High Voltage Electrical Treatments To Improve the Protein

Research Article pubs.acs.org/journal/ascecg

High Voltage Electrical Treatments To Improve the Protein Susceptibility to Enzymatic Hydrolysis Sergey Mikhaylin,*,† Nadia Boussetta,‡ Eugène Vorobiev,‡ and Laurent Bazinet† †

Institute of Nutrition and Functional Foods (INAF), Dairy Research Center (STELA) and Laboratory of Food Processing and Electromembrane Processes (LTAPEM), Department of Food Sciences, Pavillon Paul-Comtois, Université Laval, Québec City, Quebec G1V 0A6, Canada ‡ Unité Transformations Intégrées de la Matière Renouvelable, EA 4297, Centre de Recherches de Royallieu, Université de Technologie de Compiègne, BP 20529, 60205 Compiègne Cedex, France ABSTRACT: The rapidly growing global population raises important issues associated with the environmental burden imposed by agri-food and biotechnological industries to satisfy the increasing demand of high-quality food and nutraceuticals. Hence, the introduction of emergent ecoefficient technologies in the bioproduction lines is inevitable. The present study deals with environmentally sustainable high voltage electrical treatments (HVETs)pulsed electric field (PEF) and electrical arcto improve the susceptibility of β-lactoglobulin to enzymatic hydrolysis. This protein was chosen due to its high excess in dairy industry coproducts, which must be valorized. The results demonstrate that, at the optimal HVET duration of 10 min (voltage = 40 kV and pulse frequency = 0.5 Hz), the degree of hydrolysis can be improved by 80% and 66% for the PEF and electrical arc, respectively. This fact is related to the ability of HVET to induce the active sites formation in protein molecule for the nucleophilic enzymatic action, which leads to the release of bioactive and functionally active peptides. Moreover, it is possible to control the selectivity of hydrolysis by varying the HVET modes. Thus, the implication of HVET to valorize the dairy whey proteins by their enzymatic hydrolysis can significantly improve the process ecoefficiency. KEYWORDS: Whey protein, Pulsed electric field, Electrical arc, Enzymatic hydrolysis, Peptides

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INTRODUCTION The utmost problem of contemporary agri-food and biotechnologies is sustainable management of wastes and coproducts. Indeed, 1.3 billion tons of food waste is generated per year in the world, meaning an inefficient use of resources and substantial emissions, which significantly affect our ecosphere.1 Moreover, the rapidly growing population and its urbanization promote the increase of food production and, consequently, waste generation. Hence, the modern research should be oriented toward sustainable development strategies, consisting of the involvement of technologies deriving the high added value products having benefits for the human health without the major environmental burden.2 The high voltage electrical treatments (HVETs) of food constituents, considered as green ones, could be a promising alternative to conventional energy and resource consuming processes. Indeed, HVET allow improvement of the transformation of diverse food products, as well as the inactivation of pathogen microorganisms.3 Moreover, HVET stands among the emerging technologies having a high potential for commercialization in the coming years.4 Two different groups of HVET can be distinguished: pulsed electric field (PEF) and high voltage electrical discharge (electrical streamer and electrical arc). During PEF treatment, © 2017 American Chemical Society

voltage pulses are applied between two plane electrodes imposing mechanical stress (created by externally applied electric field) and producing chemically active species (reactive radicals and molecular species (e.g., H2O2 and O3)) while electrical discharge (arc) is generated between needle and plane electrodes imposing mechanical stress and producing chemically active species, shock waves, ultraviolet (UV) light and vapor cavities.3,5−7 Streamer is a prebreakdown phase when the thin current filaments are formed, and as soon as the streamer propagates to the plane electrode, the breakdown phase (called arc) occurs. The most studied phenomenon of HVET is electroporation,3 which consists of the electrical breakdown of cell membranes. The electroporation phenomenon could provoke an inactivation of microorganisms, making PEF a perspective approach for the cold or mild thermal pasteurization.3 Moreover, electroporation could induce mechanical, osmotic, viscoelastic, and hydrodynamic instabilities, which can improve food transformation efficiency (e.g., drying, freezing).8−15 Numerous studies demonstrated that the extraction of Received: September 9, 2017 Revised: October 8, 2017 Published: October 30, 2017 11706

DOI: 10.1021/acssuschemeng.7b03192 ACS Sustainable Chem. Eng. 2017, 5, 11706−11714

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ACS Sustainable Chemistry & Engineering

Figure 1. Scheme of the high voltage electrical treatment (HVET) system equipped with pulsed electric field and electrical arc treatment chambers.

