WO2024100577A1 - Method for fractioning of an edible insect - Google Patents

Abstract

The present disclosure relates to a method for extracting proteins from insects wherein said method comprises an ultrafiltration step. Moreover, the present disclosure also relates to the protein composition obtained through such process and to ingestible products or foodstuff ingredients comprising such protein composition.

Description

D E S C R I P T I O N METHOD FOR FRACTIONING OF AN EDIBLE INSECT TECHNICAL FIELD [0001] The present disclosure relates to the field of protein extraction from edible insects, specifically focusing on a method that utilizes ultrafiltration for the separation and concentration of proteins. It specifically relates to a novel method for fractioning edible insects and producing a high-quality protein extract suitable for human consumption. BACKGROUND [0002] In the next decades, due to population growth and modifications in dietary patterns in developing countries, demand for protein-rich foods will increase. If this demand is fulfilled by current protein-sources (e.g., livestock, poultry) it will exacerbate current environmental, consumers’ health, food safety and economic viability problems. So, there is a need to assess and use alternative protein sources that can both guarantee nutritional requirements and environmental sustainability (Parodi et al., 2018; van der Spiegel et al., 2013; van der Weele et al., 2019). [0003] One of such sources can be edible insects, which present both environmental (Moruzzo et al., 2021) and nutritional advantages (Payne et al., 2016; Rumpold & Schlüter, 2013; Weru et al., 2021). Compared to other animal-based protein sources, production of edible insects present lower greenhouse gas emissions and depletion of natural resources (land, water and feed) and can be fed with organic waste, contributing to a circular economy. Nutritionally, most edible insect species present very high protein content (between 40-60% on a dry matter basis) with excellent quality – similar essential amino acid profile and biological value as animal-based protein sources. Lipids are the second most abundant macronutrients (20-30% on a dry matter basis), generally presenting a high degree of unsaturation and high content in essential fatty acids ((ω3 andω6). Edible insects also present high fibre content (5-15% on a dry matter basis), which is mainly composed of chitin present in their exoskeleton. Chitin is indigestible to humans, but it can be deacetylated into chitosan which presents a wide array of applications in agriculture, biomedicine, cosmetic, water treatment, etc. [0004] The consumption of edible insects (entomophagy) is a common practice in several regions of the world (mainly in Southeast Asia, Sub-Saharan Africa and Central/South America) with estimates that more than 2 billion people from over 100 countries regularly consume more than 2000 edible insect species due to their nutritional content and sensory appeal (playing a role as food delicacies to tourists in Thailand or Mexico) (FAO, 2021; Jongema, 2017; van Huis et al., 2013). In Western countries (Europe and North America), edible insects have been traditionally neglected, but this situation has been changing in the last decade, since the publication of the book “Edible Insects: Future prospects for food and feed security” by the Food and Agriculture Organization of the United Nations in 2013 (van Huis et al., 2013). This heightened interest is visible in the scientific community (growth in the number of scientific papers working with edible insects) and in the food industry (rise in insect-rearing companies and insect-based food products present in the market as well as growth of market values of the edible insects sector) (Baiano, 2020; van Huis, 2022). [0005] Reflecting the popularity of edible insects and their potential role as alternative food sources, the European Union has already listed four edible insect species as safe for human consumption, including the yellow mealworm, Tenebrio molitor larvae (EFSA, 2023; EFSA, 2021a; EFSA, 2021b), which is one of the most popular species for human consumption in the West (Baiano, 2020; van Huis, 2022). Currently, T. molitor has been approved as safe to consume by the European Food Safety Authority in the following forms: frozen (EFSA, 2021a), freeze-dried (EFSA, 2021a), oven-dried (EFSA, 2021b) and oven-dried with UV-treatment (EFSA, 2023). [0006] Despite the advantages presented by edible insects and their popularity growth, the edible insects sector still face some challenges for their successful implementation in the food market, namely low consumer acceptance (Cunha and Ribeiro, 2019; Kröger et al., 2021; Ribeiro et al., 2022a). One of the key strategies to increase consumer acceptance is the development of appropriate products with high sensory appeal to target consumer groups. In this context, a consumer group with higher willingness to try insect-based food products is consumers that value and have high interest in the food products’ health and nutritional benefits (Menozzi et al., 2017; Orsi et al., 2019). Furthermore, high-protein insect-based functional food products are deemed as very appropriate by consumers (Ardoin and Prinyawiwatkul, 2020) and are positively sensory evaluated by consumers (Ribeiro et al., 2023a). [0007] This increased acceptance of high-protein insect-based functional products among consumers, should intensify the focus on the fractionation of edible insects, and in particular, the valorisation of the protein fraction (Baiano, 2020; Lucas et al., 2020; Nongonierma and FitzGerald, 2017; Rahman et al., 2023). To better develop these types of food products, edible insects should be incorporated with optimal nutritional, biological, and technological characteristics. The simple incorporation of insect powder (dried and ground insect) in high- protein functional products presents several disadvantages, since these would not present comparable properties (e.g., protein content or healthy benefits) to functional products already on the market, atop the more reduced sensory attractiveness. Instead, insect protein fractions should be preferred, since they present higher protein content, better technological and sensory properties, and can exhibit improved bioactive properties (e.g., antioxidant, antihypertensive or antidiabetic) (Ribeiro et al., 2023b). [0008] Currently, the most applied insect protein isolation methods consist of alkaline solubilization followed by acid precipitation or isoelectric point precipitation (Baiano, 2020; Lucas et al., 2020; Rahman et al., 2023; Ribeiro et al., 2023b). However, most obtained insect protein fractions present a protein content below 