Fabrication and Characterization of Kafirin Microparticles Made Using Ionic Gelation Vibrational Jet Flow Technology: Influence of Processing Parameters on Physicochemical Properties

TL;DR

This study explores how to make kafirin microparticles using a new method and shows how different settings affect their size and structure.

Contribution

The study introduces the use of IGVJFT for kafirin microparticle fabrication and identifies key processing parameters for optimization.

Findings

Microparticle size ranged from 522 to 937 µm, and zeta potentials from −32.8 to −12.3 mV.

Lower protein concentrations produced spherical, porous particles, while higher concentrations formed oval-shaped, matrix-like structures.

Kafirin concentration, nozzle diameter, voltage, and frequency were the most significant parameters affecting microparticle properties.

Abstract

Kafirin is a protein from sorghum grain or its high‐protein byproducts, including dried distillers’ grain with solubles (DDGS) from bioethanol production. This highly hydrophobic and slowly digestible protein has demonstrated self‐assembly properties, indicating its high potential for the manufacture of microparticles. In this study, DDGS kafirin microparticles were prepared using ionic gelation vibrational jet flow technology (IGVJFT). The effects of nozzle diameter (µm), integrated electrode voltage (V), internal frequency/vibration (Hz), gelation solution concentration (CaCl2 [% w/v]), and kafirin concentration (% w/v) were evaluated. A fractional factorial design (25−1) of 16 processing runs was applied to model the influence of processing parameters on the microparticle physicochemical properties in terms of volume‐weighted mean microparticle size (µm) and zeta potential (mV). The production runs yielded microscale particles with a volume‐weighted mean microparticle size of 522–937 µm and zeta potentials ranging from −32.8 to −12.3 mV. Scanning electron microscopy revealed differences in the morphological microstructure. At lower protein concentrations, spherical particles with porous structures formed, whereas higher concentrations produced oval‐shaped microparticles with an open, matrix‐like architecture. Overall, IGVJFT produced DDGS kafirin microparticles with reproducibility, uniformity, and high‐speed production. The kafirin concentration, nozzle diameter, integrated electrode voltage, and internal frequency were identified as the most significant parameters affecting DDGS kafirin microparticle size and zeta potential. This study provides a foundation for the use of IGVJFT by identifying its significant processing parameters required for large‐scale optimization of kafirin microparticle production.

Figures

Field‐emission scanning electron micrograms (FE‐SEM) of DDGS kafirin microparticles using secondary electron beam signal at lower resolution, illustrating the effect of production processing conditions on particle morphology. Microparticles from (A) Run 2, (B) Run 8, (C) Run 11, and (D) Run 12, all at a magnification of 500 µm, a voltage of 5.00 kV, and a field of view of 1.63 mm, using secondary electron beam electron signal.

Close‐up of DDGS kafirin microparticles from (A) Run 2, (B) Run 8, (C) Run 11, and (D) Run 12, showing microstructural texture at higher resolution: magnification 50 µm, voltage 5.0 kV, and field of view 185 µm using a secondary electron beam signal. These images highlight the impact of processing parameters of ionic gelation vibrational jet flow technology on the microstructural texture and porosity of DDGS kafirin microparticles.

Energy‐dispersive X‐ray spectroscopy (EDS) of microparticles. (A) Run 2, (B) Run 11 DDGS kafirin microparticle (larger sized), and (C) Run 12 (smaller sized) DDGS kafirin‐based microparticle. The spectra show C, N, O, and S from the kafirin matrix and Ca and Cl from CaCl2‐mediated ionic gelation, with Na detected in samples resulting from residual extraction reagents. Differences in S detection between Run 11 (larger particles and denser) and Run 12 (smaller and porous) suggest that processing conditions and morphology affect the exposure of sulfur‐containing residues at the particle surface.

Tables

TABLE 1. Factorial independent process parameters with maximum and minimum values.

		Actual values		Coded values
Factor	Independent process parameters	Min	Max	Min	Max
A	Nozzle diameter (µm)	300	450	−1	+1
B	Integrated electrode voltage (V)	200	600	−1	+1
C	Internal frequency (Hz)	900	1600	−1	+1
D	Gelation solution concentration (% w/v)	2	5	−1	+1
E	DDGS kafirin concentration (% w/v)	1	2	−1	+1

TABLE 2. Experimental levels generated using a fractional factorial design and the obtained results.

