Antioxidants In Foods Natyral Antioxidants Biology Essay

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Oxidation is a well-known chemical reaction detrimental to health and food. Natural antioxidants are well known for their health effects. Their use is getting more and more extensive in food industry and nowadays, synthetic antioxidants are substituted by natural ones. This is strongly recommended by modern nutritionists due to the health benefits associated with the consumption of antioxidant compounds present in human diet (new health claims in European Union). The oxidation of fats and oils causes serious damages in taste, odor and finally nutritional value. Thermal stressing of vegetable oils, destroys valuable components such as tocopherols and polyphenols. In this study, the addition of some natural antioxidants and its effect on the extra virgin olive oil quality was investigated. To measure antioxidant capacity, ethanolic extracts of natural antioxidants from two different plants, namely sage and oregano were added in extra virgin olive oil in concentrations of 0.02% w/w. The olive oil samples were stressed at 75 0C for 5 days and at 120 0C for 6 hours. At regular time intervals small samples were removed from the reaction vat and their peroxide value was determined. Each thermal stressing for the different mixtures was repeated twice. The state of oxidation was checked by measurement of Peroxide Value (PV) by a volumetric titration method officially accredited for the determination of peroxides produced during oxidation and thermal stressing of vegetable oils. Each determination was carried out five times. The temperature of 120 0C was used in order to accelerate olive oil oxidation and check antioxidant behavior of plant extracts at higher temperatures. Experimental results showed satisfactory antioxidant protection of extra virgin olive oil at both 75 0C and 120 0C for both natural extracts used

Word count: 282

Table of Contents

Abstract………………………………………………………………………………..2

Introduction……………………………………………………………………………4

A. Oxidation and antioxidants…………………………………………………4

Antioxidants in Foods-Natyral antioxidants………………………….……5

Antioxidants extraction from plants………………………………………..8

B. Choice of model…………………………………………………………....9

Olive Oil……………………………………………………………………9

Health effects..…………………………………………………………….10

C. Oxidation of olive oil……………………………………………………...11

Autooxidation Mechanism………………………………………………...12

About the essay………………………………………………………………….…...15

Experimental…………………………………………………………………………16

General preparatory work……………………………………………………………16

Experiment 1: Determination of Peroxide Value of thermally stressed virgin olive oil and virgin olive oil enriched with antioxidant extracts at 75 0C……………………..17

Methods………………………………………………………………………19

Results………………………………………………………………………..21

Experiment 2: Determination of Peroxide Value of thermally stressed virgin olive oil and virgin olive oil enriched with antioxidant extracts at 120 0C …………………...25

Methods………………………………………………………………………26

Results………………………………………………………………………..26

Discussion of results………………………………………………………………….30

Conclusion...……………………………….…………………………………………31

Evaluation………………………………………………………………….…………32

Bibliography………………………………………………………………………….33

Appendices…………………………………………………………………………...34

Appendix 1: Preparing aqueous starch solution 1% w/v……………………..34

Appendix 2: Preparation of aqueous standard solution of Na2S2O3 0.0100N..34

Appendix 3: Preparation of aqueous saturated KI…...……………………….35

Introduction

Oxidation and Antioxidants

Oxidation is defined as the loss of at least one electron from a substance. The electrons that are lost must then be gained by another substance. The process involving the gain of one or more electrons by a substance is called reduction. In processes where electron transfer is involved oxidation and reduction must occur simultaneously. Such types of reactions are referred to as redox reaction.

Oxidation may occur in many different types. One of the main types of oxidation is air oxidation where a molecule of oxygen (O2) oxidizes a chemical compound. An example of air oxidation is the oxidation of glucose where one molecule of glucose reacts with six oxygen molecules to produce six molecules of carbon dioxide and six molecules of water (C6H12O6 + 6O2  6CO2 + 6H2O). An oxidation reaction may also involve the creation or free radicals, which then start the commonly known "chain reactions"

In order to prohibit foods from oxidizing quickly industries all over the world use antioxidants. Antioxidants are therefore organic or inorganic compounds that decrease the rate at which a food is oxidized. Specifically, antioxidants are used in order to destroy the "chain reactions" which are created, as aforementioned, from the free radicals actualized by oxidation reactions. In order for these reactions to be destroyed antioxidants remove the free radicals thus preventing other oxidation reactions of happening. These compounds thus, prevent the presence of fatty materials. Mechanism of the way they react in oxidation of fatty lipids will be described later on this essay.

