Effects of high pressure processing (hydrostatic high pressure and ultra-high pressure homogenisation) on whey protein native state and susceptibility to tryptic hydrolysis at atmospheric pressure

Food Research International 79 (2016) 40–53

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Effects of high pressure processing (hydrostatic high pressure and ultra-high pressure homogenisation) on whey protein native state and susceptibility to tryptic hydrolysis at atmospheric pressure Claire Blayo, Océane Vidcoq 1, Françoise Lazennec, Eliane Dumay ⁎ Université de Montpellier, UMR 1208–Ingénierie des Agropolymères et Technologies Emergentes, Équipe de Biochimie et Technologie Alimentaire, CC023, Site du Triolet, 2 Place Eugène Bataillon, 34095 Montpellier Cedex 5, France

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Article history: Received 3 October 2015 Received in revised form 24 November 2015 Accepted 26 November 2015 Available online 27 November 2015 Keywords: Whey proteins High-pressure Tryptic hydrolysis Differential scanning calorimetry Ammonium sulphate precipitation Light scattering

a b s t r a c t Dispersion of whey protein isolate (WPI) prepared at 10% proteins (w/w) and pH 6.5, was pressure-processed using high hydrostatic pressure (HHP) at 300 MPa and 25 °C for 15 min, or ultra-high pressure homogenisation (UHPH) at 300 MPa and initial fluid temperature (Tin) of 24 °C. UHPH-processing was followed (or not) by rapid cooling of the processed fluid at the immediate HP-valve outlet. Short-time thermal treatment (STTT) at 75 °C for 10 s, 43 s or 110 s was studied for comparison. Processing-induced effects were investigated in terms of (i) protein denaturation by differential scanning calorimetry (DSC) and ammonium sulphate (AS) precipitation, (ii) susceptibility to tryptic proteolysis at atmospheric pressure after processing, (iii) protein particle sizes from light scattering measurement during the hydrolysis process and, (iv) residual proteins by SDS-PAGE after 135 min of hydrolysis. UHPH followed by efficient cooling at the HP-valve outlet, limited significantly (p = 0.05) protein denaturation as assessed by DSC and AS precipitation, while increasing notably protein susceptibility to tryptic attack, comparing with the non-processed sample. In opposite, neglecting the cooling step at the HPvalve outlet, led to significantly (p = 0.05) lower residual protein native state, then tryptic hydrolysis efficiency. HHP treatment led to intermediate results. The slight protein unfolding that pre-processing could induced, favoured thus the further trypsin attack. Generally speaking, high pressure (avoiding overheating) offers this potential. Process-induced protein aggregation, but also reassembly of protein fragments and peptides released during hydrolysis may affect the proteolysis efficiency. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Susceptibility of dairy proteins to enzymatic hydrolysis has been investigated using specific proteases to probe protein structural stability in their native state or after chemical modifications (Chobert, Dalgalarrondo, Dufour, Bertrand-Harb, & Haertlé, 1991; Creamer et al., 2004; Huang, Catignani, & Swaisgood, 1994; Sitohy, Chobert, & Haertlé, 2001), to evaluate protein functionality such as ligand-binding or interfacial properties upon limited proteolysis (Ipsen et al., 2001; Sponton, Perez, Carrara, & Santiago, 2014), to elaborate protein preparations with reduced antigenicity and allergenicity (Bernasconi, Fritsché, & Corthésy, 2006; Pahud, Monti, & Jost, 1985), or to produce bioactive peptides for nutritional purpose (Doyen, Husson, & Bazinet, 2013; Garcia-Mora, Peñas, Frias, Gomez, & Martinez-Villaluenga, 2015; Power, Fernández, Norris, Riera, & FitzGerald, 2014; Tulipano, Faggi, Nardone, Cocchi, & Caroli, 2015). The approach involving specific proteases allows assessing temperature or high pressure-induced protein ⁎ Corresponding author. E-mail address: [email protected] (E. Dumay). 1 Present address: Nestlé Purina PetCare, Wisbech, Cambs PE13 2RG, UK.

http://dx.doi.org/10.1016/j.foodres.2015.11.024 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

structural changes on the resulting proteolysis efficiency (Hayashi, Kawamura, & Kunugi, 1987; Kitabatake & Kinekawa, 1998). Proteolysis of purified β-lactoglobulin (β-Lg) or whey proteins has thus been studied using thermolysin, trypsin or chymotrypsin at pH 7–8, pepsin at pH 2.5–4, or other food-grade proteases (i) after high-pressure treatment, and (ii) also under high-pressure. While β-Lg resists peptic hydrolysis in physiological conditions (0.1 MPa; pH 3–4), its rate of hydrolysis greatly increased under pressure up to 300 MPa (Chobert et al., 1996), and also after β-Lg pressurisation but at higher pressure levels (Zeece, Huppertz, & Kelly, 2008). A previous pressurisation of purified β-Lg in aqueous phase at neutral pH (300–450 MPa and 25 °C for 15–20 min) increases its further proteolysis by trypsin, chymotrypsin or other proteases at pH 6.8–7.5 and atmospheric pressure, as compared to the non-processed β-Lg (Belloque, Chicόn, & Lόpez-Fandiño, 2007; Chicόn, Lόpez-Fandiño, Quirόs, & Belloque, 2006; Knudsen, Otte, Olsen, & Skibsted, 2002; Maynard, Weingand, Hau, & Jost, 1998). When conducted under pressure, proteolysis of purified β-Lg or β-Lg processed in bovine whey at neutral pH, was particularly enhanced with an optimum at 200–400 MPa depending on the protease (Belloque et al., 2007; Chicόn et al., 2006; Dufour, Hervé, & Haertle, 1995; Hayashi et al., 1987; Maynard et al., 1998; Okamoto, Hayashi,

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Enomoto, Kaminogawa, & Yamauchi, 1991; Peñas, Préstamoa, Baeza, Martínez-Molero, & Gomez, 2006; Stapelfeldt, Petersen, Kristiansen, Qvist, & Skibsted, 1996). Such proteolysis enhancement was attributed to the pressure-induced β-Lg unfolding, especially at pH 7 or 8. A more efficient hydrolysis was yet observed when β-Lg proteolysis at neutral pH using chymotrypsin or trypsin was conducted under high-pressure comparing with hydrolysis performed after β-Lg pressurisation (Belloque et al., 2007; Maynard et al., 1998; Peñas et al., 2006). The difference was attributed to an accelerated breakdown of large intermediate hydrolysis-products of high hydrophobicity, into final hydrolysates (Maynard et al., 1998). The difference could also result from β-Lg refolding after pressure release (Stapelfeldt et al., 1996). It is worth noting that pressurisation could also increase protease activities in the 200–400 MPa range (Ruan, Lange, Bec, & Balny, 1997; Van Willige & Fitzgerald, 1995). Higher pressure levels inactivated the protease. Interestingly, pressure-assisted proteolysis was accompanied by a reduction of β-Lg (or whey proteins) antigenic activity (Bonomi et al., 2003; Nakamura, Sado, & Syukunobe, 1993; Okamoto et al., 1991) and the production of WPI hydrolysates exerting antiinflammatory (Vilela, Lands, Chan, Azadi, & Kubow, 2006) or antioxydative properties (Garcia-Mora et al., 2015). Hence high-pressure processing may be proposed to develop convenience foods with health attributes (Barba, Esteve, & Frigola, 2012; Barba, Terefe, Buckow, Knorr, & Orlien, 2015). However, this knowledge has not yet been applied to high pressure homogenisation taken as a continuous process. The aim of the present study was to evaluate effects of pressureprocessing on whey protein native state and in parallel, its susceptibility to tryptic hydrolysis, both assessed after processing. The susceptibility of WPI samples to tryptic attack was probed at pH 7.0 and 37 °C, while monitoring the changes in protein particle size upon proteolysis. Pressure treatment was chosen on the basis of our previous studies: high hydrostatic pressure (HHP) at 300 MPa and 25 °C for 15 min; dynamic high-pressure (or ultra-high-pressure homogenisation, UHPH) at 250 MPa or 300 MPa followed or not by immediate cooling at the high-pressure valve (HP-valve) outlet. Short-time thermal treatments (STTT) at 75 °C for 10 s, 43 s or 110 s were also studied for comparison.

