Effect of Flavorization on Virgin Olive Oil Oxidation and Volatile Profile

Citation: Díaz-Montaña, E.J.; Aparicio-Ruiz, R.; Morales, M.T. Effect of Flavorization on Virgin Olive Oil Oxidation and Volatile Profile. Antioxidants 2023,12, 242. https://doi.org/10.3390/ antiox12020242 Academic Editor: Joaquin Velasco Received: 23 December 2022 Revised: 15 January 2023 Accepted: 17 January 2023 Published: 21 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Effect of Flavorization on Virgin Olive Oil Oxidation and Volatile Profile Enrique Jacobo Díaz-Montaña , Ramón Aparicio-Ruiz and María T. Morales * Department of Analytical Chemistry, Faculty of Pharmacy, University of Seville, Calle Profesor García González, 41012 Seville, Spain *Correspondence: [email protected] Abstract: The volatile compounds of virgin olive oil (VOO) have an important role from a sensory point of view as they are responsible for the aroma of the oil. Once the oil is obtained, auto-oxidation is the main process contributing to its deterioration, modifying the volatiles profile and aroma. The addition of aromatic herbs to VOO is a traditional technique to change the flavor and to preserve the oil. The aim of this study was to evaluate the effect on the volatile profile and sensory properties of flavoring VOO with rosemary and basil herbs and its impact on the evolution of the oxidative process during a six-month shelf-life study at 15.7 ± 3.6 ◦ C and exposed to 500 ± 100 lx of light for 12 h each day. The determination of quality parameters, volatiles concentrations and VOO sensory properties and their comparison with the flavored VOO samples showed that the addition of basil or rosemary herbs, in addition to retarding the oxidation of the oil, allowed the discrimination of the flavored samples due to the migration of compounds from herbs to the oil. The aroma of basil olive oil (BOO) samples was mainly due to β -pinene, ocimene and 1,8-cineol compounds while for rosemary olive oil (ROO) samples, their aroma was mainly due to the concentrations of camphene, β -myrcene, α -terpinolene, limonene and 1,8-cineol. From the antioxidant standpoint, the effect of the herbs was more noticeable from the third month onwards. Keywords: volatile compounds; virgin olive oil; flavored olive oil; rosemary; basil; oxidation 1. Introduction Olive oil is the main fat and one of the dietary staples in countries of the Mediterranean basin. In recent years, the consumption of olive oil has increased worldwide due to its well-known health benefits which are mainly due to the presence of antioxidant compounds, among others. Virgin olive oil (VOO) is obtained from the fruit of the olive tree ( Olea europaea L.) when it is fresh, sound and has its optimal maturity [ 1 ]. VOO, and extra virgin olive oil (EVOO) in particular, differs from other edible oils in its nutritional, health and organoleptic properties [ 1 – 3 ]; it is one of the most-studied antioxidant food sources [ 4 ]. The health properties of VOO are attributed to a high content of oleic acid (monounsaturated fatty acid) and the presence of a series of minor components such as triterpenic alcohols, phenols, tocopherols, etc. [ 1 , 5 ]. In recent years, more health properties of olive oil have been reported, including oxidative stress prevention, anti-inflammatory properties, lowering the risk of hypertension and antitumoral properties, among many others. These properties have allowed olive oil to stand out from other fats and edible oils [2]. VOO is also appreciated for its sensory characteristics; it has a pleasant aroma and delicious taste, which contribute to appetite, satiety and consumer preference [ 6 ]. Olive oil’s flavor is due to the presence of phenolic compounds which are responsible for its bitterness and pungency, and the volatile compounds, which are responsible for the aroma [ 4 , 7 ]. The biogenesis of VOO volatiles is mainly due to the oxidation of unsaturated fatty acids by the endogenous enzyme lipoxygenase, which generates volatile compounds using linoleic Antioxidants 2023,12, 242. https://doi.org/10.3390/antiox12020242 https://www.mdpi.com/journal/antioxidants Antioxidants 2023,12, 242 2 of 16 and linolenic acids as precursors [ 8 ]. C6 aldehydes and alcohols, and their corresponding esters, are mainly responsible for positive sensory perception, whereas chemical oxidation, hydrolysis and exogenous enzymes (usually from microorganisms) produce the volatiles associated with sensory defects [ 9 ]. The quality deterioration of oil is linked to food safety since oxidation products are formed when oil ages. The oxidative degradation of olive oil causes an increase in free radicals and the formation of off-flavors [ 10 ], producing the appearance of a rancid sensory defect [ 11 ] and a decrease in the antioxidant content [ 12 ]. The oxidation process is highly influenced by the