METHOD OF EXTRACTING HIGH-GRADE MELANIN FROM BLACK SOLDIER FLY (Hermetia illucens) LARVAE

Abstract

The invention discloses a method of extracting high-purity melanin from Black Soldier Fly(Hermetia illucens)larvae. The methodessentially comprises ofenzymatic breakdown of polymer exoskeleton components, subcritical water extraction, low-pH hydrolysis using formic acid, nanofiltration using graphene oxide membranes, ultrasonic treatment for metal ion removal and crystallization using supercritical CO2 to achieve melanin with purity levels greater than 95%. This method reduces the need for harmful organic solvents and treatment with acids,thereby making the process sustainable and environment friendly.

Description

[0001] PromOO 1

[0002] METHOD OF EXTRACTING HIGH-GRADE MELANIN FROM BLACK SOLDIER FLY (Hermetia illucens) LARVAE

[0003] Field of the Invention

[0004] The invention discloses a novelmethod of extracting high-grade melanin from the biomass of black soldier fly Hermetia illucens) larvae.The invention is a vast improvement over existing technologies and has applications in several industries.

[0005] Background

[0006] Melanin, is a biopolymer with multiple applications in cosmetics, pharmaceuticals, and biomaterials. It is typically extracted from natural sources. Black Soldier fly is a good source of melanin, more specifically eumelanin, which is characterized by dark brown colour and has high UV absorption properties. The existing methods for extracting melanin often involve harsh chemicals or solvents. For example, Yunilas & Nasution (2024) developed an optimized process for extracting melanin from Hermetia illucens larvae, employing boiling at 100°C followed by sequential chemical treatments with HC1 and NaOH.

[0007] Santos et al. (2024) also explored Hermetia illucens larvae extracts for dermo- cosmetic applications, using organic solvents (acetone and n-hexane) for extraction. Their methodology includes microwave-assisted extraction (MAE) and maceration techniques. Ushakova et al. (2019) used a 16-hour extraction process with 10% NaOH and acid precipitation to isolate melanin, followed by centrifugation and drying at 70°C.

[0008] RU2728458C1 describes a method for producing a covalent-linked chitosan-melanin complex, involving degreasing with diethyl ether, demineralization with HC1, and PromOO 1 treatment with NaOH at 90-100°C, followed by lyophilic drying. US11981818B2 describes a method for obtaining melanin from invertebrate biomass involving sequential demineralization, deproteination, and alkaline extraction with NaOH, followed by acid precipitation.

[0009] RU2692260C1 outlines a method for producing melanin from Black Lionches larvae using cold pressing and freezing / thawing cycles to extract melanin. Khayrova et al. (2020) developed a method of extracting chitin / chitosan-melanin complexes from Hermetia illucens using chemical extraction from pupal exuviae and imagoes and lyophilic drying.

[0010] The present invention was developed with an aim to employ environmentally sustainable, scalable method for producing high yield of high-grade melanin. Ituses Hermetia illucens larvae, which are a low-cost biomass source. The methodinvolvesenzymatic digestion in a compatible solubilizer system to remove protein contaminants and efficient and selective removal of other contaminants. Through the present there is targeted lipid removal using organic solvents, which improvesthe purity and integrity of the final product. The biochemical extraction approach is used over the existing mechanical pressing method to preserve the biochemical integrity of melanin.

[0011] The present process also differs by isolating melanin alone instead of the completebiomaterial-complex, achieving a purity of greater than 95%. Rather than drying at high temperatures, which can degrade the melanin's antioxidative properties, the invention employs treatments with subcritical water and CO2 at the specified temperatures and pressures. It differs significantly from the existing methods by avoiding prolonged high-temperature boiling or treatment with harsh chemicals or PromOO 1 radiation. High concentrations of NaOH, can degrade melanin. Thermal or mechanical degradation of the biomolecule is thus avoided. The purity of melanin obtained in the present invention, surpasses that of Ushakova et al.'s method of melanin extraction.

