# Effect of consumption temperature of whole milk on in vitro gastric digestion using a biomimetic digestion simulator

Jiajun Feng, Jeehyun Lee, Vincent Mathieu, Yann Le Gouar, Romain Jeantet, Didier Dupont, Steven Le Feunteun

► To cite this version:

Jiajun Feng, Jeehyun Lee, Vincent Mathieu, Yann Le Gouar, Romain Jeantet, et al. Effect of consumption temperature of whole milk on in vitro gastric digestion using a biomimetic digestion simulator. Food Research International, 2025, 223 part 2 (janvier 2026), pp.117869. 10.1016/j.foodres.2025.117869. hal-05372540

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https://institut-agro-rennes-angers.hal.science/hal-05372540v1

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# Effect of consumption temperature of whole milk on *in vitro* gastric digestion using a biomimetic digestion simulator

Jiajun Feng, Jeehyun Lee, Vincent Mathieu, Yann Le Gouar, Romain Jeantet, Didier Dupont, Steven Le Feunteun*

INRAE, L'Institut Agro Rennes-Angers, STLO, 35042 Rennes, France

## ARTICLE INFO

### Keywords:

*In vitro* digestion
Milk
Coagulation
Temperature
Stomach
Gastric emptying

## ABSTRACT

This study evaluates the effect of consumption temperature on the gastric digestion behaviour of whole milk using a dynamic *in vitro* model (NERDTM, computer-controlled biomimetic system). Human intragastric temperature profiles in response to cold, warm, and hot beverages were accurately reproduced to mimic the conditions encountered after consumption of milk at 4 °C, 37 °C and 60 °C. For comparison, control experiments were performed with milk at 37 °C without any pepsin addition. Results show that milk coagulated more rapidly within the stomach when consumed at 37 °C or 60 °C compared to 4 °C and 37 °C without pepsin. Moreover, the gastric emptying rate of milk proteins in the presence of pepsin was slower with milks at 37 °C and 60 °C compared with the milk at 4 °C. The hydrolysis of milk proteins also appeared to be temperature-dependant, with proteolysis increasing with higher milk temperatures towards the end of digestion. Regarding the fat fraction of milk, our data suggest that both hot (60 °C) and cold milk (4 °C) could plausibly increase the gastric emptying rate of milk fat, compared to a 37 °C milk, but further studies remain needed to verify this hypothesis. At the end of the experiments, the wet and dry masses of the residual gastric digesta was similar across the three conditions with pepsin, but significantly lower than in the absence of pepsin at 37 °C. This study enhances our understanding of how consumption temperature influences the kinetics of pepsin-induced milk coagulation and the subsequent gastric disintegration of dairy particles.

## 1. Introduction

Milk coagulates during gastric digestion due to the combined action of pepsin, the gastric protease, and the increased acidity of the stomach contents caused by gastric acid secretions. The kinetics of curd formation and its resulting structure are partly governed by milk processing steps. In particular, heat treatment affects the structure of milk proteins and their interaction with lipids, which could impact gastric emptying and overall digestion kinetics. These effects have been extensively investigated and well characterized both *in vivo* (Ahlborn et al., 2023; Barbé et al., 2013; Lacroix et al., 2008) and *in vitro* (Li et al., 2022; Mulet-Cabero et al., 2019). For instance, in a human study, Lacroix et al. (2008) found that the postprandial protein digestion kinetics were enhanced after consumption of ultra-high temperature (UHT) skimmed milk compared to pasteurized and unheated milks. Ahlborn et al. (2023) reported that heat treatment and homogenization both influence the gastric emptying of proteins in pigs in the following order: UHT

homogenized milk > pasteurized homogenized milk > pasteurized non-homogenized milk = raw milk. Using an *in vitro* approach, Ye et al. (2016a) also showed that heat-induced aggregation can hinder gastric curd formation by limiting the capacity of proteins to form a network.

In addition to processing steps, consumption practices may also affect milk digestion. In particular, there is a lack of research concerning the effect of consumption temperature on the behaviour of milk within the gastrointestinal tract, considering that milk coagulation partly depends on temperature and that intragastric temperature is significantly influenced by the temperature of the ingested meal. Indeed, different studies have reported that the intragastric temperature can drop to as low as 21 °C within 20–30 min of consuming 350–400 mL of coffee or orange juice at 4 °C, before the temperature returns to 37 °C (McArthur & Feldman, 1989; Sun et al., 1988). Conversely, consuming these beverages at high temperatures (58 °C for coffee and 50 °C for orange juice) can result in an initial intragastric temperature of approximately 44 °C, returning to 37 °C within 20–30 min (McArthur & Feldman, 1989; Sun

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: steven.le-feunteun@inrae.fr (S. Le Feunteun).</footnote>
et al., 1988). Moreover, variations in the intragastric temperature necessarily affect pepsin activity (Sholeh et al., 2019; Sun et al., 2011), with possible consequences on both the milk coagulation kinetics and the structure of the curd formed (Ahlborn et al., 2023; Horne & Lucey, 2014; Nájera et al., 2003).

In addition, consumption temperature may further affect gastric emptying rates. Fujihira et al. (2024) recently reported that elderly individuals experienced a faster gastric emptying when ingesting a hot protein-containing drink at either 37 °C or 60 °C than the same drink at 4 °C. Mishima et al. (2009) also found that consuming a hot meal (60 °C) significantly accelerated gastric emptying when compared to a meal at 4 °C, using a ¹³C-labelled acetate breath test. However, Troncon and Iazigi (1988), using quantitative scintigraphy techniques, observed that both cold (4 °C) and hot (55 °C) liquid meals had an inhibitory effect on gastric emptying, with a significant delay in gastric emptying only observed with the hot meal. Overall, it seems plausible that consuming warm liquid meals may promote faster gastric emptying than cold ones. However, the literature remains conflicting to date, probably because most studies rely on a limited number of participants and use different methods to assess gastric emptying.

Although the effect of consumption temperature on the intragastric coagulation of milk proteins and on gastric emptying may be significant, relevant in vivo studies remain limited. Yang et al. (2023) examined the differences in κ-casein hydrolysis in skimmed milk at various consumption temperatures using dynamic in vitro digestion. While the initial rate of κ-casein hydrolysis by pepsin was higher in 50 °C milk than in 37 °C and 4 °C milk, the overall hydrolysis rate of κ-casein was lowest at this temperature by the end of digestion. This study also revealed that 4 °C milk coagulated more slowly under gastric conditions resulting in easier digestion, as the curd formed was looser and softer compared to 37 °C or 50 °C. Fitzpatrick et al. (2024) recently used Magnetic Resonance Imaging (MRI) to evaluate the effect of consumption temperature of whole milk (4 °C, 37 °C and 60 °C) on its behaviour during semi-dynamic in vitro gastric digestion. The study also found that consuming cold milk delays intragastric coagulation. Furthermore, they showed that the dairy particles formed after consuming hot milk tended to remain at the bottom of the digestive vessel, whereas particles tended to float to the top after consuming cold milk. These differences suggest that the temperature at which milk is consumed may affect gastric emptying rates of lipids and potentially affect the sensation of fullness. However, the latter study used manual stepwise procedures to perform gastric mixing and emptying. More studies are thus needed using gastric mixing and emptying conditions that are more physiologically relevant.

