peptide release rates during simulated in vitro digestion

International Dairy Journal 56 (2016) 169e178

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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Commercial cheeses with different texture have different disintegration and protein/peptide release rates during simulated in vitro digestion Xixi Fang, Laurie-Eve Rioux, Steve Labrie, Sylvie L. Turgeon* STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Universit
e Laval, Qu
ebec City, Qc, G1V 0A6, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 August 2015 Received in revised form 25 January 2016 Accepted 27 January 2016 Available online 10 February 2016

Solid food disintegration in the stomach has recently been linked to food texture, which changes during digestion. This phenomenon is likely to affect the kinetics of protein digestion and therefore associated postprandial metabolic responses. Depending upon the variety, the cheese protein and lipid content as well as the texture can be modulated, illustrating complexity. Five commercial cheeses, covering a range of textural properties, were selected and characterised. Cheese particles were submitted to an in vitro digestion model to study cheese disintegration and protein/peptide release. Cheese disintegration was affected by cheese texture and composition. At the end of gastric digestion, elastic cheeses (mozzarella) were less disintegrated when compared with ripened and soft cheeses with high fat content (Camembert, aged Cheddar). The protein digestion was different amongst cheeses according to different disintegration rates. Cheese structural and textural properties, attributed to processing parameters, can be used to modulate gastro-intestinal digestion of cheese proteins. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The dynamics of food digestion may affect the kinetics of release and subsequent absorption of amino acids in the gastrointestinal (GI) tract. This could affect postprandial protein anabolic response, and some physiological functions such as satiety and muscle pro
ger-Guist'hau, 2011; Breen & Phillips, tein accretion (Boirie & Le 2011; Dangin et al., 2003; Lacroix et al., 2006). The disintegration of solid food starts in the mouth (Kong & Singh, 2008). Mastication converts ingested food pieces into a cohesive mixture consisting of small food particles, with varying particle size (0.82e3.04 mm) depending upon food structure (Jalabert-Malbos, Mishellany-Dutour, Woda, & Peyron, 2007). The disintegration of solid food in the stomach is complex. For example, proteins released out of the solid matrix may be hydrolysed into peptides. Therefore, the rate of solid food disintegration could affect the kinetics of nutrient release. Only liquids and small particles (<1e2 mm) can be emptied from the stomach into the duodenum (Dressman, 1986). After gastric digestion, proteins are further

* Corresponding author. Tel.: þ1 418 656 2131x4970. E-mail address: [email protected] (S.L. Turgeon). http://dx.doi.org/10.1016/j.idairyj.2016.01.023 0958-6946/© 2016 Elsevier Ltd. All rights reserved.

hydrolysed by pancreatic enzymes (trypsin, chymotrypsin and exopeptidases, etc.) in the small intestine. The digested products are dissolved in duodenal juices and adsorbed through the intestinal wall (Ganapathy, Ganapathy, & Leibach, 2001). These processes may be modulated by the initial form (soft gel versus solid hard food) of the food. For example, miniature pigs fed with rennet gel (a cheese model) had delayed protein digestion. After 12 h digestion, only 24.7% of the proteins in the rennet gel were recovered in the plasma, whereas up to 90% were recovered from digested ingested milk and acid gels (a yogurt model) (Le Feunteun et al., 2014). This result was explained by a very long half gastric emptying time for rennet gel (352 min) compared with milk (114 min) and acid gels (159 min) (Le Feunteun et al., 2014). The authors hypothesised that the longer gastric retention for rennet gel was related to the formation of firm aggregates upon acidification in the stomach (Le Feunteun & Mariette, 2008). The delayed protein digestion for rennet gels may also be explained by the limited accessibility of digestive enzymes to proteins in a solid substrate (Morris & Gunning, 2008). Thus, the physico-chemical state of the dairy matrix may affect the behaviour of ingested meals, which should affect the kinetic of protein digestion (Le Feunteun et al., 2014; Parada & Aguilera, 2007; Turgeon & Rioux, 2011).

