Structural, Textural, and Functional Properties of Plant-Based Meat Analogs Prepared by High-Moisture Extrusion of Soy–Wheat–Mung Bean Multi-Protein System

TL;DR

This study examines how adding mung bean protein affects the structure and texture of plant-based meat made from soy and wheat proteins using high-moisture extrusion.

Contribution

The study introduces mung bean protein as a viable addition to soy–wheat systems in high-moisture extrusion for plant-based meat analogs.

Findings

20% mung bean protein addition created the densest fibrous network in extrudates.

MBP above 40% disrupted the protein network structure.

Hydrogen and disulfide bonds were key in stabilizing the protein network.

Abstract

High-moisture extrusion (HME) is critical for plant-based meat analogs with meat-like fibrous structures. To expand HME protein sources, this study explored mung bean protein (MBP) substitution (0–50%, dry basis) effects on structural, textural and functional properties of soy protein concentrate (SPC)–wheat gluten (WG) HME products. At 20% MBP addition, the proteins formed a dense layered fibrous network, and the fibrous degree of the extrudates reached the peak. MBP > 40% disrupted the continuous protein network. The optimal rehydration for 20% MBP dried extrudates was 60 °C for 40 min, preserving fibrous texture. Protein interaction analysis indicated that hydrogen bonds and disulfide bonds played an important role in stabilizing the protein network structure. Overall, MBP can be incorporated into SPC-WG-based HME products to diversify protein sources, providing a feasible strategy for developing high-quality, nutritionally diversified plant-based meats.

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1. Introduction

According to data from the Food and Agriculture Organization (FAO), the global population is projected to exceed 10 billion by 2050. Currently, a substantial proportion of human dietary protein comes from animal sources, which gives rise to numerous issues, including excessive land and water consumption, significant greenhouse gas emissions, and animal welfare issues [1,2]. Thus, the development of sustainable protein sources is imperative, and plant-based meat analogs have garnered widespread attention as sustainable alternatives to conventional animal meat [3]. The commonly used technologies for preparing plant-based meat are low-moisture extrusion and high-moisture extrusion (HME), among which HME stands out as the main method for producing structured plant-based meat [4,5], because high-moisture-extruded plant-based meat has better taste and anisotropic fibrous structures [6,7].

Soy protein concentrate (SPC) and wheat gluten (WG) are widely used in HME for their excellent gelling and viscoelastic properties [8,9]. SPC and WG form a good protein network structure during the extrusion process [10,11], resulting in an anisotropic fibrous structure [8,12]. However, the excessive reliance on soy protein and WG limits the nutrition, texture, and flavor of plant-based meat. In addition, protein interactions during high-moisture extrusion have not yet been fully studied, which limits the broader acceptance of plant-based meat products.

Mung bean protein (MBP) is considered a promising alternative plant protein that can be used in plant-based meat production. It is rich in essential amino acids, has good emulsifying and water-holding properties, and forms a stable gel under heat treatment [13,14]. Adding MBP in high-moisture extrusion (HME) can not only enrich the protein sources of plant-based meat but also produce products with different textures and flavors [15]. Currently, research on adding MBP to SPC-WG high-moisture extrusion systems for producing plant-based meat is still limited, especially studies on the effects of MBP addition on protein structural changes and product characteristics during extrusion.

Therefore, the aim of this study is to explore the effects of adding MBP (0–50%, dry basis) on the physicochemical, structural and functional properties of SPC-WG HME plant-based meat products. By means of stop-sampling, we analyzed protein solubility and secondary structure in samples collected from different extruder zones and the final extrudates. The study examined how adding different proportions of MBP affects protein network formation and intermolecular interactions during HME. Furthermore, the effects of MBP addition on the texture and rehydration properties of the extrudates were investigated to provide experimental evidence for the optimization of multi-protein formulations in HME processing for plant-based meat production.

