JFS C: Food Chemistry and Toxicology. C: Food Chemistry & Toxicology. Introduction Melanosis, or blackspot, appears very frequently during the

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1 JFS C: Food Chemistry and Toxicology Effectiveness of Onboard Application of 4-Hexylresorcinol in Inhibiting Melanosis in Shrimp (Parapenaeus longirostris) P. M ONTERO, O. MARTÍNEZ-ÁLVAREZ, AND M.C. GÓMEZ-GUILLÉN ABSTRACT: Inhibition of melanosis in shrimp (Parapenaeus longirostris) captured at different times of year and treated with different 4-hexylresorcinol based formulations onboard ship was studied. Melanosis inhibition increased with inhibitor concentration. The 4-hexylresorcinol proved effective at extending shelf life over that of untreated shrimp at all the concentrations tested. Combining the inhibitor with citric acid, ascorbic acid, and acetic acid did not increase the extent of melanosis inhibition but did noticeably improve shrimp appearance. Adding ethylenediaminetetraacetic acid (EDTA) and sodium pyrophosphate to the formulation enhanced melanosis inhibition at all times of year. Spray application of the inhibitor formulation extended shelf life compared with dipping and dusting, but dusting resulted in the lowest melanosis levels for a given formulation composition and concentration. Keywords: melanosis, shrimp, 4-hexylresorcinol, chilled storage Introduction Melanosis, or blackspot, appears very frequently during the storage of crustaceans post mortem before the onset of spoilage. Melanosis starts in refrigerated and frozen crustaceans within just a few hours of capture, and susceptibility is often greater in autumn and winter, coinciding with molting and lower food availability. The condition is brought on by the action of polyphenoloxidase, which oxidizes phenols to quinones. Polymerization of the colorless quinones gives rise to black high-molecular-weight pigments. The pigments themselves are not dangerous to human health, but affected crustaceans are rejected by consumers because of their appearance. Refrigeration alone does not prevent, but only slows, blackspot development, because the enzyme remains active during refrigeration, storage on ice, and post-freeze thawing. Sulfite-based formulations, mainly metabisulfite, are currently used to avoid or at least delay melanosis. High concentrations of additive are sometimes required to prevent melanosis effectively. For this reason, it is relatively common for local seafood retailers to add an extra dose of metabisulfite, thereby increasing the total content in the edible portions to levels exceeding the limits established by lawmakers and regulators. Furthermore, sulfites can cause adverse reactions in certain population groups, especially asthmatics (Collins-Williams 1983; Gunnison and Jacobsen 1987). Indeed, metabisulfite is regarded as a causative agent of asthma attacks (Collins-Williams 1983), making research into alternatives to sulfite derivatives very important. Based on previous work, 4-hexylresorcinol appears to be a good alternative. Its use is permitted in the United States, Canada, Australia, and some Latin American countries. 4-Hexylresorcinol seems to be highly effective, although the proportions required to inhibit melanosis have not been clearly established. MS Submitted 5/5/04, Revised 6/18/04, Accepted 6/30/04. The authors are with Inst. del Frío (CSIC). José Antonio Novais, Madrid, Spain. Direct inquiries to author Gómez-Guillén ( cgomez@if.csic.es) Institute of Food Technologists Further reproduction without permission is prohibited Some work has cited 0.005% (McEvily and others 1991) in brown shrimp (Penaeus aztecus) and pink shrimp (Penaeus duorarum) and Otwell and others (1992) in pink shrimp (Penaeus duorarum). Other researchers have reported 0.01% (Guandalini and others 1998) in deepwater pink shrimp (Parapenaeus longirostris), whereas still others have cited considerably higher concentrations, namely, as much as 0.5% in tiger shrimp (Penaeus japonicus) (Montero and others 2001b) and 0.1% in deepwater pink shrimp (Parapenaeus longirostris) (Montero and others 2003). These differences may be due to interspecies variations, to cyclical changes in physiological susceptibility, or to melanosis inhibitor concentration and the method of