Study on Functional, Structural, Thermal properties of Insect protein source extracted from black soldier fly pre-pupae (Hermetia illucens)

Study on Functional, Structural, Thermal properties of Insect protein source extracted from black soldier fly pre-pupae (Hermetia illucens) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Study on Functional, Structural, Thermal properties of Insect protein source extracted from black soldier fly pre-pupae (Hermetia illucens) Monisha Chandran, Loganathan Manickam This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7654817/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Edible insect’s specifically black soldier fly is hailed as a naturally profitable source of alternative protein source that can be used to replace existing food and feed sources. The functional, thermal, crystallinity and structural properties of black soldier fly prepupae (BSFP) protein were investigated in this study. The protein content in BSFP flour before and after defatting was found to be increased from 37.7% to 46.97%. The functional properties of BSFP protein such as WAC (75.53%), OBC (83.78%), FC (112.78%), and EC (46.32%) shows a huge viability to use as a functional ingredient in food sectors. The endothermic peak at 170℃ in DSC reveals the high thermal stability of modified BSFP protein due to strong pH shift (Iso-electric) method. The electrostatic modification and hydrophobic interaction triggers the modified protein with enhanced physical, functional, thermal properties. The morphology of the BSFP flour sample in SEM was found to be agglomerated together due to high temperature prevailed during defatting process. The smooth globules structure depicts the protein embedded on the surface with porous structure due to freeze drying. The XRD image with 2 peaks at 2θ = 8.28° and 2θ = 19.44° depicts the BSFP protein is in crystallinity structure and the particle size was found to be 8.3434 nm. The insect protein from black soldier fly prepupae was found to have better functional and thermal properties which paved path as a commercial functional ingredient and novel protein alternate in food industry. Insect protein future foods protein characteristics acid alkali extraction pH shift method protein structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights 1. The functional characteristic of insect (BSFP) protein depicts the prominent features of alterative proteins in food sectors. 2. The thermal and structural properties exhibit the potential opportunity to use insect protein as an alternative food source. 3. The marketable exploitation of the undervalued BSFP protein capitalizes of its potential in future foods such as tailored foods, RTE, meat alternates, 3D and 4D food printings. 1. Introduction The edible insect is emerging as a sustainable and innovative nutritional source to converge the futuristic world demand and will be available at low cost. In ancient time there is a direct historical evidence for eating edible insects (Govorushko et al., 2019; Baiano 2020 ; Rumpold 2013). Edible insect protein is an innovative protein source that emanates from sustainable sources. The edible insect has various potential benefits, including increased nutritional value and reduced greenhouse gas emissions, feed conversion ratio, forest biomass waste, usage in land and water for rearing (Baiano, 2020 ; Rumpold 2013; Van Itterbeeck 2012 ; Varelas 2013). The market demand for protein from edible insect is growing rapidly it is predicted the global insect protein will reach 23% CAGR in 2027 to reach USD 400 billion. Thus the consumption of meat is increasing drastically, which will lead to increase all the factors dependent of growing livestock such as usage of feed, land and water (Gallo, 2019 ). The edible insect feed energy conversion ratio is higher than meat industry which reveals a positive sign for sustainability (Halloran et al., 2014 ). Apart from higher feed energy conversion efficiency, the insect reproducibility were also higher in rate such as yellow mealworm beetle T.molitor lays 250–1000 eggs and black soldier fly lays 500 to 900 eggs. This depicts the sustainability of insect sources with promising nutritional value with less carbon foot prints (Gere et al., 2019; Gold et al., 2020 ; Govorushko, 2019 ). The urbanization results increase in demand for food and feed sources, which exacerbates global food security issues (Baiano, 2020 ; Gere, 2019). There are already a host of new companies producing edible insects such as the black soldier fly, mealworm, cricket, locust fly, and other species (Mishyna et al., 2020). The insect protein incorporated food products such as breads, burgers, pizza and flour based biscuits are some of the existing examples (Bessa et al., 2020 ; Gonzalez et al., 2019; Montevecchi et al., 2021). However the utilization of insects is still scarce due to socio-economic influences. Processed insect-based foods may be a strategy to enhance customer preferences of edible insects because they are often regarded as filthy and having an unpleasant appearance in western culture (Van Huis, 2013). Thus, few studies have been conducted on the development of insect meal and protein processing technologies from Tenebrio molitor and H. illucens that could dispel consumer misconceptions about edible insects (Kim et al., 2020). Black soldier fly prepuape (BSFP) is an optimistic alternative source of protein for mass commercialization. The interesting fact is the BSFP will be reared on organic food waste ultimately converts into a valuable nutritional components such as protein, chitin and fats (Smets et al., 2019). The growing utilization of insects in human diets demands a greater focus on their safety risks. The assessment of edible insect safety by European food safety authority 2015 reported the need of safety with insect as any other form of food, entails the screening of microbial pathogens, parasites, toxins, heavy metals and allergens (Gravel et al., 2020). The interesting advantage of black soldier fly at adult stage was the insect does not depend on food material. The gut microbial enzymes of black soldier fly will suppress the toxin levels (Varelas et al., 2017). Few research depicts there is a chance of metal contamination in black soldier fly (Bessa et al., 2020 ). The microbial contamination can be prevented by proper hygienic processing techniques (Poma et al., 2017 ). In general there are few methods for protein precipitation such as aqueous, alkaline and pH shift method. The alkaline solubilization followed by acid precipitation method to recover protein with optimistic yield percent. Thus another common chemical method based on Osborne fractionation and enzyme-assisted method (Caligiani et al., 2018 ). Bußler et al. ( 2016 ) studied on defatted larvae of H.illucens and Tenebrio molitor for protein extraction using aqueous method using distilled water. The protein yield percent was found to be 82% for H.illucens and 83% for T.molitor using aqueous extraction method (Caligiani et al., 2018 ). Yi et al. ( 2013 ) investigated on protein fractions using aqueous extraction from edible insects. The supernatant holds 20% of total protein yield percent. Further the purity of supernatant ranges from 65 to 75 per cent. (Huang et al., 2019 ) studied on protein extraction using pH shift method on black soldier fly (H.illucens). The resulted protein exhibit with high amount of lysine and valine serves an excellent replacement of animal protein. The BSF prepupae protein contains all amino acids with highest concentration of aspartic and glutamic acid as well as lowest concentration with cystine, methionine, and tryptophan. The BSFP contains protein 30–53% and also. high amount of calcium, iron and zinc (Müller, Wolf, and Gutzeit 2017 ; Bußler et al. 2016 ; Finke et al. 2013; Liland et al. 2017 ). It is rich in lauric acid, which act as an antibacterial agent (Spranghers et al., 2017 ). The major advantage of BSFP protein was ease of protein digestibility when compared to the animal protein digestibility, additionally the cost of protein also less since it’s reared in food waste. Insect protein powders are most commonly used in pasta, cookies, burgers, patties, and healthy snacks as a source of protein. Insect protein has a promising future utility in culinary applications when utilized in a veiled forum (Huang et al., 2019 ). However, limited studies are available for novel insect protein as a functional ingredient in food sectors. Thus the study explores the possibility of valorization of insect protein in human diets as a functional component. The major aim of the study was to characterize the BSFP protein such as functional, structural and thermal properties which paves a path in incorporation of BSFP protein in food substances. 2. Materials and Methodology The Black soldier fly ( Hermetia illucens) pre-pupae were reared in NIFTEM-T using spent foods like corn cob, leftover grain waste, fruit peels and vegetable waste. All of the analytical-grade chemicals required for the experimental purpose were purchased from Sigma-Aldrich (India). 2.1 Preparation of Defatted Insect flour The BSF prepupae stage is selected for this research work. The BSFP were gently cleaned and disinfected with chlorinated water. Then BSFP was freeze-dried for 24 hours and pulverized with the particle size of < 0.40 mm (mesh size). The BSFP insect flour was defatted in a soxhlet apparatus using n-hexane as the solvent for 6 hours (AOAC 920.39). Following, the defatted insect flour is kept in vacuum-sealed pouches and stored under − 18°C (Chandran et al, 2022). 2.1.1 Nutritional profile for Raw and Defatted BSFP flour The protein in raw and defatted flour was determined (AOAC method 984.13) using Kjeldhal apparatus (Kjeltec 2300 analyser, Sweden). The lipid was assessed using soxhlet apparatus (AOAC 920.39). The crude fiber estimated using Weende’s method. The ash was estimated using muffle furnace (AOAC 942.05). The energy value was computed for the freeze dried defatted flour (Chandran et al., 2022). 