Electro-membrane fractionation of antioxidant peptides from protein hydrolysates of rainbow trout (Oncorhynchus mykiss) byproducts

Accepted Manuscript Electro-membrane fractionation of antioxidant peptides from protein hydrolysates of rainbow trout (Oncorhynchus mykiss) byproducts

Shyam Suwal, Sunantha Ketnawa, Jen-Yi Huang, Andrea M. Liceaga PII: DOI: Reference:

S1466-8564(17)30494-0 doi: 10.1016/j.ifset.2017.08.016 INNFOO 1835

To appear in:

Innovative Food Science and Emerging Technologies

Received date: Revised date: Accepted date:

3 May 2017 24 July 2017 30 August 2017

Please cite this article as: Shyam Suwal, Sunantha Ketnawa, Jen-Yi Huang, Andrea M. Liceaga , Electro-membrane fractionation of antioxidant peptides from protein hydrolysates of rainbow trout (Oncorhynchus mykiss) byproducts, Innovative Food Science and Emerging Technologies (2017), doi: 10.1016/j.ifset.2017.08.016

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ACCEPTED MANUSCRIPT Electro-membrane fractionation of antioxidant peptides from protein hydrolysates of

submitted to

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rainbow trout (Oncorhynchus mykiss) byproducts

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Innovative Food Science and Emerging Technologies

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Shyam Suwal, Sunantha Ketnawa, Jen-Yi Huang* and Andrea M. Liceaga*

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Department of Food Science, Purdue University

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745 Agriculture Mall Drive, West Lafayette, Indiana 47907, USA

*Corresponding authors: Jen-Yi Huang e-mail: [email protected], tel.: +1 765-496-6034, FAX: +1 765-494-7953 Andrea M. Liceaga e-mail: [email protected], tel.: + 765-496-2460, FAX: +1 765-494-7953

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ACCEPTED MANUSCRIPT Abstract Fish protein hydrolysates are an important source of antioxidant peptides. Electrically driven membrane fractionation called electrodialysis with filtration membrane (EDFM) is a separation technology based on molecular charge and mass, which can fractionate active peptides from

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complex hydrolysates. This work aimed to evaluate the feasibility of sequential EDFM process

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for separation of cationic (CP) and anionic (AP) peptides from rainbow trout frame protein

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hydrolysate, and determine their antioxidant properties. The concentrations of CP and AP increased in the recovery solution, reaching 156 and 85 µg/mL, respectively, after 4-hour

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treatment, with migration rates of 19.55±2.19 and 10.94±0.39 g/m2 h. The CP separation was

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approximately 50% energy efficient than AP. Both CP and AP fractions were enriched with peptides with DPPH and ABTS radical scavenging properties. The results showed that two-step

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EDFM process is feasible for recovery and concentration of antioxidant peptides from rainbow

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trout protein hydrolysate.

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Keywords Electrodialysis with filtration membrane; Fish protein hydrolysate; Cationic peptides;

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Anionic peptides; Antioxidant properties

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ACCEPTED MANUSCRIPT 1. Introduction According to the U.S. Department of Agriculture Economics report, the total sale amount and economic value of farmed rainbow trout (Oncorhynchus mykiss) exceeded 1.8 million pounds and 96 million dollars in 2015, respectively. However, fish processing industries are known to

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generate more than 30% of total biomass as fish waste or byproducts such as frame/bone, skin,

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head, tail, fin, and viscera, depending on the type of fish and processing methods (Ghaly,

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Ramakrishnan, Brooks, Budge, & Dave, 2013). The majority of the byproducts ends up in landfill sites causing environmental problems, and some in low-economic value products such as

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compost, aquaculture and animal feeds. However, fish byproducts are rich in industrially

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valuable compounds such as protein (up to 73.6% in the case of rainbow trout fish frame), oils and minerals (Torres, Rodrigo-García, & Jaczynski, 2007). Therefore, from the sustainability

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point of view, it is important to valorize fish protein byproducts in the context of environmental

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impacts as well as economic benefits of fish processing industries. Among many investigations focusing on exploring potential ways to utilize protein rich

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byproducts, the enzymatic hydrolysis for the production of protein hydrolysates that contain

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bioactive peptides (BP) has been widely studied (Li-Chan, 2015). Indeed, fish protein hydrolysates (FPH) have been found to possess peptides with various functions as well as BP

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with antioxidant, antimicrobial, antidiabetic, and antihypertensive properties (Doyen, Beaulieu, Saucier, Pouliot, & Bazinet, 2011b; Li-Chan, 2015; Malaypally, Liceaga, Kim, Ferruzzi, Martin, & Goforth, 2015; Samaranayaka, Kitts, & Li-Chan, 2010). The presence of such active components makes FPH a potential source for the production of functional foods, nutraceuticals, and biopharmaceuticals. The peptide fractions differ in molecular mass and amino acid composition, which subsequently determine their biological activities. However, hydrolysis of

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ACCEPTED MANUSCRIPT native proteins present in fish byproducts forms a complex polypeptide mixture comprised of non-hydrolyzed protein fractions, lipids, and enzymes. The choice of an appropriate fractionation method for the recovery of BP with improved bioactivities has been considered as one of the most crucial steps. Conventional pressure-driven

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membrane filtration processes such as ultrafiltration (UF) and nanofiltration (NF) are commonly

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used for enrichment processes (Bazinet, Amiot, Poulin, Tremblay, & Labbé, 2005). However,

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development of innovative, alternative fractionation processes is essential due to the lower selectivity of pressure-driven membrane filtration to the peptides with similar molecular sizes,

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and membrane fouling resulting from the use of high pressure (Bazinet, et al., 2005). Although

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some NF have shown charge selectivity to peptides to certain extent (Pouliot, Wijers, Gauthier, & Nadeau, 1999), the majority of conventional filtration techniques are not able to fractionate

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peptides on the basis of their charges, which have a major role in the bioactivities. Moreover, the

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migration efficiency and selectivity of conventional pressure driven cross flow UF have been reported to be significantly improved by adding an electric field which works as an additional

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driving force, known as cross flow electro-membrane filtration (CFEMF) (Holder, Merath,

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Kulozik, & Hinrichs, 2015; Holder, Scholz, Kulozik, & Hinrichs, 2013; Leeb, Holder, Letzel, Cheison, Kulozik, & Hinrichs, 2014). The authors found a significant increase in peptide yield

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with a 3 to 6-fold increase in ACE inhibitory activity of peptide fractions recovered from dairy protein hydrolysates using CFEMF. More recently, an electrically driven membrane process, known as electrodialysis with filtration membrane (EDFM), has been developed and patented by Dr. Bazinet (Bazinet, et al., 2005). The EDFM technique is currently being explored for its applications in BP fractionation. EDFM technique is able to selectively and effectively separate and concentrate biomolecules

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ACCEPTED MANUSCRIPT from protein hydrolysate (PH) by combing electric field and molecular cut-off of filtration membrane (Langevin, Roblet, Moresoli, Ramassamy, & Bazinet, 2012). The separation of BP by EDFM is based on the peptides’ net charge using electric potential difference as a driving force for migration, and also their sizes using filtration membrane with sieving effect (size

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exclusion). Furthermore, EDFM is considered as a greener technology as it does not require use

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of solvents during fractionation (Bazinet & Firdaous, 2009), and no fouling has been observed on

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filtration membranes except on ion-exchange membranes (Langevin & Bazinet, 2011; Suwal, Doyen, & Bazinet, 2015a; Suwal, Roblet, Amiot, & Bazinet, 2015b).

