Fatty acid composition including trans-isomers of Serbian ... - doiSerbia

Fatty acid composition including trans-isomers of Serbian biscuits Snežana Ž. Kravić1, Zvonimir J. Suturović1, Jaroslava V. Švarc-Gajić1, Zorica S. Stojanović1, Mira M. Pucarević2, Ivana R. Nikolić3 1

Department of Applied and Engineering Chemistry, Faculty of Technology, University of Novi Sad, Novi Sad, Serbia Faculty of Environmental Protection, EDUCONS University, Sremska Kamenica, Serbia 3 Department of Carbohydrate Food Engineering, Faculty of Technology, University of Novi Sad, Novi Sad, Serbia 2

Abstract An experimental study was carried out with the aim of evaluating the quality of the lipid fraction of Serbian biscuits. Total fat contents of the biscuit samples ranged between 10.2 and 24.5%. The saturated, cis-monounsaturated and cis-polyunsaturated fatty acid contents were within the ranges of 18.5−85.6%, 10.6−49.9% and 2.7−13.3% of total fatty acids, respectively. The content of trans-fatty acids (TFA) ranged from 0.0 to 42.5% and the mean was 10.2%. In a total of 34 investigated samples, 10 of them were found to be trans-free, 8 contained low level of TFA (under 2%), 4 samples contained between 2 and 10% of TFA, while 12 samples contained very high amounts of TFA (12.0−42.5%). The results obtained showed a considerable variability in fatty acid composition of biscuits which indicated that different types of fats and oils were used for production of biscuits in Serbia.

SCIENTIFIC PAPER UDC 664.68:547.392.4:543.544

Hem. Ind. 65 (2) 139–146 (2011) doi: 10.2298/HEMIND101001078K

Keywords: Trans-fatty acids • Biscuits • Gas chromatography • Mass spectrometry Available online at the Journal website: http://www.ache.org.rs/HI/

Lipids play a primary role from the nutritional point of view and exert a fundamental action in many biological processes [1]. Besides the nutritional aspects, lipids also have an important role in the processing technology of various foodstuffs. Flour, sugar, fat, water and salt are the fundamental components in a soft dough biscuit formulation [2]. Fat is the principle ingredient responsible for adding a rich quality to cookies [3] and in a biscuit formulation has numerous roles. It interacts with other ingredients to develop texture, mouth feel and overall sensation of lubricity of the product, thereby affecting the rheological properties of baked biscuits [4]. For biscuits manufacturing, the choice of a better kind of lipid often depends on technological and economic parameters, without considering the nutritional implications. For this reason, it is necessary to evaluate the quality and quantity of fat. Many reports indicate the association of dietary intake of fat and fatty acids with occurrence of some civilization diseases like coronary heart disease, obesity or some kinds of cancer. According to many studies, some saturated fatty acids (SFA) and especially some trans-fatty acids (TFA) have adverse impact on human health. Evidence from several controlled human intervention studies indicates that consumption of diets containing TFA, consistently results in increased low density lipoprotein cholesterol (LDL) and decreased

Correspondence: S.Ž. Kravić, Department of Applied and Engineering Chemistry, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia. E-mail: [email protected] Paper received: 1 November, 2010 Paper accepted: 23 December, 2010

high density lipoprotein cholesterol (HDL) of serum, compared with consumption of diets containing cis-monounsaturated (cis-MUFA) or cis-polyunsaturated (cis-PUFA) fatty acids, thereby increasing the ratio total:HDL cholesterol, which is associated with an increased risk of cardiovascular disease (CVD) [5]. Consumption of TFA predicts higher risk of coronary heart disease, sudden death, and possibly diabetes mellitus [6]. These associations are greater than it would be predicted by the effects of TFA on serum lipoproteins alone, suggesting that TFA intake may also influence other, non-lipid risk factors. TFA intake has also been suggested to influence inflammation, early development and fetal growth, cancer or allergies, although data are not always consistent [7,8]. TFA are formed from cis-unsaturated fatty acids during hydrogenation [5,9]. In nature, hydrogenation occurs in the rumen of cattle, and consequently, milk and beef have been sources of trans-fatty acids for centuries. More recent sources of trans fatty acids are industrially partially hydrogenated vegetable oils (PHVO). Such PHVO are attractive to the food industry because of their longer shelf life, oxidative stability, and semisolidity at room temperature [6]. TFA are commonly found in commercial baked goods, shortenings, some margarines and table spreads, and industrial cooking oils. There is very limited data available on the quality of fat used in Serbian foodstuffs. Although previous studies have shown the presence of significant amounts of trans fatty acids in Serbian margarines [10] the data on other foods are lacking. The increasing pressure to decrease the calories intake from fat, removal of trans-fat from foods and to supply products with much low139

