Studying the impact of phycoerythrin on antioxidant and antimicrobial activity of the fresh rainbow trout fillets

Marine cyanobacteria present a significant potential source of new bioactive compounds with vast structural diversity and relevant antimicrobial and antioxidant activities. Phycobiliproteins (PBPs) like phycocyanin (PC), phycoerythrin (PE), and water-soluble cyanobacterial photosynthetic pigments, have exhibited strong pharmacological activities and been used as natural food additives. In this study, phycoerythrin (PE) isolated from a marine strain of cyanobacterium Nostoc sp. Ft salt, was applied for the first time as a natural antimicrobial as well as an antioxidant to increase the shelf life of fresh rainbow trout i.e., (Oncorhynchus mykiss) fillets. Fresh trout fillets were marinated in analytical grade PE (3.9 μg/mL) prepared in citric acid (4 mg/mL), and stored at 4 °C and 8 °C for 21 days. Microbiological analysis, antioxidant activity and organoleptic evaluation of both control and treated fish fillets were then statistically compared. The results demonstrated noticeable (P < 0.05) differences in the microbial counts, antioxidant activity, and organoleptic characteristic values between PE-treated and non-treated groups. In addition, we observed that treating fresh fish fillets with a PE solution leads to a significant increase in shelf life by at least 14 days. Consequently, PE could be an alternative to synthetic chemical additives since it does not contain the potentially dangerous residues of the synthetic chemical additives and is thus healthier to the consumers.


Extracting and purifying analytical grade of phycoerythrin
Nowruzi et al. 18 , conducted extracting and purifying phycoerythrin (PE).Briefly, In order to procure a pellet, after centrifuging at 4000 rpm, we removed PE from 500 mL of homogenized log phase culture which was 14 days old.We suspended the pellet in 100 mL of 20 mM acetate buffer (pH 5.1), and then for 4 days, we applied the continual freezing (− 20 °C) and thawing (room temperature) method until the cell biomass turned dark purple.We obtained a crude extract by detaching cell debris and then centrifugng at 5000 rpm for 10 min.We applied Afreen and Fatma method (2018) 29 and purified the extract.Then we slowly added solid ammonium sulphate to the crude extract to achieve 65% saturation via stirring the extract continuously.After that, we let the resulting solution to stand in a cold room for 12 h, and centrifuged it at 4500×g for 10 min.We resuspended the pellets in a small volume of 50 m acetic acid-sodium acetate buffer (pH 7.1), and dialyzed it overnight.This way, we recovered the extract from the dialysis membrane and filtered it through 0.45 μm filter 30,31 .

Determination of PE content and its purity ratio
We calculated the amounts of PE, PC and APC in the various extracts obtained by measuring the absorbance at 565, 620 and 650 nm using the following equations 32 .We calculated the purity of PE at each step by calculating the purity ratio (A555/A280) where absorbance at 280 nm and 555 nm showed the concentration of proteins and PE 32 .We noted the UV-visible spectrum over the wavelength range from 250 to 700 nm applying a U-2910 spectrophotometer (HITACHI Co., Japan) at room temperature 33 .

Effect of different preservatives and temperature on the stability of the analytical grade C-PE
We tested the stability of phycoerythrin by adding the following preservatives, calcium chloride, viz.sucrose, citric acid, and sodium chloride, kept at two temperatures (4 and 8 °C).To check the effectiveness of preservatives, calcium chloride (0.4 g), sucrose (0.4 g), sodium chloride (0.4 g), and citric acid (0.4 g) were added in 25 ml phycoerythrin solution.We dissolved 5 mg of freeze dried phycoerythrin in 25 ml of sodium phosphate buffer (0.1 M, pH 7.2) one at a time and kept it at 4 °C and 8 °C to study.The stability of PE was then measured by recording its absorption spectrum and determining the percentage of degration as described by 34 .

