Extraction and physicochemical characterization of Tenebrio molitor proteins

    Extraction and physicochemical characterization of Tenebrio molitor proteins C. Azagoh, F. Ducept, R. Garcia, L. Rakotozafy, M.-E. Cuvelier, S. Keller, R. Lewandowski, S. Mezdour PII: DOI: Reference:

S0963-9969(16)30245-9 doi: 10.1016/j.foodres.2016.06.010 FRIN 6305

To appear in:

Food Research International

Received date: Revised date: Accepted date:

2 January 2016 8 June 2016 15 June 2016

Please cite this article as: Azagoh, C., Ducept, F., Garcia, R., Rakotozafy, L., Cuvelier, M.-E., Keller, S., Lewandowski, R. & Mezdour, S., Extraction and physicochemical characterization of Tenebrio molitor proteins, Food Research International (2016), doi: 10.1016/j.foodres.2016.06.010

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ACCEPTED MANUSCRIPT Extraction and physicochemical characterization of Tenebrio molitor proteins Azagoh C. a, Ducept F. a, Garcia R. ab, Rakotozafy L. ab, Cuvelier M-E. a, Keller S. a , Lewandowski R. a, Mezdour S. a*

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ª UMR Ingénierie Procédés Aliments, AgroParisTech, Inra, Université Paris-Saclay, 91300 Massy, France b Cnam, 75003 Paris, France

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Abstract

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This study focused on the extraction and physicochemical characterization of proteins from larvae and larvae meal of Tenebrio molitor. The larvae were subjected to a protein extraction process which involved a thermo-mechanical pre-treatment to produce the larvae meal. Soluble proteins from larvae and from larvae meal were subsequently extracted by solubilisation at an alkaline pH. The products obtained were then characterized and compared. The larvae and larvae meal were rich in protein (65.6% and 71.6% respectively) and displayed good essential amino acid (EAA) profiles. They contained all EAA and in sufficient quantities to meet the dietary requirements of both humans and salmon, except for a deficiency in methionine. The EAA profile of the larvae meal was also comparable to those of fish and soya meals used for feed. At pH 10 and 45 °C, the protein extraction yield of larvae (59.9%) was two-fold that of larvae meal (26.4%). The soluble proteins had protein contents on dry matter of 84% and 80% from larvae and larvae meal respectively. Molecular weights ranged from ≤14 to 100 kDa but the two soluble proteins differed. The soluble proteins had a solubility which was highly pH-dependent, with a low solubility at pH 3 to 5. Their surface charge depended on both the pH (in particular) and the NaCl concentration. The surface hydrophobicity at pH 7 of soluble proteins from larvae (670.3) was higher than that of soluble proteins from larvae meal (102.5). These soluble proteins lowered the water surface tension to 42 mN/m and 32 mN/m for the soluble proteins from larvae and from larvae meal respectively. Keywords: Insect, proteins, extraction, physicochemical properties, characterization. Chemical compounds used in this work Glycine (PubChem CID: 750); Glycerol (PubChem CID: 753); Tris(hydroxymethyl)aminomethane (PubChem CID: 4468930); Sodium chloride (PubChem CID: 5234); Ethanol (PubChem CID: 702); Monosodium phosphate (PubChem CID: 23672064); Disodium hydrogen phosphate (PubChem CID: 24203); 2-mercaptoethanol (PubChem CID: 1567); Hydrochloric acid (PubChem CID: 313); Bromophenol blue (PubChem CID: 8272); Sodium hydroxide (PubChem CID: 14798); Sodium dodecyl sulphate (PubChem CID: 3423265). *

Corresponding author: [email protected] Tel: 00 33 (0) 1 69 93 51 08 Fax: 00 33 (0) 1 69 93 50 05 AgroParisTech 1, avenue des Olympiades 91300 Massy - FRANCE

