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Synergistic interaction between exogenous and endogenous emulsifiers and its impact on in vitro digestion of lipid in crowded medium
Food Chemistry 299 (2019) 125164
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Synergistic interaction between exogenous and endogenous emulsifiers and its impact on in vitro digestion of lipid in crowded medium Shen-Zhi Wang, Hui-Qian Dai, Ke-Xian Chen, Juan Li, Zhong-Xiu Chen
T
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Molecular Food Science Laboratory, College of Food & Biology Engineering, Zhejiang Gongshang University, Hangzhou 310018, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Macromolecular crowding Lipase Synergistic interaction Lipid digestion Bile salt
Control of lipid digestibility by various food components has received great attention in recent decades. However, there is limited literature on investigating the synergistic effect of exogenous emulsifiers and endogenous sodium cholate (SC) on lipid digestion in a simulated physiological crowded medium. In this work, the synergistic interaction of Tween80 and SC according to the regular solution theory, and the hydrolysis of lipid emulsions containing tricaprylin, glyceryltrioleate or soybean oil in crowding medium was studied. The results show that emulsions stabilized by a combination of Tween80 and SC showed higher digestion rate and transformation than those with Tween80 or SC. The digestion rate could be increased by polyethylene glycols (PEGn) with varying crowding degree. The denaturation temperature of the lipase was increased in macromolecular crowded medium. This work allows for better understanding of the interaction between the amphiphiles and the macromolecular crowding effect on lipase digestion in the physiological environment.
1. Introduction Digestion and absorption of lipids in human gastrointestinal (GI) tract play an important role in body lipid homeostasis. The increasing concern regarding the controllable digestion of lipids has evoked many studies on the impact of other food components on lipid metabolism. In small intestine, lipids digestion is largely dependent on the interfacial composition of the emulsion droplets. The key intestinal components that support interfacial activation of enzyme and facilitate lipid digestion are amphiphilic molecules. As typical endogenous surfactants, bile salts help emulsifying lipids to oil droplets, altering interfacial composition to favor lipase adsorption and participating the formation of mixed micelles that can solubilize free fatty acids and other lipid nutrients and transport them to the epithelium cells (Russell, 2009). In food processing, emulsifiers has been added as stabilizing agents to ensure the taste, flavor, texture and other specific features of food. Therefore, surfactants are commonly present in many processed foods and drug formulations. In the GI tract, a surfactant-stabilized food emulsion system might suffer droplet flocculation or coalescence due to a drastic change in pH and a variation in ionic strength. The exogenous surfactant would inevitably interact with endogenous emulsifiers, such as bile salts. The digestion rate and fate of lipids depend on the oil composition, exogenous surfactant type and dosage, the interfacial microstructural composition of the formed membrane, the size
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distribution of other intestinal aggregates like micelles and vesicles, and also the binding affinity of lipase towards these interfaces (Mao & Miao, 2015; Chang & McClements, 2016). However, the impact of these dietary emulsifiers on lipid digestion is particularly complex, because they might cause the aggregation and flocculation of oil droplets and also affect the digestion performances of lipase at the same time (LopezPena & Mcclements, 2014; Beysseriat, Decker, & McClements, 2006). An early study showed that the self-assembled aggregates at emulsionwater interfaces in the upper small intestine during lipid hydrolysis facilitated the dissolution of lipolytic products into unsaturated mixed micelles (Hernell, Staggers, & Carey, 1990). These results indicate that a synergistic interaction between the endogenous amphiphiles and the exogenous surfactants in the small intestine could possibly affect the lipase-catalyzed hydrolysis of lipids. As an important surfactant abundant in human body, sodium cholate (SC) and its aggregation behavior with surfactants such as anionic (Hildebrand, Garidel, Neubert, & Blume, 2004), cationic (Manna, Chang, & Panda, 2012), nonionic (Patel, Bharatiya, Ray, Aswal, & Bahadur, 2015) and zwitterionic (Naskar, Mondal, & Moulik, 2013) have been extremely studied. Interaction of SC with dietary phospholipids also attracts extensive interests (Coreta-Gomes et al., 2015). We previously found a structural evolution in the interaction of SC with a vitamin-based bolaamphiphiles vesicle (Tian, Ge, Shen, He, & Chen, 2016). Tween80 is an aliphatic, nonionic surfactant approved by the
Corresponding author. E-mail address: [email protected] (Z.-X. Chen).
https://doi.org/10.1016/j.foodchem.2019.125164 Received 6 December 2018; Received in revised form 19 June 2019; Accepted 9 July 2019 Available online 09 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. Critical micelle concentration (CMC) measurements
U.S. Food and Drug Administration (FDA) for use in injectable, oral, and topical products. Mixed micelles of BS and Tween80 were studied as biocompatible colloidal drug carriers (Ćirin, Poša & Krstonošić, 2012). However, to the best of our knowledge, the synergistic interaction of Tween80 and SC on lipid digestion within the simulated small intestine has not been studied yet. During lipid digestion, pancreatic lipases interact with other compounds, such as polysaccharides and proteins. These macromolecules create a highly crowded environment, which constitutes the native environment of the lipase. Our research group reported that both catalytic reactions that involve small molecules and the non-covalent binding process could be affected by the “macromolecular crowding” effect. In a crowded medium, vesicle-catalyzed production of fatty acids (FAs) displayed unusual chain-length dependence (Cao et al., 2014). The binding of FAs to phospholipid bilayers and serum albumin also became unusual (Wang, Yang, Zhu, Wang & Chen, 2016; Zhu et al., 2018). Actually, the impact of hydrocolloids on lipid digestion within the gastrointestinal tract was thoroughly investigated (Qin, Yang, Gao, Yao, & McClements, 2016). Some of the dietary fibers increased the lipid digestion rate, but some inhibited lipid hydrolysis, depending on their molecular and physicochemical properties. Whether the excluded volume effect was involved was not clear. However, the macromolecular crowding effect did affect the catalytic activities of enzymes because their conformation was changed by the crowded medium (Norris & Malys, 2011; Balcells et al., 2014; Pastor et al., 2014). In this research, the synergistic effect of an exogenous emulsifier (Tween80) and the endogenous emulsifier (SC) on the lipid digestion based on regular solution theory and was studied. The impact of the crowding medium on lipid digestion in the presence of both exogenous and endogenous surfactants was also discussed. The results improve our understanding of the interaction of the surface active agents in the physiological environment, and the macromolecular crowding effects on lipase digestion.
The CMC values of SC, Tween 80 and the binary mixture were measured by monitoring the fluorescence intensity I1/I3 ratio of pyrene which was used as fluorescence probe by using Hitachi F-7000 fluorescence spectrophotometer. The concentration of pyrene was kept at 0.5 µmol L−1. The fluorescence emission spectra were obtained by employing an excitation wavelength of 334 nm. The I1 and I3 intensities in fluorescence emission spectra correspond to the first and third vibronic peaks of pyrene was located at wavelengths of ca. 373 and 384 nm, respectively. The I1/I3 ratios were plotted as a function of the concentration of binary mixture of Tween80 and SC. The CMC values were taken from the point of inter-section by fitting the premicellar and postmicellar data in linear equations. A typical example is shown in the Supporting Information section.
2.5. Particle size and ζ- potential characterization Both the particle size and ζ-potential of the emulsions were characterized by Zetasizer Nano-ZS (Malvern Instruments, Ltd, United Kingdom). The instrument uses a laser at a wavelength of 632.8 nm and detects the scattered light at an angle of 173°. Zeta potential was measured based on laser Doppler electrophoresis. All measurements were performed in a temperature-controlled chamber. Experiment duration (equilibration time) was in the range of 2 min, and each test was repeated three or more times.
