Comparative Study of in Situ and ex Situ Enzymatic Hydrolysis of Milk

Research Article pubs.acs.org/journal/ascecg

Comparative Study of in Situ and ex Situ Enzymatic Hydrolysis of Milk Protein and Separation of Bioactive Peptides in an Electromembrane Reactor Shyam Suwal, Élodie Rozoy, Mahder Manenda, Alain Doyen, and Laurent Bazinet* Institute of Nutrition and Functional Foods (INAF), Dairy Research Centre (STELA), Laboratory of Food Processing and Electromembrane Processes (LTAPEM), Department of Food Science, Université Laval, Québec, 2425 Rue de l’Agriculture, Québec, G1 V 0A6, Canada ABSTRACT: Tryptic hydrolysis of whey protein isolate was performed simultaneously during (in situ) and before (ex situ) fractionation by electrodialysis with ultrafiltration membrane (EDUF) to obtain bioactive peptides. Peptide migration to anionic (ARC−) and cationic (CRC+) peptide recovery compartments was strongly dependent on the digestion strategy used. Indeed, peptide migration to the ARC− was observed to be higher with in situ digestion while peptide migration to the CRC+ was higher in an ex situ digestion: a final peptide concentration of 103.10 ± 2.76 μg/mL was found in the CRC+ (ex situ) while it was 49.65 ± 6.13 μg/mL in the ARC− (in situ). HPLC-MS studies showed 23 major peaks that were generated by tryptic digestion of whey protein isolate. Seven of these peptides migrated to the ARC− while nine and eight peptides migrated to the CRC+ for ex situ and in situ digestions, respectively. Among them, different antihypertensive, antimicrobial, and hypocholesterolemic peptides were recovered depending on the recovery compartment such as IDALNENK and VYVEELKPTPEGDLEILLQK in ARC− and ALPMHIR, TKIPAVFK, VLVLDTDYKK, and VAGTWY in CRC+ regardless of the mode of enzymatic hydrolysis. KEYWORDS: In situ, Ex situ, Enzymatic hydrolysis, Bioactive peptides separation, Electromembrane reactor

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INTRODUCTION As the knowledge of molecules contained in food sources deepens, scientists have long started looking at food as more than just a source of calories and nutrients. The presence of bioactive molecules contained in the complex food matrix and other natural sources has attracted much attention from food scientists and the food industry. Bioactive ingredients are molecules obtained from natural sources that can exert physiological changes in microbes or in humans. They can exist as polysaccharides or oligosaccharides, polyphenolic compounds, conjugated linoleic acids, peptides, etc. The existence of such molecules in everyday food sources, making them functional foods, or the isolation of such molecules from food sources and their preparation into specialized products such as nutraceuticals can be a cost-effective alternative to increasingly very expensive drug-based health care.1 Bioactive peptides have long been known to exert a range of physiological effects in humans and other organisms acting as antihypertensive, antithrombotic, antimicrobial, antioxidative, immunomodulatory, and opioid molecules.2 The bioactivity of these peptides is inherent in their amino acid sequence that sometimes leads to multifunctionality. However, due to their low concentrations in nature and their interactions with other molecules that can potentially lead to a loss of bioactivity, bioactive compounds have limited bioavailability. These led to © 2017 American Chemical Society

several attempts to either directly synthesize the peptide sequences or isolate them efficiently from their natural sources. Direct synthesis by chemical methods or using recombinant DNA technology are, as of now, costly for large peptides and difficult to streamline, while isolation from natural sources including everyday food materials by hydrolysis and then isolation presents a cost-effective and easy to scale-up alternative. Enzymatic hydrolysis of milk/whey proteins by digestive enzymes such as trypsin has been reported to be an important phase of bioactive peptide generation.3 Moreover, following the enzymatic digestion, isolation of these peptides of specific functionality is a real challenge for food scientists and dairy industries. Membrane processes and materials play an important role in the isolation of bioactive peptides obtained from enzymatic hydrolysis of milk/whey protein. An enzymatic membrane reactor (EMR), for instance, usually uses ultrafiltration membranes (UFMs) to reuse the enzyme and also to separate the digested products.4 Pressure driven processes like ultrafiltration (UF) and nanofiltration (NF) have also been used, separately or in combination, to fractionate bioactive Received: March 2, 2017 Revised: April 24, 2017 Published: May 10, 2017 5330

DOI: 10.1021/acssuschemeng.7b00651 ACS Sustainable Chem. Eng. 2017, 5, 5330−5340

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ACS Sustainable Chemistry & Engineering

Figure 1. Schematic diagram of the electromembrane reactor for the recovery of anionic and cationic peptides. AEM, anion-exchange membrane; UFM, ultrafiltration membrane; CEM, cation-exchange membrane; ARC−, anionic peptides recovery compartment; CRC+, cationic peptides recovery compartment; P+, cationic peptides; P−, anionic peptides; P±, neutral peptides.

