
==== Front
Food Chem X
Food Chem X
Food Chemistry: X
2590-1575
Elsevier

S2590-1575(24)00645-X
10.1016/j.fochx.2024.101757
101757
Research Article
Incorporation of amylose improves rheological and textural properties of Moringa oleifera seed salt-soluble protein
Wu Yan-Hui acd1
Lu Lin-Qian ad1
Li Jie-Mei ad
Liu Xing-Long ad
Fu Zhen fuzhen13@gxu.edu.cn
ad⁎
Ren Min-Hong msminhong@126.com
b⁎⁎
a Institute of Light Industry and Food Engineering, Guangxi University, 530004 Nanning, China
b Guangxi Vocational & Technical Institute of Industry, Nanning 530001, China
c Xiangsihu College of GuangXi Minzu University, 530225 Nanning, China
d Key Laboratory of Deep Processing and Safety Control for Specialty Agricultural Products in Guangxi Universities, Education Department of Guangxi Zhuang Autonomous Region, China
⁎ Corresponding author at: Institute of Light Industry and Food Engineering, Guangxi University, 530004 Nanning, China. fuzhen13@gxu.edu.cn
⁎⁎ Corresponding author at: Guangxi Vocational & Technical Institute of Industry, Nanning 530001, China. msminhong@126.com
1 They contribute equally to the article.

21 8 2024
30 10 2024
21 8 2024
23 1017576 4 2024
18 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The interactions between corn amylose (CA) and Moringa oleifera seed salt-soluble protein (MOSP) were explored to improve the gel properties of MOSP. With increasing CA content, the MOSP-CA gel network structure was improved but the size of the gel porosity decreased firstly and then increased; the water holding retention (WHR) of MOSP-CA was decreased from approximately 94 % to 85.43 ± 2.54 %. The MOSP-CA-2.5 gel exhibited the best water holding stability (WHS), with a value of 37.1 ± 0.33 %. The MOSP-CA gel hardness increased with CA concentration, and MOSP-CA-2.5 showed relatively optimal cohesiveness, elasticity, adhesiveness, and chewiness. Meanwhile, MOSP-CA-2.5 exhibited gel strength. Incorporation of CA significantly increased the exposure of hydrophobic residues and the concentration-dependent increase in disulfide bonds in MOSP-CA gel. Thus, hydrophobic interactions, hydrogen bonds, and disulfide bonds collectively stabilized the structure of MOSP-CA gel. The findings would broaden the application of MOSP and improve the utilization value of MOSP in various industries.

Highlights

• Incorporation of amylose enhanced the gel properties of MOSP gel.

• Addition of amylose significantly increased the hardness of MOSP gel.

• Addition of amylose significantly increased the exposure of hydrophobic residues.

Keywords

Amylose
Moringa oleifera seed salt-soluble protein
Interaction
Texture
Rheological properties
==== Body
pmc1 Introduction

Plant protein sources are currently used to meet the desired properties for consumers and stimulate the development of traditional and new foods in the food industry due to the low sustainability of animal protein (Mota da Silva, Souza Almeida, & Kawazoe Sato, 2021). An increasing variety of plant proteins is being assessed and incorporated into foods that are rich in protein. One of the key functional properties of food proteins is their ability to form gels, which imparts texture to various food products. Typically, the process of thermal gelation for globular proteins involves heating, causing the proteins to denature and expose their reactive side chains. Subsequently, the denatured protein molecules reorganize and aggregate, driven by the formation of disulfide bridges, hydrogen bonds, hydrophobic interactions, and van der Waals forces. Ultimately, this leads to the formation of a cohesive three-dimensional gel network (Zhu, Huang, Guo, & Chen, 2021).

Moringa oleifera, a plant indigenous to India (Gopalakrishnan, Doriya, & Kumar, 2016), is now globally cultivated for its impressive ability to adapt to various environments. The seeds of Moringa oleifera (MOS) are a treasure trove of protein, fats, minerals, and dietary fiber, nutrients that have increasingly captured the interest of researchers (Meireles, Gomes, Lopes, Hinzmann, & Machado, 2020; Saa, Fombang, Ndjantou, & Njintang, 2019). Currently, MOS is extensively used to enhance the flavor and nutritional value of various foods, as well as to create new consumer products. Moringa seed protein is one of the main components of Moringa seeds, with a content of over 35 %. The protein composition of Moringa seeds is similar with that of legumes, grains, and oilseeds, and it contains 17 amino acids, which are close to the ideal protein essential amino acid pattern for the human body (Baptista et al., 2017; Ma, Ahmad, Zhang, Khan, & Muhammad, 2020). Therefore, it can be used as a high-quality protein alternative to other legume plant proteins, and is a new protein resource with great potential development (Gopalakrishnan et al., 2016).

Preliminary investigations have elucidated that Moringa seed salt-soluble protein (MOSP) exhibited notable elasticity and stability, culminating in the formation of a self-supporting protein gel characterized by a specific network structure (Huang, Wu, Liu, Du, & Fu, 2024; Tang et al., 2023). To extend the application scope and refine the inherent properties of the MOSP gel, requisite modifications are imperative (Mao, Wu, Zhang, Ma, & Cheng, 2020). Studies have shown that the modification of proteins solely through physical, chemical, or enzymatic methods has disadvantages such as poor modification effects and high prices. Copolymerization modification is a novel modification method that involves the formation of a mixed system with unique quality, processing, and functional properties through the interaction between two macromolecular substances. The interplay between polysaccharides and proteins presents a substantial avenue for the modification and enhancement of protein functional properties. Strategic control over the amalgamation of proteins and polysaccharides stands as a direct and promising approach to enhance gelation and structural performance (Cao & Mezzenga, 2020; Li, Wang, Hu, Wu, & Van der Meeren, 2022).