Preparation and treatment of fish fillets
Forty Fresh rainbow trout specimens (Oncorhynchus mykiss; average weight: 600 ± 650 g) were taken from the Caspian Sea and we kept them in ice while we were transporting them to the laboratory 36 .A total of 60 fillets were used for the study.First, we washed the fillets in tap water, and then divided them into two lots.The first lot contained 40 fillets, each with the size of 5 cm × 5 cm × 1 cm (width × length × thickness), and weighing 10 g, 20 fillets were used for microbiological analysis control and 20 other fillets a for sensory evaluation.The second lot contained 20 fillets each with the size of 8 cm × 8 cm × 1 cm (length × width × thickness) and weighing 25 g were used for detection of Salmonella sp.Each day of the experiment, a separate fillet of fish was used (0, 3, 7, 14 and 21 days) to evaluate the shelf life at different temperatures of storage (4 and 8 °C).According to the results of the MIC, 30 fillets were marinated in the analytical grade of the purified C-PE prepared in a citric acid solution (4 mg/mL) as preservative at an ultimate concentration of 3.9 μg/mL and the other fillets were used as control without C-PE.Then we packaged the treated fillets one at the time in polyethylene zipper bags and stored them at 4 °C and 8 °C in refrigerator for (0, 3, 7, 14, and 21 days).Extraction of each fish fillet was performed separately on the day of the experiment excepct for salmonella sp.where the fillets were used directly for testing.For extraction, sterile physiological serum (90 ml) was added to each fillet and homogenized in a Waring blender (Waring Products, Torrington, Conn., USA) for 1 min.

Total viable microorganisms count
We conducted total viable psychrophilic bacteria counts based on Mari and Antonini 37 and demonstrated microbiological loads as number of colony-forming units (cfu) per gram.

Total psychrophilic bacteria count
We conducted total viable psychrophilic bacteria counts based on Raeisi et al. 38 .Briefly, We incubated plates of psychotropic bacteria for 10 days at 7 °C, and we counted them on Cetrimide Agar (CFC, Merck, Darmstadt, Germany) after incubating them for 48 h at 25 °C.Every count in this study is demonstrated as log colonyforming unit (cfu)/g and is carried out twice.

Staphylococcal coagulase-positive bacteria count
We conducted staphylococcal coagulase-positive bacteria counts based on Junior et al. 39 .Briefly, we laid out 0.1 ml samples of serial dilutions of fish tissue homogenates on the Baird Parker Agar surface at a temperature of 35-37 °C and incubated it for 24-48 h and then through the coagulase test, found out the suspected colonies.

Enumerating total coliforms
We used Most Probable Number (MPN) method to quantitatively estimatate total coliforms 40,41 .To do so, we needed needs at least 24 h, while for for gas negative tubes we needed an additional 24 h incubation of inoculated lauryl sulphate tryptose (LST) broth at 37 °C.Every gas positive tube needed to be converyed into brilliant green bile (BGB) broth and incubated for 24 h at the same temperature, so that it confirms to the presumptive coliform results aquired from LST broth.

Total fecal count (E.coli) test
To detect the presence or absence (positive or negative) of faecal coliform, a loopful of suspension from each of the previously incubated LST samples was transferred into a test tube with Brilliant Green Lactose Bile broth (BGLB-DIFCO ™ ) and (Escherichia coli broth (EC-DIFCO ™ ), and incubated at 35 °C for 48 h and 45.5 °C for 24 h, respectively.The positive test tubes were gently agitated and examined for gas formation or effervescence.The results were recorded as positive or negative for faecal coliform 41 40 .
We apllied Sanjee and Karim 41 procedure to detect Salmonella spp.Briefly, An aliquot of 25 g of each fish fillet was dissolved in 225 mL of sterilized Lactose Broth (LB), blended, and incubated at 37 °C for 16-20 h.We restrictively enriched an aliquot of 1 mL from the incubated LB culture into the 10 mL sterilized Rappaport Vassiliadis Soya Broth and incubated it again for 24-48 h at 37 °C.Then we incubated, one loopful inoculum from the particular enrichment culture and streaked it onto the preincubated SS and XLD agar plate.Normal Salmonella spp.make pink colonies accompanied by or without black colonies on SS agar, and black centers on XLD agar.Then we spotted the suspected colonies by their biochemical, cultural, and morphological characteristics, through TSI (Triple Sugar Iron), Urease test, and Indole test 40 .The identified Salmonella spp.were serogrouped using a BD Difco™ Salmonella O Antisera in slide agglutination tests 41 .