S.Ž. KRAVIĆ et al.: FATTY ACID COMPOSITION INCLUDING trans-ISOMERS OF SERBIAN BISCUITS

er saturated fat contents have become the global concern of many international food organizations, such as the Food and Agriculture Organization (FAO), the World Health Organization (WHO) and Food and Drug Administration (FDA), etc. Therefore, the present work reports the results of fat contents and fatty acid profiles of the most consumed biscuit brands to explore the quality of fat used in the manufacturing of the biscuits. MATERIALS AND METHODS Materials Thirty-four biscuit samples of the most commonly marketed brands were purchased from various supermarkets located in northern Serbia between August 2007 and November 2009. The choice of the brands was based on the highest consumption among those available in the market. Each sample was coded with a number followed by letter (letter indicates the year of manufacture (A − 2007, B − 2008 and C − 2009). All samples were acquired and analyzed within the recommended time of consumption. A multi-standard from Supelco (Cat. No. 47885-U, Bellefonte, PA, USA) containing the methyl esters of 37 fatty acids was used to confirm the retention times and mass spectra for peak identification, as well as to confirm that the peak areas reflected the actual composition of these mixtures. The reagents used were methanol, potassium hydroxide, hydrochloric acid and n-hexane. All reagents were of analytical grade purity. Methods Total fat determination Total fat content of the homogenized biscuit samples were determined according to the Soxhlet extraction methods [11], and expressed as a percentage by mass of the product as received. Sample preparation method Simultaneous microwave-assisted extraction–esterification procedures have been carried out as described in previous investigation [12]. A homogenized biscuit sample (1.5 g), 5 ml of n-hexane and 1.2 ml of 2.0 mol/dm3 methanolic potassium hydroxide solution were placed into the extraction flask placed in the microwave oven furnished with an Allihn condenser. After leaching (5 min of microwave irradiation at 800 W), 2.4 ml of 1.0 mol/dm3 HCl was added and gently stirred. After phase separation, the upper phase containing the fatty acid methyl esters was decanted off and finally a 1.0 μl aliquot was used for GC–MS analysis. Gas chromatography – mass spectrometry analyses A Hewlett-Packard HP 5890 gas chromatograph coupled to an HP 5971A quadrupole mass spectrometer equipped with an SP-2560 fused silica capillary co140

Hem. ind. 65 (2) 139–146 (2011)

lumn (100 m×0.25 mm, 0.20 μm) coated with highly polar biscyanopropyl polysiloxane liquid phase, provided by Supelco (Bellefonte, PA, USA), was used for the specific analysis of the trans-fatty acids from the extracts. Helium was used as carrier gas for the GC–MS analysis of the FAME extracts at a constant flow rate of 0.58 cm3/min. The following temperature program was used: injector temperature 230 °C, initial temperature 100 °C (held 5 min), temperature increase 6 °C/min to 240 °C and held at this temperature for 20 min. Total run time was 50 min. The injection was carried out manually, and the volume was 1.0 μl, split ratio 1:80. The mass spectrometer was operated in the electron ionization mode with quadrupole temperature of 180 °C. Data acquisition was carried out in the scan mode (range 40−400 m/z). The instrument was tuned daily by operating software programs (AUTOTUNE) using perfluorotributylamine (PFTBA) calibration substance. Mass spectrometer parameters were adjusted so that the masses 69, 219 and 502 and their respective isotopes met the target mass – intensity criteria. The fatty acids were identified by comparing their retention times and mass spectral data to the massspectral data obtained by analysis of standard fatty acid methyl esters solution under the same conditions. Commercial data base of mass spectra “Wiley” was also used. The response factor, mean of five injection of the standard solution for each fatty acid methyl ester present in the calibration standard solution is calculated related to palmitic acid (C16:0) according to Eq. (1): Ri =

m o ,i A 16:0 m 16:0 A o ,i

(1)

where mo,i is the mass% of FAMEi in the calibration standard solution; A16:0 the peak area of C16:0 in the calibration standard solution chromatogram; m16:0 the mass% of C16:0 in the calibration standard solution; Ao,i the peak area of FAMEi in the calibration standard solution. The content of each fatty acid expressed by mass percentage is calculated according to Eq. (2):

