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Palm Lipid Emulsion Droplet Crystallinity and Gastric Acid Stability in Relation to in vitro Bioaccessibility and in vivo Gastric Emptying

It is poorly understood how the physical state of emulsified triacylglycerol (TAG) alters colloidal behavior in the gastrointestinal tract to modulate lipid digestion and absorption. We, therefore, aimed to investigate the individual and combined effects on fatty acid (FA) bioaccessibility using the dynamic TIM-1 in vitro digestion model and integrate the results with those from a human clinical study. Four 20% oil-in-water emulsions with overlapping particle size distributions contained either partially crystalline solid (palm stearin) or liquid (palm olein) lipid droplets at 37°C and either the colloidally acid-stable Tween 80 (2.2%) or acid-unstable Span 60 (2.5%) emulsifier. Experimental meals were fed to the TIM-1, and jejunal and ileal dialysates were analyzed over 6 h to measure free FA concentration. Cumulative FA bioaccessibility was significantly higher for the liquid stable emulsion compared to all others (p < 0.05), which did not differ (p > 0.05). Emulsified TAG physical state was associated with differences in overall bioaccessibility (higher for liquid state TAG) in the colloidally stable emulsions, but this difference was blunted in droplets susceptible to acidic flocculation. In contrast, human postprandial TAG concentrations did not differ significantly between the emulsions. The discrepancy may relate to differences in in vivo gastric emptying (GE) as evidenced by ultrasonography. When the in vivo differences in GE were accounted for in follow-up TIM-1 experiments, the findings aligned more closely. Cumulative FA bioaccessibility for the liquid stable emulsion no longer differed significantly from the other emulsions, and SU’s bioaccessibility was the lowest, consistent with the in vivo observations. This work highlights the potential for TAG physical state and colloidal stability to interactively alter behavior in the gastrointestinal tract with implications for FA absorption, and the importance of establishing and improving in vitro–in vivo correlations in food-nutrition research.


