# Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy

Juliane Floury, Tiago Bianchi, Jonathan Thévenot, Didier Dupont, Frédéric Jamme, Evelyne Lutton, Maud Panouille, François Boue, Steven Le Feunteun

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## To cite this version:

Juliane Floury, Tiago Bianchi, Jonathan Thévenot, Didier Dupont, Frédéric Jamme, et al. Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy. Food Chemistry, 2018, 239, pp.898-910. 10.1016/j.foodchem.2017.07.023. hal-01580650

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Submitted on 25 Apr 2025

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This file presents the 'accepted version' of the following original research article:

Floury, J., Bianchi, T., Thévenot, J., Dupont, D., Jamme, F., Lutton, E., Panouillé, M., Boué, F., & Le Feunteun, S. (2018). Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy. Food Chemistry, 239, 898-910. https://doi.org/10.1016/j.foodchem.2017.07.023
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Exploring the breakdown of dairy protein gels during *in vitro* gastric digestion

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using time-lapse synchrotron deep-UV fluorescence microscopy

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Juliane Floury¹*, Tiago Bianchi², Jonathan Thévenot¹, Didier Dupont¹, Frédéric Jamme³, Evelyne

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Lutton⁴, Maud Panouillé⁴, François Boué⁴, Steven Le Feunteun⁴.

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¹ UMR STLO, Agrocampus Ouest, INRA, 35000 Rennes, France

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² IRTA-Food Industries, Monells, Spain

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³ SOLEIL synchrotron, Gif-sur-Yvette, France

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⁴ UMR GMPA, AgroParisTech, INRA, Université Paris-Saclay, 78850, Thiverval-Grignon, France

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*Corresponding author: juliane.floury@agrocampus-ouest.fr

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**Abstract**

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A novel time-lapse synchrotron deep-UV microscopy methodology was developed that made use of the natural tryptophan fluorescence of proteins. It enabled the monitoring *in situ* of the microstructural changes of protein gels during simulated gastric digestion. Two dairy gels with an identical composition, but differing by the coagulation mode, were submitted to static *in vitro* gastric digestion. The kinetics of gel particle breakdown were quantified by image analysis and physico-chemical analyses of digesta. The results confirm the tendency of rennet gels, but not acid gels, to form compact protein aggregates under acidic conditions of the stomach. Consequently, the kinetics of proteolysis were much slower for the rennet gel, confirming the hypothesis of a reduced pepsin accessibility to its substrate. The particle shapes remained unchanged and the disintegration kinetics followed an exponential trend, suggesting that erosion was the predominant mechanism of the enzymatic breakdown of dairy gels in these experimental conditions.

**Keywords:** gastric digestion, dairy gels, pepsin, synchrotron deep-UV microscopy.
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# 1. Introduction

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Food is not only a collection of nutrients but also a complex mixture of various chemical species as the result of the particular food processing and component interactions (Jacobs & Tapsell, 2007).

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There is today a consensus about the essential role of both the micro- and macro-structural characteristics of foods on their nutritional quality (Bornhorst & Singh, 2014). The effects of food structure are often attributed to the different mechanisms of the digestion of food in the stomach, many of which are not yet fully understood. (Borreani, Llorca, Quiles, & Hernando, 2017; Fang, Rioux,

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Labrie, & Turgeon, 2016; Kong & Singh, 2008; Luo, Boom, & Janssen, 2015; Luo, Borst, Westphal, Boom, & Janssen, 2017; Norton, Wallis, Spyropoulos, Lillford, & Norton, 2014). Numerous studies have considered the mechanisms of food breakdown as a function of the different physicochemical processes occurring during gastric digestion (Drechsler & Ferrua, 2016; Van Wey & Shorten; Ye, Cui,

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Dalgleish, & Singh, 2016). However, in most published studies, the physical observations essentially arise from two aspects: the initial macroscopic features of the ingested food, and the subsequent concentration profiles of released or absorbed nutrients. The most common hypothesis is that enzyme hydrolysis is limited by the accessibility of substrates in solid food particles (Morris &

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Gunning, 2008; Nyemb et al., 2016).

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In the case of gels based on food proteins, processing is known to induce modifications of the protein molecules, which affects their digestibility. For example, *in vitro* experiments have shown that the sensitivity of whey proteins to digestive proteases is higher in filamentous gels than those with a particulate structure (Macierzanka et al., 2012). In addition to the degree of proteolysis, recent studies have also demonstrated that the protein network structure impacts on the nature of generated peptides during *in vitro* digestion of egg white protein aggregates and gels (Nyemb, Guérin-Dubiard, et al., 2014; Nyemb, Jardin, et al., 2014; Nyemb et al., 2016; Nyemb-Diop et al., 2016). Ye, Cui, Dalgleish, & Singh (2016) have also shown by using a human gastric simulator (HGS) that unheated milk forms a dense and tightly knitted structured clot during digestion, whereas

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48 heated milk results in a looser clot with larger voids. In consequence, the digestion of unheated milk
49 led to a decreased rate of casein hydrolysis.

50 The impact of the dairy gel structure on protein digestion has also been demonstrated *in vivo*, with
51 respect to gastric emptying and amino acid bioavailability (Barbé et al., 2013, 2014). In this study
52 using miniature pigs, ingestion of rennet gels (a cheese model) obtained from heated or unheated
53 milks led to a pronounced delay in protein digestion when compared to acid gels (a yogurt model)
54 and differently processed liquid milks. Furthermore, a mathematical model was developed to test
55 whether different gastric retention times could explain the experimental observations. This indeed
56 provided convincing results for the different milks and acid gels, with 90% of ingested proteins
57 recovered in the blood plasma after 12 h of digestion; the model predicted only 25% and 68% of
58 amino-acid recovery in blood plasma after 12 h in the case of the unheated and heated rennet gels,
59 respectively (Le Feunteun et al., 2014). Although these latter values need to be treated with caution,
60 longer gastric retention for rennet gels fits well with previously reported micro-densification of the
61 protein network and the associated increase in stiffness when rennet gels are acidified (Le Feunteun
62 & Mariette, 2008). It is moreover well-known that acid and rennet gels do not have the same
63 structural and physicochemical properties, which explains their different rheological behavior
64 (Aichinger et al., 2003), and different functional properties such as water holding capacity or
65 permeability (Lucey, Tamehana, Singh, & Munro, 2001; Mellema, Heesakkers, van Opheusden, & van
66 Vliet, 2000).

