Studying the impact of zein microfibers on the physicochemical and microstructural properties of bi-gels based on ι-carrageenan hydrogels and beeswax oleogels

Abstract

This research presents a novel bi-gel system formed by combining zein microfiber -reinforced carrageenan hydrogels and beeswax oleogels. The main objective is investigating the impact of the interplay between zein microfibers, ι-carrageenan hydrogels, beeswax oleogels on the properties of bi-gels. The study focused on bi-gel formulations combining beeswax oleogel and carrageenan, both plain and with zein microfibers. Different ratios of oleogel to ɩ-carrageenan hydrogel and oleogel to reinforced ɩ-carrageenan hydrogel were established: 5:95, 10:90, 15:85. The designed bi-gels exhibited semi-solid gel properties in rheological analysis, with increased oleogel content enhancing firmness, storage modulus, and loss modulus (G' < G″, p < 0.05). The incorporation of oleogel in the bi-gel substantially increased its consistency from 131 (g.s) to 668 (g.s) in the bi-gel containing 0.5% zein microfiber, 10% oleogel, and 90% hydrogel. FTIR results suggested that the bi-gels were formed through physical interactions without covalent cross-linking. Microfibers had a positive effect on the textural characteristics of bi-gels. The hardness of bi-gels increased from 13.26 to 35.12 g to 31-93-64.14 g after addition of microfibers. The BGZ10 formulation, consisting of 10% oleogel and 90% zein-reinforced hydrogel, showed the highest consistency among samples, with measurements of 668.48 ± 3.53 (g.s) and a G′ value of 291000 ± 91.27 (Pa) (P < 0.05). Additionally, the BGZ10 formulation displayed the highest complex viscosity, measuring at 47300 ± 20.73 (P < 0.05). The thermal stability of bigel considerably increased by cooperation fibers in hydrogel. The developed bi-gels demonstrate significant potential for substituting conventional solid fats and introducing distinctive visual characteristics in various food products.

Graphical abstract

Highlights

• •Bi-gels were made of ι-carrageenan, zein microfiber, and beeswax oleogel.
• •More oleogel cooperation in the bi-gel system enhanced firmness and elasticity.
• •Zein microfibers (ZMF) improved the textural and viscoelastic properties of bi-gels.
• •FTIR analysis demonstrated that bi-gels were formed through physical interactions.
• •10% ZMF bi-gel had the most uniform and elastic structure compared to 5 and 15%.

1. Introduction

Combining different substances to form distinct structures and functional materials has garnered considerable interest in materials science, including food science and engineering. Recently, bi-gels, a fusion of hydrogels and oleogels (organogels), have emerged as promising materials with diverse applications (Fasolin et al., 2023; Singh et al., 2014). Bi-gels, also known as hybrid gels, are semi-solid formulations typically prepared by high-speed mixing of oleogel and hydrogel at a specific temperature. Due to the presence of two gelled phases instead of one, these hybrid systems offer superior properties over emulsion gels. Additionally, combinations of micro-phase separated proteins and polysaccharides can generate bi-gel-like systems in a single solvent, such as water (Shakeel et al., 2021). Combining hydrogels and other components, such as beeswax oleogels, can result in synergistic effects in bi-gels. These responses can also affect the release and adsorption of bioactive ingredients (Salgueiro et al., 2013).

Hydrogels are commonly created through the entrapment of the solvent phase within the 3D network of gelling agents (Shakeel et al., 2021). Hydrogels can integrate substantial amounts of water into their 3D structures (Yang et al., 2022a). Hydrogels can be divided into two categories: physical and chemical hydrogels. The former are generally reversible and heterogeneous, with molecular entanglements holding networks together. The latter rely permanently on covalently cross-linked networks (Salgueiro et al., 2013). In recent years, hydrogels derived from natural polymers such as proteins and polysaccharides have gained popularity in the functional food industry. This is due to their low toxicity, edibility, biocompatibility, biodegradability, and affordable price (Akrami-Hasan-Kohal et al., 2020; Naji-Tabasi et al., 2023). This indicates that modifying the structures of hydrogels can elicit different reactions to environmental factors such as pH, ions, enzymes, temperature and reinforced fibers. Zein microfibers can be crucial in improving hydrogels' and bi-gels' structural integrity and functional properties. When integrated into hydrogels, zein microfibers act as reinforcing elements, enhancing the mechanical strength and stability of the gel structure. These microfibers establish a network within the hydrogel matrix, thereby boosting its overall integrity and increasing deformation resistance (Mattice and Marangoni, 2020). Therefore, it is essential to consider adjusting hydrogel structures for optimal performance in different conditions (Khalesi et al., 2020; Yang et al., 2023). Incorporating zein nanoparticles into starch-based bio-nanocomposite films has been observed to enhance the tensile strength and Young's modulus while simultaneously reducing the elongation at break of the films (Alinaqi et al., 2021). Moreover, the utilization of zein microfibers within the alginate gel system has demonstrated an increase in hydrogel degradation, resulting in accelerated compaction, improved interaction between cells and the gel, and enhanced alignment of bovine muscle precursor cells (MacQueen et al., 2019; Melzener et al., 2023; Sharma et al., 2020). It is assumed that the presence of zein microfibers can contribute to the formation of a dual-gel system with unique properties derived from the individual components. Zein, a corn protein with unique properties, has shown promising applications in forming biopolymer composites and gel systems. The presence of zein microfibers can indeed contribute to the formation of dual-gel systems with unique properties derived from individual components (Kim et al., 2010; Yuan et al., 2022). Zein can self-assemble in aqueous medium and form gels in hydroalcoholic solvents at higher concentrations makes it an excellent candidate for creating composite gel systems (Raza et al., 2023). Interestingly, while zein can form gels, it can also coat other particles, creating unique composite materials. This coating process utilizes zein's property of adsorbing to hydrophilic particle surfaces in aqueous ethanol solutions, forming aggregates and large agglomerates (Kim et al., 2010). This versatility allows for the creation of diverse dual-gel systems with tailored properties. The interactions between zein microfibers, ι-carrageenan hydrogels, and beeswax in sunflower oil oleogels can result in a complex gel network and better bi-gel structure and properties.

Bi-gels can be designed as fat replacers due to their unique biphasic structure and potential advantages over traditional fat substitutes. Fat reduction is desirable for health reasons, it can significantly affect texture, organoleptic properties, mouthfeel, appearance, and stability of dairy products This highlights the challenge in developing effective fat substitutes that can maintain product quality. Previously, Ghiasi and Golmakani (2022) explored the limitations of existing fat replacers. The biphasic system of bigels can be tailored to achieve desired properties by adjusting the oleogel fraction, making it versatile for various food applications. the selection of bi-gels as fat replacers was driven by their unique structure, versatility, and potential to overcome the limitations of existing fat substitutes. The distinction between our work and previous research is that we have utilized a reinforced bi-gel system, wherein the hydrogel component is reinforced with zein microfibers. The reinforced bi-gel system is a fruitful area of future research in fat substitution in foods in our future study (Xu et al., 2024).

