High quality fish protein hydrolysates prepared from

journal of functional foods 9 (2014) 10–17

Available at www.sciencedirect.com

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High quality fish protein hydrolysates prepared from by-product material with Fucus vesiculosus extract Sigrun M. Halldorsdottir a,b,*, Holmfridur Sveinsdottir c, Agusta Gudmundsdottir b, Gudjon Thorkelsson a,b, Hordur G. Kristinsson a,d a

Matis, Vinlandsleid 12, 113 Reykjavík, Iceland University of Iceland, Department of Food Science and Nutrition, School of Health Sciences, Saemundargotu 2, 101 Reykjavík, Iceland c Iceprotein, Haeyri 1, 550 Saudarkrokur, Iceland d University of Florida, Department of Food Science and Human Nutrition, 359 FSHN Building, Newell Drive, Gainesville, FL 32611-0370, USA b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Value added products such as fish protein hydrolysates (FPH) can be produced from fish

Received 12 February 2014

by-products. Lipid oxidation and bad taste are the major challenge in the commercializa-

Received in revised form 10 April

tion of bioactive FPH. The aim of this research was to study the production of high quality

2014

FPH from fish by-products prepared by enzymatic hydrolysis using a natural antioxidant

Accepted 11 April 2014

extracted from the Icelandic brown algae Fucus vesiculosus (Fv). FPH were produced from cod

Available online 3 May 2014

waste material; i.e. cod bone mince, in the absence and presence of an Fv extract (Fv-e). Oxidation during the FPH production was evaluated (lipid hydroperoxides and thiobarbituric

Keywords:

acid reactive substances). The FPH were sensory analyzed (generic descriptive analysis) and

Fish protein hydrolysates

in vitro antioxidant activity was evaluated. Results show that Fv-e contributed to better tasting

Fucus vesiculosus extract

FPH with regard to bitter, soap, fish oil and rancidity taste. Results from the oxidation and

Enzyme hydrolysis

antioxidant activity assays indicated a protecting effect of Fv-e during processing. © 2014 Elsevier Ltd. All rights reserved.

Lipid oxidation Antioxidant activity Sensory analysis

1.

Introduction

Consumers are increasingly searching for food products with known bioactivities to improve their health or prevent diseases. The nutraceutical and functional food market is one of the fastest growing markets in the world (Leatherhead Food Research, 2011). Fish protein hydrolysates (FPH) possess many

desirable properties such as health promoting bioactivities, making them eligible ingredients in nutraceuticals and functional food (Harnedy & FitzGerald, 2012; Kristinsson, 2007). FPH have also shown ability to work as protecting antioxidant agents in various food systems to sustain their quality e.g. in a washed fish model (Raghavan & Kristinsson, 2008) and oil-in-water emulsion systems (Samaranayaka & Li-Chan, 2008; Thiansilakul, Benjakul, & Shahidi, 2007). Even though fish supplies in the

* Corresponding author. Tel.: +354 4225000/+354 8651704; fax: +354 4225001. E-mail address: [email protected] (S.M. Halldorsdottir). http://dx.doi.org/10.1016/j.jff.2014.04.009 1756-4646/© 2014 Elsevier Ltd. All rights reserved.