The objective of this article is thus to further clarify and explore how the temperature profile of whole milk entering the stomach can impact the kinetics of gastric digestion and emptying. To achieve this, the study uses an advanced biomimetic dynamic digestion simulator that can reproduce both the biochemical and biomechanical processes involved in gastric digestion: the NERDT™ system. This system has been previously applied in different studies to investigate the fate of milk in the stomach. For example, it has been used to study the casein-to-whey protein ratio on gastric emptying, proteolysis and peptidome profile of fermented milk (Zhang, Duan, et al., 2023). In this case, the simulator was programmed to simulate the gastric conditions found in preschool children. Overall, it showed no impact of the casein-to-whey ratio on the gastric emptying rate but a significant effect on protein digestibility with higher digestibility observed with lower casein-to-whey ratio that was attributed to the formation of less protein aggregates and a softer gel network structure when whey proteins were more abundant. The NERDT™ system was also used to compare the *in vitro* digestion of skim milk when programmed with parameters simulating the young adult and elderly gastric conditions (Zhang, Iqbal, et al., 2023). Results showed that gastric emptying was slowed down and proteolysis was impaired by ageing. Again, the differences observed were attributed to structural differences in the stomach cavity with the formation of larger clusters and protein aggregates under elderly digestion. The NERDT™

system was also used to assess the effect of heat treatment on the *in vitro* digestion of yak milk (Liu et al., 2022). More clots and a slower gastric emptying rate were observed in the stomach when the milk was raw or autoclaved than when it was pasteurized or microwaved. Digestibility of raw milk was significantly lower than that of the heat-treated milks. Finally, the NERDT™ was used to investigate the gastric digestion of skimmed milk (Feng et al., 2024). The results showed that the NERDT™ was able to reproduce the kinetics of gastric acidification and emptying observed in humans, with a sieving effect that naturally led to a delayed emptying of caseins. Pepsin was shown to play a key role on the gastric coagulation of caseins and subsequent hydrolysis and emptying of dairy particles.

In the present study, the NERDT™ was used to investigate the effect of consumption temperature of whole milk on gastric digestion considering three temperature profiles, in line with milk consumption practices. Gastric digestion was then monitored by tracking the mass, structure, and biochemical properties of samples collected throughout the *in vitro* digestion experiments.

## 2. Material and methods

### 2.1. Chemicals and reagents

A pasteurized whole milk powder (WMP) containing 25 % protein, 26 % fat, and 36 % lactose (w/w) was provided by Sill Entreprises, France. The morning before use, WMP was reconstituted in osmosis water at 14 % (w/v) under magnetic stirring at room temperature for 1 h minimum. Rehydrated full-fat milk contained 3.9 % protein, 4.1 % lipid, and 5.6 % carbohydrates. Porcine pepsin (P6887-5G), with an enzymatic activity of 3200 U/mg, Pepstatin A (P5318-25MG), and orthophthalaldehyde (P0657-5G) were purchased from Sigma-Aldrich, USA. Pepstatin A was dissolved in ethanol and stored at 4 °C prior to use. Milli-Q water was used. Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (USA) and were of standard analytical grade.

### 2.2. *Dynamic in vitro* gastric digestion

#### 2.2.1. Experimental conditions and devices

The NERDT™ system was used to conduct *in vitro* gastric digestion of milk at different consumption temperatures. Four experimental conditions were considered in order to simulate those encountered after consuming whole milk: (1) at 37 °C without pepsin (no-enzyme control condition), (2) at 37 °C with pepsin (temperature control condition), (3) at 4 °C with pepsin, and (4) at 60 °C with pepsin. Each condition was tested in triplicate.

As previously described in details elsewhere (Feng et al., 2024), the NERDT™ primarily consists of (i) a J-shaped silicone human stomach model, (ii) a pneumatically controlled driving device to mimic gastric peristaltic contractions, (iii) turntable and pyloric sieving devices to control gastric emptying, and (iv) secretion and temperature control systems. It was fitted with soft pH probes (Ohmega, MMS, The Netherlands) and a thermocouple (753–652, TC Direct, France) to monitor online gastric pH and temperature, respectively. A scale (PS 6100.X2, Radwag Balance, Poland) was used to continuously measure and record the mass of digested food leaving the stomach.

#### 2.2.2. Target values based on *in vivo* observations

The *in vivo* situation that was reproduced *in vitro* corresponds to the gastric digestion of 400 mL of whole milk by healthy human adults. To define a set of NERDT™ operating parameters that mimic this *in vivo* situation, the target kinetics or values of gastric emptying, gastric pH, and pepsin activity must first be identified.

Okabe et al. (2015) observed that the gastric emptying in healthy volunteers fed with 500 mL of whole milk decayed exponentially with a half-time ($t_{1/2}$) between 50 and 60 min. Mahé et al. (1992) reported the
same trend with a $t_{1/2}$ of 25 ± 7 min for 400 mL of skimmed milk. However, skimmed milk has a calorie content approximately half that of whole milk (Collard & McCormick, 2021), and the rate of gastric emptying can be considered as inversely proportional to its caloric density (Calbet & MacLean, 1997). Therefore, the target gastric emptying kinetics consisted of an exponential decay with a $t_{1/2}$ of 50 min. For gastric acidification, we used the target values of gastric pH obtained by Malagelada et al. (1979) for an ordinary homogenized meal in humans: pH 4.0 at about $0.5 \times t_{1/2}$ and pH 2.0 at about $t_{1/2}$. For pepsin addition, we used a target pepsin activity of 2000 U/mL after $t_{1/2}$. The NERDT™ parameters that enable all these targets to be reproduced fairly were finally obtained after some preliminary experiments and calculations, using a previously described mass balance model (Feng et al., 2024). This model assumes that the NERDT™ stomach behaves like a perfectly stirred reactor.

### 2.2.3. Experimental protocol and setting of NERDT™ parameters

The digestion protocol was similar to that previously described for the digestion of skimmed milk (Feng et al., 2024), in which the full protocol was described and justified in details, with few noticeable exceptions. Firstly, the kinetics of gastric emptying and acidification were about twice longer because the considered $t_{1/2}$ for whole milk was twice as long. Secondly, the kinetics of pepsin addition remained the same over time, resulting in a twofold increase when expressed as a function of gastric emptying. Thirdly, the temperature conditions were varied in order to mimic those when milk is consumed at 4 °C and 60 °C, as explained further in Section 2.2.4.