67 The above described findings and considerations lead to several key questions: How does
68 hydrochloric acid modify the microstructure of rennet gel particles? Does the pH rapidly equilibrate
69 within such high buffering particles? Is their longer gastric retention time due to their greater
70 mechanical resistance to stomach contractions or to their higher resistance to peptic hydrolysis?
As underlined by Bornhorst, Gouseti, Wickham, & Bakalis (2016), multiple length scales are involved in food breakdown, mixing and absorption, with interrelated mechanisms between these scales.

Most of the above questions are related to the microscopic scale, for which only few methods are compatible with the monitoring of the structural changes during the course of digestion. Until recently, only a few studies have focused on acid diffusion in food matrices (Kong & Singh, 2011; Marcotte, Grabowski, Karimi, & Nijland, 2012; Van Wey et al., 2014) and the first results for enzyme diffusion (especially pepsin) in food protein gels were only published very recently (Luo et al., 2017; Thévenot, Cauty, Legland, Dupont, & Floury, 2017). In these studies, the diffusivity of fluorescently labeled pepsin (fully inactivated) in whey and in rennet protein gels was quantified, using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP), respectively. They showed that pepsin diffusivity was significantly lower in stronger gels because of the differing obstruction of the protein network. As a consequence of acid diffusion, it is noteworthy that pH gradients inside foods are very difficult to observe even though they are often put forward as an argument to explain different gastric digestion behavior (Dekkers, Kolodziejczyk, Acquistapace, Engmann, & Wooster, 2016; Kong & Singh, 2009b). Indeed, the pH inside the stomach (i) directly affects enzymatic reactions, (ii) varies over time during digestion, and (iii) has been shown to have a significant effect on the degradation and material loss of the food matrix (Van Wey & Shorten, 2014).

However, until now, such phenomena have only rarely been addressed in the published literature, probably due to a lack of appropriate techniques for measuring local pH in the course of digestion.

The present study sought to develop a novel methodology based on time-lapse synchrotron deep-UV microscopy using the natural tryptophan fluorescence of milk proteins (Fox, 1989). It would allow the monitoring in situ of the microstructural changes of protein gels during a simulated gastric digestion.

Using the pH dependence of the tryptophan emission spectra (Albani, 2007), observations of pH gradients within gel particles would be possible. Two dairy gels of identical composition but differing by the method of coagulation (induced by acid or rennet), were used in a static in vitro digestion with the purpose of distinguishing the effect of acidification from the enzymatic action of pepsin.
Concurrently, the kinetics of particle disintegration of the dairy gels during separate static *in vitro*
gastric digestion were characterized using more traditional methods: 1. particle disintegration rates,
2. degree of protein/peptide release in the soluble phase, and 3. SDS-PAGE profiles of the whole
digesta. Combining the results from these two approaches were expected to enable the
identification of the mechanisms of acid and enzymatic food particle breakdown during gastric
digestion.

## 2. Materials and Methods

### 2.1 Materials

Rennet CHY-MAX® 200 M was purchased from Chr Hansen France SAS (Arpajon, France). Pepsin from
porcine gastric mucosa (3200–4500 U/mg protein, Sigma) and other chemicals were purchased from
Sigma-Aldrich, Inc. (St. Louis, USA). Pepsin activity was measured as 2823 (±180) U/mg using
haemoglobin (Hb) as the substrate, where one unit produces a ΔA<sub>280</sub> of 0.001 per minute at pH 2.0
and at 37 °C, measured as TCA-soluble products, (also referred to as “Sigma” or “Anson” pepsin
units) (Minekus et al., 2014). Distilled water was used in all experiments. 1 mL of porcine pepsin stock
solutions were prepared extemporaneously before each digestion experiment and stored on ice
before use. The activity of pepsin stock solutions was adjusted to 78000 and 64000 U/mL for both
the microscopic observations and the static *in vitro* digestion experiments, in order to ensure 2000
U/mL in the final mixture made of dairy gel particles and gastric fluid. Ultra Low Heat (ULH) skim milk
powder (lot no. 78107; 3.5% of humidity, 34% of protein (Nx6.25), 56% of lactose, 8.5% of ash) was
purchased from LACTALIS ingredients (Société Laitière de Pontivy, Le Sourn). A stock solution of
pepstatin A (a pepsin inhibitor) was purchased from Fischer Scientific (Germany) and prepared at a
concentration of 0.5 mg / ml of MeOH.

