Advanced Materials Research
ISSN: 1662-8985, Vols. 781-784, pp 889-894
doi:10.4028/www.scientific.net/AMR.781-784.889
© 2013 Trans Tech Publications, Switzerland
Online: 2013-09-04
Influence of Astaxanthin on Pearl Oyster Pinctada Martensii
Lili Ji1, Jie Liao2, Wendong Song3*, Jianshe Liu1* and Xiaotian Ma4
1
Environmental Science and Technology College, Donghua University, Shanghai 200090,
China;
2
Zhejiang Fenix Pearl Biological Technology Co., Ltd., Zhuji 311800, China;
3
College of Petrochemical and Energy Engineering, Zhejiang Ocean University, Zhoushan
316000, China.
4
Food Science and Technology College, Guangdong Ocean University, Zhanjiang 524088,
China.
E-mail: [email protected]
E-mail: [email protected]
E-mail: [email protected]
E-mail: [email protected]
E-mail: [email protected]
Keywords: Astaxanthin, Pinctada martensii, High Performance Liquid Chromatography,
Micro-Raman spectrometry
Abstract. In the study, through the addition astaxanthin into the bait for the pearl oyster Pinctada
martensii for 3 months, we studied the accumulation and existence form of astaxanthin in pearl oyster
through qualitative and quantitative analysis which adopted High Performance Liquid
Chromatography method; the transmission of astaxanthin in shells was detected by Micro-Raman
spectrometry. The results showed: 6.870±1.356µg/g of astaxanthin existed in the control group of
Pinctada martensii, and 74.799±5.907µg/g of astaxanthin existed in the experimental group, some of
them were existent in the form of astaxanthin esters. Weak carotenoid characteristic peak occurred in
the control group, while the carotenoid characteristic peak’s intensity enhanced obviously in the
experimental group, which illustrated remarkable increase of carotenoid content in the shell. These
findings will not only provide the basis for colorful pearl cultivation via food-borne transmission but
also lay a foundation for further artificial regulation and control of pearl color.
Introduction
As is well known, color is one of the main indexes for evaluating pearls, and colorful pearls are
increasingly welcomed on today’s market, among which the gold yellow pearl with the name of
“gold-pearl” is the most expensive pearl. Therefore, the coloration mechanism of pearl and artificial
induction of pearl’s coloration has gradually become a focus of research[1-3]. At present, the
coloration mechanism of pearl is mainly attributed to such factors as internal and external, the former
are metal ion, organic coloring and the mother-of-pearl’s body color; the latter are the temperature,
pH, concentration of the cultivation water, and the trace elements’ type and content in the water.
Astaxanthin, also called “shrimp flavin’’, is a kind of carotenoids, usually used as the feed additive
in aquaculture to ensure the larva’ s survival rate and promote the colorization of the muscle and skin.
Research results showed that many marine animals are rich in astaxanthin, such as coelenterate[4],
crustacean [5], mollusk[6], echinodermata[7], fish[8] etc. Due to its strong capacity of pigment
deposition, it can let the body have gorgeous colors, and let various biological calcium carbonate
skeletons have colors. For example, Tsushima[9] adopted chemical methods to research on the
coloration mechanism of sea chestnut’s exoskeleton and spicule, result showing that the main
coloring material was carotenoid. Cusack [10] studied the color of the red calcium carbonate shells of
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some perforated shellfish brachiopoda, deduced that the color was caused by carotenoid, and also
identified two carotenoids: astaxanthin and canthaxanthin. Besides, carotenoid can make pearls color,
as an organic coloring method. Zhang[11,12] detected carotenoids in shellfish of Hyriopsis cumingii
and Pinctada martensii, and found pearl color was correlated with carotenoid concentration. And
Urmos[13] also discovered carotenoids with the mehod of Resonance Raman spectroscopy in natural
seawater pearl. So, we consider if exogenous carotenoid would make pearl color, based on this, the
present experiment, by feeding astaxanthin, studied the accumulation and existence form of
astaxanthin in Pinctada martensii through qualitative and quantitative analysis which adopted HPLC
method; the transmission of astaxanthin in shells was detected by Micro-Raman spectrometry, thus
providing the basis for studies on Pinctada martensii breeding and pearl colorization mechanism.
