Essential oil fraction, method of obtaining the same and use thereof

Abstract

The invention relates to a degradation products-free essential oil fraction, method of obtaining the same and uses thereof.

Description

[0001] “ESSENTIAL OIL FRACTION, METHOD OF OBTAINING THE SAME

[0002] AND USE THEREOF”

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] The invention relates to an essential oil fraction free of degradation products, method of obtaining the same and use thereof.

[0006] BACKGROUND

[0007] An “essential oil” (EO) is a concentrated liquid containing volatile chemical compounds. It is essential in the sense that it contains the characteristic fragrance of the plant (or other source) from which it is derived. The term essential oil was created in the 16thcentury and refers to the theory of “Quinta essentia” proposed by the famous German- Swiss alchemist and physician Paracelsus (1493-1541 ) (E. Guenther, 1950).

[0008] The benefits of essential oils have been recognized for thousands of years. Their use is described in the Bible for rituals and for healing the sick. The ancient Egyptians used essential oils for embalming, religious rites and medicinal purposes. King Tutankhamun’s tomb was found to contain 50 jars of essential oil when it was opened in 1922.

[0009] EO are typically formed by terpenes, terpenoids, phenylpropanes, phenylpropanoids and other minor constituents. Terpenes and terpenoids are generally nonpolar substances and hence soluble in lipid. However, some volatile compounds can be water soluble or partially water soluble.

[0010] Terpenes are a wide class of natural products consisting of compounds with the formula (CsHsjn for n > 2. Terpenes are major biosynthetic building blocks, comprising more than 30,000 compounds. These aromatic compounds are the primary constituents of EO and are responsible for the characteristic scent of many plants, such as cannabis, pine, and lavender, as well as fresh orange peel. In an ecological context, terpenes play an important role in the relations between organisms, for example, as attractants of pollinators or deterrents of herbivores. Terpenes are classified by the number of carbons: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), as examples. Examples of monoterpenes are limonene, myrcene, menthol, carvone, pinene, linalool; of sesquiterpenes are humulene, farnesol, a-bisabolene, betacaryophyllene; and of diterpenes are phytol, retinol, and labdane. When terpenes are modified chemically, such as by oxidation or rearrangement of the carbon skeleton, the resulting compounds are generally referred to as terpenoids. Some authors use the term "terpene" more broadly, to include terpenoids. Terpenes are used in perfumes, cosmetics, soaps, air fresheners and other products, for flavoring food and drink, and for adding scents to incense and household cleaning products.

[0011] While the major constituents of essential oils are generally mono- and sesquiterpenes, in certain plant families or genera phenylpropanoid compounds are also found, sometimes as the main component. In particular, cinnamic acid not only occurs in some cases as a constituent of essential oils, but together with p-hydroxycinnamic acid is the most important metabolic intermediate in the pathways leading to the volatile phenylpropanoids found in these essential oils. Chavicol, estragole, safrole, eugenol are examples of such phenylpropanoids.

[0012] Essential oils can be extracted in many ways. Different extraction systems deliver different EO fractions.

[0013] Due to the relatively low boiling point of their constituents, EOs can be extracted by distillation. Hydro-distillation or steam distillation have become well-established extraction processes in the essential oil industry, especially for the extraction of terpenes having a low boiling point and high vapor pressure, such as monoterpenes. In hydro-distillation, plant material is boiled in water. The EO evaporates with water vapor and is collected in a chilled condensed chamber or Florentine flask. In steam distillation, plant material is placed into a steam distillation chamber and steam at around 110-120 °C is forced to pass through it. As the volatile compounds interact with the steam, the steam flows into the chilled condensed chamber, turning back into a liquid, providing the EO. In general, hydro-distillation provides higher yield, while steam distillation provides EO with stronger biological properties.

[0014] The main limitations of traditional distillation methods are the degradation of thermally liable compounds as well as a poor reproducibility of plant’s volatile compounds profiles that contain compounds having a relatively high boiling point and low vapor pressure, such as most of sesquiterpenes that have vapor pressure below 100 Pa, or compounds that are slightly water soluble, such as most of phenypropanoids, or have a density close to that of water.

[0015] The most delicate terpenes and other compounds can decompose or degrade due to the elevated temperature, generating undesirable off-notes. Benzene, alkyl benzenes, and polycyclic aromatic hydrocarbons are known to form during terpene thermolysis (Meehan-Atrash, 2017). If lower temperature is applied to prevent decomposition or product degradation during distillation, the extraction efficiency can be negatively impacted. In particular the recovery volatile compounds having relatively high boiling points and low vapor pressure, such as most of the sesquiterpenes (es. betacaryophyllene) and some terpene alcohol (es. linalool), can be affected, resulting in essential oil that does not reflect the original plant’s EO composition. Applying deep vacuum in plant distillation to reduce the temperature at which these compounds volatilize is technically possible but challenging, considering the large amount of water that co-distill. Water has a vapor pressure higher than most of the volatile compounds present in essential oils. Furthermore, given the large water consumption in steam or hydro-distillation, recovering volatile compounds which are soluble or slightly soluble in water is difficult. In the traditional steam and hydro-distillation processes, two fractions are obtained: the first is the fraction that is insoluble in water and is decanted off from the separator in a traditional manner; the second is the water-soluble fraction which remains in the distillation water and is usually discarded with the water (hydrosol). In steam distillation the amount of steam required for oil recovery is normally comparable to the weight of the plant material. In hydrodistillation is at least 10 times the dry weight of the plant material. Since commercial essential oil-bearing botanical usually yield between 0.1 %. and 1 ,0%. of the oil, the amount of aqueous effluent exceeds that of recovered oil by a hundredfold or more. Due to the different solubilities as well as relatively high densities, various essential oil components are disproportionately partitioned into the hydrosol phase and, hence, a significant portion of valuable polar or denser volatile components are either reduced or lost. Because of this phenomenon, some volatile components can escape completely resulting in an unbalanced odor and flavor profile when compared with that of the original plant (Fleisher, 1991 ). That is the case, for instance, of slightly water-soluble phenyl ethyl alcohol, a main constituent of rose’s EO, or heavy terpenoid compounds such as camphor (density 0.99 kg / L) and eucalyptol (0.92 Kg / L) in rosemary, that mostly end up into the hydrosol fraction, or even below the hydrosol such as cinnamon (1 .03 Kg / L) and clove essential oils (1.05 Kg / L). Post-treatments of the hydrosol (cohobation or recovery with organic solvents) are often used to rebalance the EO fraction.