100

R i Ai  R i Ai

(2)

where Ri is the response factor for each fatty acid and Ai the peak area of fatty acid methyl ester in the sample solution. Statistical analysis All samples were prepared and analyzed in triplicates. The results were statistically tested using ANOVA method and the means were compared by one-factor analysis of variance with subsequent comparisons by

S.Ž. KRAVIĆ et al.: FATTY ACID COMPOSITION INCLUDING trans ISOMERS OF SERBIAN BISCUITS

Hem. ind. 65 (2) 139–146 (2011)

samples are given in Tables 1–3. The presented results represent the mean±standard deviation of three replications for each sample. Total fat contents of the samples ranged from 10.3 to 24.5% with a mean of 17.8%. The highest amount of fat was found in sample 15B (24.5%) and the lowest in

Duncan test at a significance level of 0.05, using software SPSS 15.0 (Statsoft, Tulsa, USA). RESULTS AND DISCUSSION

The total fat contents and the fatty acid composition of the lipid fraction of the examined biscuit Table 1. Fatty acid composition of biscuit samples Sample No. Fat content, g/100 g C8:0 C10:0 C12:0 C14:0 C15:0 C16:0 C16:1 C18:0 C18:1t C18:1c C18:2t C18:2c C20:0 C18:3 C22:0

1A

2A

22.0

23.0

3.8±0.13 2.7±0.11 29.3±0.09 6.6±0.08 – 28.8±0.10 – 2.6±0.09

– – 1.8±0.07 0.6±0.05 – 13.1±0.18 – 5.5±0.09 24.8±0.15 20.6±0.16 48.7±0.22 – – 5.7±0.07 5.5±0.07 – – – – – –

3A

4A

5A

6A

7A

8A

9A

10A

21.3

20.0

20.1

20.3

23.9

12.0

17.6

15.3

– – – – – 11.6±0.15 – 6.9±0.12 20.1±0.19 49.9±0.24 1.0±0.06 10.5±0.10 – – –

2.7±0.10 5.1±0.11 5.3±0.09 9.1±0.12 1.7±0.07 22.3±0.22 0.8±0.05 6.9±0.09 14.6±0.13 25.0±0.19 – 6.5±0.10 – – –

– – – – – 12.9±0.15 – 9.6±0.12 37.5±0.24 31.5±0.21 2.3±0.07 5.4±0.08 0.4±0.04 0.1±0.01 0.3±0.02

2.0±0.08 3.6±0.10 3.4±0.11 6.5±0.12 – 18.2±0.17 1.1±0.07 7.2±0.13 4.4±0.09 42.6±0.27 – 11.0±0.14 – – –

Fatty acid content (% of total fatty acid) – – – – – – – – – – – – – – – – – – – – 14.6±0.16 14.5±0.17 17.2±0.11 13.0±0.17 – – – – 6.9±0.09 6.6±0.09 15.5±0.09 5.1±0.10 41.9±0.23 39.5±0.24 25.9±0.16 35.1±0.27 31.4±0.20 31.9±0.19 35.8±0.24 35.0±0.21 0.6±0.05 0.5±0.05 0.5±0.04 1.8±0.08 4.6±0.10 7.0±0.08 4.7±0.05 9.2±0.13 – – 0.4±0.03 0.4±0.03 – – – 0.4±0.04 – – –

Table 2. Fatty acid composition of biscuit samples Sample No.

11B

12B

13B

14B

15B

16B

17B

18B

19C

20C

21C

22C

Fat content, g/100 g

11.6

24.0

18.1

12.8

24.5

19.6

20.6

22.1

12.2

23.5

20.5

12.6

Fatty acid content (% of total fatty acid) C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1t C18:1c C18:2t C18:2c C20:0 C20:1 C18:3 C22:0