world are seriously dwindling, a lot of high quality protein material is discarded or underutilized after fish processing (FAO, 2005). Utilizing these by-products in high value products such as bioactive FPH would significantly add value to the seafood industry. Lipid oxidation is the major challenge in the commercialization of bioactive fish protein ingredients whereas it is the main cause of quality deterioration in muscle foods (Ladikos & Lougovois, 1990). Studies show that oxidation can develop rapidly during hydrolysis of fish protein when a prooxidant is present (Halldorsdottir, Sveinsdottir, Freysdottir, & Kristinsson, 2014; Yarnpakdee, Benjakul, Kristinsson, & Maqsood, 2012). Moreover, oxidation products have been found to negatively affect the bioactivities of FPH (Halldorsdottir et al., 2014). As a matter of course, it is of wide importance to sustain the bioactivity throughout the process of the material if the aim is to use it as a bioactive ingredient. Antioxidant strategies have shown to be useful in preventing oxidation during hydrolysis (Halldorsdottir, Kristinsson, Sveinsdottir, Thorkelsson, & Hamaguchi, 2013; Yarnpakdee, Benjakul, Nalinanon, & Kristinsson, 2012). Moreover, natural antioxidants have shown to not only protect the fish protein hydrolysates from losing the beneficial properties during hydrolysis but also contribute to the bioactive properties of the final product (Halldorsdottir et al., 2013). Bitter taste has also been identified as a major hindrance regarding utilization and commercialization of bioactive FPH (Kim & Wijesekara, 2010). The most widespread methodology for reducing bitterness is by using enzymes that are specifically prepared with the goal of limiting the development of bitterness during hydrolysis (Kristinsson, 2006). However, it has also been suggested that oxidation products play a part in the development of bitter taste (Liu, Morioka, Itoh, & Obatake, 2000). Several compounds derived from hydroperoxides (primary oxidation products) such as oxo, epoxy, mono-, di- and trihydroxy carboxylic acids, have been identified as having bitter taste. These compounds are known to play a part in the bitter taste of foods that are rich in unsaturated fatty acids such as fish and fish products (Belitz, Grosch, & Schieberle, 1999). Cod bone mince (CBM) is a typical low-value material that is left over in abundance during cod processing in many northern countries including Iceland. It contains a lot of valuable protein that would be ideal for FPH production. However, it also contains a lot of contaminants such as pro-oxidants, phospholipids etc. These constituents can have detrimental effect during hydrolysis and can significantly reduce the quality of the resulting FPH. In this study CBM was hydrolyzed to recover bioactive FPH in the presence and absence of a natural antioxidant extracted from the Icelandic brown algae Fucus vesiculosus (Fv). Fv is rich in highly antioxidative phenolic compounds, primarily phlorotannins (Wang et al., 2012), that have shown a great capacity to inhibit oxidation during hydrolysis (Halldorsdottir et al., 2013). Phlorotannins from brown algae can have up to eight aromatic rings that are interconnected (while terrestrial plants only have three or four rings) which can to some extent explain their exceptional antioxidant capacity (Hemat, 2007). During formulation of functional food and nutraceuticals it is important to consider the synergistic effects obtained by a combination of compounds present in the raw material (Shahidi, 2004). In this study the synergistic effect of FPH and polyphenols from brown algae is taken into consideration in terms of antioxidant activity and sensory attri-

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butes. CBM and brown algae are both underutilized materials that exist in abundance and contain valuable bioactive compounds. By incorporating them together and hydrolyzing, a highly active ingredient from low value material with great potential in high value products such as functional foods and nutraceuticals could be obtained.

2.

Materials and methods

2.1.

Material

CBM was kindly provided from MPF Island (Grindavik, Iceland). MPF Island filleted fresh cod (Gadus morhua) 1–4 days after it was caught and collected the by-products including cod backbone. The cod backbone was minced in a BAADER 665 (Baader Iceland, Kopavogur, Iceland) grinder to CBM and transported on ice to the Matis laboratory. The protein content of the CBM was determined according to the Kjeldahl method (Kjeltec System, FOSS Tectator, Hoganas, Sweden). A factor of 6.25 was used to convert nitrogen to crude protein content. The enzyme Protease P “Amano” 6 was kindly provided by Amano enzyme company (Nagoya, Japan). Brown algae, Fv, used for the preparation of Fv-e, were collected during autumn in a coastal area southwest of Iceland. The algae were washed with clean seawater to remove sand and epiphytes of their surface and transported to the Matis laboratory. There they were carefully rinsed with tap water, cut into small pieces and freeze dried. The dried algal powder was stored at −20 °C prior to extraction. Chemicals were purchased from Sigma-Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland) or others reported where relevant.

