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Complex coacervate of wheat germ protein/high methoxy pectin in encapsulation of d-limonene


d-limonene is a volatile compound widely used in food flavorings, but it is chemically unstable in the presence of air, light, moisture, and high temperatures. Coacervation is a well-established technique which involves the formation of biopolymer nanocomplexes between negatively charged polysaccharides and positively charged proteins to protect bioactive components. The study shows the effect of pH, protein/polysaccharide ratio and total biopolymer concentration on complex coacervation of defatted wheat germ protein (DWGP) and apple pectin (high methoxyl pectin (HMP)) in the food industry as a carrier and delivery system for d-limonene. DWGP was used for the first time to form coacervate with apple pectin. Turbidity, ζ-potential and particle size of the complex coacervate and their individual biopolymers were examined for preliminary assays of complex coacervation. The optimum condition for DWGP/pectin complex coacervation was found to be at a ratio of 4:1 with a total biopolymer concentration of 1% and a pH of 4.0. The rheological properties of DWGP, pectin, and their coacervate were measured at various pH levels and ratios. The resulting coacervates, used to microencapsulate d-limonene, underwent spray-dried and were characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) measurements. These findings have the potential to contribute to the development of DWGP/pectin complex coacervates as effective delivery systems for volatile aromas or bioactive compounds.

Graphical Abstract


d-Limonene (C10H16) is an important flavoring ingredient used widely in food products. d-Limonene is a clear liquid that belongs to the terpene family which are unsaturated hydrocarbons. This monoterpene can be found in the essential oils of lime, orange, dill, and cumin. It is a flavoring agent with antioxidant properties that can be dissolved in alcohol, but it is insoluble in water and barely soluble in glycerin. It is unstable under environmental factors such as light, air, and moisture. Therefore, extensive research has been conducted to identify techniques that protect this compound from damage during storage and production processes. Encapsulation is one of the most effective methods for maintaining the stability of sensitive substances, ensuring their targeted release within the product, and preventing undesirable interactions with food ingredients [1].

Defatted wheat germ (DWGP) contains about 30% protein (primarily albumin and globulin) [2, 3]. It is known that wheat germ protein contains the same amount of essential amino acids as animal proteins, including lysine, methionine, and threonine [3]. Therefore, wheat germ is a rich source of natural protein, especially in defatted form [4].

Pectin can be found in plants’ cell walls, which has a polysaccharide structure. The main component of pectin is α-(1–4)-d-galacturonic acid, known as homogalacturonan (HG) [5, 6]. This structure also contains side branches with sugars such as rhamnose. Pectin is subdivided into low and high methoxy groups (< 50% and > 50%, respectively) [7]. The pectin structure makes it suitable for many applications in the food industry [8,9,10,11].

Complex coacervation, caused by electrostatic attraction between biopolymers including protein (Pr) and polysaccharide (Ps) with opposite charges (e.g., Pr–Ps, Ps–Pr, Pr–Pr), has been ultilized in developing encapsulation systems [1, 12,13,14], designing multilayer structures [15], controlling delivery systems [16, 17], as well as in food emulsions [8] and food structures [9, 10, 11, 18]. As the pH transitions from neutral to acidic, three critical pH values (pHc, pHφ1, pHφ2) and four distinct regions can be distinguished (co-soluble complexes, soluble complexes, complex coacervates, and complex dissolutions). The highest turbidity was observed between pHφ1 and pHφ2, often referred to as the optimum pH (pHopt), where the maximum amount of coacervates was produced [9, 19].

Several parameters can affect the structural properties of pectin, including biopolymer ratio, solvent properties, molecular mass, charge group density, pH, and ionic strength [20,21,22,23]. Coacervation and its functional attributes are influenced by the shape, composition, function, molecular weight, and other properties of the biopolymer. Therefore, the type of biopolymer used in the coacervate process determines the final product attributes. Wheat germ protein is used in a wide variety of food applications due to its low cost, availability, high protein content, and essential amino acid composition.