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Cheese is a complex matrix consisting of a casein network with open spaces occupied by fat and serum (Everett & Auty, 2008; Gunasekaran & Ak, 2003c). From hard to soft, each cheese type has an expected dominant textural character, induced by different composition and manufacturing steps associated with each cheese variety (Foegeding, Brown, Drake, & Daubert, 2003; Gunasekaran & Ak, 2003c). For example, the pressing applied during hard cheese manufacturing (in Cheddar, for example) increases cheese hardness due to the decrease of water content, leading to a more compact protein network (Gunasekaran & Ak, 2003c). The kneading and stretching processes during pastafilata cheese manufacture (mozzarella) creates a characteristic elastic, fibrous texture (Kindstedt, Caric, & Milanovic, 2004). The proteolytic breakdown of the cheese protein matrix during ripening (Cheddar, smear cheese, Camembert) decreases cheese hardness (Gunasekaran & Ak, 2003c; McSweeney, 2004). Therefore, the manufacturing processes affect cheese macronutrient organisation and texture (Gunasekaran & Ak, 2003a, 2003c), and consequently may modulate the kinetics of cheese disintegration and nutrient release in the GI tract. The kinetics of cheese matrix degradation and fat release were investigated in vitro for cheeses with different textures (mild Cheddar, aged Cheddar, light Cheddar, and mozzarella) in a first study (Lamothe, Corbeil, Turgeon, & Britten, 2012). The aged Cheddar cheese matrix was easily degraded compared with light Cheddar cheese. This was attributed to the light Cheddar cheese having a higher protein content, conferring a firm and cohesive cheese texture, thus limiting cheese degradation (Lamothe et al., 2012). The degradation results were well-fitted to the release of free oil, but the release of protein was not investigated. Considering that cheese structural and textural properties may also modify cheese protein digestion as it affects cheese disintegration, research linking cheese texture, cheese disintegration and protein digestion within the GI tract is essential to understand the bioaccessibility of nutritional compounds in cheese. The aim of the present study was to study the digestion profiles of commercial cheeses that vary in texture under conditions mimicking GI conditions, as well as cheese matrix degradation and nutrient release. 2. Materials and methods 2.1. Cheeses and cheese characterisation Stabilised Camembert (high curd pH resulting in higher calcium concentration in cheese and firm texture), smear cheese, young Cheddar, aged Cheddar, and string mozzarella cheeses were chosen to represent a variety of textural properties induced by different manufacturing methods. Cheeses were purchased from a local
tro, Quebec, QC, Canada) and analysed 30 ± 7 days grocery store (Me before the expiry date, except for aged Cheddar cheese, which was analysed 6 ± 1 months beforehand. The total solids (AOAC, 2008), fat (ISO/IDF, 2004), total nitrogen (ISO/IDF, 2008), ash (AOAC, 2000) and proteolysis (IDF, 1991b) of cheese were analysed. Total nitrogen was converted to protein using a factor of 6.38. The proteolysis index was determined and expressed as water soluble nitrogen as a percentage of total nitrogen (WSN/TN  100). This refers to a ripening index since intact para-casein remains insoluble when cheese is dispersed in water. Cheese texture was evaluated by Texture Profile Analysis (TPA). The measurement was carried out using a TA-XT2 Texture Analyzer (Texture Technologies Corp., Hamilton, MA, USA) at room temperature (25  C). Cylindrical cheese samples (1 cm height, 1 cm diameter) were compressed twice to 50% of the original height at a speed of 1 mm s1. Textural parameters (hardness, cohesiveness, springiness, chewiness and adhesiveness) were determined from

the TPA curve using Texture Exponent Software (Texture Technologies Corp.). The definitions of these textural parameters are given by the International Dairy Federation (IDF, 1991a). For each cheese sample, the analysis was repeated 11 times, and nine of them were chosen to measure the mean value of each repetition. 2.2. Digestion profile of cheeses at the end of oral, gastric and duodenal digestion 2.2.1. Cheese particles preparation Studied cheeses were firstly cut into pieces weighing 15 or 20 g, then milled in a coffee grinder for different durations to produce cheese particles with a median size of 2.4 ± 0.5 mm (Supplementary Table S1) (Jalabert-Malbos et al., 2007). The preparation was carried out at 10  C to limit cheese particle aggregation. The determination of cheese particle size was based on the study of Jalabert-Malbos et al. (2007) with minor adjustments according to the available instruments in our laboratory. Cheese particle size was determined using sieve stacks with apertures of 4.00, 2.36, 2.00, 1.00, 0.50 and 0.30 mm (Canadian Standard Sieve Series, WS Tyler, Saint-Catharines, ON, Canada). Cheese particles were sifted through the sieve stacks by shaking at a speed of 70 (arbitrary units found on the system) for 20 min (model 41314, Retsch Inc., Newtown, PA, USA). Cheese particle size distribution is presented in Supplementary Fig. S1, and the approximate median cheese particle size was defined as the theoretical sieve through which 50% of the particle weight can pass. For Camembert cheese, the measurement of sample size could not be done due to the viscous character. A grinding time of 20 s was selected (Supplementary Table S1) to obtain a homogenous sample. 2.2.2. In vitro digestion model The in vitro digestion was based on the yogurt model of Rinaldi, Gauthier, Britten, and Turgeon (2014) with several modifications (Fig. 1). The gastric digestion time was prolonged to 2 h, considering the solid state of the cheese (Lamothe et al., 2012). HCl and NaHCO3 were added at the beginning of gastric and duodenal digestion, respectively, to reach the targeted digestion pH for each cheese type (Supplementary Table S2). The target gastric pH was between 2 and 3, and the duodenal pH was 6e7. The speed of orbital shaking movement during GI digestion was increased to 200 rpm, with glass bead addition into the beaker to ensure adequate disintegration of the cheese matrix. Composition of the digestion fluids were described by Rinaldi et al. (2014). All the enzymes used were purchased from SigmaeAldrich (Oakville, ON, Canada): a-amylase (A3176), pepsin (P7000), lipase (L3126), bile (B8631) and pancreatin (P7545). No gastric lipase was included in our model since it is not commercially available as reported in a previous study (Minekus et al., 2014). The activities of the enzymes used (units per g of sample) were described by Rioux and Turgeon (2012). Cheese samples (9.00 ± 0.05 g) with glass beads (2.00 ± 0.05 g) were added into a beaker (8.5 cm high, 5.5 cm diameter) in a water bath to maintain a digestion temperature of 37 ± 1  C. Oral digestion was started by adding 6 mL of simulated saliva to the cheese samples, and mixing for 2 min at 75 rpm using a Caframo overhead stirrer (Caframo, Georgian Bluffs, ON, Canada) equipped with a dual blade paddle (1 cm high, 3.8 cm diameter). Gastric digestion was started by adding 6 mL of gastric juice, and mixing carried out by orbital movement. Another 6 mL of gastric juice was added after 1 h of gastric digestion to simulate the dynamic gastric juice secretion in vivo. At the end of the gastric digestion, 18 mL of duodenal juice was added, and mixing was carried out for 3 h by orbital movement. Several digestions were carried out simultaneously to obtain separately digested samples at each of the digestion times: the end

