Effects of Enzymatic Pretreatment on Yield and Volatile Composition of Citrus Peel Essential Oils

TL;DR

This study shows how enzyme pretreatment can change the chemical makeup of citrus peel essential oils, though it only slightly increases their yield.

Contribution

The novelty lies in systematically evaluating how different enzymes and buffers affect the volatile composition of citrus essential oils.

Findings

Enzymatic pretreatment increased essential oil yield slightly for orange and mandarin peels but not for clementine.

Mandarin oils showed increased sesquiterpenes and aldehydes after pretreatment, while clementine oils had higher oxygenated monoterpenes.

Limonene remained the dominant compound in all oils, with no significant shift in its proportion.

Abstract

Enzymatic pretreatment is a promising method for modulating essential oil isolation. This study 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. Essential oil yield increased slightly for orange and mandarin peels (up to approximately 2%) compared to non-enzymatic controls, while clementine yield was unaffected. Limonene remained the dominant compound in all oils, reaching 81.16% in orange, 77.50% in mandarin, and 75.29% in clementine. Enzyme pretreatment particularly affected the secondary components: mandarin peel showed increased sesquiterpenes (up to 60.52%) and aldehydes (up to 4.86%), while clementine oils exhibited higher oxygenated monoterpenes after buffer-based enzymatic treatments. These results indicate that enzyme-assisted pretreatment can modulate the volatile profile of citrus essential oils, although overall yield gains are modest. The inclusion of no-enzyme and no-pretreatment controls is crucial for the reliable assessment of enzymatic effects under laboratory conditions. This systematic approach provides insight into enzyme-assisted extraction, highlighting its potential to influence essential oil quality and composition rather than dramatically increase yield, and offers a foundation for further optimization research.

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Full text

1. Introduction

Global citrus production is approximately 134 million tons per year, with oranges accounting for about 75 million tons and mandarins—including clementines and satsumas—about 33 million tons [1]. The processing of citrus fruits generates significant amounts of waste. Citrus waste consists mainly of peel, pulp, pits, and membranes, with peel being the most important component. The percentage of peel in citrus varies but generally accounts for about 50% of the total weight of the fruit [2,3,4], which corresponds to an estimated 67 million tons of citrus waste [5]. A small proportion of citrus peel is recycled, while a large proportion is buried and incinerated, which can pollute the environment and waste resources. Due to their rich and diverse composition, citrus peels offer a variety of uses, transforming what is often considered waste into a valuable resource. Therefore, many attempts have been made to valorize citrus peels to extract value-added biologically active compounds, including polyphenols, pectin and essential oils [5,6,7,8,9]. One of the products derived from citrus peels are essential oils, which are used in various industries, the perfume and fragrance industry, detergents, food and beverages [2,10]. Citrus essential oils are a complex mixture of volatile compounds belonging to different chemical classes, such as oxygenated terpenes, terpene hydrocarbons, alcohols, aldehydes, ketones, acids and esters. Essential oils isolated from the peels of citrus fruits such as oranges (Citrus sinensis) and clementine (Citrus clementine) contain high amounts of monoterpene hydrocarbons, with limonene dominating, while oxygenated monoterpenes were found in greater amounts in mandarins (Citrus reticulata) [11,12,13,14,15]. Regardless of the production process, a considerable number of by-products are generated during the isolation of essential oils, including hydrolates and water residues, which usually contain dissolved components of the essential oils [16,17].

