Lipid extraction from insects

Abstract

Aspects of the present disclosure are directed to lipid extraction from insects. According to an aspect of the present disclosure, a method for extracting lipids includes preparing an insect biomass from dried insects (e.g., by grinding them into a powder), incubating the powder with a green solvent under suitable extraction conditions to form a lipid-containing solution and a defatted 5 biomass, separating the lipid-containing solution from the defatted biomass, and removing the solvent to recover the lipid product. The disclosure further includes a composition containing the lipid product obtained by this method, and using the composition as an anti-foaming agent in fermentation or bioprocessing media. In another embodiment, the method also includes adding a non-polar solvent to the lipid-containing solution, adding ice-cold acetone to precipitate phospholipids, and separating the phospholipid precipitate. According to another aspect, the composition containing the phospholipids can be used in nutraceutical, food-grade, pharmaceutical-grade, or cosmetic-grade applications.

Description

[0001] LIPID EXTRACTION FROM INSECTS

[0002] PRIORITY CLAIM

[0003] The instant patent application is related to and claims priority from the India provisional application entitled, “METHODS FOR LIPID EXTRACTION FROM INSECTS”, Application No.: 202441052527, Filed: 09 July 2024, which is incorporated in its entirety herewith to the extent not inconsistent with the description herein.

[0004] TECHNICAL FIELD

[0005] The present disclosure related to lipids and lipophilic constituents, such as phospholipids, derived from insects and more particularly, their extraction for various industrial applications. In particular, there is disclosed the use of environmentally friendly solvents in the extraction.

[0006] BACKGROUND

[0007] Insects are a promising source of high-quality biomolecules like proteins, fatty acids, lipids and phospholipids, sterols, bioactive compounds and even biomaterial compounds with a substantially lower ecological footprint. As insects have higher reproductive rates, less land use, lower energy consumption, less water use, and produce lower levels of greenhouse gases, for years, humankind has utilized insects in food or feed products. Consequently, insect-derived materials have found use in food and feed industries and are now being explored more broadly for high- value applications.

[0008] Lipids or oils extracted from insects are rich in essential fatty acids, phospholipids, and other valuable lipophilic nutrients, making them potential ingredient for various applications in the food, feed, cosmetics, plant nutrition, lab reagents and pharmaceutical industries. These lipids or oil have gained increasing attention in recent years as a sustainable and environmentally friendly alternative to conventional vegetable oils and animal fats. The challenge however is the extraction of lipids and lipophilic constituents from insects and their efficient separation from other components such as proteins, using optimised and sustainable methods.

[0009] Crude insect lipids typically comprise several types of lipids, such as triacylglycerols, phospholipids, sterols, glycolipids. Among the various constituents, phospholipids are an important lipid class and the content of these lipid classes in crude lipid is usually below 20% but this varies between life stage and insect species. These lipids are polar and have great significance in cosmetic formulations, nutritional supplements, and drug delivery systems due to their amphiphilic structure and bioactive properties. However, phospholipids are structurally integrated into cellular membranes and often bound to proteins or polysaccharides, making their selective extraction particularly challenging. Traditional methods often use solvents like chloroform and methanol, which effectively extract phospholipids but pose health and environmental risks due to their toxicity.

[0010] The extraction efficiency from insect biomass varies significantly with species, life stage, and rearing conditions. Solvent-based methods remain the most prevalent, particularly Soxhlet and maceration extraction using n-hexane, petroleum ether, or chloroform-methanol systems. Despite their efficiency, these solvents are unsuitable for food or pharma applications due to toxicity, flammability, and regulatory restrictions.

[0011] Traditionally, lipid extraction from insects has relied on organic solvents such as hexane, petroleum ether, or chloroform-methanol mixtures using Soxhlet or Bligh-Dyer protocols. While effective, these approaches involve long extraction cycles, require high energy input, and rely on toxic solvents that are unsuitable for food, pharmaceutical, or environmental applications.

[0012] While newer approaches like supercritical CO2 extraction and enzymatic processing are an emerging technology for extracting lipids and other biomolecules from organic sources, they require significant capital investment and complex equipment, rendering them impractical for decentralized or low-cost applications. Mechanical pressing, though scalable, results in degraded lipids that demand extensive post-purification and often yield insufficient recovery of polar lipids like phospholipids.

[0013] To overcome these challenges, green solvents have been explored as environmentally benign alternatives. Green solvents such as ethyl acetate, dimethyl carbonate (DMC), 2- methyltetrahydrofuran (2-MeTHF), CPME, D-limonene, ethanol, isopropanol, and tert-butyl alcohol have been assessed for their polarity, boiling point, toxicity, and biodegradability. Despite the proposed use of green solvents as alternatives to hazardous chemicals for lipid extraction, there remains a need in the art for suitable green solvents and corresponding methods for both lipid extraction and subsequent phospholipid precipitation.

[0014] US9375453B2 discloses method of production of polar krill oil from Euphausia superba and use of polar solvent to extract a polar krill oil comprising phospholipids. US20090028989A1 discloses a phospholipid composition obtained from a fish meal by use of an organic solvent to produce a lipid-containing liquid. US8273248B1 discloses extraction of neutral lipids from algal cells and describes methods for separating neutral lipids using amphipathic and hydrophobic solvent sets in single and multistep extraction processes.

[0015] Although solvent extraction is a widely established method for recovering lipids or oils from a variety of biological sources, such as fishmeal, krill, marine algae, plant seeds, oil-rich fruits, and microbial biomass, there remains a critical need to optimize extraction parameters and conditions specifically for insect biomass. Insects are rapidly gaining global recognition as a sustainable, nutrient-rich, and ecologically viable source of proteins, lipids, and bioactive compounds. Abutaha et al has reported higher lipid yields from mealworms (Tenebrio molitor) following extraction with green solvents, namely ethyl acetate (EtOAc) and Isopropanol (IPA), compared extraction with n-hexane. Similarly.. Ruben Smets et al has shown similar higher yields of lipids following extraction with EtOAc, ethanol (EtOH) and IPA from black soldier fly larvae (Hermetia illucens).

[0016] In spite of studies that show the effectiveness of use if green solvents for lipid extraction from invertebrates and some insects, each insect matrices differ significantly from conventional oil sources in their structural composition, lipid distribution, and association with proteins and chitin, posing unique challenges for solvent penetration and lipid selectivity, necessitating the development of environmentally sustainable and efficient extraction methods that yield high- quality oil with minimal ecological footprint.

