# First assessments of nutrient bioaccessibility with an INFOGEST semi-dynamic gastric digestion in vitro protocol adapted to model proton pump inhibitor use

Ruoxuan Deng, Françoise Nau, Tiphaine Lucas, Marie-Bernadette Maillard, Arlette Leduc, Jordane Ossemond, Maja Musse, Steven Le Feunteun

► To cite this version:

Ruoxuan Deng, Françoise Nau, Tiphaine Lucas, Marie-Bernadette Maillard, Arlette Leduc, et al. First assessments of nutrient bioaccessibility with an INFOGEST semi-dynamic gastric digestion in vitro protocol adapted to model proton pump inhibitor use. Food Research International, 2025, 203, pp.115833. 10.1016/j.foodres.2025.115833. hal-04970147

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# First assessments of nutrient bioaccessibility with an INFOGEST semi-dynamic gastric digestion in vitro protocol adapted to model proton pump inhibitor use

## Authors:

Ruoxuan Deng<fnref n="1" />,<fnref n="2" />

Françoise Nau<fnref n="1" />

Tiphaine Lucas<fnref n="2" />

Marie-Bernadette Maillard<fnref n="1" />

Arlette Leduc<fnref n="1" />

Jordane Ossemond<fnref n="1" />

Maja Musse<fnref n="2" />

Steven Le Feunteun<fnref n="1" />,*

For the purpose of Open Access, a CC-BY public copyright has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this manuscript.

<figure><img src="image_2.png" /></figure>

CC-BY 4.0 https://creativecommons.org/licenses/by/4.0

<footnote n="1">¹ INRAE, L'Institut Agro, STLO, 35042 Rennes, France.</footnote>

<footnote n="2">² INRAE, OPAALE, 35044 Rennes, France.</footnote>

<footnote>* steven.le-feunteun@inrae.fr</footnote>
## 1 Abstract

Physiologically relevant gastrointestinal digestion models for infants, adults, and the elderly are commonly used to explore the fate of food in vitro. However, no consensus protocol exists to simulate the specific conditions observed in the stomach of people using oral proton pump inhibitors (PPIs), a class of widely prescribed medications that reduce gastric acid secretion and may alter food digestion and nutrient absorption. The first objective of this study was to propose an in vitro gastric digestion protocol adapted to model PPI use. This protocol is an extension of the semi-dynamic INFOGEST protocol previously developed to mimic the digestion of an adult human (referred to hereinafter as “Standard”), with two modifications based on reported clinical effects of PPIs: (i) a final gastric pH of 4.2 and (ii) a 50% reduction in simulated gastric acid fluid volume. The second objective was to compare the release kinetics of peptides, soluble carbohydrates, lipids, and minerals during simulated gastric digestion of a mixed meal (bread, cheese, and tomato) with both standard and PPI versions of the protocol. Results demonstrated that the release of peptides, arabinose, and minerals, including calcium, magnesium, and phosphorus, was significantly (p < 0.05) reduced in the PPI model, while the hydrolyses of starch and lipids, assessed through maltose release and triacylglycerol disappearance, respectively, were not significantly affected. These findings are in agreement with the expected effects of reduced gastric acidity on pepsin activity and mineral solubility. These findings are also consistent with known or presumed side effects of PPIs such as an increased risk of hypomagnesemia, fractures, skeletal muscle loss, and vitamin B12 deficiency. In conclusion, this modified INFOGEST protocol appears to serve as a valuable tool to study the side effects of PPI use on food digestion and related nutrient bioaccessibility.

## 23 Keywords

PPI, Proteolysis, Lipolysis, Amylolysis, Mineral release, Gastric pH, Acid suppression.
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# 1 Introduction

Proton pump inhibitors (PPIs) are a class of medications (e.g., omeprazole, lansoprazole, rabeprazole, and pantoprazole) that induce a profound and prolonged reduction of gastric acid secretion (Laine et al., 2008). PPIs are prescribed for several gastric acid-related disorders, such as gastroesophageal reflux and peptic ulcer. A recent review showed that nearly 25% of adults in Western countries (North America, Europe, and Oceania) are PPI users (Shanika et al., 2023).

PPIs demonstrate robust antisecretory effects that are superior to histamine H₂ receptor antagonists (H₂RAs) (Stedman & Barclay, 2000). PPIs effectively block gastric acid secretion by irreversibly binding to and inhibiting the hydrogen-potassium ATPase pump located on the luminal surface of the parietal cell membrane (Shin & Sachs, 2008).

Although PPIs are generally considered safe for short-term use, emerging evidence suggests that PPIs are overprescribed and may be associated with several side effects (Hálfdánarson et al., 2018; Schoenfeld & Grady, 2016; Shanika et al., 2023). Side effects include reduced absorption of vitamin B₁₂ and minerals, such as calcium, iron, and magnesium (Cheungpasitporn et al., 2015; Ito & Jensen, 2010; Lam et al., 2013), increased risks of bone fractures (Cheungpasitporn et al., 2015), various inflammation and increased risk of infection (Bavishi & Dupont, 2011), skeletal muscle function loss (Vinke et al., 2020), and kidney disease (Lazarus et al., 2016). Epidemiological studies even suggest that PPI use may increase the risk of gastric cancer in the corpus/fundus (Yibirin et al., 2021). Although side effects on digestion remain controversial, at least one clinical study has shown that omeprazole administration significantly reduced dietary protein assimilation, according to reduced postprandial breath carbon-labeled carbon dioxide concentrations (from labeled egg whites) and increased urinary output of phenol and p-cresol (Evenepoel et al., 1998). Some animal experiments also demonstrated that PPIs may increase the amount of undigested protein in the lower gut of
49 chickens (González-Ortiz et al., 2022), and affect blood biomarkers of mineral, protein, and
50 lipid metabolism in horses (Mélo et al., 2014).

51 As a consequence of reduced gastric acid secretion, PPIs also have an impact on the volume of
52 gastric contents. After esomeprazole administration in 9 adults for 7 days, gastric volume was
53 reduced during the first 75 min after consumption of a liquid meal compared to meal
54 consumption without PPI (Babaei et al., 2009). According to magnetic resonance imaging (MRI)
55 studies, gastric volume was reduced by approximately 50% with 1 week of 40 mg pantoprazole
56 per day compared to placebo (Curcic et al., 2015; Steingoetter, Sauter, et al., 2015). Another
57 study found that gastric fluid volume decreased by 44% after 3 days of 40 mg of omeprazole
58 per day (de Waal et al., 2020). Beyond those volumetric considerations, the HCl concentration
59 of gastric secretion was also reduced by 43% with omeprazole or pantoprazole administration
60 in varied doses from 20 mg daily up to 40 mg twice daily (Rohof et al., 2014). This strong
61 reduction in both the volume and acidity of gastric acid secretion led to a prolonged high gastric
62 pH during the postprandial period. As reported in the literature, gastric pH is typically in the
63 range of 4-5 two hours after meal ingestion in PPI users instead of pH ~ 2 in healthy adults
64 (Armbrecht et al., 1997; Brunner et al., 1995).