Starch, a ubiquitous component in food production, plays a pivotal role in attaining optimal texture and viscosity, concurrently enhancing water-holding capacity, structural performance, and storage stability. Starch and protein, as the main components in food, often interact with each other during food processing and storage, thereby affecting the solubility, emulsification properties, foaming properties, and gelation properties of protein, as well as the rheological, gelatinization, and retrogradation characteristics of starch. Recent studies have delved into the intricate interactions between starch and protein molecules, revealing their profound effects on the structure, mechanical properties, and rheological characteristics of protein gels. Studies have shown that starch enhances gel hardness, elasticity, and stability, while its type and source significantly influence gel properties. Molecular-level interactions between starch and protein molecules are under investigation, and novel starch materials are being developed to further optimize gel performance (Hou, Li, Cui, Tao, & Han, 2021; Luo & Wang, 2022; Wu et al., 2020). Moreover, there are various types of starch and protein, and there are differences in their structure, properties, and the preparation techniques of mixed systems, which further exert different impacts on food quality. The effects of starches on the gelation characteristics of protein depending on the type of starch, the amount added, and the ratio of amylopectin to amylose (Johansson et al., 2023; Wang, Zhou, & Chen, 2022; Zhu et al., 2022a, Zhu et al., 2022b). Currently, there are still many issues related to the research on starch-protein mixed systems that urgently need further investigation; The interaction between starch and protein mainly occurs through covalent and non-covalent bonding, and the research on the formation mechanism is not deep enough and an in-depth exploration of the underlying mechanisms remains a subject of ongoing investigation (Lavoisier & Aguilera, 2019). Although there is much literature about starch or modified starch on gel properties and protein conformation, but there is little literature on the effect of amylose on the gel properties of Moringa oleifera seed salt-soluble protein that a new high-quality protein. Furthermore, the study provides a nuanced understanding of the impact of amylose on MOSP gel characteristics and broadens the application of MOSP and improve the utilization value of MOSP in various industries. Therefore, we think it is worth studying and meaningful.

Therefore, in this study, the influence of different concentrations of corn amylose (CA) on the properties and structure of MOSP gel was explored through rheology, texture analysis, and microstructure analysis using techniques such as infrared spectroscopy, Raman spectroscopy, and analysis of interaction forces. The water-holding capacity, and swelling properties of the MOSP-CA gel system were analyzed. Additionally, the potential mechanisms of interaction between MOSP and amylose in the composite gel were discussed.

2 Materials and methods

2.1 Materials

Corn amylose (CA) (99 % amylose) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. The Moringa oleifera seed salt-soluble protein (MOSP) was isolated based on our published work (Du, Wu, Xue, & Fu, 2022). The purity of the MOSP was 91.03 ± 0.74 % and all reagents were of analytical grade.

2.2 Preparation of MOSP-AM composite gel

Initially, a MOSP solution with a concentration of 22 % (w/v) was meticulously prepared. Subsequently, different concentrations (0.5 %, 2.5 %, 5 %, 7.5 % w/v) of corn amylose (CA) were judiciously added into the MOSP solution. The resulting mixture was continuously stirred and agitated with a whirlpool shaker for a period of 5 min. The pH of the blend was then adjusted to 8.0 by 1 M NaOH. The subsequent step involved subjecting the mixture to a water bath at 90 °C for a duration of 30 min, followed by prompt cooling using an ice water bath. The resulting gels were labelled as MOSP-CA-0.5, MOSP-CA-2.5, MOSP-CA-5.5, and MOSP-CA-7.5, respectively. As a control group, protein gel without CA were prepared.

2.3 Scanning electron microscopy (SEM) analysis

The gel samples were freeze-dried (frozen at −80 °C for 12 h, then lyophilization at −80 °C for 48 h) and pulverized, and then adhered to a sample holder coated with double-sided conductive adhesive. Excess powder samples were gently blown away using nitrogen, and the holder was placed in a vacuum evaporator. A thin layer of gold foil was sprayed onto the samples under 10 KV (Min, Ma, Kuang, Huang, & Xiong, 2022). The samples were then placed inside a scanning electron microscope (F16502, Phenom Company, Eindhoven, Netherlands) to observe the microstructure of the gel surface, and the images were scanned and recorded.

2.4 Water holding retention and stability measurements

The procedure of water holding retention (WHR) was based on the published method outlined by Cao et al. (2023). The gel samples were meticulously cut into small 10 × 10 × 10 mm pieces. The weight of the gel (W gel) was recorded, followed by drying the samples to a constant weight (The mass difference of sample before and after drying should not exceed 0.002 g) in a 60 °C oven. After cooling to room temperature, the weight was recorded as W dry. The WHR was calculated using the Eq. (1).(1) WHR%=Wgel−WdryWdry×100%

The procedure of water holding stability (WHS) was as followed: the gel samples were subjected to centrifugation (5418R, Eppendorf, Germany) at 10000 rpm and 20 °C for a duration of 15 min, followed by meticulous removal of the separated water. The WHS is precisely expressed in Eq. (2).(2) WHS%=M2M1×100%

M2 is the remaining gel weight after centrifugation; M1 is the gel weight before centrifugation.