– – – – – – – – 2.2±0.07 – – 5.1±0.10 – – – 0.7±0.06 – – – – 3.1±0.08 – – 6.4±0.11 2.6±0.07 2.2±0.06 2.2±0.07 0.7±0.06 – – – 1.3±0.07 2.7±0.08 – 1.0±0.07 5.0±0.09 3.4±0.08 1.5±0.06 1.7±0.08 1.9±0.10 – – – 1.1±0.07 4.8±0.10 – 0.5±0.03 6.7±0.10 9.1±0.11 10.2±0.12 11.5±0.13 2.8±0.09 0.5±0.03 0.6±0.04 – 15.7±0.14 4.7±0.09 0.7±0.04 3.5±0.09 3.6±0.08 10.1±0.11 4.3±0.09 5.0±0.09 7.3±0.12 1.2±0.09 1.3±0.10 – 6.0±0.17 9.4±0.11 1.4±0.09 2.1±0.09 5.9±0.09 0.6±0.05 – – 0.5±0.04 – – – – 1.1±0.06 – – – 0.9±0.07 – – 0.9±0.07 – – – – 1.2±0.06 – – – 32.6±0.23 25.6±0.22 27.4±0.26 32.7±0.27 34.5±0.27 42.2±0.27 11.4±0.13 31.5±0.24 20.8±0.22 34.8±0.29 41.1±0.32 21.7±0.19 1.3±0.07 – 0.1±0.01 1.1±0.08 – – – – 1.2±0.07 – – – 0.6±0.05 – 0.1±0.01 0.6±0.05 0.2±0.01 – – – 0.6±0.05 – – – 7.4±0.10 4.1±0.08 8.6±0.14 8.7±0.06 5.2±0.07 6.9±0.11 8.4±0.09 4.8±0.13 8.1±0.14 5.0±0.10 11.0±0.17 7.1±0.14 1.2±0.07 – 7.4±0.12 4.6±0.08 0.2±0.01 0.3±0.03 27.5±0.19 11.3±0.15 1.5±0.07 1.2±0.09 12.0±0.11 22.4±0.21 40.5±0.24 27.6±0.23 27.0±0.21 44.5±0.27 37.6±0.23 45.0±0.32 29.6±0.17 19.9±0.12 44.2±0.29 31.6±0.26 22.2±0.19 – – 0.6±0.03 – – – 1.1±0.07 – 1.3±0.07 – – – 7.4±0.10 11.4±0.19 7.0±0.11 9.5±0.10 13.1±0.11 10.8±0.11 5.8±0.12 9.7±0.09 6.4±0.11 11.6±0.10 7.2±0.14 4.3±0.08 – 0.2±0.02 0.4±0.04 0.4±0.03 0.4±0.03 0.3±0.02 0.4±0.03 0.3±0.02 0.3±0.03 0.4±0.03 0.8±0.04 – – – 0.1±0.01 – – – – – 0.2±0.01 0.1±0.01 – – 0.4±0.03 – 0.1±0.01 0.6±0.03 0.2±0.01 – – – 0.7±0.03 0.3±0.02 – – – – 0.2±0.02 – – – 0.4±0.04 – – – – –

141


Hem. ind. 65 (2) 139–146 (2011)

Table 3. Fatty acid composition of biscuit samples Sample No. Fat content, g/100 g

23C 13.0

24C 12.0

25C 22.2

26C 10.3

27C 20.1

28C 11.7

29C 11.8

30C 13.0

31C 13.2

32C 18.3

33C 18.4

34C 22.6

Fatty acid content (% of total fatty acid) C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1t C18:1c C18:2c C20:0 C20:1 C18:3

6.6±0.08 7.1±0.13

–

1.2±0.06 0.7±0.04 1.1±0.06 1.7±0.06 1.0±0.06

–

–

–

–

–

–

5.5±0.07 5.5±0.09 17.4±0.14 1.6±0.06 0.9±0.04 1.8±0.07 3.4±0.11 3.5±0.11 0.8±0.04 3.4±0.09

–

1.2±0.06

6.9±0.08 6.0±0.08 8.4±0.09 3.1±0.08 1.6±0.07 3.2±0.11 4.6±0.12 3.5±0.11 1.5±0.06 2.0±0.07

–

1.1±0.06

4.1±0.06 3.6±0.06 37.2±0.27 3.2±0.09 1.8±0.06 3.1±0.10 5.7±0.12 8.9±0.14 1.9±0.07 11.7±0.11 0.5±0.02 16.8±0.19 6.9±0.08 7.0±0.08 7.2±0.08 7.2±0.11 4.3±0.07 7.1±0.14 7.2±0.13 6.8±0.12 4.4±0.13 3.9±0.06 2.1±0.07 6.6±0.10 –

–

–

0.8±0.04 0.4±0.03 0.6±0.03 0.7±0.03 0.5±0.03 0.3±0.02

–

–

–

–

–

–

0.9±0.05 0.5±0.03 0.9±0.04 0.8±0.04 0.5±0.03 0.4±0.02

–

–

–

26.3±0.22 16.7±0.17 10.4±0.11 30.6±0.24 32.1±0.26 33.0±0.29 31.6±0.28 25.9±0.29 44.3±0.33 31.7±0.31 50.5±0.37 34.2±0.27 –