of the present work is to investigate the effect of enzymatic digestion techniques (in situ and ex situ) on electrodialytic parameters, peptide recovery yield, and composition in both anionic and cationic peptide fractions during and after EDUF.

peptides mainly by their molecular mass and, to some extent, by their charge by the interaction with NF membranes.3,5−7 In both cases, the extent of fouling presents a significant challenge, and both lack the selectivity. Moreover, they are unable to simultaneously fractionate cationic and anionic peptides, which may have close molecular mass but completely different bioactivity.8 Among electromembrane processes, including electrically enhanced membrane filtration systems and forced flow membrane electrophoresis, electrodialysis with filtration membrane (EDFM) was the only one to demonstrate the ability to fractionate cationic and anionic peptides even when the peptides have very close molecular weights. Electrodialysis with ultrafiltration membrane (EDUF) or more generally EDFM has now been used to isolate bioactive compounds from multitude of sources. In addition to high selectivity, the EDFM process has been considered to be a green technique as it does not require the use of any chemical solvent. Very recently, Doyen et al.9 used EDUF technology to simultaneously hydrolyze and separate bioactive peptides from whey protein isolate. However, a comparative study of protein digestion within the EDUF setup or outside of it and its effect on peptide migration profile has not been yet studied. Therefore, the aim

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MATERIALS AND METHODS

Materials. BiPro, containing mainly β-lactoglobulin (β-LG) protein, with a purity of ≥90%, was purchased from Davisco Foods International Inc. (Minnesota, USA). Bovine pancreatic trypsin (with an activity ≥7500 BAEE units/mg) was purchased from Sigma− Aldrich (St. Louis, MO, USA). A PVDF ultrafiltration (MWCO 50 kDa) membrane was purchased from Synder Filtration (California, USA). Ion exchange membranes were bought from Ameridia (New Jersey, USA). HCl and NaOH solutions were obtained from Fisher Scientific (Montreal, QC, Canada). NaCl and KCl were purchased from ACP Inc. (Montréal, QC, Canada). Electromembrane (EDUF) Reactor Design. The separation reactor consisted of an EDUF cell of MP type with 100 cm2 of membrane effective surface area manufactured by ElectroCell Systems AB Company (Täby, Sweden), four external reservoirs, each of which was connected to centrifugal pumps, and a power supply system (Figure 1). The EDUF cell was mounted with one Neosepta CMX-SB cationic membrane (ASTOM, Tokyo Japan), one Neosepta AMX-SB anionic membrane (ASTOM, Tokyo, Japan), and two polyvinylidene 5331


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ACS Sustainable Chemistry & Engineering fluoride (PVDF) UFMs (UFM-I, anode side; UFM-II, cathode side) with a molecular weight cutoff (MWCO) of 50 kDa. The configuration consisted of five compartments. The central compartment situated between the two UFMs was fed with BiPro (in case of in situ digestion) or BiPro hydrolysate (in case of ex situ digestion) solutions (2 L, 12.5 g/L). Two compartments adjacent to the feed were circulated each with aqueous KCl solution (2 L, 2 g/L) for the recovery of anionic peptides, called the anionic recovery compartment (ARC−), and cationic peptides, called the cationic recovery compartment (CRC+). Finally, the last two interconnected compartments contained electrode rinsing solution, NaCl (3 L, 20 g/ L). KCl solutions were used in the recovery compartments instead of NaCl to reduce sodium content in the recovered fractions, since one of our ultimate goals is to separate the β-lg 142−148 fraction known as an antihypertensive peptide. The solutions were circulated using four centrifugal pumps, and the flow rates were controlled using flowmeters. In permeate and the feed compartment, the solution flow rates were 1.5 L/min, while it was 2 L/min for the electrode solution. Hydrolysis and Separation Procedures. BiPro solution was prepared after overnight hydration of 25 g of BiPro in 2 L of distilled water (1.25% w/v) in a cold room at 4 °C. Tryptic hydrolysis of BiPro was carried out in two setups: one in a beaker (ex situ) with continuous stirring of the BiPro solution and the other in the EDUF system (in situ) after the pH was adjusted to 7.8. Enzymatic hydrolysis was started by the addition of 10 mL of trypsin solution at a concentration of 25 g/L (w/v) to attain the final concentration of 125 mg/L. In both in situ and ex situ digestions, the hydrolysis was performed for 120 min, and the enzymatic reaction was stopped by raising the temperature of the solution to 80 °C for 30 min. For the fractionation of peptides, a constant electric field strength of 8.22 V/cm corresponding to a constant voltage of 35 V was applied between the two electrodes. For both digestion modes, the fractionation was performed for 120 min. The system was started initially at room temperature, and the EDUF parameters were recorded every 15 min throughout the experiment. Under both in situ and ex situ conditions, the pH was maintained at pH 7.8, corresponding to the optimum pH value of trypsin, with 0.5 M NaOH using a pH meter from Thermo Scientific Orion 9206BN probe (ThermoFisher, Montreal, Qc, Canada). The pH of recovery compartments was also maintained at 7.8 by a continuous addition of NaOH and HCl by using the same type of pH meter. A total of 10 mL of samples from the hydrolysate/feed and each recovery compartment (ARC−, CRC+) was collected before applying voltage and every 30 min during the treatment. Samples were heated to 80 °C for 30 min to stop the enzyme activity. Following each EDUF treatment, the final volumes of ARC−, CRC+, and the feed/hydrolysate compartments were recovered and freeze-dried for storage after the inactivation of enzyme in the case of in situ EDUF treatment. Finally, a cleaning-in-place (CIP) procedure for the EDUF cell was performed after each run to ensure the recovery of the UFMs’ and IEMs’ performances.10 Analyses. Solution Conductivities. Conductivities of ARC−, CRC+, and feed compartments were measured every 15 min during the 120 min of EDUF with a YSI conductivity meter (model 3100) equipped with a YSI immersion probe (model 3252, cell constant K = 1 cm−1, yellow Springs Instrument Co., Yellow Springs, OH, USA). The conductivities were measured in order to evaluate the mineralization or demineralization of the solutions during the process. Peptide Concentration. Total peptide concentrations in the ARC−, CRC+, and feed solutions were determined from samples withdrawn every 30 min over a period of 120 min using BCA protein assay (Pierce, Rockford, IL, USA). The microplate was first incubated at 37 °C for 30 min and then cooled to room temperature, and the absorbance was read at 562 nm on a microplate reader (THERMOmax, Molecular devices, Sunnyvale, CA). Concentration was determined with a standard curve in the range of 5−2000 μg/mL of bovine serum albumin (BSA). Protein and Peptide Profile with HPLC. The peptide composition of the ARC−, CRC+, and hydrolysate solutions was determined by RP-