application used. However, nearly all reports have involved small-scale experimental or laboratory studies. The literature contains no studies on different methods of application, seasonal variations, or other factors that might influence blackspot development. There is a commercial formulation, Everfresh, composed of 4- hexylresorcinol and NaCl (Opta Food Ingredients, Inc., Bedford, Mass., U.S.A.; US patent nr ). However, for certain species, such as deepwater pink shrimp (Parapenaeus longirostris), the product has proved to be quite ineffective using the concentrations and application times recommended by the manufacturer, and noticeable melanosis has been reported to appear in these shrimp within 3 d, probably because this species is particularly susceptible to blackspot development (unpublished data). Sulfites are commonly used in association with another antioxidant such as citric acid or ascorbic acid. Some studies have suggested that when used on prawns (Penaeus japonicus) 4-hexylresorcinol and ascorbic acid form an effective combination (Montero and others 2001b). For all these reasons, seeking more potentially effective 4-hexylresorcinol based formulations holds considerable interest. The object of this study was, therefore, to examine the action of 4-hexylresorcinol when applied under various conditions (concentration, time of year, and method of application) and in combination with Vol. 69, Nr. 8, 2004 JOURNAL OF FOOD SCIENCE C643 Published on Web 10/7/2004

2 other compounds to determine the effectiveness of onboard treatment of shrimp caught by conventional means. Materials and Methods The experiments were performed on deepwater pink shrimp (Parapenaeus longirostris) caught by trawler off the coast of southern Spain (Cádiz). Mean shrimp weights and average temperatures at the time of capture were 4.38 ± 1 g and 19 C to 20 C in February, 6.19 ± 0.5 g and 19 C to 20 C in March, 8.03 ± 0.54 g and 20 C to 23 C in May, 9.6 ± 1 g and 26 C to 30 C in July, and 4.58 ± 0.26 g and 25 C to 26 C in October. Onboard ship, the shrimp were separated from the by-catch and washed in seawater. Processing of the shrimp delayed application of the blackspot inhibitor (dipping, dusting, or spraying) by approximately 1 h. Various concentrations of 4-hexylresorcinol (H6250; Sigma Chemical Co., St. Louis, Mo., U.S.A.) ranging from % to 1.0% (g of inhibitor per 100 g of shrimp) were used for the dipping treatments. Reagent-grade citric acid, ascorbic acid, acetic acid, ethylenediaminetetraacetic acid (EDTA) (0.0225% to 0.1%), and disodium dihydrogen pyrophosphate (PP) (1% to 3%) were also added to the solutions. The dips were made up at a seawater ice to shrimp ratio of 2:1 (v/w). The blackspot inhibitor admixture was dissolved in seawater with ice and added, and the shrimp were then immersed and left for 1 h. After treatment, the shrimp were removed, placed in perforated polystyrene boxes with a holding capacity of 2 kg, and covered with ice. The dusting and spray treatments were also performed onboard ship, and the treated shrimp were placed in perforated polystyrene boxes with a 2-kg holding capacity and covered with ice. On arrival of the trawler in port, the boxes were taken by refrigerated truck to the Instituto del Frío in Madrid where they were stored in ice at 2 C. A taste panel composed of 8 trained panelists evaluated 14 shrimp per batch every 2 d during the storage period. Panelists considered appearance, attachment of the head (carapace) to the abdomen, and presence of yellow-green coloring of the viscera. Melanosis (manifested as black spots on the shell, especially on the head) was assessed according to a visual scale from 1 to 4, in which 1 = complete absence of black spots; 2 = a few small spots on the carapace; 3 = considerable spotting on the carapace; 4 = substantial spotting over the entire shrimp. Panelists also examined the commercial viability of each batch. 