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LWT - Food Science and Technology

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Evaluation of antioxidant properties of Chlorella vulgaris and Spirulinaplatensis and their application in order to extend the shelf life of rainbowtrout (Oncorhynchus mykiss) fillets during refrigerated storage

Mir Babak Taghavi Takyara, Shabnam Haghighat Khajavia,∗, Reza Safarib

a Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iranb Caspian Sea Ecology Research Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Mazandaran, Iran

A R T I C L E I N F O

Keywords:DPPHFRAPAntioxidant capacitySensory evaluation

A B S T R A C T

The present study was undertaken to evaluate the antioxidant effect of alcoholic extract of Chlorella vulgaris andSpirulina platensis on extending the shelf life of rainbow trout (Oncorhynchus mykiss) fillet in 4 ± 1 °C. Theexperiments were performed on 5 samples (1 blank and 4 treatments) and in triplicate. The results from DPPHand FRAP assays represents the higher antioxidant activity of Chlorella (42.96% and 0.33) at concentration of200 μg/ml and Spirulina (62.46% and 0.41; 77.12% and 0.46) at concentrations of 400 and 600 μg/ml, re-spectively. The results from performed chemical tests compared to the blank sample showed that samples treatedwith algae extracts were able to control the increase in these parameters. Among the treated samples, maximumcontrol of peroxide value, thiobarbituric acid and free fatty acid, respectively pertained to the treatmentscontaining 0.1% of Spirulina (3.14, 2.93 and 2.25) and Chlorella (3.21, 3.06 and 2.28) extracts. The results ofsensory evaluation showed that except the sample containing 0.05% of Chlorella, in other samples containing thealgae extract, less negative effects on color, odor, texture and taste were observed. Consequently, Chlorella andSpirulina extracts can delay lipid oxidation process and maintain or improve sensory properties of refrigeratedfish.

1. Introduction

Fish is a valuable source of protein and contains high amounts of ω-3 polyunsaturated fatty acids (PUFAs) which play an important role in ahealthy diet (Avadi & Freon, 2015). Generally, due to the high amountsof free amino acids, volatile basic nitrogen and PUFAs in fish, they aremore perishable compared to red meat and poultry meat (Hoel,Jakobsen, & Vadstein, 2017). Fat oxidation is one of the most importantreasons for food quality deterioration which leads to changes in color,taste, nutrional value and shelf life of food (Pike & O'Keefe, 2017).Oxidation of PUFAs causes off-flavour and rancid taste due to the for-mation of aldehydes and ketones, reduces the nutrional value by de-struction of essential fatty acids, lipid soluble vitamins and has adverseeffects on human health due to the formation of free radicals (Ikape &Cheikyula, 2017; Miyashita, Uemura, & Hosokawa, 2018). Anti-oxidants, when present at lower concentrations than the substance,prolong or inhibit oxidation of oxidizable substances (Kasote, Katyare,Hegde, & Bae, 2015). The ability to generate a new radical afterscavenging the radical that is stable within intramolecular hydrogen

bonding is another property of a compound to be considered an anti-oxidant (Carocho & Ferreira, 2013). Synthetic antioxidants are mainlyphenolic compounds from which BHT, BHA, TBHQ and PG are mostcommonly used in food industry (Pelle & Compagnone, 2018). Adversehealth problems such as cancer and birth defects can be undesirableeffects of persistent consumption of these antioxidants and nowadaysthe use of natural antioxidants as an alternative to synthetic anti-oxidants is recommended (Tavakoli, Brewer, Zaraei-Jelyani, & Estakhr,2017). A chain oxidation reaction of PUFA starts by formation of peroxyradicals and continues by formation of non-radical products such asaldehydes and alkanes. Formation of aldehydes has adverse effects onfood quality, stability and functionality (Falowo, Fayemi, & Muchenje,2014). Since chain oxidation reactions start with peroxide formation,any compound which has the ability of inhibiting peroxide formationcan be considered as an antioxidant. Algae contain antioxidant com-pounds which are efficient oxygen free radical scavengers and can in-hibit the oxidation of fatty acids (Renugadevi, Valli Nachiyar, Sowmiya,& Sunkar, 2018). Antioxidant activities of phenolic compounds aremainly due to their redox properties that play an important role in

https://doi.org/10.1016/j.lwt.2018.10.079Received 17 July 2018; Received in revised form 21 October 2018; Accepted 25 October 2018

∗ Corresponding author.E-mail address: sh.h.khajavi@gmail.com (S. Haghighat Khajavi).

