https://orcid.org/0009-0008-9613-4869
Figures
Abstract
Protein-based gel matrices are increasingly explored for feed applications requiring soft or semi-solid formulations, but achieving strong gelation along with high water- and oil-holding capacities at low protein concentrations remains challenging. In this study, soy protein isolate (SPI) was modified via nitrogen-protected thermal induction followed by microbial transglutaminase (MTG) cross-linking to enhance its suitability as a gel-feed matrix. Single-factor and orthogonal experimental designs were used to investigate the effects of SPI concentration, MTG dosage, reaction temperature, reaction time, and pH on macroscopic gelation (measured as apparent viscosity), nitrogen solubility index (NSI), water-holding capacity (WHC), oil-holding capacity (OHC), and microstructure (SEM). Under optimized conditions—10% SPI, 0.3% MTG, 50 °C, 3 h, pH 7—gelation increased by approximately 373%, WHC improved by 222%, and OHC increased by 150%, while SEM confirmed the formation of a more regular three-dimensional network compared with native SPI. These results indicate that dual-modified SPI exhibits enhanced functional properties relevant to gel-feed formulations, providing a practical foundation for laboratory-scale process optimization. Further pilot-scale evaluation and biological validation are warranted to assess scalability, processing efficiency, and performance in target animal applications.
Citation: Guo Y, Qu L (2026) Nitrogen-protected thermal induction combined with Microbial Transglutaminase (MTG) cross-linking enhances functional properties and gel structure of Soy Protein Isolate relevant to gel-based feed formulation. PLoS One 21(2): e0343526. https://doi.org/10.1371/journal.pone.0343526
Editor: Lei Zhang, University of Waterloo, CANADA
Received: October 8, 2025; Accepted: February 6, 2026; Published: February 23, 2026
Copyright: © 2026 Guo, Qu. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Protein-based gel systems with tunable texture and hydration properties have attracted increasing attention in food and feed research, particularly in applications requiring soft or semi-solid matrices to accommodate physiological, nutritional, or processing constraints. [1] A wide range of gel matrices based on animal proteins, polysaccharides, and plant-derived proteins have been explored [2]; however, achieving a balance between structural stability, water and oil retention, nutritional value, and formulation flexibility under practical processing conditions remains a challenge.
In modern animal production and laboratory animal science, the weaning and early feeding period represents a critical developmental stage that strongly influences growth performance and health outcomes. Young animals, such as weaned piglets, often experience reduced feed intake, intestinal dysfunction, and transient weight loss during the transition from milk to solid feed, which can increase morbidity, mortality, and production costs [3–5]. Previous studies have shown that palatable, high-moisture gel feeds can help restore feed intake, support intestinal development, and alleviate weaning-associated stress [6–7]. The choice of an appropriate gel matrix is therefore central to the development of gel-based feed formulations.
Polysaccharide-based gelling agents (e.g., pectin, carrageenan, agar, and konjac) exhibit strong water absorption capacity but typically provide low nutritional density and limited protein content, and their digestibility for young animals may be suboptimal [8–9]. Animal-derived proteins, such as gelatin and collagen, offer favorable gelling behavior and nutritional quality but are relatively costly, which limits their large-scale application [10–11]. In contrast, plant-derived proteins—particularly soy protein isolate (SPI)—are attractive because of their high protein content, wide availability, and cost-effectiveness [12–15]. However, native SPI often exhibits insufficient gelling ability and limited water-holding capacity (WHC) and oil-holding capacity (OHC), which restrict its application in gel-based feed systems [16]. Therefore, improving SPI’s gelling capacity at low substrate concentration (which benefits gel-feed formulation and palatability) while maintaining functional properties is a key challenge [17–18].
Microbial transglutaminase (MTG) catalyzes covalent cross-linking between glutamine and lysine residues, thereby promoting intermolecular polymerization and enhancing gelation strength, water retention, and thermal stability [19–21]. MTG has been widely reported to improve the functional properties of SPI [22–24], whey proteins [25–27], fish gelatin [28–29], and other protein systems. Various pretreatment strategies, including thermal induction, ultrasonication, high-pressure processing, and pH shifting, have been used to partially unfold protein structures and expose reactive sites, which can further enhance MTG-mediated cross-linking efficiency [30–32]. However, most existing studies have focused on food processing applications [33–34], and investigations specifically targeting gel-feed formulation under low-protein conditions remain limited.
