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Correspondence Yeon Soo Han, College of Agriculture and Life Science, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Korea. Email: hanys@chonnam.ac.kr Received 25 June 2012; accepted 13 August 2012. doi: 10.1111/j.1748-5967.2012.00469.x
Abstract
This study was conducted to examine the nutritional status of the grasshopper (Oxya chinensis formosana, OCF) as human food, exploring it as an alternative edible resource. Analysis of free amino acid shows that there are various essential amino acids in addition to saturated and unsaturated fatty acids in OCF dried powder. Analysis of the mineral contents and vitamins of dried OCF indicates that it is rich in calcium, vitamin B6, and niacin. The heavy metal content of OCF recorded was low, making it safe for human consumption: OCF had plumbum at 0.010.03 mg/100 g, cadmium at 0.0020.005 mg/100 g, arsenic at 0.070.17 mg/ 100 g, and mercury at 0.00030.0007 mg/100 g. In conclusion, given its high nutritive quality in terms of proteins and fats, coupled with lower heavy metal content, it would be recommended to use the grasshopper (OCF) as substitute to the traditional sources of protein.
Key words: Oxya, chinensis formosana, grasshopper, alternative edible resources.
Introduction
Improved life standards and increased attention to health has led to new awareness regarding the importance of dietary habits to maintain health. Accordingly, interest in exploring alternative food resources that contain nutritionally functional materials from various insects has been growing (Joint FAO/WHO Expert Consultation on Protein Quality Evaluation, 1991; DeFoliart 1992; Bukkens 1997; Shantibala et al. 2012; Van Itterbeeck and van Huis, 2012). Many edible insects have been investigated and reported by other researchers in the world (Bukkens 1997; Ramos-Elorduy et al. 1997, 2011; Wu et al. 2000; Xia et al. 2012). Edible grasshoppers such as Oxya chinensis formosana, (OCF) present one such alternative. Although OCF has been used as food for a long time, no systematic analysis of OCF has been conducted and reported either in Korea or abroad. Therefore, this study attempts to examine nutritional value of OCF using various biochemical and physiochemical methods.
2012 The Authors Entomological Research 2012 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
was added to it and 12 mL of sulfuric acid was dropped along the inside wall of the ask. One mL of 30% hydrogen peroxide was added in small portions while shaking the ask. The mixture was digested following the protocols prescribed in the food code. The crude protein content was measured in terms of the amount of nitrogen released (%) as this correlated with the amount of sodium hydroxide used in the neutralization reaction as given by the formula below:
Crude fat content (g 100g food ) Fatty acid Fatty acid content (g 100g fatty acid ) = content 100 (g 100g food )
Analysis of inorganic components and heavy metals The analysed inorganic contents of the sample used in this study included inorganic metallic components such as Na, Fe, Ca, Mg, K, Mn, Cu, and Zn. The OCF (0.5100 g) was placed in a microwave digestion system (Micro Prep Q2000, Questron Technologies Corporation, Mississauga, Canada). The content was treated with nitric acid and decomposed. It was transferred to a volumetric ask and brought to a constant volume of 100 mL. This test solution was analyzed by an atomic absorption spectroscopy system (Analyst 400, Perkin Elmer, USA) and an inductively coupled plasma mass spectrometry system (ICP-MS) (Agilent 7500 series, Santa Clara, CA, USA). The standard solution and its blank test solution were treated by the same procedure. A calibration curve was drawn and the concentration of the test solution was calculated. For the phosphorus content determination, the protocol prescribed by the proximate analysis of the food code was used to prepare the test solution using Oxya chinensis formosana (0.5100 g) samples. A blank set was run under the same conditions without the samples. Phosphorus content was calculated using the following formula.
Nitrogen (%) = 0.7003 (a b ) 100 Collected sample amount (mg ) Nitrogen coefficient
a: mL of 0.05N sodium hydroxide consumed for neutralization in blank test b: mL of 0.05N sodium hydroxide consumed for neutralization in actual test Following the protocols of the standard food code, the test solution was analyzed by an amino acid automatic analyzer (S-433H, Sykam GmbH, Eresing, Germary).