[0017] The present disclosure addresses these challenges by providing a novel method for extracting lipids from insects using green solvents. This approach not only ensures a reduced environmental footprint but also offers high extraction efficiency and facilitates the isolation of specific, high-value lipid components such as phospholipids, which are often challenging to obtain selectively using conventional methods. Specifically, the disclosure seeks to overcome the limitations of prior art by providing an optimized green solvent system and process parameters tailored for diverse insect biomass, thereby yielding purified lipid products with minimal solvent residues.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0020] Figure 1 is a table depicting the percentage of fat extracted from various insect biomass samples over three days by a modified column extraction method using n-hexane as the solvent, according to an aspect of the present disclosure.

[0021] Figure 2 is a table depicting the residual fat percentage of different insect biomass samples after fat extraction using n-hexane under various biomass-to-solvent ratios and extraction durations, according to an aspect of the present disclosure.

[0022] Figure 3 is a graph showing the residual fat percentage of Bombyx mori (Multivoltine) insect biomass after fat extraction for two hours using different green solvents at 1:3 w / v, 1:5 w / v, and 1:10 w / v biomass-to-solvent ratios, according to an aspect of the present disclosure.

[0023] Figure 4 is a graph showing the residual fat percentage of Bombyx mori biomass after fat extraction using acetone at 50°C for two and three hours, according to an aspect of the present disclosure.

[0024] Figure 5A is a graph showing the residual fat percentage of Bombyx mori biomass after fat extraction using 50%, 70%, and 100% ethanol at 60°C for two hours, according to an aspect of the present disclosure.

[0025] Figure 5B is a graph showing the protein percentage of Bombyx mori biomass after fat extraction using 50%, 70%, and 100% ethanol at 60°C for two hours, according to an aspect of the present disclosure.

[0026] Figure 6 is a table depicting the residual fat percentage and protein percentage of Bombyx mori (Multivoltine) insect biomass after fat extraction using ethyl acetate at different biomass-to- solvent ratios, extraction durations, and extraction stages at 60°C, according to an aspect of the present disclosure.

[0027] Figure 7 is a table depicting the residual fat percentage and protein percentage of Bombyx mori (Multivoltine) insect biomass after fat extraction using dimethyl carbonate at different biomass-to-solvent ratios, extraction durations, and extraction stages at 60°C, according to an aspect of the present disclosure.

[0028] Figure 8A is a table depicting the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass subjected to column extraction using ethyl acetate at a 1:10 w / v biomass-to-solvent ratio, followed by processing using the pellet method, according to an aspect of the present disclosure.

[0029] Figure 8B is a table depicting the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass subjected to column extraction using ethyl acetate at a 1:10 w / v biomass-to-solvent ratio, followed by processing using rotary evaporation, according to an aspect of the present disclosure.

[0030] Figure 9A is a table depicting the percentage of fat removed from Bombyx mori (Multi voltine) insect biomass subjected to column extraction using dimethyl carbonate at a 1:10 w / v biomass-to-solvent ratio, followed by processing using the pellet method, according to an aspect of the present disclosure.

[0031] Figure 9B is a table depicting the percentage of fat removed from Bombyx mori (Multi voltine) insect biomass subjected to column extraction using dimethyl carbonate at a 1:10 w / v biomass-to-solvent ratio, followed by processing using rotary evaporation, according to an aspect of the present disclosure.

[0032] Figure 10 is a table depicting the average percentage of fat extracted from Bombyx mori (Multivoltine) insect biomass over three days using n-hexane, ethyl acetate, and dimethyl carbonate during column extraction at a 1: 10 w / v biomass-to-solvent ratio, according to an aspect of the present disclosure.

[0033] Figure 11 is a table showing the percentage of phospholipids extracted from various insect biomass samples using sequential solvent extraction with ethanol, n-hexane, and ice-cold acetone, according to an aspect of the present disclosure.

[0034] Figure 12 A is an image depicting the foaming behavior of a solution before the addition of insect oil.

[0035] Figure 12B is an image depicting the foaming behavior of the same solution after the addition of insect oil, showing a reduction in foam.

[0036] In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[0037] SUMMARY

[0038] Aspects of the present disclosure are directed to a method for lipid extraction from insects. According to an aspect of the present disclosure, a method comprises grinding dried insects to obtain an insect biomass powder; incubating the insect biomass powder with at least one green solvent capable of dissolving lipids, under suitable extraction conditions to form a lipid-containing solution and a defatted biomass; separating the lipid-containing solution from the defatted biomass; and recovering the lipid product by removing the green solvent.

[0039] In an embodiment, the insects may be selected from Bombyx mori, Samia ricini, Antheraea assamensis, Hermetia illucens, Periplaneta americana, Gryllus bimaculatus, Tenebrio molitor, Diploptera punctata, Zophobus mono, or a combination thereof.

[0040] In another embodiment, the insects may be selected from Acheta domesticus, Gryllodes sigillatus, Melanoplus sanguinipes, Sphenarium purpurascens, Schistocerca gregaria, Alphitobius diaperinus, or Ruspolia differens , Gromphadorhina portentosa, Teleogryllus emma, Oxya yezoensis, Gonimbrasia belina, Locusta migratoria, Schistocerca spp., Tettigoniidae, Thasus gigas, Liometopum apiculatum, Atta cephalotes, Lethocerus indicus, Antheraea pemyi, Atta laevigata, Magicicada spp, Cicada spp. or a combination thereof.

[0041] In yet another embodiment, the green solvent may be selected from the group consisting of dimethyl carbonate (DMC), methyl acetate, 2 -methyltetrahydrofuran (2-MeTHF), cyclopentyl methyl ether (CPME), D-limonene, p-cymene, and a-pinene, or a combination thereof. The green solvent may further be selected from ethanol, isopropanol, tert-butyl alcohol, 1 -butanol, 1- propanol, or isoamyl alcohol.

[0042] In yet another embodiment, the suitable extraction conditions may comprise a temperature below the boiling point of the green solvent.

[0043] In yet another embodiment, the separation of the lipid-containing solution from the defatted biomass is carried out by filtration or centrifugation.

[0044] According to another aspect of the present disclosure, a method for phospholipid isolation comprising adding a non-polar solvent to the lipid-containing solution to form a non-polar solvent- lipid solution, and adding a neutral or slightly polar solvent to the non-polar solvent-lipid solution to precipitate phospholipids, is disclosed.

[0045] In an embodiment, the neutral or slightly polar solvent may be selected from acetone, ethanol, isopropanol, methanol, tert-butyl alcohol, or acetonitrile

[0046] In yet embodiment, the phospholipid precipitate may be separated by filtration or centrifugation to obtain purified phospholipids.

