Total carotenoids and antioxidant activity of fillets and shells (in natura or cooked) of “Vila Franca” shrimp (Litopenaeus Schmitti) in different intervals of storage under freezing

Shrimps are sources of carotenoids, astaxanthin is the predominant, responsible for their special and desirable properties, as well as for their instability under heat treatment during the domestic preparation, industrial processing or storage under freezing. These can cause discoloration and reduce the beneficial health properties. This study aimed to evaluate the effect of heat treatment and storage under freezing (0, 45 and 90 days) on the levels of total carotenoids and stability of the antioxidant activity of ethanolic extracts of fillets and shells, raw and cooked, of the white shrimp (“Vila Franca”) Litopenaeus schmitti (Burkenroad, 1938). The antioxidant ability of the extracts was evaluated using the radicals DPPH• (2,2-diphenyl-1-picryl-hydrazyl) and ABTS+• (2,2’-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid), as well as by the iron reducing power (FRAP) test. The extracts of cooked or in natura shrimps (fillets and shells) represent dietary sources of carotenoids, displaying antioxidant activity through all the tested methods, after heat treatment and storage under freezing. The antioxidant activity of the extracts was superior to the one of ascorbic acid, mainly in the cooked fillet and shells. The samples of shrimp shells seemed a valuable source of carotenoids, whose antioxidant activity was verified even 90 days after freezing, and can be used in food products as functional natural supplement, adding value to this waste.

The white shrimp ("Vila Franca" or "caboclo" or "legitimate") [Litopenaeus schmitti (= Penaeus schmitti) (Burkenroad, 1938) (Crustacea: Decapoda: Penaeidae)], occurs in the western Atlantic, from the West Indies (23º30`N) to southern Brazil (29º45`S), and because of its size (about 20 cm long) and flavor, it is a highlighted crustacean in the cuisine of northeastern Brazil. It is handexploited, but with significant commercial interest (Santos et al., 2012). Once there is no scientific information about the total carotenoid content and antioxidant stability of this cooked or in natura shrimp, before or after freezing, it was evaluated in this study.

Harvesting and preparation of samples
A batch of 2.0 kg of white shrimp (Litopenaeus schmitti), from the coastline (geographic coordinates 8º8'12''S and 10º29'12''S) of Maceió-Alagoas/Brazil, where the water reaches high salinity (35.5 psu) and medium temperature 27.8 °C (Araújo, 2006), was acquired in May 2013, soon after collected. The samples were placed in plastic bags, kept in cooler box with ice and immediately transported to the laboratory.
This batch was split into two groups of 1.0 Kg. Group "I" was formed by fresh samples and group "II" was subjected to cooking in 1 L water during 17 min at 99.4 °C. In both groups, the residue of the head and intestines of the shrimps were removed. Only the fillets and shells from the exoskeleton carapace, plus tail and legs, were used. As the mass of fillets for the analyzes was 30g (in natura and after cooking), it was estimated that the amount of acquired sample per group (1Kg) was sufficient.
Then, the mass of appropriate aliquots of the in nature (group I) and cooked (group II) samples of the fillets (30 g) and the shells (15 g) randomically collected were measured and identified for analysis. The remaining samples (in natura and cooked) were packed in aseptic plastic bags, identified and stored in plastic containers in the freezer at -17 °C (± 1 °C), until the moment of the analysis (0, 45 and 90 days under freezing). The "time zero" samples were processed at the same day, whilst the others were stored under freezing until the interval settled for the analysis (45 or 90 days).

Extraction of carotenoids
The extracts were subjected to chemical analysis in quadruplicate.

Shrimp fillet
The carotenoid pigments were extracted from the fillets samples (30 g) through homogenization with 92.8 ° ethanol (100 mL) in a blender during 5 min. The commercial ethanol (92.8 °) was used since it enables a better extraction with less cost and it is a less toxic solvent (Santos et al., 2012).
Each crude extract was centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant (liquid phase) was stored in amber bottles, while the precipitate (16 g) was re-homogenized for 5 min with commercial ethanol until the complete removal of pigments (56 mL). The second supernatant was added to the first and the mixture was filtered (filter paper) before the final collection on a second amber vial. To avoid possible oxidation due to contact with oxygen from the air, nitrogen gas was sprayed into the vial before being closed. Then it was stored in a freezer at -17 °C (± 1 °C) until the next day when the total carotenoids and antioxidant activity were determined.

