J. Agric. Food Chem. 1997, 45, 2660−2665
2660
Characteristics of Plants and Plant Extracts from Five Varieties of Basil (Ocimum basilicum L.) Grown in Australia Katarzyna J. Lachowicz,† Gwyn P. Jones,*,† David R. Briggs,† Fred E. Bienvenu,‡ Martin V. Palmer,§ Vijay Mishra,§ and Mohamed Murray Hunter| School of Nutrition and Public Health, Deakin University, Geelong, Victoria 3217, Australia; Ovens Research Station, Agriculture Victoria, P.O. Box 235, Myrtleford, Victoria 3737, Australia; Australian Food Industry Science Centre, Private Bag 16, Werribee, Victoria 3030, Australia; and Perlis Essential Oils Sdn Bhd, 173-191 Taman Kemajuan, Jalan Raja, Syed, Alwi, Kanger, Perlis 01000, Malaysia
A horticultural trial on five varieties of basil (Ocimum basilicum L.) showed differences in morphological features, growing characteristics, and yields of essential oil produced per unit area of land. Anise basil was the most productive in terms of plant biomass, while Cinnamon basil produced the most essential oil. Gas chromatographic analysis showed considerable differences in the composition of oils among varieties. Seasonal variations had a significant effect on plant growth, essential oil yield, and composition when Reunion and Anise were grown in successive years in the same soil. All varieties were susceptible to cold temperature injury caused by ground frost. Variations in oil composition due to environmental factors appear to be of the same order of magnitude as genetic factors. Data are provided on the yield, composition, and physical appearance of extracts within the same variety obtained by hydrodistillation and with CO2. The yields were highest using supercritical CO2 followed by liquid CO2 and then water. All of the CO2 extracts were quite similar in chemical composition with respect to volatile components. The hydrodistilled oil contained a larger proportion of lower boiling point hydrocarbons and oxygenated terpenes than the CO2 extracts. The sensory evaluation of hydrodistilled oil and liquid CO2 extracts obtained with a one-stage separator under defined conditions indicated that they were quite different products and they would appear to have different end uses. Keywords: Australian basil; essential oils; plant extracts; hydrodistillation; sensory evaluation INTRODUCTION
Essential oils are volatile substances extracted from odoriferous plants. They are used as flavorings in foods and beverages and as fragrances in pharmaceutical and industrial products. The estimated world production of essential oils in 1984 was about 36 000 tonnes, and of this amount approximately 14 tonnes was from basil (Ocimum basilicum L.) (Lawrence, 1985); by 1991 the production had increased to 43 tonnes (Lawrence, 1991). There are several types of basil oil available on the world market which are obtained from different varieties of sweet basil (Simon et al., 1990). They are produced from leaves and flowering tops usually by steam distillation and have been extensively used in food for flavoring confectionary and baked goods; condiments (catsups, tomato pastes, chili sauces, pickles, vinegars); sausages, and meats (Guenther, 1952); and nonalcoholic beverages, ice cream, ices, etc. (Fenaroli, 1975). Basil oil has also found wide application in perfumery, dental, and oral products. In recent years CO2 extracts of basil have become commercially available, produced in Germany (Flavex Naturextrakte GmbH) and in the United States from imported dried materials (Hunter, 1996). The essential oil industry in Australia comprises a diverse range of products including eucalyptus, orange, * Author to whom correspondence should be addressed (telephone +61 352271369; fax +61 352272170; e-mail