[0016] In the past to extract EO from delicate flowers, such roses, a technique called “enfleurage” was used. In such technique, an odorless, bland, fixed oil or fat is spread in a thin layer on glass plates. The flower petals are placed on the fat for a few hours; then repeatedly, the oil petals are removed, and a new layer of petals is introduced. After the fat has absorbed as much fragrance as possible, known as “pomade”, the essential oil may be removed by extraction with alcohol to obtain an “absolute”. The main drawbacks of such method are the labor intensiveness, prolonged processing time and low extraction efficiency. For these reasons, such method is largely abandoned nowadays in favor of steam or hydro- distillation or solvent extraction.

[0017] In the modern perfume industry, most EOs production is accomplished by solvent extraction, using volatile solvents such as petroleum ether and hexane. In case organic solvents are utilized, the first crude extract of essential oils obtained is called “concrete”. Concrete is a viscous product containing EO along with plant waxes and pigments. Ethanol is thus utilized to further purify the concrete, through a winterization and filtration steps, into a cleaner fraction called “absolute”. The main advantage of solvent extraction over distillation is that uniform and lower temperature can be maintained during the process. As a result, extracted essential oils have a more natural odor that is unmatched by traditional water or steam distillation, which may have undergone chemical alteration by the high temperature. Example of botanicals that are extracted in such a manner are the delicate EO from Jasmine as well as the fixative resinoid benzoin. The main drawback of solvent extraction is the use of petro-derived, highly flammable solvents, which make the process unsafe and not environmentally sustainable. Furthermore, significant loss of EO can occur when removing the organic solvent by evaporation. Despite the organic solvent evaporation, residues can still be present in the finished product.

[0018] To recover delicate, thermally liable volatile compounds, mechanical means, such as “expression” or “cold pressing” are used. For instance, citrus oil is expressed by puncturing the oil glands by rolling the fruit over a trough lined with sharp projections that are long enough to penetrate the epidermis and pierce the oil glands located within outer portion of the peel. A pressing action on the fruit removes the oil from the glands, and a fine spray of water washes the oil from the mashed peel while the juice is extracted through a central tube that cores the fruit. The resulting oil-water emulsion is separated by centrifugation. A variation of this process is to remove the peel from the fruit before the oil is extracted. The main drawback of the method is that it can be applied only to few selected botanicals, such as orange and citrus, but not to plants such as rose, lavender or cannabis wherein terpenes are contained in fragile petals or outer glandular structures (es. trichomes) localized on aerial parts.

[0019] Supercritical fluid extraction (SFE), as supercritical CO2, has also been used to extract terpenes, and in general essential oils. The extract obtained by SFE presents better quality than that obtained by traditional steam distillation (Kotnik et al. 2007). However, a study performed on six cannabis chemovars demonstrated that the chemical profile of extracts obtained via SCCO2 is not representative of the original flower. A decrease of monoterpene content and an increase in terpene alcohols and sesquiterpenes as compared to the flower have been observed (Sexton et aL, 2018). An additional inconvenience of SFE is that it requires costly equipment that cannot be always available near the fields to process fresh plants, making the method suitable for extracting raw material that can be transported such as wood or spices, but unsuitable for fresh flowers.

[0020] Infusion or maceration of medicinal and aromatic plants in vegetable oils as solvents dates back to ancient civilizations. In recent years, edible commercial vegetable oils have been successfully enriched or aromatized with bioactive compounds from herbs, spices or other plant materials in order to improve their nutritional values and organoleptic qualities, and to prolong shelf-life as well. The resulting oils can be defined as “flavored oil” named as the plant extracted.

[0021] US20020164413A1 describes a method to enrich olive oil with compounds from herbs. However, the method results in a significant increase of lipid degradation products, such as aldehydes Hexanal, Heptanal, 2- Hexenal and Octanal in the flavored oil as compared to the starting carrier oil. The presence of unsaturated fatty acids in the plant material and / or in the vegetable oil carrier can led to the formation of degradation products that can lead to the generation of off-notes. In presence of oxygen or water, unsaturated free fatty acids enzymatically convert in aldehydes or alcohols which are responsible for unwanted notes, such as mature fruit, herbaceous, “burnt” or mown grass smell. Linolenic acid can convert in (E)-2-Hexenal, while linoleic acid can form n-hexanal and then n-hexanol by lipoxygenases and hydroperoxide lyases respectively (Hatanaka et aL, 1975, B. O. de Lumen, 1978; Feussner, 2002).

[0022] Maceration using vegetable oil carriers has been used to extract terpenes from cannabis inflorescences. Vegetable oil-based extracts containing terpenes, prepared by heating the plant material in the carrier oil, also showed to contain lipid oxidation products, such as hexanol and hexanal (Calvi et aL, 2018). In cannabis inflorescences 39 fatty acid species were identified, including linolenic acid (1.39-7.95 mg g-1 ) and linoleic acid (1 .04-7.87 mg g-1 ) that can convert in aldehydes and alcohols (Piovesana et aL, 2019).

[0023] US10973864B2 provides a novel and environmentally friendly method of enzyme- assisted, lipid-based extraction, showing a remarkable efficiency in extracting terpenes at low temperatures, even in their ratios as originally present in the plant. However, such method presents some limitations in obtaining an essential oil fraction with a high terpene content and no presence of degradation products.

[0024] Maceration can also be performed by using non-plant derived natural solvent, such as lipid originated from cultured yeast, fungi, algae, and bacteria, or deep eutectic solvents (NADES). NADES is a mixture of cellular components such as sugars, alcohols, amino acids, organic acids and choline derivatives, known as the third solvent in living cells, it has the extremely dissolving power for natural compounds. NADES is made by mixing hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD), which causes its melting point to be lowered by the interaction of hydrogen bonding and van der Waals interactions. Today, NADES is a sustainable new green medium, which is widely used in the food, cosmetic, agrochemical and pharmaceutical industries, among others. Currently, NADES has been widely used for the extraction of compounds, such as cumin seed essential oil and angelica essential oil.

[0025] US1 1078137B1 describes a batchwise method to recover terpenes by volatilizing them from a crude cannabis oil placed in a container in a vacuum oven. The method requires one or two hours to obtain a full volatilization of terpenes. Even if the temperature is slowly raised over that time, most delicate terpenes or other extracted compounds present in the crude oil can degrade and / or decompose in one- or two-hours heating, and off-notes can be generated and extracted along with terpenes. Furthermore, the method starts already from a crude cannabis oil concentrate that is typically obtained by means of supercritical CO2, ethanol, or butane extraction. It does not teach how to extract terpenes from a terpene-rich carrier oil. Finally, the method does not teach how to avoid the presence in the volatile fraction of off-notes derived from lipid oxidation.