The existing gap in research about hydrogel reinforcement using zein microfibers underscores the necessity for further exploration to fully grasp this composite material's potential advantages and applications. Further investigation in this realm could unveil novel possibilities and enhance the characteristics of hydrogels reinforced with zein microfibers. The study by (Lu et al., 2022) focused on the development of bi-gels composed of oleogel-in-hydrogel structures to serve as carriers for curcumin and epigallocatechin gallate (EGCG). The researchers investigated the impact of the oleogelator glycerol monostearate (GMS) content on these bi-gels' structures and delivery functionality. Similarly (Yang et al., 2022b), developed a bi-phasic gel system that incorporated a hydrogel component (κ-carrageenan) along with an oleogel component. They specifically investigated the influence of different oleogelators, namely glycerol monostearate (GMS) and beeswax (BW), on the structures of these bi-gels. The bi-gels exhibit enhanced stability and functional properties in comparison to single-phase systems. The combination of hydrogel and oleogel phases allows for more precise control over physicochemical characteristics, microstructure, and mechanical properties. For instance, increasing the oleogel proportion resulted in a reduction in hardness but an increase in cohesiveness in bi-gels. At the same time, adding κ-carrageenan increased hardness and thermal stability (Zampouni et al., 2023). Notably, the type of oleogelator employed can exert a considerable influence on the bi-gel structure and characteristics. This versatility enables the adaptation of bi-gels to specific applications. In some cases, combining of κ-carrageenan with other components can improve over systems using single hydrocolloids. For example, adding κ-carrageenan to pea protein significantly increased gel hardness and water-holding capacity, with higher synergy values observed at higher temperatures (Bartkuvienė et al., 2024).

Zein-based bi-gels exhibit unique rheological properties that can be tailored for various applications. Adjusting the zein concentration can enable stable dispersions with pronounced solid-like characteristics, especially at physiological temperatures. These gels demonstrate pseudoplastic behavior and maintain their elastic properties up to 50 °C, making them suitable for various applications. Additionally, zein gels can encapsulate different compounds, showing various release profiles depending on the interactions between the probes and the polymeric matrix (Gagliardi et al., 2020). The use of zein microfiber as a reinforcement for hydrogel systems, particularly those incorporating ι-carrageenan, represents an intriguing avenue of inquiry that is currently under-researched. While both zein and ι-carrageenan have been individually studied for their distinctive properties and applications, there is a paucity of research examining the combined potential of these two materials in hydrogel formulations.

However, to our knowledge, all the research has focused on utilizing simple hydrogels to produce bi-gels. Given this scenario, the novelty of this manuscript is to offer an overview of the application of Zein microfibers to reinforce the structural properties of ι-carrageenan hydrogels and to investigate the subsequent impacts on the Beeswax in sunflower oil oleogels. Understanding the characteristics and performance of these bi-gels can yield valuable insights into their prospective applications in sectors like food and beverage, pharmaceuticals, and cosmetics. It is hoped that the results of this study enrich the increasing knowledge of bi-gels and delve into the future possibilities of these exceptional materials by examining the interactions between components and analyzing the microstructure.

2. Materials and methods

2.1. Materials

Ɩ-carrageenan (powder, 99.8% purity, and water gel strength: 1100–1600 g/cm², SMS TX2 Analyzer, 1.5% solution was provided by GPI Co., Ltd (Canada). Zein from corn (product number Z3625, 22–24 kDa) was purchased from Sigma Aldrich (St. Louis, MO, SA). Ethanol and calcium chloride (powder, 98% purity, and Molecular Weight; 110.95 g/mol) were acquired from VWR (Radnor, PA) and Fisher Scientific, an avantorTM Company, respectively. Distilled water was bought from Kimia Chemical Company in Iran. Sunflower oil was obtained from Oila Co., and Beeswax was sourced from the Larijan countryside in Amol, Iran.

2.2. Preparation of Oleogel

The organogels were prepared using sunflower oil as a solvent and beeswax as an organogelator. The method involved combining beeswax with sunflower oil and then heating them to 85 °C in a thermostatic water bath on a plate heater (VELP Scientific, Italy). Subsequently, the system was continually heated on a laboratory stirrer (RW 20, IKA-Werke, Germany) at 3000 rpm. Mixing was continued for 5 min after the organogelator had completely dissolved. The solution was then slowly cooled from 85 to 25 °C in a thermostatic water bath (F25, Julabo, USA) and held at 4 °C for the necessary period to attain thermal equilibrium (Gómez-Estaca et al., 2019a). Different treatments have been identified based on the quantity of beeswax employed, as detailed. Composition of the Oleogels (100 gr): OB7%: 7 (gr) Beeswax added 93 (gr) sunflower Oil, OB9%: 9 (gr) Beeswax added 91 (gr) sunflower Oil, and OB11%: 11(gr) Beeswax added 89 (gr) sunflower Oil.

2.2.1. Microstructure of Oleogel

Using a glass capillary tube, an appropriate amount of molten sample was carefully dispensed onto a preheated microscope slide and covered with a coverslip to ensure a thin sample layer. The samples were left to cool to room temperature for 24 h to facilitate complete crystal development. Images were observed and captured using an inverted optical microscope (Nikon, E100, Genus, Japan) at a 40x magnification. Additionally, a polarizing optical microscope equipped with a halogen lamp supply unit (Olympus, TH4-200) and a grey-level microscope charge-coupled device (CCD) camera were utilized (Chai et al., 2022).

2.2.2. Textural properties

The textural characteristics of oleogels were assessed using a Texture Analyzer TA-XT Plus (Stable Microsystems, Surrey, UK) equipped with a 45° conic acrylic probe. Samples stored in a glass container (inner diameter = 50 mm, height = 120 mm) at refrigerator temperature (4 °C) were removed and examined 30 min prior to the tests. The analysis comprised a penetration test technique at a 3.0 mm/s penetration speed to a 23 mm depth, followed by probe extraction at a 10 mm/s speed. The Texture Exponent v.6.1.1.0 software (Stable Microsystems) on the device was applied to calculate the parameters. Hardness as the height of the first peak and negative peak of the graph indicates the adhesion force and the negative level under the curve is introduced as adhesion (Yılmaz and Öğütcü, 2015).