In recent years, it has been found that protein and polysaccharide interact with each other to form electrostatic complexes that are influenced by parameters such as pH, ionic strength, mixing ratio, biopolymer concentrations and charge density [1, 16, 24,25,26,27,28]. To the best of our knowledge, the complex coacervation between DWGP and pectin has not been reported. Furthermore, the reaction between polymers may result in the formation of a new complex coacervate that can be used to preserve flavors, essential oils, and bioactive compounds in food products. Therefore, the present study is conducted to determine the effect of different pH levels, biopolymer ratios, and total biopolymer concentrations on the development of complex coacervation. The results provide insight into how DWGP and pectin form a complex and its use in the food industry.

Materials and methods


The microwave extraction of pectin from apple pomace by mixing the dried apple pomace with HCl was performed according to the previous work [30]. After filtering, the extract was precipitated with ethanol 95% (v/v) in equal proportions. To remove mono- and disaccharides from the pectin mass, it was washed three times with ethanol 95% (v/v). It was then dried in an oven at 50 °C and kept in a desiccator. Dried pectin was stored in airtight bags (HDPE) in powder form.

DWGP was extracted by alkaline extraction and acid precipitation methods [31, 37]. Defatted DWGP suspension, was mixed with NaCl solution (0.5 N) for 30 min at room temperature, followed by centrifugation at 8582 g for 20 min at 4 °C. To precipitate the proteins, the supernatant solution was separated and the pH was adjusted to 4 with HCl (0.5 N) [32].

D-limonene and all other chemicals were analytical grade and were purchased from Sigma-Aldrich (St. Louis, USA).

Preparation of stock solutions

Apple pectin and DWGP (1% w/v) were dissolved in distilled water at ambient temperature and stirred for 2 h to prepare the stock solutions. Based on the pretreatment, initial concentrations were determined. An increase in turbidity and viscosity was observed at concentrations above 1%. To complete the hydration process, two solutions were placed in a refrigerator overnight [33].

Complex coacervation in d-Limonene encapsulation

According to our previous study, the optimal conditions for coacervation of DWGP and pectin were as follows: a mixing ratio of 4:1 w/w, Pr:Ps, with a total biopolymer concentration (CT) of 1% w/v (Jamshidian et al., 2022). Therefore, mixtures of DWGP and pectin were prepared at R = 4:1 with CT = 1% at 25 °C and pH 7.0. The solutions were then placed in an ice-water bath to gradually lower the temperature below 10 °C under constant stirring to promote complex coacervation. Afterwards, Tween 80 (10% of the total solids) was then added and stirred until completely dissolved [36]. Finally, d-limonene was gradually added to this solution at pH = 4 under ultrasonic homogenization (Iranian Ultrasonic Company, 400 W, 20 kHz, 12 mm probe diameter) at ambient temperature and 350 W for 10 min [1]. Then, the capsules were dried using a spray dryer (spray drying process creates capsules with high sphericity, which allows them to retain more compounds). The samples were spray-dried using B-191 mini spray-dryer (BÜCHI Labortechnik AG, Flawil, Switzerland) at 180 ± 10 °C inlet temperature, 80 ± 10 °C outlet temperature, 10.0 mL/min feed rate, 4 bar compressed air pressure, and 0.60 m3/h air flow rate.

Complex coacervation characteristics measuremnets

A UV–vis spectrophotometer (Jenway, 6305, London, England) calibrated to 100% transmittance (T%) with ultrapure water was used to determine the pH-dependent turbidity at a wavelength of 600 nm. Turbidity was determined by absorbance (%ABS) and the pH of the solution was adjusted with 1, 0.1, and 0.01 N HCl at room temperature (pH meter; Metrohm, 744, Herisau, Switzerland). The turbidity of the solution increased during acidification, and the pHc, pHφ1 and pHφ2 are shown graphically [34]. It has been observed that turbidity and reaction between two polymers are most prominent at pHmax [33, 35]. The zeta potential was measured in the range of pH 2 to 5.5. Z-potentials were determined using a dynamic light scattering (DLS) instrument (HORIBA, SZ-100, Minami-ku Kyoto, Japan). The diffusion coefficients (D, m2/s) as a function of pH were measured to determine the hydrodynamic diameters of the complexes (R = 4:1, CT = 1%) and their constituent biopolymer solutions. The solutions were placed in the He–Ne laser path with a wavelength of 633 nm for dynamic light scattering (DLS) measurement at different pH levels (HORIBA, SZ-100, Minami-ku Kyoto, Japan) [35, 36].