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EDFM processes have successfully been used for the recovery of bioactive peptide fractions,

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for example, antihypertensive peptides from rapeseed protein hydrolysate (He, Girgih, Rozoy, Bazinet, Ju, & Aluko, 2016b), antidiabetic and antihypertensive peptides from flaxseed protein

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hydrolysate (Doyen, Udenigwe, Mitchell, Marette, Aluko, & Bazinet, 2014), anticancer and

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antibacterial peptides from snow crab byproducts protein hydrolysate (Doyen, et al., 2011b; Suwal, et al., 2014a), antioxidant peptides from soy protein hydrolysates (Langevin, et al., 2012),

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and hypocholesterolemic peptides from beta-lactoglobulin (Doyen, Husson, & Bazinet, 2013a).

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Recently, a significant enhancement in glucose uptake was observed in salmon frame protein hydrolysate after EDFM fractionation (Roblet, et al., 2016). Although, the productivity (i.e.

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peptide migration rate) of this technique is relatively low compared to conventional membrane and electro-membrane filtrations, and is yet not available commercially, this process is very selective, and the peptides separated have shown considerably improved bioactivity (Doyen, Beaulieu, Saucier, Pouliot, & Bazinet, 2011a; Roblet, et al., 2016). More research works are needed to optimize the processes parameters depending on the type of raw materials. Moreover, this technique has not yet been used to separate BP from rainbow trout fish frame protein

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ACCEPTED MANUSCRIPT hydrolysates. Accordingly, there is a need to develop an efficient and scalable method for BP separation and purification from FPH. Moreover, previous studies have shown that rainbow trout FPH contain antioxidant peptides (Ketnawa & Liceaga, 2017; Nguyen, Jones, Kim, San MartinGonzalez, & Liceaga, 2017). The presence of such active components makes the FPH a potential

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source for the production of functional foods, nutraceuticals, and biopharmaceuticals. However,

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the concentration and purity of BP in the initial FPH is not high enough to be used without

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further fractionation (Sila & Bougatef, 2016). Therefore, the fractionation of BP from FPH using EDFM technology could significantly improve its bioactivity and thus commercial value.

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The main goal of this study is to evaluate the feasibility of two-step sequential EDFM

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process for BP separation from rainbow trout FPH, and to explore a new application of this green technology. The present work aims to (1) sequentially fractionate cationic and anionic peptides

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and determine the efficiency; (2) compare the efficiency of two different configurations for

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peptides fractionation; (3) characterize the recovered peptide fractions; and (4) evaluate the

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antioxidant capacities of the peptide fractions recovered from trout frame protein hydrolysate.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Chemicals The food-grade enzyme Alcalase® 2.4 L, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St

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Louis, MO, USA). The reagents 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

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(TROLOX), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (Ferrozine), ferric

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chloride, ethylenediaminetetraacetic acid (EDTA) were purchased from Fisher Scientific (Waltham, MA, USA). Hydrochloric acid, sodium hydroxide, potassium chloride and sodium

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sulfate were purchased from VWR International Inc. (Radnor, PA, USA). All other chemicals

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2.2 Preparation of protein hydrolysates

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were of analytical grade from Fisher Scientific (Waltham, MA, USA).

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Rainbow trout (Oncorhynchus mykiss) frames were obtained as a gift from Bell Aquaculture™ (Redkey, IN, USA). Trout frame protein hydrolysate was prepared according to

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the method of Ketnawa & Liceaga (2017). In brief, the frames were homogenized with distilled

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water at the ratio of 1:4 w/v. The homogenous solution was hydrolyzed using Alcalase® at 3 % (w/w, of trout frame protein) at 55 ºC for 15 min. After hydrolysis the protein hydrolysate

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solution was heated at 95 ºC for 15 min to stop the enzymatic reaction. The solution was centrifuged at 15,000×g for 15 min. The resulting supernatant referred to fish frame protein hydrolysates (FPH) was stored at –20 ºC until further analyses.

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ACCEPTED MANUSCRIPT 2.3 Electro-membrane fractionation process The EDFM system used in this study was designed using an ED cell (ElectroCell, Denmark) consisting of two platinized titanium electrodes. The cell was stacked with one 20 kDa ultrafiltration membrane (UFM) provided by Synder Filtration (Vacaville, CA, USA), and ion-

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exchange membranes including anion exchange membrane (AEM) and cation exchange

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membrane (CEM) purchased from Ameridia (New Jersey, USA). The effective surface area of

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membranes was 10 cm2. The electric field across the electrodes of the system was supplied using a variable 0–60 VDC power source (B&K Precision, Model 1685B).

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Two EDFM cell designs were used, namely configurations 1 and 2 for cationic and anionic

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peptides fractionation, respectively. Each configuration had four compartments as illustrated in Fig. 1. The configuration 1 (Fig. 1, bottom) was used to separate cationic peptides (CP) from the

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feed solution (FPH). The FPH solution was fed into the compartment between AEM and UFM.

