Unravelling the Deterioration Mechanism of the Coated Tofu Gel During Cold Storage: The Role of Protein Oxidation

TL;DR

This study explores how cold storage affects the quality of coated tofu by examining protein changes that lead to spoilage.

Contribution

The study reveals how protein oxidation and structural changes during cold storage correlate with quality deterioration in coated tofu.

Findings

Lower storage temperature (4°C) significantly slows protein oxidation and quality loss in coated tofu.

Protein structural changes, including loss of α-helices and β-sheets, correlate with textural and color deterioration.

Oxidative denaturation and structural reaggregation of proteins are key drivers of coated tofu spoilage during cold storage.

Abstract

Coated tofu is prone to spoilage and degradation during processing, storage, and transportation. As the material basis for gel of coated tofu, proteins determine coated tofu’s unique qualities, such as its colour, flavour, and texture. This study aimed to investigate the changes in the quality of coated tofu and the physicochemical properties of its proteins during cold storage (4 °C and 10 °C, 14 days), as well as the intrinsic correlations between these variables. Quality deterioration and protein structural changes were significantly slower at 4 °C than at 10 °C, with lower temperature effectively delaying quality loss. The results indicated that as storage time increased, the freshness of coated tofu declined, its textural properties significantly deteriorated, and the protein gel network structure became impaired. Meanwhile, the proteins underwent significant oxidative denaturation, characterized by a decrease in the free thiol group content and an increase in surface hydrophobicity. The tertiary structure exhibited unfolding and disruption, while the secondary structure transitioned from an ordered to a disordered state. Specifically, the contents of α-helixes and β-sheets decreased significantly, reaching 34.96% and 8.68%, respectively, after 14 days of storage at 4 °C. In contrast, the contents of β-turns and random coils increased to 30.11% and 26.25%, respectively, under the same storage conditions. The subunit bands of the 11S and 7S proteins gradually weakened, and the protein structure tended to loosen. Correlation analysis revealed that the oxidative denaturation, structural depolymerization, and reaggregation of proteins were highly significantly correlated with the textural breakdown and colour deterioration of coated tofu, which together contributed to the quality degradation of coated tofu during cold storage. The findings of this study provide fundamental data and technical support for the development of cold storage methods for coated tofu.

Figures

Full text

1. Introduction

As a traditional soy protein gel, tofu owes its key properties to a three-dimensional network formed by soy protein aggregation during processing, which retains abundant moisture and nutrients and forms the foundation of its distinct texture and water-holding capacity [1]. The production of tofu mainly consists of key steps including soybean soaking, grinding, filtering, coagulant addition, and moulding [2]. Processing parameters influence the formation, density, and pore size of the protein network, which in turn determine the quality attributes of tofu gels, such as texture and stability [3]. For instance, coated tofu, a specialty snack originating from the southwest region of China, is highly favoured by consumers for its unique texture characterised by crispy skin and a liquid inside after heating [4]. This distinct texture is primarily attributed to the alkaline treatment introduced during its processing, which clearly differentiates it from traditional tofu. Alkaline treatment can not only cause changes in the structure of protein networks [5], but also plays an indispensable role in the subsequent gel-sol phase transition of heat-induced soy proteins [6]. Moreover, it prompts deep hydrolysis of proteins and fats, generating small molecule peptides, free amino acids (such as glutamic acid and aspartic acid, which give an umami taste), and fatty acids [4,7], thereby enhancing coated tofu’s unique nutrition and flavour, and making it alkaline (pH > 8).

As the main structural component of tofu gel, soybean protein directly dictates the three-dimensional structural characteristics of the system through its state (including conformation, solubility, aggregation state and cross-linking pattern), which in turn modulates the physicochemical properties and sensory quality of the product, encompassing texture (hardness, springiness, chewiness), flavor and nutritional attributes [8,9,10]. Many studies have shown that quality deterioration in high-protein plant-based foods during storage is often closely related to protein oxidation, which can lead to molecular changes such as protein side chain modification, disulfide bond rearrangement, non-disulfide covalent cross-linking, and peptide chain cleavage [11]. These changes directly disrupt the gel network structure. Peptide chain cleavage reduces cross-links and leads to a loose gel network, or alternatively, induces abnormal protein aggregation that enlarges pores and prevents effective moisture retention. For example, the decrease in hardness, reduced water-holding capacity, and the development of an off-flavour of soybean products under cold storage are largely associated with protein oxidation, denaturation, and aggregation [12]. A similar phenomenon is observed in thousand-layer tofu, whose degraded texture after storage correlates with changes in the protein tertiary structure, decreased molecular flexibility, and the formation of insoluble aggregates [13]. These findings indicate that examining molecular changes in protein oxidation, degradation, and aggregation can help elucidate the mechanisms underlying the quality deterioration of high-protein gel-based foods.

