Visualization of fluorescently labeled lipase distribution characteristics at the oil–water interface

Visualization of fluorescently labeled lipase distribution characteristics at the oil–water interface | 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 Visualization of fluorescently labeled lipase distribution characteristics at the oil–water interface Xian Du, Chunxiao Zhang, Biyu Peng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5758102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract A method based on fluorescently labeled enzyme proteins was established to visualize the absorption properties of lipase at the oil–water interface, and it can be used for the effective observation of the distribution characteristics of lipase at the oil–water interface. The optimal conditions for observation include the following: oil content of 10–20% (wt%), concentration of fluorescently labelled enzyme protein of 0.25 mg/mL, reaction temperature of 25℃–30°C, emulsion dispersion and stirring time of 10 min, and emulsion resting time of 30–120 s. Based on this method, a preliminary analysis of the effects of oil and lipase species on the distribution characteristics of lipase at the oil–water interface was performed. The results reveal that differences in the distributions of lipase at the oil–water interface of various fats and oils had a certain degree of correspondence with their specificity and that the distribution characteristics of the lipases on the surface of olive oil enabled effective catalytic hydrolysis to a certain extent. This method is a more objective guide for the development of lipase application technology in the fields of tanning, fur making, glue making, detergents, sewage treatment and so on. Lipase Interfacial catalysis fluorescent labelling oil–water interface affinity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Lipase (EC 3.1.1.3) is an ester bond hydrolase[ 1 , 2 ] that can catalyze the hydrolysis of triglycerides into diglycerides, monoesters, glycerol and fatty acids under certain conditions[ 3 ]; with the advantages of mild and nontoxic conditions, it prevents the destruction of the main components at high temperatures and in strong alkalis and has been widely used in tannery[ 4 ], washing, wastewater treatment, food processing[ 5 , 6 ], pulp and paper[ 7 ], fine chemicals synthesis[ 8 ], and other industries; this enzyme is important for the improvement of product quality, reduced usage of surfactants and organic solvents, increased efficiency of sewage treatment, and enhanced recycling of oil and grease and their hydrolysis products. The role of lipases in lipid degreasing is shown in Fig. 1 . The main role played by lipases in lipid processing is the hydrolysis reaction, i.e., the breakdown of fats (mainly triacylglycerols) into fatty acids and glycerol. This process is an important part of fat metabolism, and lipase releases long-chain fatty acids from fats through an enzyme-catalyzed hydrolysis reaction. Lipase breaks down triacylglycerols into glycerol and fatty acids by binding to ester bonds in the fat molecule. The products of this reaction can be used in subsequent processes such as fat extraction, modification or synthesis of other chemicals. In leather processing, lipases are commonly used to remove excess fat from the surface of the leather, which helps to minimize hardening and improve the softness of the leather. Lipases also regulate the hydration of leather and enhance its dyeing and coating. The application of lipase in leather processing can improve the efficiency of fat processing and reduce the by-products and environmental pollution that may be caused by traditional chemical methods (e.g. acid hydrolysis). Secondly, the mild reaction conditions of lipase usually do not require high temperatures or a strongly acidic environment, making it more environmentally friendly, energy efficient and conducive to the sustainable use of resources. In addition, lipase can effectively prevent oil-water separation by improving the stability of water-in-oil emulsions. Water-in-oil emulsions (W/O emulsions) are an important emulsification system widely used in food, cosmetics, pharmaceuticals, and leather processing. Lipases change the size and distribution of lipid particles by hydrolyzing the ester bonds in fat molecules, resulting in the breakdown of larger fat particles into smaller fatty acid and glycerol molecules. These smaller molecules are better dispersed in the aqueous phase, reducing aggregation between particles. In this way, lipase helps to form more homogeneous and stable emulsions; secondly, the catalyzing action of lipase alters the interfacial properties between the fat and aqueous phases, reducing the interfacial tension and thus enhancing the stability of the emulsion. Stabilized emulsions can effectively prevent the separation of oil and water and extend the shelf life of the emulsion. Lipase enhances emulsification and facilitates emulsion formation by changing the molecular structure and size of the fat component of the emulsion. This is particularly important in leather processing, where lipase can effectively improve the stability and uniformity of degreased fats and oils in water, avoid re-layering of the emulsion, and prevent fats from re-adhering to the surface of animal skins, thus improving the quality of the leather treatment, facilitating efficient processing, and reducing the generation of wastes, thus improving the overall production efficiency. In the tanning industry, lipase, as a biocatalyst, has long been focused on improving its stability at high temperatures to adapt to the high-temperature environments common to the tanning process. However, with increasing environmental awareness and rising production costs, traditional high-temperature treatments are facing more and more challenges. High temperatures not only require large amounts of energy support, but may also bring about environmental pollution problems such as exhaust emissions and excessive use of chemical reagents. Therefore, there is an urgent need for the tanning industry to develop lipases that are suitable for efficient operation at ambient temperatures in order to achieve greener and more sustainable production methods. Lipase operating efficiently at room temperature has obvious advantages. Firstly, operation at room temperature significantly reduces energy consumption. Compared with high temperature treatment, the energy requirement during production is greatly reduced, lowering the production cost of enterprises and reducing environmental pollution. Secondly, at room temperature, lipase can more gently remove the excess fat from the leather, avoiding any surface damage or deterioration that may be caused by high temperature, and also helping to maintain the natural softness and touch of the leather. In addition, the role of ambient lipase in emulsion stabilization is also crucial. In leather processing, the homogeneity and stability of the emulsion directly affects the coating effect and the quality of the final product. Whereas emulsions are traditionally prone to delamination at high temperatures, high efficiency lipases at ambient temperatures can effectively improve emulsification and maintain long term stability of emulsions, avoiding wasteful production and inconsistent quality caused by unstable emulsions. Therefore, the efficient operation of lipase at ambient temperature not only meets the tanning industry's requirements for environmental protection, energy saving and cost control, but also improves the overall efficiency in several process steps such as degreasing, softening and emulsification. This characteristic makes ambient lipase a key breakthrough point in the development of modern tannery industry, which provides important technical support for realizing green production, improving product quality and reducing environmental burden. However, a large number of applications have shown that the catalytic hydrolysis rate of lipase on grease substrate is low in aqueous environments (50–60%) [ 9 ]. Through optimization of the selection of lipase and construction of a synergistic system including lipase, surfactant, and alkali, the hydrolysis rate of ester bonds can only be increased to approximately 70–80% [ 10 ]. Such an outcome is observed mainly because the catalytic process of lipase in the hydrolysis of fat substrates is carried out at the oil–water interface, which has the characteristic of “interface activation.” [ 11 ] [ 12 ] Lipase needs to be adsorbed onto the fat surface to make full contact with the substrate and catalyze the substrate hydrolysis. Therefore, strategy for to gaining insight into the distribution characteristics of lipase on the surface of oil and grease and their influence discipline, must be developed to further guide the efficient catalytic hydrolysis technology involving lipase on oil and grease substrate, which has long been a great concern in the industry. The lower the surface tension of oil and grease, the easier the interface adsorption by lipase and other molecules. Determination of the substrate surface tension can provide the basic properties of the oil– water interface, which can offer a reference for assessment of the adsorption capacity of lipase on the fat surface; however, directly revealing the distribution characteristics of specific lipases at the oil–water interface and the nature of the reaction presents a challenge[ 13 ]. Raman spectroscopy[ 14 ], infrared spectroscopy[ 15 ], and other spectroscopic techniques can be used for the comprehensive and molecular–level exploration of the interaction between lipases and substrates and the resulting structural and conformational changes; photon correlation spectroscopy, neutron scattering[ 16 ], and other techniques offer a strong support to the study of the size, distribution and aggregation of lipase particles in the oil–water interface, the location of adsorption position, and structural characteristics of lipase particles; mass spectrometry analysis[ 17 ], electrochemical technology[ 18 ], surface plasmon resonance[ 19 ], and other technologies; they also enable the qualitative and quantitative analyses of the interaction between lipase and a substrate and product generation, which strongly promotes the investigation of the adsorption and reaction kinetics of lipase on the surfaces of oil and grease; with the help of atomic force microscopy, high–resolution images of the adsorption of lipase on the surfaces of oil and grease and membrane formation can be obtained[ 20 ]. The development and application of these analytical techniques have strongly promoted the development and improvement of the theory of lipase catalysis at the oil–water interface. However, common shortcomings, such as high requirements for instrumentation and samples, complex analytical testing process, and difficulty in effectively guiding the development of engineering technology, are the key factors leading to the long–term challenges in achieving effective breakthroughs in the research on the role of lipase in the efficient catalysis of the hydrolysis of oil and grease substrates. Increasing attention is being paid to the search for a strategy for the analysis of the distribution characteristics of lipase on the surfaces of oil and grease[ 21 ] and their influence discipline via key process parameters [ 22 – 24 ], which can be used quickly and