Briefly, both Simulated Salivary Fluid (SSF) and Simulated Gastric Fluid (SGF) were prepared using the electrolyte concentrations recommended in Brodkorb et al. (2019). SGF consisted of a mixture of simulated acid and pepsin fluids, with final HCl concentration of 0.16 M (Hersey & Sachs, 1995) and pepsin activity of 6770 U/mL. Due to the limited volume of the model stomach, the volume of the meal was scaled down by 2 compared to the *in vivo* conditions, meaning that 200 mL of rehydrated WMP was used for each *in vitro* experiment. Before starting an experiment, 12 mL of SGF pre-warmed at 37 °C and adjusted at pH 2 was introduced into the stomach model to mimic the fasted state, assuming an *in vivo* basal gastric fluid content of 24 mL (Lydon et al., 1999). The 200 mL of whole milk were mixed with 19 mL of pre-warmed SSF without amylase (pre-heated to 37 °C using a water bath), and this mixture was introduced into the NERDT™ stomach within 2 min. The NERDT™ system was programmed to perform three gastric contractions per minute with a pylorus opening size of 2.5 mm. The flow rates of the syringe pumps and the stepwise procedure used for the tilting angle are all provided in Table S1 of the Supplementary Material.

Digested samples were collected from after the pylorus at 8, 14.5, 27, 52, 77, 102, and 152 min of gastric digestion. These time points correspond to multiples (0.125, 0.25, 0.5, 1, 1.5, 2, and $3 \times$) of the target $t_{1/2}$ of 50 min, with an additional 2 min allowed for feeding. pH measurements, dry matter (DM) determinations, and confocal microscopy observations were performed immediately after the digesta had been emptied. Other samples were frozen after addition of Pepstatin A (0.5 mg/mL) pending further analysis.

### 2.2.4. Temperature control

The digestion simulator was fitted with two heaters to maintain an ambient air temperature of 37 °C around the stomach (Power: 100–300 W, MT-31015, Shenzhen Essens Technology Co., Ltd., China). During digestion, the temperature profile of the intragastric contents was therefore determined by the temperature of the milk prior to its introduction in the NERDT™. Different temperature profiles were considered, based on human studies reporting initial intragastric temperature of about 21 °C and 44 °C following the consumption of 350–400 mL of beverages at 4 °C and 50–60 °C, respectively, with a return to 37 °C within 20–30 min (McArthur & Feldman, 1989; Sun et al., 1988). In these studies, half or more of the consumed beverages had been emptied

from the stomach within 20–30 min, hence suggesting that the delay for a return at 37 °C should be longer for foods that empty at a lower rate, as for whole milk. This is why our experimental conditions were optimized to return to 37 °C within approximately 50 min using starting intragastric temperatures of about 21 °C and 44 °C for the cold (4 °C) and the hot (60 °C) milk, respectively. To reach these targets, the milk was either pre-cooled to 10 °C or pre-warmed to 52 °C prior to introduction into the NERDT™. In the following of this paper, these conditions will be referred to as ‘4 °C milk’ and ‘60 °C milk’ for the sake of clarity, even though milk was never brought to those temperatures in practice.

### 2.3. Dry matter

A Moisture Analyzer (HE73, Mettler Toledo SAS, Versailles, France) was used to measure the DM content of the digesta collected at the stomach exit. The digesta were homogenized with a digital T18 homogeniser (ULTRA-TURRAX®, IKA, Germany) at a stirring speed of 8000 rpm. Approximately 1 g of the sample was used for each measurement. At the end of each experiment, both the wet and dry masses of any remaining digesta in the stomach of the NERDT™ were also measured.

### 2.4. Protein concentration

The 4th-derivative UV method, as proposed by Lüthi-Peng and Puhan (1999), was used to determine the protein concentration in the samples collected at the exit of the stomach. Briefly, a buffer solution containing 6 M guanidine-HCl and 0.1 M sodium acetate was prepared and the pH was adjusted to 5.0. The samples were diluted with the buffer solution by a factor of approximately 100 and vortexed, before their UV spectra were measured from 260 nm to 320 nm using a UV spectrophotometer (SPECTR 0021, SAFAS Monaco) and 1 cm quartz cuvettes. The 4th-derivative spectra were computed using the spectrometer software (SP2000V7, SAFAS Monaco). The total protein concentration of each sample was then estimated from the characteristic peak of the 4th-derivative spectra, at around 284 nm, and pre-established calibration curves prepared with rehydrated solutions of the whole milk powder (0–1.5 g/L of proteins).

### 2.5. SDS-PAGE

SDS–PAGE was performed on the samples collected at the stomach exit using 4–12 % polyacrylamide NuPAGE Novex Bis–Tris 15-well precast gels (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The samples were loaded onto the gels at a dilution factor of 40. Mark 12 Unstained Standard (STD, Invitrogen, Thermo Fisher Scientific, USA) was used as a molecular weight marker. For comparison, a solution of porcine pepsin (P6887-5G, Sigma-Aldrich) prepared at 0.5 mg/mL was also loaded. The gels were stained with colloidal Coomassie Blue (Cat. #1610787, Bio-RAD, USA) and subsequently destained using a 10 % glacial acetic acid solution for 2 h. The gels were then scanned and analysed using the Image Scanner II (Amersham Biosciences, UK).

### 2.6. R-NH₂ concentration

The concentration of primary amines (R-NH₂) in the digesta collected at the exit of the stomach was quantified using the o-phthalaldehyde (OPA) method, following the protocol described by Lorieau et al. (2018). This protocol is based on the protocol of Church et al. (1983) for microplates. The sample was diluted (1:4) with sodium tetraborate buffer (50 mM). Then, 10 µL of the diluted sample was added to each well, followed by the addition of 200 µL of the OPA reagent. The mixtures were then incubated for 10 min at 37 °C, and the absorbance was measured at 340 nm using a microplate reader (Thermo Fisher Scientific Oy, 1510, Finland). Absorbance values were then converted into R-NH₂
concentrations using a calibration curve prepared with L-methionine standard solutions (0–2 mM).

## 2.7. Triacylglycerol concentration

The triacylglycerol (TAG) content of the samples collected from the stomach exit was analysed using a UV kit (Triglycerides FS, Lot 60113624, DiaSys, Germany). A TAG standard solution (1.5 mol/L) was used to create a series of standards with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 mol/L in water. The collected samples were vortexed and diluted with water. Aliquots of 10 µL (from the samples and stock standards) were then transferred to a 96-well plate. Next, 100 µL of the kit reagent was added to each well. The mixtures were incubated for 10 min at 37 °C, and the absorbance was measured at 500 nm using a microplate reader (Thermo Fisher Scientific Oy, 1510, Finland). The results were converted into molar concentration using a calibration curve established with the TAG standard solution (0–1.5 mol/L).

