.7. Zeta potential

The zeta potential of the freeze-dried emulsions was measured following the method of Errate et al. [54]. The analysis was done by subjecting the walnut emulsions to Zeta sizer (Malvern Worcestershire, U.K; Nano S). For zeta potential, 0.01 % of the sample was suspended in KCl (0.1 mM) and pH was adjusted to neutral before measurement.

2.8. Encapsulation efficiency

Microcapsule surface oil was determined according to the method of Liu [44]. Briefly, 1 g of microcapsules was dispersed in 30 ml of hexane, followed by vigorous shaking for 30 s. The solvent was filtered (Whatman Gr. 1 paper) into a 40 ml beaker and the beaker with solvent was placed in a fume hood overnight for solvent evaporation. Microcapsule surface oil was then determined gravimetrically, after heating the beaker at 105 °C for 30 min to remove any residual solvent. Total oil content of the microcapsules was determined, using the method described by Klinkesorn [57] with some modifications. Briefly, 4 ml of water were added to 1 g of microcapsules, followed by mixing at 300 rpm for 2 min. The resulting solution was then mixed with 25 ml of hexane/isopropanol (3:1 v/v), stirred at 300 rpm for 15 min and centrifuged at 1500 × g for 2 min. The clear organic phase was collected and the aqueous phase was re-extracted with the aforementioned solvent mixture. The organic phases were pooled and filtered through anhydrous Na₂SO₄, and the solvent was allowed to evaporate overnight in a fume hood. Total oil content was determined gravimetrically, after heating at 105 °C for 30 min.

The encapsulation efficiency (EE) was calculated from the quantitative determinations in accordance with the procedure laid down by Anwar [26]:

$$\text{EE}\%=\frac{\text{Total Oil}-\text{Surface Oil}}{\text{Total Oil}}\times \text{1}00$$

Wherein the total oil represents the oil incorporated in the emulsion systems whereas the surface oil represents the amount of oil which remained on the surface of the microcapsules and was not efficiently trapped inside the microcapsules.

2.9. Hygroscopicity

Hygroscopicity was measured according to the method of Caparino [27] with slight modifications. Briefly, 1 g microencapsulated powder samples were placed in Petri dishes in separate glass desiccators containing saturated NaCl solution (75.5 % humidity) and stored for 7 days at 25 °C. The results were expressed as moisture adsorbed (g) per 100 g dry solids.

2.10. ATR-FTIR

ATR-FTIR analysis of the WO samples was done following the method laid down by Akhtar [55]. The infrared spectra (Spectrum Two, Agilent, USA) of WO microcapsules were recorded over the range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹.

2.11. X-ray diffraction

X-ray diffraction (XRD) of WO microcapsules was carried out according to the method laid down by Klinkesorn [57] using a diffractometer (X'Pert PROPAN analytical, Netherlands). The equipment was set at 40 kV and 30 mA while the samples were scanned through the angle of diffraction of 5° to 50° at a scan speed of 5°/minute.

2.12. Thermogravimetric analysis

The thermogravimetric analysis (TGA) was carried out using a thermogravimetric analyser (TGA/SDTA851e, Mettler, Germany). Briefly, 3 mg powder was placed in an aluminium pan and heated from 30 to 500 °C at the rate of 10 °C/min under the constant N₂ flow of 20 mL/min

2.13. Scanning electron microscopy (SEM)

The sample was mounted on the aluminium stub and coated with gold. The SEM images were obtained using the Philips SEM 505 (Eindhoven, The Netherlands) operating at 27 kV Akhtar et al. [56].

2.14. Statistical analysis

The results were expressed as mean ± standard deviation of triplicate determinations. Means were compared by analysis of variance (Duncan's test) at 5 % level of significance using the software Statistica 8.0 (Statsoft Inc., Tulsa, USA).