It is worth noting that alkaline treatment may significantly alter the initial state of proteins in coated tofu, such as their charge distribution, solubility, and conformation, as compared to ordinary tofu; this will affect the pathways and rates of oxidation and degradation under refrigeration conditions [14,15]. Consequently, while alkaline treatment contributes to the uniqueness of coated tofu, the resulting higher pH may also make it more sensitive to temperature and time during storage, transportation, and sale. Compared to other high-moisture, high-protein foods (e.g., meat, conventional tofu), coated tofu may be more susceptible to irreversible quality deterioration, such as souring, sliminess, and softening, thereby limiting its shelf life and marketability [16]. At present, relatively few studies have examined the mechanism of protein structure changes and quality deterioration during the refrigeration process of coated tofu, a food with high moisture and protein contents and a high pH. This has led to an inadequate theoretical basis for related preservation technologies, restricting the further promotion of this product. Therefore, investigating the changes in coated tofu protein during cold storage and clarifying its intrinsic relationship with gel network structure and quality deterioration is of great significance for elucidating the mechanism of quality deterioration and developing corresponding preservation strategies.

To this end, this study focused on the changes in the physicochemical properties, freshness, texture characteristics, microstructure, and protein physicochemical properties of coated tofu during storage (14 days) under the common temperature conditions of a refrigerator compartment (4 °C) and supermarket display cabinet (10 °C). Pearson correlation analysis was used to comprehensively explore the associations between protein oxidation and degradation indicators and key quality attributes of coated tofu, helping to clarify the molecular mechanism underlying gel network structure damage and quality deterioration of coated tofu mediated by protein oxidation, in order to provide a solid theoretical basis for the cold storage, shelf-life prediction, and quality monitoring of coated tofu. Ultimately, this study aimed to contribute to the further development of a broader market for this unique product.

2. Results and Discussion

2.1. Changes in the Freshness of Coated Tofu During Cold Storage

Since the key step in producing coated tofu is the alkali soaking treatment, the initial pH of coated tofu was 8.21 ± 0.02 (Figure 1A). The samples stored at 4 °C and 10 °C reached the lowest pH values on the 12th and 8th day, at 7.50 ± 0.05 and 7.21 ± 0.05, respectively. Subsequently, a significant increase in the pH values of all samples was observed (p < 0.05). However, throughout the entire cold storage process, the coated tofu remained alkaline (pH value > 7.00). The decrease in the pH value in the early stage of storage may be due to the acidic metabolic products of low-temperature-resistant acid-producing microorganisms [17]. In the later stage, the presence of acid-resistant spoilage microorganisms has been observed to result in the decomposition of proteins, leading to the production of alkaline substances such as ammonia and amines. This process has been shown to not only increase the pH level but also to contribute significantly to the escalation in the total volatile base nitrogen (TVB-N) value [18], as shown in Figure 1B. Specifically, after storage at 4 °C for 14 days, the TVB-N value of the coated tofu gradually increased from 2.74 ± 0.59 mg/100 g on day 0 to 13.68 ± 0.91 mg/100 g on day 14, and even reached 18.55 ± 0.53 mg/100 g at 10 °C storage. The maximum increase in the TVB-N values between adjacent time points occurred on the 10th–12th day of storage at 4 °C and the 6th–8th day of storage at 10 °C. TVB-N values exhibit a positive correlation with the degree of sample spoilage. However, on the 14th day of storage, regardless of the storage temperature, the coated tofu began to show varying degrees of off-flavour; other spoilage phenomena were not observed.

The thiobarbituric acid reaction substrate (TBARS) value is an indicator of lipid oxidation. An increase in the TBARS value can reflect the development of an unpleasant odour of coated tofu, shortening its shelf life. The TBARS values of coated tofu at the different storage temperatures all increased with increases in the storage time (Figure 1C), from 0.53 mg/kg on day 0 to 1.72 ± 0.08 mg/kg (at 4 °C) and 2.28 ± 0.07 mg/kg (at 10 °C) after 14 days of storage, respectively. The maximum increase in the TBARS values between adjacent time points occurred on the 10th–12th days of storage at 4 °C and the 6th–8th days of storage at 10 °C. Huang et al. [17] reported similar results in their study of the changes in the quality of snack tofu at different temperatures. The higher the storage temperature, the more prone lipids are to oxidation. This might be because high temperatures increase the secretion of lipase by microorganisms, accelerating the decomposition of hydrogen peroxides, and altering the oxidizability of food substrates. At the same time, protein oxidation promotes lipid peroxidation.