[0012] The invention disclosed herein describes a novelmethod of extracting high grade melanin from biomass of black soldier fly {Hermetia illucens) larvae.The present invention yields a higher level of purity (Greater than 95%) of melanin. This high grade ultra-pure melanin is suitable for use in semiconductors and medical coatings.

[0013] Summary

[0014] The invention discloses a method of extracting high-grade melanin from Hermetia illucens larvae, comprising of the steps:enzymatic treatment for targeted breakdown of Chitin; subcritical water extraction at a standardised temperature and pressure;low-pH acid hydrolysis using formic acid;nanofiltration using graphene oxide membrane;and chelating ultrasonic treatment with EDTA for removal of ions; andcrystallization using supercritical COo.Thc melanin obtained is pure, crystalline with a high purity level of greater than 95%,

[0015] Detailed Description

[0016] Hermetia illucens is a known source of Melanin. However, this melanin is bound in polymer / chitin exoskeleton. In order to extract melanin, it is imperative to break the bonds and digest the polymersby using concentrated mineral acids and other harsh treatments. In this invention, these polymersare removed from the biopolymer complex through enzymatic degradation by Chitinase. The invention has been PromOO 1 optimized for extraction of Melanin from Black Soldier Fly which is a high yield providing and sustainable source of raw material.

[0017] Targeted Breakdown of Chitin

[0018] Chitinase specifically breaks down chitin and its derivatives, major components of the BSF exoskeleton, which otherwise hinder access to melanin. Chitinase degrades and reduces the rigidity of the biomass structure. This step thusimproves the solubility of melanin without damaging melanin itself.

[0019] In addition to Chitinase, Choline Chloride and Malic Acid are added which provide a mild, non-destructive environment for melanin solubilization. Choline Chloride and Malic acid act as components of a stable Deep Eutectic solvent (DES), resulting in a significant decrease in freezing points of both components in a molar ratio of 1:2. This solvent system has low viscosity and efficient solvating properties. The resultant can be subjected to advanced means of purification to obtain high purity melanin.

[0020] Through this essential step, greater than 95% purity of melanin is achieved.Choline Chloride and Malic acid act as a green solvent system, where Choline Chloride provides stability and ensures efficient solubilization of impurities; Malic acid, being a Hydrogen-bond donor, introduces mild acidity, which is essential for dissolving metal and protein impurities and also stabilizes melanin in its solubilized state. This combination efficiently breaks down proteins and lipids in the biomass without attacking the melanin molecules. Malic acid also has a chelating effect and helps to remove metal impurities from the biomass. This further improves the purity of the final melanin product. The solvents used are eco-friendly, biodegradable, nonvolatile, and sustainable. PromOO 1

[0021] While Chitinase breaks down chitin and its derivatives in the exoskeleton parts, exposing melanin to extraction, the Choline Chloride and Malic acid DES solubilizes melanin, maintaining its structural integrity and enhances the extraction efficiency. The combination of these three is essential for the optimum extraction of melanin. Chitinase is the main enzyme in this reaction without which, the chitin barrier cannot be broken and the melanin yield is greatly reduced. Choline Chloride alone without malic acid DES system does not have the required acidity to solubilize melanin. Further, the impurities are not broken down and removed efficiently. Malic acid alone does not have the ionic strength or the solvent properties for efficient extraction of melanin. Both Choline Chloride and Malic acid are cost effective, widely available and can solubilize biological polymers and impurities.

[0022] Besides, Choline Chloride and Malic Acid, other DES systems and ionic liquids can be used for melanin extraction. These include Choline Chloride + Urea (1:2), Choline Chloride + glycerol (1:2), Betaine + Citric Acid (1: 1) and Lactic Acid + Choline Chloride (l: l).Along with Chitinase, breakdown of chitin and its derivatives from the exoskeleton of blacksoldier fly larvae is also possible through digestion by Laccase and Lipase in compatible solubilizer systems.