2. Material and methods 2.1. Reagents and purified proteins All chemicals were of analytical grade. Ammonium sulfate (1.01217.1000) was from Merck (Darmstadt, Germany). Trizma® base (T-1503), sodium dodecyl sulfate (SDS, L-4509), N-α-benzoyl-Larginine ethyl ester hydrochloride (BAEE; B4500) came from SigmaAldrich (St. Louis, MO, USA). Sodium mono- and di-phosphate (480087; 480137), glycerol (453755), methanol (528101) and acetic acid (401391) were from Carlo Erba, Milan, Italy. The whey protein isolate (WPI; Prolacta 90, lot A13004) has been industrially prepared by Lactalis Ingredients (Retiers, France) in mild conditions: milk microfiltration followed by ultrafiltration of milkmicrofiltrate, then spray-drying of the ultrafiltration-retentate. WPI composition is indicated in Section 2.2. Purified and freeze-dried bovine β-Lactoglobulin (β-Lg; L2506; non-crystallized; A and B variants; 18.4 kDa), α-Lactalbumin (α-La; L5385; 14.4 kDa), bovine serum albumin (BSA; A0281; 66 kDa) and trypsin from bovine pancreas (E.C. 3.4.21.4; T8003; 24 kDa) came from Sigma-Aldrich. The concentration of purified proteins was determined using the following weight attenuation coefficients (ε1‰) at 280 nm: 0.96, 2.09 and 0.66 L g−1 cm−1 for βLg, α-La and BSA, respectively (Farrell et al., 2004), and 1.29 L g−1 cm−1 for trypsin (Shaw, Mares-Guia, & Cohen, 1965). Coomassie blue (Brillant Blue R, B7920), ColorBurst™ Electrophoresis Marker (C1992) (8– 220 kDa) came from Sigma (St. Louis, MO, USA). Precision Plus Protein™ standards Dual Color came from Bio-Rad (Hercules, CA, USA).

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2.2. Whey protein dispersions WPI powder contained 94.9 g dry solids per 100 g, and in dry basis (w/w), 90.4% protein (N × 6.38), ~ 2% ash (including 0.25% calcium) and ~ 4% lactose. Protein constituents corresponded mainly to β-Lg and α-La (i.e., ~68.5% β-Lg and 21.5% α-La per 100 g of soluble protein at pH 6.6, as given by the producer) plus small amounts or traces of bovine serum albumin (BSA), lactoferrin and immunoglobulins. WPI dispersions were prepared at 10% proteins (w/w) in deionised water (Millipore®) and pH 6.5 ± 0.1 (spontaneous pH) by gentle magnetic stirring at 22 ± 2 °C for 2 h avoiding foam formation, then stored overnight at 4 °C to complete protein hydration. Dispersion density equaled 1027.8 g L−1 at 20 °C. WPI dispersions were equilibrated at 24 °C prior to experiments. WPI protein concentration was calculated from absorbance measurement at 280 nm (UV-2 spectrophotometer, Unicam, Thermo Optek, Montigny-le-Bretonneux, France) using an apparent weight attenuation coefficient (ε1‰) of 1.23 L g−1 cm−1. WPI “solubility index”, evaluated as the % of proteins remaining soluble vs. total proteins after centrifugation (12,061 g at 20 °C for 20 min; rotor SS34, centrifuge RC 5B plus Sorvall) of WPI dispersions prepared at ~1% proteins (w/w) in 50 mmol L− 1 sodium phosphate buffer (pH 6.5), equaled 87.6 ± 2.2%, indicating a good native state. 2.3. Processing of protein dispersions Protein dispersion prepared at 10% proteins (w/w) and pH 6.5, was submitted to one of the following technological processing: UHPH at 300 MPa with or without rapid cooling at the immediate HP-valve outlet; hydrostatic high pressure treatment at 300 MPa and 25 ± 0.5 °C for 15 min; STTT at 75 °C (water bath temperature) for 10 s, 43 s or 110 s, as detailed in the following sub-sections. 2.3.1. Ultra-high pressure homogenisation of WPI dispersions WPI dispersions equilibrated at an initial temperature (Tin) of 24 ± 0.5 °C were processed using an ultra-high pressure homogeniser (model FPG7400H, Stansted Fluid Power Ltd., Essex, UK) up to a homogenisation pressure (P1) of 250 MPa or 300 MPa. Temperature and pressure changes were followed at different locations of the homogeniser using thermocouples, manometers, pressure gauges and a fast data acquisition system (10 Hz) as already detailed (Dumay et al., 2013). The sole high-pressure valve (HP-valve or first stage) was used in the present study. Temperature and pressure were measured (i) at the inlet (T1, P1) and at the outlet (T2, P2) of the HP-valve, (ii) after rapid cooling of the processed fluid through a first cooling-device located at the immediate HP-valve outlet (T3). A second cooling device was installed just before the fluid outlet from the homogeniser (T4) to adjust the final temperature of the processed fluid at ≤30 °C. The first cooling device operated using a cryostat (Lauda RK8KS, Königshofen, Germany) set at 8 ± 0.5 °C, and was put into use or not, depending on the experiments. The second cooling device operated using a second external cryostat (Lauda RK8CS model, Königshofen, Germany) set at 14 ± 2 °C and was used in all cases. (T1) was the real inlet temperature of the fluid at the entrance of the HP-valve, including heat of compression induced by the pressure build-up in the homogeniser intensifier. (T2) resulted from the temperature jump of the fluid passing through the HP-valve gap. Indeed, the pressure drop (P1 − P2) recorded when the fluid flows through the HP-valve gap, generates mechanical forces (elongational stress plus turbulence, cavitation phenomena and impacts with solid surfaces and between particles) plus a temperature jump (T2) due to a thermal dissipation of kinetic energy as already described (Dumay et al., 2013). When the processed fluid flew through the HP-valve, its temperature was brought to (T2) for less than 0.25 s. Then, the fluid reached temperature (T3) in 1.75 s. Samples were collected at the homogeniser outlet after the first 500 mL corresponding to ~ 3-times the mean residence time of a particle in the whole homogeniser, then analysed within 1–2 h at 21 ± 1 °C or kept at 4 °C

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before further analyses. At least two independent UHPH experiments were carried out on different days. 2.3.2. Hydrostatic high pressure treatment of WPI dispersions HHP-processing was carried out at 300 ± 1 MPa for 15 min, in a high pressure vessel (ACB, Nantes, France) filled with deionised water as pressure-transmitting medium (PTM) and equipped with a hydropneumatic pump (Haskel DSHXF903 type, General Pneumatic, Villeneuve d'Ascq, France), a calibrated pressure gauge (PR801F) from Asco Instruments (Chateaufort, France; 0.15% accuracy over the 0.1– 700 MPa scale) and a pressure regulation valve. The HP cell (1 L capacity, 80 mm in diameter; 220 mm in height) was filled with deionised water at 25 ± 1 °C. Temperature in the vessel was controlled by circulation of water around the HP-cell using an external cryostat (Lauda RK8CS model). PTM temperature was recorded using a thermo-indicator (Eurotherm 842, Dardilly, France) and a J-thermocouple (1.0 mm in diameter, TC S.A., Dardilly, France) immersed in the PTM at 6 cm from the HP-chamber top. A fifty g of protein dispersion was poured into flexible polyvinylidene chloride tubing (PVDC, 35 mm in diameter, Krehalon, Eygalières, France) sealed with double knots at both ends. Before being placed in the PTM at 25 ± 1 °C, samples were previously equilibrated in a water-bath at 25 ± 0.5 °C. Pressure was raised from 0.1 to 300 MPa at a rate of 100 MPa min−1, maintained at 300 MPa for 15 min then released down to 0.1 MPa at a rate of 100 MPa min− 1 using the HP vessel calculator. PTM temperature increased by ~ 3 °C per 100 MPa during the pressure built-up and conversely decreased at the pressure release. During the pressure maintaining for 15 min (plateau), PTM temperature slowly decreased from ~ 33 to ~ 26 °C. Previous studies carried out with similar HP-cell and sample geometry, indicated that temperatures recorded at the centre of the sample cylinder or 1–2 mm below its surface remained close to that of the waterPTM during pressure-processing in the 5–35 °C temperature range (Kolakowski, Dumay, & Cheftel, 2001; Regnault, Thiebaud, Dumay, & Cheftel, 2004). At least, two independent HP-processing were performed on different days. Samples were analysed within 1–2 h at 21 ± 1 °C or kept at 4 °C before further analyses. 2.3.3. Continuous short-time thermal treatment of WPI dispersions WPI dispersion at 10% proteins (w/w) and pH 6.5, previously equilibrated at 24 ± 0.5 °C was submitted to continuous STTT by circulating through a stainless-steel tubing (2.10 mm inner Ø; 3.18 mm outer Ø) immersed in a water bath at 75 ± 0.5 °C, controlling the fluid flow rate. The residence time in this tubing was 10.2 ± 0.7 s (called “10 s”), 43.3 ± 2.9 s (called “43 s”) or 110 ± 10 s (called “110 s”). The fluid temperature checked at the tubing outlet was close to 74 ± 0.5 °C. Samples were immediately cooled with ice at the tubing outlet, then analysed within 1–2 h at 21 ± 1 °C, or kept at 4 °C before further analyses. At least, two independent runs were carried out on different days. 2.4. Differential scanning calorimetry (DSC) Calorimetric measurements were carried out using a Micro-DSC III microcalorimeter equipped with a CS32 Controller (Setaram, Caluire, France) and conducted using the Calisto Software vs 1.12 (AKTS, Siders, Switzerland). WPI dispersions at 10% proteins (w/w) and pH 6.5 were diluted to 3.65% proteins (w/w) with deionised water for DSC measurements. Ultra-pure water was used as the reference. A 850 mg of protein sample and reference was weighted into hermetic C276 hastelloy cells, then heated in the calorimeter from 22 °C to 95 °C at 1 °C min−1 heating rate (first run). After cooling at 1.5 °C min−1, sample and reference were heated again (second run) under the same heating conditions to assess possible reversibility of protein unfolding. After baseline flattening, the enthalpy change was determined from the area of endothermal peak using a straight baseline between the beginning and the end of thermal transition. The apparent enthalpy of thermal denaturation (ΔHTD, J g−1)

