https://orcid.org/0000-0002-2593-7997
Abstract
In this study, a novel frozen pasta product (direct-fried yeast-fermented frozen youtiao, DFYF-frozen youtiao) was developed, and its quality deterioration mechanism was explored from gluten. The comprehensive quality assessment revealed that DFYF-frozen youtiao essentially satisfied consumers' expectations. However, after 10 freeze–thawed cycles (FTc), its specific volume dropped, accompanied by an 83.3% reduction in gas production capacity. Meanwhile, FTc treatment resulted in a noticeable decrease in springiness and resilience, and increased the hardness and springiness by 32.8% and 44.5% respectively. The deterioration of rheological properties for DFYF-frozen youtiao dough during FTc was attributed to the loose gluten network, as evidenced by glutenin macropolymer depolymerisation, high hydrophobicity and weak thermal stability of gluten proteins. This study aims to offer technical guidance for producing palatable frozen youtiao with enhanced health attributes and understand the intricate relationships between the characteristics of gluten proteins and quality of DFYF-frozen youtiao.
Introduction
As an emerging frozen pasta product, frozen youtiao is widely popular in East and Southeast Asian countries because of its double advantage of being both delicious and time saving (Yang et al., 2020). It is preserving the enticing taste and flavour of traditional youtiao, requiring only reheating before consumption and offering substantial economic benefits. The fundamental ingredients of frozen youtiao include wheat flour, water and leavening agents, primarily chemical leavening agents. The production processes encompass mixing, proofing, moulding, frying, freezing/frozen storage and refrying. While frozen youtiao in the market caters to the demand for convenience, it also presents several drawbacks that require attention and improvement. For example, during frozen storage and transportation, temperature fluctuations can lead to the growth and recrystallisation of ice crystals, which disrupted the gluten network structure of the frozen dough and weakened yeast activity, resulting in a decrease in gas production capacity (Li et al., 2022). Consequently, youtiao prepared from frozen dough frequently exhibit undesirable alterations compared to the fresh dough, including increased hardness, irregular pore structure and diminished comprehensive acceptability of the product (Bai et al., 2022). Moreover, the prevalent reliance on chemical leavening agents coupled with the refrying process imposes significant limitations on commercially available frozen youtiao, including limited flavour and excessive oil content. Hence, the development of flavourful frozen youtiao with enhanced health attributes represents a challenging and actively pursued research area.
The majority of recent studies on youtiao focus on conventional youtiao, with only one report addressing frozen youtiao. Investigations into conventional youtiao primarily centre on three key areas: (i) exploring the generation, migration, health implications and mitigation strategies of chemical toxins (polycyclic aromatic hydrocarbons, acrylamide, 5-hydroxymethylfurfural, 3-monochloropropane-1,2-diol fatty acid esters and aluminium, etc.) (Li et al., 2016; Gong et al., 2018; Shyu et al., 2021; Qiu & Liu, 2022; Yuan et al., 2022); (ii) decreasing the oil content by selecting appropriate wheat flour varieties (Wang et al., 2023) and incorporating exogenous substances, such as water-unextractable arabinoxylans (Sun et al., 2022); (iii) seeking the method to improving the flavour quality of youtiao by measuring the main flavour substances and exploring the relationship between flavour substances and ingredients of youtiao (Du et al., 2019; Yang et al., 2020). A solitary investigation by Bai et al. (2022) explored the physicochemical properties and frying performance of frozen youtiao dough composed of wheat flour, water and chemical leavening agents.
As universally acknowledged, yeast stands out as the paramount microbial starter and leavening agent in fermented pasta products, exerting an irreplaceable role in both dough fermentation and the frying process of youtiao. The principal functions of yeast in youtiao encompass: (i) the majority of gas generated during fermentation is retained within the dough network structure, thereby augmenting the dough volume and imparting a fluffy and porous structure to youtiao (Verheyen et al., 2014); (ii) the metabolites produced by yeast fermentation, such as alcohols and organic acids, can interact with gluten to improve the ductility of dough (Wang et al., 2020a); (iii) the distinctive flavour (floral and fruity aromas, wine notes and baking flavours) of youtiao was imparted by flavour substances, including organic acids, alcohols, esters and aldehydes, generated through Maillard reaction and fermentation (Meerts et al., 2018); (iv) the inclusion of minerals, proteins, amino acids and vitamins in yeast contributes to the enhancement of the nutritional value of youtiao. Notably, there is currently no research available on the production and quality assessment of direct-fried yeast-fermented frozen youtiao.
Against this backdrop, an attempt was made to develop direct-fried yeast-fermented frozen youtiao (DFYF-frozen youtiao), and the rheological properties of DFYF-frozen youtiao dough during frozen storage were systematically analysed. Additionally, the quality assessment of DFYF-frozen youtiao was elucidated from the perspective of gluten proteins. This study was to provide technical guidance for the industrial production of flavourful frozen youtiao with enhanced health attributes. Simultaneously, the research contributes to a deeper understanding of the intricate relationships between the characteristics of gluten proteins and the overall quality of DFYF-frozen youtiao.
Materials and methods
Materials
Wheat flour (13.23% moisture; 13.11% protein; 0.48% ash) was purchased from Shanxi Longgucang Grain and Oil Technology Co., Ltd. (Shanxi, China). Yeast was provided by Guangxi Danbaoli Yeast Company, Ltd. (Guangxi, China). Soya bean oil (Yi Hai Kerry Co., Ltd., China) and salt were obtained from a local supermarket. The chemicals used were of analytical grade.