Colour is a key factor in the sensory characteristics and quality of coated tofu and is measured by the brightness value (L*), redness value (a*), and yellowness value (b*). The lower the L* value, the lower the whiteness, and the poorer the freshness and quality. The initial L* of fresh coated tofu was 86.45, a* was −0.5, and b* was 18.06 (Table 1). During storage, L* decreased significantly, while a* and b* gradually increased. The colour of the samples tended to be more yellow and red. This might be due to the oxidation of lipids and proteins, which leads to a decrease in light reflectivity [18], consistent with the TBARS results.

Overall, the freshness-related indicators changed relatively slowly between days 0–6/8 days (10 °C) and 0–10/12 days (4 °C) of cold storage. Then, with further increases in the storage time, the freshness decreased significantly. The key time points for freshness changes in coated tofu were the 6th to the 8th days (at 10 °C) and the 10th to the 12th days (at 4 °C) during cold storage.

2.2. Changes in the Texture Characteristics and Microstructure of Coated Tofu During Cold Storage

The WHC of coated tofu is related to the uniformity and strength of the gel network structure formed by proteins [19]. The water-holding capacity of coated tofu decreased over time at the different storage temperatures (Table 1). At 4 °C, the water-holding capacity slowly decreased from 86.50% to 82.51% in the preliminary phase of storage. (0–8 days). The decrease was most significant from the 12th day compared to the 10th day (p < 0.05) and dropped to 75.47% by the 14th day. After storage at 10 °C for 14 days, the water-holding capacity decreased by 19.23%. In particular, the water-holding capacity of tofu decreased by 4.52% on the 8th day compared with that on the 6th day. This might be due to the oxidative degradation of proteins, which damages the gel structure, increasing the number of pores and reducing the physically bound water [20]. A slightly higher storage temperature caused more severe damage to the gel network and a more significant decrease in the WHC.

Cold storage altered the textural properties of coated tofu, including its hardness, springiness, and chewiness (Table 1). Compared to day 0 (81.59 ± 1.38 g), the hardness of tofu decreased with increasing storage time. Coated tofu stored at 4 °C reached its lowest hardness value on day 14 (63.78 ± 0.47 g), while that stored at 10 °C showed a similar level as early as day 10 (62.07 ± 3.56 g). The chewiness and springiness followed similar trends. Unlike other types of tofu, which become harder and chewier in the early storage period due to dehydration [21,22], coated tofu did not exhibit this during cold storage. This might be because tofu gel, made with fermented soybean whey as the coagulant, has a randomly aggregated gel network structure, and thus, its texture is relatively softer [23,24], while the hardness of tofu after alkaline treatment is lower [14]. Thus, during storage, the disruption of the gel network likely has a greater effect on coated tofu than dehydration does.

To further observe the differences in the microstructure of the gel network of coated tofu at different temperatures, scanning electron microscope (SEM) images (Figure 2A,B) and corresponding binarized images (Figure 2C) of fresh samples on day 0 and samples on days 4, 8, and 12 stored at 4 °C and 10 °C, respectively, were analysed. The average pore size of the fresh samples on day 0 was 12.49 ± 2.87 μm, and the maximum pore size was 17.3 μm. The average pore sizes of the gel network structures of the coated tofu on day 14 after storage at 4 °C and 10 °C were 56.22 ± 12.47 μm and 84.85 ± 21.22 μm, respectively. The maximum apertures were 76.09 μm and 132.99 μm, respectively. The gel network of fresh coated tofu appeared relatively dense and uniformly distributed. With prolonged storage, however, the structural uniformity was disrupted, leading to the formation of larger voids. Notably, higher storage temperatures resulted in more pronounced pore enlargement. A denser and more uniform network structure is known to retain more moisture and to exhibit higher hardness and chewiness [25,26]. In contrast, when the proteins, the primary structural components of the network, undergo oxidation, the resulting damage impairs the gel matrix, leading to deterioration in both the water-holding capacity and texture. Thus, these microstructural observations help explain the decline in the water-holding capacity and texture of coated tofu during cold storage.