[0047] In an embodiment of the present disclosure, the non-polar solvent is selected from n- hexane, petroleum ether, diethyl ether, cyclohexane, chloroform, toluene, benzene, carbon tetrachloride, d-Limonene, 2-Methyltetrahydrofuran, p-Cymene and isopropyl myristate.

[0048] According to another aspect of the present disclosure, a composition comprising the purified lipid product obtained by the lipid extraction method is disclosed.

[0049] According to another aspect of the present disclosure, a composition wherein the purified lipid product is formulated for use as an anti -foaming agent in fermentation or bioprocessing media is disclosed.

[0050] According to another aspect of the present disclosure, there is provided the use of the purified phospholipids in fermentation media selected from food-grade fermentation, ethanol production, or microbial bioreactors.

[0051] According to another aspect of the present disclosure, there is provided a composition comprising the purified phospholipid product extracted by the phospholipid isolation method, wherein the composition is formulated for use in nutraceutical, food-grade, pharmaceutical-grade, or cosmetic-grade applications.

[0052] Several aspects of the present disclosure are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the disclosure can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well known structures, materials, or operations are not shown in detail to avoid obscuring the features of the disclosure. Furthermore, the features / aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness. DETAILED DESCRIPTION

[0053] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as illustrated therein being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0054] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof.

[0055] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0056] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more compositions or elements or structures or components preceded by “comprises... a” does not, without more constraints, preclude the existence of other compositions or elements or other structures or other components or additional compositions or additional elements or additional structures or additional components.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0058] Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0059] Parameters of different steps, components, and features of the embodiments are described separately but may be combined consistently with this description to enable other embodiments as will be understood by those skilled in the art.

[0060] The present disclosure provides a novel lipid composition, including valuable phospholipids, and efficient methods for extracting them, from insect biomass using environmentally friendly green solvents. This method addresses the limitations of conventional lipid extraction techniques, which often rely on hazardous organic solvents, high energy consumption, and complex purification steps. The disclosed process offers a sustainable alternative, yielding high-quality lipid products with minimal environmental impact. For the purposes of this disclosure the terms lipid, oil, fat mean the same and may be used interchangeably.

[0061] The starting material for the lipid extraction process is insect biomass. Various insect species can be utilized, including but not limited to: Bombyx mori (silkworm), Hermetia illucens (Black Soldier Fly), Tenebrio molitor (mealworm), Gryllus assimilis (cricket), Galleria mellonella (Greater Wax Moth), Zophobas morio (Superworm), Blattella germanica (German cockroach), Acheta domesticus (house cricket), Locusta migratoria (migratory locust), and Apis mellifera (honeybee). The insect biomass is typically in a dried form, such as oven-dried or freeze-dried.

[0062] The starting material may further include arthropods and / or invertebrates. Non-limiting examples of suitable insects include: super worms (Zophobas morio)', crickets (House Cricket (Acheta domesticus), Banded Cricket (Gryllodes sigillatus), and Two-spotted Cricket (Teleogryllus emma))', mealworms (Tenebrio molitor)', lesser mealworms (Alphitobius diaperinus)', black soldier fly (Hermetia illucens)', grasshoppers, locusts, or katydids (Differential grasshopper (Melanoplus differentialis), Migratory grasshopper (Melanoplus sanguinipes), Chapulines Grasshopper (Sphenarium purpur asc ens), Desert Locust (Schistocerca gregaria), Schistocerca spp., Locusta migratoria, Oxya grasshopper (Oxya yezoensis), Nsenene Katydid (Ruspolia differens), katydids of the family Tettigoniidae)', silkworms (Bombyx mori), including bivoltine and multivoltine strains; mopane worm (Gonimbrasia belina)', giant water bug (Lethocerus indicus)', Giant mesquite bug (Thasus gig as)', cicadas (Magicicada spp. and Cicada spp.); Madagascar hissing cockroach (Gromphadorhina portentosa)', edible ants including Liometopum apiculatum, Atta cephalotes, and Atta laevigata', and Chinese oak silkmoth (Antheraea pemyi)', or a combination thereof.

[0063] The lipid and protein composition of insect biomass can vary significantly depending on the species, life stage, and rearing conditions. For example, silkworm pupae contain about 25-30% fats and 55-65% proteins. Further, in the insect biomass, lipids can be distributed in different tissues and structures, such as the fat body, cuticle, and reproductive organs. Pre -treatment methods are applied to increase lipid accessibility prior to extraction. For instance, the dried insect biomass undergoes a pre-treatment step to increase the surface area and facilitate efficient lipid release. This typically involves physical disruption methods, such as grinding or pulverizing, to obtain an insect biomass powder. Other suitable physical disruption techniques may include, but are not limited to, ultrasonication, microwave treatment, electric pressing, bead milling, or a combination thereof. The chosen method aims to effectively break down the insect cellular structures, making the lipids more accessible to the green solvent. The particle size of the powder can be optimized to maximize extraction efficiency. Other pre-treatment processes may include methods such as mechanical disruption, enzymatic treatment, or ultrasound-assisted techniques, to enhance lipid accessibility and improve extraction yields.

[0064] As used herein, the term “insect biomass” refers to a powdered or ground material prepared from one or more whole dried insects in any developmental stage, comprising lipids, proteins, and structural components such as chitin.

[0065] The "leftover insect biomass" as used herein is the solid residue remaining after the oil or fat extraction from the insect biomass. This residue consists of defatted or partially defatted material and proteins.

[0066] As used herein, the term “whole insect” refers to an intact insect body in larval, pupal, or adult form, processed without prior removal of lipids or proteins.

[0067] The prepared insect biomass powder is then incubated with at least one green solvent capable of dissolving lipids under suitable extraction conditions. The term “green solvent” refers to biodegradable organic solvent derived from naturally (water and CO2) or agricultural crops or residues (terpenes) which have good solubilizing properties like conventional solvents and offer a sustainable alternative to routine or conventional solvents. Examples of suitable green solvents include, but are not limited to alcohols such as ethanol, isopropyl alcohol, tert-butyl alcohol, butanol, isoamyl alcohol; esters such as dimethyl carbonate (DMC), ethyl lactate, ethyl acetate; ketones such as acetone; ethers such as cyclopentyl methyl ether (CPME), 2- methyltetrahydrofuran (2-MeTHF); plant-derived terpenes such as D-limonene, p-cymene, a- pinene; ionic liquids (ILs) such as those based on choline chloride, choline acetate, quaternary ammonium salts, and phosphonium salts; deep eutectic solvents (DESs) formed from combinations such as choline chloride and urea, betaine and lactic acid, or other suitable hydrogen bond donor / acceptor pairs; supercritical fluids such as supercritical carbon dioxide (CO2); and blends of one or more of the aforementioned green solvents can also be used to optimize extraction selectivity and efficiency. In a specific embodiment the green solvent may be DMC.