2.2.3. Oil holding capacity (OHC) of Oleogel

Approximately 100–200 mg of oleogel was placed in 1.5 mL microtubes and the samples were centrifuged at 13,000 rpm (15,871×g) for 30 min using a micro centrifuge (Mikro 120, Hettich Zentrifugen, Andreas Hettich GmbH und Co, Tuttlingen, Germany). The oil holding capacity (OHC) was calculated as the percentage ratio between the weight of oil retained in the oleogel after centrifugation and the total weight of oil in the sample (Yang et al., 2022b). The gel weight was recorded as W₀, the variation (W) in the oil content of the sample before and after centrifugation was assessed, and the oil holding capacity was calculated using Equation (1).

$$OHC(\%)=\frac{W_0-\mathrm{W}}{W_0}\times 100$$ (1)

2.2.4. Oil loss of oleogel

Oil loss was determined by measuring the rate of oil migration over 30 days at 20 °C using the method proposed by (Bascuas et al. 2020) with some modifications. The weight of the oil released was gauged following the 30 days. A funnel and filter paper were positioned on the Erlenmeyer flask to capture the liquid oil dripping from the oleogel. The total weight of the funnel, filter paper, and flask was measured (M₁). Subsequently, 10 g of oleogel was weighed (M₃) and placed into the funnel. Samples were retrieved at specified intervals using a small, flat spatula. The funnel, filter paper, and flask containing the released liquid oil were weighed again (M₂). Results were articulated as grams of oil lost per 100 g of oleogel, calculated using Equation (2) and determined in triplicate for each sample.

$$\text{Oil}\mathrm{loss (\%)}=\frac{\mathrm{M}2-\mathrm{M}1}{\mathrm{M}3}\times 100$$ (2)

2.3. Preparation of zein microfiber

An initial 10 mL solution comprising 40% zein in 80% ethanol was dispersed, and the zein was allowed to dissolve completely over about 30 min. Subsequently, the zein fibrous network was established by introducing excess water directly into the ethanol-zein solution (Deng et al., 2018). The zein fibers underwent thorough washing to ensure comprehensive precipitation.

2.4. Preparation of hydrogel

The cold set technique (employing calcium ions) was utilized for the preparation of the ι-carrageenan hydrogel. Initially, a 1% w/v ι-carrageenan solution was prepared in hot water (80 °C), and then CaCl₂ was gradually introduced (0.5% w/v). The solution was stirred at this temperature using an IKA® C-MAG HS7 for 15 min. Then, the samples were cooled to room temperature (Zhou et al., 2021). The microfiber-reinforced hydrogel was prepared by adding 40% zein microfibers to the hydrogel system at 0.5% concentrations using the injection method. (Dai et al., 2017). The injection method results in the formation of a uniform and homogeneous mixture, which exhibits a clear appearance (Cui et al., 2020).

2.5. Preparation of Bi-gels

Bi-gels were created by independently preparing hydrogels and oleogels and combining them using a mechanical homogenizer (rotor-stator VELP). The sample preparation times were coordinated to ensure the oleogel and hydrogel preparations were completed simultaneously at similar temperatures. The hot oleogel (11% with Beeswax) was poured into the pre-gelled hydrogel. Both phases underwent homogenization using a preheated homogenizer (Ultra Turrax, IKA, Staufen, Germany) at 23,000 rpm for 3 min at ambient temperature. After cooling at room temperature for 1 h, the samples were stored overnight at 4 °C. Six ratios of oleogel to ɩ-carrageenan hydrogel and oleogel to reinforced ɩ-carrageenan hydrogel were established: 5:95 (BG5), 10:90 (BG10), 15:85 (BG15), and reinforced hydrogel 5:95 (BGZ5), 10:90 (BGZ10), 15:85 (BGZ15). The distribution of each component's mass for every formulation is presented in Table 1. Before analysis, all samples were stored at 4 °C and left to equilibrate at room temperature for 30 min. Unless stated otherwise, characterization was carried out at room temperature.

Table 1.

Composition of the bi-gels (100 gr).

Formulations	Oleogel (gr)	Hydrogel (gr)	Zein Microfiber Reinforced Hydrogel (gr)
BG5	5	95	–
BG10	10	90	–
BG15	15	85	–
BGZ5	5	–	95
BGZ10	10	–	90
BGZ15	15	–	85

2.5.1. Textural properties of Bi-gels

The texture of the bi-gels was analyzed using a Texture Analyzer TA-XT Plus (Stable Microsystems, Surrey, UK) equipped with a 45° conical acrylic probe. Approximately 40 g of bi-gels was molded in a 100 mL cylindrical glass beaker (inner diameter = 50 mm, height = 120 mm) at 4 °C, and the penetration test was done after 24 h. The gels were compressed to a depth of 10 mm at a 60 mm/min speed. Consistency (g.s), Adhesiveness (g.s), and Firmness were employed to characterize the textural features of the samples. Each test was repeated at least five times, and the results were reported as mean ± SD (Paciulli et al., 2020).

2.5.2. Rheological measurements of Bi-gels

The rheological assessment was undertaken after a 24-h storage period at 7 °C. The rheological analysis employed the Anton Paar MCR301 rheometer (Austria) with parallel plate geometry (25 mm diameter, 1 mm gap) at 7 °C. The selected temperature of 7 °C frequently represents storage conditions for many food products, dairy items, and other materials that may be stored in refrigeration. Samples were transferred to the plate's center and left to equilibrate for 5 min to ensure thermal and mechanical balance post-sample handling. Initially, a strain sweep ranging from 0.01% to 100% at a frequency of 1 Hz and 7 °C was performed to identify the Linear Viscoelastic (LVE) range. Subsequently, oscillatory frequency sweep experiments were conducted across frequencies from 0.1 to 100 Hz at 7 °C, with a constant strain of 1%. Subsequent studies employed this bi-gel system as a fat replacement and subjected it to refrigerated conditions for examination. The storage modulus (Gʹ) and loss modulus (G″) were measured relative to the angular frequency (ω) (Zhu et al., 2021).

2.5.3. Optical microscopy of Bi-gels

The formulations' homogeneity was scrutinized using a polarized optical microscope (DAFFODIL MCX100, Austria). Sudan staining (1% w/v liquid paraffin) was employed to delineate between the oleogel and hydrogel phases, ensuring the fundamental homogeneity of the formulation (Flood et al., 2016).

2.5.4. FTIR of Bi-gels

The freeze-dried samples were subjected to FTIR experiments using a PerkinElmer Fourier transform spectrophotometer (PerkinElmer, West Midlands, UK) equipped with an attenuated total reflectance sampling accessory. Spectra were captured across the 4000 to 400 cm⁻¹ range at a 4 cm⁻¹ resolution. To facilitate the analysis, the bi-gels were mixed with potassium bromide (KBr) and compressed into tablets utilizing a hydraulic press (Bollom et al., 2020).