2. Materials and Methods

2.1. Materials

SPC (composed of 84.51% protein and 7.81% moisture) and WG (composed of 78.23% protein and 3.78% moisture) were purchased from Jinshuo Guoye Co., Ltd. (Xi’an, China). MBP (composed of 84% protein and 1.05% moisture) was purchased from Dafengshou Biotechnology Co., Ltd. (Xi’an, China). Sodium dodecyl sulfate (SDS), β-mercaptoethanol (β-ME), and urea (U) were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All supplementary chemical reagents were of analytical grade. Deionized (DI) water was employed consistently throughout the experiment.

2.2. High-Moisture Extrusion Process

All protein powders were dried in a constant-temperature oven (101-3B, Supor Instrument, Shao’xing, China) at 30 °C for 12 h prior to extrusion mixing. SPC, WG and MBP were mixed at ratios of 5/0/5, 5/1/4, 5/2/3, 5/3/2, 5/4/1, 5/5/0 (w/w/w, dry basis) by a mixer (SYH-5, Yirui Dry Co., Chang’zhou, China). The experiments were performed with modifications to the previous procedure of Dou et al. [16]. The high-moisture extrudates were produced by a twin-screw extruder (Process 11, Thermo Fisher Scientific, Waltham, MA, USA). The temperatures of seven independent heating zones and the cooling die zone of the extruder were set and maintained at 40, 60, 80, 100, 120, 150, 150 and 20 °C, respectively.

Protein powder and DI water were fed at rates of 0.36 kg/h and 0.63 kg/h, respectively, with the screw speed fixed at 150 r/min. Once steady state was reached and sufficient extrudates were collected, the extruder was immediately stopped (dead-stop), then we quickly disassembled the extruder and collected samples from each zone.

2.3. Textural Property Analysis

The textural properties were determined based on the method reported by Zhang et al. [17]. Briefly, a texture analyzer (TA.XT Plus, Stable Micro Systems, Godalming, UK) was used to determine the textural properties of the samples. The texture probe cut the samples along the parallel and vertical directions, with the cutting speed and penetration depth set at 1 mm/s and 5 mm, respectively. The data of chewiness, hardness, and resilience were calculated by the texture analyzer, and the fibrous degree was fitted as the ratio of force in the vertical direction to that in the parallel direction.

2.4. Fourier Transform Infrared Spectrum Analysis

Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific, Waltham, MA, USA) was used to characterize the secondary structures of proteins during extrusion and in the extrudates [18]. The extrudate samples were freeze-dried and then ground into fine powder to ensure homogeneity. FTIR samples were prepared via the KBr disk method: precisely, 1 mg of extrudate powder was thoroughly mixed with 100 mg of KBr and compressed into a transparent disk. The infrared spectra were collected at room temperature in the range of 500 to 4000 cm^−1^ with an average of 64 scans and a resolution of 4 cm^−1^. The amide I band (1600–1700 cm^−1^) in each spectrum was estimated and analyzed using Peakfit software (ver. 4.12, SeaSolve Software Inc., Framingham, MA, USA).

2.5. Scanning Electron Microscopy

The operation was briefly described as follows: fresh samples were cross-sectioned, placed in 2.5% glutaraldehyde solution for fixation for more than 1.5 h, and rinsed three times with 0.1 mol/L phosphate buffer (P). Thereafter, gradient dehydration was performed sequentially with 50%, 70%, 90% and 100% ethanol, and the samples were then soaked in tert-butanol. Upon completion of soaking, the samples were frozen at −20 °C for 30 min and subjected to 4 h of freeze-drying. Ultimately, micrographs were taken using a scanning electron microscope (SEM, Model TM-4000, Hitachi Ltd., Japan) at a magnification of ×1500.

2.6. Visual Analysis

Fresh extrudates were torn along the middle, followed by fixation and photography for macroscopic structure observation.

