Astaxanthin extraction from golden apple snail (Pomacea canaliculata) eggs to enhance colours in fancy carp (Cyprinus carpio)

Abstract

High-value ornamental fish farming is one of the most valuable industries in the recent years, and the commercial value of ornamental fish, such as fancy carp (Cyprinus carpio) is primarily dependent on their body coloration. Therefore, a trial was conducted to investigate skin colour enhancement in fancy carp by feeding different levels (0, 25, 50, 100 and 200 mg/kg) of a dietary natural astaxanthin extracted from golden apple snail (Pomacea canaliculata) eggs, using a diet containing synthetic astaxanthin (50 mg/kg) and a control diet with no colouring added for comparison. The duration of the trial was four weeks with experimental diets containing carotenoid followed by the low carotenoid diet for other two weeks. The chromaticity of fish body skin of red and white position was assessed using the CIE L* a* b* system. Pigmentation response of skin redness (a*) of fancy carp fed with diets at 50, 100 and 200 mg/kg of natural astaxanthin supplement was higher than other treatments (P < 0.05). The lightness and yellowness (L* and b* values) indexes of carp fed with diet containing astaxanthin from golden apple snail eggs were similar compared to the other dietary treatments (P > 0.05). After two weeks of feeding diet without natural carotenoid supplement demonstrated that the group fed 50 mg/kg of natural astaxanthin still retained their redness (a*) and also had an overall tendency to improve skin colouring (L* and b*). Finally, dietary concentrations 50 mg/kg of astaxanthin extracted from golden apple snail eggs showed the highest efficiency for improving skin pigmentation in fancy carp.

Keywords:

• fancy carp
• astaxanthin
• golden apple snail eggs
• skin colour

1. Introduction

High-value ornamental species such as koi carp (Cyprinus carpio) are characterised by a wide range of colours and colour patterns (Gomelsky et al. 1996), and more than 100 different types of coloration have been developed (Paripatananont et al. 1999; Sun et al. 2012). High-quality aquaculture products must fulfil several requirements, including the adequate colour, which is among the most important fish quality parameters in the market. Colour is the first characteristic perceived and is a determinant selection criterion, directly related to the subsequent acceptance or rejection (Shahidi et al. 1998). Carotenoids, which are lipid soluble pigments, are responsible for the skin colour of ornamental fish, and they are also vital nutrients for healthy growth and reproduction. Because of fish cannot synthesise carotenoids, they must be obtained from dietary sources (Gouveia et al. 2003). Carotenoids commonly occur in freshwater food sources including β-carotene, lutein, taraxanthin, astaxanthin, tunaxanthin, α- and β-doradexanthins and zeaxanthin (NRC 1993). Many synthetic pigments (β-carotene, canthaxanthin, eaxanthin and astaxanthin) and natural sources (yeast, bacteria, algae, plants and crustacean meal) have been used as dietary supplements to enhance the pigmentation of both fish and crustaceans (Kalinowski et al. 2005).

Natural carotenoids sources are usually composed by several carotenoids in different forms, and they vary for digestibility, leading the pigmentation efficiency complicated to interpret. Given the high costs of synthetic pigments, efforts have been made to evaluate the potential of natural compounds. In the present study, we focused on astaxanthin extraction from golden apple snail (Pomacea canaliculata) eggs as it has become a serious calamity in agriculture around the world because of its strong reproductive capacity and its ability to extend its geographical range. The eggs of the apple snail are spherical and pink to red. It has been found that golden apple snail eggs and gonads resulted in high levels of astaxanthin (Wu & Yang 2008). Thus, the use of golden apple snail eggs would be benefit to aquaculture industry and private sectors who are interested to improve the colour quality in fancy carp or in other high-value ornamental fish. Therefore, the aim of the present study was to evaluate the effect of different levels of natural astaxanthin extracted from golden apple snail eggs as a dietary carotenoid source for colouring the skin of fancy carp, in comparison with synthetic astaxanthin and using also a negative-control unsupplemented diet.