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The post-treated FPH (FPH-CP) was then fed into the compartment between UFM and CEM in the configuration 2 (Fig. 1, top) to fractionate anionic peptides (AP). The final FPH (FPH-CP-

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AP) was recovered at the end of the treatment. In both of the configurations, the filtration side of

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the UFM faced towards the compartment of feed solution. Similar configurations were previously used for the fractionation of AP and CP at pH 5 and 9, respectively, from whey

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protein hydrolysate by Poulain et al. (2006). However, the authors used fresh hydrolysate solutions as feed for each configuration, unlike our study where the post-treated hydrolysate (by configuration 1) was sequentially used as the feed for configuration2. The recovery compartments separated by UFM and CEM in the configuration 1 and AEM and UFM in the configuration 2, respectively, were fed with KCl solution (2 g/L). The compartments adjacent to the electrodes were fed with electrolyte solution (Na2SO4, 20 g/L). The initial pH of FPH and

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ACCEPTED MANUSCRIPT KCl solution were found to be 7.4 and 6.4, respectively. The pH of post-treated FPH after cationic peptide fractionation (i.e. FPH-CP) was readjusted to 7.4 before feeding it to the configuration 2. Each solution of 600 mL was circulated in a closed loop from an external reservoir at a flow rate of 500 mL/min using centrifugal pumps. The fractionations were carried

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out at a constant voltage of 20 V across the electrodes, equivalent to the electric field strength of

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11 V/cm, for 4 hours. After each treatment the system was thoroughly washed for at least 4 times

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with excess of distilled water. At the end, five different peptide fractions were obtained, namely initial FPH, CP, FPH-CP (after cationic peptide separation by configuration 1), AP, and FPH-

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CP-AP (after anionic peptide separation by configuration 2). Each fraction was freeze-dried and

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stored at –20 ºC until further analyses. The pH and electrical conductivity of the feed and KCl compartments were monitored to evaluate the changes in physicochemical properties of solutions

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and determine the (de)mineralization kinetics.

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Fig. 1. EDFM cell configurations for the fractionation of fish protein hydrolysate. Configurations 1 (bottom) and 2 (upper) for separation of cationic peptides (CP) and anionic peptides (AP), respectively. FPH-CP: post-treated FPH solution using configuration 1 for CP fractionation and used as feed for configuration 2, FPH-CP-AP: post-treated FPH-CP solution using configuration 2 for AP fractionation.

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ACCEPTED MANUSCRIPT 2.4 Analysis and calculations 2.4.1 Solution pH and electrical conductivity The pH and electrical conductivity of the feed and recovery solutions were measured by a SymphonyTM benchtop meter (Model B30PCI) equipped with a pH Probe and a conductivity

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probe (cell constant K=1 cm-1) purchased from VWR International Inc. (Radnor, PA, USA).

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2.4.2 Protein concentration

The concentrations of protein in the recovery and feed solutions were determined using micro

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bicinchoninic acid (BCA) protein assay reagents (Pierce Biotechnology Inc., Rockford, IL, USA)

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following the procedure recommended by the manufacturer and using bovine serum albumin as a

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standard. The peptide migration rate was calculated using equation 1: (1)

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where CF is the final peptide concentration in KCl solution, V is the final volume of KCl

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solution, A is the effective membrane surface area (m2), and t is the treatment time (h).

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2.4.3 Degree of hydrolysis

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The degree of enzymatic hydrolysis (DH) was quantified using the trinitrobenzenesulfonic acid (TNBS) method adapted from Adler-Nissen (Adler-Nissen, 1986) with further modification by Liceaga-Gesualdo and Li-Chan (1999). Absorbance was read at 420 nm using an UV-Visible spectrophotometer (Beckmann, Irvine, CA, USA). DH was calculated using equation 2: ( )

(2)

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ACCEPTED MANUSCRIPT Hydrolysis equivalents (h) were calculated by the increase in released amino groups. For fish protein, the total hydrolysis equivalents, htot, was assumed to be 8.64 meq/g (Adler-Nissen, 1986). 2.4.4 Demineralization rate

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The demineralization and mineralization rates, R (%), were calculated from the following

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equation 3:

(3)

2.4.5 Number of charges transported

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where Ki and Kf are initial and final electrical conductivities (mS/cm) of solution.

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The total number of charges transported during electro-membrane fractionation was

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determined using equation 4, as described by (Suwal, Amiot, Beaulieu, & Bazinet, 2016): (4)

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∫

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where q is the number of charges transported (C), I is the current intensity (A) and t is the time

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(s).

2.4.6 Relative energy consumption The total amount of energy consumed associated with the applied voltage during electromembrane fractionation was determined using equation 5, as described by (Koumfieg Noudou, Suwal, Amiot, Mikhaylin, Beaulieu, & Bazinet, 2016): ∫

(5)

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ACCEPTED MANUSCRIPT where E is the energy consumed (Wh), I is the current intensity (A), and U is the applied voltage (V). The relative energy consumption was then calculated using equation 5.

Where ER (Wh/g) is the relative energy consumed, CF is the final peptide concentration in KCl

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solution, and V is the final volume of KCl solution.

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2.4.7 Peptide characterization

The total amino acid composition of freeze dried samples was analyzed by UPLC Amino acid

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Analysis Solution using the AccQ•Tag Ultra Derivatization kit with UV detection (Water

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Corporations, Milford, MA, USA) by the Danforth Center’s Proteomics and Mass Spectrometry Facility (St Louis, Missouri, USA). Samples were pre-oxidized using performic acid and subject

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to acid hydrolysis with 0.5% phenol/6M HCl in a vapor‐ phase hydrolysis vessel under vacuum.

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Cysteine (Cys), Methionine (Met) and Tryptophan (Trp) are destroyed by the acid hydrolysis.

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The pre-oxidation step using performic acid prior to the standard acid hydrolysis yields stable forms of Cysteic acid (Cya) and methionine sulfoxide (MetS), which can therefore be measured.

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The quantity (moles) of amino acids in each peptide fraction was calculated using a series of standard solution run before the samples. The relative abundance of amino acids (mole %) was

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calculated by dividing the quantity (moles) of each individual amino acid by the sum of the concentration of all amino acids and multiplied by 100 and expressed as mole %. The molecular mass of peptide fractions was determined using Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on a Voyager-DE PRO (Applied Biosystems, Foster City, CA, USA). Sinapinic acid was used as matrix as it also allows the determination of higher molecular weight peptide (>10kDa). Measurements were performed in

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ACCEPTED MANUSCRIPT linear mode of operation in the positive-ion reflection mode at an accelerating voltage of 25 kV and a delay of 575 nanoseconds. The spectrum was collected in the mass range of 500–20000 Da with 100 laser shots per spectrum. The relative abundance (%) of each peptide was determined by dividing the area of corresponding peak by the sum of the areas of all peptides detected.

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Peptides were then classified into different ranges of molecular mass.

