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Characterisation of inorganic elements and volatile organic compounds in the dried sea cucumber Stichopus japonicus
Food Chemistry 147 (2014) 34–41
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Characterisation of inorganic elements and volatile organic compounds in the dried sea cucumber Stichopus japonicus Hae-Won Lee a, Na-Lae Lim a, Kichul Cho a, Hye Young Yang a, Kyung June Yim a, Mi-Ju Kim a, Myunglip Lee a, Dong Hyeun Kim b, Hyoung Bum Koh b, Won-Kyo Jung c, Seong Woon Roh a,⇑, Daekyung Kim a,⇑ a b c
Jeju Center, Korea Basic Science Institute (KBSI), Jeju 690-756, Republic of Korea Jeju Special Self-Governing Province Ocean and Fisheries Research Institute, Jeju 690-700, Republic of Korea Department of Biomedical Engineering, and Center for Marine-Intergrated Biomedical Technology (BK21 Plus) Pukyong National University, Busan 608-737, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 14 July 2013 Accepted 19 September 2013 Available online 29 September 2013 Keywords: GC–MS ICP-MS Inorganic elements Stichopus japonicus Volatile organic compounds
a b s t r a c t The sea cucumber Stichopus japonicus lives in a variety of marine habitats and is an important cultivated edible aquatic species in East Asia. In this study, S. japonicus, collected from the sea near Jeju Island of Korea, was lyophilised or vacuum-dried and then analysed by gas chromatography–mass spectrometry (GC–MS) or inductively coupled plasma mass spectrometry (ICP-MS). The GC–MS profiles of vacuumdried and lyophilised samples differed. Based on direct injection and static headspace analysis, 37 volatile organic compounds (VOCs) were identified in vacuum-dried samples and 33 VOCs were identified in lyophilised samples. Therefore, the odour of vacuum-dried sea cucumber is thought to be due to the presence of various VOCs that are absent in lyophilised sea cucumber. According to ICP-MS analysis, the levels of 15 inorganic elements were slightly higher in lyophilised samples than in vacuum-dried samples. The results of the inorganic and organic chemical analyses provide information about the composition of dried sea cucumber. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The sea cucumber Stichopus japonicus is a cylinder-shaped invertebrate that lives in a variety of marine habitats and is an important cultivated aquatic species in China and Japan (Zhong, Khan, & Shahidi, 2007). China is the biggest cultivator of sea cucumbers and has the most sea cucumber farms (Zhong et al., 2007). The majority of countries that consume sea cucumber are in the Indo-Pacific region, including the Philippines, Malaysia, Hong Kong, Singapore, South Korea, China, and Japan (Wang et al., 2012). Nutritionally, sea cucumber has a high protein content, a low fat content, and is rich in essential amino acids, such as tryptophan, arginine, and lysine. Moreover, the body wall of sea cucumbers contains insoluble collagen and has been used as a nutritional supplement. Sea cucumbers are a rich source of polysaccharide chondroitin sulphate, which can reduce arthritis pain (Chen, 2003). Scientific reports by nutritionists and pharmacologists have supported the belief that sea cucumbers have nutritional and medicinal value (Zhong et al., 2007). Rehydrated sea cucumber can be mixed with a variety of foods (Zhong et al., 2007), and stored forms of the product, such as smoked and dried, are gener⇑ Corresponding authors. Tel.: +82 64 800 4931; fax: +82 64 805 7800 (S.W. Roh). Tel.: +82 64 800 4930; fax: +82 64 805 7800 (D. Kim). E-mail addresses: [email protected] (S.W. Roh), [email protected] (D. Kim). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.110
ated from the gonad, intestine, or the entire body of the sea cucumber. Dried sea cucumber is taken as a nutritional supplement, in the form of a capsule or tablet, and is used as a cooking ingredient in East Asia. Previous studies have analysed the inorganic element, nutrient (Chen, 2003), and antioxidant (Liu et al., 2012) composition of sea cucumber. However, a comparative analysis of VOCs and inorganic elements in sea cucumber has not been reported. Gas chromatography–mass spectrometry (GC–MS) can efficiently separate and identify organic molecules. Samples are introduced into the gas chromatograph, evaporated, and the vapour is separated through a mobile phase such as helium (Smart, Aggio, Van Houtte, & Villas-Boas, 2010). These separated compounds are analysed by mass spectrometry to generate highly reproducible mass spectra (Smart et al., 2010). Food samples can contain various volatile organic compounds (VOCs) that have a range of volatilities; therefore, such samples need to be prepared in a variety of ways to profile their VOCs (Wanakhachornkrai & Lertsiri, 2003). Inductively coupled plasma mass spectrometry (ICP-MS) has been used since 1983 for rapid and sensitive multi-element analysis of liquid and solid samples (Gabler, 2002). Most commercially available ICPMS instruments are based on quadrupole mass filters (Gabler, 2002). In this study, we analysed lyophilised and vacuum-dried samples of S. japonicus sea cucumbers collected from the sea near Jeju Island of Korea. We assessed the nutritional value of sea cucumber
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
in terms of daily value (DV) guidelines by analysing VOCs and inorganic elements by GC–MS and ICP-MS, respectively. 2. Materials and methods 2.1. Collection and preparation of sea cucumber samples Sea cucumbers (S. japonicus) were collected from the sea (location, 33°260 N, 126°680 E; water temperature, 14–15 °C) near Jeju Island, Republic of Korea on April 9, 2013. Vacuum-dried and lyophilised samples of sea cucumbers were prepared by vacuum hot plate drying (22–66 °C) for 41 h and by freeze drying for 96 h, respectively (FDI 06-05, Biocryos, Republic of Korea). The weights of three vacuum-dried sea cucumbers were 40.2, 37.0 and 38.1 g (average 38.4 g), and the weights of three lyophilised sea cucumbers were 24.7, 42.2 and 53.1 g (average 40.0 g). The whole bodies of vacuum-dried and lyophilised sea cucumber samples were homogenised by grinding for static headspace GC–MS and ICP-MS analyses. 2.2. Materials and reagents 4-Methyl-1-pentanol (97%) was purchased from Sigma–Aldrich (USA). Methanol (HPLC grade) was purchased from Merck (Germany). Nitric acid and hydrogen peroxide solution were purchased from Dongwoo Fine-Chem (electronic grade, South Korea). Water used in the ICP-MS analysis was purified by the Milli-Q purification system. Multi-element calibration standards 1, 2A, 3, and 4 were from Agilent, USA. 2.3. Sample preparation for direct injection GC–MS analysis Each 1 g of homogenised sample was extracted in 15 ml of methanol by sonication for 20 min with an ultrasonic cleaner (UC-20, Lab companion, South Korea). The internal standard 4methyl-1-pentanol was added to each sample to a final concentration of 3.98 lg kg1. 2.4. Sample preparation for static headspace GC–MS analysis The sample (1 g) and the internal standard 4-methyl-1-pentanol (10 ll) were placed into a 20 ml headspace vial with a screw neck, which was immediately sealed with a silicone blue transparent/PTFE white septum and an aluminium screw cap (Gerstel, Germany). Vials were placed in an autosampler tray for headspace sampling. Samples were heated at 80 °C for 10 min for gas saturation before GC–MS analysis.
