ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2023, 28(1): 7–15 DOI: 10.53541/jams.vol28iss1pp7-15 Received 9 September 2021 Accepted 24 February 2022 Effect of intermittent frying on the stability of vitamins A and D in commercially fortified oils Seham Al-Amri, M. Shafiur Rahman, Nasser Al-Habsi, and Mohammed Al-Khusaibi* Mohammed Al-Khusaibi*( ) mohamedk@squ.edu.om, Department of Food Science and Nutrition, College of Agricultural and Marine Scienc- es, Sultan Qaboos University. P.O.Box 34, AlKhoudh 123, Muscat, Oman Introduction Malnutrition due to micronutrients deficiency is a major global concern, which is also af-fecting Middle East countries. According to World Health Organization (WHO, 2010), deficiencies reported in the Middle East include calcium, iodine, iron, and zinc, folate, vitamin A, and vitamin D. Inter- vention strategies have been adopted to reduce the de- ficiencies among populations; including fortification of food with micronutrients (Allen et al., 2006). Vegetable oils have been proposed as a vehicle for fat-soluble vita- mins in food fortification strategies. However, fat-solu- ble vitamins are not stable during processing and their retention is questionable especially at high processing temperatures (Hrncirik, 2010). The exposure of vegeta- ble oils to high frying temperatures in the presence of أتثري القلي املتقطع على ثبات فيتامني )أ( وفيتامني )د( يف الزيوت املدعمة سهام العامري، حممد شفور الرمحن، انصر احلبسي، حممد اخلصييب * Abstract. Fat-soluble vitamins and fatty acids are very sensitive to high temperature and oxidation. In this study, stability of vitamins A and D, and physiochemical changes were investigated in two types of oils, palm olein (PO) and a blend of PO and sunflower oil (OB). The intermittent frying was performed at two different temperatures 160 and 190 °C. Batches of French fries (500 g) were fried in a deep-fat fryer for 5 cycles, a cycle per day. In each cycle, food was fried every 15 min with a total frying time of 5 h per day. Oil samples were taken at the end of each heating cycle to measure vitamins A and D concentration, free fatty acid (FFA), peroxide value (PV) and color. In PO, vitamin A was reduced by 96.7% after 25 h of frying at 160 °C and it was completely depleted after 15 h at 190 °C. In OB, the vitamin was reduced by 72.2% after 25 h of frying at 160 °C; and it was completely depleted at 190 °C at the end of frying cycles. Vitamin D was less stable, its content in PO decreased by 67.0% and 80.0% at 160 °C and 190 °C, respectively, after 5 h of frying. In OB, it was reduced by 50.0% and 67.0% at 160 °C and 190 °C, respectively, after 5 h of frying. It was found that both types of oil and frying temperatures showed significant effect on vitamin A concentration, while they did not show any effect on vitamin D. Red color increased linearly with frying time in both oils, while fried at two frying temperatures. The change in color was significantly associated with the vitamin A depletion. A low, but significant, association was found between color changes and depletion of vitamin D. In conclusion, the stability of these vitamins depended on the types of oil and temperature of frying. These were more stable in OB compared to PO, and similar stability was observed in the cases of FFA and PV. Keywords: Frying, Vitamin Depletion, Vitamin A, Vitamin D, Retinol, Cholecalciferol, Fat-Soluble vitamins. املســتخلص: تعتــر الفيتامينــات الذائبــة يف الدهــون واألمحــاض الدهنيــة حساســة جــًدا لدرجــات احلــرارة املرتفعــة واألكســدة. يف هــذه الدراســة، مت قيــاس ثبــات فيتامينــات )أ( و )د( والتغــرات الفيزيوكيميائيــة يف نوعــن مــن الزيــوت، زيــت النخيــل وخليــط مــن زيــت النخيــل وزيــت عبــاد الشــمس )يســمى الحقــا ابخلليــط(. مت اســتخدام الزيــوت يف القلــي املتقطــع علــى درجتــن حــرارة خمتلفتــن: 160 و190 درجــة مئويــة. مت قلــي دفعــات مــن البطاطــس )500 جــرام( يف مقــاة عميقــة ملــدة إمجاليــة بـــخمس دورات، دورة واحــدة يف اليــوم. يف كل دورة، مت قلــي دفعــة مــن البطاطــس كل 15 دقيقــة حيــث كان وقــت )FFA( األمحــاض