A Review of the Carotenoid, Astaxanthin, as a Pigment Source and Vitamin for Cultured Penaeus Prawn
By Todd Lorenz, Ph.D.
Carotenoids are a family of over 600 natural lipid-soluble pigments that are produced within microalgae, phytoplankton, and higher plants. They are synthesized through the isoprenoid pathway which also produces such diverse compounds as essential fatty acids, steroids, sterols, vitamins A, D, E, and K. Within the various classes of natural pigments, the carotenoids are the most widespread and structurally diverse pigmenting agents. They are responsible, in combination with proteins, for many of the brilliant yellow to red colors in plants and the wide range of blue, green, purple, brown and reddish colors of fish and crustaceans. The general distribution and metabolic pathways of carotenoids has been extensively detailed previously (Goodwin 1984, Davis 1985, Matsuno and Hirao, 1989).
The kuruma prawn, Penaeus japonicus, and tiger prawn, Penaeus monodon, are cultured worldwide, demand and production has steadily increased for these important aquaculture crustaceans. The market value of prawn is predominately based on the visual appeal of their body color. Product appearance and resulting quality implications play a significant role in maintaining the highest consumer acceptance. Most crustaceans contain a mixture of carotenoids in the carapace in addition to the blood, eyes, midgut gland, and ovary. The red carotenoid, astaxanthin, has been identified as the predominant pigment isolated from Penaeus shrimp (Katayama et al., 1971, 1972). Astaxanthin was shown to be the principal pigment in the carapace and internal organs of seven different crustacean species, accounting for 86-98% of the total carotenoids, and was shown to be responsible for the desirable body color of prawns upon cooking (Tanaka et al., 1976, Okada et al., 1994). Specifically, body color of crustaceans is dependent on the qualitative and quantitative presence of carotenoids in the hypodermal chromophores and the pigmented layer of the epidermal exoskeleton.
Crustaceans and other aquatic animals are unable to produce astaxanthin de novo, only plants and protists (bacteria, algae, fungi) are capable of synthesizing carotenoids. Therefore astaxanthin must be available in either their native habitat or manufactured diet to meet metabolic nutritional requirements (Steven, D.M. 1948). In the natural aquatic environment, astaxanthin is biosynthesized in the food chain within microalgae or phytoplankton at the primary production level. The microalgae are consumed by zooplankton, insects or crustaceans which accumulate astaxanthin, and in turn are ingested by fish which then accrue astaxanthin. The majority of the astaxanthin within the epidermal tissue is in the mono-esterified form, meaning that one of the hydroxyl groups is esterified to a fatty acid. Whereas, complexes of carotenoids and proteins called carotenoproteins and carotenolipoproteins dominate in the exoskeleton. Astaxanthin appears as a red pigment, but when complexed with various proteins, the light absorbance shifts and cause crustaceans to range in color from green, yellow, blue to brown. Thus, despite the fact that astaxanthin is the chromophore prosthetic group of the different carotenoproteins, many colors can be achieved (Muriana et al., 1993 and Nur-E-Bordan et al., 1995, Britton et al., 1981). The red color of cooked crustaceans is produced by the release of the individual carotenoid prosthetic group (astaxanthin) from the carotenoproteins when denatured by the heat of cooking. The final color and hue saturation is dependent on the amount of astaxanthin deposited.
The lack of dietary astaxanthin in cultured Penaeus monodon has been shown to be the cause of "Blue Color Syndrome". After four weeks of feeding a diet containing 50 ppm of astaxanthin, prawns with Blue Color Syndrome resume their normal greenish-brown pigmentation. Analysis of the tissues from the experimental groups verified that the astaxanthin-fed group increased in carotenoids 318% , and had a normal appearance. Those fed the commercial diet without astaxanthin had a carotenoid increase of only 14% and had a blue hue (Menasveta et al., 1993).