2. Materials and methods

2.1. Astaxanthin extraction

A total of 1000 g of fresh apple snail eggs were homogenised with 3000 ml of distilled water and 400 ml acetone under room temperature, and then the obtained homogenate was filtered (Wu & Yang 2008). Filtrate was a glycol-lipo-carotenoprotein solution, and it was stored in a 4°C dark box under close system in order to avoid contact with air, light and high temperature in the dark room. A 15 g of papain (Merck Chemicals; Cat. No.107149.1000; specific activity 30,000 USP-U/mg) was added to 1000 ml of the glycol-lipo-carotenoprotein solution and agitated for 24 h. The digested solution was filtrated to remove solid residues. A 120 ml of 10% acetic acid was dropped into the filtrate. The mixture was agitated in dark room under close system for 3 h, and then mixture was filtrated to remove the precipitate containing the digested proteins. Antioxidants, 2 g of butyl hydroxyanisole and 3 g of butyl hydroxytoluene, were added to the filtrate; the reactant was kept in a 60°C dark room and agitated for 12 h to allow the ester-astaxanthin to be hydrolysed. The thin layer chromatography (TLC) analysis was used to determine the completion of the hydrolysis reaction. After reaction was completed, the product was cooled to room temperature. An ethanol solution containing 2% Triton X-100 with a volume threefold of the product was added, and the mixture was filtrated to remove the precipitate containing lipids and saccharide compounds, and an astaxanthin containing hydrophilic solution was then obtained. The solution was concentrated and ethanol contained was evaporated under decompression. Astaxanthin contained in each concentrated solution was extracted by n-hexane. The organic phase was washed by distilled water several times and dried by anhydrous sodium sulphate. A 16.8 g of astaxanthin mixture (from 1000 g of apple snail eggs) was obtained through a decompressing concentrating process. The components of astaxanthin mixtures were analysed by TLC (0.25 mm pre-coated sheet) with a mobile phase of n-hexane:acetone (4:1). Free astaxanthin, monoester astaxanthin and diester astaxanthin were observed from the TLC graph.

2.2. Fish rearing condition and experimental diets

The homogeneous red colour fancy carps were obtained from a commercial fish farm in Ratchaburi Province of Thailand. Before the beginning of the experiment, fishes were fed with the control diet for two weeks to acclimatise them to the laboratory culturing system. At the begin of the trial, fancy carps (mixed sexes with an initial mean length 3 inches and 3 month of ages) were randomly divided into six treatments of 60 fish each (six treatments in triplicate). Each group of fish was stocked in a 0.675 m³ indoor tank with a freshwater input of 0.5 l/min. The oxygen level was more than 6 mg/l. The fish were subjected to a natural photoperiod, and the average temperature of the water was 25–27°C. The duration of the experiment was 25 days with experimental diets containing carotenoids followed by the low carotenoid diet for other 15 days.

A basal herbivorous pelleted diet consisting of commercial feed, containing 155 g/kg crude protein and 30 g/kg fat, 100 g/kg fibre, 120 g/kg moisture and 75 g/kg ash (Table 1), without added carotenoid was used (TR1 diet). Using this basal mixture, five other diets were formulated so that they contained the following: a treatment containing 50 mg/kg diet of commercial synthetic astaxanthin coated by spraying (TR2); a diet containing 25 mg/kg diet of natural astaxanthin from golden apple snail eggs (TR3); a diet containing 50 mg/kg diet of natural astaxanthin from golden apple snail eggs (TR4); a diet containing 100 mg/kg diet of natural astaxanthin from golden apple snail eggs (TR5); and a diet containing 200 mg/kg diet of natural astaxanthin from golden apple snail eggs (TR6).

Table 1. Chemical composition of the basal diet fed to fancy carp.

Item	g/kg as fed basis
Chemical composition
Dry matter	880
Crude protein	155
Crude fat	30
Crude fibre	100
Ash	75

Fish were fed the experimental pelleted diet at the rate corresponding to 3–5% of their body weight twice a day (09:00 h and 18:00 h). Chemical composition of the diets was determined using the following the AOAC International (2004) procedures: dry matter after drying at 105°C for 24 h (method 945.15); ash by combustion at 550°C for 12 h (method 967.05); crude protein (N × 6.25; method 990.03) by a Kjeldahl method after acid digestion; and fat by Soxhlet dichloromethane extraction (method 945.16). Skin colour assessment was performed by reflectance spectroscopy, with transformation into colour parameters based on tristimulus values, in accordance with the system CIE L* a* b* for lightness, redness and yellowness, respectively (Skrede 1987), using a portable Miniscan EZ HunterLab calibrated towards a white standard. The measurements were performed on both sides of the dorsal fish skin. Measurements were recorded at the beginning and at the ending of experimental feeding period.

2.3. Statistical analysis

Data were analysed using the one-way PROC ANOVA option of the PROC GLM of SAS/STAT software (SAS Institute 2004) as a completely randomised design with the six dietary treatments as main effect. When there was a significant F-value, means were compared by the Duncan’s multiple range test. Significance implies P < 0.05 unless stated otherwise.

3. Results and discussion

Carotenoids are known to have a key role in the intermediary metabolism in fish that could enhance nutrient utilisation and may finally result in improved performances (Kalinowski et al. 2005).

