Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Attenuation of Palm Stearin Emulsion Droplet in Vitro Lipolysis with Crystallinity and Gastric Aggregation Surangi H. Thilakarathna and Amanda J. Wright*
J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 09/25/18. For personal use only.
Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada ABSTRACT: Emulsions with partially crystalline solid (SE) and undercooled-liquid (LE) droplets with equivalent droplet sizes (centering ∼416 nm), surface charges (∼−56 mV), and spherical morphologies were prepared by hot microfluidization based on 10% palm stearin and 0.4% Span 60. Lipid crystallinity attenuated early gastroduodenal lipolysis in vitro (p < 0.05), both with and without inclusion of a gastric phase (p < 0.05). Gastric exposure, in particular acidic pH, led to partial coalescence of SE and flocculation and partial crystallization of LE, and it attenuated the rate and extent of lipolysis in both samples. In vitro shear conditions further impacted colloidal stability, particularly for SE, with implications for digestibility. Although lipid crystallinity consistently attenuated early lipolysis, gastric-phase SE partial coalescence had a relatively greater impact on digestibility than did droplet physical state. These findings show that a complex interplay exists among a droplet’s physical state, colloidal properties, and digestion conditions, which combine to impact emulsion in vitro lipolysis. KEYWORDS: emulsions, physical state, in vitro digestion, lipolysis, colloidal stability
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INTRODUCTION Dietary lipid digestion has wide-ranging implications for health and disease. The specific impacts of lipid physical properties (i.e., physical state, solid-fat content (SFC), polymorphism, etc.) in determining digestibility and absorption are rarely considered, but this is an area of renewed interest.1−3 Indeed, there is evidence from in vitro,4,5 animal,6−8 and human9−11 studies that fatty acid release can be slower and bioavailability can be lower for lipids with relatively higher melting temperatures or higher SFCs compared with those of lipids that are liquid at body temperature (i.e., 37 °C). For example, canola-stearin solid-lipid nanoparticles stabilized with either Poloxamer 188 or Tween 20 had slower lipolysis rates and were hydrolyzed to lower extents compared with canola-oil emulsions prepared with the same emulsifiers.12 Similarly, in whey-protein-stabilized emulsions containing different proportions of liquid soybean oil and high-melting, fully hydrogenated soybean oil, decreasing rates of lipid digestion were evidenced with increasing SFC.5 Unfortunately, in the above-mentioned and other studies, lipid-physical-property differences were confounded by various other physicochemical differences, including composition (i.e., fatty acid, triacylglycerol (TAG), or emulsifier), particle size, morphology, and surface charge. Bonnaire et al.13 uniquely addressed this by comparing the in vitro digestibility of undercooled-liquid versus crystalline-solid tripalmitin and sodium dodecyl sulfate emulsion droplets, achieved by tempering the samples differently after hot, high-pressure homogenization. Huynh and Wright14 replicated their finding that lipolysis was more extensive for the liquid droplets than for the solid droplets when a gastric phase was included in the in vitro model and also evidenced the induction of a small amount of crystallinity in the undercooled droplets. The solid and liquid particles were also found to have different morphologies, meaning that the comparison of physical state was partly confounded by differences in interfacial area. This is © XXXX American Chemical Society
important given that TAG lipolysis is an interfacial process. The same trend of attenuated early lipolysis for the liquid state was also recently observed in comparisons of undercooledliquid- and solid-cocoa-butter-emulsion droplets formulated using Span 60 and Tween 60.15 In this case, the droplets had equivalent compositions, size distributions, surface charges, and morphologies, although experiments were carried out at 25 versus 37 °C in order to maintain the differences in droplet crystallinity. In the present study, we aimed to extend our understanding of how lipid physical state impacts digestibility using emulsions with undercooled-liquid (LE) and crystalline-solid (SE) droplets, in which particle size, morphology, and surface charge were similar, at 37 °C using only food-permissible ingredients and levels.16 Initial experiments were carried out with various lipids (i.e., palm oil, fully hydrogenated cottonseed oil, fully hydrogenated canola oil, palm stearin, and blends thereof) and emulsifiers (i.e., Tween 60, Span 60, Tween 80, Span 80, soy lecithin, and blends thereof). It was challenging to find a combination in which droplets could be stabilized both as undercooled-liquid droplets and as droplets with contrastingly high SFC (data not shown). However, samples with 10 wt % palm stearin and 0.4% Span 60 were successfully tempered into undercooled-liquid and partially crystalline droplets. Span 60 was partly investigated on the basis of reports that it can delay some TAG polymorphic transitions17,18 and therefore might not promote crystallization in the undercooled system. Herein, we provide a characterization of these palm stearin−0.4% Span 60 emulsions and detail their behavior during exposure to simulated duodenal conditions (with and without a preceeding gastric phase), which aimed at Received: May 20, 2018 Revised: September 10, 2018 Accepted: September 12, 2018
