Co-solvent Selection for Supercritical Fluid Extraction of Astaxanthin and Other Carotenoids from Penaeus monodon Waste

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1 Journal of Oleo Science Copyright 2014 by Japan Oil Chemists Society doi : /jos.ess13184 Co-solvent Selection for Supercritical Fluid Extraction of Astaxanthin and Other Carotenoids from Penaeus monodon Waste Shazana Azfar Radzali 1, Badlishah Sham Baharin 1*, Rashidi Othman 2, Masturah Markom 3 and Russly Abdul Rahman 1 1 Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Malaysia 2 International Institute for Halal Research and Training (INHART), Herbarium Unit, Department of Landscape Architecture, Kulliyyah of Architecture and Environment Design, International Islamic University Malaysia, Kuala Lumpur, Malaysia 3 Department of Chemical and Process Engineering and Built Environment, National University of Malaysia, Bangi, Malaysia Abstract: In recent years, astaxanthin is claimed to have a 10 times higher antioxidant activity than that of other carotenoids such as lutein, zeaxanthin, canthaxanthin, and β-carotene; the antioxidant activity of astaxanthin is 100 times higher than that of a-tocopherol. Penaeus monodon (tiger shrimp) is the largest commercially available shrimp species and its waste is a rich source of carotenoids such as astaxanthin and its esters. The efficient and environment-friendly recovery of astaxanthins was accomplished by using a supercritical fluid extraction (SFE) technique. The effects of different co-solvents and their concentrations on the yield and composition of the extract were investigated. The following co-solvents were studied prior to the optimization of the SFE technique: ethanol, water, methanol, 50% (v/v) ethanol in water, 50% (v/v) methanol in water, 70% (v/v) ethanol in water, and 70% (v/v) methanol in water. The ethanol extract produced the highest carotenoid yield (84.02 ± 0.8 mg/g) dry weight (DW) with 97.1% recovery. The ethanol extract also produced the highest amount of the extracted astaxanthin complex (58.03 ± 0.1 μg/g DW) and the free astaxanthin content (12.25 ± 0.9 mg/g DW) in the extract. Lutein and β-carotene were the other carotenoids identified. Therefore, ethanol was chosen for further optimization studies. Key words: Penaeus monodon, astaxanthin, carotenoids, SFE, HPLC 1 INTRODUCTION In recent years, the global production of shrimp has been growing gradually and this trend is predicted to continue 1. In the last 20 years, shrimp has contributed to about 20 of the total value of exported fishery products 2. Penaeus monodon or Tiger shrimp is one of the most significant commercial species found in Malaysian waters, and in terms of production, it has continued to be the most important species for the past 5 years at a value of USD 160, Shrimp processing plants produce a large volume of waste products. The shrimp body parts that are processed for human consumption comprises only 70 of the overall shrimp landing 4. Hence, a remarkable tonnage of shrimp waste is generated, from which astaxanthin, one of the major carotenoids, can be obtained. Astaxanthin is a potential source of carotenoids for the aquaculture and poultry industries, which need enormous supplies of this carotenoid. Astaxanthin 3, 3-dihydroxy-β, β-carotene-4, 4-dione is the most valuable ketocarotenoid, both from a biotechnological and commercial point of view. It has been found in most crustaceans like shrimps, crabs, and lobsters 5. This carotenoid pigment is also found in birds like flamingo, and in insects, microorganisms, and micro-green alga Haematococcus pluvialis 5. It exhibits a vibrant red color and higher antioxidant activity compared to other carotenoids such as α-carotene, β-carotene, lutein, lycopene, canthaxanthin, and vitamin E 6, 7. Thus, it has been applied as a food colorant and in pharmaceutical products 8, 9. Recently, some studies proved that astaxanthin inhibits the invasion of cancer cells 10. Astaxanthin has been used as a carotenoid pigment in the aquaculture and poultry industries and recently, it gained attention in the nutraceutical and pharmaceutical industries 5. Nowadays, the demand for * Correspondence to: Badlishah Sham Baharin, Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, MALAYSIA badli@food.upm.edu.my Accepted March 11, 2014 (received for review October 30, 2013) Journal of Oleo Science ISSN print / ISSN online