2.7. Raman Spectrum Analysis

Samples collected from different zones were analyzed via a Raman spectrometer (DXR3xi, Thermo Fisher Scientific, Waltham, MA, USA) to estimate the disulfide bonds of proteins [17,19,20]. Firstly, the extrudates underwent freeze-drying, followed by pulverization and sieving through a 60-mesh standard sieve. Then, the apparatus parameters were configured as follows: excitation source 785 nm, output power 300 mW, spectral scanning range 400–2800 cm^−1^, and spectral resolution 2 cm^−1^. The characteristic peaks in the 500–550 cm^−1^ wavenumber interval of each spectrum were deconvoluted to determine the percentage of various disulfide bond conformations.

2.8. Rehydration Test

The fresh extruded products were cut into blocks and dried at 60 °C to constant weight. Then, the dried extrudates were placed into three separate constant-temperature water baths, with temperatures of 25, 60, and 100 °C. Finally, the weight of the extrudates was recorded regularly, and their texture characteristics were determined according to the method in Section 2.3. The rehydration rate was calculated as the ratio of the mass of rehydrated extrudates to that of fresh extrudates, multiplied by 100%.

2.9. Rheological Property Analysis

The method referred to that of Lan et al. [21] with slight modifications; 5 g of sample was placed in 20 mL of deionized water and stirred at a constant speed of 700 r/min for 2 h, followed by standing at 4 °C for 12 h. The rheometer (HakkeRS600, Thermo Fisher Scientific, Waltham, MA, USA) utilized a plate sensor with a diameter of 35.00 mm and a gap of 1.00 mm. The measurement parameters were as follows: temperature 25 °C, shear rate range of 1–100 s^−1^.

2.10. Protein Solubility Analysis

The dissolved protein mass in eight extraction solutions was measured to evaluate the effect of MBP addition on protein solubility. Eight extraction solutions were prepared according to the method of Liu et al. [22] and Zhang et al. [17]. Eight solvents were prepared accordingly: (a) 0.1 M pH 7.6 P, (b) 0.052 M sodium dodecyl sulfate (SDS) in P (PS), (c) 8.0 M urea (U) in P (PU), (d) 0.1 M β-mercaptoethanol (β-ME) in P (PM), (e) 0.052 M SDS and 8.0 M urea (PSU), (f) 0.052 M SDS and 0.1 M β-ME in P (PSM), (g) 8.0 M urea and 0.1 M β-ME in P (PUM), (h) 8.0 M urea, 0.052 M SDS, and 0.1 M β-ME in P (PSUM). Then, 0.25 g of freeze-dried and ground samples were separately added into 20 mL of extraction solution and stirred to dissolve the samples. The soluble protein concentration was determined by combining the Dumas nitrogen determination method and the Lowry method [23,24].

2.11. Statistical Analysis

All experiments were conducted with at least three replicates, and one-way ANOVA followed by Duncan’s multiple comparison test was performed on the data using SPSS software (Version 27.0), with the significance level set at p < 0.05.

3. Results and Discussion

3.1. Textural Analysis

The texture profiles of extrudates with different MBP ratios were assessed through instrumental texture analysis. Chewiness, hardness, and resilience emerged as critical determinants influencing the sensory attributes of products. As presented in Table 1, the textural properties of the extrudates were characterized. With increasing MBP ratio, the fibrous degree of plant-based meat increased first and then decreased. When the MBP addition level was 20%, the fibrous degree was 2.42 ± 0.06, potentially resulting from synergistic interactions between MBP and WG that formed a tighter structure during the cooling process. Concurrently, MBP strengthened gelling network functionality, thereby reinforcing the fibrous structure [25]. When the MBP ratio was below 20%, the fibrous degree was lower than 2.42 (e.g., 1.91 at 0% MBP). This may be because the relatively high WG content made the mixture unstable during the extrusion process, which in turn reduced the fibrillation of the extrudate [26]. In addition, an excessively high content of MBP destroyed the protein network of the system, thereby reducing the fibrillation of the plant-based meat [14].