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DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
1:250 with DI water. For both techniques, values of 1.45 and 1.33 were used as the refractive indices of PS and water, respectively. Melting and Recrystallization Behavior. The melting and crystallization behaviors of bulk PS and each batch of the emulsions were analyzed in duplicate by differential-scanning calorimetry (DSC; Q2000 model, TA Instruments, Mississauga, ON, Canada). In brief, 5−10 mg of emulsion was weighed and sealed in a preweighed alodined aluminum DSC pan (TA Instruments, Mississauga, ON, Canada). Samples were held in the DSC at 37 °C for 3 min, then heated at 5 °C/min to 80 °C, held for 3 min, and then cooled at 5 °C/min to 0 °C. To confirm complete crystallization, SE samples at 37 °C were cooled from 37 to 0 °C at 5 °C/min. Enthalpies and peakonset and maximum temperatures were determined using the system software (TA Instruments Universal Analysis 2000 software, TA Instruments, Toronto, ON, Canada). Lipid Polymorphism. The polymorphism of the SE droplets before and during gastric and duodenal digestion, as well as that of the PS tempered in a similar manner to SE, was investigated using a MultiFlex X-ray diffractometer (Rigaku, Tokyo, Japan). Scans were performed from 15 to 30° at 0.3°/min on ∼1 mL of sample placed on an X-ray-diffraction (XRD) glass slide preheated and maintained at 37 °C. The instrument was operated at 40 kV and 44 mA with copper as the X-ray source (λ = 1.54 Å) and angle slits of 0.5 and 0.5° and 0.3 mm. Peak positions were determined on the basis of Bragg’s law using MID’s Jade 9.0 software (Rigaku, Tokyo, Japan). Solid-Fat Content (SFC). The SFC of bulk PS was measured according to AOCS official method Cd 16b-93 using a Bruker Minispec PC/20 series pulsed nuclear-magnetic-resonance spectrophotometer (Bruker Spectrospin, Milton, ON, Canada). Emulsion-Droplet Morphology. Emulsion-droplet morphology was observed by light microscopy. A drop of emulsion was placed on a glass slide preheated to 37 °C, and a preheated (37 °C) coverslip was carefully placed on top, ensuring no air bubbles were present. The sample was then observed with an Olympus BH light microscope (Olympus, Tokyo, Japan) equipped with a Sony Camera (Sony Corporation, Tokyo, Japan) and Image Capture software under polarized and bright-field conditions. In Vitro Digestion during Exposure to Gastric and Duodenal Conditions. The two emulsion samples were subjected to an in vitro digestion model consisting of simulated gastric and duodenal phases, as previously described.22 All fluids, along with the amber jars used, were warmed to 37 °C prior to use. Briefly, 5 mL of emulsion sample was added to 5 mL of simulated gastric fluid (2000 U/mL pepsin and 12.6 mg/mL pyrogallol as an antioxidant) and incubated at pH 3 and 37 °C for 2 h in a shaking incubator at 250 rpm to simulate the gastric conditions. Simulated duodenal fluid (10 mL) was then added to start the duodenal phase, and the final digestion mixture consisted of 2000 U/mL pancreatic lipase, 10 mg/mL bile extract (∼10 mM bile salts), and 3.8 mg/mL phospholipids (∼8 mM). The pH of the final mixture was adjusted to 7 by dropwise addition of 1 M sodium hydroxide, and the duodenal-digestion phase was carried out for 4 h. Variations in the experimental conditions were applied in order to understand the impacts of specific factors. First only the duodenal phase was utilized (i.e., without any preceding gastric conditions). Second, glass beads (four, each 10 mm in diameter) were added to each digestion jar to change the shear conditions. Lastly, when the gastric stage was included, experiments with two different pH levels (i.e., 3 and 7), as well as in the absence and presence of pepsin, were conducted. Digestate Analysis. Duodenal digestate samples were collected at 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, and 240 min and extracted into acidic hexane. FFA concentrations were determined using a nonesterified FA kit (NEFA-HR2, Wako Diagnostics, Richmond, VA) with spectrophotometric analysis at 550 nm (Spectramax plus, Molecular Devices Corporation, San Jose, CA).22 Digestate samples were inspected visually and DSC and particle-size analyses were performed at the end of the gastric phase and during the duodenal phase. Because of extensive aggregation during the acidic gastric phase, the SE could not be sampled representatively. Data and Statistical Analysis. At least three separate experiments were carried out with freshly prepared samples and at least
addressing the hypothesis that TAG physical state, specifically, attenuates lipolytic digestibility. Experiments were also conducted with higher-impact shear conditions (i.e., inclusion of glass beads), without pepsin, and at pH 7 to determine these relative contributions to colloidal stability, crystallinity, and subsequent lipid digestion.
