Antioxidant activities and functional properties of protein and peptide fractions isolated from salted herring brine

Accepted Manuscript Antioxidant Activities and Functional Properties of Protein and Peptide Frac‐ tions Isolated from Salted Herring Brine Ali Taheri, K.H. Sabeena Farvin, Charlotte Jacobsen, Caroline P. Baron PII: DOI: Reference:

S0308-8146(13)00890-X http://dx.doi.org/10.1016/j.foodchem.2013.06.113 FOCH 14321

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

17 January 2013 3 June 2013 28 June 2013

Please cite this article as: Taheri, A., Sabeena Farvin, K.H., Jacobsen, C., Baron, C.P., Antioxidant Activities and Functional Properties of Protein and Peptide Fractions Isolated from Salted Herring Brine, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.06.113

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Antioxidant Activities and Functional Properties of Protein and Peptide Fractions

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Isolated from Salted Herring Brine.

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Ali Taheri1, K. H. Sabeena Farvin2, Charlotte Jacobsen2,Caroline P. Baron2*

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Department of Seafood Sciences, Faculty of Marine Sciences, Chabahar Maritime and Marine Sciences University,Chabahar, P.O. Box 99717-65499, Iran.

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National Food Institute, Technical University of Denmark. B. 221, Søltofts Plads, DK-2800

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Kgs, Lyngby, Denmark.

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___________________________________________________________________________

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Running Head: Antioxidant activity of protein fractions from salted herring brine

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*Corresponding author

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C.P. Baron,

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National Food Institute,

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Technical University of Denmark,

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B. 221, Søltofts Plads, DK-2800 Kgs, Lyngby,

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Denmark.

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Tel. + 45 45 25 49 19

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Fax: + 45 45 88 47 74

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Email: [email protected]

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ABSTRACT

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In the present study proteins isolated from herring brine, which is a by-product of marinated

24

herring production were evaluated for their functional properties and antioxidant activity.

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Herring brine was collected from the local herring industry and proteins were precipitated by

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adjusting the pH to 4.5 and the obtained supernatant was further fractionated by using

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ultrafiltration membranes with molecular weight cut offs of 50, 10 and 1 kDa. The obtained

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>50kDa, 50-10kDa, 10-1kDa fractions and pH precipitated fraction were studied for their

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functional properties and antioxidant activity. Functional properties revealed that >50 kDa

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polypeptides showed good emulsion activity index when compared to the other fractions.

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However all fractions had low emulsion stability index. The pH precipitated fraction showed

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the highest foaming capacity and stability at pH 10. The 50-10kDa and 10-1kDa peptide

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fractions showed good radical scavenging activity and reducing power at a concentration of

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0.5mg protein/ml. All the fractions demonstrated low iron chelating activity and did not

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inhibit oxidation in a soybean phosphatidylcholine liposome model system. However all the

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fractions were to some extent able to delay iron catalyzed lipid oxidation in 5% fish oil in

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water emulsions and the 10-50kDa fraction was the best. These results show the potential of

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proteins and peptide fractions recovered from waste water from the herring industry as source

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of natural antioxidants for use in food products.

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Keywords Herring brine, ultra filtration, peptides, antioxidant, functionality

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Introduction

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Lipid oxidation is of great concern to the food industry and consumers since it leads to the

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development of undesirable off-flavours and potentially toxic reaction products (Frankel,

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2005). One of the ways to limit this problem and increase the shelf life of food products is by

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using antioxidants. Indeed, synthetic antioxidants such as butylated hydroxyanisole (BHA),

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butylated hydroxytoluene (BHT), t-butyl-hydroquinone and propyl gallate are often used in

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the food industry. However, the use of synthetic antioxidants in food products is under strict

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regulation because of their potential health hazards (Branen, 1975; Linderschmidt et al.

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1986). Therefore, there is a growing interest to identify antioxidants from many natural

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sources. In recent years, peptides have shown real potent antioxidative activities and could

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further be investigated for potential use as food additives (Kudo et al, 2009; Zhuang et al,

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2009; Xue et al, 2009). Moreover, peptides have received considerable attention, due to their

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low molecular weight, easy absorption, and potential antioxidative, antihypertensive and

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immune modulatory effects under physiological conditions (Grimble et al, 1987; Monchi and

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Rerat, 1993; Byun et al. 2009). Antioxidant activity has been reported for protein

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hydrolysates from various fish protein sources such as tuna cooking juice (Hsu et al. 2009),

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herring press juice (Sannaveerappa et al. 2007), yellowfin sole frame (Jun et al. 2004), Alaska

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Pollack frame (Je et al. 2005 ), hoki frame (Je et al. 2005), and pacific hake muscle

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(Samaranayaka and Li-Chan, 2008).

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Herring (Clupea harengus) is one of the most important fish species in the North

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Atlantic and Baltic Sea (Zeller et al, 2011). Herring is known for its high content of long

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chain omega-3 fatty acids EPA and DHA and hence its health beneficial effects (Aro et al,

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2000). Pickling or marinating is a traditional way of processing herring in Scandinavian

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countries and is a part of the traditional heritage. This process involves two steps, the first

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step is salting of the herring in barrels and allowing to ripe for 6-12 months. During this

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ripening period the herring develop a very characteristic texture and taste. Several

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investigations have demonstrated that during ripening degradation and oxidation of protein

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take place and this is believed to be some of the main factors involved in ripening of salted

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herring (Andersen et al., 2007; Christensen et al. 2011). It has been shown that proteases

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from the intestinal and the muscle tissue participate in proteolytic degradation of the muscle

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proteins (Nielsen, 1995; Stefansson et al., 2000) which leads to an increase in soluble

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nitrogenous compounds, such as peptides and amino acids (Nielsen 1995; Nielsen &

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Børresen, 1997). The second step is removal of the brine and adding flavourings, typically

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vinegar, salt and sugar solution to which ingredients like peppercorn, bay leaves and raw

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onions are added. During this step a large quantity of brine is discarded as waste which is rich

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in amino acids, peptides and proteins fragments. Therefore, efficient recovery and utilization

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of this nutrient rich brine is an important concern for the herring industry in order to reduce

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discharge of potentially environmental unfriendly effluents and to maximize economic

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benefits.

