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INTRODUCTION
Extra virgin olive oil (EVOO) is widely known because of its benefits for human health: it has been associated with the capacity to prevent cardiovascular diseases by reducing the levels of low-density cholesterol (LDL) in serum. EVOO is more resistant to oxidative degradation than other types of olive oil, due to the presence of bioactive compounds with antioxidant capacity such as polyphenols and tocopherols1 . EVOO also contains a significant amount of oleic acid (C18:1n-9), a monounsaturated fatty acid (MUFA) which is more stable against oxidation than polyunsaturated fatty acids (PUFA).
However, EVOO lacks a group of fatty acids which are essential for human health, called n-3 PUFA. Such fatty acids play key physiological roles and can improve the lipid profile of blood and are also precursors of lipid mediators with anti-inflammatory activity in the human body2 .
Among n-3 PUFA, α-linolenic acid (ALA, C18:3 n-3) is considered essential since the human organism cannot synthesize it and must be supplied with the diet. ALA is a precursor of other n-3 PUFA such as stearidonic acid (SDA, C18:4 n-3), eicosapentaenoic acid (EPA, C20:5 n-3), and docosahexaenoic acid (DHA, C22:6 n-3). EPA and DHA are mainly found in seafoods. SDA is the product of the metabolic conversion of ALA, which is catalyzed by the enzyme Δ-6 desaturase3 .
Since the activity of this enzyme is limited in humans, the synthesis of SDA is often inefficient. Thus, a direct dietary supply of SDA could be an alternative to increase the levels of longer-chain n-3 PUFA such as EPA, by-passing the metabolic step catalyzed by the Δ-6 desaturase. Although SDA has been nutritionally less studied than ALA, it also provides important health benefits. SDA can act as a potent inhibitor of tumor growth, decreases triglyceride levels in blood, and increase the levels of EPA in patients with atopic dermatitis and acne4 , 5 .
ALA is found in seed oils of terrestrial plants such as chia (50-60% of total fatty acids), linseed (55-50%), and walnut oils (13-16%)6 , 7 , 8 . ALA and SDA are found together in a few oils, such as those from viper's bugloss ( Echium plantagineum ) (33 and 13% of total fatty acids respectively) and corn gromwell ( Buglossoides arvensis ) seeds (46 and 20% of total fatty acids respectively)9 , 10 .
The aim of this work was to enrich EVOO with n-3 PUFA through blending EVOO with n-3 PUFA-rich seed oils in different proportions and then evaluate the fatty acid profile, induction time, and organoleptic characteristics of the resulting mixtures. Commercial seed oils from chia, walnut, linseed and viper's bugloss were used as sources of n-3 PUFA, thus avoiding the use of marine oils which may provide undesired attributes to the mixtures in terms of organoleptic properties and oxidative stability.
Chile produced 25,500 tonnes of olive oil in the 2020/2021 crop season, being one of the main producers of olive oil in America ( https://www.internationaloliveoil.org/wp-content/uploads/2022/12/116-HO-2022.pdf ). Although EVOO is highly appreciated for its organoleptic characteristics and for the benefits attributed to human health, adding n-3 PUFA to this matrix may provide higher value added to this food.
MATERIAL AND METHODS
Commercial oils
All oils were purchased in the local market in Santiago, Chile and different brands were selected: “Banquete” for EVOO, “Benexia” for chia oil and “Fontevita” for linseed and walnut oils. Viper's bugloss seed oil was not available in Chile and was imported from De Wit Specialty oils (Texel, The Netherlands) (0150200 NEWmega™ Echium Oil).
Oil blends
EVOO was blended with each n-3 PUFA-rich oil mentioned above in three different proportions (95:5, 90:10 and 80:20 w/w) in 50 ml-glass flasks and then vortexed for 1 min prior to analytical determinations.
Fatty acid profiles
Fatty acid (FA) profiles of original oils and blends were obtained after derivatization to fatty acid methyl esters (FAMEs). Briefly, an aliquot of 15 mg from each sample was weighed in a test tube and 2 ml of methanol:acetyl chloride 20:1 (v/v) were added along with 1 ml of n-hexane. The tubes were capped and heated at 100 °C for 30 minutes, and after they were cooled down, 1 ml of distilled water was added. The tubes were then centrifuged (2,100 g, 4 min), and the hexane upper layer containing the FAMEs was collected for fatty acid analysis by gas chromatography coupled with flame ionization detection (GC-FID) (Agilent 6890N with a 7683B autosampler, Agilent Technologies, Santa Clara, CA, USA) as described in a previous work11 .
Peroxide value
Peroxide value (PV) was carried out only with the original oils (EVOO, chia, linseed, walnut and viper's bugloss) according to the AOCS Cd 8b-90 method.
Free acidity
This analysis was carried out only with the original oils (EVOO, chia, linseed, walnut and viper's bugloss) according to the AOCS Ca 5a-40 method.
