Food Chemistry 239 (2018) 898–910

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Food Chemistry

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Exploring the breakdown of dairy protein gels during in vitro gastricdigestion using time-lapse synchrotron deep-UV fluorescencemicroscopy

http://dx.doi.org/10.1016/j.foodchem.2017.07.0230308-8146/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: juliane.floury@agrocampus-ouest.fr (J. Floury).

Juliane Floury a,⇑, Tiago Bianchi b, Jonathan Thévenot a, Didier Dupont a, Frédéric Jamme c, Evelyne Lutton d,Maud Panouillé d, François Boué d, Steven Le Feunteun d

aUMR STLO, Agrocampus Ouest, INRA, 35000 Rennes, Franceb IRTA-Food Industries, Monells, Spainc SOLEIL Synchrotron, Gif-sur-Yvette, FrancedUMR GMPA, AgroParisTech, INRA, Université Paris-Saclay, 78850 Thiverval-Grignon, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2017Received in revised form 3 July 2017Accepted 7 July 2017Available online 8 July 2017

Keywords:Gastric digestionDairy gelsPepsinSynchrotron deep-UV microscopy

A novel time-lapse synchrotron deep-UV microscopy methodology was developed that made use of thenatural tryptophan fluorescence of proteins. It enabled the monitoring in situ of the microstructuralchanges 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 ofgel particle breakdown were quantified by image analysis and physico-chemical analyses of digesta. Theresults confirm the tendency of rennet gels, but not acid gels, to form compact protein aggregates underacidic conditions of the stomach. Consequently, the kinetics of proteolysis were much slower for the ren-net gel, confirming the hypothesis of a reduced pepsin accessibility to its substrate. The particle shapesremained unchanged and the disintegration kinetics followed an exponential trend, suggesting that ero-sion was the predominant mechanism of the enzymatic breakdown of dairy gels in these experimentalconditions.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Food is not only a collection of nutrients but also a complexmixture of various chemical species as the result of the particularfood processing and component interactions (Jacobs & Tapsell,2007). There is today a consensus about the essential role of boththe micro- and macro-structural characteristics of foods on theirnutritional quality (Bornhorst & Singh, 2014). The effects of foodstructure are often attributed to the different mechanisms of thedigestion of food in the stomach, many of which are not yet fullyunderstood (Borreani, Llorca, Quiles, & Hernando, 2017; Fang,Rioux, Labrie, & Turgeon, 2016; Kong & Singh, 2008; Luo, Boom,& Janssen, 2015; Luo, Borst, Westphal, Boom, & Janssen, 2017;Norton, Wallis, Spyropoulos, Lillford, & Norton, 2014). Numerousstudies have considered the mechanisms of food breakdown as afunction of the different physicochemical processes occurring dur-ing gastric digestion (Van Wey & Shorten, 2014; Ye, Cui, Dalgleish,& Singh, 2016). However, in most published studies, the physical

observations essentially arise from two aspects: the initial macro-scopic features of the ingested food, and the subsequent concentra-tion profiles of released or absorbed nutrients. The most commonhypothesis is that enzyme hydrolysis is limited by the accessibilityof substrates in solid food particles (Morris & Gunning, 2008;Nyemb et al., 2016).

In the case of gels based on food proteins, processing is knownto induce modifications of the protein molecules, which affectstheir digestibility. For example, in vitro experiments have shownthat the sensitivity of whey proteins to digestive proteases ishigher 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 networkstructure impacts on the nature of generated peptides duringin vitro digestion of egg white protein aggregates and gels(Nyemb et al., 2014; Nyemb et al., 2016; Nyemb-Diop et al.,2016). Ye et al. (2016) have also shown by using a human gastricsimulator (HGS) that unheated milk forms a dense and tightly knit-ted structured clot during digestion, whereas heated milk results ina looser clot with larger voids. In consequence, the digestion ofunheated milk led to a decreased rate of casein hydrolysis.

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The impact of the dairy gel structure on protein digestion hasalso been demonstrated in vivo, with respect to gastric emptyingand amino acid bioavailability (Barbé et al., 2013; Barbé et al.,2014). In this study using miniature pigs, ingestion of rennet gels(a cheese model) obtained from heated or unheated milks led toa pronounced delay in protein digestion when compared to acidgels (a yogurt model) and differently processed liquid milks. Fur-thermore, a mathematical model was developed to test whetherdifferent gastric retention times could explain the experimentalobservations. This indeed provided convincing results for the dif-ferent milks and acid gels, with 90% of ingested proteins recoveredin the blood plasma after 12 h of digestion; the model predictedonly 25% and 68% of amino-acid recovery in blood plasma after12 h in the case of the unheated and heated rennet gels, respec-tively (Le Feunteun et al., 2014). Although these latter values needto be treated with caution, longer gastric retention for rennet gelsfits well with previously reported micro-densification of the pro-tein network and the associated increase in stiffness when rennetgels are acidified (Le Feunteun & Mariette, 2008). It is moreoverwell-know that acid and rennet gels do not have the same struc-tural and physicochemical properties, which explains their differ-ent rheological behavior (Aichinger et al., 2003), and differentfunctional properties such as water holding capacity or permeabil-ity (Lucey, Tamehana, Singh, & Munro, 2001; Mellema, Heesakkers,van Opheusden, & van Vliet, 2000).

The above described findings and considerations lead to severalkey questions: How does hydrochloric acid modify the microstruc-ture of rennet gel particles? Does the pH rapidly equilibrate withinsuch high buffering particles? Is their longer gastric retention timedue to their greater mechanical resistance to stomach contractionsor to their higher resistance to peptic hydrolysis?