65 In the postprandial period, the acidic environment in the stomach is crucial for digestion
66 because the activity of digestive enzymes at the gastric stage is strongly dependent on pH. The
67 amylolytic activity of human saliva is optimal close to pH 6-7 and becomes zero at pH 3 (Freitas
68 et al., 2018), while the optimal pH for human gastric lipase and pepsin are pH 5-6 (Gargouri et
69 al., 1989; Moreau et al., 1988) and pH 2 (Piper & Fenton, 1965), respectively. A highly acidic
70 environment is, moreover, particularly important for good mineral solubilization. By increasing
71 postprandial gastric pH, PPI use may thus reduce food digestion and nutrient absorption,
72 possibly increasing the risks of previously described side effects. This area of research remains
73 largely underexplored and would benefit from a physiologically relevant *in vitro* gastric
digestion model to quantitatively evaluate the effects of PPI use on nutrient metabolism and
bioaccessibility. However, to date, there only exists a proposed protocol to model PPI use in a
static gastric digestion simulation protocol (Garvey et al., 2022).

The first aim of this study was to propose a novel semi-dynamic *in vitro* gastric digestion
protocol to model the effects of PPI use. This “PPI protocol” was adapted from the INFOGEST
semi-dynamic digestion protocol, incorporating the reported effects of PPIs on the acidity and
rate of gastric secretions in humans. Using the PPI digestion simulation protocol, the effects of
reduced gastric acidity and secretions on the digestion of macronutrients (proteins,
carbohydrates, and lipids) and the release of minerals (calcium, phosphorus, and magnesium)
were compared to the INFOGEST Standard condition from a test meal consisting of bread,
cheese, and tomato. These foods were selected for their widespread consumption in Western
countries and the inclusion of all macronutrients as well as several micronutrients.
## 2 Material and Methods

### 2.1 Material

Bread, cheese, and tomato were purchased in a local supermarket (Rennes, France). The par-baked, sliced bread loaves were made with whole-wheat flour (Pain complet tranché, Picard, France). Before each experiment, a stack of three slices of bread was completely defrosted. Each outside surfaces of the stack were toasted for 4 min (8 min total) using a toaster, and the stack was then left to cool for 1 hour at room temperature. Only the middle slice was kept for the following experiment. The water content of the baked bread was about 50% (w/w) with a macronutrient composition of 1.7% fat, 8.1% protein, and 41.8% total carbohydrate (including 3.0% sugars). The cheese was Emmental (Emmental français, President, France) containing 28% fat, 28% protein, and 1% carbohydrate (w/w). The nutrient compositions of bread and cheese were based on their food labels. Ripe vine tomatoes of similar color and size from the same truss were used in this study, with their nutrient composition taken from the Ciqual database (https://ciqual.anses.fr/#/aliments/20047/tomato-raw): 0.24% fat, 0.86% protein, and 2.49% carbohydrate (w/w).

Frozen pooled human saliva was obtained from Lee Biosolutions (ref. 991-05-P, Maryland Heights, United States). Rabbit gastric extract (RGE) containing gastric lipase (10.5 U/mg) and pepsin (400 U/mg) was purchased from Lipolytech (Marseilles, France). The enzyme inhibitors pepstatin A (Product No. P5318) and 4-bromophenylboronic acid (Product No. B75956) were obtained from Sigma-Aldrich (France). Enzyme activities and bile salt concentrations were determined according to the protocols described by Minekus et al. (2014).

The preparation of electrolyte simulated gastric fluid (eSGF) followed the recipe from (Mulet-Cabero et al., 2020). For the sake of clarity, the term ‘simulated gastric acid fluid’ (SGAF) will be used hereafter to refer to the HCl solutions prepared from eSGF (see section 2.3.2). RGE was dissolved in eSGF at pH 7 to achieve a stock solution with 1,200 U gastric lipase/mL,
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which was prepared immediately before digestion simulations and stored on ice. For the enzyme
inhibitors, 0.5 mg/mL pepstatin A stock solution and 0.1 mol/L 4-bromophenylboronic acid
were prepared in methanol. The inhibitor stock solutions were stored at -20 °C before use.

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## 2.2 Effects of PPIs on human postprandial gastric pH

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To identify *in vitro* parameters that are relevant to the physiological consequences of PPI use,
a systematic search in Medline (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) was performed
using the international names of PPIs (esomeprazole, lansoprazole, omeprazole, pantoprazole,
rabeprazole) in combination with key terms “intragastric” or “gastric”, “pH” or “intragastric
acidity”, “acid secretion”, and “24-hour” to acquire publications with postprandial gastric pH
data. Of the 151 publications retrieved, 76 were descriptions of potentially relevant clinical
trials and were retained for further analysis. After screening through these publications with
criteria of ‘written in English’ and ‘presenting gastric pH data after meal intake’, 18 papers
were finally included. When gastric pH data were in the form of a data plot with no written
values, these were extracted using a data plot digitizer (https://web.eecs.utk.edu/~dcostine/personal/ PowerDeviceLib/DigiTest/index.html). Figure 1
presents the human postprandial gastric pH values pooled over these 18 studies (Armbrecht et
al., 1997; Brunner et al., 1995; Chiverton et al., 1992; Funaki et al., 2013; Furuta, 2001; Hongo
et al., 1998; Inoue & Nakamura, 1995; Johnson et al., 2005; Labenz et al., 1996; Lee et al.,
2022; Metz et al., 2008; Müssig et al., 1997; Sugimoto et al., 2004, 2006, 2010; Varannes et al.,
1994; Verdú et al., 1994; Yi et al., 2017).

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## 2.3 Semi-dynamic *in vitro* digestion

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### 2.3.1. Experimental design

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The test meal consisted of 4 g bread, 4 g cheese, 4 g tomato, and 12 mL water (as a standalone
drink). This test meal was selected to ensure the inclusion of key nutrients: carbohydrates,
proteins, lipids, calcium, phosphorus, and magnesium. The amount of drinking water added to

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the foods was based on a 1:1 mass ratio for the sake of simplicity and to ensure enough dilution
to collect samples from the start of the digestion experiments. This meal was digested under
each of 4 semi-dynamic *in vitro* gastric digestion conditions: ‘Standard’ and ‘PPI’ conditions,
with and without digestive enzymes. The experiments performed in the absence of digestive
enzymes further referred to as control conditions (‘cStandard’ and ‘cPPI’ conditions), were
conducted to assess the effect of gastric acidity alone on the release of nutrients. All conditions
were performed in triplicate.

2.3.2 *In vitro* digestion protocol and parameters for the Standard and PPI conditions

For the Standard condition, the digestion procedure followed the recommendations of the semi-
dynamic *in vitro* digestion protocol described by the INFOGEST consortium (Mulet-Cabero et
al., 2020). Two adaptations to this protocol were made for the PPI condition. First, based on the
clinical data presented in Figure 1, a final target gastric pH of 4.2 (instead of pH 2) was
implemented. Second, the volume of SGAF was reduced by half according to the reported effect
of PPIs in humans (Curcic et al., 2015; de Waal et al., 2020; Steingoetter et al., 2015). In practice,
for the Standard condition, the HCl concentration of the SGAF was determined by the pH test
tube experiment proposed by Mulet-Cabero et al. (2020). For the PPI condition, the protocol
was modified to specify a volume ratio of SGAF to the oral digesta of 0.5:1 (instead of 1:1) and
to achieve a final target pH of 4.2 (instead of 2). According to these modifications, SGAF HCl
concentrations were 0.12 mol/L and 0.05 mol/L for the Standard and PPI conditions,
respectively.

