Effects of different additives on colorimetry and melanosis prevention of Atlantic seabob shrimp ( Xyphopenaeus kroyeri ) stored under refrigeration

The aim of this work was to evaluate the effect of the food additives, sodium chloride, sodium metabisulfite, sodium nitrite and citric acid on the anti-melanotic activity in shrimp (Xyphopenaeus kroyeri) kept under refrigeration for 13 days post-capture. A sensory panel and color measurements (L*: luminosity, a*: red-green axis saturation and b*: yellow-blue axis saturation) was conducted during storage to evaluate the development stages of melanosis. Statistical differences were found in the colorimetric indexes (L*, a* and b*) and melanosis levels in all the treatments. The best results were found in 2.5% sodium metabisulfite. However, 2% sodium chloride had similar results and presented advantages such as low cost, maintaining firmness, general appearance, flavor, microbiological control, besides not causing allergic reactions. The sodium chloride is an excellent alternative to sodium metabisulfite.


INTRODUCTION
The Brazilian fish market is in active development, with a continental extractive fishing yield of 249,600.2t in 2011, according to the Fishing and Aquaculture Ministry (MPA) (Brasil, 2011).Shrimp is among the seafood with an increase of 4.49% from 2009 (5519.7 t) to 2011 (5779.5 t).
The Xyphopenaeus kroyeri, popularly known as Atlantic seabob is among the species of shrimp produced in Sul (Ibama, 2011).
The high consumption of X. kroyeri shrimp is related to its excellent nutritional quality, freshness, variety and flavor.However, despite the sector's growth in recent decades, it still needs a lot of progress with regards to it shelf life.In general, fresh shelf life shrimp is short, since after capturing, numerous biochemical and enzymatic mechanisms are activated, starting the deterioration and consequently the loss of natural shrimp characteristics, which may cause unacceptability of the product (Martinez-Alvarez et al., 2005;Nirmal and Benjakul, 2009a;Pardio et al., 2011).
One of the biggest problems found in this crustacean is melanosis (black spots) development, a dark pigment that arises by polyphenol (PFO) biochemical action, which are able to oxidize phenolic compounds into quinones (Nirmal and Benjakul, 2011) even under cold storage.Shrimp usually have limited shelf life due to melanosis formation; although its presence is harmless to consumers, it causes drastic reduction of the value in market products and the acceptance in consumer sensory (Nirmal and Benjakul, 2009b).
The fishing industry has been using food additives that have preservation ability to maintain and/or improve the quality of the final product (Okpala et al., 2014).According to the Identity and Quality Technical Regulation (RTIQ) for fresh shrimp, Ordinance n. 456 of the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento -MAPA), sodium metabisulphite is the only preservative allowed for conservation purposes (Brasil, 2010).Among these preservatives, the citric acid prevent oxidation and reducing dehydration of crustaceans, while the sodium metabisulfite is widely used to maintain the product organoleptic properties and shelf life (Mol and Turkmen, 2010).However, the overuse of sulfite is associated with adverse reactions in people with asthma, thus its residual concentration in food is legally controlled (Gómez-Guillén et al., 2005).
Another preservative commonly used in dried shrimps is sodium chloride, which intensifies flavor attributes, hardness and reduces microorganism levels (Niamnuy et al., 2007).Nitrite is also used in curing salts in order to increase conservation of marine products by controlling microbial growth during storage, inhibiting pathogenic and deteriorating microorganisms (Lyhs et al., 1998).
In this context, the aim of this work was to evaluate the colorimetric changes and anti-melanotic action of the additives sodium chloride, sodium metabisulfite, sodium nitrite and citric acid in fresh shrimp (Xyphopenaeus kroyeri) stored under refrigeration for 13 days.

Samples collection and storage
The shrimps (X.kroyeri) used were from the Lagoa dos Patos, in Fossati et al. 75 Tramandai,RS,Brazil (29°59'05''S and 58°08'01''W).The temperature at the time of capture ranged from 20 to 23°C, and their individual weight was 5.00±0.50g.Whole shrimps were collected immediately after taken to the boats and kept on ice at 1:2 (shrimp : ice) and sent to the Teaching, Research and Meat Technology Center (Centro de Ensino, Pesquisa e Tecnologia de Carne -CEPETEC), Faculty of Veterinary Medicine, Rio Grande do Sul Federal University (UFRGS).

