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Introduction
The great demand in the international market that presents the Pacific white shrimp (Penaeus vannamei) and the growing exports of this product from Ecuador, generate a large inflow of foreign currency to the country and allow an important movement of money worldwide.
Due to the high competitiveness and the demands of the markets, the control of the quality of the product becomes increasingly rigorous, rejecting containers that do not comply with microbiological, chemical or sensory specifications. One of the most important indicators in this control, due to the immediacy of its evidence is the presence of melanosis (Martínez, 2017), which constitutes a serious problem that has resulted in great economic losses for producers and the entire marketing chain, because it generates a depreciation of shrimp quality (Loubes et al., 2009). Hence the great importance of achieving a rapid and efficient process before freezing, since melanosis, being a phenomenon of enzymatic origin, usually begins immediately after the death of the animal, although in some cases it has taken place in live specimens (Ogawa et al., 1984). For these reasons enzymatic darkening is one of the most important reactions that affect crustaceans, so its prevention is very essential (Marshall et al., 2000).
In order to avoid the melanismic phenomenon suffered by most crustaceans of commercial interest, sulphites began to be used in the 50s and from then on, they spread widely due to their effectiveness and low cost. But in recent years, numerous research studies about their possible adverse effects and cases of allergies in asthmatic or sensitive people have aroused the concern of consumers and the search for alternative treatments in the prevention of melanosis. Due to the danger of sulfites to public health, different countries have adopted regulatory measures regarding the use and residual concentration of sulfites in crustaceans (Loubes et al., 2009), considering a potential danger to human health because it can cause nausea , gastric irritation and vomiting in humans due to the destruction of thiamine and in susceptible individuals causes allergic reactions in small quantities (Hidalgo, 2011; Díaz, 2011).
Currently the shrimp P. vannamei cultivated in Ecuador is marketed treated with sodium metabisulfite, an antioxidant of proven efficacy, but over time the residual rate of metabisulfite in tissues decreases and when it becomes insufficient, melanosis mechanisms start again (Llerena, 2011), for which the export sector shows its concern to comply with product quality requirements and the maximum residual concentration of sulphites in the edible part. Another disadvantage is the harmful impact of sodium metabisulfite on the environment, analyzing the quality of water in the shrimp supply channels, shows the harmful effect in the shrimp environment, appreciated by the deterioration of water in the growth of phytoplankton, concentration of dissolved oxygen, transparency, among others (Ibarra-Menéndez & Ibarra-Menéndez, 2012).
For all the above, in the last two decades studies have focused on the search for other alternatives for the replacement of sodium metabisulfite, being the 4-hexylresorcinol the possible substitute, since its use is allowed by the health authorities of EE.UU, Canada, Australia and some Latin American countries, since it has been used in the cosmetologically and pharmaceutical industry (Martínez-Álvarez et al., 2007).
The objective of this work was to compare in the shrimp muscle the residual concentrations of the preservatives metabisulfites of sodium and 4-hexylresorcinol in shrimp P. vannamei, in turn know if they allow to maintain the quality of the product during marketing, inhibiting melanosis and that they do not present risks for the health of the consumer.
Material and Methods
Study area
P. vannamei shrimp were treated with sodium metabisulfite (MBS) in a shrimp farm located in Cojimíes, Pedernales, Manabí, Ecuador. The residual analyzes of MBS were carried out in the laboratory of a shrimp exporting company located in Coaque, Pedernales, Manabí, Ecuador, between October 2013 and January 2014.
In the experiment, a randomization design with four treatments and six repetitions was used. The treatments determined were: control (T1); immersion of the fresh shrimp in a 2 % solution of 4-hexylresorcinol (T2); immersion of fresh shrimp in a 2.5 % solution of 4-hexylresorcinol (T3); immersion of fresh shrimp in 4 % sodium metabisulfite solution (T4) and immersion of fresh shrimp in 6 % sodium metabisulfite solution (T5).
All treatments lasted 15 minutes
Sampling was done with shrimp of a commercial size of 12 g in each of the replicates. The amount in kilograms of animals used in the different dives was 4.5 kg, corresponding to the harvesting protocol of the exporting company, in the case of sodium metabisulfite. For 4-Hexylresorcinol the dosage of the chemical was carried out according to the recommendation of the manufacturer of the input.
The measurement of residual sodium metabisulfite was calculated using the modified Monier Williams method AOAC 18Th 990.28 (Horwitz, 2000) and for 4-Hexylresorcinol the technique of Nakazato et al. (2005) (HPLC-UV).