2.6. In vitro digestion model (pH-stat) The pH-stat model (Li, Hu & McClements, 2011) was employed as the in vitro digestion model in this study. The procedure used is as following:
2. Materials and methods
(i) 30.0 mL emulsion was added into a glass beaker and was placed in water bath at 37.0 °C. The pH was adjusted to 7.0 by NaOH or HCl solutions and was stirred for 10 min. (ii) 5.0 mL SC solution (187.5 mg SC dissolved in phosphate buffer, 87 mmol L−1, pH = 7.0) and 1.0 mL CaCl2 solution (188 mM) were added to the above emulsion under stirring condition and the pH of the system was adjusted back to 7.0 if required. (iii) 60 mg lipase powder was added to the above mixture. A pH-stat automatic titration unit (Metrohm, USA Inc.) was then used to automatically monitor the pH and maintain the pH at 7.0 by titrating appropriate concentrations of NaOH solution. The volume of NaOH added to the emulsion was recorded, which was used to calculate the concentration of free fatty acids (FFAs) generated from the lipolysis using the following formula:
2.1. Materials Lipase (porcine pancreas) (Item NO. P7475), tricaprylin (Item NO. T9126) and glyceryltrioleate (Item NO. T7410) were purchased from Sigma-Aldrich with purity of 99%. Polyethylene glycols (PEGn (n = 200, 2000 and 20000)), soybean oil, calcium chloride (CaCl2), polyoxyethylene dehydrated sorbitol monooleate (Tween80), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from Aladdin Corporation. Sodium Cholate (SC) was purchased from TCI Corporation with purity of 99%. 2.2. Solution preparation The stock solution of Tween80 (50 mmol L−1), SC (50 mmol L−1) and CaCl2 (188 mmol L−1) were prepared by dispersing the corresponding chemicals into the phosphate buffer solution (20 mmol L−1, pH = 7.0) and were stirred for at least 3 h. Tween80 and SC at varying molar ratio was mixed for determination the critical micelle concentration. The stock solution containing polyethylene glycols (PEG) were prepared by dispersing polymer into the phosphate buffer, which was used as the macromolecular crowded medium.
%FFA = 100 ×
VNaOH × mNaOH × MOil WOil × 2
where VNaOH is the volume of NaOH required to neutralize the FFAs produced; mNaOH is the molarity of the NaOH solution; WOil is the total weight of oil, and MOil is the molecular weight of the oil.
2.7. Thermal stability of lipase 2.3. Emulsion preparation The Differential Scanning Calorimetry measurements (DSC) of the lipase in different solutions were performed in VP-DSC instrument (MicroCalInc, Northampton, USA). All the calorimetric data were recorded by performing the heating and cooling scans from 1 to 110 °C at a scan rate of 1 °C/min. The concentration of lipase was kept at a constant of 5 mg/mL. Data analysis was done by using the Origin 7.0 software in Microcal.
Tricaprylin, glyceryltrioleate and soybean oil were used as the oil phase for emulsions. 10 wt% oil-containing stock emulsions were prepared by homogenizing the mixture of oil with the above prepared solution containing Tween80 and/or SC (20 mmol L−1) using a highspeed blender (FA25-Digita, Fluko Products, China) at 9000 rpm for 10 min. 2
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3.1.2. Particle size and ζ-potential of emulsions stabilized by SC-Tween80 mixed micelles The different emulsions containing tricaprylin, glyceryltrioleate and soybean oil were prepared with Tween80-SC mixture at the optimal ratio of 0.3, as determined by the above synergistic interaction studies. The particle size distribution and ζ-potential values of emulsions are shown in Fig. 2, respectively. It is found that the stability of emulsions stabilized by a combination of Tween80 and SC was higher than that emulsified by Tween80 or SC only. For example, the particle size distribution of tricaprylin emulsion stabilized by mixed micelles was narrower than that stabilized by single-component micelles, and its droplet size value was also much smaller (Fig. 2a1–c1). Similar trends were also observed for the emulsions containing glyceryltrioleate or soybean oil. The more narrow distribution from the combination of anionic and nonionic surfactants was expected due to better surface coverage resulting from reduced repulsion of anionic species. In terms of triacylglycerols with different chain length, the droplet size of emulsions containing glyceryltrioleate or soybean oil was significantly larger than that containing tricaprylin. Besides, for the same emulsion, emulsions stabilized by a Tween80-SC showed more negative ζ-potentials than those by Tween80 or SC only (Fig. 2 a2-c2), implying a higher stability and a good dispersity. The ζ-potential values of emulsions containing glyceryltrioleate or soybean oil were more negative than that containing tricaprylin except for the case emulsified by Tween80. And a higher ζ-potential magnitude of emulsions may indicate a better interface contact between lipase and lipids (Hsu & Nacu, 2003). It is noteworthy that when significant levels of nonionics are used, drawing conclusions from the ζ-potential can be tenuous due to the likely presence of other stabilizing mechanisms and the questionable assumption the Stern layer is located approximately at the slip plane. Therefore, though the absolute value of ζ-potential was not more than 30 mV, the emulsion was stable enough for the followed digestion experiment.
2.8. Data analysis All experiments were performed independently at least three times. The results were then reported as averages and standard deviations of these measurements. 3. Results and discussion In this paper, Tween80 was selected as the model exogenous emulsifier and SC (SC) was used as endogenous surfactant. PEGn (n = 200, 2000 and 20000) was used as crowding reagents due to its adjustable crowding degree. PEG has been universally used in foods owing to its approved safety in the body (Desbuquois & Aurbach, 1971). Lipids, tricaprylin, glyceryltrioleate and soybean oil, were used to investigate the structural aspects of oil composition on lipid digestion. 3.1. Synergistic interaction of SC and Tween80 and their effect on in vitro lipid digestion 3.1.1. Interaction between SC and Tween80 Interactions of SC and Tween80 and their mixed micelle formation in phosphate buffer solution were investigated by steady-state fluorescence measurements. The detailed results based on the regular solution theory (Haque, Das & Moulik, 1999; Ćirin et al., 2012) by Rubingh (1979) are shown in Fig. 1, where α is the mole fraction of Tween80 in solution; Xid and X1 denote the mole fraction in the ideal and real mixed micelles, respectively; CMCid and CMCex refer to the ideal and experimental CMC values of binary mixture of SC and Tween80, respectively; β represents the Rubingh synergistic interaction parameter between SC and Tween80, which indicates the magnitude of interactions operating between the unlike components in the mixed micelle state. The larger the absolute value of β, the stronger the mixing non-ideality and the synergistic effect. The values for the CMCs of pure SC and Tween80 in phosphate buffer solution are 11.8 mmol L−1 and 0.03 mmol L−1, respectively. Detailed calculation of these values was described in Supporting Information. Obviously, all β values were negative, which means that the synergistic interaction between SC and Tween80 occurred. A maximum absolute β value was found when α was 0.3, showing the strongest synergistic interaction strength at this Tween80-SC ratio. Increasing the mole fraction of the hydrophobic components (Tween80) in the mixed micelle may reduce the synergistic interaction to some extent. In addition, the large negative deviation of CMCex values from the corresponding CMCid values indicated that the thermodynamic stability of the real binary mixed micelles of SC and Tween80 were higher than that of their ideal binary mixtures. It has been reported that the CMC value of Tween80 (Bhattacharjee et al., 2010) was much smaller than that of SC (Ćirin et al., 2012). With the increase of the molar ratio of Tween80 in SC solutions ranging from 0.1 to 0.9, both CMCex and CMCid values were reduced. And the micelle mole fraction of Tween80 (X1) in mixed micelles and in the ideal state (Xid-) has a large deviation. The much lower mole fraction of SC is reflected in its small activity coefficient values. The result suggests that SC in the mixed micelle is far from the standard state. The f1 values of Tween80 are obviously higher, which represents that Tween80 in the mixed micelle is near to its standard state. In the interactions between SC and Tween 20 or Tween 60 in aqueous solution (Ćirin et al., 2012), the interaction parameter of β of Tween and SC shows the most negative value at −7.74 for αTween60 at 0.4, and −6.86 for αTween20 at 0.1. In this research, β value is −7.8 for αTween80 at 0.3. The different interaction parameter may be caused by the electrolytes (phosphate buffer) used. Based on the above observations, we speculate that a strong synergistic interaction existed in the binary system. The interaction between hydrophobic chain of Tween80 and the hydrophobic convex side of the steroid skeleton of SC contributed to the formation of binary mixed micelles in the aqueous phase.