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Available online 26 October 20180023-6438/ © 2018 Published by Elsevier Ltd.

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capturing and scavenging free radicals, oxygen suppression and per-oxide decomposition (Martins, Barros, & Ferreira, 2016). Algae are agood source of phenolic compounds (Machu et al., 2015) and there is aclose positive correlation between the amount of these compounds inextracts and their antioxidant capacity (Del Pilar Ramirez-Anaya,Samaniego-Sanchez, Castaneda-Saucedo, Villalon-Mir, & Lopez-Garciade la Serrana, 2015). There are various assays which can be used forantioxidant activity determination in vitro; among these methods,chemical assays based on free radical scavenging ability are widelyused. As examples, ABTS, DPPH and FRAP are spectrophotometric as-says which determine the antioxidant capacity of samples indirectlyand based on electron transfer reactions (Teixeira, Vale, Almeida,Ferreira, & Guimaraes, 2017). Using the above-mentioned methods candetermine the phenolic extracts efficiency on prevention of lipid oxi-dation in fish and fishery products (Maqsood, Benjakul, Abushelaibi, &Alam, 2014). Chlorella vulgaris and Spirulina platensis are among thealgae that are generally recognized as safe (GRAS) by the FDA (Bauer,Vieira Costa, Centeno da Rosa, & Santos, 2017). Chlorella vulgaris is afreshwater green microalgae that contains high amounts of chlorophyllpigments and the essential amino acids; furthermore it contains con-siderable amounts of calcium, phosphorus, iodine, manganese, iron andvitamins such as A, B1, B2, B3, B6, B12, C and E (Safi, Zebib, Merah,Pontalier, & Vaca-Garcia, 2014). Spirulina platensis is a planktonic blue-green microalgae that contains phycobilins, considerable amounts ofbeta carotene and vitamins such as A, B1, B2, B3, B6, B12, E and K. Themain active ingredient of Spirulina called phycocyanin is an antioxidantthat creates the characteristic blue-green color (Papadaki,Kyriakopoulou, Tzovenis, & Krokida, 2017).

Spirulina and Chlorella are extensively used in the medical industriesas cholesterol-reducer, cancer-protective, cardiovascular disease pre-vention, anti-aging and immune system enhancer agent (Hynstovaet al., 2018). They are also used for prevention of fat oxidation in foodproducts (Andrade, Andrade, Dias, Nascimento, & Mendes, 2018);hence, the purpose of this study was to evaluate the antioxidant prop-erties of Chlorella vulgaris and Spirulina platensis in order to extend theshelf life of rainbow trout (Oncorhynchus mykiss) fillet stored in 4 ± 1 ͦC.

2. Materials and methods

2.1. Materials

Spirulina platensis (SP) and Chlorella vulgaris (CV) powders werepurchased from Caspian Sea Ecology Research Center (Sari, Iran).Rainbow trout (Oncorhynchus mykiss) was obtained from a local market.1,1-diphenyl-2-picrylhydrazyl (DPPH), phosphate buffer, potassiumferricyanide, trichloroacetic acid, ferric chloride, potassium iodide,sodium thiosulfate, 1-buthanol, thiobarbituric acid, potassium hydro-xide, ethanol, acetic acid, chloroform, diethyl ether, starch and phe-nolphthalein were purchased from Merck Co. (Darmstadt, Germany).

2.2. Preparation of extracts

Amount of 25 g of each algae powder was mixed with 250ml of 96%ethanol and put in a Soxhlet apparatus for 24 h in order to extract thealgae active compounds. The obtained extracts were filtered withWhatman No.42 filter paper and then evaporated using a rotary va-cuum evaporator (EYELA Auto jack NAJ-100, Tokyo, Japan) at 45 °C.Finally the extracts were dried in a vacuum-drying oven at 55 °C for48 h (Saranya, Hemalatha, Parthiban, & Anantharaman, 2014).