Treatments and collection days
Shrimps were washed in running water, drained, randomly distributed into groups and subjected to five immersion treatments: T1: distilled water (no preservative -control group), T2: sodium chloride solution (2% w/v), T3: sodium metabisulfite solution (2.5% w/v) T4: Sodium nitrite solution (1% w/v) T5: citric acid solution (2% w/v).Each treatment consisted of 2.0 kg of shrimp, which were submerged in the treatments for 15 min.Afterwards, the shrimps were packaged in polyethylene bags and stored at 5°C for 13 days.
During this period, aliquots were sampled in order to perform analyzes.

Analytical measurement of colors
Determinations of the shrimp color were performed after the samples remained in an acclimatized room at 15°C for 30 min to oxygenate their surface, using a portable colorimeter Konica Minolta® (Chroma Meter CR-410), with a D65 light source, at an observation angle of 10° and 30 mm of measuring cell opening.The device was systematically calibrated with a white (L*=93.80,a*=-0.89,b*=0.95) and a black (L=1.19*,a*=1.27,b*=1.92)standard.Each sample, consisting of 10 shrimp, were subjected to readings using the CIELab system parameters (L* indicating the luminosity, a* the color and saturation at the red-green axis, and b* the color and saturation at the yellow-blue axis), established in 1967 by the International Commission on Illumination (Parisenti et al., 2011).The measurements were performed in three locations of the sample surface, and the daily averages were calculated for the first to the thirteenth days of storage.

Sensory evaluation of melanosis
A semi-trained sensory panel of 11 judges evaluated the melanosis.Visual evaluations were performed, classifying the melanosis development stages in a four-point scale: 1-without melanosis presence or absence of discoloration; 2-low color change, from mild to moderate (more than 30% of the shrimps' surface affected); 3-severe melanosis (30-70% of shrimps' surface affected in less than 50% of subjects); and 4-extremely severe darkening (70-100% of the shrimps' surface affected in most individuals), following the pictures model of Montero et al. (2004).Each judge evaluated 10 different samples from each treatment each evaluation day, performed on days 1, 2, 3, 4, 6, 7, 9 and 11.

Statistical analysis
Analysis of variance (ANOVA) was used for melanosis levels, L* (luminosity), a* (red level) and b* (yellow level).In the case of 5% statistical variation, the test for multiple comparisons of Bonferroni was performed.The correlation analysis were performed using the linear correlation Pearson (p <0.05).The statistical programs used were the Statistical Analysis System 9.0 (SAS) and the Statistical Package for the Social Sciences 18 (SPSS/PASWSTAT).