35
and then held at 300 °C for 10 min. The sample (1 ll) was injected to a split ratio of 5:1. The running time was 38 min. GC conditions for static headspace analysis were as follows: 40 °C for 2 min, then increased to 230 °C at a rate of 5 °C min1, and then held at 230 °C for 10 min. The sample (350 ll) was injected to a split ratio of 10:1. The running time was 50 min. VOCs were identified using Agilent mass hunter qualitative analysis software (version B.05.00, Agilent, USA) and the W8N50ST mass spectral library (Wiley version 8.0 and NIST version 5.0) with deconvolution algorithms. For deconvolution, the spectrum peak, signal-to-noise, and sharpness thresholds were set to 0.1%, 20%, and 25%, respectively. All GC–MS analyses were performed in triplicate. 2.6. Sample preparation for ICP-MS analysis Each sample (0.2 g) was hydrolysed at 85–170 °C for 24 h in a PTFE digestion vessel (reflux type) using 10 ml nitric acid (electronic grade) and 1 ml hydrogen peroxide (electronic grade). Blank solutions were prepared by mixing 10 ml nitric acid with 1 ml hydrogen peroxide. 2.7. ICP-MS instrumentation and conditions Inorganic elements analysis was performed using an Agilent 7700s ICP-MS (Agilent, USA) at the Jeju Center, KBSI. Radio frequency was set to 1550 W, and the rate of argon gas flow was 0.5 l min1. Acid hydrolysed samples were loaded into the ICPMS using a peristaltic pump and an autosampler. Data acquisition for elements was performed using Agilent mass hunter software for ICP-MS (Version A.01.02); specifically, 23Na, 31P, 39K, 40Ca, 7Li, 11 B, 28Si, 56Fe, and 78Se were measured in the hydrogen mode, whereas 24Mg, 27Al, 64Zn, 75As, 85Rb, and 88Sr were measured in the helium mode. Multi-element calibration standards 1, 2A, 3, and 4 were used for calibration in ICP-MS analyses. 2.8. Statistics analysis Statistical significance between the ICP-MS analyses results of the two groups (vacuum-dried and lyophilised) was determined with the independent samples t-test using Excel 2010 (Microsoft, USA). A p value <0.05 was considered significant. All analyses were carried out on triplicate samples. 3. Results and discussion 3.1. GC–MS profiles of vacuum-dried and lyophilised S. japonicus samples
2.5. GC–MS instrumentation and conditions VOC analysis was performed using an Agilent 7890A GC system coupled to an Agilent 7000B tandem quadrupole mass spectrometer (Agilent, USA) equipped with a MP2 headspace autosampler (Gerstel, Germany) at the Jeju Center, Korea Basic Science Institute (KBSI). A HP-5MS column (30 m 0.25 mm, 0.25 lm film thickness, Agilent, USA) or a DB-WAX column (60 m 0.25 mm, 0.25 lm film thickness, Agilent, West Lothian, UK) were applied to the GC–MS system for direct injection (DI) analysis and static headspace (HS) analysis, respectively. The injector, thermal AUX, and ion source temperatures were 230, 280 and 300 °C, respectively. The carrier gas was helium and the flow rate was 1 ml min1. The scan range was 20–350 m/z (scan time, 126 ms) and the ionisation energy was 70 eV. GC conditions for direct injection analysis were as follows: 40 °C for 5 min, then increased to 300 °C at a rate of 10 °C min1,
Homogenised vacuum-dried or lyophilised S. japonicus samples were analysed by GC–MS. VOCs were detected in both types of samples, and appeared in multiple peaks with significant signal intensities in the range of 20–350 Da. Total ion chromatograms showing the VOC profiles of vacuum-dried and lyophilised samples generated by GC–MS are shown in Fig. 1. The number of VOCs identified by direct injection analysis in vacuum-dried and lyophilised samples was 21 and 23, respectively (Table 1). Gas chromatography using a headspace autosampler is useful to detect VOCs with a low molecular weight (Snow & Slack, 2008). Static headspace analysis identified 17 and 10 VOCs in vacuum-dried and lyophilised samples, respectively (Table 2). The VOCs identified by direct injection analysis of vacuumdried samples included three alkanes, four alkenes, an amide, two acids, two aldehydes, an alcohol, four esters, two ketones and two miscellaneous VOCs. The VOCs identified by direct
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H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
Intensity
A
B
Retention time (min) Fig. 1. Total ion chromatograms of vacuum-dried (A) and lyophilised (B) S. japonicus samples generated by direct injection GC–MS analysis. Chromatograms were adapted using a GAUSSIAN filter. Function and GAUSSIAN width are 30 and 10 points, respectively.
Table 1 Direct injection GC–MS analysis of VOCs in vacuum-dried and lyophilised red S. japonicus samples. No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Dimethoxymethyl-(1-methylpropyl)-silane 4-Methyl-1-pentanola 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane 2-Methoxy-benzothieno[2,3-C]quinolin-6(5H)-one 5-Iodo-2-methyl-4-nitroimidazole Undecane 4-(2-Oxoethyl)-5,5-dimethyl-1,8-dioxaspiro[4,5]decane t-Butyl-2-{[(benzyloxy)carbonylamino]butyl}-3-oxoazetidine-1-carboxylate 2-Propenoic acid dodecyl ester Decanoic acid methyl ester 5,5-Dideuteriomethoxycyclohexane Pentadecanal (5S,6S,1E)-4,5-Dimethyl-6-(1-hexenyl)-3,4,5,6-tetrahydro-2H-1,4-dioxazin-2-one Methyl docosanoate Dimethyl acetal octanal 4-Amino-3-phenyl-2H-1-benzopyran-2-one Palmitic acid vinyl ester 4-(2,2,6-Trimethylcyclohexyl)-2-butanol Sulphurous acid 2-ethylhexyl nonyl ester 1-Hydroxytridecan-5-one Benzaldehyde oxime 6-Hydroxy-1,4-dichloro-5-decanone 3-Oxo-3-phenylpropionamide (1R⁄,2R⁄,6S⁄,7S⁄)-2-(tert-Butyldimethylsiloxy)-6,7,9,9-tetramethylbicyclo[4.2.1]nonan-8-one Diazomethyl-8-methoxy-5-(40 -methoxycarbonyl-30 -oxobutyl)-1,2,3,4-tetrahydronaphthalen-2-yl ketone N-(2-Cyclopropylideneethyl)-phthalimide 1,2-Benzfluorene (3b)-Cholest-5-en-3-ol 2,3,5,8-Tetrahydro-5,8-dimethyl-5,8-epoxy-1H-benzocycloheptene 3-(4-Chloro-2-methylpentyl)-cyclopentene (1R⁄,6R⁄)-6,9,9-Trimethylbicyclo[4.2.1]nonane-2,4-dione 3-Bromocholest-5-ene (Z)-2-Octenal
Concentration (lg/kg) MW
RT
VSC
162 102 194 281 253 156 236 321 240 186 116 226 211 354 174 237 282 198 320 214 121 240 163 324 358 213 216 386 188 186 194 448 126
3.13 7.55 8.32 11.19 12.2 13 16.29 18.35 20.93 23.35 24.21 24.29 24.56 25.26 25.67 26.21 26.36 27.18 27.32 28.02 28.69 28.81 31.53 31.91 31.93 32.73 32.74 33.23 33.72 34.01 34.23 35.88 40.34
16.01 3.98 1.65
(RSD) (82.21) (56.45)
0.12 2.905
(75.35) (17.34)
0.85 7.4 0.37
(43.25) (69.96) (29.39)
1.06
(54.65)
0.25
(21.37)
1.66 2.03 2.36 6.2 0.94
(80.55) (69.24) (31.15) (20.72) (73.96)
1.57 6.36
(96.36) (78.44)
0.59
(79.97)
0.77 3.09
(78.57) (104.08)
1.66 60.7
(119.55) (10.87)
Values are means (n = 3). MW, molecular weight; RT, retention time; VSC, vacuum-dried sea cucumber; LSC, lyophilised sea cucumber; RSD, relative standard deviation. a Internal standard.
LSC
(RSD)
15.87 3.98 1.23 0.3
(65.73) (90.85) (47.32)
1.79 0.17
(28.15) (22.99)
0.16 0.56 4.25 0.21 0.16 2.06
(35.68) (9.85) (17.31) (21.41) (27.05) (25.92)
4.92 8.62 0.69 0.47 0.58 2.3
(49.89) (18.46) (35.25) (26.66) (11.40) (68.82)
9.42 2.4
(41.01) (60.78)
15.14
(90.99)
0.85 11.63 50.01
(32.07) (50.16) (18.91)
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H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41 Table 2 Static headspace GC–MS analysis of VOCs in vacuum-dried and lyophilised red S. japonicus samples. No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
4-(2,2,6-Trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one Acetaldehyde N-Methoxy-D1-methanamine 2(S)-Hydroxy-c-butyrolactone Butanal Dichloro-methane Dimethyl ether [2R-[2a(E),3a]]-2-(1-Butenyl)-3-methyl-cyclobutanone Pentanal 7A-Phenyl-trans-bicyclo[4.3.0]non-2-oxime (R)-Tetrahydro-5-oxofuran-2-acetic acid Methyl-benzene 2-Methyl-3-pentanone Hexanal Heptanal (Z,E)-2,4-Nonadienal 1-Chloro-pentane Octanal 3,4-Dihydropyrrolo[1,2-A]pyrazin-1(2H)-one 4-Methyl-1-pentanola 1-Hepten-3-ol Acetic acid 3,5-Octadien-2-one (S)-(+)-4-s-Butylpyrazole
Concentration (lg/kg) MW
RT
VSC
208 44 62 102 72 129 46 138 86 229 144 92 100 100 114 138 106 128 136 102 114 60 124 124
4.45 5.17 5.57 6.27 7.2 8.21 8.42 8.78 9.4 10.4 10.42 11.01 11.37 12.13 15.1 16.4 16.93 18.1 18.41 18.74 22.34 22.44 24.35 25.67
14.84 4.93 1.11 3.68 7.2 2.79
(RSD) (38.06) (28.02) (38.48) (63.37) (48.45) (90.04)
5.81 5.07 5.64
(79.19) (53.36) (58.55)
1.46 0.27 18.2 5.11
(52.58) (26.34) (112.51) (55.38)
2 1.12 0.31 3.98 1.6
(73.74) (66.88) (145.17)
LSC
(RSD)
1.2
(49.05)
1.77
(74.84)
2.41 4.35
(85.04) (73.14)
9.41 0.72
(124.00) (51.16)
0.13
(70.43)
3.98 (58.88) 0.97 0.98 0.2
(27.55) (21.29) (81.71)
Values are means (n = 3). MW, molecular weight; RT, retention time; VSC, vacuum-dried sea cucumber; LSC, lyophilised sea cucumber; RSD, relative standard deviation. a Internal standard.
