Process optimization of thermal stability of mayonnaise based on phospholipase A2 (PLA2) and flavor protease compound enzymatic hydrolysis

Process optimization of thermal stability of mayonnaise based on phospholipase A2 (PLA2) and flavor protease compound enzymatic hydrolysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Process optimization of thermal stability of mayonnaise based on phospholipase A2 (PLA2) and flavor protease compound enzymatic hydrolysis Hetong Jin, Songyi Lin, Guangshun Jiang, Kun Liu, Fujun Guo, Zhijie Bao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7286074/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 23 You are reading this latest preprint version Abstract There have been a number of research reports on improving the stability of egg yolk emulsions at home and abroad, while relatively few studies have been conducted on the stability of high internal phase emulsions such as mayonnaise. This study aims to enhance the thermal stability of mayonnaise through composite enzymatic hydrolysis, addressing issues such as oil-phase separation and structural collapse that commonly occur during thermal processing. Comparison of phospholipase A 1 (PLA 1 ) and phospholipase A 2 (PLA 2 ) showed that PLA 2 was more effective in improving emulsion activity (EAI) and emulsion stability (ESI). Subsequently, the response surface methodology was employed to optimize the composite enzymatic digestion process of PLA 2 and flavor protease (Fla), with the optimal conditions determined as 2.3 U of PLA 2 , 92 U of Fla, a temperature of 55℃, and a duration of 2 h. Meanwhile, the effects of the primary term PLA 2 addition and the interactive terms PLA 2 addition and Fla addition as well as optimal digestion on mayonnaise were investigated. The results showed that the optimized enzymatic hydrolysis process significantly improved the thermal stability of mayonnaise, with an Lightness (L/l*) value of 25.523±0.14. Furthermore, texture and rheological analyses indicated that mayonnaise prepared under optimal enzymatic conditions exhibited greater hardness and stronger force, which is beneficial for its spreadability.This study provides theoretical basis and practical and feasible technical solutions for the production and quality improvement of mayonnaise, and helps its application in more fields, such as refrigerated and frozen foods. egg yolk liquid mayonnaise phospholipase protease thermal stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 INTRODUCTION In the food industry, mayonnaise, an emulsified condiment has become deeply integrated into the preparation of various dishes due to its unique texture and flavor [ 1 ]. From everyday sandwiches and salads to a wide range of sauces, mayonnaise not only enhances the rich and mellow taste of food products but also plays a crucial role in maintaining the structural stability of food. However, during actual production, storage, and processing, mayonnaise often faces significant challenges, with thermal instability being a key issue. Kiosseoglou V (2003) reviewed the role of egg yolk proteins in the formation of gels and emulsions and emphasized the importance of their role in the stabilization of mayonnaise [ 2 ]. When subjected to thermal processing or high temperatures, mayonnaise is highly susceptible to undesirable phenomena such as oil phase separation, structural collapse, and a sharp decline in emulsification properties. Typically, consumers reheat ready-to-eat foods in microwave ovens, a convenient and quick method that does not compromise the overall structure of the food. However, mayonnaise may exhibit significant oil separation after microwave heating, which not only severely affects product quality but also significantly diminishes the consumer's eating experience [ 3 ]. Therefore, the application of mayonnaise in ready-to-eat foods has been greatly restricted, and the development of mayonnaise products with higher thermal stability has become an urgent issue. In the complex compositional system of mayonnaise, egg yolk undoubtedly occupies a central position. Anton M et al. (2010) emphasized the important role of phospholipids and proteins in the stability of emulsions in terms of the chemical composition and action function of egg yolks [ 4 ].Mcclements, Julian D (2007) reviewed the characterization of and methods for the stability of emulsions that can be used to assess egg yolk emulsion stability [ 5 ]. To enhance the stability of egg yolk emulsions, Xu R et al. (2024) investigated the effects of heat treatment on the stability and emulsification properties of egg yolk emulsions and analyzed the mechanisms behind oil phase separation and structural collapse [ 6 ]. Guilmineau, F et al. (2005) found that EY thermal denaturation altered the emulsification function and altered the properties of O/W emulsions [ 7 ]. There are various ways to improve the stability of mayonnaise, among them Mun S et al. (2009) explored the use of modified starch and xanthan gum to improve the thermal stability of low-fat mayonnaise [ 8 ]. Primacella M et al. (2019) improved mayonnaise thermal stability by hydrolyzing and treating frozen-thawed egg yolks as anticoagulant for the preparation of mayonnaise [ 9 ]. Among these, PLA 2 has demonstrated significant potential in optimizing the properties of egg yolk emulsions by modifying egg yolk components. Guo Z et al. (2005) reviewed the application of PLA 2 in phospholipid modification, especially in functional foods and emulsion stability [ 10 ]. Gazolu-Rusanova et al. (2020) conducted a comprehensive study on the enhancement of interfacial properties and emulsion stability in egg yolk systems through PLA 2 modification [ 11 ]. Meanwhile, the effect of complex enzymatic hydrolysis different proteases with PLA 2 has gradually become the focus of research. Jiang G et al. (2024) effectively improved the thermal stability of egg yolk emulsions by enzymatic hydrolysis of egg yolk emulsion by PLA 2 together with flavored protease through synergistic effect of complex enzymatic hydrolysis. Meanwhile, it was shown that the moderate addition of flavored protease could effectively reduce the surface hydrophobicity and interfacial tension of the egg yolk and improve the emulsification stability of egg yolk protein [ 12 ]. The previous study demonstrated that PLA 2 and Fla composite enzymatic digestion a improved the thermal stability of mayonnaise relative to PLA 2 modification alone. However, the previous composite enzymatic hydrolysis was based on a fixed PLA 2 addition, but the effect of changes in PLA 2 addition in the composite enzymatic hydrolysis on the thermal stability of mayonnaise remains unclear. Consequently, this investigation aims to elucidate possible synergistic interactions between PLA 2 and flavor protease during co-digestion, with particular emphasis on their combined effects on emulsion stabilization. A Box-Benhnken central combination design experiment was used to optimize the enzymatic digestion process of egg yolk liquid by taking the L/l* value of the thermal stability of mayonnaise as the index of investigation, and the PLA 2 addition, Fla addition, enzymatic temperature and enzymatic time as the influencing factors in response surface experiments, so as to obtain the enzymatic process conditions of the egg yolk liquid when the thermal stability of mayonnaise was the best. Additionally, the effects of various factors in the enzymatic digestion process on the thermal stability of mayonnaise were analyzed to reveal potential synergistic interactions among these factors. Furthermore, the physical properties of the optimized mayonnaise were evaluated and analyzed to understand the impact of enzymatic digestion on its quality characteristics. This provides a solid theoretical foundation and practical technical solutions for innovating mayonnaise production processes, thereby promoting its broader and deeper application in the food industry. 2 MATERIALS AND METHODS 2.1 Experimental Materials Fresh eggs, sugar, salt, and Jiusan soybean oil were purchased from local supermarkets in Dalian, China. Flavored protease (500 LAPU g -1 ) was obtained from Novozymes Biologicals Ltd. (Novozymes, Denmark), while PLA 1 (10,000 U mL -1 ) and PLA 2 (10,000 U mL -1 ) were sourced from Neova Technologies Ltd. (Neova, Canada). 