2.5.5. Centrifugation and thermal stability

The stability test for the sample was conducted at 25 °C using the centrifugation method. To assess the thermal stability of the bi-gel sample, it was subjected to heating at 50 °C and 70 °C for 30 min, then cooling to 25 °C. The assessment of the solvent holding capacity (SHC) of bi-gels was employed to evaluate the ability of the bi-gel matrix to retain a significant amount of solvent when subjected to centrifugation forces (4200×g for 15 min) at room temperature (Martins et al., 2023). Two samples were selected from a simple bi-gel and reinforced bi-gel for thermal stability testing. Each bi-gel contains 10% oleogel.

2.6. Statistical analysis

The statistical assessments were performed using JMP Pro 14 software (SAS; Cary, NC, USA). Statistical distinctions were evaluated via one-way ANOVA along with Tukey's multiple comparisons test, with the significance level set at 0.05. Before the analysis process, validation of the ANOVA assumptions of independence and equal variance was conducted to ensure accuracy. This validation was carried out through triplicate tests of each treatment.

3. Results and discussion

3.1. Oleogel properties

3.1.1. Optical microscopy properties of Oleogel

The oleogel analysis showed needle-like structures consistent with beeswax-derived oleogels in Fig. 1. Beeswax crystals in sunflower oil oleogels tend to form densely packed, spherical, and white structures (Pang et al., 2020b). These distinct structures were notably visible in images A, B, and C of Fig. 1. Gelation is the process in which microcrystalline structures combine to create a three-dimensional network that can trap the oil and produce an oleogel (Pérez-Martínez et al., 2007). While waxes like candelilla and carnauba form smaller spherulitic crystals, the needle-like microcrystals in gelation are adept at trapping substantial oil volumes amidst the crystal chains (Zetzl and Marangoni, 2011). The microstructure of the gel oil is visibly influenced by the amount of beeswax added, as depicted in Fig. 1. Increasing the beeswax content leads to a more pronounced needle-like crystal structure within the oil, predominantly seen with 11% beeswax (Fig. 1C). As the wax content rises, the crystals within the network units of the oil system densify gradually. Additionally, there is a gradual increase in crystal size observed (Zhang et al., 2021). Despite differences in aggregation and crystal size within the crystal networks, these factors showed little influence on crystal morphology. The crystal structure is often presented as fine needle-shaped with closely situated crystals, consistent with the results of (Fasolin et al., 2023). Referring to Fig. 1 A, the oleogel containing 7% beeswax displayed a substandard crystalline structure with an indistinct needle-like pattern. Conversely, the dense crystal structure in Fig. 1C with 11% beeswax led to a slightly "blurred" or "fuzzy" appearance. Such a condensed crystalline network structure is known to enhance compressive resistance. The dense packing and spherical shape of beeswax crystals in sunflower oil oleogels contribute to their structural stability and oil-binding capacity. According to Pang et al. (2020b), oleogels with beeswax as the only oleogelator (Sit0/BW10) exhibited the highest hardness and maximum enthalpy change, indicating a strong and stable network structure. This is further supported by Pang et al. (2020b), which revealed that beeswax-based oleogels formed via non-covalent bonds and were stabilized with physical entanglements. Interestingly, the crystal morphology of beeswax in sunflower oil differs from that observed in other oils. For instance, Pang et al. (2020a) reported needle-shaped crystals in beeswax-based oleogels with various vegetable oils, including sunflower oil. This contradiction highlights the complexity of oleogel formation and the potential influence of specific oil-wax interactions on crystal morphology (Pang et al., 2020a, Pang et al., 2020b).

Fig. 1.

The microstructure of oleogel and formation of Beeswax crystals in different concentrations (A:7% Bees wax; B:9% Bees wax; C:11% Bees wax).

3.1.2. Textural properties

Table 2 shows that OB11% oleogel exhibited higher hardness and adhesiveness than OB7% and OB9% oleogels (p < 0.05). This result is likely due to the compact and dense cell structure of the OB11% oleogel, which is influenced by its low specific volume. The concentration of beeswax at 11% may promote denser and more interconnected crystallization dynamics compared to lower concentrations. This increased crystallization can lead to increased hardness in the gel. The optimum beeswax concentration of 11% may represent an optimum balance for the formation of a strong and cohesive gel network. This concentration of beeswax could result in the development of a structure with improved properties, such as increased hardness and adhesiveness, compared to structures with lower concentrations (Liu et al., 2019; Moghtadaei et al., 2018; Yi et al., 2017). Beeswax is an effective sunflower oil structuring agent, forming a stable oleogel network. The beeswax crystal network exhibits strong structuring performance compared to other waxes like sunflower and rice wax (Fayaz et al., 2020). The addition of beeswax modulates properties such as gel-to-sol dissociation temperature and oil adsorption capacity of the oleogel layer (Hao et al., 2022). Interestingly, there are some contradictions regarding the oxidative stability of beeswax-sunflower oil oleogels. While one study found that beeswax can improve the frying stability and oxidative resistance of sunflower oil (Tajer and Ozdemir, 2024), another study reported that beeswax-based oleogels had the lowest oxidative stability and induction period among tested samples (Sobolev et al., 2022). An addition level of less than 15% beeswax was insufficient to form stable oleogels with sunflower oil, while 10% sunflower wax was enough to create stable oleogels. The hardness and stickiness of the oleogels were influenced by the type and level of wax addition, as well as the oil's viscosity (Bartkuvienė et al., 2024).

Table 2.

Hardness, adhesiveness, oil holding capacity, and oil loss values for the oleogels with varying levels of beeswax.

Sample	Hardness (g)	Adhesiveness (g.s)	OHC (%)	Oil loss (%)
OB7%	394.24 c ±11.71	−802.33 a ±13	91.10c±0.53	0.83a±0.03
OB9%	1186.20 b ± 14.50	−1412.89 b ± 46.77	93.60b ± 0.43	0.62b ± 0.02
OB11%	1733.70 a ±39.48	−2631.45 c±9.23	95.95a±0.76	0.48c±0.01

OB7%: 7% beeswax and 93% sunflower oil; OB9%: 9% beeswax and 91% sunflower oil; OB11%: 11% beeswax and 89% sunflower oil.

Data are means ±standard deviations, n = 3. Values in the same column with different letters are significantly different (p < 0.05).

This discrepancy may be due to differences in experimental conditions or beeswax composition. In addition, OB7% showed the lowest hardness and adhesiveness, correlating with its lower beeswax concentration. The 11% beeswax oleogel was carefully chosen for its exceptional texture, sensory properties, functional attributes, cost-effectiveness, and suitability for specific applications. This decision underscores the significance of looking beyond density and considering a range of factors when formulating oleogels to precisely meet desired product specifications and align with consumer preferences (Gómez-Estaca et al., 2019b; Wang et al., 2023).