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171

Fig. 1. Schematic representation of the simulated in vitro digestion model.

of the oral (O2), after 15, 30, 60 and 120 min of gastric (G15, G30, G60, G120), and after 5 and 180 min of duodenal digestion (D5 and D180) as shown in Fig. 1. All digestion samples were first neutralised (7.00 ± 0.05) with NaOH/HCl, then, the gastric and duodenal samples were centrifuged (9800  g, 20 min, 4  C). After the centrifugation of each sample, three layers were observed: fat, supernatant (liquid), and pellet. The fat and pellet layers were weighed. Supernatants were collected and kept on ice for 10 min before freezing (80  C) for later analysis. The following treatment was applied to each sample to stop enzymatic activity. The oral digestion samples were heated (92  C, 10 min) before centrifugation, as the activity of amylase is not inhibited at pH 7. The enzymes found in the gastric sample were inactivated during the pH adjustment to pH 7. For the duodenal samples, a trypsinchymotrypsin inhibitor (SigmaeAldrich) solution was added to the supernatant (24 mg mL1 supernatant) before freezing to inhibit pancreatin activity. 2.3. Sample analysis 2.3.1. Mass balance determination The mass balance of each layer obtained after centrifugation was determined at the end of each digestion step (O2, G120 and D180). This was calculated based on the amount of cheese and simulated juices added for each of the digestion steps, and was calculated as follows:

YP or YF ð%Þ ¼ ðWP or WF =W0 þ JÞ  100

(1)

where YP represents the percentage of the digesta found in the pellet layer, YF is the percentage of the digesta found in fat layer weight, W0 is the initial cheese weight (g) before digestion, and J is the weight of simulated juices added for each digestion step (oral: 6 g; gastric: 18 g; duodenal: 38 g), WP is the pellet layer weight (g), and WF is the fat layer weight (g) after the digestion time t. The proportion of the liquid layer (YL; supernatant measured after centrifugation) was determined by difference (Eq. (2)):

YL ð%Þ ¼ 100  ðYP  YF Þ

(2)

The theoretical mass of each layer was also determined based on the amount of cheese and simulated juices added for each of the digestion steps. The amount of theoretical fat mass that may be found in the pellet was also presented. 2.3.2. Cheese disintegration The cheese disintegration (CD), representing the dispersion of cheese into the water phase during digestion, was calculated as:

CD ð%Þ ¼ ðW0  WP Þ=W0  100

(3)

where W0 is the weight (g) of initial dry cheese added at the beginning of the digestion, and Wp is the dry pellet layer weight (g) after digestion time t.

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2.3.3. Protein/peptide release The concentration of protein and peptide in supernatant (liquid layer) was determined using a bicinchoninic acid protein assay (BCA) following the supplier recommendations (http://www. piercenet.com/instructions/2160412.pdf). The term protein/peptide release refers to the intact proteins and peptides that are all together detected by the BCA method. The protein/peptide release was calculated as:

PR ð%Þ ¼ Cs  ðWS Þ=rÞ=P0  100

(4)

where Cs is the concentration (g L1) of protein/peptide in the supernatant after time t; Ws is the supernatant weight (g) after time t; P0 is the protein weight (g) in the initial cheese; and r is the density of supernatant (taken to be 1 g L1). 2.3.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis The profile of protein/peptide released in the supernatant was visualised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% acrylamide stacking gel and a 12.5% acrylamide separating gel as described previously (Rioux & Turgeon, 2012). 2.4. Statistical analysis Experiments were repeated using a completely randomised design (n ¼ 2e4). The significance of difference among cheeses was tested using the LSD test by ANOVA (SAS version 9.3, SAS Institute, Cary, NC, USA) and a significance level of 0.05 was used. The relationships between cheese composition (fat and proteolysis) and cheese disintegration were evaluated using Kendall's tau-b correlation analysis at the 0.05 significance level. 3. Results and discussion 3.1. Physicochemical properties and texture of studied commercial cheeses The selected cheese matrices have different compositions dictated by each cheese variety (Table 1). Five commercial cheeses with various compositions and texture were selected. Camembert cheese is characterised by a high moisture content. The cheese core is easily flowing at room temperature when the cheese is cut due to a high level of proteolysis. For stabilised Camembert, the pH of the curd is higher than for traditional Camembert (5.1e5.2 versus 4.5e4.7) resulting in a higher calcium concentration in the curd (Lawrence, Creamer, & Gilles, 1987). This will affect cheese texture by increasing hardness, which may be more appealing for some consumers. For smear cheese, the rind is washed several time with brine and ripening cultures. This will de-acidify the cheese surface and favour the production/release of enzymes by the surface € , & McSweeney, 2001). Young and aged microflora (Sousa, Ardo Cheddar cheeses differ in fat and moisture content. The aged Cheddar manufacturing process is usually adapted to obtain a curd

