Isolation of chitin from biomaterial

Abstract

The invention relates to methods for the isolation of chitin from arthropod or fungal biomaterial, the method comprising the steps of combining the biomaterial an alcohol or with a mixture of water and an alcohol, incubating the biomaterial to a temperature above 150 °C, and isolating the solid fraction comprising chitin.

Description

[0001] ISOLATION OF CHITIN FROM BIOMATERIAL

[0002] Field of the invention

[0003] The invention relates to the isolation of chitin and fatty acid esters from biomaterial such as insects.

[0004] Background of the invention

[0005] Insects such as Hermetia illucens (Diptera: Stratiomydae), also known as the black soldier fly (BSF), have been used as a source for e.g. protein in food and feed applications. Depending on the rearing conditions, this insect species has a very short life cycle, but it is particularly known for their voracious appetite during the larval stage. The BSF larvae excel at upcycling low value side streams into their own high- value biomass, which can, to some extent, be modified by the rearing substrate.

[0006] The potential of insects, however, is not only limited to food or feed applications as they are a source of valuable biochemicals as well. They are exceptionally rich in lipids, with lauric acid as predominant fatty acid as it can account for up to half of the total lipid fraction.

[0007] Aside from their high lipid content, insects such BSF larvae also contain significant amounts of protein and chitin. Depending on the life stage, this chitin content can vary from 5 to 10 g / 100 g dry matter. The chitin content of BSF exuviae or their cocoons, however, can even amount up to 20 % of the total dry matter. Chitin, and in particular its deacetylated derivative chitosan, are very promising bio-polymers as they are non-toxic and exhibit antitumor, haemostatic, wound-healing, antimicrobial, and antioxidant activities. In addition, the / V-acetylglucosamine backbone of the polymer can open new possibilities for the production of nitrogen-containing platform chemicals.

[0008] Mechanical procedures for the biorefinery require only a series of relatively simple unit operations, though the purity of the several derived products is low. By recovering the lipids by means of a screw press (dry fractionation) or juice press (wet fractionation), the resulting press cake will still contain residual lipids, which can account for up to 5 to 25 % of the total lipid content

[0009] The purification of the chitin matrix is complicated as it is a complex and crosslinked network of chitin fibrils, pigments, quinone species, and cuticular proteins with embedded minerals. The resulting solid fraction of industrial processes often contains residual proteins, minerals, and pigments and will likely require an additional (chemical) purification step if high-purity products are desired . Regarding chemical procedures, it is documented in the literature that pure end products can be obtained from all life stages as well as exuviae Generally, they consist of one or more acidic demineralisation steps (e.g., HCI, HCOOH), a series of consecutive alkaline deproteinization steps (e.g., NaOH), and an optional bleaching step (e.g., H2O2, NaCIO) to obtain an off-white chitin powder. Methods to isolate chitin, based on the procedure of Liu et al. (2012) Molecules 17, 4604-4611 are disclosed in EP3901179, Debasree et al. (2019) 445-457 and Smets et al. (2020) Waste Biomass. Vai. 11, 6455-6466.

[0010] US 2014275507 discloses a method to isolate chitin from fungal biomass using ammonium compounds, organic amines and alkaline silicates.

[0011] Milder methods e.g. for the isolation of fatty acids from algae have been described in Nelson & Viamajala (2016) Catalysis Today 269, 29-39. Also known are hydrothermal liquefaction for biofuel production [Peng et al. (2019) J. Anal. Appl. Pyrolysis 141, 104621] or for lignin valorisation from wood pulp or tree bark [Renders et al. (2016) ACS Sustainable Chem. Engin. 4, 6894-6904; Vangeel et al. (2019) Green Chem. 21, 5841-5851].

[0012] SUMMARY OF THE INVENTION

[0013] The lipid fraction of e.g. BSF larvae shows excellent potential as potential replacement for the two other lauric oils, being coconut and palm kernel oil. In this scope, surfactants have already been prepared from BSF lipids, which exhibited similar properties to coconut-derived and commercially available surfactants [Verheyen et al. (2018) J. Cosmetic Sci. 69, 187-202].

[0014] There is a need for high quality chitin. Using a protocol developed for the isolation of FAME from biomass, it was unexpectedly observed that the harsh reaction conditions of the transesterification reaction resulted in the removal of proteinaceous content from chitin. Simple separation steps such as filtration and sieving allow to produce preparations which contain more than 50 % chitin and less than 1 protein.

[0015] The present invention, exemplified with BSF larvae, discloses methods to isolate and enrich chitin from a biomaterial. As a side product, if present in the material, fatty acids can be equally isolated from a biomaterial. Herein, biomass is first processed in an alcohol such as methanol at high temperature. During this treatment, the larval lipids are (trans)esterified into their corresponding alkyl esters which readily dissolve in the alcohol, facilitating their recovery from the biomass. The larval proteins are presumed to undergo a significant modification to their structure or even solvolysis during this process, hereby liberating them from the complex chitin matrix.

[0016] Chitin itself is expected to be remain largely intact as this is the case for cellulose when the latter is subjected to similar conditions.

[0017] The invention is further summarised in the following statements:

[0018] 1. A method for the isolation of chitin from arthropod or fungal biomaterial, the method comprising the steps of: a) combining said biomaterial with a protic solvent, b) heating the biomaterial in the protic solvent to a temperature above 150 °C, c) isolating the solid fraction comprising chitin obtained after step b).

[0019] 2. The method according to statement 1, wherein the protic solvent is water.

[0020] 3. The method according statement 1 or 2, wherein the protic solvent comprises an alcohol and step b) is performed under conditions to allow an esterification or trans esterification reaction of fat in the biomaterial.

[0021] 4. The method according to statement 3, wherein the alcohol is a C1-C5 alcohol, such as methanol or ethanol.

