Food Chemistry 139 (2013) 777–785

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Food Chemistry

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Chemical properties of x-3 fortified gels made of protein isolate recoveredwith isoelectric solubilisation/precipitation from whole fish

Reza Tahergorabi a, Sarah K. Beamer b, Kristen E. Matak b, Jacek Jaczynski b,⇑a Oregon State University, Seafood Research and Education Center, 2001 Marine Dr., Astoria, OR 97103, USAb West Virginia University, Animal and Nutritional Sciences, P.O. Box 6108, Morgantown, WV 26506, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 August 2012Received in revised form 2 January 2013Accepted 29 January 2013Available online 9 February 2013

Keywords:Isoelectric solubilisation/precipitationProtein isolateFish processing by-productsFatty acid profileFlaxseed oilFish oilAlgae oilKrill oilFunctional food products

0308-8146/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2013.01.077

⇑ Corresponding author. Tel.: +1 304 293 1893.E-mail address: Jacek.Jaczynski@mail.wvu.edu (J. J

Protein isolate was recovered from whole gutted fish using isoelectric solubilisation/precipitation (ISP).The objective was to determine chemical properties of heat-set gels made of the ISP protein isolate for-tified with x-3 polyunsaturated fatty acids (PUFAs)-rich oils (flaxseed, fish, algae, krill, and blend). Theextent of the PUFAs increase, x-6/x-3 FAs and unsaturated/saturated FAs ratios, and the indices ofthrombogenicity and atherogenicity depended on specific x-3 PUFAs-rich oil used to fortify protein iso-late gels. Lipid oxidation in x-3 PUFAs fortified gels was minimal, although greater (P < 0.05) than controlgels (without x-3 PUFAs fortification). However, all gels were in the slightly rancid, but acceptable range.The commonly used thiobarbituric-acid-reactive-substances (TBARS) assay to determine lipid oxidationin seafood may be inaccurate for samples containing krill oil due to its red pigment, astaxanthin. Proteindegradation (total-volatile-basic-nitrogen) was greater (P < 0.05) in x-3 PUFAs fortified gels than controlgels. However, all gels were considerably below the acceptability threshold for protein degradation. Theshear stress of x-3 PUFAs fortified gels was generally greater than the control gels and the shear strainwas generally unchanged. This study demonstrates that x-3 PUFAs fortification of protein isolates recov-ered with ISP from fish processing by-products or whole fish has potential application in the develop-ment of functional foods.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Globally, fish provide 15% of the total dietary intake of animalprotein for 4.3 billion people. In 2010, the global consumption offish was 18.6 kg per capita (FAO, 2012). A global decline in marinefish stocks has become a widely disputed and publicized issue.Freeman et al. (2006) estimated that the amount of large marinefish is 10% of their original stocks prior to global industrialisation.It has also been stated that by mid-21st century most commercialfisheries may collapse (Mooney, Nichols, & Elliott, 2002). These de-clines, when coupled with increasing human population necessi-tate a development of processing strategies to maximise therecovery of functional and nutritious fish muscle proteins fromlow-value/underutilized species and fish processing by-products.The recovered proteins would be subsequently used in value-added seafood products destined for human consumption.

Isoelectric solubilisation/precipitation (ISP) allows selective,pH-induced water solubility of muscle proteins with concurrentseparation of lipids and removal of materials not intended for hu-man consumption such as bones, skin and scales. Muscle protein

ll rights reserved.

aczynski).

isolates from fish have thus far been recovered using ISP in a batchmode at the laboratory-scale (Choi & Park, 2002; Kim, Park, & Choi,2003; Kristinsson & Hultin, 2003; Undeland, Kelleher, & Hultin,2002) and pilot-scale (Mireles DeWitt, Nabors, & Kleinholz,2007). ISP processing has been applied to beef and fish processingby-products (Chen & Jaczynski, 2007a; Chen and Jaczynski, 2007b;Mireles DeWitt, Gomez, & James, 2002). Most recently, ISP hasbeen used to recover a muscle protein isolate from chicken meatby-products (Tahergorabi, Beamer, Matak, & Jaczynski, 2011;Tahergorabi, Sivanandan, & Jaczynski, 2011). ISP processing al-lows high protein recovery yields, while significantly reducingfat content (Chen & Jaczynski, 2007b; Taskaya, Chen, Beamer,& Jaczynski, 2009). Recovered protein isolates retain functionalproperties and nutritional value (Chen, Tou, & Jaczynski, 2007,2009; Gigliotti, Jaczynski, & Tou, 2008; Nolsoe & Undeland,2009; Taskaya, Chen, & Jaczynski, 2009; Taskaya, Chen, Beamer,Tou, & Jaczynski, 2009). Due to extreme pH during ISP, this tech-nology results in a non-thermal microbial reduction (Lansdowne,Beamer, Jaczynski, & Matak, 2009a, 2009b). Therefore, ISP offersseveral advantages over mechanical filleting and may be a usefultechnology to recover functional and nutritious protein isolatesfrom whole gutted fish (i.e., without prior filleting) or fish pro-cessing by-products for subsequent application in value-addedfood products.