focuses on evaluation of the HVET on the intermolecular interactions. The HVET of milk protein (β-lactoglobulin) and its further interactions with enzyme (hydrolysis) will be the main subject of the present study. The selected topic is very important from the perspective of food byproducts (e.g., whey generated by cheese and yogurt industries) valorization. Indeed, enzymatic hydrolysis leads to the release of biologically and functionally active compounds (peptides, amino acids, sugars, etc.).35−37 For instance, ACE-inhibitory activity (f(9−14), f(22−25), f(142−148), etc.), antimicrobial peptides (f(25− 40, f(92−100), etc.), hypocholesterolemic peptides (f(9−14), f(41−60), etc.) could be released by the enzymatic action on βlactoglobulin molecules. 38 The processes of enzymatic hydrolysis are reported to be advantageous, from the environmental point of view, compared to conventional pathways of food wastes valorization.39 Despite the high activity, selectivity, and specificity, enzymes are relatively expensive catalysts. Thus, the development of innovative pathways is important to improve the enzymatic hydrolysis treatments, making them economically feasible. The trypsin was chosen as a model enzyme for this study, because of its wellcharacterized β-lactoglobulin hydrolysate.40−43 This fact will allow detailed comparison of the nonpretreated with HVET pretreated β-lactoglobulin, in terms of protein susceptibility to tryptic hydrolysis.

valuable compounds from different sources (grape skin, seeds and pomace, vine shoots, fruit and vegetable peels, etc.) can be improved by the PEF.3,16−18 Hence, a large domain of valorization of food processing wastes becomes open for PEF. The electrical arc finds actually few applications in food and biomass processing, compared to the PEF.19,20 However, currently, these treatments attract more and more attention of researchers from different domains. Electrical arcs induce the production of active chemical species (H2O2, O3, OH•, H•, O•, etc.) and physical phenomena (UV light, shock waves, vapor cavities).21 The current applications of electrical arc include water depollution, electrohydraulic crushing of solids, extraction of biocompounds, and inactivation of microorganisms.6,17,22−24 Most studies dedicated to the HVET focus on the biological and physical aspects (electroporation of biological membranes, cell disintegration, product fragmentation, etc.) while the chemical aspects (intramolecular and intermolecular interactions) are very poorly explored. With a view of using HVET as a method for cold pasteurization or green extraction, multiple studies revealed that PEF had no or insignificant influence on the concentration of vitamins, polyphenols, isoprenoid compounds, and fatty acids.3 So far, only the action of a special mode of HVET, such as PEF, was a subject of investigations that focused on studying the structural, physicochemical, and functional properties of food molecules while the impact of electrical arc remains unexplored. Several studies revealed the disruption of α-helix and β-sheet structures of certain enzymes (pepsin, papain, lysozyme, etc.) by PEF, which led to their inactivation.25−29 Few studies were devoted to the influence of PEF on the structure and properties of food proteins. For instance, Xiang et al.30 and Perez and Pilosof31 demonstrated that PEF treatment of whey protein isolate could modify the protein structure, while Sui et al.32 revealed that PEF treatment had no influence on the physicochemical and functional properties of whey protein isolate, except its gelation properties. Several studies indicated the possible protein aggregation under the action of PEF, suggesting the hydrophobic and disulfide bonds as uppermost binding forces in aggregates.33,34 Given the above discussion, it is clear that deeper investigations of the impact of HVET on the molecular structure, as well as intramolecular and intermolecular interactions of food molecules are needed. The present paper

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EXPERIMENTAL SECTION

Materials. β-Lactoglobulin (98% purity) was generously donated by Davisco Foods International, Inc. (Eden Prairie, MN, USA). HCl 1.0 N and NaOH 1.0 N were obtained from FisherScientific (Nepean, Ontario, Canada). The protein was hydrolyzed with pancreatic bovine trypsin (reference No. T9201; 7500 BAEE units/mg solid) purchased from Sigma−Aldrich (St. Louis, MO, USA). Protocol. The experimental module for high voltage electrical treatments (HVETs) consisted of a pulsed high voltage power supply (Tomsk Polytechnic University, Russia) and a laboratory 1 L batch cylindrical treatment chamber with a disk electrode of 35 mm in diameter for PEF treatments or needle electrode of 10 mm in diameter for electrical arc treatments (see Figure 1). The grounded plate electrode was a stainless steel disk 35 mm in diameter. A positive pulse voltage was applied to the working (upper) electrode (see Figure 1). The high voltage pulse generator provided 40 kV−10 kA pulses. The distance between the electrodes was 5 mm, and the peak pulse voltage was 40 kV. The electrical discharges were generated by electrical breakdown in a water−protein solution. For both HVET, energy was stored in a set of low-inductance capacitors, which were charged by a 11707


