Evaluation of the process for transferring limonene from lemon to olive oil

Abstract

Lemon-flavored olive oil is one of several popular flavored oils on the market. This study aimed to examine the amount of limonene, an abundant aroma compound in lemon, that is transferred from lemon to olive oil under different flavoring conditions. First, the presence of limonene in different parts of fresh lemon tissue (flavedo, albedo, and tissues other than flavedo and albedo) was examined in olive oils flavored with each tissue. Limonene was found to be mainly distributed in the flavedo. Effects of extraction time, temperature, and quantity of the flavedo added were determined as independent variables. Results indicated that the extraction time and temperature influenced the amount of limonene transferred. In particular, the addition of increasing quantities of flavedo resulted in significant increases in the amounts of limonene transferred without increasing the oxidative degradation of the oil samples. We determined the optimal conditions for lemon-flavored olive oil preparation based on the amounts of limonene transferred.

Introduction

The intake of selected dietary antioxidants (e.g., tocopherols, carotenoids, and polyphenols) may prevent the development and progression of diseases such as coronary artery disease (Servili et al., 2009). Extra virgin olive oil (EVOO), the highest oil quality among all categories of olive oils (International Olive Council, 2019), is one of the most commonly studied antioxidant food sources containing these compounds. Therefore, EVOO is known as “the heart-friendly oil” and is often preferred as a conventional cooking oil.

Recently, aromatic herbs, spices, and vegetables have been frequently added to EVOO to produce flavored oils to improve their sensory characteristics, nutritional value, health-associated properties, and shelf-life (Antoun and Tsimidou, 1997; Gambacorta et al., 2007), which serves to attract more consumers to flavored EVOOs as a gourmet food product. According to a consumer test, lemon-flavored oil is the most preferred among oils obtained by mixing oil preparations of different flavors (onion, garlic, paprika, and lemon) to EVOO (Issaoui et al., 2019).

Lemon (Citrus limon L.) is one of the major citrus species worldwide. It has been widely used as a flavor additive in common food items, such as beverages, candies, gums, ice creams, desserts, and seasonings due to its tart flavor (González-Molina et al., 2010). Volatile compounds from lemons have been previously characterized (Ayedoun et al., 1996; Sawamura et al., 1999). Citral, a mixture of monoterpene aldehydes (neral and geranial), represents one of the main components of lemon oil. Unfortunately, its application domain is limited due to its chemical instability. Meanwhile, limonene is a relatively stable monoterpene (Fig. 1), and it is a major constituent of the essential oil from lemon peel. Further, numerous therapeutic properties of limonene, e.g., anti-inflammatory, antioxidant, anticancer, antidiabetic,antihyperalgesic, antiviral, antinociceptive, and gastroprotective effects, have been studied (Vieira et al., 2018). In fact, limonene has been used clinically to dissolved cholesterol-containing gallstones, as it is an excellent cholesterol solvent (Sun, 2007). It has also been used to relieve heartburn, as it possesses a gastric acid neutralizing effect, and it improves peristalsis. Moreover, limonene displays established chemopreventive activity against many types of cancer. Thus, limonene is a stable monoterpene with a pleasant lemon-like aroma, and shows promising applications in health and disease.

Fig. 1.

d-Limonene molecular structure.

To obtain lemon-flavored olive oil, fresh lemons are directly added to the olive mill together with fresh olives for extraction (Sacchi et al., 2017). However, this method has negative effects on oil quality, such as an increase in free fatty acids (FFAs) and dramatic decreases in total phenolics and the sensory profile of the product (Sacchi et al., 2017). There is a risk of physical instability of the product upon exposure to the acidic juice from fresh lemons, as it is an oil-in-water dispersion (Sacchi et al., 2002; Balasundram et al., 2006).