الدهنيــة احلــرة ،)القلــي الكلــي 5 ســاعات يف اليــوم. مت أخــذ عينــات مــن الزيــت يف هنايــة كل دورة لقيــاس تركيــز الفيتامينــات )أ( و )د ، قيمــة البروكســايد )PV( واللــون. وجــد أن فيتامــن )أ( نقــص بنســبة 96.7٪ بعــد 25 ســاعة مــن القلــي يف زيــت النخيــل عنــد 160 درجــة مئويــة، ونفــد متاًمــا بعــد 15 ســاعة عنــد 190 درجــة مئويــة. أمــا يف اخلليــط، فقــد اخنفــض الفيتامــن بنســبة 72.2٪ بعــد 25 ســاعة مــن القلــي عنــد 160 درجــة مئويــة. ونفــد متاًمــا عنــد 190 درجــة مئويــة يف هنايــة دورات القلــي. كان فيتامــن )د( أقــل ثبــاات، حيــث اخنفــض حمتــواه يف زيــت النخيــل بنســبة ٪67 و80٪ عنــد 160 درجــة مئويــة و190 درجــة مئويــة، علــى التــوايل، بعــد 5 ســاعات مــن القلــي. أمــا يف اخلليــط، اخنفــض بنســبة 50٪ و67٪ عنــد 160 درجــة مئويــة و190 درجــة مئويــة، علــى التــوايل، بعــد 5 ســاعات مــن القلــي. وجــد أن كا مــن نــوع الزيــت ودرجــات حــرارة القلــي هلمــا أتثــر ذو داللــة احصائيةعلــى تركيــز فيتامــن )أ(، بينمــا مل يكــن هلمــا أتثــر علــى فيتامــن )د(. زاد اللــون األمحــر خطًيــا مــع وقــت القلــي يف كا الزيتــن، بينمــا قــل عنــد درجــي حــرارة القلــي. ارتبــط التغيــر يف اللــون بشــكل كبــر بنضــوب فيتامــن أ. مت العثــور علــى ارتبــاط منخفــض، ولكــن ذو داللــة احصائيــة، بــن تغــرات اللــون ونضــوب فيتامــن د. يف اخلتــام، يعتمــد اســتقرار هــذه الفيتامينــات علــى أنــواع الزيــت ودرجــة حــرارة القلــي حيــث كانــت أكثــر اســتقرارًا يف اخلليــط مقارنــة بزيــت النخيــل حيــث لوحــظ اســتقرار مماثــا لألمحــاض الدهنيــة احلــرة وقيمــة البروكســايد. الكلمات املفتاحية: القلي، استنفاد الفيتامينات، فيتامن أ، فيتامن د، ريتينول، كوليكالسيفرول، الفيتامينات الذائبة يف الدهون. 8 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Effect of intermittent frying on the stability of vitamins A and D in commercially fortified oils oxygen and moisture (i.e. deep-fat or shallow frying) re- sults in several chemical changes. These changes result- ed in the decline of oil quality and acceptability (Al-Khu- saibi and Rahman, 2021). The peroxide value (PV) measures the concentration of hydroperoxides formed due to oxidation reactions, while free fatty acids % (FFA) measures the concentration of free fatty acids formed due to hydrolysis of triglycerides. It has been reported that peroxide value increased with the increase in fry- ing time (Park and Kim, 2016; Arsalan et al., 2017). Park and Kim (2016) reported an increase in the PV after 100 frying cycles (10 cycles per day for 10 days). They found that the degree of change in PV was associated with the type of oil (canola oil, soybean oil, lard and palm olein). The highest percentage change was found in lard and the least change was in palm olein. The increase in PV due to thermal oxidation was also reported (Moh et al., 1999; Gotoh and Wada, 2006). FFA was approved as an index of oil degradation by the 28th session of the Codex Com- mittee on Methods of Analysis and Sampling held in Bu- dapest, Hungary in 2005 (Gotoh and Wada, 2006). The FFA concentration increased with the increase on frying cycles (Sohu et al., 2020; Garg et al., 2021). Codex Ali- mentarius has set a limit for FFA in oils extracted from quick frozen French fried potatoes to be 1.5 % (Codex 2019). Different countries have set different maximum limits of FFA%. In general, it is in the range of 0.5-2.5% depending on each country’s legislations (Dobargarnes et al., 1998; Firestone, 1993). There have been extensive studies on the stability of different vitamins either in food samples or in vegetable oils during frying. Examples are vitamin E (Hou et al., 202; Hu et al., 2020; Adu et al., 2019; Santos et al., 2018b), vitamin C (Islam et al., 2021; Santos et al., 2018a), vita- min K (Omotosho 2015). The vitamins retention in dif- ferent food samples during differing food preparation methods was reviewed in several review, such as Lesko- va et al. (2006); and Fillion and Henry (1998). Several studies were conducted to study the stability of vitamin A during different cooking methods, such as boiling and baking and shallow frying (Hrncirik, 2010), storage (Fa- varo et al., 1991; Silalahi et al., 2017; Ikram et al., 2021), extrusion (Ribeiro et al., 2021; Sharma et al., 2020), heat treatments (namely pasteurization, boiling and steriliza- tion) (Sachdeva et al., 2019), boiling and repeated deep- fat frying (Favaro et al., 1992), heating at 180°C for 5 min with no food samples. Rady et al. (2019) studied the deg- radation of vitamin C and β-carotene during frying of sweet potato in soybean oil. Their sample preparation included two steps of frying (one cycle), par-frying for 60 s and finish frying for 3 min, both at 180°C. They report- ed 49% and 70% decreased in vitamin C and β-carotene, respectively. Simonne and Eitenmiller (1998) studied the stability of vitamin A, in the form of retinyl palmitate, during simulated deep-fat frying (using cotton balls) at 185 °C for 30 h of frying. They reported higher stabili- ty in palm olein than in soybean or corn oil. Favaro et al. (1991) reported high retention of vitamin A in rice and beans cooked by boiling for 40 min. However, 42% depletion was found in soybean oil subjected to four pan-frying at 115-117 °C. Riaz et al. (2009) reported high losses of vitamin A and D during extrusion. Several studies have been conducted on the stability of vitamin D during different cooking methods, such as boiling and baking and shallow frying (Hrncirik, 2010), fortification and storage (Silalahi et al., 2017; Moeller et al., 2018), pasteurization/sterilization and storage (Kaushik et al., 2014), drying (Sławińska et al., 2016). In some studies, using frying as a processing method, the concentration in the food was measured instead of oil (Omotosho, 2015; Ložnjak et al., 2018). Zareie et al. (2019) studied the stability of vitamin D3 in fortified canola oil heated in an oven at different temperatures (100 - 180 °C) for several times (5 - 30 min). The reten- tion of the vitamin was temperature and initial concer- tation dependent. The highest loss was at 180 °C (67% depletion). Loznjak et al. (2018) measured the content of vitamin D2 and D3 in sunflower oil after pan-frying for 1 minute at a temperature described as “high level” without measuring the oil temperature. The vitamin retention was 70% and 72% for vitamin D2 and D3, re- spectively. However, there is a lack of literature on its stability during deep-fat frying. The available literature discusses the stability of vitamin D during shallow frying (Jakobsen and Knuthsen, 2014; Hrncirik, 2010), which is expected to have less detrimental effect compared to deep-fat frying. For example, the concentration of po- lymerized triglycerides in oil used for shallow frying was less than 1.3%, which was negligible and it did not show excessive use of the oil as in the deep-fat frying (Jakob- sen and Knuthsen, 2014). Adu et al. (2019) showed that changes in vitamins levels of vegetable oils was greatly influenced by the nature of food being fried. Most of the studies that investigates the stability of vitamins A and vitamin D included the retention of vi- tamins at storage conditions and cooking, e.g. pan-fry- ing. In addition, studies that investigated the retention at high frying temperatures did not mimic the industrial repeated frying cycles. Both factors (i.e. high tempera- ture and repeated frying cycles) could result in higher rate of oil oxidation, which can negatively affect the sta- bility of vitamins. The aim of this study was to deter- mine the effect of intermittent frying on the stability of vitamins A and D in two vegetable oils, and to correlate the depletion of the vitamins with the physico-chemical parameters related to the quality. Materials and Methods Samples Refined bleached and deodorized palm olein (PO) and a blend of the palm olein and sunflower oil in a ratio of 40:60 (OB) were provided by Areej Vegetable oil and 9Research Paper Al-Amri, M. Rahman, Al-Habsi, Al-Khusaibi Derivatives Company (SAOC). Both oils were fortified by the manufacturer with vitamins A and D3. Par-fried French fries were purchased from the local market (Mc- Cain Alimentaire S.A., Harnes, France). The French fries were stored at -18 ºC until used for frying. Frying Intermittent frying was done by frying a batch of 500g of French fries every 15 min interval for 5 h daily for 5 days (i.e. 0, 5, 15, 20, 25 h). The sample took 7 min to get fried and 8 min were idle time until the next batch of fry- ing. The frying was done in a 12 L deep fryer (Frymaster, model No. H117-2BLCSC, U.S.A) at two temperatures (i.e. 160 and 190 °C). At the end of each 5 h of frying cy- cle, oil samples were collected in amber bottles and kept at -18 °C until further analysis. Free fatty acid and Peroxide Value Analysis Free fatty acids (FFA) were measured by titration to the American Oil Chemists’ Society (AOCS) Official Method Ca 5a-40. Recommended oil sample size according to the method was mixed with hot neutralized 95% ethyl alco- hol and phenolphthalein indicator, and the mixture was titrated with NaOH of recommended strength. The FFA % was calculated according to the following equation: Equation (1) Peroxide value (PV) were measured according to AOCS official methods Cd 8b-90. Recommend sample size was mixed with 50 ml of 3:2 acetic acid-isooctane solution. After adding 0.5 ml of KI solution and allowing the mix- ture to stand for 1 minute, it was titrated with 0.1N sodi- um thiosulfate. Titration was carried out until the disap- pearance of yellow color. Then 0.5 ml of starch indicator solution was added. Titration was carried out until the disappearance of blue color. A blank determination was conducted with reagents without sample. PV value was measure by the following equation: Peroxide value (milliequivalents peroxide/1000 g sample) Equation (2) where, S is the volume of sodium thiosulfate from sam- ple titration (mL); S is the volume of sodium thiosulfate from titration of blank; and N is the normality of sodium thiosulfate solution. All measurements were in triplicate and the mean value ± standard deviations are reported. Vitamin A and D Analysis Vitamin A was measured according to AOAC official method 2001.13 using High Performance Liquid Chro- matography (HPLC, Agilent technologies, Germany) with ZORBAX E Clipse Plus C18 column. Two grams of sample were weighed in erlenmeyer flask, added 50 mg of pyrogallic acid and added 40 ml of ethanol. Then 20 ml of 50 % KOH were added and saponified for 45 min. After saponification, 10 ml of acetic acid were added and the sample was kept at room temperature. This was made up to 100 ml with diluent tetrahydrofuran (Eth- anol, 50:50) and then 20 ul was injected in HPLC. The mobile Phase used was acetonitrile:water (70:30). The re- sults were expressed as µg/g using the following formula: Equation (3) where RF is the response factor for vitamin A; PkHT is the total test sample peak height or area of all trans and 13-cis retinol; and W is the weight of test portion (g). Vitamin D was analyzed following the AOCS official method 2002.05 HPLC (Agilent technologies, Germany) with LiChrosorb 5 RP18 column. The normal phase was ZORBAX RX-SIL (4.6x250 mm, 5 micron) from REST- EK, USA. Sample was prepared by mixing 2 gm of sam- ple, 500 mg of ascorbic acid, 50 ml of 95% ethanol and 2 ml vitamin D2 as internal standard. The mixture was sa- ponified for 30 min with 50% KOH. After saponification, 50 ml of distilled water was added through the condens- er, solution was transferred to separating funnel and 100 ml of 40% ethanol was added. The vitamin was extracted by the addition of 75 ml of hexane. After phase separa- tion, layer of hexane was removed and collected into a 250 ml separatory funnel. This was done twice and the separated layers were combined. The combined phase was washed once with 50 ml of 1M KOH, twice with 50 ml of 40% ethanol and with water for 4 times, consecu- tively. A pinch of Butyl Hydroxy Toluene (BHT) and 15 ml ethanol were added and the hexane was evaporated at 45 °C. After evaporation, 2 ml of mixture of cyclohex- ane and n-heptane were added. The extract was injected in HPLC with normal phase and vitamin D2 