ENZYMATIC EXTRACTION

What is enzyme-assisted extraction?

According to de Souza et al. (2025), enzyme-assisted hydrodistillation is a sustainable alternative that enhances oil release by degrading the cell wall. In their introduction, the same authors write that the plant cell wall has significant thermal resistance due to its structural composition, which includes cellulose, hemicellulose and lignin. According to them, enzymes such as cellulase effectively break down plant cell walls, enabling the extraction of high-quality essential oils with a lower environmental impact. They add that these enzymes can also transform compounds into new derivatives through reactions such as hydrolysis, oxidation and isomerisation, depending on the extraction conditions and the type of enzyme used.

How do the authors of the studies present the technique?

In their introduction, Penić et al. (2026) write that adding enzymes to purified water or citrate buffer during a pretreatment before hydrodistillation can improve the extraction of essential oil from citrus peels. According to the same passage, enzymes can help to break down the cell walls, facilitating the release of the essential oils and “potentially” increasing the yield and quality of the extracted oils.

According to Li et al. (2024), “the literature is limited to the optimization of enzyme-assisted extraction of REO” (REO: rosemary essential oil).

What did Mishra et al. (2005) and Khruengsai et al. (2023) test?

In the study by Mishra et al. (2005) on mandarin, Citrus reticulata Blanco (Citrus reticulata in the source), eight lots of 75 g were prepared, four of which were used for oil recovery by hydrodistillation only. The first lot, the control sample, was dipped in a buffer solution at pH 4.5 at 31–33 °C for 3.0 hours, then crushed into a pulp with a mixer, and the oil was recovered from the pulp by hydrodistillation with a Clevenger apparatus. The remaining three lots were pretreated with 0.1 %, 0.2 % and 0.3 % xylanase (“Xylananses” in the source) at the same temperature and for the same time, and the oil was recovered by the same method, hydrodistillation. According to Khruengsai et al. (2023), the optimum conditions of their “EP-MAE” method, applied to Zanthoxylum limonella (name used by the source), were a water-to-plant ratio of 11.16 mL/g, an enzyme amount of 0.68 %, a microwave time of 36.73 min and a power of 1665 W. Under these conditions the essential oil yield, 7.89 ± 0.08 mg/g, was significantly higher than those obtained by MAE (7.26 ± 0.04 mg/g) and by hydrodistillation (7.04 ± 0.03 mg/g).

What did Penić et al. (2026) test on citrus peels?

Penić et al. (2026) evaluated the effects of pectinase, cellulase, xylanase and their mixture, applied in purified water or citrate buffer before Clevenger hydrodistillation, on the yield and volatile composition of essential oils from orange, mandarin and clementine peels. The oranges, Citrus × aurantium f. aurantium (Citrus sinensis in the source), the mandarins, Citrus reticulata Blanco, and the clementines (“Citrus clementine” in the source) were purchased in a local supermarket in Zagreb, Croatia.

The peels, made of albedo and flavedo, were chopped into pieces of 1 cm × 1 cm and stored at −18 °C until analysis. The cellulase and the pectinase came from Aspergillus niger, and the xylanase from “Theryomyces” (spelling of the source), expressed in Aspergillus oryzae.

How were the peels pretreated?

The hydrodistillation of the peels was preceded either by reflux extraction without enzymes (RE), in purified water (HDW) or in citrate buffer (HDB), as control samples, or by reflux extraction with pectinase (REP), cellulase (REC), xylanase (REX) or a pectinase/cellulase/xylanase mixture (REPCX), in purified water or citrate buffer (pH = 5). For each pretreatment, 20 g of crushed peel (1 cm × 1 cm) was mixed with 250 mL; the passage does not name the liquid at that point. The peel material, in purified water or citrate buffer, was subjected to reflux extraction at 50 °C for 120 min under stirring, either without enzymes (control) or with 55 mg of pectinase, cellulase or xylanase (2.75 mg of enzyme per g of peel), or their combination (8.25 mg of total enzymes per g of peel). The pretreated peels were then hydrodistilled for 120 min in a Clevenger apparatus. The same mass of peel (20 g) and volume (250 mL) of purified water and citrate buffer (pH = 5) were also hydrodistilled without any prior pretreatment (HD).

Did the pretreatments change the yield?