[0022] 5. The method according to any one of statements 1 to 4, further comprising step d) of separating the solid fraction on particle size, and isolating particles with a size of more than 100, more than 250 or more than 500 pM.

[0023] 6. The method according to any one of statements 1 to 5, wherein the biomaterial is fungal biomaterial.

[0024] 7. The method according to any one of statements 1 to 5, wherein the biomaterial is from arthropods, crustaceans or arachnids.

[0025] 8. The method according to statement 7, wherein the arthropods are crustacean, such as prawns, crabs or lobsters.

[0026] 9. The method according to statement 7 or 8, wherein the arthropods are insects.

[0027] 10. The method according to statement 9, wherein the insects are Diptera, Coleoptera, and Lepidoptera selected from the group consisting of holometabolous insects, and hemimetabolous insects, such as Orthoptera.

[0028] 11. The method according to statement 9 or 10, wherein the biomaterial are insect larvae, puparia, pupae, imagoes, nymphs, or exuviae.

[0029] 12. The method according to statement 11, wherein the insect larvae are from Hermetia illucens, Musca domestica, Tenebrio molitor, or from Alphitobius diaperinus.

[0030] 13. The method according to any one of statements 1 to 12, wherein step b) is performed at a pH above 4, at a pH above 4,5, at a pH above 5, at a pH above 5,5 14. The method according to any one of statements 1 to 13, wherein step b) is performed under an inert atmosphere.

[0031] 15. The method according to any one of statements 1 to 13, wherein step b) is performed under an atmosphere of carbon dioxide.

[0032] 16. The method according to any one of statements 1 to 13, wherein step b) is performed under an atmosphere of hydrogen and the solvent comprises a reducing catalyst such as Pd, Pd / C or Ni.

[0033] 17. The method according to any one of statements 1 to 16, wherein step b) is performed for a period of between 1 or 2 up to 4, 5 or 7 hours.

[0034] 18. The method according to any one of statements 1 to 17, wherein step b) is performed at a temperature of above 200 °C, or above 250 °C.

[0035] 19. The method according to any one of statements 1 to 18, wherein step b) is performed at a pressure of from 6 bar, bar 8 or 20 bar up to 40 bar, 80 bar or 110 bar.

[0036] 20. The method according to any one of statements 1 to 19, wherein step b) is performed for a period of between 2 and 5 hours, at a temperature of between 200 to 260 °C, and a pressure of between 6 and 110 bar.

[0037] 21. The method according to any one of statements 1 to 20, where step c) is performed by filtration, centrifugation or decantation.

[0038] 22. The method according to any one of statements 1 to 20, where step c) is performed by filtration over a filter with a pore size of between 5 to 15 pm.

[0039] 23. The method according to any one of statements 1 to 20, where step c) is performed by sieving the solid fraction after drying over a sieve with a mesh size above 250 pm.

[0040] 24. The method according to any one of statement 1 to 23, wherein in step a) biomaterial is combined with a solvent comprising between 70 v / v % and 100 v / v % alcohol, and wherein the ratio of weight solvent vs weight biomaterial is between 7: 1 and 2: 1.

[0041] 25. The method according to any one of statements 1 to 24, wherein in step a) biomaterial is combined with pure methanol, and wherein the ratio of weight methanol vs weight biomaterial is 4: 1.

[0042] 26. The method according to any one of statements 1 to 24, wherein the biomaterial or insect larvae have a dry matter content of between 30 to 40 %.

[0043] 27. The method according to any one of statements 1 to 26, further comprising a step of isolating the liquid fraction obtained after the treatment of step b) and purifying fatty acid esters therefrom. 28. The method according to statement 27, wherein fatty acid esters are purified by solvent extraction.

[0044] 29. The method according to statement 28, wherein the solvent extraction is performed with an nonpolar solvent that is not miscible with water or with alcohol, such as hexane, petroleum ether, iso-octane, cyclohexane or heptane.

[0045] A first aspect of the invention relates to methods for the isolation of chitin and / or chitosan from arthropod or fungal biomaterial, the methods comprising the steps of: a) combining said biomaterial with an alcohol, or with a mixture of water and an alcohol, thereby obtaining a reaction mixture, b) incubating the reaction mixture of step a) at a temperature above 150 °C, c) isolating from the composition obtained in step b) the solid fraction comprising chitin and / or chitosan.

[0046] Typically in step a) said biomaterial is combined with an alcohol or with a mixture of water and an alcohol, thereby obtaining a reaction mixture consisting of biomaterial and alcohol or obtaining a reaction mixture with an alcohol concentration of at least 25 vol % alcohol.

[0047] In embodiments thereof, in step a) said biomaterial is combined with an alcohol or with a mixture of water and an alcohol, to obtain a reaction mixture with an alcohol concentration of between 80 to 90 vol % alcohol.

[0048] Typically step b) is performed for a period of between 1 and 5 hours, or for a period of between 90 minutes and 150 minutes.

[0049] Typically step b) is performed a temperature of between 200 to 300 °C, or at a temperature of between 225 to 275 °C.

[0050] Typically step b) is performed at a pressure of between 6 and 110 bar, or at a pressure of between 40 and 100 bar, or at a pressure of between 70 and 100 bar. More typically wherein step b) is performed at a pressure of between 85 and 95 bar. In specific embodiments, step b) is performed a temperature of between 225 to 275 °C, for a period of between 90 minutes and 150 minutes .

[0051] In other specific embodiments, step b) is performed a temperature of between 225 to 275 °C, at a pressure of between 40 and 100 bar, for a period of between 90 minutes and 150 minutes .

[0052] In yet other specific embodiments, the reaction mixture in step a) has an alcohol concentration between 80 to 90 vol % alcohol and step b) is performed a temperature of between 225 to 275 °C, at a pressure of between 40 and 100 bar, for a period of between 90 minutes and 150 minutes. Typically in these methods the alcohol is a C1-C5 alcohol.

[0053] Examples are one or a mixture of methanol, ethanol, or isoamylalcohol.