Sample preparation and freeze–thawed cycle treatments
The DFYF-frozen youtiao dough was formulated using 400 g of wheat flour, 5.6 g of yeast, 5.6 g of salt and 240 g of water. Initially, 400 g of wheat flour was introduced into a mixing machine (SM-25, Xinmai Machinery China Co., Ltd, Jiangsu, China). Subsequently, yeast and salt were dissolved in water and stirred for 10 min to create the dough. The dough was then allowed to rest at 35 °C with 85% relative humidity for 1 h. After proofing, the dough was cut into pieces measuring 10 cm in length, 3 cm in width and 1 cm in thickness. Finally, two pieces were stacked together, and pressure was applied to the centre using chopsticks to form the DFYF-frozen youtiao dough.
The fresh DFYF-frozen youtiao dough underwent freezing at −35 °C in a low-temperature test chamber (Zhengzhou Gemei Refrigeration Equipment Co., Ltd, Henan, China) for 2 h until the centre temperature reached −18 °C. Subsequently, it was maintained at −18 °C. Throughout the frozen storage period, a freeze–thawed cycle (FTc) was employed to replicate temperature fluctuations. Each FTc involved freezing at −18 °C for 47 h and thawing at 4 °C for 1 h. After 0, 2, 4, 6, 8 and 10 FTc, the dough was fried at 180 °C for 3 min to make DFYF-frozen youtiao. Subsequently, DFYF-frozen youtiao was cooled for 30 min and drained off the oil before the analysis of sensory evaluation, specific volume and textural properties.
For the analysis of gluten proteins, residual dough was kneaded, washed thoroughly with distilled water until all residual starch was removed, freeze-dried and subsequently passed through an 80-mesh sieve.
Characteristics of youtiao
Sensory evaluation
The assessment of DFYF-frozen youtiao quality encompassed parameters such as appearance, palatability, colour, toughness, taste and internal structure. Every test was performed by the same group of assessors (N = 100), composed of trained assessors recruited from the Sensory Evaluation Laboratory, National Engineering Laboratory for Wheat and Corn Further Processing (Zhengzhou, China). At the beginning of the sensory test, the DFYF-frozen youtiao dough after 0, 2, 4, 6, 8 and 10 FTc were fried at 180 °C for 3 min, followed by a cooling period of 30 min. Following the standard program arrangement, every assessor was assigned 10-g DFYF-frozen youtiao for evaluation. The scoring criteria (Table S1), as outlined by Wang et al. (2020b), with some modifications for the evaluation parameters and scores according to the characteristics of product.
Specific volume
The specific volume of DFYF-frozen youtiao was computed using volume divided by weight. The DFYF-frozen youtiao was weighed 30 min after frying, and the volume was ascertained through the rapeseed displacement method proposed by Li et al. (2022).
Textural properties
After cooling for 30 min at room temperature and draining off the oil, the central portion of DFYF-frozen youtiao was sectioned into 2 cm pieces for testing, and its textural properties (including hardness, springiness, chewiness and resilience) were analysed using a TA-XT Plus texture analyser (Stable Microsystems, Godalming, UK) equipped with P 50 and P 0.5 probes, as outlined by Li et al. (2022). Experimental parameters were configured as follows: a test speed of 1.0 mm s−1, pre-test and post-test speeds of 2.0 mm s−1 and 1.0 mm s−1 respectively. The trigger force was set at 5.0 g, with a 50% compression ratio and a compression time of 1 s. Hardness (g) is defined as the force necessary to attain a given deformation. Springiness (dimensionless unit – d.u.) is construed as the degree to which the sample can be recovered after being deformed by external forces. Chewiness (g) is regarded as the energy required to masticate a solid food to a state ready for swallowing and it is derived from hardness, cohesiveness and springiness. Finally, resilience (d.u.) is described as how well a food fight to regain its original position (Gonçalves et al., 2017).
Gas production capacity of DFYF-frozen youtiao dough
The gas production capacity of DFYF-frozen youtiao dough was determined using a method developed by Peighambardoust et al. (2010). A 5-g sample of dough was placed within an airtight flask submerged in a water bath. Additionally, a measuring cylinder was inverted in a beaker containing an aqueous solution with a pH of 2, and tubing was employed to connect the measuring cylinder to the flask for the collection of CO2. The water bath was maintained at a constant temperature of 40 °C to replicate the dough fermentation process. Gas volume measurements were taken at 5-min intervals throughout the experiment, with the total testing duration spanning 180 min.
Dynamic rheological properties
The dynamic rheological properties of DFYF-frozen youtiao dough were examined employing a Hacker rheometer (RheoStress 6000, HAAKE, Germany) equipped with a sample stage (25 mm diameter). Subsequent to thawing, the uniform dough was positioned on the sample stage and allowed to relax at the test temperature (25 °C) for 5 min. Following this, the dough was compressed into 2-mm slices and sealed with silicone oil to prevent moisture loss. Stress sweep tests were conducted within the linear viscoelastic region, employing a stress range of 0.1–1000 Pa and a frequency of 1 Hz (Bai et al., 2022).
Frequency sweep
The elastic modulus (G′, Pa) and viscous modulus (G′′, Pa) were derived from frequency sweep tests conducted within a range of 0.1–10 Hz, employing a strain of 0.05% and at a temperature of 25 °C (Liu et al., 2023a). The relationship between G′ and frequency sweep, as well as the strength of molecular interactions, was investigated by fitting the frequency sweep data to power law equations:
where ω represents the frequency, while the parameters z and K signify the magnitude of molecular interactions within the dough respectively.