Std	Run	FactorA (µm)	B (V)	C (Hz)	D (% w/v)	E (% w/v)	Response PS ± SD (µm)	ZP ± SD (mV)
1	1	300	200	900	2	2	784 ± 43	−17.5 ± 0.4
4	2	450	600	900	2	2	937 ± 67	−14 ± 0.12
9	3	300	200	900	5	1	694 ± 28	−28.3 ± 0.2
5	4	300	200	1600	2	1	673 ± 22	−32.8 ± 0.3
13	5	300	200	1600	5	2	754 ± 44	−18.8 ± 0.2
11	6	300	600	900	5	2	761 ± 42	−17.8 ± 0.1
8	7	450	600	1600	2	1	687 ± 22	−25.5 ± 0.5
15	8	300	600	1600	5	1	591 ± 18	−32 ± 0.6
2	9	450	200	900	2	1	673 ± 26	−21.7 ± 0.3
16	10	450	600	1600	5	2	875 ± 55	−15.1
10	11	450	200	900	5	2	967 ± 59	−12.3 ± 0.7
3	12	300	600	900	2	1	522 ± 12	−27.4 ± 0.2
6	13	450	200	1600	2	2	941 ± 66	−14.6 ± 0.6
14	14	450	200	1600	5	1	737 ± 48	−21.3 ± 0.1
7	15	300	600	1600	2	2	721 ± 41	−22.6 ± 0.1
12	16	450	600	900	5	1	653 ± 27	−24.3 ± 0.4
4K		—	—	—	—	0.6		+15.8 ± 0.5

TABLE 3. Analysis of variance (ANOVA) of the effects of significant process parameters of ionic gelation vibrational jet flow technology on the volume‐weighted mean microparticle size.

Source	F‐value a	p‐value b
Model	37.99	<0.0001	Significant
Kafirin concentration	139.37	<0.0001
Nozzle diameter	57.67	<0.0001
Voltage tension	13.84	0.0048
Internal frequency	0.0096	0.9239

TABLE 4. Analysis of variance (ANOVA) of the effects of significant process parameters of ionic gelation vibrational jet flow technology on the zeta potential.

Source	F‐value a	p‐value b
Model	61.23	<0.0001	Significant
Kafirin concentration	170.26	0.0001
Nozzle diameter	61.23	<0.0001
Internal frequency	9.86	0.0094
Voltage tension	3.41	0.0920

Full text

Introduction

1

World sustainability issues have led to greater utilization of food‐grade industrial byproducts and wastes for developing novel biomaterials, including microparticles and films (Lindgren et al. 2018; Holden et al. 2018). For the formation of microparticles for applications such as controlled release delivery of bioactives, drugs, and probiotics, hydrophobic proteins from industrial byproducts show great potential due to their abundant availability, high binding capacity, and low solubility and digestibility in aqueous environments (Wan et al. 2015). Compared to the milled protein powders, which have irregular, polydisperse fragments with uncontrolled surface chemistry, microparticles prepared through controlled structuring processes possess a controllable size and internal organization and uniform morphological functionality (Sağlam et al. 2014). Among the known microformulation morphologies, spheres are sought out because of their large surface‐to‐volume ratio, offering wide and open orientation with a designated in vivo target (Moore et al. 2022).

Kafirin is the main storage prolamin protein found in sorghum grain and in the protein‐enriched byproduct of ethanol production from sorghum, dried distillers’ grain with solubles (DDGS) (Lau et al. 2015). Recently, Shah et al. (2024) reported efficient extraction and purification methodologies for the isolation of kafirin and demonstrated its technofunctional potential as a “green” polymer to replace synthetic materials in biomaterial production. Kafirin has a high hydrophobic‐to‐hydrophilic amino acid ratio, undergoes evaporation‐induced self‐assembly, and has high levels of disulfide crosslinking, excellent gelling ability, and slow digestibility. The high hydrophobic‐to‐hydrophilic amino acid ratio is a key to kafirin's ability to self‐assemble into spherical particles. The water insolubility, in combination with exogenous factors such as protein–nonprotein interactions and endogenous factors (protein–protein), gives kafirin a unique resistance to hydration and slow digestibility (Duodu et al. 2003). In addition, kafirin protein meets key criteria, including Generally Recognized as Safe (GRAS) status, biocompatibility, biodegradability, low cost, and low immunogenicity. In our previous work (Shah et al. 2025), we compared kafirin extracted from sorghum grain to that extracted from sorghum DDGS. The results revealed that DDGS kafirin exhibited higher protein content and yield, an increased aggregated β‐sheet structure, more irregular and porous particle morphology, higher water solubility, and more active surface‐reactive segments compared to grain kafirin.

Currently, many approaches have been reported for the laboratory‐scale preparation of microparticles from prolamin proteins such as zein and kafirin. These include antisolvent precipitation, co‐precipitation, pH cycling, and solvent evaporation (Song et al. 2021). Most of these approaches have limited efficacy for commercial use due to high solvent inventories and difficulty in controlling the physicochemical properties of the resultant particles (Song et al. 2021). Technologies such as dual‐photoinitiator systems, microfluidics, electrohydrodynamic spraying, and mechanical extrusion have been developed for the fabrication of hydrogel spherical particles (Franco et al. 2011; Muir et al. 2021; Qayyum et al. 2017). For example, Muir et al. (2021) explored three microgel fabrication techniques (namely, microfluidic devices, batch emulsion, and mechanical fragmentation) by extrusion to assess their impact on the hydrogel properties. In another study, electrohydrodynamic spraying fabricated cell‐laden polyethylene glycol hydrogel microspheres, employing covalent crosslinking through Michael‐type addition for gelation (Qayyum et al. 2017).