2 S. A. Radzali, B. S. Baharin, R. Othman et al. natural carotenoids is increasing because of the consumer s concerns for food safety and strict government regulations. Normally, organic solvents such as dichloromethane and acetone are used for the extraction of astaxanthin; however, the usage of the aforementioned solvents may cause safety issues. The increasing demand for natural foods has encouraged research on the extraction of astaxanthin from natural sources. The poor yields almost 50 of the pigments are lost and considerable environmental concern of the traditional extraction methods have motivated further studies on supercritical fluid extraction SFE as an effective alternative to the traditional methods 11. SFE is used for the extraction of bioactive compounds from foods 12, 13. This method is more significant when thermo-labile compounds are present. In addition, the use of toxic solvents can be avoided, since carbon dioxide CO 2 is inexpensive and generally recognized as a safe GRAS solvent, which is easy to separate from the extract Supercritical fluids have outstanding extractive properties such as liquid-like density, high compressibility, high diffusivity, and low viscosity 17. Numerous studies have been conducted on the supercritical carbon dioxide SC-CO 2 extraction of carotenoids from crustacean and marine animal waste However, in those studies, the recovery of astaxanthin was relatively low compared to that of conventional methods, and the selectivity of the compounds extracted from the shrimp waste remained uncertain. With the addition of a suitable modifier or co-solvent to the SC-CO 2, the extraction of the solute could be enhanced by increasing the yield, solubility, and extraction rate 21. The extraction efficiency could be improved by fine-tuning the solvent selectivity, and the solubility of the carotenoids in the mixture could be increased by introducing different types and concentrations of cosolvents entrainers to the SC-CO 2. In addition, the relatively low solubility of astaxanthin in SC-CO 2 requires the addition of polar modifiers or high pressures 40 MPa to improve its dissolution in the mixture with the simultaneous enhancement in extraction yields 17, 18, 22, 23. Astaxanthin is the main carotenoid extracted from the P. monodon species 24, 25. Despite Malaysia being one of the most important producers of this species, the shrimp processing waste generated in Malaysia is not commercially exploited for the recovery of astaxanthins 1. To the best of our knowledge, there is no report on the determination of carotenoid contents from the wild Malaysian P. monodon waste. Thus, this study will offer new insights into the determination of carotenoids in the P. monodon waste from Malaysia. From a green technology point of view, the health food industry will be benefitted if this precious pigment could be extracted from an inexpensive raw material instead of it being chemically synthesized. Therefore, this study was designed to extract astaxanthins from P. monodon waste owing to the abundance of this species in Malaysia. The effects of different types and concentrations of co-solvents on the amounts of the total carotenoids extracted, astaxanthin complex extracted, and free astaxanthin content in the extract were determined. The best cosolvent will be used for further optimization of the SFE process. 2 EXPERIMENTAL PROCEDURES 2.1 Sample preparation Samples of fresh P. monodon counts kg 1 were obtained from fishermen at the Tanjung Bidara Beach, Malacca. The shrimps were immediately transported to the laboratory in an expanded polystyrene box under iced conditions 4. The waste comprising of the head and carapace were freeze-dried for 72 h using a FDU-2100 model freeze dryer Tokyo Rikakikai Co., Japan. Then, the sample was grounded into fine powder and kept at 80 until further analysis. Most of the solvents and chemicals used were of analytical grade ethanol, methanol, hexane and chloroform Fisher Scientific, USA. Ethyl acetate and acetonitrile were of high-performance liquid chromatography HPLC grade Merck, Germany. 2.2 Extractions Solvent Extraction The total amount of the carotenoids and astaxanthin complex present in the sample were determined by a solvent extraction procedure, which was performed after slight modification of a previously reported method g of the powdered, freeze-dried sample was rehydrated by adding distilled water and was extracted with a mixture of acetone and methanol 7:3, v/v at room temperature until the biomass turned colorless. The crude extract was centrifuged for 5 min at 10,000 g in a Thermo Scientific, Sorvall Biofuge Primo R Germany centrifuge machine and the supernatant was stored at 4 in the dark prior to analysis. Then, an equal volume of hexane and distilled water were added to the combined supernatants in order to extract the carotenoids. The solution was allowed to separate and the upper layer containing the astaxanthin complex was collected. The combined upper layer was then completely dried under a gentle stream of oxygenfree nitrogen and was immediately stored at 80 until subsequent analysis Supercritical fluid extraction SFE The effects of different modifiers and their concentrations on the carotenoid yield were studied by performing SFE with CO 2 on the sample. The SFE system comprised of a PU-2080 model CO 2 pump JASCO Corporation, Japan, Series III solvent pump Lab Alliance, USA, BP model back-pressure regulator BPR JASCO Corporation, Japan, extractor vessel enclosed in a FX