[0054] In yet other specific embodiments b) is performed at a pH above 4, at a pH above 4,5, at a pH above 5, or at a pH above 5,5. In the examples of the present invention, the pH of the reaction mixture is neutral or slightly basic. Under these conditions chitosan is insoluble and is co-purified with chitin.

[0055] Buffering to a pH above 4, can be of use with acidic biomaterial, to avoid that chitosan becomes soluble and discarded.

[0056] The methods can performed under an inert atmosphere, for example step b) is performed under an inert atmosphere such as hydrogen and the reaction mixture comprises a reducing catalyst such as inert Pd, Pd / C or Ni.

[0057] Alternatively, step b) is performed under an atmosphere of carbon dioxide.

[0058] The isolation of step c) can be performed by filtration over a filter with a pore size of between 5 to 15 pm, or by sieving the solid fraction after drying over a sieve with a mesh size above 250 pm.

[0059] Alternatively, step c) is performed by extraction with a solvent that removes the liquid fraction and whereby the chitin and / or chitosan remain in the solid fraction.

[0060] In these methods arthropods can be insects, including insect larvae, puparia, pupae, imagoes, nymphs or exuviae.

[0061] Typical biomaterials are insect larvae from Hermetia illucens, Musca domestica, Tenebrio molitor, or from Alphitobius diaperinus.

[0062] An example of a fungal biomaterial is Agaricus bisporus.

[0063] In step a) wherein the ratio of weight methanol vs weight biomaterial is 4: 1.

[0064] Biomaterial or insect larvae can have a dry matter content of between 30 to 40 wt %, or can be dried to a wt% below 10 or 5% /

[0065] These methods can comprise a further step d) of separating the solid fraction on particle size, and isolating particles with a size of more than 100, more than 250 or more than 500 pM.

[0066] The methods can comprise further comprise a step of isolating the liquid fraction obtained after the treatment of step b) and purifying fatty acid esters therefrom.

[0067] Herein fatty acid esters can purified by solvent extraction, typically with an nonpolar solvent that is not miscible with water or with alcohol. Examples of such solvents can be hexane, petroleum ether, iso-octane, cyclohexane or heptane. A second aspect of the invention are methods of converting chitin into chitosan comprising the step of: a) combining a composition comprising at least 25 wt % chitin with an alcohol or with a mixture of water and an alcohol thereby obtaining a reaction mixture, b) incubating the reaction mixture of step a) to a temperature above 150 °C, thereby deacetylating chitin and forming chitosan.

[0068] Typically the composition of step a) comprises at least 50 wt % or at least 75 wt % chitin.

[0069] Typically step b is performed between 225 and 275 ° C, for a period of between 1 to 5 h, at a pressure of between 80 and 100 bar.

[0070] Generally the reaction mixture in stap a) contains between 70 and 90 vol % alcohol. Embodiments of the first aspect of the invention on pressure, temperatures, time, alcohols and concentrations of alcohol are equally applicable on this second aspect of the invention.

[0071] DETAILED DESCRIPTION

[0072] Figure 1 Overview of the downstream processing steps of the obtained reaction mixture

[0073] Figure 2 Obtained fractions after the solvolytic fractionation of black soldier fly larval biomass, with from left to right: oil fraction, soluble fraction, fibrous fraction, and two powdery fractions

[0074] Figure 3 SDS-PAGE of the soluble fraction obtained after the solvolytic fractionation of black soldier fly larvae.

[0075] Figure 4 Sankey chart of the new solvolytic fractionation procedure, showing the proximate composition of the black soldier fly larvae (middle), the mass balances as well as the fraction composition (right)

[0076] Figure 5 Amino acid contents (g / 100 g dry matter) of the starting material and the fibrous fraction obtained after the solvolytic fractionation.

[0077] Figure 6 FTIR spectra of (A) commercially available chitin from shrimp and (B) the purified chitin / chitosan of black soldier fly larvae treated with subcritical methanol / water (4: 1; m:m) at 250 °C for 2 h, 89 bar. The treatment was repeated twice and showed comparable FTIR spectra and DA values. Chitin and chitosan have the following chemical structures:

[0078] Chitin can be N-deacetylated to such an extent that it becomes soluble in dilute acetic and formic acids. In chitin, the acetylated units prevail and the degree acetylation is typically 90%, while chitosan is a fully or partially N-deacetylated derivative with a typical degree of deacetylation of more than 50 %.

[0079] Upon analysis of the product of the reference material (BSF larvae), it was surprisingly found that chitin , prepared according to the methods of the present invention, became deacetylated to such an extent that it can be classified as chitosan.

[0080] Typically preparation of chitosan from chitin is performed in boiling NaOH (50 % (w / v). The present invention does provides a chitin product which is already substantially deacetylated, and requires less or no further processing to obtain chitosan.

[0081] All process parameters (temperature, pressure, time) and details on alcohols for the isolation of chitin from biomaterial are also embodiments for the methods on converting an composition comprising chitin into chitosan.

[0082] Chitin can be found in terrestrial, marine and microbial sources. The chitin content of the biomass (expressed as percentage of dry matter), however, differs substantially between sources. For whole insects, this can vary from 4 - 8 % in the larval stage to 6 - 12 % in the pupal stage. Exuviae, being the shedding after each moult, on the other hand are reported to contain a chitin content of more than 20 % for black soldier fly larvae. The exoskeleton of other insect species like butterflies or silk worms is reported to consist of 44-64 % of chitin on dry matter basis.

[0083] Marine sources of chitin are arthropods like crabs, lobsters and crustaceans, with their shells having chitin contents ranging from 15 - 60 % depending the species. Especially lobsters are characterised by a relatively high content (i.e., 50+ %). Species from the phylum Mollusca like mussels, oysters and squid are also considered a source of chitin. Still, the shells itself are largely mineralised and relatively low in their chitin content (3 - 6 %) as compared to squid pens, which are reported to contain 20 - 40 % of chitin.