Various nutritional and health benefits can be attributed to limonene. Limonene is most abundant in lemon peel, and other terpene compounds, such as α-pinene, β-pinene, and γ-terpinene, also have been reported in the peel (Vekiari et al., 2002). Lemon peel can be divided into two parts, namely albedo and flavedo (Schneider, 1968; Sadka et al., 2019). The present study was performed to produce and characterize lemon-flavored olive oils obtained by the direct addition of the lemon tissue (albedo or flavedo) in EVOO to transfer limonene to the oil. We aimed to evaluate the effects of extraction time, temperature, and quantity of the tissue added on the amounts of limonene transferred from the tissue into EVOO under these different flavoring conditions.

Materials and Methods

Materials    EVOO was prepared by Shodoshima Healthyland Co., Ltd. (Kagawa, Japan). Medium-chain triglyceride (MCT) oil (Nisshin OilliO Group, Ltd., Tokyo, Japan) was purchased from a market. Limonene, α-pinene, and β-pinene (each > 95% pure) were purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). γ-Terpinene (> 95% pure) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Chilean fresh lemons were purchased from a market.

Process of flavoring oil with lemon tissues    EVOO with no previously added lemon tissue was used as the blank oil sample. Flavored oils were prepared as follows: fresh lemon zest was grated using a citrus grater. Tissues other than flavedo and albedo were crushed using a juicer. Then, 2.5 g of grated flavedo, 7.5 g of grated albedo, or 15 g of crushed tissues other than flavedo and albedo from fresh lemons was mixed in 50 g of EVOO at 25 °C for 10 min. Thereafter, lemon tissues were crudely removed with a tea strainer, and flavored oil samples were filtered through ADVANTEC No. 131 filter paper (Advantec Toyo Kaisha, Ltd., Tokyo, Japan).

Heat treatment of flavored oil samples    Oil samples were heated at 100, 120, 130, 140, 160, or 180 °C for 10 min, then immediately cooled using ice.

Analytical procedures    FFAs, peroxide values (PVs), specific extinction coefficient (K270), and total phenolic contents (TPCs) of the oil samples were measured using an OxiTester (CDR, Ginestra Fiorentina, Italy) (Kamvissis et al., 2008). The FFA values determined using the OxiTester method were verified by comparing the results for oil samples obtained over a wide range of values with those obtained using the official analysis method (Gucci et al., 2012; Kishimoto, 2019). Oil samples were taken in cuvettes for analysis. The volume of oil samples used for measuring FFAs was 2.5 µL, 2.5 µL for PVs, 10 µL for K270, and 10 µL for TPCs.

The content of carotenoids, reported as mg/kg of oil, was determined using a UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) following a slightly modified method to that described previously (Mínguez-Mosquera et al., 1991). One hundred micrograms of each oil sample was dissolved in 1 mL of isooctane, then spectrophotometric absorption was recorded at 470 nm. Contents of oil samples were calculated using the following equation:

Carotenoids (mg/kg) = (A₄₇₀ × 10⁶)/(2 000 × 100 × d),

where A is the absorption and d is the path length of the cell (1 cm).

Total antioxidant capacity assay    Total antioxidant power of oil samples was determined using the PAO-SO test kit (Japan Institute for the Control of Aging, Nikken Seil Co., Ltd., Shizuoka, Japan), according to the manufacturer's instructions (Kishimoto and Kashiwagi, 2020). This test is based on evaluating Cu⁺ levels derived from reduction of Cu⁺⁺ by antioxidant substances in the sample. Cu⁺ forms a stable complex with bathocuproine, with optical absorption at 490 nm that can be quantified using a spectrophotometer (SH-9000Lab, Corona Electric Co., Ltd., Ibaraki, Japan). The values of antioxidant power determined for the samples were compared using a curve obtained from the standard substance provided in the kit at known concentrations used and expressed as µM.