778 R. Tahergorabi et al. / Food Chemistry 139 (2013) 777–785

The western diet is characterised by the increased dietary in-take of saturated fat, x-6 fatty acids (x-6 FAs), and trans-FAs, aswell as decreased intake of x-3 FAs. As a result, the ratio of x-6FAs to x-3 FAs is at 15–20 to 1, instead of the suggested 1 to 1 (Ea-ton & Konner, 1985; Eaton, Konner, & Shostak, 1988; Simopoulos,1991; Simopoulos, 1999a; Simopoulos, 1999b; Simopoulos,1999c). Alpha-linolenic (ALA, 18:3x-3), eicosapentaenoic (EPA,20:5x-3) and docosahexaenoic acids (DHA, 22:6x-3) are the mainx-3 polyunsaturated FAs (x-3 PUFAs), while linoleic (LA, 18:2x-6)and arachidonic acids (AA, 20:4x-6) are the main x-6 PUFAs inaquatic animals (Chen, Nguyen, Semmens, Beamer, & Jaczynski,2006). There is increasing interest in the fortification of food prod-ucts with x-3 PUFAs because of their health benefits, especially thereduction of cardiovascular disease (CVD) (Nair, Leitch, Falconder,& Garg, 1997). According to the American Heart Association, CVDhas had an unquestioned status of the number one cause of deathin the U.S. since 1921 (American Heart Association, 2009). In 2004,the Food and Drug Administration (FDA) approved a health claimfor reduced risk of CVD for foods containing x-3 PUFAs, mainlyEPA and DHA (FDA, 2004). This provided a marketing leveragefor functional foods fortified with x-3 PUFAs and initiated a devel-opment of food products addressing the diet-driven CVD. Andersonand Ma (2009) provided an up-to-date and comprehensive reviewof health benefits specific for ALA, EPA, and DHA. Since the seafoodproducts developed from the ISP-recovered fish protein isolatewould be formulated products associated with aquatic sources,they are a logical vehicle for increasing the consumption of x-3PUFAs; and therefore, addressing the diet-driven CVD withoutthe need for dietary supplements in a pill or capsule form.

The overall objective of this study was to recover a fish proteinisolate by ISP from whole gutted rainbow trout (bone-in, skin- andscale-on) as a model for fish processing by-products. The ISP-recovered isolate was subsequently used for the development ofheat-gelled functional seafood product fortified with x-3 PUFAs.Specific objectives were to determine (1) FA composition includingindices of thrombogenicity and atherogenicity, (2) lipid oxidation,(3) protein degradation, and (3) fundamental texture properties offish protein isolate gels fortified with x-3 PUFAs-rich oils (flaxseed,fish, algae, krill, and blend (flaxseed:algae:fish, 8:1:1)).

2. Materials and methods

2.1. Sample preparation and recovery of fish protein isolate withisoelectric solubilisation/precipitation

Whole gutted rainbow trout (bone-in, skin- and scale-on) werepurchased from a local aquaculture farm. Whole gutted trout wereused as a model for fish processing by-products. The fish were sub-jected to isoelectric solubilisation/precipitation (ISP) to recovermuscle protein isolate. A processing flowchart for the recovery offish protein isolate and subsequent development of heat-set gelsis shown in Fig. 1.

The fish were ground (meat grinder model 812 with 2.3 mmgrinding plates, Biro, Marblehead, OH) followed by homogenisa-tion with distilled and de-ionised water (dd H2O) at 1:6 ratio(ground fish:water, w:v) using a laboratory homogenizer (Power-Gen 700, Fisher Scientific, Fairlawn, NJ) set at speed five for fiveminutes. During the entire ISP processing, temperature was care-fully controlled at 4 �C. The processing time did not exceed60 min. The homogenisation/mixing was continued with the Pow-erGen homogenizer set at speed three during subsequent pHadjustment steps.

A 6 L aliquot of the homogenate was transferred to a beaker andthe pH was adjusted to 11.50 ± 0.05 with 5 and 0.5 M NaOH (Chen& Jaczynski, 2007a, 2007b; Tahergorabi, Beamer, et al., 2011;

Taskaya, Chen, Beamer, & Jaczynski, 2009). The 5 and 0.5 M re-agents were used for crude and fine pH adjustments, respectively,during both protein solubilisation and subsequent precipitation(pH = 5.5) (see below). Once the desired pH was obtained, the sol-ubilisation reaction was allowed to take place for 10 min, followedby centrifugation at 10,000g and 4 �C for 10 min using a laboratorybatch centrifuge (Sorvall Evolution RC Refrigerated Superspeedcentrifuge equipped with Sorvall Fiber Lite rotor SLC-6000, SorvallCentrifuges, Asheville, NC). The centrifugation resulted in threelayers: top – fish lipids, middle – fish muscle protein solution,and bottom – insolubles (bones, skin, insoluble proteins, mem-brane lipids, etc.).