Crude fat and fatty acids Both the crude fat and fatty acid content of the samples were analysed. Crude fat extraction was made under the crude fat extraction method as described by the food code. About 23 mg of sample was placed in a glass tube for hydrolysis of oil and fat that were extracted from the food, and 1 mL of internal standard solution was added to it. Following addition of 1.5 mL of 0.5N methanol sodium hydroxide and nitrogen infusion, the tube cap was closed and the content was mixed. It was warmed for about 5 min in a 100C heating block. After it was cooled, 2 mL of 14% triuoroborane methanol solution was added and nitrogen was infused again. The cap was closed immediately and the content was mixed. It was warmed for 30 min at a temperature of 100C. It was cooled to 30C. Then, 1 mL of iso-octane solution was added and nitrogen was infused. It was shaken vigorously after the cap was closed. After 5 mL of saturated sodium chloride was added and nitrogen was infused, it was shaken with the cap closed. It was then cooled to room temperature. The columns temperature was maintained at 180C for 40 min and then it was heightened to 230C at a rate of 3C/min and maintained for 15 min. The temperatures of injector and detector were set at 250C and 260C, respectively, with a ow rate of 1.0 mL/min. Fatty acid content in a 100 g sample was calculated as follows.
A 1 V 100 As S
Entomological Research 42 (2012) 284290 2012 The Authors. Entomological Research 2012 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
Clara, CA, USA) to derive the concentration of the test solution. Additionally, we analysed vitamins (A, B1, B6, C, D), niacin and energy content of our samples strictly following the respective methods described in the food code.
S: Vitamin A concentration in test solution (IU/mL) a: Total amount of test solution (mL) b: Dilution factor of test solution
Vitamin B1
Analysis of vitamins
Vitamin A Vitamin A content was measured according to the liquid chromatography quantitative analysis method under the test methods for proximate analysis prescribed by the food code. The sample (4.3833 g) was weighed precisely into a round bottom ask and 30 mL of ethanol and 1 mL of 10% pyrogallol ethanol solution were added to the sample. The content was well mixed and 3 mL of potassium hydroxide solution was added. Then, a reux condenser was attached to the ask and the content was saponied for 30 min. It was rapidly cooled down to room temperature. After 30 mL of water was added, it was transferred to an amber separatory funnel, to which the addition of 10 mL of water and 30 mL of petroleum ether (super grade) was made. The mixture was well shaken and left in that state. Then the water layer was transferred to another amber separatory funnel. The water layer was extracted twice, each time with 30 mL of petroleum ether. It was combined with the liquid that was extracted using petroleum ether and washed with 10 mL of water once and then 50 mL of phenolphthalein test solution to an extent that it did not change color. The petroleum ether layer was dehydrated by adding sodium sulfate anhydrous and all the petroleum ether extracts were combined and decompressed and dried at 40 to 50C. The residual was melted using isopropanol (super grade) and 1.0 mL of it was used as a test solution. Analysis was made by high-performance liquid chromatography (HPLC) using a Luna C18 column (4.6 250 mm, 10 mm, Phenomenex, Torrance, CA, USA) with a mobile phase of EtOH/H2O at 80 : 20 (v/v, isocratic mode) and a FL2000 uorescence detector (Waters, Milford, MA, USA), whose excitation and emission wavelengths were at 340 nm and 460 nm, respectively. The ow rate was 1.0 mL/min. For quantitative analysis, the concentration of vitamin A (IU/mL) in the test solution was calculated from a calibration curve that was derived from the peak area or height of the standard solution obtained by injecting 20 mL of the test solution and 20 mL of the standard solution. The vitamin A content (I.U/100 g) was then calculated using the following formula.