–

–

1.6±0.09 1.0±0.07 1.5±0.06 0.9±0.03 0.5±0.03 0.9±0.06

–

–

–

–

–

–

0.8±0.06 0.5±0.04 0.8±0.03

–

–

–

–

0.4±0.03 0.4±0.02

5.5±0.07 7.5±0.11 3.9±0.05 7.2±0.13 18.6±0.14 7.4±0.11 9.2±0.18 11.8±0.16 6.8±0.10 11.8±0.18 5.5±0.11 4.8±0.10 8.0±0.09 14.5±0.14 1.1±0.04

–

–

–

2.0±0.09

–

–

0.6±0.03

–

–

17.7±0.16 19.4±0.17 10.6±0.10 30.1±0.27 30.6±0.28 27.5±0.22 23.6±0.28 27.5±0.27 27.8±0.21 27.2±0.19 30.7±0.28 26.3±0.17 4.5±0.08 4.8±0.07 2.7±0.06 8.8±0.18 5.1±0.13 8.8±0.12 5.4±0.11 6.7±0.12 8.9±0.10 6.5±0.08 10.1±0.11 9.0±0.11 –

–

–

–

–

–

–

–

–

0.2±0.01 0.5±0.03 0.3±0.02 0.1±0.01 0.4±0.02 0.3±0.01 0.5±0.01 0.3±0.01 –

–

–

–

0.1±0.01

–

0.3±0.01

–

0.9±0.04 0.4±0.04 0.9±0.04 0.3±0.02 0.4±0.02 0.4±0.02 0.4±0.01 0.3±0.01

sample 26C (10.3%). These levels were comparable to Turkish, Pakistani and Italian biscuits which were reported to have 8.5−26.0%; 13.7−27.6% and 7.5−23.1%, respectively [13–15]. A high variability of fatty acid composition was observed, indicating that many different types of lipids were used for production of biscuits in Serbia. The total saturated fatty acid content was within the ranges of 18.5−85.6%. In greater detail, among saturated fatty acids, palmitic acid was the most abundant, with a mean concentration of 26.2% (range: 10.4−50.5%). The presence of high amounts of palmitic acid indicated the presence of palm oil. Also, high levels of lauric acid (C12:0) were found in samples 25C, 1A, 34C and 18B (37.2, 29.3, 16.8 and 15.7%, respectively), with respect to the other samples, indicating the presence of coconut oil or palm kernel oil. The mean content of lauric acid was 7.5%. Various studies [16,17] have suggested that saturated fatty acids with a chain length of C12:0-C16:0 are atherogenic, stearic acid is neutral, and oleic and polyunsaturated fatty acids have a lipid lowering effect. Content of atherogenic saturated fatty acids varied from 11.4 to 64.7%, mean was 36.4%. Stearic acids (C18:0) ranged from 2.6 to 18.6% and was lowest in sample 1A and highest in sample 27C. Myristic acid (C14:0) ranged from 0.6 to 10.1%, with the highest level in 11B and the lowest in 2A. Table 4 shows groups and ratio between the types of fatty acids from the composition of biscuit samples. 142

–

8.0±0.09 7.9±0.11 1.1±0.06 1.8±0.05 1.0±0.05 2.0±0.08 2.8±0.08 1.6±0.08 0.9±0.04

– – –

As may be seen from the values given, the total cis-MUFA and total cis-PUFA contents were 10.6−49.9% (average 31.2%) and 2.7−13.3% (average 7.7%), respectively. Among the cis-monounsaturated fatty acids, oleic acid (C18:1c) was the most represented, with a mean value of 30.7% (range: 10.6−49.9%). Oleic acid is considered to be responsible for lowering the LDL cholesterol levels. Myristoleic (C14:1), palmitoleic (C16:1) and eicosenoic (C20:1) acids were the other members of cis-MUFA determined at less than 1.5%. The cis-PUFA are very important for biological and nutritional values, because essential fatty acids are included in this group. Also polyunsaturated fatty acids have beneficial effects on both normal health and chronic diseases, such as regulation of lipid level [18] cardiovascular [19] and immune functions [20]. Among cisPUFA, the linoleic acid (C18:2c) acid presented the highest value ranging from 2.7 to 13.1%, mean was 7.5%. As for the linolenic acid (C18:3), their levels were present in less than 1% of total fatty acids. The low PUFA content indicated the use of solid fats, often obtained by hydrogenation of refined vegetable oils. Most naturally occurring vegetable oils are rich in unsaturated fatty acids which contain only non-conjugated double bonds in the cis configuration. The unsaturated constituents can be isomerized to the trans form during extraction and processing or as a result of oxidation, conversion during heating and by partial hydrogenation [21] for the manufacture of margarines,