3 Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste model air-circulating oven Sheldon Manufacturing, USA, model pressure transmitter Dwyer Instrument, USA and sample collector Fig. 1. The commercial grade liquefied CO was purchased from Gas Pantai Timur, Malaysia. The CO 2 was chilled to 2 using a chiller Protech Electronic, Malaysia in order to retain its liquid state before it was pumped to the extractor. The extractor comprised of a high-pressure stainless steel vessel, which was filled with the freeze-dried shrimp waste. A back-pressure regulator was used to sustain the system pressure and the flow of the SFE process was controlled by the needle valves. Seven types of co-solvents water, 50 v/v methanol in water, 50 v/v ethanol in water, 70 v/v methanol in water, 70 v/v ethanol in water, pure methanol and ethanol were studied. The parameters were set at a temperature of 60 and operating pressure of 20 MPa. The effect of co-solvent was investigated at a moderate pressure of 20 MPa and at this pressure, the rate of extraction was assumed to be moderate. A higher extraction rate might be applicable if the moderate extraction rate was effective. Besides, the experiments were carried out at 20 MPa since the maximum capacity of the co-solvent pump was 40 MPa. The co-solvent pump required a higher pressure than the CO 2 pump. Hence, if the experiment was performed at 40 MPa, the capacity of co-solvent pump must be higher than 50 MPa. The flow rate of the entrainer was set in order to achieve the desired entrainer to CO 2 concentration of 15 v/v. 5.0 g of the freeze-dried shrimp waste powder was extracted in the static mode for 15 min, followed by a dynamic extraction for 120 min 2 h. The extract fractions were collected every 20 min. All the carotenoid extracts were centrifuged at 10,000 g in a Thermo Scientific, Sorvall Biofuge Primo R Germany centrifuge machine and the supernatants were dried to completion under a gentle stream of oxygen-free nitrogen. Then, the dried extracts were stored in the dark at 80 until subsequent analysis. 2.3 Determination of total carotenoid concentration The total carotenoid concentration was determined by the spectrophotometric method which is similar to that reported previously 27. The dried carotenoid was resuspended in a known volume of ethyl acetate. Then, 50 μl of the redissolved sample was diluted with 950 μl of chloroform for spectrophotometric analysis. The carotenoid-containing solution was measured at three different wavelengths: 480 nm, 648 nm and 666 nm using a Varian Cary 50 UV-Vis spectrophotometer. The Wellburn Equation 28 as shown in Eq. 1-Eq. 3 was used to obtain the total carotenoid content in chloroform as described below: C a 10.91A A 648 C b 16.36A A 666 C x c 1000A C a 46.09C b /202 Eq. 1 Eq. 2 Eq. 3 where C a concentration at 666 nm; C b concentration at 648 nm; and C x c total carotenoid concentration at 480 nm. 2.4 Determination of the astaxanthin yield astaxanthin complex The astaxanthin yield was calculated according to the previous method 29. The resulting concentrated carotenoids were diluted in a known volume of petroleum ether and the amount of astaxanthin was quantified by measuring the absorbance at 468 nm using a Varian Cary 50 UV-Vis spectro- Fig. 1 Design of the experimental system used for the supercritical fluid extraction system with co-solvent 21). 771

4 S. A. Radzali, B. S. Baharin, R. Othman et al. photometer. In this study, the astaxanthin yield was calculated as that of the astaxanthin complex. The term complex corresponded to the mixture of free and esterified astaxanthin with other related astaxanthin derivatives. The yield of the astaxanthin complex was calculated using the following equation Eq. 4 : Astaxanthin complex yield μg astaxanthin complex/g sample A 468nm V extract Dilution factor 0.2 W sample Eq. 4 where A 468 is the absorbance at 468 nm, V extract is volume of extract, 0.2 is the A 468 of 1 μg/ml of standard astaxanthin and W sample is the sample weight in gram. 2.5 Determination of free astaxanthin and other carotenoid contents The astaxanthin content free astaxanthin in P. monodon waste extracts was determined by using the astaxanthin standard Sigma-Aldrich Corporation, USA. The astaxanthin standard was dissolved in HPLC grade acetone and was prepared in a concentration range of μg/ml. The data for the standard curve was obtained with the coefficient of determination, R The lutein and β-carotene standards Sigma-Aldrich Corporation, USA were dissolved in the HPLC grade ethyl acetate at concentrations of μg/μl. The linear regression analysis data of β-carotene and lutein also demonstrated a good linear relationship with R 2 of and 0.996, respectively. The chromatographic peaks were identified by comparing their retention times and spectra against the known standards. 