2.3.3. Experimental setup

All digestion experiments were performed using the instrument configuration illustrated in
Figure 2. Digestions were performed in a double-wall 90 mL glass vessel with a truncated cone
(Order No. 6.1418.220, Metrohm, Herisau, Switzerland) maintained at 37 °C by circulating
water under magnetic stirring (125 rpm). A titration unit (Order No. 28420010, 842 Titrando,
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Metrohm) was used to control the addition of the SGAF and monitor the resulting pH, and a syringe pump (KDS100 Legacy Fisherbrand™, Thermo Fisher Scientific, Waltham, MA, USA) was used to control the addition of the enzyme solution (RGE stock solution, kept on ice).

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About 20 min before the start of a digestion experiment, 1.305 mL eSGF at pH 2.0 and 12 mL water (to represent the drink included with the test meal) were added to the vessel and left to warm. Just before the start of the experiment, 0.29 mL RGE stock solution was also added.

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### 2.3.4. Oral phase

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#### 2.3.4.1. Food comminution

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The protocol used for the comminution of bread and cheese was as previously described by Musse et al. (2023), where complementary explanations and justification are provided.

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The crumb and the crust of the freshly re-baked bread were separated using a knife to cut the crusty bottom flat edge (thickness ~ 3.5 mm) from the middle slice of the stack of baked bread.

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The crumb was ground using a blender for 5 s at speed setting 2 (SKU No. 8010S, Waring Laboratory Science, Torrington, CT, USA) to simulate comminution. The crust was cut into rectangular pieces (approximately 5 mm × 5 mm × 3.5 mm). Crust and crumb samples were then combined in a ratio of 30:70 (1.2 g/2.8 g) to maintain the crust/crumb ratio of the original bread and were stored in a closed container until saliva was added.

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For cheese, large pieces (approximately 10 mm × 10 mm × 10 mm) were ground for 2 periods of 10 s at speed setting 2 (same blender as for bread) and the resultant particles passed through a sequence of sieves with decreasing mesh sizes (4, 2, and 1 mm). Additionally, larger cheese cubes with an edge of approximately 5 mm were produced using a knife. Cheese particles from the 4 fractions (<1 mm, 1–2 mm, 2–4 mm, and 5 mm cubes) were then combined in the following proportions: 10:40:30:20 (0.4 g/1.6 g/1.2 g/0.8 g) to reproduce the particle size distribution reported for Emmental boluses in humans (Jalabert-Malbos et al., 2007). The cheese particles were stored at -25 °C and defrosted on the day of the digestion experiment. The

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freezing of cheese particles was carried out for practical reasons and to ensure the cheese
samples were the same in all the experiments, noting that freezing is considered a good means
to preserve the quality of cheeses (Alinovi et al., 2021).

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On the day of the digestion experiment, the tomato pericarp was cut into particles of approximately 5 mm in each dimension and was stored in a closed container until saliva was added.

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### 2.3.4.2. Salivary imbibition

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To begin the digestion experiment ($t = 0$ s), 4 g bread particles, 4 g cheese particles, and 4 g tomato particles were combined and mixed with 5 mL human saliva for 30 s under gentle manual mixing with a spatula. At $t = 90$ s, the *in vitro* bolus was transferred into the digestion vessel and mixed for another 30 s with a spatula. The end of the oral phase and the start of the gastric phase occurred at $t = 2$ min.

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### 2.3.5 Gastric phase

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According to the semi-dynamic INFOGEST protocol recommendations, a gastric half-emptying time of 1 h was implemented, leading to a 2 h gastric phase that approximates gastric residence time *in vivo* after a 240-kcal meal. The addition of SGAF (0.12 mol/L HCl) was performed at a flow rate of 0.2 mL/min under Standard and cStandard conditions, allowing the digestion mixer to reach pH 2 in 2 hours. As previously explained, the concentration of HCl in SGAF and the rate of SGAF addition were adjusted to 0.05 mol/L HCl and 0.1 mL/min, respectively, under PPI and cPPI conditions in order to reach a pH of 4.2 in 2 hours. The RGE solution (kept on ice) was added using a syringe pump at 0.024 mL/min for both Standard and PPI conditions to provide 120 U/mL of lipase in the final SGF solution. Under cStandard and cPPI conditions, human saliva was replaced by water, and the RGE solution was replaced by eSGF (pH 7). Five gastric emptying (GE) steps were performed at $t = 26, 50, 74, 98, and 122$ min. At each GE step, a 15 mL plastic pipette with a cut-head was utilized to collect 9.1 mL of

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digesta for the Standard and cStandard conditions, or 7 mL for the PPI and cPPI conditions.

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These volumes ensured that 80% of the total gastric content was emptied by 2 hours under all conditions.

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For all conditions, right after a GE step, the GE sample was mixed by inversion. Then, 1 mL digesta was collected (for lipid analysis), mixed with 4-bromophenylboronic acid stock solution (50 µL/mL GE sample), and kept on ice. After 1 min to allow particles to sediment, 0.5 mL GE free liquid was collected (for carbohydrate and mineral analyses), heated at 95°C for 10 min to inactivate enzymes, and kept on ice. Finally, another 0.5 mL free liquid was collected (for protein analysis), mixed with pepstatin A stock solution (50 µL/mL GE sample), and kept on ice. All the samples were stored at -20 °C at the end of the experiments.

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## 2.4 Measurement of amino groups released

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The concentration of free primary amino (-NH₂) groups was quantified in the supernatants (10,000 g for 20 min at 4 °C) of GE samples using a previously described miniaturized o-phthalaldehyde (OPA) spectrophotometric assay (Musse et al., 2023). The measured concentration was expressed in mmol/mL.

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## 2.5 Measurement of soluble carbohydrates released

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The supernatants of GE samples were analyzed after centrifugation (Vivaspin™, Sartorius Stedim Lab Ltd, Stonehouse, UK) at 8,000 g for 30 min. Glucose, fructose, arabinose, and maltose were quantified by high-performance anion-exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) on an ICS-5000+ Dionex system (Thermo Electron SAS, Courtaboeuf, France), as previously described (Canon et al., 2021). The system was equipped with a Dionex CarboPac PA210-Fast-4 µm column preceded by a CarboPac PA210-4 µm guard column (2 × 30 mm). Samples were eluted with KOH as eluent, at a flow rate of 0.2 mL/min with the following gradient: 13 mmol/L from 0-32 min, 100 mmol/L from 32-55 min, and 13 mmol/L from 55-65 min. Data were acquired and processed by using Chromeleon7™ software

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(Thermo Scientific). These four carbohydrates were quantified using multi-standard external calibration (range: 0.1-40 mg/L, Merck, St. Quentin Fallavier, France). The measured concentration was expressed in mg/mL.