HPLC according to the method of Firdaous et al.11 adapted to the specific conditions of the feed and peptides generated during hydrolysis. The system used was an Agilent 1100 series. Peptides were analyzed with a Luna 5 μm C18 column (2 i.d. × 250 mm, Phenomenex, Torrance, CA, USA). Solvent A, TFA 0.11% (v/v) in water, and solvent B, acetonitrile/water/trifluoroacetic acid (TFA) (90%/10%/0.1% v/v), were used for elution at a flow rate of 10 μL/ min. A linear gradient of solvent B, from 3% to 60% in 85 min, was used. The detection wavelength was 214 nm, which is typically used to monitor peptide bonds.11,12 Peptide Molecular Weight. The molecular weight (MW) of proteins and peptides in recovered samples was determined by mass spectrometry (LC-MS) analyses using the ion trap method. MS analyses were performed in a scan range of 300−2200 m/z in positive polarity, with an ESI ion source type, at a dry temperature of 350 °C, a nebulizer at 30.00 psi, and dry gas of 8.00 L/min. The system used was an Agilent 1100 series (Agilent Technologies, Palo Alto, CA, USA). Peptides were analyzed with the same method, and the same column used for RP-HPLC analyses. To reduce the effect of TFA, MS was performed after infusing (10 μL/min) a mixture of 50% propionic acid and 50% isopropanol to the existing flow before the MS interface. Signals were recorded in positive mode using a 90 V fragmentation with a scan range of 300−3000 m/z.11 Peptide Migration Rate. The migration rate of each peptide, expressed in percentage, was determined from the HPLC results as previously described by Doyen et al.9 The area under the curve of each peak of HPLC chromatograms of peptide solution in the recovery compartment was compared to the area of the corresponding peak of β-LG hydrolysate obtained after 120 min of EDUF treatment (final hydrolysate). Equation 1 was used to calculate the peptide migration rate:

Tr =

A recovered × 100 Ahydrolysate

(1)

where Tr represents peptide migration (in %), Arecovered the area of a given peak in the permeate, and Ahydrolysate the area of the corresponding peak in the final hydrolysate. Statistical Analysis. The changes in peptide migration rate, electrical resistance and conductivity were subjected to repeated measures of analysis of variance (P < 0.05 as probability level for acceptance) using SigmaPlot integrated software (Systat Software, Inc. San Jose, CA, USA).

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RESULTS AND DISCUSSION EDUF Parameters. Evolution of System Electrical Resistance. The evolution of system resistance during 120 min of EDUF for ex situ and in situ experiments is presented in Figure 2. System resistance evolved in a similar fashion as a function of time for both digestion strategies used. An initial slight decrease in resistance for the first 15 min of EDUF was followed by a relatively constant resistance for the next 60 min, which was followed by a sudden rise in the system during the last 45 min of EDUF treatment. However, ANOVA indicated a significant difference (P = 0.005) in system resistance evolution with time for the ex situ and in situ experiments: the ex situ experiment exhibiting a consistently higher resistance than the in situ one throughout the 120 min EDUF except during the first 15 min of EDUF. In the ex situ experiment system, resistance varied from an initial value of 31.36 ± 2.23 Ω to a final value of 50.65 ± 4.96 Ω, while in the in situ experiment it varied from 33.07 ± 1.135 Ω to 46.40 ± 1.27 Ω. The continuous decrease in concentration of K+ and Cl− ions in the ARC− and CRC+, with time, due to their net migration into the feed and electrolyte compartments could be the reason for the increase in system resistance especially at final times of EDUF. 5332


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Figure 2. Evolution of system resistance as a function of time for ex situ and in situ experiments.