2.2. Preparation of F. vesiculosus extract (Fv-e) and quantification of phlorotannin The solvent extracts were prepared according to the method described by Wang, Jónsdóttir, and Ólafsdóttir (2009) with modifications according to Halldorsdottir et al. (2013). Briefly, 40 g of dried algal powder was extracted with 200 mL 80% ethanol (EtOH) in a platform shaker for 24 h at 200 rpm and at approximately 22 °C. The mixture was centrifuged at 2500 g for 10 min at 4 °C and filtered. The filtrate was concentrated in vacua to a small volume and the residue was suspended in a mixture of methanol (MeOH) and water (40:30, v/v) and partitioned three times with ethyl acetate (EtOAc) successively. The EtOAc soluble fraction (Fv-e) was obtained after removing the solvent, freeze dried and stored in air tight containers at −20 °C until further use. The phlorotannin content of the Fv-e was determined by the Folin–Ciocalteu method described by Wang et al. (2012) expressed as phloroglucinol equivalents (PGE).

2.3.

Fish protein hydrolysates (FPH)

The CBM was diluted in water to 3.7% (w/v) protein and adjusted to pH 8 with 2 M NaOH. It was divided in to two 1 L portions, in one Fv-e (0.16 g PGE) was added and the other one not. The material was subjected to Protease P “Amano” 6 for hydrolysis at 36 °C to achieve 20% degree of hydrolysis (%DH). The hydrolysis was executed as described by Halldorsdottir et al. (2013).The %DH with time was monitored with reference to Eq. 1 (Adler-Nissen, 1986):

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Table 1 – Sample identification. Sample name

Description

CBM CBM-FPH CBM-FPH-Fv Fd-CBM-FPH Fd-CBM-FPH-Fv

Cod bone mince Fish protein hydrolysates prepared from cod bone mince Fish protein hydrolysates prepared from cod bone mince with added Fucus vesiculosus extract Freeze dried fish protein hydrolysates prepared from cod bone mince Freeze dried fish protein hydrolysates prepared from cod bone mince with added Fucus vesiculosus extract

%DH = B × Nbase α × htotal × MP × 100

(1)

where B = volume of base used, Nbase = normality of base, α = degree of dissociation, htotal = total number of peptide bonds per mass unit and MP = amount of protein used. The degree of dissociation (α) was found by Eq. 2:

α = 10pH − pKa 1 + 10pH − pKa

(2)

where pH is the value at which the enzyme hydrolysis was performed. The pKa values were calculated according to Eq. 3 (Steinhardt & Beychok, 1964):

pKa = 7.8 + ((298 − T ) 298 × T ) × 2400

(3)

where T is the temperature in Kelvin at which the enzyme hydrolysis was performed. After hydrolysis the samples were heated to 90 °C for 10 min to inactivate the enzymes, followed by cooling on ice. Then the samples were frozen at −20 °C prior to freeze drying. Samples were taken before and after hydrolysis and after freeze drying for analysis. Samples are identified in Table 1. The protein content was determined with the Kjeldahl method (Kjeltec System, FOSS Tectator, Hoganas, Sweden). A factor of 6.25 was used to convert nitrogen to crude protein content.

2.4.

Lipid hydroperoxides

Lipid hydroperoxides were determined in all samples (Table 1) with a version of the ferric thiocyanate method as described by Undeland, Hultin, and Richards (2002). The results were expressed as mmol lipid hydroperoxide per kg of dry weight. The dry weight of wet samples was determined according to moisture content analysis.

2.5.

Thiobarbituric acid-reactive substances (TBARS)

TBARS were analyzed in all samples (Table 1) after extracting each sample with TCA (Lemon, 1975). The results were expressed as µmol of malondialdehyde (MDA) equivalents per kg of dry weight.

2.6.

