Chemical composition and amino acids content of five species of edible Grasshoppers from Mexico

S H O R T C O M M U N I C A T I O N *Corresponding author: Melo Ruiz Virginia, Dr. in Chemistry. Underutilized Food Focus in Insects. E-mail: vmelo@correo.xoc.uam.mx Received: 11 February 2015; Revised: 08 April 2015; Accepted: 14 April 2015; Published Online: 14 April 2015 Melo-Ruiz, et al.: Protein and amino acids content of Grasshoppers Emir. J. Food Agric ● Vol 27 ● Issue 8 ● 2015 655 demand in rural communities and urban cities of Mexico, and other countries in the World. MATERIALS AND METHODS Sample collection and preparation Convenience sampling of wild insects was performed in several states of Mexico, in different agroclimatic environments, and only one specie was gather in each state, even though there were more than one in the same area (Greenfield and Southgate, 2003). Collection was carried out between 7 and 9 am throughout 2012. Sphenarium histrio S, adults were manually and net collected in Mexico City, at the Xochimilco demarcation, in a grassy zone in early May. Sphenarium purpurascens C adults were harvested late June in Oaxaca, at grassland. Melanoplus femurrubrum D adults were obtained nearby an alfalfa field early August, at Huejotzingo Puebla. Taeniopoda equse B adults were gathered nearby a corn field, in early October, in Atlixco, Puebla. Schistocerca spp insects were obtained in a xerophyte thicket, late June at Actopan, Hidalgo. Insects were washed under running tap water and rinsed with distilled water and sun dried to be transported in glass containers to the University for Taxonomic Identification (Cibrián et al., 1995; Anaya Rosales et al., 2000; Morón and Terrón, 2011). Legs and wings were removed; individuals were homogenized by weight each one to 0.5 g for all samples and 0.7 g for Schistocerca spp which adult stage is much bigger, and further proceed with chemical analysis. Determination of moisture content Moisture content of samples was determined using the direct drying method. Homogenized sample by weight of each organism, (10 g) was dried in an oven at 60°C for 24 hrs. The samples were powdered in a mortar then passed through a 60 mesh size. The obtained fine powder was used for further analysis. Determination of ash Ash was obtained by incinerating at 650°C in a muffle furnace for 6 h to a constant weight, to eliminate organic matter. Determination of lipid content Lipid content determination was performed by the semi continuous solvent extraction method (AOAC Method 934.01) as follows: ten grams of the sample were extracted with 180 ml petroleum ether on a Soxhlet apparatus for 10 h. Petroleum ether was removed by evaporation and the lipid residue was weighed. All samples were analyzed by triplicate and the results are expressed as g/100g dry basis of sample. Determination of protein content Protein content of sample was determined according to the principle of the Kjeldahl method (AOAC Method 945.01). Sample (1 g) was digested with 15 ml concentrated sulphuric acid, using an electrically heated aluminum block digester. The resulting digest was diluted and then made alkaline with 50 ml 40% sodium hydroxide. This was followed by rapid steam distillation of ammonia from the diluted digest into 25 ml 4% boric acid for manual titration with 0.2N hydrochloric acid. A conversion factor of 6.25 was used to convert the measured nitrogen content to protein content. All samples were analyzed in triplicate and the results are expressed as g/100 g dry basis of sample (Osborne, 1985; Pearson, 1989). Determination of total available carbohydrate content The total available carbohydrate content of sample was determined by the Clegg-anthrone method (Peris-Torjada, 2004). Sample (1 g) was digested with 13 ml 52% perchloric acid to hydrolyze disaccharides, trisaccharides and higher oligomers to their component reducing sugars and reacted with anthrone reagent under acid condition to produce a blue/green color. Anthrone reagent was prepared by dissolving 0.1% (w/v) anthrone in diluted sulphuric acid (sulphuric acid: water in ratio of 2.3:1.0, v/v). An aliquot (1 ml) of diluted hydrolysate was mixed with 5 ml anthrone reagent. Absorbance of the reaction mixture was measured at 630 nm against a blank after incubated in boiling water for 12 min and cooled. All samples were analyzed in triplicate. Glucose (0-100 mg/L) was used to construct a standard curve for quantification and the results are expressed as g/100 g dry basis of sample. Determination of amino acids The Grasshoppers samples were dried and grind to powder, in an oven and degreased with petroleum ether. The samples for amino acid analyses were hydrolyzed in constant with 6 N HCl for 24 hr. at 110°C (Gehrke et al., 1987), evaporated under vacuum stream, the sample is then resuspended in the NA-S buffer and injected to the system. For the analysis of methionine and cysteine samples were oxidized by performic acid before hydrolysis (Moore, 1963). Amino acids were determined by HPLC