[0084] Regarding microbial sources, chitin is found in the cell wall of fungi and yeast with Ascomycetes (i.e., Saccharomyces cerevisiae), Zygomycetes and Basidiomycetes as prime examples. It is reported that dry mycelia can contain anywhere between 2 % to 60 % of chitin More specifically, dry mycelium of Aspergillus niger, an industrially relevant fungi, can contain up to 42 % of chitin.

[0085] In the methods of the present alcohol water mixtures at high temperature larval lipids are (trans)esterify the, proteins are solvolysed, a fibrous fraction rich in chitin and / or chitosan (i.e., > 50 g / 100 g dry matter) is obtained. After the treatment with alcohol, this chitin-rich fibrous fraction can easily be recovered by a simple filtration and sieving step. As a side product use of n-hexane allows to recover FAMES.

[0086] Within the chitin fraction the amino acid content is less than 1 wt% or even less than 0,5 wt%, or less than 0,25 wt %.

[0087] Further processing of the chitin fraction obtained by the present invention may include washing the fraction with alkaline or acidic aqueous solutions, washing the fraction with detergents, or treating the fraction with an aspecific protease (e.g. Proteinase K or subtilisin).

[0088] Protic solvent are solvents that have a hydrogen atom bound to an oxygen (as in a hydroxyl group -OH), a nitrogen (as in an amine group -NH or -NH-), or fluoride (as in hydrogen fluoride). In general, any solvent that contains a labile H+ is a protic solvent. The molecules of such solvents readily donate protons (H+) to solutes, often via hydrogen bonding. Water is the most common protic solvent. Other examples are water, ammonia, alcohols like methanol and ethanol, carboxylic acids like formic and acetic acid, and primary amides.

[0089] The methods of the present invention are performed in alcohol or in a mixture of water and an alcohol.

[0090] In the reaction mixture the alcohol in the reaction mixture is between 10 vol % up to pure alcohol. More typical the amount of alcohol can range from 20 vol %, 30 vol %, 40 vol % or 50 vol % alcohol up to 70 vol %, 75 vol %, 80 vol %, 90 vol % alcohol, up to pure alcohol.

[0091] All ranges of the above lower and upper ranges are herewith explicitly disclosed. In typical embodiments the amount of alcohol in the reaction mixture is between 70 and 90 vol % alcohol or between 75 and 85 vol % alcohol or between 80 and 90 vol % alcohol.

[0092] Most accurate alcohol concentrations are obtained when water and alcohol are weighed on a balance.

[0093] When the proportion of biomaterial in the reaction mixture is high and / or the water content of the biomaterial is high, the water content of the biomaterial is taken into account to obtain the desired water vs alcohol content in the reaction mixture. Thus in specific embodiments pure alcohol is added to a moist biomaterial.

[0094] For biomaterial that has been dried or that contains less than 10 % weight water or less that 5 % weight water the amount of water can be neglected, when adding alcohol or adding a mixture of alcohol and water to the biomaterial.

[0095] Step b of the methods of the present invention is performed at elevated temperatures such as above 125 °C, above 150 °C, above 175 °C, above 200 °C, above 225 °C, above 250 °C, above 275 °C, above 300°C. For practical considerations, the maximum temperature used 350 °. Explicitly disclosed herewith are all possible ranges that can be made with the above mentioned temperatures. For example between 225 °C and 275° C.

[0096] Typically, step b of the methods of the present invention is performed at a temperature of between 240 °C and 260 °C, or between 245 and 255 °C.

[0097] Step b of the methods of the present invention is generally performed for a time period of between 1 and 10 h, more typically between 1 and 5 hours.

[0098] Examples are between 1 and 5 hours, between 2 and 5 hours, between 3 and 5 hours, between 4 and 5 hours, between 1 and 4 hours, between 2 and 4 hours, between 3 and 4 hours, between 1 and 3 hours, between 2 and 3 hours, between 1 and 2 hours.

[0099] Typically, step b of the methods of the present invention is performed for between 1 and 3 hours, are between 90 and 150 minutes

[0100] Step b of the methods of the present invention is generally performed at an elevated pressure of between 10 bar and 110 bar, where it is technically possible to increase the pressure up to 180 or 200 bar. Examples of applied pressures are between 40 and 100 bar, between 50 and 100 bar, between 60 and 100 bar, between 70 and 100 bar, between 75 and 95 bar, or between 80 and 90 bar.

[0101] Alcohols used for esterification of fatty acids, and suitable in the methods of the present invention for isolating chitin are typically C1-C5 alcohols.

[0102] Cl to C5 refers to the number of carbon atoms in an alcohol. Cl alcohol: Methanol; C2 alcohol: Ethanol ; C3 alcohol: 1-Propanol, Allyl alcohol , Isopropyl alcohol, C4 alcohol: n-Butanol, Isobutanol, sec-Butanol, tert-Butyl alcohol ; C5 alcohol: 1- Pentanol, Isoamyl alcohol, 2-Methyl-l-butanol, Neopentyl alcohol, 2-Pentanol 3- Methyl-2-butanol, 3-Pentanol, tert-Amyl alcohol.

[0103] Depending on price or availability mixtures can be used.

[0104] Typically, any of methanol, ethanol, butanol or isoamyl alcohol, or mixtures thereof are used.

[0105] Preferably, methanol or ethanol are used.

[0106] After the isolation of the solid fraction, this solid fraction can be further fractionated on particle size and e.g. isolating particles with a size of more than 100, more than 250 or more than 500 pM.

[0107] The methods of the present invention are typically performed by combining the biomaterial with only alcohol or alcohol and water. In embodiments of the invention the pH can be adjusted to pH 4, pH 4,5, pH 5, or pH 5,5.

[0108] The methods of the present invention are typically performed without changing the air which is present in the reaction vessel.