injection analysis of lyophilised sea cucumber samples included five alkanes, an alkene, an amide, an imide, an acid, three aldehydes, two alcohols, five esters, and one miscellaneous VOC (Table 3). The VOCs identified by static headspace analysis of vacuum-dried samples included two alkanes, an alkene, an amine, five aldehydes, an alcohol, four ketones and two miscellaneous VOCs. The VOCs identified by static headspace analysis of lyophilised samples included an alkane, an alkene, an amine, two acids, two aldehydes, an ketone and three miscellaneous VOCs (Table 3). In total, the number of VOCs identified in vacuum-dried and lyophilised S. japonicus samples was 38 and 33, respectively. Therefore, the odour of lyophilised S. japonicus contains various VOCs that the odour of vacuum-dried S. japonicus does not. Alkenes and unsaturated alcohols are present in the odour of seafood samples (Zhang, Li, Luo, & Chen, 2010), both of which were identified in the odours of the vacuum-dried and lyophilised S. japonicus samples. The alkane undecane was detected in both types of S. japonicus samples. The presence of this hydrocarbon confirms previous observations that many aquatic organisms contaminated with petroleum contain hydrocarbons (Ogata & Miyake, 1980). Moreover, 24,851 L of oil was released into the sea near Jeju Island in 2012 year by marine pollution incidents (Jeju damage observation report 2013). This suggests that the habitat of the sea cucumbers collected may be also contaminated with petroleum. In general, the odour of vacuum-dried sea cucumber is thought to be due to the presence of various VOCs that are absent in lyophilised sea cucumber. The different drying time may cause the existence of the VOCs in the vacuum-dried and lyophilised samples (Fig. 2).
3.2. ICP-MS profiles of vacuum-dried and lyophilised S. japonicus samples Vacuum-dried and lyophilised S. japonicus samples were hydrolysed using nitric acid and hydrogen peroxide, and analysed by ICP-MS. Fifteen inorganic elements were identified in both types
of samples (Table 4 and Fig. 3). Table 4 shows the concentration of each of these inorganic elements in the vacuum-dried and lyophilised samples. In general, the concentrations of inorganic elements were slightly higher in the lyophilised samples than in the vacuum-dried samples. This may be because the vacuum-dried samples lost more moisture than the lyophilised samples during dry periods. For this reason, we feel that the levels of inorganic compounds measured in the lyophilised samples more accurately reflect the true levels in S. japonicus.
3.2.1. Alkali metals (lithium, sodium, potassium, and rubidium) Lithium is an ultratrace element and a lithium deficiency in insulin-sensitive tissue is associated with blood glucose imbalance in vivo (Hu, Wu, & Wu, 1997). Potassium and sodium promote thermal reactions of biomass (Saddawi, Jones, & Williams, 2012), and are essential nutrients that maintain the electrolyte balance in the body (Holbrook et al., 1984). However, a high sodium-topotassium ratio is strongly associated with cardiovascular and ischemic heart disease. Most processed foods have a high sodium-to-potassium ratio including bread, dry-cured meat, and canned and packaged foods (Nowson, Morgan, & Gibbons, 2003); therefore, it is recommended that natural foods with a low sodium-to-potassium ratio are consumed (Yang et al., 2011). Rubidium is mostly found in red meat and has been used as an antidepressant, and rubidium deficiency is associated with ureamia (Canavese, DeCostanzi, Bergamo, Sabbioni, & Stratta, 2008). The mean concentrations of lithium, sodium, potassium, and rubidium in vacuum-dried samples were 1.92, 93,886, 4533, and 2.35 mg kg1, respectively. The mean concentrations of lithium, sodium, potassium, and rubidium in lyophilised samples were 1.95, 134817.33, 5499, and 2.62 mg kg1, respectively. Therefore, the concentration of lithium, sodium, and potassium was higher in vacuum-dried samples than in lyophilised samples. The sodiumto-potassium ratio in vacuum-dried and lyophilised samples was 20.71 and 24.52, respectively. The daily value (DV) of sodium
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H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
Table 3 Identification of different VOCs in red S. japonicus samples by GC–MS according to the drying method and type of analysis used. Compounds Vacuum-dried
Method
Lyophilised
Method
Alkanes Dimethoxymethyl-(1-methylpropyl)-silane 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane Undecane Dichloro-methane 1-Chloro-pentane
DI DI DI HS HS
Dimethoxymethyl-(1-methylpropyl)-silane 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane Undecane 4-(2-Oxoethyl)-5,5-dimethyl-1,8-dioxaspiro[4,5]decane 5,5-Dideuteriomethoxycyclohexane Dichloro-methane
DI DI DI DI DI HS
DI DI
3-Bromocholest-5-ene Methyl-benzene
DI HS
Alkenes 1,2-Benzfluorene 2,3,5,8-Tetrahydro-5,8-dimethyl-5,8-epoxy-1Hbenzocycloheptene 3-(4-Chloro-2-methylpentyl)-cyclopentene 3-Bromocholest-5-ene Methyl-benzene
DI DI HS
Amines N-Methoxy-D1-methanamine
HS
N-Methoxy-D1-methanamine
HS
Amides 3-Oxo-3-phenylpropionamide
DI
3-Oxo-3-phenylpropionamide
DI
N-(2-Cyclopropylideneethyl)-phthalimide
DI
Imides Acids T-Butyl-2-{[(benzyloxy)carbonylamino]butyl}-3-oxoazetidine-1carboxylate
DI
Methyl docosanoate
DI
Methyl docosanoate
DI
(R)-Tetrahydro-5-oxofuran-2-acetic acid Acetic acid
HS HS
Aldehydes Pentadecanal (Z)-2-Octenal Acetaldehyde Butanal Pentanal Heptanal Octanal
DI DI HS HS HS HS HS
Pentadecanal Dimethyl acetal octanal (Z)-2-Octenal Acetaldehyde (Z,E)-2,4-Nonadienal
DI DI DI HS HS
Alcohols 4-(2,2,6-Trimethylcyclohexyl)-2-butanol 1-Hepten-3-ol
DI HS
4-(2,2,6-Trimethylcyclohexyl)-2-butanol (3.beta.)-Cholest-5-en-3-ol
DI DI
Esters 2-Propenoic acid dodecyl ester Decanoic acid methyl ester Palmitic acid vinyl ester Sulphurous acid 2-ethylhexyl nonyl ester
DI DI DI DI
Decanoic acid methyl ester Sulphurous acid 2-ethylhexyl nonyl ester
DI DI
DI DI
2-Methoxy-benzothieno[2,3-C]quinolin-6(5H)-one (5S,6S,1E)-4,5-Dimethyl-6-(1-hexenyl)-3,4,5,6-tetrahydro-2H-1,4-dioxazin2-one 1-Hydroxytridecan-5-one Diazomethyl-8-methoxy-5-(40 -methoxycarbonyl-30 -oxobutyl)-1,2,3,4Tetrahydronaphthalen-2-yl ketone
DI DI
(1R⁄,6R⁄)-6,9,9-Trimethylbicyclo[4.2.1]nonane-2,4-dione 3,5-Octadien-2-one
DI HS
Benzaldehyde oxime 2(S)-Hydroxy-c-butyrolactone Dimethyl ether (S)-(+)-4-s-Butylpyrazole
DI HS HS HS
Ketones 1-Hydroxytridecan-5-one (1R⁄,2R⁄,6S⁄,7S⁄)-2-(tert-Butyldimethylsiloxy)-6,7,9,9tetramethylbicyclo[4.2.1]nonan-8-one 4-(2,2,6-Trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one [2R-[2a(E),3a]]-2-(1-Butenyl)-3-methyl-cyclobutanone
HS HS
2-Methyl-3-pentanone 3,4-Dihydropyrrolo[1,2-A]pyrazin-1(2H)-one
HS HS
Others 5-Iodo-2-methyl-4-nitroimidazole 4-Amino-3-phenyl-2H-1-benzopyran-2-one 2(S)-Hydroxy-c-butyrolactone 7A-Phenyl-trans-bicyclo[4.3.0]non-2-oxime
DI DI HS HS
DI DI
DI, direct injection analysis; HS, static headspace analysis.
recommended by the United States Department of Agriculture (USDA) is 2400 mg. Thus, 25.56 g of vacuum-dried and 17.80 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommend DV. The level of sodium in 1 kg of dried sea cucumber was above the recommended DV. Furthermore, because sea cucumber is usually washed before cooking, it is likely that cooked sea cucumber has an even lower sodium content than that of dried sea cucumber.
