Alkali-induced aggregation behaviour of tea polyphenols and its gel strengthening mechanism to alkali-induced egg white

Abstract

The effect of black and green tea extracts with different alkali induction degrees on gel properties of alkali-induced egg white gels (EWGs) was evaluated. Results indicate that, with the increase in alkali induction time, the concentration of soluble polyphenols gradually decreases while their relative polymerisation degree increases, especially for black tea polyphenols. The addition of tea extracts, especially induced by alkaline treatment for 6, 12, and 24 h, can promote the coagulation and browning and inhibit the ‘alkali injury liquefaction’ of alkali-induced EWGs. Additionally, under alkali-induced conditions, polyphenols in tea, especially black tea, can enhance the textural properties and thermal stability of alkali-induced EWGs, this enhancement reaches its peak at 12 h of alkali induction and is consistent with protein secondary structure and interaction forces results. This research provides theoretical support for selection and induction of tea in high-gel-strength preserved eggs and other alkali-induced protein gel food processing.

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Introduction

Preserved eggs, also known as century eggs, are revered for their medicinal and nutritional benefits, making them highly popular among consumers (Xue et al., 2022). Currently, the processing of preserved eggs in China mainly follows the national standard ‘GB/T 9694-2014 Preserved Eggs.’ However, this standard does not provide specific guidelines for using tea. In recent years, researches have shown that the addition of tea can significantly improve the textural properties and thermal stability of preserved egg gels (Ganasen & Benjakul, 2011; Liu et al., 2013, 2016). As a result, tea has become an important gel fortifier in the production of preserved eggs.

However, currently, due to the lack of a unified standard, the selection and addition of tea to preserved egg processing rely solely on empirical knowledge. The main reason is that the research on the mechanism of tea in the formation of preserved EWGs has not made a breakthrough. There are three main factors that contribute to this: (i) The main bioactive substances in tea are polyphenols. Research indicates that polyphenols are stable in the pH range of 4 to 8 but undergo oxidative self-polymerisation reactions in strong alkaline environments, resulting in the formation of dimeric, trimeric, or polymeric reddish-brown or black compounds (Sioriki et al., 2021). However, the impact of alkali-induced time on the degree of polyphenol polymerisation in tea is currently not well understood. (ii) Due to differences in their production processes, different types of tea have varying compositions and levels of polyphenols. For example, in the case of green tea and black tea, polyphenols in green tea mainly consist of unoxidised catechins (Salman et al., 2022), while in black tea, they primarily include oxidised products such as theaflavins, polymerised catechins, and thearubigins (Chen et al., 2023). (iii) The molecular interaction between egg white proteins (EWPs) and polyphenols significantly influences the denaturation rate, aggregation behaviour, and reassembly process of EWPs (Xue et al., 2023). However, whether this molecular interaction is influenced by the source of tea polyphenols and the relative degree of polyphenol polymerisation requires further investigation.

In summary, the research aims to investigate the effect of alkali-induced time on the relative degree of polyphenol polymerisation in black and green tea extracts. Based on this, the study seeks to elucidate the regulatory mechanisms of alkali-induced black and green tea extracts on the aggregation behaviour, texture properties, and colour of EWGs. Furthermore, it aims to reveal the correlation between alkali-induced time, polyphenol relative polymerisation degree, and the textural properties and spatial conformation of EWGs. This research can not only provide theoretical guidance for the processing of preserved eggs in China, but also provide theoretical basis for the processing of other alkali-induced protein gel food.

Materials and methods

Materials

Fresh duck eggs (egg weight 70~75 g) were purchased from Jiangsu Gaoyou Duck Development Group Co., Ltd., Yangzhou, China. Junmei Jin black tea (caffeine, 20.93 mg g⁻¹, soluble sugars, 3.38 mg g⁻¹) and Qingshan Gao green tea (caffeine, 29.65 mg g⁻¹, soluble sugars, 19.93 mg g⁻¹) were purchased from the market, the determination methods for Caffeine and Soluble sugars were shown in Data S1. Sodium chloride (NaCl), sodium hydroxide (NaOH), hydrochloric acid, sulfuric acid, lead subacetate, caffeine, seignette salt, phenol, 3,5-Dinitrosalicylic acid, Folin-phenol, gallic acid, sodium carbonate, sodium tungstate dihydrate, phosphomolybdic acid hydrate, phosphoric acid, vanillin, ethanol absolute, urea, 2-Hydroxy-1-ethanethiol (β-Me), dibasic sodium phosphate, sodium dihydrogen phosphate, and Coomassie Brilliant Blue G-250 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Sample preparation