80.0% (Ribeiro et al., 2023b), which means they cannot compete with commercial protein concentrates or isolates. Furthermore, the application methods based on acid or isoelectric point precipitation may be challenging to apply at an industrial scale and can present some environmental disadvantages (Laroche et al., 2022). [0009] Therefore, the development of adequate protein extraction techniques is of extreme importance for incorporation of edible insects into functional products. The obtained protein extracts should have similar protein content, amino acid profile and techno-functional properties as commercially-available protein concentrates. Additionally, the developed extraction procedure should allow for a complete fractionation of the insects, namely of its fat and chitin fraction in order to increase the economic value of the developed fractionation method. [0010] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION [0011] The method described in the present disclosure seeks to address the issues identified in the prior art by introducing an innovative approach using ultrafiltration. [0012] An aspect of the present disclosure relates to a new protein isolation method applied to edible insects, based on membrane ultrafiltration. Membrane filtration techniques, such as ultrafiltration, are non-thermal technologies based on forcing a fluid through the pores of a membrane by applying pressure or an electric field (Nasrabadi et al., 2021). The obtained protein concentrates/isolates with the method of the present disclosure not only present high purity, but also improved techno-functional and sensory properties, and this process can be eco-friendlier and scaled up more easily than isoelectric precipitation. [0013] The obtained protein fraction presented a purity (protein content) above 80.0%. [0014] It was surprisingly found that the protein fraction obtained through the method of the present disclosure provides better techno functional properties, in particular, colour, foaming capacity, foaming stability, emulsifying capacity and oil binding capacity, than protein fraction obtained through isoelectric precipitation. [0015] An aspect of the present disclosure relates to a method of fractionation of an edible insect comprising a step of protein purification by ultrafiltration. [0016] In a preferred embodiment, said method comprises the steps: providing grinded, defatted insects raw material; extracting proteins from the grinded, defatted insects raw material for obtaining a soluble fraction comprising proteins and a non-soluble fraction; submitting the soluble fraction comprising proteins to an ultrafiltration in order to obtain a purified soluble fraction comprising proteins. [0017] In a preferred embodiment, the method further comprises a step of submitting the non-soluble fraction to chemical hydrolysis, decolorization and alkaline deacetylation in order to obtain chitosan. [0018] In an embodiment, the ultrafiltration is performed with a membrane comprising a pore size ranging from of 30-70 kDa. Preferably, the ultrafiltration is performed with 50 kDa membranes. [0019] In an embodiment, the extraction step is performed with an aqueous NaOH solution; preferably wherein the weight:volume ratio between the aqueous NaOH solution/ defatted insects raw material is 1:50; more preferably the aqueous NaOH solution is a 0.001 – 0.1M M NaOH solution. [0020] In an embodiment, the extraction step is performed at a temperature ranging from 40- 60°C and at a pH ranging from 11-13. [0021] In an embodiment, the extraction step comprises a centrifugation; preferably at 4000- 8000g for 30-40 min. [0022] In an embodiment, the defatting of the insect is performed with a Soxhlet apparatus; preferably during (6 hours with ethanol as a solvent and at a w:v ratio of 1:30 (weight:volume ratio between ethanol/ defatted insects). [0023] Another aspect of the present disclosure relates to a method of fractionation of an edible insect, which comprises blanching the insect, drying the blanched insect, defatting the insect, homogenizing the defatted insect with an aqueous solution of NaOH, centrifuge the resulting solution, obtaining a supernatant fraction comprising proteins and a non-soluble fraction, ultrafiltration of the supernatant fraction comprising proteins in order to obtain a purified soluble fraction comprising proteins. [0024] In an embodiment, the method further comprises a step of chemical hydrolysis and alkaline deacetylation of the non-soluble fraction. [0025] In an embodiment, the blanching is done by boiling the insect at 100°C during at least 5 minutes. [0026] In an embodiment, the drying of the blanched insect is obtained at a temperature ranging from 50°C to 100°C for 5 to 10 hours; preferably at 80°C for 7 hours. [0027] In an embodiment, the homogenization of the defatted insects is obtained in an aqueous 0.1M NaOH solution at 1:50 weight:volume ratio (weight:volume ratio between the aqueous NaOH solution/ defatted insects), at 50°C, preferably for 4 hours under constant agitation at 150 rpm, followed by a centrifugation at 3993 x g, for 30 min, and at 4°C. [0028] In an embodiment, the non-soluble fraction was subsequently treated with HCl and NaOH to obtain unbleached chitin. [0029] In an embodiment, the unbleached chitin is decolorized with KMnO4/C2H2O4. [0030] In an embodiment, the unbleached chitin is deacetylated with a NaOH solution to obtain chitosan. [0031] In a preferred embodiment, the insect is selected from the list consisting of: Tenebrio molitor larvae, Locusta migratoria, Acheta domesticus; Alphitobius diaperinus; preferably Tenebrio molitor larvae. [0032] Another aspect of the present disclosure relates to a purified soluble fraction comprising proteins obtained through the method described in any of the previous claims, wherein the purified soluble fraction comprising proteins comprises a protein content of at least 80.0 g/100g on a dry matter basis. [0033] Another aspect of the present disclosure relates to a powdered protein extract comprising the purified soluble fraction comprising proteins obtained through the method described in any of the previous claims, wherein the powdered protein extract comprises a protein content of at least 80.0 g/100g on a dry matter basis. [0034] In a preferred embodiment, the lightness colour (L*) of the powdered protein extract is of at least 60.0; measured by the CIELAB system. [0035] In a preferred embodiment, the Browning Index of the powdered protein extract is below 80; measured by the CIELAB system. [0036] Another aspect of the present disclosure relates to a foodstuff ingredient comprising the purified soluble fraction comprising proteins herein described or the powdered protein