Hem. ind. 65 (2) 139–146 (2011)

Table 4. Groups and ratio between the types of fatty acids from the composition of biscuit samples Sample

SFA

cis-MUFA

cis-PUFA

TFA

UFA

SFA/UFA

cis-PUFA/SFA

TFA/cis-FA

1A 2A 3A 4A 5A 6A 7A 8A 9A 10A 11B 12B 13B 14B 15B 16B 17B 18B 19C 20C 21C 22C 23C 24C 25C 26C 27C 28C 29C 30C 31C 32C 33C 34C

73.7 21.0 21.5 21.1 33.1 18.5 18.5 53.1 23.2 40.9 66.7 48.1 57.1 56.7 42.0 51.3 20.6 60.7 57.9 42.3 60.0 61.5 69.8 61.3 85.6 57.8 62.5 60.7 67.1 64.3 61.7 65.0 58.9 64.7

20.6 48.7 31.4 31.9 35.8 35.0 49.9 25.8 31.5 43.7 24.3 40.5 27.8 28.6 44.5 37.6 45.0 29.6 22.4 44.3 31.6 22.2 17.7 19.4 10.6 32.5 32.0 29.6 25.2 28.6 29.0 27.5 30.7 26.3

5.7 5.5 4.6 7.0 4.7 9.6 10.5 6.5 5.5 11.0 7.8 11.4 7.1 10.1 13.3 10.8 5.8 9.7 7.1 11.9 7.2 4.3 4.5 4.8 2.7 9.7 5.5 9.7 5.7 7.1 9.3 6.9 10.4 9.0

nd 24.8 42.5 40.0 26.4 36.9 21.1 14.6 39.8 4.4 1.2 nd 8.0 4.6 0.2 0.3 28.6 nd 12.6 1.5 1.2 12.0 8.0 14.5 1.1 nd nd nd 2.0 nd nd 0.6 nd nd

26.3 79.0 78.5 78.9 66.9 81.5 81.5 46.9 76.8 59.1 33.3 51.9 42.9 43.3 58.0 48.7 79.4 39.3 42.1 57.7 40.0 38.5 30.2 38.7 14.4 42.2 37.5 39.3 32.9 35.7 38.3 35.0 41.1 35.3

2.80 0.27 0.27 0.27 0.49 0.23 0.23 1.13 0.30 0.69 2.00 0.93 1.33 1.31 0.72 1.05 0.26 1.54 1.38 0.73 1.50 1.60 2.31 1.58 5.94 1.37 1.67 1.54 2.04 1.80 1.61 1.86 1.43 1.83

0.08 0.26 0.21 0.33 0.14 0.52 0.57 0.12 0.24 0.27 0.12 0.24 0.12 0.18 0.32 0.21 0.28 0.16 0.12 0.28 0.12 0.07 0.06 0.08 0.03 0.17 0.09 0.16 0.08 0.11 0.15 0.11 0.18 0.14

0.00 0.46 1.18 1.03 0.65 0.83 0.35 0.45 1.08 0.08 0.04 0.00 0.23 0.12 0.00 0.01 0.56 0.00 0.43 0.03 0.03 0.45 0.36 0.60 0.08 0.00 0.00 0.00 0.06 0.00 0.00 0.02 0.00 0.00

shortenings and a large variety of food products. Compared with cis-unsaturated fatty acids, the structure, physical properties, chemical stability and the physiological effects (atherogenic effects) of trans-fatty acids resemble those of the saturated fatty acids [5]. Nutritionists devote much attention to this category of substances because the presence of trans-isomers, in association with a scarcity of essential fatty acids, can provoke metabolic imbalance due to their ability to substitute the essential fatty acids in the synthetic pathway of phospholipids of cell membranes. This cause changes in membrane permeability and modifications of the activity of the membrane’s enzymes. It was also shown that consumption of TFA causes a two-fold increase in

the LDL/HDL ratio as compared to influence of SFA. Saturated fatty acids increase total cholesterol level, while TFA induce increase in LDL with simultaneous decrease in HDL [15]. The content of trans-fatty acids ranged from 0.0 to 42.5% of total fatty acids and the mean was 10.2%. The most frequent distribution of the total TFA content was in the 12.0−42.5% range (12 of the 34 samples were in this range), ten biscuits were found to be trans-free, eight biscuits contained a total TFA lower than 2% and four samples contained between 2 and 10% of TFA. The TFA comprised isomers of C18:1 and C18:2 acids, and trans 18:1 isomers were the major group of TFA present in all analyzed samples, ranging from 0.0 to 41.9%. 143