3.1.3. OHC Oleogel

The data presented in Table 2 illustrates an increase in OHC (Oil Holding Capacity) alongside higher beeswax concentrations, showing the highest OHC value with OB11% and the lowest with OB7%, aligning with the lowest beeswax content (p < 0.05). The higher beeswax content may enhance the stability of the oleogel structure, prevent phase separation, and contribute to increased hardness and cohesiveness. Higher wax content can promote stronger intermolecular interactions, such as crystallization, which can enhance the overall firmness and cohesiveness of the oleogel (Pino et al., 2024; Yang LiJun et al., 2014). At 11% concentration, the synergistic interactions between oil and beeswax facilitate the development of a dense and interconnected crystal network within the oleogel. This network efficiently captures and retains oil, enhancing the OHC of the gel (Fayaz et al., 2020; Principato et al., 2021).

3.1.4. Oil loss

Oil loss is an important characteristic that affects the suitability of oleogels in various food applications. The ability of oleogels to control oil migration and leakage is particularly beneficial in specific food contexts. Freshly prepared oleogels displayed no oil losses, but during the 1-month storage period, all oleogels experienced considerable oil loss. Table 2 indicates that OB11% had the lowest leakage (0.48 ± 0.1, p < 0.05) compared to other samples. The lowest oil loss in oleogels containing beeswax (OB11%) could be attributed to the unique properties of beeswax. Beeswax is known for its hydrophobic nature, which could contribute to better containment of the oil phase within the oleogels. The beeswax may form a protective barrier that helps to prevent oil leakage, enhancing the overall stability and structure of the oleogel. Additionally, the interaction between beeswax and the oil phase could create a more cohesive network that resists separation and leakage, leading to improved performance in terms of containment. According to Kim and Oh (2022), oil binding capacity was related to the crystal size and the spatial distribution (Kim and Oh, 2022). In addition, Li et al. (2021) reported that the oil binding capacity was highly correlated with the G′ values, and the strong networks with high elasticity improved the oil binding capacity (Li et al., 2021).

The oleogels have been shown to mitigate the common problem of oil loss in oil-containing products such as cookies and cream fillings (Pawar et al., 2024). This property is precious in composite food products where oil leakage between different components can negatively impact texture, appearance, and shelf life. The ability to control oil loss makes oleogels suitable for use in high-fat, soft-texture products, where maintaining structural integrity is crucial. Interestingly, the oil-binding capacity (OBC) of oleogels can be influenced by the characteristics of the oleogelators used. For instance, in cellulose nanofiber (CNF)-based oleogels, smaller CNF diameters (<20 nm) resulted in higher oil-binding capacity (∼85%) and better thermal stability (Zou et al., 2024). This enhanced OBC could be attributed to stronger hydrogen bonding interactions between CNF and oil molecules during oleogelation. Oleogels, in general, demonstrate high oil-binding capability and good structural stability. This property is crucial for preventing oil loss and maintaining the integrity of the gel structure, which could be advantageous in both food and pharmaceutical contexts. The stability and oil-binding capacity of oleogels are essential factors in their applications. Oleogels have high physical and structural stability and high oil binding capacity (Silva et al., 2022). Therefore, the 11% oleogel sample was applied for the bi-gels formulation based on its exceptional characteristics, such as high OHC, increased hardness, and minimal oil leakage.

3.2. Hydrogel component

The hydrogel is the primary structural component of the bi-gel, which is produced in this study through two straightforward methods: the use of ɩ-carrageenan hydrogelator (a simple approach) and the zein microfiber incorporated ɩ-carrageenan (a reinforced method). The hydrogel structure exerts a profound influence on the physico-chemical characteristics of the bi-gel, given that it comprises a considerable proportion of bi-gel. Consequently, the strengthening of the hydrogel's structure and properties will concomitantly enhance the bi-gel structure. The utilization of microfibers plays a pivotal role in the microstructure of hydrogel, with three samples of simple hydrogel and three samples of reinforced hydrogel were employed in the production of bi-gel. To have a comprehensive overview about the influence of microfibers on the bi-gel structure, the hydrogel component had a consistent formulation as it is described in 2.7 section.

3.3. Visual physical properties of bi-gels

Fig. 2 shows bi-gel formulations with various oleogel: bi-gel ratios. As it is shown in the picture, the addition of zein microfibers has resulted in a more formable product. The clear scrapes of the nozzle on BGZ15 (5% oleogel and 95% reinforced hydrogel) reveals the structured texture of this sample. When transitioning from the rightmost to the leftmost sample, the depth and clarity of the nozzle effect are diminished. The macroscopic observation provides indirect confirmation of the texturizing and strengthening impact of zein microfibers on bi-gel samples. It is evident that as the level of microfibers increases, the structural integrity of the sample proportionally improves (Fig. 2).

Fig. 2.

Bi-gel samples with different oleogel: hydrogel ratios BG5 (5 oleogel:95 hydrogel); BG10 (10 oleogel:90 hydrogel); BG15 (15 oleogel:85 hydrogel); BGZ5 (5 oleogel:95 reinforced hydrogel); BGZ10 (10 oleogel:90 reinforced hydrogel); BGZ15 (15 oleogel:85 reinforced hydrogel).

3.3.1. Textural properties of Bi-gel: penetration test

Table 3 outlines instrumental TA attributes for different bi-gel formulations. Notably, the firmness of the bi-gel increased as the oleogel concentration rose in reinforced hydrogels for BGZ10:90 and BGZ15:85. Firmness and consistency correlated with the structured oleogel content in the hydrogel (p < 0.05), indicating a substantial firmness elevation in bi-gels with higher oleogel ratio (BGZ10:90). Elevated oleogel content strengthened bi-gel firmness, driven by extensive hydrogen bonding reinforcing the structure, consistent with research by (Rehman et al., 2014; Singh et al., 2014; Zampouni et al., 2023). Consistency, ranged from 131 (g.s) to 668 (g.s), with BGZ10 showing superior values to BG5, whereas BG5 displayed the lowest consistency at 131(g.s). Adhesiveness increased with higher oleogel concentrations in both BG10:90 and BGZ15:85. Variances in mechanical properties across bi-gel systems are linked to oleogelator type, concentration, oil droplet attributes, and phase ratios. The concentration of oleogels and hydrogels in bi-gels, as well as the concentration of wax, specific volume, and cell structure, can affect the textural characteristics. Higher wax concentrations may result in denser textures, while lower specific volume and compact cell structures can contribute to a smoother and firmer texture. These components collectively play a significant role in determining the overall texture and consistency of the bi-gels (Shakeel et al., 2021; Zhu et al., 2021). At higher concentrations, the oleogel enhances the structural integrity of the hydrogel by providing essential support and stability. The oleogel contributes essential support and stability to the hydrogel matrix, helping maintain its overall structure and properties. This support can include improving the texture, firmness, and mechanical strength of the hydrogel, ensuring that it retains its desired characteristics and functionality. The oleogel acts as a scaffold or framework within the hydrogel, reinforcing its structure and contributing to its overall performance.