2.2 Extraction of protein from black soldier fly prepupae The protein was extracted (pH shift or Acid-alkali method) by adjusting the Iso-electric point of defatted insect flour (Huang et al., 2019 ; Bußler et al., 2016 ). The defatted insect flour was treated with NaOH (1M) solution with the ratio of 1:10 adjusted to a pH of 8.3 (Fig. 1 ). The solution was kept in an orbital stirrer at 250 rpm for 2 hours at ambient condition. The mixture was filtered and the aliquot was collected. The filtrate was centrifuged at 8000 rpm for 30min at 4℃. The recovered clear supernatant was precipitated by adjusted the pH 4.8 using 1M HCl precipitation. The precipitated sample was centrifuged again at 8000 rpm for 15min at 4℃and washed for several times. The frozen protein was lyophilized at -80℃ for 24 hours. 2.3 Characterization of insect protein 2.3.1 Protein percent yield recovery The pH shift method was used to extract the protein from BSFP. The following Eq. (1) was used to compute the yield of protein powder (Smets et al., 2020 ; Huang et al., 2019 ; Bußler et al., 2016 ). Protein yield % = \(\:\frac{w\:Freeze\:dried\:protein\:extract}{m\:Dry\:mass\:\times\:\:w\:protein}\times\:100\) (1) 2.3.2 Estimation of protein percentage using Kjeldahl apparatus The protein content was assessed in the Kjeldahl apparatus. The nitrogen-to-protein conversion factor as Kp 6.25 which is often used for insects, it was found that there is an overestimation of nitrogen content due to the presence of chitin compound. Recently, the correct Kp value for protein extracts from H. illucen s was found to be 5.71 which was used in this research (Caligiani et al., 2018 ; Nery et al., 2018 ; Janssen et al., 2017 ). 2.4 Evaluation of techno functional properties of freeze dried insect protein 2.4.1 Water absorption capacity (WAC) The protein samples were weighed 0.5g and suspended in 5ml distilled water and stirred for 5minutes. The sample was then centrifuged at 3000 rpm for 10 minutes before being weighed after the supernatant was decanted (Wang et al., 2021 ; Purschke et al., 2018 ). The following Eq. ( 2 ) was used to calculate the amount of water absorbed by the BSFP protein samples. $$\:\text{W}\text{B}\text{C}\:\left(\frac{{g}_{w}}{{g}_{DM}}\right)\:=\:\left(\frac{{W}_{0}-{W}_{1}}{{W}_{0.DM}}\right)$$ 2 Where, W 0 DM is the initial sample weight, W 0 is the tube weight with sediments, and W 1 is the final weight after decantation. 2.4.2 Oil binding capacity (OBC) The oil binding capacity termed as the amount of oil adhered to defatted BSFP flour (Batish et al., 2020 ). The protein samples 0.5g was combined with 5ml of canola oil and centrifuged at 3000 rpm for 30 minutes. The settled protein was weighed after the supernatant was decanted. The amount of oil adhered to the protein was assessed using the following Eq. ( 3 ). $$\:\text{O}\text{B}\text{C}\:\left(\frac{{g}_{w}}{{g}_{DM}}\right)\:=\:\left(\frac{{W}_{0}-{W}_{1}}{{W}_{0.DM}}\right)$$ 3 Were, W 0 DM is the initial insect protein powder, W 0 is the tube weight with sediments, and W 1 is the final weight after decantation. 2.4.3 Foaming capacity & stability The foaming capacity & stability was assessed according to the method described by Chatsuwan et al., ( 2018 ); Wang et al., ( 2021 ). The protein sample 1g was dispersed in 100 ml distilled water. The sample was adjusted to pH at 7.0 using 0.1N NaOH. The sample was agitated for 5min. The foaming volume was observed and recorded. The following Eq. (4) was used to assess the foaming capacity. Foaming stability was identified by the withstand ability of foam for 30 min. Thus the stability time period of the foam was recorded. FC % = \(\:\frac{({V}_{2}-{V}_{1})}{{V}_{1}}\times\:100\) (4) 2.4.4 Emulsifying capacity & stability The emulsifying capacity was determined as described in the method by Zielinska et al., (2018); Mshayisa and Van Wyk, ( 2021 ). 1g of protein was weighed and dispersed in 100 ml of distilled water. Following, 15ml of the protein sample was placed in a centrifuge tube and vortexed for 10 minutes. The sample solution was then vortexed constantly for 10 minutes after 15ml of maize oil was added. The emulsion was centrifuged at 3000 rpm for 10 minutes. The supernatant and residual volumes are measured. The following formula (5) was used to determine emulsion capability. Similarly, the BSFP protein (1g) was weighed and dispersed in 100ml water. The corn oil was then added to the sample solution and stirred constantly. The sample and the oil in water emulsion were both heated to 80°C / 30 minutes. The sample was cooled and centrifuged for 10 minutes / 3000 rpm and emulsion stability is recorded for 30 minutes. EC % = \(\:\left(\frac{Volume\:of\:emulsifies\:layer}{\begin{array}{c}Volume\:of\:\\\:suspension\end{array}}\right)\times\:100\) (5) 2.5 Morphology of insect protein The microstructure of protein is investigated using scanning electron microscope (VEGA3 TESCAN). The microstructure of defatted flour and protein were shown in 3 different magnifications (3000 x, 10,000 x, &15,000 x) at a accelerating voltage of 10 kV. 2.6 X-Ray Diffraction of insect protein X-ray diffraction pattern of insect protein was conducted using an XRD diffractometer (Bruker, D8 Focus, Malvern Panlytical-X‘Pert 3 ). The sample was taken in powder form and scanned over 2θ ranges between 20℃ and 80℃ at a rate of 2℃/min (Andressa Jantzen et al., 2020). 2.7 Thermal properties of insect protein Differential scanning calorimeter determines the thermal properties of BSFP protein (Mettler Toledo; DSC 3 STARe Systems). The protein is subjected to 30–600℃ (dt 1.0 s) with a nitrogen flow of 50 ml/min at heating rate of 5.0 K/min (Zhao et al., 2015). 2.8 Statistical Analysis The data analysis was performed by statistical analysis tool using the IBM SPSS software package (Version 28.0; IBM Corp, USA). The significant difference was determined by the Duncan multiple comparison test (p < 0.05) with 95% level of confidence. The OriginPro was used to generate spectra for XRD and DSC results. 3. Results and discussion 3.1 Nutritional Composition of defatted BSFP flours 3.1.1 BSFP Fat The Soxhlet extraction technique using n-hexane a solvent at 69–70℃ recovered a significant increase in lipid yield of 26.8% (P < 0.05) in the whole BSFP flour before defatting due to varying factors such as biorhythm, feed substrate, and pH. The adult larvae stop feeding once the pupal instar completes, which results in a complicated mixture of chemicals accumulating in the lipid portion of BSFP. This is due to the lack of viable mouthpart development, resulting in them to rely only on the reserves acquired during the larval and pre-pupal stages (Ravi et al., 2019 ). The observed results was slightly varied with 20.0% fat percent from BSF larvae was reported in Bußler et al. ( 2016 ) due to metamorphism and feed substrate used for rearing BSF. The Rumpold et al. (2013) reported that increased lipid content from black soldier fly has a prominent amount of EPA and DHA which can be alternate and replaced as a fish meal. Following, the defatted flour contains 4.5% (P < 0.05) of fat (Table 1 ). The efficiency may due to several factors such as solvent, treatment time, temperature. Similar findings were reported in these literatures (Smets et al., 2020 ; Abduh et al., 2018 ). Table 1 Proximate analysis of raw and defatted BSFP flour (Mean ± S.D.). Parameters Raw BSFP flour (per 100 g) Defatted BSFP flour (per 100g) Crude Protein (%) 37.8 ± 0.34 b 46.97 ± 0.37 a Fat (%) 26.8 ± 0.05 a 4.5 ± 0.03 b Carbohydrate (%) 19.1 ± 0.29 b 29.8 ± 0.09 a Ash (%) 9.0 ± 0.005 a 9.8 ± 0.02 a Moisture (%) 7.1 ± 0.29 b 8.8 ± 0.09 a Energy (kcal) 456 ± 1.55 a 419 ± 1.85 b The mean of the rows with different superscripts varies significantly (p < 0.05). 3.1.2 Protein The protein content was found to be 37.80% in BSFP flour before defatting and there was significant increase in protein concentration and yield 46.97% after defatting (Table 1 ). The protein content was calculated using 5.71 as a Kp factor for prepupae flour which gives more precise protein content (Janssen et al., 2017 ). It was observed that there is significantly difference in the protein content in before and after defatting BSFP flour samples (p < 0.05). The results were in accordance with the Bußler et al. ( 2016 ); Caligiani et al. ( 2018 ); Monisha et al. (2021). Similarly, Bußler et al. ( 2016 ) computed a protein content of 44 percent in black soldier fly larvae. The increase of protein content in defatting BSFP flour may be due defatting the protein content concentrates the thermal influence in soxhlet chamber for 6 hours. Although it is widely acknowledged that a folded protein can be disrupted by various factors (temperature, chemical denaturants, pH, force, pressure, and mutations) (Narayan et al., 2019 ). Thermal treatment causes partial unfolding of proteins thereby increase in protein percent in defatted BSFP flour, according to investigation of Purschke et al., ( 2018 ). 3.1.3 Carbohydrate The chitin compound (exoskeleton layer) is a modified polysaccharide that is predicted to be the carbohydrate. Chitin is prominent in the larvae, pupae, adult, sheddings, and cocoons of black soldier flies and also similar to protein, the chitin contains nitrogen content in its structure. The chitin content was found to be higher with 29.80% (P < 0.05) in defatted BSFP flour, which could be attributed to heat influencing the glucosamide bond breakage in the chitin structure (Table 1 ). The elevated chitin concentration in both treatments could be related to the BSF's pre-pupal stage, when the metamorphosis occurs. The chitin level of 17–18% was shown to provide similar results (Wong et al., 2019 ; Nery et al., 2018 ). The higher chitin concentration could be attributed to BSFP metamorphosis and feed substrate ingestion (Wang et al., 2017) 3.1.4 Ash Content The ash content does not significantly varies before (9.0%) and after defatting (9.8%) of BSFP flours with (P < 0.05) respectively (Table 1 ). The ash content was noted to have no notable difference in before and after defatting BSFP flours. It was predicted that BSFP had a high proportion of micronutrients such as zinc, calcium, and iron (Nery et al., 2018 ). 