4-Hexylresorcinol Shrimp caught in 2 different seasons, namely, in March and October, were dipped in different concentrations of 4-hexylresorcinol onboard ship. Melanosis progression is depicted in Figure 1a and 1b. Blackspot progression in the untreated shrimp stored in ice was the same in both seasons, attaining a score of 3 after storage for 4 d. Higher concentrations of 4-hexylresorcinol slowed melanosis development during storage in ice. In March, a concentration of 0.05% 4- hexylresorcinol delayed melanosis onset, whereas 0.5% inhibited melanosis for 11 d. Extremely high concentrations of 4-hexylresorcinol prevented melanosis from progressing over 11 d, but the heads separated from the abdomens, the viscera turned greenish yellow, and there was a slight odor of ammonia, causing the shrimp to be discarded. In October, in contrast, melanosis advanced faster, and higher concentrations of inhibitor were required to achieve the same or even a less beneficial effect. Concentrations of 0.25% 4- hexylresorcinol in October proved to be less effective at inhibiting melanosis than concentrations of 0.05% to 0.1% 4-hexylresorcinol in March. These results underscore the differing efficacy of 4-hexylresorcinol, depending on the season in which the shrimp are caught. Inhibition was less effective in October, coinciding with molting (Ogawa and others 1984). Polyphenoloxidases are known to be involved in the molt cycle and could be more active at this time of year. Studying the effectiveness of different blackspot inhibitor substances in prawns (Penaeus japonicus), Montero and others (2001b) observed that at a concentration of 0.5% applied to prawns immediately after death, 4-hexylresorcinol was the most effective inhibitor, achieving a melanosis score similar to that recorded in the present experiment after 8 d. Certain studies have been carried out on pink shrimp (Penaeus duodarum) stored in ice, finding that application of 0.005% of 4-hexylresorcinol for 1 min was more effective than the customary 1.25% of sulfite, maintaining acceptable melanosis levels for longer storage times of up to 12 d (Otwell and others 1992). Comparing treatments using the same concentrations (0.005%), these reported melanosis levels were similar to the levels observed on deepwater pink shrimp (Parapenaeus longirostris) in the present study after storage for 9 d. However, higher concentrations proved to be still more effective, achieving Statistical analysis The melanosis scores were regressed on time in storage using the Statgraphics Plus 2.1 computer program (STSC Inc., Rockville, Md., U.S.A.) for statistical processing. Linear or polynomial regressions were plotted through data dispersion, showing in all cases regression coefficients R Results and Discussion Figure 1 Melanosis scores during chilled storage for shrimp caught in (a) March and (b) October treated with various concentrations (%) of 4-hexylresorcinol. NI = no melanosis inhibitor. C644 JOURNAL OF FOOD SCIENCE Vol. 69, Nr. 8, 2004 URLs and addresses are active links at

3 very low levels of melanosis. There are other studies on the inhibitory effect of 4-hexylresorcinol on different species, such as Penaeus aztecus (Benner and others 1994), or Penaeus esculentus, Penaeus plebejus, Metapenaeus enchavoury, and Metapenaeus benettae (Slaterry and others 1995). The differences in the effectiveness of 4-hexylresorcinol reported by these researchers could be ascribable to characteristics intrinsic to the different species, capture at different times of year, different methods used to apply the blackspot inhibitor, and so on. 4-Hexylresorcinol combined with acid With a view to finding a moderate yet efficient concentration level, preliminary trials were carried out combining 0.1% and 0.25% 4-hexylresorcinol with other compounds that have been reported to exert a beneficial effect on melanosis. The inhibitor was mixed with various concentrations of citric acid, ascorbic acid, or acetic acid, singly or all 3 combined. These acids do not actually inhibit blackspot themselves, but they do enhance the appearance of shrimp treated with 4-hexylresorcinol by accentuating the natural pink coloration of the shrimp in a way that was considered highly appealing by the taste panel judges. The best formulation consisted of inhibitor combined with all 3 acids (0.5% citric acid, 0.5% ascorbic acid, and 0.3% acetic acid), which, for simplicity, will be called the ACRA formulation. Figure 2 depicts the results of treatment for shrimp caught in October. The effect on melanosis was similar to that achieved by dipping in a solution of 4-hexylresorcinol alone. Melanosis inhibition per se was