"Production of an olive oil with increased antioxidants properties, enriched with the extraction of Saffron {Crocus sativus £»)".

Saffron {Crocus sativus L.) is commonly known as the saffron crocus. Crocus is a perennial herb, which belongs to the Iridaceae family and it is widely cultivated in Iran and other countries, such as India and Greece (Rios JL et al 1996). The saffron contains various compounds, which act as carriers of multi-sensory properties, while it is widely used as a spice and food additive. It has a strong yellow-to-orange color and flavor, due to water-soluble glycosides and the volatile substance, safranal.

The saffron appears in market as dried red stigmas and powder. It has been shown that its compounds are pharmacologically active (Rios JL et al 1996). Latest pharmacological studies showed that the saffron and its active components present: anticonvulsant (Hosseinzadeh H et al 2002a), antidepressant (Hosseinzadeh H et al 2004), anti-inflammatory (Hosseinzadeh H et al 2002b) and antineoplastic effects (Abdullaev FJ et al 1996), as well as free radical scavenger activity (Zhang YX et al 1999).

Despite its use as a food colouring, it could also be used as a source of antioxidants, which, in turn, can improve the quality of foods (functional foods), drinks, beverages, and pharmaceuticals and cosmetics.

1. Methods of extraction

A) Methanolic extract: Dried powder and stigmas of Saffron {Crocus sativus L.) were used for this protocol. In detail, 40g of dried powder or stigmas were placed in a sterile volumetric flask and then 2000 ml of methanol 80%, were added. The solution was continuously under gently mixing for 24 h and then filtrated, using filter papers, in order to remove any microorganism. The filtrated solution was transferred to a flash evaporator and methanol was totally vaporized, under vacuum and low temperature. Finally, the desiccated sediment was collected into a sterile glass bottle and stored for further use. B) Ethanolic extract: Dried powder and stigmas of Saffron (Crocus sativus L.) were used for this protocol. In detail, 40g of dried powder or stigmas were placed in a sterile volumetric flask and then 2000 ml of ethanol 80%, were added. The solution was continuously under gently mixing for 24 h and then filtrated, using filter papers, in order to remove any microorganism. The filtrated solution was transferred to a flash evaporator and ethanol was totally vaporized, under vacuum and low temperature. Finally, the desiccated sediment was collected into a sterile glass bottle and stored for further use. C) Aqueous extract: Dried powder and stigmas of Saffron (Crocus sativus L.) were used for this protocol. In detail, 50g of dried powder or stigmas were placed in a sterile volumetric flask and then 1000 ml of boiled water, were added. The solution was continuously under gently. mixing for 15 min (method based on Gulcin I et al., 2004). The aqueous extract filtrated, using filter papers, in order to remove any microorganism and. then water was removed, through vaporization, using a flash evaporator. Finally, the desiccated sediment was collected into a sterile glass bottle and stored for further use.

The following table represents the yields of the three mentioned methods of extraction:

2. Enrichment of olive oil (all different types) with the saffron extracts

The use of new innovative methods, allows us to integrate a dehydrated extract in various types of olive oil, which seemed to be impossible a few years ago. This is achieved by reducing the specific weight of each grain of powder extract, to avoid its precipitation and thus to achieve a homogenous solution. The methodology is as follows: the size of each grain of the extract is being reduced by an oil mill, i.e. a mill which uses olive oil as grinding fluid. Through absorption of the olive oil inside the pores of each grain of the extract, grain and olive oil specific weight reduction is achieved. Thus, the desirable integration of the extract-powder in different types of olive oil is accomplished. Also, via the extract grinding, new charged surfaces were created, which were being repulsed electrostatically. This leads to a homogenous solution, without any sediment.

Twelve different dilutions of the extract, of both powder and stigmas, were prepared: 3 in olive oil (or pure olive oil), 3 in virgin olive oil, 3 in extra virgin olive oil and 3 in double-distilled water at the following concentrations: 0.5, 1 and 2 mg / ml of olive oil (or pure olive oil), virgin and extra virgin olive oil and double-distilled water.