Evolution of Conductivity in the Three Main Compartments. Figure 3 depicts the change in electrical conductivity of solution with time in the ARC−, CRC+, and feed compartments with time. It appeared that ex situ and in situ experiments had no significant difference in the evolution of electrical conductivity with time in all three compartments. The average demineralization rate in the ARC− compartment was calculated to be 65.78 ± 3.31%, while it was considerably lower, 44.99 ± 4.7%, in the CRC+ compartment. In contrast to recovery compartments, the electrical conductivity in the hydrolysate compartment increased with time (Figure 3c). The increase was more curvilinear than just linear as indicated by the corresponding quadratic equation on the fitted curve. ANOVA indicated no significant difference between the in situ and ex situ experiments. The plateau in the conductivity vs time curve in the feed compartment is in line with the increase in the electrical resistance observed after 75 min of treatment (Figure 2). The decreases in conductivity in the ARC− and CRC+ could be explained by the EDUF cell configuration (Figure 1). Indeed, K+ and Cl− ions from ARC− and CRC+ respectively migrated into the hydrolysate and electrolyte solutions during the treatment (Figure 3a and b). As a result, the conductivity of hydrolysate solution was increased (mineralized) in the course of treatment, as was observed previously by Firdaous et al.11 Moreover, anions and cations (especially, Na+, H+, Cl−, and OH− coming from pH adjustment) present in the hydrolysate and recovery compartments can migrate to the electrolyte solutions near the anode and cathode sides, respectively. Evolution of Total Peptide Concentration. Peptide migration into both cationic and anionic compartments for both ex situ and in situ experiments are presented in Figure 4. ANOVA indicated a significant dependence of peptide migration on the time of EDUF (P < 0.0001), recovery compartment (ARC− or CRC+, P < 0.005), and also enzymatic digestion (ex situ/in situ; P < 0.001). Peptide migration to CRC+ in both digestion strategies increased in a manner that attained a plateau at about 90 min of EDUF, while it appeared to continue to increase beyond 120 min in ARC−. These observations are similar to the ones reported by Doyen et al.9 The peptide concentration in the anionic recovery compartment (ARC−) was significantly lower (P < 0.001) for the ex situ

Figure 3. Evolution of conductivity as a function of time in the (a) ARC−, (b) CRC+, and (c) feed compartments for ex situ and in situ experiments.

experiment than the in situ throughout the 120 min of EDUF with respective final values of 54.17 ± 1.18 μg/mL and 77.93 ± 13.02 μg/mL. The opposite trend was observed for the peptide concentration in cation recovery compartment (CRC+). This could be because the ex situ experiment would have allowed time for possible peptide−peptide interaction that could happen between the relatively large number of anionic peptides (in ARC−) that were generated leading to a possible loss of charge and gain of mass, which reduce migration. In the in situ experiment, the chances for the peptides to interact were 5333


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had not attained its maxima as opposed to migration to the CRC+, which had attained a plateau after the 90 min of EDUF. In a 4 h EDUF experiment using a similar setup but with different UFM material (cellulose acetate-CA) and a higher electric field strength (14 V/cm), a significantly higher migration to the ARC− as compared to CRC+ was observed, which is in agreement with our hypothesis that if enough time is allowed, final migration to the ARC− will be higher than migration to the CRC+.9 For the CRC+, a larger final peptide migration was recorded for the ex situ experiment (103.10 ± 2.76 μg/mL) compared to the in situ (69.26 ± 7.29 μg/mL). This observation is different from the one observed for the ARC− in which in situ led to a higher migration than ex situ. This could be because there is less effect of peptide−peptide interaction among cationic peptides, as they are mainly hydrophobic rather than anionic counterparts. No electrostatic/ionic interactions are involved in the aggregation process of peptides produced from tryptic digestion of milk proteins.13 Peptide Profiles and Migration Rates. Peptide Profile in the Hydrolysate Compartment. Major peptides obtained from 120 min of digestion of β-LG by trypsin with their respective HPLC retention time, mass, potential sequence, pI, and possible source of native protein are presented in Table 1. Potential sequences, peptide location, pI, and net charges were obtained by tools from the ExPAsy Bioinformatics Resource

Figure 4. Evolution of peptide concentration in the ARC− and CRC+ as a function of time for ex situ and in situ experiments.

relatively limited because separation happened simultaneously with digestion. It was observed that, for the duration of the EDUF in our experiment, the migration rate to the CRC+ appeared to be higher than the migration rate to the ARC− (in both ex situ and in situ digestions Figure 4). This could be because of the duration of the EDUF during which the migration to the ARC−