Sensory evaluation

to international standards (ISO, 1993), participated in the sensory evaluation. The panelists were familiar with the GDA method and were experienced in sensory analysis of protein solutions. Sensory attributes (Table 2) of the fish protein solutions were defined based on a scale applied by Shaviklo, Thorkelsson, Arason, Kristinsson, and Sveinsdottir (2010). A training session was applied to synchronize the panel prior to the sensory evaluation where the panelists were trained in recognizing the sensory characteristics of the protein solutions. An unstructured scale (0–100%) was used by the trained panelists to evaluate the intensity of each sensory attribute. Table 1 illustrates the sensory attributes describing the odor and flavor of the protein solutions. Each sample, 6 mL of protein solution, was presented in a small plastic beaker that was coded with a three digit number and presented according to the latin square method.The sensory evaluation was carried out in one session with a duplicate from both sample groups. A computerized system (FIZZ, Version 2.0, 1994–2000, Biosystemes) was used for data recording.

2.7.

Oxygen Radical Absorbance Capacity (ORAC)

The ORAC of CBM-FPH, CBM-FPH-Fv, Fd-CBM-FPH and Fd-CBMFPH-Fv was evaluated according to Ganske and Dell (2006) with modifications according to Halldorsdottir et al. (2011). The ORAC value was calculated and expressed as µmol of Trolox equivalents (TE) per g of protein using a calibration curve of Trolox.

2.8.

Metal chelating ability

The metal chelating ability of CBM-FPH, CBM-FPH-Fv, Fd-CBMFPH and Fd-CBM-FPH-Fv was evaluated using the method of

Table 2 – Sensory attributes for Fd-CBM-FPH and Fd-CBM-FPH-Fv. Odor Fresh fish Sweet Earthy Sour Fermentation TMA Rancid

Odor of fresh raw fish fillet Sweet odor, fresh fruit e.g. pear and melon Earthy odor, clay, humus Sour odor, whey Fermentation odor TMA odor, trimethylamine Rancid odor

Flavor Protein solutions were prepared from Fd-CBM-FPH and FdCBM-FPH-Fv; 15 g of freeze dried protein powder was mixed with distilled water up to 250 mL. The two mixtures of protein solutions were odor and flavor evaluated with generic descriptive analysis (GDA) as described by Shaviklo, Thorkelsson, Arason, and Sveinsdottir (2012). Eight panelists, all trained according

Bitter Sour Soap Fish oil Rancid Dried fish

Bitter or pungent flavor Sour basic flavor Soapy or chemical flavor Flavor of fish oil Rancid flavor Flavor of dried fish, trimethylamine


Boyer and McCleary (1987) with modification according to Halldorsdottir et al. (2011).

2.9.

Intracellular antioxidant activity by HepG2 cell assay

HepG2 cells (ATCC 8065, American Type Culture Collection, Rockville, MD, USA) were kept in absolute medium; Minimum Essential Medium α (MEMα) supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), penicillin (50 units/mL) and streptomycin (50 µg/mL) (all from Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37 °C in a fully humidified environment under 5% CO2. Cells at passage 80–100 were used for the experiments. Cell culture medium was replaced every other day, and cells were subcultured at 3–5 days intervals before reaching 90% confluence.

2.9.1.

Cell viability assay

HepG2 cells were seeded at 6 × 104/well on black 96-well plates (BD Falcon™, Franklin Lakes, NJ, USA) in absolute medium and incubated for 24 h at 37 °C. The cells were then exposed to CBMFPH, CBM-FPH-Fv, Fd-CBM-FPH and Fd-CBM-FPH-Fv and incubated at 37 °C for 48 h. After incubation, the medium containing the test compounds was discarded and the cell viability was evaluated by exposure to 10% alamarBlue® (Invitrogen) solution in absolute medium at 37 °C in the dark for 4 h. Finally, the fluorescence was measured at excitation of 570 nm and emission of 610 nm (Page, Page, & Noel, 1993) using a POLARstar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany). Control cells were exposed to alamarBlue®. The results were given as the percentage of viable cells in the population of each concentration according to Eq. 4:

Viability (%) = (Mean fluorescence of treated cells Mean fluorescence of control cells) × 100

2.9.2.