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ACS Sustainable Chemistry & Engineering high voltage power supply. Damped oscillations were thus obtained over a total duration of 10 μs. The treatment chamber was initially filled with 300 mL of β-lactoglobulin solution (1%). Electrical treatment was applied with pulse repetition rate of 0.5 Hz (Δt = 2 s), which was imposed by the generator. The 300 mL of 1% (w/v) aqueous β-lactoglobulin solution (pH 6.5, κ = 500 μS/m) were treated at both PEF and arc modes during 1, 10, and 30 min, which corresponds to 25, 250, and 750 pulses, respectively. Each pulse has an energy of 160 J (4 kJ for 1 min, 40 kJ for 10 min and 120 kJ for 30 min of HVET). The HVET treatment did not significantly affect the pH of β-lactoglobulin solution. After each HVET, the β-lactoglobulin samples were hydrolyzed during 2 h with bovine trypsin (1% of dry β-lactoglobulin mass) at 37 °C and pH 6.8. The tryptic hydrolysis of β-lactoglobulin releases only certain peptides cleaved at carboxyl terminals of arginine and lysine, except when linked to a proline residue.44 The 10 mL samples of βlactoglobulin were taken prior to hydrolysis and after 15, 30, 60, 90, and 120 min for the further determination of the degree of hydrolysis and peptide profiles. The hydrolysis was stopped by addition of HCl (1M) until pH reached the value of 4.5 to inactivate trypsin. The HVET protein samples were compared with the control nonpretreated sample. Each treatment was repeated three times. Determination of the Degree of Hydrolysis. The degree of hydrolysis (DH) was measured by the O-phthaladehyde (OPA) assay.45 OPA reacts with primary amines. A calibration curve was done with a Phe-Gly solution (MW = 151.2 g/mol) on a range of 0−1000 μM. The reaction solution consisted of 50% borax (29.62 g/L of sodium borate·10H2O), 20% SDS (100 g/L), 2% OPA in 95% ethanol, and 0.2% β-mercaptoethanol, with the balance consisting of Milli-Q water. The pH was adjusted to 9. The measurement was performed with a Cary bio-UV-vis spectrophotometer and analyzed by Cary WinUV software (Varian, Australia Pty. Ltd.) at a wavelength of 340 nm. The real degree of hydrolysis (DHr) was calculated by subtracting the concentration of free α-amino groups of intact β-lactoglobulin (9.76%) from the theoretical degree of hydrolysis (DHh) of hydrolyzed sample. The presented real DHr (denoted below as DH) is an average value from three repetitions. Reverse-Phase Ultra Performance Liquid Chromatography (RP-UPLC) and Mass Spectrometry (MS) Analyses. Reverse-phase ultra performance liquid chromatography (RP-UPLC) analyses were performed using a 1290 Infinity II UPLC system (Agilent Technologies, Santa Clara, CA, USA). The equipment consisted of a binary pump (Model G7120A), a multisampler (Model G7167B), an in-line degasser, and a variable wavelength detector (Model VWD G7114B) that had been adjusted to 214 nm. β-Lactoglobulin hydrolyzed sample was diluted and filtered through 0.22 μm PVDF filter into a glass vial. The sample was loaded (5 μL) onto an Acquity UPLC CSH 130 1.7 μm C18 column (2.1 mm i.d. × 150 mm, Waters Corporation, Milford, MA, USA). The column was operated at a flow rate of 400 μL/min at 45 °C. The gradient consisted of solvent A (LCMS grade water with 0.1% formic acid) and solvent B (LC-MS grade ACN with 0.1% formic acid) starting at 2% B and ramping to 35% B in 40 min, then ramping to 85% B to 40.50 min, holding until 42 min, then back to initial conditions until 45 min. Each sample was run in triplicate for statistical evaluation of technical reproducibility. A hybrid ion mobility quadrupole time-of-flight (IM-Q-TOF) mass spectrometer (Model 6560 (high-definition mass spectrometry), Agilent Technologies, Santa Clara, CA, USA) was used to identify and quantify the relative abundances of the tryptic peptides. All RPUPLC-MS/MS experiments were acquired using IM-Q-TOF analysis. Signals were recorded in positive mode at extended dynamic range (2GHz, 3200 m/z with a scan range between 100 m/z and 3200 m/z). Nitrogen was used as the drying gas at 13.0 L/min and 150 °C, and was used as a nebulizer gas at 30 psig. The capillary voltage was set at 3500 V, the nozzle voltage was set at 300 V, and the fragmentor was set at 400 V. The instrument was calibrated using an ESI-L low concentration tuning mix (G1969−85000, Agilent Technologies, Santa Clara, CA, USA). Data acquisition and analysis were done using the Agilent Mass Hunter Software package (LC/MS Data Acquisition, Version B.07.00 and Qualitative Analysis for IM-MS, Version B.07.00