Among the peels studied, orange consistently produced the highest essential oil yield, 1.95 to 1.99 mL/100 g of peel. In the same passage, hydrodistillation without pretreatment (HD) gave the lowest yield, reflux extraction without enzymes in water (HDW-RE) slightly increased it, and the pretreatments with pectinase and with the enzyme mixture, in water (HDW-REP, HDW-REPCX) and in citrate buffer (HDB-REP, HDB-REPCX), led to only a marginal additional increase. Clementine peel yielded 1.46 to 1.49 mL/100 g of peel, with no improvement observed after enzymatic pretreatment compared with hydrodistillation without pretreatment or reflux extraction without enzymes. Mandarin peel showed the lowest essential oil yields (0.98 to 1.00 mL/100 g of peel), with slight increases after reflux extraction without enzymes and after selected enzymatic pretreatments.

Did they change the composition of the oils?

Orange peel oil

In orange peel oil, limonene was the dominant monoterpene, particularly after pretreatment with the enzyme mixture in water (HDW-REPCX, up to 81.16 %). Pretreatments in buffer, with or without enzymes, resulted in lower limonene content (14.15 to 61.04 %).

Mandarin peel oil

In mandarin peel oil, limonene predominated among monoterpenes, especially after reflux extraction in water without enzymes (HDW-RE, 77.50 %). Pretreatment with individual enzymes or with the mixture reduced its content (HDW-REP 62.87 %, HDW-REX 47.72 %, HDW-REC 18.84 %, HDW-REPCX 6.74 %), while buffer pretreatments with pectinase (HDB-REP, 49.65 %) or xylanase (HDB-REX, 54.42 %) partially restored it. Untreated mandarin oil (HD) contained 56.47 % limonene. Total sesquiterpenes were highest after pretreatment with the enzyme mixture in water (HDW-REPCX, 60.52 %), including β-elemene (5.84 %), (E,E)-α-farnesene (8.64 %), δ-cadinene (9.93 %) and oxygenated t-cadinol (5.25 %). The non-pretreated oil (HD) had 6.75 % and reflux in water without enzymes (HDW-RE) gave 1.77 %, while the water-based enzymatic pretreatments raised sesquiterpenes to 9.10 % (HDW-REP), 11.09 % (HDW-REC), 23.99 % (HDW-REX) and 60.52 % (HDW-REPCX). In citrate buffer, total sesquiterpenes were highest without enzymes (HDB-RE, 47.38 %) and lower with enzymes (13.64 to 37.39 %). The t-cadinol content was higher in buffer (HDB-RE 12.58 %, HDB-REP 12.47 %, HDB-REC 21.30 %) than in water (2.32 to 9.63 %). Aldehydes ranged from 0.39 % to 4.86 % in the water-based enzymatic pretreatments, the highest value following HDW-REPCX, and the untreated oil (HD) contained 2.27 % (decanal).

Clementine peel oil

In clementine peel oil, limonene predominated among monoterpenes, especially after water-based xylanase pretreatment (HDW-REX, 75.29 %), while the untreated oil (HD) contained 30.86 % and reflux in water without enzymes (HDW-RE) increased it to 39.66 %. Citrate buffer pretreatments, with or without enzymes, yielded lower total monoterpenes (78.60 to 85.56 %) and lower limonene (up to 17.43 % with HDB-REP). Oxygenated monoterpenes were abundant in all samples: trans-carveol up to 15.17 %, cis-carveol up to 10.55 %, carvone up to 16.45 %, limonene-1,2-diol up to 20.76 % and (Z)-p-mentha-1,8-diene-2-hydroperoxide up to 10.03 %. Their levels were higher after enzymatic pretreatment in buffer, whereas water-based enzymatic pretreatments favoured monoterpene hydrocarbons. Carvone rose from 6.74 % (HDB-RE) to 15.55 % with cellulase (HDB-REC), 16.45 % with pectinase (HDB-REP) and 13.07 % with the enzyme mixture (HDB-REPCX).

What do the authors conclude?

In their conclusions, Penić et al. write that these results indicate that enzymatic pretreatment can modulate the volatile profile of citrus essential oils, particularly minor components, but has a limited effect on overall yield. According to the authors, including both a no-enzyme control and a no-pretreatment control was essential for accurately assessing enzymatic effects under laboratory-scale Clevenger hydrodistillation.