Flash gas chromatography electronic nose analysis    Volatile organic compounds in the headspace of the oil samples were analyzed using a HERACLES II electronic nose (Alpha MOS, Toulouse, France). The HERACLES II was equipped with two identical gas chromatographic columns working in parallel mode: a non-polar column (MXT-5: 10 m length × 180 µm in diameter) and a polar column (MXT-WAX: 10 m length × 180 µm in diameter), which produced two chromatograms simultaneously. It was also equipped with an HS 100 auto-sampler (CTC Analysis AG, Zwingen, Switzerland) to automate sample incubation and injection. An alkane mixture (from n-hexane to n-hexadecane) was used to convert retention times into Kovats indices for calibration. For analysis, an aliquot of oil (2.0 g) was placed in a 20 mL vial and then sealed with a magnetic cap. The vial was placed in the auto-sampler, which was subsequently placed in the HERACLES shaker oven and incubated for 15 min at 60 °C with shaking at 500 rpm. A syringe was used to sample 5 mL of the headspace for injection into the gas chromatograph. The oven temperature was initially set at 40 °C (held for 10 s), then increased to 250 °C at 1.5 °C/s and held at this temperature for 60 s. The total separation time was 120 s. Data were acquired and processed using AlphaSoft software v2020 (Alpha MOS). The AroChemBase module (Alpha MOS) was used to identify volatile compounds. When using the MXT-5 column, E2-hexenal, α-pinene, β-pinene, limonene, and γ-terpinene were eluted at approximately 53, 65, 71, 77, and 81 s, respectively (Kishimoto, 2020; 2021).

Quantification of limonene, E2-hexenal, and other terpenes    To quantitate the limonene, other terpenes (α-pinene, β-pinene, and γ-terpinene), and E2-hexenal in each oil sample, we first plotted standard curves (Kishimoto, 2020; 2021). Different concentrations of limonene, other terpenes (α-pinene, β-pinene, and γ-terpinene), and E2-hexenal were prepared in MCT oil and subjected to flash gas chromatography electronic nose analysis. The contents of limonene, other terpenes (α-pinene, β-pinene, and γ-terpinene), and E2-hexenal in the oil samples were determined using standard curves with good linearity (R = 0.999). The oil samples were diluted appropriately with MCT oil. Experiments were performed in triplicate, and the results are presented as mean values.

Statistical analyses    The results are expressed as mean ± standard deviation from three replicates and were subjected to analysis of variance (ANOVA) followed by the Tukey-Kramer test using Microsoft Excel. Values of p < 0.05 were considered to be statistically significant.

Results and Discussion

Distribution of limonene in lemon tissues    The abundance of different tissue parts (flavedo, albedo, and tissues other than flavedo and albedo) per fresh lemon (97.8 ± 9.2 g, n = 5) was measured based on wet weight. The proportion of flavedo, albedo, and other tissues was 10.7 ± 2.1, 29.7 ± 3.7, and 57.6 ± 2.8%, respectively. Based on the abundance ratio, limonene content in oils flavored with the different parts of lemon tissue (flavedo, albedo, and other tissues) were evaluated. Results revealed that the limonene content in flavedo-flavored oils was obviously higher than that in other flavored oils (Fig. 2), suggesting that limonene was mainly distributed in the flavedo.

Fig. 2.

Comparison of the amount of limonene released from each lemon tissue in EVOO. The amount of limonene in the headspace of the oil samples was analyzed using a HERACLES II electronic nose. Data are expressed as mean ± standard deviation (n = 3). ^a,bMean values with different letters are significantly different (p < 0.05).