[email protected]). † Deakin University. ‡ Agriculture Victoria. § Australian Food Industry Science Centre. | Perlis Essential Oils Sdn Bhd. S0021-8561(96)00791-1 CCC: $14.00
lemon, tea tree and lavender oils and smaller quantities of sweet fennel, parsley herb, and caraway oils (Lawrence, 1985); peppermint and spearmint oils (Deer, 1993); and blackcurrant and boronia absolutes (Hunter, 1996). A range of other species, both native and introduced, are under investigation as potential essential oil crops, particularly in regions of Australia where tobacco has been traditionally grown. We have conducted preliminary studies into the yields, chemical composition, and sensory properties of essential oil from basil (O. basilicum L.) grown in northern Victoria (Lachowicz et al., 1996), and in this paper we present the results for additional varieties, viz. Cinnamon, Dark Opal, and Bush basil, and also report the effect of seasonal variation over two years for the varieties Reunion and Anise. MATERIALS AND METHODS Plants Grown at the Ovens Research Station. Seeds of four cultivars of sweet basil (O. basilicum L.) were purchased locally from a Canadian supplier (Otto Richter & Sons Ltd, Goodwood, ON). These were Anise, Cinnamon, Dark Opal, and Bush basil. Seeds of Reunion basil were provided by the Ovens Research Station, Agriculture Victoria, from their collection. Seeds were sown at the beginning of November 1994 into a mixture of pine sawdust, washed sand, horse manure, and mulch (1:1:1:1) and germinated under glass in a controlled environment with the temperature maintained between 15 and 30 °C. Seedlings were planted out on January 13, 1995, into a prepared and fertilized field (5.2% N, 8.7% P, 21.4% K, 0% Mg, and 3.5% S at 0.575 tonne/ha) at a spacing of 0.6 × 0.3 m. The total area planted was approximately 650 m2. Plants were harvested manually on April 12, 1995, at midflower stage. They were dried whole in a tobacco kiln using
© 1997 American Chemical Society
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2661
Extracts from Australian Basil
Table 1. Weather Conditions in the 1994 and 1995 Growing Seasons at Ovens Research Station, Myrtleford monthly rainfall (mm) Jan Feb March April
mean daily temp (°C)
monthly av lowest temp (°C)
av highest temp (°C)
monthly min temp (°C)
monthly max temp (°C)
1994
1995
1994
1995
1994
1995
1994
1995
1994
1995
1994
1995
45 183 30 13
166 26 0 74
N/A N/A N/A N/A
20.8 19.8 15.5 10.3
11.2 14.8 9.4 6.0
13.6 11.8 6.6 4.2
28.6 29.4 25.3 22.3
28.6 28.4 25.5 18.0
4.0 8.0 3.0 2.0
7.1 5.8 0.3 -1.4
35.0 35.5 28.5 29.0
35.0 35.8 33.9 24.3
Table 2. Vegetative Characteristics of Basil Varieties in Two Growing Seasons, 1995 and 1994 av plant ht at harvest (m)
av plant wt at harvest (kg)
mass of tissue grown per unit area of landa (kg m-2)
variety
1995
1994
1995
1994
1995
Reunion Cinnamon Anise Dark Opal Bush
0.41 0.55 0.61 0.39 0.49
0.45 -b 0.80 -
0.100 0.250 0.290 0.080 0.283
0.178 0.499 -
0.560 1.400 1.624 0.448 1.582
a
leaf color
1994 0.742 2.081 -
1995
1994
green green green dark purplish bronze light green
green green -
Calculated from plant spacing and average weight. b Not grown in 1994.