2.3. Preparation of samples

Rainbow trout with an average weight of 300–350 g were decapi-tated, deboned and filleted by hand. Four fillets with similar size of5 cm×5 cm×3 cm (length×width× thickness) were obtained from

each fish. Concentrations of 0.05% and 0.1% of each algae extract wereprepared and added to the fish fillets using a sampler (Eppendorf,Hamburg, Germany). All samples were prepared in triplicate. Treatedfillets were then separately packaged in polyethylene zipper bags andstored at 4 ± 1 ͦ C in refrigerator. Chemical and sensory analysis wereperformed at 4-day intervals to evaluate the overall quality of the fish(Pezeshk, Rezaei, & Hosseini, 2011). Four treatments and a blanksample were tested.

2.4. Antioxidant assay

2.4.1. DPPH radical-scavenging assayAccording to this method, 1.5 ml of DPPH stock solution was added

to the algae extract and mixed together. The solution was kept at roomtemperature in the dark for 30min. Then its absorbance was measuredat 517 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto,Japan). Ethanol was used as blank. The inhibition percentage of DPPHby the samples were calculated according to the below equation (1) inwhich Ac and As are the blank absorption and the sample absorption,respectively (Ortiz-Viedma et al., 2017). The stock solutions wereprepared in concentrations of 200, 400 and 600 μg/ml. The experimentswere performed in triplicate for each algae sample.

= ×I Ac AsAc

(%) – 100 (1)

2.4.2. Ferric reducing antioxidant powerIn this method, 0.5ml of each algae extract were mixed with 2.5ml

of 0.2mol/L phosphate buffer (pH=6.6) and 2.5ml of 1% potassiumferricyanide solution. The mixture was then placed in a 50 °C oven for20min. Afterwards 2.5 ml of 10% trichloroacetic acid was added andthe mixture was then centrifuged at 3000 rpm for 10min. Supernatantwas mixed with 2.5ml of distilled water and 2.5 ml of 0.1mol/L ferricchloride solution. The absorption measurements were performed at700 nm using a spectrophotometer (Ahmed, Elkhateeb, Taie, Rateb, &Fayad, 2017). Stock solutions were prepared in concentrations of 200,400 and 600 μg/ml. The experiments were performed in triplicate foreach algae sample.

2.5. Chemical analysis

2.5.1. Determination of peroxide value (PV)Amount of 5ml of fish oil was weighted in a 125ml Erlenmeyer

flask and 25ml of acetic acid:chloroform solution (3:2, v/v) was added.Afterwards, 0.5 ml of saturated potassium iodide, 30ml of distilledwater and 0.5 ml of starch reagent were added to the solution. Thereleased iodine was titrated with 0.01 N sodium thiosulphate. Peroxidevalue was calculated based on meq of H2O2 per kg of fat by the belowequation (2) in which V is the volume (ml) of sodium thiosulphate, N isthe normality of this solution and W is the weight (g) of the fish oil(Bahram et al., 2016).

=× ×PV V N

W1000

(2)

2.5.2. Determination of thiobarbituric acid reactive substances (TBARS)TBA measurement was performed using a colorimetric method. In

this experiment, 200mg of minced fish meat was placed in a 25mlvolumetric flask and filled with 1-butanol and 5ml of this solution wasplaced in a capped test tube with 5ml of TBA reagent that was added tothe solution. Test tubes were then placed in an oven at 95 °C for 2 h.Then left to cool to room temperature. The absorbance of the solutionwas measured at 530 nm using a spectrophotometer while distilledwater was used as blank. TBA value (mg of malondialdehyde per kg offish tissue) was calculated according to the below equation (3) in whichAs and Ac are the sample absorption and the blank absorption,

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respectively (Joukar, Hosseini, Moosavi-Nasab, Mesbahi, & Behzadnia,2017).

=×As AcTBA ( – ) 50

200 (3)

2.5.3. Determination of free fatty acids (FFA)In this method, 20ml of 96% ethanol:diethyl ether solution (1:1, v/

v) was added to 5 g of the fish oil sample. Then phenolphthalein wasadded to the solution. Finally 0.1 N KOH solution was gradually addedand the acid value (determined according to the AOCS official methodca 5a-40 as oleic acid (AOCS, 2012)) was calculated according to thebelow equation (4) in which V is the volume (ml) of KOH solution, N isthe normality of this solution and W is the weight (g) of the fish oilsample (Truong, Buckow, Nguyen, & Stathopoulos, 2016).

