1. INTRODUCTION
⌅The “munida” or red squat lobster (Pleuroncodes monodon), is a decapod marine crustacean with an elongated body, belonging to the family Munididae (Santamaría et al., 2018Santamaría J, Carbajal-Enzian P, Clemente S. 2018. Guía ilustrada para reconocimiento de langostinos y otros crustáceos con valor comercial en el Perú. Instituto del Mar del Perú (IMARPE), LA Punta, Callao. ). In the Peruvian sea its large biomass accompanies the anchoveta (Engraulis ringens), Castillo et al. (2020)Castillo R, Cornejo R, La Cruz L, Grados D, Cuadros G, Valdez C, Pozada M. 2020. Distribution and biomass of the pelagic, neritic, and oceanic resources in the peruvian marine ecosystem obtained through hydroacoustic cruises (2019). Bol. Inst. Mar Perú 35, 213-24. https://revistas.imarpe.gob.pe/index.php/boletin/article/view/302/288 make estimates of 2,201,712 and 1,687,044 t in summer and spring, 2019, respectively. Despite the abundant biological information on munida, studies on the extraction and quantification of its bioactive components are required in terms of value generation and commercial use.
Marine lipids are known to be the main source of polyunsaturated fatty acids (PUFA), especially ω-3 fatty acids (eicosapentaenoic acid EPA; 20:5 ω-3 and docosahexaenoic acid DHA; 22:6 ω-3) which are considered essential because of their significant influence on biochemical and physiological processes involved in human health (Narayan et al., 2006Narayan B, Miyashita K, Hosakawa M. 2006. Physiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) - A review. Food Rev. Int. 22, 291-307. https://doi.org/10.1080/87559120600694622 ).
The biological functionality of edible oils is assessed by indexes based on the fatty acid contents, on the AI pro-atherogenic and anti-atherogenic fatty acids ratio, on the TI or ratio of saturated (pro-thrombogenic) and unsaturated (anti-thrombogenic) fatty acids, and H:H index, correlating unsaturated and saturated fatty acids (Chen and Liu, 2020Chen J, Liu H. 2020. Nutritional indices for assessing fatty acids: A mini-review. Int. J. Mol. Sci. 21, 1-24. https://doi.org/10.3390/ijms21165695 ).
Besides of the referred lipids, marine crustaceans are a source of pigments such as astaxanthin (ASTX), a 40-carbon ketocarotenoid (3,3’-dihydroxy-β,β’-carotene-4,4’-dione) belonging to the xanthophyll family (Núñez-Gastélum et al., 2016Núñez-Gastélum JA, Sánchez-Machado DI, López-Cervantes J, Rodríguez-Núñez JR, Correa-Murrieta MA, Sánchez-Duarte RG, Campas-Baypoli ON. 2016. Astaxanthin and Its Esters in Pigmented Oil from Fermented Shrimp By-Products. J. Aquat. Food Prod. Technol. 25, 334-343. https://doi.org/10.1080/10498850.2013.851756 ). Natural ASTX has been referred to as a supercarotenoid with high levels of health protection and anti-inflammatory effects among other benefits (Capelli, 2018Capelli B. 2018. Natural Astaxanthin: The supplement you can feel. Algae Health Sciences, Inc., a BGG Company). In addition, it has a high antioxidant capacity associated with reduced risk of oxidative stress-generated diseases, such as cardiovascular diseases (Régnier et al., 2015Régnier P, Bastias J, Rodriguez-Ruiz V, Caballero-Casero N, Caballo C, Sicilia D, Fuentes A, Maire M, Crepin M, Letourneur D, Gueguen V, Rubio S, Pavon-Djavid G. 2015. Astaxanthin from Haematococcus pluvialis prevents oxidative stress on human endothelial cells without toxicity. Mar. Drugs 13, 2857-2874. https://doi.org/10.3390/md13052857 ).
Regarding the extraction of ASTX by solvents, it is known that its high polarity favors the process. Routray et al. (2019)Routray W, Dave D, Cheema SK, Ramakrishnan VV, Pohling J. 2019. Biorefinery approach and environment-friendly extraction for sustainable production of astaxanthin from marine wastes. Crit. Rev. Biotechnol. 39, 469-488. https://doi.org/10.1080/07388551.2019.1573798 used different organic solvents of medium polarity and their mixtures and concluded that hexane was not a good option, although its combination with acetone improved extraction efficiency. An alternative method is the supercritical fluid extraction (SFE) which offers technological and ecological advantages as well as obtaining analytes without exposure to oxygen or thermal damage. Efficient extractions of phospholipids and glycolipids from Farfantepenaeus paulensis were conducted using supercritical CO2 + 15% ethanol (Sánchez-Camargo et al., 2012Sánchez-Camargo AP, Meireles MÂA, Ferreira ALK,Cabral FA. 2012. Extraction of ω-3 fatty acids and astaxanthin from Brazilian redspotted shrimp waste using supercritical CO2 + ethanol mixtures. J. Supercrit. Fluids 61, 71-77. https://doi.org/10.1016/j.supflu.2011.09.017 ).
The objective of this study is focused on the extraction of lipids from munida using solvents hexane + isopropyl alcohol, acetone, absolute ethanol and Supercritical CO2 + ethanol and the evaluation of the quality of their bioactive lipid components (ASTX, EPA and DHA).