RESULTS AND DISCUSSION
The L* parameter began to show changes and differences among treatments from the fourth day of storage (Figure 1).The shrimp treated with sodium metabisulfite (T3) positively differed from the other treatments (P <0.05) on days 4, 5, 8, 9 and 11, showing higher L* values, hence presenting greater control of melanosis.However, it was statistically similar to the sodium chloride treatment (T2) on days 6, 7, 10, 12 and 13, and to the citric acid treatment on day 7 (p <0.05).
The lower the L* value, the higher the melanosis development will be (Yokoyama, 2007).A significantly lower value (p <0.05) of the L* parameter was related to the appearance of "black spots" in shrimps Parapenaeus longirstris (Lopez-Caballero et al., 2007).A decrease in L* value may be considered as an indicator of darkening (Martinez-Alvarez et al., 2007).Thus, the high efficiency of treatments 2 and 3 was noted with higher scores for the L* parameter over the shelf life.Rotllant et al. (2002) found significantly higher luminosity levels in shrimps Aristeus antennatus treated with sulfites-based additives compared to samples without preservatives, demonstrating that sulfites are strong reducing agents, able to whiten crustaceans.Similar results were found in this study with the treatment based on sodium metabisulphite (T3) obtaining the best luminosity results over the 13 days of evaluation.
The sodium chloride in shrimps leads to a denaturation temperature decrease of the protein.The luminosity difference (ΔL*) usually increases as the salt concentration increases, providing a fresh appearance to the product.The denaturation and myofibrillar coagulation associated with the collagen shrinkage and the low water retention capacity also causes an increase in shrimp hardness, another factor that contributes to the maintenance of the fresh product characteristics (Niamnuy et al., 2007).These characteristics also explain the results of treatment 2 (sodium chloride) in this study.
In refrigerated shrimp without additives, a gradual increase of a* values is expected to occur, due to melanosis development which causes a shrimp darkening.In a study with Norway lobsters (Nephrops norvegicus), the red intensity (a*) tended to decrease during storage, indicating that the color of this product tends to change gradually to greenish hues (Martinez-Alvarez et al., 2007).Mol and Turkemen (2010) showed that decreases in a* values suggest a darkening of the meat, becoming less red and more greenish.However, the present study showed increase of a* values (red intensity) from day 2 one (Figure 2).Treatment 3 showed higher values (p < 0.05), except at day 3, when the results were similar (p > 0.05) to treatments 1, 2 and 5, and at day 11 to treatment 2 (p> 0.05).Similar results were reported by Arancibia et al. (2015) which found increased values of a* in Litopenaeus vannamei during 14 days storage at 5°C.Different species have different values of a*, suggesting a difference in carotenoid contents between species (Benjakul et al., 2008).This would explain the differences found in the parameter a* in different studies and species.
The values of b* (yellow intensity) presented differences among treatments only on days 5, 11 and 12 (Figure 3).A gradual increase of the values in all treatments was observed confirming the occurrence of degradation and product quality loss from day 5.In a  Authors found increasing values of b* during the storage period, assuming that it occurred through antioxidant activity of the concentrated proteins and lipids (Arancibia et al., 2015;Wang, 2014).The shrimp general appearance and the melanosis development levels in the different treatments during the storage period is shown in Figure 4.
The use of melanosis inhibitor additives is essential for the fishing industry to ensure the quality of crustaceans for greater shelf life, since the low temperatures alone cannot prevent the occurrence of these black spots (Figure 4).The enzymes responsible for melanosis development remain active during refrigeration, storage on ice or after the freezing process (Pardio et al., 2011).Melanosis levels (Figure 5) showed significant differences (p <0.05) in all evaluated days and treatments.Shrimps treated with sodium metabisulfite (T3) had lower melanosis levels (1.27 ± 0.47; p <0.05) while the other treatments had average of 2.17±0.75(T1), 1.73±0.63(T2), 2.52±0.86(T4) and 2.16±0.72 (T5).
The sodium metabisulphite (T3) treatment maintained melanosis levels (1.40±0.54, 1.32±0.54, 1.17±0.42, 1.16±0.37, 1.38±0.50, 1.20±0.43, 1.27±0.44; p> 0.05) lower than the other treatments on days 1, 3, 4, 6, 7, 9 and 11 and consequently had effective control over this phenomenon.On the other hand, the melanosis levels on the sodium chloride (T2) treatment on day 9 (1.76±0.60)and 11 (1.78±0.56),was statistically superior to the others treatments.Therefore, sodium chloride (T2) may be an inexpensive and low toxicity alternative for industry as compared to sodium metabisulphite (Figure 2).A previous study suggests that sodium chloride could be used as excipient to delay the melanosis appearance when used as a spray (Montero et al., 2006).The results of this study are similar to the treatments which used low doses of the 4-hexylresorcinol to delay melanosis development.Sodium chloride seems to be efficient as some anti-melanotics, because besides preventing melanosis development, it also improves others quality parameters, such as maintaining firmness and bacterial control.
Despite the widespread use of sulfite in the food industry, some adverse health effects are related to its consumption, such as: nausea, location gastric irritation, hives and bronchospasm in sensitive asthmatic subjects, hence the importance of knowing alternative components to sulphite-derivative compounds (Machado et al., 2006;   -Alvarez et al., 2007).Different results found in melanosis studies is because of the different shrimp species, the shrimps initial condition and the difference among the types and concentrations of additives used (Gokoglu and Yerlikaya, 2008).

Martinez
Through the Pearson's correlation analysis (Table 1), significant correlations were found between L* values and melanosis levels (r =-0.8636, p <0.00001), between the L* and a* values (r=0.5279,p = 0.0005) and between the a* and b* values (r=0.7525,p <0.00001).Gokoglu and Yerlikaya (2008) also found correlation (r) between the L* values and melanosis levels in shrimps, P. longirostris immersed in water and in concentrations of grape seed extract, between a* values and melanosis levels in and between b* values and melanosis levels in the treatments.

Conclusion
The literature points out that the melanosis presence is not a public health problem, however it is responsible for significant losses in the fishing industry, reducing the shelf life of crustaceans.Therefore, the use of antimelanotics is needed for control and maintenance of the organoleptic quality of the final product.This study showed positive results regarding the variables: melanosis levels, L* (luminosity), a* (red intensity) and b* (yellow intensity) in shrimps (X.kroyeri) treated with 2.5% of sodium metabisulfite.However, 2% sodium chloride obtained satisfactory results as compared to 2.5% sodium metabisulfite.Sodium chloride, different from sodium metabisulphite, has the advantage of not allergic reactions.Furthermore, sodium chloride has other advantages, such as low cost, maintaining firmness, general appearance, flavor and microbiological control.Therefore, sodium chloride is an excellent alternative to sodium metabisulfite.The authors suggest that studies should be conducted with higher concentrations of sodium chloride, different immersion times and increased storage in order to verify its anti-melanotic action and shelf-life.