The fish muscle protein solution was collected and its pH wasadjusted to 5.50 ± 0.05 by 5 and 0.5 M HCl to precipitate the pro-teins. Once the desired pH was obtained, the precipitation reactionwas allowed to take place for 10 min. The solution with precipi-tated proteins was de-watered by centrifugation as above. The cen-trifugation resulted in two layers: top – process water, and bottom– precipitated and de-watered fish proteins isolate. The precipi-tated and de-watered fish protein isolate was collected. The finalmoisture of the isolate was adjusted to 82 g/100 g by manualsqueezing of the isolate wrapped in a cheese cloth. The isolatewas used in the preparation of fish protein isolate paste.

2.2. Preparation of fish protein isolate paste

Fish protein isolate pastes were made using the procedure de-scribed by Jaczynski and Park (2004). The ISP-recovered proteinisolates were chopped in a universal food processor (ModelUMC5, Stephan Machinery Corp., Columbus, OH) at low speed for1 min. A fish protein paste was obtained by extracting myofibrillarproteins in the fish protein isolate with 0.34 M of KCl-based saltsubstitute (AlsoSalt� sodium-free salt substitute, AlsoSalt, MapleValley, WA) and chopping at low speed for 0.5 min in the universalfood processor. This level of salt substitute was found to be opti-mum and similar to salt (NaCl) in terms of protein gelation andendothermal transitions as well as texture and colour in heat-setfish protein gels (Tahergorabi, Beamer, Matak, & Jaczynski, 2012;Tahergorabi & Jaczynski, 2012). The concentration of 0.34 M ofthe salt substitute was equivalent to 2 g of NaCl per 100 g of thefish protein isolate. The salt substitute contained 68 g of KCl per100 g of the salt substitute and L-lysine mono-hydrochloride.According to the manufacturer, the patented L-lysine derivativemasks the metallic-bitter aftertaste of KCl.

The final moisture content of the fish protein isolate paste wasadjusted to 68 g/100 g by adding functional additives at the follow-ing final concentrations: 10 g/100 g of a x-3 PUFAs-rich oil (see be-low), 3.7 g/100 g crab flavor (F-11019, Activ International, Mitry-Mory Cedex, France), 2 g/100 g of potato starch (PS) (Penbind1000 modified potato starch, Penford Food Ingredients Corp., Cen-tennial, CO), 0.5 g/100 g of microbial transglutaminase (MTGase)(Activa RM, Ajinomoto USA Inc., Teaneck, NJ), and 0.3 g/100 g ofpolyphosphate (PP) (Kena FP-28, Innophos, Cranbury, NJ). Theabove levels of functional additives were found in previous studiesas optimal for gelation of ISP-recovered fish protein isolates andconsequently physicochemical properties of heat-set gels as wellas closely resembling commercial surimi-based seafood products(Chen & Jaczynski, 2007b; Perez-Mateos, Boyd, & Lanier, 2004; Tas-kaya, Chen, Beamer, & Jaczynski, 2009; Taskaya, Chen, & Jaczynski,2009; Taskaya, Chen, & Jaczynski, 2010). A 0.5 g/100 g of titaniumdioxide (TiO2) [Titanium (IV) oxide, Sigma–Aldrich, Inc., St. Louis,MO] was also added to the paste (Tahergorabi, Beamer, et al.,2011; Taskaya et al., 2010). TiO2 is commonly added up to1 g/100 g as a whitening agent in food products. The PS, MTGase,TiO2, and PP were in a dry powder form. The crab flavor was awater-soluble liquid. The x-3 PUFAs-rich oils (see below) were

°

°

°

°

Fig. 1. A flowchart for recovery of fish protein isolate using isoelectric solubilisation/precipitation (ISP) and subsequent development of fish protein gels. The gels wereformulated to contain 68 g/100 g moisture, 0.34 M salt substitute, 10 g/100 g x-3 PUFAs oil (flaxseed, fish, algae, krill, or blend), 2 g/100 g potato starch (PS), 0.5 g/100 gmicrobial transglutaminase (MTGase), 0.5 g/100 g titanium dioxide (TiO2) 0.3 g/100 g polyphosphate (PP), and 3.7 g/100 g crab flavor. The paste pH was adjusted to 7.2.

R. Tahergorabi et al. / Food Chemistry 139 (2013) 777–785 779

added at 10 g/100 g by replacing ice/water (1:1) that is normallyadded to a fish protein-based paste such as surimi paste (Park,2005; Perez-Mateos et al., 2004). One treatment with all of theadditives except the oil was used as a control paste. The final mois-ture content of the control paste was adjusted to 78 g/100 g byadding ice/water to the paste (Park, 2005; Perez-Mateos et al.,2004). To mix all of the ingredients with the fish protein isolatepaste, chopping was applied at low speed for 1 min. Addition ofTiO2 to a protein paste results in poor quality of heat-set gel dueto the pH lowering effect of TiO2 (Park, 2005). Therefore, the finalpH of the paste in the present study was adjusted to 7.20 ± 0.05(Tahergorabi, Beamer, et al., 2011; Taskaya et al., 2010). Additionalchopping was performed at high speed under vacuum (50 kPa) forthe last 3 min. The paste temperature was controlled between 1–4 �C during chopping. The fish protein isolate pastes were preparedin 1 kg batches.