extract herein described. [0037] Another aspect of the present disclosure relates to an ingestible product comprising the purified soluble fraction comprising proteins herein described or the powdered protein extract herein described; preferably the ingestible product is a food product, a nutraceutical product, a pharmaceutical product or a dietary supplement. [0038] Another aspect of the present disclosure relates to a method of fractionation of an edible insect, which comprises: blanching the insect, drying the insect, defatting the insect, homogenizing the defatted insect with an aqueous solution of NaOH, centrifuge the resulting solution, ultrafiltration of the supernatant. [0039] In an embodiment, the blanching is done by boiling the insect at 100 °C during at least 5 minutes. [0040] In an embodiment, the drying of the blanched insect is obtained in an oven, at 50°C to 100°C for 5 to 10 hours, preferably at 80 °C for 7 hours. [0041] In an embodiment, the method comprising the defatting of the insect with a Soxhlet apparatus (6 hours with ethanol as a solvent and a w:v ratio of 1:30). [0042] In an embodiment, the solubilisation of the defatted insects is obtained in an aqueous NaOH solution (0.1M) at 1:50 weight:volume ratio, at 50°C, for 4 hours under constant agitation at 150 rpm, followed by a centrifugation at 3,913 x g for 30 min at 4 °C, for 30 min, and at 4°C. [0043] In an embodiment, the supernatant is ultrafiltrated with a 50 kDa membrane. [0044] In an embodiment, the insect is Tenebrio molitor larvae. [0045] In an embodiment, the pellet is initially treated with an aqueous solution of HCl, washed, filtrated and treated with an aqueous solution of NaOH solution, washed and filtrated to obtain unbleached chitin. [0046] In an embodiment, the unbleached chitin is decolorized with subsequent treatments with aqueous solutions of KMnO4 and C2H2O4. [0047] In an embodiment, the unbleached chitin is deacetylated, with the homogenization of the decolorized chitin samples with a strong NaOH solution. [0048] The obtained chitin and chitosan presented similar characteristics as commercial products. [0049] Chitin yield in the process was 5.28% (chitin obtained from T. molitor powder/flour). [0050] Chitosan yield the process was 3.18% (chitosan obtained from T. molitor powder/flour). [0051] In the state of the art, the colour may be measured by many methods, in the present disclosure the colour was measured by the CIELAB system (or CIE L*a*b*). BRIEF DESCRIPTION OF THE DRAWINGS [0052] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention. [0053] Figure 1: Protein molecular weight profile (according to Size Exclusion Chromatography) of different obtained protein fractions. [0054] Figure 2: Schematic representation of the protein isolation methods (membrane ultrafiltration and isoelectric precipitation), with information about each fraction's yield, protein content (PC) and protein recovery (PR) relative to the protein present on the dried larvae. [0055] Figure 3: FTIR-ATR spectra of chitin (a) and chitosan (b) from different sources: commercial, T. molitor flour/powder, A. domesticus and T. molitor pellet. DETAILED DESCRIPTION [0056] The present disclosure relates to a method of protein extraction from edible insects, in particular from the species yellow mealworm (Tenebrio molitor) larvae. The developed process also allows to obtain the fat and chitin fractions and thus it can be considered a complete fractionation method. [0057] The developed protein extraction method is based on ultrafiltration of soluble proteins. In scientific literature, the most common protein extraction methods are based on solubilization followed by acid or isoelectric point precipitation while filtration techniques are usually applied to perform purification of low molecular weight fractions. [0058] The method described in the present disclosure comprises the use of ultrafiltration membranes to selectively separate proteins from other components of edible insects, including chitin and lipids. The process begins with the collection of edible insects, which are then subjected to a pretreatment step to facilitate protein extraction. [0059] The method of the present disclosure presents several advantages over the conventional protein precipitation methods, including: - Reduced Environmental Impact: Ultrafiltration minimizes the need for chemical treatment to precipitate the proteins, making the process eco-friendlier. - Improved techno-functional properties: the protein product obtained comprises special characteristics, in particular regarding the colour, oil absorption capacity, foaming and emulsifying properties. - Scalability: the application of ultrafiltration facilitates the scalability of the protein extraction technique. EXAMPLE 1 Materials and methods Insect samples and pre-treatments [0060] The insect samples used consisted of yellow mealworm Tenebrio molitor larvae. Insect samples were provided frozen by TecmaFoods (insect rearing company in Leça do Balio, Portugal) and were submitted to a starving process of 48 h to eliminate the gut content before being euthanized. The frozen larvae were conserved at -24 °C until being submitted to a blanching treatment by immersion in boiling water (1:10, w:v) for 5 min. Blanched larvae were stored in polyethylene zip lock bags at -24 °C then dried in an electrical oven (Unox® model XF016-TG) at 80 °C for 7 h. Dried larvae were ground in a kitchen robot (Kenwood® Major Titanium with the multi-mill attachment model AT320A). [0061] Before applying the protein isolation methods, powder samples (dried and ground T. molitor) were submitted to a defatting process to improve the efficiency and quality of the protein isolation. Lipids were extracted with the Soxhlet method with ethanol as a solvent. Briefly, for each extraction, 20 g of dried and ground larva were extracted with 750 mL of ethanol in a Soxhlet extraction apparatus for 6 h. The remaining solvent was evaporated with a vacuum rotary evaporator. The cartridges with the defatted samples were dried at 95 °C for 12 h, and then the defatted fraction was stored in airtight containers at room temperature and sheltered from light until further use. The yield of the defatting step was calculated as the mean of four replicates and determined as the ratio between obtained defatted fraction and insect powder used in each extraction. [0062] The nutritional content of the powder and the defatted fraction (Table 1) were analysed for crude protein (Kjeldahl method with a nitrogen conversion factor of 6.25), total fat (Soxhlet method, with petroleum ether as a solvent), total dietary fibre (AOAC 991.43 and AOAC 985.29 methods) and ash (incineration in a muffle furnace at 550 °C).

