2020 年 26 巻 5 号 p. 605-610
Garlic-flavored olive oil, containing the garlic-derived organosulfur compound diallyl disulfide (DADS), has become popular and is widely available in markets because of its pleasant aroma. However, few studies have evaluated the process of flavoring olive oil with garlic. This study aimed to investigate the amount of DADS transferred from garlic into olive oil under different flavoring conditions. The effects of time and temperature of heating, duration for which the garlic was left standing after slicing, and amount of sliced garlic added were determined as independent variables. The results indicated that the heating temperature and duration for which the garlic was left standing strongly influenced the amount of DADS transferred. Moreover, increases in the quantity of sliced garlic resulted in improved quality and enhanced antioxidant capacity of the oil and increased the amount of DADS transferred. We determined the optimal conditions for garlic-flavored olive oil preparation by a heating process, based on the amount of DADS transferred and hence its aroma intensity.
Olive oil is one of the most valuable edible fat products worldwide. Extra virgin olive oil (EVOO), which has the highest quality among all olive oils categories, is extracted from fresh olive fruits using only physical processing with no chemical process (International Olive Council, 2019). EVOO, known as “the heart-friendly oil”, is often preferred as a conventional cooking oil and is also used as a premium edible oil because of its beneficial health effects (Servili et al., 2009).
To attract more consumers to buy edible fat products, the market is diversifying by developing new products alongside traditional ones, with flavored oils being a good example. Flavored oils are enriched in aromatic compounds, such as those obtained from spices, aromatic herbs and vegetables, which improve their nutritional value, health properties and shelf-life, and give them particular sensory notes (Antoun and Tsimidou, 1997; Gambacorta et al., 2007).
Garlic (Allium sativum L.) has long been known to possess biological activities, which include anticarcinogenic, antiatherosclerotic, antithrombotic, antimicrobial, antiinflammatory, and antioxidant effects (Augusti, 1996; Koch and Lawson, 1996; Wargovich et al., 1996; Brace, 2002; Hunter et al., 2005; Leelarungrayub et al., 2006). Therefore, garlic-derived products have recently become more popular and more widely available in the market. The characterization of garlic-flavored oils has been previously reported by Gambacorta et al. (2007), Baiano et al., (2009), González et al. (2017), and Issaoui et al. (2019). Garlic-derived organosulfur compounds, such as diallyl sulfide, diallyl disulfide (DADS), and diallyl trisulfide are responsible for the characteristic flavor of garlic and its various physiological activities (Capasso, 2013). Among these organosulfur compounds, DADS (Fig. 1) is mainly responsible for the garlic flavor in these oil products (Koch and Lawson, 1996; González et al., 2017).
Chemical structure of DADS.
Garlic-flavored olive oil is widely used in Ahijo cuisine, in which food ingredients such as prawns are rapidly fried in olive oil with garlic. Ahijo is one of the most popular Spanish tapas dishes in the world. DADS has been reported to be the dominant volatile compound released from garlic into olive oil (González et al., 2017; Issaoui et al., 2019). Therefore, because garlic flavor can satisfy consumers' sensory preferences, garlic-flavored olive oil (GFOO) is often used as a seasoning for both uncooked and cooked dishes, because of its pleasant flavor. However, to the best of our knowledge, the mechanism by which the main aroma compound, DADS, is transferred from the garlic to the olive oil remains unclear. Only a few studies have evaluated the processes of flavoring olive oil with garlic. Therefore, the present study aims to evaluate the effects of heating time, heating temperature, time left standing after slicing the garlic, and amount of sliced garlic added on the content of DADS released from garlic into EVOO, i.e., the associated GFOOs, under these different flavoring conditions.
Materials EVOO was prepared by Shodoshima Healthyland Co., Ltd. (Kagawa, Japan). Medium-chain triglyceride (MCT) oil (Nisshin OilliO Group, Ltd., Tokyo, Japan) was purchased at a market. DADS (purity > 90%) was purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Garlic cloves (Fukuchi White cultivar; Aomori, Japan) were purchased at a market.
Process of flavoring oil with garlic EVOO with no added garlic was used as blank oil samples. Flavored oils were prepared as follows: garlic cloves were sliced and left standing at room temperature for 30 min to promote allicin formation. Next, 40 g of these garlic slices were heated in 200 g of olive oil at 100 °C for 5 min.
Heat treatment of GFOO samples The GFOO samples were heated at 121 °C for 3, 5, or 10 min, and then cooled immediately using ice.
Analytical procedures Free fatty acids (FFAs), peroxide values (PVs), specific extinction coefficient (K270), and total phenolic contents (TPCs) of the oil samples were measured using an OxiTester (CDR, Ginestra Fiorentina, Italy) (Kamvissis et al., 2008). The FFA values determined using the OxiTester method were confirmed by comparing the results of oil samples obtained over a wide range of values with those obtained using the official analysis method (Gucci et al., 2012; Kishimoto, 2019). Samples of the oils were placed in cuvettes for analysis. The volumes of the oil samples were 2.5 µL for measuring FFAs, 2.5 µL for PVs, 10 µL for K270, and 10 µL for TPCs.