with BioConfirm Software). Additional search was performed using the Spectrum Mill MS Proteomics Workbench Rev B.05.00.180. The Milk and Whey protein databases were used to search for tryptic peptides from the β-lactoglobulin hydrolysate. In addition, the masses of unidentified peptides were loaded into the FindPept database (http://ca.expasy.org/tools/findpept.html) for the search of peptide, which could be derived from the β-lactoglobulin and α-lactalbumin (traces).

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RESULTS Degree of Hydrolysis. Figure 2 represents the evolution of the degree of hydrolysis (DH) with time for the β-lactoglobulin

Figure 2. Evolution of degree of hydrolysis (DH), as a function of time of β-lactoglobulin treated by pulsed electric field (PEF).

pretreated with pulsed electric field (PEF). One could see that all pretreated samples have a higher DH during enzymatic hydrolysis, compared to the control nonpretreated sample. Interestingly, the spectrophotometric analysis indicates the occurrence of new free α-amino groups of electrically treated protein, even before the addition of trypsin, suggesting the possible liberation of free amino acids and/or peptides (Figure 2, DH at 0 min). Moreover, by increasing the time of PEF pretreatment from 1 min to 30 min, the initial DH increases from 1.5% to 3.1% respectively. It is worth noting that the DH of electrically pretreated during 30 min β-lactoglobulin sample is more than 60% higher than the final DH of the control sample. Looking at the hydrolysis kinetics, it is possible to observe that, during hydrolysis, PEF-treated samples had 2−3 units of % of DH differences, compared to control ones. The final DH of treated protein after 2 h of hydrolysis is significantly higher, compared to nontreated protein. For instance, the highest final DH was observed for the 10 min (250 pulses) pretreatment of β-lactoglobulin and it is more than 80% higher than that the one of control sample. The influence of electrical arc pretreatment on DH of βlactoglobulin sample is represented in Figure 3. One could observe that the arc pretreatment increases the DH of the studied proteins, even prior to enzymatic hydrolysis. These results corroborate with those presented for the PEF treatment of β-lactoglobulin, although the PEF demonstrates a better efficiency. For instance, the nonhydrolyzed samples treated with PEF during 30 min exhibit DH values of 3.0% while those treated by electrical arc demonstrate smaller DH values (2.2%). The final DH of electrical arc pretreated samples in the best condition (10 min) is 66% higher than that of the control sample. Interestingly, the two highest DH values were reached for arc pretreatment durations of 30 min at the initial stages of hydrolysis and 10 min after 1 h of hydrolysis. However, PEF11708


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However, electrically pretreated samples (see Figures 4b and 4c) contain several LC-MS/MS peaks. The PEF treatment during 30 min released molecules detected at acquisition times of 2.2 and 3.6 min (Figure 4b). The mass spectrometry detected that the peak at 2.2 min contained principally molecules with molecular masses of 336.10, 701.21, and 775.22 Da. The matched peptides determined by the FindPept database, using β-lactoglobulin, were f(80−82) for 336.10 Da; f(32−38), f(33−39), f(152− 157), f(89−94), f(69−74) and f(70−75) for 701.21 Da; and f(108−114) and f(76−82) for 775.22 Da. However, for the αlactalbumin which could be present at trace level in the βlactoglobulin, only peptides f(47−49) and f(51−53) with a molecular weight of 336.10 Da were matched. Concerning the acquisition time of 3.6 min, the mass spectrogram demonstrated the appearance of molecules having three different molecular masses: 1004.55, 1082.67, and 1140.60 Da. The possible peptides found in database using β-lactoglobulin were f(112−120), f(38−45) and f(36−42) for 1004.55 Da; and f(104−113) and f(70−79) for 1140.60 Da. Concerning αlactalbumin, only peptide f(43−52) with molecular mass of 1140.65 Da was possible. Thus, the molecule having a molecular mass of 1082.67 remained unmatched for both proteins, which constitutes the studied sample. The PEF treatments with 1 and 10 min durations led to the liberation of the molecules at the same acquisition times as for PEF during 30 min, although the peak intensities were lower. The treatment with electrical arc during 30 min (Figure 4c) released molecules at acquisition times of 2.2, 10.9, and 31.6 min. At 2.2 min, molecules with molecular masses of 336.10 and 609.18 Da were detected. For β-lactoglobulin, the only peptide matched to this protein sequence was f(80−82) having a molecular mass of 336.10 Da, which was also found for PEF (30 min) treatment. For the α-lactalbumin, two peptides f(47− 49) and f(51−53) matched to 336.10 Da and one peptide f(26−30) matched to 609.18 Da. At 10.9 min, only one peptide with a molecular mass of 932.54 Da was detected. Three