As underlined by Bornhorst, Gouseti, Wickham, and Bakalis(2016), multiple length scales are involved in food breakdown,mixing and absorption, with interrelated mechanisms betweenthese scales. Most of the above questions are related to the micro-scopic scale, for which only few methods are compatible with themonitoring of the structural changes during the course of diges-tion. Until recently, only a few studies have focused on acid diffu-sion in food matrices (Kong & Singh, 2011; Van Wey & Shorten,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 thesestudies, the diffusivity of fluorescently labeled pepsin (fully inacti-vated) in whey and in rennet protein gels was quantified, using flu-orescence correlation spectroscopy (FCS) and fluorescencerecovery after photobleaching (FRAP), respectively. They showedthat pepsin diffusivity was significantly lower in stronger gelsbecause of the differing obstruction of the protein network. As aconsequence of acid diffusion, it is noteworthy that pH gradientsinside foods are very difficult to observe even though they areoften put forward as an argument to explain different gastric diges-tion behavior (Dekkers, Kolodziejczyk, Acquistapace, Engmann, &Wooster, 2016; Kong & Singh, 2009b). Indeed, the pH inside thestomach (i) directly affects enzymatic reactions, (ii) varies overtime during digestion, and (iii) has been shown to have a signifi-cant effect on the degradation and material loss of the food matrix(Van Wey & Shorten, 2014). However, until now, such phenomenahave only rarely been addressed in the published literature, prob-ably due to a lack of appropriate techniques for measuring local pHin the course of digestion.

The present study sought to develop a novel methodologybased on time-lapse synchrotron deep-UV microscopy using thenatural tryptophan fluorescence of milk proteins (Fox, 1989). Itwould allow the monitoring in situ of the microstructural changesof protein gels during a simulated gastric digestion. Using the pHdependence of the tryptophan emission spectra (Albani, 2007),

observations of pH gradients within gel particles would be possi-ble. Two dairy gels of identical composition but differing by themethod of coagulation (induced by acid or rennet), were used ina static in vitro digestion with the purpose of distinguishing theeffect of acidification from the enzymatic action of pepsin. Concur-rently, the kinetics of particle disintegration of the dairy gels dur-ing separate static in vitro gastric digestion were characterizedusing 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 resultsfrom these two approaches were expected to enable the identifica-tion of the mechanisms of acid and enzymatic food particle break-down during gastric digestion.

2. Materials and methods

2.1. Materials

Rennet CHY-MAX� 200 M was purchased from Chr HansenFrance SAS (Arpajon, France). Pepsin from porcine gastric mucosa(3200–4500 U/mg protein, Sigma) and other chemicals were pur-chased from Sigma-Aldrich, Inc. (St. Louis, USA). Pepsin activitywas measured as 2823 (±180) U/mg using haemoglobin (Hb) asthe substrate, where one unit produces a DA280 of 0.001 per min-ute at pH 2.0 and at 37 �C, measured as TCA-soluble products, (alsoreferred to as ‘‘Sigma” or ‘‘Anson” pepsin units) (Minekus et al.,2014). Distilled water was used in all experiments. 1 mL of porcinepepsin stock solutions were prepared extemporaneously beforeeach digestion experiment and stored on ice before use. The activ-ity of pepsin stock solutions was adjusted to 78,000 and 64,000 U/mL for both the microscopic observations and the static in vitrodigestion experiments, in order to ensure 2000 U/mL in the finalmixture made of dairy gel particles and gastric fluid. Ultra LowHeat (ULH) skim milk powder (lot No. 78107; 3.5% of humidity,34% of protein (Nx6.25), 56% of lactose, 8.5% of ash) was purchasedfrom LACTALIS ingredients (Société Laitière de Pontivy, Le Sourn). Astock solution of pepstatin A (a pepsin inhibitor) was purchasedfrom Fischer Scientific (Germany) and prepared at a concentrationof 0.5 mg/ml of MeOH.

2.2. Fluorescence intensity of dairy proteins at different pH

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 2Mhydrochloric (HCl) solution) were acquired at 37 �C using aFluoroMax-4 spectrofluorimeter (HORIBA Jobin Yvon INC, Longju-meau, France) fitted with a temperature controlled cuvette-holder. Emission spectra were recorded in the 285–800 nm rangewith an excitation wavelength of 275 nm using 1 cm � 1 cm quartzcuvettes. The Rayleigh scattering harmonic (k = 550 nm) was elim-inated from the recorded spectra and the fluorescence intensitiesmeasured above 300 nm were then summed. This analysis wasperformed to evaluate the natural variations of dairy protein fluo-rescence intensity as a function of pH applying the same conditionsas those to be used during the synchrotron deep-UV microscopyexperiments.

2.3. Preparation of gels

Two dairy gels having a similar composition (34%w/w of pro-teins) but different microstructures were prepared by rennet andacid coagulation of non-fat milk. 100 g of ULH skim milk powderwere rehydrated with water to make up to 1 kg by stirring during2 h at room temperature. Both gels were then prepared from thissolution, either by (i) acid coagulation (AG, glucono-d-lactone

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(GDL): 2%w/w) held at room temperature overnight or by (ii) ren-net coagulation (RG, rennet 1.5%vol/w) held for 1 h at 37 �C in athermostatic bath and then stored overnight at room temperature.The pH of the acid and rennet gels were 4.0 and 6.6, respectively.

