OIL”

DESCRIPTION

The present invention relates to a method for the production of an ozonized vegetable oil with a controlled degree of ozonization.

PRIOR ART

The clinical use of ozonized vegetable oils is broadly known in the art. The products of oxidation that are formed by reacting ozone with the fatty acids contained in vegetable oils have a germicidal, antimycotic and immunostimulating effect when the ozonized oils are applied topically.

By virtue also of their properties of stimulating tissue regeneration, ozonized vegetable oils can thus be used for the topical treatment of wounds, infected wounds in particular.

In order to facilitate the topical application of ozonized vegetable oils and prolong the presence of the product on the area to be treated, such oils are preferably marketed in the form of a gel or an extremely viscous liquid. Completely ozonized vegetable oils having a stable gel form can be simply produced by bubbling ozone inside a reactor in which liquid vegetable oil is placed in the absence of solvent, for example according to the method described in patent application EP2025740A1.

The peroxide value (PV) of an ozonized oil brought to complete ozonization is determined by the quantity and quality of the unsaturated fatty acids present, which give rise to different peroxidation reactions in the presence of ozone. The peroxidated products that are formed, for example peroxides, hydroperoxides and ozonides, are responsible for the wide spectrum of therapeutic action of ozonized oils.

The viscosity of the liquid, as well as the formation of a stable gel, increase with increases in the degree of ozonization of the oil, where the degree of ozonization is directly proportional to the time of contact between the ozone and the substrate. The ozonization process described in EP2025740A1 makes it possible to obtain solely ozonized olive oil which, as a stable gel, exhibits a pre-determined peroxide value, which substantially depends on the number and type of unsaturated fatty acids contained in the oil.

There is thus a felt need in this field to have a method for ozonizing vegetable oils which enables the preparation of an ozonized oil that is not only in the form of a stable gel and/or has an appropriate viscosity, but whose peroxide value can also be varied without influencing the physical state of the ozonized oil.

Vegetable oil ozonization processes are known in the art which start from emulsions of oil in water. In these systems, the water is generally present in a high amount relative to the oil: from 1 to 6 times the amount of oil and solvent together in US2,865,937 and about 2.7 times the amount of oil in US3,504,038.

However, such processes do not enable an ozonized oil in the form of a stable gel with a suitable peroxide value to be obtained.

Patent application US2006/0074129 discloses a method for obtaining ozonized oils with a peroxide value ranging from 200 to 1200 by bubbling ozone into an oil or a liquid fat to which 1 -50 vol.% of water has been added. In the example embodiments, large amounts of water are used, 3 litres of water per 10 litres of oil, equal to about 23 vol.%, along with a high ratio between the gas flow rate and the volume of oil used (200 litres/h of gas consisting of an oxygen/ozone mixture per 10 litres of oil). The peroxide value of the ozonized oils produced in the examples varies between 600-800 units for sunflower oil and 1000-1200 units for Theobroma oil. Furthermore, the ozonized oils produced, despite having a variable peroxide value, have a low viscosity (less than 350 mPa.s for ozonized sunflower oil).

In this context, the principal object of the present invention is to propose a method for the production of ozonized oil with a controlled peroxide value, preferably in the form of a stable gel. A further object of the present invention is to propose a method for the preparation of an ozonized oil which makes it possible to obtain the target peroxide value and/or viscosity value of the ozonized oil in a short time, using a limited amount of reagents per unit of volume of oil.

SUMMARY OF THE INVENTION

These and further objects, which will become more apparent in the course of the detailed description that follows, are achieved in accordance with the present invention, which, in a first aspect thereof, relates to a method for preparing an ozonized vegetable oil comprising:

(i) preparing a dispersion comprising at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, and optionally, but preferably, water; and

(ii) at least one step of bubbling a quantity Q1 of ozone into the dispersion and, optionally, adding water,

wherein:

- in step (ii) the water optionally added is in the liquid phase or vapour phase;

- the total quantity of water added to the vegetable oil in step (i) and/or step (ii) is 0.5-15.0 vol.%;

provided that the vegetable oil is not olive oil.

In a further aspect, the present invention relates to an ozonized vegetable oil that is obtained/obtainable with the above-described method and characterized by at least one of the following properties:

- it is in the form of a stable gel, in which the change in the peroxide value APV is less than or equal to approximately 100, where

APV = (PVto - PVt24)

wherein:

PVto = peroxide value measured at the end of step (b) and

PVt24 = peroxide value measured at 24 months after the end of step (b); and/or - has a peroxide value PV that is less than or equal to approximately 2000 mEq 02/Kg,

with the condition that the ozonized oil is not olive oil.