In the present study, nitrogen-protected thermal induction was employed as a pretreatment strategy to minimize oxidative modification of SPI during heating. Soy proteins contain amino acid residues (e.g., cysteine, methionine, and tyrosine) that are susceptible to oxidation under elevated temperatures, which can alter protein conformation and reduce the accessibility of lysine and glutamine residues required for enzymatic cross-linking. Nitrogen protection during thermal induction was therefore used to promote controlled protein unfolding while limiting oxidative aggregation, thereby facilitating more consistent MTG-mediated network formation.
Accordingly, this study applied a combined strategy of nitrogen-protected thermal induction followed by MTG cross-linking to modify SPI at low substrate concentration. The objectives were to (i) evaluate the effects of processing parameters on SPI gelation behavior and hydration properties, (ii) optimize formulation conditions using single-factor and orthogonal experimental designs, and (iii) characterize the resulting gels in terms of viscosity-based gelation, nitrogen solubility index (NSI), WHC, OHC, and microstructure. This work focuses on formulation efficiency and process integration under controlled conditions, rather than proposing a new biochemical mechanism, and aims to provide a practical reference for the development of plant-protein-based gel systems relevant to gel-feed formulation
2. Materials and methods
2.1. Materials
Soy protein isolate (SPI) (protein content ≥ 90%, Shanghai Xintai Industrial Co., Ltd.; stored at 4 °C in the dark, dry conditions); microbial transglutaminase (MTG) (activity 100 U/g, defined as the amount catalyzing the formation of 1 μmol hydroxamic acid at 37 °C and pH 6 in 1 min; Jiangsu Yiming Fine Chemicals Co., Ltd.; aliquoted and stored at −20 °C to avoid repeated freeze–thaw cycles); ELISA kits for soybean antigen proteins (Shanghai Jining Biotechnology Co., Ltd.; stored at −20 °C).
2.2. Instruments and equipment
RVA-3D Rapid Visco Analyzer (Newport Scientific, Australia); Avanti JXN-26 high-speed refrigerated centrifuge (Beckman Coulter, USA); VFD-2A-50 vacuum freeze dryer (Shanghai Shunzhi Instrument Manufacturing Co., Ltd., China); S-450 scanning electron microscope (Hitachi, Japan); high-purity nitrogen (≥99.99%; supplier details).
2.3. Experimental design
2.3.1. Single-factor experiments.
2.3.1.1. Effect of SPI concentration on SPI gelation. An accurately weighed 60 g amount of SPI (dry basis) was placed into a sealed reactor, immersed in a 70 °C water bath, and purged continuously with nitrogen at a flow rate of 5.0 L·min ⁻ ¹. The dry SPI was thermally induced for 1 h without external humidification. To obtain sufficient material for subsequent experiments conducted in triplicate, the thermal induction treatment was repeated multiple times under identical conditions, and the resulting modified SPI batches were combined to yield approximately 500 g of treated SPI for further use. After cooling to room temperature, the treated material was dissolved in deionized water to prepare SPI solutions at 5, 10, 15, 20 and 25% (w/w). The solution pH was adjusted to 7.0, and MTG was added at 0.3% (w/w, based on SPI mass). The mixtures were then incubated at 60 °C for 1 h with gentle agitation, then inactivated at 85 °C for 10 min. After the reaction, samples were centrifuged, washed with deionized water, freeze-dried, milled to a fine powder, and stored in airtight containers for subsequent analyses. A process flowchart for the preparation of dual modified SPI is illustrated below (Fig 1).
2.3.1.2. Effect of MTG dosage on SPI gelation. The nitrogen-protected thermally induced SPI prepared as described above was used for subsequent MTG concentration experiments. The modified SPI powder was dissolved in deionized water to prepare a 15% (w/w) SPI solution, and the pH was adjusted to 7.0. MTG was then added at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6% (w/w, based on SPI mass), respectively. The reactions were carried out at 60 °C for 1 h and subsequently inactivated at 85 °C for 10 min. After the reaction, samples were centrifuged and washed three times with deionized water (1:10, w/v) to remove unreacted components.
followed by freeze-drying, Freeze-drying was performed using a laboratory freeze dryer following a standardized protocol for all samples. Briefly, samples were pre-frozen at −60 °C for 10 h under atmospheric pressure, followed by primary drying under vacuum (1.8–5 Pa) with stepwise temperature elevation from −40 °C to 0 °C and then to 25 °C over 24 h. Secondary drying was subsequently conducted at 25–40 °C under ≤3 Pa for 8 h. The total lyophilization time was approximately 42 h.milling to a fine powder, and storage in airtight containers for subsequent analyses.