Ionic gelation vibrational jet flow technology (IGVJFT) is a new approach for forming microparticles that uses a combination of mechanical (e.g., a vibrational nozzle for particle sizing) and chemical (e.g., ionic gelation for particle curing) processes to produce particles of the desired size and shape (Wagle et al. 2020). This technology has advantages over other reported approaches for kafirin particle formation, such as low energy use, suitable particle size, reproducibility, high‐speed production, and controllability of process factors to allow optimization (Wagle et al. 2020; Mooranian et al. 2017). The key elements of this technology include a dispensing bottle, a nitrogen‐based airflow system, a particle production unit that contains a nozzle electrode with controlled voltage, frequency, and nozzle size, an airflow pump, a stroboscope, and a polymerization bath (e.g., calcium chloride dissolved in water) with a magnetic stirrer. The biopolymer solution is put into a dispensing bottle, and airflow forces it into the particle production unit. This high‐flow‐rate colloidal solution is then passed through a precisely drilled nozzle, where, with the aid of the vibrational frequency and integrated voltage, a high electric field is generated, resulting in a laminar flow beam of continuously highly charged, equal‐sized droplets. These are sprayed from the nozzle into the polymerization bath, where they are cured into stable solid particles.

In this study, DDGS kafirin microparticles are prepared using IGVJFT. This technology is expected to address weaknesses in current methodologies and improve scale‐up viability. The statistical predictive modeling is used to generate fractional factorial design runs (2^5−1^) to perform an investigation into the effects of various process parameters on properties of the DDGS kafirin microparticles obtained. The fractional factorial design enables evaluation of a relatively large number of variables in a small number of experiments by taking a fraction (portion) of the full factorial 2* ^k^

• design to form a 2* ^k^

^−^ * ^p^

• design, where k represents the number of variables and p represents the number of independent design generators chosen to fractionate the design. The advantages of this approach include analysis of each variable and investigation of their magnitudes, refinement of the model by removing nonsignificant variables, and cost‐effectiveness. In this way, a short list of those process parameters that most significantly affect the microparticle properties can be identified and used for optimization of processing variables. The microparticles from runs that produced large and small microparticles were examined for their morphological and structural properties by scanning electron microscopy (SEM) and energy‐dispersive X‐ray spectroscopy (EDS). The adjustable size, porosity, and surface charge could make DDGS kafirin microparticles a potential carrier for bioactives, probiotics, and drugs. This study is expected to yield solid microparticles with future application in the functional food and pharmaceutical industry.

Materials and Methods

2

Materials

2.1

Sodium metabisulfite, n‐hexane, methanol, and calcium chloride were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). Absolute ethanol and HCl were obtained from Thermo‐Fisher Scientific (Scoresby, VIC, Australia). The water used in this research was purified by Ultrapure Technology (Life Technologies, USA).

Extraction of Kafirin Protein

2.2

The kafirin was extracted from DDGS using the method previously described (Shah et al. 2021). The milled and sieved (duplicate × 50 g) sorghum DDGS was soaked in 250 mL of extraction solution containing 62% (v/v) absolute ethanol, 0.064 NaOH, and 0.22% Na_2_S_2_O_5_. Specifically, Na_2_S_2_O_5_ was used as a reducing agent to cleave disulfide bonds within and between kafirin molecules, thereby increasing the solubility of kafirin in the extraction solvent and overall protein yield. Although monomeric and oligomeric kafirin subunits can be isolated without reducing agents, the extraction of total kafirin (including polymeric fractions) requires a reducing environment (Shah et al. 2024).

The mixture was incubated for 1 h at 60°C with shaking at 150 rpm in a water bath (Memmert 854, Schwabach, Germany), then cooled to room temperature. The samples were then sonicated in an ultrasonic water bath (30 Hz, 60 W; Ultrasonic Cleaner, DSA, Madrid, Spain) for 5 min at 25°C. The sonicated samples were centrifuged at 1750 × g for 20 min. The supernatant containing dissolved kafirin was recovered by decantation. The vacuum evaporation (80°C) was performed to reduce the total volume of the supernatant, and the pH of the supernatant was adjusted to 5.0 using 6 N HCl. The samples were left overnight to complete the precipitation, followed by a second centrifugation at 1750 × g for 10 min at room temperature. The clear supernatant was removed, and the precipitate (kafirin) was dried overnight at 40°C in an oven (Memmert, 854 Schwabach, Germany). Three washes of absolute n‐hexane (40 mL/12 g) at 25°C for 5 h each were performed for defatting, followed by decantation of solvent. The residual hexane was removed by heating the sample overnight at 60°C in an oven (Memmert, 854, Schwabach, Germany). The final powder was milled, yielding a mean particle size of 272 µm, measured using a Mastersizer 2000 (Malvern Instruments Ltd, Malvern, UK) (Zhong et al. 2019). Protein content was 84.76 ± 0.76 g/100 g (db), measured by elemental analysis (2400, Perkin Elmer Pvt Ltd, Macquarie Park, NSW, Australia), with an extraction yield of ∼59%.