composition favourable to ageing (lower water content). This resulted in an elevated hardness. For mozzarella cheese, a stretching/cooking step that contributes to the formation of an elastic network is carried out. Therefore, cheese composition is governed by each specific manufacturing process used for each cheese. Accordingly, various water contents were observed among cheeses ranging from 30.44 to 44.59%. Camembert and aged Cheddar both contain significantly higher proportion of fat (35%). Also, the amount of protein varied from 19 to 28%, depending upon the cheese variety. Proteolysis was significantly higher for Camembert cheese (40.5%), which may subsequently affect the texture. The measured textural properties (Table 2) were modulated by cheese composition and manufacturing processes (Gunasekaran & Ak, 2003a, 2003c). The hardness was significantly different for all studied cheeses. Both young and aged Cheddar cheeses presented higher hardness values of 13.30 and 8.83 N, respectively, compared with the other cheeses (2.40e4.81 N). This parameter is important, and was recently used to predict food disintegration during gastric digestion (Bornhorst, Ferrua, & Singh, 2015). Several factors may modulate cheese hardness and other textural parameters. The composition is an important factor modulating cheese texture. The moisture content affects cheese protein content and the density of the matrix. Cheeses with high water content are generally softer (Gunasekaran & Ak, 2003b) since the protein volume fraction decreases (Lucey, Johnson, & Horne, 2003). Furthermore, higher water content is also related to higher proteolytic activity that will also affect cheese texture (Lawrence et al., 1987). Fat globules soften cheese texture by interfering with the integrity of the casein network (Everett & Auty, 2008). Biochemical reactions occurring during cheese ripening may also affect cheese texture, such as glycolysis (catabolism of lactate), lipolysis (catabolism of free fatty acids), and proteolysis (catabolism of amino acids). Lipolysis occurs during cheese ripening, but the extent depends upon the cheese variety. For several cheeses, such as Cheddar or Swiss-type cheeses, lipolysis is low and undesirable, whereas for others, such as Feta or blue cheeses, lipolysis is instrumental (Murtaza, Ur-Rehman, Anjum, Huma, & Hafiz, 2014). For mould-ripened cheese such as Camembert, the hydrolysis of lactate during ripening softens cheese texture because the increased cheese pH favours the water-holding capacity of caseins (McSweeney, 2004; Spinnler & Gripon, 2004). Proteolysis of the cheese matrix during ripening (aged Cheddar, smear cheese, Camembert) decreases cheese firmness (Gunasekaran & Ak, 2003c; McSweeney, 2004). For smear cheese, the bacterial surface ripening process leads to a higher degree of proteolysis, resulting in a decrease in hardness but an increase of the cohesiveness of cheese (Brennan, Cogan, Loessner, & Scherer, 2004; Gunasekaran & Ak, 2003a). The manufacturing processes also have an important impact upon cheese texture. During mozzarella manufacture, the kneading and stretching processes applied in hot water creates a characteristic elastic fibrous texture (Kindstedt et al., 2004) associated with high values of hardness, springiness and cohesiveness. However,

Table 1 Composition of commercial cheeses.a Parameter

Camembert

Fat (%) Protein (%) Moisture (%) Proteolysis index

35.2 19.3 42.9 40.5

± ± ± ±

0.5a 0.6c 0.9a 3.5a

Young Cheddar 30.5 24.6 37.0 4.5

± ± ± ±

0.3b 0.1b 1.7b 0.5c

Aged Cheddar 35.8 24.7 30.4 22.0

± ± ± ±

0.4a 0.2b 1.5c 0.1b

Smear cheese 30.6 23.6 37.6 17.5

± ± ± ±

1.5b 2.3b 4.7b 0.2b

Mozzarella 18.7 28.1 44.6 8.1

± ± ± ±

0.6c 0.6a 1.4a 0.3c

a The composition is expressed as percentage (w/w). Data represent the mean ± standard deviation of four independent experiments; values within a row with different superscript letters are significantly different.

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Table 2 Texture profile analysis of commercial cheeses.a Parameter

Camembert

Hardness (N) Springiness Cohesiveness Chewiness (N) Adhesiveness (N s)

2.4 0.44 0.39 0.44 0.21

a

± ± ± ± ±

0.3d 0.02c 0.02c 0.07e 0.10a

Young Cheddar 13.3 0.85 0.64 7.19 0.20

± ± ± ± ±

0.4a 0.02a 0.02b 0.17a 0.06a

Aged Cheddar

Smear cheese

Mozzarella

0.02b 0.02c 0.01d 0.08d 0.08a

3.5 ± 0.8d 0.73 ± 0.01b 0.66 ± 0.00a,b 1.7 ± 0.5c 0.18 ± 0.01a

4.8 0.77 0.69 2.4 0.25

8.83 0.45 0.28 1.10 0.35

± ± ± ± ±

± ± ± ± ±

0.8c 0.03b 0.02a 0.4b 0.02a

Data represent the mean ± standard deviation of four independent experiments; values within a row with different superscript letters are significantly different.