processing and storage conditions of the oil, which affects not only its sensory and healthy qualities [ 13 ], but also its economic value and consumer acceptability [6]. The addition of aromatic herbs, bulbs or fruits—such as oregano, basil, rosemary, thyme, garlic and lemon—to olive oil results in aromatized or flavored olive oils (FOO) [14–20] , thereby modifying their shelf life [ 12 ], and perhaps, masking undesirable sensory perceptions. The flavoring process of VOOs is varied and can include the infusion of spices into the oil, ultrasound-assisted maceration and joint malaxation of olive paste and herbs, among others [ 14 , 18 – 21 ]. Some studies have characterized the flavoring process through the detection of new sensory characteristics. Additionally, olive oil flavoring has also been studied because it enhances its health properties including antimicrobial, anticancer or antioxidant properties [22–24] . Thus, the VOO flavoring not only modifies the initial VOO sensory characteristics and health properties, but also can change its shelf life and quality parameters as consequence of the migration/transference of antioxidants and/or pro-oxidant compounds from the herbs to the oils [25,26]. Research is usually focused on characterizing flavored VOOs and only a few studies have been conducted on how these FOOs evolve over time [ 25 , 27 – 29 ]. Hence, the aims of this work were twofold: (i) to study and compare the changes produced on the profiles of volatile compounds in non-flavored olive oils and virgin oils flavored by the contact method with rosemary (Rosmarinus officinalis L.) and basil (Ocimum basilicum L.) during six months of storage at 15.7 ± 3.6 ◦ C and exposed to 500 ± 100 lx of light for 12 h each day; and, (ii) to characterize the new volatiles appearing in flavored olive oils (FOO) and to study their quantitative changes and influence on the shelf life. 2. Materials and Methods 2.1. Chemicals The reagents employed were of pure analytical grade. Acetic acid (99%), ethanol (96%) β -caryophyllene (80%), β -myrcene (90%), camphene (95%), α -pinene (98%), borneol (97%), 2-methylbutanal (95%), naphthalene (98%), p-cymene (99%), α -linalool (97%), β -farnesene (90%), β -pinene (99%), limonene (97%), 1,8-cineole (99%), octadecane (99%), γ -terpinene (97%), β -farnesene (90%), naphthalene (99%) and isobutyl acetate (<99.9%), used as internal standards, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl acetate (99.5%), butanoic acid (99%), propanoic acid (99.5%), pentanoic acid (99%), butan-1-ol (99%), butan-2-ol (99.5%), heptan-2-ol (99%), heptan-2-one (98%), (E)-2- heptenal (98%), hexanal (98%), hexan-1-ol (98%), (E)-2-hexenal (97%), 2-methyl-butan-1-ol (99.5%), 3-methyl-butan-1-ol (99.5%), 4-methyl-pentan-2-one (99%), nonanal (95%), octanal (98%), pentanal (98%), octane (99%), 1-penten-3-one (98%) and ethyl propanoate (99%) were purchased from Fluka (Zwijndrecht, The Netherlands). Ethyl butanoate (98%), heptanal (95%), (E)-3-hexen-1-ol (98%), ethyl 2-methylbutanoate (99%), 6-methyl-5-hepten-2-one (99%), octan-3-one (97%), 1-octen-3-one (50%) and pentan- 3-one (99.5%) were purchased from Panreac (Barcelona, Spain). 2.2. Samples and Storing Conditions An EVOO (var. Manzanilla) from the 2020/21 harvest was purchased from a local market (Sevilla, Andalusia, Spain). The oil was filtered and its regulated quality parameters were determined [ 30 ]. Next, the sample (VOO-0) was divided into 18 aliquots stored in 1 L glass containers, 6 of them contained only EVOO (VOO), 6 contained EVOO dressed with Antioxidants 2023,12, 242 3 of 16 dried rosemary (ROO, Rosemary Olive Oil) leaves at 5% w/wand another set of 6 samples contained EVOO dressed with dried basil (BOO, Basil Olive Oil) leaves at 5% w/wduring the whole storage conditions, respectively. The aromatic herbs, collected in September of 2021 from the countryside of Seville (Andalusia, Spain) (37.320471, − 5.979523), were treated to separate the leaves and discarded wooden parts, drying only the leaves at 60 ◦ C for 10 h [31]. The 18 samples were stored for six months at 15.7 ± 3.6 ◦ C and exposed to 500 ±100 lx of light for 12 h each day. Once per month the volatile compounds, physical–chemical parameters and sensory characteristics were determined in triplicate by sampling one bottle from each group (VOO, ROO, BOO). 2.3. Quality Parameters Free acidity (FA), peroxide value (PV) and specific extinction values (K232, K270 and ∆ K) were determined according to The International Olive Council (IOC) trade standards [32]. 