[0068] In an aspect, different insect species were subjected to lipid extraction by a modified form of a simple and effective method known as column extraction. This method eliminates the need for heavy machinery and offers a cost-effective alternative to conventional methods such as Soxhlet method, mechanical pressing, supercritical CO2, etc. In this method, the insect biomass after grinding into powder form was incubated with solvent, at a specific ratio in the range of 2:1 to 10:1 and kept undisturbed for at least 1 hour to a few days at room temperature. The mixture may be agitated or stirred during incubation to enhance mass transfer and improve extraction efficiency. During the incubation, the green solvent dissolves the lipids present in the insect biomass, forming a lipid-containing solution and leaving behind a defatted biomass.

[0069] The lipid extracted by this method was analysed by methods known in the art, in particular, by measuring the reduction in the weight of the insect biomass after fat removal and / or by measuring the weight of the fat dissolved in the solvent after separating it from the solvent by rotary evaporation. The rotary evaporation method provides an improved method of measurement with accurate data as it is easier to remove solvent traces from liquid than from solid material.

[0070] Following the incubation, the lipid-containing solution is separated from the defatted biomass. Common separation methods include filtration (e.g., using filter paper, membrane filtration) or centrifugation. The defatted biomass can be further processed or utilized for other applications (e.g., protein recovery, animal feed).

[0071] “Proximate analysis” as used herein refers to different methods to determine the approximate amounts of major components, such as proteins and fat in insect biomass.

[0072] “Proximate value” as used herein refers to the estimated or approximate fat content obtained through the methods used in the disclosure.

[0073] As used herein, the “Soxhlet method” is a process to extract organic compounds from solid samples. More specifically, the method is used to defat insect biomass, removing fats and oils to obtain a protein-rich material. This method determines the “total fat” present in the insect biomass before the defatting process.

[0074] The term “extracted fat” or extracted oil as used herein, refers to the amount of fat separated during the defatting process and is estimated by methods known in the art.

[0075] The lipid product is then recovered from the lipid-containing solution by removing the green solvent. This is typically achieved through methods such as vacuum drying or evaporation under reduced pressure, which are gentle enough to preserve the quality of the extracted lipids and allow for efficient solvent recovery and recycling. The ability to effectively recover and recycle the green solvent is a key advantage of this method, contributing to its environmental sustainability and cost-effectiveness.

[0076] Figure 1 shows the percentage of lipid or oil or fat extracted from various insect biomass sources after 1, 2 and 3 days using the modified column extraction method. The results were compared to the proximate analysis data obtained by the Soxhlet method. The values obtained by fat extraction by the present disclosure was observed to be comparable to the values obtained by conventional methods such as Soxhlet.

[0077] The methods of the disclosure showed the fat-extracted values of Bombyx mori (strain: multi voltine), Bombyx mori (strain: bivoltine), Sarnia ricini (Eri silkworm); Zophobus morio (super worms), Tenebrio molitor (mealworms), Periplaneta americana (roaches), Gryllus bimaculatus (crickets), and Hermetia illucens (black soldier fly) were close to their respective proximate values, indicating the efficiency of the method used. It is to be understood that all the insects have their own fat extraction values and cannot be compared.

[0078] The lipid or fat content in percentages may be analysed by any of the methods known in the art, for different solvents. Briefly, lipid extraction efficiency is calculated as Fat extracted / total fat xlOO. The total fat or crude fat is the amount of fat that is present in insect biomass and that is measured using Soxhlet method. The extracted oil by methods of the disclosure showed efficiency of 90-100% suggesting that the improved methods of the disclosure can be used as a better alternative to the methods known in the art.

[0079] The insect products as described herein, may be used in a wide variety of products, including but not limited to food products, supplementary food, animal feed, nutraceutical ingredients, such as omega fatty acids, vitamins, minerals, and enzymes; and pharmaceuticals such as antibiotics. The insect product may be mixed with at least one of, but not limited to, the following ingredients: flavours, colouring, seasonings, oil, fat, spices, nutrients, or vitamin supplements.

[0080] In another aspect, there is disclosed a method for extracting oils from insects using solvents. In one embodiment, the solvent is optimized for efficient extraction and selective recovery of insect oil based on solvent polarity, toxicity, and environmental impact. The present disclosure also overcomes the problem of using common solvents such as hexane, petroleum ether, chloroform, etc., that result in products containing traces of these solvents. Such products when consumed even in minute amounts can cause severe health issues such as neurological disorders. The solvent extraction methods used in the present disclosure are less hazardous in comparison to traditional methods. It is to be noted that trace remains of these solvents in the range of 10-5000 ppm, do not affect the quality or harm the final product, i.e., the extracted insect oil, thereby reducing the need for additional purification steps.

[0081] In this aspect, optimizing a cost-effective method for selectively extracting insect oil using an environmentally friendly solvent or green solvent is disclosed. The term “Green solvents” in the present disclosure is considered to comprise water, ethanol, isopropyl alcohol, tert-butyl alcohol, dimethyl carbonate, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethyl acetate; ionic liquids; deep eutectic solvents such as choline chloride, choline acetate, quaternary ammonium salt, and phosphonium salt; cyclopentyl methyl ether (CPME); 2-methyltetrahydrofuran (2- MeTHF); plant-derived terpenes such as D-limonene, p-cymene, and a-pinene; and a blend of one or more thereof. These solvents degrade more rapidly in the environment, are less toxic to mammals than many other solvents, and are consistent with the twelve principles of “green chemistry” The American Chemical Society, (2002). In particular, the bio-based solvents like dimethyl carbonate (DMC) are made from renewable crops or by-products, which has the potential to depict the sustainable approach of the chemical industry.

[0082] The extraction methods may blend specific ratios of insect biomass-to-solvent, varying conditions such as time, temperature, and stages / iterations for efficient extraction of insect oils while reducing the environmental impact and health risks associated with traditional solvents. Insect biomass may be prepared from one or more insect species, either individually or in combination. Insect biomass can be prepared from Bombyx mori (strain: multivoltine), Bombyx mori (strain: bivoltine), Samia ricini (Eri silkworm); Zophobus morio (super worms), Tenebrio molitor (mealworms), Periplaneta americana (roaches), Gryllus bimaculatus (crickets), Diploptera punctata (Beetle cockroach), and Hermetia illucens (black soldier fly) by grinding, ultrasonication, microwave, electric press, bead milling or a combination thereof. This insect biomass is mixed with solvents at specific ratios and temperatures, heated and stirred for a certain period, and centrifuged.

