Comparison of physicochemical characteristics and composition of bergamot oil seed extracted from three different cultivars

*Corresponding author: Vincenzo Sicari, Department of AGRARIA, University of Reggio Calabria, Salita Melissari 89124 Reggio Calabria (Italy). Tel.: +39 09651694370, E-mail: Vincenzo.sicari@unirc.it Received: 20 February 2016; Revised: 10 May 2017; Accepted: 14 May 2017; Published Online: 23 May 2017 R E G U L A R A R T I C L E Sicari, et al.: Composition of bergamot oil seed Emir. J. Food Agric ● Vol 29 ● Issue 6 ● 2017 471 The essential oil is used in the fragrance industry (Ricci and Rovesti 1979; Poiana et al., 1994). The bitter taste of makes it largely unsuitable for use in bergamot juice in the food industry. Citrus seed oil presents chemical and physical characteristics that make it suitable for food consumption (Ramadan and Morsel 2002). In the scientific papers, we find numerous research that have dealt with the oil of citrus seeds. The majority of the research concerns the quantitative analysis of fatty acids (French, 1962; Habib et al., 1986; Saidani et al., 2004; Anwar et al., 2008). The quality of a food grade oil depends on its fatty acid profile and above all on the ratio of saturated to unsaturated fatty acids. Furthermore, unsaturated acids can be classed as omega, ω-9, which are considered nonessential, and ω-3 and ω-6, known as essential fatty acids, since they cannot be synthesized by the human body, and must be obtained from diet (Ristic and Ristic 2003). Linoleic acid is present in all vegetable oils and is abundant in many of these, including in sunflower seeds, but also, to a lesser extent, corn oil, soybean oil and others. Linoleic acid an essential fatty acid and belongs to the ω-6 group. The seeds of the bergamot fruit, which are currently considered a waste material, may not only be a reliable source of edible oil (cooking oil, salad or for margarine) but since it is a semi-drying oil it could also have industrial uses. In this study were evaluated the chemical-physical composition of bergamot oil seed extracted from three different cultivars, in order to evaluate any differences in their composition. MATERIALS AND METHODS Chemicals Petroleum ether (40-60 °C) was of analytical grade < 98 %, Merck (Milan, Italy). Authentic standard samples of FAMEs, were from Sigma-Aldrich (Milan, Italy). β-sitosterol, campesterol, and stigmasterol were obtained from Aldrich (Milan, Italy). All other reagents of analytical grade were from Carlo Erba Reagenti (Milan, Italy). Sampling During the 2015/16 season, the Bergamot Consortium of Reggio Calabria (Italy) provided the seeds used. The seeds belonged three different cultivars (Castagnaro, Femminello and Fantastico), formerly, Femminello and Castagnaro made up virtually all commercial plantings but they have largely been replaced by Fantastico, a hybrid of Femminello and Castagnaro. All the fruit was picked on the same day and processed within 48 hours; the seeds were subsequently divided according to cultivar. After being washed with tap water, the seeds were dried at 35 °C for 24 h. Samples of the dried seeds were ground using a blender. About 100 g of the well-crushed bergamot seeds were fed into a soxhlet extractor. Petroleum ether (40-60 °C) was used as extraction solvent. After extraction, which lasted about 10 hours, the solvent was removed by distillation under vacuum. Further residual solvent was eliminated by nitrogen flow. After being weighed, the oil yields were calculated. This process was repeated for seeds from all three cultivars. Chemical and physical analysis Density and viscosity value of the oils were determined according to AOAC (2005). Iodine value For each of the three oils, 0.2 g was weighed and introduced into a pyrex glass flask. The oil was dissolved in a mixture of cyclohexane/acetic acid (30 mL). Then, 20 mL of a Wijs solution was added. The flask was closed and stored away from the light. After one hour, 15% KI (potassium iodide) solution (10 mL) and 50 mL of distilled water were added. A 0.1 N sodium thiosulphate was used to titrate the iodine liberated by the reaction. Starch solution was used as indicator (Consolidated text for olive oil analysis, Annex XVI Consleg 2003). Determination of free acidity Two grammes of each oils, was weighed accurately in a conical flask and dissolved in a mixture of diethyl ether and ethanol (1: 1) then three drops of phenolphthalein were added (Consleg 2003). The solution was titrated with 0.1 N NaOH until it became pink (end point). Results were expressed as g of oleic acid /100 g of bergamot seed oil. Determination of peroxide value The peroxide value was calculated according to the method described in the official gazette no. 1513/2003 of the EU. Fatty acid methyl esters analysis Methyl esters of fatty acids were prepared following to the Official EU Method (EEC 1991). The analysis was conducted by gas chromatography using a Fison Instrument 8000 GC with a split-splitless injector and a FID (Flame Ionization