Nutritional and Functional Properties of Defatted Flour, Protein Concentrates, and Isolates of Brachytrupes membranaceus (Orthoptera: Gryllidae) (Drury: 1773) and Macrotermes subhyalinus (Isoptera: Blattodea) (Rambur: 1842) from Burkina Faso

Simple Summary Edible insects are a source of nutrients for local populations. The present study evaluated proximal composition, fatty acid, and mineral profiles of Brachytrupes membranaceus and Macrotermes subhyalinus from Burkina Faso. The digestibility and functional properties of their defatted flours, protein concentrates, and isolates were also determined. Brachytrupes membranaceus protein concentrates and isolates showed the best nutritional values. They were more digestible and endowed with functional properties. The results revealed that defatted flours, concentrates, and isolates of proteins of Brachytrupes membranaceus and Macrotermes subhyalinus were alternative sources of minerals, proteins, essential amino acids, and essential fatty acids. They could, therefore, be used to combat protein, iron, and zinc deficiencies and for the bio-fortification of foods. Abstract Brachytrupes membranaceus and Macrotermes subhyalinus are edible insects in Burkina Faso. Our research aimed to evaluate the nutritional composition and functional properties of the defatted flours, protein concentrates, and isolates of Brachytrupes membranaceus and Macrotermes subhyalinus. Proximate and mineral composition were determined according to AOAC methods. The amino acid and fatty acid composition were determined by high-performance liquid chromatography (HPLC) and gas chromatography, respectively. The protein concentrates and isolates were obtained by solubilization, precipitation, and lyophilization. Macrotermes subhyalinus showed the highest protein (45.75 g/100 g), iron (11.76 mg/100 g), and zinc (13.18 mg/100 g) contents. The highest isoleucine and lysine contents, the best fat absorption (10.87 g/g), and foaming capacities (49.60%) were obtained with the isolate of Brachytrupes membranaceus. Consumption of Macrotermes subhyalinus could be used to fight or correct iron and zinc deficiencies. Macrotermes subhyalinus was a source of macronutrients and micronutrients, while the protein concentrates and isolates of Brachytrupes membranaceus were endowed with functional properties (fat absorption and foaming capacities).


Introduction
Protein concentrates and isolates are widely used in the food industry. Protein isolates are the most refined form of protein products, containing the highest concentration of protein. Unlike protein powders and concentrates, they do not contain dietary fiber [1]. Protein isolates are important sources of high lysine protein. They are very digestible, endowed with functional properties, and are ideal ingredients for the formulation of food products [1,2]. The main sources for protein concentrate and isolate extractions are collected in their natural habitat. The collected insects were immediately placed in coolers containing ice and transported to the laboratory. The samples were washed thoroughly in distilled water, oven-dried (50 • C), and ground to a powder for further analysis.

Preparation of Protein Concentrate and Isolates
Wolf's method with minor modifications was used to extract protein concentrates and isolates [22]. Before the extraction, the flour of the insect was defatted and stirred for 1 to 2 h at room temperature. Protein concentrates and isolates were extracted by centrifugation (10,000× g for 30 min at 4 • C) from the supernatant and pellet in acidic (pH 4.5) and alkaline (pH 11) solutions, respectively. Both protein concentrates and isolates were then washed with de-ionized water, re-dissolved in de-ionized water, neutralized to pH 7.0 with 1N NaOH at room temperature, and then freeze-dried.

Proximate and Mineral Compositions
Determination of moisture, fat, protein, and ash contents was carried out according to AOAC methods 950. 46, 960.39, 979.09, and 920.153, respectively [23]. Energy value was obtained according to the method described by Merrill and Watt [24]. The Ca, Na, K, Mg, Zn, and Fe contents were determined according to the AOAC method 999.11 [23] using an atomic spectrophotometer (Varian AA240 FS, Varian Inc., Palo Alto, CA, USA). Before the determination of mineral concentrations, the samples were incinerated at 550 • C and acid digested.

Fatty Acid Composition
The fatty acid composition was determined following the method described by IUPAC [25]. Fatty acid methyl esters were prepared following the Khan method [26]. Fatty acids, in the form of their methyl esters, were analyzed on a capillary column (60 m ID: 0.25 mm, film: 0.25 µm, J&W Scientific Co., Folsom, CA, USA) by gas chromatography (Agilent Technologies 6890N, Agilent Technologies, Inc., Santa Clara, CA, USA). The identification of the representative peaks of the fatty acid methyl esters was carried out, using reference fatty acid methyl esters, by comparing the retention distances of each peak in the chromatogram with those obtained by the standards.