2.6 HPLC Analysis The HPLC analysis was performed according to a previous method on an Agilent model 1200 series comprising a binary pump with autosampler injector, micro vacuum degassers, photodiode array PDA detector and thermostatted column compartment 31. The separation column used was a ZORBAX Eclipse XDB-C18 end capped 5 μm, mm reverse phase column Agilent Technologies, USA. The solvents used were A acetonitrile:water 9:1 v/ v and B ethyl acetate. The solvent gradient was developed as follows: 0 40 solvent B 0 20 min, solvent B min, solvent B min, 100 solvent B min and solvent B min at a flow rate of 1.0 ml/min. The injection volume was 10 μl. The column temperature was maintained at 20. The individual carotenoids were detected at the maximum absorption wavelength of the carotenoids in the mobile phase: β-carotene 454 nm, lutein 447 nm, and astaxanthin 480 nm. Compounds were identified by cochromatography with standards and by elucidation of their spectral characteristics using a PDA detector. The carotenoid peaks were detected in the range of nm. The individual carotenoid concentrations were calculated by comparing their relative proportions, as reflected by the integrated HPLC peak areas to the total carotenoid content determined by spectrophotometry. The total and individual carotenoid concentrations were expressed in terms of microgram per 1.0 g dry weight of freeze-dried matter μg/g DW. 3 RESULTS AND DISCUSSION 3.1 Carotenoid recovery The recovery mass of extracted compounds/mass of extracted compounds by acetone and methanol 7:3, v/v X 100 and yields mass of extracted compounds/mass of biomass of the carotenoids for the performed extractions are shown in Table 1. The maximum recovery 100 was obtained with acetone and methanol 7:3, v/v at room temperature. The highest total carotenoid recovery 97.1 of SFE was obtained when CO 2 -ethanol was used as the cosolvent. The total carotenoid content in the extract was μg/g under the aforementioned extraction conditions. This value was very similar to that obtained Table 1 Yield and recovery of total carotenoids from Penaeus monodon waste for all extractions. Extraction methods and solvents Total carotenoid ± (S.D.) Recovery (%) (Solvent extraction method) Acetone and methanol (7:3, v/v) 86.52± SFE, CO 2 and methanol 82.51± SFE, CO 2 and ethanol 84.02± SFE, CO 2 and water 5.00± SFE, CO 2 and 50% methanol 17.49± SFE, CO 2 and 50% ethanol 15.53± SFE, CO 2 and 70% methanol 30.74± SFE, CO 2 and 70% ethanol 51.79± *DWB-dry weight basis; Average of triplicate analyses ± standard deviation. 772

5 Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste through the solvent extraction method. Hence, the beneficial use of SC-CO 2 for the recovery of the bioactive compounds was demonstrated. The least carotenoid content was recovered when water was used as the co-solvent 5.8 for the SFE. Pure SC-CO 2 is nonpolar. Usually, a slightly polar solute like astaxanthin exhibits low solubilities in pure SC-CO 2. However, the addition of polar entrainers could increase the solubility of a less soluble solute in the solvent mixture, which in turn improves the extraction efficiency. The enhancement in solubility is possibly due to the increase of solvent density, polarity as well as the specific chemical interactions like hydrogen bonding 32. In this study, the ethanol helped to swell the pores of the biomass, which in turn improved the interaction of the SC-CO 2 with the cell. The high molecular weight of astaxanthin MW slowed down its release in SC-CO 2 due to its low volatility 33. Ethyl alcohol is also suitable to be applied as an entrainer for the extraction of the other high MW carotenoids 34, 35. Since astaxanthin was more soluble in organic solvents, which are less polar than water, the use of methanol and ethanol as the co-solvents gave better results than that obtained using water. Although the results obtained using methanol were similar to that using ethanol, the use of methanol could cause potential health issues and is intolerable for pharmaceutical and food industries. The presence of water in the sample could influence the SFE process and there had been applications of aqueous samples for SFE 36. One recent study 37 showed that the 70 methanol entrainer increased the extraction yield of essential oils