Total phenols content
Total phenols were quantified only in the original oils. Liquid-liquid extraction was performed by adding 0.25 ml of n-hexane to 500 mg of oil and vortexing for 1 min. Then, 0.25 ml of methanol/ultrapure distilled water (60:40 v/v) was added. After vortexing again, the resulting mixture was centrifuged at 2500 g during 5 min at 20 °C. The hydro-alcoholic phase was collected and the remaining n-hexane phase was extracted twice more with 0.25 ml of the methanol/water mixture each time. The hydroalcoholic extracts thus obtained were pooled and subsequently used to estimate total phenolic content (TPC), employing the Folin-Ciocalteu assay as described previously12 . TPC was estimated on the basis of a standard curve of gallic acid and expressed as mg of gallic acid equivalents (GAE) per 100 g oil.
Oxygen Radical Absorbance Capacity (ORAC)
ORAC was determined in the original oils as described in a previous work12 . 2.2’-azobis (2-amidinopropane) dihydrochloride (AAPH) and fluorescein were used as a source of peroxyl radicals and oxidizable probe respectively. ORAC was estimated on the basis of a standard curve of Trolox, using a quadratic regression equation obtained between the Trolox concentration and net area under the fluorescence decay curve. Results were reported as micromoles of Trolox equivalents per 100 g oil (μmol TE/100 g oil).
Tocopherol content
Tocopherols were quantified in the original oils. Aliquots of each sample (100 mg) were placed in 10 ml-volumetric flasks and then made up with n- hexane (HPLC quality), vortexed gently for 0.5 min and then 20 μl from the solution were injected into an HPLC equipment according to the AOCS method Ce 8-89 (AOCS, 1993). The HPLC equipment consisted of a pump (Merck-Hitachi L-7110 Merck, Darmstadt, Germany), a Rheodyne 7725i injector, a 20 μl loop, and a Merck-Hitachi 5440 fluorescence detector coupled to a computer with Clarity software. A LiChro CART Superspher Si-60 column (5 μm particle size, 4 mm i.d. x 250 mm, Merck, Germany) was used. A mobile phase of n-hexane:2-propanol (99.5:0.5 v/v) was used with a flow of 1 ml/min. The detection was performed at a wavelength of 290 and 330 nm of excitation and emission respectively. All tocopherol isoforms (α, β, γ and δ) were identified and quantified using analytical standards from Merck (Darmstadt, Germany).
Induction time
Induction time of original oils and blends was determined using Rancimat equipment (Metrohm 743 model), with a 4.0 g sample and carrying out the process at different temperatures: 80 and 100 °C were used for oils rich in n-3 PUFA and 100, 110 and 130 °C for EVOO. The air flow rate was 20 l/h. Induction time of blends was determined at 100 and 120 °C and then extrapolated to room temperature (20, 25 and 30 °C) with the software provided by the equipment.
Sensory tests
Two tests were carried out with the 80:20 (w/w) oil blends: the first one was developed to assess the sensory quality and the second one was performed to check if panelists were able to detect any organoleptic difference between EVOO and the blends (difference evaluation). The blend composed by 80% EVOO and 20% walnut oil was excluded because of its notorious “nut” flavor which made it easily distinguishable from EVOO. Tests were carried out by a trained panel (n= 12) at the Sensory Analysis Laboratory located at the Institute of Nutrition and Food Technology, University of Chile. Selection and training of panelists were carried out according to the International Standardization Organization (ISO) standards for sensory evaluation (ISO 8586:2012, Sensory analysis — General guidelines for the selection, training and monitoring of selected assessors and expert sensory assessors). Panelists were interviewed to find out if they had any food intolerance or health condition and were asked about their willingness to participate in the study. The evaluations were carried out in individual booths, designed for sensory evaluation (ISO 8589:2010, Sensory analysis — General guidance for the design of test rooms). The quality test with a scale by parameter was used with value guidelines for olive oil to analyze appearance, odor, flavor, texture, taste and strange smells (ISO 6658:2019, Sensory analysis - Methodology - General guidance). Such attributes were evaluated on a scale between 1 and 9, being 9 “optimal” and 1 “greatly diminished”. The triangular test was used to determine if panelists could find any difference among 3 samples: 2 of them being EVOO and the other one being a blend of EVOO with one of the n-3 PUFA-rich oils. In this test, the coded samples were presented in random order for all panelists to minimize bias by order of presentation (ISO 4120:2021, Sensory analysis - Methodology - Triangle test).
Statistical analyses
Determination of fatty acids profile, peroxide index, free acidity, total tocopherols, polyphenols, ORAC and induction time were performed at least in duplicate and results are shown as mean ± standard deviation. Two-way ANOVA followed by Tukey's post hoc analysis were carried out using Excel software (Microsoft® Office 2010) to identify significant differences (p<0.05) among values. Graphs depicting the relationship between induction time and percentage of n-3 PUFA (ALA, SDA) in oil blends were created using the Octave® software (GNU project 2018).