Alkali-induced treatment of black and green tea water extracts

Take 2 g of black or green tea and add 100 mL of deionised water. Boil the mixture for 10 min, then filter out the tea residue and adjust the volume to 100 mL when the solution is cooled. Then, weigh out the appropriate amounts of 5% NaOH and 4% NaCl. Dissolve these substances in the tea-infused water and mix thoroughly. Next, perform alkali-induced at different time intervals: 0, 6, 12, 24, and 48 h. In the control group, NaOH and NaCl are dissolved in deionised water.

Preparation of alkali-induced EWGs

The samples were prepared with reference to the method of Tan et al. (2021). The egg white was separated from the yolk and stirred magnetically at 800 r.p.m. for 30 min. Subsequently, 1.63 g of alkaline solution was mixed with 10 g of egg white and stirred immediately to mix evenly. Three gel samples were prepared: control gel, black tea gel, and green tea gel. The final gel contained 0.70% NaOH and 0.56% NaCl, was stirred well, and stored at 25 °C for 3 days, The experimental roadmap as shown in Figure S1.

Alkali induction on polyphenol concentration and polymerisation degree of soluble tea water extract

Polyphenols determination

The content of tea polyphenols was determined by the Folin-phenol method (Sun et al., 2022c). Dissolve 5 mg of the gallic acid to 50 mL with distilled water and dilute it to different concentrations (0.01~0.1 mg mL⁻¹). Take 0.5 mL of solution was mixed with 2.5 mL of 0.2 mol L⁻¹ Folin-phenol reagent for 5 min in the dark, then added 4 mL of 7.5% Na₂CO₃ in the solution and continued to react for 2 h. Then the absorbency-polyphenol concentration standard curve was established at 760 nm, and the alkaline solution was determined according to the above method.

Relative polymerisation degree determination

The relative polymerisation degree (FD/V) of polyphenols after alkali induction was determined using the Folin-Denis reagent. Take 2 mL of sample, add 2 mL of Folin-Denis, 4 mL of 20% Na₂CO₃, and add water to 100 mL, and measure the absorbance value (FD value) at 760 nm after 1 h. Another take 2 mL of sample, add 3 mL of 1% vanillin ethanol solution, 4 mL of concentrated hydrochloric acid, and add water to 10 mL, and the absorbance value (V value) was measured at 500 nm after 30 min.

Characterisation of the alkali-induced EWGs

Turbidity determination

The turbidity of the gel samples was determined using a UV-5200PC spectrophotometer (Shimadzu, Kyoto, Japan) according to the method of Deng et al. (2020), with minor modifications. Egg white was mixed with alkaline solution in a 10 mm cuvette, and the absorbance was measured at 600 nm. The absorbance of the gel was recorded for 24 h.

Millard browning determination

Based on the method described by Tan et al. (2022), the browning intensity of the EWGs were measured at 420 nm. Deionised water was mixed with EWGs in the ratio of 5:1. The mixture was then homogenised at 10 000 r.p.m. for 2 min and then centrifuged at 10 000 × g for 10 min; finally, the supernatant was used for Millard browning determination.

Colour determination

The colour of the gel samples was determined using a Colour Meter Pro colorimeter (Fangyuan Measuring Instrument Co., Ltd., Suzhou, China). Obtain L* (brightness, 0~100 means from black to white), a* (red-green, −a* represents greenness, +a* represents redness), and b* (yellow-blue, −b* represents blueness, +b* represents yellowness) values (Tan et al., 2022).

Texture profile analysis (TPA)

TPA of the EWPs was conducted using a TA-XT2i Plus texture analyser (Stable Micro Systems, Godalming, UK) according to the method of Tan et al. (2023) with some modifications. The EWGs (9 mm in height, 20 mm in diameter) were compressed to 50% of their original height using a P50 probe. The compression was performed at pre-test speeds, test speeds, and post-test speeds of 2.0 mm s⁻¹, with a trigger point load of 5 g.