intuitively in the practical application environment to effectively guide the construction of lipase efficient catalytic technology. Fluorescent labeling technology, as a widely used visualization method with sensitive and intuitive results, covalently combines fluorescein and proteins to render labeled proteins with fluorescent properties for their localization and tracing in cells or tissues[ 25 ]. Studies have repeatedly reported the use of fluorescent labeling technology to analyze the mass transfer behavior of enzyme proteins in animal skins[ 26 ], which promoted the development of enzyme dehairing, enzyme softening, and other technologies. However, fluorescent labeling has a potential effect on enzyme protein structure and properties, and whether ensuring that fluorescently labeled enzyme adsorption, permeation, and other behaviors within the object is consistent with those of unlabeled enzymes remains controversial[ 27 ]. To this end, our team systematically analyzed the effects of fluorescein structure, dosage, and labeling conditions on the structural characteristics and viability of the enzymes before and after fluorescent labeling and effectively avoided the remarkable effects of fluorescent labeling on the molecular weight, isoelectric point, and viability of protease by controlling the degree of binding between fluorescein isothiocyanate (FITC) and the enzyme protein[ 28 ]. This study lays the groundwork for the development of visual analysis techniques based on fluorescently labeled enzymes and proteins. In addition, in aqueous environments, oil is insoluble in water, but proteins and other substances can be easily adsorbed on the surface of oil droplets[ 29 ]. Such condition can avoid the excessive aggregation of oil droplets to a certain extent. Moreover, the dispersion stability of proteins on oil is easily affected by environmental factors and thus lays a foundation for the observation of the adsorption and distribution characteristics of lipase on oil droplet surface using labeled enzyme proteins. Therefore, this paper examined the effects of fat mass fraction, lipase dosage, temperature, stirring and dispersion time, resting time, fat type, lipase type, and other factors on the dispersion stability of fat emulsions and attempted to construct a fluorescent labeling–based method for the visualization of the distribution patterns and reaction properties of lipase at the oil–water interface. In addition, the influence of enzyme and oil types and other factors on the distribution pattern of lipase at the oil–water interface was investigated, and the results lay the foundation for the further development of an efficient catalytic technology for the deep hydrolysis of oil under the action of lipase. Materials and methods Main Instruments and Materials Main Instruments Benchtop high speed refrigerated centrifuge, Hunan Xiang Yi Lab Instru Dev Co.;APPS MV 50D Automatic protein chromatography system, Lisure Science (Suzhou) Co., Ltd༛UV–1100 UV spectrophotometer, Shanghai MAPADA Instrument Co., Ltd༛Confocal laser scanning microscope NE900, NINGBO YONGXIN OPTICS CO., LTD.༛IKA ULTRA–TURRAX Tube Drive UTTD, IKA Works Guangzhou༛THZ– 82 Incubator shakers, Guangdong Fuwa Engineering Group Co., Ltd.༛Superdex 75 prep grade, BXK16, Bestchrom (Zhejiang) Biosciences Ltd. Materials LKT lipase(400000U/g)、YMN lipase༈200000U/g༉、AKT–A lipase༈350000U/g༉、AKT–N lipase༈≥100000U/g༉,industrial grade, Chengdu Yufangda Biotechnology Co., Ltd;Fluorescein isothiocyanate, analytical pure, Sangon Biotech (Shanghai) Co., Ltd.༛Lipase labeled with fluorescein isothiocyanate, Laboratory self–made༛olive oil, analytical pure, Chengdu Kelong Chemical Co.༛Butter, mutton tallow, wild boar oil all purchased in the market༛Other general reagents are domestic analytical pure. Experimental content and methodology Effect of oil mass fraction on the distribution characteristics of lipase at the oil–water interface Weigh the appropriate amount of oil in the UTTD test tube, The oil mass fraction (wt%) were 5%, 10%, 20%, 25% and 50%, add 1.0 mL of moderately diluted labeled enzyme protein solution, the appropriate amount of Sudan IV staining solution and ultrapure water, control the total mass of 10.0 g. Use the UTTD test tube disperser to stir and disperse for 10 min, take up a drop of 10µL of liquid with a micropipette and add it onto a slide, let it stand for 120 s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Effect of enzyme protein concentration on the distribution characteristics of lipase at the oil–water interface Weigh 2.5g of olive oil in a UTTD test tube, add 1.0mL of fluorescently labeled LKT lipase with enzyme protein concentration of 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 10mg/mL respectively, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10µL droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Effect of temperature on the distribution characteristics of lipases at the oil–water interface 2.5 g of olive oil was weighed in a UTTD tube, and the temperature of the olive oil was controlled at 4℃, 25℃, 30℃ and 40℃, respectively, and 1.0 mL of 0.25 mg/mL fluorescently labelled LKT lipase at the corresponding temperatures was added, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10µL droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Effect of stirring time on the distribution characteristics of lipase at the oil–water interface 2.5 g of olive oil was weighed in the UTTD tube, 1.0 mL of fluorescent labelled enzyme protein solution with enzyme protein concentration at 0.25 mg/mL was added, add an appropriate amount of ultrapure water to control the total mass of 10.0g, and the stirring time in the UTTD tube was 1 min, 3 min, 5 min and 10 min, 10µL droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Effect of resting time on the distribution characteristics of lipase at the oil–water interface Weigh 2.5 g of olive oil in a UTTD test tube, add 1.0 mL of fluorescently labelled enzyme protein solution with enzyme protein concentration at 0.25 mg/mL, stir and disperse for 10 min with a UTTD test tube disperser, then aspirate 10 µL droplets with a micropipette and add them dropwise on a slide, and leave it for 30s, 60s, 90s and 120s, respectively, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Effect of oil type on the distribution characteristics of lipase at the oil–water interface Weigh 2.5 g of olive oil, sheep oil, lard, glycerol trioleate in a UTTD tube and add 1.0 mL of fluorescently labelled 0.25 mg/mL of LKT lipase, respectively, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10µL droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. Influence of lipase species on the distribution characteristics of lipase at the oil–water interface Weigh 2.5 g of olive oil in a UTTD tube and add 1.0 mL of fluorescently labelled 0.25 mg/mL of LKT lipase, YMN lipase, AKT–A lipase, AKT–N lipase, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10µL droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence. At the same time, take the stirred oil–water mixture and determine the acid value of the emulsion after reaction with the different lipases. Determination of acid value of oils and fats Add 2–3 drops of phenolphthalein solution and titrate with 0.05 mol/L KOH solution to determine the acid value of the reacted emulsion. Results Influence of oil mass fraction on the dispersion effect of emulsions In order to explore the visualization of lipase distribution characteristics at the oil–water interface based on fluorescently labeled enzyme proteins, the amount of olive oil in the oil-water mixture needed to be explored. Therefore, the effect of olive oil–water mixing ratio on emulsion characteristics was first examined in combination with Sudan IV dye and lipase protein after it was labeled, and the results are shown in Fig. 2 . When the dosage of olive oil was 10–20%, the dispersion of the oil phase was better, and “O/W” type oil droplets of moderate size can be formed. Influence of resting time on the stability of the mixing effect of emulsions The effect of settling time on the distribution of lipase at the oil–water interface was further examined, and the results are shown in Fig. 3 . As shown in Fig. 3 , when the emulsion was left to stand for 30–120 s, no evident aggregation of oil droplets occurred, and a uniform distribution of oil droplets and labeled enzyme proteins that were independent of each other at the interface can still be observed. Effect of stirring time on the dispersion stability of emulsions In order to investigate the effect of stirring time on the stability of oil-water mixtures, the stirring time of the oil-water mixtures was varied while the stirring speed was kept constant. The results are shown in Fig. 4 . Figure 4 reveals that a poor oil droplet dispersion effect was observed when the stirring time was 1 min despite the relatively clear oil–water interface. With the extension of stirring time, the oil–phase dispersion improved, and the uniformity of oil droplet size was gradually improved. In addition, when the stirring time was up to 10 min, the emulsion showed better stability with a clear interface, and dispersed stable oil droplets could be observed. Effect of enzyme protein concentration on the dispersion stability of emulsions The effect of various labeled enzyme proteins on the properties of oil–water interface of the olive oil was further investigated. The results in Fig. 5 shows that, at room temperature, when the concentration of the labeled enzyme protein was controlled at 0.1 mg/mL, a weak fluorescence intensity occurred on the surface of oil droplets. When the concentration of the labeled enzyme protein was increased to 0.25 mg/mL, an evident fluorescence layer transpired on the oil droplet surface. When the concentration of the labeled enzyme protein was further increased to 0.5 mg/mL, the oil–water interface of oil droplets became damaged and fuzzy. When the enzyme protein concentration was further increased to 1 mg/mL, a good thickening effect and a stable oily paste were obtained. Moreover, the independent oil droplets were difficult to observe. Effect of temperature on the mixing stability of emulsions In order to investigate the effect of temperature on the distribution of lipase at the oil-water interface, fluorescently labeled lipase was stirred and observed at different temperatures, and the results are shown in Fig. 6 . In Fig. 6 , it can be seen that at a low temperature (< 10℃), the viscosity of olive oil was large, and gradual solidification was observed (the solidification point of olive oil is about 5℃). Thus, effective dispersion in the aqueous environment was difficult to achieve, and the oil droplets were bonded to each other. As the temperature increased, the homogeneous adsorption of the marker enzyme protein at the oil–water interface was evident. However, when the temperature was extremely high (up to 40℃), it is not favorable for the observation of oil droplets. Effect of oil type on the distribution characteristics of lipase at the oil – water interface The distribution characteristics of lipase at the oil–water interface of various fats and oils were further examined, and the results are shown in Fig. 7 . Figure 7 shows that in the glycerol trioleate emulsion, lipase was fully distributed in the interior of oil droplets. Lipase can be evenly distributed on the surface of olive oil, and form a complete