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MATERIALS AND METHODS
Materials. Palm stearin (PS) and sorbitan monostearate (Span 60) were generously provided by Bunge Oils Inc. (Bradley, IL) and Croda Canada Ltd. (Vaughan, ON, Canada), respectively. The major FAs of PS were palmitic acid at ∼58% and monounsaturated oleic acid at ∼27%, on the basis of gas chromatography.19 POP (26.1 ± 0.1%), PPP (23.1 ± 0.1%), and POO (13.8 ± 0.1%) were the main TAGs present, on the basis of HPLC analysis, described below. For the in vitro gastro−duodenal experiments, pancreatin from porcine pancreas (4 × USP, containing trypsin, amylase, lipase, ribonuclease, and protease), porcine bile extract (composition as previously reported:20 hyodeoxycholic acid, 1−5%; deoxycholic acid, 0.5−7%; cholic acid, 0.5−2%; glycodeoxycholic acid, 10−15%; taurodeoxycholic acid, 3−9%; and ∼49% bile salts), pepsin (from porcine stomach mucosa with activity of 1020 U per milligram of protein), and pyrogallol (99%, ACS reagent) were purchased from SigmaAldrich (St. Louis, MO). Triacylglycerol-Composition Analysis. The TAG composition of bulk PS was determined using a Waters Alliance model 2690 highperformance liquid chromatograph with a refractive-index detector (Waters model 2410, Waters, Milford, MA). Chromatographic separation of the diacylglycerols and TAGs was achieved using a Waters xbridge C18 column (Waters Limited, Mississauga, ON, Canada; 4.6 × 250 mm internal diameter with 5 μm particle size), and identification was made by comparison with TAG-internal-standard retention times. Isocratic elution with a flow rate of 1 mL/min of degassed acetone/acetonitrile 60/40 (v/v) was applied, and the column and detector temperatures were set at 40 °C. Data were analyzed using Millenium32 (K&K Testing, LLC, Decatur, GA). Preparation and Analysis of Palm-Stearin Emulsions. A 10% (w/v) palm stearin in water emulsion was prepared using 0.4% (w/v) sorbitan monostearate (Span 60) as the emulsifier. Span 60 is a nonionic emulsifier with an HLB value of 4.7.21 In brief, to prepare 100 mL of the emulsion, 10 g of PS and 0.4 g of Span 60 were melted at 80 °C for 30 min. A coarse emulsion was prepared by adding 80 °C deionized water (DI) and mixing with a hot hand-held homogenizer at 12 000 rpm for 1 min (Ultra Turrax, IKA T18 Basic, Staufen, Germany). The emulsion was then transferred to the hot hopper of a microfluidizer (M-110EH, Microfluidics, Westwood, MA), and hot homogenization was carried out at 125 MPa for 5 passes with the piping immersed in a 95 °C water bath. Half of the hot homogenate was transferred to a glass jar warmed to 80 °C, and the sample was then directly placed at 37 °C to produce the liquid-emulsion droplets (i.e., LE, stored for a maximum of 3 days). The remainder was transferred to a glass jar previously cooled to 5 °C and then placed in an ice−water bath for 20 min before storage at 5 °C (for a maximum of 7 days) until 20−30 min before analyses when it was moved to 37 °C. This sample containing solid particles is referred to as SE. Strict temperature control was maintained throughout sample preparation, storage, and analysis, including warming utensils to 37 °C and minimizing temperature fluctuations, in order to maintain the intended differences in physical state, which were confirmed routinely by differential-scanning calorimetry, as below. Particle Size and ζ-Potential. The particle sizes of the emulsion samples were determined by laser diffraction using a Mastersizer 2000S (Malvern Instruments Inc., Southborough, MA). Particle-size distributions and volume-weighted (D4,3) and surface-weighted (D3,2) mean diameters were measured. Emulsion-droplet charges were measured using a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments Inc., Southborough, MA). In order to minimize multiple-scattering effects, emulsion samples were diluted B
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry three analytical replicates were performed for each. Normality of data was confirmed with d’Agostino−Pearson omnibus testing, and data analysis was performed either by t tests or ANOVA using GraphPad Prism (GraphPad Software, San Diego, CA) with a significance level of p < 0.05. Results are reported as means ± SD.
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RESULTS AND DISCUSSION Physical Properties, Compositional and Morphological Analyses. Different physical states were evidenced for LE and SE using DSC (Figure 1a,b). In LE there was no melting
Figure 2. Representative particle-size distributions of LE and SE. Data indicate means ± SD.
observation and particle-size analysis, LE and SE were found to be gravitationally stable during storage at 37 °C for 3 days and at 5 °C for 7 days, respectively (data not shown). Therefore, LE (stored at 37 °C) was always analyzed and used within 3 days of preparation. SE samples were stored for a maximum of 7 days at 5 °C and equilibrated at 37 °C for 30 min prior to use. LE and SE were observed under bright-field (Figure 3a,c) and polarized (Figure 3b,d) light, and both emulsions contained spherically shaped droplets. Under polarized light, the typical Maltese-cross-like pattern, as observed for birefringent crystalline lipids, were obvious for the SE droplets. Some birefringence was also evident in the LE samples, mostly at the interface, suggesting interfacial organization of the Span 60 emulsifier. Indeed, when canola-oil emulsions were similarly prepared with Span 60, the same interfacial shell was observed (Figure 3e,f), pointing to the presence of organized Span 60 at the LE-droplet interface rather than TAG crystallization. Span 60 was similarly shown to form a shell around droplets in water-in-oil emulsions containing canola oil (90%) and fully hydrogenated canola oil (8.5%).23 The impact of an emulsifier on TAG nucleation and crystal growth depends on molecular interactions and the degree of structural complementarity.18,24 In bulk cocoa butter, Span 60 was recently found to phase separate, crystallize, and effectively slow down TAG nucleation and crystal growth.25 In the present study, Span 60 and its stearyl chains evidently did not induce LE-droplet crystallization during the 3 days of storage at 37 °C, despite these TAGs being in a thermodynamically metastable state. In summary, LE and SE droplets were completely liquid and partially crystalline, respectively, but had the same size distribution, surface charge, and morphology. This eliminates these factors as confounding influences in subsequent experiments. In Vitro Duodenal Lipolysis without Exposure to a Gastric Phase. In vitro duodenal-lipolysis experiments were initially performed without the gastric phase (Figure 4a). Accordingly, both LE and SE reached the maximum achievable lipolysis by 60 min (p < 0.05), with higher lipolysis observed for LE than for SE at 5 min (p < 005), indicating a faster initial rate. The initial specific-surface-area value for SE (19.7 ± 0.2 m2/g) was slightly higher than that for LE (19.1 ± 0.1 m2/g, p = 0.0022), highlighting the dominant influence of physical state in impacting early lipolysis. The FFA-release area under the curve (AUC) was the same for LE and SE, reflecting similar extents of lipolysis over the 4 h digestion period (p > 0.05). Therefore, the liquid state facilitated lipolysis of the emulsion droplets, but did not change the ultimate
Figure 1. Representative DSC thermograms of melting and crystallization of (a) liquid (LE) and (b) solid (SE) droplets and of (c) bulk palm stearin (PS) tempered as per SE with the PS SFC curve inset. (d) X-ray diffractograms for bulk PS and SE SFC. Data indicate means ± SD, n = 3.