3. Results and discussion

3.1. Fatty acid profile

The fatty acid profile of all the encapsulated samples is presented in Table 2. Encapsulation of walnut oils in the protein-polysaccharide matrix (SPI, MD, and Pectin) under two pH systems (4.0 & 7.0) successfully retained all the omega fatty acids in comparison to the free oil (F0). The unencapsulated walnut oils showed 4.18 % saturated fatty acid content (C16:1, C18:1), 13.74 % MUFA content (C18:1), and 82.14 % of PUFA content (C18:2, C18:3). The samples prepared at pH 7.0 retained MUFA and PUFA content in the range of 32.70–33.09 % and 58.49–61.35 %, respectively with no significant differences with respect to wall material concentration. Similarly, samples prepared at pH 4.0 successfully protected the MUFA and PUFA content of the walnut oil encapsulated under different wall material concentrations. The retained MUFA and PUFA contents were in the range of 30.95–33.87 % and 58.4–60.615 %, respectively. The MUFA and PUFA content of the encapsulated samples suggested that encapsulation was successfully able to protect the essential fatty acids concentration to an effective limit. Retention of olive oil fatty acids during the encapsulation process has been reported previously [28]. Kurek [28] concluded that carbohydrates, preferably β-glucan was successfully able to maintain the PUFA and MUFA content during fish oil encapsulated by spray drying, wherein the increase in β-glucan concentration up to 50 % of the total wall material achieved the maximum retention of MUFA than PUFA that are more susceptible to degradation due to high temperature.

Table 2.

Effect of different wall material concentrations and pH on the fatty acid profile (%) of encapsulated WO microcapsules.

	Palmitic acid)	Oleic acid	Linoleic acid	Methyl Stearate
F71	5.41 ± 0.12a	33.09 ± 0.62ab	59.35 ± 1.36a	2.38 ± 0.215a
F72	5.51 ± 0.24ab	32.95 ± 0.33ab	60.49 ± 1.65a	2.24 ± 0.077a
F73	5.83 ± 0.34b	32.70 ± 0.61b	61.19 ± 1.52a	2.31 ± 0.148a
F74	5.66 ± 0.43ab	32.99 ± 1.08ab	58.49 ± 1.18a	2.39 ± 0.388a
F75	5.78 ± 0.03ab	32.73 ± 0.72b	61.35 ± 1.71a	2.28 ± 0.035a
F41	5.32 ± 0.014b	32.72 ± 0.81a	58.40 ± 1.72c	2.36 ± 0.247a
F42	6.41 ± 0.08d	32.06 ± 0.37c	60.61 ± 0.70b	2.19 ± 0.007a
F43	7.14 ± 0.32e	30.95 ± 0.36bc	59.78 ± 0.77a	2.40 ± 0.295a
F44	4.08 ± 0.06a	32.98 ± 0.02c	60.57 ± 0.56c	2.51 ± 0.450a
F45	5.58 ± 0.02c	33.87 ± 0.48ab	59.05 ± 0.28d	2.14 ± 0.063a
F0	4.18 ± 0.43a	13.74 ± 0.05d	82.14 ± 0.63d	−

Data represent the mean (n = 3) ± standard deviation (SD). Different small letters (abcd) indicate a significant difference at 5 % level. Abbreviations include F7^1-F7⁵ samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: F4¹-F4⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