2.5 Swelling rate determination

For the determination of the swelling capacity, the gel samples were meticulously cut into small 10 × 10 × 10 mm3 pieces and immersed in distilled water for 24 h, then taken out, and the surface water was carefully eliminated using filter paper. The weight of the gel before swelling was denoted as W0, while the weight after swelling was recorded as W1. The swelling rate (SR) was calculated using the following Eq. (3).(3) SR%=W1−W0W0×100%

2.6 Determination of textural properties

The textural properties of the MOSP gel were meticulously evaluated using the Texture Profile Analysis (TPA) module of a texture analyzer (Food Technology Corporation, USA) equipped with TA/0.5 cylindrical probe (12.7 mm diameter). The shape of samples was Φ10 mm × 10 mm × 10 mm. The testing parameters were as follows: 0.5 mm·s−1 compression speed, succeeded by a rising speed of 2.0 mm·s−1 post-compression. During the second compression, the sample underwent a 5-s holding period. The were compressed to a deformation of 40 % under compression, with the trigger force was set at 1.0 N.

2.7 Determination of rheological properties

The rheological properties of the gel samples were assessed with a Haake Mars III rheometer (Thermo-Fisher Scientific, Waltham, MA, USA) using a parallel metal plate with a diameter of 35 mm and a gap of 1000 μm. The linear viscoelastic region was determined by strain sweeps (0.01–5 %) at 1 Hz. The gels were equilibrated at 25 °C for 30 s before a frequency sweep was conducted from 0.1 to 10 Hz with a strain of 1 % and 10 points per decade at 25 °C. The parameters of storage modulus (G') and loss modulus (G″), and loss tangent (tan σ) were recorded.

2.8 Determination of molecular interaction forces

The intermolecular interactions of gels were determined by the treatment with protein denaturing solvents referring to the method of Zhao et al. (2023).The measurement method of molecular interaction forces between gel molecules was based on a published method (Cao et al., 2023) with slightly modification. One gram of gel sample was accurately weighed and mixed with 9 mL of denaturing solutions as follows: (1) 0.6 M NaCl (SA), (2) 0.6 M NaCl+1.5 M urea (SB), (3) 0.6 M NaCl+8 M urea (SC), and (4) 0.6 M NaCl+8 M urea+10 mM β-mercaptoethanol (SD). The solutions were mixed through the vortex oscillation (S25, IKA, Staufen, Germany) for 10 min. Subsequently, the mixture was allowed to stand at room temperature for 2 h before undergoing a 10,000 rpm centrifugation (5418R, Eppendorf, Germany) for 5 min. The protein concentration in different supernatants was determined using the Coomassie brilliant blue method, with the relevant denatured solution was taken as the blank. The percentages of ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds were calculated based on the protein concentration or concentration differences of SA, SB-SA, SC-SB, and SD-SC, respectively.

The interaction forces between molecules were expressed using the Eq. (5).(5) Percentage%=100×CdifferenceCtotal

The Cdifference represents the intermolecular force concentration of SA, SB-SA, SC-SB, and SD-SC. The Ctotal represents the total protein concentration in the composite gel.

2.9 Fourier infrared spectroscopy (FTIR) analysis

The lyophilized gel samples were meticulously mixed with potassium bromide (KBr) in a ratio of 1:100. Subsequently, the mixture was pressed under vacuum conditions. The FTIR spectroscopy involved a scanning duration of 60 s, a resolution of 4 cm−1, and 64 repeated scans within the range of 4000–400 cm−1 (Huang et al., 2024). FTIR spectrum was recorded by the TENSOR II Fourier infrared spectrometer (German BRUKER company, Shanghai, China). The acquired spectra were subjected to analysis using OMNIC software, and the secondary structure was precisely fitted using PeakFit v4.12.

2.10 Raman spectra analysis

The lyophilized MOSP gel samples was ground into a powder and a small amount of the powder was placed on a slide and gently flattened with a spoon. Utilizing an argon-ion laser with a wavelength of 785 nm and a slit setting of 1200 nm, the sample was observed through a Leica DM2700 imaging microscope (Spectra Physics Co., Mountain View, CA, USA). The experimental parameters were set as follows: excitation power at 5 %, and scanning range 400–2000 cm−1. The obtained data relevant molecular structures were calculated and analyzed using Peak Fit v4.12 software, providing detailed insights into the molecular composition and structural features of the MOSP gel. All samples were collected in triplicates.

2.11 Statistical analysis

All experiments were conducted three times independently, and the results were presented as mean ± standard deviation. Data analysis was performed using SPSS 26.0 (SPSS Inc., Chicago, IL, US) with one-way ANOVA. A significance level of p < 0.05 was applied.