## 2.8. Cumulative quantity of proteins, R-NH₂ and TAG

The flux of proteins, R-NH₂ groups and TAG leaving the stomach of the NERDT™ was calculated according to Eq. (1):

$$ \Phi_{out}^{i} = C_{out}^{i} \cdot \Phi_{out} \qquad (1) $$

with i standing for proteins, R-NH₂ or TAG, and where $\Phi_{out}^{i}$ is the flux of proteins, R-NH₂ or TAG exiting the stomach (g/min or mol/min). $C_{out}^{i}$ is the corresponding concentration (g/mL or mol/mL) in the material leaving the stomach, and $\Phi_{out}$ (mL/min) is the flux of material exiting the stomach. The values taken by $\Phi_{out}$ during digestion were predicted using a previously described mathematical model (Feng et al., 2024), which assumes that the stomach behaves as perfectly stirred reactor.

The cumulative quantity of proteins, R-NH₂ or TAG ($m_{out}^{i}$, g or mol) was then calculated using linear interpolation between experimental values, according to Eq. (2):

$$ m_{out}^{i} = \rho \cdot \sum_{j=2}^{n} \Phi_{out}^{i} \cdot \Delta t_{j} \qquad (2) $$

where $\rho$ is the density of the material emptied from the stomach, taken as 1 g/mL, and j of $\Delta t_{j}$ (time period, min) is varying from 2 to n.

## 2.9. Confocal microscopy

A confocal laser scanning microscope (CLSM, LSM880, Zeiss, Germany) was used to examine immediately the microstructure of the digesta collected from the stomach exit and the residual content in the stomach at the end of the experiment. Nile Red and Fast Green were used to stain fat (argon laser operating at 488 nm) and proteins (He—Ne laser operating at 633 nm), respectively, as follows: 200 µL of sample was transferred into an Eppendorf tube and labelled with 18 µL of 0.1 % (w/v) Nile Red and 6 µL of 1.0 % (w/v) Fast Green for 10 min at room temperature. A fraction of the labelled sample (25 µL) was then placed on a microscope slide (SWIFT BS50, Microscope Slides Kit, USA), covered with a coverslip, and observed at 10× magnification. Several image acquisitions were performed in different locations within the sample. One image per time point, considered as representative of the set of images, was later selected to be included in the paper.

## 2.10. Statistical analysis

Three datasets were analysed using R software (version 4.4). The first dataset comprised one qualitative explanatory variable (condition), one quantitative explanatory variable (time), four response variables (DM percentages, protein concentration, R-NH₂ concentration, and TAG concentration) as well as 96 individual measures. The second dataset

comprised one qualitative explanatory variable (condition), one quantitative explanatory variable (time), three response variables (cumulative quantities of emptied proteins, R-NH₂ and TAG) and 468 individual measures. The third dataset comprised one qualitative explanatory variable (condition), two response variables (wet and dry masses of the residual digesta in the stomach at the end of the experiments) and 12 individual measures. The ‘LinearModel’ function (Lê et al., 2008) was used to fit a linear model to each dataset. A one-way analysis of variance (ANOVA) was carried out and a Fisher’s exact test was performed. Differences were considered as statistically significant when the p-value was strictly below 0.05. Post hoc tests were then performed using the ‘meansComp’ function for datasets 1 and 3, and at t = 32, 52 and 152 min for dataset 2.

# 3. Results

## 3.1. Gastric temperature, pH and emptying

Fig. 1A presents the evolution of the intragastric temperature under the four conditions investigated: gastric digestion of whole milk at 37 °C without pepsin, and at 4 °C, 37 °C and 60 °C with pepsin. These temperature profiles fairly reproduced the target kinetics, with initial intragastric temperatures of about 20 °C, 37 °C and 45 °C which mimic the consumption temperatures of 4 °C, 37 °C, and 60 °C, respectively. These temperatures have previously been reported for cold, warm and hot liquid foods in vivo (McArthur & Feldman, 1989; Sun et al., 1988). The present study’s profiles are similar to those used by Fitzpatrick et al. (2024), though the return to 37 °C is slower (at around 50 min in the present study) to account for the larger milk volume, and thus the higher calorie content considered in the present study.

The pH evolution of emptied digesta is shown in Fig. 1B. The gastric pH decreased continuously from approximately 6.7 down to 2 within 80 min, and to 1.2 within 150 min, in a manner comparable to that observed in humans consuming liquid meals (Malagelada et al., 1976). No significant difference was observed in the pH evolution of emptied digesta at different consumption temperatures.

Fig. 1C and D show the cumulative wet mass of material emptied from the stomach and the evolution of gastric content volume, respectively. In Fig. 1C, the solid line shows the predictions obtained by considering the effects of simple dilution through mathematical modelling (Feng et al., 2024). In Fig. 1D, the solid line represents the target gastric volume kinetics, which corresponds to an exponential decay with a $t_{1/2}$ of 50 min starting after the 2 min of feeding. Similar gastric emptying trends were observed in Fig. 1C under different temperature conditions, closely matching the mathematical predictions. The results in Fig. 1D were calculated by subtracting the cumulative mass of emptied digesta (Fig. 1C) from the cumulative mass of material added into the stomach as predicted by mass balance modelling (milk and secretions). These results show that the gastric volume increased rapidly during feeding, before decreasing consistently with the target exponential decay. The experimentally determined $t_{1/2}$, based on the intragastric volume content, was 54.3 ± 1.5, 52.7 ± 3.9, 47 ± 2.8 and 48.3 ± 2.8 min for the milk at 37 °C without pepsin, and at 4 °C, 37 °C and 60 °C with pepsin, respectively. All these results remain very close to the target value of 52 min (i.e. $t_{1/2}$ plus the 2 min of feeding).

## 3.2. Dry matter content of the emptied digesta

Fig. 2A and B show the evolution of the DM content and the visual appearance of the digesta collected at the stomach exit. The mathematical model was also used to predict the dynamic evolution of the DM percentages based on two assumptions: (1) the NERDT™ stomach behaves as a perfectly stirred reactor; and (2) casein particles cannot exit the stomach. These hypotheses correspond to the solid and dashed lines, respectively. The evolution of the DM content during digestion was not significantly affected by the milk consumption temperature (p = 0.12).
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Evolution of the intragastric temperature (A), the pH of emptied digesta (B), the cumulative mass of digesta emptied from the stomach (C), and the volume of the gastric content (D) under the four conditions: milk at 37 °C without pepsin (▲), and at 4 °C (♦), 37 °C (●) and 60 °C (■) with pepsin. Values are means ± SD over 3 replicates. In subplot (C), the solid line represents the values predicted by modelling. In subplot (D), the solid line represents the target kinetics based on *in vivo* data.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Evolution of the dry matter content in the emptied digesta (A), their visual appearances (B), and the wet and dry masses of the residual digesta in the stomach at the end of the experiments (C) under the four conditions: milk at 37 °C without pepsin (▲), and at 4 °C (♦), 37 °C (●) and 60 °C (■) with pepsin. Values are means ± SD over 3 replicates. In subplot (A), the solid line represents the values predicted by modelling under the assumption that the stomach behaves as a perfectly stirred reactor. The dashed line further assumes that caseins do not empty from the stomach. In subplot (C), different letters indicate significant differences among the conditions for the wet (p = 0.02) and dry masses (p = 0.001).</figcaption></figure>