As the extraction process is the most important step in isolating volatile compounds, research focuses on developing new isolation methods or pretreatments in addition to those already established. Essential oils from orange, mandarin, and clementine peels are isolated by steam distillation [18], hydrodistillation [5,19,20,21,22,23,24,25], cold pressing [26], extraction with supercritical fluids [27], microwave-assisted hydrodistillation [23,28], and microwave-assisted steam distillation [15,22,29]. Although these methods have been used successfully for years, new extraction methods or pretreatments with ultrasound [22,27], salt-assisted extraction [22], or enzymes [22,30,31,32] have recently been developed to increase the yield of essential oils or improve their chemical composition. As the yield of essential oil is low, efforts are being made to increase the yield while maintaining or improving the quality of the oil. Many studies show that citrus peels have potential for environmentally friendly essential oil isolation and can be a solution that can help reduce waste costs and produce valuable new products. The addition of enzymes to purified water or citrate buffer during pretreatment prior to hydrodistillation can improve the extraction process of essential oil from citrus peels. 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. Commonly used enzymes include cellulases [22,30], xylanases [32], hemicellulases and pectinases [22,33] in water or citrate buffer (pH = 4.5). Considering the small number [22,30,32] of studies conducted on the enzymatic treatment immediately prior to the hydrodistillation of citrus essential oils, further investigations are needed. In particular, each individual enzyme, such as pectinase, cellulase and xylanase, as well as a mixture of the mentioned enzymes in water and citrate buffer should be tested at other pH values at which the mentioned enzymes show satisfactory activity. It is also important to point out that the studies that have demonstrated the isolation of essential oils from citrus peels have only used a control without pretreatment [30,32] or a pretreatment without enzymes [22]. To confirm with certainty the role of enzymes in increasing the yield of essential oils from citrus fruits, it is necessary to perform both controls, as stated by other authors who studied the effect of enzymatic pretreatment on essential oil yield from aromatic plants [34,35,36]. Chávez-González et al. [30] and Mishra et al. [32] reported the effect of enzymes on increasing the yield of citrus essential oils and altering their chemical composition despite the control by pretreatment without enzymes before hydrodistillation. Therefore, this article discusses how to reuse citrus peels for essential oil isolation and introduces the possibility of the enzymatic pretreatment of citrus peels immediately before hydrodistillation as a new method aimed at achieving the satisfactory yield and quality of citrus peel essential oil.

The aim of this study is to valorize waste citrus peels by isolating essential oils from orange, mandarin, and clementine peels. The effect of the enzymatic pretreatment of waste citrus peels on the yield and volatile composition of essential oils obtained by Clevenger hydrodistillation will be evaluated. To date, only a few studies have investigated the enzymatic pretreatment of citrus peels [22,30,32] prior to hydrodistillation, and the effects of control pretreatments—namely, hydrodistillation of citrus peels without pretreatment and pretreatment without enzymes—have not been included in the same study. This study specifically investigates the effect of individual enzymes (pectinase, cellulase, and xylanase) and their mixtures, applied in purified water and citrate buffer (pH = 5), compared to two control conditions, hydrodistillation without pretreatment and reflux pretreatment without enzymes, to determine the specific contribution of enzymatic activity to the increase in oil yield and the composition of volatile compounds. Detailed chemical analysis of isolated citrus peel essential oils will be performed using the HS-SPME GC-MS method. Statistical analysis will be used to assess the distribution of volatile compounds among peels, as well as to investigate the effect of the enzymatic pretreatment of orange, mandarin, and clementine peels immediately before hydrodistillation on the yield and volatile composition of essential oil.

2. Results and Discussion

2.1. Enzyme Activity

Cell wall degrading enzymes (pectinase, cellulase and xylanase) were used to extract the compounds from the citrus peel. The enzyme activity was measured under the applied conditions for 120 min at 50 °C, in purified water and citrate buffer (pH = 5) on a small scale using the 3,5-dinitrosalicylic acid (DNSA) assay. The enzymes were mixed with the corresponding substrates in purified water and the reaction was incubated at 50 °C for 120 min. The results showed that all enzymes were able to degrade their substrates in purified water: pectinase degraded pectin to 50.4 U/mL, cellulase degraded cellulose to 12.7 U/mL, and xylanase degraded xylan to 22.3 U/mL, as well as in citrate buffer: pectinase degraded pectin to 41.5 U/mL, cellulase degraded cellulose to 25.3 U/mL, and xylanase degraded xylan to 13.5 U/mL.

2.2. Effect of Different Hydrodistillation Pretreatments on the Yield of Citrus Peel Essential Oil

The effect of different hydrodistillation pretreatments on the yield of orange, mandarin and clementine peel essential oil is shown in Figure 1. All tested pretreatments increased the yield of orange and mandarin peel essential oils, while the yield of clementine peel essential oil was not increased by the pretreatments (Figure 1).

