334

Bulletin UASVM Agriculture, 67(2)/2010 Print ISSN 1843-5246; Electronic ISSN 1843-5386

Determination of Peroxide Value in Sunflower Halva using a Spectrophotometric Method

Vlad MUREŞAN, Sevastita MUSTE, Emil RACOLtA, Cristina Anamaria SEMENIUC,

Simona MAN, Anamaria BIROU, Carmen CHIRCU

Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur Street, 400372, Cluj-Napoca, Romania;

e-mail: vlad_muressan@yahoo.com

Abstract. Sunflower halva, popular in countries from Eastern Europe, is made of sunflower tahini, cooked sugar and soapwort root extract.

Lipid fraction in traditional sunflower halva is rich in polyunsaturated fatty acids, susceptible to peroxidation. Oxidation of the lipids is one of the main causes of lipid rich food deterioration leading to formation of off-flavour that negatively affect their quality and shelf life. In this study the initially phase of oxidation in sunflower halva was assessed, using as indicator the peroxide value (PV). The protocol followed was the one described by IDF standard which uses ammonium thiocyanate as Fe(III)-complexing agent.

Halva samples stored at room temperature, in open air conditions for four months, respectively ten months were analyzed. The PV of sunflower halva at 10 months of storage was ~ 2 times higher that the PV of sunflower halva at 4 months of storage.

The samples of sunflower seeds used for the analysis were freshly dehulled and dehulled and then stored at room temperature in open air conditions for four months. The freshly dehulled sunflower seeds had a PV of 4.14 meq O2/Kg fat, similar values with those reported in the literature. The sunflower seeds dehulled and than stored for 4 months at room temperature in open air conditions had a PV of 89.47 meq O2/Kg fat, rancid taste being detected.

Regarding the oxidative stability of sunflower halva, care must be taken on storage conditions and packaging – temperature and oxygen availability. For further studies addition of supplementary antioxidants should be considered.

Keywords: halva, sunflower seeds, lipid oxidation, peroxide value.

INTRODUCTION

Sunflower (Helianthus annuus) originates from Central and North America. It is one

of the most important oil crops. Total annual world production (2008) is some 35.6 million tons of seed from some 25 million ha, Romanian annual production (2008) being around 1.1 million tons (FAO, 2010). Most of the sunflower seed crop is crushed for oil and most of the oil is consumed by humans. A major by-product of crushing is protein-rich cake, an excellent feed for livestock. A tiny proportion of the global sunflower crop is directly eaten as “nuts” or kernels, or processed as sunflower butter or sunflower halva.

Halva is a traditional confection and has gained popularity as a versatile, quality dessert in the Mediterranean and Middle East. It consists of tahini (sesame paste), cooked sugar and Saponaria officinalis (soapwort) root extract (Eissa and Zohair, 2006). It is considered a rich source of carbohydrate, fat and protein. In some varieties of halva, cocoa powder or nuts are mixed for a richer, more flavourful taste.

335

Sunflower halva, popular in countries from Eastern Europe is made of sunflower seeds tahini, instead of sesame. Sunflower tahini as reported previously by Damir (1984) have 62.3% ether extract, 23.1% crude protein, 2.5% ash and 12.1% total carbohydrates (on a dry matter basis). Regular sunflower tahini oil fatty acid profile is made up of 11% saturated fatty acids, 20% oleic acid and 69% linoleic acid (Fernández-Martínez et al., 2007). Foods which contain high concentrations of unsaturated lipids are particularly susceptible to lipid oxidation. Oxidative stability and deterioration of oils depend on initial composition, concentration of minor compounds with antioxidant or prooxidant characteristics, degree of processing and storage conditions (Crapiste et al., 1999).

Oxidation of the lipids in fresh and processed foods is one of the main causes of deterioration, reduced stability and formation of off-flavours that negatively affect quality and storage life of food product and its consumer acceptance. The radical species formed in the peroxidation process degrade fatty acids and other components of the lipid fraction, such as carotenoids, chlorophyll pigments and tocopherols. When these natural antioxidants are present, peroxidation is delayed, so they (and other artificial ones) are commonly added to fats and oils to reduce alteration during processing and storage (Hornero-Mendez et al., 2001).