(4)

Cellular antioxidant activity

A cellular antioxidant assay was performed using HepG2 cells at a density of 6 × 104/well using black 96-well plates (BD Falcon™) in 100 µL absolute medium/well according to Wolfe and Liu (2007) and Samaranayaka, Kitts, and Li-Chan (2010) with minor modifications. Twenty-four hours after seeding, 100 µL of 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) probe (1 µM in HBSS) were added to the cells and incubated at 37 °C in the dark for 30 min. Cells were then treated with CBM-FPH, CBMFPH-Fv, Fd-CBM-FPH or Fd-CBM-FPH-Fv and incubated for 1 h at 37 °C. This was followed by the addition of 100 µL of 2,2′Azobis(2-amidinopropane) dihydrochloride (AAPH) a peroxyl radical initiator (strong pro-oxidant) to the final concentration of 500 µM AAPH in Hanks balanced salt solution (HBSS) (Invitrogen), to the cultured cells after removal of the test compounds. Oxidation in the HepG2 cells was induced by adding AAPH to the medium that generates peroxyl radicals in the cells. The control (AAPH) represents cells that had only added AAPH in the medium. Fluorescence was measured at excitation of 493 nm and emission of 527 nm and recorded using a POLARstar OPTIMA microplate reader every 10 min for 90 min after addition of AAPH. Each plate included four replicates of AAPH and control wells. Control wells contained cells exposed only to the DCFH-DA probe. The AAPH consisted of cells with DCFH-

13

DA probe and the AAPH added but in the absence of test compounds. Trolox™ (Sigma-Aldrich, St. Louis, MO, USA) (50 µM), a commercial antioxidant, was used as positive control. To determine the cellular antioxidant activity the fluorescence intensity after 60 min, run was used. Fluorescence intensity of AAPH cells were attributed the index value of 100, i.e. 100% cellular ROS generation.

2.10.

Statistical analysis

Statistical analysis was carried with one-way analysis of variance (ANOVA) followed by a Duncan’s or Holm-Sidak multiple range test for mean comparison. The following computer programs were used: SigmaSTAT for Windows Version 3.5 (Systat Software, San Jose, CA, USA) and NCSS 2000 (NCSS, Kaysville, UT, USA). In addition, panelists’ performance in sensory analysis was analyzed using PanelCheck V1.4.0 (Matforsk, Tromsø, Norway). All analyses were carried out in at least duplicates. Results were expressed as mean values ± standard deviation. Significance of differences was defined as the 5% level (p < 0.05).

3.

Results and discussion

3.1.

Lipid oxidation

The lipid hydroperoxide value was evaluated in CBM and all resulting hydrolysates. Results show that there was no significant difference (p < 0.05, n = 3) between samples (data not shown) indicating that the lipid hydroperoxide formation was already initiated in the CBM. That is in line with the said nature of the CBM, i.e. it is secondary material that contains highly pro-oxidative substances. It is likely that it is prone to photooxidation, caused by light and pigments, due to natural pigments distributed in the lipid tissue and exposure to light during processing. Photooxidation can promote the rate of autoxidation up to 1500 times (Nawar, 1996). However, primary oxidation products such as lipid hydroperoxides have not been associated with rancid odor and taste in fish products and are therefore not a significant factor regarding quality of the final product (Undeland et al., 2002). Nevertheless, these primary oxidation products can readily break down to foul smelling and rancid tasting secondary lipid oxidation products such as aldehydes that have a profound detrimental effect on the quality of fish products (Shahidi, 1997). The TBARS value was evaluated in CBM and all resulting hydrolysates (Fig. 1). Results indicate that there is significant TBARS formation during hydrolysis of CBM both in the absence and presence of Fv-e. However, there was a significant difference in the TBARS level of CBM-FPH and Fd-CBM-FPH indicating that oxidation was promoted during the freeze drying step. In contrast, no significant change was observed in the TBARS level of CBM-FPH-Fv and Fd-CBM-FPH-Fv-e, indicating a protecting effect of Fv-e during freeze drying. Antioxidants widely differ in their mechanism of action and their effectiveness is largely dependent upon the type of food systems and environmental conditions. Thus it is important to investigate specific antioxidants thoroughly in the same systems and process as they are going to be subjected to in practice (Frankel, 2007).