Among the investigated citrus peels, orange consistently produced the highest essential oil yield (1.95–1.99 mL/100 g peel). Hydrodistillation without pretreatment (HD) resulted in the lowest yield, while reflux extraction without enzymes in water (HDW-RE) slightly increased the yield. Enzymatic pretreatments with pectinase and enzyme mixtures in both water (HDW-REP, HDW-REPCX) and citrate buffer (HDB-REP, HDB-REPCX) led to only a marginal additional increase. Clementine peel yielded 1.46–1.49 mL/100 g peel, with no improvement observed after enzymatic pretreatment compared to hydrodistillation without pretreatment or reflux extraction without enzymes. Mandarin peel showed the lowest essential oil yields (0.98–1.00 mL/100 g peel), with slight increases observed after reflux extraction without enzymes and selected enzymatic pretreatments. Reported essential oil yields of citrus peels vary widely, ranging from 0.5 to 5.0% depending on the species, cultivar, harvest conditions, and extraction method [11,19,20,37,38,39,40,41]. For example, essential oil yields reported for sweet orange range from 0.7 to 2.31%, sour orange from 1.24%, mandarin from 0.7 to 4.62%, and kinow from 0.32% [11,38,39,40,41]. Meyrem et al. [19] reported essential oil yields of 0.8–1.02% for C. limonum, C. reticulata, and C. paradisi isolated by Clevenger hydrodistillation, highlighting the strong influence of biological and environmental factors. Previous studies reported increased essential oil yields after enzyme pretreatment, particularly for orange and mandarin peels [22,30,32]. For example, cellulase pretreatment of orange peel resulted in yields of 1.0–2.0 mL/100 g peel [30], while Mishra et al. [32] observed an increase in mandarin essential oil yield after xylanase pretreatment. In grapefruit, essential oil yield increased significantly when the enzyme pretreatment time was extended from 3 to 12 h (0.25 to 1.8 mL/100 g), whereas no such trend was observed for lemons, where the maximum oil yield (1.0 mL/100 g) was reached after 6 h and decreased thereafter, likely due to the thinner peel of lemon compared to grapefruit [30]. Pretreatment with a mixture of enzymes (cellulase, hemicellulase, and pectinase) before hydrodistillation increased orange essential oil yield compared to conventional hydrodistillation (3.67% vs. 1.9%) [22]. Other pretreatments, such as ultrasound or the addition of calcium chloride, also improved yields in the same study. In mandarin, essential oil yields increased after xylanase pretreatment (4.53% to 5.66%), regardless of whether only flavedo or whole peel (flavedo + albedo) was used [32]. However, several of these studies lacked controls without pretreatment and/or without enzymes, limiting the reliable assessment of the true effect of enzymatic activity [22,30,32]. Enzymatic pretreatment increased essential oil yields from orange and mandarin peels up to approximately 2%, whereas clementine peel was not significantly affected. Overall, differences in yield between the various pretreatments of the same citrus peel were minor, indicating that enzymatic pretreatment did not contribute to a substantial increase in oil content. The observed yields are comparable to those reported in other studies where no enzymes were used, further emphasizing the importance of including both enzyme-free and pretreatment-free controls to accurately evaluate the specific contribution of enzymatic pretreatment. It should be noted that this study was conducted under controlled laboratory conditions to ensure reproducibility and the accurate assessment of pretreatment effects.