2.3.1.3. Effect of reaction temperature on modified SPI gelation. After nitrogen-protected thermal induction (70 °C, 1 h) and cooling, a 15% (w/w) SPI solution in deionized water was prepared and adjusted to pH 7. MTG (0.3% w/w) was added and reactions were carried out at 30, 40, 50, 60, 70, and 80 °C for 1 h, After reaction, samples were inactivated at 85 °C for 10 min, centrifuged, washed, freeze-dried under the same conditions described above and ground for analysis.
2.3.1.4. Effect of reaction time on SPI gelation. Following nitrogen-protected thermal induction (70 °C, 1 h), a 15% (w/w) SPI solution in deionized water was prepared and adjusted to pH 7. MTG (0.3% w/w) was added and reactions were performed at 60 °C for 1, 2, 3, 4 and 5 h, respectively. After reaction, samples were inactivated at 85 °C for 10 min, centrifuged, washed, freeze-dried under the same conditions described above and ground for further testing.
2.3.1.5. Effect of pH on SPI gelation. Following nitrogen-protected thermal induction (70 °C, 1 h), a 15% (w/w) SPI solution in deionized water was prepared and adjusted to pH 4, 5, 6, 7, 8 and 9, respectively. MTG (0.3% w/w) was added and reactions were conducted at 60 °C for 1 h. Samples were inactivated at 85 °C for 10 min, centrifuged, washed, freeze-dried under the same conditions described above and ground for subsequent assays.
2.4. Analytical methods
2.4.1. Gelation (viscosity).
The gelation behavior of SPI samples was quantified by apparent viscosity using a Rapid Visco Analyzer (RVA-3D) [35]. Viscosity values are reported in kilocentipoise (kcp; 1 kcp = 1000 cP). For each measurement, 30 g of SPI dispersion at the specified concentration was prepared and transferred into standard aluminum RVA canisters.
All measurements were conducted using an identical and predefined temperature–time program to ensure comparability among samples. Samples were initially equilibrated at 20 °C, then heated to 70 °C at a constant ramping rate of 5 °C·min ⁻ ¹. The temperature was maintained at 70 °C for 20 min to allow gel structure development, followed by cooling to 25 °C at the same rate (5 °C·min ⁻ ¹).
Apparent viscosity was continuously recorded throughout the entire heating–holding–cooling cycle. The viscosity value measured at the end of the cooling stage was taken as an indicator of macroscopic gelation behavior.
2.4.2. Nitrogen solubility index (NSI).
The nitrogen solubility index (NSI) was determined for both native SPI and modified SPI samples to evaluate changes in protein solubility induced by nitrogen-protected thermal induction combined with MTG cross-linking. Protein nitrogen content was measured using the micro-Kjeldahl method according to GB/T 6432 (2018). A 1.0% (w/v) protein dispersion was prepared in deionized water and centrifuged at 5000 rpm for 10 min. The supernatant was collected for determination of water-soluble nitrogen, while total nitrogen content was measured separately. NSI was calculated as the percentage of soluble nitrogen relative to total nitrogen. All measurements were performed in triplicate. [36]
2.4.3. Water-holding capacity (WHC) and oil-holding capacity (OHC).
The swelling method was used with a custom Baumann apparatus [37]. Five grams of sample were placed in the device and allowed to absorb water (or oil) for 2 h. The sample was weighed at this point (G1), then dried in an 80 °C oven for 4 h and weighed again (G2).
WHC (or OHC) = (G1 − G2)/ initial dry sample mass (g/g).
2.4.4. Microstructure of soy protein isolate.
An appropriate amount of SPI powder was evenly spread on specimen stubs previously coated with double-sided conductive tape. Excess loose powder was gently blown off; samples were gold-coated and observed and imaged using Scanning electron microscopy (SEM) at an accelerating voltage of 15 kV [38].