3. Total antioxidant capacit of the extracts

For the estimation of total antioxidant capacity (TAC) of solutions, the Blue Cr0 ₅ assay was used according to Charalampidis et al (2009). This method is based on the use of chromium peroxide (Cr(0 ₂ ) ₂ H ₂ 0 or briefly Cr0 ₅ ) as an extremely potent oxidant. This compound is a product of the following reaction:

(NH ₄ ) ₂ Cr ₂ 0 ₇ + 4H ₂ 0 ₂ +2H ⁺ → 2Cr(0 ₂ ) ₂ H ₂ 0 + 4H ₂ 0 + ammonium salt (Reaction 1)

Chromium peroxide is a deep blue product, relatively stable in polar organic solvents with a maximum absorption wavelength at λ=569 nm in isoamyl alcohol. The acidic environment for the reaction was provided by acids, such as H ₂ S0 ₄ . When the products of the reaction 1 are mixed with an organic solvent, e.g. isoamyl alcohol, the formed Cr0 ₅ moves and dissolves in the organic phase (solution 1) of the bi-phasic solution. If the antioxidant compounds of a certain substance, added to the above solution 1 , inhibit the formation of the deep blue Cr0 ₅ . The extent of the inhibition of the colour formation in solution 1, measured by means of a spectrophotometer, represents the antioxidant capacity (or the oxidative status) of the sample under measurement. According to the above, the method has been calibrated in accordance to the concentration of a-tocopherol, as a prototype antioxidant agent.

In a 5 mL plastic capped cuvette, 1200 μί, of organic solvent (3-methylbutanol), 400 μΐ, of 0.02 M ammonium dichromate, 400 μΐ, of 0.025 M sulfuric acid solution and 20 μΐ, of the sample, were added. The cuvette was then inserted into the spectrophotometer and was incubated for a period of 3 min and the first absorbance reading was then taken. The cuvette was then removed from the photometer and 20 μΐ, of 1.6 M hydrogen peroxide solution was pipetted. After a mild stirring the cuvette was inserted again at the photometer and an absorbance reading was taken at 3 min time. For each sample there was a triplet of measurements. The same procedure was followed for blank measurements but instead of using 20 μΐ, of a sample we added 20 μΐ. of the organic solvent. All measurements were taken at a temperature of 37 °C and the spectrophotometer was set at λ = 569 nm. The antioxidant capacity of the sample is compared to that of 0.1 M a-tocopherol solution, and is measured in moles/It of a-tocopherol according to the following equation:

TACsample = [ ⁽ X-tOCOpherol] X [(AAbi _an k-AA _S a _ffl pie)/AAblank-AAstandard)]

4. Results of Total Antioxidant Capacity

The addition of the powder or the stigmas extracts from the plant Saffron (Crocus sativus L.) in different types of olive oil and in various concentrations significantly increased olive oil antioxidant properties. The following table summarizes the representative results of the methanolic extracts (solvent with the highest yield) in all types of olive oil and water.

TAC

(mM a-tocopherol)

Olive oil (or pure olive oil) (O) 14±3

a

0.5mg/ml CSL in O 41±1

lmg/ml CSL in O 60±3 ^a

2mg/ml CSL in O 66±5 ^a

Virgin olive oil (VO) 18±2 0.5mg/ml CSL in VO 32±4 ^b

lmg/ml CSL in VO 48±6 ^b

2mg/ml CSL in VO 53±5 ^b

Extra virgin olive oil (EVO) 21±1

0.5mg/ml CSL in EVO 23±3

lmg/ml CSL in EVO 40±2 ^C

2mg/ml CSL in EVO 44±2 ^C

0.5mg/ml CSL in water 16±4

lmg/ml CSL in water 49±8

2mg/ml CSL in water 54±9

a Statistically significant difference from O; Statistically significant difference from VO; ⁰ Statistically significant difference from EVO 5. Tasting

The food tasting is a process that includes the designing, analysis, description and identification of food characteristics, allowing the tester to decide on its quality.

Tools and methods

The "tools" of the tester are his senses. The environment should be simple, pleasant, sheltered from noises and odors, the tester must be isolated in order to avoid being influenced by the other testers, must use distilled water for mouth washing, must be in good physical condition, and should not be a smoker. The exemplary food test medium (spoon) and the plate must be constructed of materials that do not alter the taste or temperature.

The tastins includes the following steps:

A. Visual inspection of the surface of the food, on which we recognize immediately the color change of the various types of olive oil. The addition of the powder extract or the stigmas extract from the plant Saffron {Crocus sativus L.) in different types of olive oil and in various concentrations significantly changed the color. The higher concentration (depending on the original color of the different types of olive oil) resulted in the more intense color of the final solution. When the aqueous extract was added to food, it provided all the characteristic colorings of Saffron (Crocus sativus L), according to the final concentration.

