Chemical characterization of sunflower oil oxidized by UV and ozone with different degrees of oxidation and study of their antimicrobial action

2.1. Sunflower oil

The sunflower oil used in this work was of edible quality and supplied by the Borges Company, Spain.

2.2. UV irradiation of sunflower oil

The irradiation of sunflower oil samples was carried out in a stainless-steel photochemical reactor, equipped with a Philips TUV mercury vapor lamp, 254 nm and 11 w power. The samples were stirred at 200 rpm, the contact time was 2 and 4 h, G UV 2h and G UV 4h.

2.3. Ozonation of sunflower oil

A volume of 40 mL of sunflower oil was ozonized in a bubbling reactor at a controlled temperature of 25.0 ± 0.1 °C. The ozone gas flow rate was 30 L/h and the total ozonation time was 30 min. Ozone was generated through AQOZO equipment (CNIC, Cuba), with an initial ozone concentration of 70 mg/L, measured on an Ultrospec III spectrophotometer (Pharmacia LKB, Sweden). The reaction was carried out in the absence of solvents. The applied ozone doses were 4.3 and 25.8 g /L, for G10 and G30 ozonized oils respectively.

2.4. Determination of the peroxide value (PV)

The sample weight was 0.5 g, which was mixed with a solution of glacial acetic acid:chloroform 3:2 v/v. A volume of 0.5 mL of a potassium iodide saturated solution was added. The mixture was left in the dark for two minutes, then 30 mL of distilled water were added and the mixture was titrated with a sodium thiosulfate solution of 0.1 mol/L. The result is expressed as milligrams of active oxygen per kilogram of sample. The peroxide index value was calculated using the expression:

where:

2.5. Determination of TAG composition

TAG analysis was carried out by gas chromatography on an Agilent 6890 gas chromatograph (Palo Alto, CA) using a Quadrex Aluminium-Clad 400-65HT capillary column (30 m length, 0.25 mm inner diameter, 0.1 μm film thickness: Woodbridge, CT, USA) and a flame ionization detector. The working temperatures were 360 °C in the injector and 370 °C in the detector. The injection split ratio was 1:80, with hydrogen as the carrier gas at a rate of 50 cm/s. The method used was isothermal at 335 °C, with a pressure gradient of 100 to 180 kPa. The peaks were identified by their retention times, which were compared to those of the database obtained from the analyses of oils of known composition.

2.6. Fatty acid determination

Fatty acids were determined as their fatty acid methyl esters (FAMEs) by GC. For this purpose, sunflower oil was transesterified to its corresponding methyl esters by treating 5 mg of sample with 1.5 mL of methanol/toluene/sulfuric acid (88/10/2; v/v/v) for 1 hour at 80 ºC. This determination was made on an Agilent 6890 gas chromatograph (Palo Alto, CA) with a flame ionization detector (FID operating at 200 ºC). The silica gel capillary column used was SP-2380 (30 m x 250 μm x 0,20 μm, Bellefonte, PA). Hydrogen was used as a carrier gas at a rate of 28 cm/s. The injection volume was 1 μL.

2.7. Separation of non-polar and polar triacylglycerol fractions

The separation of the non-polar and polar lipid compounds of the oxidized samples was carried out by adsorption chromatography on a silica gel column, according to the method established by Waltking and Wessels (1981)Waltking AE, Wessels H. 1981. Chromatographic Separation of Polar and Nonpolar Components of Frying Fats, J. Assoc. Offic. Anal. Chem. 64, 1329-1330. https://doi.org/10.1093/jaoac/64.6.1329 .

The polar lipid content was determined using the following formula:

where:

2.8. Molecular exclusion HPLC analysis

The sample preparation consisted of dissolving 10 mg of sample into 1 mL of tetrahydrofuran, which was injected into the HPLC system for molecular exclusion analysis. The HPLC system consisted of a Waters 2695 separation module equipped with a 2420 ELS detector. Two stainless steel columns of 30 cm in length and 7.7 mm internal diameter, packed with a styrene-divinylbenzene copolymer with a particle diameter of 5 μm and different pore sizes were used. One had a pore size of 10 nm and the other of 50 nm (Waters Associates, USA). The columns were connected in series starting with the 50 nm column. Tetrahydrofuran was used as the mobile phase at a flow rate of 1 mL/min. Stearic acid monoglyceride was used as an internal standard for quantification (1 mg/mL).