Thermal stability determination

The thermal stability of the gel was evaluated by heating the gel (9 mm in height, 20 mm in diameter) at 90 °C for 20 min using a HH-4JS digital thermostatic water bath (Changzhou Langyue Instrument Manufacturing Co., Ltd., Changzhou, China). The degree of gel melting was observed to assess its thermal stability.

Low field nuclear magnetic resonance (LF-NMR)

The T₂ relaxation time of the gel was determined using an LF-NMR analyser (NMI20-030 V-I, Niumag Co., Ltd., Suzhou, China). 4 g of EWGs were placed in a cylindrical glass tube with a diameter of 25 mm, and the T₂ relaxation time was measured using the Carr–Purcell–Meiboom–Gill sequence. The echo time, wait time, and number of scans were set to 0.2 ms, 3000 ms, and 8, respectively. A total of 17 000 echoes were acquired for analysis (Sun et al., 2022b).

Fourier transform infrared (FTIR) spectroscopy

The secondary structure of EWGs was determined according to the method described by Fan et al. (2021) using an FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The freeze-dried gel sample powder (1 mg) was placed on the surface of the ATR crystal, and the analysis was conducted within a spectral range of 4000 to 400 cm⁻¹ at a temperature of 25 °C. The spectrum was scanned 32 times with a resolution of 4 cm⁻¹. At least three repeated measurements were taken and averaged. The spectra were analysed using Omnic 32 and Peakfit 4.12.

Protein interaction force determination

The protein interaction force was determined with a minor modification, following the method described in our previous study (Sun et al., 2023). SA (difference in protein content between S1 and S2) represents ionic bonding, SB (difference in protein content between S2 and S3) represents hydrogen bonding, SC (difference in protein content between S3 and S4) represents hydrophobic interactions, and SD (difference in protein content between S4 and S5) represents S–S bonding.

Statistical analysis

The experimental data were analysed using Excel for the mean and standard deviation of the data, SPSS software for statistical analysis of the data, one-way analysis of variance with a significant level of P < 0.05, and Origin software for graphing the data. Correlations between the TPA and the protein conformation of alkali-induced EWGs were determined using the SPSS.

Results and discussion

Polyphenol concentration and relative polymerisation degree analysis

Polyphenols are alkaline-unstable substances that easily undergo oxidative polymerisation reactions under alkaline conditions, forming quinones with strong biological activity. These quinones can then attack certain nucleophilic groups in EWP molecules, leading to the formation of covalent complexes between EWPs and polyphenols (Guo et al., 2021). Therefore, we investigated the impact of alkali-induced time on the soluble polyphenol content and the relative polymerisation degree of polyphenols (FD/V) in black and green tea extracts, as shown in Fig. 1.

From the results, the soluble polyphenol content in alkali-induced solutions of black and green tea exhibited a trend of initially decreasing and then increasing (P < 0.05), with a turning point at 24 h of alkali-induced. This phenomenon may be attributed to the fact that within the first 24 h of alkali-induced polyphenolic substances in tea extracts, they may undergo oxidative self-polymerisation reactions under alkaline conditions, forming dimers, trimers, or higher-order polymers with an increased polymerisation degree (Chen et al., 2023). This process could reduce the effective polyphenol content, aligning with the results of the study on the relative polymerisation degree of alkali-induced polyphenols. However, with further extension of alkali-induced time (48 h), some high-molecular-weight polyphenol polymers may undergo degradation under strong alkaline conditions, leading to an increase in the content of tea polyphenols (Sharma & Zhou, 2011). In addition, under strong alkaline conditions, degraded polyphenols can undergo repolymerization and form new polyphenol polymers, thus resulting in an increase in the degree of polyphenol polymerisation (Xu et al., 2020). This change is particularly significant in green tea extracts rich in monomeric polyphenols.