2.4. Synchrotron deep-UV microscopic observations during staticin vitro digestion

A full description of the beamline and imaging microscopesetup is widely available (Giuliani et al., 2009; Jamme et al.,2010; Tawil et al., 2011). The monochromatized synchrotron beamis coupled to a modified full field microscope (Axio Observer Z1,Carl Zeiss GmbH, Germany) fitted with a back-illuminated CCDPIXIS 1024-BUV camera (Princeton Instruments, USA). The focus-ing objective (Zeiss Ultrafluar 10�, Carl Zeiss GmbH, Germany) isaxially motorized by the Axio-Observer Z1 microscope. The syn-chrotron beam exciting the sample may be considered as colli-mated when entering the microscope. Based on the maximumflux available at the beamline, the excitation wavelength wasmonochromatized at 275 nm using an iHR320 monochromator(HORIBA Jobin Yvon INC, Longjumeau, France). A 300 nm dichroicmirror (Semrock, Rochester) and an emission bandpass filter cho-sen at 340/26 nm (Semrock, Rochester, USA) were used for collec-tion. As Tyrosine is weakly fluorescent around 340 nm, the signaldetected was attributed to Tryptophan fluorescence.

The maximumwidth of the field of view was 1.12 mm using the�10 objective. To enable the observation of an entire gel particle inthis field of view, samples of about 1 mm in diameter (representingaround 20 mg of the dairy gel) were prepared and dispersed with asmall spatula on a quartz coverslip on which a 5 mm height spacerhad been previously sealed using nail varnish. To optimize pepsinactivity, samples were warmed to 37 �C (PE100, Linkam, UK). Toavoid evaporation from the solution during digestion, a secondquartz coverslip was sealed on top of the spacer immediately afteraddition of simulated gastric fluids (Fig. SI1). The microscope wasthen adjusted to focus on just one particle before launching theacquisition procedure. 1024 � 1024 pixel images were automati-cally recorded using an exposure time of 5 s. Two different typesof digestions were simulated in order to distinguish the enzymaticaction of pepsin from the effects of the acid environment. For thefirst, 175 mL of 0.01 M hydrochloric acid was carefully added soto avoid any movement of the chosen particle. Micrographs weretaken at a 10 s frequency over a period of about 20 min, an incuba-tion period that enabled the stabilization within the aggregate ofacid-induced microstructural rearrangement and fluorescenceintensity variation. Following this step, 5 lL of the prepared pepsinstock solution was carefully added and image acquisition was thencontinued at a 1 min frequency for the 2 h of enzymatic phase. Sec-ondly, to study the simultaneous effect of acidification and enzy-matic reaction, 175 mL of the 0.01 M HCl solution and 5 lL of theprepared pepsin solution were carefully added to the dairy gel par-ticles. Each set of digestion experiments was performed in tripli-cate for both types of gel.

2.5. Image analysis

A set procedure for analysis of images generated by the syn-chrotron DUV microscopy was carried out using a Fiji softwarepackage (Schindelin et al., 2012). Binary images were generatedby selecting a threshold algorithm from amongst Otsu, Triangleor Yen method from the options in the Fiji software package. Sincethe image focus on the particles, and the contrast levels betweenthe fluorescent particle and the background were both differentdepending on the experiment, the selection of the segmentationmethod was made on a visual validation. The method chosenwas 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 (Regionof Interest) selection taken from the binary counterparts. The arearepresented by the ROI (denoted ‘‘Area”) and its mean fluorescenceintensity (denoted ‘‘MeanF”) were measured for each sub-image. Asmall circular ROI was also selected from a region free of particlesin order to estimate the mean gray value of the background(denoted ‘‘BackF”) in the same series of images. The corrected flu-orescence intensity of the particles (denoted ‘‘Fluo”) were esti-mated by subtracting the background fluorescence from themean particle fluorescence intensity:

Fluo ¼ MeanF� BackF ð1ÞTo estimate the digestion kinetics of the dairy gel particles

under observation, it was postulated that the ‘‘Estimated ProteinContent” in the particle was proportional to the overall fluores-cence intensity, calculated as the product of the particle area (Area)with its mean fluorescence intensity having corrected for the back-ground fluorescence (Fluo):

Estimated protein content ¼ Area� Fluo ð2ÞFor graphical representations, normalized values for all these

parameters (particle area, corrected mean fluorescence intensity,and estimated protein content) were calculated by dividing thevalue at time t by the corresponding initial value (t = 0).

2.6. Static in vitro digestions to quantify the microscopic observations

This second series of in vitro digestion experiments was carriedout to enable physicochemical analyses of samples. These wereconducted with no mechanical agitation, in order to reach condi-tions similar to those arising during the time-lapse microscopydescribed above.

In order to simulate chewing, approximately 250 (±1.0) g ofdairy gel were weighted and cut up with a spatula into smaller par-ticles (approximately � 0.5 cm) which allowed the subsequentweighing of 4.0 (±0.1) g of gel in 12 tubes (40 mL Nalgene� cen-trifuge tubes, Sigma Aldrich). These tubes were then placed on amagnetic 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 min (for the rennet gel). This procedurewas sufficient to reduce the particle size to around 1 mm for bothgels.