[0026] High-performing, continuous, deep vacuum distillation techniques, such as, molecular distillation, are applicable to extract valuable compounds from vegetable oils. However, this technique is not suitable to extract volatile compounds from solid phase material, such as plant biomass. Furthermore, if aldehydes and alcohol responsible for off-notes are present in the vegetable oils, these volatile compounds are extracted along with the targeted compound.

[0027] Solid phase micro-extraction (SPME) is a technique used in the quantitative analysis of analytes in aqueous and gaseous phases. This novel technology captures aroma molecules surrounding flower petals without touching the flower or other part of the plant. SPME has gained widespread acceptance as the technique of choice in many fields of application, including forensics, toxicology, and the analysis of flavors, fragrances, and environmental and biological matrices. However, the technology is not suitable for large scale extraction.

[0028] Among newer techniques, enzymatic pre-treatment combined with solvent-free microwave extraction (EP-SFME) has proven to perform better than hydro-distillation to extract essential oils from fresh leaves of Pelargonium graveolens L'Herit, while the use of ultrasound, always as pre-treatment before hydro-distillation, has also been positively evaluated. However, such novel treatments did not demonstrate to solve the main limitation identified for solvent-free extraction techniques, such as the low efficiency in extracting high boiling and low vapor pressure volatile compounds, without incurring in the generation of degradation products, making no use of organic solvents at any step.

[0029] A method to consistently obtain an essential oil fraction reflecting the original plant’s profile, including high boiling point terpenes and / or terpenoids as well as volatile water- soluble compounds, with no degradation products in it, that is efficient and environmentally sustainable would therefore be desirable.

[0030] There is an increasingly felt need for an improved method for obtaining a sustainable essential oil fraction, having reduced degradation products content, that is more costefficient and avoids the use of harmful solvents. Object of the present invention is the development of a more efficient and cost-effective process for obtaining an essential oil that has the advantage of avoiding the use of toxic, environment-threatening chemical substances, such as organic solvents.

[0031] SUMMARY OF INVENTION

[0032] The Applicant noted that, even if methods for obtaining essential oil fractions are known, they present several limits and need still to be improved, in particular in terms of reproducibility of plant’s volatile compound profile in the essential oil fraction, efficiency, cost-effectiveness, environmental impact, utilization of toxic or harmful solvents, presence of residual organic solvents in the products and presence of degradations products.

[0033] For example, the Applicant noted that, US10973864B2 provides a novel and environmentally friendly method of enzyme-assisted lipid-based extraction showing a remarkable efficiency in extracting and stabilizing terpenes, even in their ratios as originally present in the plant. However, such method presents some limitations in obtaining essential oil fraction with a high terpene content and no presence of carrier oil. Furthermore, the presence of unsaturated fatty acids in the carrier oil or in the starting plant can lead to the formation of degradation products during the extraction process.

[0034] The Applicant also noted that purification techniques commonly used to purify terpene concentrates and eliminate degradation products typically apply techniques that result in the use of organic solvents.

[0035] Hence, the Applicant felt that a simpler way to obtain an essential oil fraction reproducing the volatile compound fingerprint of the plant, with a high content of desirable volatile compounds and low content of degradation products would therefore be desirable and that a process that could efficiently generate such fraction, resulting in a high level of volatile compound concentration, include those having a relatively low vapor pressure, without making use of any organic solvent or costly techniques, such as chromatography, to purify terpene and eliminate degradation products, would represent a healthier and safer process for workers and consumers as well as a more environmentally friendly and convenient solution.

[0036] An object of the present invention is therefore the provision of a method for preparing an essential oil concentrated fraction, making no use of organic solvents, wherein the content of degradation products is significantly reduced, capable of attaining a high concentration of volatile compound, such as terpenes, that is efficient, environmentally friendly and cost-effective.

[0037] Therefore, the present invention relates, in a first aspect, to a method for preparing an essential oil fraction having a reduced degradation product content, comprising the steps of: a) putting in contact a biological material containing volatile compounds with lipid to form a mixture; b) agitating under vacuum the mixture of step a) at a temperature between 20 °C and 80 °C to obtain an agitated mixture; c) mechanically separating the lighter oily fraction from the agitated mixture of step b) to obtain an oily fraction; d) vacuum distilling from the oily fraction of step c) a lighter volatile fraction at a pressure below 0.01 bar, wherein the lighter volatile fraction is said essential oil fraction.

[0038] Surprisingly, the Applicant has found out that by applying the described method, it is possible to obtain an essential oil fraction that faithfully reflects that of the natural starting material, including those volatile compounds having a relatively low vapor pressure, without generating degradation products, and making no use of organic solvents at any step.

[0039] In a second aspect, here disclosed is an essential oil fraction obtainable from the method of the invention, said essential oil fraction comprising:

[0040] - more than 70% by weight, with respect to the total dry weight of the essential oil fraction, of terpenes and / or terpenoids; and

[0041] - not more than 2% of Hexanal, (E)-2-Hexenal, (Z)-4-Hexen-1 -ol, (E)-2-Hexen-1 -ol, (E)-3-Hexen-1 -ol, 1 -Hexanol, 2-Heptanone, Heptanal, Propanal, Pentanal, Hexanoic acid, Nonenal, Acetaldehyde, 2-Propenal, Butenal, Alkyl furans, Acetic acid, or Propanoic acid.

[0042] The advantages of the essential oil fraction according to the present invention has been disclosed in relation to the method according to the first aspect of the present invention and are not herewith repeated. Thanks to its compositional and purity properties, said essential oil fraction may be advantageously used for preparing pharmaceutical or nutraceutical products, cosmetics, fragrances, food or feed products, antimicrobial, antibacterial, insecticidal or biopesticides containing terpenes and / or terpenoids and / or phenylpropanoids.

[0043] In a further aspect, therefore, the present invention relates to a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide, comprising the essential oil of the invention, and to a method for preparing a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising the steps of:

[0044] - providing an essential oil fraction according to the present invention and / or preparing an essential oil fraction according to the present invention; and

[0045] - obtaining a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising terpenes and / or terpenoids and / or phenylpropanoids.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates, in a first aspect, to a method for preparing an essential oil fraction, said method comprising the steps of: a) putting in contact a biological material containing volatile compounds with lipid to form a mixture; b) agitating under vacuum the mixture of step a) at a temperature between 20 °C and 80 °C to obtain an agitated mixture; c) mechanically separating the lighter oily fraction from the agitated mixture of step b) to obtain an oily fraction; and d) vacuum distilling from the oily fraction of step c) a lighter volatile fraction at a pressure below 0.01 bar, wherein the lighter volatile fraction is said essential oil fraction.