2. MATERIALS AND METHODS
⌅2.1. Characteristics of the raw material
⌅Munida specimens were frozen on board inmediately after caught by the scientific research vessel “Humboldt” belonging to Instituto de Mar del Perú (IMARPE) in June 2019 at the area 18°6’ 20.401” S & 70°48’ 14.4” W, in front of Caleta Vila Vila (Tacna), 3.5 nm off the southern coast of Peru. Samples were placed in thermal boxes to maintain cooling until arrival to the Bioactive Compounds Laboratory of Instituto Tecnológico de la Producción (ITP). The size distribution of the specimens ranged from 10 to 16 mm cephalothorax length, mean of 12.9 ± 1.2 mm, mode of 13 mm; female specimens represented 54.4% of total samples with a mode of 13 mm, while males registered 14 mm.
2.2. Sample preparation
⌅50 kg of “munida” were placed in a cold air dryer (CV-20AN, ASAHI, Japan) at 21 °C for 27 h, then crushed in an analytical mill (A11 basic, IKA, USA) and sieved to obtain a homogeneous material between 0.50 and 0.85 mm particle size. The munida meal (MM) was packed in vacuum-sealed bags and kept at -18 °C until analysis.
2.3. Proximal chemical composition
⌅Moisture, fat, ash and protein contents were determined by duplicate determinations of fresh munida and MM according to FAO (1986)FAO. Food and Agriculture Organization. 1986. Compositional Analysis Methods. In: Manual of food quality control. 7. Food analysis: general techniques, additives, contaminants, and composition. Roma, pp 202-236. methodologies.
2.4. MM lipid extraction methods
⌅Four different lipid extraction procedures were performed using a mixture of hexane + isopropyl alcohol 60:40 (v/v) (He-I), acetone (Ac), absolute ethanol (Et) and Supercritical CO2 + ethanol as cosolvent (SC-CO2-Et).
He-I: The technique described by Sachindra et al. (2006)Sachindra NM, Bhaskar N, Mahendrakar NS. 2006. Recovery of carotenoids from shrimp waste in organic solvents. Waste Manag. 26, 1092-1098. https://doi.org/10.1016/j.wasman.2005.07.002 was followed. A mixture 60:40 hexane ACS (Fermont, Mexico) with HPLC grade isopropyl alcohol (Fisher Scientific, Spain) was used to dissolve 8 g MM sample in 50 mL tubes with 40 mL, vortexed for 2 min, sonicated at 25 °C for 10 min and centrifuged (Centrifuge 5804 R, Eppendorf, Brazil) for 40 min at 3200 g at 4 °C after 5 min resting time. The extract was filtered through Whatman Nº 42 filter paper and the residue was subjected to further extraction following the same procedure.
Ac: 8 g MM were placed in 50 mL tubes with 40 mL of Ac (ACS, Merck, 99.5% purity), vortexed for 2 min, sonicated at 25 °C for 10 min and centrifuged for 40 min at 3200 g at 4 °C after 5 min resting time. The extract was filtered using Whatman Nº 42 filter paper and the residue was treated with two additional extractions.
Et: According to the Dalei and Sahoo (2015)Dalei J, Sahoo D. 2015. Extraction and Characterization of Astaxanthin from the Crustacean Shell Waste from Shrimp Processing Industries. Int. J. Pharm. Sci. Res. 6, 2532-2537. https://doi.org/10.13040/IJPSR.0975-8232.6(6).2532-37 methodology 10 g MM were thoroughly homogenized with 100 mL Et (ACS Sharlau, Spain) for 1 hour using a magnetic stirrer. The extract was filtered through Whatman Nº 42 filter paper. Solid recovery was performed on the residue by 4 extractions until the filtrate was colorless.
SC-CO2-Et: A multi-solvent extractor Model 2802.000 (Top Industrie, France) equipped with a CO2 pump (HPFlow Pump 50 - 100), co-solvent pump (90-2491 REV L, SSI), chiller (PCPR 13.02-NED, National Lab), reactor (ø 163 x 353 mm) and a stainless-steel separator (ø 78 x 278 mm) to receive the lipid were used (Barriga-Sánchez et al., 2022Barriga-Sánchez M, Varas Condori MA, Churata Huanca AC, Aranda Pariasca DM. 2022. Supercritical Fluid Extraction of Lipids and Astaxanthin Optimization from Munida (Pleuroncodes monodon) and its Characterization. J. Aquat. Food Prod. Technol. 31, 615-624. https://doi.org/10.1080/10498850.2022.2082903 ). Pressure was manually controlled by a back pressure regulator.
The extraction of 35 g MM by SC-CO2-Et was performed following the reference parameters reported by Sánchez-Camargo et al. (2012)Sánchez-Camargo AP, Meireles MÂA, Ferreira ALK,Cabral FA. 2012. Extraction of ω-3 fatty acids and astaxanthin from Brazilian redspotted shrimp waste using supercritical CO2 + ethanol mixtures. J. Supercrit. Fluids 61, 71-77. https://doi.org/10.1016/j.supflu.2011.09.017 , 200 bar, temperature 50 °C and a solvent ratio 85/15 (CO2/ethanol) for 2 h. Evaporation of the solvent was carried out using a rotary evaporator (Laborota 4003, Heidolph) at 40 °C and the residue was stored in Ultrapure nitrogen atmosphere (Linde Peru) at -19 °C, until further analysis.
Analyses were conducted in three replicates.