3.1.3. In-vitro digestion profiles of lipid emulsions stabilized by Tween80-SC The percentage of free fatty acids (FFAs) released from emulsions was used to represent the digestion rate and conversion yield. As shown in Fig. 3, the initial digestion rates of both emulsions emulsified by Tween80-SC and SC were quite similar except for the emulsions containing glyceryltrioleate, in which lipids emulsified by SC was faster. But after some lag time, the emulsions stabilized by Tween80-SC showed higher digestion rate. By comparing the three oil fractions, emulsions containing glyceryltrioleate or soybean oil showed a higher initial rate than those containing tricaprylin. It was obvious that emulsions with more negative ζ-potential and relatively larger original size resulted in more intimate interactions between lipase and emulsion interfaces, which are favorable for the digestion. This observation was confirmed by a previous study that emulsion interface with short-chain lipid inside may be unfavorable for its contact with the lipase (Guttoff, Saberi & McClements, 2015). The overall trends of the FFAs-released curves for emulsions with pure oil composition (glyceryltrioleate or tricaprylin) were quite similar, which is different from that for the emulsions containing soybean oil. The influence of the emulsifier on the stability could be partly attributed to the lipid droplets aggregation within the GI tract. If the droplets become flocculated or coalesced within the GI tract, the surface area of lipid exposed to the lipase will be greatly reduced. This will slow down the digestion. Sometimes, the absorption of emulsifier molecules onto the droplet surface may further reduce the rate of lipid digestion by replacing lipase from the interface (Yao et al., 2013; Singh, Ye, & Horne, 2009). In this study, the overall digestion rates and the final conversion yield of lipid with Tween80-SC were larger than those emulsified by SC, demonstrating that the synergistic interaction between SC and Tween80 did promote lipid digestion. The results also revealed that the final conversion of long-chain glyceryltrioleate emulsified by Tween80-SC was higher than short-chain tricaprylin in the same system. Owing to the complicated composition of soybean oil, 3
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Fig. 1. Micellization and synergistic interaction parameters of Tween-SC binary surfactant mixtures. Plot of cmc vs αTween80 of Tween80 with SC mixtures. The broad solid line is the experimental cmc; the dotted line is ideal cmc calculated by the Clint equation. The micelle mole fraction of Tween80 (X1) in mixed micelles by regular solution theory and in the ideal state (Xid-) by applying Motomura’s equation based on excess thermodynamic quantities. The interaction parameter of β values against Tween80 molar ratio variation of the mixtures. Plot of activity coefficients of the surfactants within the micelles vs variation of αTween80. The solid line is the activity coefficients of Tween80 in mixed micelles. The dotted line is the activity coefficients of SC in a binary mixture.
Alfonso, Restrepo-SÃ nchez, NarvÃez-Cuenca & McClements, 2014) found that methyl cellulose, pectin, and chitosan inhibited lipid digestion. However, Qin’s group (Qin et al., 2016) found that at relatively low levels, the initial lipid digestion rate was only reduced by chitosan, the final conversion of lipid digestion was not affected by incorporated dietary fibers including cationic chitosan, anionic alginate, neutral locust bean gum. At relatively high dietary fiber levels, alginate and chitosan significantly inhibited lipid hydrolysis, whereas locust bean gum did not. These results imply that although some polysaccharides increases the viscosity of the digestion medium, the lipase-catalyzed hydrolysis rate does not always decrease. An excluded volume effect generated by the macromolecular surroundings could possibly compensate the decreased digestion rate. In this study, the PEGn (n = 200, 2000 and 20000) were used as the crowding reagents because of its adjustable crowded degree. The effects of crowding on the digestion of emulsions containing glyceryltrioleate and tricaprylin that are synergistically emulsified by SC and Tween80 are investigated (Fig. 4). As shown in Fig. 4 (a1-c1), without PEG, the final FFA yield of tricaprylin digestion was only about 50%, the presence of 10 wt% PEG200 increased FFA to about 80%. The initial digestion rates of glyceryltrioleate are much higher than tricaprylin
its digestion showed a more complex process.
3.2. The effects of crowded medium on the in vitro digestion of lipid emulsions synergistically emulsified by Tween80-SC 3.2.1. The influence of the “macromolecular crowding” on the in-vitro digestion of emulsions The nonspecific interactions between macromolecules and the surroundings within a crowded medium can greatly influence the equilibrium and rates of reactions in which the macromolecules participate (Minton, 2006). The “macromolecular crowding” alters the reaction rates depending on the nature of the reactions. For a diffusion-limited reaction, the diffusion of substrates decreases due to the increased viscosity and reduced mobility of the reactants. However, for a transition-state-limited reaction, the rate is increased because crowding is expected to enhance the relative abundance of the transition state complex due to an increase in the effective concentration of substrates resulting from the excluded volume effect (Mittal, Chowhan & Singh, 2015; Berezhkovskii & Szabo, 2016). Dietary macromolecules create a crowded medium for lipid digestion by lipase. However, the crowding effect was rarely concentrated. Mauricio et al (Espinal-Ruiz, Parada4
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Fig. 2. The particle size distribution and ζ-potential (mV) of three emulsions emulsified by sodium cholate (SC) and/or Tween80. The oil composition for a, b and c is tricaprylin, glycerol trioleate and soybean oil, respectively.
emulsions. However, the final conversion could not reach the same level as the medium-chain fatty acid, but still higher than control. Overall, the crowding reagents have positive effects on the digestion of emulsions. For each PEGn, the digestion rate and overall digestion extent of tricaprylin increased with increasing concentration from 0% to 10% gradually except for the initial digestion rates. The digestion rates and overall digestion extent of emulsions containing glyceryltrioleate (Fig. 4a2–c2) were more likely to depend on both the concentration and type of PEGn. These observations indicate that the digestion of emulsions could be affected by hydrophobic chain length of fatty acid and PEGn type. There was a coupling effect between crowding environment and the oil composition. Taken together, the crowed surroundings
created by PEGs promoted the digestion of lipid. It has been demonstrated that the macromolecular crowding reagents may enhance the contact possibility between the enzyme and the substrate by excluded volume effects, and consequently increase the reaction rates (Pastor et al., 2014; Agrawal, Santra, Anand, & Swaminathan, 2008). The released monoglycerides and fatty acids may also change the local microenvironment of digestion solution and alter the composition of emulsion interface, which results in different accelerated digestion rates for lipids with different fatty acid chain length.
Fig. 3. The curves for the released percentage of free fatty acids (FFAs) from three emulsions emulsified by sodium cholate (SC) and/or Tween80. The oil composition for (a), (b) and (c) is tricaprylin, glycerol trioleate and soybean oil, respectively. 5
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Fig. 4. The curves for the released percentage of free fatty acids (FFAs) from glycerol (a1, b1, c1) tricaprylin and (a2, b2, c2) trioleate emulsified by SC and/or Tween80 with or without 1%, 5% and 10% of (a) PEG200, (b) PEG2000 and (c) PEG20000, respectively.