event, confirming there was no crystalline fat present, whereas SE had a peak melting temperature of 52.6 ± 0.4 °C, indicating the presence of solid fat. There was no crystallization observed when SE was directly cooled in the DSC from 37 to 0 °C (data not shown). Also, the melting enthalpy of SE (4.5 ± 0.5 J/g) was ∼10% of that for bulk PS (44.7 ± 4.7 J/g), providing crude evidence that SE lipid droplets achieved maximum attainable crystallinity for an emulsion with a 10% lipid phase. For reference, according to pNMR analysis (Figure 1c, inset), the SFC of palm stearin at 37 °C was 33.17 ± 0.05%. Also, when PS was tempered using the same method of producing SE, it had a peak melting temperature of 54.0 ± 0.3 °C and recrystallization peaks at 30.2 ± 0.0 and 7.7 ± 0.4 °C (Figure 1c). Bulk Span 60 had a melting peak at 57.9 ± 0.7 °C and recrystallization peaks at 51.8 ± 0.4 and 44.8 ± 0.6 °C (data not shown). According to XRD, both SE and the tempered bulk PS contained the β polymorph, with a dominant reflection at 4.6 Å and weaker ones at 3.9 and 3.7 Å (Figure 1d). Figure 2 shows that the LE and SE particle-size distributions perfectly overlaid each other, with monomodal distributions centering around ∼416 nm. Values of D3,2 and D4,3 also were not significantly different (p > 0.05) between LE (0.36 ± 0.01 and 0.49 ± 0.01 μm, respectively) and SE (0.31 ± 0.03 and 0.46 ± 0.03 μm, respectively). ζ-Potential values for LE (−56.5 ± 2.7 mV) and SE (−56.6 ± 4.8 mV) were statistically similar as well (p > 0.05). Although Span 60 is a nonionic emulsifier, the highly negative droplet ζ-potential values observed may be the result of ionic impurities, including free fatty acid present in the emulsifier (acid value of 5 mg of KOH per gram, as per the manufacturer) or the palm stearin. On the basis of visual C
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Microstructures of (a,b) LE, (c,d) SE, and (e,f) 10% canola oil−0.4% Span 60 emulsion droplets under (a,c,e) bright-field and (b,d,f) polarized-light conditions.
digestibility. No obvious particle aggregation was observed for either system when digestive fluids were added, suggesting electrostatic repulsion remained.26 According to Figure 5a, slightly larger particle sizes (more so for LE) were observed by 15 min into the duodenal digestion and were likely related to coalescence, given the extensive lipolysis already achieved by this point. There also appeared to be a shift toward smaller particles in SE but not in LE, which was potentially related to differences in the rates of initial lipolysis and also to a higher tendency for LE droplets, being liquid, to coalesce during digestion and for SE particles to reduce in size as they are hydrolyzed. When LE digestate samples were analyzed by DSC there was no evidence that the undercooled droplets had crystallized (i.e., no shift in baseline, data not shown). However, with this high level of dilution and the rapid lipolysis observed, it may not have been possible to detect small amounts of solid fat. In summary, when samples of undercooled LE and partially crystalline SE (which were equivalent in particle size, morphology, and ζ-potential) were exposed to conditions simulating the upper-duodenal environment, both were rapidly and extensively hydrolyzed with slightly, but significantly, faster initial digestion of the liquid droplets. To the best of our knowledge, this is the first such investigation comparing droplets with such similar particles. The results lend strong support to the potential for kinetic differences in lipid digestion based specifically on whether
TAGs are present as liquids or solids and based on overall SFC. In Vitro Duodenal Lipolysis Following Exposure to a Gastric Phase. LE and SE were next exposed to a more physiologically relevant approach by mimicking gastric conditions before the duodenal phase. Although Span 60 is considered to be colloidally stable under mildly acidic conditions,27 the particle sizes increased for both samples (Figure 5b,c), which was likely related to decreases in droplet electrostatic repulsion in the highly acidic environment. Hydrogen-ion shielding of negatively charged emulsion droplets can decrease charge repulsion, thereby leading to droplet aggregation.14,28,29 Moreover, the gastric pH of 3.0 is below the pKa reported for stearic acid (4.3−5.5)30 such that ionization of any free stearic acid present from Span 60 or in the palm stearin is minimized. LE easily redispersed upon mixing and exposure to the duodenal fluids, indicating flocculation at the gastric stage. In contrast, SE was extensively clumped and resisted dispersal in the duodenal fluids, pointing to the likelihood of partial coalescence occurring with gastricstage aggregation (Figure 5c). According to pNMR, the palm stearin contained 33.17 ± 0.05% solids at 37 °C. This is within the range where partial coalescence is expected, especially under conditions of shear when droplet collisions are promoted.26 Other studies have evidenced that solid fat contributes to differences in gastric colloidal stability.31,32 For example, in emulsions formulated with acid-susceptible wheyD