What did Li et al. (2024) optimise on rosemary?

In the study by Li et al. (2024), rosemary was collected from Yuzhou Country, Xuchang City, Henan Province, China.

The enzyme was an acidic cellulase (>10,000 U/g), dissolved in citrate-phosphate buffer at pH 5.0. The enzyme-assisted extraction was conducted in two stages, an enzymatic pre-treatment followed by extraction by hydrodistillation. The rosemary (overground part) was pulverised to a homogeneous size (50 to 60 mesh), and 100 g of the powder was combined with 500 mL of a 2.0 % cellulase solution (citrate-phosphate buffer at pH 5.0) and incubated before hydrodistillation. In the source’s words, extraction was then conducted in a Clevenger apparatus, followed by steam distillation for 4 h; the distilled essential oil was dried with anhydrous sodium sulfate and stored at 4 °C.

Which conditions did the study vary?

In single-factor experiments, the study investigated four variables: enzyme amount (0.5 to 2.5 %), enzyme digestion pH (4.0 to 6.0), enzyme digestion temperature (35 to 45 °C) and enzyme digestion time (0.5 to 2.5 h). On these four variables, 29 trial experiments were performed using the Box–Behnken design model. The optimal conditions, with a predicted yield of 4.05 %, were validated through a pilot-scale test with an enzyme amount of 1.60 %, an enzyme digestion pH of 5.09, an enzyme digestion temperature of 46.65 °C and an enzyme digestion time of 1.69 h.

What is in the oil analysed?

Li et al. describe rosemary essential oil (REO in the source) as a transparent yellowish liquid with a specific gravity of 0.914 g/mL. A total of 37 components were identified by GC–MS, the most abundant being “1.8-cineole” (53.48 %, spelling of the source), β-pinene (20.23 %) and borneol (5.21 %). The passage does not state from which extraction the analysed oil comes.

What did Vladić et al. (2022) find before supercritical CO2 extraction of oregano?

Vladić et al. (2022) set out to establish a procedure for improving the efficiency of supercritical carbon dioxide (scCO2) extraction of Greek oregano, “Origanum vulgare L. spp. hirtum” in the source (Origanum vulgare subsp. hirtum (Link) A.Terracc. in WCVP), and the quality of the extracts obtained. Microwave and enzymatic pretreatments of the plant material were applied before the scCO2 extraction, and the enzymatic pretreatment caused a significant increase in the extraction yield. The material consisted of aerial parts of this oregano, commercial samples purchased in a supermarket in Corfu, Greece, with a mean particle size of 0.26 mm.

How was the material pretreated and extracted?

The enzyme preparation, Viscozyme, is described as a cellulolytic enzyme mixture containing a wide range of carbohydrases, including arabanase, cellulase, β-glucanase, hemicellulase and xylanase. For the enzymatic pretreatment (ENZP), ground oregano material (40 g per pretreatment) was mixed with a Viscozyme solution at a material-to-solution ratio of 1:5 (m/V) and incubated at 45 °C for 60 min. The Viscozyme solution was prepared with acetate buffer at pH 4.9, at a concentration of 8 % with respect to the plant material. All extractions of the pretreated materials and of the control without pretreatment were conducted under identical conditions, 200 bar and 40 °C. The extractions were run at 40 °C to avoid volatilisation and decomposition of thermally unstable compounds.

What happened to the yield?

The enzymatic pretreatment led to a significant, almost twofold increase in the extraction yield: 6.59 %, against 3.39 % for the control. According to the authors, exposing the plant material to cell-wall-degrading enzymes damaged and hydrolysed its structures, which facilitated the penetration of the solvent and the availability of the components, hence their faster and more efficient release. In the first 30 min of extraction, the yield with ENZP and with the pretreatment labelled MWP1 was twice as high as that of the control. The ENZP, MWP1 and MWP2 extractions were still rising after 4 h; the ENZP and MWP1 extractions were completed after an additional 1.5 h, for a total extraction time of 5.5 h and total extraction yields of 7.09 % (ENZP) and 7.92 % (MWP1).

What happened to the composition of the extracts?