Table 3.

Textural properties of Bi-Gels and reinforced Bi-Gels with zein microfibers.

Sample	Consistency (g.s)	Adhesiveness (g.s)	Firmness (g)
BG5	131.72e ± 0.83	−87.26a±2.37	13.26e±0.58
BG10	326.2c±7.26	−141.92b ± 5.45	35.12c±2.68
BG15	227.04d ± 6.84	−168.69c±4.56	24.86d ± 2
BGZ5	325.34c±2.55	−135.23b ± 3.59	31.93cd ± 2.90
BGZ10	668.48a±3.53	−401.12e± 5.24	64.14a±3.53
BGZ15	426.53b ± 6.54	−235.91d ± 8.45	49.01b ± 3

BG5: 5% oleogel and 95% hydrogel; BG10: 10% oleogel and 90% hydrogel; BG15: 15% oleogel and 85% hydrogel. BGZ5, BGZ10, and BGZ15 have similar formulations to the previous exact phrases; however, they are reinforced by zein microfibers.

Data are means ±standard deviations, n = 3. Values in the same row with different letters are significantly different (p < 0.05).

This optimal concentration range allows for synergistic interactions between the hydrogel and oleogel components. As the oleogel content increases beyond 15%, a saturation of oleogel points within the hydrogel matrix may occur. This saturation can lead to an imbalance in the composition of the hybrid gel system, disrupting the uniformity and strength of the structure. The interactions between hydrogel and oleogel components are crucial in determining the overall structural properties. Excessive oleogel content can alter these interactions, potentially weakening the bonds that contribute to the strength of the hybrid gel. This textural response occurred due to the disrupted hydrogel network resulting from the increased inclusion of oleogel.

Adding zein fiber to bi-gels could enhance the structural integrity, mechanical properties, and overall performance of the oleogels. Depending on the specific composition and concentration used, the incorporation of zein fiber may improve the texture, stability, and other characteristics of the oleogels (Phoon and Henry, 2020). The higher the interaction forces among oleogelator molecules in oleogels with elevated oleogelator content, the tighter the oleogel network, which in turn results in an increase in hardness (Gómez-Estaca et al., 2019a). Earlier studies have indicated a direct association between the oleogelator content and the firmness of the gel (Okuro et al., 2020; Shi et al., 2021; Yang et al., 2022b).

3.3.2. Optical microscopy of bi-gels

Optical microscopy plays a crucial role in studying bi-gels due to its ability to provide detailed insights into the structure, morphology, and interactions within these complex materials. The relationship between optical properties and the components oleogelator and hydrogelator in bi-gels can significantly impact the functionality and performance of the final product. Understanding how these elements interact optically can provide insights into the behavior and characteristics of the bi-gels. Fig. 3. Shows the crystal growth process, which producing strong interactions and forming supramolecular crystalline structures. These structures contain the liquid oil and form a three-dimensional network. In the case of natural waxes (beeswax), the crystal morphology depends on the chemical composition, polarity, chain length and melting point of their components. The crystals in the BG10, containing 10% oleogel: 90% hydrogel, were arranged as homogeneously distributed aggregated short rods (Fig. 3 B). This observation is in line with the earlier report using these natural waxes (Moghtadaei et al., 2018). As the amount of oleogel in the bi-gel system increases, along with the consequent rise in beeswax content, the system strengthens (Andonova et al., 2017). The researchers noted that as the amount of organogel increased, the microstructure of the bi-gels exhibited non-uniformity. They observed the presence of organogel within the bi-gels, with interconnections among organogel particles, leading to the formation of a complex matrix-in-matrix microstructure. The observed microstructure indicated the entrapment of each phase within the other. Additionally, it was discovered that the complexity of the system heightened with an increase in the proportion of organogel in the mixture (Lupi et al., 2016a). This microscopic study allowed higher particle size to be attributed to batches containing a higher proportion of the organogel. This direct correlation between the concentration of the oleogel in the bi-gels and the droplet size observed by optical microscopy has also been established by (Martín-Illana et al., 2022). Rehman et al. examined bi-gels using polarized microscopy and noted the distinct components of the formulation were distinguishable. They observed different structures, such as bi-gels or emulgels, in systems containing gelatin hydrogel and sesame/soybean oil with or without stearic acid under bright-field microscopy. The images showed the dispersion of oil droplets within the organogel in a continuous hydrogel matrix (Rehman et al., 2014). Similarly, Singh and Kumar observed a similar phenomenon in bright microscopic images of bi-gels consisting of sorbitan monopalmitate/olive oil organogel and sterculia gum/polyacrylamide hydrogel (Singh & Kumar, 2019). Martins et al. used bright-field microscopy to identify differences in bi-gel microstructure based on the organogelator proportion and the organogel/hydrogel ratio in the system, noting a more heterogeneous globular gelled structure at higher ratios (Martins et al., 2019).

Fig. 3.

Optical microscopy of bi-gels with different hydrogel at 40x: oleogel ratios

(BG5: 5%oleogel and 95% hydrogel (A); BG10: 10% oleogel and 90% hydrogel (B); BG15: 15% oleogel and 85% hydrogel (C); BGZ5 (D), BGZ10 (E), and BGZ15 (F) have similar formulations to the previous exact phrases; however, they are reinforced by zein microfibers.

