Impact of Storage Conditions and Temperature on the Physicochemical Characteristics and Microbiological Stability of Nigella Sativa Oil

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I. INTRODUCTION
Nigella sativa (black seed or black cumin), which belongs to the Ranunculaceae family, is an annual plant with many pharmacological properties.The use of Nigella sativa seeds and oil in traditional medicines dates back more than 2000 years, and the plant was described by Hippocrates as "Melanthion" i .Black seeds and their oil have a long history of use in Indian and Arab civilizations as food and medicine and have often been used as a treatment for a range of health conditions related to the respiratory system, digestive tract, kidney and liver function, cardiovascular system, immune support system, as well as for the general welfare.Nigella sativa contains many active components, such as thymoquinone (TQ), alkaloids (nigellicine and nigelledine), saponins (alpha-hederin), flavonoids, proteins, fatty acids and many others, which have positive effects in the treatment of patients with various diseases i .In ancient literature, Nigella sativa is also attributed as an analgesic, liver tonic, diuretic, appetite stimulant, analgesic and digestive remedy v .
In this paper, we will use Nigella sativa oil to show the influence of different temperatures and methods of storage of Nigella sativa oil on their physicochemical properties and their microbiological stability (sterility), as well as the influence on C.
albicans and E. coli strains using the disk diffusion method.The blank has been kept in the dark for 30 minutes.

II. METHODS AND MATERIAL
After 30 minutes, U1 was taken and then pour 15 ml of potassium iodide solution into the flask and 150 ml of distilled water."U1" has been titrated until a pale straw color is saw when it is compared to a standardized sodium thiosulphate solution.A purple color has been saw after adding around 1 ml of starch indicator to the contents of the flask.We were titrating till the solution in the flask don't loses color and becomes colorless.The titration's conclusion is marked by the blue color's absence.
The reaction involved during the iodine value test is as follows: ICl + H2O → HIO + HCl The iodine value can be calculated using the