3.2. Particle size analysis

The results of particle size analysis are presented in Table 3. The hydrodynamic diameter of particles prepared at pH 7.0 was significantly (p ≤ 0.05) higher (2.132–13.644 µm) with a broad range than pH 4.0 (3.042–4.639 µm). The higher particle size at pH 7.0 could be explained by the formation of irreversible link bridges due to agglomeration of the protein-pectin matrix at an early phase before the lyophillization process [30]. On the other hand, the lower particle size at pH 4.0 could be attributed to the charge balance caused by electrostatic equilibrium in the colloid system mainly driven by a change in pH wherein maximum electrostatic attractions occurs at pH below the isoelectric point for SPI i.e. 4.5. The charge density of SPI and pectin seem to be stoichiometrically balanced at pH 4.0 and was the electrical equivalence point (EEP) of the SPI–pectin system. These observations were in accordance with the study of Espinosa-Andrews [30] involving gum Arabic /chitosan coacervation, possibly due to the protonation of the carboxylic groups of gum Arabic. Silvana [31] reported that use of MD in combination with the wall matrix tends to lower down the emulsion droplet size. At neutral pH, amino acid residues of SPI get fully protonated with simultaneous ionization of pectin carboxyl residues that in turn lead to stronger negative surface charge with charge-to-charge repulsions between the two biopolymers. This in turn leads to a weak association of the ion pairs at pH 7.0 resulting in increased droplet sizes and coalescence [33]. SPI-MD-pectin microcapsules showed greater size, which could be due to higher emulsion viscosity [34]. In addition to this, it can also be concluded that emulsions having higher average particle size distributions and PdI values are the emulsions which had the smaller droplet sizes and conclusively the particle size distribution does not refer to the emulsion droplets but the emulsion particles overall.

Table 3.

Effect of wall material concentrations and pH on Particle size distribution, average hydrodynamic particle size, zeta potential and polydispersity index of HWO emulsions.

	Particle size (µm)	Dv 10 (µm)	Dv 50 (µm)	Dv 90(µm)	Zeta potential (mV)	PdI (%)
F71	2.13	1.60	2.03	2.48	−3.78	20.96
F72	3.25	4.37	5.61	7.21	−10.25	11.78
F73	4.41	2.23	3.01	3.92	−11.32	23.54
F74	8.62	3.27	3.54	3.73	−13.02	48.27
F75	13.64	5.87	7.58	10.18	−15.89	32.80
F41	3.04	1.94	2.45	2.96	−3.70	12.72
F42	3.23	2.07	3.09	4.31	−13.29	24.17
F43	3.50	2.25	2.95	3.73	−14.14	11.63
F44	4.31	3.28	1.04	1.60	−18.58	1.38
F45	4.63	2.12	2.78	3.49	−20.68	10.92

Abbreviations include F7¹-F7⁵ samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: F4¹-F4⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

3.3. Zeta potential

The zeta potential values obtained for both the systems showed an increase in the negative value (Table 3). The samples prepared at pH 4.0 were more electronegative (−3.70 to −20.68 mV) as compared to the ones prepared at pH 7.0 (−3.78 to −15.89 mV). This could be because pectin is a negatively charged hydrocolloid with negative ζ-potential in the range studied [35]. It is evident that the samples prepared at pH 4.0 were electrostatically more stable than at pH 7.0. The zeta potential of SPI at pH 4.5 is 0, which corresponds to its isoelectric point, below which it has a net positive charge. Therefore pH 4.0 was chosen to produce Pectin-SPI-MD emulsion that invariantly favoured the bilayer formation between positively charged SPI and negatively charged pectin around the oil droplets. The droplet surface charge could favor electrostatic repulsion to enhance emulsion stability however a higher droplet charge of F7⁵, F4⁵ corresponding to −15.89, −20.68 mV due to complexation of SPI-pectin suggested sufficient electrostatic repulsive forces to limit droplet aggregation as deciphered in ESI. During the emulsification process the protein moieties from the SPI-Pectin complex would dynamically re-align at the oil interface to expose the hydrophobic amino acid residues towards the oil phase with simultaneous exposure of negatively charged carboxylic acid groups (COOH–) outwards to the aqueous phase leading to increase of the charges compared to SPI-MD emulsion systems (F7¹, F4¹) emulsion systems of −3.7 mV. Our results also correspond well with the observation of Patharkar [35] who reported that upon increasing the pH from 3 to 7 in chickpea protein and high methoxy pectin, pectin becomes more and more negative charged, reaching a maximum plateau region