=× ×FFA V N

W28.2

(4)

2.6. Sensory evaluation

Although the 9-point scale is used in several food products as dairyand meat products, the 5-point scale was used because there is thepotential problem of “end use avoidance” associated with the 9-pointhedonic scale which means that consumers avoid expressing extremereactions; hence, the scales are sometimes reduced to 7 or even 5 points(Lawless & Heymann, 2010). Therefore, in this study the sensory eva-luation were performed using a 5-point hedonic scale rating (1=dis-like extremely to 5= like extremely) (Farouk et al., 2018). At specifiedtime intervals, 120 g of the fillet was cooked in microwave oven for2min and organoleptic properties of the samples including color, odor,texture and taste were evaluated by 6 semi-trained panelists (3 womenand 3 men, ages: 25–35 years).

2.7. Statistical analysis

All experiments were carried out in triplicate and the informationobtained was processed using the statistical program SPSS version 22(SPSS Inc., Chicago, IL, USA). All experimental data were comparedusing Duncan's multiple range tests (P < 0.05) to define the statisticalsignificance. Sensory scores were analyzed using Kruskal-Wallis andMann-Whitney non-parametric statistical tests.

3. Results and discussion

3.1. Antioxidant capacity

DPPH and FRAP assays were used to assess the antioxidant capacityof SP and CV extracts. Analyzing the obtained results shows that theincrease in concentration for both algae led to an increase in their an-tioxidant power. In concentration of 200 μg/ml no significant differ-ences were observed between the antioxidant activities of CV and SPextracts, but in concentrations of 400 and 600 μg/ml, SP extract pos-sessed significantly higher antioxidant activities in comparison to CVextract (P < 0.05). According to the obtained results, the antioxidantactivity of extracts increased in a dose-dependent manner. Higher an-tioxidant activity of SP extract compared to CV extract can be due to thehigher amounts of phenols, tannins, flavonoids and terpenoids. HighestDPPH free radical scavenging activity and FRAP value was shown by SPextract in concentration of 600 μg/ml, whereas the lowest was for bothalgae in concentration of 200 μg/ml (Table 1). Microalgae contain highlevels of phenolic compounds which contribute to the antioxidant ac-tivity of their extracts. Agregan et al. (2018) reported that antioxidantcapacity of SP extract is higher than that of CV, which can be justifiedby presence of higher levels of polyphenols in this algae and was inagreement with current study. Shalaby and Shanab (2013) reported

that high antiradical activity of methanolic extract of SP is due to thepresence of phycobilin pigments, phenolic and flavonoid compounds.Similarly, Chatzikonstantinou et al. (2017) reported that aqueous ex-tracts of Chlorella strains display varied antioxidant activities andamong them CV extract showed considerable antioxidant potential dueto its bioactive molecules.

3.2. Peroxide value (PV)

The obtained results showed that the PVs of the blank and samplescontaining 0.05% and 0.1% of CV and SP extracts increased at sig-nificantly different rates during the storage period (P < 0.05). The PVsof all the samples decreased significantly from day 12 to day 16 of therefrigerated storage. The observed reduction is due to the progress ofoxidation and the creation of secondary oxidation products. The PVs ofthe blank samples were higher than those of treated samplesthroughout this period. The samples treated with 0.05% of CV and SPextracts had similar change trends and significantly higher PVs incomparison to the samples containing 0.1% of each extracts throughoutthe refrigerated storage. Additionally, the samples treated with 0.1% ofCV and SP extracts had significantly lower PVs in comparison to othersamples during the entire storage period and except for days 4 and 8 ofstorage, no significant differences were observed in their PVs (Fig. 1).According to the results, 0.1% CV and SP treatments were the mosteffective in delaying the production of primary lipid oxidation productsduring refrigerated storage. In agreement with current study,Golmakani, Moosavi-Nasab, Keramat, and Mohammadi (2018) reportedthat within days of storage, Kilka oil oxidation was reduced by SP ex-tract to the same extent as done by BHT and PV of the SP extract wassignificantly lower than that of the blank. Similarly, Alavi and

Table 1Antioxidant activity of SP and CV extracts.