Figure 3. Evolution of the degree of hydrolysis (DH), as a function of time of β-lactoglobulin treated by electrical arc.

pretreated β-lactoglobulin showed higher DH values at 30 min pretreatment throughout the hydrolysis, except for the final DH. These differences between arc and PEF modes could be related to the variations in phenomena that occur during each pretreatment mode. Indeed, the applied high voltage electric field during PEF mode imposes mechanical stress on the protein molecules and generates some active species, which could affect the distribution of electron density in polypeptide chains and conformational modifications.46,47 The electrical arc pretreatment imposes more stress, compared to PEF pretreatment, because of the generation of vapor cavities, UV irradiation and shock waves in addition to the above-mentioned phenomena occurring at PEF mode.21 Hence, the differences in PEF and electrical arc modes affect the susceptibility of pretreated β-lactoglobulin to the tryptic hydrolysis. Liquid Chromatography with Mass Spectrometry. The RP-UPLC-MS/MS spectrum of untreated β-lactoglobulin sample does not contain any peaks, meaning that there are no substances within a mass range of 100−3200 Da, which can be separated by liquid chromatography (LC) and detected by tandem mass spectroscopy (MS/MS) (see Figure 4 a).

Figure 4. RP-UPLC-MS/MS spectrograms of β-lactoglobulin: (a) control nontreated sample, (b) sample pretreated with PEF during 30 min, and (c) sample pretreated with electrical arc during 30 min. 11709


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ACS Sustainable Chemistry & Engineering peptides from β-lactoglobulin (f(96−102), f(7−14) and f(65− 72)) and one peptide from α-lactalbumin (f(21−28)) matched a molecular mass of 932.54 Da. The mass spectrogram of one peak at 31.6 min did not reveal any peptides. Consequently, it appeared that the duration of electrical arc treatment did not affect the peptide composition of the pretreated sample. The RP-UPLC-MS/MS analysis of hydrolyzed control protein sample and pretreated with PEF and electrical arc protein samples revealed differences in the released peptides’ profiles. Indeed, the quantity of peptides released after 2 h of tryptic hydrolysis of control and pretreated during 30 min βlactoglobulin samples was not the same (Figure 5). More

Concerning the selective conversion of hydrolyzed protein, one can observe that the most abundant peptides released during control tryptic hydrolysis were f(125−138), f(92−101), f(1−8), and f(142−148) (see Table 1). Similar peptides were the most abundant in the β-lactoglobulin hydrolysate pretreated with PEF, although the conversion percentages were slightly higher than those of a nonpretreated one. However, there is a significant difference in the abundances of some other peptides in profiles of both above-mentioned hydrolysates. Indeed, the PEF-pretreated sample released 3.3 times more f(84−91) peptides located in the core of the β-lactoglobulin molecule. The β-lactoglobulin sample pretreated with electrical arc demonstrated the similar peptide profile as for the PEF pretreated sample. However, the electrical arc pretreatment seems to be the most efficient for releasing the five abovementioned peptides, since their conversion rates at this special pretreatment mode were the highest among all three analyzed hydrolysates. The differences between both pretreatment modes were in the abundances of some peptides. For instance, the chromatogram of hydrolysate pretreated with arc did not contain f(125−141) peptide, while it was detected in the PEF pretreated and control hydrolysates. This fact is related to the more preferable cutting by the trypsin of the pretreated with arc β-lactoglobulin bond 138−139 releasing the f(125−138) peptide than cutting the bond 141−142 releasing the longer peptide f(125−141). The PEF-pretreated sample released some f(125−141) peptides after tryptic hydrolysis, although in lesser quantities than a nonpretreated sample. The similar case was observed for the f(149−162) peptide. Interestingly, the PEF and arc pretreatments led to the release of f(84−101) peptide, which was not registered after 2 h of hydrolysis in the control sample. In contrast, f(92−100) was detected only for the nonpretreated β-lactoglobulin sample. It is well-known that trypsin is an efficient and selective biological catalyst. However, the multiple studies demonstrated the possibilities of nonselective catalysis by trypsin.40,41,48−50 The irregular cleavages during tryptic hydrolysis could be due to several reasons, such as the origin and contamination of trypsin (e.g., by chymotrypsin), the hydrolysis conditions, acidinduced hydrolysis of labile peptide bonds during sample preparation prior to analysis, in-source fragmentation phenomena occurring during MS analysis, partial autodigestion of