ACCEPTED MANUSCRIPT 1. Introduction

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One of the greatest challenges facing the food industry concerns the steady rise in the world’s population and the need to generate sufficient proteins to feed the nine billion people anticipated in 2050. The production of protein-rich materials is increasing but will not be sufficient to meet the needs of both humans and animals; not only that, but at present more than one billion people are suffering from protein deficiency (Ghaly & Alkoaik, 2009). Governments and several international institutions such as the Food and Agriculture Organization of the United Nations (FAO) (van Huis et al., 2013) are promoting the development of the insect industry as a new resource for animal feed and human food. Thanks to their nutritional value, insects have been assessed as an interesting food and feed alternative as legumes (soya) and algae. According to several studies, insects offer an important source of minerals and lipids and above all proteins (Rumpold & Schluter, 2013; Finke, 2012; Durst & Shono, 2010; Ng et al., 2001; Ramos-Elorduy & Pino 1990). They are often consumed because of their high protein content (DeFoliart, 1992) although not often in Europe, and indeed more than 2.5 billion people in about 130 countries consume them (Rumpold & Schlüter, 2013). 80% of edible insects belong to the orders Coleoptera (beetles), Hymenoptera (ants, bees ...), Orthoptera (grasshoppers and locusts) and Lepidoptera (caterpillars), the remaining 20% being Hemiptera (true bugs, aphids), Isoptera (termites), Diptera (flies) and others (Lavalette, 2013). Lepidoptera, Coleoptera, and Diptera are often eaten at the larval stage, while the others are generally consumed at the adult stage (Yi et al., 2013). In light of the above, insects as a protein source are an important issue, but they are required in relatively abundant quantities which mean large-scale rearing. Some research has focused on this area (Li, Zhao, & Liu, 2013; Ammattikorkeakoulu, 2013; Hardouin & Mahoux, 2003; Ekoue & Hadzi, 2000; DeFoliart, 1995; Kok, Lomaliza, & Shivare, 1988; Bondari & Sheppard, 1981). Insects such as Tenebrio molitor, Bombyx mori and Hermetia illucens which are already farmed but at a small scale or not under optimized conditions, have been chosen as good candidates (Van Huis et al., 2013). During the past year, some studies have focused on insect valorisation, using food processing options to produce ingredients. Those processes may be thermo-mechanical (combining heat treatment, grinding, pressing, supercritical CO2, centrifugation, drying) (Xia et al., 2012; Veldkamp et al., 2012; Hu, 2009), use hydrolysis (Luo, 2009) or extraction by solubilisation (Hu, 2009). Other studies on insects as a food source have focused on their nutrient contents, nutritional properties (Makkar et al., 2014; Rumpold & Schlüter, 2013; van Huis et al., 2013; Lavalette, 2013; Barker, Fitzpatrick, & Dierenfeld, 1998) or functional properties (Yi et al., 2013). Nevertheless it is still necessary to obtain a clearer understanding of the physicochemical and functional properties of these proteins. Very little research has addressed the physicochemical properties of insect proteins relative to their use by the food industry, the processing insect proteins and even less the impact of processes on the properties that affect proteins functionalities in food systems during processing, manufacturing, storage and preparation (Kinsella, 1979). Knowledge of these properties is therefore necessary to enable their successful incorporation as ingredients in food systems (Adebiyi & Aluko, 2011).

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The aim of these investigations was to study and compare the physicochemical properties of soluble proteins from Tenebrio molitor larvae and from Tenebrio molitor larvae meal, obtained using a thermo-mechanical process. The composition, molecular weight, solubility, surface charge, surface hydrophobicity and surface tension were thus studied following alkaline extraction.

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2. Materials and methods

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2.1. Chemicals

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The glycine, glycerol, tris hydrochloride (tris-HCl), sodium chloride (NaCl), ethanol, monosodium phosphate, disodium hydrogen phosphate, bovine serum albumin (BSA), and 2mercaptoethanol used during this study, were purchased from Sigma-Aldrich Chemical Co. The markers came from GE Healthcare, while the polyacrylamide gels and Bio-safe coomassie G-250 were obtained from Biorad and the Sodium dodecyl sulphate electrophoresis (SDS) from Acros Organics. Hydrochloric acid was purchased from Carlo Erba Reagents, bromophenol blue from Kuhlmann, and sodium hydroxide (NaOH) from VWR chemicals. All these chemicals were used for protein extraction or characterization procedures.