The mechanism of lipid oxidation changes significantly at elevated temperatures and depends strongly on oxygen availability. Depending on analysis conditions, differences in oxidative stability and deterioration rates can be obtained. To evaluate the oxidation of lipids various methods are used either measuring primary or secondary changes.

Peroxide value (PV) is most commonly used as an indicator of the early stages of oxidation in fats and oils. Over the past few years, numerous new methods have been developed for measuring PV. The AOAC Official Method and the American Oil Chemists’ Society (AOCS) Method (both of which determine peroxide by using iodometric titration) lack sensitivity and require large amount of lipid. To improve the drawbacks of the official methods, a number of new methods were developed.

The classical method for the determination of liberated iodine, titration with thiosulfate, has been replaced by iodine estimation as the triiodide anion (Lovaas, 1992). Another adaptation of the iodometric method is the determination of liberated iodine by coulometry (Oishi et al., 1992).

Spectrophotometric methods using the ultraviolet (UV) visible range are currently those most widely used to determine PV in food lipids, commonly using the oxidation of Fe(II) to Fe(III) ions that, once formed, can react with various reagents producing coloured complexes. These complexes include the Fe(III)-thiocyanate in the International Dairy Federation (IDF) method and Fe(III)-xylenol orange in the ferrous oxidation-xylenol orange (FOX) method. Such complexes absorb in the 400-600 nm wavelength range, and measurement is normally performed at wavelengths close to the absorption maximum.

These methods, together with others using detection in the infrared region, are displacing classical volumetric methods such as iodometry. Spectrophotometric determination has no disadvantages, except when the foodstuff contains compounds that absorb naturally in this measurement range (Hornero-Mendez et al., 2001).

The aim of this study was to asses the oxidation degree of sunflower halva and sunflower seeds, in different moments of storage using the peroxide value as indicator. To this purpose was used the protocol described in IDF Standard.

This International Standard specifies a method for determination of anhydrous milk fat and was wanted to see if this method is suitable also for vegetable oil.

336

MATERIALS AND METHODS

Samples The sunflower halva was produced by traditional technology using sunflower tahini,

cooked sugar and soapwort root extract. Halva samples were stored at room temperature, in open air conditions for four months (sample SunHa4), respectively ten months (sample SunH10). The samples of sunflower seeds used for the analysis were freshly dehulled (sample SunSeed0) and dehulled and then stored at room temperature in open air conditions for four months (sample SunSeed4).

Fat extraction For all samples, oil was extracted as follows (adapted after Folch et al., 1957): 2.5 g of

sample are homogenized with 50 ml of mix solvents (chloroform-methanol 1:1, v/v) and an ultrasound treatment is applied for 5 minutes. The sample is maintained in oven for 20 minutes at 60oC. After cooling to room temperature, 25 ml of chloroform are added followed by homogenization for 2 minutes. Sample is filtered to a Whatman no. 5 filter paper; filter is washed 2 times with 2.5 ml of chloroform. The filtrate is homogenized for one minute with 25 ml of 1M NaCl solution and stored in the refrigerator until the next day. Two phases are separated; the lower phase is separated using a separatory funnel. A spatula of anhydrous sodium sulphate is added and the sample is stored in the refrigerator for 2 hours. After filtration, the filtrate is brought to dryness using a rotary evaporator. The fat is weighed, transferred into a test tube with stopper using 10 ml of n-Hexane and stored in the refrigerator for further analysis.

IDF modified method for determination of PV The IDF Standard is ISO 3976 | IDF 74:2006 Milk fat- Determination of peroxide

value. The test is based on the co-oxidation of Fe(II) to Fe(III) by hydroperoxides from sample (fat) and the formation of the reddish Fe(III)-thiocyanate complex which is read at 500 nm to a spectrophotometer. Several modifications are introduced to enable the determination (Semeniuc, 2009; Shantha, 1994).