At $t = 8$ min, the DM content of the emptied digesta for the milk at 4 °C and for the milk at 37 °C, both with and without pepsin, was close to the model predictions accounting for simple dilution effects. The DM content of the milk at 60 °C was closer to the model predictions assuming that caseins had already coagulated and could not exit the stomach. The visual appearance of the samples collected at $t = 8$ min also suggests that casein coagulation started to take place around that time for the milk at 60 °C, as these samples were the only ones to rapidly phase separate after sample collection (Fig. 2B). Similar results were obtained at 14.5 min. At 27 min, the DM content of the emptied digesta was very similar regardless of the consumption temperature. This indicates that caseins
were coagulated under all conditions by this time. This interpretation was confirmed by the visual appearance of the collected samples, which were all translucent with a small amount of white precipitate characteristic of casein particles (Fig. 2B). From 52 to 152 min, the DM content of the emptied digesta increased gradually, except for the 37 °C condition without pepsin (Fig. 2A). This increase is due to the gastric emptying of casein particles that were previously retained within the stomach because of the combined action of pepsin and of the gastric biomechanics. This is also consistent with the increasing turbidity of the sample supernatants (Fig. 2B).

Fig. 2C shows the wet and dry masses of gastric digesta remaining in the stomach at the end of the experiments ($t = 152$ min), which consisted of finely ground chyme with no large particles under all conditions (Fig. 2B). Both the wet ($p = 0.02$) and dry masses ($p = 0.001$) of the residual material varied significantly across conditions. The wet mass of the residual digesta for the milk at 37 °C without pepsin was significantly higher than for milk at 37 °C and 60 °C with pepsin, though not for the milk at 4 °C with pepsin. The dry mass of the residual digesta for the milk at 37 °C without pepsin was significantly higher than for milk at 4 °C and 60 °C with pepsin, though not for the milk at 37 °C with pepsin. Considering that the dry mass of the meal was approximately 27.5 g, the dry mass of the residual digesta represented 24.4 ± 1.3 % of the meal dry mass for the 37 °C milk without pepsin, and 11.3 ± 3.4 %, 20.4 ± 2.6 %, and 16.2 ± 2.4 % for the milk at 4 °C, 37 °C, and 60 °C with pepsin, respectively.

### 3.3. Gastric emptying of proteins and R-NH₂ groups

Fig. 3A and B show the evolution of the protein and R-NH₂

concentrations in the emptied digesta, during which no significant differences were observed as a function of the milk consumption temperature ($p = 0.10$ and $p = 0.12$, respectively). The solid and dashed lines in Fig. 3A represent the predicted evolution of protein concentrations, based on the same assumptions as previously described. Overall, except for the milk at 37 °C without pepsin, the concentrations of proteins and R-NH₂ initially decreased and then increased, mirroring the trend observed in the DM content of the emptied digesta. Therefore, these results further confirm that the intragastric coagulation of milk proteins always occurred within the first ~25 min of digestion, and that the resulting particles formed were emptied later, after enzymatic hydrolysis and biomechanical grinding. Although the final protein and R-NH₂ concentrations were consistently higher in the presence of pepsin than the ones determined with the milk at 37 °C without pepsin, the associated standard deviations were large, which is why the observed differences were not statistically significant.

Fig. 3C shows the cumulative quantity of proteins emptied from the stomach, as estimated using Eq. 2. This variable was significantly affected by the milk consumption temperature ($p = 0.029$), with a higher final quantity of emptied protein for the milk at 4 °C with pepsin than for the milk at 60 °C. Considering that the total protein content of the milk was approximately 7.9 g, the estimated mass percentage of residual proteins in the stomach contents by the end of the experiment was 35.8 ± 8.4 %, 11.7 ± 2.3 %, 28.6 ± 8.0 % and 23.7 ± 4.4 % of the initial milk protein for the milk at 37 °C without pepsin, and the milks at 4 °C, 37 °C and 60 °C with pepsin, respectively. While these percentages are in the same order of magnitude to those measured for the residual dry mass of the meal content (Fig. 2C), they are all slightly higher on average, confirming an increased gastric retention of milk proteins over

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Evolution of the protein concentration (A), R-NH₂ concentration (B), cumulative quantity of proteins (C), and cumulative quantity of R-NH₂ (D) in the emptied digesta under the four conditions: milk at 37 °C without pepsin (▲), and at 4 °C (◆), 37 °C (●) and 60 °C (■) with pepsin. Values are means ± SD over 3 replicates. In subplot (A), the solid line represents the values predicted by modelling under the assumption that the stomach behaves as a perfectly stirred reactor. The dashed line further assumes that caseins do not empty from the stomach. In subplot (C), different letters indicate significant differences among the conditions at $t = 152$ min ($p = 0.03$).</figcaption></figure>
other milk constituents. This can also be highlighted trough the half-gastric emptying time of proteins, which was reached after about 80 min, 60 min, 88 min and 92 min with the milk at 37 °C without pepsin, and the milks at 4 °C, 37 °C and 60 °C with pepsin, respectively.

Fig. 3D presents the cumulative quantity of R-NH₂ emptied from the stomach. The observed trends were similar to those of cumulative quantity of proteins (Fig. 3C) as even unhydrolyzed proteins contain a significant quantity of primary amines. At the end of the experiments, the cumulative quantity of R-NH₂ appeared higher for the milk at 4 °C with pepsin, and lower for the milk at 37 °C without pepsin; however, these differences were not statistically significant.

### 3.4. SDS-PAGE protein patterns

Fig. 4 shows the protein profiles of the samples collected at the stomach exit analysed by SDS-PAGE under the different conditions. The intensity of the casein bands in the emptied digesta appeared to be almost saturated at *t* = 8 min except for the milk at 60 °C. From *t* = 8 min to 27 min, the intensity of the casein bands gradually decreased in all conditions, and had almost disappeared by this time for the milk at 60 °C. From *t* = 52 min to 102 min, the intensity of the casein bands increased in all conditions, likely due to the disintegration and emptying of the remaining particles. From *t* = 102 min to 152 min, the intensity of the casein bands decreased at 4 °C and 60 °C, remained constant at 37 °C and increased at 37 °C in the absence of pepsin. The major whey protein, β-Lactoglobulin (β-LG), persisted throughout the experiments for the conditions with and without pepsin consistently with previous reports (Kopf-Bolanz et al., 2014). Interestingly, small peptides were also observed from *t* = 52 min in the presence of pepsin, as revealed by the appearance of a smear at molecular weight lower than 6 kDa (in particular for milks at 37 °C and 60 °C), hence demonstrating the proteolytic effect of pepsin. Additionally, peptides smaller than 3.5 kDa could even be detected for the milk at 60 °C milk for *t* ≥ 77 min.

