Supercritical CO2 extraction of essential oil from Kabosu (Citrus sphaerocarpa Tanaka) peel
© Suetsugu et al.; licensee BioMed Central Ltd. 2013
Received: 30 November 2012
Accepted: 8 May 2013
Published: 28 May 2013
Citrus sphaerocarpa Hort. ex Tanaka is one of many popular sour citruses in Japan. Its juice processing peel residues contain a lot of useful compounds including essential oil. Our interests mainly focused on the extraction of this essential oil using supercritical carbon dioxide (SC-CO2), an environmentally benign and generally regarded as safe solvent that has many advantages such as low critical temperature, low viscosity, and easy separation from the extract. In this research, essential oil was extracted from Citrus sphaerocarpa Tanaka peel using SC-CO2 at extraction temperatures of 313 to 353 K and pressures of 10 to 30 MPa.
A maximum yield of 1.55% (by weight of wet sample) was obtained at the temperature of 353 K and the pressure of 20 MPa. The yield obtained by SC-CO2 method was over 13 times higher than that of the conventional cold-press method. Extracted essential oil was qualitatively analyzed using GC/MS, identifying 49 compounds including several non-polar and weakly polar hydrocarbons such as terpenoid, free fatty acid, and coumarin. Compared to the extracts obtained by the conventional methods, the extracts by SC-CO2 had lower content of monoterpenes and higher content of oxygenated compounds, sesquiterpenes, which strongly contribute to the aromatic characteristics of the extracts. Auraptene, a bioactive compound was also identified in the SC-CO2 extract.
Kabosu essential oil with a fresh natural fragrance was effectively extracted using SC-CO2 compared to the conventional extraction method. In addition, it was found that the extract contained higher content of aromatic components that characterize Kabosu. This work provides an important sequential method for the recovery of valuable compounds from citrus fruit waste using an environmentally friendly technique.
KeywordsCitrus sphaerocarpa Essential oil Supercritical carbon dioxide Auraptene
Citrus sphaerocarpa Hort. ex Tanaka, referred to as ‘kabosu’ in Japanese, is one of many popular sour citruses in the same genus lemon. Kabosu trees have been mainly cultivated in Oita prefecture, and the fruits are usually being harvested in August to September while they are still unripe. The fruit ranged from 100 to 150 g in weight and from 50 to 65 mm in diameter during the harvesting time. Annual production of the fruit is around 5,000 tons, about 25-30% of which are utilized for juice processing, leaving 70-75% in the fresh-fruit market. Kabosu juice contains about 5.7% citric acid and 5.0% Brix , water soluble solid content such as sugar mainly. Kabosu fruits have thin and smooth peel, possessing a very pleasant smell. Due to this characteristic flavor of kabosu, its essential oils are widely used to add distinct flavor in food, beverage, pharmaceutical, perfumery, and cosmetic industries.
The development of gas chromatographic techniques of analyses has brought about significant research progress in the chemistry of citrus essential oils. Essential oils of widely well-known citruses, sweet orange, grapefruit, mandarin, lemon, lime, bitter orange, and bergamot, had been analyzed quantitatively . Recently, the technique has also been applied to the composition analysis of essential oils obtained from different citrus varieties such as Tunisian citrus , Diamante peel , bitter orange , and commercially available citrus essential oils . Results indicated that citrus essential oil consists of mostly aromatic components such as monoterpene, sesquiterpene, and some oxygenated compounds. It is known that the monoterpenes, such as limonene, contribute a little to the aroma, while sesquiterpenes and some of the oxygenated compounds are mainly responsible for the strong fragrances and peculiar characteristic flavor of the citrus despite low contents. Many researchers have also reported about the components of kabosu peel. Sixty-six volatile components of kabosu cold-pressed oil were identified . Recently, four distinctive aroma components (citronellal, neral, linalyl acetate, and octyl acetate) like kabosu have been investigated using gas chromatography/olfactometry (GC/O) analysis . The essential oil also contains a bioactive compound called auraptene , which is considered to be a major coumarin of citrus plants, and has proven to possess anticancer activities [10, 11] and to improve metabolic syndrome .
Industrially, citrus essential oils are being collected from the byproducts of juice production processes. Trace amount of essential oil is also confirmed present in supernatant of juice. Various approaches to the extraction of citrus essential oils have been reported. Especially, steam distillation method has been employed for purposes of comparison with SC-CO2 extraction method [13, 14]. Their study showed that extraction method has an influence on the content of aroma compounds and yield of essential oil. Steam distillation method has also been reported to cause partial hydroxylation . In the extraction of natural essential oil, the use of supercritical fluid has been proposed. Of the many possible fluids of choice, carbon dioxide is the most preferred because of its low critical temperature (304 K) suitable especially for thermally labile components. Besides, it is readily available and non-toxic. Unlike the conventional organic solvent extraction, the extraction process associated with the use of SC-CO2 does not require complicated process to remove the solvent. Its presence in foods or beverages is generally regarded as safe and harmless for human consumption. With these properties, SC-CO2 has been applied to extraction of natural compounds such as essential oil [16–19], seed oil [20, 21], pigment , and bioactive compounds . In general, raw materials for SC-CO2 extraction undergo pretreatment process such as milling and drying before extraction in order to increase extraction efficiency. Excess water in the raw material has been considered to act as a barrier in the transport of the extract to the fluid . However, these pretreatment steps will expose the materials for possible air oxidation due to the increase in surface area, and furthermore enzymatic denaturation caused by disruption of cell tissues .