Materials and methods
Breeding of Pinctada martensii. The seawater was taken from Zhanjiang’s Donghai Island, and the
sand in it was mostly filtered out. Pinctada martensii with the average weight of 45±15g were derived
from the Xuwen Breeding Base, Zhanjiang. During their cultivation in the laboratory’s self-made
indoor pearl-breeding device, the water temperature was 23-28℃; pH was 8.12-8.35; the salinity was
1.020-1.025 (Seawater crystals might be used in case of salinity shortage); 24h continuous aeration
was kept to maintain the dissolved oxygen; the water was changed every week; and the total
cultivation period was 3 months. The green alga platymonas subcordiformis (50µg/L) and additive
(0.01g/L) containing 10% astaxanthin were fed pearl oysters twice a day. Meanwhile, to avoid the
pollution of water quality, the health of the pearl oyster was examined from time to time, and the dead
oysters were timely cleared away.
Standards. Astaxanthin standard substance was obtained from Dr. Company, Germany.
Astaxanthin (10mg) was dissolved in chloroform; 0.1% butylated hydroxytoluenc (BHT) was added
into the solution; the solution was subject to volumetric method with 100mL volumetric flask, and its
concentration range was prepared as 0.05-5µg/mL. Finally, the standard curve was drawn.
Sample preparation. The meat was divided into 2 groups, i.e. the control group and the
experimental group (Fig. 1). Two kinds of treatment were conducted for each group. That is, Group
A: unsaponified control group; Group B: saponified control group; Group C: unsaponified
experimental group; Group D: saponified experimental group. The oyster meat was washed; the
internal organs were gotten rid of; the tissue was smashed by stamping machine and was sealed by
tinfoil before it was refrigerated in the -80℃ refrigerator; when it was completely frozen, vacuum
freeze drying was carried out to get the freeze-dried powder. 1.0000g of such powder was taken from
each group; 30mL chloroform (analytical pure) was measured, and 0.1% BHT was added into the
solution, which was then placed in a brown bottle with nitrogen protection. After that, the bottle was
sealed and the solution in it was ultrasonically extracted for 3 hours; 2mL of KOH methyl alcohol
solution was respectively added into the extracted liquid of Group B and Group D, which was
saponified at 4℃ for 12 hours; then vacuum filtration was carried out, and the filtered liquid was
placed into the measuring flask via which the volume of the liquid was controlled as 30mL; separating
funnel was used for separating the liquid; water was added to wash the filtered liquid till its pH was
neutral.
Ultraviolet Spectrophotometer and High Performance Liquid Chromatography
determination. Firstly, 10mL of the above extracted liquid (A-D group) was taken and its
wavelength was measured by ultraviolet spectrophotometer; Secondly, taking another 10mL of the
liquid was concentrated by rotary evaporator and the concentrate matter was dissolved in 2mL methyl
alcohol (chromatographic grade); then 0.22µm filter membrane was used for filtration, thus the
sample was prepared to be detected with high performance liquid chromatography (HPLC, LC-20A,
SHIMADZU, Japan).
HPLC detection conditions included CBM-20A controller, LC-20AT liquid feeding unit, SPD-20A
UV detector, C18 chromatographic column and 476nm detection wavelength. Mobile phase:
chromatographic grade methanol: acetonitrile: water was 84:14:2. Flow rate was 1mL/min, sample
volume was 20µL, and column temperature was 30℃.
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Carotenoid in pearl oyster shell measured with Micro-Raman spectrum. The pinctada
martensii shell in the control group and the experimental group (Fig. 2) were immersed in 5% NaOCl
solution for 10 minutes to clear away the microorganism. Then they were washed by distilled water
and air dried. The front end (A) and middle part (B) of the Pinctada martensii shell were determined
as the measurement targets (Fig. 3). The test instruments made by the UK Renishaw Company
included GB’s Invia microscopic confocal laser Raman spectrometer; Ar+ exciter with the excitation
wavelength of 514nm and the output power of 20mw. The sample power was 2.5mw; laser beam spot
size was 1 µm2; the slit width was 12.5µm.