2.5. Statistical analysis
All experiments were conducted in triplicate (n = 3), and data are presented as mean ± standard deviation. For single-factor experiments, data were analyzed using one-way analysis of variance (ANOVA) to evaluate overall differences among groups. Orthogonal experimental results were analyzed using range (R) analysis to assess the relative influence of formulation factors on SPI gelation. No hypothesis-driven statistical significance testing was applied to the orthogonal design, as it was used for formulation screening and optimization purposes.
3. Results and analysis
3.1. Effect of SPI concentration on gelation of modified SPI
Fig 2 shows the dependence of gelation behavior, expressed as apparent viscosity, on SPI substrate concentration in the range of 5–25% (w/w). Gelation increased with increasing SPI concentration and reached a maximum viscosity of 10.251 ± 0.009kcp at 15%, followed by a decline at higher concentrations (20% and 25%). This trend indicates that an intermediate protein concentration favors effective MTG-mediated intermolecular cross-linking and network formation, whereas excessive protein content may restrict molecular mobility and limit the development of a continuous gel network under the tested conditions.
3.2. Effect of MTG dosage on gelation of modified SPI
Data in Fig 3 show that increasing MTG dosage from 0.1% to 0.3% (w/w) markedly enhanced the gelation behavior of SPI, as reflected by increased apparent viscosity. Further increases in MTG dosage beyond 0.3% resulted in a plateau in viscosity, indicating that additional enzyme did not lead to further improvement in macroscopic gelation. This behavior suggests that an intermediate MTG level is sufficient to promote effective intermolecular cross-linking, whereas excessive cross-linking may lead to network densification without proportional increases in bulk viscosity under the tested conditions.
3.3. Effect of reaction temperature on gelation of modified SPI
Fig 4 shows that gelation of modified SPI was strongly dependent on reaction temperature. Apparent viscosity increased with temperature and reached a maximum value of 11.563 ± 0.014kcp at 50 °C, followed by a decline at higher temperatures. This trend suggests that moderate heating promotes protein unfolding and MTG activity, facilitating effective network formation, whereas excessively low or high temperatures may limit enzymatic cross-linking efficiency or induce protein over-aggregation, thereby impairing development of an optimal three-dimensional gel network under the tested conditions.
3.4. Effect of MTG reaction time on gelation of modified SPI
As shown in Fig 5, gelation of modified SPI increased with MTG reaction time, as reflected by a gradual rise in apparent viscosity. The rate of increase diminished after 3 h, indicating that gel network formation approached a stable state beyond this time point. Although a higher viscosity value of 12.209 ± 0.092kcp was observed at 5 h, the additional increase relative to 3 h was limited. This behavior suggests that extended reaction times may result in diminishing returns in macroscopic gelation while increasing overall processing time. Therefore, a reaction time of 3 h was selected as a suitable condition for subsequent experiments, balancing gelation performance and practical processing considerations under the tested conditions.
3.5. Effect of pH on gelation of modified SPI
As shown in Fig 6, MTG-catalyzed gelation of SPI occurred effectively over a broad pH range (pH 5–8), with gelation reaching a maximum at pH 7.0. Moderate pH conditions favor both protein conformational flexibility and MTG catalytic activity, facilitating exposure of reactive lysine and glutamine residues required for intermolecular cross-linking. At lower or higher pH values, electrostatic repulsion or excessive protein aggregation may restrict effective enzyme–substrate interactions, resulting in less efficient network formation. Based on these observations, pH 7.0 was selected as an appropriate reaction condition for subsequent experiments.
3.6. Orthogonal optimization of combined nitrogen-protected thermal induction and MTG modification
Based on the trends observed in the single-factor experiments and the factor–level combinations outlined in Table 1, an orthogonal experimental design was employed to identify suitable processing conditions for the combined nitrogen-protected thermal induction and MTG modification of SPI. Gelation, expressed as apparent viscosity, was used as the evaluation index to compare the overall effects of different factor combinations.
As summarized in Table 2, the orthogonal analysis indicated that the factor combination A1B3C2D4E3 resulted in the highest gelation among the tested conditions. This combination corresponds to an SPI substrate concentration of 10% after nitrogen-protected thermal induction (A1), an MTG dosage of 0.3% (B3), a reaction temperature of 50 °C (C2), an MTG reaction time of 4 h (D4), and a reaction pH of 7.0 (E3). These conditions represent a balanced set of processing parameters that collectively favor protein unfolding, enzyme activity, and effective intermolecular cross-linking, thereby promoting the formation of a dense gel network under the constraints of the experimental design.
