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Using enzyme to improve efficiency of mechanical expressed essential oils

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According to the Pharmacopoeia, an essential oil is an odorous product, usually of complex phytochemical composition, obtained from a botanically defined raw vegetable material. Extraction methods for these interesting products include traditional ones like hydrodistillation, steam distillation and cold or hot pressing,  and modern ones such as microwave-assisted extraction (MAE), ultrasound assisted extraction (UAE), or supercritical fluid extraction (SFE) among others.  By combining some of these methods with an enzymatic pre-treatment, the yield encouragingly increases, and the cost can decrease.

Enzyme-assisted extraction (EAE) is the new frontier for the food and pharmaceutical industry in the production of EOs using the cold-pressing method.

Chemical nature of essential oils

Essential oils (EO) are volatile secondary metabolites produced by plants and soluble in organic solvents and in lipids, some of them are colorless and others range from a light yellow to a reddish orange, such as lemongrass oil, cinnamon oil, and sandal oil. Mainly, EOs are less dense than water, such as citronella oil, lime oil or orange oil, but there are some heavier than water, such as allspice oil, cinnamon oil, clove oil or garlic oil. It is estimated that of the 3000 EOs known, only 10% are used commercially. [1]

EOs are very complex natural fragrant mixtures that can contain more than 20 components at different concentrations. Terpenes, terpenoids, and aromatic and aliphatic components are the main constituents (20-70% of the total concentration), while the rest comprises the minority components.

EO are recognized for several biological activities (bactericidal, antiviral, and fungicidal) and medicinal and aromatic properties. Among their multiple uses, they are natural additives for food preservation as antioxidants with radical scavenging activity. [2] They also serve as an antimicrobial, analgesic, sedative, and anti-inflammatory drugs, spasmolytic agents, and local anesthetics. [3]

Common extraction methods for essential oils

EO can be extracted by aqueous extraction, steam distillation, with organic solvents, or by mechanical extraction (pressing).

Aqueous extraction consists of an infusion with hot boiling water, which allows the production of extracts with a moderate yield. This is due to the hydrophilic nature of the solvent and the lipophilic nature of the extract. The excess water in EOs can be removed using anhydrous salt.

Steam distillation is widely used directly in the field of industrial production of the food and pharmaceutical supply chain but is also used in scientific assays.

Steam distillation is a separation process that consists in distilling water together with other volatile and non-volatile components. The steam from the boiling water carries the vapor of the volatiles to a condenser; both are cooled and return to the liquid or solid state, while the non-volatile residues remain behind in the boiling container. If, as is usually the case, the volatiles are not miscible with water, they will spontaneously form a distinct phase after condensation, allowing them to be separated by decantation or with a separatory funnel. [4]

Solvent extraction is the most efficient method, but organic solvents are a potential hazard to the life and health for workers as well as to the environment. [5] It is the process in which a compound transfers from one solvent to another owing to the difference in solubility or distribution coefficient between these two immiscible (or slightly soluble) solvents. [6]

Mechanical pressing is safer, environmentally friendly, and it preserves valuable natural components in the resulting oils (especially cold pressing). On the other hand, this technique requires high energy consumption and a lower yield of oil extraction, because the applied mechanical force does not completely destroy cell components storing the oil. [5]

“These glands are protected by cell walls and membranes; it is necessary to degrade these structural components. The use of hydrolytic enzymes, which are fast and specific catalysts, is promising to achieve a high essential oil yield by partial hydrolysis of various cell structures. [7]

The plant cell walls and related hydrolytic enzymes

“It was the thick cell walls of cork, visible in a primitive microscope, that in 1663 enabled Robert Hooke to distinguish and name cells for the first time. [8]

The walls of neighboring plant cells, cemented together to form the intact plant, are generally thicker, stronger, and, most important of all, more rigid than the extracellular matrix produced by animal cells. In evolving relatively rigid walls, which can be up to many micrometers thick, early plant cells forfeited the ability to crawl about and adopted a sedentary lifestyle that has persisted in all present-day plants. [8]

The structure of the plant cell wall is formed by linear cellulose chains and branched hemicellulose chains immersed in a lignin matrix and features cross-linking of lignin-carbohydrate bridges, ether, and carbon-carbon bonds. [9] Cell walls consist mainly of polysaccharides such as cellulose, hemicelluloses, lignin, and pectins, but also contain a low number of glycoproteins. [10]

Cellulose is a linear chain of glucose units that can be cleaved by various cellulases. [9] Hemicelluloses consist of a large number of different mono- and oligosaccharides. Due to their specificity and regiospecificity the destruction of hemicelluloses is best performed by a mixture of enzymes. [11]