3 Results and discussions

3.1 Appearance and microstructure of MOSP gel

The appearance and internal microstructure of MOSP-CA gels were presented in Fig. 1. The MOSP-CA gels exhibited a solid, white appearance. As the CA concentration increased, a noticeable transition was observed in the appearance of MOSP-CA gels-from a smooth and bright texture to a rough and dull one. Detailed examination through SEM images revealed distinct characteristics. In the absence of CA, the MOSP gel exhibited visible connectivity, but the link with edges and corners was weak. Notably, no discernible starch particles are observed in any of the samples, indicating complete gelation of CA within the gel system. Microscopic pores appeared denser in MOSP-CA-0.5 and MOSP-CA-2.5 gels. The gel networks of MOSP-CA-2.5 gel look like honey comb and these structures appear like numerous tunnels which are responsible for imparting elastic/spongy properties to gel and facilitate holding of water (Joshi, Aldred, Panozzo, Kasapis, & Adhikari, 2014). With an increase in amylose concentration, MOSP-CA-5 displayed a gel structure with larger cavities, while MOSP-CA-7.5 exhibited a three-dimensional network of substantial skeletons characterized by rough surfaces and irregular large pores. This structural variation could be because starch gelatinization adsorbed water to increase the effective protein concentration in the continuous phase and maintain relative stability for gels with different concentrations (Johansson et al., 2023). The uneven microstructure observed in all composite gels suggested the potential gelatinization of CA as a filler through adhesion during the heating process (Min et al., 2022). These findings contributed to a comprehensive understanding of the microstructural changes induced by varying CA concentrations in MOSP gels.Fig. 1 Appearance of MOSP-CA gel and SEM image and Effect of CA on water holding rate (A), water holding stability (B) and Swelling rate of MOSP-CA gels.

Fig. 1

3.2 Water holding capacity and swelling rate of MOSP gels

Water holding capacity refers to the entrapment of water within the gel's network structure, a crucial functional property closely linked to the rheological properties of the gel (Min et al., 2022). As shown in Fig. 1, the water holding retention (WHR) (Fig. 1A) and water holding stability (WHS) (Fig. 1B) results of MOSP-CA gels were presented.

There was no significant change in the WHR of the Control group, MOSP-CA-0.5, and MOSP-CA-2.5 composite gels, with a WHR of approximately 94 %. This indicated that similar amounts of water were captured within the three-dimensional networks of these gels. However, as the CA concentration further increased, the WHR of MOSP-CA-5 and MOSP-CA-7.5 decreased to 89.76 ± 0.83 % and 85.43 ± 2.54 %, respectively. The WHR of the gels was related to the gel network structure, and the decrease in WHR was associated with the presence of inhomogeneous macroporous networks within the gel structure, which made it more difficult for water to be retained (He, Liu, Zhao, Li, & Wang, 2021).

WHS is an assessment of the ability of a gel to retain water. Water migrated through channels, and due to the formation of a more open and rough gel structure, water can easily migrate within the gel network. The Control group, MOSP-CA-2.5, and MOSP-CA-5 gel, due to the compact aggregation of proteins, exhibited a fine and uniform porous structure that was conducive to water retention. On the other hand, inhomogeneous macroporous structures weaken the ability of the gel to bind or retain water within its network. The MOSP-CA-2.5 gel exhibited the best water holding stability, with a value of 37.1 ± 0.33 %, indicating a relatively poor ability to immobilize water, but the value was notably higher than that of other plant protein gels, such as cowpea protein (Peyrano, de Lamballerie, Avanza, & Speroni, 2022), soy protein isolate (Zhao, Chen, Hemar, & Cui, 2020) and ginkgo seed protein (He et al., 2021). At low CA concentrations, there was no significant alteration in WHS, while higher CA concentrations led to a reduction in WHS. The gel's WHS was intricately linked to its microstructure and the quantity of hydrogen bonds within the gel system. From the SEM results, incorporation of CA could improve the three-dimensional MOSP gel network. The MOSP-CA-2.5 gel had a well three-dimensional gel network with honey comb structures and numerous tunnels, which is conducive to retaining water. But increasing the CA concentration, the size of the pore opening of the MOSP-CA-5 and MOSP-7.5 gel network was about increased and Addition of a higher concentrations of CA resulted in a non-uniform macroporous structure in the MOSP-CA composite gel, thereby diminishing its capacity to bind or retain water. This phenomenon aligned with findings that addition of mung bean starch could decrease the water retention capacity of flaxseed protein composite gel (Min et al., 2023). In contrast, addition of corn starch significantly improved the WHC of soy protein isolated gels, increasing with rising starch concentrations. This improvement was attributed to the increased number of hydroxyl groups forming hydrogen bonds with carboxyl and amino groups in soy protein isolate, resulting in a more compact network structure in the gel system (He et al., 2024). Additionally, the enhanced water holding capacity is a result of strong water absorption and swelling capacities of corn starch (Xu et al., 2018). These insights contribute to a nuanced understanding of the intricate relationship between CA concentration, gel microstructure, and water-holding properties.

The swelling rate (SR) serves as a crucial indicator for assessing the embedding and delivery efficiencies of gels, providing valuable insights into the physicochemical properties of gel networks. As shown in Fig. 1C, the SR of MOSP-CA-2.5 gel (12.72 ± 1.34 %) was significantly higher than that of the MOSP-alone gel. The magnitude of the gel's SR was related to the flexibility of the protein molecular chain (Hui Li, Zhao, Chen, & Mercadé-Prieto, 2016). The SR was also intricately linked to the gel network, wherein water molecules penetrated the protein gel network, interacting with the network chain and promoting network expansion. However, the MOSP-CA-5 and MOSP-CA-7.5 gels with non-uniform macropore structures exhibited less efficiency in absorbing water into their networks compared to the gels with finer pore structures like MOSP-CA-0.5 and MOSP-CA-2.5. Consequently, the contact between water and gel networks was reduced in the gels with non-uniform macropores, leading to poorer swelling properties in MOSP-CA-5 and MOSP-CA-7.5. This observation underscores the nuanced relationship between gel microstructure and swelling efficiency, providing valuable insights into the gel's water absorption dynamics.