Results and discussions
Color change of the oyster shell and meat. Pinctada martensii’s meat had different colors before
and after feeding; experimental group B’s meat color was clearly yellow which was obviously darker
than control group A (Fig. 1); control group C’s shell color was lighter than experimental group D,
and particularly, the front end of the shell was darker than the inner layer of the shell (Fig. 2). So, the
results show that food-borne astaxanthin could be involved in the metabolism of pearl oyster, leading
to mantle, adductor muscle, grill filament and kidney presenting orange, and nacre of shell presenting
golden yellow. In aquaculture, there are many studies and reports on making animal’s skin tissue
colorful by feeding astaxanthin. Page[8](Page et al., 2006) studied the pigment accumulation in
tissues of rainbow trout after feeding astaxanthin, and the study result showed that the pigment
accumulated in muscle tissue reached its peak of 47.5 mg/kg 10 weeks after feeding;
Analysis astaxanthin of oyster meat. As mollusc don’t have pigment cells to accumulate
carotenoid, most of the carotenoid existed in plasma membrane in the form of esters. For example,
Wade[14] researched the relationship between lobster’s shell color and epithelial cell to probe into
crustacea’s shell color formation, and he found that the main factor regulating and controlling
lobster’s shell color was astaxanthin esters. The present experiment, through HPLC, analyzed the
unsaponified and saponified Pinctada martensii in the control group and the experimental group, and
obtained the following conclusions: the astaxanthin standard curve was: y = 66672x + 1909.3, R2 =
0.9989; linear interval: retention time was 3.61 min; the free astaxanthin in the unsaponified control
group was 6.870±1.356µg/g; and there were other carotenoids, whose specific composition, however,
needs further identification; after saponification, the astaxanthin content was 5.199±1.145µg/g, that
is, there was free astaxanthin in the control group and there was less forms of astaxanthin esters; due
to the instability of astaxanthin’s quality, it was damaged to a certain degree during saponification,
therefore, after saponification, its content was less than that before saponification; the free astaxanthin
before saponification in the experimental group was 63.657±5.165µg/g, after saponification, the
astaxanthin content was 74.799±5.907µg/g, which showed an obvious increase (P<0.05); that is, after
feeding, the astaxanthin esters content was 11.142± 0.742µg/g; and after feeding astaxanthin for 3
months, 69.6± 4.772µg/g was accumulated in the shellfish.
Fig. 1 Color change of oyster meat before and after the feeding of astaxanthin
a: the control group, b: the experimental group
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Fig. 2 Color change of oyster shell before and after the feeding of astaxanthin
c: the control group, d: the experimental group
Fig. 3 Micro-Raman test position
Fig. 4 Chromatogram of astaxanthin
Analysis astaxanthin of oyster shell. The Raman light source will result in calcium carbonate
crystal’s vibration and electron transfer, thus the carotenoid Raman band is enhanced. Therefore,
Raman spectrum can detect the low-concentration in situ carotenoids in the shell; however, Pinctada
martensii’s fluorescence could only produce some micro-Raman spectrogram, and Zhang[12] also
got the similar result. According to the Fig. 5, the strongest peak occurred at 1085cm-1; medium
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strength peak occurred at 704cm-1, which was the typical characteristic peak of aragonite; the largest
difference between the Raman spectrogram of calcite and aragonite was the location of medium peak:
the former one was 712cm-1, and the latter one was 704cm-1; the strongest peak of calcite is 1086cm-1.
Through comparison between pinctada martensii shells before and after feeding astaxanthin, the
following findings were obtained: both of them had 3 Raman peaks caused by organics, which were
1528-1533cm-1, 1460-1462cm-1, and 1134cm-1 respectively; among them, the strongest peak occurred
at 1460-1533cm-1, and its range was the characteristic Raman spectrogram of trans-conjugation
carotenoid[15,16]; 1134 cm-1 was caused by -C-C- stretching vibration[13]. As we can know from
Fig. 9, the strength of 1528-1533cm-1 peak in the experimental group was obviously enhanced, which
illustrated that, through feeding astaxanthin, astaxanthin could transfer to shell after accumulation
and metabolization in the body, thus the shell was colorized.
Fig. 5 Carotenoid Micro-Raman spectrogram
A and B are two parts of pearl oyster; 1 and 2 are respectively control group and experimental group
Conclusion
The present research, taking pinctada martensii as its research target, discussed the transmission of
carotenoid in the body (meat and shell) of shellfish through throwing and feeding
astaxanthin-contained bait. Through HPLC and Micro-Raman analysis, the following findings were
obtained: pinctada martensii’s meat could be colorized through feeding astaxanthin; some
astaxanthin existed in the shellfish’s body in the form of stabile astaxanthin esters; and golden shell
could be generated through relevant metabolization. These findings will not only provide the basis for
colorful pearl cultivation via foodborne transmission but also lay a foundation for further artificial
regulation and control of pearl color.
Acknowledgments
This study was supported by Natural Science Foundation of Guangdong Province, China (NO.
S2012020011054).
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