2.5. Lipid yield in MM
⌅The MM lipid yield was obtained by calculations according to Equation 1.
2.6. Thin layer chromatography (TLC)
⌅The methodology of Núñez-Gastélum et al. (2016)Núñez-Gastélum JA, Sánchez-Machado DI, López-Cervantes J, Rodríguez-Núñez JR, Correa-Murrieta MA, Sánchez-Duarte RG, Campas-Baypoli ON. 2016. Astaxanthin and Its Esters in Pigmented Oil from Fermented Shrimp By-Products. J. Aquat. Food Prod. Technol. 25, 334-343. https://doi.org/10.1080/10498850.2013.851756 was applied in all samples. 1 g lipid was dissolved in 1 mL ACS petroleum ether (Tedia, USA) vortexing for 1 min. 5 µL of each sample were placed on a silica gel 60 F254 plate (Merck, Germany) pre-dried at 110 °C for 2 h. The plate was placed in a chamber saturated with 50 mL acetone: hexane (25:75, v/v) as the mobile phase.
Bands were visualized under a 254 nm UV TLC lamp (Merck) and identified by comparing the Retention Factor (Rf) value with the standard ASTX by applying Equation 2. Tests were conducted in triplicate.
2.7. Determination of total carotenoids expressed as ASTX
⌅Total carotenoids expressed as ASTX were determined in all samples according to the methodology of Sánchez-Camargo et al. (2011)Sánchez-Camargo AP, Martinez-Correa HA, Paviani LC, Cabral FA. 2011. Supercritical CO2 extraction of lipids and astaxanthin from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis). J. Supercrit. Fluids 56, 164-173. https://doi.org/10.1016/j.supflu.2010.12.009 . A standard solution of ASTX (98.6%, Dr Ehrenstorfer) was prepared by diluting 1 to 5 µg/mL of ASTX standard in hexane. 50 mg lipid sample were diluted to 10 mL in hexane. The absorbance value of each solution and the sample were measured at 472 nm (highest absorbance observed) using a UV-200 Spectrophotometer (Shimadzu, Japan) with hexane as the calibration blank. Carotenoids were expressed as µg ASTX/g lipid and µg ASTX/g MM.
2.8. Fatty acid chromatography
⌅Fatty acids were determined as described by Prevot and Mordret (1976)Prevot G, Mordret M. 1976. Utilisation des colonnes capillaires de yerre pour l´analyse des cords gras par chromatographie en phase gazeose. Rev. Fr. Corps Gras. 23, 7-8.. A gas chromatograph with a FID detector (Autosystem XL, Perkin Elmer, USA) equipped with a Supelcowax 10 column (Merck, Germany) (30 m × 0.25 mm id; film thickness: 0.25 μm) was used. Peak areas were calculated using Total Chrom Navigator software (Version: 6.2.0.0.0:B27, 2001, USA), and each fatty acid percentage was calculated by comparing the individual peak area with the fatty acid total area. The fatty acid peaks were identified by comparison with the retention times of the standard F.A.M.E. Mix C4-C24 (Supelco, Sigma-Aldrich Inc, USA).
2.9. Functional quality of MM lipid
⌅The fatty acid profile of the MM lipid was used to determine its functional quality by means of the AI and TI according to equations 3 and 4, respectively (Ulbricht and Southgate, 1991Ulbricht TLV, Southgate DAT. 1991. Coronary heart disease: seven dietary factors. Lancet 338, 985-992. https://doi.org/10.1016/0140-6736(91)91846-m ). The H:H was evaluated in accordance with equation 5 as defined by (Santos-Silva et al., 2002Santos-Silva J, Bessa RJB, Santos-Silva F. 2002. Effect of genotype, feeding system and slaughter weight on the quality of light lambs. II. Fatty acid composition of meat. Livest Prod. Sci. 77, 187-194. https://doi.org/10.1016/S0301-6226(02)00059-3 ).
Where: C12:0 (lauric acid); C14:0 (myristic acid); C16:0 (palmitic acid); C18:0 (stearic acid); C18:1ω -9 (oleic acid); C18:2ω -6 (linoleic acid); C18:3ω -3 (linolenic acid); C20:4 ω -6 (arachidonic acid); C20:5ω -3 (eicosapentaenoic acid); C22:5ω -3 (docosapentaenoic acid); C22:6 -3 (docosahexaenoic acid); MUFA (monounsaturated Fatty Acids).
2.10. Statistical analysis
⌅Minitab version 17 was used for analysis of variance and Tuckey’s comparison test for lipid yield data, ASTX contents and fatty acid profile obtained for each extraction procedure.