When the MBP ratio increased from 0 to 50%, the toughness of the extrudates increased first and then decreased, while the hardness and chewiness showed the opposite trend, decreasing first and then increasing. When the MBP ratio was 20%, the vertical values of hardness and resilience were 0.73 ± 0.02 kg, and 0.59 ± 0.01 kg/s, respectively. This suggested that the increase in MBP ratio contributed to extrudates with favorable softness and resilience. This may be because the three proteins formed a protein network structure with superior viscoelasticity, which enhanced the toughness of the composite protein extrudate. This is consistent with the results of Zhang et al. [18], who also reported that a stable viscoelastic network can be formed to improve the toughness of plant-based meat analogs.

3.2. FTIR Analysis

The amide I band (1600–1700 cm^−1^) primarily represents the C=O stretching vibration of the protein backbone, coupled with C–N stretching, C–C–N deformation, and in-plane N–H bending vibrations of plant proteins [16], which reflects the interconversion between α-helix, β-sheet, β-turn, and random coil [17]. In this study, secondary structure changes were analyzed based on the FTIR characteristic peaks of the amide I band. The spectra were deconvoluted and fitted, and the four secondary structures were quantified according to the relative area of each peak. The FTIR results of different extrusion zones are shown in Table 2. The conformational changes in the four secondary structures were inferred by calculating the corresponding protein backbone vibrations in this band via Peakfit software [27]. The results showed that in the mixing zone, with increasing MBP ratio, the content of α-helix and β-sheet increased, while the β-turn and random coil decreased significantly. This indicated that the secondary structure of the three-protein extrudate became more orderly with higher MBP levels. In the cooling zone, the contents of α-helix and β-sheet continued to decrease. This might be attributed to the instability of α-helices and β-sheets during extrusion, which led to the formation of more stable random coils, β-turns or intermolecular/intramolecular aggregations, indicating that extrusion induced the formation of ordered protein structures [28,29]. The decrease in α-helix was beneficial for the formation of the fiber network structure. Throughout the extrusion process, a decrease in the content of α-helix and an increase in the content of β-turn and coil were observed. At an MBP addition level of 20%, the α-helix and β-sheet of the SPC-WG-MBP complex underwent more pronounced conformational transitions. This might be because the hydrogen bonding interactions within the SPC-WG-MBP complexes were rearranged, contributing to the formation of a stable network structure [30].

3.3. Macro/Microstructural Evolution

The macro-and microstructures of the samples from different zones and extrudates are shown in Figure 1. From a macrostructural perspective, with increasing MBP ratio, the filamentous structure in the extrudate initially increased and then decreased. At an MBP addition level of 20%, the extrudates exhibited a distinct fibrous structure, whereas the fibrous structure gradually disappeared with further increases in MBP content. As observed in the SEM micrographs, the protein network structure of the mixing zone samples became increasingly dense with increasing MBP addition ratios. In the cooling zone, voids were observed within the microstructure, which indicated that water migration may have occurred with decreasing temperature, leading to a discontinuous microstructure. In contrast to other ratios (MBP addition: 10–40%), the micro-surfaces of samples with 0% and 50% MBP exhibited no voids, instead forming a continuous and smooth microstructure. When the MBP ratio was 20%, the results showed that proteins aggregated with each other and formed a dense fibrous structure. The 20% MBP sample exhibited a more defined fibrous structure and finer, denser voids than the 10%, 30%, or 40% ratios, demonstrating that this MBP level best promoted the development of a fibrous lamellar structure. This result is consistent with the study of Angelis et al. [31], which pointed out that appropriate addition of MBP can improve the quality of plant-based meat analogs.