2.2 Preparation of Egg Yolks and Mayonnaise 2.2.1 Preparation of Egg Yolks Fresh eggs were washed and dried. The eggshells were cracked, and the egg whites were separated from the yolks. Excess egg white was removed, and the yolk liquid was collected into a beaker after puncturing the yolk membrane with a toothpick. The yolk liquid was then stirred at 100 rpm for 30 minutes at room temperature and prepared for further use. 2.2.2 Preparation of Mayonnaise Mayonnaise was prepared according to the method described by Kim et al. (2009) with reference to Table 1 [13]. Water, sugar, salt, and the enzymatic egg yolk solution were thoroughly mixed and homogenized at 6000 rpm for 4 minutes using a homogenizer. Oil was gradually added during the homogenization process. Finally, 5% acetic acid was added, and the mixture was homogenized again at 8000 rpm for 3 minutes. Table 1 Mayonnaise recipes Ingredients egg yolk sugar salt water 5% acetic acid oil g / 100 g 8 1 1.5 8.5 3 78 2.3 Determination of Emulsifying Activity and Emulsion Stability of Egg Yolk Solution The EAI and emulsion stability ESI of the modified egg yolk solution were determined following the method of Zhang et al. (2012) with slight modifications [14].The enzymatically digested egg yolk solution was prepared as an 8% yolk mass aqueous solution. The diluted yolk solution was mixed with soybean oil at a 3:1 ratio and dispersed in a high-speed shear mixer for 2 minutes (10,000 rpm). After coarse dispersion, 20 μL of the emulsion was aspirated from the bottom and mixed with 5 mL of 0.1% SDS solution. The absorbance value A 0 was measured at 500 nm using 0.1% SDS as a blank control. The same procedure was repeated after 30 minutes of standing, and the absorbance value A t was measured at 500 nm. The EAI was calculated as follows: (1.1) T = 2.303, N is the dilution factor 250, C protein concentration in g/mL, and φ is the fraction of oil in the emulsion, which is 25%. The ESI was calculated as follows: (1.2) 2.4 Determination of Lactic Precipitation Index The lactic precipitation index was determined according to the method of Chen et al. (2018) with slight modifications [15]. A 100 mL emulsion was transferred to a measuring cylinder and allowed to stand at room temperature for 4 hours. The volume of the lower clear liquid was recorded at 15-minute intervals, and the lactic precipitation index was calculated. A curve of the lactic precipitation index over time was plotted. The lactic precipitation index was calculated as follows: (1.3) 2.5 Determination of Interfacial Tension Interfacial tension was determined using a contact angle meter according to the method of Tabor et al. (2015) [16]. The egg yolk solution was thoroughly mixed with sugar and salt and then diluted five-fold. The densities of soybean oil and the diluted sample were measured using the weighing method. Soybean oil was filled into a glass dish, and the syringe was loaded with the sample. The tension of the egg yolk solution at the oil-water interface was measured using the hanging drop method over 1600 seconds. 2.6 Determination of Three-Phase Contact Angle The three-phase contact angle of the PLA 2 -modified egg yolk solution was measured using a contact angle meter [17]. Lyophilized egg yolk powder was pressed into discs with a diameter of 1 cm and a height of 1 mm. The discs were immersed in soybean oil, removed, and placed on a slide. A 5μL water droplet was added to the surface of the discs, and images of the droplets were captured using a camera. The three-phase contact angles of different samples were obtained by measuring the angles through software, and the fitting equation was the Young-Laplace equation. 2.7 Determination of Thermal Stability of Mayonnaise The thermal stability of mayonnaise was determined by accurately weighing 10 g of mayonnaise into a 15 mL centrifuge tube for microwave heating. The microwave frequency was set to 50 Hz, and the power was 550 W. The temperature of the mayonnaise was measured using an infrared thermal imager. Heating was stopped when the temperature reached 70℃. After microwave heating, the mayonnaise was centrifuged at 6000 g for 20 minutes, and the oil separated from the upper layer was observed. 2.8 Determination of Microstructure of Mayonnaise The microstructure of microwave-treated mayonnaise was observed using fluorescence microscopy [18].The treated mayonnaise was uniformly mixed with fluorescein isothiocyanate (FITC) and Nile red (dissolved in ethanol, 1 mg mL -1 ) at ratios of 100:1 and 100:3, respectively, in the dark. The stained mayonnaise was placed on a slide and observed under a 4×objective using an inverted fluorescence microscope. 2.9 Determination of Rheological Behavior of Mayonnaise The rheological behavior of mayonnaise was tested using a DHR-2 rheometer according to the method of Primacella et al. (2019) [19]. The mayonnaise was placed on the test bench, and a 40 mm parallel plate fixture was selected with 1% strain and a 1 mm gap between the sample and the fixture. The sample was equilibrated at 25℃ for 60 seconds. Frequency scans and viscosity scans were performed on the mayonnaise. The frequency range for the frequency scan was 0.1-50 Hz, and the shear rate range for the viscosity scan was 0.1-100 s -1 . 2.10 Data Processing and Statistical Methods All data were statistically analyzed using SPSS Statistics 21. The significance of differences was determined using Duncan's multiple comparison test (p < 0.05). Correlation analysis and other image processing were performed using Origin 2021 software. 3 RESULTS AND DISCUSSION 3.1 Effect of PLA 1 and PLA 2 on yolk emulsifiability Among phospholipases, PLA 1 and PLA 2 share the same enzyme substrate and product but act at different sites. PLA 1 specifically catalyzes acyl groups at the Sn-1 position of phospholipids, whereas PLA 2 targets acyl groups at the Sn-2 position [20]. Therefore, it is necessary to further screen phospholipases that are most effective in enhancing the emulsification of egg yolk liquids. To this end, we investigated the effects of varying concentrations of PLA 1 and PLA 2 on the emulsifying properties of egg yolk liquid. Fig. 1 illustrates the effects of PLA 1 and PLA 2 on the EAI and ESI of treated egg yolk liquid, respectively. As shown in the figure, the EAI of egg yolk liquid gradually increased with higher concentrations of PLA 1 and PLA 2 , indicating that phospholipase treatment significantly enhanced the EAI. Compared to PLA 1 , the EAI of PLA 2 -modified egg yolks was significantly higher at the same concentration, reaching a maximum at 2 U g -1 of PLA 2 . Beyond this concentration, no significant increase in EAI was observed. EAI was not significantly increased. The increase in EAI may be attributed to the higher hydrophilicity of lysophospholipids produced by phospholipase hydrolysis, as their higher hydrophilic-oleophilic balance (HLB) value makes them more suitable for oil-in-water emulsions[21].As shown in Fig. 1(b), the ESI of the PLA 2 group exhibited an increasing trend with higher concentrations, whereas the PLA 1 group showed an initial increase followed by a decrease, peaking at 2 U g -1 of PLA 1 . At the same concentration, the ESI of the PLA 2 group was superior to that of the PLA 1 group. It can be concluded that, at the same concentration, PLA 2 -modified egg yolk liquid exhibited superior emulsification compared to the PLA 1 group. Therefore, PLA 2 modification was selected for further experimentation. 3.2 Effect of PLA 2 on the stability of lactic dialysis The emulsification index reflects the equilibrium state between the oil phase and the continuous phase in an emulsion system, serving as a measure of its physical stability. An increase in the emulsion precipitation index indicates faster movement of oil droplets, promoting their aggregation and reducing the emulsion's physical stability [22]. As shown in Fig. 2(a), the emulsion stability improved progressively with higher PLA 2 concentrations. During the initial minutes, the emulsion precipitation index changed more rapidly. Emulsions with higher PLA 2 concentrations maintained a low emulsification index. The enhanced emulsion stability with increased PLA 2 addition can be explained by two factors: (1) Lysophospholipids produced by PLA 2 -mediated phospholipid digestion interact with lipoproteins, increasing the viscosity of the yolk solution [23], thereby slowing oil droplet migration and improving emulsion stability; (2) The generated lysophospholipids exhibit higher hydrophilicity, enabling faster adsorption at the oil-water interface [24]. 3.3 Effect of PLA 2 on the interfacial tension of egg yolk liquid Surface tension serves as an indicator of the adsorption, unfolding, and rearrangement rates of emulsifiers at the oil-water interface, providing insights into their emulsification performance [25]. As depicted in Fig. 2(b), the surface tension of PLA 2 -modified egg yolk liquid decreased over time, indicating enhanced emulsifier adsorption at the interface. The reduced interfacial tension of the modified egg yolk liquid, compared to the control, further demonstrated that PLA 2 enzymatic digestion enhanced its emulsification properties. The interfacial tension decreased with increasing PLA 2 concentration, reaching its minimum at 4 U g -1 of PLA 2 . A notable decrease in interfacial tension occurred within the first 200 s, primarily due to the rapid adsorption of the modified egg yolk liquid at the oil-water interface. The reduction in interfacial tension of the enzyme-modified egg yolk liquid was largely attributed to the production of lysophospholipids. Lysophospholipids, which exhibit superior emulsifying properties compared to phospholipids, adsorb more rapidly at the oil-water interface and form a densely packed adsorption layer, thereby reducing the interfacial tension of the egg yolk liquid [11]. 