and uniform lipase contact layer. Meanwhile, the lipase can only form a discontinuous distribution feature on the surface of sheep oil. However, in lard emulsion, the distribution of lipase could not be observed in the lard–oil–water interface, but only in the interstices of lard droplets. Distribution characteristics of typical lipases at the oil–water interface and their catalytic hydrolysis of substrates To investigate the effect of lipase species on the distribution effect of enzyme proteins at the oil-water interface, the distribution characteristics of four typical lipases at the oil–water interface of olive oil were analyzed. Figure 8 shows that the lipase YMN had the strongest affinity for olive oil, which not only can be adsorbed on the surface of oil droplets effectively, but also dispersed into more independent droplets. In addition, a part of the enzyme proteins can penetrate the interior of the droplets, which effectively increased the contact efficiency between lipase and the substrate and thus enhanced the catalytic hydrolysis of olive oil and increased its acidic value (Fig. 8 –II). Lipase LKT can also disperse olive oil as independent oil droplets and distribute them uniformly at the oil–water interface, but it did not penetrate the interior of oil droplets. Thus, the degree of catalytic hydrolysis of the substrate was inferior to that of lipase YMN. In lipase AKT–N, the dispersion of droplets showed a poor stability, and larger droplets easily formed. Therefore, although lipase AKT–N also penetrated the interior of oil droplets, it was not as effective as lipase YMN. Lipase AKT–A which had the lowest degree of contact with the substrate, showed the poorest catalytic hydrolysis efficiency for olive oil, mainly because it failed to effectively disperse the oil droplets and can only be adsorbed on their surface. Discussion In an aqueous environment, the catalytic reaction of lipase during the hydrolysis of fat substrate was carried out at the “oil–water” interface[ 30 ]. The adsorption and permeation properties of lipase at the oil–water interface directly affect its contact efficiency with the substrate, which influences the catalytic efficiency. An intuitive analysis of the distribution characteristics of lipase at the oil–water interface and its influence law will provide a reference for the construction of an efficient lipase catalytic technology. Previous research has shown that FITC fluorescence labeling technology can be used to observe the distribution and mass transfer characteristics of enzyme proteins in different chemical reaction scenarios[ 28 ]. Therefore, after optimizing the observation conditions, this technique is attempted to be applied to analyze the adsorption behavior of lipase on the surface of lipid substrate in aqueous environment. The mixing of oil and water forms an emulsion, and to observe the distribution characteristics of lipase at the oil–water interface via fluorescence microscopy, the emulsion must have a certain degree of stability. In general, in an aqueous phase environment, proteins are easily adsorbed at the oil–water interface of the oil, form a protective film to prevent the aggregation of oil droplets, improve the stability of the emulsion, and exert a good foam, emulsification, thickening, which were effected with the changes in oil content, temperature, protein concentration, stirring intensity and resting time [ 31 – 34 ]. Firstly, the oil-to-water ratio will have a direct impact on the composition and stability of the emulsion. As shown in Fig. 1 , with the increase in oil dosage, the emulsion may gradually change from an oil–in–water (O/W) type to a water–in–oil (W/O) type, which in turn affects the stability, dispersibility, viscosity, and other emulsion properties. When the olive oil dosage was extremely low (wt%=5%), the oil droplets were small, lacked stability, showed susceptibility to agglomeration, and exhibited poor dispersion, which were not conducive to microscopic observation. In addition, fluorescence intensity in the aqueous region was strong, which indicates that most of the labeled enzyme proteins were not adsorbed at the oil–water interface. When the dosage of olive oil reached 25% or more, the oil phase was likely to aggregate and form larger oil droplets. Moreover, a non–independent “adhesion” phenomenon was observed between the oil droplets, which gradually transformed into a “W/O” emulsion (wt%=50%). As a result, a poor oil phase dispersion occurred, and observation of the oil droplet interface presented difficulty. When the dosage of olive oil was 10–20%, the dispersion of the oil phase was better, and “O/W” type oil droplets of moderate size can be formed. In addition, the stability was better, and the interface between oil and water was clearer. At this point, the uniform distribution of labeled enzyme proteins in the interface between oil and water can be observed, and the fluorescence intensity of the water–phase region was low, which was conducive to the analysis of the distribution characteristics of lipase in the oil and water interface. This condition is more conducive to the analysis of the distribution characteristics of lipase at the oil–water interface. However, emulsions are inherently unstable. Generally speaking, when stirring ceases and an emulsion is left undisturbed, various forces such as gravity, buoyancy, surface tension etc., gradually cause oil droplets to aggregate at specific positions within the liquid leading to stratification over time. Nevertheless, Fig. 2 illustrates that marker enzyme proteins adsorb onto the surface of oil droplets impeding their coalescence into larger entities within a short period. This discovery establishes a foundation for effectively observing and analyzing distribution characteristics of marker enzyme proteins at the oil-water interface. Moreover, the dispersion of the emulsion is evidently influenced by stirring intensity and other factors. Figure 3 demonstrated that, when maintaining a constant stirring speed, the dispersion stability of the emulsion is primarily affected by the duration of stirring. This could be attributed to that, with prolonged stirring time, shear force facilitates the destruction and reorganization of the oil-water interface, thereby promoting further mixing between oil and water phases and enhancing uniformity in droplet size as well as dispersion stability [ 35 ]. The alteration in protein concentration, however, will have a further impact on the dispersion of oil and the stability of emulsion. As depicted in Fig. 4 , when the concentration of the labeled enzyme protein was maintained at 0.1 mg/mL, a weak fluorescence intensity was observed on the surface of oil droplets, resulting in poor emulsion stability due to insufficient distribution of labeled proteins across the oil droplet interface. Upon increasing the concentration to 0.25 mg/mL, a distinct fluorescence layer emerged on the surface of oil droplets, indicating more uniform distribution of lipase protein at this point within the oil-water interface. Furthermore, this prevented aggregation of oil droplets and facilitated observation of lipase protein distribution at said interface. However, as we further increased the concentration to 0.5 mg/mL, excessive protein presence led to damage and blurring at the oil-water interface of these droplets; while an increase to 1 mg/mL resulted in effective thickening and formation of stable oily paste with limited visibility for individualized oil droplets. The results presented in Fig. 5 demonstrate that, at temperatures below 10°C, olive oil exhibits high viscosity and gradual solidification occurs (the solidification point of olive oil is approximately 5°C). Consequently, achieving effective dispersion in the aqueous environment becomes challenging as the oil droplets tend to bond with each other. This leads to a blurred interface between oil and water, posing difficulties for observing and analyzing the distribution characteristics of marker enzyme proteins at the interface. However, as the temperature increases, there is a decrease in the viscosity of olive oil[ 36 ], facilitating the formation of "O/W" type emulsion droplets. This enhances emulsion stability and promotes clearer formation of an oil-water interface where homogeneous adsorption of marker enzyme proteins becomes evident. Nevertheless, when temperatures reach extremely high levels (up to 40°C), enhanced Brownian motion contributes to droplet aggregation and sedimentation. Therefore, the temperature should be regulated within the range of 20–30°C, which is deemed more suitable. Then, a method based on fluorescently labeled enzyme proteins was established to visualize the reaction properties of lipase at the oil–water interface, and it can be used for the effective observation of the distribution characteristics of lipase at the oil–water interface. The optimal conditions for observation include the following: oil content of 10–20% (wt%), concentration of fluorescently labelled enzyme protein of 0.25 mg/mL, reaction temperature of 25°C–30°C, emulsion dispersion and stirring time of 10 min, and emulsion resting time of 30–120 s. Based on the developed fluorescently labeled protein observation method, the distribution characteristics of lipase at the oil-water interface of various fats and oils were further examined. Figure 6 illustrated that lipase was fully distributed within the interior of glycerol trioleate emulsion droplets, indicating its good contact properties with this substrate. Lipase could also be evenly distributed on the surface of olive oil, forming a complete and uniform lipase contact layer. However, due to the high freezing point of goat oil leading to solid formation, lipase only exhibited a discontinuous distribution feature on some solid surfaces. In lard emulsion, lipase distribution was observed in interstices between lard droplets rather than at the lard-oil-water interface. Additionally, fluorescence-labeled protein observations reveal that this lipase shows better adsorption performance at the oil-water interface of vegetable fats (such as olive oil) compared to animal fats (sheep's oil and lard) in an aqueous environment. This finding could be attributed to vegetable oils primarily containing unsaturated fatty acids [ 37 – 39 ], such as stearic and palmitic acids. Furthermore, this type of lipase demonstrated a stronger affinity for oleic acid glycol esters and achieved good affinity with oleic acid glycerides. Moreover, as shown in Fig. 7 , the adsorption performance of lipase from different sources on the oil surface exerted a large difference, which not only affected the stability of oil emulsion but also the catalytic hydrolysis performance of lipase on oil. It revealed that differences in the distributions of lipase at the oil–water interface of various fats and oils had a certain degree of correspondence with their specificity and that the distribution characteristics of the lipases on the surface of olive oil enabled effective catalytic hydrolysis to a certain extent. These results highlight how fluorescent labeling allows for more intuitive analysis of lipase distribution characteristics at the oil-water interface across various lipid substrates while providing support for further research on efficient catalysis facilitated by lipases. Conclusion A visualization method of lipase reaction at the oil-water interface based