Table 1. Nutritional content (g/100g) of T. molitor powder and defatted fraction. Analysis performed in a laboratory certified for nutritional analysis.

Protein isolation methods [0063] Two different isolation methods were applied (isoelectric point precipitation (comparative method) and membrane ultrafiltration). For both methods, the initial steps were identical and consisted of the solubilization of the defatted fractions. Defatted samples were homogenized with either NaOH 0.1 M or dH2O (distilled water) (1:50, w:v) for 4 h at 150 rpm with a temperature of 25 °C or 50 °C (Thermo Scientific MaxQ6000). After homogenization, samples were centrifuged at 3,913 x g for 30 min at 4 °C, and the obtained supernatants (S) were analysed concerning the total protein by the BCA method (Bicinchoninic acid) to determine the better solvent and temperature conditions. Then, potentiometric titration was applied to the supernatants obtained under better solubilization conditions to determine the isoelectric point (4.56 ± 0.03). [0064] For the isoelectric point precipitation isolation method, the obtained supernatant pH was adjusted to 4.56 with HCl 1 M solution and then centrifuged at 3,913 x g for 30 min at 4 °C. The obtained pellet (P), corresponding to the precipitated proteins (IP), was collected and frozen until further processing. For the membrane ultrafiltration method, the supernatants were submitted to a 50 kDa membrane (Cogent μScale – Tangential Flow Filtration, ΔP setpoint 3.0 bar) with two different fractions being collected and frozen at -24 °C: non-filtrated (>50 kDa) and permeate (< 50 kDa). The selection of the molecular weight cut-off of the membrane was based on preliminary studies as well as the results from size exclusion chromatography applied to the soluble proteins. [0065] All the fractions resulting from the initial alkaline solubilization (supernatant – S; pellet – P), isoelectric point precipitation (precipitated protein – IP) and membrane ultrafiltration (>50 kDa and <50 kDa) were freeze-dried (Telstar® LyoQuest -55 coupled with vacuum pump Ulvac® model GLD136C). Freeze-drying conditions consisted of freezing (-50 °C) for 3 h, freezing (-50 °C) with the application of vacuum (0.250 mbar) for 3 h and heat shelves (35 °C, 0.250 mbar) for 72 h. Yield, purity and protein recovery [0066] The isolation efficiency was evaluated for each obtained fraction through three parameters: yield, purity, and protein recovery. Yield pertained to the freeze-dried fraction rate obtained compared to the initial mass of T. molitor powder. Purity is the protein content of each fraction, which was calculated with the Kjeldahl method with a Nitrogen conversion factor of 6.25 (performed in duplicate). The protein recovery rate was calculated as the amount of protein present in each fraction concerning the amount of protein in the T. molitor powder (Equation 1). For each fraction, protein recovery was calculated as follows:

(Equation 1) Protein profile [0067] The protein profile of the obtained fractions (except for the pellet) was evaluated with size exclusion chromatography (SEC). The freeze-dried fraction (10 mg) was dissolved in ultrapure H2O (1 mL). NaOH 1 M (50 µL) was added to facilitate the dissolution of the IP fraction. Colour [0068] The colour of the protein fractions, as well as of the powder and defatted fraction, were assessed (six replicates) with a colorimeter (Minolta® Chroma Meter model CR-400), using the CIELAB colour space (L*a*b*). L* is the lightness value (that defines black as 0 and white as 100), the a* axis corresponds to the green–red colours (where negative values correspond to green and positive ones to red), and the b* axis corresponds to blue-yellow colours (where negative values correspond to blue and positive ones to yellow). [0069] Total colour variation in comparison to T. molitor powder was assessed with Equation 2:

(Equation 2) where the indices 0 pertain to the colour coordinates of T. molitor and 1 the colour coordinates of T. molitor defatted fraction or protein fractions. [0070] The browning index (BI) of T. molitor powder, defatted fraction and protein fractions was calculated following Equation 3 (Bußler et al., 2016):

(Equation3) where x is calculated following Equation 4:

(Equation 4) Foaming properties [0071] For foaming properties (capacity and stability), emulsifying properties (capacity and stability) and water/oil binding capacity (WBC and OBC), besides the T. molitor samples (powder, defatted and protein fractions), commercial protein concentrates (Whey – Prozis®, 71% PC; Pea protein - Bettery®, 78% PC) were also evaluated. All these evaluations were performed in duplicate. [0072] Foaming properties (Foaming Capacity – FC – and Foam Stability – FS) were evaluated according to Gravel et al. (2021) and Zielinska et al. (2018) with slight modifications.10 mL (Vi) of 2% solutions (dH2O) of each sample were homogenised (IKA Ultra-Turrax T25) at 15,000 rpm for 2 min. Total volumes were measured at 0 (V0), 5, 15, 30 and 60 minutes (Vt). Foaming capacity was calculated according to Equation 5, while foam stability according to Equation 6. (Equation 5)

(Equation 6) Emulsifying properties [0073] To evaluate the emulsifying properties (capacity and stability) a method was applied based on the studies by Zielinska et al. (2018) and Yu et al. (2021), with slight modifications. For this method, 10 mL of 1% solutions (dH₂O) were homogenized (IKA Ultra-Turrax T25) with 10 mL of vegetable oil at 15,000 rpm for 2 min. Afterwards, the solutions were centrifuged at 3000 x g for 5 min. Both the total volume (V_total) and the emulsion volume (V₀) were registered, and the emulsion activity (EA) was calculated as follows (Equation 7):

(Equation 7) [0074] The emulsions were heated in a water bath at 80 °C for 30 min, and the emulsion volume (V30) was then registered to calculate the emulsion stability (ES) (Equation 8):

(Equation 8) Water binding capacity and oil binding capacity [0075] For the evaluation of water binding capacity (WBC – Equation 9) and oil binding capacity (OBC – Equation 10), the methods outlined by Zielinska et al. (2018) were followed with slight modifications. For WBC, 0.5 g (m₁) of each sample was homogenized (stirring for 30 min at 540 rpm) with 20 mL of dH₂O (V) and then centrifuged at 8,000 g for 15 min. Then, the insoluble fraction (pellet) was weighted (m2).

(Equation 9) [0076] For evaluation of OBC, 0.5 g (mO1) of each sample was homogenized (vortex for 30 s) with 10 mL of vegetable oil (V) and then centrifuged at 8,000 g for 15 min. Then, the insoluble fraction (pellet) was weighted (m_O2).

(Equation 10) Statistical analysis [0077] All data were analysed using the software Statistical Package for Social Sciences (SPSS) - version 27 ® with all the statistical tests applied at 95% confidence level. [0078] All the results are presented with descriptive statistics (mean and standard deviation). [0079] The measured parameters (protein content, colour, WBC, OBC, FC, FS, EA and ES) were evaluated with One-Way ANOVA with samples as a factor. Post-hoc analysis was applied with Tukey's test for comparison between multiple groups. RESULTS Efficiency of isolation [0080] Total protein determined by the BCA method (Table 2), allowed to ascertain that the better conditions for T. molitor solubilisation consisted of using NaOH 0.1 M as solvent and a temperature of 50 °C. These results are expected since T. molitor protein solubility is higher at alkaline pH, particularly at pH 9-12. [0081] Table 1. Protein content measured by BCA method (mean ± SD) for soluble fractions obtained under different conditions.