DESCRIPTION OF THE FIGURES

Figure 1 shows a graph of the change in the peroxide values of the oil, as a function of the reaction time, for examples 1 -4.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, the term“dispersion” refers to a heterogeneous system consisting of a prevalent liquid phase (dispersing phase or continuous phase) in which a liquid phase that is immiscible with the dispersing phase is dispersed (dispersed phase). The drops constituting the dispersed phase can have dimensions that are greater than or equal to approximately 10 ³ pm. In the context of the present invention, the term“emulsion” refers to a dispersion in which the drops constituting the dispersed phase have dimensions of approximately 10 ³ - 1 pm.

In the context of the present invention, the term“W/O dispersion” or the term“W/O emulsion” refers, respectively, to a water-in-oil dispersion or emulsion, in which the oil constitutes the continuous phase and the water the dispersed phase.

In the context of the present invention, the term stable“dispersion (or emulsion)” refers to a dispersion (or emulsion) in which the drops constituting the dispersed phase do not give coalescence with the consequent formation of a continuous phase when the dispersion (or emulsion) is maintained at rest at 25°C for a time of approximately 5 minutes.

In the present description and in the appended claims, the term“vegetable oil” refers to a liquid mixture at 25°C and 101.3 kPa mainly consisting of esterified lipids, generally obtained from fruits and seeds of oil-bearing plants. In the context of the present invention, the terms“oleic acid”,“linoleic acid” and more in general“fatty acid” refer, respectively, to the oleic, linoleic and fatty acids present in a vegetable oil in esterified form, preferably in the form of triglycerides. The term“free fatty acid” is used in the context of the present invention to indicate a non-esterified fatty acid.

In a first aspect, the present invention relates to a method for preparing an ozonized vegetable oil comprising:

(i) preparing a dispersion comprising at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, and optionally, but preferably, water; and

(ii) at least one step of bubbling a quantity Q1 of ozone into the dispersion and, optionally, adding water,

wherein:

- in step (ii) the water optionally added is in the liquid phase or vapour phase;

- the total quantity of water added to the vegetable oil in step (i) and/or step (ii) is 0.5-15.0 vol.%;

provided that the vegetable oil is not olive oil.

The total quantity of water added to the at least one vegetable oil in steps (i) and/or (ii) is expressed as a volume percent of the total volume of the dispersion.

The total amount of oleic acid and linoleic acid is expressed as a sum of the percent amounts of the two fatty acids relative to the total amount of vegetable oil.

The method relates to the ozonization of at least one vegetable oil other than olive oil, that is, other than an oil derived by pressing fruits of olive trees and/or from olive pomace.

In a first embodiment, the method can comprise a step (i) of preparing a W/O dispersion comprising at least one vegetable oil and the total quantity of water to be used in the method. According to this embodiment, the at least one step (ii) can be carried out using anhydrous, or dry, ozone, in the absence of water vapour.

Therefore, in first embodiment, the method for preparing an ozonized vegetable oil can comprise:

(i.a) preparing a W/O dispersion comprising at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight and 0.5-15.0 vol.% of water, preferably 1.1 -12.0 vol.%, more preferably 1.1 -10.0 vol.%, even more preferably 1.5-8.0 vol.%; and

(ii.a) at least one step of bubbling a quantity Q1 of ozone into the W/O dispersion

provided that the vegetable oil of step (i.a) is not olive oil.

Step (i.a) can comprise adding the above-mentioned quantity of water, in the liquid phase, to the vegetable oil, and mixing the components with mechanical (e.g. stirrers) and/or physical (e.g. ultrasound) dispersing means normally used in the art to obtain preferably stable W/O dispersions. According to one variant, step (i.a) can be carried out by mechanically dispersing water in the vegetable oil and subsequently treating the W/O dispersion with ultrasound.