not increased, but the appearance of the shrimp was much better and very fresh-looking. Montero and others (2001b) investigated a combination of 4- hexylresorcinol with an acid (ascorbic acid or citric acid) as a blackspot inhibitor mixture in prawns (Penaeus japonicus) by dipping for 2 h; they found that the effect of each acid was intensified and melanosis levels were lower than reported here throughout the storage period. This suggested that each of these substances might suppress a different stage or step in the pigment formation process or promote the action of 4-hexylresorcinol. The presence of these acids in the inhibitor solution reduces the ph, and melanosis is more easily avoided in species in which PPO is more susceptible to inhibition at lower ph levels, such as the prawn (Penaeus japonicus) (Montero and others 2001a) than in others, such as the shrimp (Parapenaeus longirostris), in which this is not the case (Montero and others 2003). Comparing the seasonal efficacy of a moderate concentration of 4-hexylresorcinol (0.1%) in the ACRA formulation at different times of year, treatment was most effective in May (spring) and least effective in February; in October (autumn) treatment effectiveness was intermediate between these 2 extremes (Figure 3a). However, when the concentration of 4-hexylresorcinol in the ACRA formulation was 0.25% (Figure 3b), melanosis inhibition was quite similar over the 1st wk of storage in all seasons. Although there was a slight tendency toward better inhibition later in storage, in May the shrimp were discarded because of spoilage. 4-Hexylresorcinol with acids and chelating agents To improve the results obtained with the ACRA formulation that contained 0.1% 4-hexylresorcinol, the mixture was combined with various concentrations of another compound, namely, the chelating agents disodium dihydrogen pyrophosphate (PP) and ethylenediaminetretraacetic acid (EDTA) as depicted in Figure 4. Higher concentrations of chelating agent reduced melanosis, but the effect of the ACRA formulation (0.1% 4-hexylresorcinol) was heightened at all concentrations. Shrimp appearance at 9 and 11 d was better, and the viscera were less yellowish than for the formulations containing the combination of the 3 acids alone, without the chelating agents. This result is ascribed to the natures of EDTA and PP, which are metalloprotease inhibitors and, consequently, at least Figure 2 Melanosis scores during chilled storage for shrimp treated with various concentrations (%) of 4-hexylresorcinol combined with 0.5% citric acid, 0.5% ascorbic acid, and 0.3% acetic acid. NI = no melanosis inhibitor. Figure 3 Melanosis scores during chilled storage for shrimp caught at different times of year and treated with (a) 0.1% and (b) 0.25% 4-hexylresorcinol combined with 0.5% citric acid, 0.5% ascorbic acid, and 0.3% acetic acid. URLs and addresses are active links at Vol. 69, Nr. 8, 2004 JOURNAL OF FOOD SCIENCE C645

4 partially may inhibit the formation of free tyrosine and phenylalanine, substrates for the action of polyphenoloxidase (PPO), without disregarding a direct effect on the enzyme. Handling Handling and sorting of the catch before treatment is another factor that needs to be taken into account. To evaluate this factor, a shorter time (25 min) and a longer time (2.5 h) were allowed to elapse before the shrimp were treated (Figure 5). After being left to stand, the shrimp batches underwent treatment with no additives (control batch) or with the ACRA inhibitor formulation containing 0.1% 4-hexylresorcinol, the 3 acids, and the 2 chelating agents (0.025% EDTA and 1% PP). This part of the experiment was performed in February, when the shrimp are most sensitive to melanosis. The different times allowed to elapse before treatment had no substantial effect on the melanosis scores, which in both cases were better for the batch treated with inhibitor. Being able to gain this time is of great importance commercially because it permits the shrimp to be separated from the by-catch and sorted by size. Application methods The most effective inhibitor formulations (4-hexylresorcinol [0.25% and 0.1%], citric acid [0.5%], ascorbic acid [0.5%], acetic acid [0.3%], EDTA [0.025%], and PPi [1%]) were used to compare the different application methods