Table 1. Characterization of Peptides Obtained after 120 min of Tryptic Hydrolysis of Bipro peak #

Rt (min)a

obsd MWb

potential sequencec

locationc

net chargec

pIc

source of peptidec

1 2 3

11.442 24.865 28.279

4 5 6

30.600 35.282 36.630

7 8

38.151 39.610

9 10 11 12

40.272 41.107 43.697 44.735

13 14 15 16 17 18 19

45.760 48.396 52.816 53.600 53.913 56.545 57.421

20 21 22 23

59.617 60.179 61.196 65.912

573.3 916.5 673.3 949.5 1245.6 933.5 837.5 1437.7 1193.7 903.8 1635.9 696.3 1065.6 2163 1163.6 2720.2 1361.1 1200.7 2309 2030.2 2313.6 1157 1185 1477.8 1567.7 1152 1443.7 1269 1301.6 2707.4

IIAEK IDALNENK GLDIQK ndd TPEVDDEALEK LIVTQTMK ALPMHIR nd VLVLDTDYKK TKIPAVFK TPEVDDEALEKFDK VAGTWY WENGECAQKK nd LVNELTEFAK nd GSNFQLDQLQGR VGINYWLAHK nd SLAMAASDISLLDAQSAPLR VYVEELKPTPEGDLEILLQK LICDNTHITK nd LGEYGFQNALIVR DAFLGSFLYEYSR nd nd nd ETTVFENLPEK VAGTWYSLAMAASDISLLDAQSAPLR

f71−75 f84−91 f9−14 nd f125−135 f1−8 f142−148 nd f92−101 f76−83 f125−138 f15−20 f61−70 nd f66−75 nd f120−131 f118−127 nd f21−40 f41−60 f669−678 nd f421−433 f347−359 nd nd nd f230−240 f15−40

− − − nd − + + nd − + − − − nd − nd − + nd − − − nd − − nd nd nd − −

6 4.4 5.9 nd 3.8 8.8 9.8 nd 5.9 10 4 5.49 6.1 nd 4.5 nd 5.8 8.6 nd 4.21 4.3 6.7 nd 6 4.4 nd nd nd 4.3 4.2

β-LG β-LG β-LG nd β-LG β-LG β-LG nd β-LG β-LG β-LG β-LG β-LG nd BSA nd β-LG α-LAf nd β-LG β-LG Lpxg nd BSA BSA nd nd nd lactoferrin β-LG

a

Retention time. bObserved molecular weight. cAccording to ExPAsy bioinformatics resource portal. dNondefined. eImmunoglobulin gamma. fAlpha lactalbumin. gLactoperoxidase. 5334


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Figure 5. Chromatogram of initial BiPro solution before digestion by trypsin.

Portal (Swiss Institute of Bioinformatics) for bovin (Bos taurus) β-LG (UniProtKD/TrEMBL #P02754), Lactoperoxidase (UniProtKD/TrEMBL #P80025), Lactoferrin (UniProtKD/ TrEMBL #B9VPZ5), BSA (UniProtKD/TrEMBL #P02769), α-lactalbumin precursor (UniProtKD/TrEMBL #P00711), and immunoglobulin gamma Fc region receptor II precursor (IgG Fc receptor II-UniProtKD/TrEMBL Q28110) digestion by trypsin. These proteins along with other minerals, sugars, and amino acids are indicated to be present in BiPro by the manufacturer (DAVISCO). The peptide fragments labeled Nd in Table 1 are not generated by the ExPAsy Bioinformatics Resource server and it is assumed that these peptides may be generated by peptide−peptide interactions that have also been observed before.13 Of the twenty three peaks listed in Table 1, 12 were negatively charged (anionic, peak #1, 2, 4, 7, 9, 10, 15, 16, 17, 19, 22, 23), two positively charged (cationic, peak #5, 13) at the working pH (7.8), and four (peak #14, 18, 20, 21) did not have a corresponding peptide generated by ExPAsy Bioinformatics Resource Portal (hence, nd). Five of the peaks (peak #3, 6, 8, 12, 19) contained more than one possible peptide, which were either anionic, cationic, or nd. Similar to our observation mentioned above, tryptic hydrolysis of milk proteins has previously been reported to generate more anionic peptides than cationic ones.13 Figure 5 depicts the chromatograms of BiPro solution before digestion by trypsin. Peaks labeled A and B with retention times of 70.249 and 72.293, respectively, stand out in the HPLC chromatogram and correspond to proteins identified as β-LG A and β-LG B, the two major variants of β-LG protein in milk with respective molecular masses of 18 362.86 Da and 18 276.73 Da.14 The manufacturer (DAVISCO) indicated that the other proteins present in BiPro alltogether constitute less than 5% by mass and that could be why they are not visible on the chromatograms relative to the 95% β-LG composition of BiPro. Figure 6a, b, and c depict typical chromatograms of BiPro after 120 min of ex situ digestion, after 120 min of EDUF after an ex situ digestion, and after 120 min of simultaneous (in situ) digestion and EDUF treatment of BiPro, respectively. As can clearly be seen on the two chromatograms, almost all the peaks in Figure 6b appeared reduced in height, and hence area, as compared to their corresponding peaks in Figure 6a (ex situ