[0023] This enzymatic reaction was followed by hydrolysis, nanofiltration, metal ion removal and crystallization using supercritical Carbon Dioxide to give high-grade melanin.

[0024] The BSF larvae were commercially procured from Trader Hub, based in New Delhi, sourced through Amazon India.The larvae were washed to remove dirt, debris and other contaminants. They were further dried to remove the moisture content. These weretreated with Chitinase 0.5% w / v solution. To this, Choline chloride and Malic PromOO 1

[0025] Acid in the molar ratio of 1:2 were added. The larval mass and solubilizer ratio was maintained at 1: 10. This reaction was carried out at 60-70°C for 4-6 hours.

[0026] Following this, the mass was subjected to subcritical water extraction (SWE), using water at its sub-critical stage at temperatures between 150-200°Cand pressure exceeding 7.4 MPa. This process was carried out for a period of 30-60 minutes.

[0027] Thiswas followed bycontrolled acid hydrolysis using 1-2% w / vformic acidat a lowpH.During this step, the pH of this solution was maintained at 2-3 and temperature at 35-40°C for a time period of 2-3 hours.

[0028] The melanin-containing solution obtained from the previous low pH hydrolysis step was passed through ultrathin graphene oxide nano-filtration membrane. The pore size of the membrane was between 1-5 nm to ensure selective molecular size-based separation of melanin. This filtration step was carried out under pressure range of 10- 15 Bar. The flow rate optimized to maximise the separation efficiency when subjected to nanofiltration using ultrathin graphene oxide membranes. This step removed several contaminants.

[0029] To remove trace amounts of metal ions such as Iron and Zinc, which are commonly present in BSF larvae, a chelating agentsuch as EDTAwas added to the melanin intermediate, alongwithultrasonic treatment. The melanin containing intermediate solution was treated with EDTA (0.1-0.5% w / v) for 30-40 minutes. At the same time, Ultrasonic treatment at 30-40KHz was applied to enhance the chelation process. The ultrasonic treatment facilitates the removal of metal contaminants by breaking weak bonds between melanin and the metal ions. PromOO 1

[0030] The melanin intermediate, thus obtained was treated withsupercritical CChto further concentrate and crystallize the melanin. CO2 at a supercritical stage at above 31°Cand pressure greater than 7.4 MPawas used as a solvent to selectively dissolve small organic compounds and crystallize melanin. CO2 evaporates completely leaving behind ultra-pure melanin. Through this process, a high purity of melaninof greater than 95%is obtained.

[0031] In an embodiment of the invention, it is possible to use Chitinase or other enzymes or Chitinase with other enzyme in a combination for targeted breakdown of proteins. Lipase from Thermomyces lanuginosus degrades lipids that contaminate melanin extracts. This may be used in high fat sources like black soldier fly. Subtilisin, a Protease degrades protein bound to melanin granules thereby increasing purity. It is also efficient in removing proteinaceous contaminants from insect mass. Laccase from Trametes versicolor catalyses oxidative polymerisation and isolates melanin precursors. Bacillus derived Amylase removes polysaccharides present in black soldier fly gut content, which can co-precipitate with melanin. This ensures a carbohydrate free final product.