each cheese variety studied has an expected dominant textural character brought about by the distinct composition, manufacturing and ripening processes (Foegeding et al., 2003; Gunasekaran & Ak, 2003c) which may alter the cheese matrix disintegration and nutrient release during simulated in vitro digestion. 3.2. Cheese behaviour during simulated in vitro digestion In this study, controlled mixing conditions were used to study cheese particle disintegration. When a food particle is added to digestive juices, a process involving water absorption and enzymatic hydrolysis begins, allowing the food particles to solubilise and nutrients to be digested by enzymes before subsequent absorption. The theoretical liquid and pellet layer mass balance was calculated for each cheese after each digestion step (Fig. 2A). To facilitate a comparison with the experimental values, the fat portion of the calculated pellet is shown in dash lines. After oral digestion, the sum of both the pellet and the fat layer of the experimental mass balance (Fig. 2B) is expected to correspond to 60%, which is the theoretical calculated value (proportion of cheese in the oral sample) for every cheese (Fig. 2A). The theoretical and experimental data after the oral step were similar for Camembert, mozzarella and aged Cheddar. However, young Cheddar and smear cheese both have higher pellet mass, averaging 80%, indicating high water absorption after the oral step. Cheddar cheeses differed, as 13% of the mass was associated with the fat layer for aged Cheddar, attributed to the higher initial fat content compared with young Cheddar (Table 1). For Camembert cheese, the fat found in the cheese matrix was quickly released, and the fat layer represented 28% of the mass balance. During oral digestion, cheeses were mixed with simulated saliva at body temperature (37  C). At this temperature, the proportion of fat in the liquid state is higher and liquid fat may be more easily released through the cheese casein matrix without extensive dissociation of the casein matrix. Furthermore, as fat modulates cheese texture, the release during digestion may affect cheese texture, and subsequently the cheese disintegration rate. Since both aged Cheddar and Camembert contain high amounts of fat (35%, Table 1), the texture will greatly soften at 37  C and therefore, fat may be easily released. For young Cheddar, smear and mozzarella cheese, the fat layer was small (<2%) in comparison with the theoretical fat mass in the pellet suggesting that fat was still trapped within the cheese matrix at the end of oral digestion. It should be considered that the fat layer mass may be slightly overestimated (e.g., 21% for the theoretical data versus 28% experimental data for Camembert cheese). Proteins and peptides resulting from proteolysis of the milk fat globule membrane proteins may still surround fat droplets and therefore, be found in the fat layer after the centrifugation treatment and contribute to the overestimation of the mass. This hypothesis is supported by the study of Gallier, Ye, and Singh (2012) showing that the native milk fat globule membrane proteins are hydrolysed by pepsin at different rates during gastric digestion,

some peptides remaining at the interface. Water in the fat layer may also contribute to the overestimation of the mass. At the end of the gastric digestion, Camembert cheese had a lower mass balance for the sum of the pellet and fat layers compared with the other cheeses (25%, Fig. 2B). This value was lower than the theoretical value (33%, Fig. 2A). Also, the pellet layer mass was the smallest (10e20%) for Camembert and aged Cheddar cheeses compared with the other cheeses. These results suggest that Camembert and aged Cheddar cheese matrices were highly disintegrated at the end of the gastric step. Initial cheese water content (moisture) may affect the extent of cheese disintegration. Cheese with low water content has a denser protein network (Lucey et al., 2003) that may result in a low disintegration level. However, both Camembert and aged Cheddar cheeses had similar disintegration levels, although the moisture contents were significantly different. This may also be related to the softer texture (lower springiness, cohesiveness and chewiness; Table 2) associated with a higher fat content and extent of proteolysis. The degree of cheese proteolysis may affect cheese disintegration because peptides are easily dispersed in the water phase, as revealed by the high water soluble content measured through proteolysis analysis. Besides, a higher proteolysis state leads to a softer cheese matrix (Gunasekaran & Ak, 2003c) which should be disintegrated more quickly. Our results confirmed this hypothesis. Camembert and aged Cheddar cheeses, showed higher proteolysis levels than other cheeses, and were highly disintegrated (Table 1 and Fig. 2). Mozzarella cheese, showing the lowest extent of proteolysis, was resistant to the gastric environment (Table 1). Other factors may simultaneously affect cheese disintegration, such as the initial cheese fat content. Both Camembert and aged Cheddar cheeses have significantly higher amounts of fat (Table 1) resulting in a higher fat layer mass (11e14%), whereas it was small (<1%) for mozzarella cheese (Fig. 2B). This was not expected since mozzarella cheese contains large fat pools (Kindstedt et al., 2004) that are in the liquid state at the chyme temperature (37  C), and therefore are expected to be easily released from the cheese particles. Also, cheese particles are subjected to water absorption since they are immersed in gastric juice which may ease fat leakage. For higher fat cheese, such as Camembert, fat remains in a globular form that are larger than milk fat globules, suggesting coalescence (Lopez, 2005). Therefore, fat release during digestion could be facilitated since more fat is found at the surface of each cheese particle. When duodenal enzymes were added, the fat layer mass decreased for each cheese because fat (triglycerides) was hydrolysed by lipase into monoacylglycerols and free fatty acids. The fat layer mass was higher for all cheeses except mozzarella, suggesting a good accessibility of the lipase to lipid droplets. At the end of the duodenal digestion (D180), the theoretical pellet mass was around 19% for each cheese (Fig. 2A). The sum of the fat and pellet layers represents a mass balance lower than 15% for mozzarella, and young and aged Cheddar (Fig. 2B), which is in accordance with the theoretical data (Fig. 2A). However, the pellet mass was elevated for

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Fig. 2. Theoretical (A) and experimental (B) mass balance (%) of each layer isolated at the end of each digestion step (oral: O2; gastric: G120; duodenal: D180). For the theoretical data, the fat in the pellet represents the proportion of the initial cheese fat found in the pellet, and the pellet layer represents the amount of remaining cheese solid after the subtraction of the fat content found in cheese. Data represent the mean ± average deviation of two independent experiments.