3.2.2. Alkaline earth metals (magnesium, calcium, and strontium) Magnesium is an intracellular cation and is a cofactor of various enzymatic reactions related to the catabolism of food. The amount of energy needed for muscle contraction can increase when magnesium is depleted (Lukaski & Nielsen, 2002). Calcium regulates bone formation and calcium supplements are taken to reduce osteoporosis. Calcium intake is associated with bone mineral content during middle childhood (Fiorito, Mitchell, Smiciklas-Wright, &
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
39
Intensity
A
B
Retention time (min) Fig. 2. Total ion chromatograms of vacuum-dried (A) and lyophilised (B) sea cucumber samples generated by static headspace GC–MS analysis. Chromatograms were adapted using a GAUSSIAN filter. Function and GAUSSIAN width are 30 and 10 points, respectively.
Table 4 Concentrations of inorganic elements in vacuum-dried and lyophilised S. japonicus samples. Element
23
Na Mg P 39 K 40 Ca 7 Li 11 B 27 Al 28 Si 56 Fe 64 Zn 75 As 78 Se 85 Rb 88 Sr 24 31
Vacuum-dried
Lyophilised
Concentration (mg/kg)
RSD
Concentration (mg/kg)
RSD
93886.00 14241.62 2821.86 4533.00 10409.00 1.92 41.74 7.10 31.74 24.95 20.10 16.10 4.15 2.35 94.98
21.50 18.77 10.43 11.32 15.52 12.62 10.47 15.23 6.79 23.27 11.38 21.59 5.57 6.47 11.96
134817.33 19781.28 2624.55 5499.64 11237.40 1.95 56.33 7.17 32.31 23.27 17.71 15.46 4.11 2.62 112.22
5.14 4.37 7.30 3.46 13.83 19.98 2.48 36.12 14.49 12.24 9.00 18.50 6.48 5.82 6.45
Values are means (n = 3). RSD, relative standard deviation.
Birch, 2006). Strontium promotes osteomalacia in bone. However, strontium ranelate medication enhances cell replication and bone formation in vivo (Pors Nielsen, 2004). The mean concentrations of magnesium, calcium, and strontium in vacuum-dried samples were 14241.62, 10409.00, and 94.98 mg kg1, respectively. The mean concentrations of magnesium, calcium, and strontium in lyophilised samples were 19781.28, 11237.40, and 112.22 mg mg1, respectively. Therefore, the concentrations of all three of these metals were higher in lyophilised samples than in vacuum-dried samples. The recommended DVs of magnesium and calcium are 400 and 1000 mg, respectively. Vacuum-dried and lyophilised S. japonicus contained
14.24 and 19.78 g, respectively, of magnesium, and 10.41 and 11.24 g, respectively, of calcium per kg of dried weight. Therefore, 28.09 g of vacuum-dried and 20.22 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommended DV of magnesium, and 96.06 g of vacuum-dried and 88.97 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommended DV of calcium. Consequently, S. japonicus is a good source of magnesium and calcium. 3.2.3. Transition metals and post-transition metals (iron, zinc, and aluminium) Iron haemoglobin and myoglobin necessary to prevent anaemia (Pizarro, Olivares, Hertrampf, Mazariegos, & Arredondo, 2003). Zinc also functions in the immune system (Ibs & Rink, 2003). Aluminium is a non-essential metal in humans and is used in various food additives including preservatives, colouring agents, leavening agents, and anticaking agents (Soni, White, Flamm, & Burdock, 2001). The mean concentrations of iron, zinc, and aluminium in vacuum-dried samples were 24.95, 20.10, and 7.1 mg kg1, respectively. The mean concentrations of iron, zinc, and aluminium in lyophilised samples were 23.27, 17.71, and 7.17 mg kg1, respectively. The recommended DVs of iron and zinc are 18 and 15 mg, respectively; therefore, neither vacuum-dried nor lyophilised S. japonicus is a good source of these metals because an intake of more than 700–1000 g of dried sea cucumber would be needed to meet the recommended DVs of iron and zinc. 3.2.4. Metalloids (boron, silicon and arsenic) Boron influences bone health, glucose metabolism, and immune function (Hunt, Butte, & Johnson, 2005). Silicon is important for collagen formation and bone prolyl hydroxylase activity (Uthus & Seaborn, 1996). Arsenic plays a physiological role in methionine recycling and so is important for methionine metabolism.
40
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41 109 Vacuum-dried
*
Lyophilized
108 * *
Concentration (µg/kg)
107 106
*
105 104 103 102 101
1
23Na
24Mg
31P
39K
40Ca
7Li
11B
27Al
28Si
56Fe
64Zn
75As
78Se
85Rb
88Sr
Elements Fig. 3. Logarithmic graph of the concentration of inorganic elements in vacuum-dried and lyophilised S. japonicus samples as determined by ICP-MS. Bars indicate mean ± standard deviation. The concentrations of each element in vacuum-dried and lyophilised samples were compared using the independent samples t-test. ⁄Indicates p < 0.05.
The mean concentrations of boron, silicon, and arsenic in vacuum-dried samples were 41.74, 31.74, and 16.10 mg kg1, respectively. The mean concentrations of boron, silicon, and arsenic in lyophilised samples were 56.33, 32.31, and 15.46 mg kg1, respectively. The concentration of arsenic ranged from 15 to 16 mg kg1 in S. japonicus, and was previously reported to range from 160 to 2360 lg kg1 (wet weight) in seafood samples (Schoof et al., 1999). Most aquatic organisms contain organic arsenic in the form of arsenobetaine (Caumette, Koch, & Reimer, 2012). People who consume 2000 kcal per day are predicted to require 12–25 lg arsenic daily (Uthus, 1994). S. japonicus contains a sufficient level of arsenic to reach this daily requirement. 3.2.5. Nonmetals (phosphorous and selenium) Phosphorus is an essential component of ATP, DNA, RNA, and phospholipids. However, high phosphorus intake and low calcium intake can cause secondary hyperparathyroidism and bone loss (Calvo, 1993). Selenium is needed to generate selenoproteins, which protect against oxidative stress and cardiovascular disease (Tanguy, Grauzam, de Leiris, & Boucher, 2012). The mean concentrations of phosphorus and selenium in vacuum-dried samples were 2821.86 and 4.15 mg kg1, respectively. The mean concentrations of phosphorus and selenium in lyophilised samples were 2624.55 and 4.11 mg kg1, respectively. The recommended DV of phosphorus is 1000 mg, and the level of phosphorus in S. japonicus was approximately 2.6–2.8 g kg1. However, it is easy to consume too much phosphorus because phosphate additives, which can cause health risks such as hyperphosphataemia, are present in many processed foods (Ritz, Hahn, Ketteler, Kuhlmann, & Mann, 2012). The recommended DV of selenium is 70 lg, and the level of selenium in dried S. japonicus was approximately 4 mg kg1. Therefore, S. japonicus is a good source of selenium. 4. Conclusions This study has shown that the GC–MS profiles of vacuum-dried and lyophilised S. japonicus samples differ, and that the types and concentrations of VOCs varied and were higher in vacuum-dried samples than in lyophilised samples. We also used ICP-MS to
analyse the levels of various inorganic elements in S. japonicas showing slight differences in two samples. The inorganic and organic chemical analyses provide basic information about the VOC and inorganic element of dried S. japonicus.
Acknowledgments This work was supported by the project fund (C33730) to J.S.C. from the Center for Analytical Research of Disaster Science of Korea Basic Science Institute. The authors thank the Jeju Free International City Development Center and Egeonjo Co., LTD. for administrative assistance at the Jeju Center of Korea Basic Science Institute.
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Tanguy, S., Grauzam, S., de Leiris, J., & Boucher, F. (2012). Impact of dietary selenium intake on cardiac health: Experimental approaches and human studies. Molecular Nutrition & Food Research, 56(7), 1106–1121. Uthus, E. (1994). Estimation of safe and adequate daily intake for arsenic. In Estimation of safe and adequate daily intake for arsenic (pp. 273–282). Washington, DC: ILSI Press. Uthus, E. O., & Seaborn, C. D. (1996). Deliberations and evaluations of the approaches, endpoints and paradigms for dietary recommendations of the other trace elements. Journal of Nutrition, 126(9), 2452S–2459S. Wanakhachornkrai, P., & Lertsiri, S. (2003). Comparison of determination method for volatile compounds in Thai soy sauce. Food Chemistry, 83(4), 619–629. Wang, T., Sun, Y., Jin, L., Thacker, P., Li, S., & Xu, Y. (2012). Aj-rel and Aj-p105, two evolutionary conserved NF-kappaB homologues in sea cucumber (Apostichopus japonicus) and their involvement in LPS induced immunity. Fish & Shellfish Immunology, 34(1), 17–22. Yang, Q., Liu, T., Kuklina, E. V., Flanders, W. D., Hong, Y., Gillespie, C., et al. (2011). Sodium and potassium intake and mortality among US adults: Prospective data from the Third National Health and Nutrition Examination Survey. Archives of Internal Medicine, 171(13), 1183. Zhang, Z., Li, G., Luo, L., & Chen, G. (2010). Study on seafood volatile profile characteristics during storage and its potential use for freshness evaluation by headspace solid phase microextraction coupled with gas chromatography– mass spectrometry. Analytica Chimica Acta, 659(1), 151–158. Zhong, Y., Khan, M. A., & Shahidi, F. (2007). Compositional characteristics and antioxidant properties of fresh and processed sea cucumber (Cucumaria frondosa). Journal of Agricultural and Food Chemistry, 55(4), 1188–1192.
