Using sodium bicarbonate and resting temperature combined to improve the water retention and gel characteristics of low-salt chicken myofibrillary protein

Highlights

• •Water retention and cooking yield improved when resting at 30 °C for 6 h.
• •Whiteness decreased resting at 30 °C, whiteness increased when resting over 30 °C.
• •Gel strength, hardness, springiness, and chewiness were the highest when resting at 30 °C.
• •Over 30 °C destroyed gel structure, reduced water retention and texture properties.

Abstracts.

Variations in temperature induce subtle alterations in the pH of myofibrillar protein solutions containing sodium bicarbonate. To investigate the effects of sodium bicarbonate (0.4 %, w/w) and resting temperature (4–50 °C) on the gel properties of chicken myofibrillar proteins, parameters including water retention, cooking yield, gel strength, textural characteristics, chemical interactions, and microstructure were evaluated. The results demonstrated that water retention, cooking yield, gel strength, hardness, springiness, and chewiness peaked at a resting temperature of 30 °C, whereas the whiteness value was minimized. This improvement was attributed to the maximization of hydrophobic interactions and hydrogen bonding between myofibrillar protein molecules at 30 °C, which facilitated the formation of a highly ordered and dense network structure. In conclusion, the cooking yield, textural properties and microstructure of low-salt chicken myofibrillary protein with 0.4 % sodium bicarbonate were improved when the resting temperature was 30 °C for 6 h.

1. Introduction

Myofibrillar protein is the most abundant protein in muscles, accounting for 50 to 55 % of the total protein content. It is mainly composed of myosin and actin, playing an important role in the formation of gel structure during meat processing (Tan et al., 2025; Wang et al., 2022). Several factors influence the thermal gelation of myofibrillar proteins, such as temperature, pH, and ionic strength (Chen, Xu, et al., 2020; Zhang et al., 2023). Protein aggregation and crosslinking begin when the temperature exceeds 30 °C, and significant structural changes occur at around 50 °C due to myosin denaturation. The surface of myofibrillar proteins carries a large number of charges. Changes in pH and ionic strength alter the charge distribution on amino acid side chains, which affects intermolecular interactions and protein aggregation states, ultimately influencing gel structure formation (Xiao et al., 2025). Increased electrostatic repulsion can prevent excessive molecular aggregation, promote protein unfolding, and expose more hydrophobic groups. Meanwhile, charged groups bind water molecules more effectively (Wang et al., 2022; Zhang et al., 2023). These dual effects facilitate the formation of a dense, water-retaining gel network upon heating. Therefore, pH and ionic strength affect ionic bonds, hydrogen bonds, and other forces within the protein system, altering protein–protein and protein–water interactions and resulting in different gel microstructures. Macroscopically, this leads to variations in functional properties such as water retention (Liu et al., 2021).

Salt plays a dominant role in meat processing by enhancing gelatinization, improving water retention (Desmond, 2006; Kang, Hou, & Xu, 2024). The world health Organization (WHO)‌ recommends that adults' daily salt intake should be‌ no more than 5 g, excessive intake is related to hypertension and cardiovascular disease (He et al., 2020; Zoccali et al., 2025). Sodium bicarbonate is a weak electrolyte that easily decomposes at high temperatures. It has high buffering capacity and ionic strength, which can enhance protein deprotonation, increase protein repulsion, improve solubility, and ultimately enhance meat quality, making it a suitable salt substitute (Kang, Gao, et al., 2022). Chicken batter with 0.5 % sodium bicarbonate shows better water-holding capacity and texture than that with 1 % sodium chloride, because sodium bicarbonate raises the pH, induces myosin head denaturation, dissociates actomyosin complexes, and promotes the formation of β-sheet and β-turn structures (Mudalal & Petracci, 2019; Zhu et al., 2018). Li et al. (2021) found that increasing sodium bicarbonate levels improves protein solubility, exposes hydrophobic residues and sulfhydryl groups, causes Ca²⁺-ATPase inactivation and protein unfolding, leading to easier myofibrillar protein denaturation. Our previous studies (Cheng et al., 2025; Kang, Hou, & Xu, 2024) showed that higher sodium bicarbonate concentrations (0–6 g/kg) and longer resting times (0−12h) significantly increased pH, improving solubility, active sulfhydryl content, rheological properties, and texture of low-salt (1 mmol/L NaCl) chicken myofibrillar protein. At 0.4 % sodium bicarbonate, these properties reached maximum values after resting at 30 °C for 6 h. Increasing the resting temperatures from 4 °C to 50 °C slightly raised the pH from 7.75 to 8.14 (Cheng et al., 2025). However, few studies have examined how slight pH changes due to resting temperatures affect the water-holding capacity and gel characteristics of low-salt myofibrillar protein. Therefore, the study aimed to investigate the combined effects of 0.4 % sodium bicarbonate and resting temperatures (4–50 °C) on the water retention and gel structure of low-salt chicken breast myofibrillar protein.