Camembert cheese (37%; Fig. 2B). This was not expected since the pellet gained 12% in mass at the end of the duodenal digestion step compared with the end of the gastric digestion. It is hypothesised that a high amount of water may be retained in the residual pellet and thus contribute to increased pellet weight. In addition, the fatty acids which were generated by duodenal lipase action may contribute to insoluble calcium-fatty acid soap formation, as observed for other cheeses using a similar in vitro model (Lamothe et al., 2012). Stabilised Camembert cheese is known to have higher level of calcium and contained high level of lipids that may favour the formation of calcium soap. Those calcium soaps are insoluble and thus may be found in the pellet after the centrifugation step. Additional work will be necessary to confirm this hypothesis. These results suggest that cheese matrices have different behaviours attributed, in part, to the different composition and possibly to the initial texture. Young Cheddar and smear cheese both exhibited similar digestion patterns during gastric and duodenal digestion. Camembert was easily disrupted (small pellet layer mass) during gastric digestion and this may be attributed to the soft texture (low hardness) favouring a good dispersion in the chyme. In contrast, the elastic texture of mozzarella may have contributed to high pellet layer mass, limiting cheese matrix disintegration under the conditions used. Therefore, Camembert and mozzarella cheese were chosen for subsequent analysis to further understand the kinetics of cheese matrix disintegration during gastric digestion.

3.3. Camembert and mozzarella cheese behaviour during simulated in vitro digestion 3.3.1. Cheese disintegration The experimental data at the end of gastric and duodenal digestion shows a similar mass balance as seen previously in Section 3.2 for both cheeses (Fig. 3). During gastric digestion (G15 to G120), the fat layer mass was still higher for Camembert than for mozzarella, as seen previously. Also, the pellet layer mass diminished gradually over time for both cheeses, and values were higher for mozzarella. To eliminate the effect of water on the pellet layer mass, cheeses disintegration values were also presented on a dry weight basis (Fig. 4A, B). The disintegration of mozzarella was slow and gradual during gastric digestion, whereas Camembert disintegrated quickly and reached 78% after 2 min of oral digestion. At the end of the gastric step, mozzarella and Camembert reached disintegration values of 27% and 83%, respectively (Fig. 4A). This may be attributed to the cheese initial composition. Both cheeses had similar water contents but the protein-to-fat ratios were different, which subsequently affects initial cheese texture. A high protein concentration leads to a more compact structure, whereas fat softens cheese texture. Mozzarella had significantly higher hardness, springiness and cohesiveness (Table 2), suggesting higher internal forces and elastic behaviour which may reduce the capacity to disintegrate. Also, the compact protein matrix of mozzarella may limit the accessibility of cheese proteins to pepsin and,

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Fig. 3. Mass balance (%) of each layer isolated after each digestion step (oral: O2; gastric: G15, G30, G60 and G120; duodenal: D5 and D180) for Camembert and Mozzarella cheeses. Data represent the mean ± average deviation of two to four independent experiments.

Fig. 4. Disintegration (A, B) and protein/peptide release (C, D) during in vitro digestion for Camembert ( ) and mozzarella (:,-) cheese. Panels A and C refers to oral and gastric digestion and panels B and D to duodenal digestion (D5: after 5 min of duodenal digestion; D180: after 180 min of duodenal digestion). For the disintegration results, data are expressed on a dry basis. For each time point, cheeses with different letters are significantly different. Data represent the mean ± average deviation of two to four independent experiments.

therefore, reduce cheese disintegration. Cheese disintegration at the end of gastric digestion was positively correlated to initial cheese fat content (Table 3). During gastric digestion, fat may be easily dispersed into the acidic juice under agitation without disruption of the casein matrix. In addition, the release of the liquefied fat could lead to a more ‘porous’ protein matrix, which should favour the accessibility of enzymes to proteins and the degradation of the protein matrix. The aforementioned reasons

Table 3 Kendall Tau b correlation between cheese disintegration and cheese initial chemical composition.a Parameter

Disintegration G120

Disintegration D180

Fat content Proteolysis

1.000* 0.333

0.333 1.000*

a The correlation analysis was carried out for Camembert and Mozzarella cheeses; an asterisk shows correlation significant at the P ¼ 0.05 level. G120 indicates gastric digestion after 120 min; D180 indicates duodenal digestion after 180 min.