and the maximum of the endothermal peak (Tm, °C) were determined for each WPI sample. Results were the mean ± s.d. of 4–6 DSC determinations from two independent technological treatments. To quantitate processing effects in term of protein unfolding, each processing was characterised by the residual enthalpy expressed as the ΔHTD value of processed WPI dispersion relative to that of non-processed WPI dispersion ×100. DSC analysis of purified β-Lg (L2506; Sigma) at 2.5% protein (w/w) in pH 6.5, 0.05 mol L−1 phosphate buffer, was performed for comparison. 2.5. Separation of denatured WPI proteins by ammonium-sulphate precipitation Insoluble WPI proteins (unfolded and/or aggregated) were precipitated using the ammonium-sulphate (AS) procedure, as already described (Blayo, Puentes-Rivas, Picart-Palmade, Chevalier-Lucia, Lange, & Dumay, 2014), except that a full range of AS concentration (0 to 3.3 mol L− 1 in AS) was used in the present study to get the whole precipitation curves. A 1 mL of non-treated or processed WPI dispersion at 10% proteins (w/w) and pH 6.5, was deposited into polypropylene-copolymer tubes of 12 mL (PPCO, 3420–1613, Nalgene, Rochester, US). A 9 mL of 0.05 mol L−1 sodium phosphate buffer (PB; pH 6.5), prepared at the adequate AS molarity was then added to get AS final concentration in the 0–3.3 mol L−1 range. The final protein concentration in PPCO tubes equaled 1.028% (w/v). Four PPCO tubes were prepared for each AS molarity of each protein precipitation curve. After a 2 h-waiting time in a temperature-controlled cabinet at 25 ± 0.5 °C to allow protein precipitation, PPCO tubes were centrifuged (CF) at 12,061 g and 22 ± 1 °C for 20 min (SS34 rotor, RC 5B plus Sorvall centrifuge). CF-supernatants were tenfold diluted with the corresponding PB-AS solution to quantitate “soluble proteins” by absorbance measurement at 280 nm (UV-2 spectrophotometer) using the weight attenuation coefficient ε1‰ of 1.23 L g−1 cm− 1. Amounts of soluble whey proteins from CF-supernatants were expressed as g of soluble proteins per 100 g of WPI dispersion or 10 g of total protein (WPI dispersions were prepared at 10% proteins, w/w). Results were the mean of 4 protein determinations ± s.d. for each AS molarity. A protein solubility index (%) was defined as the amount of soluble proteins determined in a processed sample relative to the amount of soluble proteins in the non-processed (control) sample ×100. 2.6. In vitro tryptic digestion Tryptic digestion was carried out in vitro at atmospheric pressure using a pH-stat apparatus (Titrando STAT 902) equipped with a 807 Dosing Unit, a 801 Magnetic Stirrer, and a Micro-SC combination glass electrode, all from Metrohm® AG (Herisau, Switzerland). The pH-stat apparatus operated using the Tiamo 2.3 software (Metrohm®). Tryptic digestion of whey proteins was carried out at pH 7.0 and 37 ± 0.5 °C in a 100 mL beaker thermostated through an external water circulation. The cleavage of aminopeptides was followed during the hydrolysis process through the controlled addition of 0.02 mol L−1 NaOH solution, to keep the pH constant at 7.0. The cumulated volume of 0.02 mol L−1 NaOH (VNaOH) added to WPI dispersions was recorded by the software. In the pH-stat technique (Adler-Nissen, 1986a), the degree of hydrolysis (DH) represents the number of hydrolysed peptide bonds (or hydrolysis equivalent h, given in meqv g−1 proteins) over the total number of peptide bonds in the protein substrate (htot, given in meqv g−1 proteins) × 100 (Eq. (1)). Since hydrolysis releases protons, DH is proportional to the volume (mL) or meqv of NaOH delivered to maintain the pH constant (Eq. (2)). DH ¼ h=htot  100

ð1Þ

DH ¼ V NaOH  NNaOH  ð1=αÞ  ð1=M P Þ  ð1=htot Þ  100

ð2Þ

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where, NNaOH is the normality of NaOH solution, MP is the mass of protein (N × 6.38), (1/α) is the reciprocal of the dissociation degree of α-NH2 residues. According to (Adler-Nissen, 1986b), (1/α) equals 3.5 at 37 °C and pH 7.0, and (htot) equals 8.8 meq g− 1 protein for whey proteins. Trypsin powder was dissolved in 0.05 mol L−1 sodium phosphate buffer (PB) at pH 7.0 to get a trypsin concentration close to 7 mg mL−1. The latter stock trypsin solution was aliquotted into 2 mL Eppendorf tubes for storage at −20 ± 1 °C. Tryptic activity was checked during frozen storage over the experimentation duration using the BAEE procedure. For this 200 μL of the stock trypsin solution was 100fold diluted in PB (pH 7.0) then added to a 3 mL of 0.025 mol L− 1 BAEE prepared in 0.067 mol L− 1 NaH2PO4 buffer (pH 7.6) at 37 ± 0.5 °C. Tryptic activity was measured by UV absorption at 253 nm (UV-2 spectrophotometer) and 37 ± 0.5 °C. One BAEE unit was defined as the absorbance variation (ΔA 253 nm ) of 0.001 per min, at pH 7.6 and 37 ± 0.5 °C in a reaction volume of 3.2 mL. Using such operating conditions, tryptic activity equalled 18,525 ± 480 BAEE units per mg of trypsin over the experimentation duration. For in vitro digestion experiments, WPI dispersions (at 10% proteins, w/w, and pH 6.5) were diluted to a protein concentration of 5 mg mL−1 using deionised water (Millipore®), which corresponds to a β-Lg concentration close to 3.4 mg mL−1. A 35 mL of the latter diluted WPI dispersion was equilibrated at 37 °C for 30 min in the pH-stat beaker. At the experiment starting, the pH was adjusted to 7.0 in a 2 min-equilibration through the controlled addition of 0.02 mol L−1 NaOH by the pH-Stat. A ~250 μL of trypsin solution was then added to WPI dispersions (t = 0) to get a 0.05 mg mL−1 trypsin concentration and 1/100 (w/w) enzyme/ substrate (E/S) ratio. The pH of WPI dispersion was maintained at pH 7.0 over the 135 min of in vitro hydrolysis process. The cumulated volumes of NaOH (VNaOH) recorded by the pH-Stat software were corrected for the dilution during the hydrolysis process. Small aliquots of 200 μL were taken out of the incubation mixture at as soon as trypsin was added (t = 0 min), then after 45, 90 and 135 min of the digestion process (t = 45, 90 or 135 min) for particle size determination (Section 2.7.). At the end of incubation (t = 135 min), aliquots of the protein-trypsin mixtures were prepared in the SDS-dissociating buffer for electrophoresis (Section 2.8.), heated at 100 °C for 5 min to inactivate trypsin, then quickly frozen and stored at −20 °C until electrophoretic analysis. In vitro digestion tests were repeated 4–5 times for each independent experiment carried out on different days. Means curves obtained from 6 to 10 determinations are shown. 2.7. Particle size distribution and molecular weight estimation Particle size distribution curves were measured by photon correlation spectroscopy (PCS, or dynamic light scattering) at 25 ± 1 °C using a Nano series ZS (Malvern Instruments, Malvern, UK) equipped with a 4 mW He–Ne laser operating at 633 nm, and a photodiode detector with a detection angle of 173°, as already described (Blayo et al., 2014). In the present study, WPI samples (with or without previous tryptic digestion) were assessed at ~0.5 g L−1 and 25 °C for PCS analysis and Mw determination, using polystyrene 4-sided polished cuvettes (Sarstedt, Nümbrecht, Germany). It was checked that a previous filtration of samples through a 0.8 μm cellulose-acetate membrane (Sartorius, Goettingen, Germany) did not change the size distribution curves confirming the absence of dust or air bubbles, or loss of protein aggregates. For each protein sample, 6–9 light scattering measurements were carried out (each consisting of 13 individual runs of 10 s) changing the protein sample in the cuvette every 3 light scattering measurements. Trypsin activity in the digested WPI samples was ~ 10-fold decreased by lowering the sample temperature to 20–25 °C, allowing stable size measurements during PCS determination. Experimental data were assessed by NNLS (non-negative least square) algorithm able to fit the correlation curve to a multiple exponential decay, as recommended for biological polydisperse samples (Blayo et al., 2014). Data