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The aims of the present study were (a) to isolate different peptide fractions from herring

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brine by ultrafiltration (b) to evaluate the isolated fractions for antioxidant activities both in

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simple antioxidant assay including DPPH radical scavenging, reducing power and Fe2+

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chelating activity and in more complex systems containing lipids such as soybean

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phosphatidyl choline liposomes and emulsified fish oil and finally (c) to test the functional

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properties (emulsifying and foaming capacity) of the isolated fraction.

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Material and methods

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Samples and chemicals

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Herring brine was obtained from the herring producer Lykkeberg A/S (Horve,

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Denmark). After arrival to our laboratory the brine was divided into 800 mL buckets and 4

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stored at -30°C until use. L- phosphatidyl choline, 1,1-diphenyl-2-pycryl-hydrazyl (DPPH),

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thiobarbaturic acid (TBA), ascorbic acid, ethylene diamine tetra acetic acid (EDTA), and

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bovine serum albumin (BSA) were obtained from Sigma Aldrich (Steinheim, Germany).

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Chloroform and methanol were of HPLC grade (Lab-Scan, Dublin, Ireland). Refined non-

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deodorized fish oil without added antioxidants was donated by Maritex A/S (Sortland,

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Norway), subsidiary of TINE BA. All chemicals were of analytical grade.

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Isolation of peptide fractions from the brine

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The protein and peptides in the brine were recovered by adjusting the pH to 4.5

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followed by sequential ultrafiltration of the supernatant. The pH was chosen because pre-tests

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(2
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4.5 the brine was centrifuged (Sorvall RC 5B Plus, Dupont, Norwalk, CT, USA) at 30000 × g

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for 20 min at 4°C. The precipitate was collected, freezed dried and stored at -80°C until

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further analysis. The supernatant was placed on ice and filtered (Whatman number 4) to

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remove lipids and other insoluble matter. Subsequently, the supernatant was subjected to

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successive ultrafiltration (UF) steps using a stirred dead-end UF cell of 300 ml capacity

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(Millipore A/S, Glostrup, Denmark) with molecular cut off sizes of 50, 10 and 1kDa (Diaflo

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membranes, 76 mm diameter, Millipore Glostrup, Denmark) at 4oC with a nitrogen pressure

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of 0.4 Mpa. To remove the salt in the sample, the retentates were washed 3 times with 50 ml

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distilled water before the retentate fractions were collected (Figure 1). Thus three (retentate)

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UF-fractions were obtained >50 kDa, 50-10 kDa and 10-1 kDa, and subsequently freeze dried

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and stored at -80 °C until further analysis. The UF-fraction <1kDa was discarded as it

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contains mainly salts. The protein content (mg/ml) of each fraction was assayed using the

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BCATM kit (Thermo Sci., Pierce, Rockford, USA) and using bovine serum albumin (BSA) as

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standard. The salt content of the fractions was measured by potentiometric titration of

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chlorine ions using AgNO3 according to the AOAC standard method (AOAC 2000). Dry

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matter content was measured by freeze drying the samples for 24 hour and weighting the

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remaining materials.

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LC–MS analysis of free amino acids and total amino acids

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The precipitate and the obtained UF-fractions were analyzed for their free amino acids

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and total amino acids according to the method described by Farvin et al. (2010). For free

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amino acids, 500 mg of freeze-dried samples were added to 5 mL absolute ethanol to

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precipitate all the proteins. The mixture was homogenised using a hand operated

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homogeniser (Polytron, PT 1200CL, Kinematica AG, Switzerland) and was centrifuged

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(Sigma 4k 15, Osterode am Harz, Germany) at 2800 rpm for 10 min. The supernatant was

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collected and the precipitate was re-extracted 3 times with the same quantity of ethanol. The

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combined supernatants were evaporated to dryness under nitrogen. The residue was

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redisolved in 1 mL 0.05 N HCl and were filtered through a membrane filter with 0.2 µm

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pore size, and subsequently the amino acids were derivatized using the EZ: Fast kit from

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Phenomenex A/S (Allerød, Denmark). Sample volumes of 2 μL were injected into the HPLC

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mounted with the reversed phase column EZ: Fast AAA-MS (250 x 3.0 mm; Phenomenex

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A/S Allerød, Denmark) and eluted at 35°C with a flow rate of 0.5 mL/min. The mobile phase

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A consisted of water and B was methanol, both contained 10 mM ammonium formate. The

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gradient consisted of linear increase from 60 to 83% B in 20 min, then the column was re-

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equilibrated to 60% B until the end of the run (26 min). The eluate was transferred to the on-

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line mass spectrometer (Agilent 1100, Agilent Technology, Waldbronn, Germany) where

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amino acids were ionised using APCI with a chamber temperature of 450 °C, and mass

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spectra were obtained by positive ion mode scanning from 100 to 600 m/z. The amino acids

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were quantified based on peak areas of known concentrations of the amino acid standards. 6

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For total amino acids, a sample of 50 mg of the precipitate and UF-fractions were

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hydrolysed overnight in 2 mL 6 M HCl in sealed ampoules. The samples were appropriately

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diluted and filtered through a 0.2 µm membrane filter before derivatization of amino acids

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and analysis of amino acid content by LC-MS as described above.

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Emulsifying properties.