2.9. Minimal inhibitory concentration (MIC) of ozonized oils

Antimicrobial activity was determined by the agar dilution method according to CLSI (2012)CLSI. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 2012. Approved Standard, Ninth Edition. M07-A9. 32(2). guidelines; the final inoculum was 103 cfu/mL. Petri plates were prepared with Mueller Hinton agar containing oxidized sunflower oil samples at concentrations ranging from 5 mg/mL to 100 mg/mL. The MIC was defined as the lowest concentration of oils inhibiting visible bacterial growth after incubation for 20 h at 37 °C. Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and yeast Candida albicans ATCC10231 were used.

2.10. Statistical analysis

The mean and standard deviation of the three determinations performed in each analysis were given.

3.1. Photooxidation of sunflower oil with UV light

The irradiated sunflower oil showed an initial PV of 19.8. Irradiation produced a rapid increase in PV in the first 2 h, reaching values of 95.5 (Table 1). However, this rapid production of peroxides declined at longer exposure times, dropping to a PV of 59.0 after 4 h of exposure to UV radiation. This result shows a double effect of radiation, which induced the formation of peroxides but also destroyed them. Thus, irradiation would cause the decomposition of hydroperoxides resulting in secondary degradation products at long exposure times, producing compounds such as carboxylic acids and aldehydes, which do not contribute to PV value, which tended to decrease, as observed in Table 1.

3.2. Determination of the content of polar lipid compounds in irradiated samples

Fractionation according to the polarity of compounds is one of the techniques used for the characterization of oxidized samples (Ledea-Lozano et al., 2005Ledea-Lozano OE, Force EM, Mancheño RG, Alaiz, M, Gómez MD, Dobarganes C, Mirabal JM, Hernández Castro C, Rosado Pérez A, Vidal TC. 2005. Aplicación de Métodos Cromatograficos en el estudio de la Composición Química del Aceite de Girasol Ozonizado” OLEOZON”®. Rev. CENIC. Cien. Quím. 36, 1-14.). Its main objective is to quantify the amount of modified triacylglycerols (polar lipid fraction) and those that have not been modified (non-polar lipid fraction). In this study, the results showed that at two hours of irradiation the polar lipid fraction was 15.3%; while at four hours it was 11.4% (Table 1). This result shows that there was degradation of the oxidation products at longer exposure times, in good correlation with the results of PV. Furthermore, photooxidation was mainly affected by linoleic acid. Thus, the results in Table 1 showed that the only fatty acid that showed a decrease in its proportion was that fatty acid with a decrease of approximately 2.6% in the oxidized samples. This result agrees with the higher rates of autoxidation expected for all polyunsaturated fatty acids. The level of linoleic acid in the oils oxidized at 2 and 4 h was the same, indicating that there was no advance in the propagation of the reaction.

3.3. Influence of irradiation on TAG aggregation

The study of the impact of photooxidation on sunflower oil was completed by looking into the composition of the polar lipid fractions by molecular exclusion HPLC. This technique separated glycerolipids of different molecular weights and thus gives information on the formation of dimers and oligomers. These compounds are formed by the condensation of TAGs carrying peroxide derivatives, and their presence alters the oil’s physical properties and increases parameters such as oil viscosity. The results of the TAG aggregation of the polar fractions in the irradiated oils are shown in Figure 1. It can be observed that as a result of irradiation, there is no formation of oligomers, but only dimers, and the concentration of dimers was higher (2.41%) at 2 h of irradiation than at 4 h. No significant differences were observed in the other compounds, which were altered TAGs, DAGs, and free fatty acids. The decrease observed in dimer concentration due to the effect of prolonged irradiation was in good agreement with the decrease in peroxides observed in these samples, indicating that at 2 h the termination reactions predominate over those of propagation and no more peroxides with potential biological activity were formed.