### 3.5. Gastric emptying of milk fat

Fig. 5A shows the evolution of the TAG concentration in the emptied digesta. The TAG concentration decreased rapidly before gradually increasing in the presence of pepsin only. While the difference was not statistically significant (*p* = 0.13) across the entire dataset, the absence of pepsin at 37 °C resulted in significantly lower values than the other

<figure><img src="image_6.png" /><figcaption>Fig. 5. Evolution of the triacylglycerol (TAG) concentration (A) and of the cumulative quantity of TAG (B) in the emptied digesta under the four conditions: milk at 37 °C without pepsin (▲), and at 4 °C (◆), 37 °C (●) and 60 °C (■) with pepsin. Values are means ± SD over 3 replicates. In subplot (A), different letters indicate significant differences among the conditions at *t* = 77 min (*p* = 0.014) and *t* = 152 min (*p* = 0.022).</figcaption></figure>

conditions at both 77 min (*p* = 0.014) and 152 min (*p* = 0.022). Furthermore, this tendency is very similar to those previously described for the DM content, protein and R-NH₂ concentrations (Fig. 2A, Fig. 3A and Fig. 3B) and may therefore be attributed to the same mechanisms. Fig. 5B shows the cumulative quantity of TAG emptied from the

<figure><img src="image_7.png" /><figcaption>Fig. 4. SDS-PAGE profiles of the emptied digesta under the four conditions: milk at 37 °C without pepsin (A), and at 4 °C (B), 37 °C (C) and 60 °C (D) with pepsin. The red dashed lines indicate band cutting and repositioning within the same gel because of some inadvertent inversions during sample loadings on the gels. STD stands for the 'standard' solution containing proteins of known molecular weights, and WMP stands for the rehydrated 'whole milk powder' representing milk before digestion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
stomach, which evolved similarly across the different conditions during the first 20–30 min. After this time, the cumulative quantity of TAG for the milks at 4 °C and 60 °C with pepsin seemed to increase more rapidly than that for the milk at 37 °C with or without pepsin, although there was no significant difference in the end result (p = 0.31). Considering the milk’s TAG content was of about 11 mmol, the results of Fig. 5B would lead to a TAG half-gastric emptying time of about 85 min for the milks at 4 °C and 60 °C with pepsin, and a value greater than 152 min for the milk at 37 °C with and without pepsin. By the end of the experiment, the estimated mass percentage of residual TAG in the stomach content was 56.8 ± 10.6 %, 20.6 ± 30.0 %, 53.1 ± 11.8 %, and 25.1 ± 29.5 % of the initial milk TAG with the milk at 37 °C without pepsin, and the milks at 4 °C, 37 °C and 60 °C with pepsin, respectively. Interestingly, these values follow the order of magnitude as those measured for both the residual dry mass (Fig. 2C) and residual proteins (Fig. 3C) in the stomach. These percentages are also higher, hence suggesting a gastric emptying that is more delayed for fat than for proteins. Nevertheless, these estimates should be considered with caution because of the high variability observed between replicates, which might simply reflect the difficulty to collect and analyse a representative sample for lipid analyses (i.e. because of lipid creaming).

### 3.6. Confocal microscopy images of the digesta

Fig. 6 presents the confocal images of the samples collected at the stomach exit, and of the residual material in the stomach at the end of the experiments, under the different conditions. Before digestion, the proteins and fat globules were evenly distributed in the aqueous phase. At t = 8 min, the samples remained evenly distributed, except for the milk at 60 °C, where protein particles were already visible. At t = 14 min, different coagulated structures were observed depending on the conditions. Protein particles larger than 100 µm were observed in the milk samples at 37 °C and 60 °C with pepsin, whereas all protein particles were very small with the milk at 4 °C with pepsin. The microstructure of the digesta for the milk at 37 °C without pepsin was intermediate, indicating that coagulation had already started at t = 14 min due to the progressive acidification of the stomach contents (Fig. 1B). At t = 27 min, coalesced fat globules, either alone or covering small protein particles, appeared to dominate the emptied digesta,

except in the case of milk at 37 °C without pepsin. The small quantity of protein observed at this time point is consistent with the local minimum seen in the emptied proteins (Fig. 3A), and can therefore be attributed to the formation of large protein clots that were retained within the stomach, and thus largely absent from the digesta collected at the stomach exit. From t = 52 to 152 min, as the milk proteins were hydrolysed, an increasing number of protein-rich particles were progressively emptied together with fat-rich particles. The milk at 4 °C with pepsin also seemed to have retained lipids in the stomach for a shorter duration than the milk at 37 °C and 60 °C with pepsin (t = 152 min). For all conditions, the residual digesta in the stomach at the end of the experiments were essentially composed of protein particles with no observable fat, except for the milk at 37 °C without pepsin, in which some fat globules embedded in the protein network were detected. Compared to the milks at 37 °C without pepsin and at 4 °C with pepsin, the residual digesta also appeared to be more fragmented with the milks at 37 °C and 60 °C.

## 4. Discussion

This study investigated the effect of consumption temperature on the *in vitro* gastric digestion of whole milk using the NERDTM by considering intragastric temperature profiles representative of the conditions encountered in humans during the digestion of cold (4 °C), warm (37 °C), and hot (60 °C) liquid foods. This digestion system is particularly relevant for this purpose because it enables the reproduction of physiologically relevant biochemical and biomechanical conditions. We were able to examine not only the consequences of the whole milk consumption temperature on the intragastric milk coagulation and protein hydrolysis, but also on milk proteins and fat emptying, as well as the microstructure of the emptied digesta. Our results confirm that temperature at which whole milk is consumed has a significant impact on its gastric coagulation and further demonstrate how this may affect the gastric emptying of dairy proteins and fat.

### 4.1. Drinking whole milk hot (60 °C) instead of cold (4 °C) can delay the gastric emptying of milk proteins

With the exception of the intragastric temperature and the absence of

<figure><img src="image_8.png" /><figcaption>Fig. 6. Confocal microscopy images (magnification ×10) of the emptied digesta during gastric digestion and of the residual digesta in the stomach at the end of the experiment. Proteins appear in green and lipids appear in red. Scale bar is 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
pepsin in the control experiments at 37 °C, the same running parameters were used for all the investigated conditions. Similar changes in the wet mass of emptied material (Fig. 1C) and of gastric content volume (Fig. 1D) were observed throughout the different conditions. This demonstrates that the overall gastric emptying kinetics with the NERDTM system are predominantly governed by the 'mechanical' parameters, such as the strength and frequency of the gastric contractions, the pylorus opening size, and the turntable rotation. Indeed, the present study did not intend to modulate gastric emptying as a function of milk temperature. This is because the reported effects of the consumption temperature on gastric emptying in in vivo studies remain limited in magnitude, when observed, and because conflicting literature reports exist. For instance, while Fujihira et al. (2024) observed a higher initial gastric emptying rate with a protein drink at 60 °C and 37 °C as compared to 4 °C in elderly individuals, Sun et al. (1988) concluded that a glass of orange juice at 50 °C and 4 °C both led to a slower gastric emptying than at 37 °C in young adults. Further in vivo studies are therefore required to draw clear conclusions about the impact of beverage and meal consumption temperature on gastric emptying.