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2.4.8 Antioxidant activity measurement

2.4.8.1 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

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Scavenging activities of protein hydrolysate against DPPH radicals were determined according to the method described by Bougatef, Hajji, Balti, Lassoued, Triki-Ellouz, & Nasri

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(2009), with slight modifications by Nguyen et al. (Nguyen, et al., 2017) using a 96-well plate. A

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volume of 100 μL of each sample (1 mg/mL protein concentration) was mixed with 100 μL of

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ethanol (99.5%) and 25 μL of DPPH solution (0.02% DPPH in 99.5% ethanol). The mixture was incubated at room temperature in darkness for 30 min, and the reduction of DPPH radicals was at

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using

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UV-Visible

spectrophotometer

(Multiskan™

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measured

ThermoSciencific, Rockford, IL, USA). The control was conducted in the same manner, except

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that distilled water was used instead of sample. A lower absorbance of the reaction mixture

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indicated a higher DPPH radical-scavenging activity. Results were expressed as µmol TROLOX equivalent antioxidant capacity (µmol TE)/mg of protein in the sample based on a TROLOX standard curve.

2.4.8.2 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assay

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ACCEPTED MANUSCRIPT The ABTS radical scavenging capacity was determined according to Alemán et al. (2011) with modifications by Ketnawa and Liceaga (Ketnawa, et al., 2017). ABTS radical stock solution (7 mM ABTS in 2.45 mM potassium persulphate) was incubated in darkness at room temperature for 16 hours. Stock solution was diluted in Milli-Q water to obtain a working

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solution of ABTS-radical with an absorbance at 740 nm of 0.700±0.02. A 6 μL sample (1

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mg/mL) aliquot was mixed with 294 μL of ABTS reagent in a 96-well microplate and incubated

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in darkness at 30 ºC for 10 min. The control was conducted in the same manner, except that distilled water was used instead of sample. Absorbance at 734 nm was read by the

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spectrophotometer (Multiskan™ GO, ThermoSciencific, Rockford, IL, USA). Lower absorbance

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represents a higher ABTS scavenging activity. Results were expressed as µmol TROLOX equivalent antioxidant capacity (µmol TE)/mg of protein in the sample based on a TROLOX

2.4.8.3 Reducing power (RP)

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standard curve.

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The RP was measured according to Zhang et al. (2008) with modifications by Ketnawa and

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Liceaga (2017). A 250 µL of sample was dissolved in 0.2 M sodium phosphate buffer (pH 6.6) to have a 1 mg/mL final protein concentration and mixed with 250 µL of 1% (w/v) potassium

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ferricyanide solution. Same volume of phosphate buffer was used as blank. The assay mixture was heated to 50 ºC and incubated for 20 min followed by adding 1,000 µL of 10% (w/v) trichloroacetic (TCA). Thereafter, 100 µL of TCA mixture was mixed with 100 µL of double distilled water and 25 µL of ferric chloride (0.1% w/v), and l at 25 ºC for 10 min. Then, 250 µL of the supernatant was transferred to a 96-well microplate for absorbance determination at 740 nm. Increased absorbance of the reaction mixture indicated an increased RP. Results were

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ACCEPTED MANUSCRIPT expressed as µmol TROLOX equivalent antioxidant capacity (µmol TE)/mg of protein in the sample based on a TROLOX standard curve.

2.4.8.4 Metal chelating activity (MCA)

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MCA was measured according to Alemán et al. (2011) with modifications by Ketnawa and

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Liceaga (Ketnawa, et al., 2017). Briefly, 800 μL of sample (1 mg/mL) was mixed with 10 μL of

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2 mM FeCl2 and 20 μL of 5 mM Ferrozine. Mixture was kept at 25 ºC for 10 min prior to measuring its absorbance at 562 nm. Ethylenediaminetetraacetic acid (EDTA) was used as the

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standard metal ion chelating agent. Chelating ability was expressed as μmol EDTA

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equivalents/mg of protein (µmol EDTA/mg of protein) in the sample based on a standard curve

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of EDTA.

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2.5 Statistical analysis

Three batches of FPH were prepared and sampled for the EDFM experiments. The peptide

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concentration, electrical conductivity and pH of solution, relative energy consumed, and

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demineralization rate data of three replicates were subject to Student’s t-test. The antioxidant capacities were subject to one way analysis of variance (ANOVA) (SigmaPlot version 13, Systat

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Software, Inc., California, USA). Significant differences were declared at probability level P<0.05.

3. Results and discussion 3.1 Determination of limiting current density

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ACCEPTED MANUSCRIPT The current intensity observed as a function of applied electrical potential was plotted in Fig. 2, with 2 g/L KCl, 10 g/L of FPH and 20 g/L of Na2SO4 at solution flow rate of 500 mL/min. Both configurations exhibited the current-voltage plots with three distinct zones, namely under limiting, limiting and over limiting current zones. The limiting zone appeared after 6 V for

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configuration 1, whereas it appeared at about 12 V for configuration 2. The limiting current

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density (LCD) is associated with the phenomenon known as concentration polarization, the

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formation of thin diffusion (boundary layer) at the membrane-solution interface during ED process at higher applied voltages, which can change the local mass (ion) transport (Tanaka,

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2005). The LCD of the two electro-membrane fractionation (EDFM) configurations (1 and 2)

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was determined by the current-voltage curve as shown in Fig. 2 (Lee, Strathmann, & Moon, 2006). The LCD of configurations 1 and 2 were found to be 3.7 and 13.7 mA/cm2 at the applied

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voltages of 6 and 12 V, respectively. In addition, the over limiting currents were found after the

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voltage exceeded 8 and 17 V in configurations 1 and 2, respectively. The LCD plays a vital role in electro-membrane process as it determines the system resistance

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as well as current efficiency. The LCD depends on membrane and solution properties,

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operational parameters (e.g. flow rate), and cell design or configuration (Lee, et al., 2006). Conventionally, it is believed that an ED system working within the LCD zone can increase its

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electrical resistance and thus lower its current efficiency during operation. This is due to the depletion of ionic concentration at the membrane-solution interface, a phenomenon known as concentration polarization. On the other hand, recent studies have shown that electrodialysis efficiency (demineralization rate) (Nikonenko, et al., 2014) and peptide migration rate (Doyen, Roblet, Beaulieu, Saucier, Pouliot, & Bazinet, 2013b; Suwal, et al., 2016) can be significantly improved by working over LCD zone without increasing the relative amount of energy

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ACCEPTED MANUSCRIPT consumption. Nikonenko et al. (2014) showed that working at the over LCD zone, the mass transfer rate increased significantly during ED process due to either exalting effect of water dissociation products at ion-exchange membrane (IEM) and/or current induced electroconvection and gravitational convection. The increase in the system current intensity at over

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LCD region can thus still increase the peptide migration rate although there is no water

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dissociation at the UF membrane of the EDFM system. Therefore, in this study, the fractionation

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was carried out at 20 V, which was within the range of over LCD values for both of the

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configurations tested.

Fig. 2. Current intensity-voltage curves obtained for two configurations tested.. All data points are the mean of three replicates.