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were acquired in the 0.6–6000 nm range and analysed using the General Purpose Model (multi-modal analysis; Malvern Instruments, Ltd.) to obtain the particle size distributions in light-intensity. Particle size distributions in number frequency (%) were calculated from the distributions in light intensity. For calculation, water viscosity was taken as 0.8875 mPa s and water refractive index as 1.330 at 25 °C. Characteristics of dispersed particles were taken as for milk proteins: 0.004 and 1.36 for the imaginary and the real refractive indices, respectively (Regnault et al., 2004). Peak characteristics (minimal and maximal diameters, Peak maximum) were collected on the distribution curves. An estimation of protein molecular weight (estimated Mw) was given by the Malvern software (vs. 7.02) using a calibration curve set between the hydrodynamic radius (RH) of standard proteins and their known Mw. To characterise whey proteins, RH value was taken as half of the diameter corresponding to the Peak maximum on the distribution curves in light intensity. In a previous study (Blayo et al., 2014), we have got a good agreement between the Mw estimated by the Malvern software, the Mw calculated using an alternate of Mark–Houwink equation suitable for light scattering data, and the known protein Mw values. 2.8. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was carried in the presence of SDS without reducing agent (SDS-PAGE), using a vertical slab apparatus (Mini-Vertigel 2) and a Stavip-500 power supply both from Apelex (Massy, France). Anamed® pre-cast native Tris–Glycine gels (TG 42010; 10 cm × 10 cm; 1 mm width) prepared with a stacking gel at 4% in polyacrylamide and a separating gel at 4–20% in polyacrylamide (linear gradient) came from BLD-Apelex (Evry, France). Protein samples were diluted in pH 6.8 Tris–HCl buffer (0.1 mol L−1 final) in the presence of SDS (20 g L−1) and glycerol (150 mL L−1) then heated at 100 °C for 5 min. A 10 μL of protein mixtures at 1 mg mL−1 protein was pipetted onto the gels. Electrophoresis was carried out in pH 8.3 Tris–glycine buffer (0.025 mol L−1 Tris; 0.192 mol L−1 glycine; 1 g L−1 SDS) at a constant voltage of 90 V and 18 ± 0.5 °C for 2 h. A 6 μL of ColorBurst Electrophoresis Marker was deposited on the gels to monitor electrophoresis running. Purified β-Lg (L-2506), α-La (L-5385) and BSA (A0281) were deposited on the gels for whey protein identification. Precision Plus Protein standards from (Bio-Rad) was also used for MW determination. Electrophoresis gels were fixed and stained with a methanol/acetic acid/water (45/9.1/45) solution containing 1 g L−1 Coomassie blue then washed with a methanol/acetic acid/water (10/ 7.5/82.5) solution. The intensity of stained bands was scanned at 590 nm using a GS-300 densitometer (Hoefer Scientific Instruments, San Francisco, CA) to get a semi-quantitative determination of whey protein bands. It was checked that peak height of β-Lg and α-La bands on scans, was correlated through a 2-order polynomial equation (R ≥ 0.9961) to the amount of deposited whey proteins in the 4–10 μg range. 2.9. Statistical analysis Statistical analyses of experimental data were carried out using Fischer's and Student's tests for p = 0.05, 0.01 or 0.001. Results were the means ± s.d. of 4–6 replicates for DSC determination, 4–8 replicates for soluble protein determination at each AS molarity, 6–10 replicates for in vitro digestion tests, as indicated in the corresponding sections. Means were compared using the Unpaired t-test from SigmaPlot® 11.0 Software (Systat Software, Inc. San Jose, CA, USA). 3. Results and discussion 3.1. Evaluation of whey protein denaturation Processing-induced unfolding and denaturation of whey proteins was assessed by DSC. During DSC analysis of proteins, the disruption of protein intramolecular hydrogen bonds that is an endothermic

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Table 1 Effects of processing on whey protein native state as evaluated by differential scanning calorimetry (DSC), protein precipitation by ammonium sulphate and susceptibility to tryptic attack. Processing of WPI dispersions (10% proteins, w/w; pH 6.5)

Non-processed (control) Short-time thermal treatment (STTT) at 75 °C for 10 s Short-time thermal treatment (STTT) at 75 °C for 43 s Short-time thermal treatment (STTT) at 75 °C for 110 s UHPH at 250 MPa without immediate cooling UHPH at 300 MPa without immediate cooling UHPH at 300 MPa with immediate cooling Hydrostatic high-pressure (HHP) at 300 MPa and 25 °C for 15 min

Differential scanning calorimetry1

Soluble proteins in 1.8 mol L−1 ammonium sulphate2

Residual Tm ΔHTD (J g−1 proteins) enthalpy (%) (°C)

g of soluble protein per Protein DH at 135 min 100 g of WPI dispersion solubility of hydrolysis index (%) process (%)

100 17.85 ± 0.43 a 17.23 ± 0.39 b 96.5 c 14.05 ± 0.41 78.7 13.00 ± 0.65 d 72.8 16.98 ± 0.70 b,e 95.1 14.40 ± 0.63 c 80.7 17.09 ± 0.23 b 95.7 e 16.45 ± 0.49 92.2

7.62 ± 0.26 a 6.93 ± 0.20 b 5.35 ± 0.07 c n.d. 6.89 ± 0.13 b 5.05 ± 0.10 d 6.91 ± 0.23 b 5.70 ± 0.10 e

75.63 ± 0.22 a 76.15 ± 0.14 b 76.67 ± 0.92 b 78.34 ± 0.46 c 75.78 ± 0.13 a,d 75.07 ± 0.83 a 75.90 ± 0.15 d 75.43 ± 0.41 a

100 90.9 70.2 n.d. 90.4 66.3 90.7 74.8

Tryptic hydrolysis3

4.44 ± 0.26 a 7.72 ± 0.66 b 4.92 ± 0.57 a,c 5.36 ± 0.90 c,d 4.67 ± 0.50 a,c 5.11 ± 0.44 c 7.43 ± 0.62 b 6.02 ± 0.42 d

(a–e)

Different letters in the same column indicate significant difference for p = 0.05. Characteristics of whey protein dispersions (3.65% proteins, w/w; pH 6.5) as assessed by differential calorimetry at 1 °C min−1 heating rate: enthalpy of thermal denaturation (ΔHTD) and temperature at the maximum of enthalpic peak (Tm). Means ± s.d. of 4–6 measurements from 2 independent experiments. The residual enthalpy (%) corresponds to the ΔHTD of processed sample relative to the ΔHTD of non-processed sample ×100. 2 Solubility of whey proteins as assessed after precipitation in the presence of 1.8 mol L−1 ammonium sulphate. Means ± s.d. of 4–8 measurements from currently 2 independent experiments. The protein solubility index (%) corresponds to the amount of soluble proteins in the WPI processed sample relative to the amount of soluble proteins in WPI non-processed sample ×100. 3 Susceptibility of whey proteins to tryptic attack, as assessed by the degree of hydrolysis (DH) at 135 min of hydrolysis process. Means ± s.d. of 6–10 measurements from 2 independent experiments. 1