2. Materials and methods

2.1. Materials and ingredients

Frozen AA broiler chicken breast meat (42 days old, 2000 ± 150 g) was obtained from Zhengda Group (Qingdao, China). All chemicals were of analytical grade purity.

2.2. Myofibrillar protein extracted

The chicken myofibrillar protein was extracted referring to the way of Zou et al. (2022). Briefly, thawed the chicken and minced it. The crude myofibrillar protein was isolated by washing minced chicken three times with four volumes of phosphate-buffered saline (100 mmol/L KCl, 20 mmol/L Na₂HPO₄/NaH₂PO₄, 2 mmol//L MgCl₂, 1 mmol/L EGTA, 1 mmol/L NaN₃, pH 7.0). The resulting residue was subsequently washed twice with four volumes of 0.1 mol/L NaCl solution, filtered through four layers of gauze, and the pH of the filtrate was adjusted to 6.0 using 0.1 mol/L HCl. The final precipitate was collected as purified myofibrillar protein. The purified protein was stored at 4 °C and used within 48 h.

2.3. Myofibrillar protein solutions and gels prepared

60 mg/mL protein solution was prepared using a 1 mmol/L NaCl buffer solution (pH 7.0). Following the addition of 0.4 % sodium bicarbonate, the solution was homogenized at 2000 rpm for 10 s and subsequently rested at 4, 10, 20, 30, 40, and 50 °C for 6 h, respectively. After resting, 8 g of each treated protein solution was transferred into a 10 mL beaker and placed in the 85 °C water for 30 min, upon completion, it was cooled to 20 °C and stored at 4 °C overnight.

2.4. Water retention

Following the method of Zhao et al. (2022), an appropriate amount of myofibrillar protein gel was taken, cut into uniform squares, wrapped by filter paper, centrifuged at 5000 ×g for 15 min (LYNX 4000, Thermo Corporation, USA). After centrifugation, the sample was weighed to determine the centrifugal loss.

2.5. Cooking yield

The myofibrillar protein gel, cooled overnight, was allowed to rewarm to room temperature for 2 h. The surface moisture was then removed using absorbent paper, and the sample was weighed. Then, calculated using the following formula:

Cooking yield (%) = (1 - weight of surface moisture/weight of raw protein solution) × 100 %.

2.6. Whiteness

Whiteness of the cooking myofibrillar protein was measured using a colorimeter (CR-400 Japan). Prior to measurement, it was calibrated using a standard white plate. Then, calculated using the following formula:

$$\text{Whiteness}=100-{\left[{\left(100-{\mathrm{L}}^{\ast }\right)}^2+{\left({\mathrm{a}}^{\ast }\right)}^2+{\left({\mathrm{b}}^{\ast }\right)}^2\right]}^{1/2}.$$

2.7. Texture properties

The texture properties of the formed gels were analyzed by a texture analyzer (UK) equipped with a P36/R probe, following the method of Kang, Kong, Gao, Li, et al. (2021). Test parameters: test speed: 0.5 mm/s, compression ratio: 50 %.