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2.2. Materials

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Tenebrio molitor in the larval stage were supplied by IPV Food, Besançon, France. During rearing, the larvae had mainly been fed with wheat bran, cereal, premix and NaCl. They were received in a frozen form and were stored at -18 °C until use. Some of the larvae were freeze-dried and ground in a blender. The freeze drying was used to obtain a more stable and easier to use product. The other larvae underwent a protein isolation process which involved a thermo-mechanical method characterized by blanching with water at 90 °C for 10 min. to prevent browning, pressing with a screw press (Sovab, France) to remove part of fat and water, drying at 75 °C for 6 h and grinding in blender to obtain the larvae meal. This process was inspired by that used to produce meal from fish and legumes, obtaining a meal containing less lipids and with better storage. The larvae and larvae meal were defatted using hexane and isopropanol (at a ratio 3/2) in an ASE 200 accelerated solvent extractor (Dionex, United States) to obtain defatted larvae and defatted larvae meal which were used for the protein extraction and to determine the chemical composition. 2.3. Protein extraction process Soluble proteins were extracted from larvae and from larvae meal using solubilisation into water at an alkaline pH. The proteins were extracted under different conditions, according to a modification of the methods described by Batista (1999) and Deak, Murphy, & Johnson (2007). About 5 g of the larvae and larvae meal were weighed accurately into separate 500 mL standard beakers, and 200 mL of distilled water was added. The pH of the mixture was adjusted immediately to pH 10 using an 0.1 or 1 N NaOH solution. The mixture was stirred (magnetic stirrer, 2.5 cm) on a hot plate at a rate designed to prevent the formation of a

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vortex, for 1 h at 45 °C. The pH was monitored intermittently and maintained at 10 throughout the stirring period. After stirring, distilled water at pH 10, and NaOH were added to reach 250 ml of solution. The mixture was centrifuged at 10,000 g for 30 min. The supernatants were freeze-dried before use for the subsequent experiments. The protein content of the soluble protein fractions obtained were analysed using the Kjeldahl method with a protein-to-nitrogen conversion factor of 6.25 (AOAC, 1990). The extraction procedure was performed in triplicate (at least). The yield protein (% g/100 g as is) of solubilisation was calculated as:

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2.4. Determination of fat, water, protein and amino acid contents

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The fat content was determined using hexane and isopropanol (at a ratio of 3/2) in an ASE 200 accelerated solvent extractor (Dionex, United States) and the moisture and crude protein contents using the AOAC technique (1990). The amino acid composition reflects the quality and nutritional value of a protein (Ghaly & Alkoaik, 2009). The amino acid profile was determined using ion exchange chromatography, according to ISO international standard 13903:2005. Tryptophan was determined using reversed phase C18 HPLC with fluorescence detection at 280 nm, according to the procedure described by ISO international standard 13904:2005. The amino acid profiles of larvae meal and larvae powder were compared with data in the literature on fish and soya meal proteins used in animal feed and the requirements of humans and salmon. 2.5. Determination of molecular weight

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The soluble proteins from larvae and from larvae meal were analysed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the molecular weight (MW) distribution of the proteins. This was performed using a mini protean tetra electrophoresis cell system (Biorad, United States) and 4-15% polyacrylamide gel. The samples were dissolved in a Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 5% 2mercaptoethanol, 2% SDS, 0.01% bromophenol blue and 40% glycerol). After 2 min. of agitation and 10 min. in an ultrasonic bath, the samples were heated at 90 °C for 10 min. and centrifuged for 2 min. at 10,000 g. 10 µl of the solution was applied to the gel and the electrophoresis was run at 150 V. The gels were stained with Bio-safe Coomassie G250. 2.6. Assessment of pH-dependent soluble protein solubility Studying the protein solubility is useful when designing methods to extract proteins and evaluate their functional properties. The solubility of soluble proteins from larvae and from larvae meal was determined by making a slight modification of the method described by Morr (1985). Protein solutions at 0.1% (w/v) of the soluble proteins from larvae and from larvae meal were used to study solubility as a function of pH (2-10) at 25 °C. The mixture was stirred for 1 h (magnetic stirrer, 1 cm) on a plate at a rate designed to prevent the formation of a vortex. The pH was verified as being the same after stirring and the mixture was centrifuged

ACCEPTED MANUSCRIPT at 10,000 g for 2 min. The protein content was also determined in the supernatant using the Lowry method, with BSA as the standard protein (Lowry et al., 1951). All experiments were performed in at least duplicate for each sample.