[0048] Surprisingly, the Applicant has found out that by applying the described method, it is possible to obtain an essential oil fraction that reflects that of the original biological material, making no use of organic solvents at any step. Furthermore, the Applicant has found out that by applying the described method it is possible to obtain an essential oil fraction that includes volatile compounds having a relatively low vapor pressure.

[0049] Preferably the volatile compounds have a vapor pressure lower than 100 Pa. Even more preferably, a vapor pressure lower than 10 Pa.

[0050] Preferably, said essential oil fraction contains at least 70% of the volatile compounds having a vapor pressure lower than 100 Pa present in the starting biological material.

[0051] Furthermore, the Applicant has found out that by applying the described method it is possible to efficiently obtain an essential oil fraction that includes volatile compounds that are slightly soluble in water.

[0052] Preferably said essential oil fraction contains compounds having a solubility in water higher than 0.1 g per L.

[0053] Furthermore, the Applicant has found out that by applying the described method it is possible to efficiently recover an essential oil fraction that includes volatile compounds having a density of at least 0.92 Kg / L

[0054] Preferably said essential oil fraction contains compounds having a density higher than 1 Kg / L.

[0055] Furthermore, the Applicant has found out that by applying the described method it is possible to obtain an essential oil fraction making no use of process water.

[0056] Preferably the biological material is fresh and / or contains at least 60% of water.

[0057] Preferably, the total amount of process water to be used is less than the weight of the biological material. More preferably, less than 50% of the weight with respect to the total weight of the biological material. Even more preferably, less than 10% of the weight of the biological material.

[0058] Furthermore, the Applicant has found out that by applying the described method it is possible to obtain an essential oil fraction that has a volatile compounds content of more than 80% by dry weight, with respect to the total dry weight of the fraction.

[0059] Preferably, said essential oil fraction has a terpene content of more than 70% by dry weight, with respect to the total dry weight of the essential oil fraction, more preferably more than 75% by dry weight, even more preferably more than 80% by dry weight, even more preferably more than 85% by dry weight of the essential oil fraction.

[0060] Furthermore, the Applicant has found out that by applying the described method it is possible to obtain an essential oil fraction that has a degradation products content of less than 2%, by dry weight, with respect to the total dry weight of the fraction.

[0061] Preferably, said essential oil fraction has a degradations products content of less than 1 %, by dry weight, with respect to the total dry weight of the concentrate, more preferably less than 0.5% by dry weight.

[0062] Furthermore, the Applicant has found out that by applying the described method it is possible to obtain an essential oil fraction efficiently and consistently from a wide range of botanicals.

[0063] Preferably, said essential oil fraction has been obtained from agarwood, arnica, basil, cannabis, cistus, cardamom, chamomile, clove, cinnamon, citrus, chrysanthemum, garlic, hemp, hops, jasmine, lavender, mint, orris, neem, nutmeg, patchouli, pepper, peppermint, rose, sage, vanilla, vetiver, violet or mixtures thereof.

[0064] Within the framework of the present description and in the subsequent claims, except where otherwise indicated, all the numerical entities expressing amounts, parameters, percentages, and so forth, are to be understood as being preceded in all instances by the term "about". Also, all ranges of numerical entities include all the possible combinations of the maximum and minimum values and include all the possible intermediate ranges, in addition to those specifically indicated herein below.

[0065] Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0066] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art. As used herein, the articles “a” and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0067] As used herein, the term “essential oil” includes, but is not limited to, volatile compounds present in a biological material.

[0068] As used herein, the term “volatile compounds” includes, but is not limited to any organic compound having at 25 °C a vapor pressure of 1 Pa or more.

[0069] As used herein, the term “terpenes” includes, but is not limited to, [3-myrcene, pinene, limonene, a-terpinene, terpinen-4-ol, carvacrol, carvone, 1 ,8-cineole, p-cymene, fenchone, nerolidol, phytol, squalene, linalool, beta-caryophyllene.

[0070] As used herein, the term “lipid” includes, but is not limited to, olive oil, sunflower oil, jojoba oil, coconut oil, vegetable oil, medium-chain triglycerides, fatty acids, liposomes, glycerine, polyethylene glycol, ethyl acetate, liquid paraffin, butylene glycol, propylene glycol, ethylhexyl palmitate, eicosapentaenoic acid, docosahexaenoic acid, microcrystalline wax, mineral wax, ozokerite, polyethylene, polyoxyethylene, hydrocarbon waxes derived from carbon monoxide and hydrogen, cerosin; cetyl esters; hydrogenated vegetable oil, hydrogenated fatty acids, polyethylene glycol, or mixtures thereof, oil, fatty acids and / or triglycerides obtained from cultured yeast, fungi algae, bacteria, or mixtures thereof.

[0071] As used herein, the term “degradation products” includes, but is not limited to products generated from the oxidation, enzymatic conversion or thermal degradation of chemical compounds present in the starting biological material, such as Hexanal, (E)- 2-Hexenal, (Z)-4-Hexen-1 -ol, (E)-2-Hexen-1 -ol, (E)-3-Hexen-1 -ol, 1 -Hexanol, 2- Heptanone, Heptanal, Propanal, Hexanoic acid, Nonenal, Acetaldehyde, 2-Propenal, Butenal, Alkyl furans, Acetic acid, Propanoic acid.

[0072] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1 %, and ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. The present invention may present in one or more of the above aspects one or more of the characteristics disclosed hereinafter.

[0073] In a preferred embodiment, the biological material of step a) is a plant material, preferably the plant material is selected from the group consisting of arnica, basil, cannabis, cistus, cardamom, chamomile, clove, cinnamon, citrus, chrysanthemum, garlic, hemp, hops, jasmine, lavender, mint, orris, neem, nutmeg, patchouli, pepper, peppermint, rose, sage, vanilla, vetiver, violet or mixtures thereof.

[0074] Preferably, said biological material comprises at least 0.5% by weight, with respect to the total dry weight, of terpenes.

[0075] Preferably, said biological material comprises at least 0.5% by weight, with respect to the total dry weight, of terpenoids.

[0076] Preferably, said biological material comprises at least 0.2% by weight, with respect to the total dry weight, of phenylpropanoids.