20000, Tm slightly increased with increasing PEGn concentration from 0% to 10%. A significant increase of the denaturation temperature was observed in the cases of PEG2000. This tendency was consistent with the observed digestion extent of lipid emulsion in the corresponding crowding environment. At the same concentration of PEGn, Tm value followed the order of PEG2000 > PEG20000 > PEG200. The maximum Tm value (64.2 °C) was found when lipase was mixed with SC, Tween80 and 10 wt% PEG2000. Based on these investigations, it can be concluded that the crowding medium did affect the structure of lipase, which possibly contributed the promoted digestion of lipid. In a recent work (Zhang, Luo, Wang, Jia & Chen, 2019), we found that addition of PEG2000 results in an increase in the kinetic parameter (kcat/Km) of lipase for both tricaprylin and glyceryltrioleate. We also find that whether glyceryltrioleate or tricaprylin was used as substrate, the fluorescence intensity of lipase increased with increasing PEG concentration. Additionally, the α-helix content of PPL increased, and the loop content decreased. This conformational change parallels the change of kcat/Km and Tm. Around the catalytic site of lipase, there are several α-helices that stabilize the enzyme and maintain its activity. The increased Tm possibly came from the increased α-helix and
3.2.2. Change of the denaturation temperature of the lipase in the crowed medium Many studies have demonstrated that the macromolecular crowding environment can affect the conformations and functions of various enzymes, consequently alter their catalytic behaviors (Norris et al., 2011, Balcells et al., 2014). DSC is a common tool to study the protein stability in terms of denaturation temperature (Tm), by which the Tm along with both enthalpy change and Van’t Hoff enthalpy change can be determined (Luo, Wu, Qin, & Yu, 2012). The above results show that both the digestion rate and conversion yield were affected by PEGn. The structure of lipase could possibly be changed by the crowded medium. As shown in Fig. 5 and Table 1, with either the combination of SCTween80 or SC only, Tm of lipase was increased with increasing PEGn concentrations. But the increasing extent was differing, which may be responsible for the observed different digestion performance as shown in Fig. 4. The Tm value for pure lipase is 56.52 °C, which was increased to 56.83 and 57.57 °C in the presence of SC and Tween80-SC, respectively. A slight difference in Tm values with SC and Tween80-SC may partially explain the different digestion performance of lipase in emulsions stabilized by SC and Tween80-SC (Fig. 3). With PEG200 and
Fig. 5. The denaturation temperature of the lipase in the presence of SC and/or Tween80 with or without 1%, 5% and 10% of (a) PEG200 (b) PEG2000 and (c) PEG20000, respectively. 6
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Table 1 Impact of PEGn (n = 200, 2000 and 20000) on the thermal stability of lipase measured by DSC.a Composition Lipase Lipase + SC Lipase + SC + Tween80 Lipase + SC + Tween80 + 1%PEG200 Lipase + SC + Tween80 + 5%PEG200 Lipase + SC + Tween80 + 10%PEG200 Lipase + SC + Tween80 + 1%PEG2000 Lipase + SC + Tween80 + 5%PEG2000 Lipase + SC + Tween80 + 10%PEG2000 Lipase + SC + Tween80 + 1%PEG20000 Lipase + SC + Tween80 + 5%PEG20000 Lipase + SC + Tween80 + 10%PEG2000 a
△H(kJ/mol)
Tm(oC)
5
△Hv(kJ/mol) 4
56.52 56.83 57.57 58.69 58.76 59.16 59.91 61.95
± ± ± ± ± ± ± ±
0.29 0.41 0.13 0.13 0.054 0.21 0.41 0.17
1.539 × 10 1.310 × 105 5.308 × 104 2.855 × 104 1.815 × 104 5.182 × 104 3.426 × 103 9.883 × 103
± ± ± ± ± ± ± ±
1.268 × 10 1.205 × 104 5.274 × 103 2.457 × 103 1.092 × 103 4.898 × 103 1.804 × 103 1.503 × 103
451.3 ± 46.0 992.9 ± 142.7 1301.8 ± 165.8 862.7 ± 118.5 2224.8 ± 182.5 738.4 ± 87.9 2590.7 ± 178.7 1682.3 ± 346.6
64.20 58.72 59.36 59.87
± ± ± ±
0.10 0.24 0.13 0.31
3.554 × 104 2.722 × 104 2.733 × 104 2.888 × 104
± ± ± ±
2.491 × 103 3.483 × 103 2.22 × 103 2.227 × 103
1225.2 ± 109.7 875.3 ± 140.6 962.4 ± 90.0 2781.2 ± 218.1
Tm denotes denaturation temperature; △H refers to enthalpy change; △Hv represents Van 't Hoff enthalpy change.
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decreased loop content of lipase. Among the secondary structural components of the enzyme, loops represent a highly flexible conformation. Less loop content means less opportunity for the loop to move to the active center or binding site thereby reduces the possibility to cover the substrate binding sites, resulting in an increased possibility of substrate and enzyme combination, so that the enzyme activity is increased. The increased denaturation temperature and the transition enthalpy indicated the enzyme structure prefers to adopt a more rigid conformation, which facilitates lipid hydrolysis. Because a control experiment in sucrose solution excluded crowding-induced polarity change (Zhang et al., 2019), we conclude that a specific macromolecular crowding effect exists in the lipid digestion.
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4. Conclusions In this work, the synergistic interactions between SC and Tween80 according to regular solution theory, and their effect on the lipid’s in vitro digestion have been studied. The impact of crowding environment formed by the PEGn(n = 200, 2000 and 20000) on the lipid digestion was also investigated. The strongest synergistic interaction between Tween80 and SC was found at a mole ratio of 0.3. In addition, the crowding environment created by PEGn promoted both digestion rate and conversion of oils emulsified by a combination of Tween80-SC, especially at high PEGn concentration (10%). The denaturation temperature of the lipase increased in the presence of Tween80-SC in the crowed medium, which accounts for a more stable conformation of lipase, and therefore contributes to the enhanced digestion rates of lipids. The oil composition also showed significant impact on the digestion of emulsions, where the digestion extent of long-chain oil is higher than that of the short-chain oil. These findings improve our understanding of physiochemistry in the digestion process. It will also contribute to food formulation for regulating dietary fat digestion in the functional foods. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Authors are grateful to the financial support from the National Nature Science Foundation of China (NSFC, No. 21673207) and Nature Science Foundation of Zhejiang Province (LY19B030002). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 7
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8–13. https://doi.org/10.1016/j.bpc.2013.10.006. Patel, V., Bharatiya, B., Ray, D., Aswal, V. K., & Bahadur, P. (2015). Investigations on microstructural changes in ph responsive mixed micelles of triton x–100 and bile salt. Journal of Colloid and Interface Science, 441, 106–112. https://doi.org/10.1016/j.jcis. 2014.11.027. Qin, D., Yang, X., Gao, S., Yao, J., & McClements, D. J. (2016). Influence of hydrocolloids (dietary fibers) on lipid digestion of protein-stabilized emulsions: Comparison of neutral, anionic, and cationic polysaccharides. Journal of Food Science, 81(7), C1636–C1645. https://doi.org/10.1111/1750-3841.13361. Rubingh, D. N. (1979). Mixed micelle solutions. https://doi.org/10.1007/978-1-46157880-2_15 Solution chemistry of surfactants (pp. 337–354). Boston, MA: Springer. Russell, D. W. (2009). Fifty years of advances in bile acid synthesis and metabolism. Journal of Lipid Research, 50(Supplement), S120–S125. https://doi.org/10.1194/jlr. r800026-jlr200. Singh, H., Ye, A., & Horne, D. (2009). Structuring food emulsions in the gastrointestinal tract to modify lipid digestion. Progress in Lipid Research, 48(2), 92–100. https://doi. org/10.1016/j.plipres.2008.12.001. Tian, J. N., Ge, B. Q., Shen, Y. F., He, Y. X., & Chen, Z. X. (2016). Thermodynamics and structural evolution during a reversible vesicle-micelle transition of a vitamin-derived bolaamphiphile induced by sodium cholate. Journal of Agricultural and Food Chemistry, 64(9), 1977–1988. https://doi.org/10.1021/acs.jafc.5b05547. Wang, J., Yang, L. J., Zhu, T. T., Wang, S. Z., & Chen, Z. X. (2016). Phase transition of phospholipid vesicles induced by fatty acids in macromolecular crowding: A differential scanning calorimetry study. ActaPhysico-CheimicaSinica, 32, 2027–2038. https://doi.org/10.3866/PKU.WHXB201605033. Yao, X., Wang, N., Fang, Y., Phillips, G. O., Jiang, F., Hu, J., ... Tian, D. (2013). Impact of surfactants on the lipase digestibility of gum arabic-stabilized O/W emulsions. Food Hydrocolloids, 33(2), 393–401. https://doi.org/10.1016/j.foodhyd.2013.04.013. Zhu, T. T., Zhang, Y., Luo, X. A., Wang, S. Z., Jia, M. Q., & Chen, Z. X. (2018). Difference in binding of long-and medium-chain fatty acids with serum albumin: The role of macromolecular crowding effect. Journal of Agricultural and Food Chemistry, 66(5), 1242–1250. https://doi.org/10.1021/acs.jafc.7b03548. Zhang, Y., Luo, X. A., Wang, S. Z., Jia, M. Q., & Chen, Z. X. (2019). Catalytic behavior of pancreatic lipase incrowded medium for hydrolysis of medium-chain and long-chain lipid: An isothermal titration calorimetry study. Thermochimica Acta, 672, 70–78. https://doi.org/10.1016/j.tca.2018.12.015.