and D3 was collected. The collected sample was dried and then 0.5 ml of acetonitrile-methanol (80:20) was added and the solution was injected in HPLC in reverse phase. Vitamin D3 content was calculated using the following equation: Equation (4) where AD3 is the peak area for vitamin D3; AD2 is the peak area for vitamin D2; mD2 is the weight of vitamin D2 added to the test portion (µg); ms is the weight of test portion (g); and F is the response factor (D3/D2) at 265 nm. Color Analysis The color of oils was measured using a Lovibond Tin- tometer, UK (Seial No: 16352 and 12435) with a 5¼’’ cell. The measurement was done as per the manufacture in- structions. The cell was filled with oil sample and placed in the meter. The lamp was switched on and the viewing tube was focused until a sharp image of the aperture was observed. A set of standard color filter was adjusted to match the sample color. When both sample and stan- dard color matched, the value of red color was noted. 10 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Effect of intermittent frying on the stability of vitamins A and D in commercially fortified oils Statistical Analysis Analysis of variance (ANOVA) was used to compare between means of different independent variables fol- lowed by Least Significant Difference (LSD) using SPSS (IBM SPSS Statistics 2020). A significance probability value of 0.05 was used. Results and Discussion Changes in Free Fatty Acids and Peroxide Value The changes in the free fatty acids (FFA) of palm ole- in (PO) and oil blend (OB) are shown in Figure 1. This figure clearly shows linear increase (r2 > 0.99) in the concentration of FFA with the oil usage time. This in- crease was higher in the PO as compared to the OB at both frying temperatures (i.e., 160°C and 190 °C). The increase in FFA was well documented during deep fat frying (Warner, 2004; Tyagi and Vasishtha, 1996). From the change in the FFA, it appeared that OB degraded at lower rate as compared to PO, which indicated bet- ter stability at the frying temperatures (i.e. 160°C and 190°C). The change in FFA content during frying process could depend on many factors including frying tempera- ture, number of frying cycles, the food being fried, and replenishment of oil during frying (Karimi et al., 2017). Arsalan et al. (2017) showed different FFA in different oil blends exposed to the same frying conditions (170°C, 10 h of frying with 30 min interval). At the end of the frying process, the values of FFA ranged from 0.84 to 1.13. The Figure 1. The change in Free Fatty acids (FFA) in palm olein (PO) and oil blend (OB) at A: 160 °C and B: 190 °C Figure 2. The change in peroxide value (PV) in palm olein (PO) and oil blend (OB) at A: 160 °C and B: 190 °C 11Research Paper Al-Amri, M. Rahman, Al-Habsi, Al-Khusaibi difference in the FFA formation due to the type of oils was also reported by Azimah et al. (2017). Karimi et al. (2017) reported different values in the oil discarded by restaurants and street vendors in Kenya. The final FFA content ranged from 0.68 to 3.95. The maximum accept- ed level of FFA varied from one country to another and ranged from 0.5 to 2.5% (Dobargarnes et al., 1998; Fire- stone, 1993). Peroxide value (PV)increased significantly as a func- tion of frying temperature, frying time and types of oil. OB had lower changes in PV during frying, which was in accordance to the FFA results. Baltacıoğlu et al. (2017) reported that change in PV during frying is significantly influenced by oil type and frying temperature. With the variation of frying parameters such as the number of fry- ing cycles and frying temperature, PV values in the range 9.65 - 20 (Baltacıoğlu et al., 2017; Marinova et al., 2012). Paunović et al. (2020) reported 75 and 78% increase in per- oxide value in sunflower oil and palm olein, respectively. Degradation of vitamin A The initial vitamin A content was around 11 µg/g in PO and 13 µg/g in OB. Table 1 shows the change in the con- centration of vitamin A in PO and OB at 160 and 190 °C frying temperatures. After 25 