Detector). A Supelcowax TM-10 30 m × 0.53 mm, 0.20 μm column was used. The carrier gas was helium with a flow rate of 0.75 bar. Auxiliary Sicari, et al.: Composition of bergamot oil seed 472 Emir. J. Food Agric ● Vol 29 ● Issue 6 ● 2017 gases were air (1,5 bar) and hydrogen (1 bar). The column temperature was programmed as follows: Initially, 180 °C, rate of rise 5 °C/min up to 240 °C then and then isotherm for 10 min. The identification of the constituents was based on the comparison of their retention indices with those published data or with authentic compounds. Determination of sterols The Official EU Method (EEC 1991) was used to separate the sterol fraction using the TLC-GC technique to separate the sterol fraction. Silica gel plates were used (20 x 20 cm x 0.25 mm). The saturation of the chromatographic chamber was performed using a mixture of hexane/ ethyl ether with a ratio of 65:35; The run had a duration of 20 min. The gas chromatographic analyses were carried out using a Fisons Instruments (GC 8000) gas chromatograph, with a flame ionization detector (FID). The capillary column (25 m x 0.32 mm i.d., film thickness 0.25 μm) coated with an SE54 stationary phase. Helium was the carrier gas at a pressure of 0.76 bar and chromatographic air at a pressure of 1.52 bar. The column temperature was programmed as follows: Initially, 260 °C (15 min), with an increase of 5 °C per min. until a 270 °C maintained for 20 min. The injector and FID detector were at 280 °C and 300 °C respectively. Statistical analysis Statistical analysis was carried out using SPSS version 17.0 for windows. One-way analysis of variance (ANOVA) was used to evaluate the influence of cultivar on sterol and fatty acid content. Any significant difference between all treatments at P < 0.05, was determined using Tukey’s Test. Mean and standard deviation was determined using Microsoft Excel. RESULTS AND DISCUSSION The seeds came from bergamot fruit picked on the same day and processed within 48 hours. This fact is important since the lipid fraction obtained from citrus fruit seeds varies according to the fruit’s ripeness, as described by Saidani et al. (2011). Table 1 shows the extraction time of the oil and the yield expressed as a percentage. The oil yield is between 34.18 % and 35.01 %, similar values are reported from Saidani et al. (2004). The physico-chemical characteristics of the oils are shown in Table 2. The oils showed no significant (P > 0.05) variations regarding viscosity and density, which ranged from 0.07 to 0.07 (10-1 Pa• s) and 0.91–0.92 g/cm3, respectively. Density values are similar to those given by Habib et al. (1986) and Abdel-Rahman (1980), for Egyptian citrus seed oils (0.913–0.933 g/cm3). The iodine value is important for oils, because a high value indicates a high level of unsaturation (Knothe, 2002). The iodine values were 103.21, 102.56 and 101.37 for Castagnaro, Fantastico and Femminello seed oils, respectively. The iodine values of Citrus bergamia indicate that they belong to the semi-drying group. The viscosity was 0.07 (Pa . s) for the seed oils of all three cultivars. The density of the oils is in relation to their degree of unsaturation: A higher unsaturation level indicates a higher density (Gunstone, 2004). The density of the bergamot seed oil in the present study was 0.91333 g/mL. The acid values were 0.87, 0.62, and 0.80 for Castagnaro, Fantastico and Femminello seed oils, respectively. Similar values were found in citrus seed oils by El-adawy (1999). The peroxide values were 14.30, 11.67 and 12.38 meq/O2 kg oil, for Castagnaro, Fantastico and Femminello seed oils, respectively. Table 3 gives the fatty acid composition in bergamot seed oil. The fatty acid methyl esters indicated showed the presence of the following fatty acids: Palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidic acid. In all, six fatty acids were present, of which three were unsaturated. The major fatty acids in bergamot oil were oleic, linoleic, palmitic and linolenic which made up 95% of total fatty acids. The major fatty acid was oleic acid (31.54% for Femminello, to 33.02% for Fantastico and 34.55% for Castagnaro seed oil). The concentration of oleic acid found in the bergamot seeds is greater than found in seeds from other citrus fruits (El-Adawy et al., 1999; Anwar et al., 2008; Waheed et al., 2009 and Trandjiiska and Nguyen. 1989). The content of oleic acid in the oil is on average 33.03 %, whereas oil from citron, orange and mandarin seeds has an average oleic acid content of 22.5 %, 26.00 % and 24.89 % respectively (Fig. 1). Furthermore the quantity of linolenic acid is also greater in bergamot seed oil compared to oil from citron, orange and mandarin. However, the quantity of linoleic acid in bergamot seed oil is less than quantity found oils from the other citrus seeds (El-Adawy et al., 1999) (Fig. 1). Table 1: Extraction with petroleum ether Cultivar Time of extraction (h) Yield % Physical state at room temperature Castagnaro 8 35.01 Liquid Fantastico 8 34.18 