[0109] In a specific embodiments step b) is performed under an inert atmosphere or under an atmosphere of carbon dioxide.

[0110] In a specific embodiments step b) is performed under an atmosphere of hydrogen and the solvent comprises a reducing catalyst such as Pd, Pd / C or Ni.

[0111] In specific embodiments of the methods of the invention step c) is performed by filtration, centrifugation or decantation. Filtration can be done over a filter with a pore size of between 5 to 15 pm.

[0112] Alternatively step c) is performed by sieving the solid fraction after drying over a sieve with a mesh size above 250 pm.

[0113] The methods of the present invention have the further advantage that the lipids in the biomaterial are converted in fatty esters. While the solid fraction obtained in step c) contains the chitin, the remaining liquid fraction contains fatty acid esters. These can be purified by solvent extraction, for example with an nonpolar solvent that is not miscible with water or with alcohol, such as hexane, petroleum ether, iso-octane, cyclohexane or heptane.

[0114] An overview of the mass balances and fraction composition of black soldier flies is shown in Figure 4. The composition of the two powdery fractions (i.e., < 250 pm and 250 - 500 pm) had a similar composition in terms of chitin and ash content, and placed together in the overview. The overview shows that BSF lipids are predominantly recovered in the oil fraction of the fractionation procedure. Chitin is predominantly recovered in the fibrous fraction with a particle size greater than 500 pm.

[0115] A large portion of the BSF proteins could be recovered as small poly- or oligopeptides in the soluble fraction. The soluble fraction may contain compounds such as pigments (e.g., melanin), modified amino acids (e.g., pyroglutamic acid), organic acids (e.g., succinic acid, levulinic acid), heterocyclic N-compounds (e.g., pyrazines, pyrrolidinediones), alkylamides, and lactones.

[0116] EXAMPLES

[0117] Example 1. Sample pre-treatment of BSF larvae

[0118] Fifth instar larvae of Hermetia illucens were purchased from RADIUS (Thomas More, Geel, Belgium). The larvae were inactivated by blanching for 40 seconds in boiling water. Subsequently, excess water was drained off and the larvae were packaged under reduced atmospheric pressure (i.e., 700 mbar) in sealed pouches prior to being flash frozen and stored at -20 °C. This sample pre-treatment all served the purpose of obtaining a chemically stable product (e.g., oxidation) that could be used over the course of the duration of the experiments.

[0119] To assess the proximate composition, a portion of the larvae was freeze dried (Lyovapor L-200, Buchi) and characterised as described in the following sections. Solvolytic fractionation

[0120] Immediately prior to the fractionation, a portion of the larvae was ground while still frozen. The frozen larvae powder was then placed inside a high pressure stainless steel batch reactor (100 mL, Parr Instruments & Co., Ill, US) together with methanol to obtain a mass ratio of larvae: methanol of 1:4. Typically, 10 g of larvae was loaded into the reactor alongside 50 mL of methanol. The reactor was then sealed, flushed threefold with inert gas (i.e., N2) under continuous stirring, and pressurised with 6 bar N2 at room temperature. Subsequently, the reactor was heated to 200 - 250 °C under continuous stirring (500 RPM). Under these conditions, the reactor reaches a pressure of 45 bar (200 °C) to 90 bar (250 °C). After 2 to 5 hours, the heating mantel was removed and the reactor was cooled down to room temperature before it was depressurised. Afterwards, the reactor content was collected quantitatively by rinsing the reactor with methanol.

[0121] Subsequent processing is shown in Figure 1. First, the reaction mixture was filtered over a fritted glass filter (Por. 4, 5 - 15 pm) to recover the solid fraction. The solids were rinsed with methanol and dried in a forced-air oven at 65 °C for 3 hours. Second, the dried solids were further separated based on their particle size over analytical sieves 500 pm (250 pm and 500 pm, Retsch). This resulted in a pale brown and fibrous fraction, still showing parts of the BSF larval exoskeleton, as well as two pale brown powdery fractions.

[0122] The yellow-brown liquid fraction was subjected to a liquid-liquid extraction with n- hexane to recover the oil fraction. Using an n-hexane to methanol ratio of 1: 10 (v / v), a total of five extraction cycles were performed. All extracts were combined and dried with anhydrous MgSC after which the n-hexane was removed through a rotary evaporator (Buchi) . The resulting oil was clear and had a dark red-brown colour. The remaining black liquor, containing the soluble fraction, could be further concentrated by means of a rotary evaporator until a desired mass concentration (e.g., 7 - 10 g product / 100 g of liquid) was reached.

[0123] The above obtained fractions (Figure 2) will be referred to as fibrous fraction, powdery fractions, oil fraction, and soluble fraction.

[0124] Example 2. Fraction analysis of BSF larvae

[0125] The moisture content of all fractions was determined by drying in a forced-air oven at 105 °C for 17 hours. The mass fractions of nitrogen (wN) and carbon (wc) were analysed through elemental analysis (Flash 2000 Organic Elemental Analyzer, Interscience) according to the procedure described previously with minor modifications [Smets & van der Borght (2021) Anal. Bioanal. Chem. 413, 3119- 3130]. Briefly, around 5 mg of each fraction was weighed to the nearest 0.01 mg in tin-pressed capsules, which were then combusted inside the elemental analyser. For the oil fraction, the sample amount was increased to around 9.00 mg considering its low nitrogen content. In addition, an aliquot of the soluble fraction was first dried in the tin-pressed capsule using a heat block to remove the methanol, prior to taking the weight and the actual analysis. An analytical standard of acetanilide was used to prepare calibration curves to quantify both wNand wcof the fractions.

[0126] The ash content of the fractions was analysed by means of dry ashing.

[0127] The oil fraction was analysed by means of GC-MS as described in [Smets & van der Borght cited above]. Fatty acid methyl esters (FAMEs) were quantified through external calibration with analytical FAME standards.