Creep–recovery test
Following the frequency scanning, DFYF-frozen youtiao dough was allowed to relax for 5 min before conducting creep recovery tests. The dough was subjected to a constant stress (τ = 30 Pa) for 100 s, followed by a relaxation period of 300 s after the stress was removed.
Polymerisation degree of GMP
The polymerisation degree of GMP was determined using size-exclusion high-performance liquid chromatography (SE-HPLC) (Agilent Technology (China) Co., Ltd.), following the method of Liu et al. (2018) with slight modifications. Five milligrams of gluten protein was placed in a 2-mL centrifuge tube, mixed with 1 mL of 1% SDS (w/v) phosphate buffer (0.05 M, pH 6.9, 1% DTT added under reducing conditions), subjected to shaking in a water bath (25 °C, 2 h) and centrifuged at 13600 g for 15 min. The supernatant was then filtered through a 0.45-μm membrane and analysed using a BIOSEP SEC-4000 column (300 × 7.8 mm).
The mobile phase consisted of 50% acetonitrile containing 0.1% TFA and 50% water, with a flow rate of 0.5 mL min−1. The thermostat was set at 30 °C, and detection was performed at 214 nm. The quantities of high-molecular-weight gluten (HMW-gluten), low-molecular-weight gluten (LMW-gluten) and SDS-insoluble GMP were computed using eqns (2)–(4):
Molecular weight of gluten proteins
The determination of molecular weight and aggregation degree of gluten proteins was conducted through sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), as described by Ke et al. (2020). The detailed procedure was as follows: Initially, 1 mL of extract (comprising 2% SDS (w/v), 10% glycerol (v/v), 0.01% bromophenol blue (w/v), Tris–HCl: 0.125 M, pH 6.8) was added to a micro-tube containing 2 mg of gluten protein. Subsequently, the mixture was allowed to extract at room temperature for 3 h, followed by centrifugation at 10 000 g for 20 min at 4 °C. The supernatant was then subjected to a 100 °C water bath for 5 min, and 10 μL of the sample was loaded onto an electrophoresis gel. Electrophoresis was conducted at a constant voltage of 150 V until the bromophenol blue indicator reached the gel bottom, signalling the completion of the electrophoresis. Finally, the gel was removed for staining and decolourisation.
Free sulfhydryl (SH) content
The determination of free SH contents in DFYF-frozen youtiao dough according to the method of Liu et al. (2023b) with certain modifications. Initially, the dried sample (75 mg) was incubated with 1.0 mL Tris-glycine buffer (86.0 mmol L−1, pH 8.0, containing 10.4 g L−1 Tris, 6.9 g L−1 glycine and 12.0 g L−1 EDTA), with the addition of 4.7 g of guanidine hydrochloride. Subsequently, the Tris-glycine buffer was diluted to 10 mL, shaken in a water bath at 25 °C for 3 h and then centrifuged (9000 g, 10 min) to obtain the supernatant. Finally, 1 mL of the supernatant was mixed with 4-mL urea–guanidine hydrochloride solution and 0.05 mL of Ellman's reagent. The absorbance A412 was measured after mixing. The free sulfhydryl content was calculated according to eq. (5):
Surface hydrophobicity
The assessment of surface hydrophobicity in gluten proteins was conducted following the methodology outlined in Zhang et al. (2021b), with minor adaptations. Gluten protein (10 mg) was completely dissolved in 1 mL of 0.01 mmol L−1 phosphate buffer (pH = 7.0). The resulting mixture underwent vigorous shaking on a vortex oscillator for 10 min and was subsequently centrifuged at 4000 g for 10 min. The supernatant was then diluted with PBS (pH 7.0) to achieve protein solutions with five distinct concentrations, quantitatively determined using the Coomassie brilliant blue G-250 method. A 4-mL aliquot of the gluten protein solution was combined with 20 μL of 8 mmol L−1 8-anilinonaphthalene-1-sulfonic acid (ANS) and incubated for 15 min before transfer to a tube. The fluorescence intensity was subsequently measured using a microplate reader at excitation and emission wavelengths of 390 and 470 nm respectively. A plot of fluorescence intensity against protein concentration was constructed, with the initial slope of curve representing the surface hydrophobicity of the gluten protein.
Thermodynamic properties
The thermal stability of gluten network structure was assessed using a thermogravimetric analyser (TGA), as described by Feng et al. (2022). The specific parameters were configured as follows: a heating rate of 10 °C min−1, with the temperature increasing from 25 °C to 600 °C. The first-order derivative of TGA curve was employed to determine the pyrolysis temperature (Tp) and degradation temperature (Td).
Statistical analysis
All the experiments were replicated three times, and the results were presented as mean ± standard deviation. Statistical analyses, including one-way ANOVA and Duncan's test (P < 0.05), were conducted using SPSS 26. Significance was acknowledged at a threshold of P < 0.05.