2.3. Volatile Composition of Citrus Peel Essential Oils

Citrus essential oils (orange, mandarin and clementine) are complex mixtures of volatile compounds from chemical classes such as monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, alcohols and aldehydes. The volatile composition of orange, mandarin and clementine peel essential oils obtained after Clevenger hydrodistillation, with or without various pretreatments, was determined by HS-SPME GC-MS analysis and is presented in Tables S1–S3 in the Supplementary Materials, SM. In addition, a heat map was generated to visualize the variations in the volatile composition of citrus peel (orange, mandarin, and clementine) essential oil between pretreatments, with hierarchical clustering applied to the columns (Figure 2, Figure 3 and Figure 4). HS-SPME GC-MS analysis showed that orange, mandarin and clementine peel essential oils were richest in monoterpenes: up to 94.13%, 93.68% and 94.46%, respectively. The most abundant volatile compound was limonene, from the group of monoterpene hydrocarbons: up to 81.16%, 77.50% and 75.29% of the total peak area in orange, mandarin and clementine essential oils, respectively. An exception is mandarin essential oil, which also contains significant amounts of sesquiterpenes compared to orange and clementine essential oils. Although the volatile composition of citrus peel essential oils varies, it is consistent with other studies [7,11,15,19,22,30,38,40,42,43,44,45,46,47] and will be explained in detail below for each citrus peel.

Kruskal–Wallis analysis was used to investigate the differences between the pretreatments of citrus peels (orange, mandarin, and clementine) with or without enzymes in water or citrate buffer immediately before hydrodistillation on the volatile composition of citrus peel essential oils. The results of the Kruskal–Wallis test showed significant differences (p < 0.05) between the pretreatments (Table 1), depending on the type of citrus peel. Pretreatments performed without enzymes (HDW-RE) and with pectinase (HDW-REP), cellulase (HDW-REC), xylanase (HDW-REX), and a mixture of enzymes (HDW-REPCX) in water, as well as without enzymes (HDB-RE) and with pectinase (HDB-REP) and cellulase (HDB-REC) in citrate buffer before hydrodistillation, significantly (p < 0.05) affected the volatile composition of citrus essential oils. Their composition was comparable to the control without pretreatment (HD). Other pretreatments, such as xylanase (HDB-REX) and enzyme mixture (HDB-REPCX) in citrate buffer, did not show significant differences (p > 0.05) (Table 1).

Spearman’s rank correlation test was used to assess whether the volatile composition of citrus essential oils differed significantly between pretreatments with or without enzymes in water or citrate buffer immediately prior to hydrodistillation. Citrus peel pretreatment has a significant effect (p < 0.05) on the volatile essential oil composition of each citrus species, with positive correlations determined by the Spearman test (Figure 5a–c). It was also noted that, for a better assessment of the influence of the enzyme, a control extraction with reflux in water and citrate buffer without the addition of enzymes is needed, as well as a control without pretreatment of the citrus peel immediately before hydrodistillation.

2.3.1. Orange Peel

The most abundant compound classes in orange peel essential oil were monoterpenes (up to 94.13%), aldehydes (up to 12.51%), and sesquiterpenes (up to 10.52%) (Table S1, Supplementary Materials). Hierarchical clustering in Figure 2 shows variations in volatile composition across pretreatments. Limonene was the dominant monoterpene, particularly after pretreatment with a mixture of enzymes in water (HDW-REPCX, up to 81.16%), while pretreatments in buffer, with or without enzymes, resulted in lower limonene content (14.15–61.04%). These findings are consistent with Taktak et al. [22], who reported limonene as the main compound in orange peel essential oil, with 86.7% after Clevenger hydrodistillation and 88.3% after enzyme-assisted pretreatment with a mixture of cellulase, hemicellulase, and pectinase. However, since that study lacked a control without enzyme treatment, the role of enzymes in isolating limonene cannot be reliably assessed. Similarly, Chávez-González et al. [30] reported up to 90.18% limonene after cellulase pretreatment, but without a proper pretreatment control, limiting the assessment of enzyme effects. The slightly higher limonene yield in [30] may be due to the inactivation of endogenous enzymes before cellulase treatment in citrate buffer (pH 4.5), compared to our results with cellulase in water (HDW-REC, 73.35%) or in citrate buffer (HDB-REC, 45.86%). Other studies also confirm limonene as the main compound in orange peel oil, with relative amounts ranging from 74.4% [46] and 76.9% [11] to 88.25% [8], 96.9% [38], and up to 95.53% after microwave pretreatment before steam distillation [15]. Aldehydes were present in significant amounts, increasing from 7.42% in untreated oil (HD) to 12.51% after xylanase in buffer (HDB-REX, decanal 9.60%), while water-based enzyme pretreatments ranged from 1.22% (pectinase) to 4.82% (xylanase). Aldehydes are key aroma contributors to citrus essential oil, imparting a sweet, waxy aroma and citrus-like odor [42]. Sesquiterpenes ranged from 1.18% in buffer without enzyme (HDB-RE) to 10.52% after xylanase in buffer (HDB-REX, δ-cadinene 2.31%), and were lower in water pretreatments (0.22–1.06%), with 0.2% in untreated oil (HD). Alcohols were minor components (0.01–1.29%), regardless of pretreatment.