In this work, SC-CO2 extraction of essential oil from scraped wet peel of kabosu was carried out without any pretreatment. The optimum extraction condition was investigated based on the yield of essential oil, and the composition of the extracted oil was compared with the oil obtained by cold press and steam distillation methods. The work contributes further to the knowledge of SC-CO2 extraction of essential oil as applied to Japanese citrus.
Result and discussion
Comparison of the essential oil yield obtained by SC-CO2 and other conventional methods
The highest yield of SC-CO2 extraction reached 1.55% (by weight of wet sample), which was obtained at a temperature of 353 K and pressure of 20 MPa for 300 min. This corresponds to 66.7% recovery based on the total amount of essential oil. This yield was over 13 times higher than that obtain by cold press method, which was the lowest. Based on the results, in terms of yield or recovery of essential oil, SC-CO2 extraction method was considered to be better compared to that of the conventional cold press method. Pioana et al. have also compared the yields obtained between wet and dry materials. They reported that the essential oil obtained was about 1.9% v/w from the fresh peel and about 8.4% v/w when dried. In this case, water content of peel decreased from 80% to 19%.
GC/MS analysis of SC-CO2 extract
Effect of pressure and temperature in SC-CO2 extraction
Other than the solubility of essential oil in SC-CO2, its vapor pressure should also be considered an important factor for extraction. Higher vapor pressure of the oil at higher temperature allows its easy dissipation through the sample matrices, while higher diffusivity of SC-CO2 and lower surface tension also aid in the transport of the target compounds through the matrix and into the solvent resulting into higher extraction efficiency.
Optimum condition of kabosu peel extract
Chemical composition of essential oils
Essential oil was extracted from kabosu peel by employing SC-CO2 at various temperatures and pressures. The process was optimized, and based on the results it was suggested that the solubility of essential oil from kabosu peel in SC-CO2 was controlled by the balance between the solvent power of SC-CO2 and the vaporization power of essential oil, which are responsible for the solubility and transport properties of essential oil with SC-CO2, respectively. As a result, the maximum yield of SC-CO2 extraction was obtained at a temperature of 353 K and pressure of 20 to 30 MPa, at which the vapor pressure of essential oil and SC-CO2 density were relatively high compared to other conditions. In addition, the obtained oil contained higher amounts of volatile compounds which contribute to the specific aroma, and valuable bioactive compounds such as auraptene compared to the conventional cold-press and steam distillation methods.
Materials and chemicals
Samples of kabosu fruits were harvested in October 2010 in Oita prefecture. Its peel was scraped, placed inside an air-tight wrapping plastic bag, and then stored in a freezer at 253 K until extraction experiments were performed. No pre-drying treatment was carried out on the sample. The feed material contained water of approximately 80 wt%. Prior to extraction experiments, the sample was gently thawed to room temperature. Carbon dioxide (99.9%) was obtained from Uchimura Sanso Co., Japan.
Approximately 20 g of scraped citrus peel was minced with 500 mL of distilled water in a food processor, and placed into a 1 L round flask. The flask was then connected to a distilling receiver with Liebig condenser. Distillation was carried out at a temperature from 403 to 423 K for 24 h at atmospheric pressure. The essential oil evaporated with steam during the distillation process, and was separated from the condensates and collected in the distillate receiver. After reaching a certain level, the water was refluxed to distiller, and the distillation process continued.
Cold-pressed oil extraction
Approximately 50 g of scraped citrus peel was minced in 100 mL of distilled water and dispensed into 50 mL centrifuge tubes. These samples were centrifuged at 10,000 G for 15 min and frozen in a freezer at 253 K. The frozen samples were gently thawed to room temperature. These steps of centrifugation, freezing, and thawing were repeated three times. After these preparation procedures, the essential oil and wax layer were separated from the water layer. The essential oil was collected carefully to avoid mixing with the wax.
Yield of extracted oil
The yield was defined as the weight of extracted oils per 100 g of wet feed material.
Qualitative analysis of the extracted essential oil was carried out using GC/MS (Hewlett-Packard - 5890 series, Palo Alto, CA, USA), coupled with a mass selective detector (HP 5972). The GC conditions were as follows: The oven temperature was initially at 313 K for 3 min, then allowed to ramp up to 553 K at a rate of 278 K min-1, then to 593 K for 3 min. The injector and detector temperatures were set at 523 K. The split ratio was 12:1; with a total carrier gas (helium) flow rate of 24 mL/min; and ionizing energy of 70 eV. The injection volume was 0.02 μL. For the identification of the peaks in the chromatograms, the probability-based matching algorithm was employed for finding the most probable match in the reference library (NIST library of mass spectra and subsets, HPG 1033A). The relative composition of the components present in essential oil was calculated from the GC peak area.
All extraction experiments were duplicated. GC/MS analysis of each fraction obtained in each run was carried out in duplicate. Analysis of variance (ANOVA) was carried out using Excel Statistics 2004 to analyze the effect of temperature and pressure on the total yield of extract and composition of essential oil extract. The significance level was stated 95% with P value <0.05.
Analysis of variance
Supercritical carbon dioxide.
This work was supported by Kumamoto University Global COE Program ‘Global Initiative Center for Pulsed Power Engineering’.
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