on fluorescently labeled enzyme protein has been established, which can effectively observe the distribution characteristics of lipase at the oil-water interface under the following optimal conditions: the oil content of 10%-20% (wt%), the concentration of fluorescently labeled enzyme protein of 0.25 mg/mL, the reaction temperature of 25–30°C, the emulsion dispersion and stirring time of 10 min, and the resting time of the emulsion of 30–120 s. Based on this method, a preliminary analysis of the effects of oil and lipase species on the distribution characteristics of lipase at the oil-water interface has been carried out. Based on this method, the effects of oil and lipase species on the distribution characteristics of lipase at the oil-water interface were initially analyzed, and it was observed that the differences in the distribution of lipase at the oil-water interface of different fats and oils had a certain degree of correspondence with their specificity, and that the distribution characteristics of different lipases on the surface of olive oil had a certain degree of correspondence with their catalytic hydrolysis efficiencies. Declarations Acknowledgements The authors would like to thank all members of the National Engineering Laboratory for Clean Technology of Leather Manufacture. Author’s contributions The study design was contributed by Chun Z and Xian D; funding was obtained by Bi P and Chun Z; the experiments were performed by Chun Z and Xian D; data analysis was conducted by Xian D; the manuscript was written by Chun Z and Xian D. All authors reviewed the manuscript. Funding This study was supported by The Key R&D Program of Chengdu for Technology Innovation (2024–YF08–00060–GX); The Fund of the Central Guidance on Local Science and Technology Development (2024ZYD0218). Competing interests The authors declare no conflicts of interest relating to this work. Data availability All data supporting the findings of this study are available within the paper. References Verger R (1997) ‘Interfacial activation’of lipases: facts and artifacts. Trends Biotechnol 15:32-38. Reis P, Malmsten M, Nydén M, Folmer B, Holmberg K (2019) Interactions between lipases and amphiphiles at interfaces. J Surfactants Deterg 22:1047-1058. Haki G, Rakshit S (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17-34. Li S, Luo F, Chattha SA, Zhang C, Peng B, Mu C (2020) Surfactant-free beamhouse technology of leather manufacturing: Removing constraints for the breakdown of natural fats catalyzed by lipase. J Cleaner Prod 261:121187. Wang J, Huang XH, Wang X, Tian H, Wang L, Zhou DY, Qin L (2024) Compensatory effect of lipase on the flavor of lightly-salted large yellow croaker: integration of flavoromics and lipidomics. Food Biosci 59:103907. Yu J, Shen C, Chen H, Luo M, Zhang L, Liu Y, Xu F, Tan C-P, Cheong L-Z (2024) Lipase-catalyzed Preparation, Bioavailability and Functional properties of a DHA-enriched tuna oil. LWT 203:116341. Sundaramahalingam M, Vijayachandran P, Rajeshbanu J, Sivashanmugam P (2024) Production of lipase from Priestia endophytica SSP strain and its potential application in deinking of printed paper. Biomass Convers Biorefin 14:13861-13875. Bhavsar KV, Yadav GD (2024) Process intensification in microwave assisted lipase catalysed solvent-free synthesis of n-nonyl caprylate. J Indian Chem Soc 101:101258. Ivanova D, Costadinnova L, Todorova R (2004) The influence of some parameters in leather degreasing on the lypolytic activity of lipase. J Soc Leather Technol Chem 88:161-163. Moujehed E, Zarai Z, Khemir H, Miled N, Bchir MS, Gablin C, Bessueille F, Bonhommé A, Leonard D, Carrière F (2022) Cleaner degreasing of sheepskins by the Yarrowia lipolytica LIP2 lipase as a chemical-free alternative in the leather industry. Colloids Surf B 211:112292. Reis P, Holmberg K, Watzke HJ, Leser ME, Miller R (2009) Lipases at interfaces: a review. Adv Colloid Interface Sci 147-148:237-250. Cambillau C, Longhi S, Nicolas A, Martinez C (1996) Acyl glycerol hydrolases: inhibitors, interface and catalysis. Curr Opin Struct Biol 6:449-455. Park I, Yu H, Chang P-S (2023) Lipase-catalyzed synthesis of antibacterial and antioxidative erythorbyl ricinoleate with high emulsifying activity. Food Chem 404:134697. Chen C, Pan Y, Niu Y, Peng D, Huang W, Shen W, Jin W, Huang Q (2023) Modulating interfacial structure and lipid digestion of natural Camellia oil body by roasting and boiling processes. Food Chem 402:134198. Li K, Guo Z, Li H, Ren X, Sun C, Feng Q, Kou S, Li Q (2023) Nanoemulsion containing Yellow Monascus pigment: Fabrication, characterization, storage stability, and lipase hydrolytic activity in vitro digestion. Colloids Surf B 224:113199. Gilbert EP (2019) Small-angle X-Ray and neutron scattering in food colloids. Curr Opin Colloid Interface Sci 42:55-72. Kuang G, Wang Z, Luo X, Geng Z, Cui J, Bilal M, Wang Z, Jia S (2023) Immobilization of lipase on hydrophobic MOF synthesized simultaneously with oleic acid and application in hydrolysis of natural oils for improving unsaturated fatty acid production. Int J Biol Macromol 242:124807. Sarakhman O, Wenninger N, Rogala A, Švorc Ľ, Kalcher K, Ortner A (2023) Electrochemical flow injection approach for routine screening of lipase activity in pancreatic preparations. Talanta 260:124588. Meriaudeau F, Ferrell T, Arakawa E, Wig A, Passian A, Thundat T, Shen W-J, Patel S, Kraemer F (2001) Study of different hormone-sensitive lipase concentrations using a surface plasmon resonance sensor. Sens Actuators B 73:192-198. Liu S, Wang Y (2011) A review of the application of atomic force microscopy (AFM) in food science and technology. Adv Food Nutr Res 62:201-240. Brockman HL, Momsen WE, Tsujita T (1988) Lipid-lipid complexes: Properties and effects on lipase binding to surfaces. J AM OIL CHEM SOC 65:891-896. Ivanova MG, Svendsen A, Verger R, Panaiotov I (2002) Action of Humicola lanuginosa lipase on long-chain lipid substrates: 1. Hydrolysis of monoolein monolayers. Colloids Surf B 26:301-314. Reis P, Holmberg K, Miller R, Leser ME, Raab T, Watzke HJ (2009) Lipase reaction at interfaces as self-limiting processes. C R Chim 12:163-170. Muth M, Rothkötter S, Paprosch S, Schmid RP, Schnitzlein K (2017) Competition of Thermomyces lanuginosus lipase with its hydrolysis products at the oil-water interface. Colloids Surf B 149:280-287. Perazzo A, Gallier S, Liuzzi R, Guido S, Caserta S (2021) Quantitative methods to detect phospholipids at the oil-water interface. Adv Colloid Interface Sci 290:102392. Zhu Y, Song J, Zhang X, Gao M, Peng B, Zhang C (2023) Effect of Electrostatic Interaction between Collagen and Enzymes on Permeation of Protease into the Pelt during Leather Bating Process. J Am Leather Chem Assoc 118:428-438. Díaz-de-Quijano D, Felip M (2011) A comparative study of fluorescence-labelled enzyme activity methods for assaying phosphatase activity in phytoplankton. A possible bias in the enzymatic pathway estimations. J Microbiol Methods 86:104-107. Gao M, Song J, Zhang X, Zhang C, Peng B, Chattha SA (2023) Key mechanism of enzymatic dehairing technology for leather-making: permeation behaviors of protease into animal hide and the mechanism of charge regulation. Collagen Leather 5:1-18. Guan H, Diao X, Liu D, Han J, Kong B, Liu D, Gao C, Zhang L (2020) Effect of high-pressure processing enzymatic hydrolysates of soy protein isolate on the emulsifying and oxidative stability of myofibrillar protein-prepared oil-in-water emulsions. J Sci Food Agric 100:3910-3919. Schmid RD, Verger R (1998) Lipases: Interfacial Enzymes with Attractive Applications. Angew Chem Int Ed 37:1608-1633. Delahaije RJ, Gruppen H, Giuseppin ML, Wierenga PA (2014) Quantitative description of the parameters affecting the adsorption behaviour of globular proteins. Colloids Surf B 123:199-206. Mu T, Tan S-S, Xue Y-lJFC (2009) The amino acid composition, solubility and emulsifying properties of sweet potato protein. Food Chem 112:1002-1005. Rahmati NF, Koocheki A, Varidi M, Kadkhodaee R (2017) Adsorption of Speckled Sugar bean protein isolate at oil-water interface: Effect of ionic strength and pH. Int J Biol Macromol 95:1179-1189. Wang B, Li D, Wang L, Adhikari B, Shi JJJoFE (2010) Ability of flaxseed and soybean protein concentrates to stabilize oil-in-water emulsions. J Food Eng 100:417-426. Chen Y, Chen Y, Jiang L, Yang Z, Fang Y, Zhang WJL (2023) Shear emulsification condition strategy impact high internal phase Pickering emulsions stabilized by coconut globulin-tannic acid: Structure of protein at the oil-water interface. LWT 187: 115283 Lioumbas JS, Ampatzidis CD, Karapantsios TDJJoFE (2012) Effect of potato deep-fat frying conditions on temperature dependence of olive oil and palm oil viscosity. J Food Eng 113:217-225. Ahmad Nizar NN, Nazrim Marikkar JM, Hashim DM (2013) Differentiation of lard, chicken fat, beef fat and mutton fat by GCMS and EA-IRMS techniques. J Oleo Sci 62:459-464. Kanwal N, Musharraf SG (2024) Analytical approaches for the determination of adulterated animal fats and vegetable oils in food and non-food samples. Food Chem 460:140786. Zhang W, Zheng R, Xu X, Zhao XJFH (2024) Oil unsaturation degree dictates emulsion stability through tuning interfacial behaviour of proteins. Food Hydrocolloids 158:110588 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Jan, 2025 Reviews received at journal 25 Jan, 2025 Reviewers agreed at journal 22 Jan, 2025 Reviewers invited by journal 21 Jan, 2025 Editor assigned by journal 07 Jan, 2025 Submission checks completed at journal 07 Jan, 2025 First submitted to journal 03 Jan, 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-5758102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398907642,"identity":"882e0347-c917-4c63-aa2f-1860ab3b5b57","order_by":0,"name":"Xian Du","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Du","suffix":""},{"id":398907643,"identity":"0233d43e-6b9a-406e-99f9-5e2962e554b6","order_by":1,"name":"Chunxiao Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYDACCRBhAGNUIAkSqeUM0VpgDMY2IrTwz+4xe8xTYCcnP7v52cOv8+rkDQ4wH7zNw2CXh9OSO2fMDWcYJBszzjlmbiy77bDhhgNsydY8DMnFuLQYSOSYSXwwYE5slkgwk5bcdiDB4ACPmTQPw4HEBnxaEgzqE9sk0r9JS86pA2rh/0ZYyweDw4k9QIbkxwZmkC1seLVI3Egrk5xhcNxYQiKnTJrh2GHDmYfZjC3nGCTj1MI/I3mbNM+fajn5GenbJH/U1MnzHW9+eONNhR1OLSiAmQdMgh1MjHogYPxBpMJRMApGwSgYWQAARJdNQZKMqAIAAAAASUVORK5CYII=","orcid":"","institution":"Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Chunxiao","middleName":"","lastName":"Zhang","suffix":""},{"id":398907644,"identity":"dd714f7e-e103-45af-9b73-9f55c92dde85","order_by":2,"name":"Biyu Peng","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Biyu","middleName":"","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2025-01-03 12:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5758102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5758102/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73399179,"identity":"cc8b5e6b-33a5-4d4d-a219-ab2344fa866f","added_by":"auto","created_at":"2025-01-09 14:13:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe role of lipases in lipid processing\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/69e5e9e9a4d9323f1da301ef.png"},{"id":73399936,"identity":"dbdb8993-dd9f-42de-96bc-e2464efea2ab","added_by":"auto","created_at":"2025-01-09 14:21:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oil mass fraction (wt%) on the dispersion effect of oil–water emulsion (×40; I–Sudan VI staining; II–fluorescence