The following x-3 PUFAs-rich oils were added during prepara-tion of fish protein isolate pastes:

1) Flaxseed oil was obtained from Jedwards International, Inc.(Quincy, MA).

2) Fish oil (Omega Pure 8042TE) was obtained from OmegaPure (Reedsville, VA).

3) Algae oil (DHAS) was obtained from Martek Biosciences(Columbia, MD).

4) Krill oil (4225F) was obtained from Enzymotec USA, Inc.(Springfield, NJ).

5) Blend (Flaxseed:Algae:Fish, 8:1:1)

The added oils are good sources of x-3 PUFAs and this is whythey were selected in the present study (Kassis, Beamer, Matak,Tou, & Jaczynski, 2010; Kassis, Gigliotti, Beamer, Tou, & Jaczynski,2011; Pietrowski, Tahergorabi, Matak, Tou, & Jaczynski, 2011).Anderson and Ma (2009) provided an up-to-date and comprehen-sive review of the health benefits specific for ALA and DHA. Whenoil is homogenised with a comminuted protein-based paste, itresults in light scattering. Therefore, it improves whiteness of

780 R. Tahergorabi et al. / Food Chemistry 139 (2013) 777–785

heat-set gels (Park, 2005). This is why, besides nutraceuticalbenefits, 10 g/100 g of x-3 PUFAs-rich oil was added to the fishprotein isolate paste in the present study. The fish protein isolatepastes prepared in this manner were used to develop heat-setgels.

2.3. Preparation of fish protein isolate gels

Fish protein isolate paste was stuffed into stainless steel tubes(length = 17.5 cm, internal diameter = 1.9 cm) with screw end capsfor cooking and subsequent determination of fatty acid (FA) com-position including indices of atherogenicity and thrombogenicity,lipid oxidation, and protein degradation. To determine the funda-mental texture properties using the torsion test, the paste wasstuffed into hour-glass pre-molded stainless steel torsion tubes(length = 17.5 cm, end diameter = 1.9 cm, midsection diame-ter = 1.0 cm) with screw end caps and cooked. Since the fish pro-tein isolate pastes were prepared with MTGase, the tubes priorto cooking were incubated at 4 �C for 24 h to allow for the forma-tion of non-disulphide covalent e-(c-glutamyl)-lysine cross-linksor the ‘‘suwari’’ effect. Following incubation, the tubes were heatedin a water bath at 90 �C for 15 min. The tubes were immediatelychilled in ice slush and the fish protein isolate gels were removedfor analysis.

2.4. Fatty acid composition of heat-set fish protein isolate gels

The fatty acid (FA) composition was determined by extractinglipids with acid hydrolysis into ether followed by their methylationto fatty acid methyl esters (FAMEs) (Chen, Nguyen, Semmens,Beamer, & Jaczynski, 2007; Chen, Nguyen, Semmens, Beamer, &Jaczynski, 2008). The FAMEs were quantitatively measured usinga capillary gas–liquid chromatograph (GLC) (Model 7890Aequipped with a 7683B series injector, Agilent Technologies, SantaClara, CA) against an internal standard (C19:1). Helium was used ascarrier gas at 0.75 ml/min flow rate and a 200:1 as split ratio. Theinitial temperature of 100 �C was held for 4 min and then increasedto the final temperature of 240 �C at a heating rate of 3 �C/min. Thefinal temperature was held for 15 min. The injector and detectortemperatures were 225 and 285 �C, respectively. The data are re-ported as mean values (±standard deviation) of at least three rep-licates and the mean values are expressed as percent of a fatty acidin total fatty acids.

2.5. Indices of atherogenicity and thrombogenicity

Based on the FA composition of heat-set fish protein isolate gels,the indices of atherogenicity (IA) and thromogenicity (IT) were cal-culated. The IA relates main saturated fatty acids (SFAs) to unsatu-rated fatty acids (UFAs). SFAs favour adhesion of lipids to cells ofthe immunological and circulatory system; and therefore, are con-sidered pro-atherogenic. UFAs inhibit the aggregation of plaques,thereby reduce the risk of CVD; and therefore, are consideredanti-atherogenic (Senso, Suarez, Ruiz-Cara, & Garcia-Gallego,2007; Ulbritch & Southgate, 1991). The following equation wasused to calculate the IA:

IA ¼ ð4� C14 : 0Þ þ C16 : 0þ C18 : 0P

MUFAþP

x� 6PUFAþP

x� 3PUFA

The IT indicates the tendency to form clots in blood vessels. TheIT relates pro-thrombogenic SFAs to anti-thrombogenic FAs(Mono-UFAs, x-6 PUFAs, and x-3 PUFAs) (Senso et al., 2007;Ulbritch & Southgate, 1991). The following equation was used tocalculate the IT:

IT ¼ C14 : 0þ C16 : 0þ C18 : 0ð0:5�MUFAÞ þ ð0:5�x� 6PUFAÞ þ ð3�x� 3PUFAÞ þ x�3PUFA

x�6PUFA

2.6. Lipid oxidation of heat-set fish protein isolate gels

Oxidative rancidity of gel samples was measured by a 2-thio-barbituric acid reactive substances (TBARS) assay of malondialde-hyde (MDA) as previously described (Chen et al., 2008; Jaczynski& Park, 2003). The absorbance was measured at 535 nm using anUV/Vis spectrophotometer (model DU530, Beckman Instruments,Fullerton, CA). The TBARS values were calculated using molarabsorptivity of MDA (156,000 M�1 cm�1) at 535 nm. The TBARSvalues are reported as mean values (±standard deviation) of atleast three replicates and the mean values are expressed as mgof MDA per kg of heat-set gel sample.

2.7. Protein degradation of heat-set fish protein isolate gels

Protein degradation of gel samples was determined with totalvolatile basic nitrogen (TVBN) assay. A 10 g sample of fish proteingel was homogenised with 200 ml of dd H2O. One drop of siliconeand 2 g of magnesium oxide were added to prevent foaming. Themixture was distilled in a Micro-Kjeldahl unit and the distillatewas titrated with 0.05 M HCl. The TVBN values are reported asmean values (±standard deviation) of at least three replicates andthe mean values are expressed as mg of nitrogen per 100 g ofheat-set gel sample.

2.8. Fundamental texture properties of heat-set fish protein isolate gels

The torsion test of gel samples was performed according to Tah-ergorabi, Beamer, et al. (2011). At least seven hour-glass-shapedgels (length = 2.54 cm, end diameter = 1.9 cm, and midsectiondiameter = 1.0 cm) per treatment were glued to plastic discs andsubjected to torsional shear using a Hamman Gelometer (Gel Con-sultant, Raleigh, NC) set at 2.5 rpm. Shear stress and shear strain atmechanical fracture are fundamental texture properties indicatinggel strength and gel cohesiveness, respectively (Kim, Park, & Yoon,2005).

2.9. Statistics

The experiments were independently triplicated (n = 3). In eachtriplicate at least three measurements were performed for FA com-position, three for TBARS, three for TVBN, and eight for torsion test.Data were subjected to one-way analysis of variance (ANOVA). Asignificant difference was determined at 0.05 probability leveland differences between treatments were tested using the Fisher’sLeast Significant Difference (LSD) test (Freud & Wilson, 1997). Allstatistical analyses of data were performed using SAS (SAS Insti-tute, 2002). The data are reported as mean values ± standard devi-ation (SD).

3. Results and discussion

3.1. Fatty acid composition of heat-set fish protein isolate gels

Fatty acid (FA) composition of fish protein isolate gels fortifiedwith x-3 PUFAs-rich oils and control gels (i.e., without added x-3 PUFAs-rich oil) is presented in Fig. 2. The ratio of x-6/x-3 FAs,unsaturated/saturated FAs, as well as indices of atherogenicity(IA) and thrombogenicity (IT) are tabulated in Table 1. The fortifiedfish protein isolate gels had higher content (P < 0.05) of total x-3PUFAs (34–51% of total FAs) than the control gels (20%). However,

% F

atty

aci

d in

tota

l fat

ty a

cids

%F

atty

aci

d in

tota

l fat

ty a

cids

A

B

Fig. 2. Major fatty acids⁄ (FAs) of heat-set fish protein isolate gels with and without (control) added oil (blend was flaxseed: algae: fish, 8:1:1). ⁄Data are given as meanvalues ± standard deviation (n = 3). Different letters on data bars indicate significant differences (Fisher’s Least Significant Difference, P < 0.05) between mean values withinthe same FA. ⁄⁄(A): ALA – linolenic (18:3x3), EPA – eicosapentaenoic (20:5x3), DHA – docosahexaenoic (22:6x3), LA – linoleic (18:2x6), and AA – arachidonic (AA, 20:4x6)fatty acids. (B): x-3 – total x-3 FAs, x-6 – total x-6 FAs, SFA – total saturated FAs, and UFA – total unsaturated FAs.

Table 1The ratios* of omega-6/omega-3 fatty acids (x-6/x-3) and unsaturated/saturated fatty acids (UFAs/SFAs), index* of thrombogenicity (IT) and index* of atherogenicity (IA)determined for heat-set fish protein isolate gels with and without (control) added oil (blend was flaxseed: algae: fish, 8:1:1).

Control Flax Fish Algae Krill Blend

x-6/x-3 0.63 ± 0.00a 0.32 ± 0.00b 0.12 ± 0.00d 0.07 ± 0.00f 0.11 ± 0.01e 0.29 ± 0.00cUFAs/SFAs 1.85 ± 0.00f 8.01 ± 0.05a 2.32 ± 0.02e 3.51 ± 0.01d 3.79 ± 0.01c 6.10 ± 0.06bIT 0.41 ± 0.00a 0.05 ± 0.00e 0.21 ± 0.02b 0.12 ± 0.00c 0.11 ± 0.00c 0.08 ± 0.00dIA 0.62 ± 0.00c 0.11 ± 0.00f 0.73 ± 0.03a 0.69 ± 0.01b 0.34 ± 0.00d 0.22 ± 0.02e

* Data are given as mean values ± standard deviation (n = 3). Different letters indicate significant differences (Fisher’s Least Significant Difference, P < 0.05) between meanvalues within the same row.