RESULTS
Original oils
FA profiles of commercial oils used in this study are shown in table 1 . FA were grouped as saturated (SFA), MUFA, n-6 PUFA, n-3 PUFA, and only ALA and SDA values were reported individually. Oleic acid was the major FA (70.69% of total FA) in EVOO, whereas only a minimal amount of ALA was found (0.59% of total FA). Regarding the sources of n-3 PUFA, the highest ALA percentage was found in chia oil (63.13%), followed by linseed oil (59.25%), viper's bugloss oil (33.19%) and walnut oil (17.20%). Viper's bugloss oil was the only one containing SDA (13.93% of total FA).
Table 1 Fatty acid profile, peroxide value, free acidity, total polyphenol content (TPC), ORAC capacity, tocopherol profile and induction time of vegetable oils used in this work.
| EVOO | Walnut oil | Viper's bugloss oil | Linseed oil | Chia oil | ||
|---|---|---|---|---|---|---|
| Fatty acids (% of total FA) | ||||||
| Σ SFA | 15.78±0.01 | 8.34±0.01 | 10.49±0.01 | 8.31±0.01 | 10.24±0.01 | |
| Σ MUFA | 75.13±0.02 | 14.62±0.01 | 15.83±0.00 | 18.00±0.02 | 7.36±0.02 | |
| Σ n-6 PUFA | 8.53±0.01 | 59.85±0.00 | 26.56±0.01 | 14.45±0.00 | 19.29±0.00 | |
| α-linolenic acid (ALA) | 0.59±0.01a | 17.20±0.00b | 33.19±0.01c | 59.25±0.02d | 63.13±0.01e | |
| Stearidonic acid (SDA) | N/D | N/D | 13.93±0.00 | N/D | N/D | |
| Σ n-3 PUFA (ALA+SDA) | 0.59±0.00 | 17.20±0.00 | 47.12±0.00 | 59.25±0.02 | 63.13±0.01 | |
| Peroxide value (meq O2/kg oil) | 6.36±0.33 | 20.52±0.04 | 3.23±0.25 | 2.15±0.01 | 4.63±0.05 | |
| Free acidity (% of oleic acid) | 0.12±0.03 | 0.71±0.03 | 0.02±0.02 | 0.52±0.00 | 1.02±0.01 | |
| TPC (mg GAE/100 g oil) | 18.90±0.00 | 1.15±0.07 | 3.05±0.07 | 5.50±0.00 | 1.25±0.07 | |
| ORAC (μmol TE/100 g oil) | 358.00±1.41 | 37.00±1.41 | 37.50±0.71 | 155.00±4.24 | 39.00±0.00 | |
| Tocopherols (mg/kg oil) | ||||||
| Alpha (α) | 130.40±0.14 | 3.75±0.35 | 26.30±1.70 | 2.15±0.07 | 12.80±0.42 | |
| Beta (β) | N/D | N/D | N/D | N/D | N/D | |
| Gamma (ɣ) | N/D | 232.95±3.46 | 321.75±9.26 | 382.05±8.56 | 385.15±7.14 | |
| Delta (δ) | N/D | 19.80±0.57 | N/D | N/D | N/D | |
| Σ Tocopherols | 130.40±0.14 | 256.50±3.52 | 348.05±9.41 | 384.20±8.56 | 397.95±7.15 | |
| Induction time (h) | ||||||
| 80 °C | 17.63±0.11 | 9.46±0.44 | 16.44±0.06 | 9.92±0.15 | ||
| 100 °C | 33.51±0.24 | 3.94±0.03 | 2.36±0.10 | 3.58±0.22 | 2.10±0.18 | |
| 110 °C | 22.67±0.52 | |||||
| 130 °C | 4.75±0.11 | |||||
N/D: Not detected. Values reported as mean ± standard deviation (n=3). Different superscript letters in the α-linolenic acid row indicate significant differences (p<0.05). ΣSFA: sum of saturated fatty acids; ΣMUFA: sum of monounsaturated fatty acids; ΣPUFA: sum of polyunsaturated fatty acids.
Peroxide value (PV) was carried out to assess the primary oxidation status of oils ( Table 1 ). The highest and lowest peroxide values were found in walnut oil (20.52 meq O2/kg oil) and linseed oil (2.15 meq O2/kg oil) respectively. The peroxide value for EVOO was 6.36 meq O2/kg oil.
Free acidity indicates the percentage of free fatty acids in the oil, expressed as oleic acid percentage. The lowest acidity was found in viper's bugloss oil (0.02% oleic acid), followed by EVOO (0.12%), linseed (0.52%), walnut (0.71%) and chia (1.02%) ( Table 1 ).