### 2.2 Fluorescence intensity of dairy proteins at different pH
120 Fluorescence spectra of diluted skim milk (3.4 mg/L of proteins) with a pH varying from 7.0 down to 1.4 (adjusted with 2M hydrochloric (HCl) solution) were acquired at 37°C using a FluoroMax-4 121 spectrofluorimeter (HORIBA Jobin Yvon INC, Longjumeau, France) fitted with a temperature 122 controlled cuvette-holder. Emission spectra were recorded in the 285-800 nm range with an 123 excitation wavelength of 275 nm using 1 cm × 1 cm quartz cuvettes. The Rayleigh scattering 124 harmonic (λ = 550 nm) was eliminated from the recorded spectra and the fluorescence intensities 125 measured above 300 nm were then summed. This analysis was performed to evaluate the natural 126 variations of dairy protein fluorescence intensity as a function of pH applying the same conditions as 127 those to be used during the synchrotron deep-UV microscopy experiments. 128

129 2.3 Preparation of gels

130 Two dairy gels having a similar composition (34 %w/w of proteins) but different microstructures 131 were prepared by rennet and acid coagulation of non-fat milk. 100 g of ULH skim milk powder were 132 rehydrated with water to make up to 1 kg by stirring during 2h at room temperature. Both gels were 133 then prepared from this solution, either by (i) acid coagulation (AG, glucono-δ-lactone (GDL): 2 134 %w/w) held at room temperature overnight or by (ii) rennet coagulation (RG, rennet 1.5 %vol/w) 135 held for 1 h at 37°C in a thermostatic bath and then stored overnight at room temperature. The pH of 136 the acid and rennet gels were 4.0 and 6.6, respectively. 137

137 2.3. Synchrotron deep-UV microscopic observations during static in vitro digestion

138 A full description of the beamline and imaging microscope setup is widely available (Giuliani et al., 139 2009; Jamme et al., 2010; Tawil et al., 2011). The monochromatized synchrotron beam is coupled to 140 a modified full field microscope (Axio Observer Z1, Carl Zeiss GmbH, Germany) fitted with a back- 141 illuminated CCD PXIS 1024-BUV camera (Princeton Instruments, USA). The focusing objective (Zeiss 142 Ultrafluar 10×, Carl Zeiss GmbH, Germany) is axially motorized by the Axio-Observer Z1 microscope. 143 The synchrotron beam exciting the sample may be considered as collimated when entering the
microscope. Based on the maximum flux available at the beamline, the excitation wavelength was monochromatized at 275 nm using an iHR320 monochromator (HORIBA Jobin Yvon INC, Longjumeau, France). A 300 nm dichroic mirror (Semrock, Rochester) and an emission bandpass filter chosen at 340/26 nm (Semrock, Rochester, USA) were used for collection. As Tyrosine is weakly fluorescent around 340 nm, the signal detected was attributed to Tryptophan fluorescence.

The maximum width of the field of view was 1.12 mm using the x10 objective. To enable the observation of an entire gel particle in this field of view, samples of about 1 mm in diameter (representing around 20 µg of the dairy gel) were prepared and dispersed with a small spatula on a quartz coverslip on which a 5 mm height spacer had been previously sealed using nail varnish. To optimize pepsin activity, samples were warmed to 37 °C (PE100, Linkam, UK). To avoid evaporation from the solution during digestion, a second quartz coverslip was sealed on top of the spacer immediately after addition of simulated gastric fluids (Figure SI1). The microscope was then adjusted to focus on just one particle before launching the acquisition procedure. 1024 × 1024 pixel images were automatically recorded using an exposure time of 5 s. Two different types of digestions were simulated in order to distinguish the enzymatic action of pepsin from the effects of the acid environment. For the first, 175 µL of 0.01 M hydrochloric acid was carefully added so to avoid any movement of the chosen particle. Micrographs were taken at a 10 second frequency over a period of about 20 min, an incubation period that enabled the stabilization within the aggregate of acid-induced microstructural rearrangement and fluorescence intensity variation. Following this step, 5 µL of the prepared pepsin stock solution was carefully added and image acquisition was then continued at a 1 min frequency for the 2 h of enzymatic phase. Secondly, to study the simultaneous effect of acidification and enzymatic reaction, 175 µL of the 0.01 M HCl solution and 5 µL of the prepared pepsin solution were carefully added to the dairy gel particles. Each set of digestion experiments was performed in triplicate for both types of gel.

## 2.4. Image analysis
A set procedure for analysis of images generated by the synchrotron DUV microscopy was carried out using a Fiji software package (Schindelin et al., 2012). Binary images were generated by selecting a threshold algorithm from amongst Otsu, Triangle or Yen method from the options in the Fiji software package. Since the image focus on the particles, and the contrast levels between the fluorescent particle and the background were both different depending on the experiment, the selection of the segmentation method was made on a visual validation. The method chosen was that which allowed the clearest visualization of the contours of the particles of interest over the entire sequence of images. The original images were then annotated with the ROI's (Region of Interest) selection taken from the binary counterparts. The area represented by the ROI (denoted "Area") and its mean fluorescence intensity (denoted "MeanF") were measured for each sub-image. A small circular ROI was also selected from a region free of particles in order to estimate the mean gray value of the background (denoted "BackF") in the same series of images. The corrected fluorescence intensity of the particles (denoted "Fluo") were estimated by subtracting the background fluorescence from the mean particle fluorescence intensity:

$$183 \quad \text{Fluo} = \text{MeanF} - \text{BackF} \qquad (\text{eq. 1})$$

To estimate the digestion kinetics of the dairy gel particles under observation, it was postulated that the "Estimated Protein Content" in the particle was proportional to the overall fluorescence intensity, calculated as the product of the particle area (Area) with its mean fluorescence intensity having corrected for the background fluorescence (Fluo):

$$188 \quad \text{Estimated protein content} = \text{Area} \times \text{Fluo} \qquad (\text{eq. 2})$$

For graphical representations, normalized values for all these parameters (particle area, corrected mean fluorescence intensity, and estimated protein content) were calculated by dividing the value at time t by the corresponding initial value (t = 0).
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## 2.5. Static *in vitro* digestions to quantify the microscopic observations

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This second series of *in vitro* digestion experiments was carried out to enable physicochemical analyses of samples. These were conducted with no mechanical agitation, in order to reach conditions similar to those arising during the time-lapse microscopy described above.