According to one variant, step (i.a) can comprise:

(i.a1 ) preparing a pre-dispersion comprising a quantity Q2 of water and at least one vegetable oil, preferably at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, so as to obtain a first dispersion; and

(i.a2) mixing the first dispersion and at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, so as to obtain a W/O dispersion comprising 0.5-15.0 vol.% of water, preferably 1.1 -12.0 vol.%, more preferably 1.1 - 10.0 vol.%, even more preferably 1.5-8.0 vol.%,

with the condition that the vegetable oil of step (i.a2) is not olive oil. A quantity Q1 of ozone, preferably anhydrous ozone, is subsequently bubbled into the W/O dispersion to obtain an ozonized vegetable oil.

In one embodiment, the quantity of water Q2 can be less than or equal to approximately 60 vol.%, preferably less than or equal to approximately 50 vol.%, more preferably approximately 30-50 vol.%, even more preferably approximately 40 vol.%, wherein the quantity Q2 is expressed as a percentage of the volume of water relative to the total volume of the pre dispersion (or pre-emulsion).

The Applicant has surprisingly found that if the quantity Q2 of water pre dispersed in the at least one vegetable oil is above 60 vol.%, when the pre-dispersion is added to the vegetable oil there will be a coalescence of the dispersed phase with the formation of a continuous aqueous phase which prejudices the optimal completion of the ozonization reaction (step (ii.a)).

The pre-dispersion step (i.a1 ) can comprise adding water to the vegetable oil using mechanical (e.g. stirrers) and/or physical (e.g. ultrasound) dispersion means normally used in the art in order to obtain preferably stable dispersions.

In the pre-dispersion step (i.a1 ), use can be made of at least one vegetable oil that is identical to or different from the at least one vegetable oil to be subjected to ozonization in step (ii). In a preferred embodiment, the vegetable oil used to obtain the pre-dispersion of step (i.a1 ) can be identical to the vegetable oil ozonized in step (ii).

Step (i.a1 ) can comprise pre-dispersing the components with a mechanical stirrer at a speed of approximately 500-1500 rpm, preferably approximately 1000 rpm, until obtaining a stable pre-dispersion. Optionally, the pre-dispersion can be further treated with ultrasound after mechanical dispersion.

In a further embodiment, the method for preparing an ozonized vegetable oil can comprise a step (i) of preparing a W/O dispersion comprising the at least one vegetable oil and a smaller quantity of water than the total quantity of water to be used in the method. According to this embodiment, the at least one step (ii) can be carried out by bubbling ozone and water vapour into the W/O dispersion or adding water in the liquid phase to the vegetable oil and bubbling ozone, preferably anhydrous ozone, into the dispersion, so that the total quantity of water added in step (i) and in step (ii) is approximately 0.5-15.0 vol.%

Therefore, in this embodiment the method can comprise:

(i.b) preparing a W/O dispersion comprising at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight and water; and

(ii.b) bubbling a quantity Q1 of ozone into the W/O dispersion and adding water,

wherein:

- in step (ii.b) the water is added in the liquid phase or vapour phase and

- the total quantity of water added in steps (i.b) and (ii.b) is 0.5-15.0 vol.%, preferably 1.1 -12.0 vol.%, more preferably 1.1 -10.0 vol.%, even more preferably 1.5-8.0 vol.%, and

provided that the vegetable oil is not olive oil.

Step (i.b) can be carried out as previously described for step (i.a).

In one variant of this further embodiment, step (i.b) of the method according to the invention can comprise:

(i.b1 ) preparing a pre-dispersion comprising a quantity Q2 of water and at least one vegetable oil, preferably at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, thus obtaining a first dispersion; and

(i.b2) mixing the first dispersion and at least one vegetable oil comprising oleic acid (C18:1 ) and linoleic acid (C18:2) in a total amount greater than or equal to 35% by weight, thus obtaining a W/O dispersion,

wherein the total quantity of water added in steps (i.b1 ), (i.b2) and (ii.b) is 0.5-15.0 vol.%, preferably 1.1 -12.0 vol.%, more preferably 1.1 -10.0 vol.%, even more preferably 1.5-8.0 vol.%, and provided that the vegetable oil of step (i.b2) is not olive oil.

Steps (i.b1 ) and (i.b2) can be can be carried out as previously described for steps (i.a1 ) and (i.a2).

Step (ii.b) can comprise bubbling a quantity Q1 of ozone, preferably anhydrous ozone, into the W/O dispersion and adding water in the liquid phase.

In one embodiment, the method can comprise adding 20-30 vol.% of the total water in step (i.b) and 70-80 vol.% of the total water, preferably divided into 2-6 aliquots, more preferably aliquots of an equal amount, in step (ii.b).

