Iranian Journal of Environmental Health, Science and Engineering Iranian Association of Environmental Health (IAEH) ISSN: 1735-1979 Vol. 4, Num. 2, 2007, pp. 93-98

Iranian Journal of Environmental Health Science & Engineering,Vol.
4, No. 2, 2007, pp.  93-98
EXTRACTION OF ASTAXANTHIN ESTERS FROM SHRIMP WASTE BY CHEMICAL
AND MICROBIAL METHODS
 *1,2A. Khanafari, 2A. Saberi, 3M. Azar, 2Gh. Vosooghi, 2Sh. Jamili, 2B. Sabbaghzadeh
 1Department of Microbiological Sciences, Islamic Azad University, North of Tehran, Iran

  2Department of Marine Biology, Science and Research Center, Islamic Azad University, Tehran, Iran

  3Department of Food Science and Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
 Received 30 December 2006; revised 5 February 2007; accepted 20 March 2007
*Corresponding author-Email: khanafari_a@yahoo.com Tel: +98 21 2208
2141, Fax: +98 21 2270 0103
Code Number: se07014
INTRODUCTION 
Carotenoid pigments are the most important 
  and numerous pigments that are found in nature. 
  These compounds soluble in lipids are the factors 
  that produce yellow-red color in plant and animal products. In this group of pigments 
  astaxanthin has important applications in human and 
  animal food industries specifically neutracutical  pharmaceutical and cosmetic industries. The 
  Food and Drug Administration of the United States 
  has permitted it for use in the aqua-cultural 
  industry (Golkhoo, 2006). The main sources for this 
  pigment can be found in many favorable sea foods, 
  for example salmon, Oncoryncus mykiss, sea 
  bream, lobster and caviar. Also in birds like flamingo 
  and quails, in microorganisms, insects, crustaceans and micro-green alga (Haematococcus 
pluvialis) it can be found (Guerin, 2003).  Astaxanthin 
has effects on many of these creatures' main body function like prevention from
oxidation of essential unsaturated fatty acids, prevention from
effects of ultraviolet light, Immunological 
reactions, pigmentation, communication, and 
reproduction. There is currently considerable interest in the 
role carotenoids in delaying or preventing 
degenerative diseases such as atherosclerosis, cancer, aging 
and eye diseases. The protective effect of 
astaxanthin was even more pronounced compared to β-carotene. Furthermore, a significant
  (P<0.001) decrease in the incidence of induced colon 
  cancer in rats fed with astaxanthin versus those administered only the carcinogen, was 
  found. Dietary astaxanthin is also effective in fighting mammary tumors by > 50%,
  more than β-carotene and cantaxanthin. Astaxanthin inhibits the
  enzyme 5-α-reductase responsible for prostate growth 
  and eventually prostate cancer. Astaxanthin's anti-cancer activity might be related
  to the carotenoid's role in cell communications at gap junctions,
  which might be involved with slowing cancer cell 
  growth, the induction of xenobiotic-metabolizing 
  enzymes or by modulating immune responses against 
  tumor cells (Guerin, 2003). Among them, 
  xanthophylls including astaxanthin and cantaxanthin have 
  been shown to inhibit effectively bladder 
  carcinogenesis without any toxicity through their inhibition of 
  cell proliferation (Tanaka, 1997). The antioxidant activity of astaxanthin has
  been reported to be 10 times stronger than that of other carotenoids, namely
  zeaxanthin, lutein, canthaxanthin,
  and-carotene (Naguib, 2000). Carotenoids with no 
  pro-vitamin A property are of interest for use as 
  chemo preventives. Today with the use of methods 
  like infrared spectroscopy-IR., mass spectroscopy- 
  MS and optical rotary dispersion - ORD also 
  nuclear magnetic resonance-NMR the number of carotenoids discovered are about
  563 kinds (Martelli and Dasilva, 1993). An alternative method to de-proteinase
  crustacean's residues
  is using fermentation with Lactic acid bacteria. 
