Astaxanthin Complex as an Antioxidant in Preventing Prawn Blackening or Melanosis

Abstract

Melanosis or blackening in prawns is a common problem faced by seafood markets and industries. The formation of black spots decreases the quality of the prawn, as perceived by consumers, and thus reduces its commercial value. Controlling deterioration owing to blackening therefore poses a major challenge to food scientists. Prevention of prawn blackening is carried out mainly with the use of sulphites. The use of sulphites in food applications is a major problem because of their health-related problems in sensitive individuals. An alternative way of preventing melanosis in prawns without the use of chemicals, especially sulphites is necessary. Recently, the use of natural compounds in food applications is preferred by consumers over the use of artificial chemical additives. The main objective of this study is to perform a preliminary investigation on the inhibitory role of a natural antioxidant, astaxanthin complex against prawn melanosis as a natural alternative to chemicals.

1. Introduction

Melanosis or blackening in prawns is a common problem faced by seafood markets and industries. The black spot formation or melanosis in prawns is an enzymatic process, catalyzed by polyphenol oxidase (PPO) [1, 2]. This involves the enzymatic oxidation of phenols to quinones by PPO followed by a non-enzymatic polymerization of the quinones into dark melanins, high molecular weight pigments [3]. Although melanosis seems to be harmless to consumers, it affects the sensory characteristics, and the product shelf-life and quality [4]. Thus, melanosis in prawns reduces its commercial value. Controlling deterioration owing to blackening therefore poses a major challenge to food scientists. Prevention of prawn blackening is carried out mainly with the use of sulphites [5, 6, 7, 8, 9, 10, 11, 12, 13]. The melanosis treatment using sulphites reduced the prawn quality by decomposing trimethlylamine oxide, an osmo-regulator in prawns [14]. Sulphites liberate SO₂ gas upon exposure to moisture. Several deaths occurred among fishermen from SO₂ vapor exposure that was accumulated in the boat hold [15]. Use of sulphites in food applications is also a major problem because it causes allergies in sensitive individuals [16, 17].

4-hexylresorcinol is proposed for use as a processing aid for the prevention of melanosis in prawns and as an alternative agent to sulphites because of its low risk of toxicity. Various studies were conducted to test its suitability against the prawn melanosis [10, 18, 19, 20, 21, 22, 23, 24, 25, 26]. It has also been recognized as Generally Recognized As Safe (GRAS) by Food and Drug Administration for its application in food [27]. The use of 4-hexylresorcinol also reduces the environmental impact [28]. However, this method will always be considered as a chemical treatment irrespective of its safety, economic and environmental benefits [29]. The prevention of blackening in prawns was also investigated using oxytetracycline [30], vacuum packaging and high pressure applications [22, 31, 32], combination of high pressure treatment with the addition of chelating agents and 4-hexylresorcinol [22, 24], Modified Atmosphere packaging (MAP) [29, 33, 34], Gamma irradiation [34], and common antioxidants alone and in combination with 4-hexylresorcinol and other organic acids [24, 35, 36, 37, 38]. Treatment regarded as “natural” is often seen by consumers as more desirable than any “chemical” treatment nowadays. Therefore, various studies have been investigated to prevent prawn melanosis using natural compounds such as commercial proteases ficin and papain [39], enokitake mushroom extract [15, 40]; grape seed extract [41]; moringa extract [42]; lemon and orange peel extracts [43, 44]; lemon and pomelo peel extracts combined with copper sulphide nanoparticles [45]; green tea- and amla extracts [46]; plant polyphenols [47]; catechin [48, 49]; Annona muricata leaves extract [50]; kojic acid [22, 51, 52, 53, 54]; chitosan [55]; fully deacetylated chitosan coating incorporated with kojic acid and clove oil [56]; chamuang leaf extract [57]; avocado by-product extracts [58]; curry and moringa leaves extracts [59]; lead seed extract [60]; olive phenolic compounds extracted from olive oil by-product [61]; rosemary and green tea extracts [62]; edible brown algae extract [63]; mango seed kernel extract [64]; plant polyphenolic compounds [65] and so on.