Rheological Measurements

A Brookfield viscometer (Viscolead one L., Fungilab, spindle S00, Spain) was used to determine the viscosity of the samples (16 ml) under a shear rate of 18 to 170 s−1 at ambient temperature.


Structural properties using FTIR

The DWGP, pectin, and DWGP/ pectin coacervates were characterized by FTIR spectroscopy (a double-beam spectrometer, Bruker FTIR-Tensor 27, Ettlingen, Germany) with 64 scans at 4 cm−1 resolution over the wavelength range from 4000 to 400 cm−1 (mid-infrared region) using the KBr pellet method. The infrared spectrum of pure KBr was selected as the baseline[3].

Morphological characterization (SEM)

The morphological properties of the microcapsules were assessed using a scanning electron microscopy (SEM; Philips, Philips XI30, Netherlands). DWGP, pectin, DWGP/pectin coacervates, d-limonene, and DWGP/pectin coacervates with d-limonene were mounted onto an amorphous glass lamella and coated with a thin layer of gold prior to taking images. At least three images of typical structures were acquired at 60 and 250 magnifications using an accelerated voltage of 20 kV.

X-ray diffraction analysis (XRD)

The XRD patterns of DWGP and pectin powders and their coacervates were determined with an X-ray diffractometer (PMD Philips X-Pert, Philips Company, Netherlands) at 25 °C. The instrument was operated under Cu-K-alpha−1 radiation, a target voltage of 45 kV, a current of 30 mA, and a diffraction angle range of 10–100° (2θ) with a scan rate of 0.02/sec.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed according to a modified method described by Laemmli (1970) under reducing conditions [29]. A 5% stacking gel and a 12.5% separating gel containing 0.4% SDS were used. Samples were prepared by dissolving 2 mg/ml (DWGP) or 4 mg/ml (other) sample powder in demineralized water. 60 μl of each solution were mixed with 20 μl of the ready-to-use buffer Roti Load 1 (Carl Roth, Karlsruhe, Germany). Then, aliquots (20 μl) were loaded onto the gels. The gels were run in a Tris-Glycin running buffer containing 0.1% SDS at 90 V (20 mA). Electrophoresis was stopped when samples reached the bottom of the gels (after 1.5–2 h). Proteins were stained by Coomassie Brilliant Blue R250 in 10% acetic acid. For destaining, a solution of 10% acetic acid and 25% methanol was used. To estimate the molecular weight of samples, the protein standard Precision Plus Protein Dual Color (Bio-Rad Laboratories Inc., Hercules, CA, USA) containing weight markers from 10–250 kDa was used [29].

Statistical analysis

Experiments were performed in at least three replicates. Data were analyzed by a one-way analysis of variance (ANOVA) with a significance level of 95% (P < 0.05). All data were analyzed using SPSS and GraphPad Prism.

Results and discussion

Physicochemical properties

The composition of the biopolymers (DWGP and apple pectin) are shown in Table 1.

Table 1 Chemical features of DWGP and apple pectin

Effect of pH on the DWGP-pectin interactions

The pH of the solution plays a critical role in the formation of coacervates by adjusting the electrical charges on the surface of polymers. Phase changes occur mainly between pI (protein) and pKa (pectin) [36]. The turbidity curve of biopolymers and their complexes as a function of pH (2.0–6.0) is shown in Fig. 1. Following the reduction of the pH of the solutions during the measurement of turbidity, four sections (A, B, C, and D) as well as four critical pHs (pHc, pHφ1, pHopt and pHφ2) were identified. Part A (co-soluble section) shows that the turbidity of the solutions remains constant above pHc (6.0). Part B (soluble complex section) indicates that the turbidity of DWGP-pectin mixtures rose slightly as the pH decreased to pHφ1 (4.9) [25]. As acidification increased, the turbidity increased considerably and insoluble complexes were formed below pHφ1, likely due to the formation of insoluble coacervates of protein and pectin. A maximum turbidity is achieved at pHopt (= 4). The electrical charges of the system have been neutralized at pH = 4.85 (ζ-potential = 0), confirming the value of pHopt (C: insoluble complex region). Protein-polysaccharide interactions are generally weakened when the net negative charge of the complexes is reduced [37, 38]. Similar findings have been also reported by Ach et al. (2015) and Souza and Garcia-Rojas (2017) [39, 40].