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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human study, phase separation of a Span 80 stabilized emulsion was observed in the stomach with extensive fat layering, and there were associated effects on satiety.34 Undercooled-emulsion droplets are metastable and susceptible to the induction of crystallization. Partial crystallization was previously observed for undercooled 10% tripalmin−SDS emulsion droplets during in vitro digestion.14 In this study, when the LE digestate was analyzed by DSC at the end of the gastric phase, a small melting event was observed at 54.3 ± 0.5 °C (data not shown). Unfortunately, the extent of this crystallization is difficult to quantify. For a crude comparison, the melting enthalpy for this event was 45.4 ± 6.1% compared with the calculated result of what the SE melting enthalpy would be at an equivalent level of dilution. This suggests a significant proportion of the LE lipids crystallized in the presence of acid, which was potentially aggravated by shear leading to increased collision frequency. According to Figure 5b, some of the LE aggregation was reversible: LE droplets partly redispersed after 15 min in the duodenal phase (i.e., peaks centered around 1 and 10 μm were observed). Therefore, despite LE partial crystallization, partial coalescence in these samples was minimal compared with that of SE, potentially related to the low SFC. Assuming the crystallinity induced in LE during gastric exposure was ∼45% SFC (see above), on the basis of PS containing 33% SFC at 37 °C, the droplet solid content for this emulsion would be ∼14.8% (i.e., potentially too low for partial coalescence). Differences in the location and type of crystals formed might also play a role, given the very different nucleation conditions when SE was quench-cooled in order to crystallize.26 Interestingly, when samples of LE and aggregated SE duodenal digestates were analyzed by DSC, slightly higher end-of-melt temperatures (i.e., ∼ 58 °C) were consistently observed, compared with those drawn at the end of the gastric phase (i.e., 52−54 °C; p < 0.05). This corresponds to the melting temperature of Span 60 (i.e., 57.9 ± 0.7 °C), suggesting interfacial displacement of the emulsifier during lipolysis. Figure 4b shows that when the gastric phase was included, the initial rate of duodenal lipolysis was much faster for LE than for SE (i.e., ∼3× faster within the first 5 min, p < 0.05) and that lipolysis was significantly higher for LE than for SE up
Figure 4. Free fatty acid release during (a) duodenal digestion without the gastric phase, (b) duodenal digestion with the gastric phase, and (c) gastro−duodenal digestion with added glass beads (diameter of 10 mm). Data indicate means ± SD, n = 3. Significant differences (p < 0.05) were observed in gastro−duodenal digestion (b) up to 60 min and in gastro−duodenal digestion with added glass beads (c) up to 240 min. The lipolysis data were fitted to a first-orderkinetics model for graphing purposes. The r2 values obtained for LE and SE were, respectively, (a) 0.7771 and 0.8254, (b) 0.9231 and 0.9935, and (c) 0.9675 and 0.9825.
protein isolate and sodium caseinate, respectively, those consisting of higher solid-fat contents tended to destabilize more under gastric conditions in vitro5 and in vivo.33. In a
Figure 5. (a,b) Representative graphs showing particle-size distributions of duodenal digestion of LE and SE (a) after 15 min of duodenal digestion without gastric phase and (b) after 2 h of gastric phase and 15 min of duodenal digestion. (c,d) Visual observations of LE and SE (c) after 2 h of gastric digestion and (d) at the end of duodenal phase (4 h) following gastric phase. E
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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presence of pepsin at pH 7. However, at pH 3, both with and without pepsin, increases in particle size were observed for LE and SE, albeit to different extents (Figure 6). Whenever LE was exposed to pH 3, flocculation was visibly observed, but samples redispersed with mixing. SE aggregation was much more extensive, both with and without pepsin, at pH 3. Indeed, acidic pH was a major factor in emulsion-droplet aggregation. In terms of LE liquid-state stability, regardless of pH (3 or 7) and presence or absence of pepsin, similar levels of crystallinity were induced (p > 0.05, data not shown). Therefore, LE was vulnerable to partial crystallization independent of acidinduced flocculation. Separate experiments did show that LE crystallization was not induced by the introduction of shear (250 rpm orbital shaking for 1 h) or dilution with DI water (data not shown). The differences in the extent of gastricphase destabilization of solid versus liquid Span 60 containing emulsions point to interactions between solid-fat content, emulsifier behavior, and types of crystallinity that warrant further investigations. Static digestion models use different