Shrimp harvest method
In the northern area of Manabí, the shrimp is harvested with a bag that is collected while the pond is drained. This process is done with great care to prevent animal damage or excessive accumulation of mud and dirt mixed with the shrimp. The bag is emptied in clean baskets, tubs or bins approximately every 15 or 20 minutes. Depending on the amount to be harvested, temporary storage units (drawers’ plastic) must weigh no more than 35 to 45 pounds to allow reasonable handling. The shrimp should be treated from the shrimp farm to prevent the development of melanosis for which sodium metabisulfite should be applied.
Method of application of 4-hexylresorcinol at 2 % in shrimp farms
4.5 L. of water were placed in a tub of 50 L. capacity, adding 4.5 kg of ice and 180 mL of 4-hexylresorcinol, where the live shrimp was submerged for the respective absorption of the preservative.
Method of application of 4-hexylresorcinol at 2.5 % in shrimp
In a 50 L. capacity tank, 4.5 L. of water, 4.5 kg of ice and 225 mL of 4-hexylresorcinol were placed, then the live crustacean was submerged.
Method of application of sodium metabisulfite (4 % solution) in shrimp farming
For the application of sodium metabisulfite (4 % solution), 15.36 liters of water were placed in a 50 L. tub. Subsequently, 3.84 kg of ice was added, and the solution was stirred well, always maintaining the temperature of the water in the tub between 5-12 ºC.
Approximately 1 L. of water was placed at room temperature in a container and 800 grams of sodium metabisulfite was dissolved therein. Once the Metabisulfite is well dissolved, the solution obtained should have a pH between 4.1-4.2. This solution was added to the previously prepared tub, where a temperature of 5 °C is maintained.
The harvested shrimp was submerged in the previously prepared tub, for 15 minutes, considering that this controlled immersion was one minute for each gram of the average weight of the harvested shrimp (SLA, 2011), once the stipulated time had elapsed, the operator proceeded to remove the shrimp with a drawer (with holes) and then placed in conical (closed) drawers adding ice at the bottom and top to keep the shrimp at a temperature of 6 ± 2 °C, for transport and arrival to the processing plant.
Method of application of sodium metabisulfite (6 % solution) in shrimp farming
In a 50 L. capacity tub, 15.04 liters of water, 3.76 kg of ice and 1,200 grams of sodium metabisulfite (6 % solution) were placed, where the shrimps were submerged to absorb the preservative.
Determination of Residual Sulphite in Fresh Product using the modified Monier Williams technique
Basis
It is based on the digestion of the sample by the action of concentrated hydrochloric acid and heat, obtaining by distillation the sulfites that are collected in 3 % neutral peroxide. The titration was carried out with a solution of sodium hydroxide containing a quantity of substance 0.01 mol/L.
Sample Preparation for Analysis in Processing Plant
The crustacean sample was taken at the time it arrived at the plant and the shrimp was prepared, the head or cephalothorax and the shell removed, taking care to leave the hepatopancreas, homogenized and weighed 30 grams in a watch glass.
Digestion of the Sample
The already weighed sample was placed in a 500 mL Kjeldahl balloon, 150 mL of distilled water and 10 ml of concentrated hydrochloric acid Q.P. were added at 37 % and it was brought to a moderate boil for a period of 20 minutes.
Preparation of the Collector Dissolution in the recovery fiolas
In a 250 mL vial, 10 mL of 30 % hydrogen peroxide in 90 mL of distilled water was diluted to obtain a fresh solution of 3 % peroxide. To this solution was added 3 drops of methyl red and neutralized with a solution of sodium hydroxide containing an amount of 0.01 mol/L substance, the solution gave pinkish color change to straw yellow color. The fiola with the already neutralized solution was placed in the final part of the distillation equipment to collect the vapor condensate from the sample.
The presence of sulphites in the sample was differentiated by the color change back to pink, after 20 minutes of boiling, the fiola was removed and proceeded to the titration with the 0.01 N sodium hydroxide solution, applying the formula to obtain the results.
Calculation of results
Where:
Sodium constant: 32.03 milliequivalents of the mass mol SC.
0.01: Normality of NaOH.
1000: Conversion factor from milliequivalents to micro equivalents.
Consumption: Amount of NaOH substance 0.01 mg/L titrant.
White constant: 0.01 N NaOH consumption per reagent blank.
Weight: In grams of the shrimp sample that was introduced into the Ball.
ppm SO2: Expression of the results in mg/L.
Evaluation of Melanosis
The quality of the pigments (melanins), the decrease in their attractiveness and the commercial value (Luzuriaga et al., 1997). To evaluate melanosis, 30 units of product were left at room temperature for 6 hours, assessing hour by hour, each of the treatments with the two preservatives. The determination was made in percentages of units of related samples. The amount of shrimp in the estimated time is based on the experiences of other packers and customers in the French market.