Temperature has a direct impact on pepsin activity, which may affect the kinetics of milk protein coagulation and the physical properties of the resulting particles. This can, in turn, have an impact on gastric hydrolysis and emptying, and explain why we observed a significant difference in the residual wet and dry masses of the gastric contents at the end of the experiments (Fig. 2C). This finding aligns with Yang et al. (2023) which reveal that the total curd weight was higher for skimmed milk at 37 °C and 50 °C than for milk at 4 °C after 240 min of digestion. It also confirms that pepsin activity is an important parameter in understanding the gastric emptying kinetics of proteins and fats in whole milk.

Moreover, the threshold for statistical significance was almost reached for all quantitative kinetics (0.1 ≤ *p* < 0.13 for the results in Fig. 2A, Fig. 3A, Fig. 3B, and Fig. 5A), and all our qualitative results (Fig. 2B, Fig. 4 and Fig. 6) indicate that milk coagulated more rapidly at 60 °C than at 4 °C, and to a lesser extent than at 37 °C milk. The rapid coagulation of the 60 °C milk, around 10 min (Fig. 6) and at pH > 5.5 (Fig. 1B), can be unambiguously attributed to the higher pepsin activity at higher temperatures, given that it exhibits appreciable activity on caseins at such elevated pH (Salelles et al., 2021). Indeed, pepsin activity against haemoglobin at pH 2 is about 2-fold higher at 45 °C than at 20 °C (Brier et al., 2007), i.e. at the gastric temperatures immediately after feeding the 60 °C and 4 °C milks, respectively. These findings are in accordance with those of Yang et al. (2023), who reported a faster in vitro intragastric coagulation of skimmed milk at 50 °C than at 4 °C and 37 °C. This is also consistent with a recent semi-dynamic in vitro digestion study of whole milk which reported that consuming it at 4 °C delayed protein coagulation by 5–10 min compared to milk at 37 °C and 60 °C (Fitzpatrick et al., 2024).

Before intragastric coagulation, the milk was in a liquid state, and was therefore emptied from the stomach as predicted by the model assuming simple dilution effects. However, after coagulation, the dry mass, protein, R-NH₂, and TAG contents of the material emptied from the stomach all suddenly decreased. This explains why the faster milk coagulation at 60 °C resulted in a lower final cumulative quantity of emptied proteins than with the 4 °C milk (Fig. 3C). Although the concentration of emptied proteins was plausibly higher with the 60 °C milk towards the end of the experiment (Fig. 3A), this did not appear to be sufficient to bridge the gap. Instead, the cumulative quantities of emptied proteins evolved in a fairly parallel manner for the 60 °C and 4 °C milks during the second hour of gastric digestion. The SDS-PAGE results (Fig. 4) and the cumulative quantities of emptied R-NH₂ (Fig. 3D), which are predominantly governed by the protein and peptide content, also support this trend. The gastric emptying half-time of proteins increased in the following order: 4 °C milk (60 min) < 37 °C milk without pepsin (80 min) < 37 °C and 60 °C milks (~90 min). This ranking is consistent with the results of Yang et al. (2023), who observed lower protein contents in the remaining curd during in vitro gastric

digestion of 4 °C milk as compared to 37 °C and 60 °C milk. Therefore, our results show that intragastric coagulation occurs sooner after the consumption of whole milk at hot or warm temperature, as compared to cold milk, which may lead to a non-recoverable delay in milk proteins gastric emptying kinetics.

## 4.2. Gastric proteolysis increases with milk consumption temperature

As digestion progressed after milk coagulation, from about 30 min, the DM content (Fig. 2A), the protein (Fig. 3A) and TAG (Fig. 5) concentrations of the emptied material increased gradually. These trends are very similar to those previously reported for skimmed milk (Feng et al., 2024), where the gastric emptying of caseins and whey proteins were distinguished and discussed in detail. However, these increases occurred earlier and were more pronounced in the present study. For instance, a signature of protein hydrolysis is the appearance of a smear at molecular weights below 6 kDa in SDS-PAGE results. This appeared at around 52 min (i.e. $t_{1/2}$) for the milk at 37 °C with pepsin (Fig. 4). This phenomenon was only observed after $3 \times t_{1/2}$ (77 min), in our previous study. This is due to a two-fold increase in the rate of pepsin addition used in the current protocol, with a target pepsin activity of 2000 U/mL at $t_{1/2}$ instead of at $2 \times t_{1/2}$ in the previous study. Therefore, the comparison of the results across these two studies highlights the key role played by pepsin activity in the gastric emptying of milk particles in the late stages of gastric digestion.

Our present results further show that whole milk consumption temperature can also influence the kinetics of milk particle disintegration and hydrolysis following coagulation. The measured concentrations and quantities of R-NH₂ (Fig. 3B and D) were initially intended to assess this influence. However, the R-NH₂ concentrations and quantities that we measured were subject to too large experimental errors to enable clear conclusions to be drawn, as in the study of Fitzpatrick et al. (2024). The varying proteolytic activity of pepsin as a function of the milk temperature was better assessed using SDS-PAGE. The smears at molecular weights lower than 6 kDa were much more pronounced with the 60 °C and 37 °C milks than with the 4 °C milk. Moreover, only the 60 °C milk showed an appreciable quantity of peptides with a molecular weight lower than 3 kDa. These small peptides predominantly originate from caseins, since β-LG, the major whey protein, seemed to persist throughout the experiments under all conditions (Fig. 4). This is consistent with the report by Reddy et al. (1988) that showed that β-LG is particularly resistant to hydrolysis by pepsin.

In the presence of pepsin, more and smaller peptides were observed with increasing milk temperature in the late stages of digestion. This finding differs somewhat from the results obtained by Yang et al. (2023), who observed a higher degree of protein hydrolysis at 37–40 °C, i.e. near the optimal temperature for pepsin. Lower values were observed at both 4 °C and 48 °C, a phenomenon that was attributed to differences in the structure of the milk curd formed in the stomach. However, in that study, the initial gastric temperature was set at 4 °C and 50 °C, contrasting significantly with our more physiologically relevant initial temperatures of ~20 °C and ~45 °C, respectively. The time needed to reach 37 °C as well as the kinetics of gastric pH and of pepsin addition were also different. It is therefore highly likely that our results do not align because of the differences in the milks and protocol used.