[0128] The amino acid content of the fibrous, powdery and soluble fractions was determined via UPLC-MS after acidic and basic hydrolysis.

[0129] The chitin content of the fractions was determined after acidic hydrolysis into glucosamine as described in Smets, & van der Borght cited above.

[0130] The purity (mass fraction N-acetylglucosamine (GIcNAc) on dry matter (DM) basis) was assessed after acidic hydrolysis of the chitin into glucosamine, followed by quantification with UPLC-MS as described in our publication. The chitin yield was calculated by means of the equation below.

[0131] All analyses were performed in triplicate unless specified otherwise.

[0132] The proximate composition of the frozen starting material is shown in Table 1.

[0133] Table 1. Dry matter content of the frozen and freeze dried starting material as well as the proximate composition of the starting material

[0134] +g / 100 g as is; * g / 100 g dry matter; § g / V-acetylglucosamine / 100 g dry matter ; * sum of resp. aspartic acid with asparagine, and glutamic acid with glutamine; Example 3. Mass balance and fraction analysis of BSF larvae

[0135] Mass balances

[0136] Following the downstream processing of the reactor contents, five products were obtained. For the soluble fraction, methanol was evaporated under a flow of nitrogen gas at room temperature. The resulting dried fraction was then weighed to determine the yield of soluble products. The product yields of the solvolytic fractionation relative to the dry matter content of the starting material are shown in Table 2.

[0137] Table 2. Product yields of the solvolytic fractionation of black soldier fly biomass, mass based.

[0138] Fraction analysis

[0139] Analysis of the oil fraction with GC-MS (Table 3) shows high contents of fatty acid methyl esters (i.e., 72.55 g / 100 g oil), with methyl laurate being the most predominant FAME, followed by methyl palmitate, methyl linoleate, methyl oleate, and methyl myristate. The profile of the obtained BSF oil showed a high resemblance to those of coconut and palm kernel oil. Table 3. Fatty acid methyl ester content of the oil fraction (wFAME) and the FAME profile of the obtained oil fraction. The standards for coconut and palm kernel oil (expressed as profile) are shown as well. n.s., no signal was observed; n.a., not applicable

[0140] EPA, Eicosapentaenoic acid; FAMEs, fatty acid methyl esters

[0141] The three solid fractions (e.g., the fibrous and two powdery fractions) were analysed for their chitin content as well as for potential contaminants like amino acids and ash.

[0142] The obtained results clearly show a distinct difference between the fibrous and powdery fractions (Table 4).

[0143] The fibrous fraction, having a particle size greater than 500 pm, has a chitin content above 50 %. A small amount of amino acids were found in this fraction (0.25 g amino acids / 100 g fibrous fraction).

[0144] Most of these amino acids are bound to the matrix since only 11 - 14 % was present as free amino acids.

[0145] Ash accounting for almost a quarter of the obtained fibrous fraction and over half of the powdery fractions. The low chitin content of the powdery fractions is due the presence of fine insect cuticle particles, as observed visually.

[0146] If the larvae are not ground prior to the fractionation procedure, more chitin is likely recovered in the fibrous fraction. The present solvolytic fractionation procedure has advantages over the prior mechanical methods, namely high chitin concentration with little or no amino acids Less chemicals are required to achieve this degree of purification. The main solvent methanol and n-hexane (if FAME isolation is envisaged) can be recuperated and reused in the process.

[0147] The fibrous fraction which comprises a high quantity of ash can be treated with standard methods such as treatment with low concentrations of acids to remove these contaminants.

[0148] Table 4. Composition of the fibrous fraction (> 500 pm) and two powdery fractions (250 - 500 pm and < 250 pm) obtained through the solvolytic fractionation of black soldier fly larval biomass.

[0149] Further analysis of the obtained ashes with ICP-OES, identifies the major minerals present in the solid fractions (Table 5). It is presumed that a large portion of these minerals is bound to the carbonate ion (e.g., CaCCh), as strong effervescence occurred when concentrated acid was added to the obtained ashes. This effervescence was the strongest in the powdery fractions, especially with particle size 250 - 500 pm. The results of the ICP analysis also confirm the presence of phosphorous in the ashes, suggesting the presence of phosphate salts.

[0150] Table 5. Mineral content of the three obtained solid fractions through the solvolytic fractionation of black soldier fly larval biomass, expressed as the mass fraction of each mineral in the fibrous and powdery fractions (g / 100 g dry matter). Results represent the mean ± standard deviation of two measurements. Means in the same row with a different superscript differ significantly (p < 0.05).

[0151] Concerning the soluble fraction, several analyses were performed to determine its composition, which is expressed on dry matter basis (i.e., g / 100 g dissolved compounds) in the following section (Table 6). Analysis with GC-MS revealed a FAME content of 0.14 %, and the presence of some residual fatty acids (i.e., ± 4 %) due to incomplete transesterification. GC-MS data also revealed the presence of nitrogencontaining compounds like pyrrolidinediones and alkylamides, which may have formed as the result of amino acid decomposition. Dry ashing of the dissolved compounds resulted in a very fine, white ash which accounted for 3.14 % of the soluble compounds.

[0152] Table 6. Composition of the soluble fraction obtained through the solvolytic fractionation of black soldier fly larval biomass, expressed as the mass fraction of the dissolved compounds (g / 100 g dry matter).

[0153] The amino acid analysis of the liquid fraction is shown in Table 7. About 30 % of the soluble fraction are amino acids, of which merely 0.14 % was present as free amino acids. This indicates BSF proteins are not completely solvolyzed into individual amino acids but rather stay in solution as oligo- or polypeptides. This was also confirmed through SDS-PAGE, which showed a very dense band of small peptides located in the migration front (Figure 3). Table 7. Total amino acid content of the soluble fraction. Results are expressed as the mean value of two replicates ± standard deviation.

