Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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By: Bermúdez-Medranda, A. E. y Panta-Vélez, R. P.*

After their death, shrimps go through a process of blackening affecting their commercial value, being the cause of greater rejection of the product in the international market the melanosis in the shrimp, which is a change of color of the surface caused by enzymatic formation of compounds precursors which can spontaneously polymerize and/or react with cellular components to form insoluble pigments. A method used to prevent melanosis is the addition of preservatives in the post-harvest management of shrimp. The objective of the present study developed by researchers from the Technical University of Manabí, Ecuador was to evaluate concentrations of sodium metabisulfite at 4 and 6 % and 4-hexylresorcinol at 2 and 2.5 %, as inhibitors of melanosis.

The great demand that presents the Pacific white shrimp (Penaeus vannamei) and the growing exports from Ecuador, generate a large inflow of foreign currency. The control of the quality of the product is rigorous, rejecting containers that do not comply with microbiological, chemical or sensory specifications.

One of the indicators in this control is the presence of melanosis, a serious problem that results in great economic losses, because it generates a depreciation of shrimp quality. 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.

In order to avoid the melanismic phenomenon suffered by most crustaceans, sulphites began to be used in the 50s and spread widely due to their effectiveness and low cost. But in recent years, research studies about their possible adverse effects and cases of allergies in sensitive people have aroused the concern of consumers.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

Different countries have adopted regulatory measures regarding the use and residual concentration of sulfites in crustaceans, considering a potential danger to human health because it can cause nausea, gastric irritation and vomiting due to the destruction of thiamine.

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 in tissues decreases and when it becomes insufficient, melanosis mechanisms start again, for which the export sector shows its concern to comply with product quality requirements and the maximum residual concentration of sulphites.

Another disadvantage is the impact of sodium metabisulfite on the environment, appreciated by the deterioration of water in the growth of phytoplankton, concentration of dissolved oxygen, transparency, among others.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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.

The objective of this study was to compare in the shrimp muscle the residual concentrations of the preservatives metabisulfites of sodium and 4-hexylresorcinol in shrimp P. vannamei, and in turn know if they allow maintaining 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 Ecuador. The residual analyzes of MBS were carried out between October 2013 and January 2014.

In the experiment, a randomization design with four treatments and six repetitions was used. The treatments 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 a 4% sodium metabisulfite solution (T4) and

    • immersion of fresh shrimp in a 6% sodium metabisulfite solution (T5).

All treatments lasted 15 minutes. Sampling was done with 12g shrimp in each of the replicates. The amount of animals used in the different dives was 4.5 kg, in the case of sodium metabisulfite.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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 and for 4-Hexylresorcinol the technique of HPLC-UV.

Shrimp harvest method

The shrimp is harvested with a bag 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 and 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

To get a 2% concentration, 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. For a 2.5% concentration, 225 mL of 4-hexylresorcinol were added.

Method of application of sodium metabisulfite (4% solution)

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 were 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 were dissolved therein.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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). The shrimp is maintained at 6 ± 2°C, for transport and arrival to the processing plant.

Method of application of sodium metabisulfite (6% solution)

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

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 sample was taken at the time it arrived at the processing facilities, the 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 with 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.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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

ppmSO2 = 32.03 x 0.01 x 1000 x (Consumption – White) / Sample weight (g)



    • 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

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 and microbiological 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 threedigit 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.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

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.

Samples of shrimp treated with the two preservatives were analyzed for total aerobes and for fecal coliforms.


The results obtained in the samples of residual concentration in the shrimp muscle, comparing sodium metabisulfite and 4-hexylresorcinol, showed significant differences between treatments (Table 1).

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

Organoleptic and microbiological 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 evaluation, it was determined that there were no significant differences in the treatments in the different parameters evaluated, observing in the taste, a total average of (I like it little).

The odor presented a general average, and in the color resulted in a total average of (I do not like or dislike). There was no significant difference in any of the treatments in the microbiological analysis for total mesophilic aerobes, indicating that types and concentration of preservatives are effective in reducing the bacterial load.

There was an absence 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.


There were differences up to ten times between the control and the evaluated preservatives on the presence of melanosis. The results obtained show similar absence for both preservatives, indicating their effectiveness.


Both preservatives showed to be effective to avoid melanosis, as reported in other publications. The results obtained for the residual values indicate that the sodium metabisulfite treatment at 4 % reached values according to European regulations, so it is the alternative that provides greater economic advantage.

The use of 4-hexylresorcinol is allowed in the United States, Canada, Australia and some Latin American countries.

Effects of 4-hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)

In the present investigation the residual levels complied with the legal standard established by the European Parliament, where it establishes that the residual level of 4 -hexylresorcinol should not exceed 2 mg/kg, and is considered a generally safe additive (GRAS) because it does not leave side effects caused by sulfites, which indicates a viable and convenient alternative to metabisulfite of sodium to inhibit melanosis.

In the present investigation there were no significant differences when applying the preservatives to different concentrations, in the organoleptic aspect of color, odor and flavor, so both preservatives are an alternative in the prevention of melanosis in shrimp.


The use of 4-hexylresorcinol, an additive considered GRAS, was as effective as sodium metabisulfite in preserving shrimp from melanosis, with the advantage that consumers would appreciate a product free of allergenic additives.

*This is a summarized version developed by Ph.D. Carlos Rangel Dávalos, researcher and professor at the University of Baja California Sur México.
The original article on which is based is titled: “Effects of 4 hexilresorcinol and sodium metabisulfite on melanosis in fresh shrimps (Penaeus vannamei)” by: Bermúdez-Medranda, A. E.y Panta-Vélez, R. P.
The article was originally published on March 2019 through the Magazine Bio Ciencias under a Creative Commons license agreement.
The full version of the article can be accessed at: https://doi.org/10.15741/revbio.06.e465

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