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Short communication
Characterisation of inorganic elements and volatile organic compounds in the dried sea cucumber Stichopus japonicus Hae-Won Lee a, Na-Lae Lim a, Kichul Cho a, Hye Young Yang a, Kyung June Yim a, Mi-Ju Kim a, Myunglip Lee a, Dong Hyeun Kim b, Hyoung Bum Koh b, Won-Kyo Jung c, Seong Woon Roh a,⇑, Daekyung Kim a,⇑ a b c
Jeju Center, Korea Basic Science Institute (KBSI), Jeju 690-756, Republic of Korea Jeju Special Self-Governing Province Ocean and Fisheries Research Institute, Jeju 690-700, Republic of Korea Department of Biomedical Engineering, and Center for Marine-Intergrated Biomedical Technology (BK21 Plus) Pukyong National University, Busan 608-737, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 14 July 2013 Accepted 19 September 2013 Available online 29 September 2013 Keywords: GC–MS ICP-MS Inorganic elements Stichopus japonicus Volatile organic compounds
a b s t r a c t The sea cucumber Stichopus japonicus lives in a variety of marine habitats and is an important cultivated edible aquatic species in East Asia. In this study, S. japonicus, collected from the sea near Jeju Island of Korea, was lyophilised or vacuum-dried and then analysed by gas chromatography–mass spectrometry (GC–MS) or inductively coupled plasma mass spectrometry (ICP-MS). The GC–MS profiles of vacuumdried and lyophilised samples differed. Based on direct injection and static headspace analysis, 37 volatile organic compounds (VOCs) were identified in vacuum-dried samples and 33 VOCs were identified in lyophilised samples. Therefore, the odour of vacuum-dried sea cucumber is thought to be due to the presence of various VOCs that are absent in lyophilised sea cucumber. According to ICP-MS analysis, the levels of 15 inorganic elements were slightly higher in lyophilised samples than in vacuum-dried samples. The results of the inorganic and organic chemical analyses provide information about the composition of dried sea cucumber. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The sea cucumber Stichopus japonicus is a cylinder-shaped invertebrate that lives in a variety of marine habitats and is an important cultivated aquatic species in China and Japan (Zhong, Khan, & Shahidi, 2007). China is the biggest cultivator of sea cucumbers and has the most sea cucumber farms (Zhong et al., 2007). The majority of countries that consume sea cucumber are in the Indo-Pacific region, including the Philippines, Malaysia, Hong Kong, Singapore, South Korea, China, and Japan (Wang et al., 2012). Nutritionally, sea cucumber has a high protein content, a low fat content, and is rich in essential amino acids, such as tryptophan, arginine, and lysine. Moreover, the body wall of sea cucumbers contains insoluble collagen and has been used as a nutritional supplement. Sea cucumbers are a rich source of polysaccharide chondroitin sulphate, which can reduce arthritis pain (Chen, 2003). Scientific reports by nutritionists and pharmacologists have supported the belief that sea cucumbers have nutritional and medicinal value (Zhong et al., 2007). Rehydrated sea cucumber can be mixed with a variety of foods (Zhong et al., 2007), and stored forms of the product, such as smoked and dried, are gener⇑ Corresponding authors. Tel.: +82 64 800 4931; fax: +82 64 805 7800 (S.W. Roh). Tel.: +82 64 800 4930; fax: +82 64 805 7800 (D. Kim). E-mail addresses: [email protected] (S.W. Roh), [email protected] (D. Kim). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.110
ated from the gonad, intestine, or the entire body of the sea cucumber. Dried sea cucumber is taken as a nutritional supplement, in the form of a capsule or tablet, and is used as a cooking ingredient in East Asia. Previous studies have analysed the inorganic element, nutrient (Chen, 2003), and antioxidant (Liu et al., 2012) composition of sea cucumber. However, a comparative analysis of VOCs and inorganic elements in sea cucumber has not been reported. Gas chromatography–mass spectrometry (GC–MS) can efficiently separate and identify organic molecules. Samples are introduced into the gas chromatograph, evaporated, and the vapour is separated through a mobile phase such as helium (Smart, Aggio, Van Houtte, & Villas-Boas, 2010). These separated compounds are analysed by mass spectrometry to generate highly reproducible mass spectra (Smart et al., 2010). Food samples can contain various volatile organic compounds (VOCs) that have a range of volatilities; therefore, such samples need to be prepared in a variety of ways to profile their VOCs (Wanakhachornkrai & Lertsiri, 2003). Inductively coupled plasma mass spectrometry (ICP-MS) has been used since 1983 for rapid and sensitive multi-element analysis of liquid and solid samples (Gabler, 2002). Most commercially available ICPMS instruments are based on quadrupole mass filters (Gabler, 2002). In this study, we analysed lyophilised and vacuum-dried samples of S. japonicus sea cucumbers collected from the sea near Jeju Island of Korea. We assessed the nutritional value of sea cucumber
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
in terms of daily value (DV) guidelines by analysing VOCs and inorganic elements by GC–MS and ICP-MS, respectively. 2. Materials and methods 2.1. Collection and preparation of sea cucumber samples Sea cucumbers (S. japonicus) were collected from the sea (location, 33°260 N, 126°680 E; water temperature, 14–15 °C) near Jeju Island, Republic of Korea on April 9, 2013. Vacuum-dried and lyophilised samples of sea cucumbers were prepared by vacuum hot plate drying (22–66 °C) for 41 h and by freeze drying for 96 h, respectively (FDI 06-05, Biocryos, Republic of Korea). The weights of three vacuum-dried sea cucumbers were 40.2, 37.0 and 38.1 g (average 38.4 g), and the weights of three lyophilised sea cucumbers were 24.7, 42.2 and 53.1 g (average 40.0 g). The whole bodies of vacuum-dried and lyophilised sea cucumber samples were homogenised by grinding for static headspace GC–MS and ICP-MS analyses. 2.2. Materials and reagents 4-Methyl-1-pentanol (97%) was purchased from Sigma–Aldrich (USA). Methanol (HPLC grade) was purchased from Merck (Germany). Nitric acid and hydrogen peroxide solution were purchased from Dongwoo Fine-Chem (electronic grade, South Korea). Water used in the ICP-MS analysis was purified by the Milli-Q purification system. Multi-element calibration standards 1, 2A, 3, and 4 were from Agilent, USA. 2.3. Sample preparation for direct injection GC–MS analysis Each 1 g of homogenised sample was extracted in 15 ml of methanol by sonication for 20 min with an ultrasonic cleaner (UC-20, Lab companion, South Korea). The internal standard 4methyl-1-pentanol was added to each sample to a final concentration of 3.98 lg kg1. 2.4. Sample preparation for static headspace GC–MS analysis The sample (1 g) and the internal standard 4-methyl-1-pentanol (10 ll) were placed into a 20 ml headspace vial with a screw neck, which was immediately sealed with a silicone blue transparent/PTFE white septum and an aluminium screw cap (Gerstel, Germany). Vials were placed in an autosampler tray for headspace sampling. Samples were heated at 80 °C for 10 min for gas saturation before GC–MS analysis.