2.4. Sensory Analysis VOO-0 was evaluated according to [ 33 ] to assign the initial category of the purchased olive oil [ 30 ]. Evaluation was based on the determination of the median of the fruity attributes and the detection, or not, of sensory defects [ 33 ]. In addition, a sensory evaluation of all the samples was carried out by Quantitative Descriptive Analysis (QDA) to assess possible sensory changes occurring during storage [ 34 ]. For this purpose, a protocol was developed for the study including an evaluation sheet (Figure S1), which was created considering the different sensory attributes, and an unstructured 5 cm scale was used to evaluate the intensity of the attributes. The sensory evaluation was carried out by 8 assessors, all of whom were trained panelists, to check the consistency of the results. 2.5. Volatile Compounds Determination Solid Phase Micro Extraction-Gas Chromatography-Mass spectrometry (SPME-GC- MS, Varian 3900 GC, Bruker, Palo Alto, CA, USA; MSD 5975, Agilent Tech, Santa Clara, CA, USA) and Dynamic Head Space-Gas Chromatography with Flame Ionization Detector (DHS-GC-FID, Varian 3900 GC, Bruker, Palo Alto, CA, USA) were employed to determine the volatile compounds. 2.5.1. SPME-GC-MS For SPME-GC-MS, 2 g of oil or herb samples was placed in a 30 mL glass vial tightly capped with silicone/polytetrafluoroethylene (PTFE) septum. To reach oil–air equilibrium, the samples were left for 10 min at 40 ◦ C, after which a 1 cm SPME fiber with a 50/30 µ m film thickness with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) as a stationary phase, purchased from Supelco (Bellefonte, PA, USA), was exposed to the headspace for 40 min. The fiber was previously conditioned following the supplier instructions. Desorption of volatiles was carried out in the hot injection port by heating the fiber for 5 min at 280 ◦ C. The volatile compounds were injected into a TR-WAX capillary column (60 m × 0.25 mm i.d., 0.25 µ m; Teknokroma, Barcelona, Spain), in splitless mode, of a Varian 3900 gas chromatograph (Bruker, Palo Alto, CA, USA) coupled to a mass spectrometer, using hydrogen at 0.9 mL/min as a carrier gas. The oven was kept in isothermal mode for 10 min at 35 ◦ C and then increased by 3 ◦ C/min until 200 ◦ C. The GC–MS interface was heated to 280 ◦ C with the actual temperature reaching 180 ◦ C in MS source and 150 ◦ C in MS-quadrupole [ 35 ]. The energy used for electron impact was set at 70 eV, and data were collected in the range of 40–300 atomic mass units (amu). The signal was recorded, and the integration was carried out with Enhanced Chemstation MSD ChemStation E.02.02.1431 (Agilent Tech, Santa Clara, CA, USA). For the volatile identification the library, Wiley 7 Antioxidants 2023,12, 242 4 of 16 was used (John Wiley & Sons Limited, Hoboken, NJ, USA), and later confirmed by pure standards, when available. 2.5.2. DHS-GC-FID The dynamic headspace employed 1.5 g of samples. Once the samples were placed into a 20 mL glass vial and tightly capped with silicone/polytetrafluoroethylene (PTFE) septum, they were pre-heated for 18 min at 40 ◦ C and mixed for 15 min using a HT3 Dynamic Headspace System (Teledyne Tekmar, Mason, OH, USA). After mixing, helium employed as a portable gas, using a flow rate of 5 mL/min, moved the volatile compounds to the adsorbent trap (Tenax TATM, Supelco, Bellefonte, PA, USA). The volatiles were thermally desorbed from the trap (5 min at 260 ◦ C) and then injected with a split mode 7:1. As in the case of SPME, the injection was carried out in a TR-WAX capillary column (60 m × 0.25 mm i.d., 0.25 µ m; Teknokroma, Barcelona, Spain) of a Varian 3900 gas chromatograph (Bruker, Palo Alto, CA, USA) with a flame ionization detector (FID), using hydrogen with a flow rate of 1.5 mL/min. The oven conditions were 10 min at 35 ◦ C and then programmed to rise to 200 ◦ C at 3 ◦ C/min. The temperature of the flame ionization detector (FID) was set at 280 ◦ C, and the signal was recorded with Star Chromatography Workstation, System Control version 6 (Bruker, Palo Alto, CA, USA). Quantification was carried out using the internal standard methodological calibration procedure. The internal standard used was isobutyl acetate. 2.6. Statistical Analysis The whole dataset was imported into Excel from the instrument and the Statistica 8 package (StatSoft, Tulsa, OK, USA) was used to carry out the data treatment and the statistical analyses. A t-test was used, which allowed the selection of compounds that showed significant differences (p< 0.05) in their relative concentration among the groups. Cluster analysis of the VOO samples, using the information of the total set of volatiles, was performed by a complete standardization of the data and was implemented with the City-Block (Manhattan) distance measure and the Ward’s method linkage amalgamation rule. Multidimensional Scaling (MDS)—an unsupervised statistical procedure—was implemented to explore the level of similarity of individual samples of the flavored and unflavored oils during the six-month storage experiment. The MDS algorithm is based on the results of the Cluster Analysis [36]. 