3.3 Textural properties of MOSP gel

The assessment of MOSP-CA gel involved a comprehensive analysis of its hardness, cohesiveness, springiness, gumminess, and chewiness using a texture analyzer, and the results were summarized in Table 1. These textural attributes provided insights into the gel's firmness, internal strength, structural integrity, resilience, energy required for swallowing, and tensile resistance during chewing. Hardness indicates the strength of the gel network. Hardness of the MOSP-CA gel exhibited an increase with increasing CA concentration compared to the control group (MOSP alone). This observed trend could be attributed to that amylose facilitated the cross-linking of MOSP protein molecules. Meanwhile, the increase in the strength of the MOSP gel was partially attributed to the “filling effect”(Kong, Ogawa, & Iso, 1999). In addition, amylose plays a pivotal role in determining gel hardness due to its ability to form a three-dimensional network structure through molecular recrystallization (Biduski et al., 2018; Xu et al., 2022), thereby enhancing the MOSP gel strength. Meanwhile, the gel microstructure demonstrated that the textural firmness of MOSP-CA gels was much higher than the MOSP gels. In contrast, incorporation of starch and acetylated starch to soybean protein led to a decrease in the gel textural parameters, which was mainly due to the spacing effect and competitive adsorption of water and starch (Yu, Ren, Zhao, Cui, & Liu, 2020), meanwhile, the gelatinization behavior of starch might disrupt protein gel network formation (Fan et al., 2019).Table 1 Texture properties and interaction forces of MOSP-CA gel.

Table 1	Hardness (N)	Cohesiveness (%)	Springiness (mm)	Gumminess(N)	Chewiness (mJ)	Ionic bonding(%)	Hydrogen bonding(%)	Hydrophobic interaction(%)	Disulfide bonds(%)	
Control	5.13 ± 0.03de	0.93 ± 0.02a	1.36 ± 0.16e	5.04 ± 0.01e	9.69 ± 0.15a	7.34 ± 1.04 b	10.15 ± 0.63c	40.79 ± 0.23de	0.37 ± 0.22d	
MOSP-CA-0.5	5.25 ± 0.06d	0.63 ± 0.02cd	2.04 ± 0.04b	5.18 ± 0.01d	9.39 ± 0.06d	8.19 ± 0.70 a	11.03 ± 0.30b	40.95 ± 0.72d	0.25 ± 0.21de	
MOSP-CA-2.5	5.88 ± 0.18bc	0.77 ± 0.07b	2.23 ± 0.06a	5.56 ± 0.12a	9.51 ± 0.90ab	5.12 ± 0.30c	12.17 ± 0.70a	56.39 ± 0.44a	3.77 ± 1.6a	
MOSP-CA-5	5.95 ± 0.02b	0.63 ± 0.01c	1.85 ± 0.02c	5.44 ± 0.22abc	9.51 ± 0.90abc	4.22 ± 0.17d	8.85 ± 0.17d	47.75 ± 0.53c	1.45 ± 0.42c	
MOSP-CA-7.5	6.30 ± 0.17a	0.43 ± 0.01e	1.85 ± 0.02cd	5.48 ± 0.07ab	9.17 ± 0.04e	0.49 ± 0.15e	5.14 ± 0.15e	54.79 ± 0.32b	2.38 ± 0.69b	
Note: Different letters (a-e) in the same line indicate significant difference (p < 0.05).

Cohesiveness refers to the strength of the internal bonding that binds the matrix into clumps and inhibits the breakdown of the sample into fragments during swallowing (Joshi et al., 2014). Springiness indicates the ability of a sample to withstand deformation (Zhu et al., 2022a, Zhu et al., 2022b). The cohesiveness, springiness, gumminess, and chewiness of MOSP-CA gel displayed an initial increase followed by a decrease. Among these, MOSP-CA-2.5 gel exhibited the highest cohesiveness, springiness, gumminess, and chewiness, with values of 0.77 ± 0.07 %, 2.23 ± 0.06 mm, 5.56 ± 0.12 N, and 9.51 ± 0.90 mJ, respectively. Moderate addition of CA promoted cross-linking among MOSP molecules, resulting in a stable and dense three-dimensional gel structure that improved the gel properties of MOSP. Moreover, further increasing the addition of CA might lead to a dilution of protein concentration and hinder the cross-linking of protein molecules (Wang et al., 2023). It also effectively hindered the destruction of hydrophobic bonds and hydrogen bonds and disulfide bonds inside the gel. Consequently, these structural indicators such as cohesion, elasticity, adhesiveness, and chewiness might be compromised with excessive CA addition. Achieving an optimal amylose concentration is crucial for attaining the desired textural attributes in the MOSP-CA gel.