3.1.5 Moisture content The moisture content was found to have less than 10% (P < 0.05) which showed the freeze drying impact on flours (Table 1 ). The slight difference in moisture content before and after defatting BSFP flour is predicted due to the heat involved in soxhlet chamber which might remove the free water in the BSFP flour (Chandran et al., 2021). 3.2 Extracted Protein from defatted black soldier fly prepupae (BSFP) flour The protein was extracted by pH-shift method through modulating the Iso-electric point (pH 4.3) of protein from defatted black soldier fly prepupae (BSFP) flour. The protein content was determined using 5.71 Kp factor as nitrogen conversion factor for accurate protein concentrate in order to avoid the over estimation of nitrogen content from chitin (exoskeleton layer) N-acetylglucosamine in black soldier fly (Caligiani et al., 2018 ). The recovered biomass of protein percent was found to be 57% (semi solid bio-mass) from defatted BSFP flour with the protein content of 75% by kjeldhal analysis. The recovered protein yield is in accordance with the findings of (Bußler et al., 2016 ; Liew et al., 2023 ). The protein content may be due to denature of unstable protein while heat prevailed in soxhlet treatment during BSFP defatting. There are several factors which influence in yield percentage such as improper protein precipitation and solubility, ionic strength of acid-alkali treatment, and few amounts of proteins are insoluble at specific iso-electric point (Smets et al., 2020 ; Bußler et al., 2016 ; Huang et al., 2019 ). The protein purity was found to be 75% (P < 0.05) due to the strong acid and alkali treatment. Due to the convenience of use, the pH-shifting method has grown more popular in protein modification. With pH-shifting method, the pH is changed to an extremely alkaline or acidic level before being neutralisation (pH 7.0). This results in enhanced side-chain repulsions, which leads to the partial unfolding of proteins' secondary, tertiary, and quaternary structures, or "molten globule" structure (Pan et al., 2023 ). The purity of protein was 75% slightly decreased due to nucleic acid contamination while using strong acid and alkali (Loughrey and Matlock, 2016 ). Even though the protein recovery was high, the purity was reduced due to strong acid and alkali extraction method. The results were similar to the findings of earlier researchers Caligiani et al., ( 2018 ); Bußler et al. ( 2016 ). A globular protein structure inevitably determines the unique, very compact, highly structured configuration in which it is folded in its native state. The repetitive geometry of the hydrogen bonding sites in the polypeptide backbone gives rise to regions of regular conformation known as the α-helix and β-sheet. These regions collaborate to form the overall 3-D conformation, which is fueled and maintained by an array of parameters (Davis and Williams, 1998 ). Environmental variables such as pH, temperature, ionic strength, and solvent composition affect a protein's stability by dislodging numerous intramolecular interactions that are essential for the protein's stability and integration (Kishore et al., 2012 ). 3.3 Evaluation of techno functional attributes of BSFP protein The functional characteristics of the BSFP protein were thoroughly investigated inorder to introduce BSFP as a food ingredient or food grade material into the current food or food processing system. The chemical and topographical attributes of the protein surface are correlated to functional qualities such as wettability, dispersibility, solubility, foaming, emulsification, and oil and flavor binding (Wang et al., 2021 ). The interaction of a protein with water influences its water binding capabilities, which are generally alluded to as water absorption. The water absorption capacity of freeze-dried protein was found to be 75.53 per cent (Table 2 ). The increased WBC might be attributed to the BSFP concentrates enhanced protein composition contains higher hydrophilic groups which can adhere to water molecules. The results depicts that (BSFP) insect protein has a less water absorption capacity than plant and animal-based proteins. Few studies reported that water absorption for gluten was found to be in the range of 346.21–353.81 per cent and the commercial soy protein has increased WAC (426.82%) when compared to OBC of soya protein (90.5%) (Wang et al., 2021 ; Chatsuwan et al., 2018 ). The less absorption of water in insect protein may be due to several factors like precipitation, pH, ionic strength, concentration, isolation and drying (Bußler et al., 2016 ). The slight decline in WAC depicts the BSFP protein to have less amount of hydrophilic than hydrophobic amino groups. The oil binding capacity was found to be 83.78 per cent for black soldier fly pre-pupae. The hydrophobic components present on the surface of protein molecules which interact with oil are termed to as OBC (Batish et al., 2020 ). The foaming capacity was found to be 112.78 and the foaming stability was done for 30 min found to be 103.10 per cent. The slight increase in OBC have a positive correlation with the foaming characteristic in BSFP protein. The foaming stability will vary based on the pH of the protein sample (Chatsuwan et al., 2018 ; Wang et al., 2021 ). In the present study, the protein foaming capacity and stability was observed at the pH of 7.0. The emulsion capacity and stability of BSFP protein were found to be 46.32 and 98 per cent (Table 2 ) respectively. The emulsion stability revealed that the higher the electrostatic repulsion between oil droplets, the more stable the emulsion (Mshayisa and Van Wyk, 2021 ).The surface hydrophobicity of proteins influences their ability to emulsify, affecting the protein's inclination to absorb to the oil side of the interface. More disintegration is usually related with higher emulsion capabilities. The observed data for BSFP protein reveals a good indication of best utilization in food applications as a food ingredient and can replace soya protein. It can form highly soluble protein monomers and aggregates with outstanding emulsifying and foaming capabilities due to the redistribution of surface-active amino acid side chains. It was found that the alkaline pH-shifting method altered the native protein's structural structure more than the acidic pH-shifting process. (Pan et al., 2023 ) Table 2 Functional properties of protein isolates from BSFP (Mean ± S.D.). Parameters BSFP protein (%) WAC 75.53 ± 0.10 OBC 83.78 ± 0.03 FOC 112.78 ± 0.64 FS 103.10 ± 0.47 EC 46.32 ± 0.68 ES 98.00 ± 0.01 WAC = Water absorption capacity, OBC = Oil binding capacity, FOC = Foaming capacity FS = Foaming stability, EC = Emulsion capacity, ES = Emulsion stability. 3.4 Morphology evaluation of BSFP protein The morphology of protein from BSFP (A,B,C) was shown in the Fig. 2 . The proteins are made upon amino acids and peptides which have positive, negative and neutral charges on its surface. The BSFP protein depicts aggregation of protein, chitin along with other nucleic acids. The aggregation is predicted due to several factors such as heat involved during defatting process, pH, electrostatic force, and ionic strength (Baler et al., 2014) The smooth globules is predicted to be protein compounds which are embedded along with other compounds in aggregates due to electro static force on the protein surface in Fig. C. The protein is one of the classes of complex bio macromolecules, which has a clear hierarchy of structural levels that extends from the basic fundamental arrangement of amino acid sequences to multiprotein assemblies at the complex structures. The structural complexity of a single protein is determined by the complex interplay of electrostatic, hydrophobic, hydrogen bonding, and other interactions, and modifications to these interactions can result in major ubiquitination (Baler et al., 2014). The protein particles are observed to be in non-uniform in geometric shape and size in Fig. A, B, C. The nature of the protein compound is predicted to be smooth surface with irregular particle size and compact in structure in Fig. A. The protein powders are shown in the Fig. B. depicts the modified thermally stable proteins are scattered in surface and few complex proteins are in aggregated structure due to the aminoacids interactions in BSFP protein. Whereas, the stable globules protein scattered in heterogeneous particle sizes in Fig. A. Similar results were obtained in Xu et al., ( 2020 ); Indriani et al., ( 2020 ); Huang et al., ( 2019 ) the protein was observed to be aggregated into a complex structure due to the strong acid- alkaline extraction or pH shift method. 3.5 X-Ray Diffraction of BSFP protein The XRD pattern of insect protein was shown in Fig. 3 . The BSFP protein powder exhibited two peak intensity at 8.28 [°2θ] and 19.44 [°2θ] with d-spacing value of 10.65 [Å] and 4.56 [Å] respectively which clearly depicts the crystallinity structure. Also, two minor peaks observed at 32.72 and 35.08 [°2θ] with 2.81 and 2.55 [Å] d-spacing value. Li-Hua luo et al 2010 observed crystallinity of soy protein with peak intensity of 8.60° and 19.36°. The FWHM [°2θ] was found to be 1.1504, β and λ was 0.9; 0.15406 nm respectively. The (D) particle size of BSF protein was calculated as 8.3434 nm using Debye-Scherrer equation method. Protein crystallinity can be affected by amino acid neutralisation, pH, and the collapse of salt bridges forming between proteins (Li-Hua luo et al., 2010). It is found that very limited research done on insect protein crystallinity using X-ray diffraction. Similarly, Zhao et al., 2015 observed the peak intensity of soy protein at 9.99 and 19.60 by aqueous extraction. Thus the obtained BSFP protein is predicted to be crystalline in nature. 