technique, using an automatic auto analyzer Beckman System Gold model 6300 that includes a solvent module 126AA, post column reactor 232, detector 166, autosampler 507, a column of cation exchange resin (3x250 mm spherogel AA lithium column (sulfonated polystyrene-divinylbenzene copolymers)), and a computer work station, diluted with a gradient of buffers with a concentration of 0.2 M to 2 M of citrate and pH 3.1 to 5.6 (Beckman, 1985). Tryptophan was measured according to a colorimetric method after enzymatic hydrolysis by Pronase (Spies and Chambers, 1949; Holz, 1972). Duplicated analyses were carried out for each sample. Melo-Ruiz, et al.: Protein and amino acids content of Grasshoppers 656 Emir. J. Food Agric ● Vol 27 ● Issue 8 ● 2015 Statistical analysis Three independent analyses were done for each determination and each grasshopper, only in the determination of amino acids the analyses were done in two samples. The results are expressed as the average. RESULTS AND DISCUSSION Season for insect availability is shown in Table 1. Nomenclature and name of insects studied Table 2. Moisture and macronutrients composition Convenience manual sampling was performed in some Mexican states with different agro climatic conditions: in Xochimilco, in grassy land, in spring, with seldom rains; in Oaxaca, in grassland, during early summer, with regular heavy rains; in Puebla, some were harvested in alfalfa fields during summer with heavy rain and other species in a corn field, early autumn without rains; in Hidalgo, in an arid zone with xerophyte thicket, early summer but with exceptional heavy rain. Insects get water and food from surrounding environments, therefore characteristics of the environment might slightly change their water and macronutrient content. Almost one third part of the insect is water (Table 3). Moisture in grasshoppers ranks from 35.29% to 43.19% and dry samples from 56.81% to 64.71%. Data correspond to: Sphenarium histrio S: 37.02% and 62.96%; Sphenarium purpurascens Ch: 41.44% and 58.56%; Taeniopoda eques B: 35.29% and 64.71%; Melanoplus femurrubrum D: 39.82% and 60.18%; Schistocerca spp: 43.19% and 56.81% of water and dry matter respectively. Sphenarium purpurascens Ch, Melanoplus femurrubrum D and Schistocerca spp present the higher content of water because they were collected in rainy season and Sphenarium histrio S and Taeniopoda eques B were the lower in water because capture were on dry season and insects get water from environment. Proteins are one of the five classes of biomolecules found in cells and tissues; the others are polysaccharides, lipids, and some minerals (Brosnan and Young 2003). Macronutrient data for each species raw analyzed on dry basis was: proteins from 62.5% to 77.25% of the total weight. The determined protein content of grasshoppers was very high, from 62.5% to 77.25%, however the excess is not toxic for once the metabolism obtains the required proteins, the amino group of the amino acids is removed, the remaining part can provide energy; total lipids in dry samples were low in four species from 4.71% to 6.2%, except Shistocerca spp that content 16.0%; minerals ranked from 2.05% to 4.5% (minerals were not analyze individually); crude fiber 7.08% to 11.15%; and soluble carbohydrates (source of energy) were low but excess of proteins would bring energy to complement requirements 6.66% to 9.59%. Crude Fiber, carbohydrate polymers not hydrolysed by endogenous enzymes, were 4.84% to 9.59% (Table 4). Energetic value was calculated multiplying soluble carbohydrates and proteins by 4 and lipids by 9, to obtain the Kcal amount. There are 20 amino acids that are the basic components of proteins, and which play an important role in human metabolism. From these, 8 are essential for adults and 10 for children; their study is focused in them providing nitrogen to the organism, for proteins are the only source of this energetic income (Duffy et al., 1981). In addition to their role as substrates for the polypeptide chain formation, amino acids serve multiple and diverse Table 1: Grasshopper’s availability through the year Species/month J F M A M J J A S O N D Sphenarium histrio S x x x x x X X X X X x x Sphenarium purpurascens Ch x x x X X X X X Taeniopoda eques B X X X X X X Melanoplus femurrubrum D x x X X X X X Schistocerca spp. x x x X X X X X: Abundant, x: low, Seasonal availability depends on the biotic and abiotic conditions Table 2: Nomenclature of sample species (Morón and Terrón, 2011) Order Orthoptera Orthoptera Orthoptera Orthoptera Orthoptera Family Acrididae Acrididae Acrididae Acrididae Acrididae Genus Sphenarium Sphenarium Taeniopoda Melanoplus Schistocerca Specie histrio S purpurascens Ch eques B femurrubrum D spp Common name Grasshopper Grasshopper Grasshopper Grasshopper Grasshopper Table 3: Moisture determination of 5 Grasshopper species (%) Species Sphenarium histrio S Sphenarium Purpurascens Ch Taeniopoda Eques B Melanoplus femurrubrum D Schistocerca spp Moisture 37.04 41.44 35.29 39.82 43.19 Dry sample 