Hsu, J. P., & Nacu, A. (2003). Behavior of soybean oil-in-water emulsion stabilized by nonionic surfactant. Journal of Colloid and Interface Science, 259(2), 374–381. https:// doi.org/10.1016/s0021-9797(02)00207-2. Li, Y., Hu, M., & McClements, D. J. (2011). Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pHstat method. Food Chemistry, 126(2), 498–505. https://doi.org/10.1016/j.foodchem. 2010.11.027. Lopez-Pena, C. L., & McClements, D. J. (2014). Optimizing delivery systems for cationic biopolymers: Competitive interactions of cationic polylysine with anionic κ-carrageenan and pectin. Food chemistry, 153, 9–14. https://doi.org/10.1016/j.foodchem. 2013.12.024. Luo, J. J., Wu, F. G., Qin, S. S., & Yu, Z. W. (2012). In situ unfolded lysozyme induces the lipid lateral redistribution of a mixed lipid model membrane. The Journal of Physical Chemistry B, 116(41), 12381–12388. https://doi.org/10.1021/jp304339t. Manna, K., Chang, C. H., & Panda, A. K. (2012). Physicochemical studies on the catanionics of alkyltrimethylammonium bromides and bile salts in aqueous medium. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 415(48), 10–21. https://doi.org/10.1016/j.colsurfa.2012.09.019. Mao, L., & Miao, S. (2015). Structuring food emulsions to improve nutrient delivery during digestion. Food Engineering Reviews, 7(4), 439–451. https://doi.org/10.1007/ s12393-015-9108-0. Minton, A. P. (2006). how can biochemical reactions within cells differ from those in test tubes? Journal of Cell Science, 119(14), 2863–2869. https://doi.org/10.1242/jcs. 03063. Mittal, S., Chowhan, R. K., & Singh, L. R. (2015). Macromolecular crowding: Macromolecules friend or foe. Biochimica et. BiophysicaActa (BBA) – General Subjects, 1850(9), 1822–1831. https://doi.org/10.1016/j.bbagen.2015.05.002. Naskar, B., Mondal, S., & Moulik, S. P. (2013). Amphiphilic activities of anionic sodium cholate (nac), zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (chaps) and their mixtures: A comparative study. Colloids and Surfaces B: Biointerfaces, 112(3), 155–164. https://doi.org/10.1016/j.colsurfb.2013.07.029. Norris, M. G. S., & Malys, N. (2011). What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase. Biochemical and Biophysical Research Communications, 405(3), 388–392. https://doi.org/10.1016/j.bbrc.2011.01.037. Pastor, I., Pitulice, L., Balcells, C., Vilaseca, E., Madurga, S., Isvoran, A., ... Mas, F. (2014). Effect of crowding by Dextrans in enzymatic reactions. Biophysical Chemistry, 185,
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Synergistic interaction between exogenous and endogenous emulsifiers and its impact on in vitro digestion of lipid in crowded medium Shen-Zhi Wang, Hui-Qian Dai, Ke-Xian Chen, Juan Li, Zhong-Xiu Chen
T
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Molecular Food Science Laboratory, College of Food & Biology Engineering, Zhejiang Gongshang University, Hangzhou 310018, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Macromolecular crowding Lipase Synergistic interaction Lipid digestion Bile salt
Control of lipid digestibility by various food components has received great attention in recent decades. However, there is limited literature on investigating the synergistic effect of exogenous emulsifiers and endogenous sodium cholate (SC) on lipid digestion in a simulated physiological crowded medium. In this work, the synergistic interaction of Tween80 and SC according to the regular solution theory, and the hydrolysis of lipid emulsions containing tricaprylin, glyceryltrioleate or soybean oil in crowding medium was studied. The results show that emulsions stabilized by a combination of Tween80 and SC showed higher digestion rate and transformation than those with Tween80 or SC. The digestion rate could be increased by polyethylene glycols (PEGn) with varying crowding degree. The denaturation temperature of the lipase was increased in macromolecular crowded medium. This work allows for better understanding of the interaction between the amphiphiles and the macromolecular crowding effect on lipase digestion in the physiological environment.
1. Introduction Digestion and absorption of lipids in human gastrointestinal (GI) tract play an important role in body lipid homeostasis. The increasing concern regarding the controllable digestion of lipids has evoked many studies on the impact of other food components on lipid metabolism. In small intestine, lipids digestion is largely dependent on the interfacial composition of the emulsion droplets. The key intestinal components that support interfacial activation of enzyme and facilitate lipid digestion are amphiphilic molecules. As typical endogenous surfactants, bile salts help emulsifying lipids to oil droplets, altering interfacial composition to favor lipase adsorption and participating the formation of mixed micelles that can solubilize free fatty acids and other lipid nutrients and transport them to the epithelium cells (Russell, 2009). In food processing, emulsifiers has been added as stabilizing agents to ensure the taste, flavor, texture and other specific features of food. Therefore, surfactants are commonly present in many processed foods and drug formulations. In the GI tract, a surfactant-stabilized food emulsion system might suffer droplet flocculation or coalescence due to a drastic change in pH and a variation in ionic strength. The exogenous surfactant would inevitably interact with endogenous emulsifiers, such as bile salts. The digestion rate and fate of lipids depend on the oil composition, exogenous surfactant type and dosage, the interfacial microstructural composition of the formed membrane, the size
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distribution of other intestinal aggregates like micelles and vesicles, and also the binding affinity of lipase towards these interfaces (Mao & Miao, 2015; Chang & McClements, 2016). However, the impact of these dietary emulsifiers on lipid digestion is particularly complex, because they might cause the aggregation and flocculation of oil droplets and also affect the digestion performances of lipase at the same time (LopezPena & Mcclements, 2014; Beysseriat, Decker, & McClements, 2006). An early study showed that the self-assembled aggregates at emulsionwater interfaces in the upper small intestine during lipid hydrolysis facilitated the dissolution of lipolytic products into unsaturated mixed micelles (Hernell, Staggers, & Carey, 1990). These results indicate that a synergistic interaction between the endogenous amphiphiles and the exogenous surfactants in the small intestine could possibly affect the lipase-catalyzed hydrolysis of lipids. As an important surfactant abundant in human body, sodium cholate (SC) and its aggregation behavior with surfactants such as anionic (Hildebrand, Garidel, Neubert, & Blume, 2004), cationic (Manna, Chang, & Panda, 2012), nonionic (Patel, Bharatiya, Ray, Aswal, & Bahadur, 2015) and zwitterionic (Naskar, Mondal, & Moulik, 2013) have been extremely studied. Interaction of SC with dietary phospholipids also attracts extensive interests (Coreta-Gomes et al., 2015). We previously found a structural evolution in the interaction of SC with a vitamin-based bolaamphiphiles vesicle (Tian, Ge, Shen, He, & Chen, 2016). Tween80 is an aliphatic, nonionic surfactant approved by the
Corresponding author. E-mail address: [email protected] (Z.-X. Chen).
https://doi.org/10.1016/j.foodchem.2019.125164 Received 6 December 2018; Received in revised form 19 June 2019; Accepted 9 July 2019 Available online 09 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. Critical micelle concentration (CMC) measurements
U.S. Food and Drug Administration (FDA) for use in injectable, oral, and topical products. Mixed micelles of BS and Tween80 were studied as biocompatible colloidal drug carriers (Ćirin, Poša & Krstonošić, 2012). However, to the best of our knowledge, the synergistic interaction of Tween80 and SC on lipid digestion within the simulated small intestine has not been studied yet. During lipid digestion, pancreatic lipases interact with other compounds, such as polysaccharides and proteins. These macromolecules create a highly crowded environment, which constitutes the native environment of the lipase. Our research group reported that both catalytic reactions that involve small molecules and the non-covalent binding process could be affected by the “macromolecular crowding” effect. In a crowded medium, vesicle-catalyzed production of fatty acids (FAs) displayed unusual chain-length dependence (Cao et al., 2014). The binding of FAs to phospholipid bilayers and serum albumin also became unusual (Wang, Yang, Zhu, Wang & Chen, 2016; Zhu et al., 2018). Actually, the impact of hydrocolloids on lipid digestion within the gastrointestinal tract was thoroughly investigated (Qin, Yang, Gao, Yao, & McClements, 2016). Some of the dietary fibers increased the lipid digestion rate, but some inhibited lipid hydrolysis, depending on their molecular and physicochemical properties. Whether the excluded volume effect was involved was not clear. However, the macromolecular crowding effect did affect the catalytic activities of enzymes because their conformation was changed by the crowded medium (Norris & Malys, 2011; Balcells et al., 2014; Pastor et al., 2014). In this research, the synergistic effect of an exogenous emulsifier (Tween80) and the endogenous emulsifier (SC) on the lipid digestion based on regular solution theory and was studied. The impact of the crowding medium on lipid digestion in the presence of both exogenous and endogenous surfactants was also discussed. The results improve our understanding of the interaction of the surface active agents in the physiological environment, and the macromolecular crowding effects on lipase digestion.