The black spot formation or melanosis in prawns is an enzymatic process that involves the enzymatic oxidation of phenols to quinones by PPO. This means that PPO is an oxidative enzyme. Astaxanthin complex is a well-known antioxidant present in prawn waste. The question on why this antioxidant does not inhibit the oxidative enzyme, PPO present in prawns in vivo has never been answered or studied. The highlight of this study is to find an answer to this question. A preliminary investigation on the role of the astaxanthin complex extracted from prawn waste in inhibiting PPO activity, thus preventing prawn blackening. The success of this method will be a natural alternative to chemical treatments.

2. Methods and materials

2.1 Materials used

The materials used and the solvent extraction and UV-spectrophotometer analysis of astaxanthin complex from prawn waste are mentioned in [66]. For this investigation, only the Acetone Prawn Extracts (APE) from raw and freeze-dried prawn waste were used.

The second batch of further 15 g of raw prawn waste was added to the APE from raw prawns and kept in the shaker for 2 h. Samples were removed after 1 h and 2 h and centrifuged at 1100 rpm for 10 min. The concentration of astaxanthin complex was then calculated in these two samples using UV-spectrophotometer. The sample taken after 2 h was also analyzed by HPLC to verify the presence of astaxanthin complex in the APE.

Freeze-dried prawn waste (2.5 g, moisture content 6.48% wet basis) was mixed with 10 mL of acetone in a tightly capped flask. Then the flask was kept shaken under the same experimental conditions as mentioned in [66]. After 15 h, the APE was separated from the waste residue and centrifuged at 1100 rpm for 10 min. Then the concentration of astaxanthin complex in the APE was measured using UV-spectrophotometer. The second batch of further 2.5 g of freeze-dried prawn waste was added to this APE and kept in the shaker for further pigment extraction. After 20 h, the sample was removed and centrifuged. Then, the concentration of astaxanthin complex was measured using UV-spectrophotometer.

The presence of astaxanthin complex in the APE was confirmed by analyzing it using Waters High Pressure Liquid Chromatography (HPLC) equipped with a 996 photodiode array detector. The APE was filtered using a 0.45 μm syringe filter before HPLC analysis. The sample (20 μl) was analyzed at a wavelength of 480 nm using a Bondapak™ C₁₈ column (3.9 x 300 mm; 5 μm) at ambient temperature. Gradient method of separation was performed using mobile phase A (dichloromethane/methanol/acetonitrile/water, 5.0:85.0:5.5:4.5, v/v) and mobile phase B (dichloromethane/methanol/acetonitrile/water, 22.0:28.0:45.5:4.5, v/v) at a flow rate of 1 mL/min. The following gradient procedure was used for HPLC analysis of the APE: 0% B for 8 min; a linear gradient from 0 to 100% B for 6 min; 100% B for 51 min. The presence of free astaxanthin in the APE was confirmed by matching the elution time with that of standard astaxanthin [67].

2.2 Blackening due to PPO: confirmative tests

2.2.1 Thermal inactivation test

Heated prawn waste was prepared by heating raw prawn waste (10 g) in distilled water (50 mL) at 80°C for 30 min and the water was drained by paper filtration. Two samples of APE (5 mL each) from raw prawn waste were taken. To one sample was added 2.5 g of heated prawn waste and to the other 2.5 g of raw prawn waste. Each of the two samples was shaken under the same extraction conditions. The visual observations were recorded.

2.2.2 pH test

Two samples of APE (5 mL each) from raw prawn waste were taken. One sample was adjusted to pH 2.0 using 36% hydrochloric acid. The other sample was the control. Raw prawn waste (2.5 g) was added to each of the samples. These two samples were kept in the shaker under the same extraction conditions. The observed color change was recorded.

