Cholesterol content and fatty acid profile of chocolates mostly consumed in Turkey
Contenido de colesterol y perfil de ácidos grasos de los chocolates más consumidos en Turquía

Abstract

Cholesterol content and fatty acid profile of chocolates that are consumed in Turkey were determined by capillary gas chromatography. Chocolate samples from 20 trade marks were purchased from markets in İzmir, Turkey. As results of the analyses, average cholesterol content of the samples was 1.14 ± 0.14 mg/kg ranging between 0.963 and 1.535 mg/kg. Twenty-three types of fatty acids were determined in oil extracted from chocolates. Main fatty acids found in chocolate samples were stearic acid (39.33 ± 5.25%), oleic acid (25.99 ± 6.89%), and palmitic acid (25.56 ± 2.06%). Total saturated and total unsaturated fatty acid contents of chocolate samples were found as 69.90% and 30.05%, respectively. The fat content of the samples were in the range 31.5–67%.

Se determinó el contenido de colesterol y perfil de ácidos grasos de los chocolates que son consumidos en Turquía mediante cromatografía de gases capilar. Muestras de chocolate de 20 marcas registradas fueron adquiridas en los mercados de İzmir, Turquía. Como resultado de los análisis, el promedio en el contenido de colesterol de las muestras fue de 1,14 ± 0,14 mg/kg encontrándose en cantidades desde 0.963 hasta 1.535 mg/kg. Se encontraron 23 tipos de ácidos grasos en aceites extraídos del chocolate. Los principales ácidos grasos encontrados en las muestras de chocolates fueron ácido esteárico (39,33 ± 5,25%), ácido oleico (25,99 ± 6,89%) y ácido palmítico (25,56 ± 2,06%). El contenido total de ácidos grasos saturados y no saturados en las muestras de chocolate fue de 69,90% y 30,05% respectivamente. El contenido graso de las muestras estuvo en el rango 31,5–67%.

Keywords:

• chocolate
• cholesterol
• fatty acid profile

Palabras clave:

• chocolate
• colesterol
• perfil de ácido graso

Introduction

Cacao is derived from beans obtained from the fruit of Thebroma cacao plant. These small, flattish and purple beans of cacao are produced widely in West Africa, Brazil, and Central and South America, Sri Lanka and Indonesia (Afoakwa, Paterson, & Fowler, 2007). West Africa produces over than 70% of world cacao (Amoye, 2006). Cacao beans are completely harvested and separated by hand (Scott, 2007). Powder cacao is produced through a process of fermenting the seeds from the pods of the cacao tree. Beans are dried, roasted and crushed, then they are pressed into cakes and alkalized to form powder. Powder is homogenized with sugar, cacao butter, and milk to form chocolate (Bruinsma & Taren, 1999). There are three types of chocolates consumed all over the world; black (dark), white, and milk chocolate. White chocolate is made from cacoa butter, sugar, milk and flavoring like vanilla, whereas dark chocolate is made from cacoa butter, sugar, cacoa liquor and flavorings. Milk chocolate is made from the same ingredients as dark chocolate but milk is also added. Dark chocolate contains more cacao and less sugar than milk chocolate. It follows that any health benefits would be more pronounced in dark chocolate. As the result of above investigations, the considerable difference in fat content ranged from 6.6 to 40.0% (Daniewski et al., 1999).

Although chocolates contain high amount of lipid and sugar, it is reported that they make positive contributions to human nutrition through the provision of antioxidants and flavonoids like epicathecin, catechin, and procyanidins. Evidence from epidemiological studies suggests that a high intake of dietary flavonoids, a subgroup polyphenols, reduce the coronary heart diseases (Engler et al., 2004; Geleijnse, Launer, Hofman, Pols, & Witteman, 1999; Hertog et al., 1995). Chocolates also contain minerals like magnesium, potassium, copper, and iron (Bruinsma & Taren, 1999). It was commonly believed that chocolate comforts the liver, aids in digestion and makes the consumers strong. Also, it is known that it stimulates the kidneys, treats anemia, tuberculosis, fever, and gout.