The mean value represented 97.1% of the total trans-isomers. For the trans 18:2 isomers, only small amounts were detected in some samples. The presence of the trans fat at high amounts, especially elaidic acid, in the biscuit samples indicates that hydrogenated oil was used in the manufacturing process. These results show that the amount of trans-fatty acids varied considerably among the analyzed samples because of the differences in hydrogenation process conditions used for production of starting fats. It is known that conditions of hydrogenation process such as temperature, pressure, type and amount of catalyst influence on TFA content. Furthermore, in order to achieve biscuits with desirable characteristics, manufacturers may utilize single fats or many possible combinations of different kind of fats and oils [22,23]. A comparison of the total TFA value for our biscuits with those of the literature revealed that observed levels were lower than those found by other authors who examined Pakistani (26.7%) [14], Brazilian (20.1%) [24] and Turkish biscuits (16.4%) [13], but significantly higher than reported value of Italian biscuits (1.7%) [15]. The ratio of saturated/unsaturated fatty acids (Table 4) shows the relation between two major groups of the fat composition of examined biscuit samples. Its value varies from 0.23 to 5.94. Eight samples had a ratio under 0.5 while in the other twenty-six samples it varied from 0.69 to 5.94 which indicate a high proportion of saturated fatty acids. The prevalence of unsaturated over saturated fatty acids (smaller ratio) is considered to be positive from the nutritional point of view. The mean ratio of cisPUFA/SFA recommended by the British Department of Health is 0.45 [25]. In this study the cis-PUFA/SFA ratio ranged from 0.03 to 0.57, and the mean value was 0.19, which is much lower than the recommended value and also compared to

Hem. ind. 65 (2) 139–146 (2011)

those reported by Brazilian and Pakistani biscuits [24,14]. Only two samples (6A and 7A) had a slightly higher value than recommended. A decrease in this value indicates products that are not good for the health, in relation to cardiovascular diseases. The ratio of trans/cis-fatty acids represents the degree of formation of unnatural TFA from the natural cis forms of unsaturated fatty acids and also a higher ratio indicates the greater mixing of hydrogenated oils. The ratio varied between 0.00 and 1.18 with a mean value of 0.27. For similar biscuits, the ratio of trans/cis-fatty acids was 0.39 for Brazilian [24] and 0.95 for Pakistani products [14]. These values are higher to those found in the present study. A comparative survey of the average content of trans, saturated, cis-monounsaturated and cis-polyunsaturated fatty acids of biscuits produced in year 2007, 2008 and 2009 is given in Figure 1. It is evident that the decrease of trans-fatty acids content caused an increase in the total saturated fatty acids. The levels of total saturated fatty acids with 12, 14 and 16 carbon atoms in the biscuits of 2007 ranged from 11.6 to 64.7% (average 22.9%), of 2008 from 11.4 to 53.2% (average 40.4%) and of 2009 from 27.3 to 57.6% (average 42.9%). In relation to the biscuits from 2007, the content of TFA significantly decreases in the biscuits of 2008 (p = 0.001) and 2009 (p = 0.000) while at the same time the levels of total saturated fatty acids with 12, 14 and 16 carbon atoms significantly increases (p = 0.015 and 0.004, respectively). In most samples, trans-fatty acids were replaced by atherogenic saturated fatty acids (mainly palmitic and lauric acid). In terms of health effects, this may imply a worse overall fatty acid pattern than the animal fat- or hightrans-based products [26,17]. From a health point of view, producers should not just decrease the content

Figure 1. The average content of trans (TFA), saturated (SFA), cis-monounsaturated (cisMUFA) and cis-polyunsaturated fatty acids (cisPUFA) of biscuits produced 2007, 2008 and 2009 year (a, b and c – the mean values of the same group of fatty acids do not differ significantly (p > 0.05) if they are followed by the same letter).