[0032] Choline Chloride and Malic Acid DES can extract bio-based compounds while preserving molecular integrity of the resulting product. However, in another embodiment of the invention, chemical solubilizers other than DES can also be used for targeting specific bonds and impurities. These also result in good melanin recovery and purity. These chemical solubilizers include ionic liquids, Deep Eutectic Solvents (DES), Urea-Hydrogen peroxide mixture, Ethylenediaminetetraacetic Acid (EDTA) and Zwitterionic Surfactants, most preferably CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate). The ionic liquids include 1- PromOO 1 butyl-3-methylimidazolium chloride [BMIM] [Cl] , l-Ethyl-3-methylimidazolium

[0033] Acetate [EMIM][Ac], l-Butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4], N-Butylpyridinium Chloride [BuPy]Cl and Tetraethylammonium Bromide [TEA]Br. Among these, Urea-Hydrogen Peroxide acts as mild oxidising agent to loosen melanin aggregates. EDTA acts as a chelating agent and removes metallic impurities bound to melanin. Others solubilise melanin selectively from the larval mass. EDTA and Zwitterionic Surfactantshave not been extensively studied with respect to melanin extraction, but can significantly improve purity by removing hydrophobic or metallic impurities. These chemical solubilizers are discussed in further detail in Table 2.

[0034] In a preferred embodiment of the invention, the enzymatic breakdown was carried out using Chitinase at 0.3-0.7% w / v. Larval mass:DES ratio was maintained at 1: 10 w / v. DES system was created using Choline Chloride and Malic acid in the ratio of 1:2. This reaction was carried out at the temperature of 60-70°C for a time period of 4-6 hours. Other specific enzyme-chemical solubilizer combinations were also found to enhance the efficiency of the melanin extraction process (See Figure 7). Specifically, Chitinase-ionic liquid combination efficiently degrades chitin and its derivatives and dissolves melanin in a single step. Laccase and Deep Eutectic Solvents (DES) combination consisting of Choline Chloride and Malic Acid in 1: 1 molar ratio also preserves melanin polymer structure, while isolating it. This DES stabilizes Laccase activity, ensuring maximum efficiency of enzymatic hydrolysis and enhances melanin extraction selectivity. Being highly compatible with laccase, it facilitates selective enzymatic hydrolysis, and dissolves unwanted byproducts. It is non-toxic, biodegradable, and complementary to ionic liquid for sequential extraction.Lipase and Urea-Hydrogen Peroxide combination was found to remove fatty impurities and PromOO 1 loosen melanin granules. The chart comparing the above enzyme -solubilizer systems is given in Figure 7.

[0035] Laccase with Ionic liquid, l-butyl-3-methylimidazolium Acetate [BMIM]Ac was seen to efficiently disrupt the bonds between melanin and associated impurities (proteins, lipids, polysaccharides). It also provided a stable environment for enzymatic activity, which is critical for maintaining melanin integrity.lt has excellent biopolymer solubility, exceptional solubility for melanin precursors, is stable under enzymatic conditions and removes impurities effectively.

[0036] Example 1 Use of Enzymes for targeted lysis

[0037] Using tailored enzymes specific to the BSF composition improves extraction efficiency and reduces impurities. Table 1 below tabulates other enzymes which may be used alone or in conjunction with Chitinase for an improved process and final product. Table 1 PromOO 1

[0038] Example 2

[0039] Use of Chemical Solubilizers

[0040] Chemical solubilizers other than DES can also be used for targeting specific bonds and impurities. This results in improved melanin recovery and purity. Table 2

[0041] Example 3

[0042] Effect on yield and purity of Melanin obtained

[0043] Different combination of enzyme and chemical solubilizers were experimented with. The details are given in Table 3. The best combination with maximum yield and high purity was found to be of Chitinase along with Choline Chloride and Malic Acid DES. PromOO 1

[0044] Table 3: Effect on yield and purity of Melanin obtained using different combinations

[0045] Example 4

[0046] Melanin yield and purity in % were recorded at different temperatures, time periods and enzyme concentrations.