TPC and ORAC of analyzed commercial oils are shown in table 1 . EVOO showed the highest values of TPC (18.90 mg GAE/100 g oil) and ORAC (358.0 μmol TE/100 g oil), followed by linseed oil (5.5 mg GAE/100 g oil and 155.0 μmol TE/100 g oil respectively), whereas walnut oil showed the lowest values for both TPC (1.2 mg GAE/100 g oil) and ORAC (37.0 μmol TE/100 g oil). Regarding tocopherols, the four main structures (α, β, ɣ and δ) were quantified and results were reported in mg/kg oil ( Table 1 ). ɣ-tocopherol was found to be the main isoforms in all oils except EVOO, ranging from 232.95 mg/kg in walnut oil to 385.15 mg/kg in chia oil. β-tocopherol was not found in any of the analyzed oils. EVOO only contained α-tocopherol (130.4 mg/kg), and δ-tocopherol was found only in walnut oil (19.8 mg/kg). Total tocopherols (calculated as the sum of the four isoforms) ranged from 130.4 mg/kg for EVOO to 398.0 mg/kg for chia oil. Linseed, viper's bugloss, and walnut oils showed intermediate values for total tocopherols (384.2, 348.1 and 256.5 mg/kg respectively).
Induction times are reported in table 1 . Experimental induction times were acquired at 100, 110 and 130 °C for EVOO and at 80 and 100 °C for the other oils. EVOO showed the longest induction time at 100 °C whereas the values for n-3 PUFA-containing oils were much shorter: linseed and walnut oils had similar values; chia oil and viper's bugloss seed oil had the shortest induction times.
Oil blends
Fatty acid profiles of all oil blends are shown in table 2 . The highest ALA enrichment was achieved for the EVOO blends:chia oil and EVOO linseed oil (80:20 w/w), reaching 12.81 and 12.53% ALA of total FA respectively, with no significant differences between them. The EVOO blend containing viper's bugloss seed oil (80:20 w/w) reached an SDA value of 2.69% of total FA. ALA and SDA percentages were proportionally increased in all blends with the blending ratio at high correlation factors (R2 ≥ 0.9995).
Table 2 Fatty acid profiles of oil blends.
| Fatty acids (% of total FA) | EVOO-Walnut oil | EVOO-Viper's bugloss seed oil | EVOO-Linseed oil | EVOO-Chia oil | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 95:5 | 90:10 | 80:20 | 95:5 | 90:10 | 80:20 | 95:5 | 90:10 | 80:20 | 95:5 | 90:10 | 80:20 | |
| Σ SFA | 15.45±0.02 | 15.04±0.02 | 14.35±0.01 | 15.50±0.01 | 15.19±0.02 | 14.75±0.01 | 15.38±0.02 | 15.16±0.03 | 14.30±0.02 | 15.74±0.28 | 15.42±0.05 | 14.93±0.25 |
| Σ MUFA | 72.33±0.00 | 69.01±0.03 | 63.51±0.07 | 71.93±0.01 | 68.67±0.02 | 63.55±0.21 | 71.83±0.06 | 70.33±0.20 | 63.55±0.02 | 71.67±0.42 | 68.65±0.32 | 61.68±0.34 |
| Σ n-6 PUFA | 10.91±0.01 | 13.76±0.01 | 18.49±0.05 | 9.51±0.00 | 10.50±0.01 | 12.05±0.05 | 8.81±0.01 | 8.97±0.02 | 9.65±0.00 | 9.01±0.06 | 9.50±0.05 | 10.61±0.06 |
| α- linolenic Acid (ALA) | 1.33±0.00a | 2.22±0.01b | 3.68±0.02c | 2.35±0.00b | 4.17±0.01d | 6.97±0.11e | 4.01±0.04d | 5.57±0.21f | 12.53±0.01g | 3.61±0.04c | 6.45±0.04h | 12.81±0.11g |
| Stearidonic Acid (SDA) | N/D | N/D | N/D | 0.73±0.00a | 1.49±0.00b | 2.69±0.05c | N/D | N/D | N/D | N/D | N/D | N/ |
| Σ n-3 PUFA | 1.33±0.00 | 2.22±0.01 | 3.68±0.02 | 3.08±0.00 | 5.66±0.01 | 9.66±0.12 | 4.01±0.04 | 5.57±0.21 | 12.53±0.01 | 3.61±0.04 | 6.45±0.04 | 12.81±0.11 |
N/D: Not detected. Values expressed as mean ± standard deviation (n= 2). Data are reported as a percentage of each fatty acid / total of fatty acids. Different superscript letters in the α-linolenic acid and stearidonic acid rows indicate significant differences (p<0.05). ΣSFA: sum of saturated fatty acids; ΣMUFA: sum of monounsaturated fatty acids; ΣPUFA: sum of polyunsaturated fatty acids.
On the basis of estimated induction times at 25 °C of different blends between EVOO and chia, walnut, linseed and viper's bugloss oils (100:0, 95:5, 90:10, 80:20 and 0:100), the Octave® software was used to build curves depicting the relationship between induction time and ALA enrichment in the blends covering the whole range from 100% EVOO to 100% n-3 PUFA-rich oils ( Figure 1 ).
Figure 1 Relationship between induction time and α-linolenic acid (ALA) percentage in the blends between EVOO and n-3 PUFA-rich oils at different proportions at 25 °C. EVOO: linseed oil ( 
), EVOO: chia oil ( 
), EVOO: walnut oil ( 
), EVOO: viper's bugloss oil ( 
).