Results and discussion
The quality and yeast activity of DFYF-frozen youtiao
Sensory evaluation
The scoring criteria and results from the sensory evaluation of DFYF-frozen youtiao are presented in Table S1 and Fig. 1b. The mean score of DFYF-frozen youtiao (0 FTc) was 79.8 from 100 assessors across various attributes: colour (8.6), appearance (8), internal structure (7.6), elasticity (15.6), palatability (15.6), toughness (12.6) and taste (11.8). This result indicated that DFYF-frozen youtiao could essentially meet consumer preferences. The appearance of DFYF-frozen youtiao exhibited smooth and symmetrical, which was in accordance with that of Bai et al. (2022). Following the FTc treatment, a discernible downward trend in the mean score of DFYF-frozen youtiao was observed. The mean score of DFYF-frozen youtiao after 8 and 10 FTc was 58.2 and 54.8, respectively, which falls below the passing scores (60). The surface of DFYF-frozen youtiao exhibited roughness and small protuberances, particularly notable after 10 FTc. Furthermore, the texture of DFYF-frozen youtiao became more rigid and less palatable, and the flavour was notably diminished compared to the control (0 FTc). These phenomena, indicating a reduction in consumer acceptance of the product after FTc, can be attributed to the mechanical damage of DFYF-frozen youtiao dough caused by the growth and recrystallisation of ice crystals during FTc (Bai et al., 2022). Therefore, to ensure that the product retains a quality acceptable to consumers before reaching the retail stage, it is imperative that the product undergoes no more than 8 FTc.
Changes in the internal structure (a), sensory evaluation (b), specific volume and gas production capacity (c) and texture properties (d) of DFYF-frozen youtiao during FTc. DFYF-frozen youtiao, direct-fried yeast-fermented frozen youtiao; FTc, freeze–thawed cycles; C0, zero FTc; C10, ten FTc.
Recognising the subjective nature of sensory evaluations for DFYF-frozen youtiao, we introduced objective indicators (specific volume and texture properties), associated with the quality of pasta products, to identify the factors contributing to the decline in sensory scores. Additionally, the gas production capacity of DFYF-frozen youtiao dough was also analysed.
Specific volume, texture properties and gas production capacity
The specific volume and texture of DFYF-frozen youtiao and gas production capacity of DFYF-frozen youtiao dough are detailed in Fig. 1. As depicted in Fig. 1b and d, the specific volume (2.32 cm3 g−1) and gas production capacity (4.24 mL g−1) of DFYF-frozen youtiao (0 FTc) were slightly lower than Bai et al. (2022), but the texture properties were not different from the previous study. It is widely acknowledged that the leavening capability of yeast is lower compared to chemical leavening agents. Nevertheless, the products examined in this study exhibit satisfactory leavening properties, deemed suitable and acceptable by consumers. The discrepancy between DFYF-frozen youtiao and youtiao prepared with a chemical leavening agent may be attributed to the comparatively weaker fermentation ability of yeast when compared to a chemical leavening agent (Zolfaghari et al., 2016). With an increasing number of FTc, there was a gradual decline in the specific volume of youtiao, reaching 1.89 cm3 g−1 after 10 FTc. As illustrated in Fig. 1a, following the FTc treatment, the air holes in DFYF-frozen youtiao diminished in size and became unevenly distributed, resulting in a tighter internal structure. Notably, the hardness and chewiness of DFYF-frozen youtiao increased significantly, accompanied by a noticeable decrease in springiness and resilience. The alterations in texture closely correlate with the reduction in specific volume and changes in the internal structure of dough as previously established by Cheng et al. (2023). Furthermore, the gas production capacity of DFYF-frozen youtiao witnessed an 83.3% decline after 10 FTc, which certified that the survival rate of yeast decreased during the FTc, as shown by the decrease of air hole in the internal structure of DFYF-frozen youtiao. These variations were also in conformity to Bai et al. (2022), and consistent with the consequences of the sensory evaluation. This further indicated that ice crystal's growth and recrystallisation during FTc have compromised the fermentation activity in the dough by disrupting the yeast's gas production capacity, which resulted in a decrease in specific volume, deterioration of textural properties and consequently affects the overall acceptability of the product. Moreover, the rheological properties of dough have always been synonymous with product quality. Therefore, it is crucial to explore the changes of rheological properties and deterioration mechanism of dough during FTc for regulating the quality of DFYF-frozen youtiao.
Rheological properties of DFYF-frozen youtiao dough
The dynamic rheological attributes of dough serve as a quantitative mean to elucidate changes in dough viscoelasticity, facilitating the characterisation of dough processing performance and enabling the prediction and control of product quality. Consequently, frequency sweep and creep–recovery test of DFYF-frozen youtiao dough were constructed.
Frequency sweep
The dynamic rheological characteristics were quantified by elastic modulus (G′) and viscous modulus (G″). G′ and G″ which delineate the solid/elastic and liquid/viscous traits of samples, respectively, offering insights into the alterations in the structural interactions within the dough system (Liu et al., 2023a). The plots of G′ and G″ as functions of frequency (ω) are shown in Fig. 2a and b. Moreover, the power law model was subsequently employed for a comprehensive analysis of the frequency sweep data to obtain z, K and R2 (Table 1). The magnitude of z signifies the degree of dependence of G′ on the frequency, offering insights into the stability of molecular interactions within the dough. The dough rigidity is reflected by K value, with a noteworthy surge in K, indicating increased dough rigidity.