Microparticle Fabrication and Fractional Factorial Experimental Design

2.3

Statistical Design

2.3.1

Design‐Expert software (VII, stat‐Ease, Inc. Minneapolis, MN, USA) was used to generate a fractional factorial design (2^5 −1^ v; Table 1) for five process parameters: nozzle diameter (µm), integrated electrode voltage (V), internal frequency/vibration (Hz), gelation solution concentration CaCl_2_ (% w/v), and DDGS kafirin concentration (% w/v). Based on preliminary screening tests (data not presented), the maximum (+1) and minimum (‒1) levels of each process factor at which the technology would operate successfully to produce particles were identified and used in the design. This generated 16 processing runs (Table 2).

Formulation and IGVJFT Operation

2.3.2

The feed solutions for the IGVJFT were prepared as follows: 1 g (w/v) of DDGS kafirin was dissolved in 15 mL of 62% aqueous ethanol, to which 85 mL of water was added under stirring condition (F1 formulation); 2 g (w/v) of DDGS kafirin dissolved in 15 mL of 62% aqueous ethanol was added under stirring conditions (F2 formulation). The formulation was mixed using a high‐speed magnetic stirrer for 4 h (Alcântara et al. 2010).

The operation was performed in batch mode. The prepared formulations were loaded into a syringe connected to a specialized pump with controlled flow settings. An air‐assisted nozzle was used intermittently, along with a concentric nozzle setup, to regulate the flow during processing. The operating system was configured with levels of the factors under investigation, as shown in Table 2. During processing, the system's heating function was turned off, and the gelation solution was placed on a magnetic stirrer and continuously stirred. The formed microparticles were collected by sieving from the gelation bath. Then, the 16 resulting runs of DDGS kafirin microparticles were individually dried in a hot‐air chamber for 72 h at ∼32°C, with relative humidity maintained at 35%. The dried microparticles were stored at ambient temperature in air and moisture‐proof, sterilized glass tubes.

Microparticle Size Analysis (PS)

2.4

The volume‐weighted mean size of the microparticles was determined using a Mastersizer 2000 (Malvern Instruments Ltd., Worcestershire, UK) (Shah et al. 2025). Before measurement, microparticles were dispersed in distilled water at a concentration of 0.1% (w/v) to achieve an optimal obscuration of 10%–20%. The suspension was gently stirred for 60 s to ensure uniform dispersion without inducing particle damage. Measurements were carried out using the Hydro 2000S wet dispersion unit. Each sample was analyzed in triplicate, and the results were reported as the mean ± standard deviation (n = 3).

Zeta Potential Measurements (ZP)

2.5

The zeta potential of the microparticles was measured using a Zetasizer 3000HS (Nano S, Malvern Instruments, Worcestershire, UK). The microparticles were suspended at a concentration of 0.01% (w/v) in 0.5 mL of water (Life Technologies, USA) (Shah et al. 2025). The data were processed using the Omni SEC‐Zetasizer software (Zeta v7.11, Malvern Instruments, Worcestershire, UK) to calculate the Z‐average values. Measurements were performed in triplicate, and the results are reported in millivolts (mV).

Field‐Emission Scanning Electron Microscopy (FE‐SEM) and EDS

2.6

The surface morphology of the microparticles was examined using secondary electron imaging on a dual‐beam field‐emission scanning electron microscope (Zeiss Neon 40EsB FEBSEM, Oberkochen, Germany) (Shah et al. 2021). Before imaging, samples were stored in a desiccator, mounted on aluminum stubs with carbon tape, and coated with a 6‐nm layer of platinum using a sputter coater (208HR, Cressington, Watford, UK). Imaging was performed with a 5‐kV electron beam. The surface elemental composition was analyzed by EDS at 20 kV integrated with the FE‐SEM. Elemental distribution across different regions of the samples was mapped, and the Aztec software (Oxford Instruments, Wiesbaden, Germany) was used to identify and quantify the elements (Shah et al. 2025).

Statistical Analysis

2.7

The fractional factorial design in Design‐Expert software (VII, Stat‐Ease, Inc., Minneapolis, MN, USA) was used to generate model runs and investigate results. The volume‐weighted mean microparticle size (PS) and zeta potential (ZP) were set as responses for the statistical study. The model diagnostics, for example, half‐normal plots, Pareto charts, and analysis of variance (ANOVA), were used to assess the significance of independent process parameters deemed to have a major effect on the volume‐weighted mean microparticle size (PS) and zeta potential (ZP). Please refer to Figures S1–S6 for the complete statistical analysis.