3.4 Effect of PLA 2 on contact angle of egg yolk liquid To investigate the wettability variation of PLA 2 -modified egg yolk liquid, the contact angle was measured by depositing water droplets onto lyophilized samples using a microsyringe. It is generally believed that when the contact angle θ＞ 90°, the water droplets can not wet the sample well and the sample is hydrophobic; when the contact angle θ is close to 90°, the sample presents the best hydrophilicity and hydrophobicity; when the contact angle θ＜ 90°, the water droplets can wet the sample well , which indicates that the sample presents hydrophilicity. Fig. 2(c) shows the effect of contact angle on egg yolk before and after PLA 2 modification. As can be seen from the figure, the contact angle of unmodified egg yolk was 112.9°, indicating hydrophobicity. With increasing PLA 2 concentration, enzymatic hydrolysis generated more lysophospholipids, raising hydrophilic group content and reducing the contact angle (transitioning from hydrophobic to hydrophilic). Previous studies confirmed that lower contact angles correlate with reduced interfacial tension [26], which aligns with our findings. The shift from hydrophobic to hydrophilic behavior improves the emulsification properties of egg yolk. 3.5 Effect of PLA 2 on the thermal stability of mayonnaise Diffusing Wave Spectroscopy (DWS) is an advanced light scattering technique that quantifies the Brownian motion of tracer particles in colloidal systems. Similar to Dynamic Light Scattering (DLS), DWS analyzes temporal intensity fluctuations of scattered light, which are statistically represented by a correlation function used to derive particle dynamics. The average photon transport free range, l*, has a value that depends on the particle size, particle concentration, and refractive index in the system [27].Bonnet et al. observed that oil droplet aggregation in sodium caseinate emulsions increases the photon transport distance between scattering events, thereby reducing L/l* [28]. Thus, DWS-measured L/l* values in microwave-treated mayonnaise reflect microscopic particle dynamics and, consequently, thermal stability. Fig.3illustrates the changes in the thermal stability of mayonnaise following microwave treatment. In Fig. 3(a), the L/l* value of mayonnaise prepared through PLA 2 enzymatic hydrolysis of egg yolk significantly increased compared to the control group, indicating an enhancement in the thermal stability of the mayonnaise. There was no significant difference in the thermal stability of mayonnaise when PLA 2 was added at 2 U g -1 - 4 U g -1 . Fig. 3(b) depicts the centrifugal oil separation in mayonnaise post-heat treatment . Centrifugation separated the mayonnaise into three distinct layers: upper oil, middle emulsion, and lower aqueous phase. Microwave treatment destabilizes the emulsion, causing foam collapse and oil droplet coalescence. With the increase of PLA 2 addition, the oil precipitated at the top was decreasing, and the thermal stability of mayonnaise was enhanced, which was consistent with the results of L/l* values. When the addition amount of PLA 2 was 4 U g -1 , the oil precipitated from the top could still be seen, and the thermal stability of mayonnaise could be further improved. 3.6 Response surface optimization validation experiment After analysis using Design-Expert 13 software, the optimal enzymatic process for enhancing the thermal stability of mayonnaise was determined to be as follows: the addition of PLA 2 at 2.3 U, the addition of Fla at 92.31 U, a temperature of 54.581℃, and a duration of 1.986 hours. The theoretical L/l* value for the thermal stability of mayonnaise was calculated to be 25.506. For practical application, the enzymatic process was adjusted to the following parameters: PLA 2 was added at 2.3 U, Fla at 92 U, the temperature was set to 55℃, and the time was extended to 2 hours. Validation experiments conducted under these conditions yielded a thermal stability L/l* value of 25.523 ± 0.14, which closely aligns with the theoretical value. This similarity indicates that the response surface model established in the present experiments possesses a high degree of reliability and can be effectively utilized in actual production. After the response surface optimization of the enzymatic process conditions, the thermal stability of the optimal solution (Optimal) was compared with that of mayonnaise prepared from modified egg yolk liquid of control, Fla, PLA 2 , PLA 2 +Fla groups and commercially available mayonnaise. As can be seen from Fig. 4, the L/l* values with centrifugal oil precipitation plots indicate that the Fla group, was lower than the PLA 2 , PLA 2 +Fla and Optimal groups, while the Optimal group exhibited a similar thermal stability as the PLA 2 +Fla group. 3.7 Microstructure of mayonnaise Fig. 5 illustrates the microstructure of mayonnaise prepared from enzymatically dissolved egg yolk. The thermal stability of mayonnaise produced from enzymatically hydrolyzed egg yolk was significantly enhanced, and the aggregation of proteins along with the flocculation of oil droplets was notably reduced. In the Fla group, larger protein aggregates and oil droplets were observed compared to the other modified groups. This phenomenon may be attributed to the limited amount of peptides generated by Fla hydrolysis, which stabilized the network structure and decreased the flocculation rate of the oil droplets, thereby improving Fla's thermal stability relative to the control group, albeit not as effectively as observed in the other three groups [29]. The Optimal group exhibited a similar trend to that of the PLA 2 +Fla group, characterized by an absence of significant protein aggregation and oil droplet flocculation. Both the Optimal group and the PLA 2 +Fla group demonstrated comparable characteristics, with no apparent protein aggregation or flocculation of oil droplets, and no significant difference in thermal stability was noted between the two groups. 3.8 Effect of temperature on the stability of mayonnaise Egg yolk is sensitive to heat; when the heating temperature exceeds 64℃, the yolk proteins denature, resulting in reduced emulsification and destabilization of the emulsion, manifested as delamination and oil precipitation [30]. Fig. 6(a) illustrates the change in the L/l* value of mayonnaise at varying temperatures, while Fig. 6(b) depicts the effects of different temperatures on the appearance of mayonnaise. As shown in the figures, with increasing temperature, the L/l* value of mayonnaise gradually decreases, and signs of structural collapse and fat precipitation become apparent. When the temperature is below 60℃, the L/l* value decreases slowly as temperature rises, with the molecules within the mayonnaise undergoing irregular thermal motion [31]. This motion prevents the droplets from maintaining the maximum geometrical limit of close packing, thereby affecting the stability of the mayonnaise, which retains its original structure in this temperature range without exhibiting significant fat precipitation. However, when the temperature increases from 60℃ to 70℃, the L/l* value decreases rapidly, as the denaturation of yolk proteins adversely affects their adsorption at the oil-water interface, leading to decreased stability of the mayonnaise. As the temperature continues to rise, the L/l* value of mayonnaise continues to decline, and a significant amount of oil is observed in the control group in Fig. 6(b). In contrast, no noticeable oil droplets are present in the Optimal group and PLA 2 +Fla group, indicating that these groups can withstand higher temperatures, which is advantageous for the application of mayonnaise in refrigerated frozen food. 3.9 Effect of enzymatic digestion on the rheological behavior of mayonnaise Fig. 7 illustrates the rheological properties of mayonnaise produced from enzymatically dissolved egg yolk. The viscosity of mayonnaise from this source increased, with the Optimal group exhibiting the highest viscosity. In comparison to the PLA 2 group, the Optimal group showed an increase in viscosity with the addition of PLA 2 , while the addition of Fla resulted in a decrease. This suggests that the viscosity increase attributed to PLA 2 outweighed the viscosity reduction caused by Fla. Additionally, Fig. 7(b) and 7(c) demonstrate that the G′and G″values in the Optimal group also increased, corroborating findings from previous studies. The results of temperature scanning are presented in Fig. 7(d). As the temperature increases from 20℃ to 90 ℃, the G″value exhibits a trend of first decreasing and then increasing. A peak in G″is observed when the temperature reaches 60 to 70 ℃, which is close to the denaturation temperature of egg yolk protein and is influenced by the molecular interactions between proteins [32]. The figure indicates that the G″ values for all enzyme-digested mayonnaise samples are greater than that of the control, suggesting that the structure of mayonnaise prepared with enzyme-digested egg yolk is stronger than that of mayonnaise prepared with the control enzyme. This enhancement is attributed to the disruption of the binding between proteins and lipids in lipoproteins, the rearrangement and aggregation of yolk proteins, and alterations in the structure of yolk proteins, which collectively improve thermal stability [33]. The curve in the figure shows a gradual decrease in G″within the temperature range of 20 to 60 ℃, followed by a rapid increase at the critical temperature, indicating a transition from a liquid-like to a solid-like state, consistent with findings from Laca [34].Wu's study further demonstrated that proteins gradually transform into a gel after the peak of G″, which occurs at the critical temperature [35]. As illustrated in the figure, the control group begins to transition from sol to gel at approximately 61 ℃, while the gelation temperature of the other samples is higher. In mayonnaise, thermal denaturation exposes the hydrophobic groups that are typically buried within the protein molecule, thereby facilitating subsequent peptide bonding and the formation of a gel network structure [36]. The aggregation of proteins leads to the formation of aggregates that disrupt the network structure of the mayonnaise, resulting in oil-water separation. Enzymatic digestion enhances the gel temperature of egg yolk protein, which is beneficial for improving the thermal stability of mayonnaise. 