Changes in dynamic viscoelastic properties (a, G′; b, G″), parameter diagram of creep–recovery stage (c) and creep–recovery properties (d) of DFYF-frozen youtiao dough with different FTc. G′, elastic modulus; G″, viscous modulus; DFYF-frozen youtiao, direct-fried yeast-fermented frozen youtiao; FTc, freeze–thawed cycles; (E, F) creep stage of dough and the difference between the strain corresponding to points E and F is creep strain; (F, G) recovery stage of dough and the difference between the strain corresponding to points F and G is recovery strain.
z and K values for gluten proteins of DFYF-frozen youtiao dough fitted to frequency scan data during freeze–thawed cycles
| Freeze–thawed cycles | z | K·104 | R2 |
|---|---|---|---|
| 0 | 0.259 ± 0.006b | 1.08 ± 0.13a | 0.998 ± 0.001 |
| 2 | 0.268 ± 0.008b | 0.89 ± 0.11ab | 0.998 ± 0.000 |
| 4 | 0.277 ± 0.003b | 0.75 ± 0.03bc | 0.998 ± 0.000 |
| 6 | 0.316 ± 0.018a | 0.56 ± 0.09c | 0.989 ± 0.009 |
| 8 | 0.331 ± 0.002a | 0.58 ± 0.06c | 0.995 ± 0.000 |
| 10 | 0.329 ± 0.013a | 0.55 ± 0.03c | 0.996 ± 0.000 |
| Freeze–thawed cycles | z | K·104 | R2 |
|---|---|---|---|
| 0 | 0.259 ± 0.006b | 1.08 ± 0.13a | 0.998 ± 0.001 |
| 2 | 0.268 ± 0.008b | 0.89 ± 0.11ab | 0.998 ± 0.000 |
| 4 | 0.277 ± 0.003b | 0.75 ± 0.03bc | 0.998 ± 0.000 |
| 6 | 0.316 ± 0.018a | 0.56 ± 0.09c | 0.989 ± 0.009 |
| 8 | 0.331 ± 0.002a | 0.58 ± 0.06c | 0.995 ± 0.000 |
| 10 | 0.329 ± 0.013a | 0.55 ± 0.03c | 0.996 ± 0.000 |
Data are presented as mean ± standard deviation (n = 3). Values with different superscript lowercase letters in the same column of the same parameter represent significant differences (P < 0.05).
z and K values for gluten proteins of DFYF-frozen youtiao dough fitted to frequency scan data during freeze–thawed cycles
| Freeze–thawed cycles | z | K·104 | R2 |
|---|---|---|---|
| 0 | 0.259 ± 0.006b | 1.08 ± 0.13a | 0.998 ± 0.001 |
| 2 | 0.268 ± 0.008b | 0.89 ± 0.11ab | 0.998 ± 0.000 |
| 4 | 0.277 ± 0.003b | 0.75 ± 0.03bc | 0.998 ± 0.000 |
| 6 | 0.316 ± 0.018a | 0.56 ± 0.09c | 0.989 ± 0.009 |
| 8 | 0.331 ± 0.002a | 0.58 ± 0.06c | 0.995 ± 0.000 |
| 10 | 0.329 ± 0.013a | 0.55 ± 0.03c | 0.996 ± 0.000 |
| Freeze–thawed cycles | z | K·104 | R2 |
|---|---|---|---|
| 0 | 0.259 ± 0.006b | 1.08 ± 0.13a | 0.998 ± 0.001 |
| 2 | 0.268 ± 0.008b | 0.89 ± 0.11ab | 0.998 ± 0.000 |
| 4 | 0.277 ± 0.003b | 0.75 ± 0.03bc | 0.998 ± 0.000 |
| 6 | 0.316 ± 0.018a | 0.56 ± 0.09c | 0.989 ± 0.009 |
| 8 | 0.331 ± 0.002a | 0.58 ± 0.06c | 0.995 ± 0.000 |
| 10 | 0.329 ± 0.013a | 0.55 ± 0.03c | 0.996 ± 0.000 |
Data are presented as mean ± standard deviation (n = 3). Values with different superscript lowercase letters in the same column of the same parameter represent significant differences (P < 0.05).
The G′ and G″ values of DFYF-frozen youtiao dough exhibited a decreasing trend with the elevation of FTc, indicating a decrease in the viscoelasticity of DFYF-frozen youtiao dough during the FTc. As depicted in Table 1, a discernible trend was evident wherein the value of z exhibited a gradual elevation, accompanied by a significant reduction in the K value with the increase in FTc. Shang et al. (2023) confirmed that with the increase of B−/A− starch ratio in dough, the K value increased and the z value decreased, indicating that the increase of B starch filled the gap of gluten network structure, thus increasing the stability of gluten network structure and improving the strength of dough. Similarly, these variations in this study implied that a lower strength and unstable network structure of DFYF-frozen youtiao dough matrix have occurred during FTc, which might decrease the gas-holding capacity of DFYF-frozen youtiao dough and lead to a reduction in the specific volume of DFYF-frozen youtiao.
Creep–recovery test
The creep–recovery test serves as a comprehensive tool for quantifying the rheological behaviour of dough under constant shear stress, providing insights into the recovery level of post-deformation. As depicted in Fig. 2c, the creep–recovery curve of DFYF-frozen youtiao dough, subjected to varying numbers of FTc, predominantly exhibited two distinct periods. During the creep period, the dough strain exhibited a time-dependent increase, ultimately attaining a steady state under constant stress. Subsequently, in the recovery period, the applied stress was released, and the dough deformation partially recovered with time (Wang et al., 2021). As the number of FTc increases, the creep strain of DFYF-frozen youtiao dough during the creep period (Fig. 2d) also increases, suggesting that FTc treatment reduces the dough's rigidity. Similarly, in the relaxation period, the recovery strain of the dough exhibits an augmentation following FTc. However, in accordance with previous findings (Wang et al., 2021), the magnitude of the recovery rate in DFYF-frozen youtiao dough demonstrates an opposing trend to the variations in recovery strain, indicating that FTc treatment diminishes the dough's capacity to resist deformation.