process, is detected as an endothermic peak in DSC thermograms, which assesses the amount of residual structure in the analysed proteins (Dumay, Kalichevsky, & Cheftel, 1994). This endothermic peak is characterised by its enthalpy (ΔHTD value) and the denaturation temperature (Tm). A reduction in the protein enthalpy (ΔHTD value) as evaluated after a technological treatment indicates therefore a partial loss of the protein native structures comparing with the non-processed protein. Calorimetric enthalpies evaluated at 1 °C min− 1 heating rate for non-processed or processed WPI dispersions (3.65% proteins, w/w; pH 6.5), are indicated in Table 1 and the corresponding thermograms are shown in Fig. 1. Non-processed WPI displays a broad peak with a peak maximum at 75.63 ± 0.22 °C (Tm, Table 1) close to the denaturation temperature of purified β-Lg (A and B variants) in pH 6.5 phosphate buffer (76.67 ± 0.01 °C) at the same heating rate. Close Tm values (75.9–76.9 °C) have been previously found after extrapolation to a 0 °C min−1 heating rate for β-Lg (A and B variants) in simulated milk ultrafiltrate or for whey protein concentrates at pH 6.5 (Bernal & Jelen, 1985), or for an industrially prepared β-Lg isolate scanned at 1 °C min−1 and pH 7.0 (77.5 ± 0.4 °C; Kolakowski et al., 2001). The shoulder visible on thermograms at ~ 66 °C (Fig. 1) may correspond to the thermal denaturation of α-La holo-form in the presence of calcium (Relkin, Launay, & Eynard, 1993). Indeed, thermal denaturation of α-La depends on both heating temperature and time-duration (Chaplin & Lyster, 1986), but also on calcium concentration (Relkin et al., 1993), and thermal denaturation of α-La (with calcium) is accompanied by some renaturation (Chaplin & Lyster, 1986). Since, in the present study, WPI samples contained ~ 4.6 mol calcium per mole of α-La, a DSC signal is expected close to 65–67 °C (Fig. 1). Some renaturation of α-La could take place after the first DSC run explaining the weak enthalpy (2.61 ± 0.4 J g−1 of protein) measured at the second DSC run for non-processed WPI samples (Fig. 1). Indeed, some renaturation of α-La (or reversible unfolding) has been described upon cooling in the presence of calcium due to a stabilising effect of calcium ions on the structure of α-La holo-form (Relkin et al., 1993). Similar ΔHTD values were also observed at the second DSC run for processed samples (results not shown). Accordingly, ΔHTD values (J g−1 of proteins) calculated from the first DSC run will be discussed forward (Table 1). A ΔHTD value of 17.85 ± 0.43 J g−1 of proteins was found for nonprocessed WPI. STTT at 75 °C for 10 s induced a small although significant (p = 0.05) decrease in ΔHTD value with a residual enthalpy of 96.5% (Table 1) and thermograms close to that of non-processed samples (Fig. 1). In opposite, endothermal peaks were greatly reduced

after STTT at 75 °C for 43 s or 110 s (Fig. 1), leading to significantly (p = 0.001) lower ΔHTD values compared to non-processed sample, and residual enthalpies of 78.7% (43 s) and 72.8% (110 s) (Table 1).

Fig. 1. Thermal denaturation of whey proteins, as measured by differential scanning calorimetry (DSC). DSC analyses of 850 mg of WPI dispersions at 3.65% proteins (w/w) and pH 6.5, or purified β-Lg at 2.5% protein (w/w) in 0.05 mol L−1 phosphate buffer (pH 6.5). Thermograms of purified β-Lg (1), non-processed (control) WPI (2), or WPI dispersions previously processed by: HHP at 300 MPa and 25 °C for 15 min (3); UHPH at 300 MPa followed by immediate cooling at the HP-valve outlet (4); UHPH at 250 MPa (5) or 300 MPa (6) without immediate cooling at the HP-valve outlet; STTT at 75 °C for 10 s (7), 43 s (8) or 110 s (9). First (2) and second (10) DSC runs are shown for non-processed WPI sample. First DSC runs are shown for purified β-Lg (1) and WPI processed samples (3–9).

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Since the endothermal signals resulted mainly from the disruption of intramolecular hydrogen bonds through protein unfolding during calorimetric measurements, such ΔHTD values indicated a partial but significant process-induced loss of whey protein native state before DSC measurements. The residual enthalpy may be considered therefore as an index of protein structures that have resisted technological treatment. A significant (p = 0.05) increase in Tm values by 0.5 °C to ~ 3 °C was observed after STTT, depending on the holding time at 75 °C (Table 1). In the case of STTT for 43 s or 110 s, the marked decrease in height of the endothermal signal at 75.6 °C makes more visible the shoulder attributed to α-La at ~66 °C (Fig. 1), which suggests a greater process-induced denaturation for β-Lg than α-La. It is not excluded that α-La partly refolded after STTT before DSC measurements. Protein denaturation induced by UHPH depended on both the homogenisation pressure and the associated heating phenomena. In the case of UHPH at 250 MPa without immediate cooling at the HP-valve outlet (called UHPH 250 MPa), sample temperature reached 64.1 ± 2.4 °C in the HP-valve (T2), decreased to 56.6 °C ± 3.6 °C in 1.75 s at (T3) location, then was set to ≤ 30 °C at the homogeniser outlet (T4) using the second cooling device. Accordingly, UHPH at 300 MPa without immediate cooling at the HP-valve outlet (called UHPH 300 MPa), was accompanied by a greater temperature jump of the processed fluid passing through the HP-valve: the fluid temperature reached 75.8 ± 0.8 °C in the HP-valve (T2), decreased to 69.5 ± 3.4 °C in 1.75 s at (T3) location, before being set at ≤ 30 °C at the homogeniser outlet (T4) using the second cooling device. In comparison, the temperature jump of the processed fluid was less extensive for UHPH at 300 MPa with immediate cooling at the HP-valve outlet (called UHPH 300 MPa-C), as expected: WPI sample was brought to 72.8 ± 0.4 °C (T2) in the HP-valve, then immediately cooled down to 44.0 ± 0.2 °C (T3) in 1.75 s using the first cooling device, and got out from the homogeniser at ≤ 30 °C using the second cooling device. It must be noticed that, operating at 300 MPa without immediate cooling at the outlet of the HP-valve, corresponds to an additional holding-time (1.75 s) at a “pasteurisation” temperature of 70–76 °C, which may be desired for microbial inactivation but detrimental for functional biological compounds. UHPH 300 MPa processing (without immediate cooling at the HP-valve outlet) induced similar decrease in ΔHTD than STTT at 75 °C for 43 s (ΔHTD values non-significantly different for p = 0.05) and a residual enthalpy of 80.7% (Table 1). On the contrary, UHPH 300 MPa-C processing (with immediate cooling at the HP-valve outlet) that reduced efficiently the associated heating phenomena, induced little change in DSC thermograms compared to non-processed sample, with a significant (p = 0.05) but small decrease in ΔHTD value and residual enthalpy of 95.7% (Fig. 1; Table 1). The latter ΔHTD value did not significantly differ (p = 0.05) from that induced by STTT at 75 °C for 10 s (Table 1). The similarity of ΔHTD values obtained after STTT at 75 °C for 43 s and UHPH 300 MPa (without immediate cooling) on the one hand, and for STTT at 75 °C for 10 s and UHPH 300 MPa-C (with immediate cooling) on the other hand (Table 1), clearly illustrates the weight of heating phenomena combined with mechanical forces on protein denaturation, during UHPH at 300 MPa. Operating at lower homogenisation pressure without immediate cooling (UHPH 250 MPa) led to ΔHTD values non-significantly different (p = 0.05) from that induced by UHPH 300 MPa-C (Table 1), indicating little protein structural changes from both pressure- and temperature. Treatment by HHP at 300 MPa (15 °C, 15 min) was characterised by significantly (p = 0.01) lower ΔHTD value compared to non-processed WPI or UHPH 300 MPa-C (Table 1), indicating some irreversible pressure-induced protein unfolding. Fig. 2 displays protein precipitation induced by ammonium sulphate in the 0–3.3 mol L−1 range. A slight “salting in” effect was observed for 1.0–1.25 mol L−1 AS concentration, in the case of non-processed WPI and WPI samples treated using the mildest conditions (STTT at 75 °C for 10 s, UHPH 300 MPa-C). Such “salting in” effect was followed by a progressive “salting out” process from 1.25 to 3.3 mol L−1 AS, with a shoulder in the 1.5–2.0 mol L−1 AS range which probably corresponds

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to a different AS-sensitivity of β-Lg and α-La (Dumay & Cheftel, 1989). Generally speaking, when whey proteins have been partly denatured by processing, the corresponding AS precipitation-curves are below that obtained for native proteins as previously observed for purified βLg and α-La after heating (Dumay & Cheftel, 1989), or pressurisation (Dumay et al., 1994). However, the significant (p = 0.001) increase in protein solubility observed in the 0–1.0 mol L− 1 AS range for the mildest technological conditions (STTT at 75 °C for 10 s, UHPH 300 MPa-C) compared to the non-processed sample could result from an increase in dispersibility and hydration of WPI powder or protein assemblies after short heating or pressurisation. Solubility values obtained after protein precipitation using 1.8 mol L− 1 AS, are reported in Table 1 in parallel with ΔHTD values. Protein solubility and ΔHTD values decreased similarly, suggesting that both methods reflected mainly protein unfolding rather than aggregation. A 2-order polynomial correlation was obtained between ΔHTD values and protein solubility as determined at 1.5 mol L−1 AS (R = 0.9633) and 1.8 mol L−1 AS (R = 0.9374) (Fig. 2, inset).