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2.7. Measurement of zeta potential

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Electrostatic interactions may be involved in protein adsorption phenomena as well as van der Waals, hydrophobic, and steric interactions (Salgin, Salgin, & Bahadir, 2012). The Zeta potential was measured in order to evaluate the charge accumulated at the surface of soluble proteins, using a Zetasizer Nano system (Malvern Instruments, United Kingdom). This provides information on the electrostatic repulsion of charged particles which modifies the distribution of ions in their environment (Malhotra & Coupland, 2004). Soluble proteins from larvae and from larvae meal were dispersed in 0.01 M phosphate buffer (pH 7, 25 °C) at different NaCl concentrations (0.1, 0.2, 0.3, 0.5, and 1 M), or in water adjusted to different pH (2, 3, 3.5, 4, 4.5, 7, and 10) with 0.01 or 0.1 HCl or NaOH solutions, and then agitated for 1 h. The protein concentration of the dispersions was 0.1% (w/v). Measurements of zeta potential were performed at 25 °C in folded capillary cells (Malvern instruments, United Kingdom) without the presence of air. The refractive index was 1.450 and the absorption of the proteins was considered to be zero. The dispersant was water with a viscosity of 0.887 mPa.s, the refractive index 1.330. Each measurement was the average of six determinations and the entire experiment was performed in triplicate. 2.8. Measurement of surface hydrophobicity

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Surface hydrophobicity is often related to the functional properties of proteins, even though its measurement is not straightforward (Selmane, Vial, & Djelveh, 2010; Cheng, 2001). It was determined using 1-anilino-8-naphthalenesulfonate (ANS) as a fluorescence probe, according to the method described by Nakai (2004) with some slight modifications. Soluble proteins from larvae and from larvae meal were dispersed in 2 mM phosphate buffer at pH 7 to reach 0.1% (w/v) and were stirred for 1 hour at room temperature. After centrifugation (10,000 g, 2 min., 20 °C), the protein content was determined using Lowry method in the supernatant collected which was diluted to reach different concentrations (0.01-0.05%, w/v) and then were stirred for 10 min. Aliquots of ANS solution (15 µL, 8 mM in 0.1 M phosphate buffer, pH 7) were then added to 3 mL samples and the solutions were vortexed and equilibrated in the dark for 5 min. Relative fluorescence intensity was measured using a Safax Xenius spectrofluorometer (Safax, Monaco) in a quartz-cell (1 cm path length) at an excitation wavelength of 380 nm and an emission wavelength of 480 nm. The initial slope for fluorescence intensity versus protein concentration was calculated by linear regression analysis and used as the surface hydrophobicity. All experiments were performed at in triplicate for each sample. 2.9. Measurement of surface tension at air/water interfaces Surface tension is an important physical property that affects functional properties such as foaming (Foegeding, Luck, & Davis, 2006). Proteins are composed of both hydrophobic and hydrophilic regions and may therefore display some properties of tension-active molecules and permit the mixing of immiscible particles such as air and water. The surface tension

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between the air and water interface was measured in order to evaluate the ability of soluble proteins from larvae and from larvae meal to reduce the surface tension of water. These soluble proteins were dispersed in 2 mM phosphate buffer, pH 7 to reach 1 or 2% (w/v) and then stirred for 1 h at room temperature. After centrifugation (10,000 g, 2 min., 20 °C), the protein content was determined using the Lowry method in the supernatant collected which was diluted to reach different concentrations (0.001-1%, w/v) and then were stirred for 10 min. Surface tension was measured at 25 °C using an automated tensiometer (Tracker-TeclisIT Concept, France) based on axisymmetric drop shape analysis of an air bubble in a protein solution. The rising drop method was used with a bubble volume of about 7 µL. Measurements were taken as a function of time in order to establish the equilibrium time required to obtain a constant surface tension, and each run was performed in duplicate at least.