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In order to simulate chewing, approximately 250 (±1.0) g of dairy gel were weighted and cut up with a spatula into smaller particles (approximately ≤ 0.5 cm) which allowed the subsequent weighing of 4.0 (±0.1) g of gel in 12 tubes (40 mL Nalgene® centrifuge tubes, Sigma Aldrich). These tubes were then placed on a magnetic stirrer (Variomag Poly, Thermo Fisher Scientific Inc.) and agitated with 4 mm magnets spinning at 800 rpm for 1 min (for the acid gel) or 3 minutes (for the rennet gel). This procedure was sufficient to reduce the particle size to around 1 mm for both gels.

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The digestion was started by adding 28 mL of a 0.01 M HCl solution (pH = 2) to each of the prepared 4 g samples of dairy gel (applying the same 1:8 dilution as used during the microscopy observations) in order to reduce the pH to 3.0 - as recommended by the INFOGEST *in vitro* digestion protocol (Minekus et al., 2014), with subsequent incubation at 37 °C in a water bath. After the acidification phase of 30 min, the suspensions containing the gel particles had reached stable pH values of 3.0 (±0.5). Pepsin activity at pH 3 has been reported to be at 70% and 40% of its maximal activity (around pH 2) according to Piper & Fenton (1965) and Kondjoyan, Daudin, & Santé-Lhoutellier (2015), respectively. 1 mL of a porcine pepsin stock solution of 64000 U/mL was finally added to achieve 2000 U/mL in both final digestion mixtures (Minekus et al., 2014). The enzymatic digestion phase then took place during the following 90 min.

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During the sequential acid and enzymatic digestion phases, the tubes were inverted twice every 3 minutes to avoid sedimentation. For the acidification phase, a tube was removed and placed in ice after each time measurement, whereas during the enzymatic digestion with pepsin, the reaction was stopped in the removed tube by the addition of 10 µL of a pepstatin A solution (0.5 mg/mL MeOH) per mL of digesta before storing the tubes in ice. The 12 prepared tubes were stopped at different

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217 times along the digestion process: (i) after 1, 5, 10, 20 and 30 min for the acidification phase and
218 then (ii) after 31, 35, 40, 50, 60, 90 and 120 min for the enzymatic phase. Two batches of sub-
219 samples were prepared in parallel, one destined for biochemical analysis of the whole digested
220 samples, and the other for centrifugation and analysis of the pellet and supernatant phases. Each set
221 of digestion experiments and analyses was performed in duplicate for the acid gel and in triplicate for
222 the rennet gel.

223 2.6. Sample analysis

224 Immediately after *in vitro* digestion of the dairy gels described above, one of the two batches was
225 stored at -20°C ahead of biochemical analysis. The second batch was centrifuged for 20 min at 20,000
226 g and at 4°C. Supernatants were removed and stored at -20°C until needed for chemical analysis but
227 the pellets were immediately weighed.

228 2.6.1. Pellet mass ratio

229 The pellet mass ratio (PM, %) was defined as:

$$
230 \quad \text{PM}(t) = m_t / m_0 \times 100 \qquad (\text{eq. 3})
$$

231 where m₀ and m_t are the pellet masses (g) obtained from centrifugation before (t = 0) and during (t = t) the digestion experiment. It should be noted that m_t accounts for both the uptake of gastric juice and the loss of its solid components due to digestion and leaching processes.

234 2.6.2. Release of proteins/peptides into the supernatant

235 Supernatant from the centrifuged samples were filtered through 0.2 µm Acrodisc® syringe filters
236 (PALL Corporation, Port Washington), and the absorbance of aromatic amino acids were quantified at
237 280 nm in quartz cuvettes using a UV-visible spectrophotometer (UVmc2, Monaco SAFAS, France). A
238 0.01 M HCl solution was used as the blank. This measurement shows the release of soluble proteins
or peptides from the gel particles into the surrounding liquid phase. The protein/peptide release ratio (PR, %) was defined as:

$$241 \quad PR = (A_t - A_0) / A_0 \times 100 \quad (\text{eq. 4})$$

where A₀ and A_t are the absorbance of the supernatant before (t = 0) and during digestion at time t.

### 2.6.3. Electrophoresis

SDS-PAGE analysis was performed on the thawed whole digesta samples, without centrifugation, using 4-12 % polyacrylamide NuPAGE Novex® Bis-Tris 15-well precast gels (Novex® Life Technologies, California, USA) mounted in a Hoefer™ miniVE vertical electrophoresis system (Pharmacia Biotech, Sweden) following the manufacturer's instructions. Samples were first homogenized with a Q700 Ultrasonic Liquid Processor (Qsonica LLC., Newtown, USA) fitted with a 3.2 mm microtip probe for 45 seconds. Tubes were kept on ice during the sonication step to avoid warming. Samples for protein analysis were then diluted 8-fold with the denaturing buffer (77.975 % 0.08 M Tris-HCl pH 6.8; 20 % glycerol; 2 % SDS; 0.025 % bromophenol blue) under reducing conditions (with a 0.5 M DTT solution and 10 min at 70°C). Samples were then loaded, at a protein concentration of 15 µg of total protein/well, into the sample slots and a marker kit (Mark 12™ Unstained Standard, novex® Life Technologies, California, USA) was used for molecular weight (MW) calibration. Electrophoresis was performed at 200 V and 50 mA during 120 min. Gels were fixed in 30 % (v/v) ethanol and 10 % (v/v) acetic acid solution for 30 min and stained with Coomassie Brilliant Blue G250.
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# 3. Results

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## 3.1. Effect of pH on the fluorescence of dairy proteins

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Figure 1 shows the development of the cumulative fluorescence intensity (above 300 nm) measured by spectrofluorometry at different milk pH, using the natural pH of milk (6.5) as the reference value (100 %). The cumulative fluorescence intensity drops by about 25 % at pH 6.0, then rapidly rises to a maximum value of about 115% at pH 4.4, before decreasing again to reach a plateau value at pH 3.8, corresponding to nearly 40% lower than the reference value.