2.3.2. Mandarin Peel

The most abundant compound classes in mandarin peel essential oil were monoterpenes (up to 93.98%) and sesquiterpenes (up to 60.52%) (Table S2, Supplementary Materials). Hierarchical clustering in Figure 3 shows variations in volatile composition across pretreatments. Limonene predominated among monoterpenes, especially after reflux extraction in water without enzymes (HDW-RE, 77.50%). Pretreatment with individual enzymes or a mixture in water or buffer reduced limonene 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. These results agree with previous reports of limonene as the major compound in mandarin peel oils, ranging from 63% [46] to 97% [38], with variability due to cultivar and genetics [39]. This raises the question of whether enzymes are necessary, as water alone can substantially increase limonene yield. 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%). Non-pretreated oil (HD) had 6.75%, and water pretreatment without enzymes (HDW-RE) gave 1.77%. Water-based enzymatic pretreatments increased 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 (13.64–37.39%), while t-cadinol content was higher in buffer (HDB-RE 12.58%, HDB-REP 12.47%, HDB-REC 21.30%) than in water (2.32–9.63%). Aldehydes ranged from 0.39% to 4.86% in water-based enzyme pretreatments, with the highest after HDW-REPCX and untreated oil (HD) contained 2.27% (decanal), consistent with previous reports of decanal and octanal as the main aldehydes in mandarin peel essential oil [39,40].

2.3.3. Clementine Peel

The most abundant compound classes in clementine peel essential oil were monoterpenes (76.80–94.46%) and aldehydes (up to 11.97%) (Table S3, Supplementary Materials). Hierarchical clustering (Figure 4) shows variations in volatile composition across pretreatments. Limonene predominated among monoterpenes, especially after water-based xylanase pretreatment (HDW-REX, 75.29%), while untreated oil (HD) contained 30.86%, and water pretreatment without enzymes (HDW-RE) increased it to 39.66%. Citrate buffer pretreatments, with or without enzymes, yielded lower total monoterpenes (78.60–85.56%) and limonene (up to 17.43% with HDB-REP). Similar to orange and mandarin, limonene was the dominant monoterpene, consistent with previous reports showing limonene in clementine oils ranging from 61.31% to 95.03% depending on the extraction method [38,44,45]. 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%, (Z)-p-mentha-1,8-diene-2-hydroperoxide up to 10.03%), with higher levels after enzyme pretreatment in buffer, whereas water-based enzyme pretreatments favored monoterpene hydrocarbons. Carvone increased from 6.74% (HDB-RE) to 15.55% with cellulase (HDB-REC), 16.45% with pectinase (HDB-REP), and 13.07% with enzyme mixture (HDB-REPCX). Trans-carveol and limonene-1,2-diol also increased after buffer-based enzyme pretreatments, reaching up to 20.74% (HDW-REP). Aldehydes were abundant in all oils, increasing from 7.57% in untreated clementine oil (HD) to 11.97% after cellulase pretreatment in water (HDW-REC), with decanal predominating. The highest decanal content (8.42%) was observed after water-based enzyme mixture pretreatment (HDW-REPCX). Buffer pretreatments yielded lower aldehyde levels (5.47–9.57%). Overall, enzyme pretreatment in buffer enhanced oxygenated monoterpenes, while water-based pretreatments favored monoterpene hydrocarbons.