3.6 Thermal properties of BSFP protein The differential scanning calorimeter reveals the thermal stability of modified BSFP protein components due to strong acid- alkali Iso-electric precipitation. In order to evaluate the physical changes of samples with elevated temperatures over time, DSC employs thermal analysis. The thermogram in Fig. 4 depicts the melting point of BSFP protein powder. Initially, when the temperature increases all the moisture in the BSFP protein is removed. The sharp peak obtained 170.46 ℃ peak is the endothermic peak with Onset value of 169.74 and Endset value of 174.44. The enthalpy (ΔH) of BSF protein was found to be 90.05 J/g from the DSC curve. The Queiroz et al., 2021 reported that the endothermic peak at 200℃ for black soldier fly larvae protein, the difference is predicted to be the BSFP life stages and protein modification due to extraction methods were predicted to influence the thermal stability of protein. The thermal stability of insect protein was found to be high at 170℃, which can be commercially used as food ingredients based on its better thermal stability compared to plant and animal based protein. Some native and non-native hydrophobic interactions may be retained or even increased at high temperatures, despite the fact that many of the protein's stabilising interactions are broken when it is thermally unfolded or denatured (Kelly and Gage, 2021 ). 4. Conclusion and future scope The futuristic food like insect protein revealed the optimistic functional, thermal and structural properties of insect protein extracted from black soldier fly prepupae. The functional properties with good water absorption capacity, oil binding ability and foaming capacity revealed BSFP protein was an optimistic functional ingredient for food applications. The cost of the insect protein was comparably to be low cost sustainable sources unlike other plant based or animal protein. The thermal stability depicts the BSFP protein can be infused at high temperature processing techniques in food sectors. The BSFP protein plays a vital role in valorization and provides abundant application in future foods. Few researchers proven that the insect protein sources are better than soy protein, and animal protein. However the safety and quality measures need to be implemented from rearing stage to processed stage. Thus alternative protein from black soldier fly prepupae paves novel trend in replacing existing protein compounds. Further, research can be undertaken in insect protein digestibility, tailored protein as meat alternates and utilizing BSFP protein in food products also exploring research on other insect species which have high nutritional profile. Declarations CONFLICT OF INTEREST The authors declare that they have no conflict of interest in this presented research paper. FUNDING INTEREST The authors declare there is no known (funding) financial interests in this presented research paper. Author Contribution Monisha Chandran: Conceptualization, Design of Methodology, Conducting the experiment, Acquisition of data, Analysis and Interpretation of data, Writing the original draft, Reviewing and edit the draft Loganathan Manickam: Conceptualization, Design of Methodology, Reviewing and edit the draft, Supervision, Project administration Acknowledgement The authors acknowledge the National Institute of Food Technology, Entrepreneurship and Management-Thanjavur (NIFTEM-T) for providing the facility for the experiments. References Abduh MY, Nadia MH, Syaripudin R, Manurung, Putra. RE (2018) Factors affecting the bioconversion of Philippine tung seed by black soldier fly larvae for the production of protein and oil-rich biomass. J Asia Pac Entomol 21(3):836–842 Baiano A (2020) Edible insects: An overview on nutritional characteristics, safety, farming, production technologies, regulatory framework, and socio-economic and ethical implications. 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F (2019) Alternative solvents for lipid extraction and their effect on protein quality in black soldier fly (Hermetia illucens) larvae. J Clean Prod 238:117861 Smets R, Verbinnen B, Van De Voorde I, Aerts G, Claes J, Van Der Borght M (2020) Sequential Extraction and Characterisation of Lipids, Proteins, and Chitin from Black Soldier Fly (Hermetia illucens) Larvae, Prepupae, and Pupae. Waste Biomass Valoriz 11:6455–6466 Spranghers T, Ottoboni M, Klootwijk C, Ovyn A, Deboosere S, De Meulenaer B, Michiels J, Eeckhout M, De Clercq P (2017) and S. De Smet., Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates. Journal of the Science of Food and Agriculture . 97, 2594–2600 Van Itterbeeck J, van Huis A (2012) Environmental manipulation for edible insect procurement: A historical perspective. J Ethnobiol Ethnomed 8:1–7 Wang J, Jousse M, Jayakumar J, Fernández-Arteaga A, de Lamo-Castellví S, Ferrando M, Güell. C (2021) Black soldier fly (Hermetia illucens) protein concentrates as a sustainable source to stabilize o/w emulsions produced by a low-energy high-throughput emulsification technology. Foods 10(5):1048 Wang YS, and M. Shelomi (2017) Review of black soldier fly (Hermetia illucens) as animal feed and human food. Foods 6(10):91 Wong C-Y, Rosli S-S, Uemura Y, Ho YC, Leejeerajumnean A, Kiatkittipong W, Cheng C-K, Lam M-K (2019) and J.-W. Lim., Potential protein and biodiesel sources from black soldier fly larvae: insights of larval harvesting instar and fermented feeding medium. Energies. 12(8), 1570 Xu F, Liu W, Huang Y, Liu Q, Zhang C, Hu H, Zhang. H (2020) Screening of potato flour varieties suitable for noodle processing. J Food Process Preserv 44(3):1–10 Yi L, Lakemond CMM, Sagis LMC, Eisner-Schadler V, Van Huis A (2013) and M. A. J. S. V. Boekel., Extraction and characterisation of protein fractions from five insect species. Food Chemistry . 144(4), 3341–3348 Zielińska E, Karaś M, Baraniak. B (2018) Comparison of functional properties of edible insects and protein preparations thereof. LWT - Food Sci Technol 91:168–174 Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":158623,"visible":true,"origin":"","legend":"\u003cp\u003eExtraction of BSFP Protein using pH-shift method\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/830b25818f9596eefb6395d6.png"},{"id":100559892,"identity":"c319079f-709e-4359-9a90-d4b409cfd3fd","added_by":"auto","created_at":"2026-01-19 08:43:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":476119,"visible":true,"origin":"","legend":"\u003cp\u003eSEM Image of BSFP protein. (A) The smooth globules structure of protein embedded in surface. (B) Complex interactions of protein particles. (C) Agglomerated image of BSFP protein.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/975f836a1b39e98bb2f16506.jpeg"},{"id":100560568,"identity":"918b329d-cc14-4c57-8abb-b569333788a3","added_by":"auto","created_at":"2026-01-19 08:43:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47732,"visible":true,"origin":"","legend":"\u003cp\u003eX-Ray Diffraction image of BSFP protein\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/092b8a060e6665e1fc37cf5a.png"},{"id":100560510,"identity":"08ed2ad9-da4f-49dc-a2d7-1d0544aa5f4e","added_by":"auto","created_at":"2026-01-19 08:43:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181995,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential scanning calorimeter thermogram of BSFP Protein\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/512726a34f1707356cefcef6.jpeg"},{"id":101612045,"identity":"b93c5a4d-0327-4282-8f66-8a72fb35686d","added_by":"auto","created_at":"2026-02-01 18:24:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1844780,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/77b441f8-e75e-4c35-ad37-0173978261f1.pdf"},{"id":100560262,"identity":"4a3b1f27-b7c7-4722-b13b-bf20f5f1afa2","added_by":"auto","created_at":"2026-01-19 08:43:39","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":314967,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7654817/v1/401e765bb87c01d78a301ff5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on Functional, Structural, Thermal properties of Insect protein source extracted from black soldier fly pre-pupae (Hermetia illucens)","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. The functional characteristic of insect (BSFP) protein depicts the prominent features of alterative proteins in food sectors.\u003c/p\u003e\u003cp\u003e2. The thermal and structural properties exhibit the potential opportunity to use insect protein as an alternative food source.\u003c/p\u003e\u003cp\u003e3. The marketable exploitation of the undervalued BSFP protein capitalizes of its potential in future foods such as tailored foods, RTE, meat alternates, 3D and 4D food printings.