The essential oil is used in the fragrance industry (Ricci and Rovesti 1979;Poiana et al., 1994).The bitter taste of makes it largely unsuitable for use in bergamot juice in the food industry.Citrus seed oil presents chemical and physical characteristics that make it suitable for food consumption (Ramadan and Morsel 2002).In the scientific papers, we find numerous research that have dealt with the oil of citrus seeds.The majority of the research concerns the quantitative analysis of fatty acids (French, 1962;Habib et al., 1986;Saidani et al., 2004;Anwar et al., 2008).
The quality of a food grade oil depends on its fatty acid profile and above all on the ratio of saturated to unsaturated fatty acids.
Furthermore, unsaturated acids can be classed as omega, ω-9, which are considered nonessential, and ω-3 and ω-6, known as essential fatty acids, since they cannot be synthesized by the human body, and must be obtained from diet (Ristic and Ristic 2003).Linoleic acid is present in all vegetable oils and is abundant in many of these, including in sunflower seeds, but also, to a lesser extent, corn oil, soybean oil and others.Linoleic acid an essential fatty acid and belongs to the ω-6 group.
The seeds of the bergamot fruit, which are currently considered a waste material, may not only be a reliable source of edible oil (cooking oil, salad or for margarine) but since it is a semi-drying oil it could also have industrial uses.In this study were evaluated the chemical-physical composition of bergamot oil seed extracted from three different cultivars, in order to evaluate any differences in their composition.

Sampling
During the 2015/16 season, the Bergamot Consortium of Reggio Calabria (Italy) provided the seeds used.The seeds belonged three different cultivars (Castagnaro, Femminello and Fantastico), formerly, Femminello and Castagnaro made up virtually all commercial plantings but they have largely been replaced by Fantastico, a hybrid of Femminello and Castagnaro.
All the fruit was picked on the same day and processed within 48 hours; the seeds were subsequently divided according to cultivar.
After being washed with tap water, the seeds were dried at 35 °C for 24 h.Samples of the dried seeds were ground using a blender.About 100 g of the well-crushed bergamot seeds were fed into a soxhlet extractor.Petroleum ether (40-60 °C) was used as extraction solvent.After extraction, which lasted about 10 hours, the solvent was removed by distillation under vacuum.Further residual solvent was eliminated by nitrogen flow.
After being weighed, the oil yields were calculated.This process was repeated for seeds from all three cultivars.