4 CONCLUSION In this study, we compared the effects of PLA 1 and PLA 2 on the emulsification properties of egg yolks. Our results indicated that PLA 2 significantly improved the EAI and ESI of egg yolk liquid, leading us to select PLA 2 for further experimentation. Through a screening process, we determined that the combination of the flavored protease Fla and PLA 2 was more effective. We established the optimal enzyme digestion process via response surface optimization, which involved the addition of 2.3 U of PLA 2 , 92 U of Fla, a temperature of 55℃, and a reaction time of 2 hours. This optimized process resulted in a significant enhancement of the thermal stability of mayonnaise. Moreover, the optimal enzymatic process (2.3 U of PLA 2 and 92 U of Fla) was confirmed to be reliable. Additionally, the combined enzymatic hydrolysis improved the microstructure of mayonnaise, enhanced its resistance to high temperatures, altered its rheological behavior, and increased the gelation temperature of egg yolk protein. These findings provide theoretical and technological support for optimizing mayonnaise performance and its application in the food industry. Declarations FUNDING This study was supported by National Natural Science Foundation of China. (32001725) CONFLICT OF INTEREST This manuscript contains no conflicts of interest at the time of submission, and all authors consent to its publication. We confirm that all authors have reviewed and approved the final submitted version. Furthermore, the experimental procedures described herein represent original research that has not been previously published in whole or in part in any other journal, nor is it currently under consideration by another publication. Author Contribution H. 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Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 27 Aug, 2025 Reviews received at journal 25 Aug, 2025 Reviews received at journal 22 Aug, 2025 Reviewers agreed at journal 22 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviews received at journal 19 Aug, 2025 Reviews received at journal 19 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers invited by journal 15 Aug, 2025 Editor assigned by journal 12 Aug, 2025 Submission checks completed at journal 12 Aug, 2025 First submitted to journal 03 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7286074","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503411174,"identity":"ad50cc7d-0577-4da0-af20-1e8c1248a927","order_by":0,"name":"Hetong Jin","email":"","orcid":"","institution":"Dalian Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Hetong","middleName":"","lastName":"Jin","suffix":""},{"id":503411175,"identity":"60e3fee1-b545-4579-a867-dc21084498f8","order_by":1,"name":"Songyi Lin","email":"","orcid":"","institution":"Dalian Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Songyi","middleName":"","lastName":"Lin","suffix":""},{"id":503411176,"identity":"a01102c9-26b3-4d9f-8ad3-eba516666ccd","order_by":2,"name":"Guangshun Jiang","email":"","orcid":"","institution":"Dalian Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Guangshun","middleName":"","lastName":"Jiang","suffix":""},{"id":503411177,"identity":"b33017cd-c54e-49e7-b4ba-12be3226655c","order_by":3,"name":"Kun Liu","email":"","orcid":"","institution":"Dalian Green Snow Egg Products Development Co., Ltd Dalian","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Liu","suffix":""},{"id":503411178,"identity":"a3235c99-66d5-40af-aaa9-789cf53fec73","order_by":4,"name":"Fujun Guo","email":"","orcid":"","institution":"Dalian Green Snow Egg Products Development Co., Ltd Dalian","correspondingAuthor":false,"prefix":"","firstName":"Fujun","middleName":"","lastName":"Guo","suffix":""},{"id":503411179,"identity":"f262287f-75e6-4fd9-a19f-97e7615b3f4c","order_by":5,"name":"Zhijie Bao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBAC+xmMDQYJDAxAxHzgwIcfRGhhRGhhSzw4s4coLRAaqIXH+DAHGxFamKWbGwoeVDDk8bf3fDjMwMMgzy92AL8WNpmDQIedYSiWOHN2w+ECCwbDmbMT8GvhkUhsMEhsY0hsuJG74fAMHoYEg9sEtEiAtfxjSJx/I+fBYR42IrQYgLU0MCRuuJHDQIKWhGMMxYZnjhkAA1mCsF/sZ6Q/M/xRw5And7z58YcPP2zk+aUJaAECNgMGhv9wvxFUDgLMD4hSNgpGwSgYBSMXAAAP2Ujhje32KwAAAABJRU5ErkJggg==","orcid":"","institution":"Dalian Polytechnic University","correspondingAuthor":true,"prefix":"","firstName":"Zhijie","middleName":"","lastName":"Bao","suffix":""}],"badges":[],"createdAt":"2025-08-04 02:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7286074/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7286074/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89682202,"identity":"54510947-edb2-4bf2-9e9c-dc3ca99edabe","added_by":"auto","created_at":"2025-08-22 14:52:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67782,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u003c/sub\u003e on the emulsifying properties of liquid egg yolk. (a): Emulsifying activity. (b):Emulsion stability.\u003c/p\u003e\n\u003cp\u003eDifferent uppercase letters indicate significant intra-group differences (P \u0026lt; 0.05), and lowercase letters indicate significant differences between groups (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/28d368ccd4a27fb1e4a8fef4.png"},{"id":89683093,"identity":"bd4904fc-75db-4547-83b6-4359266e2fb4","added_by":"auto","created_at":"2025-08-22 15:00:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171875,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of PLA\u003csub\u003e2 \u003c/sub\u003eon creaming index of egg yolk emulsion. (b) Changes of interfacial tension in egg yolk after PLA\u003csub\u003e2\u003c/sub\u003e modification. (c) Changes of contact angle in egg yolk after PLA\u003csub\u003e2\u003c/sub\u003e modification.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/d9052562b02916eaffa8579d.png"},{"id":89682204,"identity":"50b63a20-9973-4b8b-b73d-c40cc5918298","added_by":"auto","created_at":"2025-08-22 14:52:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182994,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability analysis of PLA\u003csub\u003e2 \u003c/sub\u003ehydrolyzed egg yolk mayonnaise.\u003c/p\u003e\n\u003cp\u003e(a) :L/l*. (b):Centrifugal oil extraction image.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/3b3ff222aa2d7159810734f3.png"},{"id":89683094,"identity":"f44bf20f-4db0-401d-9d15-4d876ec423c2","added_by":"auto","created_at":"2025-08-22 15:00:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209334,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability analysis of hydrolyzed egg yolk for preparing mayonnaise.\u003c/p\u003e\n\u003cp\u003e(a) :L/l*. (b):Centrifugal oil extraction image.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/eea2acd4a44321518f114511.png"},{"id":89682210,"identity":"e3bc361a-7a86-409e-9781-edaa0fdcad21","added_by":"auto","created_at":"2025-08-22 14:52:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":453585,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscope image of preparation of mayonnaise from hydrolyzed egg yolk.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/dd97bd8464ce98565f9cd151.png"},{"id":89682208,"identity":"64d8713a-dd83-4308-b887-4bd047351c87","added_by":"auto","created_at":"2025-08-22 14:52:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267525,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on the stability of mayonnaise.\u003c/p\u003e\n\u003cp\u003e(a) :L/l*.(b): Appearance change.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/62534e1e20af1f63eaed82b6.png"},{"id":89682211,"identity":"0200a3d8-3c61-4b02-94de-34c8e6f297d1","added_by":"auto","created_at":"2025-08-22 14:52:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":159981,"visible":true,"origin":"","legend":"\u003cp\u003eRheological properties of hydrolyzed egg yolk for preparing mayonnaise. (a): Viscosity.(b): Storage modulus.(c): Loss modulus.(d): Temperature sweep.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/3650b03b04b3696cdb4aa0f5.png"},{"id":89684670,"identity":"e4f9e091-5864-41c6-a6b3-b19ceb5a1fb0","added_by":"auto","created_at":"2025-08-22 15:16:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2081606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/3fea3759-6050-4833-a34c-25ef89fc95b0.pdf"},{"id":89682206,"identity":"e302a55c-f2d5-4be5-b5d9-bd4fee913453","added_by":"auto","created_at":"2025-08-22 14:52:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1292373,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7286074/v1/112bb8ed68d57fa4c22510a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Process optimization of thermal stability of mayonnaise based on phospholipase A2 (PLA2) and flavor protease compound enzymatic hydrolysis","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eIn the food industry, mayonnaise, an emulsified condiment has become deeply integrated into the preparation of various dishes due to its unique texture and flavor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. From everyday sandwiches and salads to a wide range of sauces, mayonnaise not only enhances the rich and mellow taste of food products but also plays a crucial role in maintaining the structural stability of food. However, during actual production, storage, and processing, mayonnaise often faces significant challenges, with thermal instability being a key issue. Kiosseoglou V (2003) reviewed the role of egg yolk proteins in the formation of gels and emulsions and emphasized the importance of their role in the stabilization of mayonnaise [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When subjected to thermal processing or high temperatures, mayonnaise is highly susceptible to undesirable phenomena such as oil phase separation, structural collapse, and a sharp decline in emulsification properties. Typically, consumers reheat ready-to-eat foods in microwave ovens, a convenient and quick method that does not compromise the overall structure of the food. However, mayonnaise may exhibit significant oil separation after microwave heating, which not only severely affects product quality but also significantly diminishes the consumer's eating experience [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, the application of mayonnaise in ready-to-eat foods has been greatly restricted, and the development of mayonnaise products with higher thermal stability has become an urgent issue.