3.2. Particle size distribution and Mw estimation of WPI particles Fig. 3A and B shows the size distribution curves in light intensity and particle number frequency for the non-processed and processed WPI samples. As already detailed (Blayo et al., 2014; Regnault et al., 2004), the size distribution curves in light intensity reflects the presence of the largest particles and, conversely, the distribution in number frequency is sensitive to particles of small size. Peak characteristics (particle minimal and maximal diameters, Peak maximum) are shown by the distribution in light intensity or in number frequency (Fig. 3A and B) for WPI samples. The corresponding scale of estimated Mw is indicated at the top of Fig. 3A. Non-processed WPI dispersion at pH 6.5 (Fig. 3A) displays a bimodal distribution in light intensity with a first maximum at 3–5 nm (Peak 1) corresponding to Mw of ~ 9–29 kDa for globular proteins such as α-La and β-Lg, plus a second maximum at ~ 65 nm (Peak 2) corresponding to higher Mw (11.6 × 103 kDa). Peak 2 may correspond to BSA polymers and immunoglobulins assemblies present in the WPI powder as shown by SDS-PAGE (Section 3.4). The distribution curve in number frequency (Fig. 3B) was also bimodal with a major peak at 1–5 nm (Peak maximum at ~2.3 nm) and a small peak ranging at 5–15 nm (Peak maximum at ~ 7.5 nm) which indicates that globular proteins of small size and Mw (mainly α-La and β-Lg) were the majority, as expected from the given WPI composition (Section 2.2). Light scattering analyses, as a rapid and nondestructing method, will be used to assess processing-induced protein aggregation, as follows. UHPH-processing without immediate cooling or STTT at 75 °C, clearly increased whey protein particle sizes in the 40–600 nm range, as shown by the size distribution curves both in light intensity and number frequency (Fig. 3A and B). In the case of STTT, particle sizes increased as a function of holding-time at 75 °C, in both representations (Fig. 3A and B), reflecting the growing of heat-induced aggregates from 71 × 103 kDa (STTT for 10 s) to 143 × 103 kDa (STTT for 110 s) as estimated from Peak maxima (Fig. 3A). In the case of UHPH-processing without immediate cooling, size distributions were located on both sides of STTT-distributions (Fig. 3A and B), indicating also the presence of large size aggregates from 51 × 103 kDa (UHPH 250 MPa) to 202 × 103 kDa (UHPH 300 MPa) (Fig. 3A). Lower particle sizes were observed for UHPH-300 MPa-C (with immediate cooling) compared to UHPH 300 MPa (without immediate cooling), demonstrating the cooling efficiency to limit protein aggregation (Fig. 3A and B). In the case of UHPH 300 MPa-C, the size distribution in light intensity was bimodal with particles of small (20–50 nm) and large diameters (up to 600 nm) (Fig. 3A), but the distribution in number frequency (Fig. 3B) clearly displayed a main peak at ~ 24 nm instead of 142 nm for UHPH 300 MPa without immediate cooling. Particle diameters

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Fig. 2. Amounts of soluble whey proteins as determined after precipitation by ammonium sulphate (AS) at 0, 1.0, 1.25, 1.50, 1.60, 1.80, 2.0, 2.8 and 3.3 mol L−1. Amounts of soluble proteins ). (g) expressed per 100 g of WPI dispersions prepared at 10% proteins (w/w) and pH 6.5, which corresponds to10 g of total whey proteins: Non-processed WPI dispersions ( ) or 43 s ( ); UHPH 250 MPa ( ) or 300 MPa ( ) without immediate cooling at the HP-valve outlet; UHPH WPI dispersions processed by STTT at 75 °C for 10 s ( ). Mean curves from 4 to 8 measurements from currently two independent at 300 MPa with immediate cooling at the HP-valve outlet (\ \●\ \); HHP at 300 MPa and 25 °C for 15 min ( experiments are shown. The inset in Fig. 2 displays the correlation between the enthalpy of thermal denaturation (ΔHTD, J per g of whey proteins) and amount of soluble whey proteins −1 (g of soluble proteins per 100 g of WPI dispersion) as determined after precipitation by 1.50 mol L ( ) or 1.8 mol L−1 ( ) AS. A 2-order polynomial correlation was obtained between ΔHTD values and soluble proteins at 1.5 mol L−1 AS (R = 0.9633) and 1.8 mol L−1 AS (R = 0.9374) (inset).

of 20–50 nm in the distribution in light intensity correspond to Mw of ~0.7–6.3 × 103 kDa. As already explained (Dumay et al., 2013), UHPH-processing subject samples to (i) isostatic high-pressure in the pressure-intensifier to reach P1 in ~ 10 s, then (ii) to high velocity gradients and mechanical forces in the HP-valve gap resulting from the pressure drop (P1 − P2) through the HP-valve, plus (iii) a short-time temperature jump in the HP-valve due to a partial conversion of kinetic energy into heat. It is known that HHP at 150–300 MPa and 20–25 °C (i.e., isostatic high pressure) induced unfolding of mainly β-Lg among whey proteins (Huppertz, Fox, de Kruif, & Kelly, 2006). All mechanical forces (elongational stress plus turbulence, cavitation phenomena and impacts with solid surfaces and between particles) that take place in the HPvalve gap and at the gap outlet may result in both aggregation of unfold proteins through nucleation/aggregation process, and disruption of the largest aggregated particles. The temperature jump in the HP-valve may favour both protein unfolding and aggregation phenomena. Although UHPH 300 MPa-C and STTT at 75 °C for 10 s gave similar levels of protein denaturation (non-significant difference in ΔHTD values and level of protein precipitation by ammonium sulphate, Table 1), UHPH300 MPa-C induced aggregates of smaller sizes comparing with STTT at 75 °C for 10 s (Fig. 3B) probably due to shearing effects. This benefice was lost when UHPH was performed without immediate cooling at the

HP-valve outlet (Fig. 3) due to additional protein aggregation or reaggregation phenomena favoured by temperature after the HP-valve. Processing by HHP at 300 MPa and 25 °C for 15 min led to particle size distributions close to that of control samples in accordance with our previous results obtained with another batch of WPI (Blayo et al., 2014). Some particles of large size and Mw (Peak 2) were shift to lower sizes (Fig. 3), suggesting a pressure-induced dissociation process. Although, significant (p = 0.05) unfolding was observed by DSC and AS-precipitation (Table 1), light scattering did not highlight particular protein aggregation that HHP could induce comparing with the other treatments, and especially UHPH. 3.3. Partial hydrolysis of whey proteins by trypsin Fig. 4A displays hydrolysis curves of non-processed or processed WPI samples using the pH-stat method (pH 7.0; 37 °C; E/S = 1/100). It is known that trypsin activity is maximal in the typical 7.0–9.0 pH range (Adler-Nissen, 1986b). In the present study, tryptic hydrolysis was performed at pH 7.0 instead of higher pH values able to give faster proteolysis (Chobert et al., 1991; Cheison, Schmitt, Leeb, Letze, & Kulozik, 2010) to avoid superimpose protein structural changes arising at alkaline pH over that induced by processing. Fig. 4B shows tryptic activities determined at 20, 45, 90 and 135 min of digestion-time. The

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Fig. 3. Particle size distribution curves of whey proteins in light intensity (A) or light number frequency (B) determined by photon correlation spectroscopy (PCS) in absence of trypsin: ), 43 s ( ) or 110 s ( ); UHPH at 250 MPa ( ) or 300 MPa ( ) without Non-processed WPI dispersions (\ \\ \). WPI dispersions processed by STTT at 75 °C for 10 s ( ). Mean curves from 6 measurements immediate cooling at the HP-valve outlet; 300 MPa with immediate cooling at the HP-valve outlet (\ \\ \); HHP at 300 MPa and 25 °C for 15 min ( from at least two independent experimentations are shown. The corresponding Mw scale is shown in Fig. 3A.

corresponding DH values calculated at 135 min of tryptic hydrolysis as indicated in Section 2.6 are reported in Table 1. For non-processed WPI samples, DH equaled 4.44 ± 0.26 per g of whey proteins which agrees with published data (Sitohy et al., 2001). Generally speaking, the susceptibility of WPI to tryptic hydrolysis increased for all processed samples compared to the non-processed one, except in the case of UHPH at 250 MPa. Pre-processing thus induced protein structural changes that favoured trypsin accessibility and activity: the initial rate of hydrolysis clearly increased (Fig. 4A), as well as hydrolysis yields over the first 90 min of the enzymatic process (significant increase for p = 0.05) (Fig. 4B). Interestingly, WPI samples processed by UHPH at 300 MPa with immediate cooling or by STTT at 75 °C for 10 s,

both characterised by high residual enthalpy (ΔHTD) and AS-solubility values (Table 1), displayed the highest yields of proteolysis (Fig. 4) which suggested that limited protein unfolding favoured the further trypsin attack. Indeed, the corresponding DH value at 135 min was about twice that of non-processed samples (Table 1). In opposite, processing by UHPH at 250 MPa, despite resulting in high ΔHTD and AS-solubility values (Table 1), did not improve the susceptibility of whey proteins to tryptic hydrolysis compared to control samples (Fig. 4B). It must be stressed that UHPH at 250 MPa induced protein aggregation as shown by Fig. 3A and B, even though the temperature jump during UHPH did not exceed 65 °C. Such results suggested that tryptic hydrolysis of proteins may be favoured by limited but sufficient