3.4. Encapsulation efficiency

The encapsulation efficiency (EE %) varied gradually with wall material concentration and pH of the samples (Table 4). The samples prepared at pH 4.0 showed higher EE than pH 7.0. Amongst both of them, a steady increase in EE was seen with an increase in SPI-Pectin wall material concentration. The samples (F4⁴, F7⁵, F4⁴ and F4⁵) deciphered the highest EE (55.82–57.63 %). The lowest encapsulation efficiency was shown by F7¹ and F4¹ (44.69, 51.63 %). The highest EE of WO was achieved for the SPI concentration varying from 6-8 g/100 g and pectin 0.3–0.4 g/g. F4¹ and F7¹ were kinetically unstable and subsequently showed poor oil retention efficiency [37]. Several studies observed that EE of oils vary not only by emulsion pH but also by biopolymer concentration and temperature that govern the formation of stronger and much stable complexes resulting in higher EE A study conducted by Gonzalez [38] concluded that application of chia seed protein isolate and maltodextrin provided an efficient encapsulation system with EE between 59.6 -65.5 %, which is close to our results. Pectin from different sources has shown promising emulsifying activity over oil/water interface owing to electrostatic attractions between cationic protein moieties and anionic pectin galacturonic chain residues [39]. This suggests the emulsion stabilizing role of pectin that depends upon the chain-to-chain interactions of the protein-polymer wall matrices. Gharehbeglou [41] achieved an enhanced EE of 91 % upon encapsulation of oleuropein in whey protein concentrate (WPC) −pectin complex under optimum conditions (1.97 % Pectin, 8 % and WPC). The addition of pectin to MD and SPI wall material combination systems results in the formation of a double-layered emulsion system, which results in higher oil retention and minimized losses. In addition to SPI-Pectin complex formation, the incorporation of MD acts as a cryoprotectant. According to Gharsallaoui [40], EE steadily increases with the increase of the total solid concentration of the wall materials thereby supporting the overall coating structure around the core oil droplets. In general, an increase in polymer concentrations result in more intensive inter and intra chain interactions leading to a decrease in droplet mobility. Hence a change in structural orientation and conformation change in proteins induced by pectin adsorption in addition to electrostatic and gravitational force depend on the nature of inter and intrachain interactions, local charges per volume of droplets and polymer concentration.

Table 4.

Effect of different wall material concentrations and pH on the encapsulation efficiency (EE%) and Hygroscopicity of WO microcapsules.

	Hygroscopicity	Encapsulation efficiency (EE %)
F71	12.10 ± 0.50e	44.69 ± 1.15a
F72	7.31 ± 0.37d	52.06 ± 1.37b
F73	6.25 ± 0.48c	54.43 ± 0.39c
F74	4.25 ± 0.24b	55.92 ± 0.39c
F75	1.43 ± 0.30a	55.82 ± 1.78c
F41	0.21 ± 0.07a	51.93 ± 0.99a
F42	0.26 ± 0.04a	53.25 ± 0.96b
F43	1.32 ± 0.05b	55.75 ± 1.13bc
F44	3.62 ± 0.43c	57.54 ± 0.57d
F45	5.79 ± 0.59d	57.63 ± 1.16cd

Data represent the mean (n = 3) ± standard deviation (SD). Different small letters (abcd…) indicate a significant difference at 5 % level. Abbreviations include F7¹-F7⁵ samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: F4¹-F4⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