Evaluation of the organoleptic characteristics
The organoleptic analysis was carried out in fresh product immediately upon the arrival of the product to the plant. The evaluation form was presented randomly coded with a three-digit number.
For this analysis the shrimp were cooked in water at boiling temperature for 3 to 4 minutes, in individual pots, renewing the water enters each sample. After cooking, the samples underwent rapid cooling by immersion in drinking water. The beheading and peeling was done manually. The 5 treatments were evaluated by a panel of ten judges in each of the repetitions, using in the evaluation form a hedonic scale with 9 parameters to be chosen by the judges.
The four treatments in odor characteristics, color and taste against white (product without treatment) were compared in order to evaluate if changes occur in the product, due to the application of preservatives.
Microbiological Characteristics of the Product
Samples of shrimp treated with the two preservatives were analyzed according to the methodology by Maturin & Peeler (2001) for total aerobes and Feng et al. (2002) for fecal coliforms, respectively in an external laboratory.
Statistical analysis
The data obtained from the analysis were stored and processed in Excel and Infostat spreadsheets as a tool for analysis. The results were presented as the mean ± standard deviation. In all cases a Kolmogorov-Smirnov test was performed to verify normality and the Levene test for homogeneity of variance prior to the one-way ANOVA and Tukey test and for the nonparametric Kruskal-Wallis test. The significant differences were calculated in p<0.05.
Results
Evaluation of the residuals of preservatives metabisulfito de sodio and 4 hexilresorcinol
The results obtained in the samples of residual concentration in the shrimp muscle, comparing sodium metabisulfite and 4-hexylresorcinol, showed significant differences between treatments (p=0.0001). Table 1 shows the average residual concentrations of both preservatives, obtaining the lowest concentration in the 4-hexylresorcinol at 2.5 % (T3) with 1.04 ± 1.25 mg/ kg and the highest concentration in the metabisulfite of sodium at 4 % (T4) with 75.44 ± 17.39 mg/kg.
Table 1 Residual concentration of sodium metabisulfite and 4-hexilresorcinol in the shrimp muscle in the different treatments evaluated.
| Treatments | Concentración Residual (mg/kg) ( |
|---|---|
| Control (T1) | 0.00±0.00 a |
| 4-Hexilresorcinol al 2 % (T2) | 1.36±2.69 a |
| 4-Hexilresorcinol al 2.5 % (T3) | 1.04±1.25 a |
| Sodium Metabisulfite al 4 % (T4) | 75.44±17.39 b |
| Sodium Metabisulfite al 6 % (T5) | 74.50±18.17 b |
*Stockings with different letters significantly for p<0.05.
Evaluation of the organoleptic and microbio-logical characteristics
The organoleptic variables were estimated in order to establish if the antimelanoses modify the sensory characteristics of the cooked samples of the crustacean. After the evaluation of the non-parametric Kruskal-Wallis test, it was determined that there were no significant differences in the treatments in the different parameters evaluated (p>0.05) (Table 2), observing in the taste, a total average of 5.95 ± 2.00 (I like it little). In the odor I present a general average of 6.42 ± 2.03 and in the color. I present a total average of 5.46 ± 2.03 (I do not like or dislike).
There was no significant difference in any of the treatments (p=0.3673) in the microbiological analysis for total mesophilic aerobes, indicating that in the types of preservatives and their concentration, they are effective in reducing the bacterial load. Despite this, a lower initial population was observed in T4 and T3. On the other hand, shrimp treated with 6 % sodium metabisulfite (T5) showed slightly higher CFU values of aerobic mesophilic bacteria (Table 2). Likewise there was an absence of bacteria in the analysis of fecal coliforms at 45 °C in the different treatments evaluated, including the control demonstrating a good quality of raw material and management of the executed process.
Table 2 Analysis of the organoleptic and microbiological characteristics of the shrimp in the different treatments evaluated.
| Quality parameters | Treatments | P value | ||||
|---|---|---|---|---|---|---|
| Control (T1) | 4-Hexilresorcinol al 2 % (T2) |
4-Hexilresorcinol al 2.5 % (T3) |
Sodium metabisulfite al 4 % (T4) |
Sodium metabisulfite al 6 % (T5) |
||
| Flavor | 6.05±2.09 a | 5.62±2.11 a | 6.00±1.92 a | 6.39±1.80 a | 5.69±2.11 a | 0.2653 |
| Odor | 6.49±2.05 a | 6.11±2.17 a | 6.49±1.98 a | 6.87±1.85 a | 6.15±2.11 a | 0.2613 |
| Color | 5.57±2.15 a | 5.15±2.10 a | 5,52±1.89 a | 5.89±1.87 a | 5.18±2.16 a | 0.2560 |
| Aerobic mesophilic (CFU/g) | 616.67±466.55 a | 1,160.00±1,570.89 a | 291.67±358.13 a | 248.33±372.58 a | 41,428.33±79,203.91 a | 0.3673 |
*Equal letters do not show significant differences between treatments (p>0.05).