Notably, although the single-factor experiments indicated that an SPI concentration of 15% produced the highest peak viscosity, the orthogonal optimization selected a lower SPI concentration of 10% as part of the optimal factor combination. This difference reflects the distinct evaluation logic between single-factor screening and multifactor optimization. From a mechanistic perspective, at lower protein concentrations, improved enzyme diffusion and greater substrate accessibility facilitate more uniform MTG-catalyzed cross-linking [39]. In contrast, higher SPI concentrations increase system viscosity and promote protein–protein aggregation, which may hinder enzyme penetration and sterically mask reactive glutamine and lysine residues, thereby reducing effective cross-linking efficiency [40].
Although the orthogonal design suggested a theoretical optimum MTG reaction time of 4 h, a reaction time of 3 h was selected for practical validation based on the single-factor results and considerations of processing efficiency and production economics. Under these practical conditions (10% SPI, 0.3% MTG, 50 °C, 3 h, pH 7), three independent parallel experiments (n = 3) yielded an average gelation of 11.754 ± 0.008kcp for SPI modified by nitrogen-protected thermal induction followed by MTG cross-linking. Although this value is slightly lower than the theoretical maximum predicted by the orthogonal analysis, it is sufficient to achieve stable gel formation at a relatively low SPI concentration. Moreover, avoiding excessively high gel hardness is advantageous for maintaining palatability and facilitating downstream processing in gel-based feed applications.
3.7. Comparison of functionality between dual-modified SPI and native SPI
Table 3 summarizes the functional properties of native SPI and SPI subjected to combined nitrogen-protected thermal induction and MTG cross-linking. Compared with native SPI, the dual-modified SPI exhibited a pronounced enhancement in gelation behavior, with viscosity increasing from 2.487 ± 0.030 to 11.754 ± 0.008 kcp. In parallel, water-holding capacity (WHC) and oil-holding capacity (OHC) were significantly improved, increasing by 221.77% and 150.45%, respectively, while gelation increased by 372.61%.
The marked increases in WHC and OHC are consistent with the formation of a denser and more interconnected protein network, which enhances capillary retention of water and physical entrapment of oil within the gel matrix. Although protein solubility decreased by approximately 25%, this reduction is expected for covalently cross-linked protein systems and reflects the transition from a soluble protein dispersion to a structured gel network rather than a loss of functional performance [41].
3.8. Microstructure changes following dual modification
Fig 7 shows SEM micrographs of SPI samples subjected to different modification treatments. Native SPI (Fig 7a) exhibited relatively compact and fragmented lamellar structures with smooth surfaces and limited pore formation. SPI modified by MTG alone (Fig 7b) displayed lamellar fragments with visible interconnections and the presence of small internal voids, suggesting partial network formation induced by enzymatic cross-linking. In contrast, the dual-modified SPI (Fig 7c) exhibited a markedly disrupted lamellar morphology and the development of a more continuous and regular three-dimensional network structure. This microstructural evolution is consistent with the enhanced gelation behavior as well as the increased water- and oil-holding capacities observed for the dual-modified system.
4. Discussion
The functional enhancement of soy protein isolate (SPI) observed in this study can be attributed to the combined effects of nitrogen-protected thermal induction and microbial transglutaminase (MTG)-mediated cross-linking. Nitrogen protection during thermal pretreatment minimizes oxidative degradation of SPI by preventing the oxidation of amino acid residues such as cysteine, methionine, and tyrosine, which are prone to modification under elevated temperatures. This controlled process facilitates partial protein unfolding, exposing reactive glutamine and lysine residues, while minimizing unwanted oxidative aggregation. The subsequent MTG-catalyzed cross-linking enhances protein network formation by promoting covalent bonding between glutamine and lysine residues, resulting in a denser and more stable gel network. This approach significantly improved SPI’s gelation, water-holding capacity (WHC), and oil-holding capacity (OHC), which are essential properties for the successful development of gel-based feed systems.
In this study, viscosity was employed as a macroscopic indicator of gelation, providing a practical assessment of gel performance in relation to application requirements. While viscosity is an important metric, it does not capture the full rheological behavior of the gel system. Future studies should include dynamic rheological measurements, such as storage modulus (G′) and loss modulus (G″), to better understand the viscoelastic properties and structural stability of the modified SPI gels under processing conditions. These additional parameters would further elucidate the relationship between molecular cross-linking, network architecture, and the overall mechanical performance of the gel.