ENZP and MWP1 increased the abundance of oxygenated compounds in the extracts (94.67 % and 92.69 %, with no statistically significant differences among these groups) compared with the control (88.06 %). The pretreatments increased carvacrol abundance in the MWP1, MWP2 and ENZP samples compared with the control; it was highest in the MWP1 extract (80.44 %), and the MWP2 and ENZP extracts contained 76.94 % and 77.17 % carvacrol. In Table 1, which covers extracts obtained by supercritical carbon dioxide extraction at 200 bar, 40 °C and 4 h, the carvacrol row gives 74.89 for the control and 77.17 for the ENZP extract.

What do the authors conclude?

In their conclusions, the authors write that both microwave and enzymatic pretreatments intensified mass transfer and improved the extraction yield and the quality of the extracts obtained. According to the authors, the enzymatic pretreatment had the additional benefit of involving no increase in temperature over time, but required a longer pretreatment time. They also recorded a beneficial impact on total yield and carvacrol abundance after the enzymatic pretreatment.

What did de Souza et al. (2025) measure on Matourea azurea leaves?

De Souza et al. (2025) applied cellulase enzymolysis before hydrodistillation to the leaves of Matourea azurea (Linden) Colletta & V.C.Souza (written “Matourea azurea (Linden) Colletta; V.C.Souza” in the source), testing concentrations of 1.0 %, 1.5 % and 2.0 %. The commercial enzyme Celluclast 1.5 L, with a declared activity of 700 EGU/g, was used at 1 %, 1.5 % and 2 %, and a moisture test on the leaves served to compensate for the water volume in the enzymatic concentration of the system. The enzymolysis was carried out in a 50 °C bath without shaking for 30 min. The volatile oil content was determined by hydrodistillation in a Clevenger apparatus, on 75 g of fresh material (leaves).

How did the cellulase concentration act?

According to de Souza et al., all the concentrations used increased the essential oil yield, but the efficiency of enzymatic digestion decreased as the concentration increased. Since the data do not follow Michaelis–Menten kinetics, the authors exclude enzymatic saturation. According to the authors, this behaviour can be explained by enzymatic inhibition caused by cellulose degradation products, such as cellobiose and glucose, or by interactions with aromatic substances from the biomass, such as terpenes and phenols.

Did the composition of the oil change?

A qualitative difference in the composition of the oil was observed after enzymatic treatment with Celluclast 1.5 L, including the appearance of “1,5,9,9‐tetramethyl‐Z,Z,Z‐” (name truncated as the sentence writes it) at all concentrations and of p-mentha-2,8-dien-1-ol at the 1 % concentration. β-Copaen-4α-ol (20.73 %) was the major compound of the essential oil from the control sample, followed by pinocarveol (9.49 %), myrtenal (8.74 %), pinocarvone (6.31 %) and “cyclophenene” (6.30 %, spelling of the source). Overall, these compounds maintained their relative percentages across the different enzyme concentrations.

How did cellulase-assisted hydrodistillation compare with distillation in Wainer et al. (2022)?

The lavender used by Wainer et al. (2022) was Margaret Roberts lavender, which the source classifies as Lavandula × intermedia ‘Margaret Roberts’, or Lavandula × heterophylla, grown at the Margaret Roberts Herbal Centre in Hartbeespoort, South Africa, and harvested at the beginning of spring.

The study compared steam distillation, hydrodistillation and cellulase-assisted hydrodistillation, with average extraction times of 57, 51 and 49 min and an average energy consumption of 15.0, 13.4 and 30.8 kJ/g respectively. For the cellulase-assisted hydrodistillation, 80 g of plant material was pre-treated for 45 min with 1 L of distilled water and 5 mL of cellulase under magnetic stirring, the mixture being maintained between 40 °C and 45 °C, then moved to the hydrodistillation set-up. The enzyme used for the pre-treatment was Celluclast 1.5 L cellulase (Novozyme, Bagsværd, Denmark).

How long did each method take, and what did it consume?

Cellulase-assisted hydrodistillation had the shortest extraction time, but its total process time was significantly longer than that of the other methods because of the 45 min pre-treatment stage. Steam distillation and hydrodistillation, which both use only the heater and not the mixer, showed similar energy consumption, whereas cellulase-assisted hydrodistillation showed significantly higher energy consumption. The enzymatic pre-treatment stage brought the energy consumption of cellulase-assisted hydrodistillation to more than double that of conventional hydrodistillation.