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Emulsifying properties of the precipitate and the different UF-fractions were compared

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with those of sodium caseinate (standard) according to the method by Klompong et al.,

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(2007). Thirty ml of 1% protein solution (300mg) was mixed with 10 mL of rapeseed oil and

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the pH was adjusted to 7. For the pH precipitated fraction the emulsifying property was

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determined at different pH (2, 4, 6, 8 and 10). The lipid/protein fraction mixtures were

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homogenized at a speed of 14000 rpm for 1 min using a homogenizer (Polytron, PT 1200CL,

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Kinematica AG, Switzerland). An aliquot of the emulsion (50 µl) was pipetted from the

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bottom of the container at 0 and 10 min after homogenization and mixed with 5 ml of 0.1%

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sodium dodecyl sulphate (SDS) solution. The absorbance of the diluted solution was

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measured at 500 nm using a spectrophotometer (Shimadzu spectrophotometer, UV mini

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1240, Japon). The absorbance measured immediately (A0) and 10 min (A10) after emulsion

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formation were used to calculate the emulsifying activity index (EAI) and the emulsion

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stability index (ESI) according to Pearce & Kinsella, 1978.

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EAI (m2/g) =

2x 2.303x A500 0.25 x protein weight (g)

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ESI (min) = A0 x Δt/ ΔA Where ΔA = A0-A10 and Δt =10min 7

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Foaming properties

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Foaming capacity and stability of the precipitate was compared with BSA (standard)

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according to the method of Tao and Sathe (2000). 250 mg of protein samples were mixed

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with 250 ml of distilled water and the pH was adjusted to 2, 4, 6, 8 or 10. The mixture was

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homogenized at a speed of 14000 rpm for 2 min using a homogenizer (Polytron, PT 1200CL,

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Kinematica AG, Switzerland). The whipped sample was immediately transferred into a 300

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ml cylinder and the total sample volume was read at 0 min and after 60 min. The foaming

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capacity and foam stability was calculated according to the following equation.

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Foaming capacity (%) = A0 - B x 100 B

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Foam stability (%) = A60 - B x 100 B Where A0 is the volume immediately after whipping (ml) and

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A60 is the volume after 60min of whipping (ml) and B is the volume before whipping (ml).

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Evaluation of antioxidant activity of the peptide fractions

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Scavenging of ,-diphenyl--picrylhydrazyl (DPPH) free radical

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DPPH free radical scavenging capacity of the precipitate and the different UF-fractions

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was measured according to a modified method of Shimada et al. (1992). 1.5 ml of DPPH

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solution (0.1 mM in 95% ethanol) was mixed with 1.5 ml of sample solution (0.1 and 0.5mg

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protein/ml final concentration). The mixture was left for 30 min at room temperature and the

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absorbance was measured at 517 nm using a spectrophotometer (Shimadzu UV mini 1240,

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Duisburg, Germany). As control, distilled water was used instead of the sample. BHT at a

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concentration of 0.02 mg/ml was also used as a positive control for comparison. Radical

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scavenging capacity was calculated as follows:

DPPH radical scavenging capacity %  = 1197 198

A517 sample ×100 A517 control

Chelation of metal ions (Fe2+)

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Iron (II) chelating activity was evaluated by the method of Dinis, Madeira, &

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Almeida (1994) with some modification. An aliquot of the precipitate and the Uf-fractions (at

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a final concentration of 0.1 and 0.5 mg protein/ml) was made up to 3.7 ml with deionised

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water. Subsequently, 2 mM ferrous chloride (0.1 ml) was added and after 3 min, the reaction

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was inhibited by the addition of 5 mM ferrozine (0.2 ml). The mixture was shaken vigorously

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and left at room temperature for 10 min. Absorbance of the resulting solution was measured

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at 562 nm using a spectrophotometer (Shimadzu UV mini 1240, Duisburg, Germany). A

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control was made with distilled water instead of sample. EDTA (0.1 mg/ml) was used for

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comparision and run in a similar manner. The chelating capacity was calculated as follows:

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Fe2+Chelating activity %  =

Blank – Sample ×100 Blank

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Reducing power

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The reducing power was measured according to the method of Oyaizu (1986) with

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some modification. 1 mL of UF-fractions (at a final concentration of 0.1 and 0.5 mg

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protein/ml) was mixed with 1 ml of 0.2M phosphate buffer (pH 6.6) and 1 ml of potassium

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ferricyanide. The mixture was incubated at 50°C for 20 min and 1 ml 10% TCA was added to

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this mixture. An aliquot of 2 ml from this incubation mixture was mixed with 2 ml of distilled

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water and 0.4 ml of 0.1% ferric chloride. After 10 min the absorbance of the resulting

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solution was measured at 700 nm in a spectrophotometer (Shimadzu UV mini 1240,

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Duisburg, Germany). Ascorbic acid (0.02 mg/ml) was used as positive control and used for 9

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comparison. Increased absorbance (A700 nm) of the reaction mixture indicates increased

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reducing power.

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Inhibition of lipid peroxidation in a liposome model system

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Liposomes were prepared from soybean phosphatidyl choline according to the method

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described by Farvin et al, 2010. Lipid oxidation was performed in a model system containing

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0.1 mg of phosphatidyl choline liposomes per ml of phosphate buffered Saline (PBS) (3.4

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mM Na2HPO4 - NaH2PO4, 0.15 M NaCl, pH 7.0) and the UF-fractions were tested at a final

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concentrations of 0.1 and 0.5 mg protein/mL. Lipid oxidation was initiated using

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iron/ascorbate redox cycling using 50 M FeCl3 and 100 M ascorbate. The reactants were

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mixed by vortexing for 2 seconds and incubated at 37 oC in a water bath for 1 h. The

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liposome assay solution incubated with distilled water instead of the sample was used as

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control. Lipid oxidation was measured by determining the concentrations of thiobarbituric

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acid reactive substance (TBARS) formed according to the method of Buege and Aust (1978).