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the control gels were made of the ISP-recovered fish protein isolatewithout added x-3 PUFAs-rich oil. The ISP isolates normally con-tain very low amount of fat, usually below 2 g/100 g (‘‘as-is’’ basis)(Taskaya, Chen, Beamer, & Jaczynski, 2009; Taskaya, Chen, & Jac-zynski, 2009; Taskaya et al., 2009). Thus, the control gels in thepresent study had correspondingly low amount of fat. Althoughthe control gels had 20% of x-3 PUFAs in total FAs, they had verylow absolute amount of these health beneficial FAs due to thelow amount of fat in the ISP isolate. In contrast, the fortified fishprotein isolate gels had 10 g/100 of x-3 PUFAs-rich oil that wasadded during paste formulation before cooking. The highest(P < 0.05) content of x-3 PUFAs was in gels fortified with flaxseed

oil (51%), followed by blend (49%), krill (46%), algae (43%), and fishoil (34%).

Flaxseed oil is a rich source of ALA (52–55 g/100 g) (Ansorena &Astiasaran, 2004). Thus, the gels fortified with flaxseed oil and theblend (8% flaxseed oil) had the highest (P < 0.05) content of ALA(49% and 39%, respectively) compared to other x-3 PUFAs fortifiedgels (fish – 2.5%, krill – 1.2%, and algae – 0.5%). Pietrowski et al.(2011) reported similar results for surimi gels fortified with com-parable levels of x-3 PUFAs-rich oils. Singh, Chatli, Biswas, andSahoo (2011) also showed comparable data for x-3 fortified chick-en meat patties. Gels fortified with krill and fish oil had the great-est (P < 0.05) content of EPA (24 and 16%, respectively), whereas all

Fig. 3. Thiobarbituric reactive substances (TBARS) values⁄ of heat-set fish proteinisolate gels with and without (control) added x-3-rich oils. ⁄Data are given as meanvalues ± standard deviation (SD) (n = 3). The small bars of the top of data barsindicate SD. Different letters on the top of SD bars indicate significant differencesbetween mean values (Fisher’s Least Significant Difference, P < 0.05).

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other gels contained less than 3% of EPA. Krill and fish oils are verygood sources of EPA and DHA; while algal oils contain DHA as theirmain x-3 PUFA (Gigliotti, Davenport, Beamer, Tou, & Jaczynski,2011; Tou, Jaczynski, & Chen, 2007). The DHA content was thehighest (P < 0.05) in gels fortified with algal oil (41%), followedby krill (19%), fish (11%), blend (7%), and flaxseed oil (2%). As men-tioned before with the total x-3 PUFAs, although control gels (i.e.,without added x-3 PUFAs-rich oil) had 15% of DHA in total FAs;due to their very low amount of fat, they contained very low abso-lute amount of DHA. It is important to mention that EPA and DHAin krill oil are esterified in phospholipids (Gigliotti et al., 2011; Kas-sis et al., 2011). This is in contrast to all other EPA- and DHA-richoils used in the present study. Fish and algae oils have EPA andDHA esterified in triglycerides. This may have important physio-logical implications in humans and in turn contribute differentlyto the health benefits associated with these x-3 PUFAs (Amate,Gil, & Ramirez, 2001, 2002).

Gels fortified with flaxseed and blend oil had the highest(P < 0.05) content of LA (16% and 10%, respectively); while gels for-tified with krill, fish, and algae oil had under 3% of LA. Conse-quently, flaxseed and blend oil resulted in the highest (P < 0.05)content of the total x-6 PUFA (17% and 14%, respectively) in thefish protein isolate gels.

As a result of differences in FA composition between gels, theratios of x-6/x-3 FAs and un-saturated/saturated FAs (UFAs/SFAs)were also different (P < 0.05). Although significantly different(P < 0.05); gels fortified with algae, krill, and fish oil showed thelowest x-6/x-3 FAs ratio (0.07, 0.11, and 0.12, respectively), fol-lowed by gels with added blend and flaxseed oil (0.29 and 0.32,respectively). Not only is flaxseed oil is rich in ALA (i.e., x-3 PUFA),but it also has high LA (i.e., x-6 PUFA) content. The high content ofLA in flaxseed oil compared to much lower LA content in other oilsused in the present study resulted in the highest (P < 0.05) x-6/x-3FAs of gels fortified with flaxseed and blend oil. Although flaxseedoil resulted in the highest x-6/x-3 FAs ratio, it is still much lowerthan 1/1. It has been stated that in western diet the ratio of x-6/x-3 FAs ranges 15–20/1 instead of the suggested 1/1 (Simopoulos,1999b). Thus, consumption of the fish protein isolate gels fortifiedwith any of the oils used in the present study including flaxseed oilwould improve the currently high x-6/x-3 FAs ratio. The UFAs/SFAs ratio was the highest (P < 0.05) for gels with flaxseed andblend oil (8.0 and 6.1, respectively); while fortification with krill,algae, and fish oils resulted in lower (P < 0.05) ratios (3.8, 3.5,and 2.3, respectively). Although the latter three oils resulted inlower UFAs/SFAs ratios in the gels, they still contained 2–4 timesas much UFAs as SFAs.