3. Results and Discussion Rosemary and basil are usually used for VOO flavoring and to take advantage of its antioxidant properties, which could slow down the auto-oxidative process of VOO [ 37 ]. The volatile compounds of VOO vary according to different factors, such as fruit ripeness, pedoclimatic conditions, cultivation, time and conditions of storage, among others [ 38 ]. Particularly, virgin olive oil var. Manzanilla is characterized as having a different amount of alcohol dehydrogenase than other varieties, which leads to an accumulation of aldehydes. This variety is also characterized by having a low taste intensity related to bitterness and pungency, as well as a low aroma intensity of the fruity attribute, which make flavoring this oil a good option [39,40]. 3.1. Quality Parameters The initial sample (VOO-0), filtered and without storing, was classified as extra virgin olive oil once its physico-chemical and organoleptic-regulated parameters were determined [ 30 ]. VOO samples did not overstep the limits of any of the physico-chemical quality parameters during storage, but they showed a linear increase during the studied period. Conversely, the values of these parameters for the flavored samples (FOO) remained below their limits for peroxide value and K232, but they exceeded the limits for the free acidity and K270 parameters (Table S1) [ 30 ]. The free acidity, which is mainly increased due to the hydrolysis of triacylglycerides to free fatty acids, was exceeded at the fifth month due to a Antioxidants 2023,12, 242 5 of 16 higher water activity [ 31 ] and the migration of organic acids from herbs to the oil but not due to a higher oxidation. The limit for K270 parameters was exceeded by ROO samples from the third month onwards, which is not an anomalous result as it depends on the phenolic content in the oil and the bottling [12]. 3.2. Virgin Olive Oil Shelf Life The changes of the VOO samples throughout storage were first assessed, and later, the comparison with the FOO samples was carried out. More than 180 volatile compounds have been reported in VOO [ 38 ], and it is not only those with a higher concentration than their odor threshold that contribute to the aroma. In order to reduce the complexity of the system, only concentrations above the odor threshold were considered [ 38 , 41 ]. This study focused on several of the latter volatiles related to the aroma, and some of them, such as 3-pentanone or E-2-hexenal, are related to positive sensory perceptions. Others, such as E-2-heptenal or nonanal, are related to sensory defects. Table 1shows the volatile compounds identified in the initial sample (VOO-0), their concentration (mg/Kg), odor threshold (mg/Kg) and associated sensory descriptors. The concentration of volatiles attributed to sensory defects in the initial sample were below their odor thresholds [ 35 , 42 ], therefore, they did not contribute to the sample aroma, which agrees with its classification as extra virgin olive oil. Table 1. Kovats index, retention time (min), concentration (mg/Kg), odor threshold (mg/Kg) and sensory descriptors of the volatile compounds of the sample VOO-0. Volatile Compound Kovats I Rt a (min) Conc. b (mg/Kg) O. Thres. c(mg/Kg) Sensory Descriptor [38] Octane 800 4.126 0.71 ±0.05 0.94 Alkane, solvent Ethyl acetate 892 6.398 6.09 ±0.43 0.94 Sweet, aromatic Ethanol 932 7.457 29.51 ±2.09 30.00 Apple, sweet, alcohol 3-Pentanone 964 8.271 0.36 ±0.03 70.00 Sweet, fruity Isobutyl acetate (Internal standard) - 9.852 - - - 1-Penten-3-one 1016 11.548 0.16 ±0.01 0.70 Spicy, mustard Pentanal 969 12.873 27.30 ±1.93 0.24 Oily, wood, bitter, almond Hexanal 1074 14.479 0.87 ±0.06 0.08 Green, green apple, grass, oily, fatty Heptanal 1174 20.502 1.11 ±0.08 - Citrus, fatty 1-Butanol 1145 22.261 0.38 ±0.08 0.40 Sweet, oily, drug E-2-hexen-1-al 1216 23.440 2.59 ±0.05 0.42 Fruity, green, bitter almond 2-Heptanone 1170 24.159 0.27 ±0.10 0.30 Musty, soapy, cinnamon 2-Methyl-1-butanol 1210 24.949 2.50 ±0.36 - Ethereal, alcoholic, fatty 3-Octanone 1244 26.177 0.040 ±0.001 - Grass, musty, green, butter E-2-heptenal 1282 27.552 4.14 ±0.53 4.20 Soapy, fatty, almond, spicy 2-Heptanol 1288 28.299 0.01 ±0.06 0.01 Mushroom, dirt, sweet 6-Methyl-5-hepten-2-one 1347 28.643 0.15 ±0.01 1.00 Fruity, green, herb E-3-Hexen-1-ol 1366 30.060 3.30 ±0.23 1.00 Green grass Nonanal 1385 31.467 Not detected 0.15 Rancid, citrus, soapy 1-Octen-3-ol 1390 33.115 0.54 ±0.03 1.00 Musty, mushroom Acetic acid 1452 35.110 0.48 ±0.05 0.50 Bitter, winery, vinegar Propionic acid 1527 38.316 0.61 ±0.04 0.72 sweaty, pungent Butanoic acid 1628 40.340 0.56 ±0.09 0.65 Rancid, sweaty Pentanoic acid 1720 44.162 0.33 ±0.02 0.60 Spicy, rancid Hexanoic acid 1829 49.000 Not detected 0.70 Rancid, cheese Heptanoic acid 1990 53.450 Not detected 0.10 Rancid, fatty Octanoic acid 2083 55.103 Not detected 3.00 Rancid, cheese, oily, fatty Note: a, Retention time; b, Concentration; c, Odor threshold. The