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3. Results and Discussion

3.1. Determination of fat, water, protein contents

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The compositions of Tenebrio molitor larvae, larvae meal, defatted larvae and defatted larvae meal are shown in Table 1. The moisture content of the larvae was 68.8% and that of the other products was lower than 4%. The crude protein contents of defatted larvae (83.0%) and defatted larvae meal (80.8%) were higher than the crude protein content of the larvae meal (71.6%) which in turn was higher than that of the larvae (65.6%). Indeed, the removal of fat increases the crude protein content in the product. The high crude protein content of the larvae meal was superior to that of the fish meal (63.0%) (Guerreiro & Retiere, 1992) and the soya meal (55%) (Stein et al., 2008). The fat (24.5%) and crude protein contents of larvae were comparable to the data described in the literature, namely 44-69% and 23.0-47.0%, respectively (Veldkamp et al., 2012). The larvae contained more proteins than chicken (62.3%), beef (56.7%) or luncheon fish (39.0%) and contained less fat than these traditional animals (30.3%, 42.0%, and 48.3% respectively) (Li, Zhao, & Liu, 2013). 3.2. Amino acid content The protein quality of the larvae and larvae meal was estimated in terms of their amino acid composition and compared with that of other conventional resources used for livestock and salmon feeds and human foods (Table 2). The amino acid composition of larvae meal was similar to the profiles reported by Makkar et al. (2014). The amino acid content was higher than that of the larvae because it contained more proteins. Essential amino acids (EAA) contents in larvae meal were comparable to those reported for the fish and soya meals used for feeds (Makkar, 2014). Based on the recommended requirements (NRF, 1993; FAO/WHO/UNU, 1985), the proteins in larvae and larvae meal contained all EAA and more than adequate amounts for consumption by both humans and salmon, except for a deficiency in methionine. However, although the levels were more than sufficient, some EAA may be limiting factors if the levels are too high in feed. This is the case of tryptophan, found at levels too high for use in chickens as it may constitute a limiting factor with respect to weight gain (Hardouin & Mahoux, 2003; Finke, DeFoliart, & Benevenga, 1989). However, this issue can be resolved by adjustments to the feed formulation. Larvae and larvae meal were therefore found

ACCEPTED MANUSCRIPT to be rich in high-quality proteins, with a good nutritional value for use in both humans and animals such as salmon. 3.3. Alkaline extraction of soluble proteins from larvae and from larvae meal

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The protein yield and protein content after alkaline extraction are shown in Table 3. The protein yield of the larvae (59.9%) was two-fold that of the larvae meal (26.4%). The protein yield of the larvae was similar to that mentioned by Yi (2015). The lower protein yield of the larvae meal may have been due to denaturation caused by heat treatment during the thermomechanical process. Similar results had been found by Ma et al. (2011) who studied on chickpea, lentil and pea. They found that the protein solubility of these legumes, when heated at 90 °C, was lower than when they were untreated. Protein denaturation by heat treatment is often accompanied by a reduction in solubility (Alais, Linden, & Miclo, 2008). Although the protein yields were not highest, the protein contents on dry matter in the supernatant extracted from the larvae and larvae meal were highest and similar (84% and 80%, respectively). 3.4. Determination of molecular weight

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The distribution of molecular weights of the soluble proteins from larvae and from larvae meal, were estimated by SDS-PAGE using 4-15% polyacrylamide gel. The gel results revealed the same range of MW value for soluble proteins from larvae and from larvae meal. Three protein groups were found: ≤14 kDa; 14-30 kDa; 30-100 kDa (Fig. 1). However, the bands of soluble proteins from larvae and from larvae meal were different. The bands were the same below 30 kDa but differed above that value. The treatment applied the larvae produced proteins with different MW. Nevertheless, the range of MW was similar to that found by Li et al. (2013) on soluble proteins from Tenebrio molitor larvae.

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3.5. Assessment of pH-dependent soluble protein solubility The results showed that the solubility of soluble proteins was markedly pH-dependent (Fig. 2). Protein solubility increased significantly as the pH rose, to reach maximum solubility at pH 7 for the soluble proteins from both larvae (88.8%) and larvae meal (76.2%). By contrast, minimum solubility was observed at pH 3 and 4 for the soluble proteins from larvae meal and at pH 3 to 5 for the soluble proteins from larvae. This was due to a reduction in electrostatic repulsive forces between the proteins, leading to protein aggregation. This protein solubility profile was similar to those reported for several legumes and animal proteins (Adebiyi & Aluko, 2011; Selmane, Vial & Djelveh, 2010; Tian, 1998; Ma et al., 1997). Whatever the pH, the solubility of soluble proteins from larvae was higher than that of soluble proteins from larvae meal excepted at pH 5 and 6. In addition, the solubility of soluble proteins from larvae meal was higher at an alkaline pH than at an acid pH. These differences in solubility between the two soluble proteins could be attributed to the combined effect and intensity of the different treatments undergone by the larvae and larvae meal. In general, heat treatment denatures proteins by unfolding and exposing their internal hydrophobic groups. They thus acquire a random conformation which is dependent on both the intensity of the treatment applied and the forces stabilizing the structure ((Alais, Linden, & Miclo, 2008).