and bioactive compounds from Polygonum minus kesum compared to that obtained using only water or alcohol. However, in our study, the greatest yield and selectivity of the extracted carotenoids were obtained from pure ethanol. This was explained by the differences in the polarity and solubility of the targeted compounds in SC-CO 2 and co-solvent as well as the matrix characteristics. This might also be due to the fact that the solvent concentration range from v/v in the mixture was not in accordance with the polarity of the analyte. Therefore, it was most likely that astaxanthin was less soluble in the mixture. Normally, water can diminish or enhance the extraction process depending on whether it can swell the matrix, enlarge the pores, aid the fluid in accessing the analytes, and improve the flow through the matrix. Water usually improves the fluid polarity and increases the recoveries of the polar species. Nevertheless, the SFE recovery would be low when the leftover water remained in the extractor vessel, as the highly water-soluble analytes would be partitioned in the aqueous phase. In addition, carotenoids are considered lipids and therefore are insoluble in water. The water-insoluble materials would precipitate from the mixture and even if the analytes were soluble in the mixture, the leftover water would disturb the flow of analyte to fluid mixture Carotenoids extract composition The composition of the extract yield and the total and individual carotenoid contents μg/g DW in P. monodon waste for all extractions are presented in Table 2. The best results of total carotenoids TC μg/g DW, amount of astaxanthin complex μg/g DW, free astaxanthin content μg/g DW and other carotenoids were obtained when ethanol was used as the co-solvent. The yield of astaxanthin complex obtained in this study μg/g was 32.2 higher than the astaxanthin yield of P. indicus waste harvested from Indian waters μg/g reported previously 38. The existing literature showed that the range of carotenoid content in Table 2 Composition of yield of extract, total and individual carotenoid content (µg/g DW) in Penaeus monodon waste for all extractions. Extraction methods and solvents Total carotenoids Amount of astaxanthin complex (µg/g DWB) (Free astaxanthin) Lutein β-carotene Unidentified Acetone and methanol (7:3, v/v) 86.52± ±0.3 ND ND ND 16.79±0.7 SFE, CO 2 and methanol 82.51± ± ± ± ± ±1.2 SFE, CO 2 and ethanol 84.02± ± ± ± ± ±1.5 SFE, CO 2 and water 5.00± ± ± ± ± ±0.3 SFE, CO 2 and 50% methanol 17.49± ± ± ± ± ±0.9 SFE, CO 2 and 50% ethanol 15.53± ± ± ± ± ±0.1 SFE, CO 2 and 70% methanol 30.74± ± ± ± ± ±0.2 SFE, CO 2 and 70% ethanol 51.79± ± ± ± ± ±1.9 *ND- Not Detected. DWB-dry weight basis; Average of triplicate analyses±standard deviation. 773

6 S. A. Radzali, B. S. Baharin, R. Othman et al. the exoskeleton of P. monodon from waters of the Indo- Pacific region were between 23 to 331 μg/g 39. The highest carotenoid contents were obtained from the shrimps having a dark grey body color and the lowest carotenoid contents were obtained from those having a pale blue body color 39. The discrepancy in the appearance of the shrimp and the composition of the carotenoid extract was assumed to depend on the species, seasons, area of catch and other physical and environmental conditions. The amount of esterified astaxanthin was determined by the background color of the shrimp during the time of harvesting and the changed chemical form that affected the pigment s location in the chromophores 40. In addition, the physiological and morphological color changes in crustaceans were related to the rapid and slow transformations resulting from the hormonal and environmental factors 41, 42. Astaxanthin is mainly found as carotenoproteins in the exoskeleton. While cooking, the color of the pigment changes from blue-green to red because of the separation of the astaxanthin from the protein 43. The crustaceans and other animals obtain astaxanthin from their diet because the animals cannot synthesize astaxanthin and in wild shrimps, this carotenoid is derived from the conversion of other consumed carotenoids such as β-carotene and zeaxanthin 24. Therefore, the presence of astaxanthin in shrimps was dependent on its availability from the diet, ability to be digested, delivered and deposited in specific location as well as other physical and environmental conditions. The pigmentation of the shrimps is determined by the presence of carotenoid pigments mostly astaxanthin that plays a key role in influencing a consumer s choice to buy shrimps and thus would impact their market value 40. The results also proved that the TC has a positive correlation with the amounts of astaxanthin complex, free astaxanthin, and other carotenoids present in the extract Table 