Preservative effects of some selected spice oleoresins to stabilize the sunflower oil in comparison to tertiary butylhydroquinone

Abstract DPPH (1, 1‐Diphenyl‐2‐Picrylhydrazyl) radical scavenging activity was performed to find out the antioxidant activity (AA) of all the antioxidants used at a concentration of 5, 10, and 15 μl/ml. Effect of spice oleoresins (clove, black pepper, and ginger) (200 ppm) and TBHQ (200 ppm) were incorporated in stabilizing refined, bleached, and deodorized (RBD) sunflower oil heating at frying temperature (170°C) and during accelerated oxidation storage (70°C) was studied in comparison to Tert‐Butylhydroquinone (TBHQ). Antioxidant activity (AA) was found to be in the order TBHQ > clove > ginger > pepper at a concentration of 5; 10; 15 μl/ml. A direct correlation was found between AA and the effectiveness of oleoresins and TBHQ in controlling AV and PV of the sunflower oil. During both heating and storage, TBHQ was found most effective to control AV and PV followed by clove, pepper, and ginger.


| INTRODUCTION
Rancidity, simply can be said to be the subjective organoleptic appraisal of the off flavor quality of food. Rancid off flavors are concerned with the changes that result from reaction of fats/oils with atmospheric oxygen, that is, oxidative rancidity or by hydrolytic reactions catalyzed by moisture from food or from microorganisms (Lundberg, 1961). Frying is one of the most common cooking techniques used in domestic and industrial food preparation. Flavor, shelf-life, and nutrient composition of fried food and vegetable oils are altered by this process, and also some of the compounds formed may have undesirable consequences on consumers' health (Hosseini, Ghorbani, Meshginfar, & Mahoonak, 2016). Unfortunately, fats and oils are oxidative in nature even at room temperature (Potter, 1987).
Autoxidation is the major cause of rancidity of fats and oils (Allen & Hamilton, 1983).Oxidation not only decreases the stability of oils and fats during long-term storage but also negatively affects the stability of lipid containing foods, in particular those which were thermally processed like fried foodstuffs (Anwar, Jamil, Iqbal, & Sheikh, 2006). Synthetic antioxidants such as tertbutylhydroquinone (TBHQ) are often used to retard lipid oxidation in food systems. Due to their low thermal stability, the concern about their long-term effects on human health and the increasing demand by consumers for natural products, plant extracts emerged as good alternatives to synthetic antioxidants (Redondo-Cuevas, Castellano, & Raikos, 2017). Among natural antioxidants, the spices and herbal extracts, tocopherols, ascorbic acid, citric acid, and carotenoids are widely covered. It is commercially available in the market in different formulations such as oleoresin, seasoning, and flavoring agent (Upadhyay & Mishra, 2016). Natural antioxidants are effective in thermo-oxidative stabilization of fats and oils (Jaswir, Che Man, & Kitts, 2000).
In this study, we aimed to identify the natural antioxidant capacity among the used spices (black pepper, ginger, and clove) suitable for increasing the oxidative stability of commercially available sunflower oil commonly used for food applications. The effectiveness of the most potent spices as an antioxidant was evaluated by assessing the heating performance and accelerated oxidation conditions of the aforementioned vegetable oil and comparison of antioxidant capacity of spices with the synthetic antioxidant. The present world is heading toward the concept of naturalism; this work could be helpful in replacing the synthetic antioxidants. It is important to note that several spices which are abundantly found in different areas of our country can have a better market with their growing utility, if found effective.

| MATERIALS AND METHODOLOGY
Black pepper, clove, and ginger were bought from Dharan market, and oleoresins were extracted as per (Ranganna, 2007

| Methodology
The flowchart for methodology is shown in Figure 1 and Figure 2.

| DPPH radical scavenging test
Antioxidant Activity was measured by DPPH-free radical scavenging method. The DPPH radical absorbs at 517 nm and the antioxidant activity can be determined by monitoring the decrease in this absorbance. The capacity of spices oleoresins and synthetic antioxidants to scavenge the lipid soluble DPPH radical is monitored at 517 nm (Singh, Singh, Saini, & Rao, 2008).
In this method, 1 ml methanolic solution of spice oleoresin at different concentration (5, 10, and 15 μl/ml) was mixed with 4 ml of 0.004% methanolic solution of DPPH. The absorbance was measured at 517 nm after 30 min. Control (without any additive) and standards (containing synthetic antioxidants viz. Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), and Propyl Gallate (PG) in place of oleoresin) was also subjected to the same procedure for comparison. The capacity to scavenge the DPPH radical was calculated using the following equation: where A c is the absorbance of control sample, and A t is the absorbance of test sample.
Tert Butylhydroquinone (TBHQ) was used in making standard for comparison which is a modification from Singh et al. (2008). Means of the data were separated (whether they are significant or not) using LSD (least square difference) method at 5% level of significance.