Antioxidants in Foods and Natural Antioxidants

The most common antioxidants legally allowed to be used in foods are the following ones:

Ascorbic acid (vitamin C)

Butyl-hydroxy-anisol (BHA) up to 0.02 % w/w.

Butyl-hydroxy-toluene (BHT) up to 0.02 % w/w.

a-tocopherol

Esters of gallic acid (propyl-, octyl-, decyl-)

Different vegetables, fruits, cereals, etc all contain various amounts of natural antioxidants inside them. Antioxidants' stability varies with each antioxidant. Specifically, some antioxidants such as lycopene and ascorbic acid can be destroyed more easily by prolonged cooking or by long term storage. Conversely, other antioxidants such as polyphenolic antioxidants in various types of foods tend to be more stable and thus are not so easily destroyed. However, the effects of cooking, food processing and long term food storage cannot be easily determined and predicted because all three processes may affect the bioavailability of antioxidants. Characteristic examples are some carotenoids in vegetables. Finally, we may conclude that generally fresh foods, vegetables and fruits contain a greater number of antioxidants compared to processed and prolonged cooked foods. Some of the most common natural antioxidants found are shown in the table that follows.

Antioxidant compounds

Foods containing high levels of these antioxidants

Chemical Formulas

Vitamin C (ascorbic acid)

Fresh Fruits and vegetables

Vitamin E (tocopherols, tocotrienols)

Vegetable oils

Polyphenolic antioxidants (resveratrol, flavonoids)

Tea, coffee, soy, fruit, olive oil, chocolate, cinnamon, oregano

Carotenoids (lycopene, carotenes, lutein)

Fruit, vegetables and eggs

Antioxidants are used all over the world in nutritional supplements and are furthermore tested to check if they prevent diseases such as cancer and altitude sickness. Initially, studies done showed that antioxidants had positive health effects. However, clinical studies, done later on, showed that antioxidants not only had limiting positive health effects but could also be harmful if present in large quantities. Moreover, antioxidants have many important industrial uses. Specifically, antioxidants may act as preservatives in all types of different foods and may also be used to prevent degradation in both rubber and gasoline. Nowadays, industries tend to replace synthetic antioxidants, which can be harmful for an individual's health, with natural antioxidants extracted from various plants. Some of the plants examined in this essay are shown in following pictures.

Fig. 1 Oregano

Fig. 2 Sage

Fig. 3 Mountain tea (sideritis)

Antioxidants extraction from plants

The isolation of natural antioxidants from plant material is effected by extraction with various solvents such as methanol, ethanol, water and mixtures thereof. Many researchers reported that sonication or microwave combined with solvent extraction increase the efficiency of isolation of natural antioxidants and produce useful mixtures of antioxidants possessing high antioxidant capacity [4, 5]. After extraction is accomplished, solvent has to be evaporated. Using a rotary evaporator usually effects this. In this way, extracts from plant material are in solid state for easier handling and ready to be used for experiments or for addition to foods.

Choice of model

Olive Oil

"Olive oil is a fat obtained from the olive (the fruit of Olea europaea, family Oleaceae), a traditional tree crop of the Mediterranean Basin" [1] . Oil is most commonly used in cooking, but is also used for the production of cosmetics, pharmaceuticals and various kinds of soaps. The use of olive oil is very common throughout the whole world. Specifically, it was firstly used (over 5,000 years ago) in Mediterranean countries such as Spain, Malta, Greece, Italy, France and many more. Thus, as expected the three greater olive oil producers in the world are Spain, Italy and Greece which all together hold more than 75% of the global oil production. Oil can be sub-categorized as follows:

Virgin olive oil

Refined oil

Olive pomace oil

Fig. 6 Virgin olive oil

Health effects

Olive oil has a lot of positive health effects. The most important are analyzed below.