Concentration (μg/ml) Antioxidant activity

DPPH (%) FRAP (OD)

SP (200) 40.64 ± 0.04e 0.32 ± 0.01c

CV (200) 42.96 ± 0.03e 0.33 ± 0.03c

SP (400) 62.46 ± 0.01c 0.41 ± 0.01ab

CV (400) 59.54 ± 0.06d 0.38 ± 0.01bc

SP (600) 77.12 ± 0.05a 0.46 ± 0.01a

CV (600) 73.27 ± 0.03b 0.43 ± 0.00ab

OD, optical density; SP, Spirulina platensis; CV, Chlorella vulgaris.All experiments were performed in triplicate. Values are mean ± SD.Means with same letter in a column did not differ significantly (P > 0.05).

Fig. 1. Changes in the peroxide values (PV) in rainbow trout fillets duringstorage period. Blank , CV 0.05% , CV 0.1% , SP 0.05% , SP 0.1% . SP,Spirulina platensis; CV, Chlorella vulgaris. Values are given as mean ± standarddeviation (n=3). Different letters indicate significant differences (P < 0.05).

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Golmakani (2017) reported that the virgin olive oil (VOO) sampletreated with CV powder had the lowest PV, during the storage trial andusing a combination of Chlorella and citric acid had no synergistic effectagainst the oxidation of VOO samples.

3.3. Thiobarbituric acid reactive substances (TBARS)

The results of the TBARS measurements showed that TBA of theblank and samples containing 0.05% and 0.1% of CV and SP extractssignificantly increased during the refrigerated storage (P < 0.05).Within this period, TBA contents of the blank samples were higher thanthose of treated samples. The samples treated with 0.05% of CV and SPextracts had similar change trend and significantly higher TBA values incomparison to the samples containing 0.1% of each extracts during thisperiod. Additionally, the samples treated with 0.1% of SP extract hadsignificantly lower TBA values in comparison to the samples containing0.1% of CV extract and other samples during the entire storage period(Fig. 2). According to the obtained results, 0.1% CV and SP treatmentswere the most effective in lowering MDA levels during this period. Inagreement with current study, Luo et al. (2017) reported that theChinese-style sausage samples treated with 0.1%, 0.25% and 0.5% (wt/wt) SP polysaccharides (SPP) showed lower TBA values compared tothe blank sample, within refrigerated storage. Among all samples, thelowest TBA value was detected in the samples treated with 0.5% SPP,during this period. Abu-Serie, Habashy, and Attia (2018) identified thatCV extract significantly reduced the degree of lipid peroxidation andTBARS level in leukocytes compared to Ganoderma lucidum extract invitro situation. Similarly, Zeng, Fan, Zheng, Wang, and Zhang (2018)reported that malondialdehyde (MDA) content and TBA level in ery-throcytes significantly decreased after the SP protein extract was addedin vitro situation.

3.4. Free fatty acids (FFA)

The obtained results showed that the level of FFA in the blank andsamples containing 0.05% and 0.1% of CV and SP extracts increased atsignificantly different rates during the refrigerated storage (P < 0.05).For the blank samples, levels of FFA were higher than those of thetreated samples during the entire storage period. FFA levels in thesamples containing 0.05% of CV and SP extracts were higher than thosecontaining 0.1% of these algae extracts during this period. Additionally,the samples treated with 0.05% of SP extract had significantly lowerFFA levels in comparison to the samples containing 0.05% of CV extractat day 12 of storage. The samples treated with 0.1% of CV and SP ex-tracts had similar change trends and significantly lower FFA levels incomparison to the other samples throughout the refrigerated storage(Fig. 3). According to the results, 0.1% CV and SP treatments had the

highest significant effects against lipid oxidation by lowering FFA levelstill the end of refrigerated storage. In agreement with current study,Golmakani et al. (2018) reported that SP extract significantly reducedthe FFA level of Kilka oil within the storage trial but BHT was slightlymore effective than this algae extract. Similarly, Alavi and Golmakani(2017) reported that during the entire storage period, the FFA level invirgin olive oil sample containing CV powder was significantly lowerthan that of the blank.