2.3. Changes in the Protein Properties of Coated Tofu During Cold Storage

2.3.1. Protein Solubility and Free Amino Acids (FAAs)

As a key component constituting the gel structure of tofu, changes in proteins are a critical factor affecting its quality. Therefore, it is necessary to investigate protein oxidation during storage. Both the protein solubility and total FAA content reflect the extent of protein degradation and denaturation [27]. Under storage at 4 °C and 10 °C, the soluble protein content of coated tofu showed an initial increase followed by a decrease. At 4 °C solubility reached its maximum of 1.90 ± 0.03 mg/mL on day 10, then declined; at 10 °C, it increased to 2.01 ± 0.02 mg/mL by day 6 before gradually decreasing (Figure 3A). Meanwhile, the FAA content exhibited a similar trend, first increasing and then decreasing (Figure 3B). Under 4 °C storage, it gradually rose from an initial value of 256.94 ± 15.11 μg/mL to 476.57 ± 4.91 μg/mL on day 12, before declining, whereas at 10 °C, it reached a maximum of 545.95 ± 5.77 μg/mL on day 8, then decreased. In the early refrigeration stage, hydrolysis by endogenous proteases and microbial extracellular enzymes, along with cold-induced protein unfolding [28], collectively contribute to the simultaneous increase in protein solubility and FAA content. As cold storage progresses, protein oxidation and degradation intensify, leading to the formation of insoluble aggregates and a consequent decrease in protein solubility [11,29]. Concurrently, FAAs are increasingly converted via deamination, decarboxylation, and other reactions into spoilage products, with their consumption rates exceeding their production rates, resulting in reductions in their contents [30]. These changes indicate that during refrigeration, coated tofu undergoes a dynamic shift from a protein degradation-dominated phase to one dominated by oxidative aggregation and amino acid conversion. This process occurs more rapidly at higher storage temperatures (10 °C), highlighting the significant influence of storage temperature on the pathway and rate of protein degradation.

2.3.2. Surface Hydrophobicity and Free Sulfhydryl Groups

The gel structure of tofu is formed through hydrophobic interactions and disulfide bonds, making changes in surface hydrophobic regions and free sulfhydryl groups valuable indicators of alterations in the protein gel network [31]. Surface hydrophobicity reflects the relative content of hydrophobic groups on the protein molecular surface and is often associated with protein oxidation and denaturation [2]. As shown in Figure 4A, the surface hydrophobicity of coated tofu protein increased significantly (p < 0.05) during storage under both temperature conditions. At 4 °C and 10 °C, it rose from an initial value of 56.99 to 136.02 and 175.94, respectively, indicating gradual exposure of hydrophobic regions. Concurrently, Figure 4B shows that the content of free mercapto groups decreased from 2.12 ± 0.02 μmol/g (0 day) to 1.18 ± 0.03 μmol/g (4 °C, 14 days) and 0.80 ± 0.05 μmol/g (10 °C, 14 days). The more pronounced increase in surface hydrophobicity and greater decline in sulfhydryl groups at 10 °C suggest that higher storage temperatures accelerate protein unfolding and oxidation. Guo, Hu, Wang, and Liu [32] proposed that higher temperatures favour protein unfolding, thereby facilitating the exposure of hydrophobic regions. The decrease in the free sulfhydryl content is primarily attributed to the formation of disulfide cross-links via oxidation and the generation of irreversible oxidation products such as sulfenic and sulfonic acids [33]. The negative correlation between these two parameters (r = −0.993, p < 0.001) indicates that during cold storage, protein oxidation in coated tofu leads to the participation of free sulfhydryls in cross-linking, resulting in protein unfolding and exposure of internal hydrophobic regions. The exposed hydrophobic groups, in turn, promote molecular aggregation and further oxidation of sulfhydryl groups, ultimately driving the transition of soluble proteins into insoluble aggregates.

2.3.3. Fourier Transform Infrared Spectroscopy (FT-IR) and Endogenous Fluorescence Spectroscopy

FT-IR spectroscopy detects changes in molecular vibration–rotation energy levels, thereby reflecting alterations in the protein secondary structure and spatial distribution. The amide I band (1700–1600 cm^−1^) primarily originates from the C=O stretching vibration of the peptide bond (Figure 5A,B). After deconvolution and second-derivative processing, the peaks in the ranges of 1600–1640 cm^−1^ (β-sheet), 1640–1650 cm^−1^ (random coil), 1650–1660 cm^−1^ (α-helix), and 1660–1700 cm^−1^ (β-turn), respectively [34]. As storage time increased, the relative contents of ordered secondary structural components (α-helixes and β-sheets) decreased, while disordered components (β-turns and random coils) increased significantly (Figure 5C,D). Under 4 °C storage, the contents of α-helixes and β-sheets decreased by 5.39% and 10.91%, respectively, whereas β-turns and random coils increased by 9.69% and 6.61%. This trend was more pronounced at 10 °C, indicating that higher temperatures accelerate the transition from ordered to disordered protein structures. This shift mainly results from the disruption of hydrogen bonding networks due to oxidation and other factors, leading to conformational rearrangements of polypeptide chains [35].