digestion). This was expected and was due to the migration of peptides from the feed compartment to the ARC− and CRC+ (ex situ digestion), but such migration was not happening in the feed during ex situ digestion (Figure 6a). The same trend of peak area decrease was observed when comparing respective peaks in parts a and c (in situ digestion). Generally, the same numbers of peaks were observed in all three cases. Peptide Profiles and Migration Rates in the ARC− and CRC+. Figure 7a and b display typical HPLC chromatograms of the peptide fractions obtained in the ARC− ex situ and in situ, respectively, after 120 min of EDUF. In both cases, mainly seven peptides were selectively separated, and six were similar, corresponding to peak numbers 2, 4, 8, 10, 11, 12, and 16. Peaks #4 and 8 migrated to a greater extent relative to the other peaks. In general, for the ARC−, peaks in the ex situ experiment, especially peaks #2, 4, and 8, were observed to be of larger area (intensity) compared to their corresponding peaks in the in situ experiment. However, peak #6 was only present in the ARC− ex situ. Indeed, peak #6, which exhibited a marked migration in the ex situ experiment (Figure 7a), is totally missing from the ARC− of the in situ experiment. Peak #6 was composed of two peptides (with a positive and a nondefined charge, Table 1), which could be the reason why it exhibited such a complex pattern of migration. Moreover, in the in situ experiment, peak #11 with a molecular weight of 2163 was observed to have considerable migration, while such a peak was almost negligible in ex situ. Out of the seven peptides recovered in the anionic compartment (ARC−), two have proven to have important physiological roles. Peaks #2 and 16, corresponding to peptides IDALNENK (f84−91) and VYVEELKPTPEGDLEILLQK (f15−40), respectively, have demonstrated antihypertensive and hypocholesterolemic effects.15 A total of nine and eight peptides were recovered in the cationic recovery compartment (CRC+) for ex situ and in situ experiments, respectively (Figure 8a and b). Peaks #6 and 8 were markedly present in the CRC+ for both ex situ and in situ experiments, and this could be because of the positive charge they exhibited at the pH of operation (pI = 9.8 for peak 6 and pI = 10 for peak 8) and their relatively lower molecular mass (837.5 and 903.8 Da, respectively). Peaks #5 and 9 also exhibited a good migration to the CRC+ as compared to other peptides. However, peaks #6, 8, and 12 also contained other 5335


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Figure 6. Chromatograms of BiPro (a) after 120 min of ex situ digestion, (b) after 120 min of EDUF after an ex situ digestion, and (c) after 120 min of simultaneous (in situ) digestion and EDUF treatment.

MS of the ARC− sample. The peptide (peak #8) with a molecular mass of 903.8 Da has a sequence of TKIPAVFK and known to possess a hypocholesterolemic function.16 The peptide (peak #6) with the sequence ALPMHIR and molecular weight 837.5 Da was found to demonstrate an antihypertensive activity.17,18 Moreover, peptides such as IDALNENK (peak #2), VLVLDTDYKK (peak #7), and VAGTWY (peak #9) are known for their antimicrobial properties.19,20

peptides (with negative charges, and hence they were also observed in the ARC−). The power of EDUF in separating peptides of close molecular weight is illustrated by a closer look at peak #8. One of the two peptides contained in peak #8 with a molecular mass of 903.8 Da is totally missing from MS of the ARC−, while it was detected by MS in the CRC+. On the other hand, the anionic peptide present in this peak (molecular weight 1635.9 Da) is totally missing from CRC+, while it was detected in the 5336


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Figure 7. Chromatograms of peptides obtained in the ARC− after 120 min of EDUF of (a) ex situ digested BiPro and (b) in situ digested BiPro.

quantifies a total peptide concentration including peptides at smaller than 300 Da and higher than 3000 Da, which are excluded from the HPLC-MS by the method we used. In general the peptide peaks obtained for the ex situ experiment (Figure 8a) appear to be of higher areas than the ones in the in situ experiment. This is similar to the observation made for ARC− in which chromatograms for the ex situ experiment had a slightly larger area than those in the in situ experiment. In the case of total peptide migration to the CRC+ estimated by the BCA method discussed previously, it was observed that the ex situ setup led to a higher migration than the in situ setup, which is in good relation with the general observation made from the chromatograms on Figure 8.