2.8. Gel strength

Gel strength was measured according to the method of Li et al. (2019). The gel was analyzed using a texture analyzer equipped with a P/0.5 probe (UK). Test parameters: pre-test speed, 1 mm/s; test speed, 2 mm/s; post-test speed, 10 mm/s; test distance, 10 mm; trigger force, 10 g. The peak force recorded during the test was taken as the breaking force (g) of the thermally induced gel.

2.9. Chemical force

Chemical force of the myofibrillar protein gel was determined referring to the way of Yang et al. (2021).

2.10. Scanning electron microscope

3 × 3 × 3 mm³ gel cubes were prepared and fixed by immersion in a 0.1 mol/L phosphate buffer solution containing 2.5 % glutaraldehyde (pH 7.2) for more than 72 h. Following fixation, the samples were rinsed in phosphate buffer without glutaraldehyde for 10 min and subjected to a graded ethanol dehydration process. The process involved immersing the samples sequentially in 50 %, 70 %, 90 %, 95 %, and 100 % ethanol solutions for approximately 15 min each time. Afterward, samples were transferred to a 1:1 mixture of anhydrous ethanol and tert-butanol about 15 min, then by an additional 15 min immersion in pure tert-butanol. The samples were placed in a vacuum oven and dried at 30 °C for 4 h. Finally, they were sputter-coated with gold and observed using a scanning electron microscope (FEI Inc., USA).

2.11. Statistical analysis

Three independent repetitions (n = 3) were performed with different source materials and resting temperatures. All results were expressed as mean ± SE. Data were analyzed by the GLM procedure (SPSS v.26.0, USA). The means were compared using LSD procedure, significant differences (P < 0.05) were measured using Duncan's Multiple Range Test.

3. Results and discussion

3.1. Water retention

Water retention is a crucial attribute of protein gels, exerting a profound impact on the myofibrillar protein gel (Lu et al., 2025). The increase in protein water retention might also stem from alterations in the cross-linking of exposed protein residues through hydrophobic interactions and hydrogen bonding (Yang et al., 2021). The effect of sodium bicarbonate on the water retention of myofibrillar protein gel at different resting temperatures is depicted in Fig. 1. As the resting temperature rises, the water retention of myofibrillar protein gel undergoes a significant change (P < 0.05), presenting an increasing and then decreasing trend, reaching a maximum when the resting temperature at 30 °C. The reason is that as the resting temperature ascends, the decomposition of sodium bicarbonate accelerates, enhancing the pH and ionic strength of myofibrillar protein (Cheng et al., 2025). This, in turn, augments intermolecular electrostatic repulsion, leading to a variation in the spatial structure, an expansion in the intermolecular space, and increasing the amount of free water, which results in an elevation in the water retention of the gel. Nevertheless, when the temperature is elevated further, myofibrillar protein undergoes denaturation, causes a disruption of the ordered network structure between protein molecules, which subsequently reduces water retention. Previous study found that high concentrations of sodium bicarbonate can modify the pH of actinoglobulin, causing the protein molecule to unfold, increasing the degree of sulfhydryl group exposure, and enhancing water-holding properties (Chantarasuwan et al., 2011). You et al. (2024) indicated that the water retention of reduced-salt Chaozhou beef meatball significantly enhanced with the additional of sodium bicarbonate from 0 % to 0.3 %, allowed with the initial relaxation times of T₂₁ and T₂₂ to be shortened, indicating a decrease in water mobility.

Fig. 1.

Effect of sodium bicarbonate and resting temperature combined on water retention of low-sodium chicken myofibrillar protein gel. Each value represents the mean ± SE, n = 3. ^a–d Different Parameter superscripts indicate significant differences (P < 0.05).