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## 3.2. Effects of HCl addition

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The mean fluorescence intensity of a particle (i.e. Fluo in eq. 1) can be considered as the result of three factors: 1. the protein density per unit of surface, 2. the dependence of protein fluorescence on the local pH, and 3. photobleaching effects as a function of UV exposure. Figure 2A (video 1 of supplementary data) shows the typical behavior of an acid gel particle after immersion in an HCl solution at pH 2: it remained visually quite stable at the end of 18 minutes. Figure 2B shows the normalized area and mean fluorescence intensity profiles of the particle (obtained by image analysis) for the same gel particle. The area of the particle increased slightly with time whereas the mean fluorescence intensity fell slightly, but no more than 80 % of the initial fluorescence after 20 min. The change in estimated protein content with time is also shown on Figure 2B. The slight apparent loss of matter, i.e. around 13 % after 20 min, can be attributed to the loss of fluorescence due to photobleaching based on the comparison with stable samples (data not shown).

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Figure 3A (video 2 of supplementary data) shows the typical behavior of a rennet gel particle after immersion in the acidic solution at pH 2. Figure 3B shows the corresponding values obtained from image analysis. By contrast with the behavior of the acid gel particle, as soon as the rennet gel particle was in contact with the acid solution, it appears (from a careful visualization of the changing images in video 2) that its outer periphery swelled suddenly and was partly expulsed into the surrounding. However, the large majority of the particle shrank back strongly, leading to a reduction
284 in area of more than 40 % after only 1 min of immersion. Then the particle swelled slightly before
285 shrinking again at 4 min and ended up at 50 % of its initial size after 20 min. Visually, Figure 3A shows
286 that the fluorescence intensity strongly increased, especially in the central part of the particle, before
287 progressively disappearing from the outer side after 4 min. By contrast, Figure 3B shows that the
288 measured mean fluorescence of the particle followed the opposite pattern. It increased during the
289 first minute by about 20%, then decreased by more than 40 % during the progressive disappearance
290 of the inner fluorescence from 1 to 4 min, and then increased again, reaching a plateau close to its
291 initial level after 10 min. With respect to the changing estimated protein content, this decreased
292 continuously during the first 3 min, before stabilizing at nearly 50 % of its initial value.

293 **3.3. Effects of subsequent pepsin addition**

294 Figure 4 (videos 3 and 4 of supplementary data) shows the typical behavior of both gels during the
295 subsequent 2 h pepsin digestion step. Figure 4A shows that the acid gel particles were very rapidly
296 disintegrated. Indeed, although some particles were still distinguishable 20 min after pepsin addition,
297 the fluorescence level of a ROI manually drawn around the residual particle had in reality drastically
298 reduced, by about 80% and then by more than 98 % at 120 min (results not shown). Thus, the
299 automatic image processing started to fail properly identifying the remaining particles at this point.
300 The examination of the quartz cover slip at the end of the digestion experiment revealed that these
301 traces originate from a small amount of protein material adsorbed onto the lamella, a phenomenon
302 that might arise from protein modifications due to UV exposure. The disintegration process was
303 slower for the rennet gel, noting that particles of small initial size were still visible even after 2 h
304 (Figure 4B).

305 Figure 4C shows values derived from image analysis over the duration of the digestion. These results
306 confirm the observations of the images that show that the decrease of both particle area and
307 fluorescence was very rapid and especially pronounced for the acid gel. The rennet gel particle area
308 was largely unchanged during the first 10 min of digestion before it steadily fell, with only 30 % of the
initial area remaining after 2 h of pepsin digestion. The changes of the estimated protein content enable the illustration of the digestion kinetics, which can be fitted to an exponential curve. By applying such models, the protein hydrolysis rate was approximately 10 times slower for the rennet gel than for the acid gel particles.

### 3.5. Digestions in a simulated gastric solution containing both HCl and pepsin

Figure 5 (videos 5 and 6 of supplementary data) shows a typical time-lapse fluorescence imaging of both acid and rennet gels after immersion in an HCl solution at pH 2 containing also 2000 U/mL of pepsin (note that image contrasts were re-adjusted after 30 min to improve visualization). Figure 5A shows that acid gel particles were no longer observable after only 14 min of digestion. The majority of the rennet particle under similar conditions also very rapidly disappeared as well (Figure 5B). Image analysis of these microstructural observations are given graphically in Figure 5C. For the acid gel, the particle area, mean fluorescence intensity, and estimated protein content were all similar to that already observed during the pepsin phase of the sequential protocol (Figure 4C): that is a very rapid decrease in all parameters, indicative of a rapid digestion. For the rennet gel, the changes in all these values were more variable. The particle area first showed a rapid reduction up to about 25 min, followed by a much slower decrease. Its mean fluorescence decreased by more than 75% during the first 5 min, then remained stable for few minutes, before it progressively increased between 25 to 70 min before stabilizing. Two versions of the corresponding estimated protein content are given in Figure 5C for the rennet aggregate. The plain line (RG) corresponds to the results of eq. 2 on the raw data, whereas the dotted line (RG*) assumes a 40% loss of fluorescence during the first 5 min due to the effect of pH falling from 6.5 to less than 4 (Figure 1). Despite this correction, it is still clear that the great majority of the rennet gel particle (~90%) was effectively consumed within the first 20 min of digestion.
332 3.6. Physico-chemical characteristics of gastric digesta

333 Figure 6A shows that the pellet mass ratio (PM) was not greatly modified during the acidification
334 phase of the rennet gel digestion process. Over the same period, there was a slight decrease of the
335 protein/peptide release ratio (PR) in the supernatant phase (Figure 6B). The behavior of the acid gel
336 was different to that of the rennet gel, as the pellet mass ratio increased up to 150 % over the 30 min
337 following the addition of the acid solution, – over the same period, the calculated PR increased by 30
338 %.