[0083] In one embodiment, fat extraction from different insect biomass is optimised using a conventional solvent, in particular n-hexane at various ratios of concentrations and time periods of extraction. The leftover insect biomass after fat extraction may be used for further stages / iterations of extraction. After extraction, the fats dissolved in n-hexane is separated by rotary evaporation. The residual fat percentage in the leftover insect biomass post-defatting is measured by Soxhlet analysis. This result is compared with the total / crude fat present in the insect biomass, initially which is also measured by Soxhlet analysis. The lesser the residual fat percentage, the more efficient is the fat separation process.

[0084] Figure 2 shows residual fat percentage after extraction from various insect biomass using a conventional solvent, n-hexane. The extractions were carried out at different insect biomass to n-hexane ratios at 60°C for different time periods. The numbers (1+1 hrs), (1 hr), (2 hr), (1+1+1 hrs) beside the ratios depict the stages / iterations in hours. The extraction with 1:10 w / v insect biomass to solvent ratio for two hours in this analysis is standardised for all insect biomass based on a statistical approach. This optimised data of fat extraction using n-hexane is used to compare results obtained by fat extraction using green solvents in further experiments.

[0085] In another embodiment, insect biomass, in particular Bombyx mori (strain: Multivoltine) has been used for optimising fat extraction using green solvents. The use of conventional solvents such as n-hexane, petroleum ether, etc., have their disadvantages in terms of health risks. In the present disclosure, fat extraction using lesser toxic solvents or green solvents is optimised at different concentrations and time periods of extraction. The leftover insect biomass after fat extraction may be used for further stages / iterations of extractions, after which it is dried for 12-24 hours at 60°C, followed by Soxhlet analysis which gives the residual fat percentage. The lesser the residual fat percentage, the better and more efficient the fat extraction.

[0086] In an aspect of the present disclosure, fat extraction by green solvents, in particular ethanol, acetone, isopropyl alcohol, dimethyl carbonate, tert-butyl alcohol, 1 -propanol, ethyl acetate, 1- butanol and toluene at temperatures near respective boiling points are disclosed. Fig 3 shows the residual fat % value of insect biomass Bombyx mori (Multivoltine) after fat extraction for two hours using different green solvents at 1 :3 w / v, 1:5 w / v and 1: 10 w / v insect biomass to solvent concentrations at temperatures near their respective boiling points for maximum extraction.

[0087] In an embodiment, fat extraction from Bombyx mori (Multivoltine) by acetone, ethanol, ethyl acetate and dimethyl carbonate are further optimised at different concentrations, time periods and stages / iterations of extractions.

[0088] Figure 4 shows the residual fat percentage obtained after fat extraction using acetone at 50°C for two and three hours. The results show a time-dependent improvement in extraction efficiency, with a lower residual fat content observed at the longer extraction time.

[0089] Figures 5A and 5B show the residual fat and protein content, respectively, after fat extraction using ethanol at three different concentrations: 50%, 70%, and 100%, for two hours at 60°C. These results demonstrate that ethanol concentration influences both the efficiency of lipid removal and the retention of protein content in the defatted insect biomass. A higher ethanol concentration generally led to lower residual fat percentages, while maintaining desirable protein content in the final biomass.

[0090] Figure 6 shows the residual fat percentage and protein percentage values following fat extraction using ethyl acetate at 60°C. The extractions were performed at various biomass-to- solvent ratios, durations, and stages / iterations. The data show that multiple stages of extraction and optimal solvent ratios significantly improved fat removal while preserving protein content.

[0091] Figure 7 shows analogous data for fat extraction using dimethyl carbonate at 60°C. As with ethyl acetate, varying the solvent ratio, extraction time, and number of stages affected both fat removal and protein retention. Residual fat percentages decreased with successive extraction stages.

[0092] "Residual fat" indicates the remaining fat content in the insect biomass post-defatting. The most effective concentrations and extraction iterations, resulting in the lowest residual fat content, are highlighted. The protein content retained post-defatting may be used for developing proteinbased products.

[0093] In a further embodiment, fat extraction from insect biomass was conducted using column extraction with green solvents followed by two different post-processing techniques to recover the extracted lipids. In this embodiment, Bombyx mori (Multivoltine) insect biomass was subjected to column extraction using ethyl acetate and dimethyl carbonate at a 1 :10 w / v biomass-to-solvent ratio. After the column extraction step, the solvent-lipid solution was processed either by the pellet method or rotary evaporation.

[0094] Figure 8A shows the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass after column extraction using ethyl acetate at a 1:10 w / v biomass-to-solvent ratio, followed by the pellet method. In the pellet method, the lipid-solvent mixture is separated from the biomass by filtration or decantation, and the biomass is dried. The loss in dry biomass weight is used to estimate the amount of lipid extracted. The lipids remain in the solvent phase and are not isolated. The data show fat removal values across three extraction stages

[0095] Figure 8B shows the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass after column extraction using ethyl acetate at a 1:10 w / v biomass-to-solvent ratio, followed by lipid isolation using rotary evaporation. In the rotary evaporation method, the lipid- containing solvent is subjected to reduced pressure and moderate heating to remove the solvent and recover the lipid as a separate oil phase. The isolated product is insect oil.

[0096] Figure 9A shows the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass after column extraction using dimethyl carbonate at a 1:10 w / v biomass-to-solvent ratio, followed by the pellet method. In the pellet method, the biomass is separated from the solvent and dried, and the reduction in biomass weight is used to infer lipid removal.

[0097] Figure 9B shows the percentage of fat removed from Bombyx mori (Multivoltine) insect biomass after column extraction using dimethyl carbonate at a 1:10 w / v biomass-to-solvent ratio, followed by lipid isolation using rotary evaporation. In the rotary evaporation method, the solvent phase is evaporated under reduced pressure, resulting in direct recovery of the extracted lipid. The isolated product is insect oil.

[0098] Figure 10 shows the average percentage of fat extracted from Bombyx mori (Multivoltine) insect biomass using n-hexane, ethyl acetate, and dimethyl carbonate at a 1:10 w / v biomass-to- solvent ratio over a three-day column extraction process. The data represent extraction values for each solvent.