Shrimp waste (shells, tail and paws)
The carotenoid pigments were extracted from the shrimp waste (15 g) through homogenization with 92.8 ° ethanol (100 mL) in a blender during 5 min (Santos et al., 2012). Each crude extract was then centrifuged (10,000 rpm/4 °C, 10 min), and the supernatant (liquid phase) stored in amber bottles. Two more extractions were carried out with the pellet, until no pink pigment was seen -the first from 12 g of it, with the addition of 40 mL ethanol, and the second from 10 g of the remaining pellet, adding 30 mL of ethanol. The same procedures described above for the fillet samples were used.

Determination of carotenoids content
The absorbance of 1 mL of each ethanolic extract was measured at 470 nm using a spectrophotometer. The "blank" reference corresponded to 1 mL of ethanol (Schiedt and Liaaen-Jensen, 1995). The concentration of carotenes in the extract was determined using the following equation (Equation 1), wherein "A" is the absorbance at 470 nm, "vol" is the volume (mL) used in the extraction of carotenoids and "A 1% " is the absorption coefficient for 1% of the mixture of unknown carotenes at 2500:

Test of the ABTS + • radical
The antioxidant activity of the extracts, as well as of the ascorbic acid ethanolic solutions (100-275µg.mL -1 ) on the radical ABTS + • (2,2'-azino-bis-(3etilbenzotiazolina-6-acido sulfônico), was measured according to Wang and Xiong (2005). The ABTS + • stable radical originally is a blue-green chromophore. Its stock solution was prepared by the reaction of equal volumes of 2.45 mM potassium persulphate and 7 mM ABTS + •, which, after 12 hours at room temperature and dark, gets a dark blue-green color. This solution was diluted in 0.2M sodium phosphate buffer (PBS), pH 7.4, with an absorbance of 0,70 ± 0.02 at 734 nm. Then, 20 µL of the ethanolic extracts or ascorbic acid solution in different concentrations, were added to 1980 µL of the ABTS + • diluted stock solution. After vigorous homogenization for 30 s, the absorbance of the mixtures of reaction after 5 min (room temperature, dark) was measured at 734 nm. From the obtained absorbance, the percentage of inhibition (PI) was calculated according to the following equation (Equation 3):

Test of DPPH•
The sequestration of the DPPH radical was determined according to the method described by Je et al. (2009), mixing 100 µL of ethanolic solution of 0.15 mM DPPH and 100 µL of the carotenoid extracts obtained as described in 2.2. The absorbance of the mixtures, after 30 min in the dark, was measured at 517 nm using a microplate reader from Biorad ® and software MMP6. The scavenging activity was calculated using the following equation (Equation 2): The absorbance of the "blank" reference corresponds to the mixture of 100 µL of ethanol and 100 µL of total carotenoids extracts, while the "control" absorbance corresponded to the mixture of 100 µL ethanolic, 0.15 mM DPPH and 100 µL ethanol p.a.
In which, AbsA corresponds to the absorbance of the ethanolic extracts with ABTS•+ radical after 5 min of reaction, whilst AbsB corresponds to the absorbance of the diluted solution of ABTS + •.

Assay of the ferric reducing antioxidant power (FRAP)
The method used was the one described by Ahmadi, Kadivar and Shahedi (2007). Therefore, 200 µL ethanolic extracts or ascorbic acid ethanolic solutions (10-100 μg mL -1 ) were mixtured with 200 µL phosphate buffer (0.2 M, pH 6.6) and 200 µL of potassium ferricyanide (1% w/v). The mixture was incubated during 20 min (50 °C) until the addition of 200 µL trichloroacetic acid (10% w/v) for inhibiting the reaction. Then, it was centrifuged at 3000g for 10 min and an aliquot of 125 µL of the supernatant was collected and mixture with 125 µL of distilled water and 20 µL of ferric chloride solution (0.1% w/v) in a well of Elisa microplate. After 10 min at room temperature, the absorbance of the reaction was measured at 700 nm using a microplate ELISA reader and the MPM 6 Biorad ® software. As higher was the absorbance, higher the reducing antioxidant power evaluated. In the "blank" reference, distilled water was used instead of the ethanolic extract. (1)

Statistical analysis
The data were subjected to analysis of variance and the means were compared by Tukey test at the level of 5% of probability. To detect the possible relationship between methods of antioxidant activity determination, the Pearson correlation was estimated. The analyzes were performed using the statistical softwares Genes (Cruz, 2013) or SAS (Statistical Analyses System), version 9.1 (SAS, 2006).