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Fig. 1 – TBARS (µmol MDA/kg protein) formation in CBM, CBM-FPH-Fv, CBM-FPH, Fd-CBM-FPH-Fv and Fd-CBM-FPH (Table 1). Results are presented as average ± standard deviation. Data sharing the same letters are not significantly different (p < 0.05, n = 3).

A study conducted by Halldorsdottir et al. (2013) revealed that Fv-e can be useful in protecting fish protein during hydrolysis in the presence of strong pro-oxidants while these results indicate that Fv-e can be useful in protecting FPH specifically during freeze drying.

3.2.

Sensory analysis

Fd-CBM-FPH-Fv and Fd-CBM-FPH were evaluated for taste and odor with GDA (Fig. 2). Fd-CBM-FPH-Fv and Fd-CBM-FPH were significantly different with regard to taste. Both FPH had very low scores for rancid taste (0–10), indicating that the FPH had limited oxidation. However, Fd-CBM-FPH-Fv had a significantly lower rancid taste than Fd-CBM-FPH which is in line with result obtained from TBARS (Fig. 1). Interestingly, the Fd-CBM-

Fig. 2 – Radar plot of generic descriptive analysis (GDA) odor and flavor attributes (mean scores from duplicate samples) of Fd-CBM-FPH and Fd-CBM-FPH-Fv (Table 1). Asterisks indicate a significant difference between samples within an attribute and the number of asterisks indicate the level of difference: *p < 0.05; **p < 0.005; ***p < 0.0001.

FPH-Fv also received significantly lower scores compared to FdCBM-FPH for bitter taste. It is a well-known fact that the hydrolysis process often creates bitter taste in the product that restricts the practical uses of FPH (Dauksas, Slizyte, Rustad, & Storro, 2004). These results affirm the previously stated theory, that secondary oxidation products contribute to the bitter taste in FPH (Liu et al., 2000), even though present in a relatively low concentration. Additionally, Fd-CBM-FPH-Fv received significantly lower scores for soap taste and fish oil taste compared to Fd-CBM-FPH. Therefore, Fd-CBM-FPH-Fv was more favorable with regard to taste whereas bitter, soap, fish oil and rancidity tastes are not desirable attributes in foodstuff. These results highlight the potential in using Fv-e as natural antioxidant in production of high quality FPH.

3.3.