Fig. 1
figure 1

Turbidity curve of DWGP–pectin mixture during acidification (in R = 4:1 and CT = 1%) and the 4 sections and pH divisions

The turbidimetric titration curves (pH 2.0–6.0) and the appearance of DWGP (0.06%), pectin (0.04%) and their mixture (R = 4:1) at CT = 1% are presented in Fig. 2.A and B. The pectin solution was clear at all pH levels, regardless of the pH level, while the DWGP solution was cloudy at all pH levels. Turbidity was increased by decreasing the pH of the DWGP solution. The highest turbidity was observed at pH = 4.2, while the turbidity decreased as pH rose, indicating that the solubility of wheat germ protein is pH-dependent [36]. Turbidity increases as the number and size of particles increase [41]. The mixed solution with R = 4:1 at CT = 1% is actually divided into two phases, the deposition of protein particles and the clear upper phase [42, 43].

Fig. 2
figure 2

Evolution of absorbance as a function of pH for DWGP, pectin at 1% and mixture of DWGP–pectin (CT = 1% and R = 4:1) numerically (A) and visually (B)

The turbidity of the pectin was lower than that of the complex during acidification. This slight change during acidification is attributed to the polysaccharide structure [34]. Defatted wheat germ protein and apple pectin were negatively charged at pHc = 6.0. The negative charge and electrostatic repulsion of the negative charges on DWGP and pectin at pHc = 6, prevents coacervate formation [44]. Similar reports have been published by Liu et al. (2015 & 2017) [45, 46]. The DWGP-pectin mixture was also stable at pH 5.1 to 5.5, indicating that no phase separation occurred. The turbidity of the mixture (R = 4:1, CT = 1%) reached its maximum value (1.869) at pH = 4 (pHmax), which indicates the high amount of insoluble complexes and electrostatic bonding. Further decrease of pH below 4 resulted in a noticeable decrease of turbidity, due to protonation of pectin carboxyl groups and destruction of complexes. At pH 2.0 (pHφ2), separation of the complexes occurs due to the equal charge of the biopolymers [34]. Interactions between whey protein isolate and flaxseed gum also yielded in similar results [46].


The ζ-potential of homogeneous solutions of DWGP, pectin, and their mixtures, was monitored at different ratios (4:1, 2:1, and 1:1 w/w) and pH values (2.0–5.5) (Fig. 3). The surface charge of the protein ranges from a negative value (− 12.1 mv) at pH 5.5 to a positive value (12.87 mv) at pH 2, with a zero load point (IP) around 4.85. The zeta potential of proteins is pHdependent due to their amphoteric properties (containing both amino (NH2) and carboxyl (–COOH) groups simultaneously). In addition, the ζ-potential of the protein decreases with increasing pH. The decrease in ζ-potential can be attributed to the change of charges in carboxyl and amino groups with increasing pH. Furthermore, pectin is an anionic polysaccharide with a negative charge [45]. Pectin molecules are less likely to undergo carboxylate ionization as pH decreases [47]. As can be seen, the mixture of biopolymers has average values of the individual solutions.

Fig. 3
figure 3

ζ-potential (mV) of DWGP and pectin (1%, w/v), and mixture there of (CT = 1%, w/v) with different R (1:1, 2:1 and 4:1 w/w)

The zeta potential increased as R increased from 1:1 to 4:1 (Fig. 3), probably due to the higher ζ-potential of protein than pectin. When R is increased from 1:1 to 4:1, the IP increases from 3.65 to 3.93. The highest turbidity was observed near pH 4.0 in the 4:1 ratio mixture [40], confirming the electrostatic interaction between the carboxyl groups of pectin (− 10 mV) and the amine groups of protein (±5 mV) [48]. Thus, electrostatic reactions are the primary cause of coacervate formation. Pectin and protein form the most stable bond at pHmax [19, 49]. The FTIR results of protein-pectin coacervates have also demonstrated that intermolecular reactions are responsible for the formation of coacervates [36].