3.4. Raman Spectroscopy Analysis

The effects of disulfide bond changes (intramolecular and intermolecular) in extrudates and each extrusion zone on Raman spectra at different MBP ratios are shown in Figure 2. The Raman waveband ranging from 500 to 550 cm^−1^ corresponds to the disulfide bonds in plant proteins. As shown in Figure 2A,B, the intensity of this band increased first and then decreased with increasing MBP ratio, reaching a maximum at the 20% MBP level, which indicates that incorporation of MBP promotes the formation of more disulfide bonds both between and within protein molecules. The Raman curves of each extrusion zone show that as the composite protein passed through the extrusion process, the peak intensity increased, reaching a maximum in the final extrudate. This indicates that the breakage and reformation of bonds in the composite protein matrix occur in the melting zone, enhancing protein interactions and leading to a continuous and stable network structure [10]. The relative intensity of disulfide bonds was quantified. As shown in Figure 2C,D, three characteristic disulfide bond vibrations were identified: the g-g-g mode and the g-g-t mode, both representing intramolecular disulfide bonds, and the t-g-t mode, corresponding to intermolecular disulfide bonds. Further analysis of the variations in the proportion of disulfide bonds revealed that with increasing MBP ratio, the fraction of g-g-t intramolecular disulfide bonds decreased, while that of t-g-t intermolecular disulfide bonds increased. The intermolecular disulfide content reached a maximum at 20% MBP, which indicated that MBP facilitated the conversion of intramolecular disulfide bonds into intermolecular ones [32,33]. The construction of intermolecular disulfide bonds among different protein components is conducive to the formation of protein fiber network structure, thereby enhancing its fiber and viscoelastic properties, which is consistent with the research findings of Haicheng et al. [34].

3.5. Rheological Properties of Extrudates

Figure 3 and Table 3 show the steady shear flow curves and the corresponding fitted model results for the extrudates with different MBP substitution ratios. As illustrated in Figure 3, the composite system without MBP addition demonstrated the highest apparent viscosity, which was related to its high WG content, and WG can promote the unfolding and further aggregation of protein molecular chains, thus increasing the apparent viscosity [35]. Moreover, as the MBP ratio increased beyond 20%, the apparent viscosity decreased significantly. This reduction was attributed to the heat-induced denaturation of MBP, which formed a gel-like structure that decreased viscosity while improving resilience, consistent with the texture analysis results above. When the MBP addition was 10% and 20%, the apparent viscosity exhibited only a slight decrease. This indicated that at these lower ratios, MBP contributed to enhancing the viscosity of the plant-based matrix, which was beneficial to the construction of the viscoelastic network of the system.

Table 3 shows the parameters of the power law model fitted to the shear flow data of the mixed system, including the model parameters K and n, which reflect the extent of protein binding, aggregation and reaggregation [36]. The K value indicates the magnitude of the apparent viscosity, and it was observed that K decreased with increasing MBP content, consistent with the trend shown in the flow curves. The n value increased first and then decreased as the MBP ratio rose. The highest n value occurred at an MBP ratio of 20%, suggesting that this proportion promoted interactions among the protein components and encouraged the formation of a fibrous network structure. This process enhanced shear resistance, thereby increasing the n value and endowing the extrudates with more favorable apparent viscosity characteristics.

3.6. Rehydration Kinetics and Texture Preservation

The rehydration property is an important characteristic affecting the texture quality [37]. As shown in Figure 4A, the rehydration rate increased with extended rehydration time, and plant-based meat with 20% MBP showed the best rehydration effect at 60 °C for 40 min. It can be seen from Figure 4C–F that the hardness, resilience, and chewiness of plant protein extrudates decrease with increasing rehydration time. After rehydration for more than 40 min, all texture indicators were lower than those of fresh extrudates, indicating that rehydration significantly impaired the textural properties of plant-based meat. Consequently, the internal protein network structure of the extrudates is damaged, leading to a decline in textural properties [38,39]. The hardness and fiber content decreased more significantly with the increase in hydration temperature, indicating that the fiber network structure was destroyed at higher temperatures, resulting in the deterioration of the extrudate texture [40]. Furthermore, temperature and time have a minor effect on toughness and chewiness.