An amount of ~ 10 mg fat (w) is dissolved in 9.8 ml mixture of chloroform-methanol, 7:3 (v/v); 50 µl of ammonium thiocyante are added followed by 50 µl of Fe(II) solution. After 10 minutes the absorbance is measured at 500 nm against a blank using a spectrophotometer (UV-VIS 1700 Shimadzu). The blank contains all the reagents except the fat. PV is expressed as mEq O2/Kg fat using the formula:

Peroxide Value (PV) = bw

Abs 1

84.55×

× [mEq O2/Kg fat] (1)

where: w - fat weight (g); Abs - absorbance; 55.84 - atomic weight of Fe3+; b - the slope of the Fe (III) calibration curve. Calibration curve For calibration curve, a standard solution of Fe(III) 10 µg/ml was prepared. The

absorbance of Fe(III) standards [5-40 µg Fe(III)] was plotted vs. their concentrations (ISO 3976 | IDF 74, 2006; Shantha, 1994).

337

All reagents used in the method are prepared as described previously by Semeniuc (2009) and Shantha (1994). Reagents were purchased from Merck, Germany and Penta, Czech Republic and were analytical grade. Measurements were done in duplicate and mean value was used.

RESULTS AND DISCUSSION

Fig. 1 shows the calibration plot with a slope (b) of 0.0254 and a coefficient of

determination R2 = 0.9936.

y = 0,0254x + 7E-05

R2 = 0,9936

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50

Fe3+, µg

Abs

orba

nce

at 5

00 n

m

Fig. 1. Standard Fe(III) calibration curve

Peroxide values obtained for sunflower halva samples are shown in Figure 2. The peroxide value of sunflower halva at 10 months of storage (96.19 meq O2/Kg) was ~ 2 times higher that the peroxide value of sunflower halva at 4 months of storage (46.75 meq O2/Kg); rancid taste was detected in sample SunHa10. Damir (1984) reported sunflower tahini peroxide values (measured using the AOAC method) between 12–13 meq/Kg oil after 60 days of storage.

Kahraman et al. (2010) analyzed 120 sesame halva samples obtained from producers and retailers from Marmara (Turkey); 11 samples were unacceptable concerning the PV satisfactory limit by TFC (Turkish Food Codex) which stated a maximum peroxide value for sesame halva of 10 meq/Kg. It is known that oil extracted from sunflower tahini has a higher peroxide value than that extracted from sesame tahini (Damir, 1984); the antioxidant effect of sesame tahini might be due to the phenolic compound sesamoline (Glabe et al., 1957 cited by Damir, 1984).

338

0

20

40

60

80

100

120

SunHa4 SunHa10

Per

oxi

de

Val

ue

(PV

) [m

eq O

2/K

g f

at]

Fig. 2. Peroxide values of sunflower halva samples

Peroxide values are not static and care must be taken in handling and testing samples.

It is difficult to provide a specific guideline relating peroxide value to rancidity. High peroxide values are a definite indication of a rancid fat, but moderate values may be the result of depletion of peroxides after reaching high concentrations(ChromaDexTM|TechTip0011,2010)

Peroxide values of sunflower seeds samples are shown in Figure 3. The freshly dehulled sunflower seeds (sample SunSeed0) had a PV of 4.14 meq O2/Kg fat. Similar peroxide values, measured using AOCS Cd 8-53 method were previously reported by Crapiste et al. (1999): 2.45–3.92 meq/Kg for pressed sunflower oil and 3.36 meq/Kg for the extracted oil. The sunflower seeds dehulled and than stored for 4 months at room temperature in open air conditions had a PV of 89.47 meq O2/Kg fat. The PV of the sample SunSeed4 is 21 times higher than the one of SunSeed0, confirming that the dehulled sunflower seeds are more susceptible to oxidation during storage in open air conditions. Crapiste et al. (1999) reported for pressed sunflower oil stored in an open flask at 30oC for 98 days a PV of 125 meq/Kg and for the same oil stored under nitrogen atmosphere 98 days at 30oC, a PV of 4.4 meq/Kg. Hamed and Allam (2006) reported for sunflower oil stored in an air-circulating oven at 70oC for 3–4 days, peroxide values between 400 and 500 meq O2/Kg oil.