Figure 5. Number of peptides identified by RP-UPLC-MS/MS after 2 h of hydrolysis of control and pretreated during 30 min with PEF or electrical arc β-lactoglobulin samples.

peptides were identified for the control nonpretreated hydrolyzed β-lactoglobulin. A bit less peptides were released after the hydrolysis of the PEF pretreated sample, which was not significantly different from the control protein sample. However, the electrical arc pretreatment of β-lactoglobulin prior to its hydrolysis led to the release of about 40% less peptides, compared to the control and PEF-pretreated samples. These results indicated that the HVET mode of β-lactoglobulin pretreatment could affect the selectivity of tryptic hydrolysis.

Table 1. Characteristics and Relative Abundances of Major Peptides Obtained after 2 h of Tryptic Hydrolysis of βLactoglobulin: Non-pretreated (Control) and Pretreated Prior to Hydrolysis with Pulsed Electric Field (PEF) and Electrical Arc (Selective Cleavage) Relative Abundance (%) peptide sequence

MM

pI

control

84−91 142−148 1−8 125−135 92−101 125−138 92−100 84−101 125−141 76−91 41−60 15−40 149−162

915.47 837.05 933.17 1245.31 1193.41 1636.74 1065.23 2091.39 1948.16 1801.12 2314.67 2708.08 1659.85

4.4 9.8 8.8 3.8 5.9 4.0 4.2 4.7 4.3 8.2 4.3 4.2 4.5

± 0.4 ± 0.5 ± 0.8 ± 0.2 ± 1.4 ± 0.2 ± 0.5 0.0 1.6 ± 0.3 0.0 1.3 ± 0.3 0.1 ± 0.0 3.2 ± 0.8 1.6 5.5 7.6 0.4 7.8 9.8 0.9

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PEF (30 min) 8.4 ± 6.4 ± 8.6 ± 0.8 ± 9.0 ± 11.7 ± 0.0 1.8 ± 0.7 ± 0.0 0.2 ± 0.2 ± 2.9 ±

0.3 0.6 0.3 0.3 1.0 0.6 0.4 0.2 0.1 0.1 0.6

arc (30 min) 9.2 ± 7.9 ± 11.8 ± 0.4 ± 12.2 ± 11.9 ± 0.0 0.6 ± 0.0 0.7 ± 2.5 ± 1.9 ± 0.7 ±

0.8 0.8 0.3 0.1 1.1 1.2 0.3 0.0 0.7 1.1 0.0


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Table 2. Characteristics and Relative Abundances of Major Peptides Obtained after 2 h of Tryptic Hydrolysis of βLactoglobulin: Non-pretreated (Control) and Pretreated Prior to Hydrolysis with Pulsed Electric Field (PEF) and Electrical Arc (Non-selective Cleavage) Relative Abundance (%)

a

peptide sequence

MM

pI

54−60 82−89 112−136 47−50 49−68 41−57/31−47 25−40 ft=28.0a ft=28.4a

856.44 949.53 2849.28 442.32 2273.15 1944.21/1944.26 2030.32

6.0 4.4 4.0 9.9 3.9 3.9/4.7 4.2

control 0.7 ± 5.6 ± 1.9 ± 1.5 ± 1.4 ± 4.7 ± 0.0 7.7 ± 10.6 ±

0.1 0.6 0.3 0.4 0.4 0.6 1.2 0.9

PEF (30 min) ± 0.7 ± 0.2 ± 0.1 ± 0.6 0.0 3.6 ± 0.1 6.4 ± 1.2 6.2 ± 1.0 8.3 ± 1.2 2.1 0.5 0.4 1.7

arc (30 min) 3.8 ± 2.3 ± 0.0 0.0 0.4 ± 0.6 ± 4.1 ± 6.0 ± 13.3 ±

0.1 1.0

0.3 0.1 1.3 1.5 0.6

Multiple peptides were identified at this retention time.