INTRODUCTION
Excess energy intake, including dietary fat, is a leading cause of obesity (1) and a risk factor for many diseases, including cardiovascular diseases (CVD), type 2 diabetes, and some cancers (2)(3)(4). Postprandial lipemia (PPL) is recognized as a CVD risk factor (5), and lipid absorption correlates with fatsoluble vitamin absorption (6) and, more recently, endotoxemia (7). Therefore, there may be advantages to modulating lipid bioavailability (8), i.e., to promote or delay it or to change the overall absorption (9,10). Research into strategies to structure lipid-rich foods, including emulsions, that resist or enhance digestion, altering metabolism is rationalized. These include emulsification, emulsion droplet size, interfacial properties, physical lipid state, and physicochemical properties of the surrounding food matrix (11)(12)(13). Understanding the specific influence of triacylglycerol (TAG) physical state and solid fat content (SFC) at physiological temperature, i.e., 37 • C is important for designing foods that capitalize on the dynamics in the digestive tract to tailor PPL and lipid bioavailability.
In vitro digestion methods have yielded interesting insights in this area. Crystalline TAGs induce partial coalescence and different gastric structures that have been associated with attenuated in vitro lipolysis (14). Also, in vitro digestibility was higher for liquid canola-oil-based emulsions stabilized with non-ionic emulsifiers (Tween 20 and Poloxamer 188) than for high SFC emulsions with fully hydrogenated canola stearin (15). In addition, in vitro digestion kinetics were inversely associated with SFC for whey protein stabilized oil-in-water emulsions based on blends of soybean oil and fully hydrogenated soybean oil (13). Tempering has also been used to achieve differing SFCs for emulsions with identical compositions, thereby isolating the role of TAG physical state without confounding differences in fatty acid (FA) composition (16)(17)(18)(19). The rate and extent of lipolysis increased with SFC and remained constant for undercooled emulsions stabilized with Tween 80 using palm stearin and palm olein blends (16). In vitro lipolysis of partially crystallized tripalmitin (and SDS) (17,18) and palm stearin (and Span 60) (19) emulsions showed attenuated FA bioaccessibility compared to their undercooled counterparts. Similar differences were observed following consumption of the tempered palm stearin emulsions in a human study comparing PPL (20). Moreover, short-term appetite suppression was enhanced for the undercooled versus partially crystalline palm stearin emulsion (21). Aside from TAG crystallinity, the ingested emulsions were identical, and yet the droplets containing solid fat destabilized more extensively during exposure to simulated gastric conditions (19). Collectively, these studies highlight the important roles of TAG crystallinity and emulsion acid stability in determining lipid digestion and postprandial metabolism, but questions about their combined effects remain.
Emulsification in the gastrointestinal (GI) tract occurs via mechanical peristaltic forces and the addition of surfaceactive molecules that changes the interfacial area available for lipolysis (12). As an example, protein emulsifiers are digested by pepsin, whereas ionic emulsifiers experience different degrees of electrostatic repulsion depending on the gastric and luminal pH, leading to differences in emulsion stability, droplet flocculation, and potentially coalescence and phase separation (22,23). These effects can translate into altered lipid digestion profiles. For example, when olive oil emulsions with sucrose-ester surfactants were exposed to simulated gastric conditions, droplet size increased, corresponding to lower lipolysis than emulsions stabilized with Tween 80 (24). Human-level comparisons between acid-stable (Tween 60) versus acid-unstable (Span 60) emulsions indicate that gastric emptying (GE), cholecystokinin release, gallbladder contraction, PPL, and satiety all change with emulsion colloidal stability (25)(26)(27). MRI imaging revealed emulsion phase separation along with faster GE, delayed plasma lipid response, and lower satiety when participants consumed an acid-unstable palmitate-enriched olive oil-in-water emulsion (emulsified with Span 60) compared with an acid-stable emulsion (emulsified with Tween 60) (25). Steingoetter et al. showed that emulsifier acid stability, combined with solid fat to prevent emulsion redispersal, resulted in lower lipid bioavailability of oil-inwater emulsions (28), but without a treatment containing solid fat that was acid-stable, conclusions based on lipid physical state alone could not be drawn. The aim herein was to investigate the independent and combined effects of TAG crystallinity and emulsifier acid stability on TAG lipolysis and FA bioaccessibility using static and dynamic in vitro digestion methods, respectively, to compare four comprehensively characterized 20% oil-in-water emulsions containing either palm stearin (partially solid at 37 • C) or palm olein (liquid at 37 • C), and either Tween 80 (acid-stable emulsifier) or Span 60 (acid-unstable emulsifier). These emulsions were previously consumed by participants in a randomized crossover study in which different gastric microstructures were observed directly by ultrasonography and associated with different rates of GE (29).
In vitro digestion models allow gaining mechanistic insights into how food structures change in the gastrointestinal tract to differentially affect postprandial metabolism. The dynamic TIM-1 multicompartment system simulates the luminal conditions of the upper GI tract from the stomach, duodenum, and jejunum to the ileum, integrating physiological factors including meal transit time, peristaltic contractions, enzyme concentration, and pH changes (30)(31)(32). Dialysis membranes fractionate hydrolyzed and solubilized free fatty acids (FFA) from the luminal fluid (33) as a measure of bioaccessibility. Although TIM-1 results have correlated well with in vivo results (34)(35)(36), there are relatively few published reports and these exist for a relatively limited range of foods, especially in scenarios where the meals could have substantially different GE behaviors. Therefore, the secondary aim of this work was to compare the TIM-1 in vitro findings to those from an in vivo PPL study where healthy adults consumed the same four emulsions (29) in order to explore and improve the predictive value of in vitro digestion methods, addressing a recognized need in the food-nutrition community (37).