[0099] The present disclosure provides for the use of green solvents as eco-friendly alternatives to n-hexane in fat extraction that is comparable or better than n-hexane. The ratios and concentrations used here are provided as an example and the disclosure is not limited to these values. The ratio of insect biomass to solvent may range from 1:1 to 1:30 (w / v), specifically 1 :3. The mixture of insect biomass and solvent may be agitated by stirring, spinning, sonication, bubbling or other methods known in the art for specified time periods that may vary depending on the insects. After the extraction process, the supernatant now containing oil / fats dissolved in the solvent is then decanted into a separate flask followed by rotary evaporation to separate the oil / fat from the solvent, ensuring thorough removal of residual solvent. The leftover insect biomass may be subject to further stages / iterations of extraction.

[0100] The left-over insect biomass (which consists of defatted residue and majorly proteins) after the first extraction may be subject to further stages / iterations of extraction. In one embodiment the mixture is subject to three stages / iterations of extraction. The insect biomass and solvent mixture may be subject to different temperatures based on the solvent used such that the temperature shall be between the boiling point and the melting point of the solvent; and stirred for at least 5 min but may vary depending on the insect, solvent, sample solvent ratio, agitation, stage / iteration of the extraction. After the stirring stage, the solvent containing oils may be decanted, using any of the methods known in the art, specifically rotary evaporation, vacuum evaporator etc., to separate the oils from the solvent. The extracted oil may be dried in an oven for at least 1-2 days, at a temperature of at least 60°C for the removal of residual solvent. The oils will then be subject to further purification steps. The methods may include further process intensification by techniques like microwave-assisted extraction, ultrasound-assisted extraction, or pressurized liquid extraction to enhance mass transfer, reduce extraction time, and improve overall process efficiency.

[0101] The analysis showed that the fat extraction by green solvents is as effective as extraction by conventional solvents like n-hexane with residual fat percentages being less than 1% in some of the optimised conditions, thereby proving to be a better alternative to conventional solvents. The products such as insect oils / fat, and proteins obtained after fat extraction using green solvents can be used in food-grade, pharmaceuticals, and cosmetic industries.

[0102] A significant aspect of the present disclosure is the ability to selectively isolate phospholipids from the extracted lipid product. The basis of this method is solvent selectivity and polarity. Sequential extraction by selective solvents based on their polarity dissolves different components of lipids, leaving phospholipids to precipitate, thus obtaining pure phospholipids.

[0103] Phospholipids are valuable components with various applications, but their extraction and isolation can be challenging due to their amphiphilic nature and association with other biomolecules. The method for isolating phospholipids from the lipid-containing solution obtained from the initial green solvent extraction comprises a two-step precipitation process wherein a nonpolar solvent, such as n-hexane, is added to the lipid-containing solution. This forms a non-polar solvent-lipid solution. Other suitable non-polar solvents that may be used include petroleum ether, diethyl ether, cyclohexane, chloroform, toluene, benzene, carbon tetrachloride, d-limonene, 2- methyltetrahydrofuran, p-cymene, and isopropyl myristate. This is followed by the addition of a neutral or slightly polar solvent to the non-polar solvent-lipid solution. Suitable neutral or slightly polar solvents include, but are not limited to, acetone, ethanol, isopropanol, methanol, tert-butyl alcohol, or acetonitrile. The addition of this solvent causes the phospholipids to selectively precipitate out of the solution, forming a solid phospholipid product. The term ‘neutral or slightly polar solvent’ refers to an organic solvent with moderate polarity and low hydrogen bonding capacity, such as acetone, ethanol, or tert-butyl alcohol, capable of reducing lipid solubility and precipitating phospholipids when added to a lipid solution.

[0104] The specific ratios and temperatures for these precipitation steps can be optimized to maximize phospholipid purity and yield. For instance, as demonstrated in Figure 11, sequential solvent extraction using ethanol, n-hexane, and ice-cold acetone can effectively isolate phospholipids. The precipitated phospholipids can then be separated (e.g., by filtration or centrifugation) and dried to obtain a purified phospholipid product.

[0105] In an embodiment, sequential extraction by three different solvents have been used for phospholipid extraction. Solvents are selected based on their ability to interact with different lipid fractions. In this method, the different chemical properties of solvents are exploited to dissolve non-polar and neutral lipids in ice-cold conditions, allowing phospholipids to precipitate. Insect biomass, in particular Bombyx mori full-fat meal is mixed with ethanol. The mixture of insect biomass and ethanol may be agitated by stirring, spinning, sonication, bubbling or other methods known in the art for specified time periods that may vary depending on the insect. The lipids are then separated from ethanol by rotary evaporation or vacuum evaporation. These lipids are then mixed with a non-polar solvent such as n-hexane. N-hexane dissolves non-polar lipids. This step is followed by the addition of ice-cold acetone which dissolves neutral lipids and cholesterol. Phospholipids being insoluble in ice-cold acetone, precipitate. The precipitated phospholipids are dried in an oven at 60°C for 2-12 hours to remove traces of n-hexane and acetone, thus giving pure phospholipids. The total amount of phospholipid is calculated by weighing the phospholipid precipitate. The reported values of phospholipid in Bombyx mori is 2-3%, while the total amount of phospholipid obtained by this disclosure is 2.4-2.75%. This analysis shows that the disclosure can separate pure phospholipids from insects, efficiently.

[0106] The herein disclosed phospholipid extraction parameters such as insect biomass and solvent ratios, extraction time, type of solvents and stages / iterations, are not limited to these. The extraction can be done on different insect biomass including but not limited to Bombyx mori (strain: multivoltine), Bombyx mori (strain: bivoltine), Sarnia ricini (Eri silkworm); Zophobus morio (super worms), Tenebrio molitor (mealworms), Periplaneta americana (roaches), Gryllus bimaculatus (crickets), Diploptera punctata (Beetle cockroach), and Hermetia illucens (black soldier fly).

[0107] Figure 11 shows the percentage of phospholipids extracted from various insect biomass samples using sequential extraction with ethanol, n-hexane, and ice-cold acetone. The data represent the total phospholipid yield recovered by precipitation in the final acetone step. The results demonstrate that the method yields quantifiable phospholipid values across multiple insect species.