Antioxidant activity

The phlorotannin content of the Fv-e was 59.2 g PGE/100 g Fv-e as determined by the Folin–Ciocalteu method (Wang et al., 2012). During preparation of CBM-FPH-Fv, 0.3 g of Fv-e was added to 1 L of CBM solution prior to hydrolyzation. Hence the phlorotannin content in CBM-FPH-Fv was 0.16 g PGE. As previously stated, phlorotannins are postulated the main constituent of the F. vesiculosus extract responsible for its exceptional antioxidant capacity (Wang et al., 2012). The hydrolysates prepared from CBM showed ORAC values on the range 435– 528 µmol TE per g of protein (Fig. 3a). Another study that evaluated FPH prepared from different variations of a washed cod model also with Protease P Amano 6 showed values on the range 595–834 µmol TE per g of protein (Halldorsdottir et al., 2013). The difference is likely due to some extent to difference in starting material. When comparing various FPH it can be seen that FPH prepared from cod with Protease P Amano 6 have exceptionally strong ability to absorb oxygen radicals. Samaranayaka et al. (2010) reported considerably lower ORAC values, i.e. on the range from 225 to 330 μmol TE per g freeze dried sample from both crude and fractionated Pacific hake (Merluccius productus) FPH. However, if they would present their results per protein instead of per dry sample their ORAC values would probably be higher. Theodore, Raghavan, and Kristinsson (2008) reported even lower ORAC values of FPH prepared from catfish isolates in the interval 2–4 µmol TE per g of protein. FdCBM-FPH-Fv had significantly stronger ability to absorb oxygen radicals than Fd-CBM-FPH, supporting the results obtained in TBARS and sensory analysis of the protecting effect of the Fv-e during the freeze drying step and/or that the Fv-e contributes to the oxygen radical absorbance ability. The hydrolysates showed very good metal chelating ability on the range 84–94% (Fig. 3b). Several other studies have also documented that FPH are a good source of metal chelating peptides (Halldorsdottir et al., 2011; Raghavan & Kristinsson, 2008; Thiansilakul et al., 2007). Fd-CBM-FPH-Fv had significantly stronger metal chelating ability than Fd-CBM-FPH, pointing out further the protecting effect of Fv-e during freeze drying. Potentially Fv-e reactivated FPH as an antioxidative agent by acting as a secondary antioxidant via hydrogen donation (Frankel, 2007) rather than contributing to the metal chelating ability as previous findings demonstrated that, algal polyphenols such as those contained in Fv-e, mainly act as potent free radical scavengers and primary, chain-breaking antioxidants but not


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Fig. 4 – Antioxidant activity of CBM-FPH-Fv, CBM-FPH, FdCBM-FPH-Fv and Fd-CBM-FPH (Table 1) according to the HepG2 cellular antioxidant assay. HepG2 cell were treated with FPH in the concentration 1 mg/mL. Results are presented as average ± standard deviation. Data sharing the same letters are not significantly different (p < 0.05, n = 3).

Fig. 3 – The antioxidative properties of CBM-FPH-Fv, CBMFPH, Fd-CBM-FPH-Fv and Fd-CBM-FPH (Table 1) measured with two different in vitro chemical methods; (a) ORAC (TE = Trolox equivalence) and (b) iron chelating ability. Results are presented as average ± standard deviation. Data sharing the same letters are not significantly different (p < 0.05, n = 2).

as good metal chelating agents (Wang et al., 2012). However, there is conflicting data in the literature concerning the metal chelating ability of polyphenols. Polyphenols from various plant origin such as brown algae have been found to have effective ferrous ion chelating ability (Chew, Lim, Omar, & Khoo, 2008; Senevirathne et al., 2006). Their metal chelating ability has been connected to their distinctive phenolic structure and quantity and position of hydroxyl groups (Santoso, Yoshie-Stark, & Suzuki, 2004). In contrast, other studies show that metal chelating capacity plays a negligible role in the overall antioxidant activity of plant-derived phenolic compounds (Rice-Evans, Miller, & Paganga, 1996). All produced FPH showed significant ability to inhibit intracellular oxidation induced by AAPH (Fig. 4). CBMFPH-Fv showed significantly stronger intracellular antioxidant ability than CBM-FPH, indicating that the Fv-e contributed to the in vitro antioxidant activity of the FPH as assessed by the HepG2 cell assay. There was no significant difference between either version of the FPH before and after freeze drying, pointing out that the freeze drying step did not affect the in vitro intracellular antioxidant activity of the FPH. From these results it can be concluded that FPH prepared from different cod material, if not oxidized, may potentially act as an antioxidant agent in a biological system. Samaranayaka et al. (2010) also concluded that FPH may have a good potential as an an-