Emulsion Preparation
The study emulsions consisted of LS (liquid lipid-acid stable emulsifier), LU (liquid lipid-acid unstable emulsifier), SS (solid lipid-acid stable emulsifier), and SU (solid lipid-acid unstable emulsifier). To determine the effects of droplet TAG crystallinity, they were formulated with palm stearin (partially crystalline at 37 • C) or palm olein (liquid at 37 • C). They were also formulated with either Tween 80 or Span 60 to be either colloidally stable or unstable, respectively, in the presence of gastric acidity. The emulsifier concentrations were selected to achieve overlapping particle size distributions (PSDs) based on preliminary experiments. Span 60 is a relatively low HLB value (4.7) emulsifier selected based on previous observations that monomodal PSDs could be obtained for undercooled and maximally crystallized palm stearin emulsions at concentrations permissible for consumption in the corresponding human studies (19). The total weight of the emulsions was 300 g. First, 60 g of either palm olein or stearin, with either 6.6 g Tween 80 or 7.5 g Span 60, was melted together in a glass jar (oven temperature at 85 • C for 30 min to remove any lipid crystal memory), followed by the addition of 233 g boiling deionized water. A coarse emulsion was achieved using an ULTRA TURRAX R (IKA R T18 basic, Staufen, Germany) at 12,000 min −1 for 2 min and then passed through a microfluidizer (M-110EH, Microfluidics, Westwood, MA, United States) three times at 125 MPa with the machine's piping immersed in a water reservoir at ∼95 • C, using portable immersion heaters. The resulting fine emulsion with palm olein (LS and LU) was held at room temperature (21 ± 1 • C), and those with palm stearin (SS and SU) were placed in a pre-cooled jar, submerged in ice water for 20 min, and then refrigerated (5 • C) for at least 18 h to induce crystallization of the lipid droplets. Before all analyses and experiments, the emulsions were warmed to 37 • C for 1 h. All emulsions were stored and used within 7 days after confirming that PSDs and melting behavior remained consistent over this period (data not shown).

Physical Characterization of Emulsions
The FA composition of the bulk lipids and emulsifiers was determined by gas chromatography (Agilent 7890A GC) with flame ionization detection (Agilent Technologies Inc., DE, United States) (35) after extracting the total lipids using the Folch method (38). Transesterification using 14% boron trifluoride in methanol and re-suspending in hexane was performed before analysis. Laser diffraction [Mastersizer 2000S (Malvern Instruments Inc., Southborough, MA, United States)] determined the PSDs, volume-weighted, D 4,3, and surfaceweighted, D 3,2 , and mean diameters using refractive indices of 1.45 and 1.33 for the lipid and water phases, respectively. The emulsion droplet surface charge (ζ-potential) was measured by a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments Inc., Southborough, MA, United States). Emulsion SFC was measured at 37 • C 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). A differential scanning calorimeter (Q2000 model, TA Instruments, Mississauga, ON, Canada) determined the melting behavior of each emulsion, as previously described (19), by heating the samples from 37 to 85 • C at 5 • C/min. The apparent viscosity of each emulsion was determined as a function of the shear rate before and after 1 h of static gastric digestion, using a controlled strain rheometer (MCR 301, Anton Paar GmbH, Ostfildern, Germany), equipped with a concentric cylinder geometry (CC 27, 27 mm diameter), and a Peltier temperature controller to maintain 37 • C.

Exposure to Static in vitro Gastric Digestion Conditions
Static in vitro digestion experiments confirmed and compared the colloidal behavior of each emulsion during exposure to simulated gastric digestion fluid (SGF) as described previously (30). Five milliliters of each emulsion and 5 ml of SGF, containing 2,000 U/mL pepsin and 12.6 mg/mL of the antioxidant pyrogallol, were added to amber glass jars with four 10-mm glass beads and incubated in a shaking water bath [37 • C, 250 rpm (New Brunswick Scientific Co. Inc., NJ, United States)] for 2 h at pH 3. After digestion, samples were immediately analyzed for PSD, using the Mastersizer as described above. For SU and LU, the pH was adjusted to 7.0 by adding 1.0 N NaOH to assess re-dispersion (visually) and PSD.

Quantification of Free Fatty Acids
All dialysate samples were thawed at room temperature, and lipids were extracted by adding 50 µL of sample to an Eppendorf tube with 50 µL 1 M HCL and 500 µL hexane and centrifuged at 14,000 RPM for 30 min (5418 Laboratory Centrifuge, Eppendorf, Hamburg, Germany). The double-layered supernatant was added to 500 µL of hexane, and the tube was rinsed with another 500 µL of hexane. Non-esterified fatty acids (NEFA) were quantified in 96-well plates in duplicate for each TIM-1 run (performed in triplicate) according to the manufacturer instructions [Wako HR series NEFA-HR (2), FUJIFILM Wako Diagnostics, VA, United States] using an enzymatic colorimetric method assay. The cumulative FFA bioaccessibility (%) was the total accumulated lipid bioaccessibility at each time point (e.g., cumulative bioaccessibility at 60 min calculated as the sum of bioaccessibility from 0 to 30 and 30 to 60 min).