Amino Acid Composition of Defatting Flour, Protein Concentrates, and Isolates
The amino acid composition of defatted flour, protein concentrates, and isolates was determined by reverse-phase HPLC using the Pico Tag method, as described by Bidlingmeyer et al. [27]. The samples were defatted with n-hexane (Sigma Aldrich, Saint Louis, MO, USA) and hydrolyzed with 6N hydrochloric acid ((Carlo Erba, Val de Reuil, France), 37%), for 24 h at 110 • C, then filtered and derivatized with phenylisothiocyanate. The amino acid derivatives were separated by HPLC and detected by a UV detector at 254 nm after elution through a Pico Tag precolumn (Nova-Pak C18 Guard Column, 60Å, 4 µm, 3.9 mm × 20 mm, Waters Corp., Milford, MA, USA) and column (C18 PICO'TAG Column Waters (3.9 × 150 mm)), according to the conditions described by Bidlingmeyer et al. [27].

Protein Digestibility
The protein digestibility was assessed according to the methods described by Hsu et al. [28] and Satterlee et al. [29]. Ten milliliters of an aqueous protein suspension (1 mg per mL distilled water) were equilibrated at 37 • C to pH 8.0. One milliliter of three-enzyme solution (1.61 mg trypsin (Sigma aldric, Saint Louis, MO, USA), 3.96 mg chymotrypsin (MP Biomedicals LLC, Illkirch, France), and 2.36 mg peptidase (Megazyme, Bray, Ireland) per mL) was added to the protein suspension, and after exactly 10 min of incubation, the pH was recorded. The percent of protein digestibility (Y) was obtained from the following equation: digestibility = 74.33 + 53.21X X is the volume of NaOH (ml) poured at T = 10 min to maintain the pH at 8.0.

Water Absorption Capacity
The sample's water absorption capacity (WAC) was determined using the method outlined by Diniz and Martin [30]. Briefly, 0.5 g of dried sample was rehydrated with 20 mL of distilled water, stirred with a vortex mixer for 30 s, then centrifuged at 8000× g for 15 min. The difference between the final and initial weight of the protein sample was determined and the results were reported as g of water absorbed per g of protein sample.

Fat Absorption Capacity
The method of Haque and Mozaffar [31] was used to determine fat absorption capacity (FAC). Briefly, 0.5 g of dry sample was dispersed in 10 mL of vegetal oil, vortexed for 15 s, then centrifuged at 8000× g for 15 to decant the free oil. The fat absorption capacity was expressed as g of fat adsorbed per g of protein sample.

Foaming Capacity
Foaming capacity (FC) was determined according to the method of Guo et al. [32]. First, 20 mL of 1.0% protein sample solution was homogenized at 16,000 rpm for 2 min. FC was calculated from the following equation: V is the volume before whipping (mL); V0 is the volume after whipping (mL).

Statistical Analysis
Analytical determinations were performed in triplicate. The values of the different parameters have been expressed as mean and standard deviation (SD). Significant differences between the mean values (p < 0.05) were determined using ANOVA using XLSTAT software for Windows (XLSTAT 2016.02.27444).

Proximate Composition of Brachytrupes membranaceus and Macrotermes subhyalinus
Proximate composition, expressed on a dry matter basis, of Brachytrupes membranaceus and Macrotermes subhyalinus is shown in Table 1. The protein content of Brachytrupes membranaceus (26.44%) was two-fold lower than that of Macrotermes subhyalinus (45.75%). Both species exhibited similar lipid (49.56-50.12%) contents. Brachytrupes membranaceus showed higher carbohydrate and ash contents, while Macrotermes subhyalinus had a higher energy value. The protein and fat contents of Brachytrupes membranaceus were lower than those previously reported by Agbidye et al. [33]. The ash, protein, and lipid contents of Macrotermes subhyalinus were higher and lower than those reported for Macrotermes nigeriensis [34] and cricket [35], respectively.

Mineral Composition of Brachytrupes membranaceus and Macrotermes subhyalinus
The mineral compositions of Brachytrupes membranaceus and Macrotermes subhyalinus are reported in Table 2. The highest levels of iron, zinc, potassium, and sodium were obtained with Macrotermes subhyalinus. The calcium and magnesium contents of Brachytrupes membranaceus were 2.5 and 1.5-fold higher than those of Macrotermes subhyalinus, respectively. The zinc and calcium contents of Brachytrupes membranaceus were higher than those of Brachytrupes orientalis, while the iron, magnesium, potassium, and sodium contents were lower than those of the same species [36]. Shah and Wanapat [35] reported high levels of iron and zinc in crickets (11.6 and 21.5 mg/100 g, respectively). Akullo et al. [37] reported higher iron, potassium, sodium, and zinc levels in Macrotermes bellicosus. The iron and zinc contents of Macrotermes subhyalinus can cover the recommended daily intake for adults [38]. Both Brachytrupes membranaceus and Macrotermes subhyalinus could fight or correct iron and zinc deficiencies. The low sodium contents of Brachytrupes membranaceus and Macrotermes subhyalinus allow their use in low salt diets [16].