a, b, c – homogenous groups according to Tukey's post hoc test (p < 0.05). [0082] It was surprisingly found that the ultrafiltration method of the present disclosure led to fractions with a protein content above 80% (Table 3). Furthermore, there are significant differences in the protein content of the fractions obtained from ultrafiltration, with the >50 kDa fraction presenting a protein content almost twice as high as the <50 kDa fraction (81.5 ± 0.06 vs 44.2 ± 1.6). Due to this poor protein content, the <50 kDa fraction was not considered for further characterization. The protein content exhibited in the >50 kDa fraction can be considered particularly high since most studies that have recovered proteins from T. molitor reported a protein content ranging 60-80% (Anusha and Negi, 2023; Bußler et al., 2016; Gkinali et al., 2022; Gravel et al., 2021; Kim et al., 2020; Laroche et al., 2019; Purschke et al., 2018a; Yi et al., 2017; Zhao et al., 2016). Table 3. Protein content of freeze-dried protein fractions (g/ 100 g DM, mean ± SD).

a, b, c, d – homogenous groups according to Tukey's post hoc test (p < 0.05). [0083] The protein molecular weight (MW) profile of the fractions (Figure 1) explains some of the observed differences in protein content. The >50 kDa fraction presented a very similar profile as the supernatant, with only a slightly lower proportion of proteins with a MW below 13.7 kDa. On the other hand, the IP fraction presented a higher proportion of proteins with MW > 29 kDa, greater than all the other fractions (while presenting a remarkably similar profile as the >50 kDa fraction for proteins with a MW < 29 kDa). The < 50 kDa fraction can be referred to as a ‘peptides fraction’, since it is mainly composed of proteins and amino acids with very low MW (<1.2 kDa), which can also explain its relative low protein content. Both the supernatant and the >50 kDa fraction had a higher proportion of protein below < 29 kDa, which can include cuticle proteins or cockroach allergen-like protein ~15 kDa. The proteins with higher molecular weight, mostly present in the IP fraction, can correspond to melanisation-inhibiting protein (43kDa), β-glycosidase (59 kDa), trypsin-like proteinases (59kDa), melanisation-engaging types of protein (85kDa), vitellogenin-like protein (160 kDa), myosin heavy chain (225.4 kDa) or myosin-2 (223 kDa). However, it cannot be excluded that isoelectric precipitation caused some protein aggregation, which contributed to the higher proportion of molecules with a higher molecular weight. [0084] Figure 2 is a schematic representation of the protein isolation methods, with information about the fraction yield and protein recovery. Although the >50 kDa fraction presented lower protein content than the IP fraction, it led to a slightly higher protein recovery relative to the T. molitor powder (27.9% vs 26.8%). Colour [0085] Table 4 presents the colour properties of the different T. molitor samples. Overall, T. molitor processing (defatting, solubilisation, isoelectric precipitation or membrane ultrafiltration) significantly improved the colour of the samples, as observed by higher L* values and much lower BI. The supernatant presented the highest ΔE values while also exhibiting the highest L* and b* values among all the samples. Isoelectric point precipitation and membrane ultrafiltration decreased L* and b* values and increased BI, but this effect was more pronounced for the IP fraction. Nevertheless, application of protein isolation did not necessarily lead to better colour properties when compared to defatting, since the defatted fraction presented the lowest BI values. The improved colour properties of the T. molitor defatted fraction and protein fractions present an opportunity to increase consumer acceptance of insect-based products. The incorporation of insect powder into food products generally leads to a darkening/browning of the samples, and this can have a very negative impact on consumers' liking of the products (particularly with bakery-type products or snacks) (Ribeiro et al., 2019; Ribeiro et al., 2022b; Wendin et al., 2021). The better colour properties exhibited by the > 50 kDa fraction relative to the precipitate (higher L*, b* values and lower BI values), represents an additional advantage for food product incorporation, as it would guarantee that the appearance of the developed products would not be negatively affected by the incorporation of the insect protein fractions. Table 2. Colour coordinates, browning index (BI) and total colour variation (ΔE) (mean ± SD) for T. molitor powder, defatted fraction and freeze-dried protein fractions.

a, b, c, d, e, f – homogenous groups in each column according to Tukey's post hoc test (p < 0.05). Foaming properties [0086] Concerning the foaming capacity of the different samples (Table 5), all the T. molitor protein fractions presented higher FC (foaming capacity) than either the powder or the defatted fraction. In particular, it was surprisingly found that the fraction obtained by ultrafiltration (>50 kDa – 74.9 ± 2.3) presented the highest FC among all samples. The higher foam capacity exhibited by fraction obtained by ultrafiltration could be due to their higher protein and low-fat content. Furthermore, the presence of proteins with higher molecular weight in the IP fraction (Figure 1) can explain the lower FC demonstrated by this fraction when compared to the >50 kDa fraction. [0087] Regarding the foam stability (FS) (Table 5), relative to the supernatant fraction, the >50 kDa fraction presents improved FS, particularly at 15 and 30 min. Relative to the commercial samples, the whey protein concentrate presented very low FS, with no foam observed at 15 min. On the other hand, the pea protein concentrate exhibited relatively higher FS than the supernatant or >50 kDa (at 15 and 30 min), but the FC of this sample is considerably lower (Table 5). [0088] Table 5. Foaming capacity (FC) (%, mean ± SD) and foam stability (FS) (%, mean ± SD) of T. molitor powder, defatted fraction, freeze-dried protein fractions and commercial protein concentrates at different times: 5, 15, 30 and 60 minutes.