[0047] • Enzyme Concentration: 0.1%, 0.5%, 1% (See Figure 1). • Incubation Time: 2h, 4h, 6h. See Figure 2)

[0048] • Temperature: 60°C, 70°C (See Figure 1)

[0049] Example 5

[0050] Formic Acid Concentration, pH, Reaction time and temperature for Controlled acid Hydrolysis: o 1% w / v Formic Acid:

[0051] ■ It was observed that low acid concentration led to milder hydrolysis, resulting in less decomposition of the biomass. This preserved the PromOO 1 integrity of melanin but reduced the overall yield. The expected yield was in the range of 70-85%, depending on reaction time and temperature. o >3% w / v Formic Acid:

[0052] ■ It was observed that higher concentrations of Formic acid accelerated hydrolysis, breaking down melanin precursors more rapidly and potentially degrading melanin itself. This led to an increase inthe yield (up to 90-95%) but at the purity was affected. To preserve purity, robust purification steps need to be implemented.

[0053] (A) 1% w / v Formic Acid

[0054] 1. Adjust the pH of the extract to 2 using 1% w / v formic acid.

[0055] 2. Heat the solution to 150°C and maintain for 60 minutes under continuous stirring.

[0056] 3. Cool and neutralize the solution with IM NaOH or buffer to stabilize the melanin.

[0057] (B) 2% w / v Formic Acid

[0058] 1. Adjust the pH of the extract to 2 using 2% w / v formic acid.

[0059] 2. Heat the solution to 175°C and maintain for 45 minutes under continuous stirring.

[0060] 3. Cool and neutralize the solution with IM NaOH or buffer to stabilize the melanin.

[0061] (C) 3% w / v Formic Acid

[0062] 1. Adjust the pH of the extract to 2 using 3% w / v formic acid.

[0063] 2. Heat the solution to 200°C and maintain for 30 minutes under continuous stirring.

[0064] 3. Cool and neutralize the solution with IM NaOH or buffer to stabilize the melanin. PromOO 1

[0065] (D) 5% w / v Formic Acid

[0066] 1. Adjust the pH of the extract to 1.8 using 5% w / v formic acid.

[0067] 2. Heat the solution to 200°C and maintain for 25 minutes under continuous stirring.

[0068] 3. Cool and neutralize the solution with IM NaOH or buffer to stabilize the melanin. The results are summarised below:

[0069] Table 4 a.Formic Acid Concentration

[0070] • Increasing formic acid concentration beyond 3% led to increased yield but lower purity due to secondary reactions.

[0071] • Lowering acid concentration (e.g., 1%) generally required longer reaction times to achieve reasonable yields. b. Reaction Time and Temperature: PromOO 1 o Higher temperatures (closer to 200°C in subcritical water extraction) increased melanin solubilization efficiency but lead to byproduct formation if held too long. o Short reaction times at high acid concentrations yield optimal results while preserving melanin's chemical structure. c. pH Adjustment:

[0072] A lower pH (e.g., ~2) promotes better extraction but may require stringent control to avoid over-acidification that can lead to degradation.

[0073] Maximum efficiency was achieved at 35-40deg C with IM NaOH for cooling and neutralization for controlled acid hydrolysis.

[0074] Example 6

[0075] Combinations of enzymes and solubilizers including: a) Protease + glycerol + lactic acid; b) Lipase + Betaine + Citric Acid; c) Cellulase + Urea + Ethylene Glycol; d) Pectinase + Proline + Glycerol; e) Chitinase + Choline Chloride + Malic Acid were compared for yield and purity of melanin extraction. The conditions, yield and Purity of Melanin obtained are given below in Table 5.

[0076] Table 5 PromOO 1

[0077] Among the various combinations tested, Chitinase with Choline Chloride and Malic Acid (DES) combination resulted in best yield and purity of Melanin. While Protease was able to effectively break down protein, it was found to be less effective in breaking down chitin and its derivatives present in the larval skeleton. This resulted in less melanin being accessed and extracted. Lipase was able to breakdown lipids but not chitin and its derivatives or polysaccharides. This led to poor melanin recovery and residual contaminants in the resulting substance.Cellulase and Pectinase though useful for plant material did not give high yield with BSF biomass which contains minimal cellulose. These had limited impact on chitin and chitin derivatives-based exoskeleton components of insect larvae. Chitinase, Choline Chloride and Malic Acid combination was seen to hydrolyse chitin and related biomolecules and efficiently solubilize protein, lipid and metal impurities, yielding ultra-pure melanin.