The first health effect of oil arises due to the presence of monounsaturated fats. Specifically, evidence occurring from various epidemiological studies have shown that this type of fats, do not prevent, but reduce significantly, the risk of coronary heart diseases.

Another health benefit of olive oil arises from the fact that oil is able to remove/change omega-6 fatty acids without needing to involve the use of omega-3 fatty acids. Thus, omega-3 fatty acids remain unchanged during the process of displacement of omega-6 fatty acids. This way, a greater balance between omega-6 fatty acids and omega-3 fatty acids is created in an individual's organism making it significantly more stable and healthy. Finally, oil due to this property is able to lower blood sugar levels and blood pressure.

Olive oil contains wide variety of valuable antioxidants such as vitamin E, carotenoids, hydroxyltyrosol etc. Hydroxytyrosol is thought to be the main antioxidant compound in olives, and believed to play a significant role in many health benefits attributed to olive oil. Furthermore, epidemiological studies suggest that olive oil has a protective effect against certain malignant tumors in the breast, prostate, endometrium and digestive tract. Research has revealed that the type rather than the quantity of fat seems to have more implications for cancer incidence. The above mentioned reasons and the fact that olive oil is the main oil of Mediterranean diet led us to the choice of virgin olive oil in the model system to study oxidative capacity of herbs and spices [6].

Fig.7 Hydroxytyrosol

Oxidation of olive oil

Olive oil is oxidized when it comes in contact with molecular oxygen (O2). Oxidation or oxidative rancidity in olive oil has been known for many years. It was also known that certain substances retarding oxidation (antioxidants) are present in the plant issue. Oxidation products (peroxides, carbonylic compounds) have an unpleasant flavor and odor and may adversely affect the nutritional value of the oil. Essential fatty acids, such as linoleic and linolenic, are destroyed and certain fat-soluble vitamins disappear. Olive oil is relative resistant to oxidation (autoxidation), compared to other types of oils, because of the low content of polyunsaturated fatty acids and because it contains natural antioxidants. However it is very sensitive to photooxidation. The oxidation of olive oil and other lipids is affected by a number of factors such as: amount of oxygen, temperature, light ionizing radiation and presence of metals [7,8].

Fig. 8 Linoleic acid Fig. 9 Linolenic acid

Autooxidation Mechanism

The oxidation mechanism is rather complicated. It is believed that autooxidation proceeds in three stages: Initiation, propagation and termination. The following reactions describe the oil oxidation:

Initiation

RH + O2 R. + . OOH

Propagation

R. + O2 ROO.

ROO. + RH R. + ROOH

Termination

R. + R. RR

R. + ROO. ROOR Nonradical products

ROO. + ROO. ROOR + O2

Where RH= Fatty acid, R. = Fatty acid free radical, ROO. = Peroxy free radical, ROOH = Hydroperoxides

Initiation: In this stage hydrogen is abstracted from an olefinic acid molecule (RH) and a free radical is formed (R.). This radical reacts with an oxygen molecule and a peroxide radical is formed (ROO.). The peroxy radical reacts with another fatty acid molecule (RH) thus producing more peroxides (ROOH) and new free radicals. The activation energy required for this process originates either from high temperature or light or some other source. The reactions at initiation stage proceed at a slow rate. The duration of the initiation stage varies among the different fatty substances and is related to unsaturation and to the presence or absence of natural antioxidants.

Propagation: It follows the initiation stage when the oil acquires a rancid flavor. The oxidation process is now more complicated since the peroxides formed, being unstable, are easily broken forming more free radicals which in turn participate in new reactions. This chain reaction continues until either the unsaturated compound has been exhausted or the free radicals have inactivated each other.

Termination: The mutual annihilation of free radicals is known as the termination stage. The automatic termination of oxidation is difficult, since it is not likely for all free radicals formed to inactivate each other. It is possible however to accelerate the termination, before the oxidation is advanced, by adding antioxidants which may inhibit lipid oxidation by reacting with the free radicals and breaking the chain reaction. The antioxidants act as hydrogen donors resulting in the formation of antioxidants free radicals.