3.5. Sensory evaluation

The sensory properties of cooked blank and treated (containing CVand SP extracts) rainbow trout fillets were evaluated by semi-trainedpanelists during refrigerated storage. According to the obtained sensoryscores, except for texture and taste at day 4, the blank samples hadsignificantly lower color, odor, texture and taste scores in comparisonto treated samples at days 4, 8, 12 and 16 of storage. Acceptable scorewas determined as higher than 3 and therefore, the acceptability limitdropped at day 8 of storage for the blank samples. Except for textureand taste at day 8, the samples containing 0.05% of CV extract hadsignificantly lower color, odor, texture and taste scores in comparisonto other treated samples at days 8, 12 and 16 of this period. Accordingto the received scores, the samples containing 0.1% of SP and CV and0.05% of SP had significantly higher sensory scores in comparison toother samples at days 12 and 16 of storage period and no significantdifferences were observed in their scores (Table 2). At the end of thisperiod, the above-mentioned samples had firmer textures and brightercolors in comparison to the other samples (Fig. 4). This indicates anextension of shelf life up to 4 days using the above mentioned treat-ments. Except for the samples treated with 0.05% of CV extract, theother treated samples had high positive effect and the sensory proper-ties were considerably improved. The obtained sensory scores showedthat although the samples containing SP extract had dark green colorwhich is due to presence of phycocyanin and chlorophyll in this algae,the treatments had no negative effect on odor and taste scores of fishfillets. Similarly, Pezeshk et al. (2011) reported that the acceptabilitylimit was reached after 10 days for the blank samples and after 15 daysfor the samples treated with turmeric or shallot extract. In addition,Raeisi, Sharifi-Rad, Quek, Shabanpour, and Sharifi-Rad (2016) foundthat rainbow trout fillets treated with 3% shallot fruit and ajwain seedextracts showed better sensory qualities and longer shelf lives thanthose treated with 1.5% of the above-mentioned extracts. Raeisi, Quek,Ojagh, and Alishahi (2015) documented that the samples treated withcumin seed and wild mint leaf extracts had significant (P < 0.05)higher sensory scores compared to the blank samples. The addition ofalgae extracts showed considerable antioxidant activities, therefore,having high potential in prolonging the shelf life of fish while

Fig. 2. Changes in the TBA reactive substances in rainbow trout fillets duringstorage period. Blank , CV 0.05% , CV 0.1% , SP 0.05% , SP 0.1% . SP,Spirulina platensis; CV, Chlorella vulgaris. Values are given as mean ± standarddeviation (n= 3). Different letters indicate significant differences (P < 0.05).

Fig. 3. Changes in the free fatty acids (FFA) in rainbow trout fillets duringstorage period. Blank , CV 0.05% , CV 0.1% , SP 0.05% , SP 0.1% . SP,Spirulina platensis; CV, Chlorella vulgaris. Values are given as mean ± standarddeviation (n=3). Different letters indicate significant differences (P < 0.05).

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preserving or improving its quality.It is recommended that further researches utilize 9-point hedonic

scale and trained panelists; innovative sensory methods such as

quantitative descriptive analysis (QDA) (Dos Santos et al., 2015; Horitaet al., 2017) and projective methods using shopping list and word as-sociation (Pacheco et al., 2018; Pinto et al., 2018).

Table 2Sensory properties of cooked rainbow trout fillets stored at 4 ± 1 ͦ C.

Storage duration (day) blank SP (0.05%) CV (0.05%) SP (0.1%) CV (0.1%)