2.3.4. Statistical Assessment of Enzymatic Pretreatment Effects on Citrus Peel Essential Oil Composition

The ratio of volatile components in orange, mandarin, and clementine peel essential oils was generally similar across pretreatments. However, reflux extraction with enzymes in purified water and citrate buffer significantly influenced the volatile composition of citrus oils, as confirmed by Kruskal–Wallis tests (p < 0.001) and Spearman correlation analysis (Figure 5a–c, Table 1). In orange peel, enzyme-free reflux extraction in water or buffer (HDW-RE, HDB-RE) produced the highest total monoterpene content (94.13% and 89.96%) with limonene as the dominant compound. Pretreatment with a mixture of enzymes in water (HDW-REPCX) further increased the limonene content (81.16%), while enzyme treatments in buffer led to lower limonene. Spearman correlations confirmed the significant effects of HDW-REPCX (r = 0.91) and xylanase in water (HDW-REX, r = 0.95) relative to the untreated control (HD). Mandarin peel showed a similar pattern, with enzyme-free reflux extraction in water (HDW-RE) yielding the highest total monoterpenes (93.98%) and limonene (77.50%). Enzymatic pretreatments in water or buffer slightly reduced limonene but increased sesquiterpenes (up to 60.52%) and aldehydes (up to 4.86%) with the enzyme mixture in water (HDW-REPCX, r = 0.90). Oxygenated sesquiterpenes such as t-cadinol were higher after pretreatment with pectinase (HDB-REP) and cellulase (HDB-REC) in buffer, as confirmed by Spearman correlations (r = 0.53 and 0.60). Kruskal–Wallis tests confirmed the significant effects of HDW-REPCX, HDB-REP, and HDB-REC on volatile composition (p < 0.001). Clementine peel essential oil was more similar to orange than to mandarin in volatile composition, dominated by total monoterpenes (94.46%) with limonene prevailing (75.29%) after xylanase pretreatment in water (HDW-REX). Positive Spearman correlations were observed compared to enzyme-free reflux extraction in water (HDW-RE, r = 0.81) and the control without pretreatment (HD, r = 0.76), as well as with other enzyme and buffer treatments (0.76 < r < 0.97), confirming the effects on the limonene content (Figure 5c). Kruskal–Wallis tests confirmed the significant effects of xylanase and pectinase in water (HDW-REX, HDW-REP) and pectinase and cellulase in buffer (HDB-REP, HDB-REC) on volatile composition (p < 0.001, Table 1). In conclusion, enzyme-free reflux extraction produced the highest monoterpene content in all three citrus peels, while enzymatic pretreatments mainly altered the sesquiterpene and aldehyde composition. Overall, enzymes had a limited effect on major monoterpenes, and essential oil yields from orange and mandarin peels were around 2%, with no significant increase for clementine. The inclusion of both no-enzyme and no-pretreatment controls proved essential for reliably assessing the role of enzymatic pretreatment under laboratory-scale Clevenger hydrodistillation, providing a systematic understanding of enzyme effects in citrus peel extraction.

3. Materials and Methods

3.1. Chemicals

The following chemicals were used: 3,5-dinitrosalicylic acid (DNSA) (98%, Thermo Fisher Scientific, Maharahstra, India), sodium sulfite (Lach-ner, Brno, Chez Republic), sodium hydroxide (Lach-ner, Brno, Chez Republic), phenol (99+%, Thermo Fisher Scientific, Maharahstra, India), potassium sodium tartrate (Sigma-Aldrich, Buchs, Switzerland), cellulase (from Aspergillus niger) (Sigma-Aldrich, Tokyo, Japan), pectinase (from Aspergillus niger) (Sigma-Aldrich, Buchs, Switzerland), xylanase (from Theryomyces, expressed in Aspergillus oryzae) (Sigma-Aldrich, Søborg, Denmark), beechwood xylan (Biosynth, Compton, UK), sodium carboxymethyl cellulose (Sigma-Aldrich, Buchs, Switzerland), xylose (99%, Sigma-Aldrich, Buchs, Switzerland, D-(+)-glucose (99.5%, Sigma-Aldrich, Buchs, Switzerland), D-(+)-galacturonic acid monohydrate (97.0%, Sigma Aldrich, Buchs, Switzerland), C_9_-C_25_ alkanes, deuterated chloroform for NMR spectroscopy (CDCl_3_-d with 0.03% v/v TMS, 99.80%, Eurisotop, Saint-Aubin, France). Statistical analysis was performed using R software (version 3.4.0, R Development Core Team, Vienna, Austria).