AH + ROO. A. + ROOH

Where AH=antioxidant, ROO. =free radical, A. =antioxidant free radical, ROOH=hydroperoxide

The antioxidant free radicals react in two ways:

A. + A. AA

ROO. + A. ROOA

It is obvious from these two reactions that antioxidant radicals either annihilate each other or react with a peroxide free radical [7,8].

Fig. 10 Usual chemical formulas of Peroxides

About the essay

The aim of this essay was to assess the Peroxide Value of thermal treated virgin olive oil so as to check if any antioxidant capacity is observed when different ethanolic extracts from two plants (herbs and spices: oregano and sage) were added in virgin olive oil [9,10,11].

Pure virgin olive oil samples enriched with plant extracts, that are above mentioned (at a concentration of approximately 0.02% w/w), will be thermally stressed at 75 0C for 5 days in an oven. From each type of enriched olive oil, samples will be taken out every 24 hours. The peroxide value will be determined for each case. From this experiment we will evaluate if these olive oil samples, enriched with the plant extracts mentioned above, exhibit lower Peroxide Value in comparison with those of pure virgin olive oil after same time of thermal stressing. Lower peroxide values indicate antioxidant capacity of ethanolic extracts.

Afterwards the effect of higher temperatures on lipid oxidation will be investigated. Thus, samples containing plain olive oil and olive oil enriched with antioxidants will be thermally stressed at 120 0C for 6 hours. Oil samples will be taken out every two hours and their Peroxide Values will be assessed.

It has to be mentioned that all samples of olive oils will be treated in the same way at both temperatures examined in order to ensure good repeatability of the results. All measurements of lipid oxidation at 75 0C and at higher temperature of 120 0C will be perfomed in 5 replicates.

In order to achieve reproducible results thermal stressing of olive oil samples was carried out in identical beakers so as to ensure the same surface contact of thermally stressed olive oil with air oxygen. Oxidation proceeds very quickly if larger surfaces are exposed. To avoid deviations of temperature heating accurate thermometers were used, as temperature is an important factor of vegetable oil oxidation.

It has to be noted that at 120 0C both plant antioxidant material were dissolved easily in olive oil. This was attributed to this high temperature used. On the contrary only, sage was fully dissolved at 75 0C.

Experimental

General preparatory work

two plant materials were purchased from a super market: oregano and sage. All materials chosen were already grounded and dried so as to avoid presence of moisture in the samples that would cause problems with accurate weighing the analyzed samples.

Extra Virgin olive oil (Minerva S.A.) was purchased from the supermarket. The plastic bottles of this oil were in the inner side of the shelf of the super market so as to avoid photo oxidation because of their exposion to light.

Preparation of solutions:

Solution of 1% w/w starch (see Appendix 1).

Stock solution of Na2S2O3 0.0100N (equivalents/L) was prepared and used as titrating solution (see Appendix 1).

Mixture of chloroform (CHCl3)/ acetic acid (CH3COOH in a ratio of 2:3 v/v (see Appendix 2).

Saturated aqueous potassium iodide solution (see Appendix 3).

Fig.11 Acetic Acid Fig.12 Chloroform Fig. 13 Sodium Thiosulfate

Fig. 14 Starch: Amylose and Amylopectin

Experiment 1: Determination of Peroxide Value of thermally stressed virgin olive oil and virgin olive oil enriched with antioxidant extracts at 75 0C.

Aim: The aim of this experiment was to determine the peroxide values of all thermally stressed olive oil samples and measure the quantity of produced peroxides. From these findings of antioxidant capacity of the extracts will be evaluated and its effect on the protection virgin olive oil from oxidation.

Materials and apparatus

Electronic analytical weighing balance, ±0.0001 g

Six 50 mL glass beakers

Filter paper

Spatula

Temperature controlled oven

Ultrasound water bath

Rotary evaporator

Burette

Conical flasks of 250mL

Plastic Pasteur pipette

Cotton

Plants samples: oregano and sage

Volumetric cylinders and pipettes

Chemicals reagents used:

1% w/w starch aqueous solution

Sodium thiosulfate (Na2S2O3) aqueous solution of 0.0100N

Chloroform CHCl3, analytical grade

Glacial acetic acid CH3COOH, analytical grade

Ethanol, analytical grade

Deionised water from ion exchange column

Saturated aqueous potassium iodide solution.