3. RESULTS AND DISCUSSION
⌅3.1. Proximal Chemical Composition (PCC)
⌅Table 1 shows similar fat content in munida fresh samples to the data obtained by Albrecht-Ruiz and Cueto (2006)Albrecht-Ruiz M, Cueto M. 2006. Ensilje de múnida (Pleuroncodes monodon) para la obtención de pigmentos liposolubles. Boletín Investig. Inst. Tecnológico Pesq. Perú 5, 1-12 and P. planipes fresh samples (Fonseca-Rodríguez and Chavarría-Solera, 2017Fonseca-Rodríguez C, Chavarría-Solera F. 2017. Composición proximal en algunas especies de pescado y mariscos disponibles en el pacífico costarricense. Uniciencia 31, 23-28. https://doi.org/10.15359/ru.31-1.3 ) and meal data (Civera et al., 2000Civera R, Goytortúa E, Rocha S, Nolasco H, Vega-Villasante F, Balart E, Amador E, Ponce G, Colado G, Lucero J, Rodriguez C, Solano J, Flores-Tom A, Monroy J, Coral G. 2000. Uso de la Langostilla Roja Pleuroncodes planipes en la Nutrición de Organismos Acuáticos. In: Avances en Nutrición Acuícola IV. Memorías del Cuarto Simposium Internacional de Nutrición Acuícola 15-18 de Noviembre de 1998, La Paz, B.C.S, México. 349-365. https://cibnor.repositorioinstitucional.mx/jspui/bitstream/1001/2070/1/PUB-CAPITULO-Avances%20en%20Nutrición%20Acu%C3%ADcola%20IV-%20Civera-Cerecedo.PDF ) showing the higher yield oil in munida as an advantage.
| Moisture | Fat | Protein | Ashes | |
|---|---|---|---|---|
| Fresh munida (P. monodon)1 | 73.57 ± 0.07 | 6.06 ± 0.08 | 10.69 ± 0.11 | 7.59 ± 0.09 |
| Fresh munida (P. monodon)2 | 74.2 | 6.50 | 10.60 | 4.70 |
| Fresh munida (P. planipes) 3 | 83.12 ±1.66 | 1.16 ± 0.28 | 13.52 ± 1.15 | 1.51 ± 0.44 |
| Munida meal (MM)1 | 9.17 ± 0.02 | 23.16 ± 0.26 | 34.5 ± 0.12 | 15.15 ± 0.06 |
| P. planipes meal 3 | 7.83 ± 1.44 | 8.04 ± 1.42 | 40.45 ± 2.56 | 39.00 ± 1.55 |
Values in the Table are mean ±SD of duplicate analyses. 1Results obtained in the present study, 2Albrecht-Ruiz and Cueto, (2006)Albrecht-Ruiz M, Cueto M. 2006. Ensilje de múnida (Pleuroncodes monodon) para la obtención de pigmentos liposolubles. Boletín Investig. Inst. Tecnológico Pesq. Perú 5, 1-12; 3Civera et al., (2000)Civera R, Goytortúa E, Rocha S, Nolasco H, Vega-Villasante F, Balart E, Amador E, Ponce G, Colado G, Lucero J, Rodriguez C, Solano J, Flores-Tom A, Monroy J, Coral G. 2000. Uso de la Langostilla Roja Pleuroncodes planipes en la Nutrición de Organismos Acuáticos. In: Avances en Nutrición Acuícola IV. Memorías del Cuarto Simposium Internacional de Nutrición Acuícola 15-18 de Noviembre de 1998, La Paz, B.C.S, México. 349-365. https://cibnor.repositorioinstitucional.mx/jspui/bitstream/1001/2070/1/PUB-CAPITULO-Avances%20en%20Nutrición%20Acu%C3%ADcola%20IV-%20Civera-Cerecedo.PDF .
Fat is one of the most variable components in marine animals and is influenced by biotic and abiotic factors (age, catching area, time and depth of capture). Bascur et al. (2017)Bascur M, Guzmán F, Mora S, Urzúa Á. 2017. Seasonal changes in the biochemical composition of females and offspring of red squat lobster, Pleuroncodes monodon (Decapoda, Munididae), from the Southeastern Pacific. Mar Ecol. 38, 1-13. https://doi.org/10.1111/maec.12419 investigated the effect of seasonal variations and food availability to which P. monodon ovigerous females were exposed during their reproductive period (February to December) and during winter. The results indicated that these organisms adjusted their biochemical processes to ensure their survival and that of their embryos.
3.2. Thin Layer Chromatography (TLC)
⌅Table 2 shows that ASTX (Rf 0.53) was identified in all P. monodon oil extracts. The values obtained were 0.63 and 0.81, which would evidence the esterified form of this molecule (monoesters and diesters) as typical forms which are characteristic in crustaceans (Hornero-Méndez, 2019Hornero-Méndez D. 2019. Occurrence of carotenoid esters in foods. In: Mercadante AZ (ed) Carotenoid Esters in Foods: Physical, Chemical and Biological Properties. The Royal Society of Chemistry, pp 182-284). The Rf values obtained also agree with the results obtained by Dalei and Sahoo (2015)Dalei J, Sahoo D. 2015. Extraction and Characterization of Astaxanthin from the Crustacean Shell Waste from Shrimp Processing Industries. Int. J. Pharm. Sci. Res. 6, 2532-2537. https://doi.org/10.13040/IJPSR.0975-8232.6(6).2532-37 in crustacean shell residues. These authors also refer to the fact that Rf 0.99 evidences the presence of β-carotene, a molecule that would also be present in the munida lipids.
| Sample | Rf |
|---|---|
| ASTX standard | 0.53 |
| Lipid extracted by Et | 0.53, 0.63, 0.81 y 0.99 |
| Lipid extracted by Ac | 0.53, 0.63, 0.81 y 0.99 |
| Lipid extracted by He-I | 0.53, 0.63, 0.81 y 0.99 |
| Lipid extracted by SC-CO2-Et | 0.53, 0.63, 0.81 y 0.99 |
Et = absolute ethanol, He-I = hexane and isopropyl alcohol 60:40 (v/v), Ac = acetone, SC-CO2-Et = Supercritical CO2 + ethanol.