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## 2.6 Measurement of lipid hydrolysis

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GE samples were homogenized with an ultra-turrax (IKA-T25 with the axis S18N-10G/03.444838; 25,000 rpm) to break down the cheese particle and to create a suspension. A lipid extraction was then performed (Folch et al., 1957), and extracted lipids were spotted on high-performance thin-layer chromatography (HP-TLC) silica plates (10 × 20 cm, Si 60; Merck), using an Automated HP-TLC Sampler 4 (CAMAG, Muttenz, Switzerland). Lipid classes were separated with a solvent mixture of hexane/diethyl ether/acetic acid (70/30/2 v/v/v), revealed by a copper sulphate/orthophosphoric acid solution and a heat treatment for 15 min at 150 °C. The bands on the plates were scanned using a ChemiDoc Imager (Bio-Rad Laboratories, California, USA) in epi-white excitation mode. Image analysis was then performed using the software Image Lab™ (Bio-Rad Laboratories) to estimate the relative percentage of the classes of lipids (including triacylglycerols (TAG), diacylglycerols (DAG), monoacylglycerols (MAG), and free fatty acids (FFA)) in each sample.

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## 2.7 Measurement of minerals released

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The supernatants of GE samples were analyzed after centrifugation (Vivaspin™, Sartorius Stedim Lab Ltd) at 8,000 g for 30 min. After diluting the collected supernatant with a 2% HNO₃ solution, minerals (Ca, P, and Mg) were quantified by inductively coupled plasma (ICP) atomic emission spectrometry using a 7200 ICP-OES Duo (Thermo Fisher Scientific, Switzerland).

The wavelengths of the spectrometer for Ca, P, and Mg were set to 393.366 nm, 177.499 nm, and 279.553 nm, respectively. Calibration was performed before the measurement using five different levels of standard solutions that were prepared in 2% HNO₃ solution and containing Ca (0-10 mg/L), P (0-10 mg/L), or radial Mg (0-1 mg/L). The standard solutions with 2 mg/L

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Ca, 2 mg/L P, or 0.5 mg/L Mg were measured again after every 12 samples. The measured concentration was expressed in mg/mL.

The total mass of Ca, P, and Mg present in tomato, bread, and cheese were also measured. For this, about 250 mg of tomato or bread were dissolved in 9 mL 69% HNO₃ and 1 mL 30% H₂O₂, and 250 mg cheese in 6 mL 69% HNO₃ and 2 mL 30% H₂O₂. Samples were then sealed and treated in a high-performance microwave digestion system (Ethos Up, MA 182-01, ThermoFisher Scientific, Switzerland) at 210 °C and 1,800 W for 40 min to achieve complete dissolution and mineralization of the sample. Subsequently, the quantification of minerals followed the same procedure as for the supernatant of the GE samples.

## 2.8 Quantity of released nutrients

For any given GE time (t), the quantity of each nutrient released ($Q_t$) was calculated from the above-measured concentrations using the following generic equation:

$$Q_t = C_t \cdot V_t + \sum_{i=0}^{N} C_i \cdot V_i \quad (1)$$

Where $C_t$ is the concentration of the considered nutrient at time t and $V_t$ is the volume (mL) of the supernatant (estimated as described in the Supplementary material) at time t, right before the gastric emptying step. The second part of the equation represents the cumulative quantity of the nutrient emptied before time t as part of the gastric emptying steps, with N the number of preceding gastric emptying steps, $C_i$ the concentration of the nutrient and $V_i$ the volume (mL) of the supernatant of the i-th GE sample.

For each mineral, an additional calculation was performed to determine the percentage released by dividing the mass of the released mineral by the total mineral content of the original meal.

## 2.9 Statistical analysis

Means and standard deviations (SD) were calculated from the triplicate experiments. Statistical analyses were performed using R software (version 4.4.0). A linear mixed model, using least-
square means analyses, was performed to examine the effects of time, different digestion conditions, and their interaction (time × condition) on the release of nutrients (ImerTest package). The released quantities of "-NH₂ groups", "maltose", "glucose", "fructose", "arabinose", "TAG", "DAG", "MAG", "FFA", "Ca", "P", "Mg", and the released percentages of minerals "Ca%", "P%", and "Mg%" were considered as dependent variables. Fixed factors were "time" and "conditions". Effects were considered statistically significant at p < 0.05. When effects were significant, pairwise multiple comparisons of the means between conditions or between time points were carried out using Tukey's test (emmeans package).
293 **3 Results**

294 3.1 Meal composition

295 The relative contributions of cheese, bread, and tomato to the total amount of proteins,
296 carbohydrates, lipids, calcium, phosphorus, and magnesium in the test meal are presented in
297 Figure 3. The meal contained 1.5 g protein, 1.8 g carbohydrate, 1.2 g lipid, 45.8 mg calcium,
298 36.3 mg phosphorus, and 4.2 mg magnesium. Most of the proteins (75.8%) and lipids (93.5%)
299 came from cheese, while carbohydrates mainly came from bread (92.3%). Cheese was the main
300 source of calcium (96.6%), while 76.7% of phosphorus came from cheese and 21.9% from
301 bread. Bread was the main source of magnesium (60.3%), followed by cheese (35.7%) and
302 tomato (4.3%).

303 3.2 Gastric acid fluid addition, pH, and volume of gastric content

304 Figure 4 presents the kinetics of simulated gastric acid fluid (SGAF) addition, gastric pH, and
305 gastric content volume over time for all the digestion conditions. In accordance with the
306 protocol parameterizations used (see 2.3.2 and 2.3.5), and as shown in Figure 4a, the flow rate
307 and final volume of SGAF were reduced by half under PPI conditions compared with Standard
308 conditions (independent of the presence of enzymes). The volume of gastric contents therefore
309 also evolved differently between these digestion conditions (Figure 4c). Moreover, and as
310 targeted, the resulting gastric pH decreased from 5.7 to 2 in Standard and cStandard conditions,
311 and from 5.7 to 4.2 in PPI and cPPI conditions (Figure 4b). In the cStandard condition with no
312 enzymes, pH 2 was reached sooner (Figure 4b), and less gastric acid was added (Figure 4a).
313 This phenomenon was due to the higher buffering capacity of peptides generated by pepsin, as
314 compared to the equivalent undigested proteins without pepsin (Morzel et al., 2023).

315 3.3 Release of proteins and peptides during digestion
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Figure 5 presents the quantity of free amino group (-NH₂) released in the digesta supernatants during digestion. Time, condition, and their interaction (time × condition) had significant effects on the release of free amino groups (p < 0.05).

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All 4 conditions started at 0.4 mmol with no significant difference (p > 0.19). In the absence of enzymes, a significant increase was observed until 26 min but not later on (p > 0.13), with no difference (p > 0.14) between cStandard and cPPI throughout digestion. This likely reflects the release of the soluble proteins initially contained in the food particles. However, in the presence of enzymes (Standard and PPI), the released of free amino groups increased (p < 0.0001) over 120 min, with an almost linear trend. Consequently, the quantities released under the Standard and PPI conditions rapidly became higher (p < 0.02) than those under the cStandard (from 26 min) and cPPI (from 50 min), respectively.