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe edible insect is emerging as a sustainable and innovative nutritional source to converge the futuristic world demand and will be available at low cost. In ancient time there is a direct historical evidence for eating edible insects (Govorushko et al., 2019; Baiano \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rumpold 2013). Edible insect protein is an innovative protein source that emanates from sustainable sources. The edible insect has various potential benefits, including increased nutritional value and reduced greenhouse gas emissions, feed conversion ratio, forest biomass waste, usage in land and water for rearing (Baiano, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rumpold 2013; Van Itterbeeck \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Varelas 2013). The market demand for protein from edible insect is growing rapidly it is predicted the global insect protein will reach 23% CAGR in 2027 to reach USD 400\u0026nbsp;billion. Thus the consumption of meat is increasing drastically, which will lead to increase all the factors dependent of growing livestock such as usage of feed, land and water (Gallo, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The edible insect feed energy conversion ratio is higher than meat industry which reveals a positive sign for sustainability (Halloran et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Apart from higher feed energy conversion efficiency, the insect reproducibility were also higher in rate such as yellow mealworm beetle \u003cem\u003eT.molitor\u003c/em\u003e lays 250\u0026ndash;1000 eggs and black soldier fly lays 500 to 900 eggs. This depicts the sustainability of insect sources with promising nutritional value with less carbon foot prints (Gere et al., 2019; Gold et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Govorushko, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The urbanization results increase in demand for food and feed sources, which exacerbates global food security issues (Baiano, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gere, 2019). There are already a host of new companies producing edible insects such as the black soldier fly, mealworm, cricket, locust fly, and other species (Mishyna et al., 2020). The insect protein incorporated food products such as breads, burgers, pizza and flour based biscuits are some of the existing examples (Bessa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gonzalez et al., 2019; Montevecchi et al., 2021). However the utilization of insects is still scarce due to socio-economic influences. Processed insect-based foods may be a strategy to enhance customer preferences of edible insects because they are often regarded as filthy and having an unpleasant appearance in western culture (Van Huis, 2013). Thus, few studies have been conducted on the development of insect meal and protein processing technologies from \u003cem\u003eTenebrio molitor\u003c/em\u003e and \u003cem\u003eH. illucens\u003c/em\u003e that could dispel consumer misconceptions about edible insects (Kim et al., 2020).\u003c/p\u003e \u003cp\u003eBlack soldier fly prepuape (BSFP) is an optimistic alternative source of protein for mass commercialization. The interesting fact is the BSFP will be reared on organic food waste ultimately converts into a valuable nutritional components such as protein, chitin and fats (Smets et al., 2019). The growing utilization of insects in human diets demands a greater focus on their safety risks. The assessment of edible insect safety by European food safety authority 2015 reported the need of safety with insect as any other form of food, entails the screening of microbial pathogens, parasites, toxins, heavy metals and allergens (Gravel et al., 2020). The interesting advantage of black soldier fly at adult stage was the insect does not depend on food material. The gut microbial enzymes of black soldier fly will suppress the toxin levels (Varelas et al., 2017). Few research depicts there is a chance of metal contamination in black soldier fly (Bessa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The microbial contamination can be prevented by proper hygienic processing techniques (Poma et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general there are few methods for protein precipitation such as aqueous, alkaline and pH shift method. The alkaline solubilization followed by acid precipitation method to recover protein with optimistic yield percent. Thus another common chemical method based on Osborne fractionation and enzyme-assisted method (Caligiani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Bu\u0026szlig;ler et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) studied on defatted larvae of \u003cem\u003eH.illucens\u003c/em\u003e and \u003cem\u003eTenebrio molitor\u003c/em\u003e for protein extraction using aqueous method using distilled water. The protein yield percent was found to be 82% for \u003cem\u003eH.illucens\u003c/em\u003e and 83% for \u003cem\u003eT.molitor\u003c/em\u003e using aqueous extraction method (Caligiani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Yi et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) investigated on protein fractions using aqueous extraction from edible insects. The supernatant holds 20% of total protein yield percent. Further the purity of supernatant ranges from 65 to 75 per cent. (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) studied on protein extraction using pH shift method on black soldier fly \u003cem\u003e(H.illucens).\u003c/em\u003e The resulted protein exhibit with high amount of lysine and valine serves an excellent replacement of animal protein. The BSF prepupae protein contains all amino acids with highest concentration of aspartic and glutamic acid as well as lowest concentration with cystine, methionine, and tryptophan. The BSFP contains protein 30\u0026ndash;53% and also. high amount of calcium, iron and zinc (M\u0026uuml;ller, Wolf, and Gutzeit \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bu\u0026szlig;ler et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Finke et al. 2013; Liland et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is rich in lauric acid, which act as an antibacterial agent (Spranghers et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The major advantage of BSFP protein was ease of protein digestibility when compared to the animal protein digestibility, additionally the cost of protein also less since it\u0026rsquo;s reared in food waste. Insect protein powders are most commonly used in pasta, cookies, burgers, patties, and healthy snacks as a source of protein. Insect protein has a promising future utility in culinary applications when utilized in a veiled forum (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, limited studies are available for novel insect protein as a functional ingredient in food sectors. Thus the study explores the possibility of valorization of insect protein in human diets as a functional component. The major aim of the study was to characterize the BSFP protein such as functional, structural and thermal properties which paves a path in incorporation of BSFP protein in food substances.\u003c/p\u003e"},{"header":"2. Materials and Methodology","content":"\u003cp\u003eThe Black soldier fly (\u003cem\u003eHermetia illucens)\u003c/em\u003e pre-pupae were reared in NIFTEM-T using spent foods like corn cob, leftover grain waste, fruit peels and vegetable waste. All of the analytical-grade chemicals required for the experimental purpose were purchased from Sigma-Aldrich (India).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Defatted Insect flour\u003c/h2\u003e \u003cp\u003eThe BSF prepupae stage is selected for this research work. The BSFP were gently cleaned and disinfected with chlorinated water. Then BSFP was freeze-dried for 24 hours and pulverized with the particle size of \u0026lt;\u0026thinsp;0.40 mm (mesh size). The BSFP insect flour was defatted in a soxhlet apparatus using n-hexane as the solvent for 6 hours (AOAC 920.39). Following, the defatted insect flour is kept in vacuum-sealed pouches and stored under \u0026minus;\u0026thinsp;18\u0026deg;C (Chandran et al, 2022).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Nutritional profile for Raw and Defatted BSFP flour\u003c/h2\u003e \u003cp\u003eThe protein in raw and defatted flour was determined (AOAC method 984.13) using Kjeldhal apparatus (Kjeltec 2300 analyser, Sweden). The lipid was assessed using soxhlet apparatus (AOAC 920.39). The crude fiber estimated using Weende\u0026rsquo;s method. The ash was estimated using muffle furnace (AOAC 942.05). The energy value was computed for the freeze dried defatted flour (Chandran et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Extraction of protein from black soldier fly prepupae\u003c/h2\u003e \u003cp\u003eThe protein was extracted (pH shift or Acid-alkali method) by adjusting the Iso-electric point of defatted insect flour (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bu\u0026szlig;ler et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The defatted insect flour was treated with NaOH (1M) solution with the ratio of 1:10 adjusted to a pH of 8.3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The solution was kept in an orbital stirrer at 250 rpm for 2 hours at ambient condition. The mixture was filtered and the aliquot was collected. The filtrate was centrifuged at 8000 rpm for 30min at 4℃. The recovered clear supernatant was precipitated by adjusted the pH 4.8 using 1M HCl precipitation. The precipitated sample was centrifuged again at 8000 rpm for 15min at 4℃and washed for several times. The frozen protein was lyophilized at -80℃ for 24 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of insect protein\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Protein percent yield recovery\u003c/h2\u003e \u003cp\u003eThe pH shift method was used to extract the protein from BSFP. The following Eq.\u0026nbsp;(1) was used to compute the yield of protein powder (Smets et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bu\u0026szlig;ler et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProtein yield % = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{w\\:Freeze\\:dried\\:protein\\:extract}{m\\:Dry\\:mass\\:\\times\\:\\:w\\:protein}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Estimation of protein percentage using Kjeldahl apparatus\u003c/h2\u003e \u003cp\u003eThe protein content was assessed in the Kjeldahl apparatus. The nitrogen-to-protein conversion factor as Kp 6.25 which is often used for insects, it was found that there is an overestimation of nitrogen content due to the presence of chitin compound. Recently, the correct Kp value for protein extracts from \u003cem\u003eH. illucen\u003c/em\u003es was found to be 5.71 which was used in this research (Caligiani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nery et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Janssen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Evaluation of techno functional properties of freeze dried insect protein\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Water absorption capacity (WAC)\u003c/h2\u003e \u003cp\u003eThe protein samples were weighed 0.5g and suspended in 5ml distilled water and stirred for 5minutes. The sample was then centrifuged at 3000 rpm for 10 minutes before being weighed after the supernatant was decanted (Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Purschke et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The following Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was used to calculate the amount of water absorbed by the BSFP protein samples.