profiles of the volatiles changed throughout the storage, due to the modifications produced by the oxidation process. Initially VOO samples were rich in biogenically-formed C5 and C6 compounds—including 3-pentanone or E-2-hexenal—that can contribute to positive sensory perceptions [ 38 ]; the samples did not contain (or contained only at low concentrations) organic acids. After six months of storage, VOO samples presented a high number of compounds characterized by negative sensory descriptors such as acetic, propanoic and hexanoic acids, as well as other C7 and C9 compounds [ 38 ] such as heptanal or nonanal. The compounds generated throughout the storage were mainly due to the oxidation of unsaturated fatty acids. The study of the generation of sensory defects is crucial Antioxidants 2023,12, 242 6 of 16 because when a VOO reaches high intensities of sensory defects, it is classified as lampante virgin olive oil and must undergo a refining process before it can be consumed [ 25 ], which contributes to economic losses. All the studied compounds showed significant differences (t-test) (p< 0.05) during the storage, except for 1-butanol. Particularly, in VOO samples, the difference started becoming significant from the third month onwards (t-test). The behavior of each compound varied during the storage; compounds related to sensory defects increased while compounds related to positive perceptions decreased. Table 2displays the information of several representative volatile compounds, of which the concentrations greatly differed during the storage, most of them being characterized as volatiles produced by the auto-oxidation of unsaturated fatty acids. Table 2. Concentration (mg/Kg; mean ± SD) of volatile compounds of the VOO samples during the six months of storage. Compound VOO-1 VOO-2 VOO-3 VOO-4 VOO-5 VOO-6 Pentanal 38.25 ±2.71 a50.74 ±3.59 b53.43 ±3.78 b65.96 ±4.66 c67.15 ±4.75 c68.35 ±4.83 c Nonanal 0.18 ±0.04 a0.59 ±0.09 b0.62 ±0.16 b0.82 ±0.17 b1.00 ±0.21 c1.34 ±0.26 c 2-Heptanol 0.87 ±0.06 a1.06 ±0.08 a1.19 ±0.08 b1.29 ±0.09 b1.36 ±0.9 b1.43 ±0.10 b 1-Octen-3-ol 0.52 ±0.04 a0.52 ±0.04 a0.53 ±0.04 a0.55 ±0.04 a0.61 ±0.04 a0.80 ±0.06 b Butanoic acid 1.14 ±0.08 a8.26 ±0.58 b10.52 ±0.74 c21.80 ±1.54 d43.87 ±3.10 e61.66 ±4.36 f Hexanoic acid 12.90 ±0.91 a19.74 ±1.40 b38.24 ±2.70 c59.57 ±4.91 d72.94 ±5.16 d76.31 ±5.40 d Ethyl acetate 4.96 ±0.35 a3.36 ±0.24 b3.14 ±0.22 b2.82 ±0.20 b2.35 ±0.17 c2.13 ±0.15 c Ethanol 26.38 ±1.87 a24.44 ±1.73 a23.76 ±1.68 a20.81 ±1.47 b20.83 ±1.47 b20.85 ±1.46 b 1-penten-3-one 0.08 ±0.01 a0.07 ±0.01 a0.04 ±0.01 b0.03 ±0.00 c0.02 ±0.00 d0.01 ±0.00 e Note: Values with different letters ( a,b,c,d,e,f ) within the same row are significantly different (p< 0.05). Experiments were set up in two repetitions and samples for each repetition were analyzed in triplicate (n= 6). Pentanal is a volatile compound which is normally associated with a green fruity perception but it can also be related to a rancid off-flavor in olive oil undergoing oxidation [ 12 ]. Pentanal and hexanoic acid are good examples of a progressive increment during the study (Figure 1). Both compounds increased but with different slopes—higher for hexanoic acid, in part produced from hexanal oxidation, and lower for pentanal. Antioxidants 2023, 12, x FOR PEER REVIEW 7 of 17 Figure 1. Evolution of pentanal and hexanoic acid concentration (mg/Kg) during the storage in VOO samples. In addition to the compounds described in Table 2, there are other compounds (Table 1) that are also important because their sensory impact and their relationship with virgin olive oil quality as to be considered. Thus, 2-heptanone and 3-octanone, both related to sensory defects, increased their concentrations from 1.43 ± 0.01 to 11.01 ± 0.78 mg/Kg and from 0.04 ± 0.00 to 1.64 ± 0.12 mg/Kg, respectively. Other studied organic acids, such as acetic, propanoic and pentanoic acids, also increased between 5 and 30 times their initial concentrations. These results disagree with Cecchi et al. [11], who reported that the concentration of organic acids never exceeded 0.4 mg/Kg and the amount of acetic acid did not show a significant increase (p < 0.05). The concentrations of all the volatile compounds were used to perform a cluster analysis, which allowed the classification of the samples according to the storage time. Figure 2 shows that the initial sample (VOO-0) and the samples from the first three months were clustered separately from the rest of samples, which formed other clusters where the most oxidized samples were included. The first cluster, however, was split into two groups: one group joins the initial and the first month samples, showing that both samples had fewer differences; and another group joins the samples corresponding to the second and third months, showing that they had differences in their volatile content due to the