3.4. Oxidation parameters of different ozonized sunflower oils

In the present work, 3 ozonized sunflower oils were studied, differing in the dose of ozone applied to them. Therefore, the oils named G10 and G30 are partially ozonized oils, receiving doses of 4.3 and 25.8 g/L ozone. The sample named OLEOZON was the final commercial sample, which received ozone levels of 43 g/L ozone. The oxidative alterations of the oils after reacting with the amounts of ozone are shown in Table 2. When the amount of ozone applied increased, the PV of the oils increased too, with a substantial rise from G10 to G30 (from 49.6 to 159.0). The polar lipid fraction increased accordingly, which showed that ozonation is not subjected to the limitations of photooxidation and is able to increase the amount of peroxides without any apparent limitation. These high levels of oxidation affected the composition of the fatty acids. Therefore, the presence of nonanoic acid in considerable amounts (9:0) in the ozonized samples was remarkable. This fatty acid is not present in the initial oil but accumulated during the ozonation process due to lysis of ozonides formed from oleic acid, and its presence is consistent with previously published results. (Ledea-Lozano et al., 2005Ledea-Lozano OE, Force EM, Mancheño RG, Alaiz, M, Gómez MD, Dobarganes C, Mirabal JM, Hernández Castro C, Rosado Pérez A, Vidal TC. 2005. Aplicación de Métodos Cromatograficos en el estudio de la Composición Química del Aceite de Girasol Ozonizado” OLEOZON”®. Rev. CENIC. Cien. Quím. 36, 1-14.; Ledea-Lozano et al., 2019Ledea-Lozano OE, Fernández-García LA, Gil-Ibarra D, Bootello MA, Garcés R, Martínez-Force E, Salas JJ. 2019a. Characterization of different ozonized sunflower oils II. Triacylglycerol condensation and physical properties. Grasas Aceites 70, e330. http://dx.doi.org/10.3989/gya.1167182 ). Unsaturated fatty acids decreased in very appreciable amounts. In this case, both oleic and linoleic acid showed substantial decreases in all ozonized oils, although linoleic acid was the one that decreased faster (Table 2). This pattern of decrease in unsaturated fatty acids was expected due to ozone attacks on all the double bonds present in the oil, but there was no preference for those in polyunsaturated fatty acids. In the case of linoleic acid, the possibility of reacting with the added ozone was double, which explains its faster decline. This contrasts with the photooxidation of this oil, which was almost exclusively affected by linoleic acid and in a much lower proportion (Table 1). Changes in the TAG composition observed in the neutral lipid fraction corresponding to ozonized oil samples indicated that those carrying a higher number of double bonds were those that decayed faster throughout ozonation, with important decreases in the most unsaturated LLL and OLL (Table 3). In contrast, the TAGs which displayed a lower level of unsaturation (POO and StOO) experimented an increase in its proportion after the process of ozonation.

3.5. Characterization of ozonized oil by molecular exclusion HPLC

Previous studies on ozonized oils indicated that the high level of peroxidation induced by this reaction significantly altered the aggregation of TAGs, causing the presence of TAGs oligomers in considerable proportion, which altered the physical properties of the oil, increasing its viscosity (Ledea-Lozano et al., 2019bLedea-Lozano OE, Fernández-García LA, Gil-Ibarra D, Tena N, Garcés R, Martínez-Force E, Salas JJ. 2019b. Characterization of different ozonized sunflower oils I. Chemical changes during ozonization. Grasas Aceites 70, e329. http://dx.doi.org/10.3989/gya.1166182 ). This effect of ozonation was also observed in this work. Therefore, the initial sunflower oil showed a single peak corresponding to TAGs in the HPLC exclusion system used in this work (Figure 2a), and the samples with different ozone loads, G10 and G30, considerably increased their proportion of TAG dimers and smaller amounts of oligomers began to appear in the oil (Figure 2b and 2c). Finally, OLEOZON oils, which received the highest ozone load, showed a remarkable amount of polymerized TAGs (Figure 2d). The results of the composition of the different ozonized oils are shown in Figure 3, where the formation of considerable amounts of polymeric compounds can be seen, which increased the viscosity of the product. This result is consistent with the previous ones obtained for ozonized oils (Menéndez et al., 2008Menéndez S, González R, Ledea-Lozano O, Hernández F, León OS, Díaz M. 2008. Ozono, aspectos básicos y aplicaciones clínicas. La Habana, CENIC.; Uzun et al., 2018Uzun H, Kaynak EG, Ibanoglu E, Ibanoglu S. 2018. Chemical and structural variations in hazelnut and soybean oils after ozone treatments. Grasas Aceites, 69, e253. https://doi.org/10.3989/gya.1098171 ; Ledea-Lozano et al., 2019aLedea-Lozano OE, Fernández-García LA, Gil-Ibarra D, Bootello MA, Garcés R, Martínez-Force E, Salas JJ. 2019a. Characterization of different ozonized sunflower oils II. Triacylglycerol condensation and physical properties. Grasas Aceites 70, e330. http://dx.doi.org/10.3989/gya.1167182 ). In the case of OLEOZON, it was the oil that received the highest ozone load and contained a very notable proportion of dimers and oligomers, which reached 30% (Figure 3). It is important to note that the proportion of polymerized TAGs corresponds to the dose of ozone applied in any case. This result was also reported by other researchers (Guerra-Blanco et al., 2021Guerra-Blanco P, Chairez I, Poznyak T, Brito-Arias M. 2021. Kinetic Analysis of Ozonation Degree Effect on the Physicochemical Properties of Ozonated Vegetable Oils. Ozone: Sci. Engin. 43, 546-561. https://doi.org/10.1080/01919512.2020.1868972 ).