3.2. Indices of atherogenicity and thrombogenicity of heat-set fishprotein isolate gels

The index of thrombogenicity (IT) and index of atherogenicity(IA) account for different effects that single FAs have on humancardiovascular health. In particular, IT and IA indicate the probabil-ity of changes in the incidence of degenerative/pathological devel-opments associated with cardiovascular disease (CVD) (Cahu,Salen, & de Lorgeril, 2004; Ulbritch & Southgate, 1991). In the pres-ent study, the control gels made of protein isolates recovered withISP from whole gutted trout had the highest (P < 0.05) IT at 0.41,but the IA for control gels was not the highest (0.62) (Table 1). Val-fre, Caprino, and Turchini (2003) reported the IT and IA at 0.37 and0.57 for farm-raised rainbow trout, respectively. Although signifi-cantly different; the gels fortified with flaxseed, blend, and krilloil had the lowest (P < 0.05) IT and IA (flaxseed IT = 0.05 andIA = 0.11, blend IT = 0.08 and IA = 0.22, and krill IT = 0.11 andIA = 0.34); while the fortification with algae and fish oil had higher(P < 0.05) IT and IA (algae IT = 0.12 and IA = 0.69, fish IT = 0.21 and

IA = 0.73). The IT and IA determined in the present study are lowerthan those previously reported in the literature for beef and chick-en (Ulbritch & Southgate, 1991). This is indicative of likely greatercardiovascular benefits for the gels made of ISP fish protein isolatefortified with x-3 PUFAs oil. However, clinical trials would need tobe conducted to elucidate the extent of the actual benefits.

3.3. Lipid oxidation of heat-set fish protein isolate gels

The x-3 PUFAs are highly susceptible to lipid oxidation, whichcan lead to flavor and colour deterioration. Not only are endoge-nous antioxidants and FAs oxidised, but proteins are also degraded(Nolsoe & Undeland, 2009). The development of oxidative rancidityin seafood is typically measured with the thiobarbituric acid reac-tive substances (TBARS) assay (Hyldig & Nielsen, 2001; Tarladgis,Watts, Younathan, & Dugan, 1960).

The gels fortified with krill oil were the most (P < 0.05) suscep-tible to oxidation (Fig. 3). These gels had high content of EPA andDHA (Fig. 2), which may account for their high oxidation. However,gels fortified with algae oil had very slightly lower content of thetotal x-3 PUFAs. The algae oil was mainly composed of DHA, whichis even more unsaturated than krill oil; yet, the gels fortified withalgae oil showed lower (P < 0.05) oxidation. Krill oil has very highcontent of astaxanthin, a deep red pigment. Astaxanthin is also apotent antioxidant (Tou et al., 2007). The TBARS assays spectropho-tometrically determines malondialdehyde (MDA) by measuring acolour change. The colour changes to higher intensity pink as moreMDA develops due to increased oxidation. It is suggested thatTBARS assay may not be appropriate to measure oxidation of foodsamples containing krill oil because this oil is red which is veryclose to the pink colour of MDA. Astaxanthin is likely an interferingcompound for TBARS assay and probably results in erroneous over-estimation for oxidation of krill oil. In fact, astaxanthin likely re-tards oxidation of krill oil. The gels fortified with flaxseed, blend,and fish oil had lower (P < 0.05) TBARS than the gels with krilloil, but higher (P < 0.05) than the control gels. The control gelswere not fortified with x-3 PUFAs-rich oil and had only residualfat present in the ISP fish protein isolate. This most likely explainsthe low TBARS for the control gels.

Ke, Cervantes, and Robles-Martinez (1984) proposed that TBARSvalues for seafood products below 0.58 mg/kg are perceived as notrancid; 0.58–1.51 mg/kg slightly rancid, but acceptable; and above1.51 mg/kg rancid. Using these ranges, all gels in the present studywould be perceived as slightly rancid, but acceptable. These resultsare in agreement with Pietrowski et al. (2011) who studied lipidoxidation in surimi gels fortified with x-3 PUFAs-rich oils.

Fig. 5. Torsion shear stress⁄ and strain⁄ of heat-set fish protein isolate gels with andwithout (control) added x-3-rich oils (d – shear stress; s – shear strain). ⁄Data aregiven as mean values ± standard deviation (SD) (n = 3). Bars on data points indicateSD. Different letters on the top of SD bars indicate significant differences (Fisher’sLeast Significant Difference, P < 0.05).