How did the oils differ?

Oils from conventional hydrodistillation and from cellulase-assisted hydrodistillation contained more 1,8-cineole than oils from steam distillation, which contained more camphor. The retention time reported for linalool was between 17.543 and 17.554 min, and that reported for terpinolene between 17.531 and 17.541 min; the authors write that these very similar values “suggest” that the linalool peak on the chromatogram “could have been” erroneously interpreted as terpinolene.

Comparing hydrodistillation with cellulase-assisted hydrodistillation, the authors write that the results “suggested” that the enzymatic pre-treatment step gives an oil containing less camphor than without a pre-treatment step. In Table 1, the camphor content (wt%) of samples 7, 8 and 9, obtained by cellulase-assisted hydrodistillation, is 32.21, 21.73 and 19.75, against 28.34 and 30.68 for samples 1 and 2 (steam distillation) and 24.82, 36.74 and 24.97 for samples 4 to 6 (hydrodistillation). Sample 3, marked with an asterisk, has no value: the analytical equipment gave obviously incorrect results, which could not be repeated.

How did they smell?

Oils obtained by cellulase-assisted hydrodistillation were rated as the most pleasant-smelling, with an average score of 4 out of 5, and oils from steam distillation and hydrodistillation were rated equally, at an average of 3 out of 5. The cellulase-assisted hydrodistillation oils were also reported to have the weakest smell.

References

• Penić M., Grassino A.N., Aladić K., Jokić S., Jerković I. et al., « Effects of Enzymatic Pretreatment on Yield and Volatile Composition of Citrus Peel Essential Oils », Molecules 2026;31(4):657, doi:10.3390/molecules31040657 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC12943379/fullTextXML
• Li Y., Huang L., Xu Y., Cheng B., Zhao M., « Optimization of Enzyme-Assisted Extraction of Rosemary Essential Oil Using Response Surface Methodology and Its Antioxidant Activity by Activating Nrf2 Signaling Pathway », Molecules 2024;29(14):3382, doi:10.3390/molecules29143382 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC11279388/fullTextXML
• Vladić J., Duarte A.R.C., Radman S., Simić S., Jerković I., « Enzymatic and Microwave Pretreatments and Supercritical CO2 Extraction for Improving Extraction Efficiency and Quality of Origanum vulgare L. spp. hirtum Extracts », Plants 2022;11(1):54, doi:10.3390/plants11010054 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC8747452/fullTextXML
• de Souza J.C.G., de Souza L.D.S.S., Hidalgo A.D.F., de Azevedo G.T., de Souza G.L. et al., « DPPH-based Antioxidant Screening and Cellulase-assisted Hydrodistillation of Matourea azurea: Improved Essential Oil Yield and Density Functional Theory Insights Into a Rare Terpene », Chemistry & Biodiversity 2025;22(12):e01109, doi:10.1002/cbdv.202501109 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC12715992/fullTextXML
• Wainer J., Thomas A., Chimhau T., Harding K.G., « Extraction of Essential Oils from Lavandula × intermedia ‘Margaret Roberts’ Using Steam Distillation, Hydrodistillation, and Cellulase-Assisted Hydrodistillation: Experimentation and Cost Analysis », Plants 2022;11(24):3479, doi:10.3390/plants11243479 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC9785002/fullTextXML
• Mishra D., Shukla A.K., Dixit A.K., Singh K., « Aqueous Enzymatic Extraction of Oil from Mandarin Peels », Journal of Oleo Science 2005;54(6):355–359, doi:10.5650/jos.54.355 — https://www.jstage.jst.go.jp/article/jos/54/6/54_6_355/_pdf/-char/en
• Khruengsai S., Promhom N., Sripahco T., Siriwat P., Pripdeevech P., « Optimization of enzyme-assisted microwave extraction of Zanthoxylum limonella essential oil using response surface methodology », Scientific Reports 2023;13:12872, doi:10.1038/s41598-023-40142-4 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC10409809/fullTextXML
• Govaerts R. (ed.), World Checklist of Vascular Plants (WCVP), Royal Botanic Gardens, Kew — https://sftp.kew.org/pub/data-repositories/WCVP/wcvp.zip