[0108] Beyond general lipid applications, the extracted lipid product from insects, particularly from Bombyx mori, has been surprisingly found to possess excellent anti-foaming properties. Accordingly, an aspect of the disclosure comprises an insect oil composition for use as an antifoaming agent in industrial fermentation processes. Figures 12A and 12B visually demonstrate the efficacy of the insect lipid product as an anti-foaming agent, showing a significant reduction in foam height and persistence compared to control solutions. The use of insect oils is gaining attention due to their unique composition, which often includes a high proportion of unsaturated fatty acids and other bioactive compounds. These properties make insect oils potentially valuable as substrates or supplements in fermentation media to produce various bio-based products, such as biofuels, biochemicals, and food ingredients. The insect oils of the present disclosure disrupt foam lamellae by altering its surface tension. This unique application highlights the versatility and added value of the lipids extracted using the disclosed green solvent method. The formulation and specific conditions for its use as an anti-foaming agent can be further optimized based on the target application.

[0109] In an embodiment, the optimal dosage, timing, and method of introducing insect oil into the fermentation medium is disclosed. The incorporation of anti-foaming insect oil into the fermentation medium may be executed via manual or automated delivery systems built into the fermenter. It further discloses evaluation parameters and methods to analyse compatibility and potential interactions of the insect oils of the disclosure with other fermentation components, such as microorganisms, substrates, and process conditions, may be evaluated. The insect oil formulations and processes of the disclosure overcome compatibility, solubility, and dosage challenges. Encapsulation and delivery systems to enhance the stability, shelf-life, and controlled release of insect oil in different applications, ensuring consistent anti-foaming performance over time are also disclosed.

[0110] The anti-foaming properties of the insect oil can be exploited in the food industry, to prevent excessive foaming during cooking, brewing, and other food processing operations; in pharmaceutical and cosmetic industries to improve product quality, consistency, and appearance; chemical and in petrochemical industries, where foaming can occur during various processes, such as fermentation, distillation, and wastewater treatment; textile industry, where foaming can occur during dyeing, finishing, and other textile processing operations. Insect oil's anti-foaming properties make it a potential alternative to traditional chemical defoamers, offering a more sustainable and environmentally friendly solution.

[0111] The present disclosure demonstrates superior performance compared to conventional methods, as evidenced by the experimental data presented in Figures 1-12. For example, Figure 1 illustrates the high efficiency of lipid extraction using green solvents, achieving yields comparable to, or even exceeding traditional Soxhlet extraction with n-hexane, while significantly reducing environmental impact.

[0112] Figures 2-10 further detail the optimization of various parameters, including solvent type, temperature, and time, showcasing the robustness and versatility of the green solvent approach. The low residual fat percentages indicate highly efficient extraction, and the protein content analysis confirms the effective separation of lipids from the defatted biomass.

[0113] A key advantage of this green solvent-based method is the minimal or trace remains of these solvents in the final lipid product, typically in the range of 10-5000 ppm, which do not adversely affect the quality or safety of the final product. This significantly reduces the need for extensive post-extraction purification, simplifying the overall process and reducing costs. Furthermore, the disclosure provides a novel and efficient method for isolating phospholipids, which are often challenging to extract selectively. As shown in Figure 11, the sequential solvent precipitation method effectively separates phospholipids, yielding a purified product.

[0114] EXAMPLES

[0115] Example 1: Preparation of insect Biomass

[0116] In an embodiment, eight different insect samples, namely, Bombyx mori (strain: multi voltine), Bombyx mori (strain: bivoltine), Sarnia ricini (Eri silkworm); Zophobus morio (super worms), Tenebrio molitor (mealworms), Periplaneta americana (roaches), Gryllus bimaculatus (crickets), and Hermetia illucens (black soldier fly) were used to prepare the insect biomass. To prepare insect biomass, 100-150 grams of whole insects are ground to powder form using a mixer grinder. Each powdered insect biomass sample was checked for moisture content using a moisture analyser, with approximately 2 grams of the ground insect biomass used for this purpose. The moisture content is ideally maintained below 10%. If it exceeds this threshold, the biomass is dried in a hot air oven at 60°C for 2-12 hours to achieve the desired moisture level. The procedure for the preparation of insect biomass is the same for examples 2, 3 and 4.

[0117] Example 2: Modified column extraction: Preparation of insect oil / fat and estimation of fat / oil content To prepare insect oil / fat, the powdered insect biomass was mixed with n-hexane in 1:10 w / v concentrations (3 g of insect powder in 30 mL of n-hexane). The mixture was kept undisturbed in 50 mL closed falcons for 1, 2 and 3 days at room temperature. At the end of each day, the supernatant containing the solvent and the extracted oil was carefully decanted, and the solvent was evaporated using rotary evaporation. The defatted residue was subsequently placed in a hot air oven at 60 °C overnight. The experiment for each day (1, 2 and 3) was done in triplicates. The extracted fat provided in Table 1 represents the average fat percentage obtained from triplicate column extraction for each insect sample.

[0118] The amount of fat extracted was measured by two methods: rotary evaporation and pellet extraction methods.

[0119] As used herein, “rotary evaporation method" is a technique used to evaporate solvents from a solution containing the extracted fat. The solvent containing the extracted fat is placed in a round- bottomed flask and rotated under vacuum, increasing the surface area for faster evaporation. The solvent vapours are then condensed and collected, leaving behind the fat, which is weighed to determine the fat content of the sample.

[0120] In an embodiment, the supernatant undergoes rotary evaporation to remove solvent. The fat extract was dried overnight at 60°C to remove residual solvent. The difference in weight of the flask before and after drying determined the weight of the fat extracted.

[0121] The “pellet extraction method” as used herein refers to a technique for determining fat content in insect biomass after solvent extraction. The total weight of the insect biomass is measured before the fat / oil extraction process. After the extraction, the remaining solid material (pellet) is weighed. The reduction in weight of the pellet following fat extraction is used to calculate the fat content of the insect biomass.

[0122] Example 3: Fat extraction using environmentally friendly / Green solvents:

[0123] To extract fat by conventional solvents, such as n-hexane, powdered form of different insect biomass was mixed with n-hexane at different concentrations, specifically: 1:3 w / v (20 grams of insect powder in 60 mL of solvent), 1:5 w / v (20 grams of insect powder in 100 mL of solvent) and 1:10 w / v (10 grams of insect powder in 100 mL of solvent), stirred at 60°C for different time periods. The fats dissolved in n-hexane was collected. The leftover insect biomass was used for further stages / iterations of extraction.