339 In less than 1 min after the subsequent addition of pepsin to the acid gel, its PM ratio dropped from
340 150 % to around 55 % (Figure 6A), and the corresponding PR ratio soared from 42 % to 135 % (Figure
341 6B). Both the PM and PR then changed much more slowly with values of 32 % and 200 % noted at the
342 end of the enzymatic process. The behavior of the rennet gel during pepsin digestion was different,
343 as illustrated by the slower rise/fall of the PM/PR ratios, which only reached final values of 50% and
344 90%, respectively at the end of the 2-hour period.

345 Figure 6C shows the results of SDS-PAGE analyses applying the same sequential protocol. For both
346 gels, no visible changes of the bands corresponding to the main milk proteins (caseins, β-
347 lactoglobulin and α-lactalbumin) were noticeable during the first 30 min. Peptide bands did not
348 appear after HCl addition, showing that no hydrolysis occurred during the acidification phase. Figure
349 6C shows an extremely rapid and complete disappearance of casein bands only 1 min after the
350 addition of pepsin to the acid gel, whereas the casein bands vanished much more slowly for the
351 rennet gel. For both types of dairy gel, the bands corresponding to β-lactoglobulin did not change
352 much during the whole process, meaning that this protein was resistant to pepsin hydrolysis. Pepsin
353 was though able to hydrolyze α-lactalbumin, as shown by the disappearance of the corresponding
354 band during the enzymatic phase. Figure 6C also shows a progressive appearance of grey bands
355 between 3 and 10 kDa on the two different SDS-PAGE electrophoresis which can be attributed to an
356 increasing concentration of peptides produced during digestion.
## 4. Discussion

This study aimed at investigating the influence of the method of milk coagulation (i.e., acid- or rennet-induced - both commonly used in industry), on the mechanisms of dairy protein gel breakdown during gastric digestion. Although the two types of gel had the same overall composition, the different means of production led to important differences in the kinetics of protein hydrolysis during simulated static *in vitro* digestion.

### 4.1. Rennet gel undergoes important structural modifications upon acidification

Based on results from the use of synchrotron deep-UV fluorescence, the microstructural changes in acid and rennet dairy protein gels following acidification was greatly dependent on the coagulation method. For the acid-formed gel, which has an initial pH of 4, no significant changes were seen under the microscope. Nevertheless, Figure 6 showed that the pellet mass from centrifugation and the release of proteins into the supernatant during digestion both increased greatly. Visual observations also indicated that the acid gel particles became more “flaky” after immersion in the acid solution (Figure 2 and Video 1). Luo, Boom & Janssen (2015) also observed such a phenomenon during their dynamic *in vitro* digestion experiments of whey protein isolate gels with simulated gastric juice free of pepsin. This was explained by the diffusion of gastric juice into the gel matrix. As the pH inside the particle falls, it progressively reaches the isoelectric pH of caseins (pHi ~ 4.6), which may thus leads to a swelling of the particles induced by the decreased electrostatic attractions. As acidification approaches pH 3, individual casein molecules can even start to solubilize (Post el al. 2012). Both increases of the pellet mass and protein/peptide release ratios, measured during the acidification of the acid gel (Figure 6A and 6B), may thus be attributed to the increasing acidity of its components. Even if the acid gel retained an increasing amount of water during its acidification, the shape and fluorescence intensity of its particles were largely preserved based on the microscopic observations in this study: this suggests a limited molecular rearrangement of the protein network.
On the contrary, the rennet gel underwent major structural modifications during acid incubation, with an initial swelling of the particle periphery and a strong contraction of the inner part, progressively leading to a more compact particle. These results are in agreement with Barbé et al. (2014), who also observed, during a dynamic *in vitro* gastric digestion, that rennet gel formed much denser particles than the acid gel, because of pH-induced syneresis. Physicochemical analysis of the acidified rennet gel showed no significant loss of the total matter nor of protein solubilization. It is known that rennet-induced gels undergo rapid syneresis if disturbed by cutting or by wetting the gel surface, a rearrangement process that is accelerated and more extensive at high temperatures and lower pH values. This contrasts with most acid-formed gels, which do not show a great deal of spontaneous syneresis (Mellema et al., 2000; Walstra, 1993). Published work on the progressive acidification of dairy samples suggest that a number of phenomena take place as pH falls. Among them, solubilization of the colloidal calcium phosphate (CCP) nanocrystals, which usually occurs between pH ~ 5.9 and 4.9, is of interest because they are directly involved within the nano-structural organization of the protein network in rennet gels (but not in acid gels). Casein micelles contained in milk (from which the rennet protein network is formed), therefore undergo internal structural reorganization upon acidification in this pH range. This is thought to be the main cause for the observed swelling of the casein particles (Famelart, Lepesant, Gaucheron, Graet, & Schuck, 1996). On progressing below pH = 4.9, the amount of water contained by the casein particles sharply decreases because of the reinforcement of electrostatic attraction when approaching the isoelectric pH of caseins (pHi ~ 4.6) (Roefs, Walstra, Dalgleish, & Horne, 1985). Based on the available literature, it is therefore not surprising to observe an intense structural evolution, progressively leading to a compaction and densification of the protein network, when rennet gel particles are brought into a stronger acidic environment.