[0077] In a preferred embodiment, the lipid of step a) is selected from the group consisting of: vegetable oil, medium-chain triglycerides, fatty acids, liposomes, glycerine, polyethylene glycol, ethyl acetate, liquid paraffin, butylene glycol, propylene glycol, ethylhexyl palmitate, eicosapentaenoic acid, docosahexaenoic acid, microcrystalline wax, mineral wax, ozokerite, polyethylene, polyoxyethylene, hydrocarbon waxes derived from carbon monoxide and hydrogen, cerosin; cetyl esters; hydrogenated vegetable oil, hydrogenated fatty acids, polyethylene glycol, natural deep eutectic solvent or mixtures thereof.

[0078] Preferably the natural deep eutectic solvent is selected from the group consisting of: choline chloride, citric acid, lactic acid, betaine, proline, glycine, alanine, arginine, glucose, lactose, sucrose, xylose, galactose, trehalose, histidine, lidocaine, acetylcholine chloride, nicotinic acid, gallic acid, benzoic acid, coumaric acid, caffeic acid, xylitol, glucose, fructose, oxalic acid, malic acid, stearic acid, oleic, linoleic acid, urea, glycerol, sorbitol, mannitol, inositol, butylene glycol, propanediol, isosorbide or mixtures thereof.

[0079] Preferably, the lipid in step a) is a vegetable oil selected from the group consisting of: olive oil, coconut oil, sunflower oil, sesame oil, hemp seed oil, jojoba oil, avocado oil, crude palm oil, palm kernel oil or mixtures thereof. In another embodiment, the lipid is olive oil. In an embodiment, lipid is sunflower oil. In yet another embodiment, lipid is hemp seeds oil. In an embodiment, the lipid is jojoba oil. In yet another embodiment, the lipid is avocado oil. In a further embodiment, the lipid is crude palm oil. In yet another embodiment, the lipid is a palm kernel oil.

[0080] In another embodiment, the lipid is an oil, a fatty acid, a triglyceride, obtained from cultured yeast, fungi algae, bacteria or mixtures thereof.

[0081] In a preferred embodiment, the lipid has a content of unsaturated fatty acids lower than 10%, preferably lower than 5%.

[0082] Preferably, the lipid is a vegetable oil having a free-fatty acids (FFA) content below 1 %, preferably below 0.5%, even more preferably below 0.1 %.

[0083] In a preferred embodiment of the method according to the present invention, the biological material containing volatile compounds is preferably selected from a plant, an alga, a bacterium, a yeast, a fungus, a genetically engineered micro-organism, or a mixture thereof.

[0084] Preferably, said biological material containing volatile compounds is selected from the Cannabis genus of plants, wherein said biological material is pure, a hybrid or genetically modified variant thereof. Preferably, said biological material containing cannabinoids selected from the Cannabis genus of plants, belongs to the species C. sativa (hemp), C. indica and C. ruderalis.

[0085] Preferably, the biological material containing lipophilic volatile compounds has a moisture content of at least 20% of the biological material weight.

[0086] Preferably, said biological material containing lipophilic volatile compounds is newly harvested and has a moisture content of at least 50%, preferably at least 70%.

[0087] Preferably, said biological material of the method according to the invention is either fresh or dried.

[0088] In an embodiment, the biological material is newly harvested and contain high level of moisture; in such a case addition of extra water to the biological material is unnecessary.

[0089] Preferably, the biological material containing volatile compounds has a total volatile compound content of at least 0.1 % by weight, more preferably of at least 0.2 % by weight, even more preferably of at least 0.5% by weight, even more preferably of at least 1 % by weight.

[0090] In a preferred aspect, said biological material is chosen from the group consisting of buds, flowers, leaves, stalks, stems, roots and seeds or a mixture thereof. In an embodiment, the biological material includes seeds. In another embodiment, when the biological material includes seeds, no lipid is added. In a further embodiment, when the biological material includes seeds, a lipid is added. Biological material including seeds may be rich in lipid, and thus may not need the further addition of lipid.

[0091] Preferably, said essential oil fraction comprises more than 70% by weight, even more preferably more than 85% by weight, with respect to the total dry weight of the concentrate, of terpenes.

[0092] Preferably, in the essential oil fraction according to the invention the volatile compounds are selected from the group consisting of: limonene, beta-myrcene, caryophyllene, alpha-pinene, beta-pinene, pinene, alpha-terpinene, terpinen-4-ol, carvacrol, carvone, cineole, p-cymene, fenchone, cannaflavin A, cannaflavin B, nerolidol, phytol, fenchol, humulene, ocimene, eucalyptol, bisabolol, farnesene, camphor, linalool, myristic acid, p-cymene, cumene, squalene, cineole, beta-elemene, borneol, eudesmol, cadinene, methyl cinnamate, benzyl acetate, benzyl alcohol, indole, benzyl benzoate, cis-jasmone, geraniol, menthol, menthone, hexade- canoic acid, methyl oleate, guaiene, seychellene, spathulenol, patchouliol, sabinene, eugenol, myristicin, pogostone and mixture thereof.

[0093] In a preferred embodiment, the biological material of step a) is comminuted or milled before, during or after, being put in contact with lipid, to increase the surface contact. Preferably the mixture is milled under vacuum.

[0094] Preferably, said mixture of step b) is stirred for a time in the range from at least 5 seconds to less than 24 hours, more preferably from 30 to 120 minutes, even more preferably from 60 to 90 min.

[0095] Preferably, said mixture of step a) is heated to a temperature to at least 30 °C, more preferably to at least 35 °C, even more preferably to at least 40 °C.

[0096] In a further preferred aspect, a filtration step is added after step b) or step c). Preferably, said lighter oily fraction of step c) is put in contact with a new batch of biological material to be further enriched in lipophilic volatile compounds.

[0097] In an embodiment, water is added to the mixture of step a) to form a homogeneous mixture or slurry, and wherein the water that is added is five times less than the dry weight of the biological material.

[0098] In an embodiment, ultrasound / sonication, microwaves, steam explosion or cavitation may advantageously be used before or after adding lipid to the mixture to reduce the time necessary to achieve higher volatile compounds lipid-extraction yield.

[0099] In an embodiment, the mechanical separation of step c) is achieved via density separation (i.e. centrifugation) or pressing (French press) and / or filtration.

[0100] Preferably, the lipid has been neutralized and / or deodorized prior being added to the mixture.

[0101] Preferably, enzymes are added to the mixture of said step a).