2. This relationship showed that astaxanthin and its derivatives were the most predominant carotenoids, which accumulated in the P. monodon waste. Previous studies showed that the main carotenoid pigments that affect the color of P. monodon were free astaxanthin, esterified astaxanthin, and minor amounts of other pigments such as β-carotene 39, 44. Small quantities of other carotenoids such as zeaxanthin and lutein had been discovered in P. monodon 30, 45. Another interesting aspect of this study was the comparison between the carotenoid profiles obtained from the acetone and methanol 7:3, v/v and SFE extracts, using ethanol as co-solvent. The carotenoid profiles of these extracts were not similar. The acetone and methanol 7:3, v/v extract was found to have only the astaxanthin esters Fig. 2. As can be seen in Fig. 3, the free astaxanthin, astaxanthin esters, lutein and β-carotene were detected in the SFE extract using ethanol as the entrainer. The difference in the carotenoid profiles might be due to the difference in the solubility and polarity of the individual carotenoid content in the extracts. The final carotenoid extract was collected from the hexane fraction in the solvent extraction method. Slightly polar carotenoids such as lutein and free astaxanthin were difficult to dissolve in the nonpolar hexane. The polarity of carotenes β-carotene was slightly below those of the xanthophylls lutein, astaxanthin. In addition, the free carotenoids were relatively polar, while the monoesters and diesters were relatively non-polar 46. Therefore, the variation in the procedure and the different types of solvents used for extraction also affected the determination of astaxanthin. Both the HPLC profiles of the carotenoid extracts indi- Fig. 2 HPLC analysis of carotenoids extracted using solvent from Penaeus monodon waste, astaxanthin esters (AE). 774

7 Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste Fig. 3 HPLC analysis of carotenoids extracted using SFE with ethanol as a co-solvent, at 60 and 20 MPa from Penaeus monodon waste (1) Free astaxanthin, (2) Lutein, (3) β-carotene and astaxanthin esters (AE). cated that the free astaxanthin and its derivatives were the main pigments present in the P. monodon species. The results obtained in this study were in good agreement with those of the previous study 30 which stated that about of the carotenoid content in P. monodon, Metapenaeus dobsonii, Parapenaeopsis stylifera and P. indicus obtained from shallow waters off the Indian coast was contributed by astaxanthin and its esters. The carotenoid composition and accumulation level vary with the extraction method and environmental factors 47, 48. The astaxanthin content in the SFE extract of the Haematococcus pluvialis microalgae was significantly influenced by the properties of the solvent, which was used as the modifier CONCLUSIONS In this study, a rapid, selective and environment-friendly SFE technique was proposed for the extraction of carotenoids from shrimp waste. The presence of ethanol as the entrainer at 60 and 20 MPa gave the highest yield of the total carotenoids μg/g DW with 97.1 recovery. The ethanol extract produced the highest value for the amount of astaxanthin complex extracted μg/g DW and the free astaxanthin content in the extract μg/g DW. Hence, ethanol was preferred to methanol, which is also highly toxic to humans. Therefore, the recovery of astaxanthin from P. monodon waste using SFE with the addition of ethanol as the entrainer has great potential to be developed for the pilot scale production of astaxanthins. ACKNOWLEDGMENT The authors would like to thank Universiti Putra Malaysia UPM for awarding the Research University Grants Grant No RU that made this study possible. We wish to acknowledge the Department of Chemical and Process Engineering and Built Environment, National University of Malaysia UKM for the SFE equipment. Thanks also to Herbarium Laboratory, KAED, International Islamic University Malaysia IIUM for the facilities provided for the HPLC analysis. References 1 Kongkeo, H. Cultured aquatic species information programme: Penaeus monodon. FAO Fisheries and Aquaculture Department, Rome. Available online from Accessed on March 16, CAC. Discussion Paper on Risk Management Strategies for Vibrio spp. in Seafood. Food and Agriculture Organization / World Health Organization, Rome, Italy Mazuki, H. National Aquaculture Sector Overview. Malaysia. National Aquaculture Sector Overview Fact Sheets. FAO Fisheries and Aquaculture Department. Available online from Accessed on August 31, Sachindra, N. M.; Bhaskar N.; Siddegowda G. S.; Sathisha D.,Suresh P. V. Recovery of carotenoids from ensiled shrimp waste. Bioresource Technol. 98,

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