labeled enzyme protein microscopy)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/b166810e6acea5d8b9d00d47.png"},{"id":73399182,"identity":"88664665-b73a-4921-a7a3-4e6d425c48aa","added_by":"auto","created_at":"2025-01-09 14:13:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resting time (s) on the mixing effect of oil–water emulsions （×40）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/09c5648e541b4403dc95b22d.png"},{"id":73399937,"identity":"ac822a74-f43d-477e-b02e-ae08e5b50f99","added_by":"auto","created_at":"2025-01-09 14:21:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of dispersion time (min) on the mixing effect of oil–water emulsion （×40）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/37ad7caf7666874ec0c9a857.png"},{"id":73399193,"identity":"b96b54f6-5f51-48ee-b8af-5b1e8bee564b","added_by":"auto","created_at":"2025-01-09 14:13:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":160747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of labeled enzyme protein concentration (mg/ml) on the mixing effect of oil–water emulsion （×40）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/d4ececd412ddafef325d9933.png"},{"id":73399201,"identity":"0350f039-780d-4117-8789-239abca0d767","added_by":"auto","created_at":"2025-01-09 14:13:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":287646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of temperature (℃) on mixing effect of oil–water emulsion （×40）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/cc46348f1870043fda0163f2.png"},{"id":73399203,"identity":"3a31f34d-4a0c-4db1-9af0-9ec06ab2d737","added_by":"auto","created_at":"2025-01-09 14:13:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":325232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution characteristics of lipase at different oil–water interface (×40)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/ef8c13cbad6bfa14c6bc466f.png"},{"id":73399189,"identity":"7493b2ab-8494-4006-ba10-a286156cb590","added_by":"auto","created_at":"2025-01-09 14:13:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":250753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edistribution characteristics of typical lipase at the oil–water interface of olive oil (×40); II–effects of different lipases on olive oil acid value\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/f25c2112af24b9c1f64f3755.png"},{"id":73401282,"identity":"f8b97db7-3690-4a14-af83-7162e64c77fb","added_by":"auto","created_at":"2025-01-09 14:37:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3479896,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5758102/v1/38d5dc44-2e6f-4669-bc90-5213f6b3ac70.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visualization of fluorescently labeled lipase distribution characteristics at the oil–water interface","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLipase (EC 3.1.1.3) is an ester bond hydrolase[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] that can catalyze the hydrolysis of triglycerides into diglycerides, monoesters, glycerol and fatty acids under certain conditions[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; with the advantages of mild and nontoxic conditions, it prevents the destruction of the main components at high temperatures and in strong alkalis and has been widely used in tannery[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], washing, wastewater treatment, food processing[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], pulp and paper[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], fine chemicals synthesis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and other industries; this enzyme is important for the improvement of product quality, reduced usage of surfactants and organic solvents, increased efficiency of sewage treatment, and enhanced recycling of oil and grease and their hydrolysis products. The role of lipases in lipid degreasing is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe main role played by lipases in lipid processing is the hydrolysis reaction, i.e., the breakdown of fats (mainly triacylglycerols) into fatty acids and glycerol. This process is an important part of fat metabolism, and lipase releases long-chain fatty acids from fats through an enzyme-catalyzed hydrolysis reaction. Lipase breaks down triacylglycerols into glycerol and fatty acids by binding to ester bonds in the fat molecule. The products of this reaction can be used in subsequent processes such as fat extraction, modification or synthesis of other chemicals.\u003c/p\u003e \u003cp\u003eIn leather processing, lipases are commonly used to remove excess fat from the surface of the leather, which helps to minimize hardening and improve the softness of the leather. Lipases also regulate the hydration of leather and enhance its dyeing and coating. The application of lipase in leather processing can improve the efficiency of fat processing and reduce the by-products and environmental pollution that may be caused by traditional chemical methods (e.g. acid hydrolysis). Secondly, the mild reaction conditions of lipase usually do not require high temperatures or a strongly acidic environment, making it more environmentally friendly, energy efficient and conducive to the sustainable use of resources.\u003c/p\u003e \u003cp\u003eIn addition, lipase can effectively prevent oil-water separation by improving the stability of water-in-oil emulsions. Water-in-oil emulsions (W/O emulsions) are an important emulsification system widely used in food, cosmetics, pharmaceuticals, and leather processing. Lipases change the size and distribution of lipid particles by hydrolyzing the ester bonds in fat molecules, resulting in the breakdown of larger fat particles into smaller fatty acid and glycerol molecules. These smaller molecules are better dispersed in the aqueous phase, reducing aggregation between particles. In this way, lipase helps to form more homogeneous and stable emulsions; secondly, the catalyzing action of lipase alters the interfacial properties between the fat and aqueous phases, reducing the interfacial tension and thus enhancing the stability of the emulsion. Stabilized emulsions can effectively prevent the separation of oil and water and extend the shelf life of the emulsion.\u003c/p\u003e \u003cp\u003eLipase enhances emulsification and facilitates emulsion formation by changing the molecular structure and size of the fat component of the emulsion. This is particularly important in leather processing, where lipase can effectively improve the stability and uniformity of degreased fats and oils in water, avoid re-layering of the emulsion, and prevent fats from re-adhering to the surface of animal skins, thus improving the quality of the leather treatment, facilitating efficient processing, and reducing the generation of wastes, thus improving the overall production efficiency.\u003c/p\u003e \u003cp\u003eIn the tanning industry, lipase, as a biocatalyst, has long been focused on improving its stability at high temperatures to adapt to the high-temperature environments common to the tanning process. However, with increasing environmental awareness and rising production costs, traditional high-temperature treatments are facing more and more challenges. High temperatures not only require large amounts of energy support, but may also bring about environmental pollution problems such as exhaust emissions and excessive use of chemical reagents. Therefore, there is an urgent need for the tanning industry to develop lipases that are suitable for efficient operation at ambient temperatures in order to achieve greener and more sustainable production methods.\u003c/p\u003e \u003cp\u003eLipase operating efficiently at room temperature has obvious advantages. Firstly, operation at room temperature significantly reduces energy consumption. Compared with high temperature treatment, the energy requirement during production is greatly reduced, lowering the production cost of enterprises and reducing environmental pollution. Secondly, at room temperature, lipase can more gently remove the excess fat from the leather, avoiding any surface damage or deterioration that may be caused by high temperature, and also helping to maintain the natural softness and touch of the leather. In addition, the role of ambient lipase in emulsion stabilization is also crucial. In leather processing, the homogeneity and stability of the emulsion directly affects the coating effect and the quality of the final product. Whereas emulsions are traditionally prone to delamination at high temperatures, high efficiency lipases at ambient temperatures can effectively improve emulsification and maintain long term stability of emulsions, avoiding wasteful production and inconsistent quality caused by unstable emulsions.\u003c/p\u003e \u003cp\u003eTherefore, the efficient operation of lipase at ambient temperature not only meets the tanning industry's requirements for environmental protection, energy saving and cost control, but also improves the overall efficiency in several process steps such as degreasing, softening and emulsification. This characteristic makes ambient lipase a key breakthrough point in the development of modern tannery industry, which provides important technical support for realizing green production, improving product quality and reducing environmental burden.\u003c/p\u003e \u003cp\u003eHowever, a large number of applications have shown that the catalytic hydrolysis rate of lipase on grease substrate is low in aqueous environments (50\u0026ndash;60%) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Through optimization of the selection of lipase and construction of a synergistic system including lipase, surfactant, and alkali, the hydrolysis rate of ester bonds can only be increased to approximately 70\u0026ndash;80% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Such an outcome is observed mainly because the catalytic process of lipase in the hydrolysis of fat substrates is carried out at the oil\u0026ndash;water interface, which has the characteristic of \u0026ldquo;interface activation.\u0026rdquo; [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Lipase needs to be adsorbed onto the fat surface to make full contact with the substrate and catalyze the substrate hydrolysis. Therefore, strategy for to gaining insight into the distribution characteristics of lipase on the surface of oil and grease and their influence discipline, must be developed to further guide the efficient catalytic hydrolysis technology involving lipase on oil and grease substrate, which has long been a great concern in the industry.