3.3. Lipid extraction yield
⌅The highest efficiency in lipid extraction from the MM sample was obtained using Et as solvent. Likewise, Xie et al. (2018)Xie D, Mu H, Tang T, Wang X, Wei W, Jin J, Wang X, Jin Q.2018. Production of three types of krill oils from krill meal by a three-step solvent extraction procedure. Food Chem. 248, 279-286. https://doi.org/10.1016/j.foodchem.2017.12.068 reported the advantage of Et in SC-CO2 extraction compared to 3 solvents per step working with krill meal. This result could be explained by the SC-CO2 increased polarity and its ability to dissociate protein-phospholipid complexes (Hardardottir and Kinsella, 1988Hardardottir I, Kinsella JE. 1988. Extraction of Lipid and Cholesterol from Fish Muscle with Supercritical Fluids. J. Food Sci. 53, 1656-1658. https://doi.org/10.1111/j.1365-2621.1988.tb07808.x ). However, Ali-Nehari et al. (2012)Ali-Nehari A, Kim S-B, Lee Y-B, Chun B-S. 2012. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J. Chem. Eng. 29, 329-336. https://doi.org/10.1007/s11814-011-0186-2 reported higher efficiency in krill meal lipid extraction using hexane (16.2% lipids) compared to SC-CO2-Et (12.2%), although Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 reported higher yields with Et (16.33%) in comparison to hexane yields (12.18%), explaining that alcoholic solvents are more efficient for krill meal lipid extraction.
3.4. Content of carotenoids expressed as ASTX
⌅Carotenoid values expressed as ASTX and extracted under the conditions established in the present study ranged from 2998.01 to 4238.65 µg /g lipid. The highest values were obtained using Ac and SC-CO2-Et (Table 3). The higher efficiency of Ac compared to Et for extracting ASTX from crustacean lipids has been demonstrated by Dalei and Sahoo (2015)Dalei J, Sahoo D. 2015. Extraction and Characterization of Astaxanthin from the Crustacean Shell Waste from Shrimp Processing Industries. Int. J. Pharm. Sci. Res. 6, 2532-2537. https://doi.org/10.13040/IJPSR.0975-8232.6(6).2532-37 and Xie et al. (2018)Xie D, Mu H, Tang T, Wang X, Wei W, Jin J, Wang X, Jin Q.2018. Production of three types of krill oils from krill meal by a three-step solvent extraction procedure. Food Chem. 248, 279-286. https://doi.org/10.1016/j.foodchem.2017.12.068 . The lower polarity of Ac facilitates its penetration through the hydrophobic mass surrounding the pigment and favors its miscibility (Dalei and Sahoo, 2015Dalei J, Sahoo D. 2015. Extraction and Characterization of Astaxanthin from the Crustacean Shell Waste from Shrimp Processing Industries. Int. J. Pharm. Sci. Res. 6, 2532-2537. https://doi.org/10.13040/IJPSR.0975-8232.6(6).2532-37 ).
| Extraction methods | Yield (g lipids/100 g MM) | Content of carotenoids (µg ASTX /g lipid) |
|---|---|---|
| He-I | 17.29 ± 0.45 c | 2998.01 ± 81.54 c |
| Ac | 14.71 ± 0.29 d | 4238.65 ± 21.04 a |
| Et | 22.93 ± 0.71 a | 3443.23 ± 126.30 b |
| SC-CO2-Et | 18.90 ± 0.36 b | 4086.71 ± 80.11 a |
He-I = hexane and isopropyl alcohol 60:40 (v/v), Ac = acetone, Et = absolute ethanol, SC-CO2-Et = Supercritical CO2 + ethanol. Data are shown as mean ± standard deviation. Different letters in the same column indicate significant difference (p < 0.05). Tukey test (p < 0.05) was used for the comparison of means. All experiments were carried out in duplicate.
All ASTX values obtained in MM by means of the proposed treatments exceeded those reported in residues of Farfantepenaeus paulensis, a species belonging to the genus Penaeus, (1074 µg ASTX/g lipid) (Sánchez-Camargo et al., 2011Sánchez-Camargo AP, Martinez-Correa HA, Paviani LC, Cabral FA. 2011. Supercritical CO2 extraction of lipids and astaxanthin from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis). J. Supercrit. Fluids 56, 164-173. https://doi.org/10.1016/j.supflu.2010.12.009 ) and those obtained in krill oil extracted with SC-CO2-Et (86.2 µg ASTX/g lipid) and hexane (103.2 µg ASTX/g lipid) by Ali-Nehari et al. (2012)Ali-Nehari A, Kim S-B, Lee Y-B, Chun B-S. 2012. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J. Chem. Eng. 29, 329-336. https://doi.org/10.1007/s11814-011-0186-2 . These results suggest not only the affinities of the solvent and extraction conditions but that munida would represent a source of higher contents of ASTX compounds compared to similar species.
Typically, the choice of solvent is made according to the polarity of the target compound. Routray et al. (2019)Routray W, Dave D, Cheema SK, Ramakrishnan VV, Pohling J. 2019. Biorefinery approach and environment-friendly extraction for sustainable production of astaxanthin from marine wastes. Crit. Rev. Biotechnol. 39, 469-488. https://doi.org/10.1080/07388551.2019.1573798 reported improved ASTX extraction efficiency when using hexane combined with other solvents, although in the present work the use of He-I mixture extracted the lowest ASTX values indicating that isopropyl alcohol did not improve ASTX extraction efficiency (Table 3).