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Moreover, the amount of free amino groups released was higher under Standard condition than under PPI condition from 74 min onwards (p < 0.001). After 2 h, it reached 1.4 mmol for Standard vs. 1.2 mmol for PPI (p = 0.0001), while it reached 0.6 mmol for both cStandard and cPPI (p = 0.14).

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### 3.4 Release of soluble carbohydrates during digestion

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Figure 6 presents the mass of soluble carbohydrates released in the digesta supernatants during digestion. Time, condition, and their interaction (time × condition) had significant effects on the release of soluble carbohydrates (p < 0.05). In all conditions (Standard, cStandard, PPI, and cPPI), the mass released for all sugars was significantly higher at 122 min compared to 2 min (p < 0.005).

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The quantity of maltose and glucose released (Figures 6a and 6b), which predominantly come from bread starch hydrolysis, increased rapidly in the presence of saliva, leading to much higher (p < 0.005) values from 2 min onwards in the presence of enzymes (Standard or PPI) than in

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their absence (cStandard or cPPI). No significant differences were found between Standard and PPI conditions (p > 0.14 for maltose and p > 0.37 for glucose), nor between cStandard and cPPI conditions (p > 0.60 for maltose and p > 0.13 for glucose). Over time, the release of maltose plateaued from 26 min in Standard (p > 0.88) and cPPI (p > 0.12) conditions, and from 50 min in PPI (p > 0.24) and cStandard conditions (p > 0.59). The release of glucose plateaued from 50 min in all conditions: Standard (p > 0.08), PPI (p > 0.26), cStandard (p > 0.09), and cPPI (p > 0.21).

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Regarding the mass of fructose and arabinose released (Figures 6c and 6d), which predominantly come from tomato and wheat bran in the bread, the difference between the conditions with enzymes (Standard and PPI) and without (cStandard and cPPI) was much smaller. For fructose, no significant difference (p > 0.08) was observed between PPI and cPPI, while significant differences (p < 0.05) were found at 26 and 74 min between Standard and cStandard. For arabinose, no statistical difference was observed at any time between Standard and cStandard (p > 0.24, except at 50 min) or between PPI and cPPI (p > 0.30). When comparing Standard and PPI conditions, the mass of fructose and arabinose released were higher (p < 0.05) in the Standard condition from 50 min onward. Similarly, this trend was observed when comparing the two corresponding control conditions (p < 0.05). Over time, the release of fructose did not significantly increase from 50 min onward in Standard (p > 0.06), cStandard (p > 0.18), and cPPI (p > 0.12), and from 26 min onward in PPI condition (p > 0.71). No significant increase in arabinose release was observed from 98 min onward in Standard (p > 0.08), and cStandard (p > 0.54), and from 50 min in PPI condition (p > 0.13) and cPPI (p > 0.35).

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3.5 Lipid hydrolysis during digestion
<figure><img src="image_3.png" /></figure>

The compositional evolution of the main categories of lipids in the GE samples during digestion is presented in Figure 7. Time significantly affected TAG hydrolysis (p < 0.001), while condition and its interaction with time did not.

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

From 2 to 122 min, the relative percentage of TAG decreased from 86.3 ± 2.2% to 69.1 ± 4.6% in Standard condition (p = 0.0001), and from 86.6 ± 0.5% to 67.8 ± 5.1% in the PPI condition (p = 0.0008), while the relative percentage of FFA increased from 2.6 ± 0.3% to 8.8 ± 1.1% in Standard condition (p = 0.0001), and from 2.6 ± 0.1% to 6.8 ± 1.4% in PPI condition (p = 0.002).

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

In addition, lipid analysis at 2 and 122 min under control conditions (no enzyme) showed no change in the main categories of lipids over time (data not shown). Although few statistical differences were observed for lipids fractions at certain time points (Figure 7), the overall extent and kinetics of lipid hydrolysis were similar in both Standard and PPI digestion conditions.

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

### 3.6 Release of soluble minerals during digestion

<figure><img src="image_15.png" /></figure>

The mass and relative percentages of calcium (Ca), phosphorous (P) and magnesium (Mg), released in the digesta supernatants during digestion are presented in Figure 8. Time, condition, and their interaction (time × condition) had significant effects on the release of soluble minerals (p < 0.05). Starting from similar amounts at 2 min, there was a progressive release of all minerals under all conditions. The amount of released Ca, P, and Mg was always significantly higher (p < 0.005) under Standard than PPI condition, and also under cStandard than cPPI condition, i.e., regardless of the presence of enzymes.

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

Regarding the released mass and percentage of Ca, there was no significant difference (p > 0.28) between Standard digestion and its control, and only a small difference (significant from 74 min onward, p < 0.01) between PPI digestion and its control. Over time, within each condition, the increase was significant (p < 0.05), except between 98 and 122 min in cPPI condition (p =

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>
386 0.08). At the end of the digestion, the percentage of Ca released was 56.4 ± 0.5%, 58.3 ± 0.2%,
387 41.3 ± 1.1%, and 37.1 ± 0.1% for Standard, cStandard, PPI, and cPPI conditions, respectively.

388 Regarding P, the released mass and percentage were significantly higher in the presence of
389 enzymes than in their absence from t = 26 min onward (p < 0.05). Over time, within each
390 condition, they increased (p < 0.05), except between 98 and 122 min in cPPI condition (p =
391 0.08), and between 50 and 74 min, 74 and 98 min, and 98 and 122 min in cStandard condition
392 (p > 0.13). The final percentages of P released were 38.7 ± 1.0%, 30.4 ± 0.5%, 30.5 ± 0.5%,
393 and 21.1 ± 0.2% for Standard, cStandard, PPI, and cPPI conditions, respectively.

394 The release profiles of Mg were very similar to those of P for all conditions. Significantly higher
395 released mass and percentages (p < 0.001) were observed in the presence of enzymes than in
396 their absence: from 74 min onward in Standard (vs. cStandard condition), and from 50 min
397 onward in PPI (vs. cPPI condition). Over time, within each condition, the release increased (p
398 < 0.05), except between 50 and 74 min, 74 and 98 min, and 98 and 122 min in Standard
399 condition (p > 0.06), and between 50 and 74 min, 74 and 98 - 122 min, and 98 and 122 min in
400 cStandard condition (p > 0.06). At the end of the digestion, the percentage of Mg released was
401 83.9 ± 3.1%, 73.3 ± 1.2%, 72.1 ± 0.8%, and 59.5 ± 0.7% for Standard, cStandard, PPI, and cPPI
402 conditions, respectively.