  Lactic fermentation can also be a process to 
  obtain carotenoids especially astaxanthin (Armenta-Lopez, 2002). 
The objective of this study was the extraction 
  of astaxanthin esters pigment by chemical and microbial methods and comparing them with 
  each other regarding their effectiveness and also studying the effects of intervening factors 
  on microbial extraction.
MATERIALS AND METHODS 
All data reported in this study are from 
  triplicate measurements.
Microorganism and culture media 
Two species of Lactobacillus named Lactobacillus plantarum (PTTC 1058) and Lactobacillus 
  acidophilus (PTTC 1643) were kindly provided from the microbiology 
  laboratory of North-Tehran branch of Islamic Azad University. They were sub-cultured on MRS 
  broth and MRS agar media (peptone from casein 10.0 
  g, yeast extract 4.0 g, meat extract 8.0 g, D (+) glucose 20.0 g, tween 80 1.0 g, 
di-ammonium hydrogen citrate 2.0 g, sodium acetate 5.0 
g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g (MERCK). MRS Broth was mixed with 15 
g/L agar to solidify the medium and incubated at 
35-37°C in the presence of 5% CO2 for 48-72h.
Preparation of shrimp waste 
Shrimp waste from processing of Penaeus semisulcatus, comprising of head and 
  carapace, was collected from a shrimp processing 
  landing centers situated at Persian Gulf in south of 
  Iran and transported to the laboratory under iced condition. The yield of dried shell was 
  determined by weighing after dried at 50°C in oven for 
  24h. Samples were stored at two temperatures, of 
  25°C and -20°C until use. The material was thawed 
  in running water before use and homogenized in a laboratory mixer.
Chemical extraction of astaxanthin 
Astaxanthin was extracted by mixing 5g 
  shrimp waste powder homogenate, 50 mL of hexane 
  and 5 mg of glass beads and vortexed for 30 
  seconds, place in the 50°C water bath for 10 
  minutes. Aqueous and organic layers were separated 
  by 3000 rpm for 5 minutes. This step repeat until 
  the hexane is colorless. At the final step 6 mL of 
  di-methyl sulfoxide (DMSO) was added to the tube and vortex vigorously and place in the water 
  bath for 10 minutes and vortex again. Concentrated carotenoid was subjected to Thin 
  Layer Chromatography (TLC) using silica gel 60 F MERCK TLC paper (Thomason, 1998).
Microbial extraction of astaxanthin 
After adding 5 mL of MRS broth containing 
  each of the two Lactobacillus sp. to the 
  fermentative medium culture (100 mL distilled water + 10 g 
  of shrimp waste powder and incubating for 3 days 
  at 30°C, in the presence of 5% CO2. The fermentative culture medium was filtered with Wattman 
  filter paper No.41 and centrifuged at 3000 rpm for 
  5 minutes. Three groups of solvents (Group (1): petroleum ether: Acetone: Di-ethylamine 
  (10:4:1), Group (2): Hexane: Acetone (3:1), Group 
  (3): benzene: ethyl acetate (1:1)) were added to extraction astaxanthin. 
  Concentrated carotenoid was subjected to TLC using silica gel 60 
  F MERCK TLC paper. 
Optimization of conditions 
  for carotenoid extraction  
The conditions for extraction were optimized 
  with the effect of a combination of process variables  
  such as lactose sugar 1%, yeast extract 1%. The composition of lactose sugar 1%, yeast extract 
  coolage (-20oC) and were added to 
  fermentation medium and their interactions on the 
  response variable was determined. Each at three 
  equidistant levels, and the response variable was 
  the carotenoid yield. In total, combinations of 
  factors were used. The extraction of carotenoid and 
  the determination of their concentration were 
  carried out as explained earlier.
Purification of Astaxanthin by TLC 
Concentrated carotenoid extract by chemical 
  and microbial methods was subjected to TLC using silica gel 60 F MERCK TLC paper. 
  Concentrated carotenoid extract was spotted on TLC 
  plates along with standard astaxanthin and eluted with 
  a mobile phase of petroleum ether: acetone: di-ethylamine (10:4:1), hexane: acetone 
  (3:1), benzene: ethyl acetate (1:1) (Lorenz Todd, 
  1998).