The results of SC-CO2-Et extraction are in agreement with Routray et al. (2019)Routray W, Dave D, Cheema SK, Ramakrishnan VV, Pohling J. 2019. Biorefinery approach and environment-friendly extraction for sustainable production of astaxanthin from marine wastes. Crit. Rev. Biotechnol. 39, 469-488. https://doi.org/10.1080/07388551.2019.1573798 research data on the significant improvement in this extraction technology to recover ASTX using Et as cosolvent. Also, Sánchez-Camargo et al. (2011)Sánchez-Camargo AP, Martinez-Correa HA, Paviani LC, Cabral FA. 2011. Supercritical CO2 extraction of lipids and astaxanthin from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis). J. Supercrit. Fluids 56, 164-173. https://doi.org/10.1016/j.supflu.2010.12.009 reported 15% Et as cosolvent to substantially improve ASTX extraction, and highlighted the advantage of the solubilization of polar compounds such as phospholipids and glycolipids.
According to Capelli (2018)Capelli B. 2018. Natural Astaxanthin: The supplement you can feel. Algae Health Sciences, Inc., a BGG Company the recommended daily intake of ASTX (4 mg) can be provided by one gram of munida lipid obtained by Ac and SC-CO2-Et extractions (4238.65 and 4086.71 µg/g lipid, respectively); nevertheless, considering Ac toxicity, the use of SC-CO2-Et is considered the best extraction option for safety concerns among the extraction methods evaluated.
3.5. Fatty acids
⌅Table 4 shows the fatty acid profile of munida lipids obtained by the different lipid extraction methods. Among the saturated fatty acids (SFA) C16:0 represented the highest percentage in all the extracts while C18:1 ω-9 was the most abundant among monounsaturated fatty acids (MUFA) and its quantity was not affected by the extraction method applied.
| Fatty acids | Extraction Procedures | |||
|---|---|---|---|---|
| He-I | Ac | Et | SC- CO2-Et | |
| C14:0 (Myristic) | 4.04±0.17 b | 4.38±0.15 ab | 4.48±0.03 a | 4.38±0.00ab |
| C14:1 (Myristoleic) | 0.19±0.00 | Nd | Nd | Nd |
| C15:0 (Pentadecaenoico) | 0.41±0.01 a | 0.42±0.01 a | 0.42±0.00 a | Nd |
| C16:0 (Palmitic) | 21.76±0.40 b | 21.99±0.23 ab | 21.95±0.02 ab | 22.71±0.01 a |
| C16:1 (Palmitoleic) | 7.14±0.16 c | 7.80±0.10 a | 7.54±0.01 ab | 7.19±0.03 bc |
| C17:0 (Heptadecaenoic) | 2.76±0.01 ab | 2.79±0.13 ab | 2.60±0.11 b | 2.96±0.02 a |
| C17:1 (Cis-10-Heptadecenoic) | 0.84±0.02 a | 0.17±0.01 c | 0.36±0.00 b | 0.16±0.00 c |
| C18:0 (Estearic) | 2.96±0.01 a | 2.69±0.01 b | 2.67±0.01 a | 2.92±0.02 a |
| C18:1 ω-9 (Oleic) | 12.92±0.05 a | 13.15±0.03 a | 12.89±0.02 a | 12.9±0.02 a |
| C18:1 ω-7 (Vaccenic) | 4.36±0.04 a | 4.40±0.01 a | 4.36±0.02 a | 4.46±0.01 a |
| C18:2 ω-6 (Linoleic) | 1.43±0.00 a | 1.38±0.00 b | 1.43±0.00 a | 1.38±0.01 b |
| C18:3 ω-6 (γ-Linolenic) | 0.74±0.01 b | 0.81±0.00 a | 0.73±0.01 b | 0.74±0.01 b |
| C18:3 ω-3 (α-Linolenic) | 0.99±0.00 a | 1.00±0.00 a | 1.00±0.02 a | 0.89±0.00 b |
| C18:4 ω-3 (Stearidonic) | 5.71±0.35 a | 5.81±0.00 a | 5.81±0.02 a | 5.36±0.01 a |
| C20:0 (Arachidic) | 0.47±0.00 a | 0.46±0.00 a | 0.38±0.00 b | 0.47±0.02 a |
| C20:1 ω-9 (Eicosaenoic) | 0.92±0.00 a | 0.96±0.02 a | 0.83±0.00 b | 0.85±0.01 b |
| C20:2 (Eicosadienoic) | 0.26±0.06 a | Nd | Nd | 0.22±0.01 b |
| C20:3 ω-6 (Eicosatrienoic) | Nd | Nd | Nd | 0.66±0.00 |
| C20:3 ω-3 (Eicosatrienoic) | 0.64±0.01b | 0.58±0.01c | 0.70±0.03a | Nd |
| C20:4 ω-6 (Araquidonic) | 0.31±0.01 | Nd | Nd | Nd |
| C20:5 ω-3 (EPA) | 11.32±0.07 b | 11.26±0.07 b | 11.82±0.02 a | 11.31±0.01 b |
| C22:6 ω-3 (DHA) | 19.83±0.27 a | 19.96±0.22 a | 20.04±0.04 a | 20.43±0.01 a |
| SFA | 32.39±0.60 a | 32.73±0.24 a | 32.51±0.16 a | 33.44±0.03 a |
| MUFA | 26.38±0.15 a | 26.48±0.05 a | 25.98±0.03 b | 25.56±0.02 c |
| PUFA | 41.23±0.75 a | 40.79±0.30 a | 41.51±0.13 a | 41.00±0.05 a |
| ∑ ω-3 | 38.48±0.70 a | 38.60±0.30 a | 39.36±0.13 a | 37.99±0.04 a |
| EPA + DHA | 31.15±0.35 a | 31.21±0.29 a | 31.85±0.06 a | 31.75±0.03 a |
Et = absolute ethanol, He-I = hexane and isopropyl alcohol 60:40 (v/v), Ac = acetone, SC-CO2-Et = Supercritical CO2 + ethanol. Data are shown as mean ± standard deviation. Different
letters in the same row indicate significant difference (p < 0.05),
Tukey test (p < 0.05) was used for the comparison of means. All
experiments were carried out in duplicate.