3.4 Rheological properties of MOSP gel

3.4.1 Strain sweep analysis

As depicted in Fig. 2, the impact of strain amplitude (γ) on the storage modulus (G') and loss modulus (G") of MOSP-CA gels is noticeable. In the linear viscoelastic region, G' values were much larger than G" values of all samples, indicating the MOSP and MOSP-CA gels had a dominant solid-like behavior or elastic response (Lei, Zhao, Li, Wang, & Wang, 2022). In addition, as γ increased, G' and G" remained stable in the linear viscoelastic region. Beyond a critical strain amplitude (γc), a sharp decrease in G' and G" occurred, indicating a transition from linear rheology to nonlinear rheology. Therefore, the MOSP-CA gels belong to Type I (inter-cycling strain-thinning) network (Hyun, Kim, Ahn, & Lee, 2002). Addition of CA significantly enhanced the G' and G" values of MOSP gel. As CA concentration increased, G' and G" initially increased and then declined. MOSP-CA-2.5 exhibited the highest G' and G", aligning with superior elastic properties in gel texture. At higher γ values, G' and G" curves for all samples were broken, indicating gel network disruption and fracture. Moreover, when γ was further increased, G" surpassed G', suggesting a more liquid-like behavior in the gel system due to network destabilization (Hyun et al., 2011).Fig. 2 Rheological properties of MOSP-CA gels. A, B: Strain sweep; C, D: Frequency sweep.

Fig. 2

To better compare the gel strength among different MOSP-CA gels, the complex modulus (G*) values were examined to determine the critical strain (γc) values of different gels (Fig. 3B). As CA concentration increased, γc initially raised and then decreased. MOSP-CA-2.5 had the highest γc value (2.85 %), suggesting its prolonged linear viscoelastic region and ability to maintain similar properties even under significant deformations. γc values for MOSP-CA-5 and MOSP-CA-7.5 was decreased to 2.45 %, equivalent to the control group, indicating that excessive amylose hindered the interaction between proteins molecules and weakened the formation of a dense gel structure (Lei et al., 2022).Fig. 3 FTIR (A, B, C, D) and Raman spectrum (E) and disulfide bond composition (F) of MOSP-CA gel. A: the infrared spectrum, B: the proportion of peak area of the amide II band, C: the MOSP-CA secondary structure, D: the peak-splitting fitting process of the secondary structure.

Fig. 3

3.4.2 Dynamic oscillatory analysis

A dynamic frequency sweep (0.1–10 Hz) was further applied to investigate the viscoelastic properties of the MOSP-CA gels (Fig. 2C and D). G' increased with rising CA concentration, maintaining stability and independence with increased frequency, suggesting a distinctive three-dimensional network structure in the composite gel. However, at 7.5 % CA concentration, G' in MOSP-CA-7.5 gel decreased and displayed frequency dependence. All composite gels exhibited solid-like behavior within the linear viscoelastic region (G' > G”). The MOSP-CA-2.5 showed significantly highest G' and G" values compared to the other gels, suggesting that addition of appropriate amount of CA could improve the strength and viscoelasticity of MOSP gels, which were well confirmed by the SEM results. The physical cross-linking between MOSP and amylose molecules could impacting gel strength (Wijaya, Patel, Setiowati, & Van der Meeren, 2017; Zheng, Van der Meeren, & Sun, 2024). Additionally, the higher swelling capacity of amylose led to increased protein molecule concentration, strengthening hydrophobic interactions within the protein gel matrix (Wang et al., 2019). However, further increasing the addition of CA might lead to a dilution of protein concentration and hinder the cross-linking of protein molecules and hamper the molecular interaction forces (Wang et al., 2023), thereby influencing the gel strength.

3.5 Molecular interaction analysis

Protein gelation relies on various forces, including hydrogen bonding, ionic bonding, hydrophobic interaction, and covalent bonding, to provide structural support and influence gel properties. The interaction forces in MOSP-CA gels were presented in Table 1. With increasing CA concentration, the trend of hydrophobic force in the gels initially increased and then decreased. The hydrophobic force in MOSP-CA-2.5 reaching a maximum value of 56.39 %; Ionic bonds were decreased with increasing CA concentration, suggesting that there may be an electrostatic shielding effect present in the MOSP-CA gel, where the addition of CA likely suppresses the interactions between the charged amino acid side chains of MOSP, resulting in a decrease in the proportion of ionic bonds. Zhu et al. (2021) argued that hydrogen bonds played a more significant role in controlling the gel network within protein hydrogels. Upon the addition of CA, the hydrogen bonding in the gel system decreased and continues to decline as the CA content increased. What's more, the percentage of hydrophobic interactions and disulfide bonds significantly increased in MOSP-CA gels, promoting the formation of dense and uniform gel networks. The addition of CA increased protein surface hydrophobicity and sulfhydryl group content, exposing more hydrophobic groups, the results would be further analyzed in subsequent FTIR and Raman spectroscopy studies. Thus, the gel structure of all gel samples primarily relied on hydrophobic interaction and hydrogen bonds.

3.6 FTIR analysis

The structural alterations induced by CA on MOSP were examined through FT-IR spectra within the range of 4000–400 cm−1 (Fig. 3A). The protein amide I peak position occurred in the region between 1600 and 1700 cm−1 (principally C <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.019444,-0.019444)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg> O stretch) and the amide II band was at 1548 cm−1 (C—N stretch coupled with N—H bending mode). There was a broad absorption peak region at 3600-3000 cm−1, which could be attributed to the stretching vibration of O—H bonds and hydrogen bond of the hydroxyl groups (Yang, Liu, Li, & Tang, 2019).