[0154] The chitin content of the soluble fraction, expressed as the mass fraction of N- acetylglucosamine, was found to be 0.19 g / 100 g dissolved compounds. As the soluble fraction was free of insoluble material, this / V-acetylglucosamine was presumed to be present either as such or in the form of oligosaccharides, which might suggest a partial solvolysis of the chitin polymer.

[0155] Example 4. Glucosamine determination of BSF larvae

[0156] Chitin isolated from BSF larvae as detailed in the above examples was characterised by Fourier-transform infrared spectroscopy (FTIR) using KBr pellets (Thermo Scientific Nicolet iS5). Absorbance values were measured between 4000 and 400 cm’x. IR spectra were recorded by accumulating 64 scans with a resolution of 2 cm’1. The resulting spectra were processed using Thermo Scientific OMNIC 7.3 software. Commercially available o-chitin from shrimp (Sigma-Aldrich) was also analysed as a reference.

[0157] The degree of acetylation (DA) was determined according to the equation described by Brugnerotto et al. (2001) Polymer 42(8), 3569-3580, where A1320 and A1420 are the values of absorbance at 1320 (baseline from 1276 to 1348 cm’1) and 1420 cm’1(baseline from 1402 to 1478 cm’1) respectively.

[0158] 41320 / 0.3822

[0159] DA [%] = / 41420

[0160] 0.03133 Figure 1 shows the difference of the absorbance at these wavelengths indicating the conversion of chitin to chitosan.

[0161] Table 8. Degree of acetylation of various chitin preparations.

[0162] Example 5. Water content of biomaterial

[0163] To evaluate whether the methods of the present invention are both suitable for fresh moist as well as dry biomass, fresh black soldier fly (BSF) larvae was dried through freeze drying for 48 h. When the dried biomass was processed, the amount of added water was adjusted to have the same water content in the reaction mixture as with moist fresh biomaterrial. After processing, the purity (mass fraction N- acetylglucosamine (GIcNAc) on dry matter (DM) basis) was assessed after acidic hydrolysis of the chitin into glucosamine, followed by quantification with UPLC-MS as described in Smets et al. cited above. The similar purity of the end products clearly demonstrate that the prior drying step does not influence the purification.

[0164] Table 9. Chitin isolation from fresh and dried larvae.

[0165] Example 6. Comparative examples

[0166] Using the same starting material, equipment and analysis methods as in example 1- 3 BSF larvae were processed using alternative methods. Condition 0 refers to the experimental conditions as detailed in examples 1 to 3.

[0167] Table 10. chitin purification isolation of BSF larvae using alternative reaction conditions. Condition 1. Treatment with 1 M HCI at 100 °C for 2 h only resulted in a chitin content of the purified product of 36 %. This experimental procedure is the first step of the method Liu et al. (2012) cited above.

[0168] This treatment at atmospheric pressure with 1 M HCI is therefore unable to generate a purity similar to the methods of the present invention with an alcohol and water. In addition, sample workup was cumbersome due to lipid hydrolysis into fatty acids, which together with proteins in the sample clogged filters.

[0169] Condition 2. Treating larvae with boiling methanol (no added water) at atmospheric pressure at 65°C results in to purify chitin from the larval biomass (i.e., < 10 %), indicating the relevance of the temperature.

[0170] The use of dried larvae with pure methanol added and reacted at 250 °C for 2 h, 89 bar results in a yield of 20.52 g GIcNAc 100 g dry matter

[0171] Condition 3. This condition is the prior art procedure of defatting, acid and alkaline treatment. Although a high purity was obtained, the procedure was time consuming (24+ h in total) and large quantities of waste were produced.

[0172] Condition 4. Treating with water only (no added alcohol) at 250 °C gives unsatisfactory yield in terms of chitin purification and practical considerations. Hydrolysed lipids drastically hindered product workup, as the filter clogged and a sticky sample sticks to the walls of reactor to filters, to bottles, etc., resulting in significant product loss.

[0173] Example 7. Alternative arthropod biomaterials

[0174] Using the same method, equipment and analysis methods as in example 1-3, other arthropods biomaterials were processed. The biomaterial was frozen, freeze dried, and homogenised.

[0175] Condition 0 refers to the experimental conditions as detailed in examples 1 to 3.

[0176] Condition 5. Larvae of the yellow mealworm (a holometabolous insect species) yielded a high chitin content comparable to BSF larvae.

[0177] Condition 6. Fully developed crickets (a hemimetabolous insects) yielded a high chitin content comparable to BSF larvae. Condition 7. Shrimp waste (crustacean) is at present the state of the art industrial source of chitin. Shrimp waste is known to contain a high content of minerals. However, chitin content increased from 9.99 g / lOOg dry matter to 43.82 g / dry matter.

[0178] Condition 8. Exuviae or insect moults are a side stream from insect rearing companies. Processing this biomass using the conditions for black soldier fly equally allow a substantial enrichment in chitin.

[0179] Table 11. Chitin isolation of various arthropod biomaterials.

[0180] Example 8. Chitin isolation from fungal biomaterial Using the same method, equipment and analysis methods as in example 1-3, mushrooms (Agaricus bisporus) were processed.

[0181] The fungal material was frozen, freeze dried, and homogenised.

[0182] Condition 0 refers to the experimental conditions as detailed in examples 1 to 3. Table 12. Chitin isolation of mushrooms.

[0183] The conditions used for black soldier fly larvae were applied on a representative species of fungi, namely Agaricus bisporus. While processed at 150 °C, problems as mentioned in US2014275507 have not been encountered using methanol and water.

[0184] Example 9. Use of different alcohols in the isolation of chitin from BSF

[0185] Using the same starting material, equipment and analysis methods as in example 1- 3, BSF larvae were processed with the exception that different alcohols were used (+ formic acid in condition 13). Condition 0 refers to the experimental conditions as detailed in examples 1 to 3.

[0186] Table 13. Use of different alcohols in the isolation of chitin from BSF.