Particle size

To gain a deeper understanding of the interaction between DWGP and pectin, the particle size distribution of the two biopolymers was analyzed using DLS during the preparation of complex coacervates [43]. This parameter is used to investigate the formation and deveolopment of protein-polysaccharide complexes. As shown in Table 2, the particle size of homogeneous solutions of DWGP and pectin (1%), as well as their 4:1 mixture (CT = 1%), was measured at three pH levels (5.5, 4.0, and 2.5). As pH decreased from 5.5 (50.70 nM) to 4.0 (170.53 nM), larger aggregates were formed. Protein size is increased at pH 4.0 due to the repulsion reduction and the accumulation of DWGP [50]. As acidification increases, it increases the positive and identical charges, which improves the solubility of proteins.As acidification increases, it increases the positive and identical charges, which improves the solubility of proteins. This in turn results in increased solubility and a decrease in large and insoluble particles. Meanwhile, pectin particle size remained constant as acidification progressed [16].

Table 2 Particle size of DWGP and pectin (1%) and DWGP–pectin mixture (Pr:Ps 4:1) at different pH values

The complex size was 474.27 mm at pH 5.5, which was smaller than that of pectin. The particle size of pectin has a profound effect on the size of soluble complex particles. It has been demonstrated that the contraction of molecules can reduce the size of complex particles. In the range of soluble complexes (pH ~ 5.5), there is no correlation between particle size and turbidity, and this is caused by the increase in molecular weight of the compounds. Therefore, the results of this section are slightly different from those of the turbidity measurement section. The particle size increased to 474.27 nm at pH 5.5. This is consistent with the results reported in the insoluble complexes formation section. The formation of insoluble complexes reached its maximum level at pH 4 where the maximum diameter of the particles (623.37 nm) was also detected (Table 2). The insoluble complexes are possibly to be decomposed and separated at acidic pH values due to the seperation of pectin carboxyl groups.

Rheological properties

Viscosity is the ratio of shear stress to shear rate that describes the substance's resistance to flow. Viscosity is determined by the concentration of biopolymers, their interaction with solvents, as well as their structural properties, including their size, shape, and flexibility.

The viscosity of polysaccharide solutions is higher than that of protein solutions. Figure 4 illustrates the viscosity characterization of pectin and DWGP solution (1%), along with their complex coacervation at CT = 1% and Pr:Ps, 4:1. As can be seen, pectin exhibits non-Newtonian behavior characterized by a decrease in dynamic viscosity as a function of increasing applied shear stress under a shear stress less than 50 s−1. The shear-thinning behavior of pectin is attributed to the weakening of the intermolecular forces between its molecules with increasing shear rates [51, 52]. In fact, the apparent viscosity of the pectin solution decreased rapidly with increasing shear rates, from 15 to about 50 s−1, but remained almost constant at very high shear rates. At low shear rates (less than 50 s−1), a shear thinning non-Newtonian behavior is observed, while at higher shear rates, approximately between 50 and 170 s−1, Newtonian behavior is detected [52]. DWGP exhibits non-Newtonian and time-independent behavior. The effects of pH on coacervates was examined at three pH levels: the pH at the onset of coacervate formation (4.9), the pH optimal for coacervate formation (4), and the pH at the beginning of coacervate degradation (3.5). To investigate the effect of the proteintopolysaccharide ratio on flow behavior, coacervates with ratios of 1:1, 2:1, and 4:1 were used at optimal pH (Fig. 4-B). Complex coaservates showed a shear-thinning behavior at different pH levels (Fig. 4-A). The shear thinning behavior is caused by the breaking or reconstruction of the structure of the coacervates when it is subjected to shear force [36, 53]. The coacervate has a higher viscosity at pH = 4 than other pHs, indicating a denser structure caused by electrostatic interactions between protein and pectin molecules [36, 54]. The viscosity of coacervates appears to be directly related to the strength of electrostatic bonds in polyelectrolytes. When the pH is higher or lower than the optimal pH, coacervate exhibits a less dense structure, resulting in lower apparent viscosity, which is highly pH dependent [36]. Similar findings have been reported for chitosan-arabic gum coacervate [47], sodium caseinate-tragacanth gum [55], carboxymethyl cellulose-arabic gum [56], whey protein-linseed gum [46], and whey protein-high methoxy pectin [57].