Adjustment of TIM-1 Gastric Emptying Rate
Additional TIM-1 digestions were performed by modifying the fed-state lipid protocol by adjusting the GE rates to those observed for the same emulsions in our previous human study. As previously reported, gastric ultrasound was used to visualize and measure the gastric antrum cross-sectional area at baseline and postprandially (29) from which we quantified GE. Specifically, the percentage GE volume after consuming those same four emulsions was determined at 0, 30, 60, 90, 120, 150, 180, 240, 300, and 360 min, converted to the fraction of meal remaining in the stomach, and used to determine the GE half time T 1/2   using Eq. 1 (40): where F is the meal fraction remaining in the stomach, T 1/2 is the GE half time, and β is a parameter defining the shape of the curve. A total of 80 min is the standard TIM-1 GE T 1/2 used in the initial experiments. The in vivo (40) GE T 1/2 ratio observed for LS:SU was 1.55 (i.e., 233 and 150 min for LS and SU, respectively). Thus, the LS emulsion was subsequently digested in the TIM-1 using an adjusted GE T 1/2 of 124 min.

Data and Statistical Analysis
All experiments were performed in at least triplicate, with analytical duplicates, using samples prepared within 7 days.  (39): where C asymp is the asymptotic plateau representing the maximum bioaccessible FFAs, k is the rate constant of FFA release per unit time (i.e., rate of lipolysis), and t c is the induction time (i.e., the time to reach effective lipolysis where half the total amount of FFAs are released) (39). Initial values were C asymp = 40; k = 0.00; and t c = 0.1, followed by the software performing 1,000 iterations. All time course data were analyzed by two-way repeatedmeasures ANOVA (RM-ANOVA) with time (seven levels: 0, 30, 60, 90, 120, 180, 240, 300, and 360 min) and treatment (four levels: LS, LU, SS, and SU) as the two factors followed by Bonferroni's multiple comparison testing when treatment and/or time × treatment interactions were significant. AUC, 6 h % bioaccessibility, rate constant (k), and induction time (t c ) values were analyzed by one-way ANOVA. Data were normally distributed (passed the Shapiro-Wilk test), there were no missing values, and statistical significance was set at p < 0.05. The hourly TIM-1 FFA and AUC values were correlated with the hourly and AUC postprandial TAG response of 15 healthy male adult participants who consumed the same test emulsions across four study visits following an overnight fast, separated by least 6 days (29) using Pearson's correlation analysis.

Emulsion Composition and Physical Properties
Compared to palm olein, palm stearin contained significantly more palmitic acid (C16:0) (58.7 ± 0.0 versus 36.4 ± 0.1%) and less oleic acid (C18:1c9) (26.8 ± 0.1 versus 45.2 ± 0.0%) ( Table 1), leading to SFC differences between the liquid and solid emulsion droplets ( Table 2). The 20% palm stearin oil-inwater emulsions possessed ∼9.3% SFC, representing ∼44-49% of the emulsified TAGs. The emulsions, stabilized with Span 60 which melts at ∼56 • C, trended to higher SFC than the Tween 80 emulsions, but the values were not significantly different (p > 0.05). The melting behavior of SS and SU (Figure 1) confirms the presence of crystallinity which was maximal based on the absence of crystallization peak when the emulsions were cooled directly from 37 • C (data not shown). Also, statistically similar (p < 0.05) peak melting temperatures of 52.2 ± 0.1 and 52.0 ± 0.1 • C were observed for SS and SU, respectively. DSC also confirmed the absence of crystallinity in LS and LU as no melting events were observed.