Given the same total quantity of water added and ozone used, this embodiment enables an ozonized olive oil with a higher peroxide value to be obtained. Advantageously, furthermore, the peroxide value increases in a more regular manner as the reaction time increases when part of the water is added in step (ii.b), enabling a better control of the properties of the product, in particular the peroxide value and viscosity.

In a further embodiment, the method can comprise adding the total quantity of water necessary for carrying out the method exclusively in step (ii) in the form of water vapour. According to this embodiment, the method for preparing an ozonized vegetable oil can comprise bubbling a quantity Q1 of ozone and water vapour into the vegetable oil, wherein the total quantity of water added to the oil is 0.5-15.0 vol.%, preferably 1.1 -12.0 vol.%, more preferably 1.1 -10.0 vol.%, even more preferably 1.5-8.0 vol.%, with the condition that the vegetable oil is not olive oil.

In the different embodiments of the method described above:

- the total quantity of water added in step (i), in step (ii) and/or in any sub steps is approximately 0.5-15.0 vol.%, preferably approximately 1.1 -12.0 vol.%, more preferably approximately 1.1 -10.0 vol.%, even more preferably approximately 1.5-8.0 vol.%; and/or - the water can be purified water, preferably water for injection, more preferably sterile water for injection, as defined in the 12th ed. of the Official Pharmacopeia of the Italian Republic; and/or

- the at least one vegetable oil can comprise a total of approximately 35- 85% by weight, preferably approximately 50-80% by weight, more preferably approximately 55-75% by weight, of oleic acid (C18:1 ) and linoleic acid (C18:2); and/or

- the at least one vegetable oil can be selected in the group consisting of peanut oil, corn oil, sunflower oil, rapeseed oil, sesame oil, rice oil, wheat germ oil, hemp seed oil, soybean oil, linseed oil, almond oil, neem oil, hazelnut oil, grapeseed oil, pumpkin seed oil and mixtures thereof; and/or

- the at least one vegetable oil used to prepare the pre-dispersion of steps (i.a1 ) and/or (1.b1 ) can be selected in the group consisting of olive oil, olive pomace oil, peanut oil, corn oil, sunflower oil, rapeseed oil, sesame oil, rice oil, wheat germ oil, hemp seed oil, soybean oil, linseed oil, almond oil, neem oil, hazelnut oil, grapeseed oil, pumpkin seed oil and mixtures thereof; and/or

- the quantity of ozone Q1 can be less than or equal to approximately 10.0 l/h of ozone per litre of oil, preferably less than or equal to approximately 7.5 l/h, more preferably approximately 1.0 - 7.5 l/h, even more preferably approximately 1.1 - 7.0 l/h, even more preferably approximately 1.5 - 6.5 l/h. In a further preferred variant, the quantity Q1 can be approximately 2.0

- 4.0 l/h of ozone per litre of oil; and/or

- at least one of steps (i.a), (i.a1 ), (i.a2), (i.b), (i.b1 ) and (i.b2), preferably all of said steps, can be carried out at a temperature of approximately 20- 25°C and/or at a pressure of approximately 101.3 kPa; and/or

- the dispersion of at least one of steps (i.a), (i.a1 ), (i.a2), (i.b), (i.b1 ) and (i.b2), preferably of all of said steps, can be a stable dispersion, it can preferably be an emulsion, even more preferably it can be a stable emulsion. Because of its great instability, ozone is generally produced in situ by means of ozone generators known in the art, from a supply gas consisting of air or pure oxygen. The main methods for producing ozone are irradiation of the gas flow with UV light and corona discharge. In the method of the invention, an ozone generator that employs corona discharge can preferably be used.

Advantageously, before the supply gas reaches the ozone generator, said gas can be conveyed to at least one drying unit to reduce the quantity of water present in the gas itself, thus producing a dried supply gas. The presence of water in the supply gas significantly reduces the capacity of an ozone generator.

In a preferred embodiment, the dried supply gas flowing to the ozone generator can be dry air with a dew point less than or equal to approximately -50°C, preferably less than or equal to approximately -50°C and greater than or equal to approximately -100°C.

In one embodiment, the ozone generator supplied with dry air as described above can produce a flow of dry air with an ozone concentration of approximately 1 -15 vol.%, preferably approximately 3 vol.%, relative to the total volume of dry air.