Fig. 15 Burette Fig. 16 Conical flask

Method:

Blank olive oil sample

Pure olive oil sample was the thermally stressed. 40.00 g of virgin olive oil was weighed in analytical balance in a 50 mL glass beaker and placed in an oven under constant thermal heating at 75 0C for 5 days. Samples were taken out every 24 hours and their peroxide value was determined.

Oil samples enriched with plant extracts

For each sample (two in total) the following procedure was followed:

40.00 g of virgin olive oil was weighed in analytical balance in a 50 mL glass beaker. 0.0100 g of one of the plant antioxidative extracts was added (0.02 % w/w) and the beaker was placed in an oven of 75 0C. Samples were taken out every 24 hours and their Peroxide Value was determined.

Peroxide Value Method

H+The most common method to determine peroxides produced during thermal stressing is the Peroxide Value (PV). PV is measured in miliequivalents of molecular oxygen per kilogram of oil (meq O2/ Kg oil). The official method of PV assessment [12] is based on a volumetric determination of iodide that peroxides release from a saturated solution of potassium iodide (KI). Chemistry of the method is described by following reactions:

2KI + 2RCOOH 2HI + 2RCOOK

ROOH + 2HI ROH + I2 + H2O

2 Na2S2O3 + I2 Na2S4O6 + 2NaI

V x T x 10002-5 g of olive oil sample was weighed in analytical balance in 250 mL conical flasks. 25 mL of 2:3 (v/v) CHCl3: CH3COOH solution was added with a 25 volumetric cylinder. The oil was well mixed and after 1.00 mL of saturated KI solution [13] was added by pipette of 1.00 mL. The whole solution was stirred for approximately one minute, sealed with cotton and was kept in dark for 5 minutes. Afterwards 75 mL of deionised water was added with 100 mL volumetric cylinder. The sample was titrated with aqueous solution of Na2S2O3 0.0100N. Titration continues up to the point that the yellow color of titrated solution disappears. At this point 4-5 drops of 1 % w/w starch solution was added with a plastic Pasteur pipette. During the titration a deep blue color is formed [14]. Titration continues until the decolorisation of titrated solution. Volume of consumed aqueous solution of Na2S2O3 is recorded. Peroxide Value is calculated from the following mathematical equation:

m

PV=

Where:

V = volume in mL of the standard solution of aqueous solution of Na2S2O3 0.0100N consumed for the olive oil sample titration.

T = title of standard aqueous solution of Na2S2O3 equal to 0.0100N

m = weight of olive oil sample in g

Results:

PV for blank sample (pure virgin olive oil) is presented in the following table:

Sample

mass (g)

V(mL) Na2S2O3

PV

Average PV

SD

%SD

1 day

1

5.14

8.200

15.95

15.48

?

?

2

5.11

7.900

15.46

3

5.09

7.800

15.32

4

5.04

7.650

15.18

5

5.07

7.850

15.48

2 days

1

5.07

8.900

17.55

17.49

?

?

2

5.16

9.200

17.83

3

5.03

8.650

17.20

4

5.08

9.050

17.82

5

5.02

8.550

17.03

3 days

1

5.05

10.50

20.79

20.85

?

?

2

5.02

10.45

20.82

3

5.11

10.70

20.94

4

5.08

10.55

20.77

5

5.09

10.65

20.92

4 days

1

5.00

12.85

25.70

25.86

?

?

2

5.11

13.30

26.03

3

5.08

13.20

25.98

4

5.03

12.95

25.75

5

5.05

13.05

25.84

5 days

1

5.07

15.50

30.57

30.54

?

?

2

5.02

15.25

30.38

3

5.05

15.40

30.50

4

5.14

15.80

30.74

5

5.03

15.35

30.52

PV for oil sample enriched with oregano extract are presented in the following table. PV curves for olive oil sample (blank) and oil enriched with oregano extract are presented in Graph 1.a:

Sample

mass (g)

V(mL) Na2S2O3

PV

Average PV

SD

%SD

1 day

1

5.04

7.300

14.48

14.78

?