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The amount of TBA-reactive substances expressed as MDA released per mg phospholipid

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(PL) was calculated using the molar extinction coefficient of MDA as 1.56 x 105.

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Antioxidant activity of peptide fractions in 5% fish oil-in-water emulsion

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5 % oil in water emulsion was prepared with 1% citrem as emulsifier. In brief: 5 g of

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citrem and 25 g fish oil were weighed into a glass beaker and mixed together by magnetic

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stirrer. 470 ml of buffer (Imidazole-Acetate, 10mM, pH 7) was measured into a one litre

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beaker and the precipitate and UF- fractions at a concentration of 1g/ml were dissolved into

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the buffer. A pre-homogenisation was done for 3 min using an ultra turrax (T1500, Ystral,

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Dottingen, Germany) by adding the oil/citrem mix slowly over 1 min and further mixing for 2

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min. After the pre-homogenisation step the emulsion was prepared by a high pressure 10

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homogenizer (Total pressure of 800 bar, Panda 2K Homogeniser from Niro Soavi S.p.A,

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Parma, Italy). A control without antioxidant and an emulsion with 0.2g/ml of BHT were also

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made for comparison. Subsequently, 400 ml of the emulsions was poured into 500 ml sterile

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blue capped bottles in duplicate and FeSO4 solution (100 μM) was added in order to induce

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oxidation. Bottles were kept on a magnetic stirring plate at 20 °C for 48h in the dark. The

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sampling was done from the same bottle after 0, 12, 24 and 48 hours. The samples for

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chemical analysis were transferred to separate brown glass bottles, flushed with nitrogen and

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stored at -80 oC until analyses. Peroxide value (PV), Anisidine value (AV) and loss of

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tocopherols was used to assess antioxidant activity.

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Analysis of Peroxide Value (PV) and Anisidine Value (AV)

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Lipids from the emulsions were extracted by chloroform: methanol (1:1 v/v) as

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described by Bligh, & Dyer (1959). PV was measured directly on the Bligh and Dyer extract

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according to the method described by the international IDF standards (1991). Anisidine value

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was measured directly of the on the Bligh and Dyer extract according to the method of AOCS

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(1994).

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Determination of tocopherol content

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Tocopherol content in the fish oil emulsion was determined using an Agilent 1100

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series HPLC (Agilent Technologies, Palo Alto, CA, USA), equipped with a fluorescence

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detector. About 2 g of the chloroform extract from the Bligh and Dyer were evaporated under

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nitrogen and redissolved in 2 ml of n-heptane and an aliquot (40 µL) was injected onto a

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Spherisorb s5w column (250 mm 9 4.6 mm) (Phase Separation Ltd, Deeside, UK). Elution

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was performed with an isocratic mixture of n-heptane/2-propanol(100: 0.4; v/v) at a flow of 1 11

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ml/min. Detection was done using a fluorescence detector with excitation at 290 nm and

268

emission at 330 nm and according to AOCS (1994). Results were expressed in μg tocopherol

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per g of lipid.

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Statistical Analysis

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All results are given as mean values of triplicates with indication of standard

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deviation unless otherwise stated. The results were analysed using two-way analysis of

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variance and followed by tukey and Bonferroni post test using Graphpad prism 5 (Graphpad

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Softwarer Inc., San Diego, USA) and with a level of significance of at least p<0,05.

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Results and Discussions

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Composition of fractions

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The protein, salt content and dry matter of the herring brine was 49.2± 2.2 mg/ml and

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20.7±0.6% and 23.78± 0.2% respectively. The protein, salt and dry matter content of the

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different fractionations is given in Table 1. The precipitate collected after pH adjustment and

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fractions >50kDa, 50-10 kDa, 10-1 kDa showed lower salt content when compared to the

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herring brine. The salt content of the <1kDa fraction was 23.5%, which also illustrates that

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the washing procedure of the retentate was successful in reducing the salt content of the

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fractions. The <1 kDa fraction which contained mostly salt was discarded.

286 287

Emulsifying and foaming properties

288 289

Emulsion activity index (EAI) measures the area of oil–water interface stabilized by a unit

290

weight of protein (Wu et al, 1998). Higher indices represent smaller number of dispersed fat

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droplets. It also represents the ability of polypeptides for being adsorbed at the oil water 12

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interface (Pacheco Aguilar et al 2008). The EAI values for the different fractions at pH 7 are

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given in Figure 2a. EAI of >50 kDa fraction was markedly higher than the other fractions;

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followed by the pH precipitated fraction. The EIA of 50-10kDa and 10-1kDa fractions

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showed significantly lower EAI compared to the other fractions. However none of the

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fractions were as effective as sodium caseinate. The 50-10kDa and 10-1kDa fractions mainly

297

consist of short peptides and free amino acids. Thus, there was a clear indirect correlation

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between EAI and the free amino acid content of these fractions. A direct relationship between

299

surface activity and peptide length has previously been reported (Souissi et al 2007). The

300

smaller peptides often have reduced emulsifying properties.

301 302

Emulsion stability index (ESI) measures an emulsion’s ability to resist breakdown (Wu et al,

303

1998). Higher ESI values indicate more stable emulsions. ESI of the different fractions at pH

304

7 is shown in Figure 2b. The ESI value for the different fractions ranged between 1 and 2

305

min, which was significantly (p<0.05) lower when compared to sodium caseinate, which had

306

an ESI of 41.8 min. This may be due to the low molecular weight peptides present in the

307

herring brine. It has been reported that a higher content of larger molecular weight peptides or

308

more hydrophobic peptides contribute to the stability of the emulsion (Mutilangi et al 1996).