The CMC values of SC, Tween 80 and the binary mixture were measured by monitoring the fluorescence intensity I1/I3 ratio of pyrene which was used as fluorescence probe by using Hitachi F-7000 fluorescence spectrophotometer. The concentration of pyrene was kept at 0.5 µmol L−1. The fluorescence emission spectra were obtained by employing an excitation wavelength of 334 nm. The I1 and I3 intensities in fluorescence emission spectra correspond to the first and third vibronic peaks of pyrene was located at wavelengths of ca. 373 and 384 nm, respectively. The I1/I3 ratios were plotted as a function of the concentration of binary mixture of Tween80 and SC. The CMC values were taken from the point of inter-section by fitting the premicellar and postmicellar data in linear equations. A typical example is shown in the Supporting Information section.
2.5. Particle size and ζ- potential characterization Both the particle size and ζ-potential of the emulsions were characterized by Zetasizer Nano-ZS (Malvern Instruments, Ltd, United Kingdom). The instrument uses a laser at a wavelength of 632.8 nm and detects the scattered light at an angle of 173°. Zeta potential was measured based on laser Doppler electrophoresis. All measurements were performed in a temperature-controlled chamber. Experiment duration (equilibration time) was in the range of 2 min, and each test was repeated three or more times.
2.6. In vitro digestion model (pH-stat) The pH-stat model (Li, Hu & McClements, 2011) was employed as the in vitro digestion model in this study. The procedure used is as following:
2. Materials and methods
(i) 30.0 mL emulsion was added into a glass beaker and was placed in water bath at 37.0 °C. The pH was adjusted to 7.0 by NaOH or HCl solutions and was stirred for 10 min. (ii) 5.0 mL SC solution (187.5 mg SC dissolved in phosphate buffer, 87 mmol L−1, pH = 7.0) and 1.0 mL CaCl2 solution (188 mM) were added to the above emulsion under stirring condition and the pH of the system was adjusted back to 7.0 if required. (iii) 60 mg lipase powder was added to the above mixture. A pH-stat automatic titration unit (Metrohm, USA Inc.) was then used to automatically monitor the pH and maintain the pH at 7.0 by titrating appropriate concentrations of NaOH solution. The volume of NaOH added to the emulsion was recorded, which was used to calculate the concentration of free fatty acids (FFAs) generated from the lipolysis using the following formula:
2.1. Materials Lipase (porcine pancreas) (Item NO. P7475), tricaprylin (Item NO. T9126) and glyceryltrioleate (Item NO. T7410) were purchased from Sigma-Aldrich with purity of 99%. Polyethylene glycols (PEGn (n = 200, 2000 and 20000)), soybean oil, calcium chloride (CaCl2), polyoxyethylene dehydrated sorbitol monooleate (Tween80), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from Aladdin Corporation. Sodium Cholate (SC) was purchased from TCI Corporation with purity of 99%. 2.2. Solution preparation The stock solution of Tween80 (50 mmol L−1), SC (50 mmol L−1) and CaCl2 (188 mmol L−1) were prepared by dispersing the corresponding chemicals into the phosphate buffer solution (20 mmol L−1, pH = 7.0) and were stirred for at least 3 h. Tween80 and SC at varying molar ratio was mixed for determination the critical micelle concentration. The stock solution containing polyethylene glycols (PEG) were prepared by dispersing polymer into the phosphate buffer, which was used as the macromolecular crowded medium.
%FFA = 100 ×
VNaOH × mNaOH × MOil WOil × 2
where VNaOH is the volume of NaOH required to neutralize the FFAs produced; mNaOH is the molarity of the NaOH solution; WOil is the total weight of oil, and MOil is the molecular weight of the oil.
2.7. Thermal stability of lipase 2.3. Emulsion preparation The Differential Scanning Calorimetry measurements (DSC) of the lipase in different solutions were performed in VP-DSC instrument (MicroCalInc, Northampton, USA). All the calorimetric data were recorded by performing the heating and cooling scans from 1 to 110 °C at a scan rate of 1 °C/min. The concentration of lipase was kept at a constant of 5 mg/mL. Data analysis was done by using the Origin 7.0 software in Microcal.
Tricaprylin, glyceryltrioleate and soybean oil were used as the oil phase for emulsions. 10 wt% oil-containing stock emulsions were prepared by homogenizing the mixture of oil with the above prepared solution containing Tween80 and/or SC (20 mmol L−1) using a highspeed blender (FA25-Digita, Fluko Products, China) at 9000 rpm for 10 min. 2
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3.1.2. Particle size and ζ-potential of emulsions stabilized by SC-Tween80 mixed micelles The different emulsions containing tricaprylin, glyceryltrioleate and soybean oil were prepared with Tween80-SC mixture at the optimal ratio of 0.3, as determined by the above synergistic interaction studies. The particle size distribution and ζ-potential values of emulsions are shown in Fig. 2, respectively. It is found that the stability of emulsions stabilized by a combination of Tween80 and SC was higher than that emulsified by Tween80 or SC only. For example, the particle size distribution of tricaprylin emulsion stabilized by mixed micelles was narrower than that stabilized by single-component micelles, and its droplet size value was also much smaller (Fig. 2a1–c1). Similar trends were also observed for the emulsions containing glyceryltrioleate or soybean oil. The more narrow distribution from the combination of anionic and nonionic surfactants was expected due to better surface coverage resulting from reduced repulsion of anionic species. In terms of triacylglycerols with different chain length, the droplet size of emulsions containing glyceryltrioleate or soybean oil was significantly larger than that containing tricaprylin. Besides, for the same emulsion, emulsions stabilized by a Tween80-SC showed more negative ζ-potentials than those by Tween80 or SC only (Fig. 2 a2-c2), implying a higher stability and a good dispersity. The ζ-potential values of emulsions containing glyceryltrioleate or soybean oil were more negative than that containing tricaprylin except for the case emulsified by Tween80. And a higher ζ-potential magnitude of emulsions may indicate a better interface contact between lipase and lipids (Hsu & Nacu, 2003). It is noteworthy that when significant levels of nonionics are used, drawing conclusions from the ζ-potential can be tenuous due to the likely presence of other stabilizing mechanisms and the questionable assumption the Stern layer is located approximately at the slip plane. Therefore, though the absolute value of ζ-potential was not more than 30 mV, the emulsion was stable enough for the followed digestion experiment.