When the method comprises bubbling water vapour and ozone, the flow of ozone can be humidified downstream of the ozonizer by means of humidification devices which are in themselves known in the art. Alternatively, the ozone can be supplied to step (ii) as dry or anhydrous ozone and the water vapour as an additional flow on its own.

The method can be carried out in a system comprising at least one bubble column reactor of the type normally employed for gas-liquid reactions. Such reactors are in themselves known in the art and therefore they are not further described in detail.

Since the ozonization reaction of a vegetable oil is exothermal, the at least one step of bubbling ozone can preferably be carried out under conditions of cooling in order to prevent an uncontrolled temperature increase in the reaction environment from causing deterioration of the oil or triggering combustion reactions.

In the context of the present invention, the expression“under conditions of cooling” refers to conditions in which the reaction heat generated by the ozonization of the vegetable oils is removed from the reaction environment.

Therefore, the bubble column reactor can comprise an external cooling compartment (or jacket), of the type known in the art, in which a cooling fluid circulates, generally water or mineral or silicone oil.

The temperature of the cooling fluid supplied to the reactor jacket can be approximately 15-25°C, preferably approximately 18°-22°C, the temperature being measured at a pressure of approximately 101 .3 kPa.

In one embodiment, the at least one step of bubbling ozone can be carried out at a temperature less than or equal to approximately 35°C, preferably less than or equal to approximately 30°C, more preferably approximately 20-30°C.

The at least one step of bubbling ozone can be carried out at ambient pressure or, preferably, under vacuum, at a pressure preferably less than or equal to approximately 51 kPa.

The at least one step of bubbling ozone can have a variable duration.

In one embodiment, said step can be carried out until obtaining an ozonized oil in the form of a gel. In one embodiment, the at least one step of bubbling ozone can be carried out until obtaining an ozonized vegetable oil in the form of a stable gel, in which the change in the peroxide value APV is less than or equal to approximately 100, preferably 70-90, where APV = (PVto - PVt24)

wherein:

PVto = peroxide value measured at the end of the at least one ozonization step and

PVt24 = peroxide value measured at 24 months after the end of the at least one ozonization step. In one embodiment, the at least one step of bubbling ozone can be carried out until obtaining an ozonized oil with a peroxide value PVto less than or equal to approximately 2000 mEq 02/Kg, preferably approximately 350 - 2000 mEq 02/Kg, more preferably approximately 350 - 1000 mEq 02/Kg, even more preferably approximately 400 - 800 mEq 02/Kg.

The at least one step of bubbling ozone can be carried out until obtaining an ozonized vegetable oil in the form of a stable gel with a PVto as specified above.

The method according to the invention can comprise at least one step (ii) lasting approximately 14 - 24 hours, preferably approximately 18-24 hours, at the end of which the ozonized oil obtained can have at least one, preferably all, of the characteristics described above.

At the end of the at least one step of bubbling ozone, the flow of ozone can be interrupted and the ozonized vegetable oil removed from the reaction environment.

Alternatively, in order to obtain an ozonized vegetable oil with the desired characteristics, the method according to the invention can comprise a further step selected from:

(1 ) bubbling oxygen into the ozonized oil; and

(2) dispersing 0.5-15.0 vol.% of water in the ozonized vegetable oil.

The method according to the invention enables an ozonized vegetable oil with a controlled viscosity and peroxide value to be obtained, wherein said oil is preferably in the form of a stable gel.

It has been surprisingly found that, by appropriately modifying the conditions under which the at least one step (ii) is carried out within the operating parameters described above, it is possible to obtain an ozonized vegetable oil in the form of a stable gel and with high values of PVto in a short time, i.e. considerably reducing the duration of the at least one step of bubbling ozone compared to the ozonization processes normally used in the art. A further advantage of the ozonization method according to the invention lies in the fact that for the same PVto of the ozonized oil a smaller amount of reagent is used than in the ozonization processes known in the art.

In a further aspect, the present invention relates to an ozonized vegetable oil obtained/obtainable with the method described above and characterized by at least one of the following properties:

- it is in the form of a stable gel, in which the change in the peroxide value APV is less than or equal to approximately 100, preferably 70-90, where APV = (PVto - PVt24)

wherein:

PVto = peroxide value measured at the end of step (b) and

PVt24 = peroxide value measured at 24 months after the end of step (b); and/or

- has a peroxide value PVto less than or equal to approximately 2000 mEq

02/Kg, preferably approximately 350 - 2000 mEq 02/Kg, more preferably approximately 350 - 1000 mEq 02/Kg, even more preferably approximately 400 - 800 mEq 02/Kg.