The digestion was started by adding 28 mL of a 0.01 M HCl solu-tion (pH = 2) to each of the prepared 4 g samples of dairy gel(applying the same 1:8 dilution as used during the microscopyobservations) in order to reduce the pH to 3.0 – as recommendedby the INFOGEST in vitro digestion protocol (Minekus et al.,2014), with subsequent incubation at 37 �C in a water bath. Afterthe acidification phase of 30 min, the suspensions containing thegel particles had reached stable pH values of 3.0 (±0.5). Pepsinactivity at pH 3 has been reported to be at 70% and 40% of its max-imal activity (around pH 2) according to Piper and Fenton (1965)and Kondjoyan, Daudin, and Santé-Lhoutellier (2015), respectively.1 mL of a porcine pepsin stock solution of 64,000 U/mL was finallyadded to achieve 2000 U/mL in both final digestion mixtures(Minekus et al., 2014). The enzymatic digestion phase then tookplace during the following 90 min.

During the sequential acid and enzymatic digestion phases, thetubes were inverted twice every 3 min to avoid sedimentation. Forthe acidification phase, a tube was removed and placed in ice aftereach time measurement, whereas during the enzymatic digestionwith pepsin, the reaction was stopped in the removed tube bythe addition of 10 lL of a pepstatin A solution (0.5 mg/mL MeOH)per mL of digesta before storing the tubes in ice. The 12 preparedtubes were stopped at different times along the digestion process:

Video 1.

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(i) after 1, 5, 10, 20 and 30 min for the acidification phase and then(ii) after 31, 35, 40, 50, 60, 90 and 120 min for the enzymatic phase.Two batches of sub-samples were prepared in parallel, one des-tined for biochemical analysis of the whole digested samples,and the other for centrifugation and analysis of the pellet andsupernatant phases. Each set of digestion experiments and analy-ses was performed in duplicate for the acid gel and in triplicatefor the rennet gel.

2.7. Sample analysis

Immediately after in vitro digestion of the dairy gels describedabove, one of the two batches was stored at �20�C ahead of bio-chemical analysis. The second batch was centrifuged for 20 minat 20,000g and at 4 �C. Supernatants were removed and stored at�20 �C until needed for chemical analysis but the pellets wereimmediately weighed.

2.7.1. Pellet mass ratioThe pellet mass ratio (PM, %) was defined as:

PMðtÞ ¼ mt=m0 � 100 ð3Þwhere m0 and mt are the pellet masses (g) obtained from centrifu-gation before (t = 0) and during (t = t) the digestion experiment. Itshould be noted that mt accounts for both the uptake of gastric juiceand the loss of its solid components due to digestion and leachingprocesses.

2.7.2. Release of proteins/peptides into the supernatantSupernatant from the centrifuged samples were filtered

through 0.2 mm Acrodisc� syringe filters (PALL Corporation, PortWashington), and the absorbance of aromatic amino acids werequantified at 280 nm in quartz cuvettes using a UV–visible spec-trophotometer (UVmc2, Monaco SAFAS, France). A 0.01 M HClsolution was used as the blank. This measurement shows therelease of soluble proteins or peptides from the gel particles intothe surrounding liquid phase. The protein/peptide release ratio(PR, %) was defined as:

PR ¼ ðAt � A0Þ=A0 � 100 ð4Þwhere A0 and At are the absorbance of the supernatant before (t = 0)and during digestion at time t.

2.7.3. ElectrophoresisSDS–PAGE analysis was performed on the thawed whole

digesta samples, without centrifugation, using 4–12% polyacry-lamide NuPAGE Novex� Bis–Tris 15-well precast gels (Novex� LifeTechnologies, California, USA) mounted in a HoeferTM miniVE verti-cal electrophoresis system (Pharmacia Biotech, Sweden) followingthe manufacturer’s instructions. Samples were first homogenizedwith a Q700 Ultrasonic Liquid Processor (Qsonica LLC., Newtown,USA) fitted with a 3.2 mmmicrotip probe for 45 s. Tubes were kepton ice during the sonication step to avoid warming. Samples forprotein analysis were then diluted 8-fold with the denaturing buf-fer (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 DTTsolution and 10 min at 70 �C). Samples were then loaded, at a pro-tein concentration of 15 mg of total protein/well, into the sampleslots and a marker kit (Mark 12TM Unstained Standard, novex� LifeTechnologies, California, USA) was used for molecular weight(MW) calibration. Electrophoresis was performed at 200 V and50 mA during 120 min. Gels were fixed in 30% (v/v) ethanol and10% (v/v) acetic acid solution for 30 min and stained with Coomas-sie Brillant Blue G250.

3. Results

3.1. Effect of pH on the fluorescence of dairy proteins

Fig. 1 shows the development of the cumulative fluorescenceintensity (above 300 nm) measured by spectrofluorometry at dif-ferent milk pH, using the natural pH of milk (6.5) as the referencevalue (100%). The cumulative fluorescence intensity drops by about25% at pH 6.0, then rapidly rises to a maximum value of about115% at pH 4.4, before decreasing again to reach a plateau valueat pH 3.8, corresponding to nearly 40% lower than the referencevalue.

3.2. Effects of HCl addition

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 den-sity per unit of surface, 2. the dependence of protein fluorescenceon the local pH, and 3. photobleaching effects as a function of UVexposure. Fig. 2A (Video 1 of Supplementary data) shows the typ-ical behavior of an acid gel particle after immersion in an HCl solu-tion at pH 2: it remained visually quite stable at the end of 18 min.Fig. 2B shows the normalized area and mean fluorescence intensityprofiles of the particle (obtained by image analysis) for the samegel particle. The area of the particle increased slightly with timewhereas the mean fluorescence intensity fell slightly, but no morethan 80% of the initial fluorescence after 20 min. The change inestimated protein content with time is also shown on Fig. 2B.The slight apparent loss of matter, i.e. around 13% after 20 min,can be attributed to the loss of fluorescence due to photobleachingbased on the comparison with stable samples (data not shown).