\u003c/p\u003e\u003cp\u003eIn the complex compositional system of mayonnaise, egg yolk undoubtedly occupies a central position. Anton M et al. (2010) emphasized the important role of phospholipids and proteins in the stability of emulsions in terms of the chemical composition and action function of egg yolks [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].Mcclements, Julian D (2007) reviewed the characterization of and methods for the stability of emulsions that can be used to assess egg yolk emulsion stability [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To enhance the stability of egg yolk emulsions, Xu R et al. (2024) investigated the effects of heat treatment on the stability and emulsification properties of egg yolk emulsions and analyzed the mechanisms behind oil phase separation and structural collapse [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Guilmineau, F et al. (2005) found that EY thermal denaturation altered the emulsification function and altered the properties of O/W emulsions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. There are various ways to improve the stability of mayonnaise, among them Mun S et al. (2009) explored the use of modified starch and xanthan gum to improve the thermal stability of low-fat mayonnaise [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Primacella M et al. (2019) improved mayonnaise thermal stability by hydrolyzing and treating frozen-thawed egg yolks as anticoagulant for the preparation of mayonnaise [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong these, PLA\u003csub\u003e2\u003c/sub\u003e has demonstrated significant potential in optimizing the properties of egg yolk emulsions by modifying egg yolk components. Guo Z et al. (2005) reviewed the application of PLA\u003csub\u003e2\u003c/sub\u003e in phospholipid modification, especially in functional foods and emulsion stability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Gazolu-Rusanova et al. (2020) conducted a comprehensive study on the enhancement of interfacial properties and emulsion stability in egg yolk systems through PLA\u003csub\u003e2\u003c/sub\u003e modification [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Meanwhile, the effect of complex enzymatic hydrolysis different proteases with PLA\u003csub\u003e2\u003c/sub\u003e has gradually become the focus of research. Jiang G et al. (2024) effectively improved the thermal stability of egg yolk emulsions by enzymatic hydrolysis of egg yolk emulsion by PLA\u003csub\u003e2\u003c/sub\u003e together with flavored protease through synergistic effect of complex enzymatic hydrolysis. Meanwhile, it was shown that the moderate addition of flavored protease could effectively reduce the surface hydrophobicity and interfacial tension of the egg yolk and improve the emulsification stability of egg yolk protein [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe previous study demonstrated that PLA\u003csub\u003e2\u003c/sub\u003e and Fla composite enzymatic digestion a improved the thermal stability of mayonnaise relative to PLA\u003csub\u003e2\u003c/sub\u003e modification alone. However, the previous composite enzymatic hydrolysis was based on a fixed PLA\u003csub\u003e2\u003c/sub\u003e addition, but the effect of changes in PLA\u003csub\u003e2\u003c/sub\u003e addition in the composite enzymatic hydrolysis on the thermal stability of mayonnaise remains unclear.\u003c/p\u003e\u003cp\u003eConsequently, this investigation aims to elucidate possible synergistic interactions between PLA\u003csub\u003e2\u003c/sub\u003e and flavor protease during co-digestion, with particular emphasis on their combined effects on emulsion stabilization. A Box-Benhnken central combination design experiment was used to optimize the enzymatic digestion process of egg yolk liquid by taking the L/l* value of the thermal stability of mayonnaise as the index of investigation, and the PLA\u003csub\u003e2\u003c/sub\u003e addition, Fla addition, enzymatic temperature and enzymatic time as the influencing factors in response surface experiments, so as to obtain the enzymatic process conditions of the egg yolk liquid when the thermal stability of mayonnaise was the best. Additionally, the effects of various factors in the enzymatic digestion process on the thermal stability of mayonnaise were analyzed to reveal potential synergistic interactions among these factors. Furthermore, the physical properties of the optimized mayonnaise were evaluated and analyzed to understand the impact of enzymatic digestion on its quality characteristics. This provides a solid theoretical foundation and practical technical solutions for innovating mayonnaise production processes, thereby promoting its broader and deeper application in the food industry.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cp\u003e2.1 Experimental Materials\u003c/p\u003e\n\u003cp\u003eFresh eggs, sugar, salt, and Jiusan soybean oil were purchased from local supermarkets in Dalian, China. Flavored protease (500 LAPU g \u003csup\u003e-1\u003c/sup\u003e) was obtained from Novozymes Biologicals Ltd. (Novozymes, Denmark), while PLA\u003csub\u003e1\u003c/sub\u003e (10,000 U mL\u003csup\u003e-1\u003c/sup\u003e) and PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(10,000 U mL\u003csup\u003e-1\u003c/sup\u003e) were sourced from Neova Technologies Ltd. (Neova, Canada).\u003c/p\u003e\n\u003cp\u003e2.2 Preparation of Egg Yolks and Mayonnaise\u003c/p\u003e\n\u003cp\u003e2.2.1 Preparation of Egg Yolks\u003c/p\u003e\n\u003cp\u003eFresh eggs were washed and dried. The eggshells were cracked, and the egg whites were separated from the yolks. Excess egg white was removed, and the yolk liquid was collected into a beaker after puncturing the yolk membrane with a toothpick. The yolk liquid was then stirred at 100 rpm for 30 minutes at room temperature and prepared for further use.\u003c/p\u003e\n\u003cp\u003e2.2.2 Preparation of Mayonnaise\u003c/p\u003e\n\u003cp\u003eMayonnaise was prepared according to the method described by Kim et al. (2009) with reference to Table 1 [13].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eWater, sugar, salt, and the enzymatic egg yolk solution were thoroughly mixed and homogenized at 6000 rpm for 4 minutes using a homogenizer. Oil was gradually added during the homogenization process. Finally, 5% acetic acid was added, and the mixture was homogenized again at 8000 rpm for 3 minutes.\u003c/p\u003e\n\u003cp\u003eTable 1 Mayonnaise recipes\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eIngredients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eegg yolk\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003esugar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003esalt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003ewater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e5% acetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eoil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eg / 100 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e2.3 Determination of Emulsifying Activity and Emulsion Stability of Egg Yolk Solution\u003c/p\u003e\n\u003cp\u003eThe EAI and emulsion stability ESI of the modified egg yolk solution were determined following the method of Zhang et al. (2012) with slight modifications [14].The enzymatically digested egg yolk solution was prepared as an 8% yolk mass aqueous solution. The diluted yolk solution was mixed with soybean oil at a 3:1 ratio and dispersed in a high-speed shear mixer for 2 minutes (10,000 rpm). After coarse dispersion, 20 \u0026mu;L of the emulsion was aspirated from the bottom and mixed with 5 mL of 0.1% SDS solution. The absorbance value A\u003csub\u003e0\u003c/sub\u003e was measured at 500 nm using 0.1% SDS as a blank control. The same procedure was repeated after 30 minutes of standing, and the absorbance value A\u003csub\u003et\u003c/sub\u003e was measured at 500 nm.\u003c/p\u003e\n\u003cp\u003eThe EAI was calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"207\" height=\"30\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(1.1)\u003c/p\u003e\n\u003cp\u003eT = 2.303, N is the dilution factor 250, C protein concentration in g/mL, and \u0026phi; is the fraction of oil in the emulsion, which is 25%.\u003c/p\u003e\n\u003cp\u003eThe ESI was calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"163\" height=\"30\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(1.2)\u003c/p\u003e\n\u003cp\u003e2.4 Determination of Lactic Precipitation Index\u003c/p\u003e\n\u003cp\u003eThe lactic precipitation index was determined according to the method of Chen et al. (2018) with slight modifications [15].