3.2. Cooking yield

As showed in Fig. 2, a significant (P < 0.05) variation of cooking yield with increasing resting temperature, reaching a maximum when the resting temperature at 30 °C. Previous studies have manifested that sodium bicarbonate elevates the pH of myofibrillar protein solutions with the rise in temperature, with solubility attaining a maximum when the resting temperature at 30 °C (Cheng et al., 2025; Kang, Yao, et al., 2024). Due to that solubility reached its peak at this temperature, which boosted the ability of proteins to bind water, led to the cooking yield was maximum. It has been shown that the cooking yield responds to the water-holding properties of protein gels, and protein denaturation leads to a decline in the ability to bind water, which in turn leads to a decrease in their cooking yields (Zhu et al., 2023). Kang, Shang, et al. (2022) reported that sodium bicarbonate increased the net negative charge of myofibrillar protein, caused swelling and dissolution of myofibrils, and thereby significantly enhanced the cooking yield of corned beef.

Fig. 2.

Effect of sodium bicarbonate and resting temperature combined on the cooking yield of low-sodium chicken myofibrillar protein. Each value represents the mean ± SE, n = 3. ^a–d Different Parameter superscripts indicate significant differences (P < 0.05).

3.3. Whiteness

As showed in Fig. 3, the whiteness of myofibrillar protein gel decreased significantly and then increased significantly (P < 0.05) as increasing the resting temperature, reaching the lowest value when the resting temperature at 30 °C. The reason is possible that the increase in resting temperature elevates the pH and the electrostatic repulsion between protein molecules, thereby influencing the agglutination behavior of proteins and reducing the lightness (Wu et al., 2024). Wu et al. (2022) revealed that sodium bicarbonate was capable of reducing the whiteness value of PSE meat myofibrillar protein gel. However, the whiteness continued to increase as the resting temperature continued to rise to 50 °C, which might be ascribed to the denaturation of proteins, thereby increasing the whiteness value of the gel. It has been shown that the whiteness is affiliated with the protein denaturation, with a high degree of denaturation resulting in a high whiteness value (Kang, Zhang, Li, Li, et al., 2021).

Fig. 3.

Effect of sodium bicarbonate and resting temperature combined on the whiteness of low-sodium chicken myofibrillar protein gel. Each value represents the mean ± SE, n = 3. ^a–d Different Parameter superscripts indicate significant differences (P < 0.05).

3.4. Gel strength

Gel strength serves as an indicator of the quality of the meat product. A high gel strength value indicates that the protein gel possesses a superior gel network structure. It can be discerned from Fig. 4 that the myofibrillar protein gel strength significantly increased initially and then decreased significantly (P < 0.05) with rising the resting temperature, attaining the highest value when the resting temperature at 30 °C. The results indicated that the gel network structure formed by the protein when the resting temperature at 30 °C was the densest. It has been demonstrated that gels formed under neutral conditions exhibit a more homogeneous structure compared to those induced under acidic conditions. Poor-quality gels were typically caused by the local aggregation of proteins, which formed an inhomogeneous network. Conversely, an increase in pH promoted an increase in electrostatic repulsion and hinders local aggregation (Feng & Hultin, 2001). This evidence revealed that sodium bicarbonate was capable of undergoing decomposition at elevated resting temperatures, which elevated the pH of the protein (Wu et al., 2024). The observed decrease in gel strength when the resting temperature at 30 °C might be attributed to the fact that, at this resting temperature, the protein has already initiated the process of denaturation, which disrupts the structural integrity of the protein and impedes the formation of a compact network during heat-induced gelation (Chen, Zhou, et al., 2020).

Fig. 4.

Effect of sodium bicarbonate and resting temperature combined on the gel strength of low-sodium chicken myofibrillar protein gel. Each value represents the mean ± SE, n = 3. ^a–d Different Parameter superscripts indicate significant differences (P < 0.05).