Results and Discussions

3

IGVJFT Operation

3.1

This study is a continuation of our previous work that investigated the physical chemistry of DDGS kafirin and compared it with kafirin made from original grain (Shah et al. 2021). The findings revealed that DDGS kafirin, due to the high heat treatment used for the manufacture of biofuels, underwent alterations in its protein secondary structure, notably the unfolding of some α‐helices, followed by their realignment into β‐sheet structures. This structural modification contributes to enhanced solubility and the formation of irregular surface morphologies with internal pores, in contrast to the ordered, self‐assembled kafirin seen in grain kafirin in water–ethanol (Shah et al. 2021). These results suggest that grain kafirin self‐assembles spontaneously, relying on intrinsic molecular interactions, including van der Waals forces, hydrogen bonding, capillary action, and π–π interactions, without requiring external stimuli (Wang and Padua 2012). The formation of larger particles is likely due to heat‐induced disruption of disulfide (S─S) bonds during bioethanol production, which could have facilitated the reassociation of polypeptides into compact micro‐aggregates, contributing to the formation of larger particles.

Given these structural changes, we employed jet flow technology, assisted by a gelation bath, to assemble DDGS kafirin microparticles. In our Drug Development and Research laboratory, a considerable number of successful pharmacological approaches to prepare biomaterials using jet flow technology have been put forth (Wagle et al. 2020; Mooranian et al. 2018, 2020). There is, however, less literature available on the preparation of biomaterials that require binary solvents, for example, DDGS kafirin, which dissolves in water–alcohol mixtures. A proper understanding of the colloidal behavior of DDGS kafirin protein plays a paramount role in producing particles with higher efficiency. Thus, this research combines formulations and engineering process parameters of this technology to gain insights into DDGS kafirin microparticle preparations.

In this study, the selected parameter levels (based on preliminary studies), as listed in Table 2, are used to prepare DDGS kafirin microparticles. These selected parameters produced a beam of highly charged particles without aggregations. This design of experiment approach investigates five independent process parameters across 16 runs (2^5−1^), where five represents the number of independent process parameters examined at two levels, and one represents the number of independent design generators chosen to fractionate the design for the preparation of DDGS kafirin microparticles. The five parameters were nozzle diameter (µm), DDGS kafirin concentration (%, w/v), integrated electrode voltage (V), internal frequency (Hz), and gelation solution concentration (% w/v), and their impact was assessed on the volume‐weighted mean particle size and zeta potential of DDGS kafirin microparticles.

Volume‐Weighted Mean Particle Size of DDGS Kafirin Microparticles

3.2

All experimental runs yielded particles in the micrometer range, ranging from 967 ± 59 µm to 522 ± 12 µm, with a mean of 748 ± 14 (Table 2). Run 8 (591 ± 18 µm) and Run 12 (522 ± 12 µm) resulted in smaller‐sized DDGS kafirin microparticles. Analysis of the experimental results by ANOVA indicated that the significant process parameters associated with the volume‐weighted mean particle size are kafirin concentration (F = 139.37, p < 0.0001) > nozzle diameter (F = 57.67, p < 0.0001) > integrated electrode voltage (F = 13.84, p < 0.0001) (Table 3). These variations in DDGS kafirin particle size, produced by ionic gelation and vibrational jet flow technology, could influence the functional behavior of the microparticles. For example, smaller particles generally hydrate more rapidly and allow faster diffusion of entrapped compounds, whereas larger particles tend to retain water more slowly and may support a more prolonged release behavior (Siepmann and Siepmann 2008).

According to the ANOVA results, DDGS kafirin concentration is the most significant process parameter influencing microparticle size. The increase in microparticle size with increasing kafirin concentration is consistent with higher dispersion viscosity and stronger intermolecular interactions (Wang and Padua 2010). For example, at 2% (w/v), Run 2 (937 µm) and Run 11 (967 µm) likely exhibited greater resistance of kafirin droplets to jet disruption, which in turn might have favored the formation of large‐sized droplets. Compared to this, 1% formulations consistently produced smaller particles, indicating finer jet disintegration. Such interpretation is further supported by the diagnostic plot (Figure S5), which indicates that a smaller increase in kafirin concentration (w/v) leads to a significant increase in the volume‐weighted mean particle size.

The influence of nozzle diameter is based on particles per given volume. This indicates that a larger nozzle diameter produced a jet with a greater cross‐sectional area, a key parameter for subsequent droplet formation. As illustrated in the half‐normal (Figure S1) and Pareto analysis (Figure S3), nozzle diameter generated a significant effect, with a larger nozzle diameter producing larger droplets due to increased jet cross section. It is well established in the literature that a larger nozzle produces a broader laminar jet with reduced curvature, which could lead to the formation of small perturbations along the surface of the droplet (Mooranian et al. 2017). Consequently, this tends to favor the preparation of particles with higher volumes (Verma and Daya 2017). Furthermore, the integrated electrode voltage has a statistically significant main effect on the volume‐weighted mean particle size (p = 0.0048), with main effects and diagnostic plots shown in Figures S1, S3, and S5, indicating a decreasing trend in particle size with increasing voltage.

The results of the volume‐weighted mean particle size of DDGS kafirin fabricated by IGVJFT are consistent with the zein particle literature, wherein an orthogonal design, OA_16_ (4^5^), was used to assemble prolamin protein zein by ultrasonic‐assisted dialysis technology (Liu et al. 2016). It is important to note that the internal frequency, although central to droplet uniformity, did not significantly modulate particle size over the examined range of 900–1600 Hz. This suggest the dispersion behaviour of DDGS kafirin may be attributed to its strong cross‐linking and hydrophobic nature (Xiao et al. 2015; Taylor and Taylor 2018), which might have attenuated the influence of vibrational perturbations in the examined range.