Color0 3.69 ± 0.03Aa 3.89 ± 0.04Aa 3.92 ± 0.04Aa 3.93 ± 0.03Aa 3.94 ± 0.04Aa

4 3.22 ± 0.01Bb 3.56 ± 0.03Aa 3.67 ± 0.02Aa 3.60 ± 0.02Aa 3.65 ± 0.03Aa

8 2.43 ± 0.02Bc 3.11 ± 0.01Ab 2.65 ± 0.01Bb 3.42 ± 0.00Ab 3.35 ± 0.01Ab

12 1.67 ± 0.01Bd 2.71 ± 0.02Ac 2.56 ± 0.03Bc 3.21 ± 0.01Ac 3.15 ± 0.02Ac

16 1.32 ± 0.00Bd 2.65 ± 0.01Ac 1.61 ± 0.01Bc 2.55 ± 0.01Ac 2.36 ± 0.02Ac

Odor0 3.98 ± 0.02Aa 3.83 ± 0.03Aa 3.86 ± 0.02Aa 3.88 ± 0.01Aa 3.90 ± 0.01Aa

4 3.25 ± 0.00Ba 3.61 ± 0.02Aa 3.54 ± 0.01Aa 3.73 ± 0.02Aa 3.70 ± 0.00Aa

8 2.21 ± 0.01Cb 2.85 ± 0.02Ab 2.61 ± 0.02Bb 3.22 ± 0.02Ab 3.16 ± 0.02Ab

12 1.67 ± 0.01Cc 2.59 ± 0.00Ac 2.36 ± 0.01Bc 2.80 ± 0.03Ac 2.83 ± 0.01Ac

16 1.11 ± 0.03Cc 2.14 ± 0.01Ac 1.85 ± 0.00Bc 2.51 ± 0.01Ac 2.45 ± 0.01Ac

Texture0 3.84 ± 0.01Aa 3.86 ± 0.01Aa 3.88 ± 0.02Aa 3.87 ± 0.03Aa 3.91 ± 0.02Aa

4 3.21 ± 0.02Ab 3.52 ± 0.00Ab 3.41 ± 0.01Ab 3.75 ± 0.01Ab 3.72 ± 0.01Ab

8 2.17 ± 0.01Bc 3.28 ± 0.01Ac 3.16 ± 0.01Ac 3.51 ± 0.00Ac 3.55 ± 0.01Ac

12 1.35 ± 0.02Bd 2.72 ± 0.02Ac 2.38 ± 0.03Bd 2.68 ± 0.02Ad 3.26 ± 0.04Ad

16 1.12 ± 0.02Bd 2.42 ± 0.01Ac 1.75 ± 0.02Bd 2.35 ± 0.01Ad 2.77 ± 0.01Ad

Taste0 3.90 ± 0.02Aa 3.88 ± 0.03Aa 3.86 ± 0.01Aa 3.95 ± 0.02Aa 3.91 ± 0.00Aa

4 3.23 ± 0.01Ab 3.47 ± 0.01Ab 3.52 ± 0.02Aa 3.61 ± 0.01Ab 3.55 ± 0.03Ab

8 2.45 ± 0.01Bc 3.18 ± 0.00Ac 3.24 ± 0.01Ab 3.41 ± 0.01Ac 3.36 ± 0.01Ac

12 1.35 ± 0.00Cd 2.87 ± 0.02Ac 2.85 ± 0.03Bc 2.85 ± 0.04Ad 2.93 ± 0.02Ad

16 1.12 ± 0.03Cd 1.86 ± 0.01Ac 1.80 ± 0.01Bc 2.27 ± 0.02Ad 2.22 ± 0.01Ad

SP, Spirulina platensis; CV, Chlorella vulgaris. The capital letters represent the differences between various treatments during storage period while the lower case lettersindicate the variation of treatment for different samples at the same day.

Fig. 4. Trout fillets during 16 days of storage at 4 ± 1 ͦ C. SP, Spirulina platensis; CV, Chlorella vulgaris.

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4. Conclusions

Implementation of a safe and efficient preservation method forhighly perishable foods including fish and seafood products is of greatimportance. Many natural antioxidants from different plant sourcespossess similar or even higher antioxidant activities compared tocommon synthetic ones. Due to the destructive effects of freezer storageon food quality, refrigeration is preferred; hence, there is a need for safenatural preservatives which maintain their quality during refrigeratedstorage. The current study demonstrate that rainbow trout filletstreated with 0.1% extracts of SP and CV significantly had lower che-mical indices and obtained higher sensory scores. Consequently, naturalextracts from SP or CV can be used at industrial-scale as biopreserva-tives in order to delay lipid oxidation process, maintain or improvesensory properties and extend the shelf life of refrigerated fish andfishery products.

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