3.5. Hygroscopicity

Hygroscopicity represents the water adsorption capability and is a critical quality index of biopolymers utilized for oil encapsulation susceptible to lipid peroxidation. The higher the hygroscopicity index of the encapsulated oils, the higher is the risk of lipid oxidation. The hygroscopicity values of all the samples varied greatly with variation in the concentration of the wall materials as well as pH of the system (Table 4). The samples having higher concentrations of MD showed higher values of hygroscopicity, which simultaneously decreased on increasing SPI and pectin concentrations. However, WO microcapsules at pH 7.0 showed higher hygroscopicity than pH 4.0. The hygroscopicity values were still much lower than reported by Serfert [47] for encapsulation of drumstick oil in whey protein isolate and maltodextrin. Lower hygroscopicity of samples prepared at pH 4.0 can be attributed to the decreased solubility of the protein-pectin matrix existing in a very strong association. The limited availability of water binding groups leads to a simultaneous decrease in the hygroscopicity of the complexes. The other probable reason for the lower hygroscopicity at pH 4.0 can be attributed to the presence of hydrophobic amino acid side chains in SPI promoting strong electrostatic interaction between SPI. However, increase in hygroscopicity at pH 7.0 can be attributed to the formation of weaker complexes that can be easily solubilized.

In general, the degree of methylation of carboxyl group side chains determines the hydrophilic activity of the pectin molecules. Carboxyl groups as such possess an increased affinity to bind water molecules however this interaction is particularly diminished by the presence of methoxyl groups which are mostly hydrophobic in nature. However, the water sorption ability of the pectin molecules doesn’t depend only on the presence these groups but their distribution and arrangement, the degree of crystallinity and the presence of secondary structures (inter-chain bonds) are additional factors that influence water binding by the biopolymer. In addition to this, presence of methoxyl groups increases the intermolecular voids leading to the penetration of water inside the polymer matrix while as SPI also possesses several hydrophilic charged peptide groups as well as major hydroxyl groups that are supplemented by the serine, threonine, and tyrosine residues present in the protein fractions which tend to bind water molecules. The higher values of F7¹ and F4¹ can also be attributed to the presence of a higher concentration of MD which being more hydrophilic adsorbs higher amounts of moisture thus leading to an increase in hygroscopicity. Hygroscopicity depends on various intrinsic and extrinsic factors of the encapsulant and the matrix including composition, carrier material type and microcapsule size [43].

3.6. Thermogravimetric analysis

The thermogravimetric analysis (TGA) is presented in Fig. 1. The temperature of maximum obliteration of the given samples with consequent weight loss determines the temperature at which the given substance undergoes maximum degradation rate (Td). From the TGA curve, Td signifies the maximum slope of the change in weight of the given substance of which the thermogram is obtained. TGA thermograms of samples obtained at pH 4.0 (F4²-F4⁵) indicated a two-step mass loss (Fig. 1A) except F4¹ which indicated a single step of mass loss. The Td value of F4¹ was 220 °C whereas the rest of the samples (F4²-F4⁵) exhibited two Td values, the first one being at 230 °C and the second mass loss step at about 490–550 °C respectively. The two-step mass loss feature determines the moisture loss at the first step followed by dehydration as well as decomposition of the biopolymer at the second step. Similar Td values were obtained from samples prepared at pH 7.0 with F7¹ undergoing a single step mass loss. However, samples containing the highest SPI-pectin wall material concentration at both the pH systems (4.0 & 7.0) exhibited the highest thermal stability. The lower Td values of F4¹ and F7¹ might be due to the loose complex formed between MD and SPI and this thermal behavior was probably due to the fact that with the insufficient chain length, SPI could not form a crust strong enough to protect the core material against high temperature. Another reason for the lower thermal stability of F7¹ and F4¹ may be due to lower thermal resistance of unbound materials adsorbed at the surface, which undergo decomposition at the much earlier stage than SPI-Pectin conjugates that are stabilized by intermolecular electrostatic interactions and can withstand much higher temperature. It was also evident that incorporation of WO also had a profound effect in decreasing weight loss of the biopolymer matrix due to reduction in evaporation of water and other volatile substances owing to its hydrophobic nature. The presence of oil droplets inside the protein polymer matrix induces creaming effect, which in turn limits the water diffusion and evaporation. The second weight loss step was observed for all the formulations developed both at pH 4.0 and 7.0 possibly associated with degradation and denaturation of highly associated protein fractions. A study conducted by Setiowati [43] concluded that whey protein isolate (WPI) when used in combination with pectin was able to withstand dry heating many folds. Similar observations were made by Liu [44], who observed that combination of pectin with WPI not only prevents the droplet flocculation with improvement in oil stabilization but reduces the heat-induced protein aggregation thereby slowing down the denaturation process.