Data on colour parameters (L*, a*, b*) of fancy carp skin fed the experimental diets for 25 days are reported in Table 2. Data on the feeding trial show that supplementing astaxanthin from golden apple snail eggs as natural carotenoid source led a significant (P < 0.05) improvement of the skin redness of fancy carp. In particular, there was a positive increase of a* values when carp fed astaxanthin from golden apple snail eggs at 50 and 100 mg/kg, compared to the control diet and the diet supplemented with synthetic astaxanthin. It was found that dietary carotenoid level could have an effective influence on the fish immune system, therefore on fish health and growth as well as reproduction (Yuangsoi et al. 2010). Moreover, Nakano et al. (1999) reported that supplementing astaxanthin in fish diet resented in a reduction of oxidative stress in fish. It has also observed that fish with a high level of carotenoids are more resistant to bacterial and fungal diseases (Shahidi et al. 1998). Results on the other skin colour parameters (lightness and yellowness) after the feeding period indicated that there were significant differences (P < 0.05) among fish groups; however, data indicated an unpredictable colour pattern of L* and b* values because of astaxanthin was clearly visible only in all shades of red. These results are in agreement with the previous findings of Yagiz et al. (2010). Therefore, dietary inclusion of astaxanthin extracted from apple snail eggs demonstrated its effectiveness in accelerating skin red colour deposition in fancy carp already at low rate (50 mg/kg diet). Astaxanthin esters were also found to be more efficiently utilised for deposition and coloration of the skin than free astaxanthin in red sea bream (Nakazoe et al. 1984). In other species such as red porgy (Kalinowski et al. 2005) and Japanese ornamental koi carp (Sun et al. 2012), both unesterified or esterified forms of astaxanthin determined similar levels of astaxanthin in the skin.

Table 2. Colour parameters (L*, a*, b*) of fancy carp (C. carpio) skin fed the experimental diets for 25 days.

	Parameters
Experimental treatment	L*	a*	b*
TR1 control diet without astaxanthin	1.70 ± 0.518^ab	–1.309 ± 0.394^c	5.201 ± 0.973^a
TR2 commercial synthetic astaxanthin 50 mg	–0.87 ± 0.282^b,c	–1.204 ± 0.545^c	–0.552 ± 0.636^b
TR3 astaxanthin from golden apple snail eggs 25 mg	–2.05 ± 0.745^c	–0.724 ± 0.389^c	4.333 ± 0.370^a
TR4 astaxanthin from golden apple snail eggs 50 mg	3.667 ± 0.310^a	0.482 ± 0.2.188^a,b	–1.249 ± 0.877^b
TR5 astaxanthin from golden apple snail eggs 100 mg	1.38 ± 0.387^a,b,c	1.748 ± 0.310^a	0.0890 ± 0.125^b
TR6 astaxanthin from golden apple snail eggs 200 mg	1.96 ± 0.323^a,b	0.614 ± 0.387^a,b	0.898 ± 0.010^b

Note: Values are expressed as means ± SEM. Values in same column with different superscripts are significantly different (P < 0.05).

The results on fancy carp skin colour parameters after 15 days of dietary astaxanthin suspension are reported in Table 3. The skin redness (a*) values of carp after suspended supplementary astaxanthin were similar (P > 0.05) among dietary groups, whereas the lightness (L*) and yellowness (b*) values decreased positively and linearly from the control diet to the diet including the higher natural astaxanthin level (P < 0.05). In fact, less yellowness (b*) means that white zones are more pure. Our data are also confirmed by previous studies conducted by Nakazoe et al. (1984) and Yuangsoi et al. (2010) which stated that fish cannot synthesise their own colour pigments; therefore, they must be derived from feed intake, thus after the 15 days’ suspension of supplementary astaxanthin a decrease in values of skin colours would be markedly noticed.

Table 3. Colour parameters (L*, a*, b*) of fancy carp (C. carpio) skin after 15 days of dietary astaxanthin suspension.

	Parameters
Experimental treatment	L*	a*	b*
TR1 control diet without astaxanthin	9.838 ± 0.262^a	1.311 ± 0.881	3.940 ± 0.163^a
TR2 commercial synthetic astaxanthin 50 mg	7.330 ± 0.247^ab	1.695 ± 0.749	4.772 ± 0.139^a
TR3 astaxanthin from golden apple snail eggs 25 mg	3.234 ± 0.138^b	–0.211 ± 0.748	0.277 ± 0.158^a
TR4 astaxanthin from golden apple snail eggs 50 mg	3.785 ± 0.385^b	0.699 ± 0.4.27	–8.689 ± 0.795^b
TR5 astaxanthin from golden apple snail eggs 100 mg	2.434 ± 0.483^b	–0.409 ± 0.153	–7.6213 ± 0.158^b
TR6 astaxanthin from golden apple snail eggs 200 mg	1.842 ± 0.468^b	0.944 ± 0.386	–2.628 ± 0.1.58^b

Note: Values are expressed as means ± SEM. Values in same column with different superscripts are significantly different (P < 0.05).

In conclusion, from our trial, it was found that monoester astaxanthin and diester astaxanthin extracted from golden apple snail eggs can be effectively used as natural carotenoid source to enhance the skin red colour in fancy carp also at low dietary inclusion level (50 mg/kg) with equal capability comparing to commercial synthetic astaxanthin source at the same concentration, nevertheless synthetic astaxanthin was not effective for accelerating the other pigments in fancy carp.

Acknowledgements

The authors would like to acknowledge all assistant students involved in this research. This work was supported by the Animal Science Faculty of the Silpakorn University, Thailand.