Considering the critical role of the gluten network structure in maintaining dough strength, the decline in the dough's resistance to deformation during FTc can be attributed to the weakening of gluten network structure (Meerts et al., 2017). The consequence of creep–recovery test, viewed from an alternative perspective, corroborates the degradation of the gluten network structure, consistent with the findings outlined in Frequency sweep section. Gluten protein, as a pivotal determinant influencing dough viscoelasticity, assumes a crucial role in the development of dough. Consequently, the changes of gluten protein during FTc were further studied.
Changes in molecular weight distribution and GMP depolymerisation of gluten proteins during FTc
The solubility of gluten proteins in SDS and the depolymerisation of glutenin macropolymer (GMP) serve as indicators of changes in gluten protein fractions, providing insights into the degree of crosslinking within the gluten network structure. Consequently, SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC) were conducted to investigate the SDS solubility and GMP depolymerisation of gluten proteins within DFYF-frozen youtiao dough throughout FTc.
SE-HPLC
Figure S1 illustrates the typical molecular weight distribution curves of SDS-extractable proteins derived from gluten proteins in DFYF-frozen youtiao dough throughout FTc. In accordance with the categorisation proposed by Liu et al. (2018), SDS-soluble proteins comprise three fractions: wheat gluten polymers (F1), monomeric proteins (F2) and peptides and amino acids (F3). SDS-high molecular weight (HMW) proteins consist of F1, while SDS-low molecular weight (LMW) proteins are constituted by F2 and F3. The contents of SDS-HMW, SDS-LMW and glutenin macropolymer (GMP) were calculated and are presented in Fig. 3c.
Changes in molecular weight distribution and free sulfhydryl content of gluten protein during FTc: (a) SDS-PAGE reduced bands; (b) SDS-PAGE non-reduced bands; (c) variations in the proportion of SDS-HMW, SDS-LMW and GMP; (d) variations of free sulfhydryl content. FTc, freeze–thawed cycles; C0–C10, the number of FTc.
Remarkably, the content of SDS extractable protein significantly increased and GMP content decreased with the increase of FTc. After 10 FTc, there were 3.58% and 9.31% increase in the SDS-HMW and SDS-LMW contents, respectively, while GMP content decreased from 34.97% to 22.08%. This observation aligns with the results of Zhang et al. (2021a), who demonstrated an increase in monomer and polymer proteins along with a decrease in GMP content after five FTc. During dough preparation, the interaction between moisture and flour prompts the high-molecular-weight and low-molecular-weight glutenin subunits to form an organised fibrous macromolecular polymer known as GMP, which has an important effect on gluten protein networks and dough characteristics (Feng et al., 2021). During the FTc, the generation of ice crystals weakens the covalent bonds among glutenin molecules. This leads to a gradual transition from a compact fibrous structure to a looser one, transforming the protein from a large polymer into smaller, more loosely structured polymers, along with the release of free peptides or amino acids (Wang et al., 2015). Additionally, Yu et al. (2020) reported that GMP depolymerisation of gluten protein during frozen storage resulted in reduced volume and lower quality of bread. Through correlation analysis between bread quality and dough components, Wang et al. (2017) demonstrated that the depolymerisation of GMP played a predominant role in the reduction of bread volume during FTc. Therefore, the compromised dynamic rheological properties of DFYF-frozen youtiao dough and the deterioration of sensory quality in DFYF-frozen youtiao may be attributed to the depolymerisation of GMP.
SDS-PAGE
To further elucidate the reasons for GMP depolymerisation, we employed reduced and non-reduced SDS–polyacrylamide gel electrophoresis (SDS-PAGE) to characterise changes in the molecular weight of gluten proteins. In comparison with the non-reduced bands (Fig. 3b), the reduced bands (Fig. 3a) displayed no distinct migration of protein bands. However, the intensity of reduced bands with molecular weight between 25 and 35 kDa exhibited a slight enhancement with increasing FTc. This phenomenon may be attributed to the partial depolymerisation of high-molecular-weight protein polymers initially concentrated in the gel, resulting in the formation of lower molecular weight proteins after FTc. This observation aligns with the results derived from SE-HPLC.
Upon comparing Fig. 3a and b, it is evident that the presence of dithiothreitol (DTT), a reducing agent that cleaves disulphide bonds, led to the absence of bands corresponding to high-molecular-weight gluten polymers. This observation suggests that gluten protein aggregation primarily relies on extra-chain disulphide bonds, aligning with findings from previous studies (Wang et al., 2014; Lamacchia et al., 2016). Consequently, the FTc treatment may disrupt the gluten network structure by cleaving disulphide bonds. To validate this hypothesis, the free sulfhydryl content and surface hydrophobicity of gluten in DFYF-frozen youtiao dough were measured.