Fig. 1.

Thermogravimetric analysis of encapsulated WO microcapsules under two pH systems. A. Includes samples F4¹-F4⁵ prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0. B. Includes samples F7¹-F7⁵ prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0.

3.7. X-ray diffraction

The XRD diffractograms of formulations (F7¹-F7⁵, F4¹-F4⁵) at both the pH systems (4.0 and 7.0) are shown in Fig. 2. The formulations comprising different combinations of soy protein isolate (SPI) maltodextrin (MD) and pectin prepared at pH 4.0 and 7.0 displayed a characteristic peak at 20°. The F7¹, F7², and F7³ depicted a lower intensity peak value than the rest of the samples. The crystalline structure of pectin molecule depicted several characteristic diffraction peaks between 20° and 60° (Fig. 2C) other than the characteristic peaks of SPI at 9° and 20°, which are in agreement with a previous study [45], [46]. However, MD depicted a single diffraction peak at 20°. After the preparation of SPI-MD-pectin powders incorporated with WO by formation of Pickering emulsions, the characteristic peaks of pectin almost disappeared and a typical amorphous XRD pattern was formed, which clearly indicated the formation of SPI-MD-pectin complex in the solid-state dispersion. The reduction of peak in case of pectin and SPI after incorporation of WO by emulsification and lyophilization may have led to the destruction of the native crystalline packing structure of the pectin and disruption of the ordered structure of the protein and polysaccharide complex during the process of encapsulation, which includes emulsification, homogenization, and ultrasonication of the emulsion systems before lyophilization [47].

Fig. 2.

X ray diffractogram of A: WO encapsulated at pH 4.0. B: WO encapsulated at pH 7.0. C: Individual wall and core material. Abbreviations include F7¹- F7⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: F4¹ −F4⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

3.8. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR)

FTIR technique utilized to analyze different bond formation and presence of various functional groups. The carboxyl groups of pectin and MD existed in close association with the amide group of SPI due to the fluctuation of the emulsion pH prior to lyophilisation [47]. As shown in Fig. 3C, the characteristic absorption peak depicting the presence of MD can be observed at 1000 cm⁻¹ corresponding to the presence of C-O-C stretching absorption. The band of MD at 1300 cm⁻¹ corresponds to the stretching of C-O group. The FTIR spectrum of SPI depicted typical absorption bands with sharp peaks at 1700 cm⁻¹ and 1600 cm⁻¹ signifying the presence of amide I and II, respectively [17]. The presence of the peak at 1500 cm⁻¹ depicts the stretching of N-H bonds whereas, the peak at 1100 cm⁻¹ is assigned to C-H stretching vibration of peptide bonds [48]. The peak at 1100 cm⁻¹ depicts the presence of carboxyl group of pectin. After complexation of SPI-MD pectin complex incorporated with WO, the presence of a characteristic peak at 1500 cm⁻¹ depicts the amide bond formation between NH₂ and COO − of SPI and pectin [27]. The microcapsules loaded with WO at both the pH systems (4.0 and 7.0) showed characteristic bands at 3000 cm⁻¹ (CH) corresponding to vibrational stretching of the cis-olefinic double bands. The band at 2925 cm⁻¹ corresponds to asymmetric vibrations and 2855 cm⁻¹ correspond to symmetrical methylene vibrations (CH of CH₂). The band corresponding to typical ester absorption vibrations at 1743 cm⁻¹ (CO) represents triacylglycerols and is typical of oils with a high degree of unsaturation. The band at 1165 cm⁻¹ corresponds to the stretching vibration of the C-O ester groups and the bending vibration of the CH₂ group, which is typical of vegetable oils [49]. The interactions between the protein-polysaccharide complex are mediated mainly through non-covalent hydrogen and hydrophobic interactions, which alter the tertiary and secondary structure of complex proteins as evidenced by the changes in characteristic absorption bands [50].