Notably, the addition of CA did not lead to the emergence of new peaks in FTIR spectrum, which was almost identical to the gel formed by MOSP alone, indicating that no new functional groups were formed between MOSP and CA. In the FTIR spectrum, the amide II and I bands, representing CO stretching vibration, C—N stretching vibration, or N—H bending vibration, displayed two distinct peaks in the 1500–1600 cm−1 and 1600–1700 cm−1 ranges, respectively. Comparative analysis of the relative peak area of the amide II band revealed a concentration-dependent increase with CA addition (Fig. 3B). However, at a CA concentration of 7.5 %, the area was decreased. Thus, an increase in the content of C—N or N—H bonds could be a significant factor in the stabilization and enhancement of the three-dimensional network formation of MOSP gels by CA. The secondary structure of proteins is primarily composed of β-sheets, random coils, α-helices, and β-turns. The absorption peak ranges of these four components in the spectrum are 1600–1540 cm−1, 1640–1650 cm−1, 1650–1660 cm−1, and 1660–1700 cm−1, respectively. Examining the secondary structure components revealed notable changes in MOSP-CA compared to the control group (Fig. 3C). The β-turn and β-sheet contents in MOSP-CA significantly increased, while random coil content exhibited an initial decrease followed by an increase. Conversely, α-helix content decreased. This may be due to the positive correlation between the relative content of β-sheet structure and the hardness of the gel, while the relative content of α-helix structure may have a negative impact on the gel strength.

The absorption peak at 1660 cm−1 may also be attributed to the stretching vibration of glucose rings in starch molecules. The absorption peak near 1650 cm−1 is believed to be due to water molecules adsorbed in the amorphous region, which corresponds to the bending vibration of water molecules and can indicate the presence of bound water in starch (Sun et al., 2020). In addition, a strong and broad absorption peak was observed in the range of 3200–3600 cm−1 for all gels. This absorption peak in these regions represents the intermolecular hydrogen bonding of proteins through N—H stretching vibrations, or the O—H stretching vibrations of starch and proteins, indicating the formation of hydrogen bonds (He et al., 2021). Simultaneously, the absorption peak in this region shifts towards a longer wavelength with increasing concentration of CA. This rightward shift in wavelength indicates a decrease in hydrogen bonding interactions, suggesting that the role of O—H stretching vibrations is not significant in the gel formation of MOSP with CA, which was similar with the results of Mung bean starch–flaxseed protein composite gel (Liu et al., 2017) and lotus root amylopectin-whey protein isolate gel (Min et al., 2022). This also aligned with findings from molecular interaction analysis. All samples exhibited a strong and narrow absorption peak at 1400 cm−1, which was correlated with the O—H tensile vibration of starch. Additionally, MOSP-CA gels presented a new weak peak at 1025 cm−1, which was due to the characteristics of the amorphous region of starch, resulting in random coil structure structures.

3.7 Raman spectroscopy analysis

Raman spectroscopy is frequently utilized for the analysis of protein secondary structures and the investigation of specific structural features and their trends. Alterations in the structure, partial chemical bonds, and hydrophobic environment characteristic peaks of gels can be observed through Raman spectroscopy. The Raman spectrum of the MOSP-CA gels is illustrated in Fig. 3E, and the specific attributions and descriptions of characteristic peaks are detailed in Table S1. The Raman spectra of all gel samples prominently exhibited several characteristic peaks, signifying that the addition of CA induced changes in MOSP's disulfide bonds, amide II, I band, and certain amino acid groups.

The vibrational contraction amplitude of the peak near 1003 cm−1 indicates changes in phenylalanine, while the peak at 760 cm−1 represents alterations in the microenvironment of tryptophan. The Fermi resonance ratio (I850/I830) at 850 cm−1 and 830 cm−1 reflects changes in aromatic amino acids such as tyrosine. Based on the observed variations in the absorption peaks of the vibrations of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, it is speculated that there is stretching, breakage, and reconstruction of the tertiary structure of the gel.

CA addition induced nuanced changes in the MOSP-CA gel structure, impacting phenylalanine, tryptophan, and tyrosine residues, which was elucidated in Table 2. These changes provided insights into the extension, fracture, and reconstruction of the gel's tertiary structure. The addition of CA reduced the peak intensity of phenylalanine at 1003 cm−1 from 672.22 to 534.35 (MOSP-CA-0.5), indicating that phenylalanine was in an exposed state. However, with further increasing CA concentration, the I1003 values increased, suggesting a transition of phenylalanine from an “exposed state” to an “embedded state.”Table 2 MOSP-CA side chain group band intensity.

Table 2	Phe (I1003)	Trp (I760/I1003)	Tyr (I850/I830)	-CHn (I1450/I1003)	
Control	672.22 ± 3.27a	0.03 ± 0.01b	0.40 ± 0.06c	11.69 ± 0.20a	
MOSP-CA-0.5	527.25 ± 9.73c	0.08 ± 0.05a	0.68 ± 0.04b	5.15 ± 0.28bc	
MOSP-CA-2.5	534.35 ± 8.93b	0.12 ± 0.05b	1.00 ± 0.06a	5.65 ± 0.85b	
MOSP-CA-5	373.37 ± 9.90d	0.04 ± 0.01b	0.78 ± 0.07b	4.55 ± 0.62d	
MOSP-CA-7.5	181.75 ± 7.71e	0.02 ± 0.01a	0.70 ± 0.18b	4.27 ± 0.15d	
Note: Different letters (a-e) in the same line indicate significant difference (p < 0.05).