forced-air circulation at 28 °C for 8 days. The leaves and stems were separated manually and the leaves used for further analysis. The moisture content of the dried leaves was as follows: 12.1% for Dark Opal; 11.9% for Anise; 12.6% for Cinnamon; 12.5% for Bush; and 11.8% for Reunion basil. CO2 Extraction Procedures. Prior to extraction with carbon dioxide (CIG, Melbourne), the dried leaves were ground in a coffee grinder. The particle size distribution of the ground leaves was as follows: 4 mm, 1.7%; 2 mm, 10.0%; 1 mm, 31.7%; 0.5 mm, 34.0%; 0.29 mm, 9.4%; 0.15 mm, 5.3%; 80% of methylchavicol together with very small amounts of linalool (Reunion); oil that contained approximately equal proportions of linalool and methyl cinnamate together with small amounts of methylchavicol (Cinnamon); and oil with approximately equal proportions of linalool and methylchavicol (Anise). The major constituents, linalool and methylchavicol, in Reunion oil from plants grown in 1995 and 1994 were present in similar proportions (Table 9), indicating a resistance to weather-induced changes in composition. On the other hand, Anise oil obtained from the 1995 crop contained almost the reverse proportion of methylchavicol and linalool (27.8% linalool and 40.9% me-
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2663
Extracts from Australian Basil Table 7. Components (Percent by Gas Chromatography) in Hydrodistilled Oil and CO2 Extracts Obtained from Bush Basil
Table 8. Components (Percent by Gas Chromatography) in Hydrodistilled Oil and CO2 Extracts Obtained from Opal Basil
CO2
R-pinene camphene β-pinene sabinene myrcene R-terpinene limonene 1,8-cineole (Z)-β-ocimene γ-terpinene (E)-β-ocimene p-cymene fenchone pentadecane camphor linalool linalyl acetate endo-fenchol hexadecane β-caryophyllene methylchavicol heptadecane R-terpineol borneol octadecane geraniol nonadecane eicosane methyl cinnamate heneicosane eugenol docosane tricosane a
CO2
hydrodistilled
27 °C, 68 atm
40 °C, 82 atm
40 °C, 150 atm
0.6 0.2 0.5 0.2 0.4 0.5 3.0 0.2 0.5 52.0 0.2 0.7 0.3 0.9 -
0.1 -a 0.2 0.2 0.3 1.5 0.2 0.1 0.8 40.2 0.2 0.4 1.0 0.6 0.5 0.8 1.2 0.6 0.6 0.5 0.3
0.1 0.1 0.1 0.2 1.0 0.3 0.1 0.2 0.7 32.5 0.2 0.6 15.4 1.6 0.1 1.3 0.9 1.0 0.8 0.5
0.1 0.1 0.1 0.3 1.4 0.2 0.1 0.7 41.1 0.3 0.5 0.6 0.4 0.5 0.5 1.1 0.5 0.4 0.1 0.2
R-pinene camphene β-pinene sabinene myrcene R-terpinene limonene 1,8-cineole (Z)-β-ocimene γ-terpinene (E)-β-ocimene p-cymene fenchone pentadecane camphor linalool linalyl acetate endo-fenchol hexadecane β-caryophyllene methylchavicol heptadecane R-terpineol borneol octadecane geraniol nonadecane eicosane methyl cinnamate heneicosane eugenol docosane tricosane a
Not detected.
thylchavicol) compared with the 1994 crop (43.7% linalool and 27.3% methylchavicol), and the content of methyl cinnamate decreased from 11.2% in 1994 to 3.4% in 1995 (Table 9). These preliminary data, if confirmed in longer studies, indicate that some varieties of basil are more prone to exhibit season alterations in oil composition than others and would be of concern to commercial producers when consistency of product is important. The genus Ocimum exibits a wide variety of morphological characteristics, and 15 distinct varieties or subvarieties of O. basilicum L. have been identified (Lawrence et al., 1980). The oil composition seems to fall into four main types which predominate in one or two components. However, even with carefully selected strains of basil there appears to be an approximately 10% variation in the composition of the principal oil components such as methylchavicol, linalool, methyleugenol, and geraniol (Lawrence, 1988). The results presented here, with Anise for example, would seem to indicate that environmental factors can be more important than genetic factors in controlling essential oil composition, although of course they are not independent of each other. Yields and Composition of Extracts Obtained Using Liquid CO2 and Supercritical CO2. We did not attempt to control the extractive conditions to optimize the yields and composition of our extracts because we were mainly interested in the effect of plant variety and growing season on volatile terpenoid constituents. We were also interested in the effect of carbon dioxide solvent power and employed three densities in the expectation that our extracts would contain
hydrodistilled
27 °C, 68 atm
40 °C, 82 atm
40 °C, 150 atm
0.8 0.1 0.9 0.4 0.4 5.5 0.3 57.4 0.1 2.5 0.8 0.4 0.7 0.7 -
0.2 -a 0.3 0.1 0.2 0.2 2.8 1.0 45.9 2.6 2.6 0.6 0.5 0.3 0.9 0.4 2.3 0.3
0.1 0.3 0.2 0.2 0.2 2.7
0.2 0.3 0.2 0.2 0.2 3.1
0.1 52.7 0.1 2.5 2.4 0.8 0.4 0.3 0.9 0.3 0.2 2.2 -
0.1 46.9 0.1 2.6 2.9 0.8 0.4 0.3 0.9 0.2 2.3 0.3
Not detected.