Process of flavoring olive oil with lemon flavedo    We examined the effect of extraction time at 25 °C on the amounts of limonene transferred from lemon flavedo to EVOO, and showed that the limonene content reached a maximum concentration after 10 min (Fig. 3A). We then examined the effect of extraction temperature on flavoring of EVOO with the flavedo. The limonene content of various oil samples after flavoring at different temperatures was significantly different (Fig. 3B), and it was observed that the maximum amounts were found in oils prepared at 25 °C. When the EVOO was flavored at temperatures greater than 30 °C, the limonene content was lower than that in those flavored at 25 °C. We also examined the effect of the quantity of the flavedo used in the flavoring process. Thus, limonene content was determined in oils prepared with different quantities of the flavedo. Limonene content increased in proportion to the quantity of flavedo added (Fig. 3C). Higher limonene content is found in other foods (NTP, 1990). However, greater than 20 g of flavedo was excluded from this extraction process due to physical limitations. From these results, we finally determined the optimum flavoring process for maximizing the limonene content in olive oil, in which 20 g of flavedo should be added to 50 g of EVOO at 25 °C for 10 min. In addition to the limonene content, the concentrations of other terpenes (α-pinene, β-pinene, and γ-terpinene) in oils flavored under optimum conditions were 0.14 ± 0.01, 1.14 ± 0.06, and 0.80 ± 0.03 ppm, respectively. The results support a previous study showing that β-pinene is the next abundant terpene compound in lemon-flavored olive oils (Sacchi et al., 2017).

Fig. 3.

Changes in the content of limonene transferred from lemon flavedo to EVOO under different conditions. Effect of the extraction time (A), temperature (B), and the quantity of lemon flavedo added (C). Data are expressed as mean ± standard deviation (n = 3). ^a–dMean values with different letters are significantly different (p < 0.05).

Table 1 shows the main quality indices for olive oil as set by the International Olive Council (2019), TPCs, carotenoids, and total antioxidant powers measured in unflavored oil and oils flavored with different quantities of lemon flavedo. It has been reported that olive milling together with fresh lemons can promote the FFA levels of lemon-flavored olive oil because of citric acid, which is the major acid in fresh lemon juice, resulting in a more acidic environment during malaxation with a consequent increase in triglyceride hydrolysis (Sacchi et al., 2017). In this study, the FFA levels of the flavedo-flavored oils were the same as those in the unflavored oil, probably because of the presence of smaller amounts of citric acid from the flavedo than that from the juice of fresh lemons. The level of PVs, which is correlated to the presence of primary oxidation compounds, and the spectrophotometric index K270, which is indicative of the presence of carbonyl compounds as a secondary oxidation compound (Malheiro et al., 2009), were slightly greater in flavored oils compared to the unflavored oil. These quality parameters increased according to the quantity of added flavedo compared with those of the unflavored oil, suggesting that the process of flavoring oil would slightly promote primary and secondary oxidation of lipids. The TPC values in flavored oils decreased according to the quantity of added flavedo compared with that of the unflavored oil. This decrease in TPCs of the oils might be due to moisture from the flavedo, which could also promote partition toward the aqueous phase of phenolic compounds during processing (Sacchi et al., 2002; Balasundram et al., 2006). In contrast, the content of carotenoids, which possess antioxidant activity (Stahl and Sies, 2003), was increased in flavored oils compared with that in unflavored oil. This is because citrus species are a source of carotenoids, which are divided into two groups, carotenes (nonpolar carotenoids, including α-carotene, β-carotene, and lycopene), and xanthophylls (polar carotenoids, including lutein, zeaxanthin, and β-cryptoxanthin) (Kato, 2021). The total antioxidant power of flavored oils was greater than that of unflavored oil. Given that limonene also has antioxidant activity (Shah and Mehta, 2018), flavored oils with low phenolic content could exhibit an enhancing effect on the antioxidant activity.

Table 1. Chemical parameters of unflavored and lemon flavedo-flavored olive oil.