trypsin resulted in generation of pseudotrypsin, etc.43,48,50,51 The release of the most abundant peptides from the nonselective action of trypsin is shown in Table 2. One could observe that the hydrolysis of control and pretreated βlactoglobulin samples by trypsin released different quantities of nonspecific peptides. The most abundant nonspecific peptides of the control sample were released at the acquisition times of 28.0 and 28.4 min. At 28.0 min, the RP-UPLC-MS/MS spectra revealed the presence of several possible compounds having molecular masses of 734.93, 857.42, 1028.91, 1715.18, and 2572.27 Da. However, these compounds do not correspond to any peptide sequence of β-lactoglobulin or α-lactalbumin, according to the FindPept database. At 28.4 min, the peptides having molecular masses of 867.09, 1041.51, and 1734.51 Da were identified, matching to f(13-21), f(110−125), and f(86− 100) peptide sequences of β-lactoglobulin. The profile nonspecific peptides of PEF pretreated protein looks quite similar to the control one. The most abundant peptides were released at the same acquisition times of 28.0 and 28.4 min. However, at 28.4 min, there were more compounds of 743.93 and 2601.27 Da identified by MS/MS analysis, although they did not match to β-lactoglobulin or α-lactalbumin. Concerning the arc-pretreated β-lactoglobulin sample, it contained the most abundant peptides at the same times as control and PEFpretreated samples and the molecular masses of the released compounds were identical as for the PEF-pretreated sample. However, there is an influence of the pretreatment technique on the release of certain nonspecific peptides. Indeed, the 949.53 Da peptide matched to the f(82−89) of β-lactoglobulin demonstrated a very high abundance in the control sample, two times less abundance in the arc-pretreated sample and just traces in the PEF-pretreated sample. In contrast, the 1944.21 and 1944.26 Da peptides matched to the f(41−57) or f(31−47) of β-lactoglobulin had relatively high abundance in the control sample, lesser abundance in the PEF-pretreated sample, and traces in the arc-pretreated sample. Interestingly, the 2030.32 Da peptide was registered in both pretreated samples but was absent in the control sample.

meaning an increase in applied energy. These results could be explained from the mechanisms of HVET action (see Figure 6).

Figure 6. Scheme of high voltage electrical pretreatments, followed by enzymatic hydrolysis of a whey protein solution.

Actually, the mechanical stress that is created by the high voltage electric pulses can induce mechanical instabilities, resulting in changes of flexibility and conformation of protein molecules47 and highly reactive chemical species can interact with the polar or charged groups in the polypeptide chains, leading to their dissociation with the liberation of peptides or amino acids. Moreover, the additional phenomena occurring during electric arc pretreatment (vapor cavities, UV light, and shock waves) could promote the redistribution of electron density in the excited molecules, their conformational modifications, and further dissociation. However, the PEF pretreatment mode leads to the release of higher quantities of peptides from the β-lactoglobulin, compared to the electric arc mode. The RP-UPLC-MS/MS spectra corroborate the OPA essays, indicating the appearance of new molecules after both HVET modes. In addition, the chromatograms of PEF and electric arc modes had differences in peptide profiles, suggesting variations in the pathways of the protein cleavage. Note that RP-UPLC-MS/MS analysis performed for the studied samples has certain limits, such as analyzed mass range and difficulties in the detection of peptides having a relatively high content of hydrophobic amino acids. Thus, some

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DISCUSSION The high voltage electrical pretreatment demonstrated very promising features. The OPA essay revealed the generation of free primary amino groups after both tested pretreatment modes (PEF and electric arc) prior to hydrolysis. Moreover, the concentration of the molecules possessing these free primary amino groups increased as the pretreatment duration increased, 11711