The sensory analysis of blends (80:20 w/w) between EVOO and linseed (Blend A), chia (Blend B) and viper's bugloss oils (Blend C) was carried out by a trained panel (n= 12). Regarding the quality assessment, no statistically significant differences between the three samples were found in the evaluation and overall quality was described as “good” (scores between 7.7 and 7.9) ( Table 3 ). Only Blends A and B scored lower than 7.5 for flavor, but all other attributes were above 7.7. Regarding the evaluation of differences, using the triangular test, between Blends A, B and C with the control oil (EVOO), only four panelists found differences among the mixtures and the control oil ( Table 4 ).
Table 3 Quality test applied to the blends (80:20 w/w) EVOO:linseed oil (sample A), EVOO:chia oil (sample B) and EVOO:viper's bugloss oil (sample C) carried out by a trained panel (n= 12). The score for each attribute ranged from 1 (“greatly diminished”) to 9 (“optimal”).
| Appearance | Aroma | Flavor | Texture | Strange Smell | Strange flavor | Overall quality | Panelists answers | |
|---|---|---|---|---|---|---|---|---|
| Sample A | 8.4±0.5 | 8.1±0.5 | 6.7±1.3 | 8.0±1.0 | 8.5±0.5 | 7.8±0.8 | 7.7±1.0 | Good quality, typical of the product and with a bitter and spicy taste |
| Sample B | 8.3±0.6 | 8.1 ±0.5 | 6.9±1.4 | 8.1±0.8 | 8.5±0.5 | 8.0±0.8 | 7.8±1.0 | Good quality, typical of the product and with a slight spicy and bitter taste |
| Sample C | 8.0±1.0 | 8.1±0.7 | 7.6±1.4 | 8.0±1.0 | 8.9±0.2 | 7.5±.4 | 7.9±1.1 | Good quality, a little spicy and slightly bitter taste |
Table 4 Results of the triangular test according to each panelist's answers for the 80:20 blends: (A) EVOO: linseed oil; (B) EVOO: chia oil; (C) EVOO: viper's bugloss oil. Three oil samples were provided to each panelist, two of them being EVOO and the other one being a blend. If panelists could detect any difference between EVOO and the blend, they were asked to justify why.
| Panelist number | Blend | Difference detected Yes= 1; No= 0) | Panelists answers |
|---|---|---|---|
| n= 1 | A | 1 | Slightly darker color and fresher flavor |
| B | 1 | Spicy flavor | |
| C | 0 | - | |
| n= 2 | A | 1 | Non-intense flavor |
| B | 0 | - | |
| C | 1 | Slightly stronger and a little more dense | |
| n= 3 | A | 0 | - |
| B | 1 | Softer taste and less dense than the other two samples | |
| C | 0 | - | |
| n= 4 | A | 0 | - |
| B | 0 | - | |
| C | 1 | Different in color and taste (more spicy) | |
| n= 5 | A | 0 | - |
| B | 1 | More bitter and spicy | |
| C | 0 | - | |
| n= 6 | A | 0 | - |
| B | 0 | - | |
| C | 1 | A little more intense | |
| n= 7 | A | 0 | - |
| B | 0 | - | |
| C | 0 | - | |
| n= 8 | A | 1 | Slightly softer aroma |
| B | 1 | Different taste | |
| C | 0 | - | |
| n= 9 | A | 0 | - |
| B | 0 | - | |
| C | 1 | A slight bitterness at the end | |
| n= 10 | A | 0 | - |
| B | 0 | - | |
| C | 0 | - | |
| n= 11 | A | 1 | Less spicy and bitter |
| B | 0 | - | |
| C | 0 | - | |
| n= 12 | A | 0 | - |
| B | 0 | - | |
| C | 0 | - | |
| Hits | |||
| A | 4 | ||
| B | 4 | ||
| C | 4 | ||
DISCUSSION
Original oils
The FA profiles of all analyzed oils are in agreement with the expected values for each FA. It is known that oleic acid is the main FA in EVOO (up to 80% of total FA) and that its ALA percentage is lower than 0.9%13 . ALA values range between 50 and 60% of total FA in chia oil, 55 and 60% in linseed oil, 30 and 35% in viper's bugloss seed oil, and 13 and 16% in walnut oil6 , 14 , 15 , 16 , 17 . The percentage of SDA found in viper's bugloss seed oil (13.93%) is also within the expected range (11-15% of total FA)18 .