[0102] Preferably, said enzymes are one or more independently selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases, cellulase, hemicellulase, xylanase, glucanase, beta-glucanase, pectinase, glucuronyltransferase, lipase, amylase, alpha-amylase, beta-amylase, phospholipase, arabanase, galacto-, beta-mannanase, protease, esterase, phytase, cannabinoid synthase, THCA synthase, CBCA synthase and terpene synthase.

[0103] In an embodiment, said enzyme is cellulase. In another embodiment, said enzyme is beta-glucosidase. In another embodiment, said enzyme is hemicellulase. In another embodiment, said enzyme is xylanase. In yet another embodiment, said enzyme is glucanase. In yet another embodiment, said enzyme is pectinase. In still another embodiment, said enzyme is amylase. In yet another embodiment, said enzyme is lipase or phospholipase. In said another embodiment, said enzyme is glucuronosyltransferase or alcohol dehydrogenase. In yet another embodiment, said enzyme is arabinanase. In still another embodiment, said enzyme is phytase. In a further embodiment, said enzyme is protease. In yet another embodiment, said enzymes is esterase. In yet another embodiment, said enzymes is a cannabinoid synthase. In still another embodiment, said enzyme is THCA synthase. In yet another embodiment said embodiment is CBDA synthase. In another embodiment, said enzyme is CBCA synthase. In a preferred aspect, the lipid of step a) has been obtained making no use of organic solvent.

[0104] Preferably, in step a) the pH of the agitated mixture of step b) varies in the range from 4 to 6. Preferably it is adjusted by means of citric acid and / or ascorbic acid and / or ascorbyl palmitate.

[0105] In a further embodiment, at least 50% of the terpenes contained in the biological material are extracted into the essential oil fraction; at least 50%, preferably at least 70% of the terpenoids; at least 70% of the diterpenoids and at least 50%, preferably 70% of monoterpenes contained in the biological material are extracted into the essential oil fraction.

[0106] In a still further embodiment at least 70% of the sesquiterpenes and at least 50% of the monoterpenes contained in the biological material are extracted into the essential oil fraction.

[0107] In an embodiment, the two main terpenes (better volatile compounds) in the biological material are limonene, beta-myrcene, caryophillene alpha-pinene, beta-pinene, pinene, alpha-terpinene, terpinen-4-ol, carvacrol, carvone, cineole, p-cymene, fenchone, cannaflavin A, cannaflavin B, nerolidol, phytol, fenchol, humulene, ocimene, eucalyptol, bisabolol, farnesene, camphor, linalool, myristic acid, p-cymene, cumene, squalene, cineole, beta-elemene, borneol, eudesmol, cadinene, methyl cinnamate, benzyl acetate, benzyl alcohol, indole, benzyl benzoate, cis-jasmone, geraniol, menthol, menthone, hexade- canoic acid, methyl oleate, guaiene, seychellene, spathulenol, patchouliol, sabinene, eugenol, myristicin, pogostone and mixture thereof.

[0108] Preferably, said vacuum distillation is carried out in at least one equipment selected from the group consisting of: short path equipment, a wiped-film equipment, and thin- film equipment, even more preferably a wiped-film equipment.

[0109] Short path and thin-film equipment are well-known vacuum distillation equipment. Short path equipment is those vacuum distillation equipment in which the gas phase in the applied fine vacuum only has to travel over a very short path between the receiver and the condenser, whereas thin-film equipment are those vacuum distillation equipment in which the material to be distilled is spread or wiped onto the surface of the receiving cylinder surfaces by a paint roller. A Wiped-film equipment is a particular type of thin-film equipment where the material is wiped onto the receiving cylinder surfaces by a blade. Such wiped-film equipment is for example available from VTA GmbH.

[0110] In a further preferred embodiment of the invention the vacuum distillation can be coupled with column distillation to further fractionate and purify different essential oil fractions.

[0111] The method according to the present invention comprises the step of separating from said vacuum distillation a distillate containing the essential oil.

[0112] Preferably a step of condensation of the vapor generated from the mixture agitated under vacuum of step b) is added between step b) and step c), wherein the condensed vapor contains an essential oil fraction enriched in monoterpenes and / or a hydrosol enriched in water soluble compounds.

[0113] In an additional embodiment of the invention the essential oil fraction is further purified by washing it with water.

[0114] In a further aspect, the present invention relates to a degradation products-free essential oil fraction obtainable by means of the process according to the first aspect of the invention, said essential oil fraction:

[0115] - comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of terpenes and / or terpenoids;

[0116] - comprising not more than 2% of Hexanal, (E)-2-Hexenal, (Z)-4-Hexen-1 -ol, (E)-2- Hexen-1 -ol, (E)-3-Hexen-1 -ol, 1 -Hexanol, 2-Heptanone, Heptanal, Propanal, Pentanal, Hexanoic acid, Nonenal, Acetaldehyde, 2-Propenal, Butenal, Alkyl furans, Acetic acid, Propanoic acid;

[0117] - being obtained without making use of an organic solvent, and

[0118] - being obtained from biological material containing one of more of said degradation products in an amount above 0.1 % in dry weight.

[0119] Thanks to the specific conditions of the method according to the invention, an essential oil fraction is indeed obtained, showing an unexpectedly high level of volatile compounds. In a preferred aspect the essential oil fraction contains at least a volatile compound having a vapor pressure higher than 100 Pa and at least a volatile compound having a vapor pressure lower than 100 Pa. In a still further aspect, the present invention relates also to an essential oil fraction being obtained without making use of an organic solvent, wherein said organic solvent is selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, petroleum ether, propane, toluene, m-xylene, o-xylene, and p-xylene heptane.

[0120] In a still further aspect, the present invention relates also to a degradation products- free essential oil:

[0121] - comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of volatile compounds;

[0122] - being obtained without making use of an organic solvent, and

[0123] - being obtained from plant material containing one of more of said pesticides in an amount above 0.01 ppm.

[0124] In a still further aspect, the present invention relates also to an essential oil fraction comprising more than 70% by weight, with respect to the total dry weight of the fraction, of terpenes and / or terpenoids and not more than 0.001 % per cent by weight of one or more organic solvents selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, petroleum ether, propane, toluene, m-xylene, o-xylene, and p-xyleneheptane.

[0125] The Applicant has noted that the combination of a high content of mono-terpenes and sesquiterpenes, and the absence of degradation products and organic solvents content is particularly surprising compared to the prior art essential oil fractions, in which a high sesquiterpenes content is usually achieved by means of extraction or purification treatments that lead to an increase of degradation products or that involve the use of organic solvents, the elimination of which may result troublesome, expensive and not in compliance with regulatory requirements.