Nd: no detected.
A predominant presence of long-chain PUFA acids was observed, and ranged from 40.79 to 41.51% with high values of EPA and DHA. No significant differences (p > 0.05) regarding the extraction methods were observed. The values reported in the present study exceeded those obtained by Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 using Ac as solvent in three krill species as well as by Ali-Nehari et al. (2012)Ali-Nehari A, Kim S-B, Lee Y-B, Chun B-S. 2012. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J. Chem. Eng. 29, 329-336. https://doi.org/10.1007/s11814-011-0186-2 in krill oil. The later one found higher efficiency by using SC-CO2-Et compared to hexane. These high long-chain PUFA contents are considered essential for membrane fluidity and inflammatory mediator functionality and show potential benefits in neuronal development and cardiovascular health (Janssen and Kiliaan, 2014Janssen CIF, Kiliaan AJ. 2014. Long-chain polyunsaturated fatty acids (LCPUFA) from genesis to senescence: The influence of LCPUFA on neural development, aging, and neurodegeneration. Prog. Lipid Res. 53, 1-17. https://doi.org/10.1016/j.plipres.2013.10.002 ).
The high C16:0 and C20:5 ω-3 values in munida lipid samples were similar to those reported by Ali-Nehari et al. (2012)Ali-Nehari A, Kim S-B, Lee Y-B, Chun B-S. 2012. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J. Chem. Eng. 29, 329-336. https://doi.org/10.1007/s11814-011-0186-2 in oily extracts of krill, in residues of Farfantepenaeus paulensis (Sánchez-Camargo et al., 2011Sánchez-Camargo AP, Martinez-Correa HA, Paviani LC, Cabral FA. 2011. Supercritical CO2 extraction of lipids and astaxanthin from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis). J. Supercrit. Fluids 56, 164-173. https://doi.org/10.1016/j.supflu.2010.12.009 ) and in Litopenaeus vannamei; while the C22:6 ω-3 contents were higher in the samples of the present study.
The EPA+DHA contents in P. monodon showed no significant differences, and the values obtained by the extraction techniques ranged from 31.15 to 31.85%. ω-6 fatty acids were not detectable in some cases, as in the case of C20:3 ω-6 and C20:4 ω-6. C18:2 ω-6 was the omega-6 fatty acid with the highest content. The results obtained in the present study show similarity to those reported by Ali-Nehari et al. (2012)Ali-Nehari A, Kim S-B, Lee Y-B, Chun B-S. 2012. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J. Chem. Eng. 29, 329-336. https://doi.org/10.1007/s11814-011-0186-2 and Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 for krill oil.
3.6. Functional quality of MM lipid
⌅No differences were observed among the AI values of the MM lipids obtained with all the solvents used in the present study (Table 5), although these values were lower than those calculated from studies carried out by Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 and Sánchez-Camargo et al. (2012)Sánchez-Camargo AP, Meireles MÂA, Ferreira ALK,Cabral FA. 2012. Extraction of ω-3 fatty acids and astaxanthin from Brazilian redspotted shrimp waste using supercritical CO2 + ethanol mixtures. J. Supercrit. Fluids 61, 71-77. https://doi.org/10.1016/j.supflu.2011.09.017 for Euphausia superba and Penaeus paulensis respectively. This would be advantageous in the case of munida, considering that Turan et al. (2007)Turan H, Sönmez G, Kaya Y. 2007. Fatty acid profile and proximate composition of the thornback ray (Raja clavata, L. 1758) from the Sinop coast in the Black Sea. J. Fish Sci. 1, 97-103. https://doi.org/10.3153/jfscom.2007012 refer to AI values and also thrombogenicity indexes (TI) close to zero, which are considered favorable for preventing coronary heart disease.