Exposure to Simulated Gastric Fluid
Gastric stability was determined visually (Figure 4) and by measuring particle size (Figure 2B-E) before and after 2 h SGF static in vitro digestion exposure. As intended, LS and SS did not visibly flocculate or coalesce (Figure 4), nor did shifts occur in the PSD. There were no visual changes in LU observed with SGF exposure (Figure 4), but droplet size increased, with the monomodal peak shifting to the right (Figure 2C). SU visibly destabilized, coalescing into large phase-separated aggregates (Figure 4), and preventing representative sampling for PSD measurements.
The viscoelastic properties of each emulsion determined before and after 1 h of static gastric digestion ( Figure 5) reveal shear-thinning, most obviously for SU and SS and LU both before and after digestion. In contrast, the LS emulsion and LS gastric digestate have Newtonian flow profiles (Figures 5A,B). Values of apparent viscosity at a shear rate of 56.23 s −1 were compared since this is within the range of physiological relevance in the GI tract (42). SS had a significantly higher apparent viscosity compared to LS (p = 0.038) and LU (p = 0.037) before digestion ( Figure 4A). No significant differences were observed in the digestate's apparent viscosity at 56.23 s −1 after 1-h digestion, although non-significant increases occurred for SU and LU, consistent with the anticipated flocculation of these Span-stabilized emulsions at acidic pH (Figures 5C,D).

TIM-1 Free Fatty Acids
The absolute and cumulative FA bioaccessibility (calculated based on the jejunal and ileal dialysate samples) for each emulsion was collected over the 6-h TIM-1 digestion (Figure 6). According to the RM-ANOVA, LS has a significantly higher overall bioaccessible FA concentration than LU (p = 0.005), SS (p = 0.023), and SU (p = 0.0003). LS also had a significantly higher overall bioaccessibility than LU (p < 0.0001), SS (p < 0.0001), and SU (p < 0.0001). Differences were specifically observed for LS compared to all other treatments at 180, 240, and 300 min, based on FFA concentration (Figure 6A), and at 180,   240, 300, and 360 min (p < 0.05), based on cumulative FA bioaccessibility ( Figure 6B).
Area under the curve values of the FFA concentration data were not significantly different (p = 0.07), although LS was at least 52% higher than all other emulsions. AUC values of the cumulative bioaccessibility results were significantly different (p = 0.03), but there were no pairwise differences based on posthoc testing (p > 0.05). There were neither differences in terms of bioaccessible fraction nor in the rate constant values obtained from the shifted logistic model (Figure 7) (p < 0.05). At 6 h, LS had at least 24 and 39% higher bioaccessible fraction and rate constants (k), compared to all other emulsions. Similar induction times were observed (p > 0.05) due to the TIM-1 pre-set GE rate. Regardless of the analysis method, there were no significant differences in FA concentration, AUC, or time point differences among LU, SS, and SU (p > 0.05). Postprandial TAG values did not return to baseline by 6 h for two of the emulsions (LS and LU), and lipolysis was incomplete after 6-h TIM-1 for all of the emulsions ( Figure 6A).

Correlation Between TIM-1 and Human Data
The TIM-1 FFA values were positively correlated with postprandial TAG concentration (p < 0.0001, R 2 = 0.74) (Figure 8C), but the overall TIM-1 and human study trends did not align (Figures 8A,B).

Adjusted Gastric Emptying TIM-1 Free Fatty Acids
TIM-1 GE T 1/2 was set to 124 min for LS and was kept at the default 80 min for SU. This difference was intentionally proportional to the observed longer GE in vivo for LS versus SU (29). Figure 9A shows the bioaccessibility data with this adjustment in GE. According to Figure 9B, TIM-1 FA cumulative bioaccessibility and AUC values for LS were not significantly different from all other treatments. With this adjustment in GE, TIM-1 FA cumulative bioaccessibility and AUC values for LS were not significantly different from all other treatments ( Figure 9B).

DISCUSSION
This study aimed to determine the in vitro lipolysis and FA bioaccessibility of four lipid emulsions formulated to contain partially solid or liquid oil droplets at 37 • C, and to either be susceptible to flocculation or remain dispersed in the acidic environment of the stomach. This is one of the first studies to control for the individual and combined effects of emulsion TAG crystallinity and acidic colloidal stability. The use of the TIM-1 digestion model offered a rare opportunity to directly compare the bioaccessibility findings from this leading digestion simulator with PPL results from a human study in which 15 healthy men consumed the same test emulsions, namely SS, SU, LS, and LU (29), and to explore strategies to modify the in vitro digestion protocol toward the aim of physiological relevance.