Total carotenoids
The total carotenoid content of the studied samples (in natura or cooked, time "zero" or after 45 and 90 days of storage under freezing), are shown in Table 1.
The average concentrations of the carotenoids in fillet samples (raw and cooked), in each interval studied, were not statistically different (p<0.05) amongst them. So, apparently the heat treatment did not affect the stability of carotenoids in fillets of "Vila Franca" shrimp, which is a positive feature since this is the most consumed part of the shrimps and with the highest nutritional value for the human diet.
These results corroborate the ones obtained in the studies of Dutra et al. (2012), who worked with Murcote tangerine juice subjected to heat treatment (88 and 100 °C by 16 e 44 s respectively), and observed that none significant difference occurred in its content of lutein, zeaxanthin, β-cryptoxanthin and β-carotene. Also, according to Choubert, Brisbarre and Baccaunaud (2011), due to the degradation of carotenoids, the salmon frozen storage time is often limited by oxidation and discoloration of the fish. However, the content of carotenoids was not reduced in whole rainbow trout (Oncorhynchus mykiss) previously fed with astaxanthin (100 mg L -1 ) and canthaxanthin (80 mg L -1 ), when they were frozen stored (-18 °C) for 6 months, probably because the protection against oxidation is on the skin of these fishes. On the other hand, studies of Andersen et al. (1990) using wild salmon and rainbow trout from aquaculture, both packed in transparent vacuumskin packaging during storage of 6 months under freezing chamber (-17 °C), showed that fillets of rainbow trout prior to storage had higher content of carotenoids identified as astaxanthin (9.1 mg kg -1 ) than wild salmon fillets (4.9 mg kg -1 prior to storage), although in the last ones it remained virtually constant during the six months, but decreased along the time in the first ones. Rancidity developed faster in fillets of wild salmon as compared to the ones of rainbow trout. This suggests the role of astaxanthin as a sacrificial protector against radical processes.
In the present work, the carotenoid content in raw shells was 7 times higher than in the raw fillets, and regarding to the storage under freezing, the carotenoids of the raw shells remained markedly more stable, with a concentration ca. 7.25 to 8.56 times higher than in the frozen raw fillet. Additionally, the cooking decreased the total content of carotenoids of shrimp shells in 15% at all intervals of storage analyzed, although it still remained high compared to the fillets. Despite the loss resulting from the cooking, the shells still represent an important source of carotenoids. Sowmya et al. (2014) also reported that shrimp waste is the major source of carotenoid astaxanthin. Seabra et al. (2014) found 42.74 µg/g of total carotenoids in fresh waste of Pacific white shrimp (Litopenaeus vannamei), which is a concentration higher than in the present study.
The shrimp shells, with very low commercial value, represent a source of environmental pollution, and generate additional costs for its disposal, reducing the profit margin of the production system. They are usually discarded by the packing houses, without any technological use and correspond to 28.6% of the waste crustacean (Meyers, 1986). Shrimp waste is processed as animal feed and as raw material protein in diets for aquaculture (Sudaryono;Tsvetnenko;Evans, 1996). To increase the market value of this disposal, many alternatives have been applied for use of this material: proteins and amino acids (Mandeville;Yaylayan;Simpson, 1992), dyes (Nguyen, 2013), flavoring (Pan, 1990), chitin and chitosan (Coward-Kelly; Agbogbo; Holtzapple, 2006). As natural carotenoids have higher antioxidant properties than the synthetic ones (Levin;Yeshurun;Mokady, 1997), and the demand of consumers for natural products is increasing, the use of shells shrimps in the preparation of functional food represent an alternative of great added value for such residue. Perdigão et al. (1995), reported that specimens of raw shellfish shells resulted in a minimal extraction pigments compared to the boiled samples. Moreover, Becerra et al. (2014) found similar results to the ones of the present study -after 15 min of boilling, the content of astaxanthin in the shrimps was significantly reduced in comparison to that in raw crustacean, and the authors concluded that this decrease could be related to the degradation and partial solubilization of the carotenoprotein complex. In this regard, Niamnuy, Devahastin and Soponronnarit (2008b) found a significant reduction on the protein (myofibrillar, sarcoplasmic and stromatic) content in cooked shrimp, suggesting that some of these could have suffered denaturation and degradation.
Compared to time zero, the concentration of total carotenoids in the treatments of raw and cooked fillet, after 45 days under freezing, also showed no significant difference (p<0.05). After 90 days of freezing, however, statistically significant losses (p<0.05), respectively of 39% and 28%, were observed in this content. In relation to the total carotenoid content in shells (raw and cooked) stored for 45 days under freezing, there are significant losses (p<0.05) of 15.41% and 13% respectively. After 90 days under freezing, the losses reached 28.6% and 13%, respectively. Becerra et al. (2014) observed a 74% reduction in the level of astaxanthin after 60 days of storage of shrimps salted under the sun as compared to the content found at time zero.
Some studies suggest that to obtain the beneficial effects of astaxanthin is needed a daily intake of about 4 mg of the carotenoid (Parisi et al., 2008;Satoh et al., 2009). The astaxanthin capsules commercialized in some countries have concentration varying between 4 and 20 mg (Seabra;Pedrosa, 2010). Considering the total content of carotenoids on 15 g samples of raw shells of "Vila Franca" shrimp, their level corresponded to 0.498 mg (4,15 µg x 120 mL extract), i.e., 33.2 µg g -1 shrimp waste, so that about 675 g of these shrimp shells could create a capsule with a highest dose of astaxanthin (90% of the content of carotenoids in shell). Thus, these products can be used as natural food additives for human or animal use.