3.2. Plant Material

The samples of oranges (Citrus sinensis), mandarins (Citrus reticulata) and clementines (Citrus clementine) were purchased in a local supermarket in Zagreb (Croatia). The fruits were washed under running water to remove any dirt and then carefully peeled with a kitchen knife so as not to damage the active substances in the peel. The peels (consisting of albedo and flavedo) were chopped with a knife into pieces of 1 cm × 1 cm and packed in polyethylene bags and stored at −18 °C until analysis.

3.3. 3,5-Dinitrosalicylic Acid (DNSA) Assay for the Determination of Enzyme Activity

Enzyme activity was determined using a colorimetric DNSA method as described in [34] with modifications. The enzymes (cellulase, pectinase and xylanase) were added to purified water or citrate buffer (pH = 5) to a final concentration of 0.22 mg/mL. Subsequently, 10 mg of the corresponding substrate (cellulose, pectin or xylan, respectively) was added to 1 mL of the enzyme solution and the reaction was incubated for 120 min at 50 °C in a thermoshaker (Biosan, Riga, Latvia, TS-100) at 900 rpm. The sample was then purified by centrifugation (5 min, 10,000 rpm). Sample aliquots (600 μL final volume) were then mixed with 600 μL DNSA reagent containing 10.0 g/L DNSA, 0.5 g/L sodium sulfite, 10 g/L sodium hydroxide and 2 mL/L phenol. The mixtures were incubated at 95 °C for 15 min before adding 200 μL of a 40 g/L potassium sodium tartrate solution (1.4 mL final volume). Samples were cooled on ice for 5 min and then absorbance was measured at 575 nm UV–Vis spectrophotometer (Perkin Elmer, Lambda 1, Waltham, MA, USA, SAD). Product concentrations were calculated from calibration curves generated with the corresponding reducing sugars (glucose, galacturonic acid and xylose, respectively). If necessary, the original samples were diluted accordingly to obtain absorbance values in the range of the calibration curves, and the dilutions were considered when calculating enzymatic activity. One unit of enzymatic activity was defined as the amount of enzyme releasing 1 μmol of reducing sugar per minute under the specified assay conditions.

3.4. Isolation of Essential Oil from Citrus Peel by Hydrodistillation

3.4.1. Citrus Peel Pretreatment

The hydrodistillation of citrus peels (orange, mandarin, and clementine) was preceded by various pretreatments: (i) reflux extraction without enzymes (RE) in purified water (HDW) or citrate buffer (HDB) as control samples, and (ii) reflux extraction with enzymes (pectinase, REP; cellulase, REC; xylanase, REX; pectinase/cellulase/xylanase, REPCX) in purified water or citrate buffer (pH = 5).

For each pretreatment, 20 g of crushed citrus peel material (1 cm × 1 cm) was mixed with 250 mL modifications. Specifically, the citrus 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 enzymes: 55 mg pectinase, cellulase, or xylanase (2.75 mg enzyme per g citrus peel), or their combination (8.25 mg of total enzymes per g of citrus peel material). All pretreatment experiments were performed in parallel determinations.

Table 2 lists the abbreviations and their meanings for citrus peel pretreatments with and without enzymes, followed by Clevenger hydrodistillation.

3.4.2. Hydrodistillation Procedures

Citrus peels pretreated by reflux extraction with or without enzymes (see Section 3.4.1) were transferred to a round-bottom flask of a Clevenger apparatus and hydrodistilled for 120 min as described in a previous protocol [35]. The same mass of citrus peel (20 g) and volume (250 mL) of purified water and citrate buffer (pH = 5) were also subjected to hydrodistillation without any prior pretreatment (HD, Table 2).

After hydrodistillation, the essential oils obtained were stored in dark glass vials at 4 °C until further analysis, which included the determination of volatile compounds by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC-MS). The essential oil yield (%) was calculated as the volume of essential oil separated per mass of citrus peel during hydrodistillation and converted to a basis of 100 g of citrus peel (orange, mandarin, and clementine). All hydrodistillation experiments were performed in parallel determinations.