309

Small peptides and amino acids are less efficient in reducing the interfacial tension due to

310

lack of unfolding and reorientation at the interface as the large peptides do (Gbogouri et al

311

2004). Several studies have reported that emulsion stability is reduced with decreasing size of

312

peptides (Gbogouri et al 2004, Klompong et al 2007, Kristinsson and Rasco 2000).

313

Emulsion activity index and emulsion stability index of the pH precipitated fraction at

314

different pH values were also measured because this fraction was available in sufficient

315

quantity, whereas this was not the case for the other UF fractions (Fig 2 a & b ). There was no

316

significant difference in the EAI of the pH precipitated fraction at different pH values.

13

317

However, it was significantly lower than that of sodium caseinate (p<0.001) at all pH values

318

except at pH 4 where there was no significant (p< 0.05) difference between the two.

319

Maximum EAI was at pH 2 (100.8 ± 2.16 m2/g protein) and minimum was at pH 6 (85.98±

320

0.9 m2/g protein). ESI of this fraction at different pH showed a maximum stability at pH 2.

321

Then there was a drastic reduction in ESI as the pH increased. Maximum stability was 60.74±

322

21.1 min at pH 2 and minimum was 0.46± 0.13 min at pH 6. In the case of sodium caseinate

323

the maximum emulsion stability was observed at pH 2, 8 and 10 and the ESI was lowest at

324

pH 4. A similar trend was observed in the foaming properties for this fraction at different pH

325

values. The pH precipitated fraction showed maximum foaming capacity (92.5 ± 3.5%) at pH

326

10 and minimum (40.0 ± 7.0%) at pH 6. The foam stability showed also a maximum at pH

327

10 (32.5 ± 3.5%) and a minimum at pH 6 (17.5 ± 3.5%). BSA showed the lowest foaming

328

capacity and foam stability at pH 4. Sodium caseinate and BSA having isolelectric point at

329

pH 4.6-4.7 showed a decrease in emulsifying and foaming properties when pH was near to

330

their isolelectric pH. When pH moves away from the isoelectric point, the net charge of the

331

protein molecules increase, which weakens the hydrophobic interactions and increases

332

protein flexibity. This enhances their emulsifying and foaming properties (Lawal et al 2007).

333

The functional properties such as emulsification and foaming are affected by the solubility of

334

proteins (Wilding and Lilliford 1984). At or near to the isoelectric point the solubility of

335

proteins is decreased, and this may be another reason for the lower value for functional

336

properties near the isoelectric point (pH 6).

337 338

In vitro antioxidant activity of the isolated fractions

339 340

The free radical scavenging capacity of the precipitate and the UF- fractions were tested by

341

their ability to scavenge the stable DPPH radical. In radical form DPPH gives strong

14

342

absorption band at 517 nm and as the electron becomes paired off in the presence of a free

343

radical scavenger, the absorption vanishes (Desai, Wadekar, Kedar & Patil, 2008). The DPPH

344

radical scavenging activity showed a concentration dependency and increased with increasing

345

protein concentration (Table 4). At 0.1mg /ml concentration there was no significant

346

difference between the fractions except for 10-1kDa fraction which showed significantly (p<

347

0.05) higher radical scavenging activity when compared to other fractions. Also at 0.5mg /ml

348

the low molecular weight fraction 10-1k Da showed highest DPPH radical scavenging

349

activity followed by 50-10kDa fraction. There was no significant difference between radical

350

scavenging activity of >50kDa fraction and the pH precipitated fraction. However, none of

351

the fractions were as effective as BHT, which showed 79.8 % activity at a concentration of

352

0.2mg/ml. The result of our study is in accordance with some of the earlier studies. Indeed,

353

Peng et al, (2009) reported that the DPPH radical scavenging activity of 0.1-2.8 kDa fraction

354

of whey protein hydrolysate was higher than >40KDa, 2.8–40KDa and <0.1kDa fractions. A

355

study on the antioxidant activity of different fractions of yoghurt peptides by Farvin et al

356

(2010) also showed highest DPPH radical activity in the 3-10kDa and <3kDa fractions. Yang

357

et al, 2008 showed that the 3kDa fraction from protein hydrolysates of cobia skin showed the

358

highest DPPH radical scavenging activity. The results of the present study reveal that the

359

herring brine possibly contained peptides/amino acids, which acts as electron donors or could

360

react with free radicals to convert them to more stable products and terminate the free radical

361

chain reaction.

362 363

It has been recognized that transition metal ions, such as Fe2+ and Cu2+ are involved in

364

many oxidation reactions in vivo by catalyzing the generation of reactive oxygen species such

365

as hydroxyl radical and superoxide anion (Stohs, & Bagachi, 1995). Hydroxyl radicals react

366

rapidly with the adjacent biomolecules and induce severe damage. Therefore, the chelation of

15

367

metal ions also indirectly contributes to some antioxidant activity. The Fe2+ chelating activity

368

of the different fractions was negligible when compared to the chelating activity of EDTA at

369

0.2mg/ml concentration (Table 4). In addition, increasing protein concentration from 0.1

370

mg/ml to 0.5 mg/ml did not affect the metal chelating activities of the fractions. The lower

371

molecular fraction 10-1kDa seemed to be the best metal chelator, however it was still

372

inefficient compared to EDTA. Some studies have reported good metal chelating activities of

373

low molecular weight protein fractions, and increase metal chelating activity with increasing

374

degree of hydrolysis, which was not the case in our study (Klompong et al. 2007; Farvin et al.

375

2010).