Table 9. Chemical Composition (Percent by Gas Chromatography) of Oil Extracted from Two Varieties of Basil Grown in Successive Years Reunion
R-pinene camphene β-pinene myrcene R-terpinene limonene 1,8-cineole (Z)-β-ocimene γ-terpinene (E)-β-ocimene p-cymene fenchone camphor linalool linalyl acetate endo-fenchol β-caryophyllene methylchavicol R-terpineol borneol citral citronellol octadecane geraniol methyl cinnamate eugenol a
Anise
1994
1995
1994
1995
0.3 0.1 0.3 0.1 0.04 0.3 2.6 0.1 0.6 1.5 0.5 87.2 -
0.3 -a 0.3 0.3 2.4 0.2 2.2 1.0 1.5 0.2 0.4 82.6 -
0.2 0.2 0.2 0.4 0.03 0.3 1.6 0.03 0.1 1.2 43.7 0.2 27.3 0.4 0.4 11.2 -
0.2 0.2 0.2 0.3 1.3 0.4 0.2 2.5 27.8 40.9 0.4 0.7 0.4 0.1 0.2 3.4 -
Not detected.
differing proportions of monoterpenes, which have the highest solubility, followed by oxygenated terpenes, sesquiterpenes, waxes, etc. The batch extractions were conducted for a relatively short time (1 h), and under these conditions the extraction could not have been
2664 J. Agric. Food Chem., Vol. 45, No. 7, 1997
Lachowicz et al.
Table 10. Yields of Oil (Percent w/w) Obtained by Hydrodistillation and Total Extracts Obtained by Liquid CO2 and Supercritical CO2
variety
extraction conditions supercritical CO2 liquid CO2 27 °C, 68 40 °C, 82 40 °C, 150 atm, 1 h atm, 1 h hydrodistillation atm, 1 h
Reunion Cinnamon Anise Dark Opal Bush
1.1 1.2 0.6 0.9 0.3
0.75 1.54 0.63 0.86 0.42
0.37 0.79 0.29 0.44 0.27
1.16 1.90 1.23 1.27 0.63
exhaustive. The solvent to sample ratio (by mass) we used was only 12.5:1, whereas others (Reverchon, 1992; Reverchon et al., 1993) using similar herbaceous matrices have shown that asymptotic yields are generally only obtained with higher ratios (>25:1) and that higher absolute yields are obtained when the plant material is comminuted to a smaller average particle size than that which we employed. Dried leaves of all varieties were also extracted with CO2 under different temperature and pressure conditions, resulting in products which were of two typess liquids (supercritical CO2 extracts obtained at 82 atm) and semisolids (liquid CO2 extracts and supercritical CO2 extracts obtained at 150 atm). The liquid CO2 extracts were light or dark yellow (orange), while the supercritical CO2 products obtained at 82 atm were yellow and those obtained at 150 atm were brownish yellow or yellow-brown. The yields were highest using supercritical CO2 at 150 atm (Table 10), and this may be due to additional coextracted lipids and waxes (Reverchon, 1992; Reverchon et al., 1993). Tables 4-8 show the composition of extracts from five varieties. The GC peak areas of the identified components in the various extracts range from just 90% of the total areas because some of the extracts contained a very large number of small peaks of