levels and types of shear (i.e., orbital, horizontal, or rotary shaking; stirring; physical impact via a solid matrix (glass beads); etc.) to mimic gastric mixing and motility,37−39 although achieving physiological relevance is difficult. To facilitate mixing and add impact forces,37 glass beads (diameter of 10 mm) were added to each sample jar. Doing so exaggerated the differences in the colloidal properties between SE and LE at the end of the gastric phase. Specifically, LE remained dispersed, but even larger densely packed aggregates of SE were observed visually. The initial SFC of the SE droplets, decreased droplet electrostatic repulsion, and increased shear forces during gastric exposure potentially enhanced the collision frequency and capture efficiency, thereby increasing the susceptibility of SE for partial coalescence.26 The formed SE aggregates resisted redispersal throughout duodenal digestion. In terms of lipolysis, the presence of the beads attenuated early fatty acid release in both LE and SE (p < 0.05). Also, LE lipolysis was significantly higher than that for SE throughout the digestion (Figure 4c, p < 0.05). At 45 min, for example, it was ∼90% higher. Therefore, the higher impact forces resulted in attenuated lipolysis of the SE particles but had relatively minimal impact on LE. This was presumably related to massive differences in available surface area during duodenal digestion and underscores the critical role that aggregation induced during the gastric phase can play in lipid digestibility. In summary, this study investigated the differences in digestive lipolysis in vitro between partially crystalline solid emulsion droplets and undercooled-liquid-emulsion droplets based on the emulsifier Span 60 with equivalent compositions, sizes, charges, and morphologies. Early lipolysis was attenuated by the presence of TAG crystallinity in SE and by particle destabilization, which was aggravated by the presence of solid fat, during exposure to simulated gastric conditions. Moreover, the observed differences in early-stage lipolysis support the general hypothesis that solid fat is less readily digested than liquid oil, pointing to possible kinetic differences, even if lipolysis is ultimately completed. Indeed, although lipid digestion in healthy adult humans is generally considered to be complete,40 there is in vivo evidence pointing to differences in digestion kinetics based on physical state. For example, rises in plasma9,11 and chylomicron10 TAG were slower during the postprandial period when lipids with solid fat or higher solidfat contents at 37 °C, were consumed, even though similar
to 60 min (p < 0.05). This is despite the partial crystallinity observed in LE. The droplet specific surface area for LE (3.99 ± 0.05 m2/g) was significantly greater than that for SE (2.42 ± 0.14 m2/g) after 15 min of duodenal digestion (p = 0.0005). This aligns well with differences in the slopes of the lipolysis curves up to 15 min of digestion (i.e., LE = 1.63 ± 0.17 > SE = 1.17 ± 0.10% lipolysis per minute; p = 0.0262). Similar levels of lipolysis were reached for SE and LE by 1.5 and 4 h (p > 0.05), although lipolysis was, overall, relatively low for both samples. Compared with Figure 4a when the gastric phase was omitted, LE and SE duodenal lipolysis was ∼60% lower (Figure 4b). Incomplete digestion can occur with in vitro methods because of insufficient enzyme activity or the interfacial accumulation of products of digestion. However, the above results without the gastric phase support that the conditions utilized were capable of fully hydrolyzing these samples. Rather, the results point to differences in interfacial area as being a critical factor. Gastric-phase destabilization, including with the presence of solid fat,35 was previously shown to contribute to attenuated lipid digestion.36 In this study, the fact that LE partially crystallized during the gastric phase allows the comparison of duodenal lipolysis for completely liquid (0% SFC), dispersed droplets (i.e., LE without gastric phase, Figure 4a); partially crystalline (∼15% SFC), partially aggregated droplets (i.e., LE after gastric phase, Figure 4b); partially crystalline (∼33% SFC), dispersed droplets (i.e., SE without gastric phase, Figure 4a); and partially crystalline (∼33% SFC), highly aggregated, partially coalesced droplets (i.e., SE after gastric phase, Figure 4b). Specifically, the partially crystalline (∼15% SFC) LE had a comparatively lower rate and extent of lipolysis during early duodenal digestion compared with those of both the LE and SE systems that remained dispersed because they were never exposed to the gastric phase (p < 0.05). This points to the substantial impact of colloidal destabilization, amplified by the presence of solid fat, on digestibility. In summary, welldispersed liquid droplets were hydrolyzed more readily than those containing solid fat. Moreover, the presence of solid fat in the emulsions was associated with more extensive gastricphase destabilization, specifically partial coalescence, and associated attenuated lipolysis. In vivo, these systems would be expected to have different rates of gastric emptying,33 although such dynamics could not be controlled for using the current model. Role of Gastric Parameters in Determining Duodenal Lipolysis. To further investigate gastric-phase destabilization, samples were also incubated without pepsin and at pH 7, instead of at pH 3. In the case of pH 7 with pepsin, SE showed no visible aggregation. Particle-size analysis (Figure 6a,b) also showed that LE and SE were colloidally unchanged in the