Cholesterol Content and Fatty Acid Profile of Brachytrupes membranaceus and Macrotermes subhyalinus
The cholesterol content was 0.98 and 1.47 g/100 g of oil for Macrotermes subhyalinus and Brachytrupes membranaceus, respectively ( Table 3). The cholesterol content of Macrotermes subhyalinus was higher than that of Macrotermes bellicosus [39].

Proteins Contents of Protein Concentrates and Isolates
Protein contents of the protein isolate ranged from 88.68 to 89.32%, while the protein concentrates varied from 50.96 to 63.82% (Table 4). Both the protein concentrates and isolates of Brachytrupes membranaceus had 1.92 and 3.37-fold higher protein contents than those of the defatted flour. A similar increase in protein contents of the protein concentrates and isolates has been reported for Schistocerca gregaria and Apis mellifera isolates [42]. The difference in the protein content increase in the isolates and concentrates compared to the defatted flour of Brachytrupes membranaceus and Macrotermes subhyalinus could be due to diverging extraction methods. Indeed, the alkaline extraction-isoelectric precipitation method improves the protein content [4]. The production of protein concentrates and isolates consists of aqueous solubilization of proteins and carbohydrates at neutral or alkaline pH and the selective recovery of the solubilized protein. Factors such as pH, presence (absence) of salts and their concentration, ionic strength of the medium, net charge, and electrostatic repulsions influence proteins' yield and extraction properties [43].

Amino Acid Composition of Brachytrupes membranaceus and Macrotermes subhyalinus
The amino acid composition of Brachytrupes membranaceus and Macrotermes subhyalinus is shown in Tables 5 and 6, respectively. Both species contained all essential amino acids. Lysine (9.18-13.91 g/100 g protein), isoleucine (8.31-9.54 g/100 g protein), phenylalanine + tyrosine (6.82-8.43 g/100 g protein) and threonine (5.50-6.23 g/100 g protein) were the most abundant essential amino acids in defatted flour, protein concentrates and isolates of Brachytrupes membranaceus. Threonine (9.90-10.47 g/100 g protein), leucine (8.43-9.44 g/100 g protein), and phenylalanine + tyrosine (6.22-8.92 g/100 g protein) were the predominant essential amino acids in defatted flour, protein concentrates and isolates of Macrotermes subhyalinus. Séré et al. [44] reported a similar increase in leucine, isoleucine, and lysine content in the protein isolates of Carbula marginella and Cirina butyrospermi. Leucine and valine were limiting amino acids in defatted flour, protein concentrates, and isolates of Brachytrupes membranaceus, while lysine was a limiting amino acid in defatted flour of Macrotermes subhyalinus [45]. Essential amino acid contents of Macrotermes subhyalinus were higher than those of Macrotermes bellicosus and Macrotermes nigierensis [46]. Interestingly, protein concentrates and isolates from Brachytrupes membranaceus and Macrotermes subhyalinus had higher lysine levels than those recommended by FAO for the daily requirement of adults [45]. Lysine levels are generally low in most cereal proteins, which are staple foods in developing countries. It is also the limiting amino acid in most diets [47]. The high lysine values in the concentrates and isolates of Brachytrupes membranaceus and Macrotermes subhyalinus suggest that they can be used as dietary supplements.

Protein Digestibility
Protein digestibility ranged from 79.92% to 82.57%; from 82.31 to 83.37% and from 84.17 to 85.24% for the defatted flour, protein concentrates, and isolates, respectively. The protein isolates of both Brachytrupes membranaceus and Macrotermes subhyalinus exhibited the highest protein digestibility (Table 7). Oibiokpa et al. [46] reported similar digestibility for Gryllus assimilis, Cirina forda, Melanoplus foedus and Macrotermes nigeriensis. The high digestibility associated with the isolate fractions could be explained by the reduction in the proteolytic enzyme inhibitor during the extraction process.

Water Absorption Capacity of Brachytrupes membranaceus and Macrotermes subhyalinus
The water absorption capacity of Brachytrupes membranaceus and Macrotermes subhyalinus varied from 3.68% to 1.72% for defatted flour, from 4.68% to 4.11% for protein concentrates, and from 4.51% to 3.43% for protein isolates, respectively (Tables 8 and 9). Both protein concentrates and isolates of Brachytrupes membranaceus had the highest water absorption capacity. The highest protein contents of both protein concentrates and isolates of Brachytrupes membranaceus could be due to the higher protein content of Brachytrupes membranaceus than Macrotermes subhyalinus. The water absorption capacity of defatted flour of Brachytrupes membranaceus was higher than that of the whole insect of Acheta domesticus (2.03 g/g) [48]. The water absorption capacity of the protein isolates of Brachytrupes membranaceus was higher than that obtained with the isolates of Schistocerca gregaria and Gryllodes sigillatus [21]. The highest water absorption capacities were obtained for protein concentrates of Brachytrupes membranaceus and Macrotermes subhyalinus. This could be because concentrates include carbohydrates that absorb water too [4]. The differences in water absorption obtained in the present study could be related also to the content of hydrophilic amino acids, the presence of non-protein components, and the type, quality, and conformation of the proteins [49,50].  12.20 ± 0.28 a 11.80 ± 0.28 b 3.60 ± 0.56 a a,b,c Means in the same row with different superscripts are significantly different (p < 0.05).