B. First contact of the nasal with the surface of the food in order to perceive odors. (The tester has to smell the food before tasting it). In our sample, the sensation resulting from the smell of the sample is the pleasant and characteristic odor of Saffron (Crocus sativus L), as well as, the familiar smell of the different types of olive oil.

C. Second contact of the nasal after rotation of the test medium in order to appreciate the flavors that are not easily released. We can now recognize the characteristics of the various types of olive oil that remind us of fresh vegetables, fruits and even flowers, depending of course on the species of olives, the geographic location of the olive groves, the methods of farming and harvesting, how ripe or unripe were the olives when collected, and the storage procedure, along with the characteristic odor of Saffron (Crocus sativus L.) with slight scent of iodine.

D. Test - E. ingestion and analysis of food, by breathing hard a small amount of air from the mouth and for short time periods, positioning our tongue horizontally to assist the proper development and evaluation of flavors in our mouth.

Humans receive tastes through sensory organs called taste buds, concentrated on the top of the tongue. Only four flavors are perceived by the taste buds of the tongue. The distinctive sweet taste of Saffron (Crocus sativus L.) and that of the various types of olive oil becomes perceptible on the anterior part of the tongue, while the salty one, detection of which would be a negative characteristic, from the anterior and sides of the central axis. The sour taste, that is very discreet and in perfect balance in our sample, because of the high quality of the used types of olive oil combined with Saffron (Crocus sativus L), becomes perceptible from the sides and posterior part of the tongue, whereas the bitter taste, slightly stronger in our sample due to the combination of two slightly bitter products in it, is perceived mainly when swallowing. Finally, senses similar to touch are perceived on the tongue surface, in the inner side of the cheeks, and the gums and they inform us about viscosity, oily, temperature and volume of our sample.

F. Duration of taste perception. The duration of taste perception is very long due to the oily texture, the density and intense flavor of Saffron {Crocus sativus L), presented in our sample.

G. Description and assessment of the food. The fruity taste of the various types of olive oil combined with the slightly bitter taste of Saffron {Crocus sativus L), travel our senses in the flora of olive groves with the perennial olive trees and the strong aroma of fields, where the Saffron {Crocus sativus L.) grows.

It is estimated as a product of incomparable flavor which can accompany any recipe. It is also a healthy product with increased antioxidant properties.

6. References

Rios JL, Recio MC, Giner RM, Manez S. An update review of saffron and its active constituents. Phytother Res 1996; 10: 189-193.

Hosseinzadeh H, Khosravan V. Anticonvulsant effects of aqueous and ethanolic extracts of Crocus sativus L. stigmas in mice. Arch Irn Med 2002; 5: 44-47.

Hosseinzadeh H, Karimi Gh, Niapoor M. Antidepressant effects of Crocus sativus stigma extracts and its constituents, crocin and safranal, in mice. Acta Hort 2004; 650: 435^145. Hosseinzadeh H, Younesi HM. Antinociceptive and anti-inflammatory effects of Crocus sativus L. stigma and petal extracts in mice. BMC Pharmacol 2002; 2: 1- 8.

Abdullaev FJ. Biological effects of saffron. Biofactors 1993; 4: 83-86

Escribano J, Alonso GL, Coca-Prados M, Fernandez JA. Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro. Cancer Lett 1996; 100: 23-30.

Zhang YX, Sugiura M, Saito H, Shoyama Y. Acute effects of Crocus sativus L. on passive avoidance performance in mice. Biol Pharmacol Bull 1994; 17: 217— 221 / Abe K, Sugiura M, Ymaguchi S, Shoyama Y, Saito H. Saffron extract prevents acetaldehyde-induced inhibition of long-term potentiation in the rat dentate gyrus in vivo. Brain Res 1999; 851 : 287-289.

Gulcin, I.; Sat, I.G.; Beydemir, S.; Elmastar, M.; Kufrevioglu, O.I. Comparisan of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechs L.). Food Chem. 2004, 87, 393-400.

Charalampidis PS, Veltsistas P, Karkabounas S, Evangelou A. Blue Cr05 assay: A novel spectrophotometric method for the evaluation of the antioxidant and oxidant capacity of various biological substances. Eur J Med Chem 2009; 44: 4162-4168.