Gelling properties of EWGs

Colour of alkali-induced EWGs

From Table 1, it can be observed that the alkali-induced EWG in the control group appears pale yellow, while the addition of tea extracts, particularly black tea extracts, promotes the development of an appealing reddish-brown colour in the alkali-induced EWGs within a relatively short period. And shown in Table 1, comparing with the control group, the L* values of the tea extract groups significantly decreased (P < 0.05), indicating that the gels in the control group were more transparent, consistent with the turbidity results. This may be attributed to the fact that tea polyphenols accelerate the aggregation rate of EWPs more than the rate of denaturation (Xue et al., 2023), resulting in lower transparency of the gels in the tea extract groups compared to the control group. Additionally, as illustrated in Table 1, the addition of tea extracts, particularly green tea extracts, can promote the formation of final Maillard reaction products, thereby reducing the transparency of the alkali-induced EWGs.

In Table 1, the a* value of the gel in the tea extract groups significantly increased compared to the control group (P < 0.05), with the control group showing negative values and the tea extract groups showing positive values. This indicates that the gels with the tea extracts exhibit more red characteristics. On the other hand, the b* value of the gel in the tea extract groups significantly decreased compared to the control group (P < 0.05), but both were positive values. This suggests that both the gel in the tea extract group and the control group exhibit yellow characteristics, but the yellow characteristics are more pronounced in the control group. The colour difference between the control and tea extract groups in alkali-induced EWGs may result from two main factors. Firstly, under alkaline conditions, tea polyphenols undergo oxidative polymerisation, forming theaflavins and thearubigins, which alters the gel colour (Chen et al., 2021). Secondly, the formation of final Maillard reaction products during EWGs formation under alkaline conditions also affects the gel's colour.

TPA analysis

The texture properties of the gel are important indicators for evaluating preserved egg quality. The effects of alkali-induced black and green tea extracts on the hardness, chewiness, and springiness of EWGs are shown in Table 1. The hardness and chewiness of the gel in the black tea group were higher than those in the green tea group. With an increase in alkali-induced time, there was no significant change in the springiness of the EWGs in both the black tea and green tea groups. However, their hardness and chewiness showed a trend of initially increasing and then decreasing (P < 0.05), with both reaching peak values at 12 h of alkali-induced. This may be attributed to the fact that an appropriate polyphenol content and relative polymerisation degree are beneficial for improving the textural properties of alkali-induced gels.

As Balange & Benjakul (2009) discovered, oxidised polyphenols can significantly enhance the network structure and organisation of protein gels. However, when the alkali-induced time was extended to 24 and 48 h, there was a minor decrease in hardness and chewiness, indicating that excessive polyphenol polymerisation might not be conducive to further improvement of EWP gel properties.

Preserved eggs with high thermal stability are more easily processed into various food products, such as preserved egg lean meat porridge, preserved egg tofu, etc., which helps enrich the application forms and types of preserved egg products. As shown in Figure S2, significant dissolution occurred in the gel of the control group after heat treatment, while the gel in the tea extract groups, especially the gel samples from the black tea group at 12 h of alkali-induced treatment, maintained the original quality of the gel better. These results indicate that tea extracts, especially black tea extracts induced for 12 h with alkali, can significantly enhance the thermal stability of alkali-induced EWGs, consistent with the results of the texture profile analysis.

LF-NMR analysis

The changes in T₂ relaxation time of alkali-induced EWGs induced by different alkali-induced times for black tea and green tea are shown in Table 1. It can be seen that the A₂₁ and A₂₃ for the control group, black tea group, and green tea group samples are smaller, and the A₂₂ is greater than 98%, indicating that the moisture in the alkali-induced EWGs primarily exists in the form of weakly bound water.

According to Table 1, compared to the control group, both T₂₁ and T₂₂ of the alkali-induced EWGs with tea extracts significantly decreased (P < 0.05). This indicates that the addition of tea extracts can restrict the mobility of protons in the EWGs and increase the binding capacity between proteins and water molecules (Hills, 2006). Comparing the black tea and green tea groups, the relaxation time of the gel water in the black tea group is shorter than that in the green tea group, indicating that alkali-induced black tea extracts enhance the binding capacity of alkali-induced EWGs to water, making protein aggregation easier (Quan et al., 2019). This is consistent with the results obtained from the gel texture analysis. The reason for the reduced water mobility within the alkali-induced EWGs upon adding tea extracts may be attributed to the strong hydrophilicity of the active hydroxyl groups in tea polyphenols and the amphiphilic nature of proteins. The hydrophilic groups of tea polyphenols and the hydrophobic groups of proteins can form tight structures through hydrogen bonding, restricting the free movement of water molecules (Xue et al., 2021).