Differences Based on Triacylglycerol Crystallinity
The above results support that emulsion droplet crystallinity attenuates in vitro intestinal lipolysis, evidenced by the lower lipolysis of the two solid lipid emulsions (SU and SS) compared to LS. The lower bioaccessibility for SS compared to LS, specifically, provides the strongest indication that SFC was predominantly responsible for the differences since these emulsions were both stable during gastric digestion. Similar observations have been reported showing that acid-stable emulsions formulated with canola oil had higher TAG hydrolysis compared with those containing crystalline TAG (based on canola stearin, SFC ∼12.4%) (15). For example, Guo et al. observed lower lipolysis for emulsions that contained crystalline TAG (13). In that case, the lipolysis rate was proportional to blend SFC at 37 • C, with the highest and lowest rates observed for emulsions containing  (29)] and correlation analyses between TIM-1 6-h bioaccessible FFA AUC (g/L*min, n ≥ 3) and human study 6-h plasma TAG AUC (mmol/L*min, n = 15) (B) and between TIM-1 bioaccessible FFA (g/L, n ≥ 3) and human study plasma TAG data over 6 h (C). Error bars represent SEM. LS, liquid lipid-acid stable emulsifier; LU, liquid lipid-acid unstable emulsifier; SS, solid lipid-acid stable emulsifier; SU, solid lipid-acid unstable emulsifier; TAG, triacylglycerol; FFA, free fatty acid; AUC, area under the curve.
the liquid oil and hardstock, respectively (13). Similarly, Day et al. observed higher in vitro lipolysis with emulsions containing liquid canola oil stabilized with both 1% sodium caseinate and 0.25% monoglyceride compared to an emulsion containing partially crystalline TAG (6% hydrogenated vegetable oil and 14% canola oil) (43). In the same report, a significant 2-h increase in human whole blood TAG was observed for the liquid state system, compared with a more modest, non-significant increase for the emulsion with crystalline TAG. In contrast, Wan et al. recently reported that 20% palm stearin versus palm olein emulsions stabilized with sodium caseinate had a significantly greater extent of FA release at the end of a 2-h static in vitro digestion (44). According to the authors, the enhanced lipolysis for PS was attributed to the penetration of solid fat crystals at the oil-water interface, allowing greater lipase access and easier removal of digestion products (44). However, this study also with palm-based lipids and using a relatively more complex in vitro digestion method supports the conclusion that emulsion TAG crystallinity generally has an attenuating influence on digestibility.

Differences Based on Emulsifier Acid Stability
Another important bioaccessibility observation is that emulsion acid stability impacted digestibility in the absence of TAG crystallinity. This was evidenced by the higher bioaccessibility for LS compared to LU which we speculate is attributed to LS's higher interfacial area upon reaching the duodenal phase and lower digestate viscosity. The finding agrees with a human study where an acid-stable emulsion showed higher postprandial-labeled lipid chylomicrons compared to acid-unstable emulsions (25). In the current work, exposure to SGF led to increases in particle size (but not phase separation) for LU, while LS was unchanged. Therefore, LU is expected to have relatively larger particles and a correspondingly lower interfacial area for lipolysis when it enters the duodenal compartment (12). In vitro, (45) in vivo animal, (46) and human (47) studies confirm that a smaller particle size is correlated with higher lipolysis.
Beyond particle size, the composition and nature of the interface affect the ease of lipase binding and hydrolysis (12); thus, Span 60 and Tween 80 may affect lipase activity differently. Although palm olein is completely a liquid at body temperature (i.e., melting point ∼28 • C) (48), small amounts of crystallinity were observed for LS and LU, related to the emulsifier FA tails at the interface. Tween 80 contains predominantly oleic acid, while Span 60 contains equal proportions of palmitic and stearic acids which may interact differently with the droplet TAG. We previously observed the presence of a crystalline shell for Span 60 palm stearin and canola oil emulsions (19), which may contribute to the differences in lipase binding and microstructural changes during digestion. Otherwise, LU and LS possessed similar properties initially, yet TIM-1 FA bioaccessibility was greater for LS than LU, supporting the conclusion that, in the absence of TAG crystallinity, resistance to acidic flocculation led to enhanced FA bioaccessibility.