Tests for determination of antioxidant activity
There is no universal method by which the antioxidant activity can be accurately quantified (Prior;Xianli;Schaich, 2005;Frankel;Meyer, 2000). This fact establishes the need to evaluate such parameter by different techniques, such as the DPPH• and ABTS + • radicals scavenging assays or the Ferric Reducing Antioxidant Power (FRAP) test, which already have been used with shrimp protein hydrolysates (Binsan et al., 2008;Benjakul et al., 2009;Faithong et al., 2010).

Test of DPPH• radical
The ability of the studied extracts as scavengers of free radical DPPH•, expressed as a percentage of the antioxidant activity is shown in Table 2. As for the total Raw shrimp shells, in comparison to raw fillets of "Vila Franca" shrimp, according to the DPPH test, had an increase of its antioxidant activity of only 13.6%, and this arise ca. 16% in cooked fillet. Meenata et al. (2011) found strong DPPHscavenging activity in shrimp residues. After cooking the shells, there was a decrease of 19.5% in comparison to the in natura samples, but no significant difference (p<0.05) was observed regarding to the antioxidant activity of the extracts of raw or cooked fillets. Freezing this material for 90 days decreased the antioxidant activity by approximately 24% in relation to the time zero. But the frozen cooked material, even after 90 days, still retained the free DPPH• radical scavenging activity in percentage higher to 50%.