tioxidant agent, based on their study on the intracellular antioxidant capacity of Pacific hake FPH evaluated in a Caco-2 cell model. However, it should be stressed that when comparing results obtained in cell based assays it is important to consider the properties, sensitivity and growth status of the applied cells as well as the exposure dose and time of the antioxidant substance (Cheli & Baldi, 2011). Even though in vivo animal and human clinical trials are the ideal model to evaluate the actual effect that takes place in the body, in vitro cultured cell models have shown to be a very useful tool for the screening of possible bioactive compounds. Cell models are also more biologically relevant than chemical antioxidant activity assays because they account for some aspects of uptake, metabolism, and location of antioxidant compounds within cells (Wolfe & Liu, 2007). However, the chemical assays can be very useful when determining different antioxidative mechanisms of antioxidant compounds e.g. do they scavenge free radicals and/ or chelate metals. Although there was no significant correlation between results obtained from chemical and cell antioxidant assays, both types showed a general tendency toward higher antioxidant activity of FPH containing Fv-e. Halldorsdottir et al. (2014) showed that highly oxidized FPH had a negative effect on intracellular antioxidant activity assessed in a HepG2 cell model while results obtained in chemical assays showed a negligible impact. Other studies also indicate a difference in results obtained in chemical and cell based antioxidant assays (Eberhardt, Kobira, Keck, Juvik, & Jeffery, 2005; Girard-Lalancette, Pichette, & Legault, 2009). In cell based assays some peptides and polyphenols are not absorbed and therefore do not show the same intensity of antioxidant activity as in chemically based methods; hence a diversity of results can be observed. These differences highlight the importance of being aware of the shortcomings of the in vitro assays when evaluating antioxidant activity within biological models. Amino acid composition and sequence as well as chain length of peptides are among factors that establish the properties of FPH. The exact mechanism of action of peptides as antioxidants is not known but

16


it has been suggested that certain amino acids play a profound role in increasing antioxidant capacity of peptides such as hydrophobic amino acids, aromatic amino acids and histidine (Kim & Wijesekara, 2010). As previously discussed, the antioxidant capacity of the FPH observed in this study was very high compared to FPH from other sources indicating that cod bone mince, the Amano P protease, along with other conditions e.g. DH, T, pH etc., generate FPH with strong antioxidant activity. In this study an aim was to resemble an economically relevant industrial process for the food industry and therefore the FPH were not fractionated and further analyzed with regard to molecular weight and peptide sequence. However, for future research it will be interesting to perform such investigations both for the purpose of finding which peptides are giving the most profound properties and also to add to the understanding of the mechanism behind antioxidant capacity of FPH.

4.

Conclusion

Results from TBARS indicate that oxidation took place during freeze drying of hydrolyzed CBM in the absence of Fv-e, whereas Fv-e inhibited oxidation during freeze drying of hydrolyzed CBM. The Fv-e contributed to better tasting FPH with regard to bitter, soap, fish oil and rancidity taste. That is very positive because so far the unpleasant taste of FPH has been a huge hindrance in their practical use. According to antioxidant assays, both cod bone mince hydrolysates with and without Fv generally exhibited strong antioxidant activity. An overall tendency toward higher antioxidant activity of FPH containing Fv-e was observed indicating that Fv-e had a protective effect on the antioxidant capacity of the FPH during processing and/or Fv-e contributed to the antioxidant activity of the final product. Chemical antioxidant assays indicated a protecting effect of Fv-e during freeze drying whereas the cell assay indicated a protecting effect of Fv-e during hydrolysis. These results clearly demonstrate that by adding Fv-e, a highly active natural antioxidant extracted from Icelandic brown algae, to CBM, a lowvalue by-product material, prior to hydrolyzation can be an effective approach to recover high quality FPH. Results indicate that FPH produced in the combination of polyphenols from brown algae are highly eligible as functional food and nutraceutical ingredients. This study contains valuable insights for fish manufacturers and producers of functional food and nutraceuticals who want to produce high quality bioactive FPH from by-product material.

Acknowledgments This project was funded by the AVS research fund under the Ministry of Fisheries in Iceland (project no. R-047-07). The financing of this work is gratefully acknowledged. The authors would also like to thank associate professor Kazufumi Osako from Tokyo University of Marine Science and Technology for his support and Amano Enzyme Inc. for kindly providing Protease P Amano 6.

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