The alterations in the vibrational peaks of tryptophan and tyrosine groups differ from those of phenylalanine. Compared to the MOSP alone gel, the I760/I1003 and I850/I830 values of MOSP-CA-0.5 increased from 0.03 and 0.40 to 0.08 and 0.68, respectively. With the increase in CA concentration, the ratio of tryptophan to tyrosine initially increased and then decreased. MOSP-CA-2.5 exhibited the highest ratio, 0.12 and 1.00 for tryptophan and tyrosine, respectively, indicating increased exposure of tyrosine to the molecular surface.

Protein disulfide bonds play a crucial role in stabilizing the tertiary structure of proteins. The examination of Raman spectra reveals that the region associated with disulfide bonds exhibited a small wavenumber, and its absorption peak was not prominently evident. Consequently, the content and stretching vibration of protein disulfide bonds exerted minimal influence on the MOSP gel's structure, aligning with the results of intermolecular forces. The wavelength range spanning 510–545 cm−1 was meticulously analyzed by peak fitting, elucidating three primary configurations of disulfide bonds. Specifically, the Raman bands at 500–510 cm−1, 515–525 cm−1, and 535–545 cm−1 correspond to deflection-deflection-deflection (g-g-g), reverse-deflection-deflection (t-g-g), and reverse-deflection-reverse (t-g-t) conformations of disulfide bonds, as visually presented in Fig. 3F.

Among these configurations, the t-g-t conformation emerged as the predominant component of the disulfide bond, believed to be the bond type primarily contributing to intermolecular forces. Notably, as the concentration of CA increased, the t-g-t configuration attained its maximum value in both MOSP-CA-2.5 and MOSP-CA-5, each reaching 44 %. The intricate interplay of disulfide bond configurations, particularly the prevalence of the t-g-t conformation, emerges as a key factor in understanding the mechanical properties, such as hardness, exhibited by the MOSP-CA gel. Considering the inherent properties of the gel, it is reasonable to posit that this specific disulfide bond configuration significantly contributed to the observed hardness of the gel.

3.8 Proposed composite gel forming mechanism

Based on the above discussion and analysis, the general mechanism of the influence of CA on MOSP gel was speculated as shown in Fig. 4. Before heating, the MOSP proteins are internally curled and folded into spherical shapes, while the amylose exists in granular form. After heating, the MOSP proteins in the system unfolded, with a decrease in internal helical structure and an increase in folding. Simultaneously, the CA particles expanded upon heating, exposing their internal helical structure and increasing the number of available interaction sites. During the cooling process, CA was locked into the protein aggregation network framework, providing support for its formation. In this process, CA promoted hydrophobic interactions in the protein gel, as well as the formation of a small number of hydrogen bonds and ionic bonds, resulting in a more ordered and cross-linked network gel structure that provided greater gel elasticity and rigidity. Heat treatment led to partial gelatinization of starch within the protein network, forming an interpenetrating network (Lavoisier & Aguilera, 2019), and the gel formation driven by intermolecular forces was related to changes in microstructure (Makshakova & Zuev, 2022). The integrity level of the individual component networks and the increase in local component concentration in the gel positively contributed to the improvement of gel properties. This understanding shed light on the intricate interplay of molecular interactions and structural changes during the gel formation process.Fig. 4 Formation mechanism of MOSP-CA gel.

Fig. 4

4 Conclusions

The incorporation of CA significantly improved the intrinsic properties of MOSP gel, with the 2.5 % concentration optimizing water retention and swelling. However, higher CA levels at 7.5 % led to larger pores, reducing water-locking and stability. The gel's hardness increased with CA concentration, while other properties showed a peak at 2.5 %. The gel's resistance to disruption was highest at this concentration, attributed to strengthened hydrophobic interactions, hydrogen bonds, and disulfide bonds. Additionally, the concentration-dependent increase in disulfide bonds in MOSP-CA gel, primarily comprising the t-g-t configuration, contributed significantly to the observed gel structure. In conclusion, the study provides a nuanced understanding of CA's impact on MOSP-CA gel characteristics, crucial for tailoring applications in the food and pharmaceutical industries.

CRediT authorship contribution statement

Yan-Hui Wu: Writing – original draft, Methodology, Investigation, Data curation. Lin-Qian Lu: Software, Methodology, Investigation. Jie-Mei Li: Validation, Investigation. Xing-Long Liu: Methodology, Investigation. Zhen Fu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization. Min-Hong Ren: Writing – review & editing, Software.

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.

Appendix A Supplementary data

Characterization of characteristic peaks of Raman spectra.

Image 1

Data availability

Data will be made available on request.

Acknowledgements

This work was supported by The Basic Ability Enhancement Program for Young and Middle-aged Teachers of Guangxi (2023KY0014 , 2023KY1315 ) (People's Republic of China) and the Project of Bama County for Talents in Science and Technology (20210031 ) (People's Republic of China).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2024.101757.
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