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Fig. 4. In vitro tryptic hydrolysis of whey proteins using the pH-stat method at the following conditions: 5 mg mL−1 whey proteins; pH 7.0; 37 °C; E/S = 1/100. (A) Hydrolysis curves of non-processed WPI dispersions (●). WPI dispersions previously processed by STTT at 75 °C for 10 s ( ), 43 s ( ) or 110 s ( ); UHPH at 250 MPa ( ) or 300 MPa ( ) without immediate cooling at the HP-valve outlet; UHPH at 300 MPa with immediate cooling at the HP-valve outlet (○); HHP at 300 MPa and 25 °C for 15 min ( ). Mean curves from 6 to 10 measurements from at least two independent experimentations are shown. (B) Hydrolysis yields as given by volumes of added NaOH at 20 min (■), 45 min ( ), 90 min ( ) and 135 min ( ) of the digestion process. Means from 6 to 10 measurements from at least two independent experimentations are shown; different letters (a–o) on the bars indicate significant difference for p = 0.05.

pressure- and/or temperature-induced unfolding, before the setting of rapid protein nucleation/aggregation that mechanical forces could create through the HP-valve during UHPH-processing. WPI samples the most exposed to heat (STTT at 75 °C for 43 s or 110 s; UHPH at 300 MPa without immediate cooling) displayed an intermediate behaviour with an increase in the initial hydrolysis rate,

but a trend to reach the hydrolysis yield of non-processed samples at 135 min of the enzymatic process (Fig. 4). Increasing heating effects did not improve therefore the final efficiency of tryptic proteolysis. It is tricky to compare the present results with published data usually performed in a static mode at temperature ≥80 °C for longer heating times (≥ 10 min), and resulting to the formation of great insoluble protein

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materials (Kim et al., 2007; O'Loughlin, Murray, Kelly, FitzGerald, & Brodkorb, 2012). Indeed, depending on processing and/or hydrolysis conditions, all thermal treatments did not result in an increase in trypsin efficiency. Nevertheless, it looks like heating may increase the initial rate of whey protein hydrolysis but not the final hydrolysis yield (O'Loughlin et al., 2012), which agrees with the present results. Processing WPI dispersions by hydrostatic high-pressure (HHP-300MPa) increased both the initial hydrolysis rate (Fig. 4A) and hydrolysis yields over the 135 min of hydrolysis process (significant increase for p = 0.05; Fig. 4B) comparing with control samples. Pre-pressurisation thus facilitated the further tryptic attack of whey proteins due to some irreversible protein structural changes. The corresponding proteolysis curve was located between that observed for the most hydrolysed samples (UHPH 300 MPa-C; STTT at 75 °C for 10 s) and the moderately hydrolysed ones (STTT at 75 °C for 43 s or 110 s; UHPH 300 MPa) (Fig. 4A). Similarly, the corresponding level of protein denaturation as assessed by DSC or AS-precipitation, ranged between those of the two latter sample series (Table 1). In addition, samples treated by HHP displayed the lowest particle sizes and MW (Fig. 3A and B). It is worth noting that the studied physical treatments may act differently on protein native structures, involving pressure, temperature, high velocity gradients and shocks, or their combination at different levels. However, it can be concluded that high pressure at 300 MPa (isostatic/HHP or dynamic/ UHPH) improved the further WPI susceptibility to tryptic hydrolysis, but that excess of heating associated to UHPH-processing opposed the pressure-induced improvement, probably due to excessive protein denaturation (unfolding followed by aggregation) masking the accessibility of trypsin sites. Few comparative studies are available on protein digestibility carried out after processing by high pressure or heat. Higher in vitro digestibility of whey proteins (gels at 14% proteins and pH 7.0) upon successive pepsin–trypsin hydrolysis was yet observed after pressurisation at 400 MPa for 30 min than heating at 80 °C for 30 min (He, Mu, & Wang, 2013). 3.4. SDS-PAGE patterns of WPI samples exposed to tryptic attack SDS-PAGE provides additional information about whey proteins that resist tryptic attack as assessed after 135 min of enzymatic process, as shown by Figs. 5 and 6A–L. SDS-PAGE of non-processed WPI (nonsubjected to tryptic hydrolysis) (Figs. 5 and 6A) shows (i) a series of minor bands: a weak band for immunoglobulins (Ig; ~200 kDa); traces of lactoferrin (Lf; 76 kDa); a clear band for bovine serum albumin monomer (BSA; 66 kDa); a multiple band (32–40 kDa) corresponding to di/ trimers of β-Lg (and/or β-Lg and α-La) and probably resulting from

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oxidation through disulphide bonds (Grácia-juliá et al., 2008; Patel, Singh, Havea, Considine, & Creamer, 2005), plus (ii) the major bands of β-Lg (18.4 kDa) and α-La (14.4 kDa), as expected according to the WPI composition given by the producer (Section 2.2). After tryptic attack, the changes in peak heights were accurately revealed through electrophoresis gel scanning (Fig. 6D): Ig band was ~twice reduced, Lf became no more visible, BSA monomer remained quasi-unaffected, the di/trimer band was markedly reduced, β-Lg band was reduced down to ~ 83% of its initial height while α-La appeared less affected with a decrease by b10% of its initial height. Indeed, it is known that α-La in the presence of calcium ions resists tryptic attack at pH 7.0 and 37 °C (Cheison, Leeb, Toro-Sierra, & Kulozik, 2011). SDS-PAGE performed at 135 min of the hydrolysis process did not yet allow identification of the released small peptides. Other methods based on massspectroscopy analysis are needed for that purpose (Cheison, Brand, et al., 2011; Maynard et al., 1998). SDS-PAGE patterns displayed little difference between processed and non-processed samples before tryptic attack (all results are not shown) except in the case of STTT-samples. In this case, blue-stained aggregates that SDS could not dissociated appeared at the protein deposit and from the deposit down to the di/trimer band (Fig. 5), mainly for 43 s or 110 s of heating-time at 75 °C. Protein aggregation increased with heating-time, as expected, resulting partly from –SH/S–S exchanges between whey proteins (Considine, Patel, Anema, Singh, & Creamer, 2007). Indeed, in the present dissociating and non-reducing electrophoresis conditions, SDS dissociated protein aggregates generated through hydrophobic interactions but not aggregates linked by disulphide bonds. After tryptic attack of STTT-samples, heat-induced aggregates disappeared from the electrophoresis gels while some peptides appeared beyond the α-La band (Fig. 5), indicating the formation of intermediate peptides in the 8–12 kDa range, still present after 135 min of hydrolysis. Ig, Lf, BSA, and the di/trimer band were markedly reduced, while the α-La band decreased by ~ 20% of its initial height (Fig. 6E, H and K). Smaller amount of residual β-Lg was observed after STTT at 75 °C for 10 s (~ 70% by height) (Fig. 6E) compared to 43 s or 110 s (~ 78–80% by height) (Fig. 6H and K), which indicates that increased heating-times reduced the further trypsin efficiency towards β-Lg in accordance with DH results. After UHPH at 300 MPa with immediate cooling, SDS-PAGE patterns displayed only few protein aggregates visible at the protein deposit or as running traces (Fig. 5), indicating limited oxidative protein aggregation. After trypsin attack of UHPH 300-C samples, Ig, Lf and di/trimer bands were markedly reduced or disappeared, BSA band was quasi-nonaffected and α-La band decreased by ~10% of its initial height (Fig. 6C

Fig. 5. Gel electrophoresis of whey proteins (SDS-PAGE). Whey proteins without previous tryptic digestion: non-processed WPI (1, 14); WPI processed by STTT at 75 °C for 10 s (3, 10), 43 s (5) or 110 s (7); UHPH at 300 MPa with immediate cooling at the HP-valve outlet (12). Whey proteins after tryptic hydrolysis: non-processed WPI (9, 15); WPI processed by STTT at 75 °C for 10 s (4, 11), 43 s (6) or 110 s (8); UHPH at 300 MPa with immediate cooling at the HP-valve outlet (13). ColorBurst™ Electrophoresis Marker (2), purified β-Lg and α-La (16) and purified BSA (17) from Sigma are shown.