Analysis of gel-strengthening mechanisms

EWP aggregation behaviour during alkali-induced

It can be obtained from Fig. 2, compared to the control group, the addition of tea extracts increased the turbidity of the sample solutions especially black tea extracts, indicating that the addition of tea extracts promotes the aggregation of EWPs, thereby reducing the transparency of alkali-induced EWGs. Additionally, it can be observed from the Fig. 2 that under different alkali-induced times, the turbidity of the solution during the formation of alkali-induced EWG in both the control group and the sample group showed a trend of initially decreasing, then increasing, and then decreasing again (P < 0.05). The reason for the decrease in turbidity of the alkali-induced egg white solution may be that under alkaline conditions, EWPs first completely denature, binding water becomes free water, and viscous proteins become a thin, transparent solution. This is also the phase before the formation of EWGs induced by alkali, during which the denaturation rate of EWPs is greater than the aggregation rate. However, the addition of tea extracts accelerates the transition of this phase, causing the turbidity of the solution to rapidly enter the rising phase, accelerating the formation of alkali-induced EWGs. This may be because polyphenols in tea oxidise to form quinones under alkaline conditions, which then attack certain nucleophilic groups in protein molecules to form EWP-polyphenol covalent complexes, as shown in Fig. 3, thus promoting the aggregation of EWPs and increasing turbidity. Additionally, different alkali-induced times induce the formation of polyphenol polymers with different degrees of polymerisation. In an alkali-induced environment, the reactivity of polyphenol substances with EWPs is influenced by the degree of polyphenol polymerisation.

In addition, the turbidity of alkali-induced EWGs in control group and sample group showed a gradually decreasing trend after 360 min. However, compared to the control group, the turbidity reduction of samples induced for 6, 12, and 24 h is more gradual. The reason for this may be that an appropriate alkali-induced time can promote the formation of a stable and dense gel structure between polyphenols and EWPs (Fig. 3), thereby better inhibiting the ‘alkali injury liquefaction’ of alkali-induced EWGs caused by strong alkali corrosion in the later stages.

EWP-polyphenol covalent analysis

FTIR analysis

This study employed FTIR technology to qualitatively characterise the grafting of polyphenols from black and green tea extracts with EWPs under alkali-induced conditions. As shown in Figure S3, the characteristic absorption peaks of the control group's alkali-induced EWGs are located at 1632.51 cm⁻¹ (amide I band, representing C–O stretching, hydrogen bonding, and COO–) and 1521.23 cm⁻¹ (amide II band, representing C–N stretching and N-H bending modes) (Sun et al., 2022a). From Figure S3, it can be observed that there are different degrees of shifts in the characteristic peaks of the amide I and amide II bands after tea synergistic alkali induction. The tea-fortified alkali-induced EWGs exhibit a blueshift in the amide I band and a redshift in the amide II band. This indicates that covalent crosslinking occurs between EWPs and polyphenols through C–O or C–N bonds, forming EWP-polyphenol covalent complexes under alkali-induced conditions (Fig. 3), and is consistent with the observed turbidity changes in the samples.

The influence of black and green tea on the gel secondary structure under different alkali-induced times is shown in Table 2. β-sheet and α-helix are the main forms of protein secondary structure, and they can influence the textural properties of EWGs. In general, β-sheet structures can form a strong network structure, thereby increasing the hardness and chewiness of the gel (Tan et al., 2023). Previous studies have shown that an increase in β-sheet and a decrease in α-helix can lead to protein aggregation (Berhe et al., 2015; Nyemb et al., 2016). As observed from Table 2, there is no significant change in the random coil structure. β-turn and α-helix show an overall decreasing trend with increasing alkali-induced time (P < 0.05), while β-sheet initially increases and then decreases with increasing alkali-induced time (P < 0.05). The gel induced by the synergistic alkali-induced treatment with black tea extract for 12 h has the highest content of β-sheet and the lowest content of α-helix. This indicates that at this time, the gel has optimal hardness and chewiness, consistent with the TPA results. This may be attributed to the rich active hydroxyl groups in tea polyphenols, which can interact with EWPs, causing protein chain unfolding and resulting in changes in the gel's secondary structure (Ai et al., 2019).

