6The role of oxygen and waterin the extra-virginolive oil processBruno ZanoniDepartment of Agricultural, Food and Forestry System Management,University of Florence, Florence, Italy

Abstract

Oxygen and water play critical roles in the extra-virgin olive oil process. Some oxi-dation is essential for the development of positive sensory notes such as grassy andfruity flavours (the LOX pathway). At the same time, excessive oxidation leads to oilspoilage and the formation of sensory defects like rancidity. Similarly, the presenceof water is necessary for enzymatic reactions that modify the polarity of phenoliccompounds, allowing them to be transferred to the oil phase. However, excess waterin the oil can spur enzymatic or even microbial degradation during oil storage withirreversible loss of sensory and nutritional quality. Suitable adjusting of the balanceof oxygen and water is one of the most important control tools in the extra-virginolive oil process.

6.1 The conflicting roles of oxygen

In the box titled ‘Balance is everything’ concluding Chapter 3, it was pointed outhow water and oxygen play conflicting roles in the extra-virgin olive oil process,being, at the same time, the main factors of desirable and undesirable changes. Fora good control of the extra-virgin olive oil process, experts need to understand theconditions favouring / inhibiting the wanted / unwanted outcome.

Figure 6.1 represents a simplified scheme of oxidative reactions in the olive oilprocess. Unsaturated fatty acids, both free and esterified in triglycerides, react withoxygen under the action of lipoxygenase, forming hydroperoxides that are the ini-tiators of either desirable or undesirable changes.

The Extra-Virgin Olive Oil Handbook, First Edition. Edited by Claudio Peri.© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

70 CH06 THE ROLE OF OXYGEN AND WATER IN THE EXTRA-VIRGIN OLIVE OIL PROCESS

Triglycerides

Free fatty acids

Hydroperoxides

A cascade ofenzymaticreactions involvingisomerases,dehydrogenases,esterases,…leading to

aldehydes,alcohols, esters,and the fruity,grassy flavour

Autoxidation,photoxidation,thermoxidationleading to

aldehydes,ketones and therancid flavor

In olive duringharvesting andpost-harvesting

In olive paste,especially

duringmalaxation

In oil duringstorage

Lipase

Lipoxydase

The undesirableevolution

The desirableevolution

Anti-oxidants

Figure 6.1 The complex role of oxygen in the extra-virgin olive oil process.

The preferred reactant of the desirable changes is α-linolenic acid and the overallcascade reaction is called the lipoxygenase (LOX) pathway.

Lyases, enzymes that catalyse the breaking of chemical bonds by means otherthan hydrolysis and oxidation, break fatty acid molecules into shorter chain pieces.Alcohol-dehydrogenases cause the aldehydes to be reduced to alcohols. Acetyl-transferase catalyses the esterification of alcohols with acetic acid.

Hexenyl-acetate or, more precisely, a series of C6 compounds such as trans-2-hexenal, trans-2-hexen-1-ol and cis-3-hexenyl acetate, which are the possible prod-ucts of these reactions, are among the compounds of the most appreciated grassyand fruity flavours of extra-virgin olive oil.

6.2 THE ROLE OF WATER IN THE TRANSFORMATION OF PHENOLIC COMPOUNDS 71

All the reactions of the desirable transformations are enzyme-catalysed, so theycan only take place in the olive paste, during the milling process. Malaxing is thecritical operation for the formation of these flavour compounds and their transferfrom the aqueous phase to the oil phase. In fact, these molecules are soluble enoughin lipids for favourable oil-water partitioning.

If the initial oxidation of α-linolenic acid is inhibited, for instance, by operatingunder strictly inert atmospheric conditions, the LOX pathway is not initiated andimportant flavour notes will not develop.The undesirable changes also start from hydroperoxides but continue with a mech-

anism of autoxidation accelerated by light exposure (photoxidation) or high temper-ature (thermoxidation), if enough oxygen is available.

Such an autoxidative mechanism continues with formation of a mixture of highlyreactive OH* and O* radicals. The molecules break at the double bond with forma-tion of oxidized molecules, mainly aldehydes and ketones, with a perceivable rancidflavour.The undesirable changes can start from olives and olive paste if the olives have

been damaged mechanically or by pests. In this case, the good flavour componentsare overpowered by the bad ones and quality is irreversibly lost. The dotted linessuggest that reactants of the undesirable evolution can also come from products ofthe desirable evolution in an accelerated autocatalytic degradation path.

Autoxidation is inhibited by the absence of oxygen; photoxidation is inhibitedby the absence of light; thermoxidation is inhibited by storing the oil at optimumtemperature and enzymatic reactions are inhibited by elimination of enzymesand water. These considerations are the basis of suitable storage conditions, as isthoroughly discussed in Chapter 15. Antioxidants, especially phenolic compounds,intercept and neutralize reactive radicals, thus avoiding their involvement inoxidative degradation.

6.2 The role of water in the transformationof phenolic compounds

About 40 structurally different phenolic compounds have been identified in virginolive oils (Cicerale et al. 2009). The various olive cultivars have very differentamounts of phenolic compounds, but the distribution of the different phenolic cate-gories in the overall phenolic fraction is rather similar. Figure 6.2 presents a simpli-fied scheme of a possible evolution of phenolic compounds in the extra-virgin oliveoil process. The figure emphasizes the role of water in the process.The formulas in Figure 6.2 are drawn with slight differences from the formulas in

Chapter 3. They indicate the three-dimensional arrangement of atoms. Solid wedgedbonds point above the plane, while dashed wedged bonds point below the planeof the paper. The wavy symbol indicates unknown or unspecified stereochemistry.These are called “stereochemical” or “spacial” formulas.The first molecule in Figure 6.2 represents the most abundant phenolic com-

pounds in extra-virgin olive oil: oleorupein and ligstroside. They are a combination