Fig. 3A (Video 2 of Supplementary data) shows the typicalbehavior of a rennet gel particle after immersion in the acidic solu-tion at pH 2. Fig. 3B shows the corresponding values obtained fromimage analysis. By contrast with the behavior of the acid gel parti-cle, as soon as the rennet gel particle was in contact with the acidsolution, it appears (from a careful visualization of the changingimages in Video 2) that its outer periphery swelled suddenly andwas partly expulsed into the surrounding. However, the largemajority of the particle shrank back strongly, leading to a reduction

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

902 J. Floury et al. / Food Chemistry 239 (2018) 898–910

in area of more than 40% after only 1 min of immersion. Then theparticle swelled slightly before shrinking again at 4 min and endedup at 50% of its initial size after 20 min. Visually, Fig. 3A shows thatthe fluorescence intensity strongly increased, especially in the cen-tral part of the particle, before progressively disappearing from theouter side after 4 min. By contrast, Fig. 3B shows that the measuredmean fluorescence of the particle followed the opposite pattern. Itincreased during the first minute by about 20%, then decreased bymore than 40% during the progressive disappearance of the innerfluorescence from 1 to 4 min, and then increased again, reachinga plateau close to its initial level after 10 min. With respect tothe changing estimated protein content, this decreased continu-ously during the first 3 min, before stabilizing at nearly 50% of itsinitial value.

Video 2.

Video 3.

Video 4.

3.3. Effects of subsequent pepsin addition

Fig. 4 (Videos 3 and 4 of Supplementary data) shows the typicalbehavior of both gels during the subsequent 2 h pepsin digestion

step. Fig. 4A shows that the acid gel particles were very rapidly dis-integrated. Indeed, although some particles were still distinguish-able 20 min after pepsin addition, the fluorescence level of a ROImanually drawn around the residual particle had in reality drasti-cally reduced, by about 80% and then by more than 98% at 120 min(results not shown). Thus, the automatic image processing startedto fail properly identifying the remaining particles at this point.The examination of the quartz cover slip at the end of the digestionexperiment revealed that these traces originate from a smallamount of protein material adsorbed onto the lamella, a phe-nomenon that might arise from protein modifications due to UVexposure. The disintegration process was slower for the rennetgel, noting that particles of small initial size were still visible evenafter 2 h (Fig. 4B).

Fig. 4C shows values derived from image analysis over theduration of the digestion. These results confirm the observationsof the images that show that the decrease of both particle area

Video 6.

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and fluorescence was very rapid and especially pronounced forthe acid gel. The rennet gel particle area was largely unchangedduring the first 10 min of digestion before it steadily fell, withonly 30% of the initial area remaining after 2 h of pepsin diges-tion. The changes of the estimated protein content enable theillustration of the digestion kinetics, which can be fitted to anexponential curve. By applying such models, the protein hydrol-ysis rate was approximately 10 times slower for the rennet gelthan for the acid gel particles.

3.4. Digestions in a simulated gastric solution containing both HCl andpepsin

Fig. 5 (Videos 5 and 6 of Supplementary data) shows a typicaltime-lapse fluorescence imaging of both acid and rennet gels afterimmersion in an HCl solution at pH 2 containing also 2000 U/mL ofpepsin (note that image contrasts were re-adjusted after 30 min toimprove visualization). Fig. 5A shows that acid gel particles wereno longer observable after only 14 min of digestion. The majorityof the rennet particle under similar conditions also very rapidlydisappeared as well (Fig. 5B). Image analysis of these microstruc-tural observations are given graphically in Fig. 5C. For the acidgel, the particle area, mean fluorescence intensity, and estimatedprotein content were all similar to that already observed duringthe pepsin phase of the sequential protocol (Fig. 4C): that is a veryrapid decrease in all parameters, indicative of a rapid digestion. Forthe 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 fluorescencedecreased by more than 75% during the first 5 min, then remainedstable for few minutes, before it progressively increased between25 to 70 min before stabilizing. Two versions of the correspondingestimated protein content are given in Fig. 5C for the rennet aggre-gate. The plain line (RG) corresponds to the results of Eq. (2) on theraw data, whereas the dotted line (RG⁄) assumes a 40% loss of flu-orescence during the first 5 min due to the effect of pH falling from6.5 to less than 4 (Fig. 1). Despite this correction, it is still clear thatthe great majority of the rennet gel particle (�90%) was effectivelyconsumed within the first 20 min of digestion.

Video 5.

3.5. Physico-chemical characteristics of gastric digesta

Fig. 6A shows that the pellet mass ratio (PM) was not greatlymodified during the acidification phase of the rennet gel digestionprocess. Over the same period, there was a slight decrease of theprotein/peptide release ratio (PR) in the supernatant phase(Fig. 6B). The behavior of the acid gel was different to that of therennet gel, as the pellet mass ratio increased up to 150% over the30 min following the addition of the acid solution, – over the sameperiod, the calculated PR increased by 30%.

In less than 1 min after the subsequent addition of pepsin to theacid gel, its PM ratio dropped from 150% to around 55% (Fig. 6A),and the corresponding PR ratio soared from 42% to 135%(Fig. 6B). Both the PM and PR then changed muchmore slowly withvalues of 32% and 200% noted at the end of the enzymatic process.The behavior of the rennet gel during pepsin digestion was differ-ent, as illustrated by the slower rise/fall of the PM/PR ratios, whichonly reached final values of 50% and 90%, respectively at the end ofthe 2-h period.