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eA 100 mL emulsion was transferred to a measuring cylinder and allowed to stand at room temperature for 4 hours. The volume of the lower clear liquid was recorded at 15-minute intervals, and the lactic precipitation index was calculated. A curve of the lactic precipitation index over time was plotted.\u003c/p\u003e\n\u003cp\u003eThe lactic precipitation index was calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"436\" height=\"30\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;(1.3)\u003c/p\u003e\n\u003cp\u003e2.5 Determination of Interfacial Tension\u003c/p\u003e\n\u003cp\u003eInterfacial tension was determined using a contact angle meter according to the method of Tabor et al. (2015) [16].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe egg yolk solution was thoroughly mixed with sugar and salt and then diluted five-fold. The densities of soybean oil and the diluted sample were measured using the weighing method. Soybean oil was filled into a glass dish, and the syringe was loaded with the sample. The tension of the egg yolk solution at the oil-water interface was measured using the hanging drop method over 1600 seconds.\u003c/p\u003e\n\u003cp\u003e2.6 Determination of Three-Phase Contact Angle\u003c/p\u003e\n\u003cp\u003eThe three-phase contact angle of the PLA\u003csub\u003e2\u003c/sub\u003e-modified egg yolk solution was measured using a contact angle meter [17]. Lyophilized egg yolk powder was pressed into discs with a diameter of 1 cm and a height of 1 mm. The discs were immersed in soybean oil, removed, and placed on a slide. A 5\u0026mu;L water droplet was added to the surface of the discs, and images of the droplets were captured using a camera. The three-phase contact angles of different samples were obtained by measuring the angles through software, and the fitting equation was the Young-Laplace equation.\u003c/p\u003e\n\u003cp\u003e2.7 Determination of Thermal Stability of Mayonnaise\u003c/p\u003e\n\u003cp\u003eThe thermal stability of mayonnaise was determined by accurately weighing 10 g of mayonnaise into a 15 mL centrifuge tube for microwave heating. The microwave frequency was set to 50 Hz, and the power was 550 W. The temperature of the mayonnaise was measured using an infrared thermal imager. Heating was stopped when the temperature reached 70℃. After microwave heating, the mayonnaise was centrifuged at 6000 g for 20 minutes, and the oil separated from the upper layer was observed.\u003c/p\u003e\n\u003cp\u003e2.8 Determination of Microstructure of Mayonnaise\u003c/p\u003e\n\u003cp\u003eThe microstructure of microwave-treated mayonnaise was observed using fluorescence microscopy [18].The treated mayonnaise was uniformly mixed with fluorescein isothiocyanate (FITC) and Nile red (dissolved in ethanol, 1 mg mL\u003csup\u003e-1\u003c/sup\u003e) at ratios of 100:1 and 100:3, respectively, in the dark. The stained mayonnaise was placed on a slide and observed under a 4\u0026times;objective using an inverted fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e2.9 Determination of Rheological Behavior of Mayonnaise\u003c/p\u003e\n\u003cp\u003eThe rheological behavior of mayonnaise was tested using a DHR-2 rheometer according to the method of Primacella et al. (2019) [19].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe mayonnaise was placed on the test bench, and a 40 mm parallel plate fixture was selected with 1% strain and a 1 mm gap between the sample and the fixture. The sample was equilibrated at 25℃\u0026nbsp;for 60 seconds. Frequency scans and viscosity scans were performed on the mayonnaise. The frequency range for the frequency scan was 0.1-50 Hz, and the shear rate range for the viscosity scan was 0.1-100 s\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e2.10 Data Processing and Statistical Methods\u003c/p\u003e\n\u003cp\u003eAll data were statistically analyzed using SPSS Statistics 21. The significance of differences was determined using Duncan\u0026apos;s multiple comparison test (p \u0026lt; 0.05). Correlation analysis and other image processing were performed using Origin 2021 software.\u003c/p\u003e"},{"header":"3 RESULTS AND DISCUSSION","content":"\u003cp\u003e3.1 Effect of PLA\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eand PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon yolk emulsifiability\u003c/p\u003e\n\u003cp\u003eAmong phospholipases, PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u003c/sub\u003e share the same enzyme substrate and product but act at different sites. PLA\u003csub\u003e1\u003c/sub\u003e specifically catalyzes acyl groups at the Sn-1 position of phospholipids, whereas PLA\u003csub\u003e2\u003c/sub\u003e targets acyl groups at the Sn-2 position [20].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTherefore, it is necessary to further screen phospholipases that are most effective in enhancing the emulsification of egg yolk liquids. To this end, we investigated the effects of varying concentrations of PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon the emulsifying properties of egg yolk liquid.\u0026nbsp;Fig. 1 illustrates the effects of PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon the EAI and ESI of treated egg yolk liquid, respectively. As shown in the figure, the EAI of egg yolk liquid gradually increased with higher concentrations of PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u003c/sub\u003e, indicating that phospholipase treatment significantly enhanced the EAI. Compared to PLA\u003csub\u003e1\u003c/sub\u003e, the EAI of PLA\u003csub\u003e2\u003c/sub\u003e-modified egg yolks was significantly higher at the same concentration, reaching a maximum at 2 U g \u003csup\u003e-1\u003c/sup\u003e of PLA\u003csub\u003e2\u003c/sub\u003e. Beyond this concentration, no significant increase in EAI was observed. EAI was not significantly increased. The increase in EAI may be attributed to the higher hydrophilicity of lysophospholipids produced by phospholipase hydrolysis, as their higher hydrophilic-oleophilic balance (HLB) value makes them more suitable for oil-in-water emulsions[21].As shown in Fig. 1(b), the ESI of the PLA\u003csub\u003e2\u003c/sub\u003e group exhibited an increasing trend with higher concentrations, whereas the PLA\u003csub\u003e1\u003c/sub\u003e group showed an initial increase followed by a decrease, peaking at 2 U g \u003csup\u003e-1\u003c/sup\u003e of PLA\u003csub\u003e1\u003c/sub\u003e. At the same concentration, the ESI of the PLA\u003csub\u003e2\u003c/sub\u003e group was superior to that of the PLA\u003csub\u003e1\u003c/sub\u003e group. It can be concluded that, at the same concentration, PLA\u003csub\u003e2\u003c/sub\u003e-modified egg yolk liquid exhibited superior emulsification compared to the PLA\u003csub\u003e1\u003c/sub\u003e group. Therefore, PLA\u003csub\u003e2\u003c/sub\u003e modification was selected for further experimentation.\u003c/p\u003e\n\u003cp\u003e3.2 Effect of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon the stability of lactic dialysis\u003c/p\u003e\n\u003cp\u003eThe emulsification index reflects the equilibrium state between the oil phase and the continuous phase in an emulsion system, serving as a measure of its physical stability. An increase in the emulsion precipitation index indicates faster movement of oil droplets, promoting their aggregation and reducing the emulsion\u0026apos;s physical stability [22].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eAs shown in Fig. 2(a), the emulsion stability improved progressively with higher PLA\u003csub\u003e2\u003c/sub\u003e concentrations. During the initial minutes, the emulsion precipitation index changed more rapidly. Emulsions with higher PLA\u003csub\u003e2\u003c/sub\u003e concentrations maintained a low emulsification index. The enhanced emulsion stability with increased PLA\u003csub\u003e2\u003c/sub\u003e addition can be explained by two factors: (1) Lysophospholipids produced by PLA\u003csub\u003e2\u003c/sub\u003e-mediated phospholipid digestion interact with lipoproteins, increasing the viscosity of the yolk solution [23], thereby slowing oil droplet migration and improving emulsion stability; (2) The generated lysophospholipids exhibit higher hydrophilicity, enabling faster adsorption at the oil-water interface [24].\u003c/p\u003e\n\u003cp\u003e3.3 Effect of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon the interfacial tension of egg yolk liquid\u003c/p\u003e\n\u003cp\u003eSurface tension serves as an indicator of the adsorption, unfolding, and rearrangement rates of emulsifiers at the oil-water interface, providing insights into their emulsification performance [25].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eAs depicted in Fig. 2(b), the surface tension of PLA\u003csub\u003e2\u003c/sub\u003e-modified egg yolk liquid decreased over time, indicating enhanced emulsifier adsorption at the interface. The reduced interfacial tension of the modified egg yolk liquid, compared to the control, further demonstrated that PLA\u003csub\u003e2\u003c/sub\u003e enzymatic digestion enhanced its emulsification properties. The interfacial tension decreased with increasing PLA\u003csub\u003e2\u003c/sub\u003e concentration, reaching its minimum at 4 U g\u003csup\u003e-1\u003c/sup\u003e of PLA\u003csub\u003e2\u003c/sub\u003e. A notable decrease in interfacial tension occurred within the first 200 s, primarily due to the rapid adsorption of the modified egg yolk liquid at the oil-water interface. The reduction in interfacial tension of the enzyme-modified egg yolk liquid was largely attributed to the production of lysophospholipids. Lysophospholipids, which exhibit superior emulsifying properties compared to phospholipids, adsorb more rapidly at the oil-water interface and form a densely packed adsorption layer, thereby reducing the interfacial tension of the egg yolk liquid [11].