Intermolecular forces of alkali-induced EWGs

In general, the stronger the interaction between protein molecules, the better the gel texture and thermal stability of the protein gels. Therefore, we analysed the intermolecular forces of proteins in alkali-induced EWGs. As shown in Table 2, both SB and SC in the tea group gels exhibit a gradual decrease, while SA initially decreases and then increases with alkali-induced time, and SD initially increases and then decreases, with all showing inflection points at 12 h. From Table 2, it can be concluded that the main forces in alkali-induced EWGs are disulfide bonds and ionic bonds, which is consistent with the results of studies by Tan et al. (2023) and Zhao et al. (2016). Additionally, the addition of tea extracts can promote the formation of protein disulfide bonds, which may be due to phenolic substances in tea extracts reacting with proteins, leading to the formation of more disulfide bonds between cysteine residues in proteins.

TPA and protein conformation correlation analysis

Through correlation analysis of the hardness, springiness, and chewiness of alkali-induced EWGs with protein interactions (Table 3) using SPSS software, it was found that the hardness of the black tea group gel is negatively correlated with SB (P < 0.05) and highly positively correlated with SD (P < 0.01). The springiness of the gel is negatively correlated with SC (P < 0.05), and chewiness is negatively correlated with SB (P < 0.05) and highly positively correlated with SD (P < 0.01). In the green tea group gel, hardness is negatively correlated with SB (P < 0.05) and highly positively correlated with SC and SD (P < 0.01). The springiness of the gel is negatively correlated with SC (P < 0.05), and chewiness is highly negatively correlated with SB and SC (P < 0.01) and highly positively correlated with SD (P < 0.01). This suggests that the addition of tea extracts can enhance the strength of alkali-induced EWGs by reducing hydrogen bonding and hydrophobic interactions between proteins while increasing disulfide bonds between proteins.

Simultaneously, according to the correlation analysis in Table 3, the hardness and chewiness of the black tea group gel are highly positively correlated with β-sheet (P < 0.01). The springiness of the gel is negatively correlated with the α-helix (P < 0.05) and highly negatively correlated with the β-turn (P < 0.01). In the green tea group gel, hardness is highly positively correlated with the β-sheet (P < 0.01), highly negatively correlated with the α-helix (P < 0.01), and significantly negatively correlated with the β-turn (P < 0.05). The springiness of the gel is negatively correlated with the β-turn (P < 0.05), and chewiness is highly positively correlated with the β-sheet (P < 0.01) and highly negatively correlated with the α-helix (P < 0.01). This indicates that the addition of tea extracts can enhance the strength of alkali-induced EWGs by reducing α-helix and β-turn interactions between proteins while increasing β-sheet interactions.

Conclusion

Alkali-induced black and green tea extracts have different effects on EWGs. Research has found that alkali-induced treatment of black tea extracts for 12 h has a significant impact on the gel properties of alkali-induced EWGs. The gel exhibits better colour, high chewiness, and high thermal stability. This effect may be attributed to the further oxidation of polyphenols in black tea, forming aldehyde compounds with larger molecular weights under alkaline conditions. These compounds may facilitate interactions between proteins, leading to the formation of a more stable three-dimensional network structure. In contrast, the impact of alkali-induced green tea extracts on protein gel is relatively minor, despite their higher polyphenol content compared to black tea. In summary, these findings contribute to a better understanding of the mechanisms of tea polyphenols with different compositions and polymerisation degrees on the formation process of preserved egg protein gel, at the same time, our study can also provide theoretical basis for the processing of other alkali-induced protein gel food.

Author contributions

Baochang Li: Writing – original draft; conceptualization; methodology; formal analysis. Ruipeng Ma: Formal analysis. Xuhua Yang: Formal analysis. Mohammed Obadi: Writing – review and editing. Bin Xu: Formal analysis. Jun Sun: Resources; supervision; project administration.

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

Ethics approval was not required for this research.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References