The migration rate of each peptide increased as a function of time as shown in Table 2. In the anionic recovery compartment, the migration rate of the peptide corresponding to peak #4 was found to be significantly lower in the in situ experiment than that in the ex situ one (P = 0.043). The maximum rate of migration was observed for this peptide for ex situ (17.46 ± 1.25%) and in situ (9.49 ± 3.19%) digestion. The migration rate of all other peptides was comparable except for peak #6, which was absent in the in situ one. On the other hand, the migration rates were relatively higher for all the cationic peptides in ex situ experiments as compared to that in situ. The maximum migration rate of 15.86 ± 1.05 and 8.74 ± 2.86% was observed for peak #6 (corresponding to the antihypertensive peptide ALPMHIR) in ex situ and in situ experiments, respectively. The migration rate of specific peptides were different from the peptide migration observed by the BCA method for the ARC− (reported in the section Peptide Concentration) where it was observed that there was a significantly higher (P < 0.005) total peptide migration in the in situ experiment compared to the ex situ experiment. This could be because the BCA method

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CONCLUSION A novel method of simultaneous protein hydrolysis and bioactive peptide separation in an electromembrane reactor (electrodialysis with ultrafiltration membrane) was investigated in the present study. Evolution of electrodialytic parameters such as solution electrical conductivity and system resistance 5337


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Figure 8. Chromatograms of peptides obtained in the CRC+ after 120 min of EDUF of (a) ex situ digested BiPro and (b) in situ digested BiPro.

was not affected by the type of electrodialytic reactor (in situ or ex situ). On the other hand, the peptide migration rate was found to be affected by the mode of enzymatic hydrolysis and separation. Peptide migration to the ARC− was higher in an in situ digestion than ex situ digestion, while peptide migration to the CRC+ was higher in an ex situ setup than an in situ setup. HPLC-MS studies identified twenty three major peaks that were generated on whey protein isolate digestion by trypsin. The migration of these peaks to the ARC− or C+RC, or both, illustrated the power of EDUF in isolating bioactive peptides from complex mixtures that contain peptides with close mass/ charge characteristics but with no apparent bioactivity. Among the peptides recovered in the anionic compartment, one peptide (IDALNENK) is known to have a hypocholesterolemic effect and one (VYVEELKPTPEGDLEILLQK) is antihypertensive. The EDUF technique also separated cationic peptides such as VLVLDTDYKK and VAGTWY, known as peptides having antimicrobial activities. In addition, in cationic recovery compartments, peptides with amino acid sequences ALPMHIR

and TKIPAVFK are well-known to possess antihypertensive and hypocholesterolemic effects, respectively. To our knowledge this is the first report that compares the ex situ and in situ enzymatic digestion of protein in an EDUF system. Though EDUF is attractive for preparative-scale separations, because it readily separates components of a sample in space and in time, research on the industrial scale is lacking. To this end, a coupling of EDUF with a pressure driven process can be investigated for increased efficiency.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 418 656 2131 ext. 7445. Fax: +1 418 656 3353. Email: [email protected]. Website: http://www. laurentbazinet.fsaa.ulaval.ca/publications/. ORCID

Shyam Suwal: 0000-0001-5513-328X Laurent Bazinet: 0000-0002-6818-3558 Notes

The authors declare no competing financial interest. 5338


Research Article


Table 2. Comparison of Migration Rate (%) of Peptides Recovered in ex Situ and in Situ Digestions in ARC− and CRC+ time (min) anionic recovery compartment (ARC−)

ex situ

in situ

cationic recovery compartment (CRC+)

ex situ

in situ

a

peak no.

Rt (min)a

mass [M + H]+

2 4 6 8 10 12 16 2 4 6 8 10 11 12 16 1 3 5 6 7 8 9 13 15 1 3 5 6 7 8 9 13

25.2 31.3 37.7 39.7 41.2 44.8 54.1 25.4 31.4 37.7 39.8 41.3 43.9 44.9 54.2 12.2 28.5 35.3 36.7 38.3 39.5 40.4 45.9 53 12.3 28.6 35.4 36.7 38.3 39.3 40.4 45.9

916.5 1245.6 1437.7 1635.9 1065.6 2720.2 2313.6 916.4 1245.6 1437.7 1635.9 1065.6 2906.3 2720.2 2313.6 573.3 949.5 933.5 837.5 1193.7 903.8 696.3 1200.7 2030.2 573.3 949.5 933.5 837.5 1193.7 903.8 696.3 1200.7

30 0.34 3.98 1.91 1.07 0.20 0.00 0.14 0.28 1.10 Nd 0.92 0.00 0.10 0.00 0.07 1.71 2.75 5.61 6.33 2.16 4.05 1.37 1.78 3.20 0.00 0.38 2.01 3.27 0.45 1.79 0.04 0.00

60

± ± ± ± ± ± ± ± ±

0.02 0.21 0.79 0.16 0.01 0.00 0.04 0.08 0.08

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.00 0.03 0.00 0.01 0.41 0.26 0.23 0.42 0.18 0.20 0.12 1.06 1.94 0.00 0.03 1.36 0.28 0.09 0.29 0.01 0.00

0.76 7.63 4.06 2.10 0.52 0.32 0.29 0.75 3.77 Nd 2.21 0.19 0.44 0.48 0.26 2.88 4.47 9.11 10.40 3.44 6.56 2.24 4.00 4.22 0.92 1.10 4.16 6.90 1.33 4.03 0.26 1.10