Total antioxidant capacity assay The antioxidant power of the oil samples was evaluated using the PAO-SO Test kit (Japan Institute for the Control of Aging, Nikken SEIL Co., Ltd., Shizuoka, Japan), according to the manufacturer's instructions. This test is based on evaluating Cu+ levels derived from the reduction of Cu++ by the action of antioxidant substances in the sample. Cu+ forms a stable complex with bathocuproine, whose typical optical absorption at 490 nm can be determined using a spectrophotometer. The values of antioxidant power detected in the samples were compared using a curve generated from the standard substance provided in the kit used at known concentrations and expressed in µM.
Electronic nose apparatus and analysis Volatile organic compounds in the headspace of the oil samples were analyzed using a HERACLES II electronic nose (Alpha MOS, Toulouse, France) (Kishimoto and Kashiwagi, 2018). The HERACLES II was equipped with two identical gas chromatographic columns working in parallel mode: a non-polar column (MXT-5: 10 m length × 180 µm diameter) and a polar column (MXT-WAX: 10 m length × 180 µm diameter), which produced two chromatograms simultaneously. It was also equipped with an HS 100 auto-sampler (CTC Analysis AG, Zwingen, Switzerland) to automate sample incubation and injection. An alkane mixture (from n-hexane to n-hexadecane) was used to convert retention times into Kovats indices for calibration. For analysis, an aliquot of oil (2.0 g) was placed in a 20-mL vial, and then sealed with a magnetic cap. The vial was placed in the auto-sampler, which was subsequently placed in the HERACLES's shaker oven and incubated for 15 min at 60 °C with shaking at 500 rpm. A syringe was used to sample 5 mL of the headspace for injection into the gas chromatograph. The oven temperature was initially set at 40 °C (held for 10 s), then increased to 250 °C at 1.5 °C/s and held at this temperature for 60 s. The total separation time was 120 s. Data were acquired and processed using AlphaSoft software v.16 (Alpha MOS). The AroChemBase module (Alpha MOS) was used to identify the volatile compounds.
Quantification of DADS To determine the amount of DADS in each oil sample, we first plotted a standard curve. Different concentrations of DADS were prepared in MCT oil and subjected to flash gas chromatography electronic nose analysis. The amounts of DADS in the sample oils were determined from the standard curve.
Statistical Analysis The results are expressed as mean ± standard deviation from three replicates and subjected to analysis of variance (ANOVA) followed by the Tukey-Kramer test using Microsoft Excel. Values of p < 0.05 were considered to be statistically significant.
Flash gas chromatography electronic nose analysis Organosulfur compounds, mainly DADS as the typical compound, present in garlic-flavored oil were analyzed as described by Issaoui et al. (2019). In the present study, DADS was considered the major component in the flavored oil. Fig. 2 shows a representative gas chromatogram of the headspace gases obtained from MCT oil containing a standard DADS, GFOO and EVOO samples after heating at 100 °C for 5 min using the non-polar column (MXT-5). The peak for DADS was eluted at a retention time of 83 s and was detected as a major volatile compound on a gas chromatogram of the GFOO sample. Two peaks were eluted at retention times of 16 and 17 s on a gas chromatogram of the GFOO sample but not on that of an EVOO sample after heating. The specific volatile compounds might be released from garlic in EVOO.
A representative chromatogram of headspace gases obtained from a GFOO sample (A) and an EVOO sample after heating (B). The box shows merged peaks with MCT containing a standard DADS sample.
Quantification of DADS A standard curve for DADS was used to determine its concentration in the oil samples. The content of DADS was correlated linearly with peak area between the concentrations of 0 and 1 000 ppb. The linear correlation coefficient (R = 0.999) indicated that this standard curve allowed the DADS content in oils be quantified with high accuracy.
Process of flavoring olive oil with garlic We examined the effect of heating time at 100 °C on the amount of DADS released from garlic in EVOO (Fig. 3), which showed that the DADS content reached a maximum value after 5 min. We then examined the effect of heating temperature for flavoring EVOO with garlic. The DADS contents after flavoring at different temperatures were significantly different (Fig. 4A), and the maximum value was observed in GFOO prepared at 100 °C. When EVOO was flavored at temperatures greater than 120 °C, the DADS content was lower than in those flavored at less than 100 °C.
The effect of heating time at 100 °C on the amount of DADS released from garlic in EVOO. The amount of DADS in the headspace of the oil samples was analyzed using a HERACLES II electronic nose. Data are expressed as mean ± standard deviation (n = 3). a–dMean values with different letters are significantly different (p < 0.05).