Chemical and physical analysis
Density and viscosity value of the oils were determined according to AOAC (2005).

Iodine value
For each of the three oils, 0.2 g was weighed and introduced into a pyrex glass flask.The oil was dissolved in a mixture of cyclohexane/acetic acid (30 mL).Then, 20 mL of a Wijs solution was added.The flask was closed and stored away from the light.After one hour, 15% KI (potassium iodide) solution (10 mL) and 50 mL of distilled water were added.A 0.1 N sodium thiosulphate was used to titrate the iodine liberated by the reaction.Starch solution was used as indicator (Consolidated text for olive oil analysis, Annex XVI Consleg 2003).

Determination of free acidity
Two grammes of each oils, was weighed accurately in a conical flask and dissolved in a mixture of diethyl ether and ethanol (1: 1) then three drops of phenolphthalein were added (Consleg 2003).The solution was titrated with 0.1 N NaOH until it became pink (end point).Results were expressed as g of oleic acid /100 g of bergamot seed oil.

Determination of peroxide value
The peroxide value was calculated according to the method described in the official gazette no.1513/2003 of the EU.

Fatty acid methyl esters analysis
Methyl esters of fatty acids were prepared following to the Official EU Method (EEC 1991).The analysis was conducted by gas chromatography using a Fison Instrument 8000 GC with a split-splitless injector and a FID (Flame Ionization Detector).A Supelcowax TM-10 30 m × 0.53 mm, 0.20 µm column was used.The carrier gas was helium with a flow rate of 0.75 bar.Auxiliary gases were air (1,5 bar) and hydrogen (1 bar).The column temperature was programmed as follows: Initially, 180 °C, rate of rise 5 °C/min up to 240 °C then and then isotherm for 10 min.The identification of the constituents was based on the comparison of their retention indices with those published data or with authentic compounds.

Determination of sterols
The Official EU Method (EEC 1991) was used to separate the sterol fraction using the TLC-GC technique to separate the sterol fraction.Silica gel plates were used (20 x 20 cm x 0.25 mm).The saturation of the chromatographic chamber was performed using a mixture of hexane/ ethyl ether with a ratio of 65:35; The run had a duration of 20 min.
The gas chromatographic analyses were carried out using a Fisons Instruments (GC 8000) gas chromatograph, with a flame ionization detector (FID).The capillary column (25 m x 0.32 mm i.d., film thickness 0.25 µm) coated with an SE54 stationary phase.Helium was the carrier gas at a pressure of 0.76 bar and chromatographic air at a pressure of 1.52 bar.The column temperature was programmed as follows: Initially, 260 °C (15 min), with an increase of 5 °C per min.until a 270 °C maintained for 20 min.The injector and FID detector were at 280 °C and 300 °C respectively.

Statistical analysis
Statistical analysis was carried out using SPSS version 17.0 for windows.One-way analysis of variance (ANOVA) was used to evaluate the influence of cultivar on sterol and fatty acid content.Any significant difference between all treatments at P < 0.05, was determined using Tukey's Test.
Mean and standard deviation was determined using Microsoft Excel.