Fat Absorption Capacity of Brachytrupes membranaceus and Macrotermes subhyalinus
The values of fat absorption capacity of the defatted flour, protein concentrates and isolates varied from 2.03% to 10.87% (Tables 8 and 9). Protein isolates of Brachytrupes membranaceus had the highest fat absorption capacity (10.87%), while the defatting flour of Macrotermes subhyalinus had the lowest one (2.03%). The fat absorption capacity of the defatting flour of Brachytrupes membranaceus was higher than that of Gryllodes sigillatus (2.82 g/g) and Schistocerca gregaria (Zielińska et al. [21]), but similar to that of Acheta domesticus [48]. Torruco-Uco et al. [51] reported values lower than that of Brachytrupes membranaceus with Sphenarium purpurascens (2.79 g/g at 60-70 • C). The fat absorption capacity of the protein isolate of Brachytrupes membranaceus was higher than that obtained with the isolates of Gryllodes sigillatus and Schistocerca gregaria [21].
The low-fat absorption capacity of the isolate of Macrotermes subhyalinus could be due to its low hydrophobic amino acid content (25.77 mg/100 g protein) compared to the protein isolate of Brachytrupes membranaceus, which had a high hydrophobic amino acid content (33.59 mg/100 g protein). Although the protein isolate of Brachytrupes membranaceus had the highest fat absorption capacity, it had a low hydrophobic amino acid content (33.59 mg/100 g protein) compared to that of the concentrates of Brachytrupes membranaceus (40.34 mg/100 g protein). This could be explained by the location of hydrophobic amino acid residues on the protein surface of the protein isolate of Brachytrupes membranaceus [52]. The fat absorption capacity is the ability of proteins to physically bind to fat through capillary attraction. It is due to the presence of electrostatic interactions, hydrophobic forces, and hydrogen bonds, which are the forces involved in lipid-protein interactions [53]. Knowledge of oil absorption capacity is important in food technology, as it imparts certain characteristics to the product, such as flavor retention palatability enhancement, and an increase in shelf life by reducing humidity and fat loss [54].

Foaming Capacity of Brachytrupes membranaceus and Macrotermes subhyalinus
The foaming capacity ranged from 12.2% to 30%, from 11.8% to 39.4%, and from 3.6% to 49.6% for defatted flour, and protein isolates and concentrates, respectively (Tables 8  and 9). The foaming capacity of the protein isolates of Brachytrupes membranaceus was 13.77-fold higher than that of protein isolates of Macrotermes subhyalinus. The foaming capacity of defatted flour of Brachytrupes membranaceus was higher than that reported by Zielińska et al. [21] with whole insects of Schistocerca gregaria (22.33%). The foaming capacity of the Brachytrupes membranaceus protein isolates was higher and lower than that of Schistocerca gregaria (32.00%) and protein Gryllodes sigillatus isolates (99.00%), respectively [21]. Although the defatted flour and the protein concentrates of Macrotermes subhyalinus had high protein contents compared to those of Brachytrupes membranaceus, these proteins were not endowed with functional properties. This could be explained by the fact that the protein concentrates and isolates contained higher levels of hydrophobic amino acids (40.34 and 33.59 mg/100 g protein, respectively). Foaming capacity depends on proteins and other components, such as carbohydrates, the location of amino acid residues on the surface of the protein, and surface hydrophobicity [55].

Conclusions
The present study determined the nutritional and functional properties of defatted flours, concentrates, and protein isolates of Brachytrupes membranaceus and Macrotermes subhyalinus.
Macrotermes subhyalinus was a rich source of proteins, lipids, iron, and zinc. Defatted flours, protein concentrates, and protein isolates of Brachytrupes membranaceus and Macrotermes subhyalinus were sources of essential amino acids. Protein concentrates and isolates of Brachytrupes membranaceus have high fat absorption and foaming capacities. Brachytrupes membranaceus and Macrotermes subhyalinus can be recommended as nutritional and functional supplements. Tasting tests could be set up to assess the flavor and acceptability of protein concentrates and isolates of edible insects.