As a result of the previous studies, it was reported that cocoa powder and chocolate have been shown to have antioxidant potential and to inhibit LDL oxidation in vitro and increase the antioxidant capacity of plasma. Extended-term consumption of cocoa and chocolate is known to increase the antioxidative capacity of plasma. On the other hand, chocolate has a high fat content which is postulated to have a hypercholesterolemic effect on human health (Arab, 2003; Fuhrman & Aviram, 2001).

Consumption of foods like chocolates having high amount of saturated fatty acids has been known as an important factor for the development of coronary heart disease and increasing cholesterol content of blood. But, it is thought that some saturated fatty acids may not deserve this reputation. The saturated fatty acids like lauric (12:0), myristic (14:0), and palmitic (16:0) acids definitely raise blood cholesterol content. On the other hand, stearic acid (18:0) has been considered as neutral effect on blood cholesterol (Cardwell, 2004; Connor, 1999).

Chocolate contains fat in the form of cacao butter which is composed of stearic (35%), palmitic (25%), and oleic (35%) acids and small amount of lineoleic acid (Steinberg, Bearden, & Keen, 2003). As saturated fatty acids content is high, chocolate consumption has always been a concern among consumers about LDL and cholesterol (Kritchevsky, 1994).

Cholesterol is the dominant sterol of milk (95% of total sterol) (Collins, McSweeney, & Wilkinson, 2003) and as milk is one of the major components of the chocolate, it was commonly thought that consumption of chocolate increases the saturated fatty acids intake and increases the cholesterol level of the plasma (Kris-Etherton & Mustad, 1994; Mursu et al., 2004). However, clinical researches indicated that chocolate consumption has a beneficial effect on the serum lipids (Mursu et al., 2004). Wan et al. (2001) reported that consumption of dark chocolate increases the serum concentration of HDL cholesterol by 4%. Although several articles pertaining to chemical composition of different cocoa samples have been published, those about fatty acid compositions and cholesterol content of cocoa and chocolates are rather limited in the literature. Therefore, the objective of this study was to determine the fatty acid composition and cholesterol content of chocolates consumed in Turkey by capillary gas chromatography.

Materials and methods

Sampling

Chocolates under 20 different trademarks were purchased from local markets in İzmir, Turkey, in their own packages. The sampling of chocolate was based on the amount of their production and consumption. Chocolate samples were kept under refrigerator conditions at 4 ± 1 °C until they were analyzed.

Reagents

Diethyl ether and methanol were from Riedel (Riedel-de Haën, Germany), and KOH and hexane was from Merck (Darmstadt, Germany). Reference standard of cholesterol and standard fatty acid methyl ester mixtures were purchased from Sigma Chemical (St. Louis, MO).

Lipid extraction and preparation of fatty acid methyl esters

Lipids were extracted with diethyl ether as described by Renner (1993). Chocolates were melted in a beaker in a water bath at 40 ± 1 °C under vacuum. Then diethyl ether (Merck) was added on melted chocolate and mixed well. After waiting for 5 min, mixture was filtered through filter paper (Whatman No 2) from a funnel. Filtrate was centrifuged for 2 min at 6000 rpm to remove the undesired particles originated from chocolate. Liquid phase of diethyl ether and oil was taken into centrifuge test tube and diethyl ether was removed using rotary evaporator (Rv 05-St, IKA Labortechic, Sweden) at 40 ± 1 °C. Then, the sample was flushed with nitrogen to remove the remained ether from oil chocolate. Fatty acid methyl esters were prepared according to AOCS (1997). Sample (∼200 mg) was weighed into a stoppered-glass centrifuge vial. About 0.5 ml of 2 N methanolic KOH and 2.5 ml of pure hexane were added into the tube. The tube was shaken well for about 30 s and centrifuged for 2 min at 6000 rpm. Upper phase was taken into a vial to be analyzed by gas chromatography (GC).