Antioxidant activity of the analytical grade of C-PE
The antioxidant activity of the analytical grade of C-PE was evaluated according to the 2, 2 diphenyl-1-dipicrylhydrazyl (DPPH) method described by Afreen and Fatma 29 with little modifications.Briefly, we purified 710 μg/

Sensory evaluation
According to the results of microbial count, ten panelists assessed f the fish fillets quality marinated in the C-PE analytical solution without PE for control fillets on the days of 0th, 3rd, 7th, 14th.Their ensory characteristics like odor, color, texture, appearance, flavor, and overall acceptability was measured by using 5-point hedonic scale according to Fadıloglu and Çoban 42 .We heated the fillets of rainbow trout in an oven using an oven bag for 10 min at 180 °C just before serving them.

Statistical study
We ascertained the effect of the categorical variables "additive", "temperature", and "storage time", we also ascertained s their interactions, for each numeric parameter studied by SPSS

Purification of PE and effects of temperature and stabilizers in its stability
The purity ratio of PE extracted from a marine cyanobacterial strain Nostoc sp.Ftsalt (MG549320), successevily with acetate buffer (pH-5.1),ammonium sulfate, and dialyze, was reported in Table 1 and Fig. 1.Conforming to the food grade requirement and after every purification step, the purity and concentration of PE were increased (Table 1).During the successive purification steps, purity ratio rose from 1.54 up to 4.88 (Table 1) and reached the analytical grade standard.Therefore, the purity from crude extract to purified PE grew by nearly four times,which suggested that the method is effective in securing high analytical purity of PE.The purity ratio of phycolibiproteins (PBPs) ibetween 0.7 to 2.9 is regarded to be food grade, the PBPs whose purity ratio ranges between 3.0 to 3.9 are the reagent grade more than 4.0 purity ratio is the analytical grade.
Data showed that the four additives, particularly citric acid, improved the C-PE stability remarkably when it was stored for 14 days at 4 °C in comparison with the control (Fig. 2A).However, during a long period (21 days) of storage at 4 °C, only calcium chloride, citric acid, and sodium chloridesignificantly improved C-PE stability, where citric acid was the more stabilizing agent.In addition, at high temperature (8 °C), the four stabilizing agents significantly improved C-PE stability for all storage times compared to the control, always with citiric acid as the most stabilizing agent, but the effect is less marked than at 4 °C (Fig. 2B).
To conclude, the purified C-PE at a concentration of 565 µg/mL was more stable in citric acid solution (4 mg/ mL) at both temperatures of 4 and 8 °C after it was stored for 21 days.Moreover, after 7 days of storage at 4 °C, the C-PE retained its color when dissolved in citric acid (Fig. 3).However, when the C-PE solution did not have t any preservative i.e., control, it showed discoloration after 7 days and completely discolored at 8 °C, but there was e no discoloration in the C-PE solution at 4 °C even after 21 days when stored in preservatives and particularely in citric acid.

Evaluating MIC of the purified C-PE
The MICs of the purified C-PE, prepared in the citric acid as a stabilizer agent (CA+) or in water (CA−), against showed that the MIC of the purified C-PE in solution in citric acid was 3.9 µg mL −1 against the two bacterial model E. coli ATCC No. 25922 CA+ and Staphylococcus aureus PTCC No. 1112 CA+ .However, the MIC was two times higher (7.81 µg mL −1 ) against the two bacterial model in water solution (CA−) (Table 2).This shows that adding citric acid as a stabilzer agent enhances the stabitiy of the C-PE and, therefore, its antibacterial potential.
Consequently, the purified C-PE at 3.9 µg/mL, corresponding to the MIC value, prepared in a citric acid solution (4 mg/mL) was further used to evaluate its antimicrobial and antioxidant potentials to increase the shelf life of fresh rainbow trout (Oncorhynchus mykiss) fillets.