Meanwhile, fluorescence spectroscopy (Figure 5E,F) revealed a gradual decrease in fluorescence intensity under both storage temperatures, accompanied by a red shift in the maximum emission wavelength (λmax). The fluorescence quenching intensity at 4 °C and 10 °C increased from 79.97 a.u. on 0 day, then dropped to 48.45 a.u. and 43.06 a.u. on the 14th day, respectively. At 4 °C, λmax shifted from an initial 350 nm to 358 nm, while at 10 °C, it shifted to 367 nm. The decrease in fluorescence intensity, together with the red shift of λmax, indicates that originally buried tryptophan residues gradually became exposed to the polar aqueous environment, resulting in fluorescence quenching [4,36,37]. This change further confirms the unfolding and disruption of the protein tertiary structure. In conclusion, the increase in the disorder of the secondary structure disrupts the local hydrogen bonds and spatial conformations of proteins, thereby promoting the development of the tertiary structure and the exposure of the hydrophobic domain. The collapse of the tertiary structure further accelerates the disordered rearrangement of peptide chains and the process of molecular aggregation. These results are consistent with the previously observed increase in surface hydrophobicity and decrease in the free sulfhydryl content during the cold storage of coated tofu, further demonstrating the transition of proteins from an ordered native structure toward a disordered aggregated state during storage.

2.3.4. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

The changes in the primary structure of proteins and the formation of protein aggregates can be determined through SDS-PAGE. Figure 6 shows the subunit composition of fresh coated tofu, including α (67 kDa), α’ (71 kDa), and β (50 kDa) of the 7S protein and acidic subunit A (35 kDa) and basic subunit B (20 kDa) of the 11S protein [36,37,38]. With increases in the storage time, all subunit bands of the proteins at 7S and 11S gradually weakened under the different storage temperatures, and the weakening rate of the protein subunit bands at 10 °C was faster than that at 4 °C. At 4 °C, both the A and B subunits of the 11S protein began to weaken significantly from the 12th day. At 10 °C, significant weakening was observed until the 8th day, and the bands basically disappeared by the 14th day, indicating that a higher storage temperature will accelerate the oxidative degradation of proteins. The weakening of protein bands can be attributed to two effects: one is covalent bond breakage and the structural damage directly caused by protein oxidation [25]; the second is enzymatic hydrolysis triggered by endogenous proteases secreted by microbial reproduction [39]. In addition, aggregate bands appeared at the top of the separation gel, and the colour of these aggregate bands deepened over time. This indicates that protein oxidation promotes the formation of intermolecular disulfide bonds and further protein aggregation, inducing the formation of insoluble aggregates, which thus cannot enter the electrophoresis gel [40]. Kong and Chang [41] also observed a similar phenomenon in their study of protein structure changes during the storage of soy protein.

Lee et al. [25] argued that protein bands and subunits are related to the quality characteristics of tofu, including its texture, water-holding capacity, and gel properties, and the changes in coated tofu during cold storage are consistent with this. There are two free thiol groups on the 11S protein [42]. With increases in the storage time, the 11S protein decreased, which is also consistent with the decrease in the content of free thiol groups in coated tofu. The 7S protein is related to the water-holding capacity of the gel network [43]. There are also reports indicating that 11S gel has a higher WHC and higher hardness [42].

2.3.5. Pearson Correlation Analysis

To further explore the mechanism of quality deterioration of coated tofu during cold storage, Pearson correlation analysis was conducted on protein indicators (protein solubility, FAAs, surface hydrophobicity, free thiol groups, α-helix, β-sheet, β-turn, and random coil) and macroscopic quality indicators (pH, volatile basic nitrogen, malondialdehyde, L* value, a* value, b* value, water-holding capacity, hardness, elasticity and chewiness). As shown in Figure 7, the pH value of coated tofu was only significantly related to the protein solubility (r = −0.844, p < 0.01) and the total amount of FAAs (r = −0.892, p < 0.01). Microbial reproduction produces organic acids, reducing the pH value. Meanwhile, proteases break down macromolecular proteins, which not only enhances protein solubility but also increases the total amount of FAA [44]. Microorganisms utilize FAAs to metabolize and produce ammonia, causing the pH value to rise. At this time, amino acids are consumed in large quantities, and proteins denature and aggregate due to long-term refrigeration, resulting in a decrease in solubility [45]. FAAs, as the products of protein decomposition, not only serve as intermediate carriers for quality deterioration, but here, they also exhibited a significant positive correlation with the spoilage index TVB-N (r = 0.741, p < 0.05). The accumulation of FAAs provides nitrogen sources for microorganisms, accelerating ammonia production metabolism, and promoting spoilage.