h of frying cycles at 160 °C, content of the vitamin in PO decreased to 0.36 µg/g, which represented 97% reduction of the initial content. At 190 °C, the vitamin was completely destroyed after 15 h, which can be attributed to the higher tempera- ture of frying. Simonne and Eitenmiller (1998) reported higher retention of vitamin A in palm olein followed by corn oil and least in soybean oil exposed to simulated frying at 185°C for 30 min. Omotosho (2015) reported that vitamin A was greatly reduced in cocoyam fried in Table 1. Change in vitamin A concentration (µg/g) in palm olein and oil blend used for frying at 160 °C and 190 ° C Frying time (h)/Temperature Palm Olein Oil Blend 160 °C 190 °C 160 °C 190 °C 0 11.05 ± 0.04a 11.03 ± 0.01a 13.07 ± 0.09a 10.01 ± 0.07a 5 5.06 ± 0.04b 1.05 ± 0.01b 9.15 ± 0.05b 4.6 ± 0.24b 10 1.69 ± 0.05c 0.32 ± 0.02c 6.9 ± 0.09c 2.43 ± 0.35c 15 0.86 ± 0.02d ND 5.83 ± 0.11d 1.31 ± 0.17d 20 0.42 ± 0.00e ND 4.59 ± 0.06e 0.68 ± 0.03e 25 0.36 ± 0.00e ND 3.63 ± 0.06f ND Values are expressed as mean ± SD of triplicate readings Values with different superscript letters in a column are significantly different at p = 0.05 Table 2. The effect of oil type, temperature, and their interaction on vitamin A depletion. Source Type III Sum of Squares df Mean Square F Sig. oil 115.55 1 115.55 8.296 0.005 Temperature 120.93 1 120.93 8.682 0.004 oil*Temperature 36.28 1 36.28 2.605 0.111 Table 3. Change in vitamin D concentration (µg/g) in palm olein and oil blend used for frying at 160 °C and 190 ° C. Frying time (h)/Temperature Palm Olein Oil Blend 160 °C 190 °C 160 °C 190 °C 0 0.054 ± 0.001a 0.054 ± 0.006a 0.063 ± 0.002a 0.057 ± 0.001a 5 0.024 ± 0.002b 0.006 ± 0.001b 0.030 ± 0.002b 0.018 ± 0.001b 10 0.011 ± 0.001c ND ND ND 15 0.004 ± 0.001d ND ND ND 20 ND ND ND ND 25 ND ND ND ND Values are expressed as mean ± SD of triplicate readings Values with different superscript letters in a column are significantly different at p = 0.05 12 SQU Journal of Agricultural and Marine Sciences, 2023, Volume 28, Issue 1 Effect of intermittent frying on the stability of vitamins A and D in commercially fortified oils canola oil (0.034 µg/g) and was completely lost when the product was fired in soya or vegetable oils. Silalahi et al. (2017) reported 49% reduction of vitamin A concentra- tion when fortified PO was heated at 180 °C for 5 min. Fat-soluble vitamins are sensitive to heat and oxidation, therefore these can be partially or completely lost during food processing. The vitamins depletion was influenced by several factors, such as high cooking temperature, exposure time, and presence of oxygen (Hrncirik, 2010; Silalahi et al., 2017). Extreme of these factors are present in the deep-fat intermittent frying. The rate of depletion of vitamin A in OB was lower than that in PO. After 25 h of frying cycles at 160 °C, content of the vitamin decreased from 13 µg/g to 4 µg/g representing 72% decline. The effect of high tempera- ture of frying (i.e. 190 °C) was evident and complete de- pletion of the vitamin was detected after 25 h of frying cycles. Similar to the free fatty acids and peroxide val- ues, it can be seen that PO had higher increase in FFA and PV. This may explain the higher rate of vitamin de- pletion in the oil. Pignitter et al. (2016) compared the stability of vitamin A in soybean oil and palm oil and they found better stability in the palm oil. This was at- tributed to the superior oxidative stability of palm oil compared to soybean oil, which was also supported in this work as evidenced in the changes of FFA and PV. A two-way ANOVA examined the effect of frying tem- perature and oil type on vitamin A depletion (Table 2). Vitamin A depletion was significantly influenced by oil type (p = 0.005) frying temperature (p = 0.004). Degradation of Vitamin D Table 3 shows the concentration of vitamin D during the 25 h frying cycles in both oils. The Vitamin D content of PO decreased by 67% after 5 h of frying at 160°C and 80% at 190 °C. After 20 h of frying cycles, vitamin D content was completely