Fig. 4
figure 4

Flow curve of DWGP/apple pectin coacervates (CT = 1%—Pr:Ps = 4:1) between apparent viscosities and shear rate (A) and different ratios of protein to pectin at optimal pH (= 4) (B)

FTIR analysis

The infrared spectra of DWGP, apple pectin, complex coacervate and complex coacervate with D-limonene are shown in Fig. 5. The FTIR spectroscopy of proteins provides useful information about their secondary structure. The secondary structure of proteins is primarily determined by their amide I region, which exhibits the stretching vibration of carboxyl groups [58]. The FTIR spectrum of DWGP exhibits a strong band at1666 cm−1 corresponding to the vibrational state of the 1st type of amide, characterized mainly by C = O stretching vibrations and partially by N–H peptide bonds. In addition, the peak observed at 1554 cm−1 is associated with the second type of amide structure (N–H and C–N stretching shape changes) and the peak at 1273 cm−1 corresponds to the third type of amide structure (N–H and C–N stretching shape changes) [59].

Fig. 5
figure 5

FTIR spectra of DWGP, pectin, DWGP/pectin coacervates, d-limonene and DWGP/pectin coacervates with d-limonene

It is believed that the peaks in the wave number of 1450 and 1416 cm−1 reflect vibrations in the CH2 and CH3 groups. Aliphatic amino acids and proteins show C–H stretching vibrations in the range of 2850–3320 cm−1 [60]. Apple pectin presented broad peaks at wave numbers of 3388 and 2941 cm−1, which represent negatively charged carboxylic groups (O–H stretching bands and C–H vibrational bands, respectively). Furthermore, in accordance with other researchers, the peak at 1749 cm−1 was attributed to the methyl ester (COOCH3) and undissociated carboxylic acid (COOH) groups [61]. Based on previous studies, the peaks at 1630 cm−1 are caused by the asymmetric stretching vibration of the carbonyl group of the carboxylate ion (–COO) and the amide I structure (C = O and C–N stretching) [62, 63]. It is thought that the absorption bands around 1300 and 1450 cm−1 are related to the asymmetric stretching vibrations of the carboxylic group, while the band at 1020 cm−1 is related to the main chain of the polysaccharide structure (C–O–C)[64, 65].

The spectrum of complex coacervate shows almost equal peaks for both biopolymers, but due to the higher protein-to-pectin ratio (1:4), the spectrum of the complex is more similar to that of pectin. The weaker and broader –OH stretching vibration at wave number 3000–3500 cm−1 indicates that a limited hydrogen interaction occurred between pectin and protein during coacervate formation. There are significant differences in the amine and carboxyl regions of the coacervate spectrum compared to protein and pectin, which indicates electrostatic interactions between them. It is consistent with the results of Sun et al. [66] for complex coaservate of propylene glycol alginate. According to Gilsenan et al. [67], hydrogen bonding contributes significantly to the formation of pectin-gelatin coaservates. Moreover, Guerrero et al. [68] demonstrate the formation of hydrogen bonds in soy protein isolate-agar coacervate. The spectra of DWGP and apple pectin coacervate exhibit two peaks at 1657 and 1548 cm−1, which are caused by stretching vibrations of –COO– and NH3+ groups. The peaks associated with the first, second, and third amide types shift from 1666, 1554, and 1273 cm−1 in protein to 1657, 1548, and 1270 cm−1 in coacervate, respectively, and their intensities decrease significantly. The electrostatic interactions between the apple pectin negative charge and the DWGP positive charge may account for these changes. In general, hydrogen bonds and electrostatic interactions play a major role in the formation of complex coacervates of DWGP and apple pectin.

In D-limonene, there are three broad peaks associated with the C–H stretching band at wave numbers 2967, 2922, and 2837 cm−1, while the C–O stretching band is associated with the peak at wave number 1642 cm−1. A strong peak at 886 cm−1 suggests a change in the C–H bond of the ether group, whereas the peak at 798 cm−1 reveals a bending of the C–H bond of the phenol group [69,70,71]. Coasarvate has a peak at wave numbers of 1657 cm−1 and 1548 cm−1 associated with stretching vibrations of the –COO– and NH3+ groups, respectively, as well as peaks corresponding to the 1st, 2nd, and 3rd types of amides at wave numbers of 1657, 1548, and 1270, respectively. The spectrum of the capsule does not reveal any d-Limonene indicator peaks (wave numbers 886 and 798 cm−1), which indicates that microencapsulation of d-Limonene was successful. Several studies have addressed this issue previously [72, 73].