using shrimp caught in July (Figure 6a and 6b). In the dust treatment, addition of 0.3% acetic acid was omitted. For all the methods of application, melanosis was very low at both inhibitor concentrations, with the lowest melanosis scores being recorded for dusting. In all cases, shelf life ended when the head turned greenish yellow. This spoilage indicator was very pronounced in the shrimp treated by dusting, less so in the shrimp treated by dipping, and very slight in the shrimp treated by spraying. Furthermore, spraying heightened the natural color of the shrimp, resulting in a particularly good appearance. Dipping caused the most mechanical damage (separation of the heads), but dusting represented the greatest danger to crewmen, Figure 4 Melanosis scores during chilled storage for shrimp treated with a mixture of 0.1% 4-hexylresorcinol, 0.5% citric acid, 0.5% ascorbic acid, and 0.3% acetic acid combined with different concentrations of ethylenediaminetetraacetic acid (EDTA) (%) and disodium dihydrogen pyrophosphate (%). Figure 5 Melanosis scores during chilled storage for shrimp allowed to stand for 25 min and 2.5 h and then treated with a formulation composed of 0.1% 4-hexylresorcinol, 0.5% citric acid, 0.5% ascorbic acid, 0.3% acetic acid, 0.025% ethylenediaminetetraacetic acid (EDTA), and 1% disodium dihydrogen pyrophosphate. I = with melanosis inhibitor; NI = no melanosis inhibitor. Figure 6 Melanosis scores during chilled storage for shrimp treated by dipping, dusting, or spraying using a formulation composed of (a) 0.1% and (b) 0.25% 4-hexylresorcinol combined with 0.5% citric acid, 0.5% ascorbic acid, 0.3% acetic acid (except for the dust treatment), 0.025% EDTA, and 1% disodium dihydrogen pyrophosphate. C646 JOURNAL OF FOOD SCIENCE Vol. 69, Nr. 8, 2004 URLs and addresses are active links at

5 and residual levels of inhibitor components in shrimp treated by dusting can vary more broadly and in some cases may be quite high (Montero and others 2003). Having all these factors in mind, spraying could be the best application method. Further studies should examine residual levels of inhibitory substances in shrimp produced by the different treatments. Acknowledgments This research was funded by a project jointly underwritten by the Consejería de Agricultura y Pesca (Junta de Andalucía) (Dept. of Agriculture and Fisheries of the Government of the Autonomous Region of Andalusia) and the Consejo Superior de Investigaciones Científicas (CSIC) (Spanish Board of Scientific Research). References Benner RA, Miget R, Finne G, Acuff GR Lactic acid/melanosis inhibitors to improve shelf life of brown shrimp (Penaeus aztecus). J Food Sci 59(2): Collins-Williams C Intolerance to additives. Anal Allergy 51: Guandalini E, Ioppolo A, Mantovani A, Stacchini P, Giovannini C Hexylresorcinol as inhibitor of shrimp melanosis: efficacy and residues studies; evaluation of possible toxic effect in a human intestinal in vitro model (Caco- 2); preliminary safety assessment. Food Addit Contam 15(2): Gunnison A, Jacobsen D Sulphite hypersensitivity. A critical review. Critical Rev Toxicol 17: McEvily AJ, Iyengar R, Otwell S Sulfite alternative prevents shrimp melanosis. Food Technol Chicago 45(9):80 6. Montero P, Ávalos A, Pérez-Mateos M. 2001a. Characterization of polyphenoloxidase of prawns (Penaeus japonicus). Alternatives to inhibition: additives and high-pressure treatment. Food Chem 75: Montero P, López-Caballero ME, Pérez-Mateos M. 2001b. The effect of inhibitors and high pressure treatment to prevent melanosis and microbial growth on chilled prawns (Penaeus japonicus). J Food Sci 66(8): Montero P, Gómez-Guillén MC, Zamorano P, Martínez-Álvarez O Estudios de los agentes conservantes e inhibidores de la melanosis en crustáceos. Ed. Junta de Andalucia. Sevilla, Spain: Consejería de Agricultura y Pesca. Ogawa M, Perdigão NB, Santiago ME, Kozima TT On physiological aspects of blackspot appearance in shrimp. Nipp Suis Gakkai 50: Otwell WS, Iyengar R, McEvily AJ Inhibition of shrimp melanosis by 4- hexylresorcinol. J Aqua Food Prod Technol 1(1): Slattery SL, Williams DJ, Cusak A A sulphite-free treatment inhibits blackspot formation in prawns. Food Austr 47(11): URLs and addresses are active links at Vol. 69, Nr. 8, 2004 JOURNAL OF FOOD SCIENCE C647