Changes in the content of DADS released from garlic in EVOO under different conditions. (A) Effect of the heating temperature. (B) Effect of the time left standing after slicing the garlic. (C) Effect of the amount of sliced garlic added. Data are expressed as mean ± standard deviation (n = 3). a–dMean values with different letters are significantly different (p < 0.05).
When a garlic clove is cut, alliin, an odorless sulfur-containing amino acid derivative, reacts with the enzyme alliinase to form allicin, which breaks down into the strongly odorous DADS (Koch and Lawson, 1996). Therefore, the length of time the garlic is left standing after slicing is important for the formation of DADS. The contents of DADS were determined in GFOO prepared with garlic left standing for different times after slicing at room temperature to check if they increased in proportion to the length of time after slicing. The DADS content reached a maximum after a standing time of 45 min (Fig. 4B).
We also examined the effect of the amount of sliced garlic used in the flavoring process. The contents of DADS were determined in GFOO prepared with different amounts of sliced garlic to check if they increased in proportion to the amount added (Fig. 4C).
Table 1 shows the main quality indices for olive oil as set by the International Olive Council (2019), TPCs, and antioxidant capacities measured in unflavored olive oil and olive oil flavored with different amounts of sliced garlic. The level of FFAs in the GFOOs decreased compared with that in the unflavored oil. The values of PVs and spectrophotometric index K270, which are indicative of the presence of carbonyl compounds (Malheiro et al., 2009), were also lower in GFOOs than in the unflavored oil. The TPC values were also lower in the GFOOs. These decreases were similar to those observed in GFOO in a study by Baiano et al. (2009). These quality parameters decreased according to the amount of garlic added compared with the unflavored oil, suggesting that the process of flavoring oil with garlic might prevent lipid oxidation. These observations might be explained by interactions between the olive oil and garlic during the flavoring process, which result in the formation of bonds between polyphenols and components, such as thiol, present in garlic (Negishi et al., 2002). The enhancing effect on the antioxidant activity of polyphenols by thiol has been previously reported (Fujimoto et al., 2013). Although the phenolic content in the GFOOs was low, the values of antioxidant power for the GFOOs were higher than those for the unflavored oil, possibly because of the presence of lipid-soluble garlic compounds, such as alliin, allicin, allyl cysteine, and allyl disulfide, which possess antioxidant activity (Prasad et al., 1995; Chung, 2006). Therefore, GFOOs with a low phenolic content could have a long shelf-life (Baiano et al., 2009).
| Amount of sliced garlic added | ||||
|---|---|---|---|---|
| Unflavored | 40 g | 80 g | 120 g | |
| FFA (%)* | 0.26 ± 0.03a | 0.21 ± 0.03b | 0.18 ± 0.02b | 0.18 ± 0.02b |
| PV (meq O2/kg )* | 15.1 ± 1.1a | 13.1 ± 0.1b | 12.5 ± 0.3b | 11.6 ± 0.6b |
| K270* | 0.117 ± 0.009a | 0.095 ± 0.002b | 0.088 ± 0.05b | 0.088 ± 0.03b |
| TPC (mg/kg )* | 292 ± 11a | 228 ± 9b | 194 ± 13c | 175 ± 9c |
| Antioxidant power (µM)** | 4 414 ± 800c | 5 630 ± 929c | 7 533 ± 1135b | 13 694 ± 558a |
From these results, we finally determined the optimum flavoring conditions for maximizing the concentration of DADS in olive oil. Garlic cloves were sliced, then left to stand for 45 min at room temperature. Later, 120 g of these garlic slices were heated in 200 g of EVOO at 100 °C for 5 min. The results indicated that the concentration of DADS in the oil was 204 ppb, which was the highest value among GFOOs prepared previously.
Evaluation of thermal stability of GFOO When preparing garlic-derived products, a thermal process is needed to ensure that any Clostridium botulinum spores that may be present in the garlic are destroyed. Therefore, a heat treatment at 121 ° for at least 3 min was adopted as the minimum standard to provide a “botulinum cook” (Stumbo et al., 1975). We examined changes in the DADS content in GFOO at 12 °C and found that this treatment resulted in a loss of 21% (Fig. 5).
Behavior of DADS in GFOO when heated at 121 °C. Data are expressed as mean ± standard deviation (n = 3). a–dMean values with different letters are significantly different (p < 0.05).
In this study, we demonstrated that different flavor processing conditions can significantly affect the DADS content in GFOOs. These differences in the DADS content allow the effects of flavoring conditions (heating time and temperature, time left standing after slicing the garlic, and the amount of sliced garlic) to be determined. We have provided an optimum procedure for flavoring olive oil with garlic by a heating process to obtain maximum DADS content. Further detailed investigation of garlic-derived organosulfur compound composition in GFOO for flavor evaluation and health benefits is now required.
Acknowledgements We would like to thank Philip Creed, PhD, from the Edanz Group (www.edanzediting.com/ac) and Editage (www.editage.com) for English language editing.