Morphological properties (SEM)

An electron microscope image of pectin, DWGP, complex coacervate dried with a spray dryer, d-Limonene, and complex coacervate containing d-Limonene is presented in Fig. 6. DWGP has a spherical appearance characterized by a rough and cluster-shaped surface. A polar group is believed to be responsible for the roughness of the surface of globular proteins. Furthermore, it is arranged in clusters with dense three-dimensional networks, similar to milk proteins images at the isoelectric point. The surface of apple pectin is characterized by irregular polygons with grooves. The morphology of pectin powder showed a large number of columns/sheets interspersed with small fragments. In the SEM micrograph of the complex coacervate, a non-porous, non-uniform layer is evident with aggregation while limonene was encapsulated (Fig. 6. E). When the particles are being dried, water rapidly evaporates from their surfaces, resulting in wrinkles on their surfaces. As compared to pectin and protein, coacervate has a networked and porous structure, which indicates electrostatic interactions between pectin and protein. Coacervates contain vesicles of different sizes on their surface, which have the potential to trap sensitive compounds [63].

Fig. 6
figure 6

SEM micrographs of the apple pectin (A), DWGP (B), DWGP/pectin coacervates (C), d-limonene (D) and DWGP/pectin coacervates with d-limonene (E) at 60 and 250 × magnification, 20 kV

According to the XRD diagram (Fig. 7), d-limonene has an amorphous structure. Coaservates containing d-limonene are less amorphous (semi-crystalline) than pure d-limonene. As mentioned earlier, the DWGP/ apple pectin complex coacervate is composed of a non-porous and non-uniform layer. The absence of cracks or porosity on the surface of the particles indicates that the wall material has to some extend covered the core material, demonstrating the success of the coating process. A major contributing factor to this problem is the use of the spray drying method [63].

Fig. 7
figure 7

X-ray diffraction comparison of pectin (A), DWGP (B), their complex coacervate (C), d-limonene (E) and complex coacervate with d-limonene

X-ray diffraction analysis (XRD)

X-ray diffraction is used to determine the degree of crystallinity and the type of structure of materials. A spectrum with sharp peaks indicates crystalline character, whereas a spectrum with broad peaks indicates an amorphous structure. X-ray diffraction patterns of apple pectin, DWGP, complex coacervates of DWGP/pectin, d-Limonene and complex coacervates of DWGP/pectin with d-Limonene are shown in Fig. 7.

There are four sharp peaks found in DWGP, at 19.5, 22, 26.5 and 29 degrees (2Ɵ) (Fig. 7B), which indicates its crystal structure. X-ray difraction patterns of the protein showed two peaks at 10 and 22°, indicating the presence of α-helix and β-sheet structures, respectively. A lack of a peak in the x-ray pattern of DWGP at 10° confirms the FTIR results and the fact that DWGP’s structure contains more β-sheet than α-helix. It was found that pectin has amorphous structure along with semi-crystalline structures in 21 and 28° (2Ɵ), respectively, which indicates lack of molecular order [74].

The X-ray diffraction pattern of complex coacervate shows a peak at 20° (Fig. 7C), which indicates its amorphous structure. The disappearance of protein and pectin peaks in coacervate indicates that electrostatic interactions and hydrogen bonds have formed between pectin and protein, leading to changes in its crystal structure, as confirmed by FTIR. The structure of the complex coacervate of DWGP/apple pectin is generally amorphous.

According to X-ray diffraction analysis of the complex, the sharp peak of d-limonene (20°) was eliminated, suggesting that d-limonene was fully incorporated. Furthermore, the removal of protein and pectin peaks in coacervate suggests that electrostatic interactions and hydrogen bonds have been formed between pectin and protein, resulting in changes in its crystal structure. In general, DWGP/pectin complex coacervates have an amorphous structure. The XRD patterns of the loaded samples revealed only one concentrated peak at 22°, indicating that the capsules had become amorphous following the addition of d-limonene (Fig. 7E). As a result of these structural changes in the amorphous capsule matrix, molecular mobility increases, resulting in a decrease in encapsulated compound retention.