[0126] The other advantages of the essential oil fraction according to the present invention have been disclosed in relation to the method according to the first aspect of the present invention and are not herewith repeated. In a second aspect, here disclosed is an essential oil fraction obtainable from the method of the invention, said essential oil fraction comprising:

[0127] - more than 70% by weight, with respect to the total dry weight of the essential oil fraction, of terpenes and / or terpenoids; and

[0128] - not more than 2% of Hexanal, (E)-2-Hexenal, (Z)-4-Hexen-1 -ol, (E)-2-Hexen-1 -ol, (E)-3-Hexen-1 -ol, 1 -Hexanol, 2-Heptanone, Heptanal, Propanal, Hexanoic acid, Nonenal, Acetaldehyde, 2-Propenal, Butenal, Alkyl furans, Acetic acid, or Propanoic acid.

[0129] Preferably, the essential oil fraction obtainable from the method of the invention comprises not more than 0.001 % per cent by weight of one or more organic solvents selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, petroleum ether, propane, toluene, m-xylene, o-xylene, and p-xylene heptane.

[0130] Thanks to its compositional and purity properties, said essential oil fraction may be advantageously used for preparing pharmaceutical or nutraceutical products, cosmetics, food or feed products, antimicrobial, antibacterial, insecticidal or biopesticides containing one or more cannabinoids.

[0131] In a further aspect, therefore, the present invention relates to a a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide, comprising the essential oil of the invention, and to a method for preparing a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising the steps of:

[0132] - providing an essential oil fraction according to the present invention and / or preparing an essential oil fraction according to the present invention; and

[0133] - obtaining a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising said essential oil fraction.

[0134] As delined above, the method of the present invention has surprisingly allowed the development of a more efficient and cost-effective process for obtaining an essential oil fraction with a high terpene content, which avoids the use of toxic, environmentthreatening chemical substances, such as organic solvents and costly techniques to purify terpene and eliminate degradation products.

[0135] Further features and advantages of the invention will appear more clearly from the following d non-limiting and exemplary examples.

[0136] EXPERIMENTAL PART

[0137] Example 1

[0138] 455 g of fresh cannabis inflorescences strain Blue Dream having a moisture content of 78% and an essential oil content of 2.3% on dry matter weight was put in contact with 150 g of refined sunflower oil and milled for 30 seconds with a kitchen robot (Moulinex Companion) using blades rotating at high speed (>4000 rpm). The mixture was transferred in a vacuum reactor (0.2 mbar) and stirred at 60 rpm for 2 hours at 40 °C. After 2 hours the mixture was separated via lab centrifugation (5,000 rpm for 15 min) and 129 g of a lighter oily fraction was collected. The oily fraction was vacuum distilled at 0.001 bar and 75 °C, utilizing a KDL-5 by UIC Gmbh. A lighter volatile fraction (1.1 g) was obtained, and a sample was sent out for GC-MS analysis along with a sample of the starting plant material.

[0139] The same experiment was performed without applying vacuum to the mixture in the reactor.The plant material was analyzed for its organoleptic and aromatic features through the solid-phase microextraction (SPME) technique coupled to GC-MS.

[0140] The essential oil samples obtained from the experiment under vacuum and without applying vacuum were diluted with heptane at two different dilutions to optimize the analysis of the compounds present in traces and in larger amount. Analysis was performed utilizing an Agilent 7820A (Agilent Technologies, USA) coupled to a mass spectrometer (MS) Agilent 5977E at electric ionization with single quadrupole equipped with automatic sampler Gerstel MPS2 XL. The GC method was as follows: Agilent DB- Wax Ul polar capillary column, length 60 m, internal diameter 0.250 mm, thickness of the film 0.5; carrier gas Helium 5.5; carrier flow at 1 .2 ml / min; starting temperature 40 °C, 5 °C / min up at 200 °C, 10 °C / min up to 240 °C; Splitless injection (1 min.); injected volume 1 pl; temperature of the injector = 250 °C. The mass spectrometer operated with an electron ionization of 70 eV, in scan mode in the m / z range 29-330, at 4.5 scans sec-1. The recognition of the molecules was carried out using the commercial reference standard where available; for the other molecules, the linear retention indices were calculated after having injected under the same analytical conditions a mixture of linear alkanes. Identification of the compounds was obtained by comparing the mass spectrum of each peak with those present in the Database (NIST Mass Spectral Library).

[0141] Table 1 : Plant Material and Essential oil fractions compositions.

[0142] Surprisingly, the method of the invention resulted not only in an essential oil fraction that faithfully reflects the terpene fingerprint of the plant material, including low vapor pressure compounds such as sesquiterpene beta-caryophillene or terpene alcohol linalool, but also a high purity, making no use of organic solvents. Additionally, the essential oil fraction obtained by applying vacuum also contained a significantly lower amount of degradation products as compared to the fraction obtained without applying vacuum. Example 2

[0143] A test equivalent to the previous test performed under vacuum with the only difference of adding to the mixture an enzymes cocktail, including cellulase, hemicellulase and pectinase in the dosage of 5% on plant material dry content was performed.

[0144] After mixing for 2 hours, 148 g of a lighter oily fraction were recovered by centrifugation. After vacuum distilling the oily fraction through the molecular wiped film distillatory, 1 .9 grams of a volatile fraction were recovered.

[0145] Table 2:

[0146] It can be appreciated that the addition of enzymes significantly increased the essential oil yield, while maintaining low the presence of degradation products. Example 3

[0147] 208 g of fresh lavender buds having a moisture content of 52% and an essential oil content of 5.3% on dry matter weight was put in contact with 150 g of refined sunflower oil and milled for 30 seconds with a kitchen robot (Moulinex Companion) using blades rotating at high speed (>4000 rpm). The mixture was transferred in a vacuum reactor (0.2 mbar) and stirred at 60 rpm for 2 hours at 40 °C, after adding 5 g of the same enzyme cocktail, as per the previous test, and 100 g of water. After 2 hours the mixture was separated via lab centrifugation (5,000 rpm for 15 min) and 141 g of a lighter oily fraction was collected. The oily fraction was vacuum distilled at 0.001 bar and 75 °C, utilizing a KDL-5 by UIC Gmbh. The lighter volatile fraction obtained (4.8 g) was sent out for GC-MS analysis along with a sample of the starting plant material. Table 3:

[0148] ANNEX

[0149] Table 4: Vapor Pressure of Selected volatile compounds

Claims

CLAIMS1. A method for preparing an essential oil fraction, said method comprising the steps of: a) putting in contact a biological material containing volatile compounds with a lipid to obtain a mixture; b) agitating under vacuum the mixture of step a) at a temperature between 20 °C and 80 °C to obtain an agitated mixture; c) mechanically separating the lighter oily fraction from the agitated mixture of step b) to obtain an oily fraction; and d) vacuum distilling from the oily fraction of step c) a lighter volatile fraction at a pressure below 0.01 bar, wherein the lighter volatile fraction is said essential oil fraction.