incipient progression of the oxidative process. Figure 1. Evolution of pentanal and hexanoic acid concentration (mg/Kg) during the storage in VOO samples. Antioxidants 2023,12, 242 7 of 16 In addition to the compounds described in Table 2, there are other compounds (Table 1) that are also important because their sensory impact and their relationship with virgin olive oil quality as to be considered. Thus, 2-heptanone and 3-octanone, both related to sensory defects, increased their concentrations from 1.43 ± 0.01 to 11.01 ± 0.78 mg/Kg and from 0.04 ± 0.00 to 1.64 ± 0.12 mg/Kg, respectively. Other studied organic acids, such as acetic, propanoic and pentanoic acids, also increased between 5 and 30 times their initial concentrations. These results disagree with Cecchi et al. [ 11 ], who reported that the concentration of organic acids never exceeded 0.4 mg/Kg and the amount of acetic acid did not show a significant increase (p< 0.05). The concentrations of all the volatile compounds were used to perform a cluster analysis, which allowed the classification of the samples according to the storage time. Figure 2shows that the initial sample (VOO-0) and the samples from the first three months were clustered separately from the rest of samples, which formed other clusters where the most oxidized samples were included. The first cluster, however, was split into two groups: one group joins the initial and the first month samples, showing that both samples had fewer differences; and another group joins the samples corresponding to the second and third months, showing that they had differences in their volatile content due to the incipient progression of the oxidative process. Antioxidants 2023, 12, x FOR PEER REVIEW 8 of 17 Figure 2. Cluster analysis of the volatile compounds of the VOO samples. In the second cluster, the samples corresponding to the fourth month appeared separately from the other two more altered samples. The separation between the sample of the third month and the samples of the fourth, fifth and sixth months is in accordance with the information obtained from the t-test. 3.3. Comparison between Unflavored and Flavored Samples The results of the study of the volatile compounds in VOO samples were compared with those obtained in FOO samples. The addition of rosemary and basil altered the content of some volatiles due to their antioxidant activity and the migration of volatile compounds. These changes varied not only depending on whether the sample was flavored or not, but also depending on the type of flavoring agent. The volatile composition of both vegetal materials was first identified to evaluate the potential migration of volatiles from the herbs to the oil. Table 3 shows the volatile compounds identified in the two vegetal materials: 23 in rosemary and 26 in basil. The presence of volatiles of the herbs in the FOO samples allowed the confirmation of the migration of several of them, but not all the basil and rosemary volatiles migrated from the herb to the oil; some volatiles remained in the herbs and others were already present in VOO, not all of which influenced the aroma. Table 3. Volatile compounds identified in rosemary and basil herbs by SPME-GC-MS. Rosemary Compounds Volatile Compound * Kovats I ** Molecular Formula Molecular Weight Fragments (m/z) Octane 800 C8H18 114.23 43 85 57 71 56 Pentanal 935 C5H10O 86.13 44 29 58 41 27 α-pinene 1035 C10H16 136.23 93 92 79 43 94 Camphene 1080 C10H16 136.23 93 121 41 79 39 m-menthene 1150 C10H18 138.25 95 138 67 p-menthene 1152 C10H18 138.25 139.1 97.1 95.1 113.1 83.1 β-myrcene 1158 C10H16 136.23 93 69 94 79 77 Figure 2. Cluster analysis of the volatile compounds of the VOO samples. In the second cluster, the samples corresponding to the fourth month appeared separately from the other two more altered samples. The separation between the sample of the third month and the samples of the fourth, fifth and sixth months is in accordance with the information obtained from the t-test. 3.3. Comparison between Unflavored and Flavored Samples The results of the study of the volatile compounds in VOO samples were compared with those obtained in FOO samples. The addition of rosemary and basil altered the content of some volatiles due to their antioxidant activity and the migration of volatile compounds. These changes varied not only depending on whether the sample was flavored or not, but Antioxidants 2023,12, 242 8 of 16 also depending on the type of flavoring agent. The volatile composition of both vegetal materials was first identified to evaluate the potential migration of volatiles from the herbs to the oil. Table 3shows the volatile compounds identified in the two vegetal materials: 23 in rosemary and 26 in basil. The presence of volatiles of the herbs in the FOO samples allowed the confirmation of the migration of several of them, but not all the basil