Prolamin proteins (zein and kafirin) have demonstrated the capacity to form spherical particles in aqueous alcohol (Wang and Padua 2012). This means that disordered kafirin protein can be converted into an organized structure without external guidance but relies on weak internal interactions, such as van der Waals, capillary, and hydrogen bonds (Chen et al. 2022). The self‐assembly of the kafirin protein is based on the evaporation‐induced mechanism, which involves two or more solvents, with one solvent evaporating faster. As a result, the polarity of the solution changes, which drives the self‐assembly of the solute (Wang and Padua 2010; Dehcheshmeh and Fathi 2018; Argos et al. 1982; Matsushima et al. 1997). DDGS kafirin, because of structural transformation (Duodu et al. 2001), results in the formation of large aggregates with less shape using the solvent‐induced technique (Shah et al. 2021). This experimental study utilized IGVJFT to prepare particles. The overall process includes three steps: (a) preparation of DDGS kafirin formulations; (b) laminar liquid jet flow breakdown of particles by electrostatic charge generated by the electrode voltage at specific internal frequency/vibration, which accumulates charge on the surface of the protein/alginate dispersions; and (c) polymerization of this highly charged laminar flow jet beam of particles upon contact with the gelation solution (CaCl_2_ in water).

Zeta Potential of Microparticles

3.3

Zeta potential is a physicochemical parameter that assesses the magnitude of surface charge of a particle and indicates the potential stability of particles against aggregation. Zeta potential values were determined for all experimental runs and are presented in Table 2. The zeta potential value of DDGS kafirin colloidal suspension was +15.8 ± 0.5 mV. However, compared to previous studies, DDGS kafirin protein has a less positive zeta potential value than those from zein (+22.8 ± 1.3 mV) and kafirin protein (+19.38 mV) (Wenjuan et al. 2018; Yan et al. 2021; Ji et al. 2018). This less positive charge might be because of heat‐induced structural transformation resulting from the distillation process, which has shown unfolding of α‐helix followed by realignment and reorganization into β‐sheets (Duodu et al. 2001; Shah et al. 2021).

The zeta potential values of the DDGS kafirin microparticles ranged from ‒32.8 ± 0.9 mV to ‒12.3 ± 0.7 mV (Table 2). The zeta potential range of ‒32.8 ± 0.9 mV to ‒12.3 ± 0.7 mV provides insight into the electrostatic stability of the kafirin microparticles, where surface charges generate repulsive forces rather than the counterpart Van der Waals attraction and prevent particle aggregation. Although DDGS kafirin dispersion showed positive zeta potential (+15.8 ± 0.5 mV), the microparticles prepared using jet flow technology assisted with CaCl_2_ exhibited net negative values upon redispersion in water. Kafirin is a highly hydrophobic protein with limited hydration in water (Shah et al. 2024). Upon suspension of DDGS kafirin in water, only the outermost surface becomes hydrated, which might have buried acidic residues within aggregated protein domains (Xiao et al. 2015). As a result, the surface is dominated by neutral or weakly cationic groups, which could have led to a positive zeta potential. The IGVJFT produces droplets that solidify while they undergo shear, vibration, and Ca^2+^‐induced aggregation (Wagle et al. 2020; Shah et al. 2025). During this microparticle production process from droplet to solid particle formation, some hydrophobic regions of kafirin might have packed into the particle interior, while some polar and ionizable residues may have migrated toward the surface. This re‐arrangement could lead to exposure of glutamic and aspartic acid residues at the particle surface. So when the DDGS kafirin microparticles made using IGVJFT are resuspended in water for zeta‐potential measurement, the exposed acidic residues are deprotonated at the near‐neutral pH of water, which could result in negatively charged carboxylate groups at the slipping plane.

Furthermore, this indicates that the overall negative charge (anionic groups) of the formulated system was significantly higher than the positive charge (cationic groups), subsequently resulting in a net negative charge (Song et al. 2021). The ANOVA data suggest the significance of the process parameters for zeta potential, as follows: DDGS kafirin concentration (F = 170, p < 0.0001) > nozzle diameter (F = 146.41, p < 0.0001) > internal frequency (F = 3.84, p < 0.00920) (Table 4). It is evident that lower DDGS kafirin concentration resulted in greater ionic repulsive force (more negative zeta potential) than attractive force. The stability of the particle is a balance between attractive and repulsive forces (Guo et al. 2017; Ohshima 2015); that is, the DDGS kafirin colloidal system will be more stable when repulsive forces (higher negative) are stronger than attractive forces (Yan et al. 2021). The more negative zeta potential is created by steric repulsion or electrostatic interactions, which influence the stability of particles by decreasing the van der Waals force (Ohshima 2015; Midekessa et al. 2020). Overall, the study indicates that a higher density of DDGS kafirin within the droplets might have enhanced intermolecular interactions such as hydrophobic interactions and disulfide‐mediated crosslinking, and these interactions could reduce the availability of surface‐oriented ionizable groups. As a result, particles prepared from higher levels of DDGS kafirin concentration carried a lower net negative charge.