\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e3.4 Effect of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon contact angle of egg yolk liquid\u003c/p\u003e\n\u003cp\u003eTo investigate the wettability variation of PLA\u003csub\u003e2\u003c/sub\u003e-modified egg yolk liquid, the contact angle was measured by depositing water droplets onto lyophilized samples using a microsyringe. It is generally believed that when the contact angle \u0026theta;＞\u0026nbsp;90\u0026deg;, the water droplets can not wet the sample well and the sample is hydrophobic; when the contact angle \u0026theta; is close to 90\u0026deg;, the sample presents the best hydrophilicity and hydrophobicity; when the contact angle \u0026theta;＜\u0026nbsp;90\u0026deg;, the water droplets can wet the sample well , which indicates that the sample presents hydrophilicity. Fig. 2(c) shows the effect of contact angle on egg yolk before and after PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003emodification. As can be seen from the figure, the contact angle of unmodified egg yolk was 112.9\u0026deg;, indicating hydrophobicity. With increasing PLA\u003csub\u003e2\u003c/sub\u003e concentration, enzymatic hydrolysis generated more lysophospholipids, raising hydrophilic group content and reducing the contact angle (transitioning from hydrophobic to hydrophilic). Previous studies confirmed that lower contact angles correlate with reduced interfacial tension [26],\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewhich aligns with our findings. The shift from hydrophobic to hydrophilic behavior improves the emulsification properties of egg yolk.\u003c/p\u003e\n\u003cp\u003e3.5 Effect of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eon the thermal stability of mayonnaise\u003c/p\u003e\n\u003cp\u003eDiffusing Wave Spectroscopy (DWS) is an advanced light scattering technique that quantifies the Brownian motion of tracer particles in colloidal systems. Similar to Dynamic Light Scattering (DLS), DWS analyzes temporal intensity fluctuations of scattered light, which are statistically represented by a correlation function used to derive particle dynamics. The average photon transport free range, l*, has a value that depends on the particle size, particle concentration, and refractive index in the system [27].Bonnet et al.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eobserved that oil droplet aggregation in sodium caseinate emulsions increases the photon transport distance between scattering events, thereby reducing L/l* [28].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThus, DWS-measured L/l* values in microwave-treated mayonnaise reflect microscopic particle dynamics and, consequently, thermal stability.\u003c/p\u003e\n\u003cp\u003eFig.3illustrates the changes in the thermal stability of mayonnaise following microwave treatment. In Fig. 3(a), the L/l* value of mayonnaise prepared through PLA\u003csub\u003e2\u003c/sub\u003e enzymatic hydrolysis of egg yolk significantly increased compared to the control group, indicating an enhancement in the thermal stability of the mayonnaise. There was no significant difference in the thermal stability of mayonnaise when PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas added at 2 U g \u003csup\u003e-1\u003c/sup\u003e - 4 U g \u003csup\u003e-1\u003c/sup\u003e. Fig. 3(b) depicts the centrifugal oil separation in mayonnaise post-heat treatment . Centrifugation separated the mayonnaise into three distinct layers: upper oil, middle emulsion, and lower aqueous phase. Microwave treatment destabilizes the emulsion, causing foam collapse and oil droplet coalescence. With the increase of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eaddition, the oil precipitated at the top was decreasing, and the thermal stability of mayonnaise was enhanced, which was consistent with the results of L/l* values. When the addition amount of PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas 4 U g \u003csup\u003e-1\u003c/sup\u003e, the oil precipitated from the top could still be seen, and the thermal stability of mayonnaise could be further improved.\u003c/p\u003e\n\u003cp\u003e3.6 Response surface optimization validation experiment\u003c/p\u003e\n\u003cp\u003eAfter analysis using Design-Expert 13 software, the optimal enzymatic process for enhancing the thermal stability of mayonnaise was determined to be as follows: the addition of PLA\u003csub\u003e2\u003c/sub\u003e at 2.3 U, the addition of Fla at 92.31 U, a temperature of 54.581℃, and a duration of 1.986 hours. The theoretical L/l* value for the thermal stability of mayonnaise was calculated to be 25.506. For practical application, the enzymatic process was adjusted to the following parameters: PLA\u003csub\u003e2\u003c/sub\u003e was added at 2.3 U, Fla at 92 U, the temperature was set to 55℃, and the time was extended to 2 hours. Validation experiments conducted under these conditions yielded a thermal stability L/l* value of 25.523\u0026nbsp;\u0026plusmn;\u0026nbsp;0.14, which closely aligns with the theoretical value. This similarity indicates that the response surface model established in the present experiments possesses a high degree of reliability and can be effectively utilized in actual production.\u003c/p\u003e\n\u003cp\u003eAfter the response surface optimization of the enzymatic process conditions, the thermal stability of the optimal solution (Optimal) was compared with that of mayonnaise prepared from modified egg yolk liquid of control, Fla, PLA\u003csub\u003e2\u003c/sub\u003e, PLA\u003csub\u003e2\u003c/sub\u003e+Fla groups and commercially available mayonnaise. As can be seen from Fig. 4, the L/l* values with centrifugal oil precipitation plots indicate that the Fla group, was lower than the PLA\u003csub\u003e2\u003c/sub\u003e, PLA\u003csub\u003e2\u003c/sub\u003e+Fla and Optimal groups, while the Optimal group exhibited a similar thermal stability as the PLA\u003csub\u003e2\u003c/sub\u003e+Fla group.\u003c/p\u003e\n\u003cp\u003e3.7 Microstructure of mayonnaise\u003c/p\u003e\n\u003cp\u003eFig. 5 illustrates the microstructure of mayonnaise prepared from enzymatically dissolved egg yolk. The thermal stability of mayonnaise produced from enzymatically hydrolyzed egg yolk was significantly enhanced, and the aggregation of proteins along with the flocculation of oil droplets was notably reduced. In the Fla group, larger protein aggregates and oil droplets were observed compared to the other modified groups. This phenomenon may be attributed to the limited amount of peptides generated by Fla hydrolysis, which stabilized the network structure and decreased the flocculation rate of the oil droplets, thereby improving Fla\u0026apos;s thermal stability relative to the control group, albeit not as effectively as observed in the other three groups [29].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe Optimal group exhibited a similar trend to that of the PLA\u003csub\u003e2\u003c/sub\u003e+Fla group, characterized by an absence of significant protein aggregation and oil droplet flocculation. Both the Optimal group and the PLA\u003csub\u003e2\u003c/sub\u003e+Fla group demonstrated comparable characteristics, with no apparent protein aggregation or flocculation of oil droplets, and no significant difference in thermal stability was noted between the two groups.\u003c/p\u003e\n\u003cp\u003e3.8 Effect of temperature on the stability of mayonnaise\u003c/p\u003e\n\u003cp\u003eEgg yolk is sensitive to heat; when the heating temperature exceeds 64℃, the yolk proteins denature, resulting in reduced emulsification and destabilization of the emulsion, manifested as delamination and oil precipitation [30].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eFig. 6(a) illustrates the change in the L/l* value of mayonnaise at varying temperatures, while Fig. 6(b) depicts the effects of different temperatures on the appearance of mayonnaise. As shown in the figures, with increasing temperature, the L/l* value of mayonnaise gradually decreases, and signs of structural collapse and fat precipitation become apparent. When the temperature is below 60℃, the L/l* value decreases slowly as temperature rises, with the molecules within the mayonnaise undergoing irregular thermal motion [31].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThis motion prevents the droplets from maintaining the maximum geometrical limit of close packing, thereby affecting the stability of the mayonnaise, which retains its original structure in this temperature range without exhibiting significant fat precipitation. However, when the temperature increases from 60℃ to 70℃, the L/l* value decreases rapidly, as the denaturation of yolk proteins adversely affects their adsorption at the oil-water interface, leading to decreased stability of the mayonnaise. As the temperature continues to rise, the L/l* value of mayonnaise continues to decline, and a significant amount of oil is observed in the control group in Fig. 6(b). In contrast, no noticeable oil droplets are present in the Optimal group and PLA\u003csub\u003e2\u003c/sub\u003e+Fla group, indicating that these groups can withstand higher temperatures, which is advantageous for the application of mayonnaise in refrigerated frozen food.