Other studies of Blue Disease in farmed tiger shrimp P. monodon attribute the nutritional deficiency to carotenoids. The majority of carotenoids present in wild P. monodon were astaxanthin, astaxanthin esters and small amounts of (-carotene. A total carotenoid concentration of 26.3 ppm was isolated from the exoskeleton of wild shrimp, and farmed shrimp which are supplemented with sufficient astaxanthin. Whereas, specimens displaying Blue Disease had total carotenoid concentrations of only 4-7 ppm in the exoskeleton. Upon cooking, shrimp with Blue Disease assume a pale yellow color rather than the bright red coloration characteristic of wild shrimp (Howell and Matthews, 1991)
The effect of dietary carotenoids on pigmentation has been studied by feeding 100 ppm of various carotenoids ( (-carotene, canthaxanthin and astaxanthin) to Penaeus japonicus. After 8 weeks, it was found that all three carotenoids were deposited in the tissues, however groups fed dietary astaxanthin had the highest levels of tissue astaxanthin (16.5 mg/kg body wt.) This result was 23% higher than those fed canthaxanthin and 43% higher than those fed (-carotene. Thus, the tests demonstrated that astaxanthin was the most effective carotenoid source for pigmentation, in agreement with studies conducted on trout and Atlantic salmon. A second experiment focused on specific feeding levels for astaxanthin from 50 to 400 ppm over an eight-week period. The study established that as dietary astaxanthin increases up to 200 ppm, deposition also increases to a maximum of 29.1 mg/kg body wt. However, dietary levels above 200 ppm did not lead to an increase of tissue concentrations, indicating a saturation point is attained (Yamada et al., 1990).
Similar feeding trials with 100 ppm astaxanthin, 100 ppm canthaxanthin and a mixture of the carotenoids have been conducted. The results revealed that astaxanthin-supplemented diets allowed 128% higher accumulation of carotenoids within the carapace than canthaxanthin and 135% higher than the astaxanthin-canthaxanthin mixture (Negre-Sadargues et al., 1993).
It has been demonstrated that there is a significant decrease in mortality of adult shrimp fed a carotenoid-enriched diet in comparison with individuals receiving carotenoid-free diets. A survival rate of 91% was observed with individuals fed a diet supplemented with 100 ppm astaxanthin compared to 57% in the control group without astaxanthin after 4-8 weeks of growth (Yamada et al., 1990).
Chien and Jeng (1992) studied the effects of various regimes of carotenoid-supplemented diets in P. japonicus using astaxanthin and (-carotene. They found that after one month astaxanthin at 100 mg/100g diet was the most effective pigment for optimal coloration of prawns resulting in deposition of 107, 46 and 74 mg/kg of tissue in the heads, flesh and shells, respectively. In contrast, (-carotene at 100 mg/100g diet resulted in deposition of 12, 4, and 6 mg/kg of tissue in the head, flesh and shells, respectively. Survival was higher in prawns fed the astaxanthin diet, and a positive correlation between survival and pigment concentration of tissues suggested that the carotenoids functioned as an intracellular oxygen reserve. This permitted the crustaceans to survive under hypoxic conditions common in pond cultures. Shrimp fed astaxanthin at 100 mg/kg diet had an average survival rate of 77% in contrast to shrimp supplemented with (-carotene which averaged 40% (Chien 1996).
High quality broodstock maturation diets are an essential key for successful and sustained production of nauplii. Most maturation diets depend on fresh or frozen natural feeds such a squid, krill, mussels and polychaete worms. However, under sustained conditions, a general decline of nauplii quality and larval performance is observed. The degradation is associated with a loss of pigmentation and bleaching of the ovaries of mature females and larval egg yolks. Consequently, there is low larval feeding rates in Z1, high levels of larval Z1 deformities, and very reduced survival to larval stage zoea II. This condition has been termed "Pigment Deficiency Syndrome" (PDS). Paprika has been found to be somewhat effective as a pigmentation source for American lobsters (D'Abramo, 1983), and has been successfully used as a carotenoid source to reverse the deleterious effects of PDS in High Health broodstock (Wyban 1996). In one experiment, a group of broodstock afflicted with PDS was utilized to test the inclusion of paprika carotenoids in the diets. After four weeks under simulated commercial conditions, nauplii quality improved dramatically with the mean ZII survival rate increasing from 25% to 83% (p<0.01). The percentage of larvae with full guts increased from 49% before paprika to 96% (p<0.01), and percentage of deformed larvae decreased from 21% to 4% after four weeks of carotenoid supplementation. There was no significant difference between ablated and unablated females within the treatment groups (Wyban, 1996).