The nozzle diameter also showed an influence of the zeta potential, possibly because a larger nozzle diameter produced thicker droplets (Section 3.2), which might have lower shear at the point of ejection. This lower extent of interfacial shear‐driven rearrangement during droplet breakup might have influenced the orientation and exposure of more charged residues on the particle surface (Villermaux 2007). As is evident from the diagnostic plot (Figure S6), an increase in the nozzle diameter increased the zeta potential of the system. Another possible explanation might be that slower jet disruption promotes the formation of a more compact surface, where buried residues contribute less to surface charge (Eggers 1997).

Although the influence of internal frequency was lower than that of the other parameters, it remained statistically significant (F = 3.84, p = 0.0092). The data indicate that increasing the frequency from 900 to 1600 Hz might have introduced stronger periodic disturbances to the jet, thereby improving the efficiency of particle breakup. Consistent with this, microparticles produced at 1600 Hz exhibited more negative zeta potential than those formed at 900 Hz. The possible explanation might be that higher levels of internal vibration might have reduced protein–protein interactions during the particle production process, which could have allowed more deprotonated carboxyl groups to remain exposed at the particle interface (Norde 2008).

The influence of electrode voltage may relate to the electrohydrodynamic environment at the droplet interface. Increasing the applied voltage enhances interfacial polarization and electrical stress, which can alter charge distribution between the forming droplets and the surrounding CaCl_2_ solution, thereby affecting the final surface charge of the microparticles (Jaworek 2007). Indeed, voltage did not exceed the Bonferroni significance threshold (Figure S4); however, the data indicated a consistent trend. As an example, many particle formulations processed at an integrated voltage of 600 V displayed a slightly more negative zeta potential than those compared to 200 V under similar other conditions. For example, Run 7 with an integrated electrode voltage of 600 V showed a zeta potential value of −25.5 mV, whereas Run 4 (200 V) showed −32.8 mV. This shift indicated that the applied electric field affects interfacial charge organization during ionic gelation through the partial rearrangement at the droplet surface (Ohshima 2015; Midekessa et al. 2020). Overall, while the magnitude of the effect is small (Figures S2, S4, and S6), the measured values indicate voltage‐dependent change at least at the surface of microparticles.

Microparticle Surface Morphology and Elemental Composition

3.4

FE‐SEM micrographs at a lower resolution (500 µm) revealed differences in microparticle morphology between the selected runs (Figure 1A–D). A near‐oval‐shaped DDGS kafirin‐based microparticle was seen from Runs 2 and 11 (Figure 1A,C), whereas Runs 8 and 12 produced nearly spherical DDGS kafirin‐based microspheres (Figure 1B,D). These findings agree with previous literature on zein (Coelho et al. 2021), in which the authors reported that a higher zein concentration resulted in microparticles with structural deformation. This behavior could be attributed to the increased viscosity and stronger intermolecular interactions at the higher concentration of prolamin protein, which might limit molecular mobility during droplet solidification (Wang and Padua 2012). As the solvent diffuses out, restricted chain rearrangement might promote the formation of internal voids, which could lead to deformation (Li et al. 2017).

Field‐emission scanning electron micrograms (FE‐SEM) of DDGS kafirin microparticles using secondary electron beam signal at lower resolution, illustrating the effect of production processing conditions on particle morphology. Microparticles from (A) Run 2, (B) Run 8, (C) Run 11, and (D) Run 12, all at a magnification of 500 µm, a voltage of 5.00 kV, and a field of view of 1.63 mm, using secondary electron beam electron signal.

The surface texture of microparticles was also resolved at higher magnification (50 µm). The DDGS kafirin‐based microparticles (Runs 2 and 11, and Runs 8 and 12; Table 2) showed nonhomogeneous surface texture (Figure 2A–D). Despite this, fewer microphase cracks in Runs 2 and 11 microparticles were seen when compared to Runs 8 and 12. These cracks are a characteristic feature of protein microparticles with amphiphilic surfaces, including zein, when the colloidal dispersion is highly dense (Wang and Padua 2010; Wang and Padua 2012). Such findings are similar to previous studies, in which one study reported an increase in the concentration of zein large particles with inhomogeneous morphology (Li et al. 2017).

Close‐up of DDGS kafirin microparticles from (A) Run 2, (B) Run 8, (C) Run 11, and (D) Run 12, showing microstructural texture at higher resolution: magnification 50 µm, voltage 5.0 kV, and field of view 185 µm using a secondary electron beam signal. These images highlight the impact of processing parameters of ionic gelation vibrational jet flow technology on the microstructural texture and porosity of DDGS kafirin microparticles.