Differences Based on the Combined Effects of Triacylglycerol Crystallinity and Emulsifier Acid Stability
It is not surprising that different lipolysis trends are observed for liquid versus solid TAGs depending on the emulsifier present. The differences in digestibility between liquid-and solid-state droplets are attributed to restricted lipase access when less mobile crystalline TAG is present at an emulsion interface (15). However, direct evidence of these differences is rare, given that flocculation and partial coalescence can occur in the presence of solid fat. For example, within the acid-unstable emulsions (LU and SU), bioaccessibility over time did not differ significantly, although LU had a 15% higher cumulative FFA release at the end of the 6-h digestion compared to SU. In the previous studies, liquid oil-in-water emulsions with acid-unstable emulsifiers were subsequently redispersed, while those containing crystalline TAG did not (19,28). In this study, SU and LU (20) were susceptible to acid-induced colloidal changes, and when the digestate pH was raised to 7 at the start of the duodenal phase, partial redispersal occurred for LU, but SU did not redisperse (data not shown). Comparing SS (resisted acidic flocculation) and SU (partial coalescence and phase separation in the gastric phase) indicates minimal to no differences in droplet digestibility based on TAG crystallinity. This is somewhat surprising since SU presumably had a lower interfacial area available for lipolysis, but conceivably this factor is less significant for crystalline TAG.
Overall, based on the TIM-1 results, both TAG crystallinity (LS > SS) and acid instability (LS > LU) have the potential to attenuate lipolysis independently. Nonetheless, the combination of crystalline TAGs and acid-unstable emulsifier (SU) resulted in the greatest gastric instability (i.e., complete separation of the water and lipid phases into clumps which resisted redispersion) and the trend toward the lowest bioaccessibility compared to all other emulsions.

TIM-1 and Human Data Comparison
The positive correlation between human postprandial TAG concentration and the TIM-1 FFA values is not surprising given that both TIM-1 FFA values are strongly time-dependent. However, it is very important that the overall trends from the TIM-1 and human studies point to different conclusions about the role of TAG crystallinity and emulsion acid stability. In vivo, no significant differences were observed in TAG iAUC (as an indicator of overall TAG absorption), but TIM-1 bioaccessibility was higher for LS. The trends in TIM-1 AUC values based on FFA concentration also do not agree with those based on the human postprandial TAG AUC. Therefore, the human study results do not concur with the observed higher TIM-1 bioaccessibility for LS. However, both in vitro and in vivo models suggest that acidic flocculation in the presence of TAG crystallinity has an attenuating influence. The TIM-1 FFA AUC value for SU was 50, 26, and 20% lower than LS, SS, and LU, respectively, but only LS was statistically different from the other treatments. As previously reported, human PPL AUC TAG data for SU trended to be 29, 32, and 22% lower compared to LS, SS, and LU, respectively, with no appreciable differences between LS and SS or SU (29). These PPL differences are of a similar magnitude as where statistical significance was previously observed using a very similar human study design (20), pointing to implications of nuanced differences in emulsion physical properties for PPL. This is especially exciting given the wide-ranging implication PPL has for metabolism, e.g., CVD risk, satiety, inflammation related to lipopolysaccharide absorption, beta-oxidation, and interactions with gut microbiota.
There are many reasons why the results from in vitro and in vivo methods may not align precisely. In this case, differences in GI transit may help to explain the apparent discrepancies. GE is controlled by a complex feedback mechanism via gastrointestinal hormones (49), which, along with antral contraction, can be influenced by food properties (37). GE is a major factor that controls the rate of lipolysis in vivo (14). In the human study, ultrasonography confirmed the differences in gastric structure and GE rate between the emulsions (29). Thus, we exploited this in vivo data to explore whether adjusting the TIM-1 GE rate would achieve better alignment between the in vitro and in vivo findings.