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• The main advancement of the HVET is the substantial increase in the degree of hydrolysis (DH) of pretreated βlactoglobulin, compared to the nonpretreated protein. Indeed, the pretreatment with electric arc mode improves the further tryptic hydrolysis of β-lactoglobulin by 66%, while the PEFpretreated samples demonstrated 80% better efficiency, compared to the nonpretreated sample. Moreover, the application of electric arc to the protein solutions was performed for the first time. • The HVET of a protein solution could liberate peptides even prior to enzymatic hydrolysis, due to disruption of the noncovalent bonds of peptides associated with protein molecules or peptide bonds within the protein molecule. • The peptide profile of the nontreated sample was different, compared to the electrically pretreated one. Moreover, there were differences in peptide abundances between PEF and electric arc modes, because of the variations in phenomena occurring in each studied mode. Electric arc seems to affect the structure of β-lactoglobulin more severely, compared to PEF facilitating the access of trypsin to the active substrates in polypeptide chains. Hence, the selectivity of hydrolysis of βlactoglobulin pretreated with electric arc is higher, compared to that pretreated with PEF and the nonpretreated one. Indeed, the β-lactoglobulin pretreated with electrical arc releases ∼40% less peptides, compared to the PEF-pretreated sample. The future works should be directed to the profound studies of structural changes of protein molecules that undergo the high voltage electrical pretreatments to explain the mechanisms of action proposed in the present study. Moreover, it is interesting to study HVET with durations of >30 min in order to explore the possibility of peptide release without enzymatic action. In addition, the biological, functional, and nutritional properties of hydrolysates obtained after HVET should be studied, since the phenomena occurring during PEF and arc modes could modify the properties of released peptides and amino acids.

peptides released during HVET could remain undetected by RP-UPLC-MS/MS. The application of HVET as a pretreatment technique prior to enzymatic hydrolysis seems to be more advantageous, compared to high hydrostatic pressure, showing a low efficiency, according to Maynard et al.52 However, carrying out enzymatic hydrolysis at simultaneous high hydrostatic pressures, as well as microwave-assisted hydrolysis, demonstrated significant improvement of the hydrolysis efficiency related to the structural rearrangements favorable for the protein/enzyme interactions.42,52,53 The peptide profile after tryptic hydrolysis of control and electrically pretreated β-lactoglobulin demonstrated significant differences in the relative abundances of multiple peptides cleaved via selective and nonselective pathways. Generally, comparing the relative abundances of the most prevalent peptides (e.g., f(84−91), f(1−8), f(92−101), f(125−138)) detected after hydrolysis, the electric arc pretreatment results in their higher abundances, compared to PEF pretreated and control samples. Moreover, the electric arc pretreatment leads to the release of fewer numbers of peptides after 2 h of hydrolysis (32) versus the PEF pretreatment (48) and control sample (51). These results suggest that the structural modifications of the polypeptide chains caused by the action of electric arc on the protein molecules favor the cleavage of a restricted number of bonds while the tryptic cleavage of PEF pretreated and control samples released a greater variety of peptides having lesser relative abundances. One could speculate that electrical arc promotes better unfolding of the protein molecule, decreasing the steric and energy barriers for the reaction of trypsin with the appropriate substrate. Indeed, UV light, which is an inherent phenomenon of electric arc pretreatment, can affect the distribution of electronic density in protein molecules via photochemical pathways, leading to harmful consequences for their structure. For instance, the UV light excitation of the aromatic residues (Phe, Tyr, and Trp) affects the electron ejection from their side chains.54 Once excited, these electrons could be captured by disulfide bonds, which eventually leads to the formation of free thiol groups in the protein.54 Moreover, other arc-inherent phenomena (vapor cavities and shock waves) could also promote stronger structural modifications of the treated proteins, compared to the PEF mode. Hence, the electrical arc unfolding of protein molecules is favorable for the release of the greater amounts of active sites for the nucleophilic attack by the aspartic residues located in the hydrophobic pocket of the trypsin. However, one could notice that the PEF mode leads to the higher DH after 2 h of proteolysis, compared to electric arc mode, which could be due to the fact that PEF pretreatment affects a greater amount of protein molecules. This could be explained by the more uniformly distributed electrical field between two plate electrodes (PEF) than between needle and plate electrode (electric arc). Indeed, the uniform distribution of electrical field leads to the better distribution of applied high voltage mechanical stress imposed on the protein molecules, facilitating their further hydrolysis.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (+1) 418-656-2482. E-mail: sergey.mikhaylin@fsaa. ulaval.ca. ORCID

Sergey Mikhaylin: 0000-0002-2366-7284 Laurent Bazinet: 0000-0002-6818-3558 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) is acknowledged. Authors gratefully thank Jacinthe Tibodeau, Veronique Perreault, and Oleksii Parniakov for their kind assistance in the preparation and analysis of protein samples.

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CONCLUSION The results obtained in this study demonstrate several innovative features in the application of various modes of high voltage electric treatments (HVETs) as a pretreatment technique for the improvement of the enzymatic hydrolysis of β-lactoglobulin:

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