[0187] The above overview illustrates that all of the above alcohols are suitable in the methods of the present invention.

[0188] In the specific conditions of temperature, time, pressure, water content and starting material and butanol provide an optimal chitin yield.

Claims

CLAIMS1. A method for the isolation of chitin and / or chitosan from arthropod or fungal biomaterial, the method comprising the steps of: a) combining said biomaterial with an alcohol, or with a mixture of water and an alcohol, thereby obtaining a reaction mixture, b) incubating the reaction mixture of step a) at a temperature above 150 °C, c) isolating from the composition obtained in step b) the solid fraction comprising chitin and / or chitosan.

2. The method according to claim 1, wherein in step a) said biomaterial is combined with an alcohol or with a mixture of water and an alcohol, thereby obtaining a reaction mixture consisting of biomaterial and alcohol or obtaining a reaction mixture with an alcohol concentration of at least 25 vol % alcohol.

3. The method according to claim 1 or 2, wherein in step a) said biomaterial is combined with an alcohol or with a mixture of water and an alcohol, to obtain a reaction mixture with an alcohol concentration of between 80 to 90 vol % alcohol.

4. The method according to any one of claims 1 to 3, wherein step b) is performed for a period of between 1 and 5 hours.

5. The method according to any one of claims 1 to 4, wherein step b) is performed for a period of between 90 minutes and 150 minutes.

6. The method according to any one of claims 1 to 5, wherein step b) is performed a temperature of between 200 to 300 °C.

7. The method according to any one of claims 1 to 6, wherein step b) is performed a temperature of between 225 to 275 °C.

8. The method according to any one of claims 1 to 7, wherein step b) is performed at a pressure of between 6 and 110 bar.

9. The method according to any one of claims 1 to 8, wherein step b) is performed at a pressure of between 40 and 100 bar.

10. The method according to any one of claims 1 to 9, wherein step b) is performed at a pressure of between 70 and 100 bar.

11. The method according to any one of claims 1 to 10, wherein step b) is performed at a pressure of between 85 and 95 bar.

12. The method according to any one of claims 1 to 11, wherein step b) is performed a temperature of between 225 to 275 °C, for a period of between 90 minutes and 150 minutes .

13. The method according to any one of claims 1 to 12, wherein step b) is performed a temperature of between 225 to 275 °C, at a pressure of between 40 and 100 bar, for a period of between 90 minutes and 150 minutes .

14. The method according to any one of claims 1 to 13, wherein the reaction mixture in step a) has an alcohol concentration between 80 to 90 vol % alcohol and step b) is performed a temperature of between 225 to 275 °C, at a pressure of between 40 and 100 bar, for a period of between 90 minutes and 150 minutes.

15. The method according to any one of claims 1 to 15, wherein the alcohol is a Ci- C5 alcohol.

16. The method according to claim 15, wherein the alcohol is one or a mixture of methanol, ethanol, or isoamylalcohol.

17. The method according to any one of claims 1 to 16, wherein step b) is performed at a pH above 4, at a pH above 4,5, at a pH above 5, or at a pH above 5,5.

18. The method according to any one of claims 1 to 17, wherein step b) is performed under an inert atmosphere.

19. The method according to claim 18, wherein step b) is performed under an inert atmosphere such as hydrogen and the reaction mixture comprises a reducing catalyst such as inert Pd, Pd / C or Ni.

20. The method according to any one of claims 1 to 17, wherein step b) is performed under an atmosphere of carbon dioxide.

21. The method according to any one of claims 1 to 20, wherein step c) is performed by filtration over a filter with a pore size of between 5 to 15 pm, or by sieving the solid fraction after drying over a sieve with a mesh size above 250 pm.

22. The method according to any one of claims 1 to 20, wherein step c) is performed by extraction with a solvent that removes the liquid fraction and whereby the chitin and / or chitosan remain in the solid fraction.

23. The method according to any one of claims 1 to 22, wherein the arthropods are insects, and wherein the biomaterial are insect larvae, puparia, pupae, imagoes, nymphs or exuviae.

24. The method according to claim 23, wherein the insect larvae are from Hermetia illucens, Musca domestica, Tenebrio molitor, or from Alphitobius diaperinus.

25. The method according to any one of claims 1 to 22, wherein the fungal biomaterial is of Agaricus bisporus.

26. The method according to any one of claims 1 to 25, and wherein the ratio of weight methanol vs weight biomaterial is 4: 1.

27. The method according to any one of claims 1 to 26, wherein the biomaterial or insect larvae have a dry matter content of between 30 to 40 wt %.

28. The method according to any one of claims 1 to , further comprising step d) of separating the solid fraction on particle size, and isolating particles with a size of more than 100, more than 250 or more than 500 pM.

29. The method according to any one of claims 1 to 28, further comprising a step of isolating the liquid fraction obtained after the treatment of step b) and purifying fatty acid esters therefrom.

30. The method according to claim 29, wherein fatty acid esters are purified by solvent extraction, typically with an nonpolar solvent that is not miscible with water or with alcohol.

31. The method according to claim wherein the solvent is hexane, petroleum ether, iso-octane, cyclohexane or heptane.

32. A method of converting chitin into chitosan comprising the step of a) combining a composition comprising at least 25 wt % chitin with an alcohol or with a mixture of water and an alcohol thereby obtaining a reaction mixture, b) incubating the reaction mixture of step a) to a temperature above 150 °C, thereby deacetylating chitin and forming chitosan.

33. The method according to claim 32, wherein the composition of step a) comprises at least 50 wt % or at least 75 wt % chitin.

34. The method according to claim 32 or 33, wherein step b is performed between 225 and 275 ° C, for a period of between 1 to 5 h, at a pressure of between 80 and 100 bar.

35. The method according to any one of claims 32 to 34, wherein the reaction mixture in stap a) contains between 70 and 90 vol % alcohol.

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