Furthermore, both TVB-N and TBARS were extremely significantly negatively correlated with ordered secondary structures (α-helix: r = −0.916, p < 0.01; r = −0.929, p < 0.01/β-sheet: r = −0.923, p < 0.01; r = −0.925, p < 0.01) and free thiol groups (r = −0.981, p < 0.001; r = −0.989, p < 0.001) and exhibited extremely significant positive correlations with disordered structures (β-turn: r = 0.816, p < 0.05; r = 0.837, p < 0.01/random coil: r = 0.956, p < 0.001; r = 0.946, p < 0.001) and surface hydrophobicity (r = 0.988, p < 0.001; r = 0.994, p < 0.001). This indicates that the disintegration of the ordered structure of proteins and the exposure of hydrophobic regions accelerated the degradation of proteases, leading to the accumulation of TVB-N. On the other hand, this provided more oxidation interfaces for lipids, promoting an increase in the TBARS content. The a* value and b* value were positively correlated with disordered structures and surface hydrophobicity. This may be related to the transformation of the protein secondary structure to disordered conformations such as β-turns/random coils. These structural changes expose more hydrophobic groups, resulting in enhanced surface hydrophobicity and subsequently triggering protein aggregation or oxidation reactions [13,46], eventually leading to a change in colour. Conversely, the maintenance of the water-holding capacity and texture characteristics (hardness, elasticity) was highly dependent on free thiol groups and ordered secondary structures (such as β-sheets), highlighting the decisive role of the complete gel network in moisture retention and the maintenance of quality characteristics. Similar results have been found in other studies [36,37].

Based on the above analysis, the quality deterioration of coated tofu during cold storage is essentially a synergistic deterioration process that is driven by microbial action, with protein structure disorder as the key factor, ultimately leading to lipid oxidation and protein spoilage. Therefore, controlling the denaturation and hydrolysis rate of proteins and maintaining their ordered secondary structure and the integrity of the gel network are key to extending the shelf life of coated tofu and maintaining its quality.

3. Conclusions

This study revealed the intrinsic correlations and mechanisms between quality deterioration and protein oxidative degradation of coated tofu during storage at 4 °C and 10 °C for 14 days. Prolonged storage significantly increased the TVB-N and TBARS values, while the water-holding capacity and texture properties (hardness, springiness, chewiness) decreased markedly, indicating reduced freshness, a damaged protein gel network, and gradual quality deterioration. Freshness was stable initially (0–8 d at 10 °C, 0–12 d at 4 °C) but declined rapidly after days 6–8 (10 °C) and 10–12 (4 °C), with faster and more severe deterioration at 10 °C. Meanwhile, proteins underwent significant oxidative denaturation, the free sulfhydryl content decreased, the surface hydrophobicity increased, the secondary structure shifted from ordered α-helixes/β-sheets to disordered β-turns/random coils, and all protein subunit bands weakened, resulting in a loose structure. Pearson correlation analysis showed strong correlations between protein oxidation and quality indicators, confirming protein oxidation close association with lipid oxidation, colour change, and texture deterioration, and verifying that protein structural changes are key to quality loss. Thus, controlling microbial activity, slowing protein denaturation/hydrolysis, and maintaining an ordered secondary structure and gel network integrity are key to extending the shelf life of coated tofu and maintaining its quality.

4. Materials and Methods

4.1. Materials

This study used commercially available soybeans from Anhui Province. The BCA protein Assay Kit and Bradford protein assay Kit were purchased from BIOISCO Biotechnology Co., Ltd. (Lianyungang, China). The reagents used for electrophoresis were purchased from Shanghai Beyotime Biotech Inc. All other chemicals were analytical grade and bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

4.2. Preparation of Coated Tofu

The preparation of coated tofu was performed in the Key Laboratory of Soybean Processing and Safety of Hunan Province (Shaoyang, China), with reference to the method described in Xie et al. [4]. The detailed process is shown in Figure 8. In short, after cleaning and removing impurities from the soybeans, they were soaked for 8 to 12 h. Then, the soybeans were placed in the integrated pulping equipment (SJJ-20, Cooked pulp integrated machine, Kangdeli Intelligent Technology Co., Ltd., Haining, Zhejiang, China) at a soybean water ratio of 1:8 (w/v) for grinding, boiling of soy milk, and filtering, to obtain cooked soy milk. The soy milk was heated to 85 °C, and the soybean clear fermentation liquid provided by the laboratory (fermented soybean whey–soy milk = 1:4, v/v) was added and stirred evenly. After standing for 15 min, the solidified soy milk gel was crushed and then poured into a mould to be pressed into shape (with a pressure of 0.5 Mpa). The formed tofu was cut into blocks (3 cm × 3 cm × 1.5 cm) and left to stand at 4 °C for 8 h. Finally, the formed tofu was soaked in 2% sodium bicarbonate solution (containing 0.8% sodium chloride, 1:2, w/v, 25 °C) for 10 h. Subsequently, the surface moisture was drained from the tofu, and the tofu was put in sealed boxes and stored in refrigerators at 4 °C (refrigerator temperature) or 10 °C (supermarket display cabinet temperature). Samples were taken every 2 days for testing.