PV is one of the most used parameters to measure primary oxidation of oil. A high PV is indicative of a high concentration of peroxides and hydroperoxides in the oil matrix and therefore, the lower the PV, the better the quality of the oil. According to the Chilean Food Sanitary Regulation, the upper limit for PV in EVOO is set at 20 meq O2/kg oil ( https://www.minsal.cl/reglamento-sanitario-de-los-alimentos/ ). For edible oils other than olive oil, the Chilean Food Sanitary Regulation is more restrictive and sets the upper limit for PV at 10 meq O2/kg oil. Commercial oils from chia, linseed, walnut and viper's bugloss oils should therefore be below this limit, as has been described in previous works19 , 20 , 21 . In this study, PV for chia, linseed and viper's bugloss oils were found to be below 10 meq O2/kg oil, but the value for walnut oil was 20.52 meq O2/kg oil. This value is actually high for a commercial oil according to the Chilean regulation and may be due to inadequate transport or storage conditions. The Codex Alimentarius standards set the PV at 10 and 15 meq O2/kg oil for refined and cold pressed/virgin oils respectively ( http://www.fao.org/3/y2774e/y2774e04.htm ). This way, according to these standards, all oils except walnut oil were within the expected range.
Among the oils rich in n-3 PUFA used in this study, only the viper's bugloss is refined, whereas oils from chia, linseed and walnut oils were obtained by cold pressing according to the manufacturer. The upper limit of free acidity for refined and cold pressed oils set by the Codex Alimentarius is 0.6 and 4.0 mg KOH/g oil respectively ( http://www.fao.org/3/y2774e/y2774e04.htm ), which in terms of oleic acid percentage, are translated into 0.3 and 2.0% oleic acid (free acidity calculated as % oleic acid that can be expressed as mg KOH/g oil divided by 2). Free acidity values for all tested oils in this work fell within the acceptable range established by the Codex Alimentarius and also in accordance with values reported in the literature19 , 22 . Free acidity of viper's bugloss seed oil was below the upper limit (0.25% of oleic acid) set by the Chilean sanitary food regulation for refined edible oils ( https://www.minsal.cl/reglamento-sanitario-de-los-alimentos/ ).
The oxidation stability of oils depends on several factors, including their FA profile, the amount of antioxidant compounds in its composition, and the extraction and storage conditions (light, temperature and exposure to atmospheric oxygen)23 . Phenolic compounds are minor but highly bioactive components in EVOO. These components have been reported to provide protective effects against atherogenicity and plasmatic metabolic alterations caused by a high-fat diet1 , 24 . Furthermore, they are thought to play a key role in EVOO protection against aging-associated cognitive impairment and neurodegenerative pathologies25 and there is existing evidence of other beneficial effects (antimicrobial, antioxidant and anti-inflammatory activities) of phenolic compounds contained in EVOO26 . The value of TPC in EVOO analyzed in this study (18.9 mg GAE/100 g oil) is within the expected range, according to previous works which reported values between 7 and 29.7 mg GAE/100 g oil in commercial EVOO from different varieties, harvest season, and origin (Spain, Greece, Portugal, Italy and Argentina)27 and between 2.6 and 31.6 mg GAE/100 g oil in Spanish and Italian EVOO from different varieties28 . EVOO used in this study can be classified as “low TPC content” as it contained less than 20 mg GAE/100 g oil25 . The phenolic content of EVOO depends on several factors, but mainly on the olive variety, fruit maturity, oil production, and storage conditions1 . In the case of the EVOO used in this study, the manufacturer only indicated that it is produced “from a variety of olive types”. However, in Chile the most important olive variety used for EVOO production is “Arbequina” (50% of the total production area in the country), which is a variety characterized by low TPC25 . EVOO used in this work was likely extracted mostly from “Arbequina” olives, which may explain its TPC value. EVOO obtained from “Arbequina” has a soft aroma and taste, making it suitable in countries as Chile, where consumers prefer soft organoleptic attributes for olive oils. TPC in linseed oil (5.50 mg GAE/100 g oil) is within the expected range according to previous references, where this value was found to be between 1.1 and 11.5 mg GAE/100 g oil29 , 30 . The lowest TPC was found in walnut and chia oils, with similar values that those found in other seed oils such as sunflower and rapeseed29 .
ORAC is one of the most used methods to measure the antioxidant capacity of foods, and it is generally recognized that foods with high ORAC values have increased antioxidant capacity compared to matrixes with low ORAC values. Regarding ORAC in EVOO, values found in the literature cover a wide range. A study reported ORAC values between 20 and 1,060 μmol TE/100 g oil in EVOO from Italy and Spain and different olive varieties28 and other work found ORAC values between 146 and 497 μmol TE/100 g oil in Spanish EVOO from the Picual variety31 . Thus, the value found in the EVOO used in our study (358 μmol TE/100 g oil) falls within the expected range. Commercial walnut oils were reported to have ORAC values 92 and 560 μmol TE/100 g oil32 , whereas values between 58.4 and 98.4 μmol TE/100 g oil were found in fresh cold pressed walnut oils33 . The ORAC value found in the walnut oil used in this study is lower than these values (37.0 μmol TE/100 g oil), which may relate to its high PV, thus indicating an increased oxidative degradation and, consequently, a lower ORAC value than expected.