RESULTS AND DISCUSSION
The seeds came from bergamot fruit picked on the same day and processed within 48 hours.This fact is important since the lipid fraction obtained from citrus fruit seeds varies according to the fruit's ripeness, as described by Saidani et al. (2011).
Table 1 shows the extraction time of the oil and the yield expressed as a percentage.The oil yield is between 34.18 % and 35.01 %, similar values are reported from Saidani et al. (2004).
The physico-chemical characteristics of the oils are shown in Table 2.The oils showed no significant (P > 0.05) variations regarding viscosity and density, which ranged from 0.07 to 0.07 (10 -1 Pa• s) and 0.91-0.92g/cm 3 , respectively.Density values are similar to those given by Habib et al. (1986) and Abdel-Rahman (1980), for Egyptian citrus seed oils (0.913-0.933 g/cm 3 ).
The iodine value is important for oils, because a high value indicates a high level of unsaturation (Knothe, 2002).The iodine values were 103.21, 102.56 and 101.37 for Castagnaro, Fantastico and Femminello seed oils, respectively.The iodine values of Citrus bergamia indicate that they belong to the semi-drying group.The viscosity was 0.07 (Pa .s) for the seed oils of all three cultivars.
The density of the oils is in relation to their degree of unsaturation: A higher unsaturation level indicates a higher density (Gunstone, 2004).The density of the bergamot seed oil in the present study was 0.91333 g/mL.
Table 3 gives the fatty acid composition in bergamot seed oil.The fatty acid methyl esters indicated showed the presence of the following fatty acids: Palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidic acid.In all, six fatty acids were present, of which three were unsaturated.The major fatty acids in bergamot oil were oleic, linoleic, palmitic and linolenic which made up 95% of total fatty acids.The major fatty acid was oleic acid (31.54% for Femminello, to 33.02% for Fantastico and 34.55% for Castagnaro seed oil).
The concentration of oleic acid found in the bergamot seeds is greater than found in seeds from other citrus fruits (El-Adawy et al., 1999;Anwar et al., 2008;Waheed et al., 2009 andTrandjiiska andNguyen. 1989).The content of oleic acid in the oil is on average 33.03 %, whereas oil from citron, orange and mandarin seeds has an average oleic acid content of 22.5 %, 26.00 % and 24.89 % respectively (Fig. 1).Furthermore the quantity of linolenic acid is also greater in bergamot seed oil compared to oil from citron, orange and mandarin.However, the quantity of linoleic acid in bergamot seed oil is less than quantity found oils from the other citrus seeds (El-Adawy et al., 1999) (Fig. 1).In edible oils, the ratio of unsaturated to saturated fatty acids plays an important role in human nutrition.The total mean of unsaturated fatty acids in bergamot seed oil was 72.94% of total fatty acids.Fatty acid composition of bergamot seed oil varies only slightly between cultivars.
The concentration of bergamot seed oil fatty acids shows that food use of this oil may have beneficial effects on consumer health.In fact, the ratio of unsaturated and saturated fatty acids can reduce the risk of heart diseases (Bhatnagar and Durrington 2003;Von Schacky 2003).In fact, high levels of saturated fatty acids result in an increase of the LDL (low density lipoprotein) (Holub 2002;Nilsen et al., 2001;Bhatnagar and Durrington, 2003).
Of the saturated fatty acids, palmitic acid ranged between 23.04 % and 21.03 % and stearic acid ranged between 3.09 and 3.87 %.
The palmitic acid content is lower than that found in oil from grapefruit, orange and mandarin seeds (El-Adawy et al., 1999;Anwar et al., 2008).While arachidic acid ranged between 0.38 % and 0.44%, which is similar to earlier investigations into various citrus seed oils (El-Adawy et al., 1999;Anwar et al., 2008).The fatty acid profile of bergamot seed oil (Table 3) shows a slightly higher percentage of unsaturated fatty acids compared to saturated fatty acids which is similar to earlier investigations into various citrus seed oils (Anwar et al., 2008).
The ratio between saturated and unsaturated fatty acids was 1:2.67; 1:2.88 and 1:2.80 in Fantastico, Castagnaro and Femminello respectively.These results are slightly below than those found by Moussa (1990).
The content of linoleic acid is lower in bergamot seed oils than in other citrus seed oils.Other authors have found that the of linoleic acid content in orange, lemon and grapefruit oils may be 40% of the total fatty acids (Anwar 2008;Spiller et al., 1991).
Linolenic acid can reach 10% of total acids found in bergamot seed oils.This agrees with its high presence in other citrus seed oils (Anwar 2008).
The total essential fatty acid content (linoleic and linolenic acids), which have positive health benefits, ranged between 38.39-41.09% (Table 3) which concord with those reported for Egyptian citrus seed oils (40.8-43.0%)(Anwar et al.,   2008).Fig. 1 gives a comparison with other citrus essential fatty acids.
Table 4 shows the composition of the sterol fraction.
In all bergamot seed oils analized, the steol present in the greatest quantity was β-sitosterol, which agrees with earlier investigations in to other varieties of citron seed oil (Lazos and Servos, 1988;Matthausa and Özcan, 2012).The oils from the three different bergamot cultivars showed very similar amount of β-sitosterol: Between 79.67 and 81.68 %.
The quantity of β-sitosterol is slightly greater than that found in other citrus seed oils.In those oils analized by Matthausa and Özcan (2012), the content of β-sitosterol was 71.7 % in orange seed oil (Citrus aurantifolia), 75.1 % in grapefruit seed oil (Citrus paradisi) and 74.9 % in lemon seed oil (Citrus lemon) (Fig. 2).
Among other sterols identified, after β-sitosterol, campsterol is present in the greatest quantity.The highest percentage, of 12.4 % was found in the Fantastico cultivar, whereas Femminello and Castagnaro showed percentages of 11.52 and 10.87 % respectively.The quantity of campesterol present in these oils is very similar to that found in other citrus seed oil (Matthausa and Özcan, 2012), except for oil from Citus sinensis which has a percentage of 8.4 %.The amount of 2,4-methylencholesterol found in bergamot seed oil was 0.62, 0.55 and 0.48 % for Fantastico, Femminello and Castagnaro cultivars respectively.Compared to oils from other citrus fruit, this sterol is present in greater quantity ( (Matthausa and Özcan, 2012).The concentration of the sterols identified (stigmasterol, ∆ 5 -avenasterol and cholesterol) is very similar in oil from all other cultivars in the study, as can be seen in the Table 4.
In bergamot seed oil, β-sitosterol is present in a higher percentage than in oils derived from other seeds.