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{W}\\text{B}\\text{C}\\:\\left(\\frac{{g}_{w}}{{g}_{DM}}\\right)\\:=\\:\\left(\\frac{{W}_{0}-{W}_{1}}{{W}_{0.DM}}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere, W\u003csub\u003e0 DM\u003c/sub\u003e is the initial sample weight, W\u003csub\u003e0\u003c/sub\u003e is the tube weight with sediments, and W\u003csub\u003e1\u003c/sub\u003e is the final weight after decantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Oil binding capacity (OBC)\u003c/h2\u003e \u003cp\u003eThe oil binding capacity termed as the amount of oil adhered to defatted BSFP flour (Batish et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The protein samples 0.5g was combined with 5ml of canola oil and centrifuged at 3000 rpm for 30 minutes. The settled protein was weighed after the supernatant was decanted. The amount of oil adhered to the protein was assessed using the following Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{O}\\text{B}\\text{C}\\:\\left(\\frac{{g}_{w}}{{g}_{DM}}\\right)\\:=\\:\\left(\\frac{{W}_{0}-{W}_{1}}{{W}_{0.DM}}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWere, W\u003csub\u003e0 DM\u003c/sub\u003e is the initial insect protein powder, W\u003csub\u003e0\u003c/sub\u003e is the tube weight with sediments, and W\u003csub\u003e1\u003c/sub\u003e is the final weight after decantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Foaming capacity \u0026amp; stability\u003c/h2\u003e \u003cp\u003eThe foaming capacity \u0026amp; stability was assessed according to the method described by Chatsuwan et al., (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); Wang et al., (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The protein sample 1g was dispersed in 100 ml distilled water. The sample was adjusted to pH at 7.0 using 0.1N NaOH. The sample was agitated for 5min. The foaming volume was observed and recorded. The following Eq.\u0026nbsp;(4) was used to assess the foaming capacity. Foaming stability was identified by the withstand ability of foam for 30 min. Thus the stability time period of the foam was recorded.\u003c/p\u003e \u003cp\u003eFC % = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{({V}_{2}-{V}_{1})}{{V}_{1}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (4)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Emulsifying capacity \u0026amp; stability\u003c/h2\u003e \u003cp\u003eThe emulsifying capacity was determined as described in the method by Zielinska et al., (2018); Mshayisa and Van Wyk, (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 1g of protein was weighed and dispersed in 100 ml of distilled water. Following, 15ml of the protein sample was placed in a centrifuge tube and vortexed for 10 minutes. The sample solution was then vortexed constantly for 10 minutes after 15ml of maize oil was added. The emulsion was centrifuged at 3000 rpm for 10 minutes. The supernatant and residual volumes are measured. The following formula (5) was used to determine emulsion capability. Similarly, the BSFP protein (1g) was weighed and dispersed in 100ml water. The corn oil was then added to the sample solution and stirred constantly. The sample and the oil in water emulsion were both heated to 80\u0026deg;C / 30 minutes. The sample was cooled and centrifuged for 10 minutes / 3000 rpm and emulsion stability is recorded for 30 minutes.\u003c/p\u003e \u003cp\u003eEC % = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\frac{Volume\\:of\\:emulsifies\\:layer}{\\begin{array}{c}Volume\\:of\\:\\\\\\:suspension\\end{array}}\\right)\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (5)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Morphology of insect protein\u003c/h2\u003e \u003cp\u003eThe microstructure of protein is investigated using scanning electron microscope (VEGA3 TESCAN). The microstructure of defatted flour and protein were shown in 3 different magnifications (3000 x, 10,000 x, \u0026amp;15,000 x) at a accelerating voltage of 10 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.6 X-Ray Diffraction of insect protein\u003c/h2\u003e \u003cp\u003eX-ray diffraction pattern of insect protein was conducted using an XRD diffractometer (Bruker, D8 Focus, Malvern Panlytical-X\u0026lsquo;Pert\u003csup\u003e3\u003c/sup\u003e). The sample was taken in powder form and scanned over 2θ ranges between 20℃ and 80℃ at a rate of 2℃/min (Andressa Jantzen et al., 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Thermal properties of insect protein\u003c/h2\u003e \u003cp\u003eDifferential scanning calorimeter determines the thermal properties of BSFP protein (Mettler Toledo; DSC 3 STARe Systems). The protein is subjected to 30\u0026ndash;600℃ (dt 1.0 s) with a nitrogen flow of 50 ml/min at heating rate of 5.0 K/min (Zhao et al., 2015).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe data analysis was performed by statistical analysis tool using the IBM SPSS software package (Version 28.0; IBM Corp, USA). The significant difference was determined by the Duncan multiple comparison test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with 95% level of confidence. The OriginPro was used to generate spectra for XRD and DSC results.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Nutritional Composition of defatted BSFP flours\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 BSFP Fat\u003c/h2\u003e \u003cp\u003eThe Soxhlet extraction technique using n-hexane a solvent at 69\u0026ndash;70℃ recovered a significant increase in lipid yield of 26.8% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the whole BSFP flour before defatting due to varying factors such as biorhythm, feed substrate, and pH. The adult larvae stop feeding once the pupal instar completes, which results in a complicated mixture of chemicals accumulating in the lipid portion of BSFP. This is due to the lack of viable mouthpart development, resulting in them to rely only on the reserves acquired during the larval and pre-pupal stages (Ravi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The observed results was slightly varied with 20.0% fat percent from BSF larvae was reported in Bu\u0026szlig;ler et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) due to metamorphism and feed substrate used for rearing BSF. The Rumpold et al. (2013) reported that increased lipid content from black soldier fly has a prominent amount of EPA and DHA which can be alternate and replaced as a fish meal. Following, the defatted flour contains 4.5% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of fat (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The efficiency may due to several factors such as solvent, treatment time, temperature. Similar findings were reported in these literatures (Smets et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Abduh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProximate analysis of raw and defatted BSFP flour (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRaw BSFP flour\u003c/p\u003e \u003cp\u003e(per 100 g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDefatted BSFP flour\u003c/p\u003e \u003cp\u003e(per 100g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Protein (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbohydrate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy (kcal)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e456\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e419\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe mean of the rows with different superscripts varies significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Protein\u003c/h2\u003e \u003cp\u003eThe protein content was found to be 37.80% in BSFP flour before defatting and there was significant increase in protein concentration and yield 46.97% after defatting (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The protein content was calculated using 5.71 as a Kp factor for prepupae flour which gives more precise protein content (Janssen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It was observed that there is significantly difference in the protein content in before and after defatting BSFP flour samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results were in accordance with the Bu\u0026szlig;ler et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); Caligiani et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); Monisha et al. (2021). Similarly, Bu\u0026szlig;ler et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) computed a protein content of 44 percent in black soldier fly larvae. The increase of protein content in defatting BSFP flour may be due defatting the protein content concentrates the thermal influence in soxhlet chamber for 6 hours. Although it is widely acknowledged that a folded protein can be disrupted by various factors (temperature, chemical denaturants, pH, force, pressure, and mutations) (Narayan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thermal treatment causes partial unfolding of proteins thereby increase in protein percent in defatted BSFP flour, according to