Assessment of oxidative deterioration of soybean oil at ambient and sunlight storage

Se lleva a cabo un estudio para conocer las diferencias en la oxidación del aceite de soja conservado a temperatura ambiente y a la luz solar durante un periodo de 180 días. Los cambios en la oxidación fueron evaluados mediante medidas periódicas del índice de peroxidos, color, acidez libre, índice de refracción (RI), índice de p-anisidina, dienos conjugados e índice de yodo. Al final del periodo de almacenamiento, los niveles de acidez e índice de peroxidos fueron 1,77 y 2,90% y 20.52, 41.89 meq/kg de aceite, respectivamente para los aceites a temperatura ambiente y a la luz solar. Los valores iniciales de RI (40°C), p-anisidina, dienos conjugados y trienos conjugados a los 180 días fueron 1.4647 y 1.4659; 36.13 y 50.40; 23.97 y 41.49; y 13.81 y19.35, respectivamente. Por el contrario, el índice de iodo (g I2 /100 g aceite) disminuyó desde 138.0 to 126.18 y 118.04, respectivamente. Los resultados de los distintos parámetros analizados demostraron que la magnitud de la degradación oxidativa de las muestras de aceite de soja expuestas a la luz solar fue en todos los casos, significativamente superior (P < 0.05) a la que ocurre a temperatura ambiente.


INTRODUCTION
Lipid oxidation is considered a principal mean of deterioration in the quality of foodstuffs.It not only imparts rancid and undesirable flavors to fat products, but also it generates reactive oxygen species, which are linked to carcinogensis, inflammation, aging and cardiovascular disorders (Pezzuto and Park, 2002;Siddhuraju and Becker, 2003;Min, 1998).Lipid oxidation also influences the chemical, sensory, and nutritional properties of edible oils and fatty foods and thus plays an important role in determining their use and shelf-life (Dobarganes and Ruiz, 2003;Anwar et al., 2003).
It is an established fact that different variables involved in oil shelf-life, such as processing, storage conditions, light exposure, type of packaging material, availability of oxygen, and addition of antioxidants do affect the quality and characteristics of vegetable oils/ fats and lipid containing products.However, studies on shelf-life or storage of edible oils at room temperature, which may approximate real storage conditions, are inadequate when compared to investigations on accelerated oxidative conditions.The practical studies under ambient conditions require prolonged experiments and there are also difficulties in drawing general conclusions from limited numbers and specific composition and quality of the oils tested (Polvillo et al., 2004).
Auto-oxidation through the free radical chain reaction via the attack on the alpha methylene of the carbon double bonds of the unsaturated fatty acids is a slow process and requires considerable time to produce a sufficient quantity of peroxides (the main initial product of auto -oxidation) and develop unpleasant flavors in lipid containing products (Naz et al., 2004;Lawson, 1995;Shahidi and Wanasundara, 1997).On the other hand, photo-oxidation which involves the direct attack of the extremely electrophilic singlet oxygen on the unsaturated fatty acids, resulting in the generation of peroxy radicals and, ultimately, hydroperoxides is a very fast reaction.The reaction rate of the photooxidation also known as singlet oxygen oxidation is at least 1000-1500 times faster than that of common triplet oxygen oxidation (Cuppett et al., 1997).Photosensitized oxidation during food processing and preservation has been demonstrated to display highly detrimental effects on quality of foods due to an accelerated rate of reaction.The susceptibility of vegetable oils or fats and lipid containing products to auto-, and photo-oxidation generally increases due to inadequate means of processing, packing and storage (Xiangqing et al, 2004).
In Pakistan, there are a huge number of shopkeepers and retail whole-sellers who intentionally keep their oils and fats (packed in tin containers, polyethylene terephthalate (PET) bottles and PET bags etc.,) outside their shops, thus directly exposing them to sunlight, for the purposes of advertising or due to insufficient space in their shops.Such lack of control in the practice for storage and shipping of vegetable oils and fats prompts the commencement of photo-oxidation in a silent way.It is, therefore, important to evaluate and quantify the effects of sunlight on the extent of oxidative degradation affecting the quality of oils and fats.The present study was planned with the primary objective of investigating and evaluating the extent of oxidative deterioration and the shelf-life of soybean oil stored under ambient and sunlight conditions.Efforts were also made to compare and correlate the oxidative alterations of the oil investigated under both sets of storage conditions by establishing orderly relationships between the results of different indicative parameters.

Materials
Samples of freshly prepared refined, bleached and deodorized (RBD) soybean oil (SBO) were obtained from the "Deodorization Unit" of United Industries Limited, Kashmir Road, Faisalabad, Pakistan.The samples were collected in 150 mL translucent polyethylene terephthalate (PET) bottles, carefully sealed and immediately transferred to the experimental Lab. and kept under refrigeration (4 °C), until used for trial.All the chemicals and reagents (analytical grade) used were from Merck (Darmstadt, Germany), or Sigma-Aldrich Chemical Co.(St.Louis, MO), unless stated otherwise.