CONCLUSIONS
Citrus seed oils are a source of comestible oil appropriate for human consumption.The results indicate that the oils extracted from bergamot seeds of all three cultivars are valuable source of essential fatty acids.
The viscosity, density and iodine value do not show significant differences between the three cultivars.The oil had a good oxidative stability as shown by acidity and peroxide value.
It is potentially suitable for human composition although preconditions are necessary for food use, due to its high oxidability.Composition is dominated by β-sitosterol, as are many other vegetable oils.
The seeds of the bergamot fruit, which are currently considered a waste material, may not only be a reliable source of edible oil, but since it is a siccative oil it could also have industrial uses.

Fig 1 .
Fig 1.Comparison of the oleic, linoleic and linolenic acid, of bergamot, orange, citron and tangerine oil seeds.The content of the fatty acids present in the oil of bergamot are the average value of the concentration of the three cultivars.While the content of the same in the other citrus fruits are those obtained by El-Adawy et al. (1999).

Fig 2 .
Fig 2.Comparison between the β-sitosterol content of bergamot, lemon, orange and grapefruit oil seeds.The β-sitosterol content in bergamot oil seed is the average of that contained in the three cultivars object of the experimentation.While the content of the same in the other citrus fruits is that reported byMatthausa and Özcanb, 2012).

Table 2 : Physicochemical quality parameters evaluated in the bergamot seed oil samples Cultivar Density at 25°C (g cm −3 ) Viscosity 10 −1 Pa• s Iodine value mg g -1 oil Acidity g/100g Peroxide meq O 2 kg −1 oil
Values are mean±SD of three sample seed oils, analyzed in triplicate, different letters indicate significant differences.differences were evaluated by one-way analysis of variance (ANOVA) test completed with a multicomparison Tukey's test, **P<0.05compared with the positive control

Table 3 : Fatty acid composition (%) of bergamot seed oil
Values are mean±SD of three sample seed oils, analyzed in triplicate, different letters indicate significant differences, differences were evaluated by one-way analysis of variance (ANOVA) test completed with a multicomparison Tukey's test, **P<0.05compared with the positive control