and rosemary volatiles migrated from the herb to the oil; some volatiles remained in the herbs and others were already present in VOO, not all of which influenced the aroma. Table 3. Volatile compounds identified in rosemary and basil herbs by SPME-GC-MS. Rosemary Compounds Volatile Compound * Kovats I ** Molecular Formula Molecular Weight Fragments (m/z) Octane 800 C8H18 114.23 43 85 57 71 56 Pentanal 935 C5H10O 86.13 44 29 58 41 27 α-pinene 1035 C10H16 136.23 93 92 79 43 94 Camphene 1080 C10H16 136.23 93 121 41 79 39 m-menthene 1150 C10H18 138.25 95 138 67 p-menthene 1152 C10H18 138.25 139.1 97.1 95.1 113.1 83.1 β-myrcene 1158 C10H16 136.23 93 69 94 79 77 α-terpinene 1184 C10H16 136.23 121 93 136 91 77 Limonene 1208 C10H16 136.23 93 68 136 121 67 1,8-cineole 1222 C10H18O 154.25 43 81 108 γ-terpinene 1262 C10H16 136.23 93 91 136 77 121 m-cymene 1267 C10H14 134.22 93 135 121 133 119 3-octanone 1285 C8H16O 128.21 43 57 29 72 99 α-terpinolene 1297 C10H16 136.23 93 121 136 91 79 3-octanol 1390 C8H18O 130.23 59 83 55 41 101 Tetrahydrolinalool 1397 C10H22O 158.28 73 69 55 43 129 Acetic acid 1452 C2H4O260.05 43 45 60 15 42 L-camphor 1498 C10H16O 152.23 152 108 81 95 109 Bornyl acetate 1610 C12H20O2196.29 95 43 93 β-caryophyllene 1618 C15H24 204.35 91.1 93.1 133.1 79.1 41.1 Borneol 1642 C10H18O 154.25 95 93 96 94 97 2,6-dimethyloctane 1727 C10H22 142.28 57 43 71 Octadecane 1800 C18H38 254.50 71 85 99 70 113 Basil compounds Volatile compound * Kovats I ** Molecular formula Molecular weight Fragments (m/z) 2-methylbutanal 864 C5H10O 86.13 41 29 57 3-methylbutanal 912 C5H10O 86.13 44 41 43 58 27 Pentanal 935 C5H10O 86.13 44 29 58 41 27 2,6-dimethyl-2-octene 936 C10H20 140.27 69 70 41 Hexanal 1088 C6H12O 100.16 44 41 56 43 27 β-pinene 1118 C10H16 136.23 121.1 136.1 135.1 Limonene 1208 C10H16 136.23 93 68 136 121 67 1,8-cineole 1222 C10H18O 154.25 43 81 108 β-ocimene 1245 C10H16 136.23 93.1 91.1 79.1 92.1 77.1 γ-terpinene 1262 C10H16 136.23 93 91 136 77 121 p-cymene 1280 C10H14 134.22 119 134 91 120 117 6-methyl-5-hepten-2-one 1340 C8H14O 126.20 43 41 55 69 39 Tetrahydrolinalool 1397 C10H22O 158.28 73 69 55 43 129 Acetic acid 1452 C2H4O260.05 43 45 60 15 42 Copaene 1510 C15H24 204.35 105 91 41 119 93 Dihydrolinalool 1525 C10H20O 156.26 109 69 73 41 43 α-linalool 1544 C10H18O 154.25 71 93 55 69 80 β-farnesene 1674 C15H24 204.35 41 69 93 81 67 Aristolene 1697 C15H24 204.35 105 161 91 Pentanoic acid 1698 C5H10O2102.13 60 27 29 41 39 Naphthalene 1718 C10H8128.17 128 127 51 64 126 2,6-dimethyloctane 1727 C10H22 142.28 57 43 71 α-curcumene 1777 C15H22 202.33 119 132 41 α-bergamotene 1779 C15H24 204.35 93 119 41 69 55 β-ionone 1955 C13H20O 192.30 177 43 41 178 135 Ethyl vanillin 2565 C9H10O3166.17 137 166 138 81 27 * Identification of the rosemary and basil volatile compounds by mass spectrometry using the WILEY 7 (John Wiley & Sons Limited, NJ) library, and later confirmed by pure standards analysis. ** Kovats Index reported in literature [38]. Throughout the storage, several volatiles showed the same trend in FOO and VOO, such as pentanal, nonanal or ethyl acetate, but the rate of generation or degradation due to the oxidation varied between the different samples. As previously described, pentanal increased in VOO from the first month (38.25 ± 2.71 mg/Kg) to the sixth month ( 68.35 ±4.83 mg/Kg ), Antioxidants 2023,12, 242 9 of 16 while in ROO, it increased from 33.90 ± 2.40 mg/Kg to 84.98 ±6.00 mg/Kg , and in BOO it increased from 36.34 ±2.57 mg/Kg to 70.86 ± 5.01 mg/Kg. The concentrations in the first months of storage were similar and increased to higher values; in the case of FOO samples, this was likely due to the migration of pentanal from the herbs to the oil and not only due to the auto-oxidation process. The concentration of nonanal, in concordance with [ 43 ], increased during the storage even in flavored samples, but with different trend. Nonanal increased its concentration 10.91% in ROO and 34.93% in BOO, but these increments were lower than in VOO. Thus, nonanal—an aldehyde produced by the auto-oxidation of monounsaturated oleic acid—is a good example of the protective role of the herbs. While the concentration of nonanal increased significantly (p< 0.05) in VOO samples, it did not vary significantly (p< 0.05) in FOO samples; the concentration slope was higher in VOO than in FOO samples (Figure 3). In addition, the increase in the nonanal concentration was significant in VOO samples from the second month onwards, while the increase was delayed until the fourth month in FOO samples, especially in the case of ROO. Antioxidants 2023, 12, x FOR PEER REVIEW 10 of 17 in the first months of storage were similar and increased to higher values; in the case of FOO samples, this was likely due to the migration of pentanal from the herbs to the oil and not only due to the auto-oxidation process. The concentration of