ACCEPTED MANUSCRIPT 3.6. Measurement of zeta potential

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Measurement of the zeta potential of the larvae and larvae meal proteins showed that this potential was markedly affected by the pH and ionic strength (Fig. 3a). The same trend was observed regarding soluble proteins from larvae and from larvae meal. Malhotra & Coupland (2004) also found similar results in a study on soy protein isolates. At 0 M of NaCl, the zeta potential decreased when the pH increased, with positive values at an acid pH and negative values at an alkaline pH. This curve trend could be explained by protonation at an acidic pH, and deprotonation at an alkaline pH, of the amino acids in the protein (Bouzid et al., 2008). At pH 2, the larvae and larvae meal proteins had zeta potentials of 32 and 30 mV respectively. At pH 7, the zeta potential for soluble proteins from larvae (-30 mV) and that of soluble proteins from larvae meal (-25 mV) were close to or equal to the threshold absolute value of 30 mV described as the stability boundary (Carneiro-da-Cunha et al., 2011). These results reveal that soluble proteins from larvae experienced more electrostatic repulsions than those from larvae, so that they were less sensitive to aggregation (or more soluble) when compared to those from larvae meal (or less soluble). This confirmed the results obtained relative to solubility. Soluble proteins from both samples were more soluble and stable in an alkaline solution (pH 10). If a suspension has a high absolute zeta potential value, particles will tend to repel each other and will have a tendency not to flocculate. However, if the potential is low, then particles will tend to aggregate because there is no force that prevents them from flocculating. The zeta potential approached zero at pH 3.5 and pH 3 for soluble proteins from larvae and from larvae meal respectively, which was indicative of the low solubility. The zeta potential as a function of ionic strength at pH 7 displayed a diminution towards 0 mV when the ionic strength was increased from 0.1 to 1 M (Fig. 3b). This phenomenon was observed because when the salt concentration rises, salt ions attract some water molecules, which reduces the number of water molecules available for water-protein interactions. This causes an aggregation of proteins, so that the zeta potential tends towards to zero. This result of the zeta potential evolution versus NaCl concentration was also found by Salgin, Salgin, & Bahadir (2012). They found that the zeta potential of BSA was -11 mV at 0.1 M. This was lower than the zeta potentials of soluble proteins from larvae (-18 mV) and from larvae meal (-22 mV). 3.7. Measurement of surface hydrophobicity The results of surface hydrophobicity measurements are presented in Fig. 4 and Table 4. The evolution of relative fluorescence intensity was linearly proportional to the protein concentration, and the slope corresponded to the surface hydrophobicity of the proteins. Soluble proteins from larvae displayed a surface hydrophobicity (670.3) that was higher than that of soluble proteins from larvae meal (102.5). The solubility of soluble proteins displayed the same trend. Wagner, Sorgentini, & Añón (2000), working on soy protein isolates, found that surface hydrophobicity rose in line with an increase in solubility, explaining that aggregating proteins are more hydrophobic and buried hydrophobic zones inside the structure of the proteins, thus the resulting in soluble aggregates with low surface hydrophobicity.