4.3. pH Value

According to the method described in Huang et al. [17], a whole block of coated tofu was selected, crushed, and weighed to 5.0 g. Subsequently, 45 mL of distilled water should be added to the sample, and the mixture should be thoroughly mixed until a uniform suspension is obtained. The pH value of the supernatant after homogenization was measured using a digital pH meter (S-3C, Shanghai LIDA Instrument Factory, Shanghai, China).

4.4. Total Volatile Basic Nitrogen (TVB-N)

The detection of TVB-N was based on the method described in Kang et al. [18]. 10.0 g of the mashed coated tofu was obtained and added to 100 mL of distilled water. It was then homogenised, left for 30 min, filtered through double-layer filter paper, and 10 mL of the filtrate was obtained. Then, 10 mL of the filtrate was obtained and 5 mL of magnesium oxide (10 g/L) added. A Kjeldahl nitrogen analyser (UDK139, Beijing Yingsheng Hengtai Technology Co., Ltd., Beijing, China) was used for distillation. The distillate was collected and titrated with 0.01 mol/L sulfuric acid solution. The TVB-N content was expressed as mg/100 g.

4.5. Thiobarbituric Acid Content (TBARS)

Determination of the TBARS content was based on the method described in Huang et al. [17], with slight modifications. First, 5.0 g of mashed coated tofu was obtained, and 50 mL of trichloroacetic acid (TCA) mixture (containing 0.1% EDTA-Na2) was added. The sample was then shaken at 50 °C for 30 min. After cooling, it was filtered with double-layer filter paper. Then, 5 mL of filtrate was obtained and 5 mL of a 15% TCA + 0.375% TBA mixture containing 0.25 M HCl was added. The 90 °C water bath was used for the reaction for 30 min. The cooled solution’s absorbancy was measured at 532 nm using a spectrophotometer (UV-1240, Shimadzu, Tokyo, Japan).

4.6. Colour Analysis

Colorimetric analysis was performed on coated tofu samples using the CIE L* a* b* method (CR-400 colorimeter, Konica Minolta, Tokyo, Japan). The L*, a*, and b* values of each sample were measured at three different positions. Prior to use, the colorimeter was calibrated using a standard whiteboard.

4.7. Water Holding Capacity (WHC)

The WHC was determined by referring to the method outlined in Gao et al. [36], with minor adjustments. 3 g of the coated tofu sample was obtained from the centre of the tofu and placed in a centrifuge tube with absorbent cotton. It was then counted at 4000 g and 4 °C for 15 min, before being weighed to obtain the mass. The WHC was then calculated using Equation (1):

$$[eqn]$$

4.8. Textural Attributes

The determination of the texture was performed with reference to Lee et al. [25], with modifications, involved cutting the central part of the tofu into 1.0 cm × 1.0 cm × 1.5 cm pieces. The alterations in the texture of the coated tofu during storage were ascertained by means of a texture analyser (LS-5, AMETEK, Inc., Berwyn, IL, USA). A P35 cylindrical flat-bottom probe was selected. The TPA mode of the texture analyser was selected, with a compression amount of 40% and a 5 s intermediate stay. The trigger force was 0.05 N. The hardness, springness, and chewiness of the samples were tested; five repeated measurements were performed for each sample.

4.9. Microstructure

SEM was performed according to the method described in Li et al. [47], with slight modifications. The coated tofu samples were cut into cubes with a side length of about 3 mm. They were fixed with 2.5% glutaraldehyde solution for 24 h. After fixation, they were rinsed 5 to 10 times with PBS buffer (0.1 mol/L, pH 7.2) and then subjected to gradient dehydration with ethanol concentrations of 30%, 50%, 70%, 90%, and 100% for 10 min each. The samples were then subjected to freeze-drying for a period of 15 h. Prior to observation, gold was sprayed onto each sample for a duration of 45 s. The samples’ morphology was subsequently subjected to scanning electron microscope analysis (TESCAN MIRA LMS, Brno, Czech Republic).