Test of ABTS + • radical
The ability of the extracts of carotenoids as scavengers of free ABTS + • radical, expressed as a percentage is shown in Table 3. There was no statistically significant difference (p>0.05) between scavenging activity percentages of the extracts obtained at time zero of in natura fillets in relation to shells (in natura or cooked). After 45 days under freezing, however, significant reductions were observed (p<0.05), in this activity with respect to time zero, corresponding to 26.03% for crude fillet; 16.40% for the cooked fillet; 24.28% for the cooked shrimp shells and 22.37% for the in natura shells. These values differ from those expressed by DPPH• test, but are similar to ones of other research where freezing range time could interfere in the content of carotenoids and antioxidant activity if vacuum packaging material and the preparation of the fish skin was not specific to prevent lipid oxidation in long time storage (Andersen et al., 1990).
Regarding the comparison of the extracts obtained from raw fillet and cooked shells stored under freezing for 45 days, there was no significant difference (p<0.05) in ABTS + • sequester activity, but the extracts of the cooked fillets and raw shells showed significant difference between them (p<0.05).
The ABTS + • sequester activity of the extracts obtained from samples under 90 days of freezing suffered significant losses (p<0.05) from that of extracts obtained at time zero, it means, reduction of 76.37% in raw fillet, 60.72% in cooked fillet, 76.76% in raw shells and 69. 97% in cooked shells, highlighting the cooked fillet, which presented the lowest percentage of losses (60.72%). This suggests that the usual form of shrimp consumption provides some antioxidant activity of its bioactive compounds.
When the results of this antioxidant activity, measured by ABTS + • sequestration, were expressed in terms of equivalents µg mL -1 ascorbic acid antioxidant activity (standard curve y = 0.0046x -0.008; R² = 0.9903), the same profile was observed, and to a content of 4.153 µg mL -1 total carotenoids (Table 1), the antioxidant activity obtained was similar to the one of 200 µg mL -1 ascorbic acid (Table 4).
* Averages followed by the same lower case letter in the same column, and capitalized case letter in equal lines, do not differ statistically at 5% probability (Tukey test).  Despite significant losses (p<0.05) of antioxidant activity in each extract obtained from the samples stored 90 days under freezing, the superiority of the antioxidant potential of them compared to ascorbic acid was observed mainly for the cooked fillet and shells, it means, for carotenoid contents of 0.405 µg mL -1 and 2.545 µg mL -1 , respectively, corresponding antioxidant activities of ascorbic acid of 52 µg mL -1 and 53 µg mL -1 were obtained (Table 4).
Results from Sowmya and Sachindra (2012) evidenced the high antioxidant activity of astaxanthin extracted from shrimp waste. Weeratunge and Perera (2016) also reported that the carotenoid astaxanthin exhibits a high degree of antioxidant activity. These results confirm the literature information that the antioxidant potential of astaxanthin-predominant carotenoid of shrimps, exceeds the antioxidant benefits of vitamin C (Palozza;Krinsky, 1992).

Ferric reducing antioxidant power (FRAP) assay
The FRAP results for the extracts of fillet and shells of "Vila Franca" shrimp are shown in Table 5. The cooked shrimp shells showed a statistically significant decrease (p<0.05) of the FRAP after different freezing intervals (0, 45 and 90 days), i.e., 28.1%, 47.91% and 40%, respectively. Then, temperature and interval of freezing-storage could cause physico-chemical changes in compounds responsible for this antioxidant activity.
The cooked fillet, in turn, also showed a reduction of this effect in relation to the raw fillet at time zero (@8%) and after 90 days of frozen storage (@6.9%), although at 45 days of storage that potential was not statistically changed by cooking. In fact, the FRAP decreased over the freezing time for all treatments, but these losses were only statistically significant (p<0.05) in extracts of cooked shrimp shells studied 45 days after freezing (48.55% reduction), and from the crude shells 45 and 90 days after freezing, i.e., respectively 28.99% and 37.86%.
When these results were expressed in terms of equivalent µg mL -1 ascorbic acid antioxidant activity (standard curve y = 0.0078x -0.0234; R² = 0.998), the same profile was observed, it means, for a carotenoid content of 4.1533 µg mL -1 (Table I), the equivalent FRAP expressed in terms of ascorbic acid was 46.27 µg mL -1 (Table 6).

CONCLUSIONS
According to the present study, the ethanolic extracts of the in natura (fillet and shells) "Vila Franca" shrimp (L. schmitti) are good sources of carotenoids, with outstanding antioxidant activity. Heat treatment resulted in significant decay of the total carotenoid content and antioxidant activity observed by DPPH radical sequestration and through iron reducing power (FRAP) in cooked shells. This change in antioxidant activity was not detected by the ABTS method. Freezing led to losses in the total carotenoid content as well as in the antioxidant activity (measured by the three methods) in the extracts of shrimp shells (with higher concentrations of carotenoids), probably due to oxidation of the compounds during the storage. Even though, the shells of "Vila Franca" shrimp still represent a valuable source of carotenoids, predominantly astaxanthin, whose antioxidant activity was detected even after 90 days of freezing. Despite significant losses of antioxidant activity in each extract obtained from the samples stored 90 days under freezing, the superiority of the antioxidant potential of them compared to ascorbic acid was observed mainly for the cooked fillet and shells. Studies should be conducted to ensure better use of the active compounds of the crustacean, allowing the use of the product as a functional food, which assists in the primary prevention of diseases also generated by oxidative stress.