nonanal, in concordance with [43], increased during the storage even in flavored samples, but with different trend. Nonanal increased its concentration 10.91% in ROO and 34.93% in BOO, but these increments were lower than in VOO. Thus, nonanal—an aldehyde produced by the auto-oxidation of monounsaturated oleic acid— is a good example of the protective role of the herbs. While the concentration of nonanal increased significantly (p < 0.05) in VOO samples, it did not vary significantly (p < 0.05) in FOO samples; the concentration slope was higher in VOO than in FOO samples (Figure 3). In addition, the increase in the nonanal concentration was significant in VOO samples from the second month onwards, while the increase was delayed until the fourth month in FOO samples, especially in the case of ROO. Figure 3. Variation in the concentration (mg/Kg) of nonanal in VOO, ROO and BOO samples throughout the storage. Other compounds that showed the same behavior in the three types of samples (VOO, ROO, BOO) were 2-heptanol and ethyl acetate. Thus, 2-heptanol showed an increasing trend, reaching the highest value in VOO in the sixth month. The concentration of ethyl acetate, however, decreased, with BOO samples having the highest amount (4.52 ± 0.32 mg/Kg); the difference between ROO and BOO could be due to a greater antioxidant protection of BOO. On the other hand, compounds such as ethanol, 1-penten-3-ol (associated to positive sensory descriptors) and 1-octen-3-ol (responsible for the musty/mushroom sensory perception) [9] showed different behavior at least in one of the FOO samples. Ethanol showed a completely different behavior between FOO and VOO samples; as an average of FOO samples, its initial concentration was 19.17 ± 4.83 mg/Kg and increased to 24.32 ± 0.51 mg/Kg, but without significant differences (p < 0.05). Volatile 6-methyl-5-hepten-2-one showed a different behavior in each sample. In VOO, the concentration increased significantly (p < 0.05) due to the oxidation process [43], while the variations were not significant (p < 0.05) in FOO samples. In ROO samples, the concentration of 6-methyl-5-hepten-2-one showed a downward trend, whereas it did not change in BOO samples due to the decrease in its initial concentration which was compensated for by its transfer from the herb to the oil. In relation to organic acids, the FOO samples showed large differences with VOO samples. Pentanoic and hexanoic acids maintained the same concentration as in the initial sample (VOO-0), indicating that there was a slowing down of the oxidative process that generated them due to the migration of antioxidant compounds. The concentration of Figure 3. Variation in the concentration (mg/Kg) of nonanal in VOO, ROO and BOO samples throughout the storage. Other compounds that showed the same behavior in the three types of samples (VOO, ROO, BOO) were 2-heptanol and ethyl acetate. Thus, 2-heptanol showed an increasing trend, reaching the highest value in VOO in the sixth month. The concentration of ethyl acetate, however, decreased, with BOO samples having the highest amount ( 4.52 ±0.32 mg/Kg ); the difference between ROO and BOO could be due to a greater antioxidant protection of BOO. On the other hand, compounds such as ethanol, 1-penten-3-ol (associated to positive sensory descriptors) and 1-octen-3-ol (responsible for the musty/mushroom sensory perception) [ 9 ] showed different behavior at least in one of the FOO samples. Ethanol showed a completely different behavior between FOO and VOO samples; as an average of FOO samples, its initial concentration was 19.17 ± 4.83 mg/Kg and increased to 24.32 ±0.51 mg/Kg , but without significant differences (p< 0.05). Volatile 6-methyl-5-hepten-2-one showed a different behavior in each sample. In VOO, the concentration increased significantly (p< 0.05) due to the oxidation process [ 43 ], while the variations were not significant (p< 0.05) in FOO samples. In ROO samples, the concentration of 6-methyl-5-hepten-2-one showed a downward trend, whereas it did not change in BOO samples due to the decrease in its initial concentration which was compensated for by its transfer from the herb to the oil. In relation to organic acids, the FOO samples showed large differences with VOO samples. Pentanoic and hexanoic acids maintained the same concentration as in the initial Antioxidants 2023,12, 242 16 of 16 38. Morales, M.T.; Aparicio-Ruiz, R.; Aparicio, R. Chromatographic methodologies: Compounds for olive oil odor Issues. In Handbook of Olive Oil: Analysis and Properties, 2nd ed.; Aparicio, R., Harwood, J., Eds.; Springer: New York, NY, USA, 2013; pp. 261–309. 39. Genovese, A.; Caporaso, N.; Leone, T.; Paduano, A.; Mnea, C.; Perez-Jimenez, M.A.; Sacchi, R. 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Phytochemistry 2021 ,185, 112687. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.