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ACCEPTED MANUSCRIPT 3.2 Electrodialysis parameters 3.2.1 Electrical conductivity and demineralization rate The parameters such as electrical conductivity, pH, and temperature of the feed and recovery solutions were measured over the course of fractionation (Fig. 3). Fig. 3a shows that the

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electrical conductivity of the feed (FPH) solution in configuration 1 remained unchanged during

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the separation process (P=0.746). The demineralization rate of FPH was determined to be only

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4% after 4-hour treatment. For configuration 2, the electrical conductivity decreased linearly from 2151 to 1700 µS/cm with the demineralization rate of 19%, which was approximately 4.8

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times higher than that of configuration 1 (Table 1). Similarly, the conductivity of the recovery

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(KCl) solution decreased steadily with a higher rate compared to feed in both configurations (Fig. 3b). The conductivity decreased significantly faster in configuration 2 (from 3210 to 990

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µS/cm) compared to that of configuration 1 (from 3272 to 1699 µS/cm) throughout the treatment

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(P=0.008). Likewise, the demineralization rate of the recovery solution was found to be significantly lower in configuration 1, of 45%, compared to that of configuration 2, of 69%

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(Table 1).

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Table 1. Comparison of system performance of cationic (configuration 1) and anionic (configuration 2) peptides separation Parameters

Configuration 1

Degree of hydrolysis, DH (%)

Demineralization rate (%)

29.09±0.47 FPH KCl

Number of charges transported (C) Peptide migration rate (g/m2 h) Relative energy consumption (W h/g of peptides) *

Configuration 2

3.99±0.53*a 45.34±7.31a 1506±109a 19.55±2.19a

19.47±4.23b 69.31±7.27b 1872±225a 10.94±0.39b

116.01±16.24a

219.48±34.64b

Mean of 3 replicates ± standard deviation Values in row with different letters are significantly different based on Student t-test (P<0.05).

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ACCEPTED MANUSCRIPT 3.2.2 pH The pH of the feed (FPH) solution decreased significantly from the initial value of 7.4 to 5.5 in configuration 1, while it decreased slightly to 7.1 for FPH-CP solution in configuration 2 (Fig. 3c). The decrease in pH is due to the dissociation of H2O molecules at the interface of AEM-feed

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solution ((Doyen, et al., 2013b). In the configuration 1, the proton (H+) ions generated from H2O

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dissociation migrate to feed solution which was adjacent to the AEM and eventually reduced its

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pH. The dissociation of H2O molecules was the result of concentration polarization. Since the LCD zone of configuration 1 is lower than that of configuration 2 (Fig. 2), H2O dissociation

US

started earlier and at faster rate in configuration 1 than in configuration 2. Therefore,

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significantly lower pH of FPH solution (configuration 1) compared to FPH-CP (configuration 2) was due to a higher rate of H2O splitting at AEM-feed interface.

M

In contrast to the feed, the pH of the recovery solution increased slightly during the first half

ED

hour and remained unchanged thereafter, indicating no or minimum level water dissociation at the CEM-solution interfaces. Indeed, it has been established that water dissociation rate is

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significantly higher at AEM interface than that of CEM during both EDFM and ED processes

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(Doyen, et al., 2013b; Zabolotskiy, But, Vasil'eva, Akberova, & Melnikov, 2017). No water dissociation occurs at the interfaces of UFM-solution. Therefore, in summary, configuration 2

AC

seems to favor electro-convective ion transfer, especially through CEM, resulting in a higher demineralization rate. However, configuration 1 favors water splitting at the AEM-surface, resulting in a decrease in feed (FPH) solution pH.

20

ACCEPTED MANUSCRIPT b. a.

3500 3500

3000 Conductivity (µS/cm)

2500 2000 1500 Configuration 1 Configuration 2

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2500 2000 1500 1000

Configuration 1 Configuration 2

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Current intensity (A)

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2500 Configuration 1 Configuration 2

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1500

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500

0

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Time (h)

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Fig. 3. Evolutions of electrical conductivity (a, b) and pH (c, d) in feed (left), i.e. FPH for configuration 1 and FPH-CP for configuration 2, and recovery (right) solutions, electrical current

21

ACCEPTED MANUSCRIPT of the system (e) and total number of charges transported (f) during fractionation of fish protein hydrolysate.

3.2.3 Temperature

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The fractionation process was initiated at room temperature. The temperature of the solutions

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increased rapidly within the first hour and was stabilized at about 33 ºC after 2 hours in all

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compartments (data not shown). The increase in temperature can be attributed to the Joule’s (Ohmic) heating effect due to the applied electric potential across the electrodes and the pump

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used to circulate the solution in the system. Firdaous, et al. (2009) also observed an increase in

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solution temperature during EDFM process of alfalfa white protein hydrolysate.

M

3.2.4 Electrical current and number of charges transported

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The electrical current in the system decreased steadily over the course of the fractionation experiments in both configurations tested (Fig. 3e). While the initial current intensities (t=0) of

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configurations 1 and 2 were comparable, their current intensities decreased at different rates

CE

(P=0.015) during the EDFM process. The current intensity decreased significantly (P=0.002) by more than 56%, from 0.16 to 0.07 A during the 4-hour cationic peptide separation (configuration

AC

1), while it decreased only by 25% (from 0.14 to 0.10 A) during the anionic peptide separation (configuration 2) (P=0.012). The average current intensity over the length of the study remained higher (maintaining lower system resistance) in configuration 2 compared to configuration 1. The decrease in current intensity of the system could be because of the formation of concentration polarization layer at the membrane-solution interface, resulting in water molecule dissociation as explained above. The formation of concentration polarization layer and water

22

ACCEPTED MANUSCRIPT dissociation has been found to increase the membrane electrical resistance, especially of AEM (Doyen, et al., 2013b; Suwal, et al., 2015b). The higher demineralization rates of both feed (FPH-CP) and recovery solutions in configuration 2 (Table 1) were in line with the values of current intensity. This is associated with

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a higher electro-convection in the over LCD region, which generates more vortices at the

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depleted (dilute) side of the membrane (i.e. at the interface of CEM-KCl solution). The vortices

membrane-solution

interface.

These

phenomena

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lead to a continuous mixing and supply ions (K+ in this case) to depleted layer (dilute side) of the increase

the

counter

ions

transfer

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(demineralization) rate during ED process and delay or diminish the formation of concentration

AN

polarization layer (Nikonenko, et al., 2014).

Contrary to demineralization rate, no significant difference (P=0.065) was observed in the

M

total number of charges transported (t=4 hours) during the EDFM process between the two

ED

configurations (Fig. 3f). The theoretical values of number of charges transported are proportional to the total ions transported, including minerals as well as peptides (Suwal, et al., 2014a).