?

2

5.07

7.450

14.69

3

5.12

7.650

14.94

4

5.18

7.800

15.06

5

5.09

7.500

14.74

2 days

1

5.05

8.200

16.24

16.37

?

?

2

5.15

8.550

16.60

3

5.02

8.100

16.13

4

5.08

8.350

16.44

5

5.12

8.400

16.41

3 days

1

5.07

9.750

19.23

19.44

?

?

2

5.17

10.20

19.73

3

5.02

9.600

19.12

4

5.09

9.900

19.45

5

5.13

10.10

19.69

4 days

1

5.01

12.15

24.25

24.50

?

?

2

5.04

12.30

24.41

3

5.02

12.20

24.30

4

5.12

12.70

24.81

5

5.08

12.55

24.71

5 days

1

5.04

14.80

29.37

29.60

?

?

2

5.10

15.25

29.90

3

5.08

15.05

29.63

4

5.02

14.70

29.28

5

5.14

15.30

29.77

Graph 1.a

PV for oil sample enriched with sage extract is presented at following table.

PV curves for olive oil (blank) sample and oil enriched with sage extract are presented in Graph 1.b:

Sample

mass (g)

V(mL) Na2S2O3

PV

Average PV

SD

%SD

1 day

1

5.25

7.300

13.91

13.92

?

?

2

5.03

6.950

13.82

3

5.08

7.050

13.88

4

5.09

7.100

13.95

5

5.12

7.200

14.06

2 days

1

5.11

8.100

15.85

15.64

?

?

2

5.03

7.700

15.31

3

5.05

7.800

15.45

4

5.08

8.000

15.75

5

5.15

8.150

15.83

3 days

1

5.04

9.500

18.85

18.91

?

?

2

5.02

9.350

18.63

3

5.15

9.800

19.03

4

5.08

9.600

18.90

5

5.17

9.900

19.15

4 days

1

5.28

12.60

23.86

23.54

?

?

2

5.17

12.20

23.60

3

5.08

11.85

23.33

4

5.03

11.65

23.16

5

5.12

12.15

23.73

5 days

1

5.02

14.40

28.69

28.80

?

?

2

5.08

14.65

28.84

3

5.04

14.55

28.87

4

5.15

14.80

28.74

5

5.09

14.70

28.88

Graph 1.b

PV for oil sample enriched with mountain tea extract are presented at following table. PV curves for olive oil (blank) sample and oil enriched with mountain tea extract are presented in Graph 1.c:

Sample

mass (g)

V(mL) Na2S2O3

PV

Average PV

SD

%SD

1 day

1

5.01

7.100

14.17

14.45

?

?

2

5.05

7.250

14.36

3

5.12

7.500

14.65

4

5.07

7.350

14.50

5

5.15

7.500

14.56

2 days

1

5.02

8.500

16.93

17.05

?

?

2

5.14

8.750

17.02

3

5.08

8.700

17.13

4

5.05

8.600

17.03

5

5.17

8.850

17.12

3 days

1

5.14

10.45

20.33

20.15

?

?

2

5.02

9.950

19.82

3

5.05

10.15

20.10

4

5.07

10.25

20.22

5

5.10

10.35

20.30

4 days

1

5.05

12.60

24.95

25.02

?

?

2

5.12

12.85

25.10

3

5.02

12.50

24.90

4

5.15

12.95

25.15

5

5.08

12.70

25.00

5 days

1

5.02

14.55

28.98

29.07

?

?

2

5.08

14.80

29.13

3

5.15

15.05

29.22

4

5.07

14.65

28.90

5

5.12

14.90

29.10

Graph 1.c

Experiment 2: Determination of Peroxide Value of thermally stressed virgin olive oil and virgin olive oil enriched with antioxidant extracts at 120 0C.