2.2.3 Astaxanthin complex as an antioxidant: a confirmative test

The APE containing astaxanthin complex without the addition of prawn waste was kept at an ambient temperature as a negative control. Two samples of the APE (5 mL each) from raw prawn waste were prepared. One sample was enriched with 2 mL of astaxanthin standard (0.1 mg/mL) and the other 2 mL of ascorbic acid (0.1 mg/mL). Raw prawn waste (2.5 g) was added to both samples and shaken under the same extraction conditions. The visual observations of the color change were recorded.

3. Results and discussion

3.1 UV-spectrometric analysis of astaxanthin complex

Part of the results are published in [66]. Figure 1 shows the concentration of astaxanthin complex in the APE recovered from raw and freeze-dried prawn waste. The concentration of astaxanthin complex in the APE obtained from raw prawn waste was increased with increasing extraction time until 15 h. When the second batch of raw prawn waste was added to the APE after 15 h, the concentration of astaxanthin complex in the APE decreased sharply within hours. At the same time, dark red pigments and bubbles were formed in the APE. Two hours later, the APE completely turned black.

Figure 1.

Recovery of astaxanthin complex extracted from prawn waste.

These dark red pigments are quinones, which are formed by the enzymatic oxidization of tyrosine by PPO. These quinones are later non-enzymatically oxidized into black melanin pigments. The bubbles consisted of CO₂, a by-product of the melanosis pathway [3]. The concentration of astaxanthin complex extracted from freeze-dried prawn waste increased with time after the addition of the second batch of freeze-dried shrimp waste into the APE (Figure 1). Thus, freeze-drying inactivated the PPO activity.

The APE recovered from raw prawn waste kept at ambient temperature did not show any color change for more than a month. It was expected that some PPO would also be extracted along with astaxanthin complex into the APE. However, the concentration of PPO in the APE was negligible compared to the concentration of PPO in raw prawn waste. Therefore, the astaxanthin complex inactivated PPO in the APE and prevented blackening.

The addition of raw prawn waste into the APE increased the concentration of PPO; PPO acted on the prawn tyrosine present in the APE as well as in the added prawn waste. This initiated the melanosis pathway and the APE turned black. Acetone is a polar solvent. Thus, the effect of the blackening reaction was spread through the entire solvent system. Eventually, the pigment extract turned black owing to the melanin pigments formed at the end of the blackening process. Hence, the astaxanthin complex present in the APE was not enough to inactivate PPO from the added raw prawn waste a second time.

3.2 HPLC analysis of astaxanthin complex

Figure 2 shows the HPLC analysis of the APE obtained from raw prawn waste before and after the addition of the second batch of raw prawn waste. In Figure 2A, the peak of free astaxanthin present in the APE appeared at a retention time of 19.98 minutes same as standard astaxanthin (Appendix 7.14). This confirmed the presence of free astaxanthin in the APE. According to [68], the other peaks at retention times 13.02, 14.67 and 24.44 min were assumed to be esterified forms of astaxanthin complex under the same operating conditions. Because of the unavailability of astaxanthin ester standards, the astaxanthin complex present in the APE was not further identified for this study. In Figure 2B, HPLC analysis of the blackened solvent showed the absence of free as well as ester forms of astaxanthin peaks. The pigment was degraded during melanosis.

Figure 2.

HPLC analysis of astaxanthin complex. (A) APE obtained from raw prawn waste, (B) blackened APE.

3.3 Visual observations

Pure acetone with heated prawn waste turned red because of the extraction of the astaxanthin complex from heated prawn waste into the solvent (Figure 3A). The APE with added raw prawn waste showed melanosis, but the APE added with heated prawn waste did not show any color change (Figure 3B). This is because heat treatment above 60°C or boiling prawns for 3 minutes inactivates PPO [69, 70]. pH and thermal inactivation tests confirmed that the black color formation in the APE was due to PPO present in the added raw prawn waste (Figure 3D).

Figure 3.

Prawn blackening due to PPO. (A) Raw prawn waste added into pure acetone – no color change; (B) boiled prawn waste added to the crude pigment extract—no color change; (C) Raw prawn waste added to the crude pigment extract adjusted to pH 2.0—no color change; (D) Raw prawn waste added to the pigment extract—black color formation.