\u003c/p\u003e \u003cp\u003eThe lower the surface tension of oil and grease, the easier the interface adsorption by lipase and other molecules. Determination of the substrate surface tension can provide the basic properties of the oil\u0026ndash; water interface, which can offer a reference for assessment of the adsorption capacity of lipase on the fat surface; however, directly revealing the distribution characteristics of specific lipases at the oil\u0026ndash;water interface and the nature of the reaction presents a challenge[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Raman spectroscopy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], infrared spectroscopy[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and other spectroscopic techniques can be used for the comprehensive and molecular\u0026ndash;level exploration of the interaction between lipases and substrates and the resulting structural and conformational changes; photon correlation spectroscopy, neutron scattering[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and other techniques offer a strong support to the study of the size, distribution and aggregation of lipase particles in the oil\u0026ndash;water interface, the location of adsorption position, and structural characteristics of lipase particles; mass spectrometry analysis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], electrochemical technology[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], surface plasmon resonance[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and other technologies; they also enable the qualitative and quantitative analyses of the interaction between lipase and a substrate and product generation, which strongly promotes the investigation of the adsorption and reaction kinetics of lipase on the surfaces of oil and grease; with the help of atomic force microscopy, high\u0026ndash;resolution images of the adsorption of lipase on the surfaces of oil and grease and membrane formation can be obtained[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The development and application of these analytical techniques have strongly promoted the development and improvement of the theory of lipase catalysis at the oil\u0026ndash;water interface. However, common shortcomings, such as high requirements for instrumentation and samples, complex analytical testing process, and difficulty in effectively guiding the development of engineering technology, are the key factors leading to the long\u0026ndash;term challenges in achieving effective breakthroughs in the research on the role of lipase in the efficient catalysis of the hydrolysis of oil and grease substrates. Increasing attention is being paid to the search for a strategy for the analysis of the distribution characteristics of lipase on the surfaces of oil and grease[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and their influence discipline via key process parameters [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which can be used quickly and intuitively in the practical application environment to effectively guide the construction of lipase efficient catalytic technology.\u003c/p\u003e \u003cp\u003eFluorescent labeling technology, as a widely used visualization method with sensitive and intuitive results, covalently combines fluorescein and proteins to render labeled proteins with fluorescent properties for their localization and tracing in cells or tissues[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Studies have repeatedly reported the use of fluorescent labeling technology to analyze the mass transfer behavior of enzyme proteins in animal skins[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which promoted the development of enzyme dehairing, enzyme softening, and other technologies. However, fluorescent labeling has a potential effect on enzyme protein structure and properties, and whether ensuring that fluorescently labeled enzyme adsorption, permeation, and other behaviors within the object is consistent with those of unlabeled enzymes remains controversial[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To this end, our team systematically analyzed the effects of fluorescein structure, dosage, and labeling conditions on the structural characteristics and viability of the enzymes before and after fluorescent labeling and effectively avoided the remarkable effects of fluorescent labeling on the molecular weight, isoelectric point, and viability of protease by controlling the degree of binding between fluorescein isothiocyanate (FITC) and the enzyme protein[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This study lays the groundwork for the development of visual analysis techniques based on fluorescently labeled enzymes and proteins. In addition, in aqueous environments, oil is insoluble in water, but proteins and other substances can be easily adsorbed on the surface of oil droplets[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Such condition can avoid the excessive aggregation of oil droplets to a certain extent. Moreover, the dispersion stability of proteins on oil is easily affected by environmental factors and thus lays a foundation for the observation of the adsorption and distribution characteristics of lipase on oil droplet surface using labeled enzyme proteins.\u003c/p\u003e \u003cp\u003eTherefore, this paper examined the effects of fat mass fraction, lipase dosage, temperature, stirring and dispersion time, resting time, fat type, lipase type, and other factors on the dispersion stability of fat emulsions and attempted to construct a fluorescent labeling\u0026ndash;based method for the visualization of the distribution patterns and reaction properties of lipase at the oil\u0026ndash;water interface. In addition, the influence of enzyme and oil types and other factors on the distribution pattern of lipase at the oil\u0026ndash;water interface was investigated, and the results lay the foundation for the further development of an efficient catalytic technology for the deep hydrolysis of oil under the action of lipase.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMain Instruments and Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eMain Instruments\u003c/h2\u003e \u003cp\u003eBenchtop high speed refrigerated centrifuge, Hunan Xiang Yi Lab Instru Dev Co.;APPS MV 50D Automatic protein chromatography system, Lisure Science (Suzhou) Co., Ltd༛UV\u0026ndash;1100 UV spectrophotometer, Shanghai MAPADA Instrument Co., Ltd༛Confocal laser scanning microscope NE900, NINGBO YONGXIN OPTICS CO., LTD.༛IKA ULTRA\u0026ndash;TURRAX Tube Drive UTTD, IKA Works Guangzhou༛THZ\u0026ndash; 82 Incubator shakers, Guangdong Fuwa Engineering Group Co., Ltd.༛Superdex 75 prep grade, BXK16, Bestchrom (Zhejiang) Biosciences Ltd.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMaterials\u003c/h3\u003e\n\u003cp\u003eLKT lipase(400000U/g)、YMN lipase༈200000U/g༉、AKT\u0026ndash;A lipase༈350000U/g༉、AKT\u0026ndash;N lipase༈\u0026ge;100000U/g༉,industrial grade, Chengdu Yufangda Biotechnology Co., Ltd;Fluorescein isothiocyanate, analytical pure, Sangon Biotech (Shanghai) Co., Ltd.༛Lipase labeled with fluorescein isothiocyanate, Laboratory self\u0026ndash;made༛olive oil, analytical pure, Chengdu Kelong Chemical Co.༛Butter, mutton tallow, wild boar oil all purchased in the market༛Other general reagents are domestic analytical pure.\u003c/p\u003e\n\u003ch3\u003eExperimental content and methodology\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect of oil mass fraction on the distribution characteristics of lipase at the oil\u0026ndash;water interface\u003c/h2\u003e \u003cp\u003eWeigh the appropriate amount of oil in the UTTD test tube, The oil mass fraction (wt%) were 5%, 10%, 20%, 25% and 50%, add 1.0 mL of moderately diluted labeled enzyme protein solution, the appropriate amount of Sudan IV staining solution and ultrapure water, control the total mass of 10.0 g. Use the UTTD test tube disperser to stir and disperse for 10 min, take up a drop of 10\u0026micro;L of liquid with a micropipette and add it onto a slide, let it stand for 120 s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of enzyme protein concentration on the distribution characteristics of lipase at the oil\u0026ndash;water interface\u003c/h2\u003e \u003cp\u003eWeigh 2.5g of olive oil in a UTTD test tube, add 1.0mL of fluorescently labeled LKT lipase with enzyme protein concentration of 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 10mg/mL respectively, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10\u0026micro;L droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of temperature on the distribution characteristics of lipases at the oil–water interface\u003c/h3\u003e\n\u003cp\u003e2.5 g of olive oil was weighed in a UTTD tube, and the temperature of the olive oil was controlled at 4℃, 25℃, 30℃ and 40℃, respectively, and 1.0 mL of 0.25 mg/mL fluorescently labelled LKT lipase at the corresponding temperatures was added, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10\u0026micro;L droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e\n\u003ch3\u003eEffect of stirring time on the distribution characteristics of lipase at the oil–water interface\u003c/h3\u003e\n\u003cp\u003e2.5 g of olive oil was weighed in the UTTD tube, 1.0 mL of fluorescent labelled enzyme protein solution with enzyme protein concentration at 0.25 mg/mL was added, add an appropriate amount of ultrapure water to control the total mass of 10.0g, and the stirring time in the UTTD tube was 1 min, 3 min, 5 min and 10 min, 10\u0026micro;L droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of resting time on the distribution characteristics of lipase at the oil\u0026ndash;water interface\u003c/h2\u003e \u003cp\u003eWeigh 2.5 g of olive oil in a UTTD test tube, add 1.0 mL of fluorescently labelled enzyme protein solution with enzyme protein concentration at 0.25 mg/mL, stir and disperse for 10 min with a UTTD test tube disperser, then aspirate 10 \u0026micro;L droplets with a micropipette and add them dropwise on a slide, and leave it for 30s, 60s, 90s and 120s, respectively, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of oil type on the distribution characteristics of lipase at the oil\u0026ndash;water interface\u003c/h2\u003e \u003cp\u003eWeigh 2.5 g of olive oil, sheep oil, lard, glycerol trioleate in a UTTD tube and add 1.0 mL of fluorescently labelled 0.25 mg/mL of LKT lipase, respectively, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10\u0026micro;L droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of lipase species on the distribution characteristics of lipase at the oil\u0026ndash;water interface\u003c/h2\u003e \u003cp\u003eWeigh 2.5 g of olive oil in a UTTD tube and add 1.0 mL of fluorescently labelled 0.25 mg/mL of LKT lipase, YMN lipase, AKT\u0026ndash;A lipase, AKT\u0026ndash;N lipase, add an appropriate amount of ultrapure water to control the total mass of 10.0g, stir and disperse with a UTTD test tube disperser for 10 min, 10\u0026micro;L droplets were aspirated with a micropipette, added onto a slide, left for 120s, and then observe the state of the oil and water under CLSM (Confocal laser scanning microscope). FITC was excited under an argon ion laser at a wavelength of 488 nm to produce green fluorescence.\u003c/p\u003e \u003cp\u003eAt the same time, take the stirred oil\u0026ndash;water mixture and determine the acid value of the emulsion after reaction with the different lipases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of acid value of oils and fats\u003c/h2\u003e \u003cp\u003eAdd 2\u0026ndash;3 drops of phenolphthalein solution and titrate with 0.05 mol/L KOH solution to determine the acid value of the reacted emulsion.