demand in rural communities and urban cities of Mexico, and other countries in the World.

Sample collection and preparation
Convenience sampling of wild insects was performed in several states of Mexico, in different agroclimatic environments, and only one specie was gather in each state, even though there were more than one in the same area (Greenfield and Southgate, 2003).Collection was carried out between 7 and 9 am throughout 2012.Sphenarium histrio S, adults were manually and net collected in Mexico City, at the Xochimilco demarcation, in a grassy zone in early May.Sphenarium purpurascens C adults were harvested late June in Oaxaca, at grassland.Melanoplus femurrubrum D adults were obtained nearby an alfalfa field early August, at Huejotzingo Puebla.Taeniopoda equse B adults were gathered nearby a corn field, in early October, in Atlixco, Puebla.Schistocerca spp insects were obtained in a xerophyte thicket, late June at Actopan, Hidalgo.Insects were washed under running tap water and rinsed with distilled water and sun dried to be transported in glass containers to the University for Taxonomic Identification (Cibrián et al., 1995;Anaya Rosales et al., 2000;Morón and Terrón, 2011).Legs and wings were removed; individuals were homogenized by weight each one to 0.5 g for all samples and 0.7 g for Schistocerca spp which adult stage is much bigger, and further proceed with chemical analysis.

Determination of moisture content
Moisture content of samples was determined using the direct drying method.Homogenized sample by weight of each organism, (10 g) was dried in an oven at 60°C for 24 hrs.The samples were powdered in a mortar then passed through a 60 mesh size.The obtained fine powder was used for further analysis.

Determination of ash
Ash was obtained by incinerating at 650°C in a muffle furnace for 6 h to a constant weight, to eliminate organic matter.

Determination of lipid content
Lipid content determination was performed by the semi continuous solvent extraction method (AOAC Method 934.01) as follows: ten grams of the sample were extracted with 180 ml petroleum ether on a Soxhlet apparatus for 10 h.Petroleum ether was removed by evaporation and the lipid residue was weighed.All samples were analyzed by triplicate and the results are expressed as g/100g dry basis of sample.