a, b, c - homogenous groups in each column according to Tukey's post hoc test (p < 0.05). A, B - homogenous groups in each row of FS results according to Tukey's post hoc test (p < 0.05). Emulsifying properties [0089] Concerning the emulsifying properties (Table 6), it was not possible to observe an overall improvement according to a higher level of protein isolation as it occurred for the foaming properties. Nevertheless, the supernatant and the >50 kDa fractions presented improved emulsifying properties concerning the T. molitor powder and defatted fraction. The >50 kDa fraction presented the highest EC (emulsifying capacity) of all samples, significantly higher than the commercial whey protein concentrate. Furthermore, although this fraction presented lower emulsion stability than the commercial pea protein concentrate, this difference was not statistically significant. On the other hand, the IP fraction presented very poor emulsifying properties related to capacity and stability (it presented the lowest emulsion stability of all the tested samples). Protein denaturation that occurs when proteins are precipitated might explain the relatively poorer techno-functional properties of the IP fraction in comparison to the >50 kDa fraction. Water and oil binding capacity [0090] Except for the pellet, all the samples presented very low WBC (water bind capacity) (Table 6). In particular, the supernatant and the >50 kDa fractions exhibited a non-existent WBC, meaning that both samples dissolved completely when homogenized with water. On the other hand, for OBC (oil binding capacity), different behaviours between the samples were observed. As it occurred for WBC, the pellet exhibited the highest OBC. However, it was also possible to observe that the solubilisation of T. molitor protein increased its OBC, with the supernatant and >50 kDa fractions exhibiting higher OBC than the T. molitor powder and the defatted fraction. Furthermore, both fractions also presented higher OBC than the IP fraction, although all insect protein fractions presented superior OBC than commercial protein concentrates (whey or pea protein). [0091] The protein fractions demonstrate a high OBC, which may be extremely important for future food product development. OBC enhances sensory properties such as mouthfeel and flavour retention. As such, T. molitor protein fractions obtained through the present method could can be incorporated into food products where the physical entrapment of oil/fat is critical (e.g., ground meat products, doughnuts, and baked products). Akin to other edible insect species, T. molitor is mostly incorporated into baked goods, snacks and meat products, and although these products can present acceptable nutritional and techno-functional properties, their sensory properties can be negatively perceived by consumers. Several other studies have demonstrated that defatted fractions present better sensory properties than the respective powder. The high protein content and excellent techno-functional properties make the >50 kDa fraction an improved ingredient to develop functional products (e.g., protein bars or shakes) with high nutritional value. Table 6. Emulsifying capacity and stability after 30 minutes, water binding capacity and oil binding capacity (%, mean ± SD) of T. molitor powder, defatted fraction, freeze-dried protein fractions and commercial protein concentrates.

a, b, c, d, e, f – homogenous groups in each column according to Tukey's post hoc test (p < 0.05). EXAMPLE 2 (Chitin extraction and chitosan synthesis) Materials and methods [0092] The freeze-dried pellet (insoluble fraction) resulting from the alkaline solubilisation applied to defatted T. molitor was used as a substrate for chitin extraction. Chitin extraction and chitosan production [0093] For chitin extraction, conventional chemical extraction consisting of acid (demineralization) and alkali (deproteinization) hydrolysis was applied. Briefly, pellet samples were initially treated (1:20, w:v) with an aqueous solution of 1M HCl (37%) under constant stirring at 50 °C for 3 h. The demineralized samples were filtered by vacuum filtration through a 60-68 μm pore size paper filter and washed with distilled water until pH neutrality was achieved. For deproteinization, the demineralized samples were treated (1:20, w:v) with an aqueous solution of 1 M NaOH solution under constant stirring at 95 °C for 3 h. The obtained samples were filtered and washed as mentioned above. Next, the samples were dried for 4 h at 95 °C (air-flow lab incubator, Binder® APT. line series model ED115). [0094] After chitin extraction, a decolorization step based on an oxidation-reduction reaction was performed. Briefly, the chitin samples were subsequently treated with aqueous solutions of 0.5% KMnO4 and 0.5% C2H2O4, under constant agitation at 200 rpm for 1 h at room temperature with filtration and washing between treatments as mentioned above. Afterwards, the samples were dried for 4 h at 95 °C (air-flow lab incubator, Binder® APT. line series model ED115). [0095] Chitin yield was calculated relative to the pellet and relative to the T. molitor flour/powder used for defatting and subsequent alkaline solubilisation. [0096] For chitosan production, an alkaline deacetylation procedure was applied, consisting of the homogenization (1:50, w:v) of the different decolorized chitin samples with a 50% (w:w) NaOH solution at 100 ºC for 4 h under constant stirring. The deacetylated samples were filtrated and washed as mentioned before. The obtained chitosan samples were dried for 4 h at 95 ºC (air-flow lab incubator, Binder® APT. line series model ED115). [0097] Chitosan yield was calculated relative to the pellet, to the chitin and relative to the T. molitor flour/powder used for defatting and subsequent alkaline solubilisation. Chitin and chitosan characterisation [0098] Both chitin and chitosan were structurally characterized by Fourier-transform infrared spectroscopy - with attenuated total reflectance (FTIR-ATR), with a Bruker spectrometer (Alpha, Bruker Optic GmbH) using a diamond crystal. The samples were placed directly on the crystal prism, and two spectra per sample were acquired with 64 scans per spectrum at a spectral resolution of 4 cm^-1 in the spectral region of 4000-400 cm^-1. [0099] The viscosity-average molecular weight (Mw) of chitosan was determined through a viscosimetric method according to Malm & Liceaga (2021), with slight modifications. An Ubbelohde Dilution Viscometer (Xilem-SI analytics, Germany) with a capillary size of 0.53 mm was used in a water bath at a constant-temperature (25 ºC). For each chitosan sample, solutions ranging from 1.5 mg/mL to 5.5 mg/mL were prepared. The solvent system used for the dilution of the samples consisted of equal proportions of 0.1 M acetic acid solution and 0.2 M NaCl solution. The Mw of the different chitosan samples was estimated by the Mark- Houwink equation (Equation 11): (Eq.11) where [η] is the intrinsic viscosity K = 1.81 x 10-3 and α = 0.93 are constants dependent on the type of the solvent-solute system and the temperature used [31]. [00100] The obtained chitin and chitosan from the pellet resulting from alkaline solubilisation of defatted T. molitor were compared with chitin and chitosan obtained from T. molitor, house cricket (Acheta domesticus) (obtained under the same extraction conditions, and with the application of previous defatting), as well as commercially available chitin (CAS: 1398-61-4) and chitosan (CAS: 9012-76-4, low viscosity) powder. RESULTS Chitin and chitosan yield [00101] Chitin extraction yield relative to the pellet was 9.60 ± 1.39%, while chitosan yield was 6.20 ± 0.26% relative to the pellet and 60.09 ± 2.14% relative to the chitin. Performing estimations based on defatting and solubilisation yields (Figure 2), from 100 g of T. molitor it would be possible to obtain 3.81g of chitin and 2.46g of chitosan, which are slightly lower values than chitin extraction/chitosan synthesis directly from either T. molitor (4.77g and 3.82g, respectively) and or A. domesticus (5.34g and 3.77g, respectively). Chemical composition (FTIR-ATR analysis) [00102] The chemical composition of the insect-based chitins is extremely similar to the one presented by the commercial shrimp chitin (Figure 3a). The chitins analysed in this study presented three peaks at around 1651-1652 cm^-1 , 1620-1622 cm^-1 , and 1550-1551 cm^-1, which are characteristic peaks of the α-allomorphic form (Jang et al., 2004). These peaks are ascribed to the C=O secondary amide stretch (Amide I) and the N-H bend and CN stretch (Amide II), respectively. [00103] Evaluating the different FTIR-ATR spectra of the insect-based chitosan samples and the commercial shrimp chitosan sample, a similarity is also visible (Figure 3b). Peaks around 1650 and 1590 cm⁻¹, corresponding to C=O in the NHCOCH3 group (Amide I band) and the amine (NH2) in the NHCOCH3 group (Amide II band), are characteristic for chitosan (Erdogan & Kaya, 2016). The data acquired from the FTIR-ATR spectra of the different chitosan samples confirmed the presence of these bands at around 1644-1587 cm^-1, 1648-1584 cm^-1 and 1656- 1582 cm^-1 for A. domesticus, T. molitor (flour/powder) and T. molitor (pellet) chitosan, respectively. The presence of these two bands indicates the formation of chitosan, and the weakened band at 1644-1656 cm^-1 (C=O) in the NHCOCH3 group (Amide I band) suggests successful deacetylation (Figure 3b). This band intensity is attributed to the degree of N- deacetylation, which increases when the intensity is lower (Glab et al., 2021). Molecular weight [00104] Molecular weight (Mw) is one of the most important chitosan parameters, helping dictate its industrial application due to its influence on the physicochemical and biological properties. Although non-significant statistical differences were found in the average molecular weight between all samples, the chitosan extracted from T. molitor pellet (201.52 ± 87.25 kDa) presented the lowest Mw (Table 7), being classified as medium molecular weight. However, chitosan from T. molitor flour/powder (302.58 ± 50.29 kDa) and the commercial shrimp chitosan (292.38 ± 41.56 kDa) were also classified as medium molecular weight, while A. domesticus chitosan (332.58 ± 73.37 kDa) was characterized by a high molecular weight (Mohan et al., 2022). It is known that high molecular weight chitosan can be applied in wastewater treatment, the papermaking industry, as a foliar treatment agent, as well as in wound healing, while low molecular weight chitosan is usually preferred in the pharmaceutical industry due to its role in drug delivery and higher antimicrobial activity (Kou et al., 2022; Mohan et al., 2022; Mohan et al., 2020; Petronela, 2017). [00105] Table 7. Average molecular weight (kDa; mean ± SD) of the different chitosan samples (n = 2).