120a

90

± ± ± ± ± ± ± ± ±

0.03 0.25 0.54 0.24 0.06 0.21 0.08 0.12 0.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.32 0.08 0.18 0.12 0.01 0.25 0.47 0.39 0.67 0.21 0.14 0.19 0.40 0.83 0.07 0.22 2.91 0.81 0.40 0.66 0.04 0.63

1.24 12.41 5.46 3.45 0.85 0.61 0.46 1.33 7.74 Nd 4.01 0.57 0.97 1.31 0.81 3.86 6.08 12.08 11.77 4.76 8.69 3.07 7.56 6.83 1.34 2.04 5.86 9.78 2.22 4.60 0.68 3.11

± ± ± ± ± ± ± ± ±

0.05 0.61 2.83 0.03 0.11 0.23 0.08 0.19 0.20

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.29 0.20 0.21 0.24 0.21 0.64 0.72 4.74 0.46 0.38 0.32 2.65 1.93 0.12 0.41 4.10 1.23 0.74 0.71 0.13 1.48

2.15 ± 17.46 ± 14.18 ± 5.35 ± 1.31 ± 1.49 ± 0.91 ± 1.62 ± 9.49 ± Nd 3.96 ± 0.94 ± 1.37 ± 2.00 ± 1.00 ± 4.42 ± 7.03 ± 13.82 ± 15.86 ± 5.55 ± 9.81 ± 3.62 ± 10.63 ± 9.06 ± 1.32 ± 2.05 ± 4.62 ± 8.74 ± 1.99 ± 5.11 ± 0.75 ± 3.45 ±

0.09a 1.25a 3.41a 0.39a 0.17a 0.19a 0.16a 0.76a 3.19b 1.87a 0.55a 1.02a 1.42a 0.70a 0.20a 0.62a 0.71a 1.05a 0.33a 0.33a 0.22a 2.54 1.42a 0.30b 0.59b 1.43b 2.86b 0.32b 1.43b 0.25b 0.20b

Superscripts a and b indicate statistically significant difference (P < 0.005) in the migration rate of a peptide for the two digestion methods.

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ACKNOWLEDGMENTS

(1) Jackson, C.-J. C.; Paliyath, G. Functional Foods and Nutraceuticals. In Functional Foods, Nutraceuticals, and Degenerative Disease Prevention; Paliyath, G., Bakovic, M., Shetty, K., Eds.; WileyBlackwell: Oxford, 2011; pp 11−43. (2) Nagpal, R.; Behare, P.; Rana, R.; Kumar, A.; Kumar, M.; Arora, S.; Morotta, F.; Jain, S.; Yadav, H. Bioactive peptides derived from milk proteins and their health beneficial potentials: an update. Food Funct. 2011, 2 (1), 18−27. (3) Korhonen, H. Milk-derived bioactive peptides: From science to applications. J. Funct. Foods 2009, 1 (2), 177−187. (4) Kitts, D. D.; Weiler, K. Bioactive Proteins and Peptides from Food Sources. Applications of Bioprocesses used in Isolation and Recovery. Curr. Pharm. Des. 2003, 9 (16), 1309−1323. (5) Pouliot, Y.; Gauthier, S.; Groleau, P. E. Membrane-Based Fractionation and Purification Strategies for Bioactive Peptides. In Nutraceutical Proteins and Peptides in Health and Disease; CRC Press: Boca Raton, FL, 2005; pp 639−658. (6) Saxena, A.; Tripathi, B. P.; Kumar, M.; Shahi, V. K. Membranebased techniques for the separation and purification of proteins: An overview. Adv. Colloid Interface Sci. 2009, 145 (1−2), 1−22. (7) Fernández, A.; Zhu, Y.; FitzGerald, R. J.; Riera, F. A. Membrane fractionation of a β-lactoglobulin tryptic digest: effect of the membrane characteristics. J. Chem. Technol. Biotechnol. 2014, 89 (4), 508−515. (8) Zhao, Y.; Li, B.; Liu, Z.; Dong, S.; Zhao, X.; Zeng, M. Antihypertensive effect and purification of an ACE inhibitory peptide

We would like to thank Diane Gagnon for her technical assistance.

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REFERENCES

ABBREVIATIONS

EDUF: electrodialysis with ultrafiltration membrane CEM: cation-exchange membrane AEM: anion-exchange membrane IEM: ion-exchange membrane UFM: ultrafiltration membrane UF: ultrafiltration NF: nanofiltration PVDF: polyvinylidene fluoride MWCO: molecular weight cutoff MW: molecular weight BCA: bicinchoninic acid ARC−: anionic peptides recovery compartment CRC+: cationic peptides recovery compartment pI: isoelectric point WPI: whey protein isolate TFA: trifluoroacetic acid 5339


Research Article

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Comparative Study of in Situ and ex Situ Enzymatic Hydrolysis of Milk

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