Spectrophotometer assay 
The synthetic and concentrated astaxanthin 
  was dissolved separately in acetone and hexane. λMax was estimated with UV-VIS scanning spectrophotometer, UV 2101 pc, SHIMADZU 
  by measuring the absorbance at 200 - 500 nm.
Infrared analysis 
The fraction of TLC paper which had 
  a fluorescence characteristic stronger than others and its Rf value being around astaxanthin Rf 
  value was scrapped and dissolved in acetone and 
  the peaks were obtained by IR assay (Nageswara Rao, 2005).
NMR analysis 
The fluorescence fraction was obtained from 
  the TLC was dissolved in acetone and the NMR spectrum was obtained. The NMR equipment 
  had V: 300 MHz, B 0:7 T (Bruker) (Negewara Rao, 
  2005).
Statistical analysis 
All determinations were carried out in 
  triplicate. All data are expressed as mean ±SD. Data 
  were analyzed by an analysis of variance (P < 0.05) and the means separated.
Results  
Extraction of astaxanthin esters from a 
  certain Persian Gulf shrimp species waste 
  (Penaeus Semisulcatus), purification and identification 
  of this pigment by chemical and microbial methods was investigated. The total carotenoid 
  and fractions were compared with synthetic 
  astaxanthin by TLC. The solvent extracted carotenoid was 
  in the form of a paste with an orange-red color. Results of TLC indicated that in the 
  chemical method extract, the orange-colored 
  astaxanthin forms the major band which migrates slowly. 
  In microbial extraction method, the highest number of carotenoid fractions (in TLC method) 
  from waste was obtained when the carotenoids were extracted with hexane, followed by solvent 
  Group (2): hexane: acetone (3:1) (Table 1). 
Group (1): petroleum ether: Acetone: Diethyl amine (10:4:1), Group 
  (2): Hexane: Acetone (3:1), Group (3): benzene: ethyl acetate (1:1)). The 
  highest number of carotenoid fractions (in TLC method) was obtained followed 
  by solvent Group (2): Hexane: Acetone (3:1)
The lowest carotenoid yield was obtained with 
  two other solvents, Groups 1 and 3. Rf values 
  varied slightly useing standards to confirm the 
  carotenoids (Table 2) (Lorenz Todd, 
  1998). Results of TLC analysis indicated that in microbial extraction 
  of astaxanthin nine to eleven different 
  carotenoids bands were produced. These fractions 
  were fluorescence property under UV cabinet at 366 nm. Results showed that microbial method 
  of extraction of astaxanthin was more effective 
  than chemical method. Lactobacillus plantarum (PTTC 1058) had ability to determined 
  fractions in TLC method like Lactobacillus 
    acidophilus (PTTC 1643). No significant difference 
  was observed in carotenoid content between astaxanthin extraction 
  by Lactobacillus plantarum (PTTC 1058) and Lactobacillus acidophilus (PTTC 1643) 
  (P<0.05) (Table 3). 
No significant difference was observed in carotenoid content 
between astaxanthin extraction by Lactobacillus plantarum (PTTC 1058) and Lactobacillus 
acidophilus (PTTC 1643) (P<0.05) Optimization of conditions for 
carotenoid extraction showed that, when lactose was 
added to fermentation medium, it gave a higher 
carotenoid yield than added yeast extract (Fig. 1). But 
the composition of both of them had a decreasing effect on the carotenoid yield. Results 
obtained from this study showed that the coolage at (- 
20oC) not only does not have an amplifying effect on 
the production of astaxanthin but also slightly 
reduces this effect (Fig. 1). IR spectrum result 
showed that, a characteristic absorbance at 1736 
cm-1 was assigned to C=O bond and the other peak in 
the spectrum belong to C-H, O-H and methyl groups were the same as in the molecule of 
pure astaxanthin. Results obtained from NMR 
analysis showed, a characteristic absorbance was 
assigned to methyl group in 2.07 ppm area, 
methyl-allyle groups at 1.21 ppm area and methyl-vinyl 
groups at 1.59 ppm area. Also O-H group and vinyl 
group were observed at 2.07 and 5.39 ppm area. 