Protein fractionation was performed according to the classical method of Osborne (1924) with minor modifications [65]. The distribution and protein content of protein fractions in DWGF are presented in Table 3. The globulin fraction had the highest protein content followed by glutelin, albumin, and prolamine. A large amount of carbohydrates and pigments were extracted into the albumin and prolamine fractions causing their low protein content. The water-soluble albumin was the predominant protein fraction, accounting for 34.5% of the total proteins, which was higher than that in wheat flour and rice [66, 67].

Table 3 Distribution and protein content of protein fractions of DWGPa [65]

To check for the formation of high molecular weight material as an indication of complex coaservation, SDS-PAGE was performed on heat treated DWGP-Pectin mixtures. Reducing conditions were chosen, so that only complex are detected. DWGP-Pectin mixtures are expected to have high molecular weight above 100 kDa [29, 68, 69]. Figure 8 shows SDS-PAGE gel of the DWGP-Pectin mixtures. The lane above the letter corresponds to the protein standard while lanes above numbers contain the investigated samples. Spery-drying of DWGP-Pectin mixtures as one of the step in coaservate preparation did not have any significant influence on the molecular weight of the protein (compare Fig. 1, lanes A1 and 2).

Fig. 8
figure 8

SDS-PAGE of DWGP:pectin (4:1 w/w and CT = 1% w/v). In gel (A), lane 1 corresponds to mixtures in start point (0 h); lanes 3–7 correspond to passing of time

At the beginning, intensely colored bands were visible at 14 kDa, 18 kDa, 37 kDa and 75 kDa, corresponding to monomeric and dimeric, and BSA, respectively (Fig. 8, lanes 1) [29, 70]. No protein containing substances with a molecular weight above 75 kDa were detected at first. Upon passing of time, the characteristic protein bands from DWGP fractions became paler as is described in literature [29, 71, 72].

Substances with a molecular weight above 100 kDa were detected after passing of time (Fig. 8, lanes A4). These substances appeared in the form of polydispersed bands. Due to the polymeric nature of pectin, its molecular weight is not as well defined as that of DWGP. This leads to the formation of complex with a broad molecular weight distribution. After more passing of time (Fig. 8, lanes A7), intensive immobilized bands were seen at the gel injection point. This indicates protein rich substances which were excluded from intrusion into the gel pores because of them being too large in molecular weight and/or volume (> 250 kDa) [73, 75].

SDS-PAGE of separately incubated protein and pectin did not indicate any formation of high molecular weight material (Fig. 9). Therefore, self-polymerization of the single substances did not take place. Only intraction of protein and pectin together led to the formation of high molecular weight species. However, some characheristics of complex coacervate of the wheat germ protein/high methoxy pectin was evaluated here, other properties of the coacervates such as surface activity, molecular structure [76,77,78], application in drinks and novel algorithms [79,80,81,82,83,84,85] needs to be studied in future trends.

Fig. 9
figure 9

SDS-PAGE of individually investigated DWGP and pectin samples. Lane 1: DWGP, lane 2: pectin. The pectin sample does not show inherent protein. The lane above letter D contains the protein standard


The use of wheat germ protein, as a novel plant protein in coacervation with apple high methoxy pectin for encapsulation of d-limonen was investigated. In order to investigate the molecular interactions between DWGP and pectin, turbidimetric analysis, zeta potential, and particle size were measured at different pH levels. Soluble complexes were observed at pH = 6.0 followed by phase separation at pHφ1 = 4.9. The maximum biopolymer interaction was found at pH = 4.0 and CT 1% (w/v). When anionic pectin interacted with cationic protein fragments, complex soluble coacervates were formed between pHc and pHφ1. Rheological properties were measured. The complexation process was confirmed by physical properties such as FTIR, SEM, and XRD. This study provides valuable information for the design of food ingredients with specific functions based on DWGP and pectin complex. The DWGP-pectin complex cocervate is used in foods for the protection and controlled release of flavors or other sensitive compounds.

Availability of data and materials

All data are presented in the manuscript.


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I would like to express my very great appreciation to Research Institute of Food Sciecne and Technology for its valuable and constructive assistance during the planning and development of this research work.


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Jamshidian, H., Rafe, A. Complex coacervate of wheat germ protein/high methoxy pectin in encapsulation of d-limonene. Chem. Biol. Technol. Agric. 11, 60 (2024).

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