3.5. Texture properties

The effect of sodium bicarbonate on the texture of myofibrillar protein gel at different resting temperatures is presented in Table 1. It was discovered that the hardness, springiness, cohesion, and chewiness of myofibrillar protein gel changed significantly with the increasing resting temperature (P < 0.05), and the hardness, springiness, and chewiness all reaching their maximum values when the resting temperature at 30 °C, while cohesion reached its minimum value at the same resting temperature. It has been asserted that increased solubility can lead to form a homogeneous and ordered three-dimensional network structure of the gel during heat induction process, which can enhance the springiness and hardness of gels (Xiong et al., 2010). Sodium bicarbonate can solubilize some salt-soluble proteins, thereby increasing the number of sites in the polypeptide chain that are capable of interacting with each other during the heating process, which in turn enables the formation of elastic, stiff and stable protein gels (Cheng et al., 2025). In this study, the decomposition of sodium bicarbonate was accelerated with the rising resting temperature, resulting in an enhanced solubility of proteins. This led to a similar effect on the hardness, springiness, and chewiness of the myofibrillar protein gel, which presented an upward trend up to the resting temperature at 30 °C. However, when the resting temperature was elevated, the solubility of the gel decreased due to the denaturation of the proteins and the alteration of the protein network structure, which led to a reduction in the textural properties of the gel. Li et al. (2014) found that an increase in the number of disulphide bond cross-links and an increase in the water content are both significant factors contributing to an increase in the stiffness of gels.

Table 1.

Effect of sodium bicarbonate and resting temperature combined on the texture properties of low-sodium chicken myofibrillar protein gel.^a–e

Temperature (°C)	Hardness (g)	Springiness	Cohesiveness	Chewiness (g.mm)
4	157.96 ± 7.12cd	0.836 ± 0.003c	0.438 ± 0.017b	75.13 ± 5.16b
10	164.94 ± 4.92c	0.864 ± 0.007b	0.429 ± 0.046bc	79.32 ± 15.48b
20	224.28 ± 9.22b	0.873 ± 0.014b	0.401 ± 0.025bc	89.83 ± 13.11b
30	240.32 ± 4.84a	0.890 ± 0.009a	0.333 ± 0.005d	103.02 ± 20.29a
40	145.44 ± 9.46d	0.870 ± 0.003b	0.380 ± 0.039cd	69.30 ± 17.80c
50	94.39 ± 8.96e	0.803 ± 0.007d	0.521 ± 0.026a	38.73 ± 6.12d

Each value represents the mean ± SE, n = 3.

^a–eDifferent Parameter superscripts indicate significant differences (P < 0.05).

3.6. Chemical force

Myofibrillar protein undergoes significant structural alterations during heat treatment, which can influence the interaction forces between protein molecules (Yang et al., 2021). Fig. 5 shows the effect of sodium bicarbonate on the chemical force of myofibrillar fibrin gels at different resting temperatures. The ionic bonding, hydrogen bonding, hydrophobic interactions, and disulfide bonding of myofibrillar protein gel changed significantly as rising the resting temperature. Among the various chemical forces, both ionic and disulfide bonds reached the lowest values when the resting temperature at 30 °C, while hydrophobic interactions and hydrogen bonds achieved their highest values at the same resting temperature. Hydrophobic interactions and disulphide bonding constitute the majority, suggesting that these two forces play a crucial role in the gel formation process. The decrease in disulphide and ionic bonds might be due to the decomposition of sodium bicarbonate as rising the resting temperature, which raises the pH and enhances the repulsive forces between the molecules, resulting in the partial rupture of the ionic and disulfide bonds (Wu et al., 2024). However, as the resting temperature continued to increase, the proteins undergo denaturation and the molecules started to aggregate, which leading to an increase in the concentration of these substances. It has been shown that proteins exposed hydrophobic groups and sulfhydryl groups during heat-induced gelation, with sulfhydryl groups interconnecting to form disulfide bonds and hydrophobic groups aggregating through hydrophobic interactions, and that these interactions contributed to the formation of gel networks (Liu et al., 2014). Furthermore, it has been hypothesized that at temperatures exceeding 45 °C, non-covalent bonding serves as the primary driving force for protein aggregation. Conversely, at temperatures exceeding 75 °C, disulfide bonding is the predominant mechanism maintaining protein structure (Ko et al., 2007). It can be witnessed that the disulphide bonds show a gradual increase in concentration after the elimination of the influence of pH (Xu et al., 2019).