Total viable microorganisms and psychrophilic bacteria
The results of the microbiological analysis, such as total viable microorganisms and psychrophilic bacteria, in trout fillet samples marinated with or without a solution of the purified C-PE (3.9 µg/mL) and stored for (0, 3, 7, 14 and 21) days at 4 and 8 °C which were presented in the Fig. 4. As shown in Fig. 4A, total viable microorganism counts in trout fillets, marinated in acitric acid soultion without C-PE (Control), increased significantly from 14 days of storage at 4 °C to reach a maximum on day 21.The curve of total viable microorganisms in fillet samples marinated in the C-PE (Fig. 4A) followed the same pattern as the control curve except that the increase in the count of these microoganisms was far less important on the 7 and 14 days.However, on day 21 the total viable microorganism counts reached the value of the control.At intenser heat (8 °C), the significant decline in the count of total viable microorganisms in treated fillets was observed only on day 7 in comparison with the control (Fig. 4B).
For psychrophilic bacteria, a similar effect than total viable microorganisms was observed at 4 °C, with a significant decrease of psychrophilic bacteria counts in treated trout fillets on days 7 and 14 in comparison with the the control (Fig. 4C).However, at 8 °C this difference was observedearlier on day 3 and up to only 7 days of storage (Fig. 4D, Supp S2).
In conclusion, the presence of C-PE at 3.9 µg/mL induced a significant reduction in overall viable microorganisms, and psychrophilic bacteria counts in the trout fillets at 4 °C.up to 14 days storing However, at 8 °C this reduction remained significant in only up to 7 days of storage.www.nature.com/scientificreports/

Staphylococcal coagulase-positive bacteria and total coliforms
The outcomes of C-PE impact at 3.9 µg/mL on Staphylococcal coagulase-positive bacteria and total coliform counts in trout fillets stored up to 21 days at 4 and 8 °C were presented in Fig. 5.As shown in Fig. 5A, the Staphylococcal coagulase-positive bacteria counts in untreated trout fillets (control) increased significantly until reaching a plateau after 7 days of incubation at 4 °C.However, in treated fillets, the count of bacteria remained stable at the level of the starting value and significantly considerably lower than the control throughout the entire period of storage (up to 21 days) at 4 °C.Although at 8 °C, the Staphylococcal coagulase-positive bacteria counts remained stable in treated and untreated trout fillets throughout the storage period, it was generally much lower in treated fish (Fig. 4B).
As for total coliforms, their level in untreated trout fillets remained stable but very high during the period of storage at 4 °C (Fig. 5C).Nevertheless, in treated fillets a slightly significant increase was observed after 14 days of storage, but the level always remained remarkably lower than the control throughout the entire period of storage (Fig. 5C).At 8 °C, the level of total coliforms in the control increased significantly after 7 days of storage and then dropped significantly until it reached its initial level in 21 days (Fig. 5D).In the treated trout fillets, the same pattern of the curve was observed, except that the level of total coliforms always remained significantly lower compared to the control.
In conclusion, the presence of C-PE at 3.9 µg/mL induced a significant reduction of Staphylococcal coagulasepositive bacteria and total coliform levels in the trout fillets up to 21 days of storage at 4 °C.However, when the  temperature reached to 8 °C the above mentioned reduction remained significant up to21 days of storage for Staphylococcal coagulase-positive bacteria, but only up to 14 days of storage for total coliforms.The presence of E. coli in the treated fillets and control during all periods of storage was confirmed by biochemical test (positive indole reaction).The E. coli found in the treated fillets throughout storage periods at 4 and 8 °C was less than the control, with zero E. coli in treated fillets at 4 °C on days 14 and 21 was recorded compared to E. coli in the control.Moreover, the presence of Salmonella spp. in the treated fillets and control was confirmed by biochemical test (negative urease reaction, negative indole test, TSI Test and H 2 S production).The presence of Salmonella spp. was detected at 4 °C in the control only on day 21 of storage.However, they were not found in treated fillets at the same temperature during the period of storage.At 8 °C, the presence of Salmonella spp. in the control was detected during the whole storage period, however, it was only found on days 0, 3 and 7 in treated trout fillets.The results of the serological analysis on the basis of polyvalent antisera showed that the species of Salmonella spp.detected belonged to groups of R, S, T, U, P and Q.