403 **4 Discussion**

4.1 A physiologically based semi-dynamic protocol to study the effects of PPI use

404 The semi-dynamic *in vitro* digestion protocol from the INFOGEST consortium (Mulet-Cabero
405 et al., 2020) has become a critical resource to investigate the kinetics of food disintegration and
406 the release of nutrients. This protocol was specifically designed to incorporate crucial kinetic
407 aspects associated with the gastric phase of digestion, including gradual acidification, fluid and
408 enzyme addition, and gastric emptying. Nevertheless, suboptimal digestive conditions
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necessitate the development of tailored *in vitro* protocols. The use of PPIs, which induces a
significant reduction in gastric acidity, represents one such condition. A model adapted from
the static INFOGEST protocol has already been proposed to simulate the use of PPIs (Garvey
et al., 2022). However, to the best of our knowledge, no semi-dynamic *in vitro* digestion model
currently exists that mimics digestion under PPI conditions, as proposed in this study.

To adapt the INFOGEST semi-dynamic *in vitro* protocol to the case of proton pump inhibitor use, only two recommendations were modified. First, we propose to target a final gastric pH of 4.2 (instead of pH 2), as inferred from the linear regression of the clinical data presented in Figure 1. Second, we propose to reduce the volume of simulated gastric acid fluid (SGAF) by 50% to be consistent with the reported effect of PPIs on gastric contents volume in humans (Curcic et al., 2015; de Waal et al., 2020; Steingoetter et al., 2015).

With these two modifications, the proposed “PPI protocol” effectively generated a pH profile
(Figure 4b) comparable to the pattern observed in the pooled *in vivo* data (Figure 1 and Figure
S1 in Supplementary material). Moreover, to reach the target final pH of 4.2, the HCl
concentration of the SGAF also had to be reduced by 42% as compared to the INFOGEST
conditions (Standard condition). This extent of decrease is in very good agreement with the 43%
PPI-associated reduction of the HCl molarity of human gastric secretions estimated by (Rohof
et al., 2014). These findings further confirm the relevance of the PPI semi-dynamic *in vitro*
gastric digestion protocol, developed as part of this study, to reproduce the main physiological
consequences of PPI intake.

4.2 Gastric proteolysis was reduced under the PPI condition

The present study shows reduced gastric digestion of proteins under the PPI condition compared
to the Standard condition (Figure 5), as a consequence of higher gastric pH (Figure 4b). Indeed,
it is well established that pepsin activity is highly dependent on pH, with optimal activity at low
values. For instance, the optimal pH of porcine pepsin is typically reported to be approximately 2 when hemoglobin is used as a substrate, while its activity on egg white or pea proteins is negligible at pH 6 and exhibits a gradual increase down to pH 1 (Salelles et al., 2021). Similarly, the hydrolysis of bovine serum albumin by human pepsin is optimal at pH 2, with a 30% reduction at pH 4 and a 60% reduction at pH 5 (Piper & Fenton, 1965). However, caseins, the primary protein component of the test meal in this study, have been shown to be particularly susceptible to pepsin from pH 1 to 5, with a notable reduction in activity at pH 6 only (Salelles et al., 2021). These data suggest that casein hydrolysis was already underway at the initiation of the experiments, thereby explaining why a statistically significant difference emerged as early as t = 26 min between the conditions with and without enzymes (Standard vs. cStandard, and PPI vs. cPPI), despite the gastric pH remaining above 4.5 in all conditions. This may also explain why a significantly higher proteolysis under Standard condition in comparison to PPI condition was observed so late, i.e. after 74 min of digestion, when the pH was below 3 under Standard condition and above 4.2 under PPI condition (Figure 1). However, the increasing gap between Standard and PPI conditions during the second part of the digestion may also be attributed to the pH-dependence of pepsin activity on wheat gluten, the secondary protein component of the meal (around 21%, Figure 3), for which a ten-fold increase has been reported between pH 4.5 and pH 2 (Crévieu-Gabriel et al., 1999). These findings also suggest that a more pronounced reduction in proteolysis may be expected under PPI conditions if the primary protein components of the meal were not caseins.

The known or suspected side effects of PPIs include a reduction in muscle mass and function (Vinke et al., 2020), an increase in colonic fermentation of undigested proteins (Evenepoel et al., 1998), and a reduction in the absorption of vitamin B₁₂ when bound to dietary proteins (Ito & Jensen, 2010), associated with deficiencies in this vitamin (Lam et al., 2013). The various effects observed *in vivo* may be explained by the reduced gastric proteolysis under PPI
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conditions, as suggested by the present study. This highlights the value of *in vitro* models in attempting to explain phenomena observed *in vivo*. However, additional research is required to determine with absolute certainty the causal relationships proposed by the present study.

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4.3 The release of fructose and arabinose, but not amylolytic products, was reduced under PPI condition

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Salivary α-amylase, which is active at pH > 3 (Freitas et al., 2018), hydrolyses starch into limit dextrin, maltose, and some glucose (Jacobsen et al., 1972). The rapid release of maltose and glucose observed under both Standard and PPI (Figure 6a and 6b), but not under cStandard and cPPI, indicates that the enzymatic hydrolysis of starch started immediately upon the addition of saliva to the test meal. Moreover, despite the observed difference in acidification kinetics between the Standard and PPI conditions, the gastric pH remained above 3 for a minimum of 74 min in both cases (Figure 4b). And yet, Freitas et al. (2018) demonstrated that α-amylase from human saliva is capable of releasing up to 80% of the total bread starch content in less than 30 min, even when the gastric pH drops to pH 2 within that time. This may explain why starch hydrolysis and subsequent maltose and glucose release were already well advanced within the first 26 min or 50 min of digestion, with no significant difference in the amount of maltose and glucose released between the Standard and PPI conditions.

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The quantity of maltose released reached a plateau at 26 min of digestion under Standard condition and at 50 min under PPI condition (Figure 6a), indicating that all the digestible starch had been hydrolyzed by those timepoints. This is also corroborated by the observation that both plateaus reached the same amplitude. However, it is noteworthy that the plateau was reached later under PPI condition compared to Standard condition. This may indicate an enhanced accessibility of the amylase to its substrate under Standard condition, possibly due to more extensive acid-mediated breakdown of the bread particles, or improved mixing efficiency

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associated with the higher gastric fluid volume. Further investigations would be needed to gain
a deeper understanding of this observation.

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The level of glucose plateaued at 50 min for both Standard and PPI conditions. The glucose
release could be partly attributed to the starch hydrolysis, facilitated by the persistence of local
pH values above 3 inside the particles, but it likely also resulted from a non-enzymatic process.

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In addition to starch hydrolysis, a proportion of the released glucose may be due to the
solubilization of entrapped glucose, which likely originated from the tomato according to the
composition of the test meal. This interpretation is further supported by the release of fructose,
which increased up to 26 min for PPI or 50 min for Standard condition (Figure 6c), likely
originating solely from tomato. Furthermore, it can be concluded that the solubilization of
fructose was favored by acidic conditions, as the released quantity was consistently higher in
Standard conditions compared to PPI conditions, with and without enzymes. This may even
explain why the final quantity of released fructose in cStandard was higher than the final
quantity of released glucose in the same conditions (approximately 70 mg vs. 60 mg), as mature
tomatoes generally contain more fructose than glucose, by approximately 15% (Kasim & Kasim,
2015).