In addition to revealing these structural modifications, the deep-UV fluorescence technique enabled a study of acidification within the aggregates at the microscale: this arises from monitoring changes in the fluorescence intensity of a particle as the experiment proceeds. Indeed, the environmental pH
has a known large impact on the tryptophan fluorescence intensity of dairy proteins (Herbert et al 1999). Figure 1 shows that the natural fluorescence intensity of dairy proteins increases by approximately 20% when reducing the pH from 6.5 to around 4.5, before abruptly losing around a third of intensity when implementing a further decrease of the pH from 4.5 to 4. This dependency of the protein fluorescence intensity to pH can indeed explain the spatial variations of fluorescence intensity observed in the rennet particle during the first minutes of acidification (Figure 3A). From 1 to 3 min, the highly fluorescent zone observed would correspond to a region of the aggregate where the pH is still between 5.5 and 4.5 (i.e. the plateau in Figure 1). Meanwhile the dark outer layer of the particle would correspond to regions where the pH is less than 4. In other words, these spatial variations of fluorescence reflect the pH gradients within the particle. The subsequent increase of the particle fluorescence, beyond 4 min of exposure to acid conditions, can only be attributed to the progressive shrinking of the particle with a corresponding rise in protein concentration: this is confirmed by the observed corresponding decrease of its area whilst the estimated protein content remains constant at about 55 % (Figure 3B). This latter value is a little less than the ~ 65 % expected from the pH effects on fluorescence (Figure 1). This slight discrepancy can be attributed to the effects of both photobleaching (as for the acid gel, Figure 2B) and the previously reported small loss of matter from the outer periphery immediately after the addition of HCl. Overall, these latter results illustrate the great potentiality of the developed methodology to study gastric digestion of foods, and more especially in order to investigate the evolution of pH gradients within protein particles.

427

## 4.2. Hydrolysis kinetics of the rennet gel by pepsin is much slower than that of the acid gel.

Three methods of study: using DUV fluorescence (synchrotron microscopy), electrophoresis (SDS-PAGE) and the analysis of the pellets and supernatants after centrifugation – they all show that the acid gel particles were broken down much more rapidly than rennet gel particles during the pepsin phase of the 2-part digestion protocol. Acid gel particles were hydrolysed just a few minutes after the
addition of pepsin (Figure 4A and 6C), with a corresponding very high rate of protein/peptide release (Figure 6B). In contrast, the particles of the rennet gels were still visible even after 2 h of digestion (Figure 4B), with a corresponding much slower rate of protein/peptide release and a lower final concentration of this digestion product (Figure 6B). For both types of gel, it is also noteworthy that the initial morphology of particles upon pepsin digestion remained almost unchanged during the simulated gastric phase observed using the DUV fluorescence imaging (Figure 4A and 4B). This suggests that the predominant mechanism was surface erosion, an interpretation consistent with the observed exponential model (Figure 4C), which were previously shown to adequately describe the breakdown of solid food particles during gastric digestion (Kong & Singh, 2009a, 2009b, 2011).

It was postulated that the internal bonds between the casein micelles were weakened by the penetration of the acid solution into the acid-formed gel particle, hence facilitating pepsin hydrolysis, which was rapid. The behavior of the rennet gel was completely different, due to the restructuring and subsequent contraction of the particles (syneresis) during the acidification stage. In other words, instead of weakening the internal bonds, the acid solution increased the resistance of the rennet gel protein network to pepsin action, either by limiting the diffusion of pepsin within the gel particle, and/or by reducing accessibility of the protein substrate to the enzyme active site (because of tightened and strengthened protein strands and nodes). This latter explanation fits in better with other published work (Kong & Singh, 2008). Moreover, it is well-known that during the aging of rennet gel particles merge and reorganize, resulting in the formation of straighter and denser strands, thus with more bonds per casein micelles, and hence stronger interactions (Mellema et al., 2000; Mellema, Walstra, van Opheusden, & van Vliet, 2002). This phenomenon increases if a progressive acidification occurs during this aging phase (Le Feunteun and Mariette, 2008). Ye et al. (2016) reported that the intense coagulation of milk in the acidic environment of the stomach probably strongly hinders the gastric fluid from diffusing into the particles formed. Luo et al. (2015) compared the kinetics of digestion of egg white protein with whey protein isolate solutions and gels using simulated gastric conditions. They showed that the barrier of the protein gel structure did not
460

simply slow down the reaction rates (compared to soluble protein digestion), but it also altered the mechanisms of enzymatic hydrolysis. They postulated that the difference in activity observed stemmed from the steric hindrance of gel structure to the diffusion of pepsin and its hydrolysates.

461

462

463

Indeed, Thévenot et al. (2017) showed that the diffusion coefficient of pepsin (of about 100 µm²/s in water), decreased from 51 to 21 µm²/s in rennet gels as the casein concentration was increased from

464

465

3.2 to 13 % w/w. Luo et al. (2017) also reported that pepsin hydrolysis in 15 to 20 % w/w whey protein gels was constrained to a 2 mm layer at the surface of the gel, even after 6 h of incubation in a gastric solution. They suggested that since pepsin had only a limited penetration depth, digestion

466

467

468

processes were largely constrained by the exposed surface area of the gel particles, and hence by the surface erosion rate. All of these results support the argument that the principal mode of pepsin

469

470

action during static *in vitro* digestion experiments is the hydrolysis of the proteins located at the surface of the particles. Thus for whey protein gels, Luo *et al.* (2017) concluded that the mode of gel

471

472

digestion was determined by the combined effect of diffusion limitation, hydrolysis rate and microstructure transformation.

473

474

When acid and pepsin were simultaneously added to gel particles, the kinetics of breakdown for both gels followed an exponential model with well-preserved particle shapes (Figure 5A and B), suggesting that their digestion was again dominated by a surface erosion process. The hydrolysis rate of rennet gel particles was still slower than for acid gel, but the difference was greatly reduced. As illustrated by the residual particle in Figure 5B (which resisted digestion well even after 120 minutes), acid-

475

476

477

478

479

induced syneresis of the rennet particle also occurred after immersion within the HCl + pepsin solution. However, by the time this process had ended, most of the initial particle was lost (~ 90 %)

480

481

because of the presence of pH-activated pepsin ready to hydrolyze the protein network from the beginning of the digestion process. Such mechanisms may also be enhanced by the swelling of the

482

483

outer layer of the rennet gel particle when brought in contact with acid. Indeed, if the periphery of the particle swells, protein substrate should be more readily in contact with the pepsin, and thus more accessible to the enzyme action. Acid diffusion and enzymatic reactions appear to act together,

485
486 thereby accelerating the hydrolysis rate of the rennet gel to almost the same level as that observed
487 for the acid gel.