3.6. Analysis of the behavior of polymers formed in aged ozonized sunflower oil

A molecular exclusion HPLC analysis of ozonized sunflower oil samples stored at 5 ºC for one and three months (Figure 4) was also carried out. The initial ozonized oil contained 68% non-polymerized TAG and amounts of dimers and oligomers at around 15%. The aged sample showed a significant increase in TAGs, with levels close to 80% at the expense of a notable decrease in the polymerized ones, which remained at around 10% for both groups (dimers and oligomers, Figure 4). This means that during storage, the polymerized TAGs tend to decompose, probably due to the breakage of the peroxide bonds that exist between them. This should involve a loss in viscosity values, which has been previously reported in previous storage trials (Menéndez et al., 2008Menéndez S, González R, Ledea-Lozano O, Hernández F, León OS, Díaz M. 2008. Ozono, aspectos básicos y aplicaciones clínicas. La Habana, CENIC.). Peroxidic species formed during the ozonization of sunflowers are metastable species that tend to decay with storage time. This decay involves loss in viscosity and, most likely, loss in antimicrobial activity, so it is important to store this product under proper storage conditions in order to avoid these peroxide decompositions, with low temperature storage, and the absence of humidity.

3.7. Determination of the antimicrobial activity of oxidized sunflower oil

The samples irradiated with UV light did not show germicidal activity within the minimum inhibitory concentration (MIC) range established in this work (Table 4), whose upper limit was 100 mg of oxidized oil per mL of sample. MIC values higher than that are not of interest because of the high amount of oil is necessary to obtain any effect. In general, in the case of experiments with UV light, the results indicate that as the irradiation time increases, the peroxidic species formed disappear, so the total amounts of active peroxides accumulated are too low to achieve the necessary inhibitory activity.

Table 4 shows the minimum inhibitory concentration values obtained for ozonized sunflower oil against one Gram-positive bacterium (Staphylococcus aureus ATCC 25923), two Gram-negative (Escherichia coli ATCC 25922 and Pseudomona aeruginosa ATCC 27853) and one yeast (Candida albicans ATCC 10231). The results show that as the applied dose of ozone and the content of polar compounds increased in the ozonized oil, the MIC value decreased, which means that the oil displayed higher antimicrobial activity. This indicated that the peroxides formed during that process are responsible for the biocidal effect and that a longer ozonation times involves the presence of higher amounts of compounds with biocidal activity. This result is consistent with the criterion that the mixture of peroxy compounds formed by ozonation constitutes the active pharmaceutical ingredient in these oils and is in good agreement with previous results (Poznyak et al., 2018Poznyak T, Blanco PG, Martínez AP, Oria IC, Cuevas CLS. 2018. Ozone Dosage is the Key Factor of Its Effect in Biological Systems. In Jan Derco, Marian Koman (Eds.) Ozone in Nature and Practice. IntechOpen, London. Chapter 3.). The MIC values obtained for each microorganism are related to their morphological and physiological characteristics. Growth inhibition was observed in smaller amounts of ozonized sunflower oil in the case of Gram-positive bacteria and yeast. A greater resistance was observed for the two Gram-negative bacteria. Similar results have been shown for bacteria and yeast treated with ozonized oils (Sechi et al., 2001Sechi LA, Lezcano I, Nunez N, Espim M, Duprè I, Pinna A, Molicotti P, Fadda G, Zanetti S. 2001. Antibacterial activity of ozonized sunflower oil (Oleozon). J. Appl. Microbiol. 90, 279-284. https://doi.org/10.1046/j.1365-2672.2001.01235.x ; Ugazio et al., 2020Ugazio E, Tullio V, Binello A, Tagliapietra S, Dosio F. 2020. Ozonated oils as antimicrobial systems in topical applications. Their characterization, current applications, and advances in improved delivery techniques. Molecules 25, 334. https://doi.org/10.3390/molecules25020334 ; Higa et al., 2022Higa B, Cintra BS, Álvarez CM, Ribeiro AB, Ferreira JC, Tavares DC, Enriquez V, Martinez LR, Pires, RH. 2022. Ozonated oil is effective at killing Candida species and Streptococcus mutans biofilm-derived cells under aerobic and microaerobic conditions. Medical Mycol. 60, myac055. https://doi.org/10.1093/mmy/myac055 ). This evaluation shows that even at low doses, ozonized sunflower oil has a remarkable germicidal character that is not present in autoxidized oils.