3.3.3. Rheological measurement of bi-gels

Rheological properties were studied to provide more information about the viscoelasticity and structure of bi-gels, which consequently makes it suitable for application in the food industry. With this in mind, the rheological properties of bi-gels enhance performance in specific use cases by providing tailored characteristics (Du and Meng, 2024). Fig. 4 shows the storage (G′) and loss modulus (G″) of bi-gels against angular frequency. The elastic modulus consistently exceeded the viscous modulus in all bi-gel samples, maintaining a solid viscoelastic behavior without crossover across frequency ranges. At lower frequencies, the elastic and viscous moduli were closely aligned, but as frequency rose, the gap between them widened, with the elastic modulus overtaking the viscous modulus. The incorporation of oleogel into the bi-gels resulted in an enhancement of their elasticity compared to BG5. The values of G′ and G″ increased with increasing oleogel concentration. Notably, BGZ10 exhibited the highest storage and loss modulus, indicating a superior quasi-solid property among the samples. Bi-gels with higher elastic moduli typically exhibit enhanced interactions and stability in the linear viscoelastic range. The frequency-dependent nature of both loss and storage moduli suggested an increase with rising frequency, which is characteristic of weak gel behavior. The loss tangent (tan δ = G''/G′) serves as a valuable indicator of viscoelasticity, with values below one indicating solid viscoelastic behavior. Based on the information provided, bi-gel samples enhanced with zein microfibers exhibit superior rheological properties compared to plain samples. Specifically, the BGZ10 sample demonstrates higher elastic properties than all other samples (G' = 291000 ± 91.27; P < 0.05). Across all samples, tan δ remained below one, demonstrating a dominance of elastic behavior over viscous and gel-like properties. The ratio of oleogel to hydrogel emerged as a key factor influencing the rheological behavior of the bi-gels. Studies have demonstrated that an increase in the oleogel fraction results in enhanced firmness, cohesiveness, viscosity, and stability (Lupi et al., 2016b; Yang et al., 2022b). With the dominance of the hydrogel, the rheological and textural attributes were dependent on the robust framework provided by the oleogel fractions (Mazurkeviciute et al., 2018; Zulfakar et al., 2018). Just like in an emulsion gel, incorporating oleogel can serve as an "interacting filler," enhancing the viscoelasticity of bi-gels (Lupi et al., 2016a). According to (Wakhet et al., 2015), as both the oleogel and hydrogel existed in a semi-solid state, it was expected that the filler effect would be more pronounced for the bi-gels system compared to emulgels. The increase in oleogel content resulted in a tighter packing of the dispersed phase, further reinforcing the structuring of the bi-gels. A higher G′ value typically indicated greater resistance to deformation, and a higher amount of oleogel could create bi-gels with stronger mechanical properties (Table 4). As depicted in Fig. 4, reinforced samples exhibited higher elastic characteristics than simple hydrogel samples at specific oleogel levels. Among the reinforced samples, BGZ10 demonstrated the highest rheological properties, correlating with its elevated oleogel content. This is also shown in Table 3 indicating that this sample exhibited the highest consistency. Generally, a higher G′ value indicated that the material was more resistant to deformation, and a higher oleogel fraction could produce bi-gels with stronger mechanical properties. The G′ and G″ of bi-gels tended to increase with frequency (0.01–10 Hz) and the increasing trend became more significant for bi-gels with higher oleogel fractions, suggesting that both the G′ and G″ of the bi-gels were frequency-dependent. The tan δ values (G''/G′) of all bi-gels were lower than 1 but higher than 0.1, representing a typical weak gel behavior (Zhu et al., 2021). Consequently, more oleogel molecules within the bi-gels at the interface would interact with water molecules, essentially serving as active filler (Budai et al., 2023).

Fig. 4.

Rheological measurements of bi-gels with different oleogel:hydrogel ratios (BG5: 5%oleogel and 95% hydrogel; BG10: 10% oleogel and 90% hydrogel; BG15: 15% oleogel and 85% hydrogel. BGZ5, BGZ10, and BGZ15 have similar formulations to the previous exact phrases; however, they are reinforced by zein microfibers.

Table 4.

Dynamic rheological parameters of bi-gels with different oleogel:hydrogel ratios by frequency sweep test at f = 1 Hz, $\dot{\gamma }$ = 5%, Pa and 7 °C.

Con. (%)	G' (Pa)	G" (Pa)	η∗ (Pa.s)	Tan δ
BG5 BG10	86400e±33.66 99600e±45.31	16900f±13.84 19400e±28.41	14000f±3.87 16100 e±15.24	0.20 b ± 0.02 0.20 b ± 0.02
BG15	114000d ± 35.77	21900d ± 21.55	18500 d ± 2.85	0.19 b ± 0.03
BGZ5	167000c±59.37	33800c±33.28	27100 c±31.86	0.20 b ± 0.02
BGZ10	291000a±91.27	59800a±8.75	47300 a±20.73	0.21 a±0.03
BGZ15	201000b ± 19.40	47500b ± 6.38	32900 b ± 11.13	0.24 c±0.02

BG5: 5%oleogel and 95% hydrogel; BG10: 10% oleogel and 90% hydrogel; BG15: 15% oleogel and 85% hydrogel. BGZ5, BGZ10, and BGZ15 have similar formulations to the previous exact phrases; however, they are reinforced by zein microfibers.

The data is represented as means ± standard deviations, based on n = 3. Values in the identical column that are labeled with different letters are deemed significantly different (p < 0.05).

3.3.4. FTIR of bi-gels

The FTIR spectra (at 4000–400 cm⁻¹) of bi-gels were scrutinized to evaluate the interactions between the oleogel and hydrogel components within the bi-gel system. This examination is crucial for understanding the molecular-level interactions and compatibility between the two gel components in the bi-gel system. Fourier transform infrared (FTIR) analysis in attenuated total reflectance (ATR) mode within a specific wavenumber range is commonly utilized to identify the functional groups and comprehend the chemical interactions among the components in bi-gel systems (Wakhet et al., 2015). Functional groups in molecules generally absorb infrared radiation within the range of 4000 to 1500 cm⁻¹. This spectral range is commonly utilized to assess the lipophilic and hydrophilic characteristics of colloidal mixtures (Rehman and Zulfakar, 2017).

No new peaks or shifts were identified in the bi-gels samples with varying oleogel fractions, indicating a lack of chemical affinity between the carrageenan hydrogel and BW-based oleogel. A broad band in the range of 3690–3000 cm⁻¹ was observed in all the bi-gels, likely associated with O–H stretching vibrations involved in hydrogen bonding within the carrageenan-based hydrogel and the heads of fatty acyl molecules in the oleogel. ι-carrageenan had a band at 846 cm⁻¹ associated with the sulfate group at C₂ in 3,6-anhydrogalactose and a characteristic band at 1257 cm⁻¹ related to O=S=O asymmetric stretching (Fig. 5). The band at 2918 cm⁻¹ was attributed to CH₂ stretching and the broad band at 3428 cm⁻¹ was attributed to vibrational stretching associated with free, intermolecular and intramolecular bound hydroxyl groups. There were other characteristic bands of ι-C including 1069 cm⁻¹ for S=O symmetric stretching (Jayaramudu et al., 2013; Maciel et al., 2016; Wang et al., 2021). The primary spectrum of FTIR for beeswax typically includes characteristic peaks at around 2800-3000 cm⁻¹ (stretching vibrations of CH₂ and CH₃ groups), 1740 cm⁻¹ (ester carbonyl group), 1460 cm⁻¹ (bending vibrations of CH₂ and CH₃ groups), and 720-750 cm⁻¹ (scissoring vibrations of CH₂ groups) (Fig. 5) (Pang et al., 2020b). As the quantity of oleogel increased, there was a tendency for the OH stretching vibration intensity to diminish, hinting at a reduction in hydrogen bonding within the formulations (Behera et al., 2015b). The FTIR spectra of natural beeswax/sunflower oil-based bi-gels revealed broadened peaks within the 2600-3000 cm⁻¹ range associated with a greater presence of hydroxyl groups fostering hydrogen bonding within the bi-gels. Carrageenan based bi-gels showed larger number of free hydroxyl groups as compared to the other two. Similarly (sahoo et al.,2015), also reported stronger hydrogen bonding in acacia gum based bi-gels as compared to guar gum based bi-gels. The peak detected at 1740 cm⁻¹ is linked to the C=O stretch of the ester carbonyl functional group present in triglycerides (Fasolin et al., 2021). The vibrations of ester C=O bonds, as discussed by (Behera et al., 2015a; Paul et al., 2018) are influenced by the specific chemical structure of the polar head from the lipid blend. As previously noted, the intricate nature of the bi-gel might impede the identification of vibrational modes from the hydrogel confined within the oleogel matrix. The distinctive peak at 1448 cm⁻¹, solely found in oleogels and bi-gels samples, is associated with the bending vibrations of the CH₂ and CH₃ aliphatic groups, reflecting the concentration of oleogels (Rehman et al., 2014). The sequence of BG index peaks, arranged from highest to lowest frequency encompasses 3386 cm⁻¹ (N-H bond stretching bands) 2847 cm⁻¹ and 2916 cm⁻¹ (aliphatic C-H stretching absorption bands), 1734 cm⁻¹ (Amide I), and 1150 cm⁻¹ (Amide II stretching absorption bands). It is necessary to explain that peak amide I is related to the stretching vibration bands of the carbonyl C=O group and peak amide II is related to the bending vibrations coupled with the stretching vibrations C-N of the peptide chain (Loza-Rodríguez et al., 2023).