\u003c/p\u003e\n\u003cp\u003e3.9 Effect of enzymatic digestion on the rheological behavior of mayonnaise\u003c/p\u003e\n\u003cp\u003eFig. 7 illustrates the rheological properties of mayonnaise produced from enzymatically dissolved egg yolk. The viscosity of mayonnaise from this source increased, with the Optimal group exhibiting the highest viscosity. In comparison to the PLA\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egroup, the Optimal group showed an increase in viscosity with the addition of PLA\u003csub\u003e2\u003c/sub\u003e, while the addition of Fla resulted in a decrease. This suggests that the viscosity increase attributed to PLA\u003csub\u003e2\u003c/sub\u003e outweighed the viscosity reduction caused by Fla. Additionally, Fig. 7(b) and 7(c) demonstrate that the G\u0026prime;and G\u0026Prime;values in the Optimal group also increased, corroborating findings from previous studies.\u003c/p\u003e\n\u003cp\u003eThe results of temperature scanning are presented in Fig. 7(d). As the temperature increases from 20℃\u0026nbsp;to 90\u0026nbsp;℃, the G\u0026Prime;value exhibits a trend of first decreasing and then increasing. A peak in G\u0026Prime;is observed when the temperature reaches 60 to 70\u0026nbsp;℃, which is close to the denaturation temperature of egg yolk protein and is influenced by the molecular interactions between proteins [32].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe figure indicates that the G\u0026Prime;\u0026nbsp;values for all enzyme-digested mayonnaise samples are greater than that of the control, suggesting that the structure of mayonnaise prepared with enzyme-digested egg yolk is stronger than that of mayonnaise prepared with the control enzyme. This enhancement is attributed to the disruption of the binding between proteins and lipids in lipoproteins, the rearrangement and aggregation of yolk proteins, and alterations in the structure of yolk proteins, which collectively improve thermal stability [33].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe curve in the figure shows a gradual decrease in G\u0026Prime;within the temperature range of 20 to 60\u0026nbsp;℃, followed by a rapid increase at the critical temperature, indicating a transition from a liquid-like to a solid-like state, consistent with findings from Laca [34].Wu\u0026apos;s study further demonstrated that proteins gradually transform into a gel after the peak of G\u0026Prime;, which occurs at the critical temperature [35]. As illustrated in the figure, the control group begins to transition from sol to gel at approximately 61\u0026nbsp;℃, while the gelation temperature of the other samples is higher. In mayonnaise, thermal denaturation exposes the hydrophobic groups that are typically buried within the protein molecule, thereby facilitating subsequent peptide bonding and the formation of a gel network structure [36].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe aggregation of proteins leads to the formation of aggregates that disrupt the network structure of the mayonnaise, resulting in oil-water separation. Enzymatic digestion enhances the gel temperature of egg yolk protein, which is beneficial for improving the thermal stability of mayonnaise.\u003c/p\u003e"},{"header":"4 CONCLUSION","content":"\u003cp\u003eIn this study, we compared the effects of PLA\u003csub\u003e1\u003c/sub\u003e and PLA\u003csub\u003e2\u003c/sub\u003e on the emulsification properties of egg yolks. Our results indicated that PLA\u003csub\u003e2\u003c/sub\u003e significantly improved the EAI and ESI of egg yolk liquid, leading us to select PLA\u003csub\u003e2\u003c/sub\u003e for further experimentation. Through a screening process, we determined that the combination of the flavored protease Fla and PLA\u003csub\u003e2\u003c/sub\u003e was more effective. We established the optimal enzyme digestion process via response surface optimization, which involved the addition of 2.3 U of PLA\u003csub\u003e2\u003c/sub\u003e, 92 U of Fla, a temperature of 55℃, and a reaction time of 2 hours. This optimized process resulted in a significant enhancement of the thermal stability of mayonnaise. Moreover, the optimal enzymatic process (2.3 U of PLA\u003csub\u003e2\u003c/sub\u003e and 92 U of Fla) was confirmed to be reliable. Additionally, the combined enzymatic hydrolysis improved the microstructure of mayonnaise, enhanced its resistance to high temperatures, altered its rheological behavior, and increased the gelation temperature of egg yolk protein. These findings provide theoretical and technological support for optimizing mayonnaise performance and its application in the food industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis study was supported by National Natural Science Foundation of China. (32001725)\u003c/p\u003e\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\u003cp\u003eThis manuscript contains no conflicts of interest at the time of submission, and all authors consent to its publication. We confirm that all authors have reviewed and approved the final submitted version. Furthermore, the experimental procedures described herein represent original research that has not been previously published in whole or in part in any other journal, nor is it currently under consideration by another publication.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH. J.：Writing - Original Draft; Validation Verification; Formal analysis.S. L.：Conceptualization; Supervision; Project administration.G. J.：Methodology; Visualization.K. L.: Investigation; Resources.F. G.： Methodology; Resources.Z. B.：Writing - Review \u0026amp; Editing; Supervision; Funding acquisition.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciate the National Natural Science Foundation of China (32001725) for funding this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ.A Depree \u0026amp; G.P Savage.(2001).Physical and flavour stability of mayonnaise.Trends in Food Science \u0026amp; Technology,12(5-6),157-163.\u003c/li\u003e\n\u003cli\u003eV Kiosseoglou.(2003).Egg yolk protein gels and emulsions.Current Opinion in Colloid \u0026amp; Interface Science,8(4),365-370.\u003c/li\u003e\n\u003cli\u003eRaffaella Inchingolo,Vladimiro Cardenia \u0026amp; Maria Teresa Rodriguez‐Estrada.(2013).The effects of microwave heating on edible oils and lipid‐containing food.Lipid Technology,25(3),59-61.\u003c/li\u003e\n\u003cli\u003eAnton, M. , \u0026amp; Gandemer, G. . (2010). 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Characterization of fractions.Food Hydrocolloids,24(4),434-443.\u003c/li\u003e\n\u003cli\u003eDan Wu,Jian Xiong,Pei Li,Yan Zhang,Fan Li,Tao Yin \u0026amp; Qilin Huang.(2024).Dual enhancement effects of different yeast extract on gel properties and saltiness perception of low-salt surimi gel from silver carp.Food Hydrocolloids,152,109925-.\u003c/li\u003e\n\u003cli\u003eLiyan Huang,Tong Wang,Zhaopeng Han,Yanli Meng \u0026amp; Xiaoming Lu.(2016).Effect of egg yolk freezing on properties of mayonnaise.Food Hydrocolloids,56,311-317.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"egg yolk liquid, mayonnaise, phospholipase, protease, thermal stability","lastPublishedDoi":"10.21203/rs.3.rs-7286074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7286074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere have been a number of research reports on improving the stability of egg yolk emulsions at home and abroad, while relatively few studies have been conducted on the stability of high internal phase emulsions such as mayonnaise. This study aims to enhance the thermal stability of mayonnaise through composite enzymatic hydrolysis, addressing issues such as oil-phase separation and structural collapse that commonly occur during thermal processing. Comparison of phospholipase A\u003csub\u003e1 \u003c/sub\u003e(PLA\u003csub\u003e1\u003c/sub\u003e) and phospholipase A\u003csub\u003e2\u003c/sub\u003e (PLA\u003csub\u003e2\u003c/sub\u003e) showed that PLA\u003csub\u003e2 \u003c/sub\u003ewas more effective in improving emulsion activity (EAI) and emulsion stability (ESI). Subsequently, the response surface methodology was employed to optimize the composite enzymatic digestion process of PLA\u003csub\u003e2\u003c/sub\u003e and flavor protease (Fla), with the optimal conditions determined as 2.3 U of PLA\u003csub\u003e2\u003c/sub\u003e, 92 U of Fla, a temperature of 55℃, and a duration of 2 h. Meanwhile, the effects of the primary term PLA\u003csub\u003e2 \u003c/sub\u003eaddition and the interactive terms PLA\u003csub\u003e2\u003c/sub\u003e addition and Fla addition as well as optimal digestion on mayonnaise were investigated. The results showed that the optimized enzymatic hydrolysis process significantly improved the thermal stability of mayonnaise, with an Lightness (L/l*) value of 25.523±0.14. Furthermore, texture and rheological analyses indicated that mayonnaise prepared under optimal enzymatic conditions exhibited greater hardness and stronger force, which is beneficial for its spreadability.This study provides theoretical basis and practical and feasible technical solutions for the production and quality improvement of mayonnaise, and helps its application in more fields, such as refrigerated and frozen foods.\u003c/p\u003e","manuscriptTitle":"Process optimization of thermal stability of mayonnaise based on phospholipase A2 (PLA2) and flavor protease compound enzymatic hydrolysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 14:52:41","doi":"10.21203/rs.3.rs-7286074/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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