Determination of fatty acid composition by gas chromatography

The instrumentation used for the analyses was as follows: a Hewlett-Packard GC (model 6890) equipped with Supelco SP-2380 fused silica capillary column (60 m × 0.25 mm i.d., 0.2 μm film thickness; Supelco, Bellefonte, PA) and a flame ionization detector. The injection volume was 2 μl. The temperature of GC oven was programmed from 100 to 220 °C at the rate of 4 °C/min. The injector and detector temperatures were 300 °C. Nitrogen was used as the carrier gas and the flow rate was 1 ml/min. The split ratio was set at 1:100 (Dönmez, Seçkin, Sağdıç, & Şimşek, 2005).

The identification of the peaks was achieved by retention times and by comparing them with authentic standards analyzed under the same conditions. Peak areas of duplicate injections were measured with a HP computing integrator.

Determination of cholesterol

Cholesterol was determined by the procedure described by Fletouris, Botsoglou, Psomas, and Mantis (1998). One gram of chocolate was taken into a test tube and 5 ml of 2 N KOH was added into it. The tube was shaken well for 15 s then kept in a water bath at 80 °C for 30 min and shook at 5 min intervals. The tube was cooled down under tap water and 1 ml of distilled water and 5 ml of hexane were added, then shaken for 1 min and centrifuged for 1 min at 2000 rpm. Upper phase was taken into a vial and analyzed by Hewlett-Packard GC (model 6890).

For preparation of cholesterol standards, the stock solution (2 mg/ml) was prepared by dissolving 20 mg of reference standard (Sigma Chemical Company, St. Louis, MO) with hexane in a 10 ml volumetric flask. Working solutions were prepared by appropriately diluting aliquots from the stock solution with hexane to obtain solutions in the range of 10–80 μg/ml.

GC conditions used for analyses were as follows: ZB-1 silica capillary column (30 m × 0.25 mm i.d., 0.1 μm film thickness; Phenomenex). Oven temperature was set at 285 °C, injection port temperature at 300 °C, and flame ionization detector temperature at 300 °C. The flow rates were 2 ml/min for nitrogen, 30 ml/min for hydrogen, and 300 ml/min for air. The injection volume was 2 μl with a split ratio of 20:1.

The concentration of cholesterol (C) in analyzed samples was calculated according to the equation C = M × V × 2.5, where M is the computed mass (ng) of the analytic in the injected extract (1 μl), V the dilution factor, if any, that was applied.

Statistical analysis

Microsoft Excel Software (Washington) and Statistical Analysis Software (SAS, 2001) were used to perform for all statistical analysis.

Results and discussion

Fatty acid composition of chocolate samples

Fatty acids found in chocolate samples were given in Tables 1 and 2 under two separate groups of “saturated fatty acids” and “unsaturated fatty acids”, respectively.

Table 1. Saturated fatty acid composition of chocolate consumed in Turkey (% of total fatty acid). Sample number = 20.
Tabla 1. Composición del ácido graso saturado en el chocolate consumido en Turquía (% del total de ácidos grasos). Número de muestras: 20.

	Mean	SD
C6	0.23	0.17
C8	0.24	0.10
C10	0.97	1.71
C11	1.22	1.93
C12	0.02	0.03
C13	0.44	0.35
C14	0.03	0.04
C15	0.12	0.10
C16	25.56	2.05
C17	0.34	0.15
C18	39.33	5.25
C20	1.22	0.28
C22	0.04	0.02
C24	0.15	0.05

Table 2. Unsaturated fatty acid composition of chocolate consumed in Turkey (% of total fatty acid).
Tabla 2. Composición de ácidos grasos no saturados en el chocolate consumido en Turquía (% del total de ácidos grasos).

Sample, n = 20	C15:1	C17:1	C18:1	C18:2	C18:3	C20:1	C20:3	C22:6	C24:6
Mean	0.15	0.25	25.99	3.34	0.01	0.26	0.01	0.03	0.01
SD	0.08	0.09	6.89	1.28	0.01	0.04	0.01	0.02	0.01

As seen in Table 1, 14 different types of saturated fatty acids were determined in oil phase of chocolates and it was also reported that 69.95% of total fatty acids were composed of saturated fatty acids (Table 3). Stearic acid (C18:0) is the major saturated fatty acid (39.33% ± 5.25%) whereas it was followed by palmitic acid (25.56% ± 2.06%) which is the second major saturated fatty acid of chocolate samples. Also, caproic (C6:0), caprilic (C8:0), capric (C10:0), undecanoic (C11:0), lauric (C12:0), myristic (C14:0), pentadecanoic (C15:0), margaric (C17:0), arachidic (C20:0), behenic (C22:0), and lignoseric (C24:0) acids were determined at low amounts in oil content of chocolate samples.