[0124] To extract fat by green solvents, powdered insect biomass sourced from Bombyx mori (strain: multivoltine) was used to optimise the fat extraction process. The optimization involved testing three different ratios of powdered insect biomass to solvent: 1:3 w / v (20 grams of insect powder in 60 mL of solvent), 1:5 w / v (20 grams of insect powder in 100 mL of solvent) and 1:10 w / v (10 grams of insect powder in 100 mL of solvent). Furthermore, ten different solvents were used for fat extraction, including acetone, isopropanol, tert-butyl alcohol, ethyl acetate, dimethyl carbonate, 1-propanol, 1-butanol, toluene, ethanol, and n-hexane. The results obtained using n-hexane as a conventional solvent, has been used as a benchmark for evaluating the fat extraction efficiency of eco-friendly solvents.

[0125] In order to estimate the extracted fat / oil content, the dried defatted residue was subjected to the Soxhlet method to determine the amount of fat left in the defatted residue. A lower leftover fat value indicated greater solvent efficiency in fat removal.

[0126] Example 4: Extraction of phospholipids using a combination of solvents:

[0127] In this embodiment, 100 grams of Silkworm (Bombyx mori) full-fat meal in a conical flask is mixed with 300 mL of absolute ethanol and stirred at 150 rpm at 25°C for 15-30 minutes. The solvent is decanted, and the extracted fat / oil is separated from the solvent using rotary evaporation. This fat / oil is mixed with 150 mL of n-hexane in a beaker in ice-cold condition. The mixture is then continuously stirred on a magnetic stirrer while adding 300 mL of ice-cold acetone simultaneously. The neutral and non-polar lipids dissolve in n-hexane and ice-cold acetone, leaving phospholipids to precipitate. The n-hexane and acetone fractions are removed. The beaker now contains precipitated phospholipids, which is then dried in a hot air oven at 60°C for 2-12 hours to remove traces of acetone and n-hexane. The weight of the beaker before extraction and after drying gives the weight of the phospholipid extracted.

[0128] Example 5: Use of insect oil as a de-foaming agent in fermentation

[0129] During the fermentation process, foam generation is a common issue. This foam can interfere with crucial analytical parameters such as pH and dissolved oxygen, and it can also lead to filter clogging within the fermenter, leading to operational issues. To address this, the addition of 0. 1 ml of sterile insect oil directly to the fermenter not only eliminates existing foam but also prevents further foam formation.

[0130] The present disclosure is not merely restricted to these embodiments but illustrates the use of green solvents in defatting silkworm pupae and other invertebrates, using lesser known or non- classical fat extraction methods such as column extraction, and similar approaches to get the maximum fat extracted.

[0131] REFERENCES

[0132] Nael Abutaha, Fahd A Al-Mekhlafi; Green Solvent-Based Extraction of Lipids and Proteins from Tenebrio molitor: A Sustainable Approach and Cytotoxic Activity; Food Sci Anim Resour 2025; 45(3):807-820 Ruben Smets, Peter Goos, Johan Claes, Mik Van Der Borght; Optimisation of the lipid extraction of fresh black soldier fly larvae (Hermetia illucens) with 2-methyltetrahydrofuran by response surface methodology; Separation and Purification Technology; Volume 258, Part 2, 1 March 2021, 118040

Claims

CLAIMS1. A method for extracting lipids from insects, the method comprising: a. grinding insects in a dried form to obtain an insect biomass powder; b. incubating the insect biomass powder with at least one green solvent capable of dissolving lipids, under suitable extraction conditions to form a lipid-containing solution, and a defatted biomass; c. separating the lipid-containing solution from the defatted biomass; and d. recovering the lipid product by removing green solvent.

2. The method of claim 1, wherein the insects are selected from Bombyx mori, Samia ricini, Antheraea assamensis, Hermetia illucens, Periplaneta americana, Gryllus bimaculatus, Tenebrio molitor, Diploptera punctata, Zophobus morio or a combination thereof.

3. The method of claim 1, wherein the insects are selected from: Acheta domesticus, Gryllodes sigillatus, Melanoplus sanguinipes, Sphenarium purpurascens, Schistocerca gregaria, Alphitobius diaperinus, or Ruspolia differens , Gromphadorhina portentosa, Teleogryllus emma, Oxya yezoensis, Gonimbrasia belina, Locusta migratoria, Schistocerca spp., Tettigoniidae, Thasus gigas, Liometopum apiculatum, Atta cephalotes, Lethocerus indicus, Antheraea pemyi, Atta laevigata, Magicicada spp, Cicada spp. or a combination thereof.

4. The method of claim 1, wherein the solvent is selected from dimethyl carbonate (DMC), methyl acetate, 2-methyltetrahydrofuran (2-MeTHF), cyclopentyl methyl ether (CPME), D- limonene, p-cymene, a-pinene, or a combination thereof.

5. The method of claim 1, wherein the solvent is selected from ethanol, isopropanol, tertbutyl alcohol, 1 -butanol, 1 -propanol, or isoamyl alcohol, or a combination thereof.

6. The method of claim 4, wherein the solvent is DMC.

7. The method of claim 1, wherein the suitable extraction conditions comprise a temperature below the boiling point of the green solvent.

8. The method of claim 1, wherein the separation of the lipid-containing solution from the defatted biomass is carried out by filtration or centrifugation.

9. The method of claim 1, wherein the green solvent is removed by vacuum drying or evaporation under reduced pressure.

10. A method of isolating phospholipids from insects, the method comprising a. adding a non-polar solvent to the lipid-containing solution of claim 1, to form a nonpolar solvent-lipid solution; b. adding a neutral or slightly polar solvent to the non-polar solvent-lipid solution to precipitate phospholipids.

11. The method of claim 10, wherein the neutral or slightly polar solvent is selected from acetone, ethanol, isopropanol, methanol, tert-butyl alcohol, or acetonitrile.

12. The method of claim 10, further comprising separating the phospholipid precipitate by filtration or centrifugation to obtain purified phospholipids.

13. The method of claim 10, wherein the non-polar solvent is selected from n-hexane, petroleum ether, diethyl ether, cyclohexane, chloroform, toluene, benzene, carbon tetrachloride, d-Limonene, 2 -Methyltetrahydrofuran, p-Cymene and isopropyl myristate.

14. A composition comprising the purified lipid product obtained by the method of claim 1.

15. The composition of claim 14, wherein the purified lipid product is formulated for use as an anti-foaming agent in fermentation or bioprocessing media.

16. The use of purified phospholipids obtained by the method of claim 10, wherein the fermentation media is selected from food-grade fermentation, ethanol production, or microbial bioreactors.

17. A composition comprising the purified phospholipid product extracted by the method of claim 10, wherein the purified phospholipid product is formulated for use in nutraceutical, foodgrade, pharmaceutical-grade, or cosmetic -grade applications.

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