The variation in free sulfhydryl content of gluten proteins during FTc
Disulphide bonds, which are stable covalent linkages that play a crucial role in stabilising the spatial structure of peptide chains, are essential for maintaining the structure, function and regulation of protein activities (Fu et al., 2020). Free sulfhydryl (SH), functioning as a key group within gluten proteins, typically participates in the aggregation behaviour of gluten proteins through the formation of disulphide bonds. Consequently, alterations in SH content can serve as an indicator of breakage of disulphide bonds (Zhao et al., 2020). The aforementioned data indicated that FTc have inhibited the aggregation of gluten proteins (Fig. 3a–c). To investigate the underlying mechanisms, we quantified the SH content of gluten proteins (Fig. 3d). The free SH content of gluten proteins in DFYF-frozen youtiao dough exhibited a significant increase with the rise in the number of FTc. After 10 FTc, the free SH content increased from 3.86 to 5.69 μmol g−1, which aligns with the results reported by Wang et al. (2017), who attributed the main cause of the deterioration in bread quality to GMP depolymerisation resulting from the breakage of disulphide bonds. The temperature fluctuations induced by FTc trigger water migration and recrystallisation of ice crystals, which in turn accelerate the compression between gluten proteins and intermolecular forces. These intense physical effects lead to alterations in protein structure and the cleavage of disulphide bonds (Zhao et al., 2013). In addition, the three-dimensional gluten network is inherently stabilised by covalent disulphide bonds and further fortified by non-covalent interactions, including hydrogen bonds, ionic bonds and hydrophobic interactions (Liu et al., 2018). Compared with other intra/inter-molecular interactions, disulphide bonds constitute crucial forces that uphold the three-dimensional network structure of gluten proteins and play a pivotal role in determining their functional properties (Wang et al., 2015). Therefore, FTc treatment leads to the localised breakage of disulphide bonds, which emerges as a likely primary factor contributing to GMP depolymerisation and deterioration of gluten network structure.
As reported, sulfhydryl oxidase (Du et al., 2020) and protein disulphide isomerase (Fu et al., 2020) could improve the aggregation degree of gluten network structure by catalysing the formation of disulphide bonds between cysteine residues of proteins. Therefore, in our future study, in order to improve the rheological properties of DFYF-frozen youtiao dough, enzymes such as sulfhydryl oxidase and protein disulphide isomerase, which promote the formation of disulphide bonds, could be used to strengthen the gluten network.
Alterations in surface hydrophobicity of gluten proteins in DFYF-frozen youtiao dough during FTc
Surface hydrophobicity, which was assessed using fluorescence spectroscopy as outlined by Zhang et al. (2021b), serves as a pivotal indicator of tertiary structure of gluten proteins and intermolecular interactions among protein molecules. The hydrophobic side chains in proteins possess the capability to absorb the fluorescence of incident light in the ultraviolet region. Thus, the initial slope of the curve in the relationship between fluorescence intensity and protein concentration can serve as a representative measure of the surface hydrophobicity of gluten proteins. As depicted in Fig. 4a, it is evident that the surface hydrophobicity of gluten protein increased by 18.1% after 10 FTc. Notably, in the spatial structure of proteins, the majority of non-polar amino acid side chains are typically situated within the protein molecule, constituting a hydrophobic core. Meanwhile, polar amino acids are distributed on the protein surface in a hydrophilic environment (Jiao et al., 2019). However, during the FTc, the mechanical forces exerted by ice crystal aggregation disrupt the structural integrity of gluten proteins. As a result, the observed increase in surface hydrophobicity of gluten protein is likely due to the destruction of the original molecular structure of the protein and exposure of the hydrophobic groups, which is caused by FTc treatment.
Changes in surface hydrophobicity (a) and thermodynamic properties (b: Tp and Td; c: WI and WII) of gluten proteins during frozen storage. Tp, pyrolysis temperature; Td, degradation temperature; WI, weight loss in stage I; WII, weight loss in stage II.
Changes in thermodynamic properties of gluten proteins in DFYF-frozen youtiao dough during FTc
To assess the structural stability of gluten proteins in DFYF-frozen youtiao dough, thermogravimetric analysis (TGA) was conducted to investigate the variation in pyrolysis temperature (Tp), degradation temperature (Td) and weight loss of gluten proteins in DFYF-frozen youtiao dough during FTc (Feng et al., 2022). The thermogravimetric curves and derivative thermogravimetric profiles are presented in Fig. S2. As depicted in Fig. S2, the weight loss of gluten proteins primarily occurred during two distinct stages (temperature ranges of 50–150 °C and 250–350 °C). In stage I (temperature range of 50–150 °C), the observed weight loss (WI) is attributed to the evaporation of both free water and bound water during heating process; while the weight loss in stage II (250–350 °C) (WII) primarily results from the thermal decomposition of proteins, involving the breakdown of covalent bonds such as S–S, O–N, O–O bonds and peptide bonds of amino acids (Nawrocka et al., 2016).
As depicted in Fig. 4b and c, the WI of gluten proteins exhibited a gradual increase, while the Tp of proteins gradually decreased during FTc. This phenomenon can be attributed primarily to water evaporation, signifying a reduction in the moisture retention capacity of the gluten network structure during FTc, in accordance with findings reported by Huang et al. (2023). After 10 FTc, the degradation temperature (Td) of gluten protein decreased from 320.5 °C to 318.7 °C, and WII increased from 73.76% to 75.16%. This indicated that FTc treatment disrupted the molecular structure of gluten proteins by cleaving disulphide bonds. Consequently, the formation of the gluten network structure in DFYF-frozen youtiao dough was inhibited, ultimately resulting in a diminished thermal stability of gluten proteins. The increase in hydrophobicity and the reduction in thermal stability of gluten proteins collectively illustrated the loose structure of gluten network during FTc. In summary, the deterioration of rheological properties in DFYF-frozen youtiao dough during FTc was attributed to the weakening of gluten network structure, which manifested by GMP depolymerisation, heightened hydrophobicity and reduced thermal stability of gluten proteins.