376

The reducing power assay is often used to evaluate the ability of natural antioxidants to

377

donate electrons or hydrogen (Dorman et al. 2003). We used the ferric reducing antioxidant

378

assay, which is based on the ability of an antioxidant to reduce Fe3+ to Fe2+ in a redox linked

379

colorimetric reaction, which involves one electron transfer. Different studies have reported

380

that there is a direct correlation between antioxidative activities and reducing power of

381

certain protein hydrolysates and peptides (Duh et al. 1999; Bougatef et al. 2009). The

382

reducing power of the different fractions at 0.1mg/ml and 0.5mg/ml is shown in Table 4. In

383

general, the reducing power was found to be very low for all the fractions at the

384

concentrations tested and it showed a concentration dependency. There was no significant

385

difference between the different fractions even though the lower molecular weight fractions

386

showed higher reducing power when the concentration was increased to 0.5mg/ml. None of

387

the fractions were as efficient as ascorbic acid.

388 389

The ability of the different fractions of herring brine to inhibit lipid oxidation was tested in

390

a liposome model system and compared with that of BHT. This method was used because it

391

mimics the biological membrane and it has been used extensively for in vitro lipid

16

392

peroxidation studies (Duh et al., 1999; Westerlund et al., 1996). Similar to metal chelating

393

activity and reducing power all the fractions showed poor inhibition of lipid oxidation in the

394

liposome model system (Table 4). In this system the >50kDa fraction was better than the

395

other fractions and there was a reduction in TBARS formation as the concentration increases

396

in all fractions except for the pH precipitated fraction. All the fractions were significantly

397

(p<0.05) less effective than BHT. This was not totally unexpected because in biphasic

398

systems like liposome the solubility of the antioxidant and the location of the antioxidant is

399

crucial for it to have an effective antioxidant activity.

400 401

Free and total amino acid content

402 403

The contents of free amino acids (expressed as g/100 g dry weight) in the pH

404

precipitated fraction and the different UF-fractions are shown in the Table 2. It was found

405

that 50- 10kDa fraction had the highest content of free amino acids followed by 10-1 kDa and

406

the pH precipitated fraction. The predominant free amino acids in the 50-10 kDa fraction

407

were Arg, Lys, Ala, which constituted about 12.4, 11.5, 11.4, % of the total free amino acids

408

respectively and was followed by Glu, Leu , Asp, Ser and His. The major free amino acids in

409

10-1 kDa and pH precipitated fractions were Lys and Arg followed by Leu, Glu, Ser and His.

410

The >50kDa fraction contained the least free amino acids. The major free amino acids in this

411

fraction were Lys, Glu, Ala and Leu which were followed by Arg and Gly.

412

The total amino acid compositions of the different fraction were expressed as % of total

413

amino acids and are shown in the Table 3. All the fractions contained high levels of Lys, His,

414

Asp, Glu and Ala. The 10-1kDa, 50-10kDa fractions showed high levels of Arg, Ser and Pro

415

while the >50kDa and the pH precipitated fractions showed high content of Leu and Val. The

416

fractions showed typical amino acid profile of muscle proteins. 17

417

Several amino acids have been reported to show antioxidant activity (Karel, et al. 1966;

418

Marcuse, 1960 & 1962). His, Thr, Lys, and Met were reported to have antioxidant activity in

419

sunflower oil emulsions (Riison et al. 1980). Good antioxidant activity was reported for His

420

and Trp in both linoleic acid and methyl linoleate emulsions (Marcuse, 1962). His exhibits

421

strong radical-scavenging activity due to the presence of the imidazole ring (Yong and Karel,

422

1978). The higher radical scavenging activity of the 10-1kDa and 50-10kDa fractions may be

423

due the presence of higher amounts of His both as free form and in peptide form. However,

424

His also has a strong tendency to invert to a pro-oxidative effect at higher concentrations

425

(Marcuse, 1962). Moreover the antioxidant role of His in different lipid oxidation system is

426

not consistent. Erickson et al. (1990) found that His stimulated the oxidation of flounder

427

sarcoplasmic reticulum while Karel et al. (1966) reported that His inhibited lipid oxidation in

428

a freeze dried model system. The ability of His to accelerate Fe-dependent peroxidation has

429

also been reported and proposed to be due to the chelation of the iron at the imidazole ring,

430

which was found to enhance the prooxidative activity of iron (Winkler et al. 1984; Din et al.

431

1988). This is in agreement with the poor performance of these fractions in liposomes were

432

the oxidation is induced by iron/ascorbate.

433 434

Antioxidant activity of the isolated fractions in 5% oil–in-water emulsions

435

In order to further assess the antioxidant potential in food emulsions, all fractions

436

were tested at a protein concentration of 1g/ml in 5% fish oil in water emulsion. The results

437

of PV, AV and tocopherol loss are shown in Figure 4. Peroxide value increased in all samples

438

as the storage progressed. The emulsions containing the different fractions were able to delay

439

the PV development for the first 12h of storage and thereafter PV increased significantly in

440

all fractions. At the end of the storage period, i.e. 48h there was no significant difference

18

441

between the control and the tested fractions while BHT showed a significantly lower PV

442

throughout the entire storage period (Figure 3a). In contrast to PV, the anisidine values and

443

the tocopherol loss revealed that all fractions except the pH precipitated fractions were

444

efficient antioxidants and were comparable to BHT (Figure 3b & 3c). The 10-50kDa fraction

445

resulted in the lowest anisidine value and the highest tocopherol content and was not

446

significantly (p<0.05) different from BHT during the entire period of storage. Based on the

447

assay performed we can speculate that the antioxidant activity of the 10-50kDa fraction is due

448

to its radical scavenging activity. Chen et al. (1996) proposed that the antioxidant activity of

449

peptides depended on amino acid compositions and their sequences. Under certain

450

experimental conditions, some amino acids such as Gly, Met or Trp have been reported to

451

accelerate oxidation even though they have been reported to act as antioxidants in other

452

studies (Marcuse, 1962; Matsushita, & Ibuki, 1965). Moreover, as mentioned above the

453

herring brine fractions had poor iron chelating ability which might be due to the fact that the

454

fractions might contain already some iron chelated to peptides and therefore are unable to

455

chelate more metals. Poor performance in iron induced oxidation towards liposome model

456

system when compared to 5% oil-in-water emulsions maybe explained by the difference in

457

the bi-phasic system used and the location of the antioxidant at the interface oil/water which

458

has been showed to be essential. However, the fractions were tested at much higher

459

concentration in the oil–in-water emulsion (1g/ml) compared to the liposome model system

460

(0.5g/ml) and this could explain the difference between the liposome assays and the oil-in-

461

water emulsions assay. More investigations are needed to further characterize the peptides

462

present in herring brine and their potential antioxidant activity.