PT

Despite of the significant variations in the demineralization rates, the similarity in the number of

CE

charges transported is due to the differences in peptide migration rates between the two

AC

configurations, which is further discussed in Section 3.3.

3.3 Protein migration rate and energy consumption The degree of hydrolysis (DH) of fish protein was found to be 29.09% (Table 1). The change in the peptides concentration of the feed and recovery solutions over the course of fractionation is shown in Fig. 4. The concentrations of both cationic (CP, in configuration 1) and anionic (AP, in configuration 2) peptides increased gradually in the recovery (KCl) solutions but at different

23

ACCEPTED MANUSCRIPT rates. The CP concentration was more than 1.8 times higher than AP (P=0.002). The final concentrations of CP and AP were found to be 156 and 85 µg/mL, respectively. Similarly, the migration rate of CP was found to be considerably higher, of 19.55±2.19 g/m2 h compared to that of AP, of 10.94±0.39 g/m2 h (Table 1) (P=0.002). As compared to the present study, Poulin et al.

T

(2006) found considerably lower peptide migration rates for both CP and AP using similar feed

IP

concentration, EDFM configurations, and effective membrane surface area. However, it is

CR

difficult to draw conclusions from the differences between the results because of several other parameters, such as applied voltage (20 vs 5 V), solution pH (7.4 vs 5 and 9), UF membrane

US

material (polyethersulfone vs cellulose acetate), and raw material (FPH vs whey protein). To our

AN

best knowledge, the peptide migration rate obtained here is the highest compared to other studies using EDFM process which have been reported so far. Previously, Koumfieg Noudou et al.

M

(2016) observed the migration rate of 16.2 and 7.8 g/m2 h for CP and AP, respectively during a

ED

simultaneous separation process of a feed solution with a much higher protein concentration (4%) at an applied electric field strength of 4.8 V/cm. In this study, we applied a significantly

PT

lower feed protein concentration (1%) but a higher electric field strength of 11 V/cm. In contrast

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to the demineralization rates, a greater peptide flux in configuration 1 could simply be due to a higher concentration of such cationic peptides (with net positive charge) in the feed (FPH)

AC

solution at pH 7.4.

The peptide concentration of the feeds did not change significantly during the EDFM treatment in configuration 1 (FPH, P = 0.603) and configuration 2 (FPH-CP, P=0.955), as shown in Fig. 5b. In addition, there was no difference observed in the peptide concentration of the feed solutions between the two configurations (P=0.330). This is due to the fact that only a small fraction, about 0.94−1.3%, of the total peptides present in the feed solution (FPH) were

24

ACCEPTED MANUSCRIPT transported to the recovery solution after 4 hours of treatment. This is why the peptide concentration in the feed during EDFM process are generally ignored. No study has shown any significant change in the peptide concentration in the feed solution after EDFM fractionation except one with a lower (0.5% w/v) initial feed concentration with the configuration with two

T

cell pairs and simultaneous AP and CP separation (He, Girgih, Rozoy, Bazinet, Ju, & Aluko,

IP

2016a). However, the peptide concentration in the feed of configuration 2 was slightly lower

CR

than that recovered after the treatment using configuration 1, i.e. FPH-CP, which was due to the dilution of FPH-CP solution by water present in the ED system (dead volume) after cleaning

US

process. The average peptide concentration of the feeds of configurations 1 and 2 was found to

AN

be about 10 mg/mL (1%).

The amount of energy consumed during the EDFM process is critical for its industrial

M

application for large-scale production. The relative amount of energy consumed during the

ED

EDFM process for CP separation was 116 W h/g of peptides, which was 1.9 times lower compared to that of AP (219 W h/g of peptides) (Table 1). The relative energy consumed are in

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the range of the values observed previously for cationic peptide separation from snow crab

CE

protein hydrolysate using similar cell configuration and UF membrane of same MWCO (20 kDa) (Suwal, et al., 2014b), while are considerably lower as compared to another study with similar

AC

configuration but with pre-demineralized hydrolysate (Suwal, et al., 2016). In an another study a significantly lower energy consumption was observed as compared to the present study using cell configuration for the simultaneous separation of peptides from snow crab protein hydrolysate and UF membrane of MWCO of 100 kDa (Koumfieg Noudou, et al., 2016). Therefore, the energy consumption is one of the major factor but it depends on various ED parameters and type of biomass used.

25

ACCEPTED MANUSCRIPT b.

a.

16 FPH FPH-CP-AP

14

0.15

0.10

0.05

12 10 8 6 4

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CP AP

Protein concentration (mg/mL)

2

0.00

0

0

1

2

3

4

0

1

Time (h)

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Protein concentration (mg/mL)

0.20

2

3

4

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Time (h)

Fig. 4. Evolution of protein concentration in recovery (a) and feed (b) solutions during

AN

US

fractionation of fish protein hydrolysate.

3.4 Amino acid analysis

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The relative abundance of amino acids was calculated for each peptide fraction and depicted

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in Fig. 5a. Gly (19.16%) was the most abundant amino acid followed by Glu (11.86%), Ala (9.55%), Asp (8.81%), Leu (6.43%), and Pro (6.34%) in the FPH. The presence of all other

PT

amino acids was below 5%. The abundance of positively charged amino acids such as Arg (9.92

CE

%) and Lys (9.76%) was higher in CP than other fractions. Similarly, negatively charged amino acid such as Glu (16.12%) was concentrated in the AP fraction. In addition, aromatic amino acid

AC

(Phe) and branched chain amino acid (Leu) were also concentrated by more than 1.4 times in the AP fraction. Moreover, the total relative abundance of hydrophobic (Gly, Ala, Val, Ile, Leu, Phe, MetS), and essential (His, Val, Ile, Leu, Arg, MetS, Phe, Thr) amino acids increased in CP (5 and 25%) and AP (15 and 26%) fractions (Table 2). Roblet et al. {, 2016 #1775} observed a significant increase in the relative abundance of Phe in AP, and Met and Cys in CP fractions after EDFM treatment of salmon frame protein hydrolysate. Moreover, the authors reported an increase in the relative abundance of amino acids such as Arg, Gly, Tyr, and His in both AP and 26

ACCEPTED MANUSCRIPT CP fractions. In the present study, Cys and Tyr were not detected or only at very low concentration. The abundance of Gly, Ala, and Pro was found to be higher in FPH-CP-AP fraction compared to initial FPH. This result is interesting because these amino acids, especially Gly, are important biomolecules as they are involved in many metabolic reactions, and act as

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inhibitory neurotransmitter in the spinal cord and brain stem cells, as well as anti-inflammatory,

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cytoprotective and immune modulating substances (Gundersen, Vaagenes, Breivik, Fonnum, &

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Opstad, 2005). In contrast to the previous work on Alaska Pollock frame protein hydrolysate where aromatic amino acids were absent (Hou, Li, Zhao, Zhang, & Li, 2011), a considerable

US

amount (3.5 %) of these amino acids were present in the rainbow trout frame protein

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hydrolysates.