2.8. Data analysis All experiments were performed independently at least three times. The results were then reported as averages and standard deviations of these measurements. 3. Results and discussion In this paper, Tween80 was selected as the model exogenous emulsifier and SC (SC) was used as endogenous surfactant. PEGn (n = 200, 2000 and 20000) was used as crowding reagents due to its adjustable crowding degree. PEG has been universally used in foods owing to its approved safety in the body (Desbuquois & Aurbach, 1971). Lipids, tricaprylin, glyceryltrioleate and soybean oil, were used to investigate the structural aspects of oil composition on lipid digestion. 3.1. Synergistic interaction of SC and Tween80 and their effect on in vitro lipid digestion 3.1.1. Interaction between SC and Tween80 Interactions of SC and Tween80 and their mixed micelle formation in phosphate buffer solution were investigated by steady-state fluorescence measurements. The detailed results based on the regular solution theory (Haque, Das & Moulik, 1999; Ćirin et al., 2012) by Rubingh (1979) are shown in Fig. 1, where α is the mole fraction of Tween80 in solution; Xid and X1 denote the mole fraction in the ideal and real mixed micelles, respectively; CMCid and CMCex refer to the ideal and experimental CMC values of binary mixture of SC and Tween80, respectively; β represents the Rubingh synergistic interaction parameter between SC and Tween80, which indicates the magnitude of interactions operating between the unlike components in the mixed micelle state. The larger the absolute value of β, the stronger the mixing non-ideality and the synergistic effect. The values for the CMCs of pure SC and Tween80 in phosphate buffer solution are 11.8 mmol L−1 and 0.03 mmol L−1, respectively. Detailed calculation of these values was described in Supporting Information. Obviously, all β values were negative, which means that the synergistic interaction between SC and Tween80 occurred. A maximum absolute β value was found when α was 0.3, showing the strongest synergistic interaction strength at this Tween80-SC ratio. Increasing the mole fraction of the hydrophobic components (Tween80) in the mixed micelle may reduce the synergistic interaction to some extent. In addition, the large negative deviation of CMCex values from the corresponding CMCid values indicated that the thermodynamic stability of the real binary mixed micelles of SC and Tween80 were higher than that of their ideal binary mixtures. It has been reported that the CMC value of Tween80 (Bhattacharjee et al., 2010) was much smaller than that of SC (Ćirin et al., 2012). With the increase of the molar ratio of Tween80 in SC solutions ranging from 0.1 to 0.9, both CMCex and CMCid values were reduced. And the micelle mole fraction of Tween80 (X1) in mixed micelles and in the ideal state (Xid-) has a large deviation. The much lower mole fraction of SC is reflected in its small activity coefficient values. The result suggests that SC in the mixed micelle is far from the standard state. The f1 values of Tween80 are obviously higher, which represents that Tween80 in the mixed micelle is near to its standard state. In the interactions between SC and Tween 20 or Tween 60 in aqueous solution (Ćirin et al., 2012), the interaction parameter of β of Tween and SC shows the most negative value at −7.74 for αTween60 at 0.4, and −6.86 for αTween20 at 0.1. In this research, β value is −7.8 for αTween80 at 0.3. The different interaction parameter may be caused by the electrolytes (phosphate buffer) used. Based on the above observations, we speculate that a strong synergistic interaction existed in the binary system. The interaction between hydrophobic chain of Tween80 and the hydrophobic convex side of the steroid skeleton of SC contributed to the formation of binary mixed micelles in the aqueous phase.
3.1.3. In-vitro digestion profiles of lipid emulsions stabilized by Tween80-SC The percentage of free fatty acids (FFAs) released from emulsions was used to represent the digestion rate and conversion yield. As shown in Fig. 3, the initial digestion rates of both emulsions emulsified by Tween80-SC and SC were quite similar except for the emulsions containing glyceryltrioleate, in which lipids emulsified by SC was faster. But after some lag time, the emulsions stabilized by Tween80-SC showed higher digestion rate. By comparing the three oil fractions, emulsions containing glyceryltrioleate or soybean oil showed a higher initial rate than those containing tricaprylin. It was obvious that emulsions with more negative ζ-potential and relatively larger original size resulted in more intimate interactions between lipase and emulsion interfaces, which are favorable for the digestion. This observation was confirmed by a previous study that emulsion interface with short-chain lipid inside may be unfavorable for its contact with the lipase (Guttoff, Saberi & McClements, 2015). The overall trends of the FFAs-released curves for emulsions with pure oil composition (glyceryltrioleate or tricaprylin) were quite similar, which is different from that for the emulsions containing soybean oil. The influence of the emulsifier on the stability could be partly attributed to the lipid droplets aggregation within the GI tract. If the droplets become flocculated or coalesced within the GI tract, the surface area of lipid exposed to the lipase will be greatly reduced. This will slow down the digestion. Sometimes, the absorption of emulsifier molecules onto the droplet surface may further reduce the rate of lipid digestion by replacing lipase from the interface (Yao et al., 2013; Singh, Ye, & Horne, 2009). In this study, the overall digestion rates and the final conversion yield of lipid with Tween80-SC were larger than those emulsified by SC, demonstrating that the synergistic interaction between SC and Tween80 did promote lipid digestion. The results also revealed that the final conversion of long-chain glyceryltrioleate emulsified by Tween80-SC was higher than short-chain tricaprylin in the same system. Owing to the complicated composition of soybean oil, 3
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Fig. 1. Micellization and synergistic interaction parameters of Tween-SC binary surfactant mixtures. Plot of cmc vs αTween80 of Tween80 with SC mixtures. The broad solid line is the experimental cmc; the dotted line is ideal cmc calculated by the Clint equation. The micelle mole fraction of Tween80 (X1) in mixed micelles by regular solution theory and in the ideal state (Xid-) by applying Motomura’s equation based on excess thermodynamic quantities. The interaction parameter of β values against Tween80 molar ratio variation of the mixtures. Plot of activity coefficients of the surfactants within the micelles vs variation of αTween80. The solid line is the activity coefficients of Tween80 in mixed micelles. The dotted line is the activity coefficients of SC in a binary mixture.
Alfonso, Restrepo-SÃ nchez, NarvÃez-Cuenca & McClements, 2014) found that methyl cellulose, pectin, and chitosan inhibited lipid digestion. However, Qin’s group (Qin et al., 2016) found that at relatively low levels, the initial lipid digestion rate was only reduced by chitosan, the final conversion of lipid digestion was not affected by incorporated dietary fibers including cationic chitosan, anionic alginate, neutral locust bean gum. At relatively high dietary fiber levels, alginate and chitosan significantly inhibited lipid hydrolysis, whereas locust bean gum did not. These results imply that although some polysaccharides increases the viscosity of the digestion medium, the lipase-catalyzed hydrolysis rate does not always decrease. An excluded volume effect generated by the macromolecular surroundings could possibly compensate the decreased digestion rate. In this study, the PEGn (n = 200, 2000 and 20000) were used as the crowding reagents because of its adjustable crowded degree. The effects of crowding on the digestion of emulsions containing glyceryltrioleate and tricaprylin that are synergistically emulsified by SC and Tween80 are investigated (Fig. 4). As shown in Fig. 4 (a1-c1), without PEG, the final FFA yield of tricaprylin digestion was only about 50%, the presence of 10 wt% PEG200 increased FFA to about 80%. The initial digestion rates of glyceryltrioleate are much higher than tricaprylin
its digestion showed a more complex process.
3.2. The effects of crowded medium on the in vitro digestion of lipid emulsions synergistically emulsified by Tween80-SC 3.2.1. The influence of the “macromolecular crowding” on the in-vitro digestion of emulsions The nonspecific interactions between macromolecules and the surroundings within a crowded medium can greatly influence the equilibrium and rates of reactions in which the macromolecules participate (Minton, 2006). The “macromolecular crowding” alters the reaction rates depending on the nature of the reactions. For a diffusion-limited reaction, the diffusion of substrates decreases due to the increased viscosity and reduced mobility of the reactants. However, for a transition-state-limited reaction, the rate is increased because crowding is expected to enhance the relative abundance of the transition state complex due to an increase in the effective concentration of substrates resulting from the excluded volume effect (Mittal, Chowhan & Singh, 2015; Berezhkovskii & Szabo, 2016). Dietary macromolecules create a crowded medium for lipid digestion by lipase. However, the crowding effect was rarely concentrated. Mauricio et al (Espinal-Ruiz, Parada4
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Fig. 2. The particle size distribution and ζ-potential (mV) of three emulsions emulsified by sodium cholate (SC) and/or Tween80. The oil composition for a, b and c is tricaprylin, glycerol trioleate and soybean oil, respectively.
emulsions. However, the final conversion could not reach the same level as the medium-chain fatty acid, but still higher than control. Overall, the crowding reagents have positive effects on the digestion of emulsions. For each PEGn, the digestion rate and overall digestion extent of tricaprylin increased with increasing concentration from 0% to 10% gradually except for the initial digestion rates. The digestion rates and overall digestion extent of emulsions containing glyceryltrioleate (Fig. 4a2–c2) were more likely to depend on both the concentration and type of PEGn. These observations indicate that the digestion of emulsions could be affected by hydrophobic chain length of fatty acid and PEGn type. There was a coupling effect between crowding environment and the oil composition. Taken together, the crowed surroundings
created by PEGs promoted the digestion of lipid. It has been demonstrated that the macromolecular crowding reagents may enhance the contact possibility between the enzyme and the substrate by excluded volume effects, and consequently increase the reaction rates (Pastor et al., 2014; Agrawal, Santra, Anand, & Swaminathan, 2008). The released monoglycerides and fatty acids may also change the local microenvironment of digestion solution and alter the composition of emulsion interface, which results in different accelerated digestion rates for lipids with different fatty acid chain length.