		Quantities of lemon flavedo added
Analytical parameters	Unflavored	5 g	10 g	20 g
FFA (%)*1	0.09 ± 0.02a	0.10 ± 0.01a	0.10 ± 0.02a	0.10 ± 0.02a
PV (meq O /kg)1*1	5.3 ± 0.3a	6.1 ± 0.2ab	6.45 ± 0.2b	6.7 ± 0.5b
K270*1	0.056 ± 0.003a	0.084 ± 0.003b	0.093 ± 0.08bc	0.103 ± 0.011c
TPC (mg/kg)*1	302 ± 9a	173 ± 3b	171 ± 3b	165 ± 2b
Carotenoids (mg/kg)*2	0.550 ± 0.013d	0.605 ± 0.015c	0.658 ± 0.016b	0.698 ± 0.015a
Total antioxidant power (µM)*3	8 249 ± 620bc	8 092 ± 191c	9 280 ± 312ab	9 733 ± 372a
E2-hexenal (ppm)*4	17.1 ± 0.7b	19.0 ± 1.8ab	20.6 ± 1.2ab	21.4 ± 1.1a

*1  These chemical parameters were measured using an OxiTester.

*2  This parameter was determined using a spectrophotometer.

*3  This parameter was determined using the PAO-SO test kit.

*4  This amount was determined using an electronic nose. Data are expressed as mean ± standard deviation (n = 3).

^a–d  Mean values in the same row with different superscript letters are significantly different (p < 0.05).

E2-hexenal, which is produced by the lipoxygenase (LOX) pathway during the olive milling process and is known to be quantitatively the most abundant volatile in virgin olive oil, contributes considerably to the aroma of olive oil and is related to the positive green sensory note of olives (Angerosa et al., 2004). It has been reported that a general decrease in volatile compounds produced from the LOX pathway, including E2-hexenal, was observed in lemon-flavored olive oil, since the LOX pathway can be strongly inhibited by the low pH of the juice from fresh lemons (Baylac and Racine, 2003; Sacchi et al., 2017). In contrast, the E2-hexenal content of flavedo-flavored oils increased according to the quantity of added flavedo compared with that of the unflavored oil (Table 1). These observations may be explained by the differences in the flavoring process. In this study, the flavoring process does not require stimulation of the LOX pathway due to the direct maceration of the flavedo, which contains E2-hexenal (Reuss et al., 2020), in EVOO; therefore, this results in an increase in the E2-hexenal content.

Evaluation of thermal stability of lemon flavedo-flavored oil    Oils are used as a seasoning not only for uncooked dishes but also for cooking or frying of food. Thus, it is important to evaluate the thermal stability of flavored oils. The limonene content in the oil samples treated at different temperatures (from 100 to 180 °C) for 10 min was evaluated (Fig. 4). Heat treatment at 100 and 120 °C of the flavored oils was not detrimental for limonene. Limonene showed high resistance to heat treatment at 100 and 120 °C (100 and 97%, respectively), whereas, when heating temperatures exceeded 120 °C, the limonene content in flavored oils was dramatically reduced. The percentages of the limonene content in flavored oils treated at 130, 140, 160, or 180 °C were reduced to 26, 18, 17, or 15%, respectively. These results may help to select heating temperatures that maintain the limonene content in flavored oils for domestic cooking.

Fig. 4.

Behavior of limonene in flavedo-flavored oil when heated at different temperatures for 10 min. Relative content of limonene in oil samples heated at each temperature to its initial content. Data are expressed as mean ± standard deviation (n = 3). ^a–cMean values with different letters are significantly different (p < 0.05).

Conclusions

In this study, we demonstrated that different processing conditions during flavoring can significantly affect the limonene content of lemon flavedo-flavored olive oils. These differences in the limonene content allow the effects of flavoring conditions (extraction time, temperature, and quantity of added flavedo) to be determined. We determined the optimum procedure to obtain maximum amounts of limonene transferred from the flavedo to EVOO. Our maceration technique shows ease of handling compared to the conventional flavoring process of adding fresh lemons to olive milling. Moreover, our method has advantages in that it can maintain the oil quality and increase the content of E2-hexenal, a green sensory note, following processing. These findings may help to produce high-quality olive oils with lemon aroma for use in domestic cooking.

Acknowledgements    We would like to thank Editage (www.editage.com) for the English language editing.

Conflict of interest    There are no conflicts of interest to declare.

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