The purified C-PE antioxidant activity
We assessed free radical scavenging potential of the purified C-PE at three concentrations (3.9, 7.8, and 15.6 µg/ mL) by the DPPH radical scavenging activity method in treated fillets compared to control at 4 °C during 3, 7, and 14 days of storage.As shown in Fig. 6, the purified C-PE in solution in citric acid preserved its antioxidant potential for all concentrations tested by scavenging the DPPH to 80% up to 7 days of incubation.However, for a longer period (14 days) its antioxidant potential dropped slightly but remained around 70%.In the treated trout fillets with the C-PE, the DPPH radical scavenging activity increased significantly throughout the storage period in dose dependent manner compared to the control, and it remained significantly similar to that of the C-PE in solution in the citric acid (Fig. 6).
In conclusion, from the low concentration corresponding to the MIC (3.9 µg/mL), the C-PE significantly enhanced the antioxidant potential of the treated trout fillets compared to the control and up to 14 days of storage.This antioxidative potential increased significantly with the dose to reach a value of 70% at 15.6 µg/mL on day 14.

Results of sensory evaluation
Sensory values (color, odor, flavor, and texture) of tout fillets stored at different periods with C-PE at 3.9 µg/mL (up to 14 days) and in the control without C-PE (up to 7 days) were presented in Fig. 7.In the absence of C-PE, the color, odor, flavor, and texture of trout fillets deccreased significantly from 3 days of storage to reach a score of 3 (acceptable) on day 7 (Fig. 7).However, for the treated trout fillets, all of these sensory values remained at a high score level of 5 (extremely good) during the first 3 days of storage, and they substantially decreased to the score of 3 (acceptable) only on day 14 (Fig. 7).Moreover, after 7 days of storage of trout fillets in a C-PE solution of 3.9 µg/mL, the panelists considered the odor, flavor, and texture of fillets to be good.
In conclusion, at low concentration (3.9 µg/mL), corresponding to the MIC, C-PE significantly preserved the sensory quality parameters (color, odor, flavor and texture) of treated trout fillets to a good value in comparison with the control up to 7 days of storage.