Fig. 3.

ATR-FTIR of A: WO encapsulated at pH 4.0. B: WO encapsulated at pH 7.0. C: Individual wall and core material. Abbreviations include F7¹-F7⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: F4¹-F4⁵ are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

3.9. Scanning electron microscopy (SEM)

The SEM is a technique used to visualize the external morphology or microstructure [8]. The SEM micrographs of all the samples encapsulated using different concentrations of MD, SPI and Pectin produced at pH 4.0 and 7.0 are presented in Fig. 4. Micrographs of the samples F7¹ and F4¹ show an oily consistency with multiple dents and folds across the microcapsule area. The irregular dents and folds were probably related to the amount of unencapsulated oil present at the surface since the inner core was not able to retain much of the oil. Peres [51] also reported similar morphological features in spray dried liposomes encapsulated using MD as wall material. The probable reason for this irregular morphology may be rapid water transfer during the lyophilization process. The external morphology of other samples (F7²-F7⁵, F4²-F4⁵) showed flaked structures, characteristic of freeze-dried oil microcapsules. The samples comprising pectin in combination with SPI and MD showed a typical layered structure with oil globules buried inside the pectin and MD matrix. In these samples, the oil is entrapped inside the SPI-Pectin bond matrix layered with MD, which acts as a cryoprotectant. More profound wall characteristics were shown in samples containing an increasing amount of pectin wherein round-shaped walnut oil globules can be seen embedded in the wall matrix. The samples prepared at pH 4.0 showed a more uniform coverage of the oil globules due to the underlying phenomenon of electrostatic interaction between the cationic SPI and anionic pectin biopolymer, which preferably takes place at pH lower than the isoelectric pH of SPI [52]. These charge-based interactions result in the formation of a compact interfacial layer around the oil droplets forming an effective barrier resisting the oozing out of oil droplets. Hence, the addition of pectin has a profound effect on the morphological characteristics of WO microcapsules [53]. Similar results were reported by Silvana [31] upon the encapsulation of flaxseed oil in multilayer emulsion systems.

Fig 4.

Scanning electron microscopy (SEM) of encapsulated WO microcapsules. Abbreviations include A (a-e) are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 7.0: B (a-e) are samples prepared with different concentrations of soy protein isolate (SPI), Maltodextrin (MD) and pectin (Pec) at pH 4.0.

4. Conclusion

In this study, Himalayan walnut oil (WO) Pickering emulsions were developed by complexation of soy protein isolate and maltodextrin complexation at two pH systems (4.0 &7.0). Emulsion developed at pH 4.0 depicted better oil stabilizing properties due to much greater electrostatic interactions between the polymers. Encapsulated WO powder comprising increased Soy protein- pectin concentration effectively retained higher oil content. Formulations at pH 4.0 exhibited lower hygroscopicity values thereby decreasing oil peroxidation. X-ray diffraction findings depicted Pickering emulsions of WO using SPI-pectin complexes changed the crystallinity of the resulting microcapsules. Thermo-gravimetric analysis results revealed that Pickering emulsions developed at pH 4.0 could be relatively more heat resistant. Moreover, SPI-high methyl pectin Pickering emulsion at pH 4.0 were used to produce WO microcapsules with high encapsulation efficiency and improved thermal stability. Our results showed that SPI-high methyl pectin Pickering emulsion properties can be applied to prepare microcapsules to produce DHA and PUFA rich supplements for infant food formulation and pharmaceutical industries.

CRediT authorship contribution statement

Gazalla Akhtar: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. F.A. Masoodi: Supervision, Resources, Project administration. Sabeera Muzaffar: Writing – review & editing, Software, Investigation, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.