Paprika contains the xanthophylls beta-carotene, beta-cryptoxanthin, capsanthin and capsorubin, some of which can apparently be slowly converted to astaxanthin after a lag time. (D'Abramo 1983, Latscha 1991, Wyban, 1996). Preliminary trials demonstrate that supplementation with NatuRose natural astaxanthin yields superior results compared to paprika, as there is no lag time for biosynthetic conversion and it can be directly utilized for metabolic purposes. NatuRose natural astaxanthin is now exclusively used in High Health broodstock to improve larval quality, survival and allow sustained nauplii production (Jim Wyban, personal communication).
Presently, combinations of krill oil/meal, crawfish oil, Phaffia yeast, and synthetic astaxanthin are used as carotenoids in shrimp feeds. The natural sources have low astaxanthin concentrations, from 1500 ppm in the oils to 4000 ppm in Phaffia. The quantities required in the feeds for efficient pigmentation adds unwanted bulk and ash to the final feed. Consumer demand for natural products makes synthetic pigments much less desirable. However, an algal source of astaxanthin from Haematococcus pluvialis (NatuRose™ has also gained wide acceptance in the aquaculture markets as a "concentrated" form of krill and crawfish. NatuRose contains 15,000-20,000 ppm of astaxanthin in an esterified form, the more stabilized structure found in krill and crawfish (Yamaguichi et al., 1983). Approximately 85% of the carotenoid fraction is astaxanthin esters, the remaining 15% consists of free astaxanthin, lutein and (-carotene which are also beneficial as pigments and provitamin A activity.
The use of carotenoids as pigments in aquaculture species is well documented, and it appears their broader functions include a role as an antioxidant and provitamin A activity as well as enhancing immune response, reproduction, growth, maturation and photoprotection. An extensive body of data stresses the vital role of carotenoids in the physiology and overall health and concludes that carotenoids are essential nutrients that should be included in all aquatic diets (Craik 1985, Torrissen 1990, Grung et al., 1993). Linear and ponderal growth rates are less with P. japonicus fed a carotenoid-free diet compared to groups fed astaxanthin-supplemented diets. Additionally, supplementation of the diet with astaxanthin decreases the period of postlarval development by inducing quantitative variations of molting hormones (Petit 1993).
Carotenoids can also be characterized by their capacity to interact with the chemically reactive species of oxygen, singlet oxygen. Astaxanthin is approximately 10 times stronger than other carotenoids, including (-carotene, in terms of antioxidant activity and 100 times greater than vitamin E (alpha-tocopherol). Astaxanthin also showed strong activity as an inhibitor of lipid peroxidation mediated by active forms of oxygen and has been proposed as the "super vitamin E" (Miki 1991, Ranby and Rabek 1978).
Summary
Crustaceans cannot synthesize carotenoids, thus it must be supplied in their diet. Astaxanthin is the optimal carotenoid for the proper pigmentation of Penaeus shrimp. A nutritional deficiency of astaxanthin in the diet causes Blue Color Syndrome.
Additional benefits of this essential carotenoid include roles as an antioxidant and provitamin A activity, as well as enhancing immune response, reproduction, growth, maturation, photoprotection, and defense against hypoxic conditions common in pond cultures. Astaxanthin dramatically improves the nauplii quality and zoea survival of shrimp broodstock.
One first strategy would be to supplement shrimp diets with 100-200 ppm of NatuRose™ astaxanthin two months prior to harvest to achieve a total body carotenoid content in excess of the critical threshold of 30-40 mg/kg. A second strategy consists of supplementing 50-100 ppm astaxanthin during the entire culture period. To significantly improve nauplii quality and Zoea survival, broodstock should be supplemented with 150 ppm of astaxanthin.
References
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