| Solvent | Crustacean | AI | TI | H:H |
|---|---|---|---|---|
| He-I1 | Pleuroncodes monodon | 0.56 | 0.20 | 1.81 |
| Ac1 | 0.59 | 0.20 | 1.77 | |
| Et1 | 0.59 | 0.19 | 1.78 | |
| SC-CO2-Et1 | 0.61 | 0.21 | 1.73 | |
| Ethanol2 | Euphausia superba | 1.96 | 0.24 | 1.32 |
| Acetone2 | 2.85 | 0.31 | 1.14 | |
| Ethanol3 | Euphausia superba | 1.24 | 0.17 | 1.64 |
| Supercritical CO2 + ethanol4 | Penaeus paulensis | 0.97 | 0.40 | 2.16 |
1Our data; calculated based on the results of: 2Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 , 3Xie et al. (2018)Xie D, Mu H, Tang T, Wang X, Wei W, Jin J, Wang X, Jin Q.2018. Production of three types of krill oils from krill meal by a three-step solvent extraction procedure. Food Chem. 248, 279-286. https://doi.org/10.1016/j.foodchem.2017.12.068 , 4Sánchez-Camargo et al. (2012)Sánchez-Camargo AP, Meireles MÂA, Ferreira ALK,Cabral FA. 2012. Extraction of ω-3 fatty acids and astaxanthin from Brazilian redspotted shrimp waste using supercritical CO2 + ethanol mixtures. J. Supercrit. Fluids 61, 71-77. https://doi.org/10.1016/j.supflu.2011.09.017 . He-I = hexane and isopropyl alcohol 60:40 (v/v), Ac = acetone, Et = absolute ethanol, SC-CO2-Et = Supercritical CO2 + ethanol, AI: atherogenicity index, TI: thrombogenicity index, H:H: Hypocholesterolemia: hypercholesterolemia ratio.
The AI and TI values obtained in the present study are slightly higher than those obtained by Lopes et al. (2014)Lopes LD, Böger BR, Cavalli KF, dos S. Silveira-Júnior JF, Osório DVCL, de Oliveira DF, Luchetta L, Tonia IB. 2014. Fatty acid profile, quality lipid index and bioactive compounds of flour from grape residues. Cienc. Investig. Agrar. 41, 215-234. https://doi.org/10.4067/s0718-16202014000200009 for grape pomace oil (AI= 0.18-0.32, TI= 0.06-0.17). Studies by Pinto et al. (2020)Pinto RHH, Menezes EGO, Freitas LC, et al. 2020. Supercritical CO2 extraction of uxi (Endopleura uchi) oil: Global yield isotherms, fatty acid profile, functional quality and thermal stability. J. Supercrit. Fluids. 165, 104932. https://doi.org/10.1016/j.supflu.2020.104932 on Endopleura uchi oil reported AI values similar to those obtained in our work, although their TI values were higher (AI=0.44, TI=1.32) than ours.
Regarding the use of solvents, the highest H:H ratio was observed in the extracted munida lipids using the He-I mixture (1.81). This value exceeded those obtained by Xie et al. (2017)Xie D, Jin J, Sun J, Liang L, Wang X, Zhang W, Wang X, Jin Q. 2017. Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Food Chem. 233, 434-441. doi:10.1016/j.foodchem.2017.04.138 and Xie et al. (2018)Xie D, Mu H, Tang T, Wang X, Wei W, Jin J, Wang X, Jin Q.2018. Production of three types of krill oils from krill meal by a three-step solvent extraction procedure. Food Chem. 248, 279-286. https://doi.org/10.1016/j.foodchem.2017.12.068 , although it was lower than that obtained by Sánchez-Camargo et al. (2012)Sánchez-Camargo AP, Meireles MÂA, Ferreira ALK,Cabral FA. 2012. Extraction of ω-3 fatty acids and astaxanthin from Brazilian redspotted shrimp waste using supercritical CO2 + ethanol mixtures. J. Supercrit. Fluids 61, 71-77. https://doi.org/10.1016/j.supflu.2011.09.017 for Penaeus paulensis when using SC-CO2-Et. Low H:H values are considered unfavorable as they may induce an increase in cholesterolemia (Santos-Silva et al., 2002Santos-Silva J, Bessa RJB, Santos-Silva F. 2002. Effect of genotype, feeding system and slaughter weight on the quality of light lambs. II. Fatty acid composition of meat. Livest Prod. Sci. 77, 187-194. https://doi.org/10.1016/S0301-6226(02)00059-3 ); while high values like 2.66 in uxi (Endopleura uchi) are recommended by Pinto et al. (2020)Pinto RHH, Menezes EGO, Freitas LC, et al. 2020. Supercritical CO2 extraction of uxi (Endopleura uchi) oil: Global yield isotherms, fatty acid profile, functional quality and thermal stability. J. Supercrit. Fluids. 165, 104932. https://doi.org/10.1016/j.supflu.2020.104932 .
Our results suggest that the SC-CO2-Et lipid extraction method applied in Peruvian marine species of commercial importance contributes to quantifying carotenoid pigments and essential fatty acids (EPA and DHA) among other bioactive comounds. Argopecten purpuratus (scallops), Romaleon setosum, Cancer porteri, Platymera gaudichaudii, Paralomis longipes (crabs) Loxechimus albus (sea urchin) and many other fish species represent a promising dietary source.
4. CONCLUSIONS
⌅Higher ASTX contents were obtained from munida using Ac and SC-CO2-Et, followed by Et extractions. On the other hand, the contents of SFA, PUFA, omega-3, EPA+DHA in munida lipids with all extraction solvents showed no significant differences. PUFA were the most predominant in the lipid. Among SFA and MUFA, C16:0 and C18:1 ω-9 were prevalent. The sum of C20:5 ω-3 and C22:6 ω-3 varied from 31.15 to 31.85%.
The functional quality indexes AI and TI for munid lipid were favorable; while the H:H values were low. The results of the present study suggest that it is an important source of lipids which contain ASTX, EPA and DHA. The extraction of munida lipids with Et or SC-CO2-Et in further studies are suggested for possible application in the food industry.