Determination of protein content
Protein content of sample was determined according to the principle of the Kjeldahl method (AOAC Method 945.01).Sample (1 g) was digested with 15 ml concentrated sulphuric acid, using an electrically heated aluminum block digester.The resulting digest was diluted and then made alkaline with 50 ml 40% sodium hydroxide.This was followed by rapid steam distillation of ammonia from the diluted digest into 25 ml 4% boric acid for manual titration with 0.2N hydrochloric acid.A conversion factor of 6.25 was used to convert the measured nitrogen content to protein content.All samples were analyzed in triplicate and the results are expressed as g/100 g dry basis of sample (Osborne, 1985;Pearson, 1989).

Determination of total available carbohydrate content
The total available carbohydrate content of sample was determined by the Clegg-anthrone method (Peris-Torjada, 2004).Sample (1 g) was digested with 13 ml 52% perchloric acid to hydrolyze disaccharides, trisaccharides and higher oligomers to their component reducing sugars and reacted with anthrone reagent under acid condition to produce a blue/green color.Anthrone reagent was prepared by dissolving 0.1% (w/v) anthrone in diluted sulphuric acid (sulphuric acid: water in ratio of 2.3:1.0,v/v).An aliquot (1 ml) of diluted hydrolysate was mixed with 5 ml anthrone reagent.Absorbance of the reaction mixture was measured at 630 nm against a blank after incubated in boiling water for 12 min and cooled.All samples were analyzed in triplicate.Glucose (0-100 mg/L) was used to construct a standard curve for quantification and the results are expressed as g/100 g dry basis of sample.

Determination of amino acids
The Grasshoppers samples were dried and grind to powder, in an oven and degreased with petroleum ether.The samples for amino acid analyses were hydrolyzed in constant with 6 N HCl for 24 hr.at 110°C (Gehrke et al., 1987), evaporated under vacuum stream, the sample is then resuspended in the NA-S buffer and injected to the system.For the analysis of methionine and cysteine samples were oxidized by performic acid before hydrolysis (Moore, 1963).Amino acids were determined by HPLC technique, using an automatic auto analyzer Beckman System Gold model 6300 that includes a solvent module 126AA, post column reactor 232, detector 166, autosampler 507, a column of cation exchange resin (3x250 mm spherogel AA lithium column (sulfonated polystyrene-divinylbenzene copolymers)), and a computer work station, diluted with a gradient of buffers with a concentration of 0.2 M to 2 M of citrate and pH 3.1 to 5.6 (Beckman, 1985).Tryptophan was measured according to a colorimetric method after enzymatic hydrolysis by Pronase (Spies and Chambers, 1949;Holz, 1972).Duplicated analyses were carried out for each sample.

Statistical analysis
Three independent analyses were done for each determination and each grasshopper, only in the determination of amino acids the analyses were done in two samples.The results are expressed as the average.

RESULTS AND DISCUSSION
Season for insect availability is shown in Table 1.Nomenclature and name of insects studied Table 2.