Tocopherols are bioactive compounds whose concentration in EVOO is greatly dependent on several parameters such as olive variety, geographical location, or agronomical conditions, among others. α-tocopherol is the most abundant among all tocopherols in EVOO (>98.5% of total tocopherols)34 , 35 . Previous studies have reported total tocopherols in EVOO within a wide range, between 75 and 312 mg/kg34 , 35 , 36 , which is in agreement with our results. Values between 268 and 436 mg/kg total tocopherols have been previously reported in walnut oils extracted with solvents from walnuts of different origins, with γ-tocopherol being more abundant than the rest (≥80% total tocopherols), followed by δ, α and a small amount of β (<1.5% total tocopherols)17 . The same trend was found in the walnut oil used in this work, where γ-tocopherol accounted for 90.8% of total tocopherols. Walnut oil was the only one among assayed oils where δ-tocopherol was detected (7.7% of total tocopherols), which was in a lower proportion than values reported in other studies for commercial walnut oils (11.5% and 9.2%) and authors did not find β-tocopherol37 , 38 . Tocopherol amounts of 499 and 541.5 mg/kg were found for crude and commercial linseed oils respectively, and in both cases γ-tocopherol was the most abundant isoform (more than 94% of total tocopherols), followed in much lower proportion by α-tocopherol (less than 6%)38 , 39 . In our study, γ-tocopherol accounted for 99.4% of total tocopherols. Values between 238 and 427 mg/kg of total tocopherols were reported in a previous study of chia seed oils, with γ-tocopherol reported as the most abundant (>85% of total tocopherols)6 . β-tocopherol was not found, which is in agreement with our results. Tocopherol content was significantly higher (p ≤ 0.05) in oils obtained by solvent extraction compared to by pressing. Total tocopherols in our chia oil was 398 mg/kg, which can be considered a high value as this oil is produced by cold pressing. It has been described that γ-tocopherol is the main isoform in seed oils from the Boraginaceae species, such as those from the Echium genus. Actually, a high amount of tocopherols (531 mg/kg) was found in seed oil from Echium vulgare , from which 90% represented γ-tocopherol and 10% α-tocopherol40 . The commercial seed oil from E. plantagineum used in our study was refined and tocopherols were added afterwards as antioxidants, however the profile was similar to those found in some types of crude oils.
As expected, EVOO showed the highest induction time among all tested oils in this work, because of its low content of PUFA and high ORAC and TPC. Among n-3 PUFA-rich oils, walnut and linseed oils showed similar induction times. Walnut oil contains a lower percentage of ALA than linseed oil and therefore it would be expected that induction time of walnut oil was higher than that of linseed oil. The similar induction times between walnut and linseed oils found in this study may be due to the high PV found in walnut oil. Although chia and linseed oil have a similar percentage of ALA, linseed oil had a higher ORAC, which probably resulted in an induction time much higher than that of chia oil. Induction time of viper's bugloss seed oil showed low values which can be explained by its high SDA content, a more unsaturated n-3 PUFA than ALA. Previous studies have reported similar values for induction time than those obtained in this work using the same experimental conditions (100 °C and 20 L/h air flow): 4.2 h for commercial walnut oil, 2.4-3.0 h for chia oils, and 3.8-4.7 h for linseed oils 19 , 37 , 41 , 42 . Induction times of cold-pressed linseed oil at 80 °C and 20 L/h air flow was reported to be between 14 and 18.3 h, which is in agreement with the value found in this work42 . Regarding viper's bugloss seed oil, an induction time of 5.15 h at 90 °C and 20 L/h air flow has been reported43 . Although we did not measure induction time at 90 °C, we estimated it to be 4.72 h according to the Rancimat equipment software, which is consistent with the referenced value.
Oil blends
The percentage of ALA in the EVOO blends with n-3 PUFA-rich oils (95:5, 90:10, 80:20 w/w) was proportional to the blending degree ( Table 2 ), and they were also significantly different in the three blend proportion within EVOO and each single n-3 PUFA-rich oil. The same trend was observed for SDA in the EVOO blends with viper's bugloss seed oil. The highest ALA percentages (12.53 and 12.81%) were found in the 80:20 blends of EVOO with linseed oil and chia, with no significantly differences between them. This was also expected as linseed and chia were the richest sources of ALA assayed in this work.