investigation of Purschke et al., (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Carbohydrate\u003c/h2\u003e \u003cp\u003eThe chitin compound (exoskeleton layer) is a modified polysaccharide that is predicted to be the carbohydrate. Chitin is prominent in the larvae, pupae, adult, sheddings, and cocoons of black soldier flies and also similar to protein, the chitin contains nitrogen content in its structure. The chitin content was found to be higher with 29.80% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in defatted BSFP flour, which could be attributed to heat influencing the glucosamide bond breakage in the chitin structure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The elevated chitin concentration in both treatments could be related to the BSF's pre-pupal stage, when the metamorphosis occurs. The chitin level of 17\u0026ndash;18% was shown to provide similar results (Wong et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nery et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The higher chitin concentration could be attributed to BSFP metamorphosis and feed substrate ingestion (Wang et al., 2017)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Ash Content\u003c/h2\u003e \u003cp\u003eThe ash content does not significantly varies before (9.0%) and after defatting (9.8%) of BSFP flours with (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The ash content was noted to have no notable difference in before and after defatting BSFP flours. It was predicted that BSFP had a high proportion of micronutrients such as zinc, calcium, and iron (Nery et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5 Moisture content\u003c/h2\u003e \u003cp\u003eThe moisture content was found to have less than 10% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) which showed the freeze drying impact on flours (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The slight difference in moisture content before and after defatting BSFP flour is predicted due to the heat involved in soxhlet chamber which might remove the free water in the BSFP flour (Chandran et al., 2021).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Extracted Protein from defatted black soldier fly prepupae (BSFP) flour\u003c/h2\u003e \u003cp\u003eThe protein was extracted by pH-shift method through modulating the Iso-electric point (pH 4.3) of protein from defatted black soldier fly prepupae (BSFP) flour. The protein content was determined using 5.71 Kp factor as nitrogen conversion factor for accurate protein concentrate in order to avoid the over estimation of nitrogen content from chitin (exoskeleton layer) N-acetylglucosamine in black soldier fly (Caligiani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The recovered biomass of protein percent was found to be 57% (semi solid bio-mass) from defatted BSFP flour with the protein content of 75% by kjeldhal analysis. The recovered protein yield is in accordance with the findings of (Bu\u0026szlig;ler et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liew et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The protein content may be due to denature of unstable protein while heat prevailed in soxhlet treatment during BSFP defatting. There are several factors which influence in yield percentage such as improper protein precipitation and solubility, ionic strength of acid-alkali treatment, and few amounts of proteins are insoluble at specific iso-electric point (Smets et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bu\u0026szlig;ler et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The protein purity was found to be 75% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) due to the strong acid and alkali treatment. Due to the convenience of use, the pH-shifting method has grown more popular in protein modification. With pH-shifting method, the pH is changed to an extremely alkaline or acidic level before being neutralisation (pH 7.0). This results in enhanced side-chain repulsions, which leads to the partial unfolding of proteins' secondary, tertiary, and quaternary structures, or \"molten globule\" structure (Pan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The purity of protein was 75% slightly decreased due to nucleic acid contamination while using strong acid and alkali (Loughrey and Matlock, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Even though the protein recovery was high, the purity was reduced due to strong acid and alkali extraction method. The results were similar to the findings of earlier researchers Caligiani et al., (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); Bu\u0026szlig;ler et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A globular protein structure inevitably determines the unique, very compact, highly structured configuration in which it is folded in its native state. The repetitive geometry of the hydrogen bonding sites in the polypeptide backbone gives rise to regions of regular conformation known as the α-helix and β-sheet. These regions collaborate to form the overall 3-D conformation, which is fueled and maintained by an array of parameters (Davis and Williams, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Environmental variables such as pH, temperature, ionic strength, and solvent composition affect a protein's stability by dislodging numerous intramolecular interactions that are essential for the protein's stability and integration (Kishore et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Evaluation of techno functional attributes of BSFP protein\u003c/h2\u003e \u003cp\u003eThe functional characteristics of the BSFP protein were thoroughly investigated inorder to introduce BSFP as a food ingredient or food grade material into the current food or food processing system. The chemical and topographical attributes of the protein surface are correlated to functional qualities such as wettability, dispersibility, solubility, foaming, emulsification, and oil and flavor binding (Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The interaction of a protein with water influences its water binding capabilities, which are generally alluded to as water absorption. The water absorption capacity of freeze-dried protein was found to be 75.53 per cent (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The increased WBC might be attributed to the BSFP concentrates enhanced protein composition contains higher hydrophilic groups which can adhere to water molecules. The results depicts that (BSFP) insect protein has a less water absorption capacity than plant and animal-based proteins. Few studies reported that water absorption for gluten was found to be in the range of 346.21\u0026ndash;353.81 per cent and the commercial soy protein has increased WAC (426.82%) when compared to OBC of soya protein (90.5%) (Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chatsuwan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The less absorption of water in insect protein may be due to several factors like precipitation, pH, ionic strength, concentration, isolation and drying (Bu\u0026szlig;ler et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The slight decline in WAC depicts the BSFP protein to have less amount of hydrophilic than hydrophobic amino groups. The oil binding capacity was found to be 83.78 per cent for black soldier fly pre-pupae. The hydrophobic components present on the surface of protein molecules which interact with oil are termed to as OBC (Batish et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The foaming capacity was found to be 112.78 and the foaming stability was done for 30 min found to be 103.10 per cent. The slight increase in OBC have a positive correlation with the foaming characteristic in BSFP protein. The foaming stability will vary based on the pH of the protein sample (Chatsuwan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, the protein foaming capacity and stability was observed at the pH of 7.0. The emulsion capacity and stability of BSFP protein were found to be 46.32 and 98 per cent (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) respectively. The emulsion stability revealed that the higher the electrostatic repulsion between oil droplets, the more stable the emulsion (Mshayisa and Van Wyk, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).The surface hydrophobicity of proteins influences their ability to emulsify, affecting the protein's inclination to absorb to the oil side of the interface. More disintegration is usually related with higher emulsion capabilities. The observed data for BSFP protein reveals a good indication of best utilization in food applications as a food ingredient and can replace soya protein. It can form highly soluble protein monomers and aggregates with outstanding emulsifying and foaming capabilities due to the redistribution of surface-active amino acid side chains. It was found that the alkaline pH-shifting method altered the native protein's structural structure more than the acidic pH-shifting process. (Pan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFunctional properties of protein isolates from BSFP (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBSFP protein (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e75.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e83.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFOC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e112.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e103.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e46.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e98.