Discussion
Fish is a major source of unsaturated fatty acids, and high-quality proteins for humans; yet, it is extremely vulnerable to both oxidation and microbiological deterioration.In order to protect the health of consumers, the International Commission on Microbiological Specifications for Foods established the maximal restriction of 7.0 log CFU/g for aerobic microorganisms in fish 43 .the Shelf life can be increased through freezing and cold storage, yet, the quality of the fish is not entirely preseved 44 .Moreover, coating and using different chemical preservatives for increasing the shelf life of fresh fish fillets have been investigated 7,42,[45][46][47] .However, the excessive use of chemical preservatives can generate unfavorible results on human health as well as on the nutrition amount of food.Hence, there is an urgent need to search for new natural agents, which are are not harmful and which are active against several food-borne pathogens and, as a result, can help in the preservation of food without altering its quality.The PBPs (PE, PC, and APC), covering 60% of the total cellular proteins and around 20% of the whole dry mass of the cyanobacteria, are among the value-added bioactive products isolated from cyanobacteria, which have given promising results as an ecological approach for inhibiting bacterial contamination of foods and thus increasing their shelf life 48 .The commercial production of PBPs was chiefly limited to the cyanobacterial genus Arthrospira (Spirulina) 49 50-57 .Recently, other cyanobacterial species belonging to the  genus Anabaena 58 59 , Synechococcus 60 , Phormidium 58 , Lyngbya 61 , and the marine red algae Amphiroa anceps 62 and Porphyridium cruentum 63 have been explored.Lately, Limrujiwat et al. (2022) reported that six isolated cyanobacteria, Phormidesmis sp.RK02, Leptolyngbya sp.SCOM01, Leptolyngbya sp.SWL01, Leptolyngbya sp.LKK14, Nostoc sp.SW02, and Scytolyngbya sp.LKK05, made phycobiliprotein content with more than 400 mg g protein -1.when the highest yield (31.9%) was secured from Nostoc sp.SW02, with the ratio of phycoerythrin: phycocyanin: allophycocyanin 3.4:1.9:1.0.
In this study, we purified the PE from a marine cyanobacterial strain Nostoc sp.Ftsalt (MG549320) at analytical grade (C-PE) and evaluated its antimicrobial and antioxidant activities for the first time to increase the shelf life of fresh rainbow trout (Oncorhynchus mykiss) fillets.However, analytical grade C-PE is extremely sensitive to temperature 64 and, therefore, for its use as an antimicrobial in the food industry and to make the process commercially viable, there is an essential need for research about a suitable preservative to enhance its stability 65 .Therefore, to investigate thermal stability of PBPs purified from a cyanobacterial strain Oscillatoria sp.BTA-170, Sharma et al. 66 several monosaccharides like lactose, glucose, and fructose, were tested.Their results showed that glucose was found to be the most essential supplement enhancing the half-life of PE from 5.57 to 15.77 h at 85 °C.In this study, four industrial stabilizing agents (citric acid, calcium chloride, sodium chloride, and sucrose) were investigated at a soultion of 4 mg/mL.Our results showed that among these stabilizers, citric acid (4 mg/ml) was found to be suitable for C-PE stability for 14 days at 4 °C.Adding citric acid as a stabilizing agent improved the antibacterial potential of C-PE, hence its MIC (3.9 µg/mL) against Echerichia coli ATCC n °25,922 and Staphylococcus aureus PTCC n °1112, was halved from the one found in water (7.81 µg/mL).
Therefore, analytical grade C-PE (3.9 μg/mL) prepared in citric acid (4 mg/mL) has been used as a functional ingredient to upgrade the shelf life of fresh trout fillets and promote the consumer health.To increase the shelf life, the main microorganisms accountable for the decomposition of fresh trout fillets kept at refrigerated temperatures must, therefore, be reduced.The ICMSF 43 recommended that the flesh total viable microorganism's count should not exceed 10 6 cfu/g wet weight.Our results showed that the overall pratcial microorganism's counts in the trout fillets marinated in a C-PE solution and stored at 4 °C and 8 °C were below this maximum acceptable limit, until days 14 and 21, however it was until days 14 and 7 for control fillets, respectively.In addition, the total psychrophilic bacteria counts at 4 °C and 8 °C were below the maximum acceptable limit, 10 7 cfu/g 43 , up to day 14 for treated fillets, but it was only until day 7 for the control.Concerning staphylococcal coagulase-positive bacteria, their total counts in the control fillets at 4 °C were below the maximum acceptable limit, 10 3 cfu/g 43 67 , up to day 7, however, for treated fillets, it was until day 14.
The limited number of the total coliforms can also be useful to specify the efficcay of safety measures throughout handling and of food 41,67 .In this analysis, the whole coliforms count in treated fillets span from 0.2 to 5 MPN/g at 4 °C, and from 0.93 to 11 MPN/g at 8 °C.However, in control fillets it ranged from 24 to 25 MPN/g at 4 °C and from 0.2 to 25 MPN/g at 8 °C.Moreover, C-PE also reduced the fecal coliforms count significantly at both temperature from 5 MPN/g in control fillets to 0-1 MPN/g in treated fillets.Similary, C-PE decreased the count of the more precise index of fecal contamination, that is Escherichia coli remarkably with zero colony recorded in treated fillets at 4 °C on days 14 and 21, compared to 5 colonies in the control.The existence of E. coli in foods means that the latter is contaminated with pollution of faecal origin and that it may, therefore, contain pathogenic microorganisms such as Salmonella spp.0 cfu/25 g is the utmost microbiological limitation for the Salmonella count is that dissociates the products with good quality from those which have bad quality. 