Figure 6. Gastric-phase impact on (a) LE and (b) SE particle-size distributions: -·-· with pepsin at pH 3, without pepsin at pH 3, - - with pepsin at pH 7, ○ undigested LE, □ undigested SE. F
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
(11) Robinson, D. M.; Martin, N. C.; Robinson, L. E.; Ahmadi, L.; Marangoni, A. G.; Wright, A. J. Influence of interesterification of a stearic acid-rich spreadable fat on acute metabolic risk factors. Lipids 2009, 44 (1), 17−26. (12) Nik, A. M.; Langmaid, S.; Wright, A. J. Digestibility and βcarotene release from lipid nanodispersions depend on dispersed phase crystallinity and interfacial properties. Food Funct. 2012, 3 (3), 234. (13) Bonnaire, L.; Sandra, S.; Helgason, T.; Decker, E. A.; Weiss, J.; McClements, D. J. Influence of lipid physical state on the in vitro digestibility of emulsified lipids. J. Agric. Food Chem. 2008, 56 (10), 3791−3797. (14) Huynh, S.; Wright, A. J. Tripalmitin-Sodium Dodecyl Sulfate Emulsion Droplet Liquid vs. Solid State Impacts in vitro Digestive Lipolysis. J. Am. Oil Chem. Soc. 2018, 95 (March), 161−170. (15) Hart, S. M.; Lin, X. L.; Thilakarathna, S.; Wright, A. J. Emulsion droplet crystallinity attenuates early in vitro digestive lipolysis and beta-carotene bioaccessibility. Food Chem. 2018, 260, 145−151. (16) Health Canada. Food Additives, 2016. Government of Canada. https://www.canada.ca/en/health-canada/services/food-nutrition/ food-safety/food-additives.html. (17) Aronhime, J. S.; Sarig, S.; Garti, N. Mechanistic considerations of polymorphic transformations of tristearin in the presence of emulsifiers. J. Am. Oil Chem. Soc. 1987, 64 (4), 529−533. (18) Aronhime, J. S.; Sarig, S.; Garti, N. Dynamic control of polymorphic transformation in triglycerides by surfactants: The button syndrome. J. Am. Oil Chem. Soc. 1988, 65 (7), 1144−1150. (19) Anderson, B. M.; MacLennan, M. B.; Hillyer, L. M.; Ma, D. W. L. Lifelong exposure to n-3 PUFA affects pubertal mammary gland development. Appl. Physiol., Nutr., Metab. 2014, 39 (6), 699−706. (20) Hur, S. J.; Decker, E. A.; McClements, D. J. Influence of initial emulsifier type on microstructural changes occurring in emulsified lipids during in vitro digestion. Food Chem. 2009, 114 (1), 253−262. (21) Griffin, W. C. Classification of surface-active agents by “HLB”. J. Soc. Cosmet. Chem. 1949, 1 (5), 311−326. (22) Lin, X.; Wright, A. J. Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility. Food Hydrocolloids 2018, 76, 49−59. (23) Tran, T.; Green, N. L.; Ghosh, S.; Rousseau, D. Encapsulation of water-in-oil emulsion droplets within crystal spheroids. Colloids Surf., A 2017, 524, 1−7. (24) Douaire, M.; Di Bari, V.; Norton, J. E.; Sullo, A.; Lillford, P.; Norton, I. T. Fat crystallisation at oil-water interfaces. Adv. Colloid Interface Sci. 2014, 203, 1−10. (25) Sonwai, S.; Podchong, P.; Rousseau, D. Crystallization kinetics of cocoa butter in the presence of sorbitan esters. Food Chem. 2017, 214, 497−506. (26) Fredrick, E.; Walstra, P.; Dewettinck, K. Factors governing partial coalescence in oil-in-water emulsions. Adv. Colloid Interface Sci. 2010, 153 (1−2), 30−42. (27) Croda Europe Ltd (2009) Span and Tween, www.Croda.Com/ Europe. (28) Comas, D. I.; Wagner, J. R.; Tomás, M. C. Creaming stability of oil in water (O/W) emulsions: Influence of pH on soybean proteinlecithin interaction. Food Hydrocolloids 2006, 20 (7), 990−996. (29) Mantovani, R. A.; Cavallieri, Â . L. F.; Netto, F. M.; Cunha, R. L. Stability and in vitro digestibility of emulsions containing lecithin and whey proteins. Food Funct. 2013, 4 (9), 1322. (30) Quast, K. The use of zeta potential to investigate the pKa of saturated fatty acids. Adv. Powder Technol. 2016, 27 (1), 207−214. (31) Golding, M.; Wooster, T. J. The influence of emulsion structure and stability on lipid digestion. Curr. Opin. Colloid Interface Sci. 2010, 15 (1−2), 90−101. (32) Golding, M.; Wooster, T. J.; Day, L.; Xu, M.; Lundin, L.; Keogh, J.; Clifton, P. Impact of gastric structuring on the lipolysis of emulsified lipids. Soft Matter 2011, 7 (7), 3513. (33) Steingoetter, A.; Radovic, T.; Buetikofer, S.; Curcic, J.; Menne, D.; Fried, M.; Schwizer, W.; Wooster, T. J. Imaging gastric structuring of lipid emulsions and its effect on gastrointestinal function: A