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWAC\u0026thinsp;=\u0026thinsp;Water absorption capacity, OBC\u0026thinsp;=\u0026thinsp;Oil binding capacity, FOC\u0026thinsp;=\u0026thinsp;Foaming capacity FS\u0026thinsp;=\u0026thinsp;Foaming stability, EC\u0026thinsp;=\u0026thinsp;Emulsion capacity, ES\u0026thinsp;=\u0026thinsp;Emulsion stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Morphology evaluation of BSFP protein\u003c/h2\u003e \u003cp\u003eThe morphology of protein from BSFP (A,B,C) was shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The proteins are made upon amino acids and peptides which have positive, negative and neutral charges on its surface. The BSFP protein depicts aggregation of protein, chitin along with other nucleic acids. The aggregation is predicted due to several factors such as heat involved during defatting process, pH, electrostatic force, and ionic strength (Baler et al., 2014) The smooth globules is predicted to be protein compounds which are embedded along with other compounds in aggregates due to electro static force on the protein surface in Fig. C. The protein is one of the classes of complex bio macromolecules, which has a clear hierarchy of structural levels that extends from the basic fundamental arrangement of amino acid sequences to multiprotein assemblies at the complex structures. The structural complexity of a single protein is determined by the complex interplay of electrostatic, hydrophobic, hydrogen bonding, and other interactions, and modifications to these interactions can result in major ubiquitination (Baler et al., 2014). The protein particles are observed to be in non-uniform in geometric shape and size in Fig. A, B, C. The nature of the protein compound is predicted to be smooth surface with irregular particle size and compact in structure in Fig. A. The protein powders are shown in the Fig. B. depicts the modified thermally stable proteins are scattered in surface and few complex proteins are in aggregated structure due to the aminoacids interactions in BSFP protein. Whereas, the stable globules protein scattered in heterogeneous particle sizes in Fig. A. Similar results were obtained in Xu et al., (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); Indriani et al., (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); Huang et al., (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) the protein was observed to be aggregated into a complex structure due to the strong acid- alkaline extraction or pH shift method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.5 X-Ray Diffraction of BSFP protein\u003c/h2\u003e \u003cp\u003eThe XRD pattern of insect protein was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The BSFP protein powder exhibited two peak intensity at 8.28 [\u0026deg;2θ] and 19.44 [\u0026deg;2θ] with d-spacing value of 10.65 [\u0026Aring;] and 4.56 [\u0026Aring;] respectively which clearly depicts the crystallinity structure. Also, two minor peaks observed at 32.72 and 35.08 [\u0026deg;2θ] with 2.81 and 2.55 [\u0026Aring;] d-spacing value. Li-Hua luo et al 2010 observed crystallinity of soy protein with peak intensity of 8.60\u0026deg; and 19.36\u0026deg;. The FWHM [\u0026deg;2θ] was found to be 1.1504, β and λ was 0.9; 0.15406 nm respectively. The (D) particle size of BSF protein was calculated as 8.3434 nm using Debye-Scherrer equation method. Protein crystallinity can be affected by amino acid neutralisation, pH, and the collapse of salt bridges forming between proteins (Li-Hua luo et al., 2010). It is found that very limited research done on insect protein crystallinity using X-ray diffraction. Similarly, Zhao et al., 2015 observed the peak intensity of soy protein at 9.99 and 19.60 by aqueous extraction. Thus the obtained BSFP protein is predicted to be crystalline in nature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Thermal properties of BSFP protein\u003c/h2\u003e \u003cp\u003eThe differential scanning calorimeter reveals the thermal stability of modified BSFP protein components due to strong acid- alkali Iso-electric precipitation. In order to evaluate the physical changes of samples with elevated temperatures over time, DSC employs thermal analysis. The thermogram in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts the melting point of BSFP protein powder. Initially, when the temperature increases all the moisture in the BSFP protein is removed. The sharp peak obtained 170.46 ℃ peak is the endothermic peak with Onset value of 169.74 and Endset value of 174.44. The enthalpy (ΔH) of BSF protein was found to be 90.05 J/g from the DSC curve. The Queiroz et al., 2021 reported that the endothermic peak at 200℃ for black soldier fly larvae protein, the difference is predicted to be the BSFP life stages and protein modification due to extraction methods were predicted to influence the thermal stability of protein. The thermal stability of insect protein was found to be high at 170℃, which can be commercially used as food ingredients based on its better thermal stability compared to plant and animal based protein. Some native and non-native hydrophobic interactions may be retained or even increased at high temperatures, despite the fact that many of the protein's stabilising interactions are broken when it is thermally unfolded or denatured (Kelly and Gage, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion and future scope","content":"\u003cp\u003eThe futuristic food like insect protein revealed the optimistic functional, thermal and structural properties of insect protein extracted from black soldier fly prepupae. The functional properties with good water absorption capacity, oil binding ability and foaming capacity revealed BSFP protein was an optimistic functional ingredient for food applications. The cost of the insect protein was comparably to be low cost sustainable sources unlike other plant based or animal protein. The thermal stability depicts the BSFP protein can be infused at high temperature processing techniques in food sectors. The BSFP protein plays a vital role in valorization and provides abundant application in future foods. Few researchers proven that the insect protein sources are better than soy protein, and animal protein. However the safety and quality measures need to be implemented from rearing stage to processed stage. Thus alternative protein from black soldier fly prepupae paves novel trend in replacing existing protein compounds. Further, research can be undertaken in insect protein digestibility, tailored protein as meat alternates and utilizing BSFP protein in food products also exploring research on other insect species which have high nutritional profile.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest in this presented research paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eINTEREST\u003c/p\u003e \u003cp\u003eThe authors declare there is no known (funding) financial interests in this presented research paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMonisha Chandran: Conceptualization, Design of Methodology, Conducting the experiment, Acquisition of data, Analysis and Interpretation of data, Writing the original draft, Reviewing and edit the draft Loganathan Manickam: Conceptualization, Design of Methodology, Reviewing and edit the draft, Supervision, Project administration\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the National Institute of Food Technology, Entrepreneurship and Management-Thanjavur (NIFTEM-T) for providing the facility for the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbduh MY, Nadia MH, Syaripudin R, Manurung, Putra. 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LWT - Food Sci Technol 91:168\u0026ndash;174\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Insect protein, future foods, protein characteristics, acid alkali extraction, pH shift method, protein structure","lastPublishedDoi":"10.21203/rs.3.rs-7654817/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7654817/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Edible insect’s specifically black soldier fly is hailed as a naturally profitable source of alternative protein source that can be used to replace existing food and feed sources. The functional, thermal, crystallinity and structural properties of black soldier fly prepupae (BSFP) protein were investigated in this study. The protein content in BSFP flour before and after defatting was found to be increased from 37.7% to 46.97%. The functional properties of BSFP protein such as WAC (75.53%), OBC (83.78%), FC (112.78%), and EC (46.32%) shows a huge viability to use as a functional ingredient in food sectors. The endothermic peak at 170℃ in DSC reveals the high thermal stability of modified BSFP protein due to strong pH shift (Iso-electric) method. The electrostatic modification and hydrophobic interaction triggers the modified protein with enhanced physical, functional, thermal properties. The morphology of the BSFP flour sample in SEM was found to be agglomerated together due to high temperature prevailed during defatting process. The smooth globules structure depicts the protein embedded on the surface with porous structure due to freeze drying. The XRD image with 2 peaks at 2θ = 8.28° and 2θ = 19.44° depicts the BSFP protein is in crystallinity structure and the particle size was found to be 8.3434 nm. The insect protein from black soldier fly prepupae was found to have better functional and thermal properties which paved path as a commercial functional ingredient and novel protein alternate in food industry.","manuscriptTitle":"Study on Functional, Structural, Thermal properties of Insect protein source extracted from black soldier fly pre-pupae (Hermetia illucens)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 08:25:58","doi":"10.21203/rs.3.rs-7654817/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29679ebd-0177-43e9-abfa-ce4102b6b728","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-01T18:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 08:25:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7654817","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7654817","identity":"rs-7654817","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}