Vitamin B1 content was measured according to the liquid chromatography quantitative analysis method under the test methods for proximate analysis prescribed by the food code. A sample of 3.1717 g was weighed precisely and 0.1N HCl 50 mL was added for homogenization. Its pH was set at 45 by addition of 2N sodium acetate and then it was brought to a constant volume of 50 mL with distilled water. It was poured into a centrifuge tube for centrifugal separation at 3000 rpm for 15 min, and 4 mL of its supernatant was mixed with 3 mL of 15% NaOH and 3 mL of 1% K3Fe(CN)6 in a test tube. It was neutralized with 3 mL of 3.75N HCl. It was passed through a Sep Pak C18 cartridge (MeOH/5 mM ammonium acetate, 30 : 70 (pH 5) mL) and used as a test solution for HPLC analysis. The analysis was made using a Luna C18 column (4.6 250 mm, 10 mm, Phenomenex) with a mobile phase of MeOH/5 mM ammonium acetate at 30 : 70 (v/v, isocratic mode) and a FL2000 uorescence detector (Waters), whose excitation and emission wavelengths were at 374 nm, 450 nm, respectively. The ow rate was 0.7 mL/min. Vitamin B1 content (mg/100 g) of the sample was calculated using the following formula.
Vitamin B2 Vitamin B2 content was measured according to the liquid chromatography quantitative analysis method under the test methods for proximate analysis prescribed by the food code. 8.0266 g of the sample was precisely weighed and 25 mL of distilled water was added to it for homogenization. Then the content was brought to a constant volume of 50 mL. It was sonicated for 30 min and placed into a centrifuge tube for centrifugal separation at 3000 rpm for 15 min. The supernatant was ltered by a 0.45 mm lter and used as a test solution for HPLC analysis. The analysis was made using a Luna C18 column (4.6 250 mm, 10 mm, Phenomenex) with a mobile phase of MeOH/10 mM NaH2PO4 at 35 : 75 (v/v, isocratic mode) and a FL2000 uorescence detector
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(Waters), whose excitation and emission wavelengths were at 444 nm and 530 nm, respectively. The ow rate was 0.8 mL/min.
Vitamin B2 100 ab ( riboflavin, FMN, FAD ) = S 1000 l e Collected samp (mg 100 g ) amount (g )
S: Vitamin B2 concentration in test solution (mg/mL) a: Total test solution amount (mL) b: Dilution factor of test solution
50 mL volumetric ask. After the container was washed with a small amount of 5% metaphosphoric acid, the content was brought to a constant volume of 50 mL. Then it was centrifugally separated at 3000 rpm for 15 min and its supernatant was appropriately diluted with 5% metaphosphoric acid solution. It was used as a test solution for HPLC analysis. The analysis was made using a Luna C18 column (4.6 250 mm, 10 mm, Phenomenex) with a mobile phase of 0.05 M KH2PO4 solution (pH 3, isocratic mode) and a 486 UV detector (Waters) at 254 nm. The ow rate was 0.5 mL/min.
Vitamin B6 Vitamin B6 content was measured according to the liquid chromatography quantitative analysis method under the test methods for proximate analysis prescribed by the food code. A sample of 8.0266 g was weighed precisely and 25 mL of distilled water was added to it for homogenization. Then the content was brought to a constant volume of 50 mL. It was sonicated for 30 min and placed into a centrifuge tube for centrifugal separation at 3000 rpm for 15 min. The supernatant was ltered by a 0.45 mm lter and used as a test solution for HPLC analysis. The analysis was made using a Luna C18 column (4.6 250 mm, 10 mm, Phenomenex) with a mobile phase of MeOH/10 mM NaH2PO4 at 35 : 75 (v/v, isocratic mode) and a FL2000 uorescence detector (Waters), whose excitation and emission wavelengths were at 290 nm and 396 nm, respectively. The ow rate was 1.0 mL/min.