ns – non-significant differences according to One-Way ANOVA (p < 0.05). EXAMPLE 3 (comparative example) [00106] Initially, the method was tested at a laboratory scale. For the lab-scale test, consecutive batches of 100 g (total 200 g) of euthanized Tenebrio molitor larvae (frozen) were blanched for 5 min at 100 ml of boiling water (for each batch of 100 g). Afterwards, insects were dried in an oven at 80°C for 7 hours and ground. The dried and ground insects were defatted with the Soxhlet method for 6 hours with ethanol as a solvent (18.5g of dried and ground insect to 700 mL of solvent in each extraction, in a total of 3 extractions), while the fat was recovered after treatment with rotary evaporator. [00107] The defatted insects (40 g) were homogenized with 2000 ml of an aqueous solution of NaOH 0.1 M and subsequently centrifuged under optimal solubilization conditions (1:50 w:v ratio, pH 13, 50 °C, 4 hours, 3,913 x g for 30 min at 4 °C). [00108] For the isoelectric point precipitation, the supernatant pH was adjusted to 4.56 and then centrifuged. The pellet (precipitated protein) was freeze-dried and further characterized. [00109] It was obtained 10.9 g of precipitate (9.6 g of protein) and 15.6 g of fat. [00110] Table 8 describes the protein concentrate obtained with the precipitation method. [00111] Table 8. Protein concentrate obtained with the precipitation method.

EXAMPLE 4 [00112] Initially, the method was tested at a laboratory scale. For the lab-scale test, consecutive batches of 100 g (total 200 g) of euthanized Tenebrio molitor larvae (frozen) were blanched for 5 min at 100 ml of boiling water (for each batch of 100 g). Afterwards, insects were dried in an oven at 80°C for 7 hours and ground. The dried and ground insects were defatted with the Soxhlet method for 6 hours with ethanol as a solvent (18.5g of dried and ground insect to 700 mL of solvent in each extraction, in a total of 3 extractions), while the fat was recovered after treatment with rotary evaporator. [00113] The defatted insects (40 g) were homogenized with 2000 ml of an aqueous solution of NaOH 0.1M and subsequently centrifuged under optimal solubilization conditions (1:50 w:v ratio, pH 13, 50 °C, 4 hours, 3,913 x g for 30 min at 4 °C). [00114] The supernatant (1430 mL) was filtrated with a 50 kDa membrane. Both the not- filtrated fraction (> 50 kDa; 410 mL) and permeate (< 50 kDa; 1020 mL) were recovered and freeze-dried (> 50 kDa – 12.3g; < 50 kDa – 10.2g). According to results the > 50 kDa fraction had similar yields and purity (protein content) as the precipitated protein. [00115] Table 9 describes the protein concentrate obtained with the ultrafiltration method (laboratorial scale) of the present disclosure. Table 9. Protein concentrate obtained through the ultrafiltration method (laboratorial scale) of the present disclosure