| Antioxidant activity
The antioxidant activities of the used antioxidants sources {(clove, black pepper, ginger) oleoresins, and TBHQ)} used were calculated by DPPH free radical scavenging method, at concentration of 5, 10, and 15 μl/ml. TBHQ showed the highest antioxidant activity (AA) in comparison to the other spices oleoresins. The order of AA was found to be, TBHQ > clove > ginger > black pepper. The values of AA were subjected to analysis of variance (ANOVA) for statistical analysis between individual samples at different concentration.
Significant difference (p ≥ .05) was not found between them. ANOVA was also done between different samples at individual concentration. No significant difference was found for 10 and 15 μl/ml of concentration.
Significant difference was found (p ≤ .05) at 5 μl/ml. The AA was found to increase steadily with increase in the concentration, as shown in Figure.3.
As the concentration of the test solution was increased the value for antioxidant activity increases almost at the same pattern.
In a similar type of study performed by Sanjel (2010) the increasing pattern in antioxidant activity with increasing concentration of spices oleoresins and TBHQ was observed. The results were even similar to Wagensteen, Samuelsen, and Malterude (2004). Concentrationdependent antioxidant activity was found in Zanthoxylum (Upadhyaya & Ashok, 2010); Black pepper and capsicum (Singh et al., 2008); TBHQ (Sanjel, 2010) in their study.

Sunflower oil
RBD sunflower oil was estimated for its important physico-chemical property and the values so obtained are given in Table 1. According to AOCS (1967), specification for sunflower oil for the parameters, that is, saponification value must be in the range (188)(189)(190)(191)(192)(193)(194), peroxide value less than value 10 can be consumed, FFA value ≤ 0.25, Kries test ≤ 3.0.
During cold test, the oil remained perfectly clear. From the analysis the used sunflower oil was of good quality, that is, was clear with no mass of crystal fats on the surface and was free of rancid. Saponification value was 188.3, peroxide value was 0.627, FFA was 0.082, acid value was 0.164, and Kries test 0.083 within the range.

| Effect of heating (at frying temperature, 170°C) on AV of different samples
Acid value (AV) was found to be increasing with the increase in heating time for all samples except for TBHQ at 30 and 60 min. Control exhibited the highest AV at 0, 30, 60, and 90 min followed by pepper, ginger, clove, and TBHQ, respectively. At the final stage of heating (90 min), the AV for blank, clove, pepper, ginger, and TBHQ were 0.65 ± 0.0026, 0.28 ± 0.002, 0.58 ± 0.0015, 0.46 ± 0.001, and 0.25 ± 0.001, respectively, maximum value was found for blank and least for TBHQ as shown in Figure 4.

| Effect of heating (at frying temperature 170°C) on PV of different samples
Peroxide value (PV) was found to be increasing with the increase in heating time for all samples, higher PV was observed for control followed by pepper, ginger, clove, and TBHQ, respectively, but at 0 min

| CONCLUSIONS
The natural antioxidants from plants have been solitary studied for their antioxidant potential in the vegetable oil replacing synthetic antioxidants. The antioxidant activity was found to be in the order TBHQ > clove > ginger > pepper, by DPPH radical scavenging activity Method. The effectiveness of the used oleoresins and TBHQ to control AV and PV was found in the order TBHQ > clove > ginger > pepper. A direct correlation was found between antioxidant activity and TBHQ in controlling AV and PV of the sunflower oil. Clove was found to be the most effective to stabilize sunflower oil among the used spices oleoresins with respect to TBHQ.

ACKNOWLEDGMENTS
We are very much indebted to our parents and family members for their constant encouragement, love and inspiration throughout the work. Without their moral support, this work would never have seen the light of day. We are grateful to Mr. Som Raj Shrestha for his help in this research work.