III. RESULTS AND DISCUSSION
In this paper, the influence of storage conditions on the physicochemical properties of Nigella sativa oil during 15 days, as well as their microbiological stability, was investigated.Physicochemical analysis of Nigella sativa oil was performed after 7 and 15 days.
Among the parameters, the influence of oil incubation at 37°C, storage in a light and dark bottle, and finally the influence of storage in a refrigerator at a temperature of about 4°C on the iodine, peroxide and acid numbers were examined.was examined.Many government rules require that the packaging of food products include a statement regarding the purity of the fats, which is an important factor to measure, because of that the iodine value is the best indicator of the purity of saturated/unsaturated lipids.
Farhan et al in their study obtained high iodine values (122.7 mg/100 g) where they showed a high degree of unsaturation (86%) for Saudi BCSO with about 60% dominant linoleic acid (C18:2).ix Unlike them, we got a low iodine number, which shows that our oil is less reactive, more stable and softer than theirs.
Figure 1.Impact of storage conditions on the iodine value (mg I2/100 g) of Nigella sativa oil collection A total of 5 samples Nigella sativa.The oil is obtained by cold pressing from the seeds of the Egyptian Nigella sativa.The samples were stored in a refrigerator at ± 4 °C, incubator +37°C, in a dark bottle that was sun-exposed, and in a bright bottle that was sun-exposed.The microbiological stability and physical and chemical analysis were performed immediately upon receipt in the laboratory and in a period of 7 and 15 days.Determination of the iodine value The iodine number is the number of milligrams of iodine adsorbed per gram of adsorbent vi .Pipette 0.16g of a Nigella sativa sample that has been dissolved in 15 ml chloroform into a "U1"-labeled iodination flask.Iodine monobromide reagent (25 ml) should be added to the flask.The flask's contents should be completely combined.The flask is then let to stand for a half-hour period in the dark.In another iodination flask, has been created a blank by adding 15 ml of chloroform to the flask.25 ml of Iodine Monobromide Reagent has been added to the blank, and the contents of the flask was thoroughly mixed.
following formula: IN -Iodine number (value) (mg I2/100 g oil) a -amount of 0.1 M sodium thiosulfate solution (ml) used for the blank test b -amount of 0.1 M sodium thiosulfate solution (ml) used for the main test, p -measured amount of sample (g) U2 -oil sample at room temperature Determination of the peroxide value The sample was treated in solution with a mixture of acetic acid and a suitable organic solvent and then with a solution of potassium iodide.The liberated iodine was titrated with a standard solution of sodium thiosulfate.Peroxide values are expressed either in milliequivalents of peroxide/kg or in millimoles of peroxide/L. 1 g of sample was added to a 250 mL Erlenmeyer flask, then 10 mL of the appropriate solvent mixture was added, followed by 0.2 mL of saturated potassium iodide solution, freshly prepared, and allowed to react for 60 seconds ± 1 second with manual vigorous stirring.After that, 20 mL of distilled water was added and titrated with the appropriate sodium thiosulfate solution (0.01 or 0.1 mol/L) using 1 mL of starch solution from purple to slightly yellow or colorless end point.The indicator should be added towards the end of the titration, but while a pale straw color is still present.During the titration, the flask was shaken until the blue color disappeared.A blind test was carried out under the same conditions.Determination of the acid value Acid number is a measure of the free fatty acid concentration.It measures the amount of unreacted acid remaining in the biodiesel fuel.It can also be expressed as an indicator of oxidized fuel.The acid value is defined as mg of NaOH required to neutralize the free fatty acids in 1 g of vegetable oil vii .A 3 g sample of Nigella sativa oil was weighed into a conical flask, then 50 ml of ethanol and ethyl ether (1:1) were added to another flask (150 ml).After that, 2-3 drops of the phenolphthalein indicator solution were added and neutralized by adding 0.1 M KOH until a light pink solution was formed.The neutralized ethanol was then added to the flask containing the sample.The burette was filled with 0.1 M KOH (standardized).The titration was started by adding a few drops of phenolphthalein indicator.The titration was carried out with vigorous mixing of the flask in order to obtain an accurate result.The titration is complete when the color of the solution changes to white-pink.Determination of microbiological stability of oilFive samples of Nigella Sativa oil were brought to the microbiological laboratory, the first of which was seeded on a non-selective medium CPS to test the sterility of the samples.After 24 hours, there was no growth on the substrates, and the sample is considered sterile.Other samples were used to test the effect of oil on pathogenic microorganisms.Testing the sensitivity of the Candida albicans reference strain (ATCC 90028) to Nigella sativa oil From a pure culture of the resistant strain of Candida albicans (ATCC 90028), which had previously grown on a solid medium, we made an inoculum in a physiological solution so that it contained 1x10⁶ to 5x10⁶ CFU/ml (0.5 MCFarland).After 15 minutes, we dipped a sterile swab (drained on the wall of the test tube) into the inoculum, and with the swab we covered the entire surface of the Muller-Hinton agar to which we added 2% glucose and methylene blue each time in all three directions, and left the plate open for a few minutes to dry the surface of the agar.After that, we dropped 4 drops of each oil onto the seeded and dried surfaces of M-H agar.We left the inverted M-H substrates in a thermostat for incubation for 24 hours at 37˚C.We worked on 1 MH agars for Nigella sativa oil.Testing the sensitivity of the reference E. coli strain (ATCC 25922) to Nigella Sativa oil From a pure bacterial culture of the reference strain E. coli (25922), which had previously grown on a solid medium, we made an inoculum in a physiological solution so that it contains 1-2x10⁸ CFU/ml (0.5McFarland).After 15 minutes, we dipped a sterile swab (drained on the wall of the test tube) into the inoculum, and with the swab we covered the entire surface of the Muller-Hinton agar each time in all three directions, and we left the plate open for a few minutes to let the agar surface dry.We repeated the procedure a total of 4 times and left the inverted M-H substrates in a thermostat for incubation for 24 hours at 37˚C.

Figure 1 .
Figure 1.shows the impact of storage conditions on the iodine value (mg I2/100 g) of Nigella sativa oil.From the graphic results, it is clearly noticeable that the iodine value changed over the storage of 15 days in relation to the freshly analyzed oil.The iodine value for fresh oil was 14.56 mg I2/100 g respectively.During storage of the oil in the incubator at 37 o C, the iodine value increased significantly, namely 27.72 and 98.95 mg I2/100 g.However, by storing the oil in a bright and dark bottle for 7 days, much higher values were obtained, which were above 90 mg I2/100 g.Better values were obtained by keeping the oil in a light bottle for 15 days compared to keeping it in a dark bottle.The highest recorded iodine value was for an oil sample that was kept at fridge temperature for 7 days and this value was 101.79 mg I2/100 g of Nigella sativa oil where we can conclude that the oil became more reactive, less stable, softer and more sensitive to oxidation.

Figure 2 .
Figure 2. Impact of storage conditions on the peroxide value (mmol O2/kg) of Nigella sativa oil

Figure 3 .Figure 3 .
Figure 3. Impact of storage conditions on the acid value (mg KOH/g) of Nigella sativa oil

Figure 4 .
Figure 4.The effect of Nigella sativa on the growth of C. Albicans Results: In our test, Nigella sativa samples kept in the sun in dark and light bottles are completely eliminated and prevented the growth of the reference E. coli strain.Other samples did not show sensitivity to Nigella sativa oil, and inhibition zones did not appear.Other authors also showed a positive effect of Nigella sativa on the elimination of E. Coli xi , xii , xv , xvi .

Figure 5 .Figure 6 .
Figure 5.The effect of Nigella sativa on the growth of E. coli (The sample is stored in a dark bottle)

Table 1 .
Initial physicochemical characteristics of Nigella Article 17. Quality requirements for edible unrefined vegetable oils and edible cold-pressed vegetable oils viii .