Vitamin C Vitamin C content was measured according to the liquid chromatography quantitative analysis method under the test methods for proximate analysis prescribed by the food code. A sample of 2.3295 g was weighed and the same amount of 10% metaphosphoric acid solution was added to it. The content was suspended for 10 min and an adequate amount of 5% metaphosphoric acid was added to it for homogenization. The homogenized sample was transferred into a
Entomological Research 42 (2012) 284290 2012 The Authors. Entomological Research 2012 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
Vitamin D amount in 100g sample D ( IU ) P S = sa 100 Pst Collected sample amount (g ) Vitamin D content in 100g sample S = Collected sample amount (g ) S 100 Collected sample amount (g ) 1000
S: Concentration of Vitamin D standard solution (IU/mL) Pst: Peak height or area of Vitamin D solution Psa: Peak height or area of sample Niacin Niacin content was measured according to the colorimetric method of Kenichi reaction under the test methods for proximate analysis prescribed by the food code. A precise 2.0214 g of the sample, whose niacin content was equal to 400 mg, was placed into a 100 mL beaker and 20 mL of 6N hydrochloric acid solution was added to it. Then, it was leached for 60 min in a boiling water bath and hydrolyzed. It was cooled with owing water and transferred to a 100 mL volumetric ask. After it was ltered or centrifugally separated with 100 mL of water, the remainder or supernatant was used as a sample solution. Next, 25 mL of the sample solution was put into a sedimentation tube and two droplets of phenolphthalein were added. Then the content was neutralized with 20 mL of 6N until it became slightly acidic. Two millilitres of saturated zinc sulfate solution was added to this neutralized solution and a few droplets of amyl alcohol were dropped as an antifoaming agent. Then the content was shaken and mixed until the sediment of zinc hydroxide was formed and neutralized by the addition of 3 M sodium hydroxide solution and 1N sodium hydroxide solution. 2N zinc sulfate solution was added to it until its pH reached 6.5 and it was brought to
a constant volume of 50 mL by the addition of water. It was occasionally shaken and mixed and left in that state for 10 min. It was ltered or centrifugally separated and used as a test solution. For color development, each of two test tubes was lled with 5 mL of test solution, and 2 mL of cyanogen bromide solution was added to each. They were put into reaction for 15 min in a water bath at 60 to 70C and cooled in ice water: 1 mL of para amino acetophenone solution was added to one, with the other as a control solution, 10 mL of water was added to both. The above color-developed solution in a cooled state was left in a dark room for 15 min and absorbance (A) of the control solution was measured at a wavelength of 420 nm. The calibration curve was derived by adding 4 mL of water to 1 mL of the niacin standard solution. Niacin content was equal to X (mg) derived from a point corresponding with A, which was niacin content in the 5 mL solution that appeared in the calibration curve.
1 100 Total niacin amount = X 40 in sample (mg 100 g ) Collected sample 1000 amount (g )
Free Amino Acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Cysteine Methionine Glutamic acid
Content (mg/100 g) 287.8 42.9 50.6 39.2 25.4 65.7 19.6 2.3 132.6
Free Amino Acid Aspartic acid Glutamine Asparagine Lysine Arginine Phenylalaine Tyrosine Tryptophan Histidine Proline
Content (mg/100 g) 20.0 94.2 55.1 545.0 23.4 63.9 1.6 75.7 106.4
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Table 2
Composition of fatty acids in dried OCF powder Saturated fatty acid (%) Unsaturated fatty acid (%)