Fig. 6C shows the results of SDS-PAGE analyses applying thesame sequential protocol. For both gels, no visible changes of thebands corresponding to the main milk proteins (caseins, b-lactoglobulin and a-lactalbumin) were noticeable during the first30 min. Peptide bands did not appear after HCl addition, showingthat no hydrolysis occurred during the acidification phase. Fig. 6Cshows an extremely rapid and complete disappearance of caseinbands only 1 min after the addition of pepsin to the acid gel,whereas the casein bands vanished much more slowly for the ren-net gel. For both types of dairy gel, the bands corresponding to b-lactoglobulin did not change much during the whole process,meaning that this protein was resistant to pepsin hydrolysis. Pep-sin was though able to hydrolyze a-lactalbumin, as shown by thedisappearance of the corresponding band during the enzymaticphase. Fig. 6C also shows a progressive appearance of grey bandsbetween 3 and 10 kDa on the two different SDS-PAGE elec-trophoresis which can be attributed to an increasing concentrationof peptides produced during digestion.

Fig. 2. (A) Typical sequence of fluorescence images of the acid gel (AG) after immersion in hydrochloric acid at pH 2. The yellow line denotes the ROI’s selection with Otsuthresholding method (FIJI software package). The scale bar is 100 mm. (B) The corresponding normalized evolution of the particle area (����), its mean fluorescence intensity(�����), and its protein content (—), all derived from the image analysis of the ROI selections. (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

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4. Discussion

This study aimed at investigating the influence of the method ofmilk coagulation (i.e., acid- or rennet-induced - both commonlyused in industry), on the mechanisms of dairy protein gel break-down during gastric digestion. Although the two types of gel hadthe same overall composition, the different means of productionled to important differences in the kinetics of protein hydrolysisduring simulated static in vitro digestion.

4.1. Rennet gel undergoes important structural modifications uponacidification

Based on results from the use of synchrotron deep-UV fluores-cence, the microstructural changes in acid and rennet dairy proteingels following acidification was greatly dependent on the coagula-tion method. For the acid-formed gel, which has an initial pH of 4,no significant changes were seen under the microscope. Neverthe-less, Fig. 6 showed that the pellet mass from centrifugation and therelease of proteins into the supernatant during digestion bothincreased greatly. Visual observations also indicated that the acidgel particles became more ‘‘flaky” after immersion in the acid solu-tion (Fig. 2 and Video 1). Luo et al. (2015) also observed such a phe-nomenon during their dynamic in vitro digestion experiments ofwhey protein isolate gels with simulated gastric juice free of pep-sin. This was explained by the diffusion of gastric juice into the gel

matrix. As the pH inside the particle falls, it progressively reachesthe isoelectric pH of caseins (pHi � 4.6), which may thus leads to aswelling of the particles induced by the decreased electrostaticattractions. As acidification approaches pH 3, individual caseinmolecules can even start to solubilize (Post, Arnold, Weiss, &Hinrichs, 2012). Both increases of the pellet mass and protein/pep-tide release ratios, measured during the acidification of the acid gel(Fig. 6A and B), may thus be attributed to the increasing acidity ofits components. Even if the acid gel retained an increasing amountof water during its acidification, the shape and fluorescence inten-sity of its particles were largely preserved based on the micro-scopic observations in this study: this suggests a limitedmolecular rearrangement of the protein network.

On the contrary, the rennet gel underwent major structuralmodifications during acid incubation, with an initial swelling ofthe particle periphery and a strong contraction of the inner part,progressively leading to a more compact particle. These resultsare in agreement with Barbé et al. (2014), who also observed, dur-ing a dynamic in vitro gastric digestion, that rennet gel formedmuch denser particles than the acid gel, because of pH-inducedsyneresis. Physicochemical analysis of the acidified rennet gelshowed no significant loss of the total matter nor of protein solu-bilization. It is known that rennet-induced gels undergo rapidsyneresis if disturbed by cutting or by wetting the gel surface, arearrangement process that is accelerated and more extensive athigh temperatures and lower pH values. This contrasts with most

Fig. 3. (A) Typical sequence of fluorescence images of the rennet gel (RG) after immersion in hydrochloric acid at pH 2. Yellow lines correspond to ROI’s selections withTriangle thresholding method (FIJI software package). The scale bar is 100 mm. (B) The corresponding normalized evolution of the particle area (����), its mean fluorescenceintensity (�����), and its protein content (—), all derived from the image analysis of the ROI selections. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

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acid-formed gels, which do not show a great deal of spontaneoussyneresis (Mellema et al., 2000; Walstra, 1993). Published workon the progressive acidification of dairy samples suggest that anumber of phenomena take place as pH falls. Among them, solubi-lization of the colloidal calcium phosphate (CCP) nanocrystals,which usually occurs between pH � 5.9 and 4.9, is of interestbecause they are directly involved within the nano-structural orga-nization of the protein network in rennet gels (but not in acid gels).Casein micelles contained in milk (from which the rennet proteinnetwork is formed), therefore undergo internal structural reorgani-zation upon acidification in this pH range. This is thought to be themain cause for the observed swelling of the casein particles(Famelart, Lepesant, Gaucheron, Graet, & Schuck, 1996). On pro-gressing below pH = 4.9, the amount of water contained by thecasein particles sharply decreases because of the reinforcementof 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 toobserve an intense structural evolution, progressively leading toa compaction and densification of the protein network, when ren-net gel particles are brought into a stronger acidic environment.