TIM-1 Free Fatty Acids Bioaccessibility With Adjusted Gastric Emptying
According to ultrasound imaging, the acid-stable emulsions (LS and SS) remained dispersed in the stomach, as intended, and had significantly faster GE than SU and LU, which extensively flocculated and phase-separated in the stomach (29). In vivo GE half times were significantly higher for the acid-stable emulsions (∼300 and 262 min for LS and SS versus ∼184 and 190 min for LU and SU) (29). As above, the TIM-1 applies a pre-set GE rate based on a half time of 80 min (TNO, Zeist, Netherlands) (50) and lacks feedback mechanisms to adjust motility and meal transit. As such, the TIM-1 experiments may have limited ability to detect digestibility differences that occur in vivo based on differences in gastric structuring and subsequent GE. To explore this, a second set of TIM-1 experiments was conducted, taking into consideration the in vivo GE rate differences. When the TIM-1 GE T 1/2 was adjusted for LS, its results no longer differed from the other three emulsions, and the results for SU remained lower than all other emulsions as observed in vivo. This confirms that the GE rate is a plausible main explanation for the observed lack of precise alignment between the in vitro TIM-1 and in vivo human results. This finding urges caution in comparing the results for meals where gastric structures and emptying are expected to differ. There are reports of an agreement between TIM-1 bioaccessibility and postprandial TAG response (35,36,51), and differences in the alignment of in vitro and TIM-1 findings may relate to differences in the form of lipids being tested (i.e., emulsified or not and SFC) and the degree of associated structural changes in the stomach. In this case, when GE differences were accounted for, TIM-1 bioaccessibility more closely reflected the outcomes observed in vivo. Effectively, this refinement in in vitro transit corrected for an apparent major augmentation of lipid bioaccessibility for LS. In vivo, the lipid phase of the LS (and SS) emulsions emptied the stomach earlier than the separated lipids from SU and LU, triggering the ileal brake which led to a decrease in gastric motility (29), altering lipid absorption kinetics in a way that the TIM-1 cannot mimic. Incidentally, although PPL did not differ significantly between the emulsions (p > 0.05), substantial differences were observed in satiety hormones and participant ratings.
The improved in vitro-in vivo correlation with adjusted GE half times highlights the utility of non-invasive ultrasonography to obtain physiologically relevant GE rates to improve the predictability of in vitro digestion models. Other reasons in vitro results may not mirror human findings, which arise due to various pre-and post-absorptive processes (52). For example, while TIM-1 fractionates the absorbable (i.e., bioaccessible) lipids from the digestate, it cannot account for potential systemic differences in in vivo clearance of absorbed TAG. Moreover, both the in vitro and the in vivo results point to the need for longer experimental protocol postprandial TAG values since the two liquid emulsions had not returned to baseline by 6 h, and TIM-1 lipolysis was incomplete for all of the emulsions at that timepoint.

CONCLUSION
This study investigated the individual and combined effects of TAG physical state and colloidal acid stability of palmbased oil-in-water emulsions on in vitro lipid bioaccessibility using a computer-simulated dynamic digestion model TIM-1. The findings were compared to an acute postprandial TAG study in which healthy men consumed the same emulsions. Overall, the combination of an acid-stable emulsifier and liquid oil (i.e., the LS emulsion) led to the highest TIM-1 digestibility, although there were no differences in the shifted logistic model parameters. The trends observed based on AUC values from the in vitro and in vivo methods did not concur. Specifically, TIM-1 did not indicate the same treatment differences observed in vivo. It was hypothesized that this was because of in vivo GE differences between the emulsions. Indeed, when the TIM-1 GE rate was adjusted to reflect the differences in emptying observed in vivo, the in vitro TIM-1 FFA response more closely resembled the human lipemic response, with the combination of solid lipid and acid-unstable emulsifier leading to the lowest digestibility both in vivo and in vitro. This provides insight into the importance of considering gastric meal structures and corresponding differences in GE in future in vitro digestion studies. Overall, emulsion TAG properties can be tailored for different gastrointestinal behaviors and in vitro and in vivo methods should be integrated for a comprehensive understanding of how food properties influence metabolic response.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the University of Guelph Human Research Ethics Board. The participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
SH and AW designed the research and analyzed the data. SH, RC, YL, NR, ZZ, and PN conducted the research. SH, MR, and AW interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.