Discussion  
Use of a mixture of polar and non-polar 
  solvents for extraction of carotenoids from shrimp 
  waste produces the highest yield. The extraction 
  yield differed significantly (p< 0.05) between 
  solvents. Solvent Group 2 gave significantly 
  (p< 0.05) higher yield than Group 1 and 3.  Britton et al., (1985) recommended the use of water miscible polar organic solvents, usually acetone, methanol or ethanol, for extraction of carotenoids from tissues containing water. Delgado-Vargus et al., (2000) discussed the advantages and disadvantages 
  of various organic solvents for extraction of carotenoids and suggested that polar solvents are generally good extraction media for xanthophylls but not for carotenes. For wet tissues, use of non-polar solvents is not recommended as 
  their penetration through the hydrophobic mass 
  that surrounds the pigment is limited 
  (Delgado-Vargus et al., 2000). De Ritter and Purcell (1981) postulated that complete 
  extraction of carotenoids from plant tissues could be achieved with samples of low moisture content by use of slightly polar plus non-polar solvents. Although the 
  results obtained are for the waste from the 
  species Penaeus semisulcatus, it would be applicable 
  to waste from other species of shrimps. The 
  residue available after carotenoid extraction may be 
  used for the preparation of chitin/chitosan, thus 
  having an integrated approach for efficient utilization 
  of shrimp waste. Lactic fermentation is a simple 
  and environmentally friendly method to extract 
  highly unstable caretoneid pigments 
  (Armenta-López, 2002). In this study, the pigment extracted 
  from certain amount of shrimp powder 23.128 mg/g 
  was calculated. Shahidi and Synowiecki (1991) reported that the carotenoid content in the shells of 
  snow crab, Chinocetes opilio, was 14 mg/g. The carotenoid content in blue crab, Callinectes 
  sapidus, was 4.63 mg/g (Felix-Valenzuela et 
    al., 2001). Thin-layer chromatographic separation 
  of carotenoid extracts from Penaeus 
    semisulcatus yielded eleven distinct bands at 
  Rf=0.33 corresponded to astaxanthin,  while yellow 
  bands at Rf=0.75 and 0.99 corresponded to 
  astaxanthin di-esters and â-carotene, respectively, as 
  indicated by the TLC of standards. The orange bands 
  at Rf=0.51, 0.40, 0.87 and at 0.25 corresponded to 
astaxanthin monoester, 
cantaxanthin, echinenone and Lutein respectively, as quoted in the 
literature (Lorenz Todd, 1998). The results 
indicated that astaxanthin, astaxanthin monoester and 
di-ester, and â-carotene are the major pigments in 
the Penaeus semisulcatus, while Lutein also 
could be separated from the shrimp waste extract 
using TLC. The NMR and IR methods of carotenoid extracts indicate that astaxanthin and its 
esters were the major carotenoids in the extract 
from Penaeus semisulcatus. The total content 
of astaxanthin and its esters was 23.128 mg/g. Astaxanthin and its esters have been found to 
be the major carotenoids in the marine crustaceans 
(Shahidi et al., 1998). In the marine crab, accumulation 
of astaxanthin, â-carotene and zeaxanthin has been 
reported (Matsuno, Watanabe, and Nagata, 1974). Zeaxanthin and lutein 
were found to be major pigments in fresh water 
mullets (Matsuno, Nagata, and Chiba, 1975). Matsuno 
and Maoka (1988) reported that 
astaxanthin contributes 3339 g/100 g of total carotenoids in 
meat and shell of marine crab Paraalithodes brevipes, 
from Japanese waters. The present study indicates 
that the marine shrimp, Penaeus 
semisulcatus, accumulates astaxanthin esters, as 
major carotenoid and microbial method of extraction 
of astaxanthin is more effective than chemical 
method.
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