3.5. Headspace Solid-Phase Microextraction (HS-SPME) with Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Headspace solid-phase microextraction (HS-SPME) was performed with the PAL Auto Sampler System (PAL RSI 85, CTC Analytics AG, Schlieren, Switzerland) using the fiber covered with a layer of carbon wide range/polydimethylsiloxane (Carbon WR/PDMS) (Agilent Technologies, Palo Alto, Santa Clara, CA, USA). The fiber was conditioned prior to extraction according to Agilent Technologies’ instructions. Gas chromatography–mass spectrometry (GC-MS) was performed using an Agilent Technologies model 8890A gas chromatograph (Palo Alto, CA, USA) coupled to a model 5977E mass selective detector. The compounds were separated on a HP-5MS column (Agilent Technologies, Santa Clara, CA, USA) 30 m × 0.25 mm with a stationary phase (5% diphenyl/95% dimethylpolysiloxane) and a film thickness of 0.25 μm. The following GC operating conditions were used: 250 °C injector temperature; 300 °C detector temperature; column temperature program: 2 min isothermal at 70 °C, followed by a temperature gradient from 70 °C to 200 °C at 3 °C/min and further retention at constant temperature for 15 min. The carrier gas was helium with a flow rate of 1.0 mL/min; the MSD (EI mode) was operated at 70 eV; the mass range was set from 30 to 300 amu. The compounds were identified by comparing their retention indices (RIs), based on the retention times of C_9_–C_25_ alkanes, with those in the literature (National Institute of Standards and Technology) [48] and their mass spectra with those from the Wiley 9 (Wiley, New York, NY, USA) and NIST Chemistry WebBook [49] mass spectral libraries. Percent composition was calculated using the normalization method (without correction factors). The HS-SPME/GC-MS was performed in duplicate, and the results obtained were expressed as the average percentage of the peak areas.

3.6. Statistical Analysis

The data were first tested for normality using the Shapiro–Wilk test. As the null hypothesis of normality was rejected, indicating that the data significantly deviated from a normal distribution (p < 0.05), nonparametric statistical methods were applied. Differences among pretreatments prior to Clevenger hydrodistillation across citrus types were assessed using the Kruskal–Wallis test, a nonparametric alternative to one-way analysis of variance based on ranked data. This test was used to evaluate the null hypothesis that there were no statistically significant differences in the median values of the analyzed variables among pretreatments. The analyzed variables included the relative abundances (% of total peak area) of individual volatile compounds identified in the essential oils, as well as grouped chemical classes of volatiles. Spearman’s rank correlation test was employed to evaluate whether the relative abundances of volatile compounds and chemical classes in the essential oils differed significantly among pretreatments within each citrus type. This nonparametric correlation method was selected to assess the strength and direction of monotonic relationships between pretreatments and volatile composition without assuming normal data distribution. Spearman’s correlation coefficients (r), ranging from −1 to +1, were calculated, and their statistical significance was evaluated at a p-value threshold of 0.05. In addition, a heatmap was generated using the pheatmap package [50] in R to visualize variation in the relative abundances of volatile compounds across pretreatments. Hierarchical clustering was applied to the columns based on similarity measures to identify patterns and relationships among pretreatments. All statistical analyses were performed using R software (R Development Core Team) in R Studio (version 1.0.12), and statistical significance was determined at p < 0.05.

4. Conclusions

This study demonstrated that enzymatic pretreatments in purified water or citrate buffer slightly increased essential oil yields from orange and mandarin peels up to ~2%, while no increase was observed for clementine. Limonene remained the dominant compound in all peels (up to 81.16% in orange, 77.50% in mandarin, and 75.29% in clementine), while mandarin peel also showed higher sesquiterpenes (up to 60.52%) and aldehydes (up to 4.86%) after enzymatic pretreatment. 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. Inclusion of both no-enzyme and no-pretreatment controls was essential for accurately assessing enzymatic effects under laboratory-scale Clevenger hydrodistillation, providing a systematic understanding of enzyme-assisted citrus peel extraction.