levels of lipemia were eventually achieved. Differences in lipiddigestion kinetics may also impact the release locations of encapsulated molecules, the induction of hormonal feedback loops related to satiety, and the profile of the lipemic response.31,34,41 The current results also support that in vitro digestion experimental parameters, in particular the inclusion of a gastric phase and impact shear conditions, influence the results obtained. The use of a static model lacking an oral phase and gastric lipase are limitations of the work. Additionally, although comparing crystalline droplets to undercooled droplets of identical composition enables a unique comparison minimizing confounding influences, the situation does not exactly resemble that of typical liquid dietary lipids, given the thermodynamic instability of the LE system and the fact that partial crystallization of LE was induced. Further investigations are required to better understand how lipid physical properties impact colloidal and lipolytic behavior during digestion, including through validation with human research.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: 519-824-4120 ext. 54697. E-mail: ajwright@uoguelph. ca. ORCID
Amanda J. Wright: 0000-0001-6178-1961 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) McClements, D. J.; Decker, E. A.; Park, Y.; Weiss, J. Designing food structure to control stability, digestion, release and absorption of lipophilic food components. Food Biophys 2008, 3 (2), 219−228. (2) McClements, D. J.; Decker, E. A.; Park, Y. Controlling lipid bioavailability through physicochemical and structural approaches. Crit. Rev. Food Sci. Nutr. 2008, 49 (1), 48−67. (3) Michalski, M. C.; Genot, C.; Gayet, C.; Lopez, C.; Fine, F.; Joffre, F.; Vendeuvre, J. L.; Bouvier, J.; Chardigny, J. M.; RaynalLjutovac, K. Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism. Prog. Lipid Res. 2013, 52 (4), 354−373. (4) Thilakarathna, S. H.; Rogers, M.; Lan, Y.; Huynh, S.; Marangoni, A. G.; Robinson, L. E.; Wright, A. J. Investigations of in vitro bioaccessibility from interesterified stearic and oleic acid-rich blends. Food Funct. 2016, 7 (4), 1932−1940. (5) Guo, Q.; Bellissimo, N.; Rousseau, D. The Physical State of Emulsified Edible Oil Modulates Its in Vitro Digestion. J. Agric. Food Chem. 2017, 65 (41), 9120−9127. (6) Bergstedt, S. E.; Hayashi, H.; Kritchevsky, D.; Tso, P. A comparison of absorption of glycerol tristearate and glycerol trioleate by rat small intestine. Am. J. Physiol. 1990, 259 (3), G386−G393. (7) Wang, X.; Wang, T.; Spurlock, M. E.; Wang, X. Effects of triacylglycerol structure and solid fat content on fasting responses of mice. Eur. J. Nutr. 2016, 55, 1545. (8) Kaplan, R. J.; Greenwood, C. E. Poor Digestibility of Fully Hydrogenated Soybean Oil in Rats : A Potential Benefit of Hydrogenated Fats and Oils 1 − 3. J. Nutr. 1998, 128 (5), 875−880. (9) Berry, S. E. E.; Miller, G. J.; Sanders, T. A. B. The solid fat content of stearic acid − rich fats determines their postprandial effects. Am. J. Clin. Nutr. 2007, 85, 1486−1494. (10) Hall, W. L.; Fiuza Brito, M.; Huang, J.; Wood, L. V.; Filippou, A.; Sanders, T. a. B.; Berry, S. E. E. An Interesterified Palm Olein Test Meal Decreases Early-Phase Postprandial Lipemia Compared to Palm Olein: a Randomized Controlled Trial. Lipids 2014, 49 (9), 895−904. G
DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry randomized trial in healthy subjects. Am. J. Clin. Nutr. 2015, 101 (4), 714−724. (34) Marciani, L.; Faulks, R.; Wickham, M. S. J.; Bush, D.; Pick, B.; Wright, J.; Cox, E. F.; Fillery-Travis, A.; Gowland, P. A.; Spiller, R. C. Effect of intragastric acid stability of fat emulsions on gastric emptying, plasma lipid profile and postprandial satiety. Br. J. Nutr. 2009, 101 (06), 919. (35) Day, L.; Golding, M.; Xu, M.; Keogh, J.; Clifton, P.; Wooster, T. J. Food Hydrocolloids Tailoring the digestion of structured emulsions using mixed monoglyceride e caseinate interfaces. Food Hydrocolloids 2014, 36, 151−161. (36) Mackie, A. Food: more than the sum of its parts. Curr. Opin. Food Sci. 2017, 16, 120−124. (37) Kong, F.; Singh, R. P. A Model Stomach System to Investigate Disintegration Kinetics of Solid Foods during gastric digestion. J. Food Sci. 2008, 73 (5), 202−210. (38) Eldemnawy, H. Y.; Wright, A.; Corredig, M. A Better Understanding of the Factors Affecting In vitro Lipolysis Using Static Mono-compartmental Models. Food Dig. 2015. (39) O'Sullivan, C. M.; Davidovich-Pinhas, M.; Wright, A. J.; Barbut, S.; Marangoni, A. G. Ethylcellulose oleogels for lipophilic bioactive delivery − effect of oleogelation on in vitro bioaccessibility and stability of beta-carotene. Food Funct. 2017, 8 (4), 1438−1451. (40) Mu, H.; Høy, C. E. The digestion of dietary triacylglycerols. Prog. Lipid Res. 2004, 43, 105−133. (41) Steingoetter, A.; Buetikofer, S.; Curcic, J.; Menne, D.; Rehfeld, J. F.; Fried, M.; Schwizer, W.; Wooster, T. J. The Dynamics of Gastric Emptying and Self-Reported Feelings of Satiation Are Better Predictors Than Gastrointestinal Hormones of the Effects of Lipid Emulsion Structure on Fat Digestion in Healthy AdultsA Bayesian Inference Approach. J. Nutr. 2017, 147 (4), 706−714.
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DOI: 10.1021/acs.jafc.8b02636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