The improvements in WHC and OHC observed in the modified SPI gels indicate that the gel network can effectively retain water and oil, which is critical for maintaining gel stability and functionality in gel-based feed formulations. High WHC is associated with better moisture retention, dimensional stability, and resistance to syneresis, while enhanced OHC reflects improved interactions between the protein matrix and lipid components, which is beneficial for encapsulating hydrophobic nutrients. If WHC and OHC are insufficient, the gel may suffer from water separation, oil leakage, or structural collapse, compromising its stability during processing and storage. Therefore, the enhanced WHC and OHC observed in this study suggest that the dual-modified SPI gels are suitable for applications where moisture retention and lipid encapsulation are critical.
The observed decrease in solubility after modification is consistent with the formation of an insoluble protein network due to covalent cross-linking. This transition from a soluble protein form to a gel matrix improves texture retention and structural integrity, which are desirable for gel-based formulations. However, the reduction in solubility may affect the release of nutrients and digestibility, and future studies should address these aspects through in vitro digestion assays or controlled feeding trials.
In terms of industrial application, the dual-modified SPI system offers several advantages, including the use of commercially available raw materials (SPI and MTG), well-established processing methods, and a low-protein formulation that reduces costs. This system is flexible in terms of formulation and can be easily adapted for various gel-based feed applications. However, further validation is needed at the pilot scale to assess the economic feasibility and scalability of this process, including considerations such as enzyme consumption, nitrogen usage, and overall throughput [42–43].
Compared to traditional gel-based feed systems, such as those based on polysaccharides or animal proteins (e.g., gelatin and collagen) [44–46], the SPI-based system developed in this study offers advantages in terms of sustainability, raw material availability, and the ability to fine-tune gel properties through enzymatic cross-linking. While the gel strength of SPI may not match that of collagen-based systems, the modified SPI gel system’s sustainability, formulation flexibility, and tunability position it as a complementary option for gel-feed formulations, rather than a direct replacement for existing systems. Future work should explore the performance of this system under more complex processing and application conditions, as well as conduct biological and nutritional validation to confirm its efficacy in animal feed applications [47].
5. Conclusions
This study demonstrates that nitrogen-protected thermal induction combined with microbial transglutaminase (MTG) cross-linking significantly enhances the functional properties of soy protein isolate (SPI), improving gelation, water-holding capacity (WHC), and oil-holding capacity (OHC). The optimized conditions—nitrogen purge at 5.0 L·min ⁻ ¹, thermal treatment at 70 °C for 1 h, followed by a SPI concentration of 10% (w/w), MTG dosage of 0.3% (w/w), reaction temperature of 50 °C, reaction time of 3 h, and pH 7—resulted in a 372.61% increase in gelation, a 221.77% improvement in WHC, and a 150.45% enhancement in OHC. SEM analysis confirmed the formation of a regular three-dimensional network structure, which supports the proposed mechanism of enhanced network formation.
These results highlight the potential of the dual-modified SPI system for gel-feed formulations, particularly in applications requiring moisture and lipid retention. The observed improvements in gelation, WHC, and OHC suggest that the modified SPI gels possess desirable functional properties for industrial and feed applications. However, further validation at the pilot scale is needed to evaluate the scalability, enzyme consumption, and cost-effectiveness of the process. Additionally, future studies should investigate the biological performance of the modified SPI gels in feeding trials to confirm their suitability for animal feed applications.
While the modified SPI gels offer advantages in sustainability, raw material availability, and formulation flexibility compared to traditional gel systems (e.g., polysaccharides and animal proteins), the gel strength of SPI may not exceed that of collagen-based systems. Nonetheless, the SPI-based system provides a complementary option for gel-feed formulations, which can be fine-tuned via enzymatic cross-linking. This study provides a valuable framework for further optimization and industrial-scale implementation of SPI-based gel systems in feed applications, offering a promising avenue for the development of cost-effective, plant-protein-based gel matrices.
Supporting information
S1 Data. This file provides the source experimental data for Figs 2–6 and Tables 2 and 3 in the main manuscript.
https://doi.org/10.1371/journal.pone.0343526.s001
(XLSX)
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