Analysis of the stored samples
The magnitude of oxidative alterations taking place in the soybean oil samples subjected to ambient and sunlight storage was determined by the measurement of changes in the refractive index (RI), color, free fatty acid contents (FFA), peroxide value (PV), iodine value (IV), para-anisidine (panisidine) value, conjugated diene and triene contents.Analyses were carried out periodically after every 15 days (Anwar et al., 2003).A fresh soybean oil sample (storage period 0 days) was also analyzed for the above mentioned physicochemical characteristics, selected as indicative of oxidative deterioration.

Determination of conjugated dienes and trienes
Conjugated dienes and trienes, in terms of the measurement of specific extinctions at 232 and 268 nm were determined using spectrophotometer (U-2001, Hitachi Instrument Inc. Tokyo, Japan).Oil samples were diluted with iso-octane to bring the absorbance within limits (0.2-0.8) and ε 1% 1cm , was calculated following an IUPAC method (IUPAC, 1987).

Determination of p-anisidine value
The determination of p-anisidine value of the oil samples was carried out following an IUPAC method (IUPAC, 1987).The oil samples dissolved in isooctane were allowed to react with p-anisidine solution in acetic acid (0.25% w/v) for 10 minutes to produce a colored complex and the absorbance value was noted at 350 nm by using spectrophotometer (U-2001, Hitachi Instruments Inc. Tokyo, Japan).

Statistical analysis
Duplicate samples of soybean oil for each treatment/period were taken.Each sample was analyzed individually in triplicate.The data are reported as mean (n = 2 ϫ 3) ± SD (n = 2 ϫ 3).Statistical analyses of the data were performed by Analysis of Variance (ANOVA) using STATISTICA 5.5 (Stat Soft Inc, Tulsa, Oklahoma, USA) software.A probability value of P ≤ 0.05 was considered to denote the statistically significant differences.