Aim: The aim of this experiment was to determine the peroxide values of all thermally stressed olive oil samples and measure the quantity of produced peroxides at a much higher temperature. From these findings of antioxidant capacity of the extracts will be evaluated and its effect on the protection virgin olive oil from oxidation.

. Materials and apparatus

Electronic analytical weighing balance, ±0.0001 g

250 mL spherical flasks

Mercury thermometers

Burette

Conical flasks of 250mL

Plastic Pasteur pipette

Cotton

Volumetric cylinders and pipettes

Spatula

Chemicals reagents used:

1% w/w aqueous starch solution

Sodium thiosulfate (Na2S2O3) aqueous solution of 0.0100N

Chloroform (CHCl3) , analytical grade

Glacial acetic acid (CH3COOH) , analytical grade

Deionised water from ion exchange column

Saturated potassium iodide ( aqueous solution).

Method

60,00 g of plain virgin olive oil and virgin olive oil plus 0.0120 g of the natural antioxidant were added in spherical flask and the whole was thermally stressed on heating mantles at 120 0C for 6 hours (3 oil samples in total). To calculate temperature mercury thermometers were used. Oil samples are taken away every two hours and PV was determined as described in experiment 1.

Results:

The results recorded from the sample containing the plain olive oil are presented at following table

Sample

mass (g)

V(mL) Na2S2O3

PV

Av. PV

SD

%SD

2h

1

5.03

13.15

26.14

26.42

?

?

2

5.21

13.90

26.68

3

5.03

13.20

26.24

4

5.04

13.30

26.39

5

5.12

13.65

26.66

4h

1

5.15

27.05

52.52

52.51

?

?

2

5.12

26.90

52.54

3

5.02

26.50

52.79

4

5.09

26.75

52.58

5

5.05

26.30

52.10

6h

1

1.37

8.500

62.04

62.62

?

?

2

1.42

8.900

62.68

3

1.80

11.25

62.50

4

1.30

8.200

63.10

5

1.68

10.55

62.80

Results:

Results for oil sample enriched with oregano extract are presented at following table.

PV curves for olive oil sample (blank) and oil enriched with oregano extract thermally stressed at 120 0C are presented in graph 3.a:

Sample

mass (g)

V(mL) Na2S2O3

PV

Av. PV

SD

%SD

2h

1

5.01

10.10

20.16

20.59

?

?

2

5.16

10.80

20.93

3

5.15

10.70

20.78

4

5.03

10.25

20.38

5

5.07

10.50

20.71

4h

1

5.10

23.30

45.69

45.77

?

?

2

5.16

23.70

45.93

3

5.12

23.45

45.80

4

5.08

23.25

45.77

5

5.05

23.05

45.64

6h

1

1.36

7.100

52.21

52.60

?

?

2

1.30

6.850

52.70

3

1.38

7.300

52.90

4

1.31

6.900

52.67

5

1.41

7.400

52.48

Graph 3.a

120 0C

Results for oil sample enriched with sage extract are presented at following table.

PV curves for olive oil sample (blank) and oil enriched with sage extract thermally stressed at 120 0C are also presented in graph 3.b:

Sample

mass (g)

V(mL) Na2S2O3

PV

Av. PV

SD

%SD

2h

1

5.03

12.65

25.15

25.47

?

?

2

5.20

13.50

25.96

3

5.06

12.80

25.30

4

5.07

12.85

25.35

5

5.12

13.10

25.59

4h

1

5.07

26.10

51.48

51.38

?

?

2

5.05

25.90

51.29

3

5.11

26.35

51.57

4

5.14

26.50

51.56

5

5.02

25.75

51.30

6h

1

1.70

10.50

61.77

61.65

?

?

2

1.55

9.550

61.61

3

1.20

7.400

61.67

4

1.87

11.45

61.23

5

1.63

10.10

61.96

Graph 3b.

120 0C

Results for oil sample enriched with mountain tea extract are presented at following table. PV curves for olive oil sample (blank) and oil enriched with mountain tea extract thermally stressed at 120 0C are also presented in graph 3.c:

Sample

mass (g)

V(mL) Na2S2O3

PV

Av. PV

SD

%SD

2h

1

5.05

14.85

29.41

29.59

?