Fig. 3. The curves for the released percentage of free fatty acids (FFAs) from three emulsions emulsified by sodium cholate (SC) and/or Tween80. The oil composition for (a), (b) and (c) is tricaprylin, glycerol trioleate and soybean oil, respectively. 5
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Fig. 4. The curves for the released percentage of free fatty acids (FFAs) from glycerol (a1, b1, c1) tricaprylin and (a2, b2, c2) trioleate emulsified by SC and/or Tween80 with or without 1%, 5% and 10% of (a) PEG200, (b) PEG2000 and (c) PEG20000, respectively.
20000, Tm slightly increased with increasing PEGn concentration from 0% to 10%. A significant increase of the denaturation temperature was observed in the cases of PEG2000. This tendency was consistent with the observed digestion extent of lipid emulsion in the corresponding crowding environment. At the same concentration of PEGn, Tm value followed the order of PEG2000 > PEG20000 > PEG200. The maximum Tm value (64.2 °C) was found when lipase was mixed with SC, Tween80 and 10 wt% PEG2000. Based on these investigations, it can be concluded that the crowding medium did affect the structure of lipase, which possibly contributed the promoted digestion of lipid. In a recent work (Zhang, Luo, Wang, Jia & Chen, 2019), we found that addition of PEG2000 results in an increase in the kinetic parameter (kcat/Km) of lipase for both tricaprylin and glyceryltrioleate. We also find that whether glyceryltrioleate or tricaprylin was used as substrate, the fluorescence intensity of lipase increased with increasing PEG concentration. Additionally, the α-helix content of PPL increased, and the loop content decreased. This conformational change parallels the change of kcat/Km and Tm. Around the catalytic site of lipase, there are several α-helices that stabilize the enzyme and maintain its activity. The increased Tm possibly came from the increased α-helix and
3.2.2. Change of the denaturation temperature of the lipase in the crowed medium Many studies have demonstrated that the macromolecular crowding environment can affect the conformations and functions of various enzymes, consequently alter their catalytic behaviors (Norris et al., 2011, Balcells et al., 2014). DSC is a common tool to study the protein stability in terms of denaturation temperature (Tm), by which the Tm along with both enthalpy change and Van’t Hoff enthalpy change can be determined (Luo, Wu, Qin, & Yu, 2012). The above results show that both the digestion rate and conversion yield were affected by PEGn. The structure of lipase could possibly be changed by the crowded medium. As shown in Fig. 5 and Table 1, with either the combination of SCTween80 or SC only, Tm of lipase was increased with increasing PEGn concentrations. But the increasing extent was differing, which may be responsible for the observed different digestion performance as shown in Fig. 4. The Tm value for pure lipase is 56.52 °C, which was increased to 56.83 and 57.57 °C in the presence of SC and Tween80-SC, respectively. A slight difference in Tm values with SC and Tween80-SC may partially explain the different digestion performance of lipase in emulsions stabilized by SC and Tween80-SC (Fig. 3). With PEG200 and
Fig. 5. The denaturation temperature of the lipase in the presence of SC and/or Tween80 with or without 1%, 5% and 10% of (a) PEG200 (b) PEG2000 and (c) PEG20000, respectively. 6
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Table 1 Impact of PEGn (n = 200, 2000 and 20000) on the thermal stability of lipase measured by DSC.a Composition Lipase Lipase + SC Lipase + SC + Tween80 Lipase + SC + Tween80 + 1%PEG200 Lipase + SC + Tween80 + 5%PEG200 Lipase + SC + Tween80 + 10%PEG200 Lipase + SC + Tween80 + 1%PEG2000 Lipase + SC + Tween80 + 5%PEG2000 Lipase + SC + Tween80 + 10%PEG2000 Lipase + SC + Tween80 + 1%PEG20000 Lipase + SC + Tween80 + 5%PEG20000 Lipase + SC + Tween80 + 10%PEG2000 a
△H(kJ/mol)
Tm(oC)
5
△Hv(kJ/mol) 4
56.52 56.83 57.57 58.69 58.76 59.16 59.91 61.95
± ± ± ± ± ± ± ±
0.29 0.41 0.13 0.13 0.054 0.21 0.41 0.17
1.539 × 10 1.310 × 105 5.308 × 104 2.855 × 104 1.815 × 104 5.182 × 104 3.426 × 103 9.883 × 103
± ± ± ± ± ± ± ±
1.268 × 10 1.205 × 104 5.274 × 103 2.457 × 103 1.092 × 103 4.898 × 103 1.804 × 103 1.503 × 103
451.3 ± 46.0 992.9 ± 142.7 1301.8 ± 165.8 862.7 ± 118.5 2224.8 ± 182.5 738.4 ± 87.9 2590.7 ± 178.7 1682.3 ± 346.6
64.20 58.72 59.36 59.87
± ± ± ±
0.10 0.24 0.13 0.31
3.554 × 104 2.722 × 104 2.733 × 104 2.888 × 104
± ± ± ±
2.491 × 103 3.483 × 103 2.22 × 103 2.227 × 103
1225.2 ± 109.7 875.3 ± 140.6 962.4 ± 90.0 2781.2 ± 218.1
Tm denotes denaturation temperature; △H refers to enthalpy change; △Hv represents Van 't Hoff enthalpy change.
doi.org/10.1016/j.foodchem.2019.125164.
decreased loop content of lipase. Among the secondary structural components of the enzyme, loops represent a highly flexible conformation. Less loop content means less opportunity for the loop to move to the active center or binding site thereby reduces the possibility to cover the substrate binding sites, resulting in an increased possibility of substrate and enzyme combination, so that the enzyme activity is increased. The increased denaturation temperature and the transition enthalpy indicated the enzyme structure prefers to adopt a more rigid conformation, which facilitates lipid hydrolysis. Because a control experiment in sucrose solution excluded crowding-induced polarity change (Zhang et al., 2019), we conclude that a specific macromolecular crowding effect exists in the lipid digestion.
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4. Conclusions In this work, the synergistic interactions between SC and Tween80 according to regular solution theory, and their effect on the lipid’s in vitro digestion have been studied. The impact of crowding environment formed by the PEGn(n = 200, 2000 and 20000) on the lipid digestion was also investigated. The strongest synergistic interaction between Tween80 and SC was found at a mole ratio of 0.3. In addition, the crowding environment created by PEGn promoted both digestion rate and conversion of oils emulsified by a combination of Tween80-SC, especially at high PEGn concentration (10%). The denaturation temperature of the lipase increased in the presence of Tween80-SC in the crowed medium, which accounts for a more stable conformation of lipase, and therefore contributes to the enhanced digestion rates of lipids. The oil composition also showed significant impact on the digestion of emulsions, where the digestion extent of long-chain oil is higher than that of the short-chain oil. These findings improve our understanding of physiochemistry in the digestion process. It will also contribute to food formulation for regulating dietary fat digestion in the functional foods. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Authors are grateful to the financial support from the National Nature Science Foundation of China (NSFC, No. 21673207) and Nature Science Foundation of Zhejiang Province (LY19B030002). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 7
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