488 The remarkable resistance of the small residual rennet particle to pepsin digestion nevertheless
489 opens questions on the *in vivo* reality and possible consequences of any implied mechanisms.
490 Indeed, gastric juice is secreted by the stomach wall, and must spread throughout the gastric
491 contents to enable pepsin hydrolysis. Since the small hydrogen ions diffuse more rapidly than pepsin,
492 the *in vivo* timeframe of pH-induced effects and of pepsin action on a stomach content made up of
493 rennet particles might lie somewhere in between the purely sequential and purely simultaneous
494 digestion protocols simulated in this study. Observations showing the rapid formation of an acidified
495 rennet particle that is highly resistant to pepsin hydrolysis therefore constitutes a likely mechanism
496 which can explain the pronounced slowdown of amino-acid absorption observed during *in vivo*
497 digestions in mini-pigs of rennet gels when compared to acid gels (Barbé et al., 2014).

498 5. Conclusion

499 Time-lapse synchrotron deep-UV fluorescence microscopy enabled the study in real time and with a
500 high resolution of the changes in food particles during the digestion process. It showed the
501 propensity of rennet gels, (but not acid gels), to undergo large microstructural modifications
502 (syneresis), leading to compact protein aggregates in the acidic conditions of gastric digestion. The
503 resulting structure of the particle reduces the accessibility of pepsin to the protein substrate, which
504 most probably prolongs the gastric phase, as first suggested from previous *in vivo* results. The shape
505 of particles remained unchanged during the simulated digestion process, and the kinetics of
506 disintegration were adequately fitted to an exponential model, suggesting that erosion was the
507 predominant mechanism of the breakdown of dairy gels during the simulated gastric digestion based
508 on static hydrodynamic conditions. These findings help describe how the food structure can affect
509 subsequent digestion kinetics. In this respect, the time-lapse deep-UV microscopy is well adapted to
510 such studies. There is now a real need to develop or adapt methods that will allow the monitoring of
the changes in the micro- and nanostructure of food in the course of its digestion: this will allow a better understanding of the mechanism of enzyme action during digestion of food matrices.

## 513 ACKNOWLEDGMENTS

The authors are grateful to the team running the SOLEIL synchrotron that provided beam time and efficient support, under project proposals n°20140065 and 20150452. They also acknowledge Tiago Bianchi who awarded a mobility PhD grant from the National Institute for Agricultural and Food Research and Technology (INIA) – Spanish Ministry of Economy and Competitiveness.

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655
656 **Figure captions**

657 Figure 1. pH dependence of the fluorescence intensity of dairy proteins, under the same conditions
658 as those used for deep-UV microscopy (Cumulative fluorescence intensity above 300 nm with an
659 excitation at 275 nm)

660 Figure 2. (A) Typical sequence of fluorescence images of the acid gel (AG) after immersion in
661 hydrochloric acid at pH 2. The yellow line denotes the ROI's selection with Otsu thresholding method
662 (FIJI software package). The scale bar is 100 µm. (B) The corresponding normalized evolution of the
663 particle area (●●●●), its mean fluorescence intensity (— —), and its protein content (—), all derived
664 from the image analysis of the ROI selections.

665 Figure 3. (A) Typical sequence of fluorescence images of the rennet gel (RG) after immersion in
666 hydrochloric acid at pH 2. Yellow lines correspond to ROI's selections with Triangle thresholding
667 method (FIJI software package). The scale bar is 100 µm. (B) The corresponding normalized evolution
668 of the particle area (●●●●), its mean fluorescence intensity (— —), and its protein content (—), all
669 derived from the image analysis of the ROI selections.

670 Figure 4. Typical sequence of fluorescence images of the (A) Acid Gel and (B) Rennet Gel during the
671 pepsin digestion phase at pH 2. Yellow lines correspond to ROI's selections with Yen thresholding
672 method (FIJI software package). The scale bar is 100 µm. (C) Corresponding changes with time of the
673 particle area, its mean fluorescence intensity, and its protein content, all derived from image analysis
674 of the ROI selections. All values have been normalized to unity at time zero. The thin dotted lines
675 represent the exponential fittings of the data.

676 Figure 5. Typical sequence of fluorescence images of the (A) Acid Gel and (B) Rennet Gel particles as a
677 function of digestion time in a simulated gastric juice: Hydrochloric and pepsin solution at pH 2. The
678 yellow lines correspond to ROI's selections with Triangle thresholding method (FIJI software
679 package). The scale bar is 100 µm. The image contrast was adjusted after 30 min of observation. (C)
Corresponding changes with time of the particle area, mean fluorescence intensity, and protein content obtained by image analysis of the ROI selections. All values have been normalized to unity at time zero. RG* corresponds to the corrected protein content when correcting for the 40% loss of fluorescence intensity previously observed during the acidification of the rennet gel.

Figure 6. Changes with time of (A) pellet mass ratio (%), (B) Protein/peptide release (%), (C) SDS-PAGE protein profile for both the acid (AG) and rennet (RG) gels. The first 30 min correspond to the acidification phase (with an HCl solution at pH 2), before pepsin addition.

Figure S1. Experimental set up used for tryptophan fluorescence imaging.
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<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure3
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<figure><img src="image_7.png" /><figcaption>Figure4
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<figure><img src="image_8.png" /><figcaption>Figure5
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<figure><img src="image_9.png" /><figcaption>Figure6
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