43.Our results revealed that the Salmonella count of the control fillets at 4 °C were below the maximum acceptable limit, until day 14, however, it was until day 21 for treated fillets.Nevertheless, at 8 °C Salmonella spp.were detected both in control and treated fillets.The presence of Salmonella spp.found in fillets treated with C-PE after storage for 7 days at 8 °C may be due to contamination during the various stages of fish processing such as transport and handling or when it is washed or iced.In conclusion, the results obtained in this work clearly showed that the C-PE purified from a marine cyanobacterial strain Nostoc sp.Ftsalt (MG549320) significantly inhibits the increase of the count of most bacteria in harge of spoiling fresh trout fillets and thus prolongs their shef life in the refrigerator for at least 7 days.This is congruent with the earlier research, which have shown the antibacterial scope of cyanobacterial PE against various bacterial species.Indeed, Afreen and Fatma 29 reported that PE exhibited antibacterial activity against Gram-negative as well as Gram-positive bacteria, with the strongest pre-emptive activity observed at 0.1 mg/mL against Pseudomonas aeruginosa MTCC2543, E. coli 25,922, and S. aureus MTCC902.Similarly, Sarada et al. 68 manifested that PC from Spirulina platensis showed excessive antibacterial activity against Klebsiella pneumoniae, E. coli, S. aureus., and P. aeruginosa.Recently, Nowruzi et al., 18 demonstrated that PE exhibited also a strong inhibitory activity against Bacillus subtilis, and Candida albicans.
Bacterial contamination is one of the main resons that the fish and fish-products quality drops during storage, but it is not the only cause.Given their high content in polyunsaturated (26%) and monounsaturated (50%), fatty acids 69 , fish and fish-products are also very sensitive to lipid peroxidation during storage, which leads to a degradation in quality and the formation of unwanted odors.Many studies have been conducted that describe the extraction and purification of PE and PC from various algal and cyanobacterial species, the evaluation of their physicochemical properties 30,[70][71][72][73][74][75][76][77] their application as colorant in foods 34,[78][79][80] , and their antioxidant potentiality but they were not added to the foods [81][82][83] .Nevertheless, as far as we are concerned, studies reporting the antioxidant activity of C-PE in food matrices to expent their shelf life are scarce.The antioxidant activity of PE purified from a marine cyanobacterial species Halomicronema sp.R31DM, was evaluated by three in vitro assyas namely, DPPH-radical scavenging activity, reducing power assay, and ferric-ion reducing ability of plasma assay 19 .The outcomes of the research revealed that PE exhibited substantial antioxidant activity probed by these 3 tests and the DPPH-scavenging activity of PE at 100 µg was considered to be similar to the positive control ascobic acid (80 µg), manifesting 100% scavenging.The protein sequences of the and-subunits of PE has 33 and 37 charge residues, respectively that have a capacity for sequestration of free radicals and metal ions and also the presence of linear tetrapyrrols in their structure.They are rich in doubles bonds and have the reducing ability, and both contribute to the total antioxidant activity of PE 19 .Our results showed, that for the first time the C-PE significantly enhanced the antioxidant potential in dose dependent manner in the treated trout fillets in comparison with the control, and up to 14 days of storage.The outcomes are congruent with other analyses that demonstrated that PE exhibeted greater free radical scavenging activity (IC 50 = 0.023 mg/mL) than DPPH (IC 50 = 0.043 mg/mL), and Superoxide anion radical (SOR) assay (IC 50 = 0.553 mg/mL) 29 .El Baky et al. 84 reported that the antioxidant potential of Spirulina is close (75.3% of DPPH radical scavenging activity) to that of the reference chemical antioxidant BHT (95% of DPPH radical scavenging activity).Similary, Shalaby andShanab 85 noted that extracts of Spirulina platensis at 200 mg/mL exhibeted a DPPH radical scavenging activity up to 95.3%.These results have been confirmed and refined using encapsulated Spirulina platensis and a C-PC extract in dried noodles (1.5%, w/w) as an antioxidant in yogurts 86 , and cookies 84 , respectively.The results of these studies showed that yogurts supplemented with Spirulina and cookies supplemented with C-PC exhibited higher antioxidant activity throughout shelf life compared to products without the addition.
Moreover, human fish and fish-products eating is mainly reliant on the sensory attributes 87 , in which, color, odor, flavor, and texture are considered as the dominant factors for these foods quality.Because of their inherent bright color, easy availability, nontoxic proteinaceous nature, potential free radical scavenging ability, and water solubility, PBPs are extensively applied as a natural protein food coloring 88 .For example, the fillets of rainbow trout are highly sensitive to lipid oxidation throughout refrigerated storage, that cause the in quality and formation of undesirable odor.During this study, we demonstrated that marinated fish fillets in C-PE significantly preserved their sensory parameters (color, odor, flavor and texture) to a good value compared to the control up to 7 days of storage.In addition, panelists preferred treated fish fillets with addition of C-PE until day 14 with acceptable mark.Hadiyanto et al. 51 conversely, noted that dried noodles enriched by using phycocyanin extracted from Spirulina sp. at a high concentration (1.5%, w/w) were least favored by lecturers in term of colour, throughout sensory assay, yet showed the highest antioxidant capacity estimated by the DPPH method.