Table 3. Fatty acid ratio of chocolate consumed in Turkey (%).
Tabla 3. Razón de ácidos grasos en el chocolate consumido en Turquía (%).

Sample (n)	TSFA	MUFA	PUFA	TUFA
20	69.95	26.65	3.40	30.05
Note: TSFA, total saturated fatty acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; TUFA, total unsaturated fatty acids.

According to Lipp et al. (2001), palmitic, stearic, oleic, linoleic, and arachidic acid contents of cocoa butter are 26.23%, 35.76%, 33.60%, 2.68%, and 0.93%, respectively. Cacao butter contains 24–30% palmitic acid, 32–37% stearic acid, 31–37% oleic acid, 2–5% linoleic acid, and 1–2% arachidic acid, whereas, chocolates contain averagely 21% oil and their monounsaturated and saturated fatty acids concentrations are 36% and 52%, respectively. Also, chocolates contain 3.8% lauric acid, 2% myristic acid, 24% palmitic acid, 20.5% stearic acid, 0.5% linoleic acid, and 9% linolenic acid.

Lipp and Anklam (1998) reported that cacao butter consumed in Brazil contains palmitic (25.6%), stearic (36%), oleic (34.6%), and linoleic (2.6%) acids, whereas cacao butter consumed in Ivory Coast is averagely composed of palmitic (25.8%), stearic (36.9%), oleic (32.9), and linoleic (2.8%) acids.

According to Denke (1994), one third of chocolate fat is composed of stearic acid (18:0), whereas other vegetable oils generally contain 1–3% stearic acid. The remainder part contains one third palmitic acid and one third oleic acid. Our results resemble with those of Lipp et al. (2001) and Denke (1994). The results of Mursu et al. (2004) were lower than our results. These differences may be come from properties of raw chocolate and raw milk, origin, feeding, harvesting, season, and production method. As it can be seen from Table 1, the percent of C6:0 determined between 0 and 0.62% and statistical analyses showed that there is no important difference between samples (p > 0.05). C10:0 and C11:0 percent of samples showed important differences (p < 0.05). The percent of C10:0 changed between 0.01% and 5.29% while the percent of C11:0 changed between 0.02% and 7.54%. This important difference may be come from the production type properties of milk. The percent of C8:0 in the samples were importantly affected by sample (p < 0.05). The amounts of C12:0 in the samples are very similar. Therefore, there was no statistical difference between the samples. The concentration levels of C13:0, C14:0, C15:0, and C24:0 showed statistical differences between the samples (p < 0.05). The C16:0 concentration changed between 22.95% and 31.55%. There were about 10% differences between the samples respect to C16:0 levels. The range of C18:0 changed between 26.70% and 45.91%. There is an important difference between the the lowest and highest levels of C18:0. Statistically, these differences were found to be important (p < 0.05). Although the amounts of C20:0 were very similar, the differences are found to be important (p < 0.05). In C22:0, the amounts of samples were very similar and there are no differences between the samples statistically (p > 0.05). There was a difference between the C24:0 amount of the samples. These amounts were very similar and there were no differences statistically (p > 0.05).

The total unsaturated fatty acid concentration is 30.05%. For all samples, the type of sample affected all unsaturated fatty acids concentration statistically (p < 0.05). Average unsaturated fatty acids content of chocolate samples were given in Table 2. As seen, major unsaturated fatty acid was C18:1 (25.99% ± 6.89) whereas the second was C18:2 (3.34 ± 1.28%). C15:1, C17:1, C18:2, C18:3, C20:1, C22:6, and C24:6 were also determined in chocolate, but their average amounts were extremely low. In the Table 3, the results for the fatty acid ratios of chocolates are categorized as total saturated (TSFA), monounsaturated (MUFA), polyunsaturated (PUFA), and total unsaturated fatty acids (TUFA).