AC

CE

PT

ED

M

Table 2. Summary of the composition (mole %) of total amino acids in initial protein hydrolysate (FPH), post-treated hydrolysate by configuration 1 (FPH-CP), post-treated hydrolysate by configuration 2 (FPH-CP-AP), and cationic (CP) and anionic (AP) peptide fractions Group of Peptide fractions (mole %) amino acids FPH FPH-CP FPH-CP-AP CP AP * HAA 56.93 56.36 62.09 60.24 65.80 PCAA 12.88 11.99 11.53 21.59 4.58 NCAA 20.81 23.10 24.74 12.59 24.21 BCAA 15.40 14.66 15.29 19.08 22.48 AAA 3.50 2.72 2.33 2.22 5.43 EAA 32.68 31.35 27.29 40.71 41.16 *Hydrophobic amino acids (HAA): alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, proline, methionine and cysteine; positively charged amino acids (PCAA): arginine, histidine, lysine; negatively charged amino acids (NCAA): aspartic, glutamic, threonine, serine; Branched chain amino acids (BCAA): leucine, isoleucine, valine aromatic amino acids (AAA): phenylalanine, tryptophan and tyrosine; essential amino acids (EAA): histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

27

CE

AC ED

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15

T

IP

10

CR

5

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0

AN

M

H is S er A rg G ly A s M p et S G lu Th r A la P ro Ly s Ty r V al I le Le u P he

Amino acid composition (mole %)

ACCEPTED MANUSCRIPT

a.

25

20

FPH FPH-CP FPH-CP-AP CP AP

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ACCEPTED MANUSCRIPT b.

50

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40

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FPH FPH-CP FPH-CP-AP CP AP

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30

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20 10

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Relative abundance (%)

60

0

-1 -1 .5 .5 -2 -2 .5 .5 -3 -3 .5 .5 -4 -4 .5 .5 -5 -1 0 5 1 1 2 2 3 3 4 4

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0 .5

ED

Molecular weight (kDa)

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Fig. 5. Amino acid composition and relative abundance expressed in mole fraction (mole %) (a) and distribution of peptide molecular weight (b) in different peptide fractions recovered by

CE

EDFM process. The peptides with molecular weight bigger than 10 kDa were not detected.

AC

The distribution of peptides sizes is shown by relative abundance (%) of peptides as a function of their molecular weight (Fig. 5b). The results showed that the untreated hydrolysate is mainly composed of peptides (63%) with molecular size ranging from 1 to 3 kDa. The peptides of 1.5 to 2 kDa were present in a highest quantity (25%) and the abundance decreased with increasing size. The peptides of more than 5 kDa were present in a significant quantity (6%). As expected, the cationic and anionic peptide fractions were found to consist of smaller peptides.

29

ACCEPTED MANUSCRIPT Both CP and AP fractions were mainly composed of peptides smaller than 1 kDa with the relative abundances of 52 and 33%, respectively. Interestingly, the peptide molecular weight profile of post-treated hydrolysates (i.e. FPH-CP and FPH-CP-AP) changed significantly compared to that of untreated sample. These results are an indicative of peptide-peptide

T

interactions during EDFM treatment which needs further investigations. The peptide-peptide

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interaction leading to bigger aggregates was found under electric field during isoelectric focusing

CR

of whey protein hydrolysate (Groleau, Morin, Gauthier, & Pouliot, 2003). In contrast, Roblet et al. (2016) observed only a slight difference in molecular weight profile of peptides after EDFM

US

treatment on salmon frame protein hydrolysate. It should be noted that Roblet et al. (2016) used

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the ultra-filtered (UF of 1 kDa) hydrolysate, therefore it contained mainly the smaller peptides of less than 800 Da, resulting in less interaction compared to the bigger peptides obtained in our

3.5 Antioxidant capacity

ED

M

study.

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The antioxidant capacity of untreated FPH and the peptide fractions recovered by EDFM

CE

process was determined by free radical scavenging activities (DPPH and ABTS), reducing power (RP), and metal ion chelating activity (MCA). Several antioxidant activities with different

AC

mechanisms were analyzed to confirm their dependency on peptide properties such as charge (i.e. cationic, anionic), amino acid composition and molecular weight (size). In this study, the DPPH and ABTS antioxidant activities of 14.78±0.80 and 140.47±3.77 µmol TE/mg of protein, respectively, were observed for the control (untreated FPH) (Fig. 6). After 4 hours of EDFM treatment, the DPPH and ABTS activities of both cationic and anionic peptide fractions increased compared to untreated FPH, as illustrated in Fig 7. DPPH activities of

30

ACCEPTED MANUSCRIPT both post-treated FPH fractions recovered from the EDFM treatments using configurations 1 and 2 (i.e. FPH-CP and FPH-CP-AP, respectively) increased to 36.01±0.12 and 46.99±0.64 µmol TE)/mg of protein. Furthermore, CP and AP fractions increased in DPPH activities to 34.81±1.23 and 28.26±0.54 µmol TE/mg of protein, respectively. Similarly, ABTS activities of

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CP and AP fractions increased to 282.35±4.05 and 345.55±3.14 µmol TE/mg of protein,

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respectively. The considerable increment (2–2.5 times) in the DPPH and ABTS activities of both

CR

CP and AP fractions showed that during the EDFM treatment they were enriched with the peptides that possess electron or hydrogen donating ability.

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Compared with the FPH, the increases in the DPPH and ABTS activities of the CP and AP

AN

fractions can be attributed to the increase in the concentration of hydrophobic amino acids, and decrease in the peptides size after EDFM treatment using 20 kDa UF membrane, as shown in

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Fig. 5b. The results indicated some non-covalent peptide-peptide (electrostatic) interactions

ED

under the applied electric field during EDFM process, forming smaller/larger peptides. The effect of applied electric field of 0–60 V during EDFM process on peptide-peptide interactions

PT

has not yet been reported and is beyond the scope of this study. Further work on changes in

CE

protein conformation during EDFM process is needed. The antioxidant activities are directly related to amino acid composition, molecular weight, and hydrophilicity of separated peptide

AC

fractions which explain the observed differences between DPPH and ABTS activities in the present study (Xiong, 2010). The ABTS∙+ radical is soluble in water and organic solvents, enabling the determination of antioxidant capacity of both hydrophilic and lipophilic compounds/samples. On the other hand, DPPH scavenging activity was found to be positively related to the hydrophobicity of the peptide fractions.