In addition to revealing these structural modifications, thedeep-UV fluorescence technique enabled a study of acidificationwithin the aggregates at the microscale: this arises from monitor-ing changes in the fluorescence intensity of a particle as the exper-iment proceeds. Indeed, the environmental pH has a known largeimpact on the tryptophan fluorescence intensity of dairy proteins(Herbert, Riaublanc, Bouchet, Gallant, & Dufour, 1999). Fig. 1shows that the natural fluorescence intensity of dairy proteinsincreases by approximately 20% when reducing the pH from 6.5to around 4.5, before abruptly losing around a third of intensitywhen implementing a further decrease of the pH from 4.5 to 4.

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This dependency of the protein fluorescence intensity to pH canindeed explain the spatial variations of fluorescence intensityobserved in the rennet particle during the first minutes of acidifi-cation (Fig. 3A). From 1 to 3 min, the highly fluorescent zoneobserved would correspond to a region of the aggregate wherethe pH is still between 5.5 and 4.5 (i.e. the plateau in Fig. 1). Mean-while the dark outer layer of the particle would correspond toregions where the pH is less than 4. In other words, these spatialvariations of fluorescence reflect the pH gradients within the par-ticle. The subsequent increase of the particle fluorescence, beyond4 min of exposure to acid conditions, can only be attributed to theprogressive shrinking of the particle with a corresponding rise in

Fig. 4. Typical sequence of fluorescence images of the (A) Acid Gel and (B) Rennet Gel duwith Yen thresholding method (FIJI software package). The scale bar is 100 mm. (C) Corresits protein content, all derived from image analysis of the ROI selections. All values have bfittings of the data. (For interpretation of the references to colour in this figure legend,

protein concentration: this is confirmed by the observed corre-sponding decrease of its area whilst the estimated protein contentremains constant at about 55% (Fig. 3B). This latter value is a littleless the �65% expected from the pH effects on fluorescence(Fig. 1). This slight discrepancy can be attributed to the effects ofboth photobleaching (as for the acid gel, Fig. 2B) and the previ-ously reported small loss of matter from the outer peripheryimmediately after the addition of HCl. Overall, these latterresults illustrate the great potentiality of the developed methodol-ogy to study gastric digestion of foods, and more especially inorder to investigate the evolution of pH gradients within proteinparticles.

ring the pepsin digestion phase at pH 2. Yellow lines correspond to ROI’s selectionsponding changes with time of the particle area, its mean fluorescence intensity, andeen normalized to unity at time zero. The thin dotted lines represent the exponentialthe reader is referred to the web version of this article.)

Fig. 5. Typical sequence of fluorescence images of the (A) Acid Gel and (B) Rennet Gel particles as a function of digestion time in a simulated gastric juice: Hydrochloric andpepsin solution at pH 2. The yellow lines correspond to ROI’s selections with Triangle thresholding method (FIJI software package). The scale bar is 100 mm. The image contrastwas adjusted after 30 min of observation. (C) Corresponding changes with time of the particle area, mean fluorescence intensity, and protein content obtained by imageanalysis 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 offluorescence intensity previously observed during the acidification of the rennet gel. (For interpretation of the references to colour in this figure legend, the reader is referredto the web version of this article.)

J. Floury et al. / Food Chemistry 239 (2018) 898–910 907

4.2. Hydrolysis kinetics of the rennet gel by pepsin is much slower thanthat of the acid gel.

Three methods of study: using DUV fluorescence (synchrotronmicroscopy), electrophoresis (SDS-PAGE) and the analysis of thepellets and supernatants after centrifugation – they all show thatthe acid gel particles were broken down much more rapidly thanrennet gel particles during the pepsin phase of the 2-part digestionprotocol. Acid gel particles were hydrolysed just a few minutes

after the addition of pepsin (Figs. 4A and 6C), with a correspondingvery high rate of protein/peptide release (Fig. 6B). In contrast, theparticles of the rennet gels were still visible even after 2 h of diges-tion (Fig. 4B), with a corresponding much slower rate of protein/peptide release and a lower final concentration of this digestionproduct (Fig. 6B). For both types of gel, it is also noteworthy thatthe initial morphology of particles upon pepsin digestion remainedalmost unchanged during the simulated gastric phase observedusing the DUV fluorescence imaging (Fig. 4A and B). This suggests

Fig. 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 mincorrespond to the acidification phase (with an HCl solution at pH 2), before pepsin addition.

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that the predominant mechanism was surface erosion, an interpre-tation consistent with the observed exponential model (Fig. 4C),which were previously shown to adequately describe the break-down of solid food particles during gastric digestion (Kong &Singh, 2009a; Kong & Singh, 2009b; Kong & Singh, 2011).