ACCEPTED MANUSCRIPT 3.8. Measurement of surface tension at air/water interfaces

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The surface tension evolution versus time for soluble protein solutions at different concentrations (0.003-0.1%) are showed on the Fig. 5. These kinetics showed curves with a lag time (where the surface tension is close to the water value of 72 mN/m) followed by a decrease of surface tension down to a constant value. This shape curve is in agreement with those described by Beverung, Radke, & Blanch (1999) which also divided the curve into three stages: (1) Diffusion and protein interfacial affinity; (2) Conformational rearrangement of interfacial film; (3) Relaxation of the absorbed layer and possible build-up of multilayers. The lag time of soluble proteins from larvae is longer than the lag time of soluble proteins from larvae meal. The lag time is longer when the protein concentration is lower, and similarly, the equilibrium time is longer when the protein concentration is lower. At higher concentrations (0.1%), there is no lag time, and the surface tension decreasing quicker. The surface tension evolution depended on the protein and its concentration. Fig. 6 showed that the surface tension at the equilibrium of soluble larvae and larvae meal protein solutions fell when the concentration increased. This result was anticipated, because in general, a low surface tension value is observed when the protein concentration rises (Amine et al., 2014; Guillermic, 2011; Kinsella, 1981). The surface tension of soluble larvae meal protein solution was lower than that of soluble larvae meal protein solution for all concentrations. This means that the soluble proteins from larvae meal caused a greater reduction in water surface tension than those from the larvae. At a 1% concentration, the surface tension of the larvae protein solution (42 mN/m) was low and comparable to that of proteins in general (42-57 mN/m, Foegeding, Luck, & Davis, 2006; Bos & Vliet, 2001; Kitabatake & Doi, 1982). However, that of the protein solution of larvae meal was 32 mN/m, which was lower than the classic surface tension of proteins. This difference between the two surface tension values may have been due to the molecular configuration of the proteins, their surface charge and surface hydrophobicity induced by the protein treatments.

4. Conclusion

Soluble proteins from larvae and from larvae processed into larvae meal using a thermomechanical process were characterized for their physicochemical properties. The determination of crude protein contents showed that both were rich in proteins (65.6% and 71.6%, respectively) and these values were superior to fish meal (63%) and soya meal (55%). Defatting increased the crude protein content to 80% or more. These proteins from larvae meal and larvae displayed a good nutritional quality. They contained all EAA and more than adequate quantities to meet the dietary requirements of humans and salmon, except for a deficiency in methionine. The EAA contents of larvae meal were also comparable to those of fish and soya meals used for livestock. Soluble proteins extraction at pH 10 and 45 °C produced a soluble proteins content of 80%, and the protein yield of larvae (59.9%) was two-fold that of larvae meal (26.4%), due to the thermo-mechanical process. The MW range for soluble proteins was inferior to 100 kDa but the two soluble proteins differed.

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The solubility study of soluble proteins at 25 °C revealed a strong pH-dependent effect and low solubility at pH 3 and 4 for the soluble proteins from larvae meal and at pH 3 to 5 for the soluble proteins from larvae. However, the solubility of soluble proteins from larvae was higher than that of soluble proteins from larvae meal, as they did not contain the same type proteins. Moreover, the surface charge was close to zero when the pH trend to the low solubility pH and the surface charge of soluble proteins from larvae was slightly higher (thus facilitating aggregation) than that of soluble proteins from larvae meal whatever the pH and NaCl concentration. The surface hydrophobicity of soluble proteins from larvae was higher than that of soluble proteins from larvae meal. This could be explained by the aggregation of soluble proteins from larvae meal which buried the hydrophobic zones inside and led to low surface hydrophobicity. The surface tension of soluble proteins from larvae (42 mN/m) was comparable that of conventional proteins, while that of soluble proteins from larvae meal (32 mN/m) was lower. This difference might have been due to the different in molecular configuration of the proteins, their surface charge and surface hydrophobicity. All these findings provide data on characterization of the physicochemical properties of proteins from insects and, the impact of processes. They improved the knowledge of these systems, thus facilitating efforts to achieve industrial application. Further studies are needed with respect to the identification of insect proteins, their isolation and determination of their technological suitability as ingredients in food and feed preparations. These studies must also continue to focus on physicochemical properties such as interfacial rheology and functional properties. This will enable a clearer understanding of the relationships between these properties and the development of extraction and production processes to facilitate the use in feed and food industries.

ACCEPTED MANUSCRIPT Acknowledgments

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This work received support by the French National Research Agency (ANR) through its DESIRABLE project (Designing an insect biorefinery to contribute to a more sustainable agro-food industry). The authors would like to thank to Tania Roux and Audrey Baustier for their technical assistance.

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ACCEPTED MANUSCRIPT Fig. 1: Distribution molecular weight of soluble proteins from larvae and from larvae meal determined by SDS-PAGE using 4-15% polyacrylamide gel (sample from the left to right: markers, soluble proteins from larvae and from larvae meal). Fig. 2: Effect of pH at 25 °C on the soluble proteins from larvae (●) and from larvae meal (▲).