## 4.3. Consumption temperature can affect the gastric emptying of milk fat

The concentration of TAG in the emptied digesta initially decreased and then gradually increased, at least in the presence of pepsin (Fig. 5). The decrease in TAG concentration may be attributed to milk coagulation, whereby milk fat becomes trapped in the forming protein network. It is also likely that a proportion of milk fat tended to cream at the surface of the gastric content, thereby contributing to the prolonged retention of dairy fat within the stomach. However, the observed trend remained similar to those observed for protein and primary amine
(Fig. 3A and B), which suggests that milk coagulation was the predominant mechanism behind those results. This is also supported by the confocal images acquired at 14 min (Fig. 6), which show milk particles containing a significant proportion of fat, aligning with prior research findings (Mulet-Cabero et al., 2019).

In the presence of pepsin, the material emptied from the stomach between 27 and 152 min was composed of numerous small particles covered with fat (Fig. 6). This is consistent with previous reports showing that entrapped lipids may accumulate at the periphery of the eroding protein network (Luo et al., 2021), a phenomenon that may be promoted by gastric peristalsis (Ye et al., 2016b, 2017). By the end of the experiment, the residual digesta in the stomach consisted primarily of protein particles of varying sizes. The observation that the residual digesta consisted of larger and more swollen particles at 4 °C and at 37 °C without pepsin as compared to the milks at 37 °C and 60 °C, may be related to the formation of looser dairy curds that are less prone to syneresis at lower temperatures and/or without pepsin. However, the most notable finding was that almost no fat was visible by confocal microscopy in the residual particles, except in the case of the milk at 37 °C without pepsin where some embedded lipids could be observed (Fig. 6). This observation can be paralleled with the lower TAG release of the 37 °C milk without pepsin (Fig. 5), which provides further evidence that pepsin activity can promote the release of dairy fat from the milk particles. However, the absence of visible fat in the residual digesta appears to contradict the results of Fig. 5, from which it was estimated that the residual fat should account for between 20.6 % and 56.8 % of the initial milk fat content. This discrepancy is likely due to the difficulty of collecting a representative ~25 mL sample of residual digesta from the stomach (Fig. 2C) for confocal observations. Fat and fat-rich particles tend to phase separate; consequently, a fat-enriched sample is collected from the surface, and a fat-depleted sample is collected from the sedimented particles. Milk fat might also have adhered to the lipophilic walls of the silicone stomach during digestion. The absence of fat in the confocal images of the residual digesta should thus be considered with caution. Overall, our results seem to indicate that drinking milk at 60 °C or 4 °C could accelerate the gastric emptying of milk fat compared to drinking milk at 37 °C, but further studies are clearly needed to confirm or refute this finding.

In a previous study on the effect of whole milk consumption temperature on gastric digestion (Fitzpatrick et al., 2024), lipid quantification by MRI showed that the particles formed with 60 °C milk tended to remain at the bottom of the digestion vessel. In contrast, particles formed with 4 °C milk tended to cream in the middle of the digestion experiment; the particles formed with 37 °C milk and pepsin showed an intermediate behaviour. While it was not possible to visually observe whether a similar phenomenon occurred during our experiments, this could potentially explain why lipids appeared more abundant in the confocal images of 60 °C and 37 °C milks compared to the 4 °C milk from 52 min onwards (Fig. 6). It should also be noted that gastric lipase was not used in the present work nor in the study of Fitzpatrick et al. (2024). However, gastric lipolysis contributes 10–30 % of lipid hydrolysis throughout the gastrointestinal tract (Gallier et al., 2013), and the interaction between gastric lipase and pepsin in gastric digestion of milk is not fully understood (Pan et al., 2021). Consequently, further studies are needed to better investigate the effect of consumption temperature on the gastric emptying and the hydrolysis of whole milk fat.

## 4.4. Limitations of the study

This study presents several inherent limitations. Even if the NERDT™ is among the most physiologically relevant devices to mimic gastric digestion, this system cannot reproduce all the mechanisms that take place *in vivo*. In particular, we cannot ensure that the reduced gastric emptying rate of milk proteins we observed for the 60 °C milk would not be compensated *in vivo* by more intense gastric contractions and/or an increased emptying rate towards the small intestine. The effects we

observed on the gastric emptying of milk protein and lipid could also be outweighed by a direct effect of meal temperature on gastric emptying *per se*, a putative mechanism that could not be simulated in experiments because of the current conflicting *in vivo* reports. Another limitation is that gastric lipase was not used for financial reasons, while its activity could modify the behaviour of lipid structures during gastric digestion, notably with regards to lipid creaming. Despite these limitations, the present study was carefully designed to be as physiologically relevant as possible, using cutting-edge experimental tools and methods. It thus represents significant advances in an original area of research that has been little explored until now, opening up new avenues for further *in vitro* and *in vivo* investigation.

## 5. Conclusions

This study has investigated the effect of consumption temperature on the dynamic gastric digestion of whole milk using the NERDT™ system. Our results confirm that milk coagulates faster in the stomach when consumed at a high or warm temperature (37 °C or 60 °C) as compared to a cold temperature (4 °C). They further show that the earlier milk coagulation of hot milk may significantly delay the gastric emptying of milk proteins until the late stages of digestion. Milk consumption temperature can also influence the gastric emptying of milk fat, notably because it is partly entrapped within the protein particles. Our results even suggest that drinking hot (60 °C) or cold milk (4 °C) could accelerate gastric emptying as compared to milk at 37 °C. However, this latter statement clearly needs to be re-evaluated in future studies, as does the influence of gastric lipase, which has yet to be investigated. Even though the main effects of milk consumption temperature take place during the early stages of gastric digestion, this study demonstrates that they are likely to have repercussions over the whole postprandial period. It remains to be confirmed whether these effects will further affect the fate of proteins and lipids in the small intestine. Provided that *in vivo* studies suggest a possible impact of consumption temperature on gastric emptying, and that our current results indicate it is likely the case for the protein and lipid of milk, another interesting area for future research relates to the possible influence of meal temperature on satiety. More *in vitro* and *in vivo* studies are therefore required to verify this hypothesis, and to improve our understanding of all the consequences of food consumption temperature on digestion and related repercussions.

### CRediT authorship contribution statement

**Jiajun Feng:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. **Jeehyun Lee:** Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. **Vincent Mathieu:** Writing – review & editing, Validation. **Yann Le Gouar:** Validation, Investigation. **Romain Jeantet:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Didier Dupont:** Writing – review & editing, Validation, Supervision, Project administration, Conceptualization. **Steven Le Feunteun:** Writing – review & editing, Validation, Supervision, Project administration, Conceptualization.

### 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.

### Acknowledgment

The authors would like to thank Eurial, France for providing milk samples. Jiajun Feng was supported by the China Scholarship Council (CSC grant #202006920043). This study was performed in the frame of the International Associated Laboratories (LIA)'Foodprint' between
L'Institut Agro Rennes-Angers, INRAE (France) and Soochow University
(China).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodres.2025.117869.

Data availability

Data will be made available on request.

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