The ozonized vegetable oil obtained/obtainable with the method according to the invention has demonstrated bactericidal and/or antimycotic activity and can therefore have application in the treatment, preferably the topical treatment, of infections in humans and animals caused by bacteria and/or fungi.

Measurement methods

Amount of oleic and linoleic acid: the percentage of fatty acids is determined by gas-liquid chromatography (GLC).

Acid value: the acid value is quantitatively determined using method ISO 660-2009

Peroxide value: iodometric titration with sodium thiosulphate of the iodine liberated by the reaction of the peroxides with potassium iodide (compliant with AOAC method No. 28.022). Apparatus: Mettler Toledo G20 titrator provided with an internal burette and DMM 47-SC combined electrode (platinum - pH) and LabX ^® titration software. Titrating solution: sodium thiosulphate 0.1 N (EXAXOL, Italy). Prepare a solution (“Sol. A”) by mixing glacial acetic acid (#Cat.33209, SIGMA Aldrich) and chloroform (#Cat.132950-1 L, SIGMA Aldrich) in a proportion of 3:2 v/v, under gentle stirring. Before taking the measurement, remove the electrode from the liquid it is preserved in and wash the electrode with deionised water for a few seconds. Weigh about 4g of potassium iodide (#Cat.30315, SIGMA Aldrich) into the beaker of the titrator; add 30ml of “Sol. A” under stirring. Set the timer of the instrument to 4 minutes and start it, maintaining the solution under stirring. After 3 min. and 35” have elapsed, add 25ml of deionised water to the solution and proceed with the automatic determination of the blank value (in the absence of the sample) with sodium thiosulphate. The blank determination makes it possible to calculate the relative error in the peroxide value due to changes in potential caused by the presence of any impurities in the reagents. The value in mmol of the titrant used for blank determination is memorised by the instrument as B[1] To determine the peroxide value of the sample, exactly weigh about 2g of sample into the beaker of the titrator, recording the amount of sample weighed (p) on the instrument, add 30ml of “Sol. A” and stir until the sample is completely dissolved. Add about 4g of Kl to the solution to be titrated, set the timer of the instrument to 4 minutes and start it, maintaining the solution under stirring. After 3 min. and 35” have elapsed, add 25ml of deionised water to the solution and proceed with the automatic titration of the iodine liberated from the sample with sodium thiosulphate. The result of the titration is positive if the instrument is able to record a minimum of 11 points on which to build the curve mapping the changes in potential and identify the curve inflection point. The instrument directly provides the peroxide value in meq/Kg O2. The peroxide value is calculated as the arithmetic mean of three measurements performed on the same sample. Wash the electrode with chloroform between measurements. Examples 1 and 2

2 vol.% of purified water (Italian Official Pharmacopeia) was added to an almond oil in a bubble column reactor provided with a water-cooled jacket, the water being pre-emulsified in a suitable amount of oil.

In example 1 , all of the water was added at the beginning of the reaction, before starting to bubble the ozone. In example 2, 25% of the total water was added before ozonization began and the remaining 75% of the total water was added once the reaction had begun, as indicated in table 1.

A flow of dry air containing approximately 3 vol.% of ozone was bubbled into the oil. The temperature of the reactor cooling fluid was set at approximately 18°C. The temperature of the reaction environment was kept below 30°-31 °C thanks to the cooling fluid.

Samples of oil were taken from the reactor at different times during the ozonization reaction and at the end of the reaction, approximately 24 hours after the start of bubbling, and the peroxide value was determined for each sample. The change in the peroxide value of the oils of examples 1 and 2, as a function of the reaction time, is shown in figure 1.

Examples 3 and 4

A sunflower oil was ozonized according to the procedure described in examples 1 and 2.

In example 3 all of the water was added at the beginning of the reaction, before starting to bubble the ozone. In example 4, 25% of the total water was added before ozonization began and the remaining 75% of the total water was added once the reaction had begun, as indicated in table 1. The change in the peroxide value of the oils of examples 3 and 4, as a function of the reaction time, is shown in figure 1. Table 1