[00116] Additionally, the insoluble fraction (pellet) (22.07 g, freeze-dried) was treated in order to obtain chitin and chitosan. As such, acid hydrolysis (adding 441.4 ml of 1M HCl) followed by alkaline hydrolysis (adding 441.4 ml of 1M NaOH) were applied in order to obtain unbleached chitin. Decolorization (133.5 mL KMnO40.5% (w/v)/133.5 mL C2H2O40.5% (w/v)) was applied to untreated chitin in order to obtain decolorized chitin (2.12g). Chitin was then deacetylated under aggressive alkaline conditions (106 mL of 50% (w/v) NaOH solution) in order to produce chitosan (1.37g). [00117] The present disclosure discloses a method to obtain high-value protein concentrates from T. molitor with membrane ultrafiltration (cut-off 50 kDa), while comparing the obtained fractions with the commonly applied alkaline solubilisation followed by isoelectric point precipitation method. The >50 kDa fraction presented high protein content (> 80.0 and both fractions attained similar protein recovery rates). Notably, the >50 kDa fraction exhibited superior techno-functional properties, including better colour, foaming, emulsifying properties, and oil binding capacity than the IP fraction. The >50 kDa fraction also had better techno-functional properties than commercial whey, and pea protein concentrates. These results demonstrate the potential of the method comprising ultrafiltration herein disclosure to obtain high-quality T. molitor protein fractions, that could be further incorporated into functional food products. [00118] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. [00119] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable. [00120] The following dependent claims further set out particular embodiments of the disclosure. REFERENCES Anusha, S., & Negi, P.S. (2023). Characterization and techno-functional properties of Tenebrio molitor larvae protein concentrate. Food Bioscience, 5,: 102882. https://doi.org/10.1016/j.fbio.2023.102882 Ardoin, R., & Prinyawiwatkul, W. (2020). Product appropriateness, willingness to try and perceived risks of foods containing insect protein powder: A survey of U.S. consumers. International Journal of Food Science & Technology, 55(9), 3215-3226.

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Claims

C L A I M S 1. Method of fractionation of an edible insect comprising a step of protein purification by ultrafiltration.

2. Method according to the previous claims wherein said method comprises the steps: providing grinded, defatted insects raw material; extracting proteins from the grinded, defatted insects raw material for obtaining a soluble fraction comprising proteins and a non-soluble fraction; submitting the soluble fraction comprising proteins to an ultrafiltration in order to obtain a purified soluble fraction comprising proteins.

3. Method according to the previous claim further comprising a step of submitting the non- soluble fraction to chemical hydrolysis, decolorization and alkaline deacetylation in order to obtain chitosan.

4. Method according to any of the previous claims wherein the ultrafiltration is performed with a membrane comprising a pore size ranging from of 30-70 kDa.

5. Method according to the previous claim wherein the ultrafiltration is performed with 50 kDa membranes.

6. Method according to any of the previous claims 2-5 wherein the extraction step is performed with an aqueous NaOH solution; preferably wherein the weight:volume ratio between the aqueous NaOH solution/ defatted insects raw material is 1:50; more preferably the aqueous NaOH solution is a 0.001 – 0.1M NaOH solution.

7. Method according to any of the previous claims 2-6 wherein the extraction step is performed at a temperature ranging from 40-60°C and at a pH ranging from 11-13.

8. Method according to any of the previous claims 2-7 wherein the extraction step comprises a centrifugation; preferably at 4000-8000g for 30-40 min.

9. Method according to any of the previous claims 2-8 wherein the defatting of the insect is performed with a Soxhlet apparatus; preferably during 6 hours with ethanol as a solvent and at a w:v ratio of 1:20.

10. Method of fractionation of an edible insect, which comprises: blanching the insect, drying the blanched insect, defatting the insect, homogenizing the defatted insect with an aqueous solution of NaOH, centrifuge the resulting solution, obtaining a supernatant fraction comprising proteins and a non-soluble fraction, ultrafiltration of the supernatant fraction comprising proteins in order to obtain a purified soluble fraction comprising proteins.

11. Method according to the previous claim further comprising a step of chemical hydrolysis and alkaline deacetylation of the non-soluble fraction.

12. Method according to the previous claim 10 wherein the blanching is done by boiling the insect at 100°C during at least 5 minutes.

13. Method according to the previous claim 10 wherein the drying of the blanched insect is obtained at a temperature ranging from 50°C to 100°C for 5 to 10 hours; preferably at 80°C for 7 hours.

14. Method according to the previous claim 10 wherein the homogenization of the defatted insects is obtained in an aqueous 0.1M NaOH solution at 1:50 weight:volume ratio, at 50°C, preferably for 4 hours under constant agitation at 150 rpm, followed by a centrifugation at 3993 x g, for 30 min, and at 4°C.

15. Method according to the previous claim 10 wherein the non-soluble fraction was subsequently treated with HCl and NaOH to obtain unbleached chitin.

16. Method according to the previous claim wherein the unbleached chitin is decolorized with KMnO₄/C₂H₂O₄.

17. Method according to any of the previous claims 15-16 wherein the unbleached chitin is deacetylated with a NaOH solution to obtain chitosan.

18. Method according to any of the previous claims wherein the insect is selected from the list consisting of: Tenebrio molitor larvae, Locusta migratoria, Acheta domesticus; Alphitobius diaperinus; preferably Tenebrio molitor larvae.

19. Purified soluble fraction comprising proteins obtained through the method described in any of the previous claims, wherein the purified soluble fraction comprising proteins comprises a protein content of at least 80.0 g/100g on a dry matter basis.

20. Powdered protein extract comprising the purified soluble fraction comprising proteins obtained through the method described in any of the previous claims, wherein the powdered protein extract comprises a protein content of at least 80.0 g/100g on a dry matter basis.

21. Powdered protein extract according to the previous claim 20 comprising a lightness colour (L*) is of at least 60.0; measured by the CIELAB system.

22. Powdered protein extract according to the previous claims 20-21 comprising a browning index is bellow 80; measured by the CIELAB system.

23. Foodstuff ingredient comprising the purified soluble fraction comprising proteins according to the previous claim 19 or the powdered protein extract according to any of the previous claims 20-22.

24. Ingestible product comprising the purified soluble fraction comprising proteins according to the previous claim 19 or the powdered protein extract according to any of the previous claims 20-22.

25. Ingestible product according to the previous claim wherein the ingestible product is a food product, a nutraceutical product, a pharmaceutical product or a dietary supplement.