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Fig. 3: The pH effect at 0 M NaCl (a) and NaCl effect at pH 7 (b) on the zeta potential of soluble proteins from larvae proteins (●) and from larvae meal proteins (▲).

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Fig. 4: Relative fluorescence intensity at 480 nm versus soluble protein concentrations from larvae (●) and from larvae meal (▲) at pH 7. Fig. 5: Evolution of surface tension of soluble proteins from larvae and from larvae meal versus time at pH 7, 25 °C. Soluble protein concentrations from larvae (a) 0.003, (c) 0.01, (e) 0.1 % and from larvae meal (b) 0.003, (d) 0.01, (f) 0.1.

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Fig. 6: Surface tension at equilibrium versus soluble protein concentrations from larvae ( ●) and from larvae meal (▲) at pH 7, 25 °C.

ACCEPTED MANUSCRIPT Table 1 : Composition (g/100 g) of Tenebrio molitor larvae, larvae meal, defatted larvae, and defatted larvae meal (mean ± S.D., n = 3).

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% Dry matter Crude proteins Fat 65.6 ± 1.7 24.5 ± 0.5 71.6 ± 1.7 14.6 ± 0.9 83.0 ± 3.4 80.8 ± 2.2 -

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Moisture (%) 68.8 ± 0.1 3.9 ± 0.1 2.0 ± 0.1 2.0 ± 0.1

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Sample Larvae Larvae meal Defatted larvae Defatted larvae meal

ACCEPTED MANUSCRIPT Table 2: Amino acid composition (g/16 g nitrogen) of Tenebrio molitor larvae and larvae meal versus fish meal, soya meal, and juvenile Chinook salmon and human dietary protein requirements. Larvae meal

Fish meal a

Soya meal a

Essential amino acids Histidine Isoleucine Leucine Lysine Methionine Phenylalanine+ tyrosine Threonine Tryptophan Valine Cysteine+Cystine

2.5 3.5 6.2 5.4 0.7 7.0 3.2 1.2 5.4 0.6

2.9 4.7 8.0 6.3 1.4 9.5 4.3 1.2 8.5 0.8

2.4 4.2 7.2 7.5 2.7 7.0 4.1 1.0 4.9 0.8

3.1 4.2 7.6 6.2 1.3 8.6 3.8 1.4 4.5 1.4

Non-essential amino acids Arginine Glycine Glutamic acid Aspartic acid Proline Serine Alanine

4.2 4.2 8.3 6.4 4.4 4.0 6.6

5.4 5.5 10.6 7.8 6.0 4.6 8.4

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6.2 6.4 12.6 9.1 4.2 3.9 6.3

Requirement for juvenile Chinook Salmon b

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7.6 4.5 19.9 14.1 6.0 5.18 4.54

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*Methionine plus cystine; a Makkar et al., 2014; b NRF, 1993.

1.8 2.2 3.9 5.0 4.0* 5.1 2.2 0.5 3.2

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1.9 2.8 6.6 5.8 2.5 * 6.3 3.4 1.1 3.5

ACCEPTED MANUSCRIPT Table 3: % Protein yield and protein content of the supernatant from larvae and larvae meal. Larvae % protein yield % protein content 83.7 ± 3.8

26.4 ± 2.7

80.4 ± 0.9

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59.9 ± 6.1

Larvae meal % protein yield % protein content

ACCEPTED MANUSCRIPT Table 4: Surface hydrophobicity and solubility of soluble proteins from larvae and from larvae meal. Surface hydrophobicity 670.3

% Solubility at pH 7 88.8±2.3

Soluble proteins from larvae meal

102.5

76.2±6.9

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Samples Soluble proteins from larvae

ACCEPTED MANUSCRIPT Highlights The larvae meal obtained by thermo-mechanical process and larvae were rich in proteins.

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The proteins from larvae and from larvae meal contained all essential amino acids and more than adequate quantities to meet the dietary requirements of humans and salmon, except for a deficiency in methionine.

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Soluble proteins from larvae and from larvae meal contained 80% of proteins.

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At pH 7 and 25 °C, the physicochemical properties of soluble proteins from larvae were higher than these of soluble proteins from larvae meal.