2. The method according to claim 1 , wherein the biological material is a plant material, preferably the plant material is selected from the group consisting of agarwood, arnica, basil, cannabis, cistus, cardamom, chamomile, clove, cinnamon, citrus, chrysanthemum, garlic, hemp, hops, jasmine, lavender, mint, orris, neem, nutmeg, patchouli, pepper, peppermint, rose, sage, vanilla, vetiver, violet or mixtures thereof.

3. The method according to any one of claims 1 or 2, wherein the lipid of step a) is selected from the group consisting of: vegetable oil, medium -chain triglycerides, fatty acids, liposomes, glycerine, polyethylene glycol, ethyl acetate, liquid paraffin, butylene glycol, propylene glycol, ethylhexyl palmitate, eicosapentaenoic acid, docosahexaenoic acid, microcrystalline wax, mineral wax, ozokerite, polyethylene, polyoxyethylene, hydrocarbon waxes derived from carbon monoxide and hydrogen, cerosin; cetyl esters; hydrogenated vegetable oil, hydrogenated fatty acids, polyethylene glycol, fatty acid or triglycerides obtained from cultured yeast, fungi, algae, bacteria, natural deep eutectic solvent or mixtures thereof, preferably the natural deep eutectic solvent is selected from the group consisting of: glycerol, sorbitol, mannitol, inositol, butylene glycol, propanediol, isosorbide, choline chloride, citric acid, lactic acid, betaine, proline, glycine, alanine, arginine, histidine, lidocaine, acetylcholine chloride, nicotinic acid, gallic acid, benzoic acid, coumaric acid, caffeic acid, xylitol, glucose, fructose, oxalic acid, malic acid, stearic acid, oleic, linoleic acid, urea, glucose, lactose, sucrose, xylose, galactose and trehalose, or mixtures thereof.

4. The method according to any one of claims 1 to 3, wherein the lipid in step a) is a vegetable oil selected from the group consisting of: olive oil, coconut oil, sunflower oil, sesame oil, hemp seed oil, hemp seeds oil, jojoba oil, avocado oil, crude palm oil, palm kernel oil or mixtures thereof.

5. The method according to any one of claims 1 to 4, wherein the biological material of step a) is comminuted or milled before, during or after, being put in contact with lipid, to increase the surface contact, preferably wherein the mixture is milled under vacuum.

6. The method according to any one of claims from 1 to 5, wherein the mixture is agitated for a time in the range from at least 5 seconds to less than 24 hours.

7. The method according to any one of claims from 1 to 6, wherein water is added to the mixture of step a) to form a homogeneous mixture or slurry, and wherein the water that is added is five times less than the dry weight of the biological material.

8. The method according to any one of claims from 1 to 7, wherein a step of condensation of the vapor generated from the mixture agitated under vacuum of step b) is added between step b) and step c), wherein the condensed vapor contains an essential oil fraction enriched in monoterpenes and / or a hydrosol enriched in water soluble compounds.

9. The method according to any one of claims from 1 to 8, wherein, a filtration step is added after step c) or step d).

10. The method according to any one of claims from 1 to 9, wherein said lighter oily fraction of step c) is put in contact with a new batch of biological material to further enrich it in volatile compounds.1 1. The method according to any one of claims from 1 to 10, wherein ultrasound / sonication, microwaves, steam explosion or cavitation are applied before or after adding the lipid to the biological material of step a) to reduce the time necessary to achieve higher volatile compounds lipid-extraction yield.

12. The method according to any one of claims from 1 to 1 1 , wherein the mechanical separation of step c) is achieved via density separation (i.e. centrifugation) or pressing (French press) and / or filtration.

13. The method according to any one of claims from 1 to 12, wherein the lipid of step a) has been neutralized and / or deodorized prior being added to the mixture.

14. The method according to any one of claims from 1 to 13, wherein the lipid of step a) has been obtained making no use of organic solvent.

15. The method according to any one of claims from 1 to 14, wherein enzymes are added to the mixture of step a), wherein said enzymes are preferably one or more independently selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases, cellulase, hemicellulase, xylanase, glucanase, beta-glucanase, pectinase, glucuronyltransferase, lipase, amylase, alphaamylase, beta-amylase, phospholipase, arabanase, galacto-, beta-mannanase, protease, esterase, phytase, cannabinoid synthase, THCA synthase, CBCA synthase and terpene synthase.

16. The method according to any one of claims from 1 to 15, wherein the pH of the agitated mixture of step b) varies in the range from 4 to 6., wherein the pH is preferably adjusted in the range from 4 to 6 by means of citric acid and / or ascorbic acid and / or ascorbyl palmitate.

17. The method according to any one of claims 1 to 16, wherein at least 50% of the terpenes contained in the biological material are extracted into the essential oil fraction.

18. The method according to any one of claims 1 to 17, wherein at least 50% of the terpenoids contained in the biological material are extracted into the essential oil fraction.

19. The method according to any one of the claims 1 to 18, wherein the essential oil fraction contains at least a volatile compound having a vapor pressure higher than 100 Pa and at least a volatile compound having a vapor pressure lower than 100 Pa.

20. An essential oil fraction obtainable from the method according to the method of any one of claims 1 to 19 comprising- more than 70% by weight, with respect to the total dry weight of the essential oil fraction, of terpenes and / or terpenoids; and- not more than 2% of Hexanal, (E)-2-Hexenal, (Z)-4-Hexen-1 -ol, (E)-2-Hexen-1 -ol, (E)-3-Hexen-1 -ol, 1 -Hexanol, 2-Heptanone, Heptanal, Propanal, Pentanal, Hexanoic acid, Nonenal, Acetaldehyde, 2-Propenal, Butenal, Alkyl furans, Acetic acid, or Propanoic acid.21 . The essential oil fraction according to claim 20, comprising not more than 0.001% per cent by weight of one or more organic solvents selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, petroleum ether, propane, toluene, m-xylene, o-xylene, and p-xylene heptane.

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