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Similar to fructose, the release of arabinose occurred via a non-enzymatic process, as no
difference was noted between the conditions with and without enzymes. Moreover, the process
was promoted by an acidic pH below 3.0-3.5, as it only occurred in Standard and cStandard
conditions from 74 min of digestion (Figure 6d). This suggests that arabinose in the test meal
originated from arabinoxylans and pectin, which are present in the bran and germ fractions of
cereal grains and in the cell wall of tomato, respectively (Izydorczyk & Biliaderis, 1995; Van
Buren, 1991). This interpretation is consistent with previous studies that have reported an acid-
induced degradation of these compounds at pH ≤ 3 (Krall & McFeeters, 1998).

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Arabinose has been reported to inhibit sucrase activity, regulate postprandial glucose, and modulate gut microbiota (Krog-Mikkelsen et al., 2011; Li et al., 2019). More generally, soluble fibers are also considered to be more beneficial to gut microbiota than insoluble fibers (Guan et al., 2021). Lower gastric release of soluble fibers, such as arabinose, may help explain the lower abundance of gut commensal microbes and decreased microbial diversity observed in PPI users (Jackson et al., 2016). However, more studies on high-fiber substrates are needed to test this hypothesis.

4.4 Gastric lipolysis was not significantly affected under PPI conditions

The absence of any significant difference in lipid digestion between PPI and Standard conditions (Figure 7) can be explained by the activity profile of rabbit gastric lipase (RGL), which may be considered to be not so much pH-dependent in the range of pH from 6 to 3.5. RGL exhibits a pH optimum in the pH range from 6 to 4, depending on the substrates, with a reduced but non-zero activity at pH 3 (Moreau et al., 1988). However, human gastric lipase (HGL) has a pH activity profile shifted about 1 pH unit higher than RGL. For instance, the optimal pH of HGL on long-chain TAG is approximately 5, compared to 4 for RGE, and around 6 for short and medium-chain TAG, compared to 5 for RGE, (Gargouri et al., 1989; Moreau et al., 1988). In humans, we may thus hypothesize that the sustained higher postprandial gastric pH induced by PPI medications could promote greater gastric lipid hydrolysis. This hypothesis is supported by findings in Equidae, where higher rates of lipid absorption were observed under PPI conditions (Mélo et al., 2014). Nevertheless, additional investigations in humans are necessary to substantiate or invalidate this hypothesis.

4.5 The release of minerals was reduced under PPI conditions

Emmental cheese accounted for 96.6% of the total Ca content of the test meal (Figure 3). In cheese, calcium is predominantly present as micellar Ca, namely as colloidal calcium phosphate
531 (Guinee, 2009). The majority of micellar Ca is solubilized when the pH decreases from 532 approximately 6.5 to 2 (Le Graet & Gaucheron, 1999), which explains the progressive release 533 of Ca during gastric digestion as the pH decreased under all conditions, as well as the higher 534 release observed in the more acidic Standard conditions (Figure 8a). The lack of difference 535 between Standard and cStandard conditions indicates that acidification is the primary factor 536 influencing Ca release. However, the significantly higher Ca release under PPI than under cPPI 537 conditions also suggests a possible positive effect of proteolysis on Ca release.

538 The proportion of P contributed by cheese and bread to the test meal was 76.7% and 21.9%, 539 respectively (Figure 3). In Emmental cheese, organic protein-bound P accounts for 540 approximately one-third of the total P content, with the remaining two-thirds being inorganic 541 P, which is typically present in the form of calcium phosphate within casein micelles (Lucey & 542 Fox, 1993). In bread, organic P originates from whole-grain wheat (70-75%), while inorganic 543 P originates from the whole-grain wheat and phosphate additives (Saha et al., 1994). The release 544 of inorganic P is a non-enzymatic process that is enhanced in an acidic environment (Le Graet 545 & Gaucheron, 1999). This may explain, at least in part, the progressive release of P under 546 Standard and PPI conditions, with or without enzymes (Figure 8c), as well as the higher release 547 in Standard conditions compared to PPI conditions. However, the lower release of P in the 548 absence of enzymes indicates that acidification was not the sole cause of P release. The 549 discrepancy between digestion conditions and their corresponding no-enzyme controls may be 550 attributed to the organic form of P, derived from casein, which can be released along with the 551 protein fraction into the supernatant during digestion by pepsin.

552 In contrast to Ca and P, the primary source of Mg was bread, accounting for 60% of the total, 553 followed by cheese, accounting for 35.7% (Figure 3). Magnesium in whole grains is complexed 554 with phytic acids, proteins, and fibers (Brouns, 2021; Oury et al., 2006), while in cheese, it is 555 primarily present in casein micelles (Le Graet & Gaucheron, 1999). The release kinetics of Mg
during gastric digestion were highly similar to those of P (Figure 8). The release was slower
under PPI conditions than under Standard conditions, with gastric acidification identified as a
key influencing factor. The acceleration of Mg release from both fibers and caseins in acidic
conditions has been documented (Camire & Clydesdale, 1981; Le Graet & Gaucheron, 1999).
Furthermore, as with P, the contrasting trends in Mg release in the presence and absence of
gastric enzymes suggest that proteolysis by pepsin also contributed to Mg release.

The findings of this study demonstrated that both PPI-induced gastric hypoacidity and the
subsequent reduction in pepsin activity can constrain the release of minerals (Ca, P, and Mg).
These *in vitro* data align with clinical findings on reduced mineral absorption in PPI users, and
may explain some of the side effects observed. With regard to Ca and P, which are critical for
bone health, our findings align with the reported correlation between chronic PPI use and an
increased risk of bone fractures (Ito & Jensen, 2010). This correlation has been attributed to
insufficient Ca and P absorption during prolonged PPI use, although further research is needed
to confirm this hypothesis (Philippoteaux et al., 2024). Additionally, the reduced gastric release
of Mg under PPI conditions may contribute to the development of Mg deficiency, which is
particularly relevant given that hypomagnesemia (serum magnesium concentration <
0.7 mmol/L) is a common side effect of PPI use (Gommers et al., 2022; US Food and Drug
Administration, 2011).

## 4.6 Limitations of the study and perspectives

The adaptation of the INFOGEST semi-dynamic digestion protocol, proposed and tested in the
present study, offers a relevant approach to investigate the consequences of PPIs on digestion,
given the widespread use of these drugs. Nevertheless, this study represents a first investigation
with inherent limitations. A significant limitation of the present study is that the intestinal phase
of digestion was not included. Consequently, it was not possible to ascertain whether the
580 activity pancreatic enzymes might compensate for the consequences observed during the gastric
581 phase. It can thus be hypothesized that the side effects of PPI use on protein digestion, sugar
582 and mineral release may be less pronounced than suggested by the current results. The most
583 prominent hypothesis for the impact of PPI use on mineral absorption, particularly calcium, is
584 that reduced gastric acidity impairs intestinal absorption (Kopic & Geibel, 2013). Other
585 questions pertain to the impact of PPI use on the fate of other foods and food components, such
586 as soluble fibers and vitamins. For example, given the relatively low fiber content of the test
587 meal in this study, it is pertinent to ascertain whether gastric acidity, even under Standard
588 conditions, is sufficient to facilitate the hydrolysis of fibers and the release of beneficial sugars,
589 such as arabinose, when consuming high-fiber foods.