Several enzymes are known to attack and degrade lignin, such as laccase, lignin peroxidase, and manganese peroxidase. [9]

Enzymatic degradation as extraction pretreatment

In nature, white-rot fungal enzymes can destroy not only lignin, but also all the main components of lignocellulose, including cellulose and hemicellulose. The enzymes expressed by brown-rot fungi are able to cause lignin oxidation, depolymerization, demethylation of lignin methoxy groups, and the removal of cellulose and hemicelluloses from plant cell walls. [9]

Enzyme-assisted extraction is a method employing enzyme(s) to treat the sample before being extracted. [12]

Partially purified enzymes can be obtained from a previously isolated and identified pectinase producer Aspergillus terreus, amylase producer Aspergillus niger, lignocellulase producer Aspergillus fumigatus, and cellulase producer Bacillus massiliensis, respectively, and were used for the enzyme pre-treatment. [13]

Mechanical expression and enzyme-assisted extraction

Cold pressing is used for extracting EOs from plants of the genus Citrus: the whole fruits or their peals move in a system covered with thousands of sharp and tiny spikes; glands break, open, and release their content. To isolate essential oil from this mixture, it is filtered and centrifuged to allow obtaining pure essential oil. Mechanical expression provides high quality products with characteristic fragrances nearly identical to the starting fruit because there are no structural changes in the fingerprint of the product. [14]

The yield is due to the pressing forces and the surface contact, much of the product can remain inside the glands and be lost with the filtration.

Enzyme pretreatment gave more than 50% higher yield than control in terms of weight of extracted essential oil. [13]

The results of many investigations showed that the weight in gram percentage and volume percentage per gram obtained by partially purified enzyme‐assisted extraction were indeed higher than those obtained in the control (sample treated with denatured enzymes, or without enzyme pretreatment). The increase in specific gravity in all pretreated samples indicates the possible extraction of some extra components during the enzyme pretreatment.

The relative increase in the percentage purity of components and the increase in the number of chromatographic peaks using gas-chromatography paired with mass-spectrometry (GC_MS) in the samples pretreated with single or mixed enzymes, show the help of the enzymes in jointly extracting some additional components to the main components and improve the overall extraction yield. [13]


[1] Haro-González JN, Castillo-Herrera GA, Martínez-Velázquez M, Espinosa-Andrews H. Clove Essential Oil (Syzygium aromaticum L. Myrtaceae): Extraction, Chemical Composition, Food Applications, and Essential Bioactivity for Human Health. Molecules. 2021.

[2] Ramadan, M.M.; Ali, M.M.; Ghanem, K.Z.; El-Ghorabe, A.H. Essential oils from Egyptian aromatic plants as antioxidant and novel anticancer agents in human cancer cell lines.2015.

[3] Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils—A review. Food Chem. Toxicol. 2008.

[4] Steam Distillation, Academic Accelerator: Encyclopedia, Science News & Research Reviews.

[5] Kumar SPJ, Prasad SR, Banerjee R, Agarwal DK, Kulkarni KS, Ramesh KV. Green solvents and technologies for oil extraction from oilseeds. Chem Cent J. 2017.

[6] Hongzhang Chen, Lan Wang, Posttreatment Strategies for Biomass Conversion. Technologies for Biochemical Conversion of Biomass, 2017

[7] Vovk H, Karnpakdee K, Ludwig R, Nosenko T. Enzymatic Pretreatment of Plant Cells for Oil Extraction. Food Technol Biotechnol. 2023.

[8] Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

[9] Martínez AT, Ruiz-Duenas FJ, Martinez MJ, del Rio JC, Gutierrez A. Enzymatic delignification of plant cell wall: From nature to mill. Curr Opin Biotechnol. 2009.

[10] Kalia VC. Rashmi, La lS, Gupta MN. Using enzymes for oil recovery from edible seeds. J Sci Ind Res (India). 2001.

[11] Ricochon G, Muniglia L. Influence of enzymes on the oil extraction processes in aqueous media. Oilseeds and fats, crops and lipids. 2010.

[12] Sowbhagya HB, Purnima KT, Florence SP, Appu Rao AG, Srinivas P. Evaluation of enzyme-assisted extraction on quality of garlic volatile oil. Food Chem. 2011.

[13] Amudan, Rajalakshmi & Kamat D, & Kamat S. Enzyme‐assisted extraction of essential oils from Syzygium aromaticum. South Asian Journal of Experimental biology. 2011.

[14] Başer, K.H.C., Buchbauer, G. Handbook of Essential Oils: Science, Technology, and Applications, Second ed. CRC Press, Boca Raton, FL. 2015.

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