Conclusion
Phycoerythrin with high purity (C-PE) was extracted from a marine cyanobacterial strain Nostoc sp.Ftsalt (MG549320).The addition of citric acid as a stabilizing agent improved its stability and antibacterial potential.At low concentration (3.9 µg/mL), this purified C-PE improved the microbiological quality of fresh rainbow trout fillets refregired at (4 °C), by significantly reducing the load of the main bacteria responsible for the spoilage on these fillets and extending their shelf life to at least 14 days.In addition, the purified C-PE remarkably enhanced the antioxidant potential in dose dependent manner in the treated trout fillets in comparison with the control, and up to 14 days of storage.The use of C-PE increased the antioxidant and microbiological shelf life of refrigerated fresh rainbow trout fillets and amplifies the sensory adequacy of these products when compared with those nonmarinated.Therefore, increasing the shelf life of fresh rainbow trout fillets would make it possible for trout producers in Iran to increase the length of time in the supply chain and make it more feasible for processors to maintain fresh, never-frozen rainbow trout fillets which can be sold in the local retail market or be exported.

Figure 1 .
Figure 1.The general chemical structure of PE binds covalently to proteins via two thiol bonds to form PBPs and the UV-visible absorbance spectrum (below) of the purified analytical grade (C-PE) from Nostoc sp.Ft salt (MG549320).

Figure 2 .
Figure 2. Stability of the purified C-PE in four stabilizer agents (calcium chloride, sodium chloride, citric acid, and sucrose) compared to control (water) in different storage times (3 to 21 days) at 4 °C (A) and 8 °C (B).The data were expressed as means ± SE of three independent experiments in triplicate each.The letters refer to comparisons within each day and treatment.The same letter indicates that data are not considerably different from one another (P < 0.05, ANOVA followed by Tukey's grouping tests).

Figure 4 .
Figure 4. Total viable microorganisms count (cfu/g) of the treated rainbow trout fillets (orange curve) and control (blue curve) at 4 °C (A), and 8 °C (B) and total psychrophilic bacteria counts of the treated rainbow trout fillets (orange curve) and control (blue curve) at 4 °C (C), and 8 °C (D).The data were expressed as means ± SE of three independent experiments in triplicate each.The same letter indicates that the data are not very unlike each other (P < 0.05, ANOVA followed by Tukey's grouping tests).

Figure 5 .
Figure 5. Staphylococcal coagulase-positive bacteria counts of the treated rainbow trout fillets (orange curve) and control (blue curve) at 4 °C (A), and 8 °C (B) and total coliforms of the treated rainbow trout fillets (orange curve) and control (blue curve) at 4 °C (C), 8 °C (D).The data were expressed as means ± SE of three independent experiments in triplicate each.The same letter indicates that data are not remarkably different from one another (P < 0.05, ANOVA followed by Tukey's grouping tests).

Figure 6 .
Figure 6.Free radical scavenging potential of analytical grade C-PE at different concentrations (3.9, 7.8, and 15.6 µg/mL) in water (control) and in citric acid (citric acid + C-PE), and of treated rainbow trout fillets with C-PE in citric acid (Treated fillets + Citric acid + C-PE) after 3, 7 and 14 days of storage at four °C.The data were expressed as means ± SE of three independent experiments in triplicate each.The same letter indicates that the data are not remarkably different from one another (P < 0.05, ANOVA followed by Tukey's grouping tests).

Figure 7 .
Figure 7. Sensory value of the rainbow trout fillets without (C) and with adding C-PE (+ PE) after 3, 7, and 14 days of storage at 4 °C.The data were expressed as means ± SE of three independent experiments in triplicate each.The same letter indicates that the data are not remarkably different from one another (P < 0.05, ANOVA followed by Tukey's grouping tests).
StatementAll methods were conducted congruent with relevant guidelines and regulations.Authors have not done do any experiments on humans and/or the use of human tissue samples.All experimental protocols and panelists involved in the study were accepted by ethics committee of Tehran medical sciences, Islamic Azad University, Tehran, Iran.Moreover evaluating panel sampled the rainbow trout (Oncorhynchus mykiss) fillets.The full name of the ethics committee that approved the study is Dr Fahimeh Nemati and Dr Sarvenaz Falsafi.