35
and then held at 300 °C for 10 min. The sample (1 ll) was injected to a split ratio of 5:1. The running time was 38 min. GC conditions for static headspace analysis were as follows: 40 °C for 2 min, then increased to 230 °C at a rate of 5 °C min1, and then held at 230 °C for 10 min. The sample (350 ll) was injected to a split ratio of 10:1. The running time was 50 min. VOCs were identified using Agilent mass hunter qualitative analysis software (version B.05.00, Agilent, USA) and the W8N50ST mass spectral library (Wiley version 8.0 and NIST version 5.0) with deconvolution algorithms. For deconvolution, the spectrum peak, signal-to-noise, and sharpness thresholds were set to 0.1%, 20%, and 25%, respectively. All GC–MS analyses were performed in triplicate. 2.6. Sample preparation for ICP-MS analysis Each sample (0.2 g) was hydrolysed at 85–170 °C for 24 h in a PTFE digestion vessel (reflux type) using 10 ml nitric acid (electronic grade) and 1 ml hydrogen peroxide (electronic grade). Blank solutions were prepared by mixing 10 ml nitric acid with 1 ml hydrogen peroxide. 2.7. ICP-MS instrumentation and conditions Inorganic elements analysis was performed using an Agilent 7700s ICP-MS (Agilent, USA) at the Jeju Center, KBSI. Radio frequency was set to 1550 W, and the rate of argon gas flow was 0.5 l min1. Acid hydrolysed samples were loaded into the ICPMS using a peristaltic pump and an autosampler. Data acquisition for elements was performed using Agilent mass hunter software for ICP-MS (Version A.01.02); specifically, 23Na, 31P, 39K, 40Ca, 7Li, 11 B, 28Si, 56Fe, and 78Se were measured in the hydrogen mode, whereas 24Mg, 27Al, 64Zn, 75As, 85Rb, and 88Sr were measured in the helium mode. Multi-element calibration standards 1, 2A, 3, and 4 were used for calibration in ICP-MS analyses. 2.8. Statistics analysis Statistical significance between the ICP-MS analyses results of the two groups (vacuum-dried and lyophilised) was determined with the independent samples t-test using Excel 2010 (Microsoft, USA). A p value <0.05 was considered significant. All analyses were carried out on triplicate samples. 3. Results and discussion 3.1. GC–MS profiles of vacuum-dried and lyophilised S. japonicus samples
2.5. GC–MS instrumentation and conditions VOC analysis was performed using an Agilent 7890A GC system coupled to an Agilent 7000B tandem quadrupole mass spectrometer (Agilent, USA) equipped with a MP2 headspace autosampler (Gerstel, Germany) at the Jeju Center, Korea Basic Science Institute (KBSI). A HP-5MS column (30 m 0.25 mm, 0.25 lm film thickness, Agilent, USA) or a DB-WAX column (60 m 0.25 mm, 0.25 lm film thickness, Agilent, West Lothian, UK) were applied to the GC–MS system for direct injection (DI) analysis and static headspace (HS) analysis, respectively. The injector, thermal AUX, and ion source temperatures were 230, 280 and 300 °C, respectively. The carrier gas was helium and the flow rate was 1 ml min1. The scan range was 20–350 m/z (scan time, 126 ms) and the ionisation energy was 70 eV. GC conditions for direct injection analysis were as follows: 40 °C for 5 min, then increased to 300 °C at a rate of 10 °C min1,
Homogenised vacuum-dried or lyophilised S. japonicus samples were analysed by GC–MS. VOCs were detected in both types of samples, and appeared in multiple peaks with significant signal intensities in the range of 20–350 Da. Total ion chromatograms showing the VOC profiles of vacuum-dried and lyophilised samples generated by GC–MS are shown in Fig. 1. The number of VOCs identified by direct injection analysis in vacuum-dried and lyophilised samples was 21 and 23, respectively (Table 1). Gas chromatography using a headspace autosampler is useful to detect VOCs with a low molecular weight (Snow & Slack, 2008). Static headspace analysis identified 17 and 10 VOCs in vacuum-dried and lyophilised samples, respectively (Table 2). The VOCs identified by direct injection analysis of vacuumdried samples included three alkanes, four alkenes, an amide, two acids, two aldehydes, an alcohol, four esters, two ketones and two miscellaneous VOCs. The VOCs identified by direct
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H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
Intensity
A
B
Retention time (min) Fig. 1. Total ion chromatograms of vacuum-dried (A) and lyophilised (B) S. japonicus samples generated by direct injection GC–MS analysis. Chromatograms were adapted using a GAUSSIAN filter. Function and GAUSSIAN width are 30 and 10 points, respectively.
Table 1 Direct injection GC–MS analysis of VOCs in vacuum-dried and lyophilised red S. japonicus samples. No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Dimethoxymethyl-(1-methylpropyl)-silane 4-Methyl-1-pentanola 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane 2-Methoxy-benzothieno[2,3-C]quinolin-6(5H)-one 5-Iodo-2-methyl-4-nitroimidazole Undecane 4-(2-Oxoethyl)-5,5-dimethyl-1,8-dioxaspiro[4,5]decane t-Butyl-2-{[(benzyloxy)carbonylamino]butyl}-3-oxoazetidine-1-carboxylate 2-Propenoic acid dodecyl ester Decanoic acid methyl ester 5,5-Dideuteriomethoxycyclohexane Pentadecanal (5S,6S,1E)-4,5-Dimethyl-6-(1-hexenyl)-3,4,5,6-tetrahydro-2H-1,4-dioxazin-2-one Methyl docosanoate Dimethyl acetal octanal 4-Amino-3-phenyl-2H-1-benzopyran-2-one Palmitic acid vinyl ester 4-(2,2,6-Trimethylcyclohexyl)-2-butanol Sulphurous acid 2-ethylhexyl nonyl ester 1-Hydroxytridecan-5-one Benzaldehyde oxime 6-Hydroxy-1,4-dichloro-5-decanone 3-Oxo-3-phenylpropionamide (1R⁄,2R⁄,6S⁄,7S⁄)-2-(tert-Butyldimethylsiloxy)-6,7,9,9-tetramethylbicyclo[4.2.1]nonan-8-one Diazomethyl-8-methoxy-5-(40 -methoxycarbonyl-30 -oxobutyl)-1,2,3,4-tetrahydronaphthalen-2-yl ketone N-(2-Cyclopropylideneethyl)-phthalimide 1,2-Benzfluorene (3b)-Cholest-5-en-3-ol 2,3,5,8-Tetrahydro-5,8-dimethyl-5,8-epoxy-1H-benzocycloheptene 3-(4-Chloro-2-methylpentyl)-cyclopentene (1R⁄,6R⁄)-6,9,9-Trimethylbicyclo[4.2.1]nonane-2,4-dione 3-Bromocholest-5-ene (Z)-2-Octenal
Concentration (lg/kg) MW
RT
VSC
162 102 194 281 253 156 236 321 240 186 116 226 211 354 174 237 282 198 320 214 121 240 163 324 358 213 216 386 188 186 194 448 126
3.13 7.55 8.32 11.19 12.2 13 16.29 18.35 20.93 23.35 24.21 24.29 24.56 25.26 25.67 26.21 26.36 27.18 27.32 28.02 28.69 28.81 31.53 31.91 31.93 32.73 32.74 33.23 33.72 34.01 34.23 35.88 40.34
16.01 3.98 1.65
(RSD) (82.21) (56.45)
0.12 2.905
(75.35) (17.34)
0.85 7.4 0.37
(43.25) (69.96) (29.39)
1.06
(54.65)
0.25
(21.37)
1.66 2.03 2.36 6.2 0.94
(80.55) (69.24) (31.15) (20.72) (73.96)
1.57 6.36
(96.36) (78.44)
0.59
(79.97)
0.77 3.09
(78.57) (104.08)
1.66 60.7
(119.55) (10.87)
Values are means (n = 3). MW, molecular weight; RT, retention time; VSC, vacuum-dried sea cucumber; LSC, lyophilised sea cucumber; RSD, relative standard deviation. a Internal standard.
LSC
(RSD)
15.87 3.98 1.23 0.3
(65.73) (90.85) (47.32)
1.79 0.17
(28.15) (22.99)
0.16 0.56 4.25 0.21 0.16 2.06
(35.68) (9.85) (17.31) (21.41) (27.05) (25.92)
4.92 8.62 0.69 0.47 0.58 2.3
(49.89) (18.46) (35.25) (26.66) (11.40) (68.82)
9.42 2.4
(41.01) (60.78)
15.14
(90.99)
0.85 11.63 50.01
(32.07) (50.16) (18.91)
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H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41 Table 2 Static headspace GC–MS analysis of VOCs in vacuum-dried and lyophilised red S. japonicus samples. No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
4-(2,2,6-Trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one Acetaldehyde N-Methoxy-D1-methanamine 2(S)-Hydroxy-c-butyrolactone Butanal Dichloro-methane Dimethyl ether [2R-[2a(E),3a]]-2-(1-Butenyl)-3-methyl-cyclobutanone Pentanal 7A-Phenyl-trans-bicyclo[4.3.0]non-2-oxime (R)-Tetrahydro-5-oxofuran-2-acetic acid Methyl-benzene 2-Methyl-3-pentanone Hexanal Heptanal (Z,E)-2,4-Nonadienal 1-Chloro-pentane Octanal 3,4-Dihydropyrrolo[1,2-A]pyrazin-1(2H)-one 4-Methyl-1-pentanola 1-Hepten-3-ol Acetic acid 3,5-Octadien-2-one (S)-(+)-4-s-Butylpyrazole
Concentration (lg/kg) MW
RT
VSC
208 44 62 102 72 129 46 138 86 229 144 92 100 100 114 138 106 128 136 102 114 60 124 124
4.45 5.17 5.57 6.27 7.2 8.21 8.42 8.78 9.4 10.4 10.42 11.01 11.37 12.13 15.1 16.4 16.93 18.1 18.41 18.74 22.34 22.44 24.35 25.67
14.84 4.93 1.11 3.68 7.2 2.79
(RSD) (38.06) (28.02) (38.48) (63.37) (48.45) (90.04)
5.81 5.07 5.64
(79.19) (53.36) (58.55)
1.46 0.27 18.2 5.11
(52.58) (26.34) (112.51) (55.38)
2 1.12 0.31 3.98 1.6
(73.74) (66.88) (145.17)
LSC
(RSD)
1.2
(49.05)
1.77
(74.84)
2.41 4.35
(85.04) (73.14)
9.41 0.72
(124.00) (51.16)
0.13
(70.43)
3.98 (58.88) 0.97 0.98 0.2
(27.55) (21.29) (81.71)
Values are means (n = 3). MW, molecular weight; RT, retention time; VSC, vacuum-dried sea cucumber; LSC, lyophilised sea cucumber; RSD, relative standard deviation. a Internal standard.