We hypothesized that blending EVOO with relatively low proportions of n-3 PUFA-rich plant oils would increase the amount of ALA (and SDA where applicable) but with a minimal decrease in the induction time of the blends because of the protective effect of the highly antioxidant EVOO matrix. However, it was found that even low amounts of added ALA in the blends led to sharp decreases in induction time ( Figure 1 ). This could be due to the minimal amount of ALA available in EVOO (<1% of total fatty acids), and when the proportion of ALA increased in the blends, the higher unsaturation degree of this n-3 PUFA exceeded the antioxidant capacity of the EVOO matrix, as ALA was barely available in EVOO ( Table 1 ). The sharpest fall of induction time was shown in the blend between EVOO and viper's bugloss seed oil, which contains lower percentages of ALA than the blends with linseed and chia seed oils, but is the only blend containing SDA, which may also affect the induction time of the blend as SDA is more unsaturated than ALA. In order to verify that these marked decreases of the induction time were due to the incorporation of n-3 PUFA (ALA and SDA) into the EVOO matrix, a commercial sunflower oil was blended with EVOO at 95:0, 90:10 and 80:20 w/w ratios. The sunflower oil contained linoleic acid (C18:2n-6) at 57.02% of total fatty acids, a similar percentage that ALA in linseed oil ( Table 1 ). Although linoleic acid is also a C18-PUFA, it contains only 2 double bonds in its hydrocarbon chain and therefore its unsaturation degree is lower than that of ALA and SDA. After measuring the induction time of the EVOO sunflower oil blends, a graph was created using the Octave® software depicting the induction time of the blends vs linoleic acid percentage ( Figure 2 ). This time, induction time decreases were less pronounced than those shown in figure 1 . Linoleic acid was naturally available in EVOO (8.53% of total fatty acids), and it seems that higher percentages of this PUFA (up to 15.53% of total fatty acids in the EVOO:sunflower seed oil 80:20 w/w) does not affect the induction time of the blend as much as the addition of ALA to the blend. This may indicate that the degree of unsaturation of PUFA incorporated into EVOO significantly influences the oxidative stability of the blends (i.e., the higher the degree of unsaturation of added PUFA, the sharper the decrease in induction time).
Figure 2 Relationship between induction time and linoleic acid percentage in the blends between EVOO and sunflower oil at different proportions at 25 °C.
Sensory analysis includes several tools to measure human response to food or food ingredients by testing parameters such as appearance, aroma, flavor and texture. In many cases, sensory analysis is the last step of the innovation process44 . Sensory food analyses are carried out by trained panels to achieve more accurate results than employing non-trained individuals. In this work, a sensory panel was subjected to various training tests, prior to the evaluation, which allows optimizing the internal consensus between the evaluators and the repeatability of the technique45 . The three blends between EVOO and chia, linseed and viper's bugloss oils (80:20 w/w) were selected for the sensory analysis because of their highest enrichment in n-3 PUFA among all tested proportions in each case. The blend with walnut oil was discarded because of the high PV found in this oil, which was above the upper limit set by the Chilean Food Sanitary Regulation (10 meq O2/kg oil). Twelve trained panelists carried out the quality test, which is considered an adequate number of people for analyzing food products46 . All blends were described as “good” by the panel ( Table 3 ). Regarding results of the difference test between the control oil (EVOO) and the blends, there were no remarkable differences between samples. As at least eight affirmative answers out of twelve is required to conclude that there is a significant difference, the evaluated blends did not show any significant difference with the EVOO. This is a relevant finding since EVOO is widely accepted by consumers in many countries because of its organoleptic characteristics. Our results imply that enriching EVOO with 20% of oils rich in ALA and/or SDA leads to a significant increase of such n-3 PUFA without modifying the organoleptic characteristics of EVOO. Thus, a new product, as similar as possible to the original, but with an ingredient that provides an additional health benefit for consumers is obtained. These products should be labeled as a “mix of vegetable oils” in case they are marketed, listing first the oil with the highest proportion, according to the Chilean Food Sanitary Regulation.
Commercial availability of vegetable oils rich in n-3 PUFA to obtain the blends with EVOO described in this work is another important issue to consider. Whereas walnut, linseed and chia oils can be found in Chile, viper's bugloss seed oil is not currently available and had to be purchased abroad. Blends between EVOO and linseed or chia oils provide the highest proportion of ALA among all assayed vegetable oils in this study, and organoleptic properties of the 80:20 blends (w/w) are not significantly different from those of EVOO, according to the results of this study. Additionally, current market prices make linseed oil an interesting option to be blended with EVOO, because of its current lower cost compared with chia oil. However, despite the fact that viper's bugloss seed oil is not commercially available in Chile, there is a potential for this plant species to be grown in the country to extract and commercialize this nutritionally relevant oil (besides ALA, it also provides SDA) for specific food formulations as is the case of the blends described in this work.
CONCLUSIONS
When adding oils rich in n-3 PUFA (ALA and/or SDA) to EVOO in small proportions, the induction time of the blend was markedly decreased. The degree of unsaturation of FA had a powerful influence on induction time, even more than the total content of n-3 PUFA in the blends. Enriching EVOO with a PUFA that is originally found in the oil, such as linoleic acid, led to a less pronounced decrease in the induction time of the blend. This could be due to the fact that EVOO already contains linoleic acid in its structure, while it lacks ALA and SDA. When introducing these n-3 PUFA, the antioxidant properties of EVOO become weak and are hardly effective against the pro-oxidant capacity of ALA and SDA. Blending EVOO in the proportion 80:20 w/w with chia, linseed and viper's bugloss oils maintains the organoleptic characteristic of EVOO, while providing an extra benefit to potential consumers. However, the induction time of the blends is markedly decreased compared to EVOO, and this fact must be considered when marketing them. Addition of some extra antioxidants might be advised.