Evaluation of melanosis
There were significant differences between the control and the evaluated preservatives (p<0.05) (Figure 1) on the presence of melanosis in shrimp. The results obtained in the concentrations studied show similar results of absence, indicating that the two preservatives are effective for the control of melanosis in shrimp. The control or control sample (T1) showed an average of 7.27 ± 4.64 % melanization in the shrimp from the fifth hour of exposure in the resistance test.
Discussion
In the research carried out with the two treatments, both in concentration and type of preservative showed to be effective to avoid enzymatic darkening or melanosis in shrimp of the species Penaeus vannamei, which is related to that found by McEvily et al. (1991), Guandalini et al. (1998) and Loubes et al. (2009), who tested concentrations of sodium metabisulfite and 4-hexylresorcinol in Penaeus aztecus, Penaeus duorarum, Parapenaeus longirostris and Pleoticus muelleri, determining that they are suitable agents to avoid melanization.
The results obtained for the residual values of the preservatives indicate that the sodium metabisulfite treatment at 4 % reached adequate values and according to European regulations of maximum allowed (Directive 95/2/EC). (Table 3), so it is the alternative that provides greater economic advantage, taking into account the lower consumption of sodium metabisulfite compared to treatment 5 of 6 % sodium metabisulfite.
Table 3 Directive 95/2/EC of the European Parliament and the council. Maximum residual dose of sodium metabisulfite in the edible parts of fresh shrimp.
| Up to 80 units | 150 mg/kg |
| Between 80 and 120 units | 200 mg/kg |
| More than 120 units | 300 mg/kg |
The use of 4-hexylresorcinol is allowed in the United States, Canada, Australia and some Latin American countries (Martínez-Álvarez et al., 2007). In the present investigation the residual level of the preservatives complied with the legal standard of residuality in the shrimp muscle, established by Directive 2006/52/EC of the European Parliament of July 5, 2006, where it establishes that the residual level of 4 -hexylresorcinol should not exceed 2 mg/kg ( European Union 2006), and is considered a generally safe additive (GRAS) because it does not leave side effects caused by sulfites, which indicates a highly viable and very convenient alternative to metabisulfite of sodium to inhibit melanosis.
In the present investigation in the analyzed shrimps there were no significant differences when applying the preservatives to different concentrations, in the organoleptic aspect of color, odor and flavor. Similar situation registered Loubes et al. (2009), where samples of prawns treated with 4-hexylresorcinol did not reveal changes in taste, tenderness and fibrosity in comparison to sodium metabisulfite and if a juicier mass when the applied treatment was 4-HR at 0.01 %. When no significant differences were observed in this study, both preservatives are the alternative to use in the prevention of melanosis in shrimp, without causing organoleptic changes in shellfish.
In the analysis of mesophilic aerobes presented lower values in the treatment with 4 % sodium metabisulfite. Fecal coliform bacteria indicated absence in all treatments. These results were similar to Yokoyama (2007), who found that between the treatment with sodium metabisulfite and 4-hexylresorcinol there were no significant differences in the counting of mesophilic aerobic bacteria, while a smaller initial population was observed in the treatments with the antimelanosicos, in comparison with the control, equal situation mentions it Montero, López-Caballero & Perez-Mateos (2001), where it indicates that the preservatives used in this essay have certain antimicrobial effect. In turn Yokoyama (2007) did not observe variation in the recovery of faecal coliforms throughout the development of his research, which cannot be compared with the present study due to the fact that no presence of this bacterium was registered.
Conclusions
The use of 4-hexylresorcinol, an additive considered GRAS, was as effective as sodium metabisulfite in preserving shrimp from melanosis during this study. The treatments carried out with 4-hexylresorcinol showed average values of residuals according to European regulations, with the advantage that there are no side effects caused by sulfites, indicating as a highly viable and convenient alternative, considering at the same time that consumers would appreciate a product free of allergenic additives.
It is recommended that the export sector of the shrimp processing industry look for the alternative of use for 4-hexylresorcinol, with a comfortable price that can be accessible to exporters since they are expensive and limit their use in these companies.