EDS showed the presence of C, N, O, Cl, and Ca elements on the surface of Run 11 (large size) DDGS kafirin‐based microparticles (Figure 3B). The elemental profile of Run 12 (small‐spherical) DDGS kafirin‐based microparticles shows the presence of C, O, Na, S, Cl, and Ca on the surface (Figure 3C). The findings indicate that the concentration‐dependent resulting morphology influences the surface elemental composition of the particles. For example, Run 11, produced at a higher DDGS kafirin protein concentration, yielded a denser structure, whereas Run 12 particles, which were prepared at a lower concentration of DDGS kafirin, showed a more open structure (Table 2; Figure 2). Although kafirin contains sulfur‐bearing amino acids (cysteine and methionine) (Taylor and Taylor 2018; Shah et al. 2024), their detection by EDS depends on whether these residues are exposed at the outermost segments, that is, 1–2 µm (which is the maximum penetration depth of EDS). In Run 11, the absence of detectable S indicates burial of cysteine‐rich domains within the interior, consistent with the denser, more compact morphology of these larger particles, as evident from SEM imaging. Conversely, S appeared in Run 12, which suggests that lower kafirin concentration and a more porous structure resulted in greater surface exposure of sulfur‐containing residues.

Energy‐dispersive X‐ray spectroscopy (EDS) of microparticles. (A) Run 2, (B) Run 11 DDGS kafirin microparticle (larger sized), and (C) Run 12 (smaller sized) DDGS kafirin‐based microparticle. The spectra show C, N, O, and S from the kafirin matrix and Ca and Cl from CaCl2‐mediated ionic gelation, with Na detected in samples resulting from residual extraction reagents. Differences in S detection between Run 11 (larger particles and denser) and Run 12 (smaller and porous) suggest that processing conditions and morphology affect the exposure of sulfur‐containing residues at the particle surface.

The presence of Ca and Cl on particle surfaces indicates crosslinking when the droplets enter the CaCl_2_ gelation bath. Such ions provide particle rigidity and resistance to rapid disintegration in aqueous media. The detection of Na in Run 12 might be due to residual sodium from the extraction reagents (NaOH and Na_2_S_2_O_5_), which could also reflect a slightly higher exposure of hydrophilic or ionizable segments in more porous, low‐concentration particles. Taken together, the elemental profile indicated that formulation with the higher DDGS kafirin concentration resulted in the bigger particles, which tend to bury sulfur‐ and nitrogen‐rich regions, while processing conditions that prepared smaller DDGS kafirin particles increased the exposure of sulfur‐ and nitrogen‐rich groups at the interface. In addition, detection of Ca and Cl at the surface further supports the interpretation of zeta potential results, as these ions are expected to interact with deprotonated carboxyl groups and contribute to the overall electrostatic potential of the formulated microparticles.

Furthermore, C, O, S, and N elements have previously been shown to be present on the surface of zein protein with a water contact angle of 126° (Dong et al. 2013), and other seen elements suggest some surface segments are made of those elements that belong to the bottom left corner of the periodic table. The absolute measurements of the surface elemental profile by EDS are difficult, as X‐rays can penetrate very little into the inner part of the particle, but not more than 2 microns. However, EDS has routinely been used for surface elemental profiling of biomaterials, including those for zein and kafirin (Dong et al. 2013; Shah et al. 2021; Mooranian et al. 2016; Mooranian et al. 2016; Small et al. 1998). EDS data therefore indicate that the processing parameters of IGVJFT, including DDGS kafirin concentration, manipulate morphological and chemical properties of formulated microparticles and thereby influence hydration behavior, mechanical integrity, and capacity to act as a delivery vehicle.

Conclusion

4

Using a fractional factorial design, the study aimed to identify the significant process parameters of the IGVJFT that affect the volume‐weighted mean particle size and zeta potential of DDGS kafirin microparticles. The fractional factorial design showed that DDGS kafirin concentration, nozzle diameter, integrated voltage, and internal frequency are the most important process parameters that influence the volume‐weighted mean microparticle size and the zeta potential of the microparticles. With this approach, microparticles with different sizes, stabilities, and morphologies were formed by tuning the process parameters of the IGVJFT. The microparticles produced using the IGVJFT comprise three sequential stages: (a) preparation of DDGS kafirin formulation; (b) disruption of the laminar jet into droplets under the combined influence of vibrational frequency and electrode charging; and (c) rapid ionic crosslinking of charged droplets upon contact with gelation solution of CaCl_2_, which causes solidification. Further studies should utilize the most important process parameters identified here to fine‐tune microparticle preparations using IGVJFT. Notably, the tunability demonstrated in this study offers a pathway to design DDGS kafirin microparticles as solid carriers for applications in the nutraceutical and pharmaceutical industries.

Author Contributions

Umar Shah: methodology, investigation, formal analysis, visualization, software, writing – original draft. Rewati Bhattarai: writing – review and editing. Hani Al Salami: writing – review and editing. Chris Blanchard: writing – review and editing. Stuart K. Johnson: conceptualization, supervision, funding acquisition, project administration, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Information: jfds70958‐sup‐0001‐SuppMat.docx