Table 5
Heavy metal C14:1 C16:1 C18:1 C18:2 C18:3 C20:1 C20:2 C20:3 C20:4 C20:5 C22:1 0.021 0.880 0.917 2.124 0.007 0.003 0.008 0.002 0.003 0.008 Pb Cd As Hg
C20:0
0.101
Table 6
Items of Analysis Azoxystrobin, Bifenthrin, Butachlor, Chlorfenapyr, Chloruazuron, Chlorothalonil, Cyuthrin, Cypermethrin, Deltamethrin, Dichlouanid, Dicofol, Difenoconazole, Endosulfan(Total), Fenarimol, Fenoxanil, Fenpropathrin, Fenvalerate, Fipronil, Flufenoxuron, Flutolanil, Fthalide, Halfenprox, Indoxacarb, Iprodione, Isoprothiolane, Kresoxim-methyl, L-Cyhalothrin, Lufenuron, Nuarimol, Paclobutrazole, Penconazole, Permethrin, Probenazole, Procymidone, Pyridaben, Pyridaryl, Teuthrin, Tetraconazole, Tetradifon, Thiuzamid, Triadimefon, Vinclozolin, Bitertanol, Buprofezin, Cadusafos, Chlorpyrifos, Chlorpyrifos-methyl, Cyprodinil, Diazinon, Diniconazole, Edifenphos, EPN, Ethoprophos, Fenitrothion, Fenthion, Fludioxonil, Furathiocarb, Hexaconazole, Iprobenfos (IBP), Malathion, Metalaxyl, Methidathion, Parathion, Pendimethalin, Phenthoate, Phorate, Phosalone, Pirimiphos-methyl, Pyrazophos, Tebuconazole, Tebufenpyrad, Tebupirimfos, Terbufos, Tolclofos-methyl, Triumizole, Acetamiprid, Boscalid, Carbendazim, Clothianidin, Cyazofamid, Cymoxanil, Diethofencarb, Diubenzuron, Dimethomorph(E,Z), Imidacloprid, Mepanipyrim, Pencycuron, Pyraclostrobin, Pyrimethanil, Tebufenozide, Teubenzuron, Thiacloprid, Tricyclazole, Trioxystrobin, Carbaryl, Carbofuran, Fenobucarb (BPMC), Fluquinconazole, Isoprocarb, Methiocarb, Methomyl, Thiamethoxam
3.973 (70.7%)
Table 3 Mineral Na Fe Ca Mg K Mn P Zn Cu
Mineral content in dried OCF powder Content (mg/100 g) 115.7 6.8 84.4 84.6 902.5 2.2 545.5 14.6 6.2
Vitamin content in dried OCF powder Content (mg/100 g) A B1 B2 B6 C D 0.0478 0.7421 2.5076 2.0200
Crude fat content of dried OCF was 9.02 g/100 g. Table 2 shows the composition of fatty acids in dried samples of OCF. Although its lipid content was lower than that of livestock products such as beef, pork, and chicken, OCF was
rich in unsaturated fatty acids (7174%) than the saturated fatty acids (2629%). The ability to supply more unsaturated fatty acids and less saturated fatty acids in any dietary preparation makes grasshoppers superior to the livestock products. The content of a-Linolenic acid (C18:3), an omega-3 fatty acid with an 18-carbon chain and three cis double bonds, accounted for over 50% of unsaturated fatty acids and that of a-Linolenic acid (C18:2) made up 23% of unsaturated fatty acids. These linolenic acids are essential fatty acids, categorized as Vitamin F. The mineral contents of dried samples of OCF dried powder are as presented in Table 3. Results indicated that the grasshopper was rich in calcium (2530 %) and iron (23%).
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Since deciencies in calcium and iron are not uncommon in humans, grasshoppers could be a good preventive dietary measure for such deciencies in addition to phosphorus and vitamins (Table 4) (Fontaneto et al. 2011). Table 5 presents the heavy metal content of OCF. Although there are no established regulatory limits for toxic heavy metal contents of grasshopper, their intake is still much lower than that of livestock products and, therefore, a less strict standard is expected to be applied to grasshoppers. As shown in Table 6, its heavy metal content is not high enough to be problematic but a clean breeding environment is considered necessary. Similarly, the contents of 102 kinds of residual pesticides investigated were undetectable; grasshoppers are, therefore, regarded to have no relevant safety problem.
Acknowledgements
This research was supported by the Hampyeong County.
References
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