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of trans-fats, but the saturated fatty acids with 12, 14 and 16 carbon atoms also, and at the same time should increase content of unsaturated fats and stearic acid, which does not affect level of cholesterol in blood [27]. But the use of increased amounts of C18:0 is very limited. Challenges to the food industries in replacing trans-fatty acids are to supply product that provide equivalent functionality, are economically feasible and do not greatly increase saturated fatty acids content. Production of fat blends that are either low in trans or regarded as trans-free and at the same time with the lower degree of saturated fatty acids, therefore requires modification of the base natural fats and oils. Daily intake of trans fatty acids is limited (about 2 g/day), and it is known that amount is less than 1% of the energy balance from the total fatty acid content [28]. This study monitored their impact on the health effects in human medicine. CONCLUSION

The results obtained in this study showed a considerable variability in fatty acid composition of biscuits which indicated that hydrogenated, interesterified or blended fats and oils were used for production of biscuits in Serbia. Biscuits contain considerable amounts of fat, mainly composed of oleic, palmitic, elaidic, stearic and linoleic acids. The contents of trans-fatty acids were remarkably reduced in 2009 compared with 2007. The monitoring of TFA in biscuits between years suggested that artificially produced PHVO, the major source of trans-fats, has been replaced with currently available alternatives of trans-free fats. However, this resulted in a significant increase (p = 0.004) in the content of atherogenic saturated fatty acids. Therefore, it would be necessary to keep monitoring and inspecting content of atherogenic fatty acids in biscuits, as well as in other food products.

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Acknowledgements

The presented research was funded by the Ministry of Science and Technological Development of the Republic of Serbia through project TR20023. REFERENCES [1] [2]

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IZVOD SASTAV MASNIH KISELINA UKLJUČUJUĆI TRANS IZOMERE KEKSA U SRBIJI

Snežana Ž. Kravić1, Zvonimir J. Suturović1, Jaroslava V. Švarc-Gajić1, Zorica S. Stojanović1, Mira M. Pucarević2, Ivana R. Nikolić3 1

Katedra za primenjene i inženjerske hemije, Tehnološki fakultet, Univerzitet u Novom Sadu, Novi Sad, Srbija Fakultet za zaštitu životne sredine, EDUCONS Univerzitet, Sremska Kamenica, Srbija 3 Katedra za inženjerstvo ugljenohidratne hrane, Tehnološki fakultet, Univerzitet u Novom Sadu, Novi Sad, Srbija 2

(Naučni rad) Iako ga potrošači ne doživljavaju kao masnu hranu, keks sadrži značajne količine masti. S obzirom da se keks koristi u svakodnevnoj ishrani, sprovedeno je ispitivanje s ciljem procene kvaliteta lipidne frakcije keksa. Analizirane su 34 uzorka keksa različitih proizvođača u periodu od avgusta 2007. do novembra 2009. godine. Sadržaj masti kretao se u intervalu od 10,2 do 24,5%, a prosečna vrednost je iznosila 17,8%. Rezultati su pokazali značajne razlike u pogledu sastava masnih kiselina u keksu, što ukazuje da se različite vrste masti koriste za proizvodnju keksa u Srbiji. Najzastupljenije masne kiseline su bile: oleinska, palmitinska, elaidinska, stearinska i linolna sa prosečnim sadržajem od 30,7; 26,2; 9,9; 7,6 i 7,5%, redom. Ukupan sadržaj zasićenih, cis-mononezasićenih i cis-polinezasićenih masnih kiselina kretao se u opsegu 18,5−85,6%, 10,6−49,9% i 2,7−13,3%, redom. Sadržaj trans-masnih kiselina (TFA) kretao se u veoma širokom intervalu, od 0,0 do 42,5%, a prosečna vrednost je iznosila 10,2%. U 10 od ukupno 34 ispitivana uzorka nije detektovano prisustvo TFA, osam je sadržalo male količine TFA (ispod 2%), četiri uzorka je sadržalo 2−10% TFA, dok je u 12 uzoraka sadržaj TFA bio veoma visok (12,0−42,5%). Ustanovljeno je da je količina trans-masnih kiselina u keksu značajno smanjena od 2007. do 2009. godine. Međutim, to je rezultiralo značajnim povećanjem sadržaja aterogenih zasićenih masnih kiselina u keksu; prosečna vrednost iznosila je 22,9, 40,4 i 42,9% u 2007, 2008 i 2009. godini, redom. Konzumiranje proizvoda sa visokim sadržajem aterogenih zasićenih masti može imati za posledicu znatno lošije zdravstvene efekte u odnosu na proizvode sa većim udelom animalnih ili trans-masti. Stoga bi trebalo i dalje pratiti sadržaj aterogenih masnih kiselina (zasićenih i trans masnih kiselina) u keksu, kao i drugim prehrambenim proizvodima.

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Ključne reči: Trans-Masne kiseline • Keks • Gasna hromatografija • Masena spektrometrija