72 CH06 THE ROLE OF OXYGEN AND WATER IN THE EXTRA-VIRGIN OLIVE OIL PROCESS

OO

OCH3

O

R = OH, oleuropeinR = H, ligstroside

Enzymatic hydrolysisof glycosidic bond

Hydrolysis of the ester bond

R = OH, oleuropein aglycone

R = OH, hydroxytyrosol Elenoic acid

OR

O

OO

HO

HO

HOModeratelysoluble in water

Insoluble in water

Soluble in water

HO

OH

R

HO

O

O

OCH3

HO

O

O

R

HO

OH

O

OCH3

HO

HOO

O

Figure 6.2 A simplified picture of the transformation of phenolic compounds in the extra-virginolive oil process.

of elenoic acid esterified with a β-pyranose molecule on one side and tyrosol (in thecase of ligstroside) or hydroxytyrosol (in the case of oleuropein) on the other side.

Oleorupein and ligstroside are moderately soluble in water and almost insolublein oil. Therefore, their concentration in oil is always very low (a few mg/kg).

Oleuropein and ligstroside are found in the olive fruit but also in a very highamount in the olive leaves. They confer resistance to disease and insect infestation.They are present in relatively high concentrations in olive mill wastewater, with asignificant antimicrobial and phytotoxic effect, which makes waste disposal moredifficult.

An endogenous β-glucosidase causes the formation of oleuropein and ligstrosideaglycones; they are esters of elenoic acid with tyrosol or hydroxytyrosol. The secondmolecule in Figure 6.2 represents oleuropein aglycone.

6.2 THE ROLE OF WATER IN THE TRANSFORMATION OF PHENOLIC COMPOUNDS 73

In this first transformation, the elimination of the glucopyranose moiety causesa change in the polarity of the molecule, which becomes almost insoluble in waterbut moderately soluble in oil. This change in polarity allows some oleuropein agly-cone to be transferred to the oil phase, mainly during the olive paste malaxation.Aglycones of oleorupein and ligstroside and their various derivatives are the mostabundant phenolic compounds in extra-virgin olive oil with concentrations in therange of 100 to 300 ppm or more. They are important in determining the bitternessand pungency sensations and the antioxidant power of the extra-virgin oil, which isessential for oil stability and health-promoting properties.This point is worthy of a comment. The enzymatic reaction transforming oil-

insoluble oleuropein into a slightly oil-soluble aglycone has a decisive impact onextra-virgin olive oil quality. If the transformation of oleuropein to the aglycone orthe solubility of the aglycone in the oil were excessive, the antioxidant potentialwould be enhanced, but the bitter taste would be excessive. If, on the other hand, thetransformation of oleuropein and the oil-solubility of the aglycone were excessivelylow, the oil would be less bitter, but also less stable. Only a very precise tuning of theenzymatic reaction and of the polarity of the molecules can assure a suitable balanceof oil taste, stability, and health-promoting properties.The final step of phenolic compounds transformation consists in the hydrolysis

of the ester bond linking elenoic acid to tyrosol or hydroxytyrosol. Hydroxytyrosolis one of the most powerful in vivo antioxidants. It has immunostimulant, antibioticand neuroprotective effects.

Again, the critical point is the change in polarity, hydroxytyrosol being muchmore polar than the oleuropein aglycones. Therefore, hydroxytyrosol can be formedonly in the presence of water because it derives from an enzymatic reaction and alsobecause it is soluble in water. This is a real problem because excessive water is arisk factor for oil stability, possibly favouring negative enzymatic activities such as,for example, lipolysis, or microbial growth. In conclusion, despite its antioxidantpotential, hydroxytyrosol is considered as an indication of oil degradation insteadof quality.

It can also be observed that in heating, cooking or frying, the antioxidants inextra-virgin olive oil are for the most part preserved due to the protection from oleu-ropein and ligstroside aglycones. Free tyrosol and hydroxytyrosol have much lowerstability to heat.

A further comment may illustrate the extraordinary complexity and balance inextra-virgin olive oil. In ancient times, as well as up to the 1940s, the production ofoil was carried out in very poor operating conditions: long processing times, muchcontact with air, ineffective separation of the oil from the water and the solid compo-nents of the olive paste. Nowadays, these factors are considered as the worst possibleconditions for oil quality. Oils were frequently rancid and very few of them wouldhave met the standards of extra-virgin olive oil. However, even at that time, oliveoils showed striking health benefits (Colomer et al. 2008). Besides oleic acid andother particular components of the triglyceride fraction, was this due to a high con-centration of hydroxytyrosol? Very probably so.

74 CH06 THE ROLE OF OXYGEN AND WATER IN THE EXTRA-VIRGIN OLIVE OIL PROCESS

References

Cicerale, S., Conlan X.A., Sinclair A.J. and Keast, R.S.J. (2009) Chemistry andhealth of olive oil phenolics. Critical Reviews in Food Science and Nutrition49 (3), 218–236.

Colomer, R., Lupu, R., Papadimitropoulou, A. et al. (2008) Giacomo Castelvetro’ssalads. Anti-HER2 oncogene nutraceuticals since the 17th century? Clinical andTranslational Oncology 10, 30–34.

Further reading

García-Rodríguez, R., Romero-Segura, C., Sanz, C. et al. (2011) Role of polyphenoloxidase and peroxidase in shaping the phenolic profile of virgin olive oil. FoodResearch International 44 (2), 629–635.

Servili, M., Selvaggini, R., Esposto, S. et al. (2004) Health and sensory properties ofvirgin olive oil hydrophilic phenols: agronomic and technological aspects of pro-duction that affect their occurrence in the oil. Journal of Chromatography 1054,113–127.