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explain why cheese disintegration was affected by cheese fat content. Cheese proteolysis level did not significantly affect cheese disintegration at the end of the gastric digestion. However, as seen previously (Section 3.2), cheese proteolysis may strongly modulate cheese disintegration. The duodenal proteolytic enzymes, trypsin and chymotrypsin, hydrolyse proteins and peptides and can further contribute to degradation of the cheese matrix. A static in vitro model was used in this study, therefore, the impact of gastric emptying on the phenomena occurring during this step cannot be differentiated. At the end of the duodenal digestion, both cheeses were no longer different, suggesting that the duodenal proteolytic enzymes are capable of disrupting the mozzarella casein matrix (Fig. 4B). The addition of duodenal enzymes decreased the disintegration (dispersion of cheese into the water phase) value to 53.16% as shown in Fig. 4B (compared with 75.59% at the end of gastric digestion as shown in Fig. 4A) for Camembert, suggesting an increase of insoluble matter. As explained previously (Section 3.2), high calcium concentration in cheese may reduce bioaccessibility of specific fatty acids by precipitation as calcium soap, as observed for Cheddar cheese (Lamothe et al., 2012), therefore increasing the proportion of insoluble matter. Cheese disintegration at the end of duodenal digestion correlated negatively to cheese proteolysis (Table 3). This was unexpected since water soluble nitrogen may be easily released into the liquid phase compared with intact caseins and, therefore, contribute to increased cheese disintegration. The reduction in cheese disintegration for Camembert cheese (weight gain) has affected the correlation results. 3.3.2. Protein/peptide release Matrix disintegration during GI digestion induces a reduction in the cheese particle size, and substrates can be released and available for the enzymes. Also, enzymes may diffuse within the cheese particles, hydrolysing the substrates, and contributing to matrix disintegration. At the end of oral digestion, the amount of protein/ peptide released into the liquid layer was high for Camembert (16%) compared with mozzarella cheese (2%) (Fig. 4C). The visualisation of the protein found in the liquid layer by SDS-PAGE showed that lower amounts of caseins could be visualised on the gel for mozzarella cheese (Fig. 5). Less caseins may be solubilised during the oral step for mozzarella, attributed to the low proteolysis level (Table 1) and to the elastic texture that may prevent cheese protein/ peptide release and limit access to enzymes. The protein/peptide release kinetics during gastric digestion was in accordance with the disintegration kinetics (Fig. 5). For

Camembert cheese, proteins were mostly released within the first 15 min, and were slowly released during the rest of the gastric digestion. Concomitantly, low molecular weight peptides increased over time. Unlike Camembert, proteins in mozzarella were released continuously during the whole gastric digestion process corresponding to the slow disintegration process. The fast protein/peptide release for Camembert provided sufficient time to complete the hydrolysis of the soluble proteins at G120. At the end of gastric digestion, no bands corresponding to caseins were visible. Results were in accordance with previous work showing that caseins in Emmental water-soluble extract were rapidly hydrolysed by pepsin at pH 2 (Parrot, Degraeve, Curia, & Martial-Gros, 2003). Few changes in caseins band intensity were observed during gastric digestion for mozzarella (Fig. 5). During the entire gastric digestion, cheese protein/peptide release and hydrolysis were slow and continuous. The bands of caseins were still intense for mozzarella cheese, indicating that the caseins were not totally hydrolysed at the end of gastric digestion. This result may be explained by the slow disintegration, which gradually released cheese proteins. Our results show that cheese disintegration varied amongst cheeses during gastric digestion, which affected the protein digestion profile during this step. Gastric digestion profile is important for the subsequent digestion steps since it controls the rate at which the partially digested nutrients appear in the intestinal tract (Gaudichon et al., 1994; Lacroix et al., 2006; Le Feunteun et al., 2014). At the end of duodenal digestion, the total protein/peptide release was similar amongst studied cheeses (Fig. 5). Trypsin and chymotrypsin hydrolysed proteins and solubilised peptidic fragments quickly, suggesting that cheese proteins are highly bioaccessible. It is worthwhile to note that the BCA assay used in this experiment may underestimate the exact protein/peptide release, since di-, tri-, and tetra-peptides and amino acids were hardly detected, except if containing a cysteine, tyrosine and/or tryptophan residue (Wiechelman, Braun, & Fitzpatrick, 1988). The SDSPAGE showed that duodenal enzymes quickly degraded the released proteins and peptides into amino acids during duodenal digestion. All bands corresponding to caseins and peptides were not visualised for all cheeses during duodenal digestion, even after 5 min of duodenal digestion. This suggests that as soon as the proteins were released, they were quickly hydrolysed into small peptides and amino acids. Although gastric emptying was not taken into account in this study, the data suggest that dairy proteins are highly bioaccessible, independent of the cheese matrix studied. The faint residual bands on the gel corresponded to the

Fig. 5. SDS-PAGE of the liquid layer for Camembert and Mozzarella cheese during in vitro gastro-intestinal digestion. Lane O2 shows the relative composition of released proteins at the end of oral digestion. Lanes G15, G30, G60, G120 show the related composition of released proteins after 15, 30, 60, and 120 min gastric digestion. Lanes D5, D180 show the related composition of released proteins after 5, 180 min duodenal digestion. Lane STD corresponds to the standard proteins measured, b-lac: b-lactoglobulin; a-lac: a-lactalbumin.

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enzymes added in our model of in vitro digestion (Rioux & Turgeon, 2012).