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of oil mass fraction on the dispersion effect of emulsions\u003c/h2\u003e \u003cp\u003eIn order to explore the visualization of lipase distribution characteristics at the oil\u0026ndash;water interface based on fluorescently labeled enzyme proteins, the amount of olive oil in the oil-water mixture needed to be explored. Therefore, the effect of olive oil\u0026ndash;water mixing ratio on emulsion characteristics was first examined in combination with Sudan IV dye and lipase protein after it was labeled, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. When the dosage of olive oil was 10\u0026ndash;20%, the dispersion of the oil phase was better, and \u0026ldquo;O/W\u0026rdquo; type oil droplets of moderate size can be formed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of resting time on the stability of the mixing effect of emulsions\u003c/h2\u003e \u003cp\u003eThe effect of settling time on the distribution of lipase at the oil\u0026ndash;water interface was further examined, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, when the emulsion was left to stand for 30\u0026ndash;120 s, no evident aggregation of oil droplets occurred, and a uniform distribution of oil droplets and labeled enzyme proteins that were independent of each other at the interface can still be observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of stirring time on the dispersion stability of emulsions\u003c/h2\u003e \u003cp\u003eIn order to investigate the effect of stirring time on the stability of oil-water mixtures, the stirring time of the oil-water mixtures was varied while the stirring speed was kept constant. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e reveals that a poor oil droplet dispersion effect was observed when the stirring time was 1 min despite the relatively clear oil\u0026ndash;water interface. With the extension of stirring time, the oil\u0026ndash;phase dispersion improved, and the uniformity of oil droplet size was gradually improved. In addition, when the stirring time was up to 10 min, the emulsion showed better stability with a clear interface, and dispersed stable oil droplets could be observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of enzyme protein concentration on the dispersion stability of emulsions\u003c/h2\u003e \u003cp\u003eThe effect of various labeled enzyme proteins on the properties of oil\u0026ndash;water interface of the olive oil was further investigated. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that, at room temperature, when the concentration of the labeled enzyme protein was controlled at 0.1 mg/mL, a weak fluorescence intensity occurred on the surface of oil droplets. When the concentration of the labeled enzyme protein was increased to 0.25 mg/mL, an evident fluorescence layer transpired on the oil droplet surface. When the concentration of the labeled enzyme protein was further increased to 0.5 mg/mL, the oil\u0026ndash;water interface of oil droplets became damaged and fuzzy. When the enzyme protein concentration was further increased to 1 mg/mL, a good thickening effect and a stable oily paste were obtained. Moreover, the independent oil droplets were difficult to observe.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of temperature on the mixing stability of emulsions\u003c/h2\u003e \u003cp\u003eIn order to investigate the effect of temperature on the distribution of lipase at the oil-water interface, fluorescently labeled lipase was stirred and observed at different temperatures, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, it can be seen that at a low temperature (\u0026lt;\u0026thinsp;10℃), the viscosity of olive oil was large, and gradual solidification was observed (the solidification point of olive oil is about 5℃). Thus, effective dispersion in the aqueous environment was difficult to achieve, and the oil droplets were bonded to each other. As the temperature increased, the homogeneous adsorption of the marker enzyme protein at the oil\u0026ndash;water interface was evident. However, when the temperature was extremely high (up to 40℃), it is not favorable for the observation of oil droplets.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of oil type on the distribution characteristics of lipase at the oil\u003c/b\u003e\u0026ndash;\u003cb\u003ewater interface\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe distribution characteristics of lipase at the oil\u0026ndash;water interface of various fats and oils were further examined, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows that in the glycerol trioleate emulsion, lipase was fully distributed in the interior of oil droplets. Lipase can be evenly distributed on the surface of olive oil, and form a complete and uniform lipase contact layer. Meanwhile, the lipase can only form a discontinuous distribution feature on the surface of sheep oil. However, in lard emulsion, the distribution of lipase could not be observed in the lard\u0026ndash;oil\u0026ndash;water interface, but only in the interstices of lard droplets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDistribution characteristics of typical lipases at the oil\u0026ndash;water interface and their catalytic hydrolysis of substrates\u003c/h2\u003e \u003cp\u003eTo investigate the effect of lipase species on the distribution effect of enzyme proteins at the oil-water interface, the distribution characteristics of four typical lipases at the oil\u0026ndash;water interface of olive oil were analyzed. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows that the lipase YMN had the strongest affinity for olive oil, which not only can be adsorbed on the surface of oil droplets effectively, but also dispersed into more independent droplets. In addition, a part of the enzyme proteins can penetrate the interior of the droplets, which effectively increased the contact efficiency between lipase and the substrate and thus enhanced the catalytic hydrolysis of olive oil and increased its acidic value (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026ndash;II). Lipase LKT can also disperse olive oil as independent oil droplets and distribute them uniformly at the oil\u0026ndash;water interface, but it did not penetrate the interior of oil droplets. Thus, the degree of catalytic hydrolysis of the substrate was inferior to that of lipase YMN. In lipase AKT\u0026ndash;N, the dispersion of droplets showed a poor stability, and larger droplets easily formed. Therefore, although lipase AKT\u0026ndash;N also penetrated the interior of oil droplets, it was not as effective as lipase YMN. Lipase AKT\u0026ndash;A which had the lowest degree of contact with the substrate, showed the poorest catalytic hydrolysis efficiency for olive oil, mainly because it failed to effectively disperse the oil droplets and can only be adsorbed on their surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn an aqueous environment, the catalytic reaction of lipase during the hydrolysis of fat substrate was carried out at the \u0026ldquo;oil\u0026ndash;water\u0026rdquo; interface[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The adsorption and permeation properties of lipase at the oil\u0026ndash;water interface directly affect its contact efficiency with the substrate, which influences the catalytic efficiency. An intuitive analysis of the distribution characteristics of lipase at the oil\u0026ndash;water interface and its influence law will provide a reference for the construction of an efficient lipase catalytic technology. Previous research has shown that FITC fluorescence labeling technology can be used to observe the distribution and mass transfer characteristics of enzyme proteins in different chemical reaction scenarios[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, after optimizing the observation conditions, this technique is attempted to be applied to analyze the adsorption behavior of lipase on the surface of lipid substrate in aqueous environment.\u003c/p\u003e \u003cp\u003eThe mixing of oil and water forms an emulsion, and to observe the distribution characteristics of lipase at the oil\u0026ndash;water interface via fluorescence microscopy, the emulsion must have a certain degree of stability. In general, in an aqueous phase environment, proteins are easily adsorbed at the oil\u0026ndash;water interface of the oil, form a protective film to prevent the aggregation of oil droplets, improve the stability of the emulsion, and exert a good foam, emulsification, thickening, which were effected with the changes in oil content, temperature, protein concentration, stirring intensity and resting time [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirstly, the oil-to-water ratio will have a direct impact on the composition and stability of the emulsion. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with the increase in oil dosage, the emulsion may gradually change from an oil\u0026ndash;in\u0026ndash;water (O/W) type to a water\u0026ndash;in\u0026ndash;oil (W/O) type, which in turn affects the stability, dispersibility, viscosity, and other emulsion properties. When the olive oil dosage was extremely low (wt%=5%), the oil droplets were small, lacked stability, showed susceptibility to agglomeration, and exhibited poor dispersion, which were not conducive to microscopic observation. In addition, fluorescence intensity in the aqueous region was strong, which indicates that most of the labeled enzyme proteins were not adsorbed at the oil\u0026ndash;water interface. When the dosage of olive oil reached 25% or more, the oil phase was likely to aggregate and form larger oil droplets. Moreover, a non\u0026ndash;independent \u0026ldquo;adhesion\u0026rdquo; phenomenon was observed between the oil droplets, which gradually transformed into a \u0026ldquo;W/O\u0026rdquo; emulsion (wt%=50%). As a result, a poor oil phase dispersion occurred, and observation of the oil droplet interface presented difficulty. When the dosage of olive oil was 10\u0026ndash;20%, the dispersion of the oil phase was better, and \u0026ldquo;O/W\u0026rdquo; type oil droplets of moderate size can be formed. In addition, the stability was better, and the interface between oil and water was clearer. At this point, the uniform distribution of labeled enzyme proteins in the interface between oil and water can be observed, and the fluorescence intensity of the water\u0026ndash;phase region was low, which was conducive to the analysis of the distribution characteristics of lipase in the oil and water interface. This condition is more conducive to the analysis of the distribution characteristics of lipase at the oil\u0026ndash;water interface.\u003c/p\u003e \u003cp\u003eHowever, emulsions are inherently unstable. Generally speaking, when stirring ceases and an emulsion is left undisturbed, various forces such as gravity, buoyancy, surface tension etc., gradually cause oil droplets to aggregate at specific positions within the liquid leading to stratification over time. Nevertheless, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates that marker enzyme proteins adsorb onto the surface of oil droplets impeding their coalescence into larger entities within a short period. This discovery establishes a foundation for effectively observing and analyzing distribution characteristics of marker enzyme proteins at the oil-water interface.\u003c/p\u003e \u003cp\u003eMoreover, the dispersion of the emulsion is evidently influenced by stirring intensity and other factors. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e demonstrated that, when maintaining a constant stirring speed, the dispersion stability of the emulsion is primarily affected by the duration of stirring. This could be attributed to that, with prolonged stirring time, shear force facilitates the destruction and reorganization of the oil-water interface, thereby promoting further mixing between oil and water phases and enhancing uniformity in droplet size as well as dispersion stability [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe alteration in protein concentration, however, will have a further impact on the dispersion of oil and the stability of emulsion. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, when the concentration of the labeled enzyme protein was maintained at 0.1 mg/mL, a weak fluorescence intensity was observed on the surface of oil droplets, resulting in poor emulsion stability due to insufficient distribution of labeled proteins across the oil droplet interface. Upon increasing the concentration to 0.25 mg/mL, a distinct fluorescence layer emerged on the surface of oil droplets, indicating more uniform distribution of lipase protein at this point within the oil-water interface. Furthermore, this prevented aggregation of oil droplets and facilitated observation of lipase protein distribution at said interface. However, as we further increased the concentration to 0.5 mg/mL, excessive protein presence led to damage and blurring at the oil-water interface of these droplets; while an increase to 1 mg/mL resulted in effective thickening and formation of stable oily paste with limited visibility for individualized oil droplets.\u003c/p\u003e \u003cp\u003eThe results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrate that, at temperatures below 10\u0026deg;C, olive oil exhibits high viscosity and gradual solidification occurs (the solidification point of olive oil is approximately 5\u0026deg;C). Consequently, achieving effective dispersion in the aqueous environment becomes challenging as the oil droplets tend to bond with each other. This leads to a blurred interface between oil and water, posing difficulties for observing and analyzing the distribution characteristics of marker enzyme proteins at the interface. However, as the temperature increases, there is a decrease in the viscosity of olive oil[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], facilitating the formation of \"O/W\" type emulsion droplets. This enhances emulsion stability and promotes clearer formation of an oil-water interface where homogeneous adsorption of marker enzyme proteins becomes evident. Nevertheless, when temperatures reach extremely high levels (up to 40\u0026deg;C), enhanced Brownian motion contributes to droplet aggregation and sedimentation. Therefore, the temperature should be regulated within the range of 20\u0026ndash;30\u0026deg;C, which is deemed more suitable.\u003c/p\u003e \u003cp\u003eThen, a method based on fluorescently labeled enzyme proteins was established to visualize the reaction properties of lipase at the oil\u0026ndash;water interface, and it can be used for the effective observation of the distribution characteristics of lipase at the oil\u0026ndash;water interface. The optimal conditions for observation include the following: oil content of 10\u0026ndash;20% (wt%), concentration of fluorescently labelled enzyme protein of 0.25 mg/mL, reaction temperature of 25\u0026deg;C\u0026ndash;30\u0026deg;C, emulsion dispersion and stirring time of 10 min, and emulsion resting time of 30\u0026ndash;120 s.\u003c/p\u003e \u003cp\u003eBased on the developed fluorescently labeled protein observation method, the distribution characteristics of lipase at the oil-water interface of various fats and oils were further examined. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrated that lipase was fully distributed within the interior of glycerol trioleate emulsion droplets, indicating its good contact properties with this substrate. Lipase could also be evenly distributed on the surface of olive oil, forming a complete and uniform lipase contact layer. However, due to the high freezing point of goat oil leading to solid formation, lipase only exhibited a discontinuous distribution feature on some solid surfaces. In lard emulsion, lipase distribution was observed in interstices between lard droplets rather than at the lard-oil-water interface. Additionally, fluorescence-labeled protein observations reveal that this lipase shows better adsorption performance at the oil-water interface of vegetable fats (such as olive oil) compared to animal fats (sheep's oil and lard) in an aqueous environment. This finding could be attributed to vegetable oils primarily containing unsaturated fatty acids [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], such as stearic and palmitic acids. Furthermore, this type of lipase demonstrated a stronger affinity for oleic acid glycol esters and achieved good affinity with oleic acid glycerides. Moreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the adsorption performance of lipase from different sources on the oil surface exerted a large difference, which not only affected the stability of oil emulsion but also the catalytic hydrolysis performance of lipase on oil. It revealed that differences in the distributions of lipase at the oil\u0026ndash;water interface of various fats and oils had a certain degree of correspondence with their specificity and that the distribution characteristics of the lipases on the surface of olive oil enabled effective catalytic hydrolysis to a certain extent.\u003c/p\u003e \u003cp\u003eThese results highlight how fluorescent labeling allows for more intuitive analysis of lipase distribution characteristics at the oil-water interface across various lipid substrates while providing support for further research on efficient catalysis facilitated by lipases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA visualization method of lipase reaction at the oil-water interface based on fluorescently labeled enzyme protein has been established, which can effectively observe the distribution characteristics of lipase at the oil-water interface under the following optimal conditions: the oil content of 10%-20% (wt%), the concentration of fluorescently labeled enzyme protein of 0.25 mg/mL, the reaction temperature of 25\u0026ndash;30\u0026deg;C, the emulsion dispersion and stirring time of 10 min, and the resting time of the emulsion of 30\u0026ndash;120 s. Based on this method, a preliminary analysis of the effects of oil and lipase species on the distribution characteristics of lipase at the oil-water interface has been carried out. Based on this method, the effects of oil and lipase species on the distribution characteristics of lipase at the oil-water interface were initially analyzed, and it was observed that the differences in the distribution of lipase at the oil-water interface of different fats and oils had a certain degree of correspondence with their specificity, and that the distribution characteristics of different lipases on the surface of olive oil had a certain degree of correspondence with their catalytic hydrolysis efficiencies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e The authors would like to thank all members of the National Engineering Laboratory for Clean Technology of Leather Manufacture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e The study design was contributed by Chun Z and Xian D; funding was obtained by Bi P and Chun Z; the experiments were performed by Chun Z and Xian D; data analysis was conducted by Xian D; the manuscript was written by Chun Z and Xian D. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003eThis study was supported by The Key R\u0026amp;D Program of Chengdu for Technology Innovation (2024\u0026ndash;YF08\u0026ndash;00060\u0026ndash;GX); The Fund of the Central Guidance on Local Science and Technology Development (2024ZYD0218).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no conflicts of interest relating to this work.\u003c/p\u003e\n\u003ch1\u003eData availability\u003c/h1\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVerger R (1997) \u0026lsquo;Interfacial activation\u0026rsquo;of lipases: facts and artifacts. 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J Food Eng 113:217-225.\u003c/li\u003e\n\u003cli\u003eAhmad Nizar NN, Nazrim Marikkar JM, Hashim DM (2013) Differentiation of lard, chicken fat, beef fat and mutton fat by GCMS and EA-IRMS techniques. J Oleo Sci 62:459-464.\u003c/li\u003e\n\u003cli\u003eKanwal N, Musharraf SG (2024) Analytical approaches for the determination of adulterated animal fats and vegetable oils in food and non-food samples. Food Chem 460:140786.\u003c/li\u003e\n\u003cli\u003eZhang W, Zheng R, Xu X, Zhao XJFH (2024) Oil unsaturation degree dictates emulsion stability through tuning interfacial behaviour of proteins. Food Hydrocolloids 158:110588\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":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lipase, Interfacial catalysis, fluorescent labelling, oil–water interface, affinity","lastPublishedDoi":"10.21203/rs.3.rs-5758102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5758102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA method based on fluorescently labeled enzyme proteins was established to visualize the absorption properties of lipase at the oil\u0026ndash;water interface, and it can be used for the effective observation of the distribution characteristics of lipase at the oil\u0026ndash;water interface. The optimal conditions for observation include the following: oil content of 10\u0026ndash;20% (wt%), concentration of fluorescently labelled enzyme protein of 0.25 mg/mL, reaction temperature of 25℃\u0026ndash;30\u0026deg;C, emulsion dispersion and stirring time of 10 min, and emulsion resting time of 30\u0026ndash;120 s. Based on this method, a preliminary analysis of the effects of oil and lipase species on the distribution characteristics of lipase at the oil\u0026ndash;water interface was performed. The results reveal that differences in the distributions of lipase at the oil\u0026ndash;water interface of various fats and oils had a certain degree of correspondence with their specificity and that the distribution characteristics of the lipases on the surface of olive oil enabled effective catalytic hydrolysis to a certain extent. This method is a more objective guide for the development of lipase application technology in the fields of tanning, fur making, glue making, detergents, sewage treatment and so on.\u003c/p\u003e","manuscriptTitle":"Visualization of fluorescently labeled lipase distribution characteristics at the oil–water interface","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 14:13:34","doi":"10.21203/rs.3.rs-5758102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-27T16:44:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-25T08:20:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28121202929700194810938327388729082706","date":"2025-01-22T12:03:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-22T01:29:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-07T13:24:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-07T13:18:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2025-01-03T11:58:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"54f5c92b-9dcc-405a-a06d-c621987aad03","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T19:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-09 14:13:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5758102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5758102","identity":"rs-5758102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}