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Fig. 6. (A–L): Electrophoresis runs of whey proteins (SDS-PAGE). WPI samples not subjected to previous tryptic hydrolysis: non-processed WPI (A); WPI processed by STTT at 75 °C for 10 s (B) or by UHPH at 300 MPa with immediate cooling at the HP-valve outlet (C). WPI samples after tryptic hydrolysis (+ Trypsin): non-processed WPI samples (D); WPI samples processed by HHP at 300 MPa (G), STTT at 75 °C for 10 s (E), 43 s (H) or 110 s (K); UHPH at 300 MPa with immediate cooling at the HP-valve outlet (F); UHPH at 250 (I) or 300 MPa (L) without immediate cooling at the HP-valve outlet. Purified β-Lg, α-La and BSA from Sigma (J). MW values from Precision Plus Protein standards from Bio-Rad are indicated (C). Identification of electrophoretic peaks: Immunoglobulins (1); lactoferrin (2); bovine serum albumin monomer (3) and polymers (3′); protein aggregates (4); β-Lg (5); α-La (6).

and F), as for non-processed WPI samples (Fig. 6D), but β-Lg was much more hydrolysed (β-Lg band decreased down to ~ 67% of its initial height) (Fig. 6D and F). UHPH at 250 MPa or 300 MPa without immediate cooling led to higher amounts of residual β-Lg upon tryptic hydrolysis compared to UHPH 300 MPa-C, and smaller amounts of residual α-La and BSA (Fig. 6F, I and L), suggesting different susceptibility of whey proteins after processing depending on UHPH conditions. In the opposite, HHP at 300 MPa, led to similar amounts of residual β-Lg, α-La, and BSA upon tryptic hydrolysis than UHPH 300 MPa-C (Fig. 6G and F). It looks like that β-Lg hydrolysis was favoured by pre-processing at 300 MPa (isostatic or dynamic high-pressure with a temperature control), but limited by associated heating phenomena (UHPH without immediate cooling). Conversely, heating favoured the further tryptic hydrolysis of α-La and other minor whey proteins. The positive effect of hydrostatic high-pressure favouring β-Lg hydrolysis by trypsin agrees with published results (Knudsen et al., 2002; Maynard et al., 1998; Stapelfeldt et al., 1996). However, the low amount of residual β-Lg (~ 15%) obtained by Knudsen et al. (2002) after pre-pressurisation (300 MPa, 25 °C, 15 min) of purified β-Lg A using close proteolysis conditions (E/S ratio = 0.5/100; pH 7.5; 37 °C) than in the present study,

may result from hydrolysis performed within a shorter time after pressure release (30 min instead of 1.5–2 h in the present study). Indeed, it is known that some β-Lg refolding takes place after pressure release (Dumay et al., 1994; Kolakowski et al., 2001). Protein refolding during a standing time after pressure release may reduce the further trypsin efficiency (Stapelfeldt et al., 1996). 3.5. Particle size of WPI particles exposed to tryptic attack The change in protein particle size upon tryptic hydrolysis was monitored as soon as trypsin was added (t = 0) to WPI dispersions, then at t = 45, 90 and 135 min of the digestion process. Distribution curves in particle number frequency (%) that are sensitive to particles of small sizes are presented in Fig. 7A–H. Trypsin addition to WPI dispersion was accompanied by an apparent increase of particle sizes up to 30–50 nm (Peak maxima) particularly visible when samples displayed particles of small size in absence of trypsin (non-processed sample, Fig. 7A; HHP 300 MPa-sample, Fig. 7E). This phenomenon probably resulted from the adsorption of the positively charged trypsin (trypsin pI is close to 8.0) to the negatively charged whey proteins at neutral

C. Blayo et al. / Food Research International 79 (2016) 40–53

Fig. 7. (A–H): Particle size distribution curves of proteins in number frequency as determined by PCS. WPI samples were collected at 0 min (

51

), 45 min (

), 90 min (

) and

135 ( ) of the tryptic digestion process for non-processed WPI dispersions (A); WPI dispersions processed by STTT at 75 °C for 10 s (B), 43 s (C) or 110 s (D); HHP at 300 MPa and 25 °C for 15 min (E); UHPH at 250 MPa (F) or 300 MPa (G) without immediate cooling at the HP-valve outlet; UHPH at 300 MPa followed with immediate cooling at the HP-valve outlet (H). The corresponding samples non-subjected to tryptic hydrolysis (WO Tryp;\ \\ \) are indicated.

pH, resulting in floc formation. Indeed, immediate flocculation clearly appeared as a white cloud at trypsin addition when using WPI dispersions of high protein contents (N5 mg mL−1) (Results not shown). Generally speaking, particle sizes decreased towards small sizes (1–10 nm) for all samples at 45 min of tryptic attack compared to 0 min, the magnitude of this trend depending on pre-processing and protein denaturation–aggregation. This decrease in particle sizes upon

tryptic proteolysis was a marker for the shortest heating-time at 75 °C (10 s compared to 43 s and 110 s; Fig. 7B–D), or in the case of minimal associated heating effects (comparing UHPH at 300 MPa with or without immediate cooling; Fig. 7F–H). It is tempting to correlate the protein particle size decrease with trypsin efficiency. In fact, the observed particle sizes result from both protein splitting and re-association of the released protein fragments and peptides into peptide-aggregates

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through hydrophobic interactions and/or electrostatic attractions as previously suggested (Creusot & Gruppen, 2008; Otte, Lomholt, Halkier, & Qvist, 2000; Pouliot et al., 2009). At 90 min and 135 min of enzymatic process, particle sizes no more decreased for most samples, or even increased again probably due to peptide re-association as observed for HP-sample (Fig. 7E). It looks like that the formation of large size peptide aggregates slow down the hydrolysis process explaining moderate final DH values in spite of an increased initial hydrolysis rate (Fig. 4). It should be interesting to characterise tryptic hydrolysates depending on pre-processing, and determine amount and composition of aggregated material generated by trypsinolysis, in further studies. 4. Conclusion The best trypsinolysis efficiency was obtained in the case of the less denaturing treatments: UHPH at 300 MPa followed by efficient cooling or STTT at 75 °C applied with the shortest residence time (10 s). The slight protein unfolding that pre-processing could induce, thus favoured the further accessibility of trypsin to hydrolysis sites and the final proteolysis yield. Processing-induced protein aggregation, but also reassembly of protein fragments and peptides released during hydrolysis seem to affect proteolysis efficiency. Additional studies could be designed to characterise peptides and peptides aggregates released during hydrolysis in terms of amounts and size, hydrophilic/hydrophobic nature, and electrostatic attraction/repulsion. UHPH-processing applied with a temperature control appears beneficial to increase protein digestibility for a nutritional purpose, and must be explored to produce protein hydrolysates with peculiar techno-functional or biological properties, or reduced antigenicity. It remains to optimise proteolysis efficiency by adding the enzyme (trypsin or other food-grade protease proposed at an industrial scale) to the protein mixture just before or immediately after UHPH-processing to take advantage of the processing-induced structural changes before protein refolding occurs. In view of processimplementation, UHPH offers the advantage of a continuous pressureassisted tool also able to reduce the fluid microbial load down to a pasteurisation level (Picart et al., 2006), which may be useful to conduct a protein digestion process after pressure treatment. Acknowledgements The research, including the salary of one of us, C. Blayo, benefited from the financing of the European Union's Seventh Framework Programme (FP7/2009-2012) under grant agreement no. 232603, FUNENTECH EU. References Adler-Nissen, J. (1986a). Methods in food protein hydrolysis. In J. Adler-Nissen (Ed.), Enzymic hydrolysis of food proteins (pp. 110–131). London: Elsevier Applied Science Publishers Ltd. Adler-Nissen, J. (1986b). Some fundamental aspects of food protein hydrolysis. In J. AdlerNissen (Ed.), Enzymic hydrolysis of food proteins (pp. 9–24). London: Elsevier Applied Science Publishers Ltd. Barba, F. J., Esteve, M. J., & Frigola, A. (2012). High pressure treatment on physicochemical and nutritional properties of fluid foods during storage: A review. Comprehensive Reviews in Food Science and Food Safety, 11(3), 307–322. Barba, F. J., Terefe, N. S., Buckow, R., Knorr, D., & Orlien, V. (November 2015). New opportunities and perspectives of high pressure treatment to improve health and safety attributes of foods. A review Food Research International, 77, 725–742. Belloque, J., Chicόn, R., & Lόpez-Fandiño, R. (2007). Unfolding and refolding of β-lactoglobulin subjected to high hydrostatic pressure at different pH values and temperatures and its influence on proteolysis. Journal of Agricultural and Food Chemistry, 55(13), 5282–5288. Bernal, V., & Jelen, P. (1985). Thermal stability of whey proteins — A calorimetric study. Journal of Dairy Science, 68(11), 2847–2852. Bernasconi, E., Fritsché, R., & Corthésy, B. (2006). Specific effects of denaturation, hydrolysis and exposure to Lactococcus lactis on bovine β-lactoglobulin transepithelial transport, antigenicity and allergenicity. Clinical and Experimental Allergy, 36(6), 803–814. Blayo, C., Puentes-Rivas, D., Picart-Palmade, L., Chevalier-Lucia, D., Lange, R., & Dumay, E. (December 2014). Binding of retinyl acetate to whey proteins or phosphocasein

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