3.4. Role of cheese texture on the disintegration rate Cheese textural characters are expected to play an important role in the rate of cheese disintegration. Kong and Singh (2009) tested this hypothesis for a variety of common foods (peanut, beef jerky, almond, fried dough, ham, carrot, etc.), and Guo, Ye, Lad, Dalgleish, and Singh (2014) studied different protein gels. An in vitro digestion model was used in both studies. The food products/gels exhibited different disintegration rates and profiles depending upon the initial texture and textural change during digestion. Several factors were shown to impact food disintegration. For example, ham steak with initial low hardness was disintegrated very quickly, whereas almonds with initial high hardness were disintegrated slowly (Kong & Singh, 2009). Our results also showed that cheese disintegration may be dominated by different mechanisms depending upon the texture (Fig. 4). Camembert cheese presented the lowest hardness, springiness and chewiness compared with mozzarella (Table 2). The cohesiveness was higher than for aged Cheddar cheese but lower than for young Cheddar, smear cheese, and mozzarella cheese (Table 2). The disintegration of Camembert was faster from the beginning of the digestion. The disintegration of mozzarella cheese was slow and continuous during gastric digestion due to the compact and elastic matrix (Table 2). The disintegration kinetics during gastric digestion were not studied for aged Cheddar, smear cheese, and young Cheddar cheeses. In this study, commercial cheeses, varying in texture, presented different disintegration profiles during gastric digestion. During digestion, food texture may change and affect the disintegration rate. During a static soaking process, the water absorption phenomenon (the humidity of almond was increased from 3.3 to 30.7%) contributed to a significant decrease in almond hardness, resulting in an increase in the disintegration rate (Kong & Singh, 2009). These authors also demonstrated that acidity and temperature have a significant effect on the food disintegration rate related to texture softening. Results showed that the half-time of carrot disintegration decreased from 3.3 to 1.5 h when the digestion pH was modified (from 5.5 to 1.8). The half-time of disintegration and the hardness of carrots were correlated under the stated digestion conditions, suggesting that changing the food texture affected food disintegration. The addition of pepsin (1%) increased the disintegration rate of beef jerky by 21%. However, in the case of carrots, pepsin addition did not improve the disintegration rate indicating the pepsin hydrolysis had limited effect on carrot disintegration since carrots contain few proteins (<1%, compared with ~50% protein for beef jerky). The model of Kong and Singh (2009) was also used by Bornhorst and Singh (2013) to describe the disintegration of different types of bread samples. These authors demonstrated that different types of bread exhibited different disintegration related to water absorption capacity (Bornhorst & Singh, 2013). The acid had limited effect on disintegration whereas amylase hydrolysis softened breads and accelerated disintegration. Therefore, the food disintegration mechanism may change during gastric digestion due to various factors such as water adsorption, acidic and enzymatic hydrolysis (Kong & Singh, 2009; Lentle & Janssen, 2011). In addition to these reports, the present study revealed that the cheese disintegration level at the end of the gastric digestion increased (reduction in the pellet layer mass) and tended to be different amongst cheese varieties (Fig. 2). We suggest that acidic conditions and pepsin hydrolysis were involved in cheese texture modification during digestion and decreased the

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forces that hold the cheese matrix together. However, it is not possible to distinguish the effect of each individual factor. 4. Conclusions Commercial cheeses, varying in texture, exhibited different cheese disintegration and protein digestion patterns during gastric digestion. Both cheese texture and cheese composition affected cheese digestibility. Cheese disintegration at the end of gastric digestion was higher when initial cheese hardness, cohesiveness, and chewiness were lower. Soft cheese, such as Camembert, needed less force to break apart the cheese matrix. The disintegration of Camembert was fast from the beginning of digestion (oral). Pepsin hydrolysis contributed to a decrease in the cohesive forces that held the matrix together, resulting in an increase in cheese disintegration. Mozzarella, with higher hardness and cohesiveness values, was disintegrated slowly and continuously during gastric digestion. The fat content and the extent of proteolysis affected cheese textural characteristics, which were shown to affect cheese disintegration. In addition, during digestion, fat and nitrogen release may help degrade the cheese matrix and accelerate cheese disintegration. During duodenal digestion, the addition of duodenal juice along with enzymatic reactions played an important role in disintegration and protein digestion. Our results suggest that the adjustment of cheese processing parameters, resulting in structural and textural changes to cheese, can be used to modulate the digestion of cheese proteins. This could affect postprandial protein metabolic response and physiological functions. Future research is needed to relate the different nutrient release rates to metabolic responses, such as satiety and muscle protein accretion. Acknowledgements This project was supported by grants from: 1) the Canadian Agri-Science Clusters Initiative (Dairy Farmers of Canada, Agriculture and Agri-Food Canada and the Canadian Dairy Commission); 2) the Canadian Foundation for Innovation to establish a platform to study food structure for optimal nutritional uses (FCI 25496); 3)
bec e Nature et Technologies (2012Le fonds de recherche du Que
re de l'Economie, VN-164567); and 4) the Ministe de l'Innovation et de l'Exportation (MEIE) (PSR-SIIRI-748). The authors wish to acknowledge the scholarship support for X. Fang from the ‘FAST’ program funded through a CREATE-NSERC project. The authors also wish to thank D. Gagnon and D. Buchner Sanchez Olivera for technical support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.idairyj.2016.01.023. References AOAC. (2000). Ash of cheese. Method no. 935.42. In W. Horwitz (Ed.), Official methods of analysis of AOAC International. Gaithersburg, MD, USA: Association of Official Analytical Chemists International. AOAC. (2008). Loss on drying (moisture) in cheese. Method no. 926.08. In W. Horwitz (Ed.), Official methods of analysis of AOAC International. Gaithersburg, MD, USA: Association of Official Analytical Chemists International.
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