Fig. 5.

FTIR measurements of bi-gel with different amount of oleogel:hydrogel ratios (BG5: 5%oleogel and 95% hydrogel; BG10: 10% oleogel and 90% hydrogel; BG15: 15% oleogel and 85% hydrogel (BGZ5, BGZ10, and BGZ15 have similar formulation to the previous same phrases, however, they are reinforced by zein microfibers).

As shown in Fig. 5, pure bi-gels (BG5-BG10-BG15) and also reinforced bi-gels (BGZ5-BGZ10-BGZ15) did not change in peak amide I and II compared to its powder sample significantly. Additionally, small shifts in the bands on spectra of the fibers were observed. For instance, the band at 3500 cm⁻¹ on FTIR spectrum BG10 (referring to the carbonyl stretch, C= O, attributed characteristic amide groups) blue shifts to 3386 cm⁻¹. The slight change in blue shift in amide I is due to the breaking and unfolding of the beta sheets of the increase oleogels in bi-gels (Sagiri et al., 2015). In other words, no chemical changes occurred during the production process of bi-gels.

3.3.5. Centrifugation and thermal stability

Based on rheological and textural properties, BG10 and BGZ10 were selected for stability measurements. BG10 and BGZ10 were completely stable after centrifugation at 25 °C, and no alteration was observed in the bi-gels (the data was not shown). However, the BG10 sample formed two distinct phases after thermal treatments (50 °C and 70 °C for 30 min and then cooling) (Fig. 6A–C). Increasing temperature from 50 to 70 °C led to more instability in BG10. This phase separation likely results from differences in thermal expansion or stability between carrageenan hydrogel and beeswax oleogel, which are sensitive to temperature changes. The BGZ10 did not show any phase separation under the same conditions. Zein microfibers appear to have a stabilizing effect, preventing the physical and chemical changes observed in the control sample (Fig. 6B–D). The capacity of zein microfibers to withstand high temperatures may be attributed to their molecular structure and interactions with carrageenan and beeswax, which reinforce the physicochemical stability of the system. Notably, incorporating zein-based into κ-carrageenan systems markedly enhances their physicochemical stability in diverse environmental conditions (Chen et al., 2024). This improvement can be attributed to the formation of supplementary junction zones within the gel network, which has been observed due to complementary interactions between the fibers and the κ-carrageenan double helix (Derkach et al., 2018). The molecular structure of zein and its interactions with carrageenan and beeswax results in a versatile and stable system for utilization in the food industry. These interactions result in enhanced physicochemical properties and augmented stability, rendering zein fiber-reinforced bi-gel systems promising candidates for diverse applications in the food and pharmaceutical industries.

Fig. 6.

A) Two distinct phases are observed following the heating of the control sample (BG10) at 50 °C for 30 min, after which it is subsequently cooled. B) The BGZ10 sample did not exhibit two phases during the same procedure. C) Two distinct phases are observed following the heating of the control sample (BG10) at 70 ᵒC for 30 min, after which it is subsequently cooled. D) The BGZ10 sample did not exhibit two phases during the same procedure (at 70 ᵒC for 30 min).

4. Conclusion

The research was centered on developing advanced bi-gels utilizing carrageenan reinforced with zein microfiber hydrogel and beeswax oleogel, achieved through a high shear technique. These bi-gels exhibited semi-solid gel properties, with increased oleogel content enhancing firmness, storage modulus, and loss modulus. This unique combination exhibits significant potential for utilization in low-fat food products and as delivery systems for lipophilic nutraceuticals. By leveraging the synergistic properties of carrageenan, zein microfibers, and beeswax oleogel, these bi-gels offer a promising solution for enhancing product texture, stability, and functionality while catering to the growing demand for healthier food options and effective delivery systems for nutraceuticals. Characterization involved textural (Penetration test), rheological measurements, optical microscopy, and FTIR analyses. Bi-gels with varying oleogel content showed different properties, with less than 20% oleogel content displaying an O/W type and smooth texture. The amount of oleogel affects elasticity, adhesiveness, deformability, recovery, and adhesion properties of the bi-gel. The penetration tests revealed that the printing extrusion process was subject to disruptive effects. FTIR analysis revealed bi-gels were formed through physical interactions without covalent cross-linking. Bi-gels have broad applications in the food, pharmaceutical, and cosmetic industries, showing potential for controlled drug release. Further investigation is imperative to tackle stability and bioequivalence concerns for the optimal utilization in controlled delivery platforms. Bi-gels hold significant promise for various applications, addressing stability and bioequivalence concerns is crucial for their optimal utilization in controlled delivery platforms. By outlining specific research directions, we can pave the way for the advancement of bi-gel technology, ensuring that these innovative materials can meet both industry standards and consumer expectations. Through collaborative efforts across disciplines, the potential of bi-gels can be fully realized, leading to improved products in the food, pharmaceutical, and cosmetic sectors. In light of the findings of our investigation, it seems that reinforced bi-gels could represent a promising avenue for further research, including the potential use of this material as a fat substitute.

CRediT authorship contribution statement

Mojtaba Rezaei: Investigation, Formal analysis, Visualization, Writing – original draft. Sara Naji-Tabasi: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing. Behrouz Ghorani: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing, Funding acquisition. Bahareh Emadzadeh: Validation, Methodology, Supervision, Writing – review & editing.

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.

Handling Editor: Professor Aiqian Ye

Data availability

The data that has been used is confidential.