463

Conclusions

464

In conclusion this investigation revealed the presence of some antioxidant peptides in

465

the salted herring brine. The isolation procedure for the peptides and proteins in this study 19

466

showed that the UF fractionation works well for herring brine and it can effectively eliminate

467

high content of salt content of the fractions. The functional properties of the isolated fractions

468

were lower than that of the sodium caseinate and BSA. The lower molecular weight fractions

469

showed good radical scavenging properties. However all the fractions were low in reducing

470

power and iron chelating activity which might explain their poor performance in iron induced

471

oxidation system such as liposomes. This suggests that components of the herring brine

472

fractions primarily act as free radicals scavengers with the fraction between 50 and 10 kDa

473

being the most potent. Even though PV data showed some protection up to 12 h, AV and

474

tocopherol loss showed that all the fractions were effective in preventing oxidation in 5% fish

475

oil in water emulsion. Further purification and characterisation of these fractions are needed

476

to explain the discrepancies in the antioxidant activities of the peptides in simple model

477

system compared to more complex emulsion systems. In emulsions systems several factors

478

are known to be important in relation to oxidation and these include for example the type,

479

properties, location of the prooxidants/antioxidant and especially their location at the

480

interface lipid/water. Finally, one should not forget that the classic in-vitro tests for

481

measuring antioxidant activity are not always reliable to demonstrate antioxidant activities in

482

food and tests in food matrices are always recommended.

483

Acknowledgments

484

Authors are very grateful to The Technical University of Denmark for supporting this

485

research.

486

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660 27

661

Figure Captions

662

Figure 1: Illustration of the separation sequence used for herring brine fractionation

663

into different fractions

664

Figure 2: Functional properties of different herring brine fractions (1% protein) at pH

665

7 a) emulsion activity index and b) emulsion stability index.

666

Figure 3: Emulsifying and foaming properties of herring brine fractions measured at

667

different pH values (2, 4, 6, 8 and 10) and using a) emulsifying activity, b)

668

emulsifying stability, (with sodium caseinate as reference), c) foaming capacity and d)

669

foaming stability (using BSA as reference).

670

Figure 4: Antioxidant effect of different herring brine fractions (1g/ml) in 5% fish

671

oil-in-water emulsions using FeSO4 (100 M) as initiator. With a) Peroxide value b)

672

Anisidine value and c) tocopherol loss.

28

673 674

Table. 1. Protein, salt and dry matter content of the 675 different fraction of herring brine. Protein Salt content Dry matter 676 (mg/ml) (%) (%) pH 18.5±0.13 22.6±0.4 25.11±0.3677 Precipitated Fraction 678 >50 kDa 37.8±0.1 2.3±0.1 7.9±0.7 10-50kDa 2.3±0.1 1.7±0.3 2.1±0.0 679 1-10kDa 1.1±0.0 1.5±0.0 2.1±0.6 <1 KDa 7.4±0.2 23.5±0.1 22.2±0.5 680 681 682 683

29

684 685

Table 2.Free Amino acid composition expressed in g/100g dry weight and in % of total amino acids (in brackets) of different herring brine fractions

686

Lys Arg Ala Leu Met Phe Pro Thr Tyr Asp Ser Glu Val His Trp Ile Gly total

pH >50kDa 50-10kDa 10-1 kDa precipitate d fraction 0.61 (9.6) 0.53 (13.9) 1.26 (11.5) 0.97 (12.4) 0.67 (10.6) 0.24 (6.4) 1.37 (12.4) 0.93 (11.9) 0.43 (6.8) 0.36 (9.5) 1.26 (11.4) 0.55 (6.9) 0.56 (8.9) 0.36 (9.5) 0.87 (7.9) 0.72 (9.2) 0.16 (2.5) 0.10 (2.6) 0.46 (4.2) 0.30 (3.8) 0.27 (4.3) 0.17 (4.5) 0.27(2.5) 0.25 (3.1) 0.22 (3.5) 0.09 (2.4) 0.28 (2.5) 0.26 (3.4) 0.22 (3.5) 0.13 (3.4) 0.40 (3.7) 0.26 (3.3) 0.20 (3.2) 0.13 (3.4) 0.31(2.8) 0.22 (2.8) 0.44 (6.9) 0.20 (5.3) 0.73 (6.7) 0.45(5.7) 0.47(7.4) 0.20(5.1) 0.68 (6.2) 0.50 (6.4) 0.69(10.9) 0.44 (11.7) 0.95 (8.6) 0.67 (8.5) 0.33(5.2) 0.18 (4.7) 0.59 (5.4) 0.51(6.5) 0.45(7.0) 0.20 (5.2) 0.66 (6.0) 0.49 (6.2) 0.06(1.0) 0.02 (0.6) 0.05 (0.4) 0.02 (0.3) 0.17 (2.7) 0.21(5.4) 0.33 (3.0) 0.34(4.4) 0.38 (6.0) 0.23 (6.2) 0.51(4.6) 0.40 (5.1) 6.34(100) 3.79 (100) 10.99(100) 7.84 (100)

687 688

30

689 690

Table 3. Amino acid composition (expressed as % of total amino acids) of different herring brine fractions