Among the fatty acids classes, saturated fatty acids were predominating following by MUFA and PUFA (Table 3). The abundance of fatty acids decreased in average, in order: C18:0 > C18:1 > C16:0 > C18:2 >C11 = C20 > C10:0 > C13:0 > C17:0 > C17:1 > C8:0 > C6:0 > C15:1 = C24:0 > C15:0 > C22:0 > C22:6 = C14:0 > C12:0 > C18:3 = C20:3 = C24:6.

Cholesterol contents of the chocolate samples

Cholesterol is a sterol (a combination of steroid and alcohol) found in the cell membranes of all body tissues, and is transported in the blood plasma of all animals. Trace amounts of cholesterol are also found in plant membranes. The heart foundation recommends that total blood cholesterol levels should be below 4 mmol/l to reduce the risk of heart disease and other problems. The recommended blood level is LDL < 2.5 mmol/l, HDL > 1.0 mmol/l, trigycerides < 2.0 mmol/l, respectively (Pharmaceutical Society of Australia July 2003, www.psa.org.au).

Percentage recovery was determined by adding a known concentration of cholesterol to selected samples during extraction. The amounts added were roughly 50% of the actual concentrations of the samples. The concentration of cholesterol standard in the mixture was then determined in a way similar to the sample analysis. Recovery rates >90% were achieved for the compound analyzed.

As results of our research, average cholesterol content of the samples was 1.14 ± 0.14 mg/kg between 0.96 ± 0.002 and 1.535 mg/kg (Table 4). The cholesterol concentrations of brain, kidney, lamb, liver, veal, chicken, butter are 39.6, 235.3, 80.3, 6.0, 36.0, 10.0, 9.8, and 24.0 mg/kg, respectively. As it can be seen when compared with other widely consumed foods like meat, milk and dairy products, it can be understood that cholesterol content in chocolate is very low.

Because the cholesterol content of chocolate is low, it can be stated that chocolate and cocoa consumptions do not have a negative effect on blood cholesterol level. In their studies, Kris-Etherton et al. (1993) and Kris-Etherton and Mustad (1994) reported that cocoa butter does not raise blood cholesterol level. Blood cholesterol concentration of consumers who were provided with 280 g or 46 g chocolate per day did not increase during the experiments (Kris-Etherton, Derr, Mustad, Seligson, & Pearson, 1994). Samman, Lai, and Sullivan (2000) also stated that a chocolate bar of 25 g could be safely included in a cholesterol-lowering diet of consumers.

Table 4. Fat content (%) and cholesterol level (mg/kg) of samples.
Tabla 4. Contenido graso (%) y nivel de colesterol (mg/kg) de las muestras.

Sample, n = 20	Fat	Cholesterol
Mean	36.91	1.14
SD	10.22	0.14

The fat level of samples varied between 31.5% and 67% with an average value of 36.9% ± 10.22%. There were important statistical differences in the concentration of fat (p < 0.05). These differences may come from the type of chocolate, fat level of milk or cacao fat concentration used. In previous studies, the cholesterol contents of some products correlated with their fat contents and positive correlation was found (Piironen, Toivo, & Lampi, 2002).

There was no any relationship between fat and cholesterol concentration of samples (r < 0.4). Because of fat concentration of milk, fat in the production and production method, a specific trend or correlation between cholesterol and individual fatty acids was not observed.

Conclusions

In the present study, we have measured the cholesterol content and fatty acid profile of quite high consuming 20 Turkish chocolates. Using our chromatographic conditions, we obtained satisfactory separation and identification of all analyzed compounds. The fatty acid profile showed that stearic acid (C18:0) were predominant whereas oleic acid (C18:1) was major unsaturated fatty acid. Recent recommendations suggest a polyunsaturated/saturated ratio of about 1 for fatty foods. In our study, any sample does not meet this criterion due to the nature of chocolate oil. Although chocolate contains milk, we found that average cholesterol content of the chocolate samples was too low (1.14 ± 0.14 mg/kg).