590 Building upon the *in vitro* protocol proposed here, future studies should thus incorporate the
591 intestinal phase of digestion to assess nutrient bioaccessibility and bioavailability throughout
592 the entire gastrointestinal tract. Expanding this approach to other foods and food components
593 will also be valuable. *In vitro* studies, being highly complementary to *in vivo* studies, may help
594 elucidate the mechanisms behind the side effects of PPI use and inform strategies to mitigate
595 these effects, promoting the health of individuals on PPI medication.

596 **5 Conclusion**

597 We have proposed and developed a novel adaptation of the INFOGEST semi-dynamic *in vitro*
598 gastric digestion protocol to model the effects of proton pump inhibitors (PPIs) on the
599 postprandial gastric pH, and on the flow rate and the acidity of gastric secretions. The model
600 was used to assess the effects of gastric conditions associated with PPI use on the digestion of
601 a test meal comprising cheese, bread, and tomato. The present study demonstrates that the
602 hydrolysis of proteins and the release of certain sugars (fructose and arabinose) and minerals
603 (Ca, P, and Mg) are likely to be reduced during gastric digestion in individuals using PPIs. One
hypothesis, yet to be confirmed, could be that the gastric hypo-acidity observed under PPI conditions may decrease pepsin activity and reduce the solubilization of certain minerals and sugars. In addition, although the size of food particles was not quantified in this study, gastric hypoacidity may also impair their disintegration. These observations are, furthermore, consistent with some known or presumed side effects of PPIs, including hypomagnesemia, bone fractures, muscle loss, and vitamin B₁₂ deficiency. Future research building on this *in vitro* protocol could investigate the effects of PPIs on nutrient bioaccessibility and bioavailability throughout the gastrointestinal tract, providing insights into the mechanisms underlying PPI side effects and informing strategies for their mitigation.
613 Acknowledgments

614 This project received funding from the European Union's Horizon 2020 research and

615 innovation program under the Marie Skłodowska-Curie grant agreement No 899546 and

616 Bretagne region (France).
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<figure><img src="image_26.png" /></figure>

891

892 Figure 1. Postprandial gastric pH in human clinical trial participants administered PPIs. Data
893 were pooled from 18 published clinical trials (Armbrecht et al., 1997; Brunner et al., 1995;
894 Chiverton et al., 1992; Funaki et al., 2013; Furuta, 2001; Hongo et al., 1998; Inoue & Nakamura,
895 1995; Johnson et al., 2005; Labenz et al., 1996; Lee et al., 2022; Metz et al., 2008; Müssig et
896 al., 1997; Sugimoto et al., 2004, 2006, 2010; Varannes et al., 1994; Verdú et al., 1994; Yi et al.,
897 2017). Linear regression was performed between gastric pH and time after meal ingestion (pH
898 = -0.0057 × t + 5.24, R = 0.45).
<figure><img src="image_27.png" /><figcaption>Figure 2. Illustration of the instrument configuration used to perform the *in vitro* semi-dynamic gastric digestion simulations.</figcaption></figure>
<figure><img src="image_28.png" /></figure>

Figure 3. Relative contributions of cheese, bread, and tomato to the total amount of proteins, carbohydrates, lipids, calcium, phosphorus, and magnesium in the test meal substrate. Mineral content in each food item was measured in this study, while protein, carbohydrate, and lipid content for cheese and bread were obtained from food labels, and for tomato, from the Ciqual database (https://ciqual.anses.fr/#/aliments/20047/tomato-raw).
<figure><img src="image_29.png" /><figcaption>Figure 4. Dynamic evolution of the added volume of simulated gastric acid fluid (a, SGAF), the gastric pH (b), and the gastric content volume (c) during digestion under standard digestion condition (dark blue line, ‘Standard’), control standard condition without enzymes (light blue dotted line, ‘cStandard’), PPI digestion condition (red line, ‘PPI’), and control PPI condition without enzymes (orange dotted line, ‘cPPI’). Data are means ± SD of 3 replicates.</figcaption></figure>
<figure><img src="image_30.png" /></figure>

Figure 5. Quantity of free amino (-NH₂) groups in gastric digesta supernatants during simulated digestion of a test meal comprising bread, cheese, and tomato, under standard digestion condition (dark blue bar, ‘Standard’), control standard condition without enzymes (light blue bar, ‘cStandard’), PPI digestion condition (red bar, ‘PPI’), and control PPI condition without enzymes (orange bar, ‘cPPI’). Data are means ± SD of 3 replicates. The symbols ‘*’, ‘#’, and ‘+’ indicate significant differences (p < 0.05) between Standard and PPI, Standard and cStandard, and PPI and cPPI, respectively. Results of the statistical analysis comparing changes over time within each condition are presented in the section 3.3.
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>
Figure 6. Mass of maltose (a), glucose (b), fructose (c), and arabinose (d) in gastric digesta
supernatants during simulated digestion of a test meal comprising bread, cheese, and tomato,
under standard digestion condition (dark blue bar, ‘Standard’), control standard condition
without enzymes (light blue bar, ‘cStandard’), PPI digestion condition (red bar, ‘PPI’), and
control PPI condition without enzymes (orange bar, ‘cPPI’). Data are means ± SD of 3
replicates. The symbols ‘*’, ‘#’, ‘+’, and ‘†’ indicate significant differences (p < 0.05) between
Standard and PPI, Standard and cStandard, PPI and cPPI, and cStandard and cPPI, respectively.
Results of the statistical analysis comparing changes over time within each condition are
presented in the section 3.4.
<figure><img src="image_35.png" /><figcaption>Figure 7. Evolution of the lipid composition, including triacylglycerol (TAG), free fatty acid (FFA), diacylglycerol (DAG), and monoacylglycerol (MAG), in the gastric digesta samples during simulated digestion of a test meal comprising bread, cheese, and tomato, under standard digestion condition (blue bar, ‘Standard’) and PPI digestion condition (red bar, ‘PPI’). Data are means ± SD of 3 replicates. The symbol '*' indicates significant differences (p < 0.05) between Standard and PPI at a given time. Results of the statistical analysis comparing changes over time within each condition are presented in the section 3.5.</figcaption></figure>
<figure><img src="image_36.png" /></figure>

942

Figure 8. Mass (a, c, e) and percentages (b, d, f) of calcium (a, b), phosphorus (c, d) and magnesium (e, f) in gastric digesta supernatants during simulated digestion of a test meal comprising bread, cheese, and tomato, under standard digestion condition (dark blue bar, ‘Standard’), control standard condition without enzymes (light blue bar, ‘cStandard’), PPI digestion condition (red bar, ‘PPI’), and control PPI condition without enzymes (orange bar, ‘cPPI’). Data are means ± SD of 3 replicates. The symbols ‘*’, ‘#’, ‘+’, and ‘†’ indicate significant differences (p < 0.05) between Standard and PPI, Standard and cStandard, PPI and cPPI, and cStandard and cPPI, respectively. Results of the statistical analysis comparing changes over time within each condition are presented in the section 3.6.