Lys Arg Ala Cys Leu Met Phe Pro Thr Tyr Asp Ser Glu Hyp Val His Trp Ile Gly total

pH >50kDa 5010-1 precipitated 10kDa kDa fraction 11.5 12.4 14.3 15.2 3.9 2.6 7.3 6.7 7.8 9.4 8.9 7.6 0.6 0.6 0.1 0.0 10.1 9.6 5.8 5.1 0.0 0.0 0.0 0.0 3.1 5.5 2.4 1.6 4.3 4.9 5.1 6.2 3.6 3.4 3.3 2.5 2.5 1.9 0.9 0.8 7.7 10.5 7.8 8.7 3.4 4.7 7.6 5.1 14.5 9.5 15.4 16.3 0.3 0.1 0.4 0.0 6.9 6.2 5.3 5.7 11.5 10.9 12.6 16.3 0.0 0.0 0.0 0.0 5.0 5.5 2.8 2.3 3.1 2.3 0.0 0.0 100 100 100 100

691 692 31

693 694 695 696

Table.4. In vitro antioxidant activity of isolated herring brine fractions.

697 698

Results are the mean values ± Standard deviation. Samples followed by the same letter are not significantly different in Bonferroni post test using 0.05 level of significance. Comparison between different protein concentration: a, b, and fractions: v,w, x, y.

699

Fractions

DPPH radical scavenging (%)

Protein concentration (mg/ml) 0.1 0.5 pH precipitated fraction >50 kDa 50-10kDa 10-1kDa BHT (0.2 mg/mL) EDTA(0.2 mg/mL) Ascorbic acid(0.2 mg/mL)

10.9±0.1av 10.4±0.7av 10.8±1.8av 15.1±0.9aw -

36.1±2.2bv 37.2±3.4bv 54.2±0.9bw 69.1±1.7bx 79.8 ± 1.0y -

Fe2+cheating activity (%)

Protein concentration(mg/ml) 0.1 0.5 6.1±0.2ax 5.0±0.9aw 3.4±0.5av 6.2±0.2ax

6.3±0.0avw 5.1±0.8av 5.1±0.5bv 7.9±0.8bx

Reducing power (OD at 700nm) Protein concentration (mg/ml) 0.1 0.5 0.05±0.0 av 0.02±0.0 av 0.06±0.0 av 0.08±0.0av

0.14±0.0bv 0.07±0.0av 0.24±0.0bv 0.34±0.0bv

93.7 ± 0.2y 2.3 ± 0.3w

700 701 702 32

Liposome system (µmoles of MDA formed/mg PL after 1h) Protein concentration (mg/ml) 0.1 0.5 9.3±1.1av 8.4±0.3av 9.3±0.0av 9.6±0.4av

9.5±0.7awx 7.8±0.4aw 8.8±0.3aw 8.9±0.4aw 2.43±0.99v

703 704

Figure 1:

Salted Herring Brine

705 706 707 708 709

Adjusting pH 4.5 Centrifugation (Supernatant)

pH precipitated Fraction (Precipitate)

UF 50 KDa

710 (Permeate)

>50 KDa Fraction (Retentate)

711 712 713 714 715

UF 10 KDa (Permeate)

50-10 KDa Fraction (Retentate)

UF 1 KDa 10-1 KDa Fraction (Retentate)

716 717

High salt Fraction (Permeate Discarded)

718 719

33

722

723

724

725

34 m

a na te

ca se i

kD

10 -1

30

So di u

0

D a

50

10 k

50

a

100

>5 0k D

io n

fr ac t

(a) Emulsion stability Index

200

50 -

d

ta te

pi

pr ec i

a

na te

ca se i

10 -1 kD

Emulsion activity index 150

So di um

0k D a

a

0k D

>5

io n

fr ac t

50 -1

ita te d

pr ec ip

721

pH

pH

720

Figure 2

pH7 pH7

40

(b)

20

10 0

400

(a ) pH 2 pH 4

300

pH 6 pH 8

200

pH 10

100

0 p H p re c ip ita te d fra c tio n

E m u l s io n s t a b il it y i n d e x ( m in )

Figure 3

E m u l s if y i n g a c t iv i t y i n d e x ( m 2 /g )

726

100

(b )

pH 4

80

pH 6

60

pH 8 pH 10

40

20

0 p H p re c ip ita te d fra c tio n

S o d iu m c a s e in a te

(c ) 80

150

pH 2

S o d iu m c a s e in a te

(d ) pH 2

pH 4 pH 6

100

pH 8 pH 10

50

F o a m in g s t a b ilit y ( % )

F o a m in g c a p a c it y ( % )

pH 2

pH 4

60

pH 6 pH 8

40

pH 10

20

0

0 p H p re c ip ita te d fra c tio n

p H p re c ip ita te d fra c tio n

BSA

727 35

BSA

728

Figure 4 (a)

PV (meq/kg oil)

25

Control pH precipitated fraction >50kDa 50-10kDa 10-1kDa BHT

20 15 10 5 0 0

12 24 Storage time in hours

Anisidine Value

50

48

(b)

Control pH precipitated fraction >50kDa 50-10kDa 10-1kDa BHT

40 30 20 10 0

Total Tocopherol (ug/g lipid)

0

12 24 Storage time in hours

250

48

(c)

Control pH pre cipitate d fraction

200

>50kDa

150

50-10kDa 10-1kDa

100

BHT

50 0 0

12 24 Storage time in hours

48

729 730 36

Research Highlights 

50-10kDa and 10-1kDa fractions showed good radical scavenging activity



All the fractions had low iron chelating activity



All fractions delayed the development of PV and showed low AV and tocopherol loss in emulsions



The fraction 50-10kDa showed the best antioxidant potential in oil-in-water emulsion