0

10

20

30

40

50

60

70

80

90

100

SunSeed0 SunSeed4

Per

oxi

de

Val

ue

(PV

) [m

eq O

2/K

g f

at]

Fig. 3. Peroxide values of dehulled sunflower seeds samples

339

CONCLUSIONS

Lipid fraction in traditional sunflower halva is rich in polyunsaturated fatty acids, susceptible to peroxidation. Sunflower seeds types with high content of saturated fatty acids, high palmitic (>25%), high stearic (>25%) (Fernández-Martínez et al., 2007) might be an alternative.

Regarding the oxidative stability of sunflower halva, care must be taken on storage conditions and packaging (temperature and oxygen availability).

Even if sunflower halva is likely to possess antioxidant characteristics since it contain large concentrations of polyphenolic compounds (Damir and Abdel-Nabey, 1990), addition of supplementary antioxidants should be considered.

The original approach was to modify the IDF spectrophotometric method - as these is a quick, precise method and requires a small amount of sample for determination of PV in sunflower halva and sunflower seeds.

REFERENCES

1. Crapiste, G. H., M. I. V. Brevedan and A. A. Carelli (1999). Oxidation of sunflower oil during storage. JAOCS, 76, 12: 1437-1443.

2. Damir, A. A. (1984). Utilization of sunflower seeds in tahina and halawa processing. Food Chemistry, 14:83-92.

3. Damir, A. A. and A. A. Abdel-Nebey (1990). Quality characteristics of sunflower Halawa. Die Nahrung, 34, 6:491-497.

4. Eissa, A. H. and A. Zohair (2006). Quality and safety of halawa modified with mushroom. J. Sci. Food Agric, 86:2551-2559.

5. Fernández-Martínez, J. M., B. Pérez-Vich, L. Velasco and J. Domínguez (2007). Breeding for specialty oil types in sunflower. HELIA, 30, 46:75-84.

6. Folch, J., M. Lees and G. H. Sloane Stanley (1957). A simple method for the isolation and purification of total lipids from animal tissues. The Journal of Biological Chemistry, 226:497-509.

7. Hamed, S. F. and M. A. Allam (2006). Application of FTIR Spectroscopy in the determination of antioxidant efficiency in sunflower oil. Journal of Applied Sciences Research, 2(1):27-33.

8. Hornero-Mendez, D., A. Perez-Galvez and M. I. Minguez-Mosquera (2001). A rapid spectrophotometric method for the determination of peroxide value in food lipids with high carotenoid content. JAOCS, 78, 11:1151-1155.

9. Kahraman, T., G. Issa, G. Ozmen and S. Buyukunal (2010). Microbiological and chemical quality of tahini halva. British Food Journal, 112, 6:608-616.

10. Lovaas, E. (1992). J. Am. Oil Chem. Soc. 69: 777-783, cited in “Application of FTIR Spectroscopy in the determination of antioxidant efficiency in sunflower oil”. Journal of Applied Sciences Research, 2(1):27-33 (2006).

11. Oishi, M., K. Onishi, K. Nakagomi, S. Uchiyama and S. Suzuki (1992). J.AOAC Int. 75:507-510, cited in “Application of FTIR Spectroscopy in the determination of antioxidant efficiency in sunflower oil”. Journal of Applied Sciences Research, 2(1):27-33 (2006).

12. Semeniuc, C. A. (2009). Researches concerning lipolytic and oxidative degradations of milk powders during storage. PhD Thesis USAMV Cluj-Napoca.

13. Shantha, N. C. and E. A. Decker (1994). Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. Journal of AOAC Int., 77:421-424.

14. ChromaDexTM, (2010). Tech Tip 0011-Fats and oils. http://www.chromadex.com/Literature/ TechTips/FatsAndOilsTechTip0011.pdf (1.06.2010).

15. FAO (2010). Food and Agriculture Organization of the United Nations – FAOSTAT. http://faostat.fao.org (1.06.2010).

16. International Standard ISO 3976 | IDF 74:2006. Milk fat – Determination of peroxide value.