The addition of prawn waste into the APE at pH 2.0 did not show any color change because the enzymatic activity of PPO was inactivated in an acidic environment [69]. But the pigment extract (pH 7.8) without pH adjustment showed melanosis because PPO present in the added raw prawn waste caused blackening (Figure 3C).

The APE samples enriched with standard astaxanthin, and ascorbic acid did not turn black with the addition of raw prawn waste. The enzymatic activity of PPO in these two samples was fully suppressed in an antioxidative environment containing standard astaxanthin and ascorbic acid respectively. Blackening of the APE was thus prevented and the original red color of the APE was retained. Thus, antioxidants standard astaxanthin and ascorbic acid inactivate the oxidative enzyme, prawn PPO.

The APE enriched with ascorbic acid (4 times the original astaxanthin complex concentration of the APE) turned black after two months. But the sample enriched with standard astaxanthin (4 times the original astaxanthin complex concentration of the APE) retained its original red color for more than six months (Figure 4). Standard astaxanthin was therefore more active as an antioxidant against prawn PPO than was ascorbic acid (weight basis). The proposed mechanism of astaxanthin as an antioxidant is that the pigment scavenges the oxygen free-radicals that are formed during the melanosis pathway [71].

Figure 4.

Astaxanthin complex shows more powerful antioxidant activity against prawn PPO than ascorbic acid.

The APE kept at room temperature without the addition of raw prawn waste did not change color because the prawn PPO was not introduced in this sample. Similarly, the standard astaxanthin-enriched APE upon the addition of raw prawn waste did not show any blackening because there was more astaxanthin present in this sample to inactivate PPO from the added raw prawn waste. In other words, increasing the concentration of astaxanthin complex decreases the PPO activity in prawns at a lab scale. However, PPO inhibitors other than astaxanthin complex may also be present in the APE. Therefore, an in-depth study is required to identify the effective compound(s) present in the APE that inhibits melanosis in prawns. This study will constitute a base for further research on the astaxanthin complex in preventing blackening in prawns in vivo.

4. Conclusions

The black color formation in the APE added with raw prawn waste is due to PPO activity. When the APE was enriched with astaxanthin, it inhibited the prawn blackening caused by PPO for more than 6 months and proved to be more powerful than that of ascorbic acid in the lab study. Astaxanthin complex present in prawns is a well-known antioxidant. The question is why this antioxidant does not stop the oxidative enzyme, PPO activity in prawns in vivo. The answer to this question is because the concentration of astaxanthin complex in prawns may not be sufficient to completely inhibit the PPO activity in vivo. If astaxanthin complex-enriched feed is offered in prawn culture [72, 73, 74, 75, 76, 77, 78], there is a chance that the enriched astaxanthin in prawn tissues may inactivate prawn PPO, thereby preventing prawn melanosis after the harvest. Further research will test this hypothesis.

5. Recommendations

This preliminary study of the antioxidant role of astaxanthin complex extracted from raw prawn waste recommends a field study of astaxanthin-enriched feed in prawn culture against prawn blackening in vivo and optimizing the concentration of astaxanthin complex in prawn feed to stop the prawn PPO activity. Astaxanthin complex for prawn feed enrichment can be recovered from properly stored prawn waste before its conversion into chitin. The success of this method not only prevents prawn melanosis in vivo but may also eliminate the post-harvest chemical treatments against prawn blackening, the treatment time and the associated costs. This method will be more acceptable than chemical treatments to many consumers.

Acknowledgments

Many thanks to the Department of Primary Industries, Fisheries Conservation Technology Unit, NSW, Australia for supplying raw Eastern School prawns for the research. I also thank the Australian Government for the financial support by providing the International Postgraduate Research Scholarship to undertake this research.

Notes/thanks/other declarations

Parts of this chapter were previously published in the author’s doctoral thesis: Karuppuswamy R. Extraction and characterization of major valuable compounds from prawn waste [Internet]. UNSW Sydney; 2008. Available from: http://hdl.handle.net/1959.4/43329