Moisture and macronutrients composition
Convenience manual sampling was performed in some Mexican states with different agro climatic conditions: in Xochimilco, in grassy land, in spring, with seldom rains; in Oaxaca, in grassland, during early summer, with regular heavy rains; in Puebla, some were harvested in alfalfa fields during summer with heavy rain and other species in a corn field, early autumn without rains; in Hidalgo, in an arid zone with xerophyte thicket, early summer but with exceptional heavy rain.Insects get water and food from surrounding environments, therefore characteristics of the environment might slightly change their water and macronutrient content.Almost one third part of the insect is water ( Proteins are one of the five classes of biomolecules found in cells and tissues; the others are polysaccharides, lipids, and some minerals (Brosnan and Young 2003).Macronutrient data for each species raw analyzed on dry basis was: proteins from 62.5% to 77.25% of the total weight.The determined protein content of grasshoppers was very high, from 62.5% to 77.25%, however the excess is not toxic for once the metabolism obtains the required proteins, the amino group of the amino acids is removed, the remaining part can provide energy; total lipids in dry samples were low in four species from 4.71% to 6.2%, except Shistocerca spp that content 16.0%; minerals ranked from 2.05% to 4.5% (minerals were not analyze individually); crude fiber 7.08% to 11.15%; and soluble carbohydrates (source of energy) were low but excess of proteins would bring energy to complement requirements 6.66% to 9.59%.Crude Fiber, carbohydrate polymers not hydrolysed by endogenous enzymes, were 4.84% to 9.59% (Table 4).
Energetic value was calculated multiplying soluble carbohydrates and proteins by 4 and lipids by 9, to obtain the Kcal amount.
There are 20 amino acids that are the basic components of proteins, and which play an important role in human metabolism.From these, 8 are essential for adults and 10 for children; their study is focused in them providing nitrogen to the organism, for proteins are the only source of this energetic income (Duffy et al., 1981).
In addition to their role as substrates for the polypeptide chain formation, amino acids serve multiple and diverse x x x X X X X X: Abundant, x: low, Seasonal availability depends on the biotic and abiotic conditions functions in human health, such as: leucine, regulator of protein turnover, transporter of nitrogen in the brain, and translation regulator; arginine, regulator of enzyme activity and precursor of signal transducer; glutamine, substrate of protein synthesis, for hepatic and renal gluconeogenesis, control for acid-base balance; phenylalanine, regulator for enzyme activity; tryptophan, neurotransmitter; alanine, nitrogen transporter (Duffy et al., 1981;Pencharz, 2012).These are only some of them.
In pleasurable taste sensation "umami taste", glutamine is responsible for enhance the sensory qualities of various foods (Table 5).
The amino acids content calculated as 16 g N reflected that regarding essential amino acids, tryptophan is the limiting one, compared with the standard (FAO/WHO/ UNU 2007) in all species of grasshoppers, sulphur amino acids was also lower in Sphenarium purpurascens Ch and Schistocerca spp and lysine in Melanoplus femrrubrum D, in nonessential amino acids glutamic acid with an important role in nutrition is high in all species but Shpenarium histrio S.

CONCLUSIONS
Protein is the fundamental component for cellular and organ functions, thus its deficiency has adverse effects on human health.By incorporating grasshoppers and other insects to daily diets, most people would contain not only enough protein and amino acids, but also sufficient nonprotein energy to permit the optimal use of dietary protein.
Chemical composition could slightly change, according location, environment conditions and time after insect hatch.Grasshoppers found worldwide, wherever any vegetation grows, range in size, from one to five inches in length, are the most popular edible insect, dating back at least to Old Testament times, since then grasshoppers, raw or in different preparations, as snacks or as a culinary staple are intake, they are affordable to all social groups, therefore, insects may represent a good option to improve food security worldwide.

Author contributions
V. M. R. was involved in overall planning and supervision.H. S. T. made major contributions to the review paper.
Table 3).Schistocerca spp: 43.19% and 56.81% of water and dry matter respectively.Sphenarium purpurascens Ch, Melanoplus femurrubrum D and Schistocerca spp present the higher content of water because they were collected in rainy season and Sphenarium histrio S and Taeniopoda eques B were the lower in water because capture were on dry season and insects get water from environment.

Table 5 . Amino acid composition (g/16g Nitrogen) of the 5 grasshopper species Essential amino acids FAO Pattern 1973 Species/amino acids Sphenarium histrio S Sphenarium purpurascens Ch Taeniopoda eques B Melanoplus femurrubrum D
Asparagine and glutamine can be synthesized from aspartic acid and glutamic acid.Samples were analyzed in duplicate T. Q. B. was involved in overall laboratory work.K. S. H. was involved in overall laboratory work and review paper.R. D. G. was involved in overall laboratory work and review paper.C. C. C. was involved in overall planning, supervision and review paper.