Fibrous degree is the most important indicator for evaluating the quality of plant-based meat products. As shown in Figure 4F, the fibrous degree initially increased with the extension of rehydration time. This was due to the water absorption and swelling of proteins, which supported the fibrous network system [41]. Compared with the condition at 100 °C, the time to reach the maximum was longer at 25 °C and 60 °C, with less significant degradation of fibrous degree. Notably, at 60 °C, the fibrous degree was better preserved and ultimately higher than that observed at 25 °C. Therefore, based on comprehensive consideration, the optimal rehydration condition for dried extrudates with 20% MBP content is determined to be 60 °C for 40 min.

3.7. Protein Solubility of Samples

Protein solubility analysis is commonly employed in plant-based meat research to infer the dominant intermolecular interactions involved in the formation of protein complexes [42,43]. Measuring protein solubility with a single chemical reagent can be misleading. In view of this, three chemical reagents were used in this study to disrupt the potential covalent and non-covalent interactions between proteins. U was utilized to break hydrogen bonds; SDS is a well-known reagent for disrupting hydrophobic interactions; β-ME was used as a mild reducing agent to cleave disulfide bonds in proteins. P was used as the background electrolyte for extracting native proteins. These three chemical reagents can be used either individually or in combination, with the specific aim of distinguishing the relative importance of different molecular interactions [44].

As shown in Figure 5, P-treated samples showed slight variation in protein solubility among different zones and low overall solubility. Adding U, β-ME, and SDS significantly increased protein solubility. This was because U, β-ME, and SDS disrupt hydrogen bonds, hydrophobic interactions and disulfide bonds between proteins, thus improving protein solubility [16,17]. When only one extraction solution was used, proteins extracted with urea had the highest solubility, indicating that hydrogen bonds played an important role in the protein network structure [45]. When several extraction solutions were used simultaneously (PUSM and PUM), the protein solubility in each zone increased. This result indicated that the protein maintained a stable fibrous network structure through disulfide bonds and hydrogen bonds, so disrupting these interactions can significantly enhance protein solubility [38,39]. In addition, the protein solubility in the melting zone, cooling zone, and extrudate first increased and then decreased, reaching the highest protein solubility when the MBP proportion was 20%. This result indicates that these reagents disrupted the hydrogen bonds and disulfide bonds between proteins, thereby enhancing the solubility of the extrudate [46]. Furthermore, the protein solubility of the cooling zone samples treated with PU and PM showed an increasing trend and reached the maximum when the MBP substitution ratio was 20%. These results indicated that hydrogen bonds and disulfide bonds were formed in the multi-protein system at the cooling zone, and the extraction reagents disrupted these hydrogen bonds and disulfide bonds between protein molecules, thereby increasing the solubility of the extrudates. According to FTIR and Raman spectroscopic analysis, the transformation of protein secondary structure at this stage may promote the formation of more hydrogen bonds and intermolecular disulfide bonds, thereby contributing to the development of fibrous structures. Studies have shown that hydrogen bonds and disulfide bonds play an important role in maintaining the structural stability and fibrous integrity of proteins, which is consistent with the findings of our study [17].

4. Conclusions

This study explored the effects of MBP substitution on SPC-WG HME plant-based meats to expand protein sources and improve quality. MBP substitution showed dose-dependent textural effects: 20% MBP optimized fibrous degree, chewiness, and resilience. Excessive MBP (≥40%) or WG (0% MBP) reduced fibrous degree and increased hardness via network imbalance or WG over-aggregation. Structural analyses confirmed 20% MBP induced beneficial protein changes and dense layered fibers—key for meat-like texture. The rehydration capacity of extrudate was maximized at 20% MBP, supporting practical processing. Hydrogen bonds and disulfide bonds stabilize the protein network structure; 20% MBP promotes the formation of such bonds, thereby enhancing the protein system’s cross-linking and improving the fibrous degree of the extrudates. In summary, 20% MBP in SPC-WG blends optimized HME product properties.