4.10. Soluble Protein Content

The soluble protein content was measured with reference to Guo et al. [11], with slight modifications. After crushing the whole block of coated tofu and weighing 3 g, and 27 mL of PBS buffer (0.1 mol/L, pH 8.0) was added, the centrifugation was then used at 4000 g for 20 min. The bicinchoninic acid (BCA) assay was used to determine the protein content, with bovine serum albumin as the standard.

4.11. The Free Amino Acids (FAAs)

The FAAs were determined according to the method described by Li et al. [1], with slight modifications. A 2 g sample of coated tofu was crushed and boiled in 50 mL of distilled water for 15 min. The extract was then filtered and diluted to a final volume of 100 mL. An aliquot (400 μL) of the filtrate was mixed with 3.6 mL of deionized water, 1 mL of 2% ninhydrin solution, and PBS (0.1 mol/L, pH 8.0). The mixture was heated in a boiling water bath for 15 min and then cooled to room temperature. After cooling, the volume was adjusted to 15 mL with distilled water, and the absorbance was measured at 570 nm using a UV–Vis spectrophotometer (UV-1240, Shimadzu, Tokyo, Japan).

4.12. Surface Hydrophobicity

The surface hydrophobicity was measured with reference to the method described in Li et al. [27], with modifications. First, 0.1 g of freeze-dried coated tofu sample was dissolved in 10 mL of PBS buffer (0.01 mol/L, pH 8.0), mixed at 25 °C for 1 h, and then placed at 7200 g for 10 min in a centrifuge. Fluorescence intensity of the samples was measured using the 8-Aniline-1-naphthalenesulfonic acid (ANS) probe method on a G9800A fluorescence spectrophotometer (Cary Eclipse, Agilent, Santa Clara, USA). Excitation wavelength (λ_ex_) 370 nm, Emission wavelength (λ_ex_) 490 nm, Slit width 5 nm.

4.13. Free Sulfhydryl Groups

The free sulfhydryl groups were determined according to the method described in Huang et al. [31], with slight modifications. The whole block of coated tofu was crushed and freeze-dried. Then, a 0.1 g sample of the freeze-dried coated tofu was obtained, and 10 mL of Tris-Glycine buffer (containing 10.4 g/L Tris, 6.9 g/L Glycine, 1.2 g/L EDTA, pH 8.0) was added. It was then homogenized and centrifuged at 12,000× g (4 °C, 15 min). Next, the samples were stored in the dark for a period of 15 min, after which the absorbances were measured at a wavelength of 412 nm using a spectrophotometer (UV-1240, Shimadzu, Tokyo, Japan).

4.14. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

This analysis was performed with reference to the method described in Gao et al. [36], with slight modifications. First, the appropriate quantity of freeze-dried coated tofu was obtained, following which it was then mixed evenly with KBr at a ratio of 1:50. Then, the powder was pressed into a thin sheet with a thickness of 1 mm. The analysis of each piece was conducted by means of Fourier transform infrared spectroscopy (Nicolet iS5, Thermo Fisher Scientific, Waltham, MA, USA) within the range of 4000–500 cm^−1^.

4.15. Fluorescence Spectrometry

The coated tofu’s fluorescence intensity was measured using a spectrophotometer (G9800A, Cary Eclipse, Agilent, Santa Clara, CA, USA), following the method in Yu et al. [37]. The excitation wavelength was set at 295 nm, the emission wavelength range was 320–400 nm, and the slit width was 5 nm.

4.16. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed as described in Liu et al. [22], with modifications. 1 g of coated tofu sample was dissolved in 9 mL of 5% (w/v) SDS solution. The mixture was incubated at 85 °C for 1 h before being centrifuged at 7200× g for 10 min at 4 °C. The mixture was then diluted to 2 mg/mL. 40 μL of this diluted sample was added to 10 μL of 5× protein loading buffer, heated in boiling water for 5 min, cooled, and centrifuged (10,000× g, 5 min). The gel sample was then loaded onto the electrophoresis system (5% polymerisation gel + 10% separation gel) for electrophoresis. Electrophoresis apparatus (DYCZ-24DN, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) conditions: 80 V was applied initially to the concentrating gel. When the current reached the separating gel, we increased the voltage to 120 V and maintained it until the completion of electrophoresis. The gel was subjected to staining with Coomassie Brilliant Blue R-250 staining solution for 45 min. Decolorization was performed until there was no colour in the gel blank area.

4.17. Statistical Analysis

All experiments were performed at least three times. Data are presented as means ± standard deviations, and probability values of p < 0.05 were considered statistically significant. One-way analysis of variance was used to determine the significance of any differences between means, followed by Duncan’s test. SPSS version 25.0 (SPSS Inc., Chicago, IL, USA) was used for all analyses.