It was postulated that the internal bonds between the caseinmicelles were weakened by the penetration of the acid solutioninto the acid-formed gel particle, hence facilitating pepsin hydrol-ysis, which was rapid. The behavior of the rennet gel was com-pletely different, due to the restructuring and subsequentcontraction of the particles (syneresis) during the acidificationstage. In other words, instead of weakening the internal bonds,the acid solution increased the resistance of the rennet gel proteinnetwork to pepsin action, either by limiting the diffusion of pepsinwithin the gel particle, and/or by reducing accessibility of the pro-tein substrate to the enzyme active site (because of tightened andstrengthened protein strands and nodes). This latter explanationfits in better with other published work (Kong & Singh, 2008).Moreover, it is well-known that during the aging of rennet gel par-ticles merge and reorganize, resulting in the formation of straighterand denser strands, thus with more bonds per casein micelles, andhence stronger interactions (Mellema, Walstra, van Opheusden, &van Vliet, 2002; Mellema et al., 2000). This phenomenon increasesif a progressive acidification occurs during this aging phase (LeFeunteun & Mariette, 2008). Ye et al. (2016) reported that theintense coagulation of milk in the acidic environment of the stom-ach probably strongly hinders the gastric fluid from diffusing intothe particles formed. Luo et al. (2015) compared the kinetics of

digestion of egg white protein with whey protein isolate solutionsand gels using simulated gastric conditions. They showed that thebarrier of the protein gel structure did not simply slow down thereaction rates (compared to soluble protein digestion), but it alsoaltered the mechanisms of enzymatic hydrolysis. They postulatedthat the difference in activity observed stemmed from the sterichindrance of gel structure to the diffusion of pepsin and its hydro-lysates. Indeed, Thévenot et al. (2017) showed that the diffusioncoefficient of pepsin (of about 100 mm2/s in water), decreased from51 to 21 mm2/s in rennet gels as the casein concentration wasincreased from 3.2 to 13% w/w. Luo et al. (2017) also reported thatpepsin hydrolysis in 15–20%w/w whey protein gels was con-strained to a 2 mm layer at the surface of the gel, even after 6 hof incubation in a gastric solution. They suggested that since pepsinhad only a limited penetration depth, digestion processes were lar-gely constrained by the exposed surface area of the gel particles,and hence by the surface erosion rate. All of these results supportthe argument that the principal mode of pepsin action during staticin vitro digestion experiments is the hydrolysis of the proteinslocated at the surface of the particles. Thus for whey protein gels,Luo et al. (2017) concluded that the mode of gel digestion wasdetermined by the combined effect of diffusion limitation, hydrol-ysis rate and microstructure transformation.

When acid and pepsin were simultaneously added to gel parti-cles, the kinetics of breakdown for both gels followed an exponen-tial model with well-preserved particle shapes (Fig. 5A and B),suggesting that their digestion was again dominated by a surfaceerosion process. The hydrolysis rate of rennet gel particles was still

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slower than for acid gel, but the difference was greatly reduced. Asillustrated by the residual particle in Fig. 5B (which resisted diges-tion well even after 120 min), acid-induced syneresis of the rennetparticle also occurred after immersion within the HCl + pepsinsolution. However, by the time this process had ended, most ofthe initial particle was lost (�90%) because of the presence ofpH-activated pepsin ready to hydrolyze the protein network fromthe beginning of the digestion process. Such mechanisms may alsobe enhanced by the swelling of the outer layer of the rennet gelparticle when brought in contact with acid. Indeed, if the peripheryof the particle swells, protein substrate should be more readily incontact with the pepsin, and thus more accessible to the enzymeaction. Acid diffusion and enzymatic reactions appear to acttogether, thereby accelerating the hydrolysis rate of the rennetgel to almost the same level as that observed for the acid gel.

The remarkable resistance of the small residual rennet particleto pepsin digestion nevertheless opens questions on the in vivoreality and possible consequences of any implied mechanisms.Indeed, gastric juice is secreted by the stomach wall, and mustspread throughout the gastric contents to enable pepsin hydroly-sis. Since the small hydrogen ions diffuse more rapidly than pep-sin, the in vivo timeframe of pH-induced effects and of pepsinaction on a stomach content made up of rennet particles mightlie somewhere in between the purely sequential and purelysimultaneous digestion protocols simulated in this study. Obser-vations showing the rapid formation of an acidified rennet parti-cle that is highly resistant to pepsin hydrolysis thereforeconstitutes a likely mechanism which can explain the pro-nounced slowdown of amino-acid absorption observed duringin vivo digestions in mini-pigs of rennet gels when compared toacid gels (Barbé et al., 2014).

5. Conclusion

Time-lapse synchrotron deep-UV fluorescence microscopyenabled the study in real time and with a high resolution of thechanges in food particles during the digestion process. It showedthe propensity of rennet gels, (but not acid gels), to undergo largemicrostructural modifications (syneresis), leading to compact pro-tein aggregates in the acidic conditions of gastric digestion. Theresulting structure of the particle reduces the accessibility of pep-sin to the protein substrate, which most probably prolongs the gas-tric phase, as first suggested from previous in vivo results. Theshape of particles remained unchanged during the simulateddigestion process, and the kinetics of disintegration were ade-quately fitted to an exponential model, suggesting that erosionwas the predominant mechanism of the breakdown of dairy gelsduring the simulated gastric digestion based on static hydrody-namic conditions. These findings help describe how the food struc-ture can affect subsequent digestion kinetics. In this respect, thetime-lapse deep-UV microscopy is well adapted to such studies.There is now a real need to develop or adapt methods that willallow the monitoring of the changes in the micro- and nanostruc-ture of food in the course of its digestion: this will allow a betterunderstanding of the mechanism of enzyme action during diges-tion of food matrices.

Acknowledgments

The authors are grateful to the team running the SOLEIL syn-chrotron that provided beam time and efficient support, under pro-ject proposals n�20140065 and 20150452. They also acknowledgeTiago Bianchi who awarded a mobility PhD grant from the NationalInstitute for Agricultural and Food Research and Technology (INIA)– Spanish Ministry of Economy and Competitiveness.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2017.07.023.

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