RESULTS AND DISCUSSION
Table 1 shows the changes in the refractive index (RI) and colour of the SBO samples subjected to ambient and sunlight storage.The RI values of the SBO investigated increased very slowly under both sets of conditions during the early storage period of 75 days.Later on, there was a more pronounced increase in the RI of samples stored under sunlight as compared with its counterpart and this change was noted to become more distinct during the ending weeks of storage.However, both the storage conditions did not exhibit any significant (P > 0.05) effect on the RI values of soybean oils.At the end of the storage period i.e., 180 days, the overall increase in RI of SBO stored under ambient and sunlight conditions followed an order of 1.001:1.002,respectively.
The color values of the SBO samples subjected to ambient and sunlight storage decreased slowly with the passage of time, but, data did not show any significant (P > 0.05) differences in discoloration of oil during the preliminary period of storage (ca. up to 105 days).However, with time , the changes in colour values of SBO became significant (P < 0.05).At the end of the experimental period of storage i.e., 180 days, the overall decrease in color (red + yellow) of SBO stored under ambient and sunlight conditions followed an order of 0.80:0.60+ 0.75:0.50,respectively.Literature revealed that oxidation may lead to the discoloration of color and an increase in RI of the oils due to oxidative deterioration and increased conjugation (Sherwin, 1978;McGinely, 1991).
Table 2 shows the extent of changes in free fatty acid (FFA) contents (% as oleic acid) and iodine value (IV) of the SBO samples subjected to ambient and sunlight storage.In the SBO investigated, FFA, which are mainly the product of hydrolysis, often parallel to oxidative damage (McGinely, 1991), increased in a typical fashion under both storage conditions.However, the increase was more pronounced in case of the SBO samples being exposed to sunlight as compared with those stored at ambient.After 180 days of storage , the FFA contents of SBO samples subjected to ambient and sunlight storage was noted to be increased from 0.02% (initial value) to a level of 1.77 and 2.91%, respectively.The statistical analysis of the data demonstrated the variations in FFA contents of SBO under both conditions to be significant (P < 0.05).At the end of 180 days of storage, the overall order of increase in FFA of SBO subjected to ambient and sunlight storage was 1.00:1.65,respectively.Higher values of FFA for SBO F. ANWAR, S. ALI SHAHID CHATHA AND A. IJAZ HUSSAIN subjected to sunlight storage as compared with that of ambient might be attributed to the elevated rate of hydrolysis and photo-oxidation of the oil under accelerated temperatures.The iodine value (IV) of the SBO samples subjected to ambient and sunlight storage decreased under both conditions, but did not exhibit any significant (P > 0.05) difference up to a period of ca.135 days.However, during the later storage period (after 135 days), the statistical analysis of the data showed the variations in IV in SBO stored at ambient and sunlight condition to be significant (P ≤ 0.05).After the storage period of 180 days under ambient and sunlight, the initial level of IV in SBO (138.00 g of I/100 g of oil) was decreased to 126.18 and 118.04 g of I/100 g of oil and the decline in values followed an overall order of 0.91:0.86,respectively.A considerably higher decline in iodine values of the SBO samples subjected to sunlight storage as compared with those at ambient might be attributed to the higher loss of unsaturated fatty acids as a result of the elevated rate of photooxidative degradation.Iodine value can be characterized by a decrease in the total unsaturated contents of the oil and thus is looked upon as an important indicator of deterioration of the oils (Naz et al., 2004).
Table 3 depicts the extent of changes in the peroxide value (PV) and p-anisidine value of the soybean oil subjected to ambient and sunlight storage.The PV, which measures hydroperoxide products, is a good indicator of the primary oxidation products of the oils (McGinely, 1991).A typical pattern of rise in PV of soybean oil samples stored under both conditions was observed.It was noted that the PV of the SBO samples subjected to sunlight storage increased at a significantly (P < 0.05) higher rate as compared with those at ambient.After the completion of the storage period of 180 days, the level of PV for the SBO samples stored under ambient and sunlight reached 20.52 and 41.89 meq /kg of oil, respectively.The overall order of increase in the PV of SBO samples subjected to ambient and sunlight storage was noted to be 1.00:2.04,respectively.Literature reports also revealed a higher rate of formation of primary oxidation products in light-exposed oils (Khan and Shahidi, 2000).P-anisidine value, which generally reflects the magnitude of aldehydic secondary oxidation products in oils (McGinely, 1991), for SBO stored at ambient and sunlight conditions changed from 1.10 to a level of 36.13 and 50.40, respectively.The differences in the p-anisidine values between the SBO stored under both conditions were statistically significant (P < 0.05).After 180 days, the overall order of increase in p-anisidine values of SBO samples subjected to ambient and sunlight storage was noted to be 1.00:1.39,respectively.A higher level of p-anisidine value for the SBO samples exposed to sunlight as compared with its counterpart might be attributed to the high rate of formation of secondary oxidation products in the former.Literature also revealed a higher extent of formation of secondary oxidation products in lightexposed oils which might be due, in part, to the enhanced rate of oxidative deterioration (Khan and Shahidi, 2002).The conjugated diene (CD) and conjugated trienes (CT) contents, measured in terms of specific extinctions at 232 and 268 nm, respectively, for soybean oil samples subjected to ambient and sunlight storage are shown in Table 4.The estimation of CD and CT is a good measure of the oxidative state of oils (McGinely, 1991).The periodical analysis of the SBO samples subjected to both the storage practices revealed a typical pattern of rise in the CD and CT contents.After the F. ANWAR, S. ALI SHAHID CHATHA AND A. IJAZ HUSSAIN   41.49 and 13.81, 19.35, respectively.The statistical analysis showed a rise in the CD and CT contents of the SBO, stored under both conditions to be significantly (P < 0.05) different.After 180 days, the overall order of increase in CD and CT of SBO samples subjected to ambient and sunlight storage was noted to be 1.00:1.73and 1.00:1.40,respectively.A higher value of CD and CT in the light-exposed soybean oil samples as compared with those stored at ambient might be attributed to the accelerated rate of photo-oxidation and exposure to sunlight radiations that might speed up the oxidative alterations (Khan and Shahidi, 2002;Pan et al., 2004).
The results of different parameters investigated in the present analysis strongly suggest that photosensitized oxidation of soybean oil proceeds rapidly.It has resulted in a marked increase in the magnitude of oxidative alterations of the oil investigated.Furthermore, it could be predicted from the present findings on soybean oil that the extent of oxidative deterioration with regards to sunlight exposure is more pronounced in terms of generation of FFA, PV and conjugated dienes.The oxidation threat to oils can be minimized by improving packing, shipping, storage, and using effective antioxidants.

Table 1 Relative change in refractive index and color of soybean oil (SBO) subjected to ambient and sunlight storage SP Refractive index (40 °C) Color (1-in. cell) (days) SBO-AS SBO-SS ANOVA SBO-AS SBO-SS ANOVA SBO-AS SBO-SS ANOVA
Values are mean ± SD for two SBO samples, analyzed individually in triplicate NS: Non significant *Significant SP: Storage period SBO-AS: Soybean oil subjected to ambient storage SBO-SS: Soybean oil subjected to sunlight storage

Table 4 Relative change in conjugated diene and conjugated triene contents in soybean oil (SBO) subjected to ambient and sunlight storage SP Conjugated dienes ε 1% 1cm(λ232) Conjugated trienes ε 1% 1cm(λ268) (days) SBO-AS SBO-SS ANOVA SBO-AS SBO-SS ANOVA
Soybean oil subjected to ambient storage SBO-SS: Soybean oil subjected to sunlight storage 180 day storage period, the initial level of CD (0.08) and CT (0.04) for the SBO subjected to ambient and sunlight storage jumped to the values of 23.97,