depleted. At 160 °C, vitamin D was more stable in PO than the OB. While 100% depletion was found after 10 h of frying in the blend, around 80% de- crease was detected in the PO. In both oils, vitamin was completely lost after 5 h of frying at 190 °C. Compared to vitamin A depletion, vitamin D was less stable and 100% depletion was detected after 10 h of frying cycles. It must be noted that studies on the vitamin D reten- tion in food staff during cooking are limited; especially during frying. Hrncirik (2010) reported 40% decrease in vitamin D content after shallow frying in fortified liquid margarine at 180 °C. On the other hand, 70% retention of the vitamin was reported in sunflower oil followed by pan-frying at “high-level temperature” as described by Table 4. The effect of oil type, temperature, and their interaction on vitamin D depletion. Source Type III Sum of Squares df Mean Square F Sig. oil .012 1 .012 3.595 0.064 Temperature .010 1 .010 2.846 0.098 oil*Temperature .012 1 .012 3.611 0.063 Table 5. Correlation between red color of oils and vitamins A and D content Vitamin Pearson correlation p-value A - 0.814 < 0.001 D - 0.250 0.034 Figure 3. The change in red color in palm olein (PO) and oil blend (OB) at A: 160 °C and B: 190 °C 13Research Paper Al-Amri, M. Rahman, Al-Habsi, Al-Khusaibi the authors without measuring the exact value (Ložn- jak and Jakobsen, 2018). Mattila et al. (1999) reported high vitamin D3 retention in baked fish (78-100%) and vitamin D retention in pan-fried mushrooms (80-100%). The effect of cooking parameters on vitamin D reten- tion was influenced by the heating conditions and the type of food being cooked (Jakobsen & Knuthsen, 2014; Ložnjak, & Jakobsen, 2018). Table 4 shows the effect of oil type and frying on vitamin D depletion. Oil type and frying temperature did not show any significant effect on vitamin D depletion. It must be noted that this might be due to the limited number of data resulting from the fast depletion of the vitamin. Color Analysis Red color has been always reported as an indicator of the degradation of frying oils (Jurid et al., 2020; Fauzi- ah et al., 2000). The red color was initially high in fresh palm olein (≈ 2.6 units) compared to the blended oil (≈ 0.8 units). PO has been reported to have higher values than other oils and the values reported in this study are in agreement with literature (Fauziah et al., 2000; Low et al., 1998; Tarmizi et. al., 2016). After the 5 days fry- ing cycles, the red color changed by 182% in plam olein and by 250% in the oil blend at frying temperature of 160 °C. Although the change in color in palm olein has been reported to be relatively faster than sunflower oil (Razali et al., 1999), this may not be true in the case of blended oil. It can be noticed that the change was ob- served higher in the OB compared to the PO (Figure 3). Pearson’s correlation was calculated to measure the as- sociation between vitamin content and red color change (Table 5). A strong negative association (r = -0.814, p < 0.001) can be noticed between Vitamin A content and red color. Low association (r = 0.250, p = 0.034) was found between vitamin D content and red color (-0.250). Conclusion The intermittent frying used in this study caused linear increase in the free fatty acids content (FFA) and per- oxide value, which showed the deterioration of oil by frying protocol. The depletion of vitamin A was time, temperature and oil type dependent. Vitamin A showed higher stability compared to vitamin D, which depleted completely after 15 h of frying time in PO at 160 °C. The red color of oils was negatively associated with vitamin A concentration, while the association with vitamin D concertation was not significant. The strategies of oil fortifications as an intervention method need to be re- assessed in order to make sure that the goals of inter- vention could be achieved. Based on the maximum limit of FFA, frying in the oil blend at 160 °C resulted in the least depletion of vitamin A. 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