[0078] The solubilizers used except Choline Chloride-Malic Acid DES, also had several limitations. Glycerol is a mild solvent and Lactic acid which is of lower acidity were unable to dissolve complex impurities. Betaine enhanced solubilization, but citric acid was found to be insufficient for complete impurity removal. Urea and Ethylene Glycol being mild solvents failed to remove protein and metal impurities. Proline and PromOO 1 glycerol formed a weak solvent system for black soldier fly larval mass. It was seen that excess solvents reduced the strength and purity of the yield.

[0079] A comparative performance summary of the different combinations is given in Table

[0080] 6 and Figures 5 and 6. Table 6

[0081] In the above set up, the following were observed:

[0082] Protease + Glycerol + Lactic Acid o Significant chitin residue and metal impurities remain, lowering purity. Lipase + Betaine + Citric Acid o Residual lipids and proteins reduce overall purity.

[0083] Cellulase + Urea + Ethylene Glycol o Residual chitin and proteins compromise purity.

[0084] Pectinase + Proline + Glycerol o High levels of chitin and metal contaminants remain.

[0085] Chitinase + Choline Chloride + Malic Acid PromOO 1 o Ultra-pure melanin suitable for advanced applications.

[0086] Example 7

[0087] Combinations of enzymes and solubilizers including: a) Chitinase + l-Butyl-3- methylimidazolium chloride ([BMIM]Cl)Ionic Solution; b) Laccase + DES (Choline Chloride + Malic acid in 1: 1 molar ratio); c) Lipase + Urea + Hydrogen Peroxide were compared for Dissolution efficiency of chitinous exoskeleton and other biomolecules, Preservation of Melanin and Impurity removal. Acomparison of these in percentage (%) are given in Figure 7.

[0088] In light of the above disclosure, it can verily be seen that while the most preferred embodiment is being claimed, several embodiments and replacements of essential components of the invention are possible while maintaining the same function of the invention and quality of the final product. The embodiments and examples being described are illustrative and not exhaustive.

[0089] Brief Description of Figures

[0090] Figure 1: Melanin purity vs Temperature at different enzyme Concentrations.

[0091] The graph shows the purity of Melanin obtained in the final product when subjected to different temperatures and Chitinase enzyme concentrations of 0.1%, 0.5% and 1.0%. It is seen that melanin purity changes at different enzyme concentrations and temperature. Higher purity of melanin is obtained at temperature of 70°C. Highest purity is obtained using enzyme concentration of 0.5%. Thus, the optimal condition of 0.5% Chitinase at 70°C was standardised.

[0092] Figure 2: Melanin yield vs Incubation time at different enzyme Concentrations. PromOO 1

[0093] This plot compares melanin yield at different enzyme concentrations (0.1%, 0.5%, 1.0%) across varying incubation times. It is seen that the yield improves with longer incubation times for all enzyme concentrations. However, at 0.5% enzyme concentration, the yield reaches a maximum of 70% at 6 hours. It is seen that 4-6 hours of incubation with 0.5% enzyme concentration provided the best yield.

[0094] Figure 3: Yield vs Purity at Different Process Steps.

[0095] This chart illustrates the yield and purity at various stages of the process. The process includes enzymatic action for removal of Chitin and its derivatives, acid precipitation for removal of proteins and metal ions, washing for eliminating lipophilic contaminants and Drying. The yield decreases through the process (from 70% to 40%), but purity increases significantly, reaching 99.9%. Yield drops significantly after the initial extraction, emphasizing the importance of careful process control.Purity increases progressively, peaking at the washing stage, where trace contaminants are removed.