31

ACCEPTED MANUSCRIPT In contrast, we found no significant increase in the RP and MCA of the CP, AP and FPH-CPAP fractions compared to the control FPH at the same protein concentration of 1 mg/mL, as shown in Fig. 6 (P>0.05). From these results, both CP and AP fractions showed a reduction in metal chelation ability. The protein concentration (1 mg/mL) and degree of hydrolysis (29%) of

T

samples might not be high enough to reduce metal ions or form strong complex. Besides, ferric

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reducing ability was found to be partially related to hydrophobicity, as well as protein

CR

concentration (Ketnawa, et al., 2017). Therefore, the absence of MCA in separated peptide fractions could not only be due to the peptide hydrophilicity, but also the size and the low protein

US

concentration tested. Although negatively charged peptides and positively charged metal ions

AN

should theoretically exert electrostatic interactions, the concentration tested in this study may not be high enough. In our previous studies, the DPPH and ABTS activities of FPH were found to be

M

associated with degree of hydrolysis (DH), hydrophobicity, protein concentration and peptide

ED

solubility (Ketnawa, et al., 2017; Nguyen, et al., 2017). In addition, a positive correlation has been observed between antioxidant properties (DPPH, RP, and MCA) and the concentration of

PT

peptides derived from protein of rice bran (Zhang, Wang, Zhang, & Zhang, 2014). In fact, the

CE

antioxidant capacity of peptides depend on several factors including the concentration, amino

2015).

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acid composition, structure, charge as well as molecular size of peptides (Aluko, 2015; Li-Chan,

32

ACCEPTED MANUSCRIPT

FPH FPH-CP FPH-CP-AP CP AP

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c

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300

d

a

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200

100 c b

ac d

a

bd

bcd abc a

b

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b b

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Antioxidant Activity

400

0

RP

M

DPPH

ABTS

a

a

a b

c

MCA

ED

Fig. 6. Antioxidant capacities of initial (untreated) FPH, post-treated FPH (FPH-CP, FPH-CP-

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AP), and fractionated cationic (CP) and anionic (AP) peptides. DPPH, ABTS and RP are expressed as µM TROLOX/mg protein, and MIC is expressed as µmol EDTA/mg protein. Bars

AC

CE

with different letters are significantly different (P<0.05).

4. Conclusion

This is the first study carried out for the selective fractionation of cationic followed by anionic peptides with antioxidant activities from rainbow trout frame protein hydrolysates without any pretreatment using a two-step sequential EDFM process. This approach is particularly advantageous in order to explore the biological activity of different peptide fractions before performing a simultaneous fractionation of AP and CP. The EDFM cell 33

ACCEPTED MANUSCRIPT configuration 2 for AP separation allowed higher demineralization of protein hydrolysate solution, and performed better in terms of system resistance compared to the configuration 1 for CP separation. On the other hand, the CP migration rate was significantly higher than AP resulting in a significantly lower energy requirement for CP than AP. The sequential

T

fractionation strategy was proved useful as it showed the highest peptide migration rate

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observed so far using EDFM. Both the CP and AP fractions recovered from hydrolysate

CR

using EDFM showed a more than two-fold increase in antioxidant activity. Therefore, this study has demonstrated that EDFM is an effective technology for recovery of antioxidant

AN

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peptides from enzymatic hydrolysates of fish protein byproducts.

Acknowledgement

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The authors would like to thanks for financial support from Indiana Agriculture and Rural

ED

Communities under AgSEED Funds - Agricultural Research and Extension Leading to Economic Development and College of Agriculture, Purdue University. The authors would like

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PT

to thank Bell Aquaculture™ for providing the fish byproducts used in this study.

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Holder, A., Merath, C., Kulozik, U., & Hinrichs, J. (2015). Impact of diffusion, transmembrane pressure

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International Dairy Journal, 46, 31-38.

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Hou, H., Li, B., Zhao, X., Zhang, Z., & Li, P. (2011). Optimization of enzymatic hydrolysis of Alaska pollock frame for preparing protein hydrolysates with low-bitterness. LWT - Food Science and

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Technology, 44(2), 421-428.

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Antigenicity of Fish Frame Protein Hydrolysates. Food and Bioprocess Technology, 10(3), 582591.

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Koumfieg Noudou, V. Y., Suwal, S., Amiot, J., Mikhaylin, S., Beaulieu, L., & Bazinet, L. (2016). Simultaneous electroseparation of anionic and cationic peptides: Impact of feed peptide

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concentration on migration rate, selectivity and relative energy consumption. Separation and Purification Technology, 157, 53-59.

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Langevin, M.-E., & Bazinet, L. (2011). Ion-exchange membrane fouling by peptides: A phenomenon governed by electrostatic interactions. Journal of Membrane Science, 369(1-2), 359-366.

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ACCEPTED MANUSCRIPT Manuscript submitted to Innovative Food Science and Emerging Technologies

Electro-membrane fractionation of antioxidant peptides from protein hydrolysates of rainbow

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Shyam Suwal, Sunantha Ketnawa, Jen-Yi Huang* and Andrea M. Liceaga*

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trout (Oncorhynchus mykiss) byproducts

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Department of Food Science, Purdue University

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745 Agriculture Mall Drive, West Lafayette, Indiana 47907, USA

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*Corresponding authors: Jen-Yi Huang

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e-mail: [email protected], tel.: +1 765-496-6034, FAX: +1 765-494-7953

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Andrea M. Liceaga

   

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Highlights

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e-mail: [email protected], tel.: + 765-496-2460, FAX: +1 765-494-7953

A two-step electrically driven membrane fractionation (EMF) method was developed. Two configurations for cationic and anionic peptides fractionation were tested. Cationic peptides recovery process was more energy efficient than anionic peptides. DPPH and ABTS activities increased more than 2 folds in recovered peptides.

Industrial Relevance The electro-membrane fractionation developed in this study is a two-step process, which is able to selectively separate antioxidant peptides from enzymatic protein hydrolysates based on charge and size.

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ACCEPTED MANUSCRIPT With this particular raw material as the feed, we have shown that using this approach lead to the highest peptide migration rate and a significant improvement in antioxidant activities of both peptide fractions. In addition, this technique is very selective, and environmental friendly as it requires no use of solvent and consumes less energy compared to conventional chromatographic techniques, and thus can

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be used as a green technology for the fractionation of bioactive peptides from a complex mixture of

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protein hydrolysates.

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