injection analysis of lyophilised sea cucumber samples included five alkanes, an alkene, an amide, an imide, an acid, three aldehydes, two alcohols, five esters, and one miscellaneous VOC (Table 3). The VOCs identified by static headspace analysis of vacuum-dried samples included two alkanes, an alkene, an amine, five aldehydes, an alcohol, four ketones and two miscellaneous VOCs. The VOCs identified by static headspace analysis of lyophilised samples included an alkane, an alkene, an amine, two acids, two aldehydes, an ketone and three miscellaneous VOCs (Table 3). In total, the number of VOCs identified in vacuum-dried and lyophilised S. japonicus samples was 38 and 33, respectively. Therefore, the odour of lyophilised S. japonicus contains various VOCs that the odour of vacuum-dried S. japonicus does not. Alkenes and unsaturated alcohols are present in the odour of seafood samples (Zhang, Li, Luo, & Chen, 2010), both of which were identified in the odours of the vacuum-dried and lyophilised S. japonicus samples. The alkane undecane was detected in both types of S. japonicus samples. The presence of this hydrocarbon confirms previous observations that many aquatic organisms contaminated with petroleum contain hydrocarbons (Ogata & Miyake, 1980). Moreover, 24,851 L of oil was released into the sea near Jeju Island in 2012 year by marine pollution incidents (Jeju damage observation report 2013). This suggests that the habitat of the sea cucumbers collected may be also contaminated with petroleum. In general, the odour of vacuum-dried sea cucumber is thought to be due to the presence of various VOCs that are absent in lyophilised sea cucumber. The different drying time may cause the existence of the VOCs in the vacuum-dried and lyophilised samples (Fig. 2).
3.2. ICP-MS profiles of vacuum-dried and lyophilised S. japonicus samples Vacuum-dried and lyophilised S. japonicus samples were hydrolysed using nitric acid and hydrogen peroxide, and analysed by ICP-MS. Fifteen inorganic elements were identified in both types
of samples (Table 4 and Fig. 3). Table 4 shows the concentration of each of these inorganic elements in the vacuum-dried and lyophilised samples. In general, the concentrations of inorganic elements were slightly higher in the lyophilised samples than in the vacuum-dried samples. This may be because the vacuum-dried samples lost more moisture than the lyophilised samples during dry periods. For this reason, we feel that the levels of inorganic compounds measured in the lyophilised samples more accurately reflect the true levels in S. japonicus.
3.2.1. Alkali metals (lithium, sodium, potassium, and rubidium) Lithium is an ultratrace element and a lithium deficiency in insulin-sensitive tissue is associated with blood glucose imbalance in vivo (Hu, Wu, & Wu, 1997). Potassium and sodium promote thermal reactions of biomass (Saddawi, Jones, & Williams, 2012), and are essential nutrients that maintain the electrolyte balance in the body (Holbrook et al., 1984). However, a high sodium-topotassium ratio is strongly associated with cardiovascular and ischemic heart disease. Most processed foods have a high sodium-to-potassium ratio including bread, dry-cured meat, and canned and packaged foods (Nowson, Morgan, & Gibbons, 2003); therefore, it is recommended that natural foods with a low sodium-to-potassium ratio are consumed (Yang et al., 2011). Rubidium is mostly found in red meat and has been used as an antidepressant, and rubidium deficiency is associated with ureamia (Canavese, DeCostanzi, Bergamo, Sabbioni, & Stratta, 2008). The mean concentrations of lithium, sodium, potassium, and rubidium in vacuum-dried samples were 1.92, 93,886, 4533, and 2.35 mg kg1, respectively. The mean concentrations of lithium, sodium, potassium, and rubidium in lyophilised samples were 1.95, 134817.33, 5499, and 2.62 mg kg1, respectively. Therefore, the concentration of lithium, sodium, and potassium was higher in vacuum-dried samples than in lyophilised samples. The sodiumto-potassium ratio in vacuum-dried and lyophilised samples was 20.71 and 24.52, respectively. The daily value (DV) of sodium
38
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
Table 3 Identification of different VOCs in red S. japonicus samples by GC–MS according to the drying method and type of analysis used. Compounds Vacuum-dried
Method
Lyophilised
Method
Alkanes Dimethoxymethyl-(1-methylpropyl)-silane 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane Undecane Dichloro-methane 1-Chloro-pentane
DI DI DI HS HS
Dimethoxymethyl-(1-methylpropyl)-silane 1,3-Dimethoxy-1,1,3,3-tetramethyl-disiloxane Undecane 4-(2-Oxoethyl)-5,5-dimethyl-1,8-dioxaspiro[4,5]decane 5,5-Dideuteriomethoxycyclohexane Dichloro-methane
DI DI DI DI DI HS
DI DI
3-Bromocholest-5-ene Methyl-benzene
DI HS
Alkenes 1,2-Benzfluorene 2,3,5,8-Tetrahydro-5,8-dimethyl-5,8-epoxy-1Hbenzocycloheptene 3-(4-Chloro-2-methylpentyl)-cyclopentene 3-Bromocholest-5-ene Methyl-benzene
DI DI HS
Amines N-Methoxy-D1-methanamine
HS
N-Methoxy-D1-methanamine
HS
Amides 3-Oxo-3-phenylpropionamide
DI
3-Oxo-3-phenylpropionamide
DI
N-(2-Cyclopropylideneethyl)-phthalimide
DI
Imides Acids T-Butyl-2-{[(benzyloxy)carbonylamino]butyl}-3-oxoazetidine-1carboxylate
DI
Methyl docosanoate
DI
Methyl docosanoate
DI
(R)-Tetrahydro-5-oxofuran-2-acetic acid Acetic acid
HS HS
Aldehydes Pentadecanal (Z)-2-Octenal Acetaldehyde Butanal Pentanal Heptanal Octanal
DI DI HS HS HS HS HS
Pentadecanal Dimethyl acetal octanal (Z)-2-Octenal Acetaldehyde (Z,E)-2,4-Nonadienal
DI DI DI HS HS
Alcohols 4-(2,2,6-Trimethylcyclohexyl)-2-butanol 1-Hepten-3-ol
DI HS
4-(2,2,6-Trimethylcyclohexyl)-2-butanol (3.beta.)-Cholest-5-en-3-ol
DI DI
Esters 2-Propenoic acid dodecyl ester Decanoic acid methyl ester Palmitic acid vinyl ester Sulphurous acid 2-ethylhexyl nonyl ester
DI DI DI DI
Decanoic acid methyl ester Sulphurous acid 2-ethylhexyl nonyl ester
DI DI
DI DI
2-Methoxy-benzothieno[2,3-C]quinolin-6(5H)-one (5S,6S,1E)-4,5-Dimethyl-6-(1-hexenyl)-3,4,5,6-tetrahydro-2H-1,4-dioxazin2-one 1-Hydroxytridecan-5-one Diazomethyl-8-methoxy-5-(40 -methoxycarbonyl-30 -oxobutyl)-1,2,3,4Tetrahydronaphthalen-2-yl ketone
DI DI
(1R⁄,6R⁄)-6,9,9-Trimethylbicyclo[4.2.1]nonane-2,4-dione 3,5-Octadien-2-one
DI HS
Benzaldehyde oxime 2(S)-Hydroxy-c-butyrolactone Dimethyl ether (S)-(+)-4-s-Butylpyrazole
DI HS HS HS
Ketones 1-Hydroxytridecan-5-one (1R⁄,2R⁄,6S⁄,7S⁄)-2-(tert-Butyldimethylsiloxy)-6,7,9,9tetramethylbicyclo[4.2.1]nonan-8-one 4-(2,2,6-Trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one [2R-[2a(E),3a]]-2-(1-Butenyl)-3-methyl-cyclobutanone
HS HS
2-Methyl-3-pentanone 3,4-Dihydropyrrolo[1,2-A]pyrazin-1(2H)-one
HS HS
Others 5-Iodo-2-methyl-4-nitroimidazole 4-Amino-3-phenyl-2H-1-benzopyran-2-one 2(S)-Hydroxy-c-butyrolactone 7A-Phenyl-trans-bicyclo[4.3.0]non-2-oxime
DI DI HS HS
DI DI
DI, direct injection analysis; HS, static headspace analysis.
recommended by the United States Department of Agriculture (USDA) is 2400 mg. Thus, 25.56 g of vacuum-dried and 17.80 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommend DV. The level of sodium in 1 kg of dried sea cucumber was above the recommended DV. Furthermore, because sea cucumber is usually washed before cooking, it is likely that cooked sea cucumber has an even lower sodium content than that of dried sea cucumber.