R. Tahergorabi et al. / Food Chemistry 139 (2013) 777–785 783

3.4. Protein degradation of heat-set fish protein isolate gels

The formation of total volatile basic nitrogen (TVBN) indicatesprotein degradation due the release of trimethylamine and ammo-nia from the protein by spoilage microorganisms (Benjakul, Vises-sanguan, Riebroy, Ishizaki, & Tanaka, 2002; Gram & Huss 1996).The TVBN content of control gels was 4.3 mg N/100 g, whereas gelsfortified with x-3 PUFAs-rich oils had higher (P < 0.05) TVBN (7.1–7.6 mg N/100 g) (Fig. 4). TVBN content of 30 mg N/100 g is re-garded as the fish acceptability limit (Sikorski, Kolakowska, & Burt,1990). All gels in the present study had TVBN well below this limit.This indicates that the difference between control and fortified gelsdid not have a practical meaning. Balange, Benjakul, and Maqsood(2009) also reported similar results for the mackerel mince andmackerel surimi. They concluded that TVBN was reduced by thealkaline washing process. Lansdowne et al. (2009a, 2009b) demon-strated that ISP processing of fish results in microbial reductiondue to alkaline and acidic pH. The alkaline pH (11.5) used in thepresent study during ISP likely inactivated spoilage microorgan-isms that normally increase TVBN; and thus, contributed to lowTVBN of the gels.

3.5. Fundamental texture properties of heat-set fish protein isolate gels

A torsion test is considered as an objective method to measuremechanical properties of protein-based gels (Hamman & Lanier,1987; Kim, Hamman, Lanier, & Wu, 1986; Kim et al., 2005). Thistest allows determination of shear stress and strain at mechanicalfracture that correlate with gel strength and cohesiveness, respec-tively. Gel strength and cohesiveness are critical quality parame-ters for restructured gelled products.

The shear stress and strain of heat-set fish protein isolate gelswith and without x-3 PUFA fortification are shown in Fig. 5. Theseresults show that fortification with x-3 PUFA-rich oil within theranges tested in the present study generally improved gel strength,but did not seem to change gel cohesiveness. The addition of algae,flaxseed, and fish oil increased (P < 0.05) shear stress, while krilland blend oil resulted in similar (P > 0.05) shear stress. The effectof oil on gel strength probably depends on the type of oil (unsatu-ration, length of FA chain, etc.) added to the protein isolate. Krill oilhas high content of phospholipids and this factor might have con-tributed to the differences in shear stress between gels fortifiedwith krill oil and other oils in the present study study. Pietrowskiet al. (2011) reported similar results for surimi gels fortified withx-3 PUFA-rich oils. Xiong (1992) and Foegeding, Xiong, and Brekke

Fig. 4. Total volatile basic nitrogen (TVBN) values⁄ of heat-set fish protein isolategels with and without (control) added x-3-rich oils. ⁄Data are given as meanvalues ± standard deviation (SD) (n = 3). The small bars of the top of data barsindicate SD. Different letters on the top of SD bars indicate significant differencesbetween mean values (Fisher’s Least Significant Difference, P < 0.05).

(1991) demonstrated that strength of gel made with myofibrillarproteins improves when fat is added. It has been reported that fi-nely comminuted fat globules behave as protein co-polymers fill-ing the voids within the protein gel matrix; and thus, result inreinforcement of the composite gel (Ziegler & Foegeding, 1990).Wu, Xiong, Chen, Tang, and Zhou (2009) found that gel strength in-creases with the concentration of added lipids. In contrast to theresults of the present study, Wu et al. (2009) reported no difference(P > 0.05) for gels made of porcine protein with the addition of10 g/100 g of lard and peanut oil. This might have been causedby the differences between aquatic and terrestrial animal proteinsas well as different types of oil used in the two studies.

4. Conclusions

Isoelectric solubilisation/precipitation (ISP) at alkaline pH wasused to recover protein isolate from whole gutted rainbow trout(bone-in, skin- and scale-on). Fortification of the ISP-recovered fishprotein isolate with x-3 polyunsaturated fatty acids (PUFAs) richoils (flaxseed, fish, algae, krill, and blend) resulted in increased(P < 0.05) content of alpha-linolenic (ALA, 18:3x-3), eicosapentae-noic (EPA, 20:5x-3) and docosahexaenoic acids (DHA, 22:6x-3) inthe cooked protein isolate gels. The extent of the PUFAs increase,x-6/x-3 FAs and unsaturated/saturated FAs ratios, as well as theindices of thrombogenicity and atherogenicity depended on spe-cific x-3 PUFAs-rich oil used to fortify the protein isolate gels. Lipidoxidation and protein degradation were slightly higher (P < 0.05) inthe x-3 PUFAs fortified gels than the control gels. However, all gelswere within acceptable range. The shear stress of the x-3 PUFAsfortified gels was generally greater than the control gels and shearstrain was generally not altered by the fortification.

This study indicates a potential application for protein isolatesrecovered with ISP from fish processing by-products or whole fishwithout prior filleting. Fortification of the ISP protein isolate withx-3 PUFAs-rich oils allows the development of a functional sea-food product.

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