[0096] Figure 4: Purity vs Incubation Time and Enzyme Concentration.

[0097] This plot demonstrates the critical balance between enzyme concentration and incubation time, confirming the optimized parameters (0.5%, 4-6 hours). It is seen that Purity improves consistently with incubation time for all enzyme concentrations.lt is seen that the 0.5% enzyme concentration achieved the highest purity (-97%) at 6 hours, outperforming other concentrations.

[0098] Figure 5: UV-Vis Spectra across combinations; FTIR spectra across combinations. PromOO 1

[0099] Figure 6: HPLC Chromatograms across Combinations; LC-MS Spectra across combinations.

[0100] Combinations of enzymes and solubilizers including: a) Protease + glycerol + lactic acid; b) Lipase + Betaine + Citric Acid; c) Cellulase + Urea + Ethylene Glycol; d) Pectinase + Proline + Glycerol; e) Chitinase + Choline Chloride + Malic Acid were compared for yield and purity of melanin extraction. A comparison of their UV-Vis peaks, FTIR Spectra, HPLC Chromatograms and LC-MS Spectra are shown in

[0101] Figures 5 and 6.

[0102] Figure 7: Comparison of Different Methods for Melanin Processing. Figure 7 shows a comparison of different enzyme- solubilizer systems used successfully for melanin extraction. These include Chitinase- l-Butyl-3- methylimidazolium chloride ([BMIM]Cl)Ionic Liquid, Laccase-Choline Chloride + Malic Acid DES and Lipase-Urea-Hydrogen Peroxide. The Dissolution Efficiency, Preservation of Melanin obtained and impurity removal in percentage (%) are shown in this figure.

Claims

PromOO 1We Claim:

1. A method of extracting high-grade melanin from black soldier fly (Hermetia illucens) larvae, comprising of the steps: washing and drying of the Hermetia illucens larvae; enzymatic treatment of these larvae using Enzyme - Solubilizer system; subcritical water extraction at specific temperature and pressure; low-pH acid hydrolysis using formic acid; nanofiltration using graphene oxide membrane; chelating ultrasonic treatment with EDTA for removal of ions; and crystallization using supercritical CO2.

2. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein the enzyme- solubilizer system is among Chitinase-Choline Chloride-Malic Acid, Chitinase-l-Butyl-3- methylimidazolium chloride ([BMIM]C1), Laccase- Choline Chloride - Malic Acid and Lipase-Urea-Hydrogen Peroxide.

3. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein the enzyme- solubilizer system is Chitinase-Choline Chloride-Malic Acidin.

4. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the larval mass and solubilizer ratio is maintained at 1: 10.PromOO 15. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein the enzyme Chitinase is added at 0.5% w / v.

6. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein for enzyme Chitinase, Choline Chloride and Malic acid are added in a molar ratio of 1:2.

7. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the reaction is carried out at 60- 70°C for 4-6 hours.

8. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the intermediate containing melanin is subjected to subcritical water extraction (SWE) using water at its sub-critical stage at temperatures betweenl50-200°Cand pressure above 7.4 MPa for a period of 30-60 minutes.

9. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the intermediate containing melanin is subjected to controlled acid hydrolysis using 1-2% w / v formic acidat pH of 2-3. temperature at 35-40°C for a time period of 2-3 hours.

10. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the intermediate containing melanin is passed through ultrathin graphene oxide nano-filtration membrane, pore size between 1-5 nm, under pressure range of 10-15 Bar.PromOO 111. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the intermediate containing melanin is treated with EDTA (0.1-0.5% w / v) for 30-40 minutes, alongwith Ultrasonic treatment at 30-40KHz.

12. The method of extracting high-grade melanin from black soldier fly Hermetia illucens larvae as claimed in claim 1, wherein, the intermediate containing melanin is treated withsupercritical CCbat temperature above 31°Cand pressure greater than 7.4 MPato give high-grade melanin.

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