3.2.2. Alkaline earth metals (magnesium, calcium, and strontium) Magnesium is an intracellular cation and is a cofactor of various enzymatic reactions related to the catabolism of food. The amount of energy needed for muscle contraction can increase when magnesium is depleted (Lukaski & Nielsen, 2002). Calcium regulates bone formation and calcium supplements are taken to reduce osteoporosis. Calcium intake is associated with bone mineral content during middle childhood (Fiorito, Mitchell, Smiciklas-Wright, &
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41
39
Intensity
A
B
Retention time (min) Fig. 2. Total ion chromatograms of vacuum-dried (A) and lyophilised (B) sea cucumber samples generated by static headspace GC–MS analysis. Chromatograms were adapted using a GAUSSIAN filter. Function and GAUSSIAN width are 30 and 10 points, respectively.
Table 4 Concentrations of inorganic elements in vacuum-dried and lyophilised S. japonicus samples. Element
23
Na Mg P 39 K 40 Ca 7 Li 11 B 27 Al 28 Si 56 Fe 64 Zn 75 As 78 Se 85 Rb 88 Sr 24 31
Vacuum-dried
Lyophilised
Concentration (mg/kg)
RSD
Concentration (mg/kg)
RSD
93886.00 14241.62 2821.86 4533.00 10409.00 1.92 41.74 7.10 31.74 24.95 20.10 16.10 4.15 2.35 94.98
21.50 18.77 10.43 11.32 15.52 12.62 10.47 15.23 6.79 23.27 11.38 21.59 5.57 6.47 11.96
134817.33 19781.28 2624.55 5499.64 11237.40 1.95 56.33 7.17 32.31 23.27 17.71 15.46 4.11 2.62 112.22
5.14 4.37 7.30 3.46 13.83 19.98 2.48 36.12 14.49 12.24 9.00 18.50 6.48 5.82 6.45
Values are means (n = 3). RSD, relative standard deviation.
Birch, 2006). Strontium promotes osteomalacia in bone. However, strontium ranelate medication enhances cell replication and bone formation in vivo (Pors Nielsen, 2004). The mean concentrations of magnesium, calcium, and strontium in vacuum-dried samples were 14241.62, 10409.00, and 94.98 mg kg1, respectively. The mean concentrations of magnesium, calcium, and strontium in lyophilised samples were 19781.28, 11237.40, and 112.22 mg mg1, respectively. Therefore, the concentrations of all three of these metals were higher in lyophilised samples than in vacuum-dried samples. The recommended DVs of magnesium and calcium are 400 and 1000 mg, respectively. Vacuum-dried and lyophilised S. japonicus contained
14.24 and 19.78 g, respectively, of magnesium, and 10.41 and 11.24 g, respectively, of calcium per kg of dried weight. Therefore, 28.09 g of vacuum-dried and 20.22 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommended DV of magnesium, and 96.06 g of vacuum-dried and 88.97 g of lyophilised S. japonicus would need to be consumed daily to ingest the recommended DV of calcium. Consequently, S. japonicus is a good source of magnesium and calcium. 3.2.3. Transition metals and post-transition metals (iron, zinc, and aluminium) Iron haemoglobin and myoglobin necessary to prevent anaemia (Pizarro, Olivares, Hertrampf, Mazariegos, & Arredondo, 2003). Zinc also functions in the immune system (Ibs & Rink, 2003). Aluminium is a non-essential metal in humans and is used in various food additives including preservatives, colouring agents, leavening agents, and anticaking agents (Soni, White, Flamm, & Burdock, 2001). The mean concentrations of iron, zinc, and aluminium in vacuum-dried samples were 24.95, 20.10, and 7.1 mg kg1, respectively. The mean concentrations of iron, zinc, and aluminium in lyophilised samples were 23.27, 17.71, and 7.17 mg kg1, respectively. The recommended DVs of iron and zinc are 18 and 15 mg, respectively; therefore, neither vacuum-dried nor lyophilised S. japonicus is a good source of these metals because an intake of more than 700–1000 g of dried sea cucumber would be needed to meet the recommended DVs of iron and zinc. 3.2.4. Metalloids (boron, silicon and arsenic) Boron influences bone health, glucose metabolism, and immune function (Hunt, Butte, & Johnson, 2005). Silicon is important for collagen formation and bone prolyl hydroxylase activity (Uthus & Seaborn, 1996). Arsenic plays a physiological role in methionine recycling and so is important for methionine metabolism.
40
H.-W. Lee et al. / Food Chemistry 147 (2014) 34–41 109 Vacuum-dried
*
Lyophilized
108 * *
Concentration (µg/kg)
107 106
*
105 104 103 102 101
1
23Na
24Mg
31P
39K
40Ca
7Li
11B
27Al
28Si
56Fe
64Zn
75As
78Se
85Rb
88Sr
Elements Fig. 3. Logarithmic graph of the concentration of inorganic elements in vacuum-dried and lyophilised S. japonicus samples as determined by ICP-MS. Bars indicate mean ± standard deviation. The concentrations of each element in vacuum-dried and lyophilised samples were compared using the independent samples t-test. ⁄Indicates p < 0.05.
The mean concentrations of boron, silicon, and arsenic in vacuum-dried samples were 41.74, 31.74, and 16.10 mg kg1, respectively. The mean concentrations of boron, silicon, and arsenic in lyophilised samples were 56.33, 32.31, and 15.46 mg kg1, respectively. The concentration of arsenic ranged from 15 to 16 mg kg1 in S. japonicus, and was previously reported to range from 160 to 2360 lg kg1 (wet weight) in seafood samples (Schoof et al., 1999). Most aquatic organisms contain organic arsenic in the form of arsenobetaine (Caumette, Koch, & Reimer, 2012). People who consume 2000 kcal per day are predicted to require 12–25 lg arsenic daily (Uthus, 1994). S. japonicus contains a sufficient level of arsenic to reach this daily requirement. 3.2.5. Nonmetals (phosphorous and selenium) Phosphorus is an essential component of ATP, DNA, RNA, and phospholipids. However, high phosphorus intake and low calcium intake can cause secondary hyperparathyroidism and bone loss (Calvo, 1993). Selenium is needed to generate selenoproteins, which protect against oxidative stress and cardiovascular disease (Tanguy, Grauzam, de Leiris, & Boucher, 2012). The mean concentrations of phosphorus and selenium in vacuum-dried samples were 2821.86 and 4.15 mg kg1, respectively. The mean concentrations of phosphorus and selenium in lyophilised samples were 2624.55 and 4.11 mg kg1, respectively. The recommended DV of phosphorus is 1000 mg, and the level of phosphorus in S. japonicus was approximately 2.6–2.8 g kg1. However, it is easy to consume too much phosphorus because phosphate additives, which can cause health risks such as hyperphosphataemia, are present in many processed foods (Ritz, Hahn, Ketteler, Kuhlmann, & Mann, 2012). The recommended DV of selenium is 70 lg, and the level of selenium in dried S. japonicus was approximately 4 mg kg1. Therefore, S. japonicus is a good source of selenium. 4. Conclusions This study has shown that the GC–MS profiles of vacuum-dried and lyophilised S. japonicus samples differ, and that the types and concentrations of VOCs varied and were higher in vacuum-dried samples than in lyophilised samples. We also used ICP-MS to
analyse the levels of various inorganic elements in S. japonicas showing slight differences in two samples. The inorganic and organic chemical analyses provide basic information about the VOC and inorganic element of dried S. japonicus.
Acknowledgments This work was supported by the project fund (C33730) to J.S.C. from the Center for Analytical Research of Disaster Science of Korea Basic Science Institute. The authors thank the Jeju Free International City Development Center and Egeonjo Co., LTD. for administrative assistance at the Jeju Center of Korea Basic Science Institute.
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