Schematic model of DFYF-frozen youtiao dough
Based on the aforementioned findings, we propose a clear schematic model delineating the mechanism underlying the deterioration in rheological properties of DFYF-frozen youtiao dough from the perspective of structural dynamic changes of gluten proteins during FTc. As illustrated in Fig. 5, as the length of GMP becomes shorter, the number of SDS-HMW and SDS-LMW fragments increases, and the gluten network structure becomes looser with the increase of FTc. GMP is characterised by a fibrous structure with robust intermolecular binding capabilities, limited fluidity and exceptional elasticity (Feng et al., 2021). SDS-HMW and SDS-LMW components mainly comprise wheat gluten polymers, monomeric proteins, amino acids and peptides, which fill the interstices surrounding GMP, forming a gluten network structure crucial for sustaining the dough's viscoelasticity. With an increase in FTc, disulphide bonds underwent breakage and transformation into free SH, leading to GMP depolymerisation. Sequentially, the contents of SDS-HMW and SDS-LMW gradually intensified (Fig. 3). The elevation in monomer protein, amino acid and peptide content resulted in the exposure of hydrophobic region of gluten protein, inducing a loose gluten network, an augmentation in the hydrophobicity of gluten protein and a reduction in thermal stability (Fig. 4). The less dense and unstable gluten network contributes to deviations in the rheological properties of DFYF-frozen youtiao dough and adversely affects the comprehensive quality of DFYF-frozen youtiao. In conclusion, the proposed schematic model further elucidates the potential impact of gluten protein changes on dough properties during FTc.
Schematic illustration delineating the mechanism underlying the deterioration in rheological properties of DFYF-frozen youtiao dough from the perspective of structural dynamic changes in gluten proteins during FTc. DFYF-frozen youtiao, direct-fried yeast-fermented frozen youtiao; FTc, freeze–thawed cycles.
Conclusions
In this investigation, a novel frozen fried pasta product (DFYF-frozen youtiao) was developed, and its quality during FTc was analysed. The findings revealed that FTc had a detrimental impact on the quality of DFYF-frozen youtiao, resulting in a decreased specific volume, firmer texture and a more compact internal structure. The dynamic rheological properties of DFYF-frozen youtiao dough deteriorated, leading to reduced dough strength, which, in turn, negatively impacted the specific volume and textural properties of DFYF-frozen youtiao. Notably, the contents of SDS-extractable protein and free SH in DFYF-frozen youtiao dough exhibited an increase. Concurrently, the surface hydrophobicity of gluten proteins intensified, while thermal stability diminished. These variations in gluten structure reflected GMP depolymerisation and a more relaxed configuration of the gluten network, contributing to the deterioration of rheological properties in DFYF-frozen youtiao dough and ultimately diminishing the quality of the DFYF-frozen youtiao. In conclusion, to preserve product quality to a standard acceptable to consumers before the retail stage, it is critical that the product is subjected to no more than 8 FTc. Subsequently, to augment the viscoelasticity of DFYF-frozen youtiao dough and improve the overall quality of DFYF-frozen youtiao, we will further strengthen the gluten network through catalysing the formation of disulphide bonds or facilitating the correct folding of proteins to establish a stable conformation.
Acknowledgments
This work was supported by National Natural Science Foundation of China [32202097]; Science and Technology Innovation in the Central Plains-Youth Talent Recruitment Project [2023HYTP044]; Key Research Projects of Higher Education Institutions in Henan Province [23B550005]; National Technical System for Wheat Industry In China [CARS-03]; Training Plan Program for Young Backbone Teachers of Henan University of Technology in 2023 [21421232] and Postdoctoral Research Start-Up Fund of Henan University of Technology [21450086].
Author contributions
Shenying Zhang: Conceptualization; writing – original draft; methodology. Mengge Niu: Formal analysis. Wenqian Dang: Methodology; formal analysis; investigation. Jiajing Han: Visualization. Mei Liu: Conceptualization; writing – review and editing; supervision. Ying Liang: Conceptualization; supervision. Lulu Yin: Methodology. Haojia Zhu: Methodology. Ying Huang: Methodology. Xueling Zheng: Project administration; supervision. Chong Liu: Writing – review and editing. Limin Li: Methodology; resources.
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethical approval
Ethics approval was not required for this research.
Peer review
The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/ijfs.17377.
Data availability statement
Data will be made available on request.
References
This reference described the impact of adding an appropriate amount of sanxan on the quality of steamed bread during FTc, offering insights into explaining the reason for the decreased specific volume of DFYF-frozen youtiao during FTc.
This reference focused on the effects of gluten network structure on the rheological properties of dough, providing a theoretical basis for our viewpoints in this study.
This reference highlighted the physicochemical alterations of wheat gluten proteins in frozen storage from the perspectives of gluten, which contributed to explaining the quality deterioration mechanism of DFYF-frozen youtiao during FTc from the perspectives of gluten proteins in this study.
This reference used sensory evaluation in the process of investigating the effect of water addition and noodle thickness on the surface viscosity of frozen cooked noodles, which provided some theoretical support for the establishment of sensory evaluation scoring criteria of DFYF-frozen youtiao in this study.