Fig. 5.

Effect of sodium bicarbonate and resting temperature combined on the chemical force of low-sodium chicken myofibrillar proteins gel. Each value represents the mean ± SE, n = 3. ^a–d Different Parameter superscripts indicate significant differences (P < 0.05).

3.7. Microstructure

As showed in Fig. 6, the surface morphology of myofibrillar protein gel under different conditions showed remarkable differences. More planar were found when the resting at 30 °C, and the network structure was discovered to be more homogeneous and dense compared with the other samples. Following, the gel network became increasingly rough when the resting temperature at 50 °C, with debris being observed on its surface. The regular gel structure was more conducive to the accommodation of water molecules, and the binding area with water was more ideal (Yang et al., 2021). This is in accordance with the results of water retention in this experiment (Fig. 1). It has been indicated that particle size and disulphide bond content play a vital role in the microstructure of myofibrillar protein gel, and that a reduction in particle size and an increase in disulphide bond content can lead to a denser gel structure (Cheng et al., 2025; Wen et al., 2017). Furthermore, low solubility and protein aggregation can cause the gel network structure to become irregular and of poor quality (Guo et al., 2019). Cheng et al.(2025) reported that as the temperature ascends, the net negative charge of the protein increases, the intermolecular repulsion intensifies, its solubility elevates, and the particle size decreases. Consequently, the network structure of the protein gel is the densest when the resting temperature at 30 °C. However, when the resting temperature at 50 °C, the protein undergoes denaturation and its structure is disrupted. This leads to a deterioration of the gel state.

Fig. 6.

Effect of sodium bicarbonate and resting temperature combined on the microstructure of low-sodium chicken myofibrillar proteins gel.

4. Conclusion

The results demonstrated that myofibrillar protein held at a resting temperature of 30 °C exhibited the highest water retention, cooking yield, gel strength, and most favorable microstructure, indicating an enhanced capacity to form a stable gel under these conditions. This improvement can be attributed to the thermal decomposition of sodium bicarbonate at elevated temperatures, which gradually increases the pH of the protein solution. The elevated pH enhances the net negative charge on protein molecules, strengthens electrostatic repulsion, and thereby improves protein solubility. As a result, the proteins are able to bind more water, leading to improved water retention and cooking yield. Moreover, increased hydrophobic interactions and hydrogen bonding contribute to the formation of a uniform and dense gel network, resulting in maximum hardness, springiness, and chewiness at 30 °C. Microstructural analysis confirmed the presence of a more compact protein matrix at this temperature. In contrast, when the resting temperature was raised to 50 °C, protein denaturation occurred, disrupting the gel network and impairing gel quality. Therefore, a resting temperature of 30 °C was determined to be optimal for gel formation in this study.

CRediT authorship contribution statement

Tao Zhang: Visualization, Supervision, Resources, Investigation, Formal analysis. Yan-Ping Li: Visualization, Supervision, Software, Methodology, Formal analysis, Data curation. Chen-Lu Xu: Writing – original draft, Supervision, Software, Resources, Methodology, Investigation, Funding acquisition, Data curation. Peng-Lei Yao: Writing – original draft, Visualization, Software, Resources, Investigation, Formal analysis. Xue-Hua Zhang: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Investigation. Zhuang-Li Kang: Writing – original draft, Validation, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that there are no financial interests or personal relationships that will influence the work reported in this paper.

Data availability

The data that has been used is confidential.