?

2

5.12

15.30

29.88

3

5.08

15.05

29.63

4

5.02

14.60

29.08

5

5.14

15.40

29.96

4h

1

5.05

28.80

57.03

57.41

?

?

2

5.09

29.15

57.27

3

5.12

29.60

57.81

4

5.02

28.70

57.17

5

5.16

29.80

57.75

6h

1

5.14

30.90

60.12

60.41

?

?

2

5.07

30.60

60.36

3

5.09

30.75

60.31

4

5.02

30.50

60.76

5

5.10

30.85

60.49

Graph 3c.

120 0C

Discussion of results:

Results from experiment 1 (75 0C) showed antioxidant capacity of extracted plant material added into virgin olive oil. More specifically sage extracts showed the lowest peroxides values in all 5 days, i.e. the highest antioxidant capacity compared to other plant extracts added in virgin olive at the low temperature of 75 0C and for the same time of thermal stressing. Oregano and mountain tea extracts also showed satisfactory antioxidant capacity. Specifically in days 2, 3, 4 oregano extracts displayed a lower peroxide value than mountain tea extracts whilst in days 1 and 5 the opposite happened.

In experiment 2 a higher temperature of 120 0C was chosen. At this temperature peroxide values were significantly increasing along the time of thermal heating, as oxidation was accelerated and completed in 6 hours instead of 5 days needed for the low temperature of 75 0C thermal stressing. In this experiment we saw that not all plant extracts that show antioxidant protection at lower temperatures (75 0C) display the same kind of protection in higher temperatures (120 0C) also. Specifically, we saw that oregano extracts displayed a significantly lower peroxide value compared to mountain tea and sage extracts. Mountain tea now, did not display any kind of protection after 2 and 4 hours, however, after 6 hours it did display antioxidant protection with its peroxide value being lower than that of the sage extracts. Sage extracts finally, in all three time intervals, displayed a satisfactory antioxidant protection.

Conclusions

Both virgin olive oil samples enriched with plant extracts showed antioxidant capacity at 75 0C. Sage extracts were the most resistant to oxidation at this temperature compared to those with oregano and mountain tea.

At 120 0C however, oregano showed the higher antioxidant capacity compared to that of sage and mountain tea.

Peroxide values may vary a lot as time progresses. For example in experiment 2, mountain tea extracts did not display any protection after 2 and 4 hours, however, after 6 hours they not only displayed antioxidant protection but this protection was better than that of sage extracts.

Volumetric measurements for PV determination were very reproducible based on analysis of 5 replicates with % standard deviation (% SD) varying from (0.1% to 1,1%)

Not all plant extracts that show antioxidant protection at lower temperatures (75 0C) display the same kind of protection in higher temperatures (120 0C) also.

Generally we may conclude that the best antioxidant of these three is oregano.

Evaluation:

The experiment was proved to be successful as most of extracts showed antioxidant capacity at low and high temperatures. Titrations were of precision and reproducible as PVs calculated were very close, thanks to purity of reagents, accurate weighing through use of analytical balance, careful preparation of reagents and stock solutions and accurate titration of sample thanks to precision to instrumentation used and the experience of the analyst.

Future work has to focus on following fields:

Test more plant antioxidants extracts

Add different quantities of antioxidant plant material and check which is the optimum and more effective concentration to apply (effect of concentration)

Test with similar method different types of oils such as kernel oil, soya bean oil, sunflower oil, refined oil etc and try to compare results

Try to keep the temperature of the environment where the experiment took place as stable as possible, i.e. avoid opening windows, doors etc.

Due to the limited number of flasks available the flasks used in the first experiment had to be cleaned and then re-used in experiment two. Thus, we can suspect that some flasks could not be perfectly clean and therefore affect the results recorded.

Finally the oven used wasn't pro-heated(δεν τον είχα προθερμάνει) at 75 and 120 degrees at experiments one and two and therefore the first samples taken in each experiment were in the oven less than the other samples at the desired temperatures as the oven needed some time in order to reach the optimum temperatures (75 0C and 120 0C).

Flasks were not all the same type?

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