Stability of E- and Z-Ajoene in Home-Made Mayonnaise

Abstract

Application of ajoene to mayonnaise and its stability were studied. Addition of 5, 10, 15, 20, 25 and 30% garlic oil containing ajoene to mayonnaise recovered E-ajoene 47.7–62.4 μg/10g mayonnaise and Z-ajoene 42–74 μg/10g mayonnaise. After one month storage 99% E- and Z-ajoene remained. In 20% substituted mayonnaise 72% and 69% E- and Z-ajoene respectively were obtained at 80^oC. After three days incubation under UV-light and fluorescent light, 92% and 98% E-ajoene; 88% and 98% Z-ajoene respectively remained. Sensory evaluation demonstrated that, 20% substituted mayonnaise was the highest score among others.

Keywords:

• Garlic
• E- and Z-ajoene
• UV-light stability
• Light stability
• Temperature stability
• Storage stability; Sensory evaluation

In general, there has been an increase in the incidence of preferring to use homemade mayonnaise rather than commercially produced one and a demand by consumers for reducing tartness, and hence acidity of the product. The latter is commonly achieved by the use of either a mixture of vinegar and lemon juice or lemon juice alone.[1] The adverse health effects associated with over consumption of certain types of lipids has led to a trend within the food industry toward the development of reduced-fat products. It was a major challenge to produce reduced-fat food, which has the same appearance, texture, stability, and flavor as their full-fat counterparts.[2]

Garlic (Allium sativum) has been advocated as a remedy for the treatment and prevention of a number of diseases, including atherosclerosis and cancer. However, several clinical studies conducted during the last three decades have shown contradictory lipid-lowering activity and beneficial effects on cancer incidence.3-5 Therapeutic actions of garlic, although still discussed, have been attributed primarily to its organosulphuric compounds, but more detailed studies on chemically defined garlic components are needed to clarify more its pharmacological properties.[3] Ajoene (Figure 1) derived from garlic is a well-established antiplatelet agent, and its inhibitory effect on platelet aggregation has been extensively studied and documented both by in vivo and in vitro experiments.6-10 Recent evidence showed that ajoene inhibits proliferation and induces apoptosis of several cancer cell lines, which ultimately leads to the reduction of tumor growth in vivo. [11] Allicin and ajoene were also reported to significantly decrease cholesterol biosynthesis in rat hepatocytes and HepG2 cells by inhibiting HMG-CoA reductase and late steps of the MVA pathway, which leads to the accumulation of the precursor lanosterol.12-14 In increasing consumer choice, self-medication, and the quest for ‘natural’ therapy, herbal products are used increasingly as alternatives to traditional drugs or as supplements to the diet.[15] Several spices—particularly garlic, clove and ginger—are used extensively in Indian diet and medicine.[16] Oil-macerated garlic product is widely used as a health food in Europe but rare in the United States and Japan.[17] Consumer's demand for more natural food products, presenting health benefits, has increased over the years. Besides the nutritional aspects, an appealing appearance with positive sensorial characteristics is also desired.[18] As a result of this trend, the use of natural ajoene derived from garlic in food product will be preferred, while the synthetic ajoene is perceived as undesirable ingredient.

Figure 1 Formation of ajoene from alliin upon crushing garlic.

Incorporation of both E- and Z-ajoene in the mayonnaise, which has not yet been examined, could provide both nutritional and medicinal health benefits and is appealing to health-conscious consumers. The purpose of this study was to apply ajoene in homemade mayonnaise and to investigate the concentration, stability, pH value, and sensorial properties.

MATERIALS AND METHODS

Materials

All chemicals were obtained from Sigma, Aldrich (Tokyo, Japan), Wako (Osaka, Japan) Nacalai Tesque, Inc. (Kyoto, Japan). Japanese garlic (Allium sativum L), egg and rice vinegar were obtained from a local market of Japan. Rice oil was obtained from Fine Foods Co., Ltd. (Osaka, Japan). The chemicals and solvents used were of guaranteed grade.

Preparation of E- and Z-ajoene from Japanese Garlic

Fresh Japanese garlic: Preparation and purification of E-and Z-ajoene from Japanese garlic was followed by previous method.[19] In brief, garlic cloves were cut into 3-4-mm thick slices, and then ground to mix oil using SMT High-Flex disperser apparatus (SMT Co., Tokyo, Japan) mechanically driven at 700 rpm by a drill press. To minimize frictional heating of the sample during the grinding process, the tissue grinder was chilled prior to and during the grinding process with an ice bath. Garlic samples were mixed with oil (0.25 kg/L) directly in the disperser apparatus. Then the mixture was stored at 80^oC for 4 h to allow complete ajoene formation. Ten ml samples were extracted with ethyl acetate and analyzed by HPLC.

Mayonnaise Preparation

Mayonnaise was prepared according to previous method.[20] with a slight modification. In brief, 150 ml of rice oil, 14.5 g egg yolk, and 4.5 ml vinegar (6% v/v). In case of substituted mayonnaise, rice oil was replaced by garlic oil containing E- and Z-ajoene (64.3 and 297.8 mg/10 ml, respectively) at a level of 5, 10, 15, 20, 25, and 30% of the total oil used and were referred to as the 5A, 10A, 15A, 20A, 25A, and 30A formulations, respectively. The mayonnaise preparation was as follows: The shells of eggs were cracked and the egg white and yolk were harvested separately into sterile containers. The egg yolk was homogenized by using SMT High-Flex disperser apparatus (SMT Co., Tokyo, Japan) mechanically driven at 700 rpm by a drill press. To minimize frictional heating of the sample during the grinding process, the tissue grinder was chilled prior to and during the grinding process with an ice bath. The oil was gradually added (75 ml) during continuous mixing. To thin the mixture and to prevent it from curdling, 2.5 ml of vinegar were mixed. Then, the remaining oil was added gradually and finally another 2 ml of vinegar. Control did not contain any ajoene containing garlic oil. The mayonnaise was dispensed into sterile containers and refrigerated (4^oC).

HPLC Analysis of the Concentration of Ajoene in Mayonnaise

Mayonnaise samples were analyzed by Si-HPLC according to previous method[19]. In briefly, Si-HPLC using a LiChrospher Si 60 column (250 mm × 4.0 mm, Kanto Chemical Co., Ltd., Tokyo). The eluent was n-hexan/2-propanol (85:15, v/v) at a flow rate of 1.0 ml/min, and the eluate was monitored at 240 nm. The HPLC system consisted of an L-6200 intelligent pump, an L-4200 UV-Vis detector and a D- 2500 chromato-integrator (Hitachi Co., Ltd., Tokyo, Japan).

pH Measurements

The pH values of liquid mayonnaise were measured at 25^oC using a Horiba pH meter F-12 (Horiba Co., Japan). Three replicate readings were taken for each pH measurements of samples.

Storage Stability

Mayonnaise was stored at 4^oC to determine its storage stability and the ajoene concentration was analyzed by HPLC after 15, and 30 day periods. The data were expressed as an average of three replications.

Temperature Stability

Mayonnaises were heated at 40, 60, and 80^oC for three days to check the temperature stability. The concentrations of ajoene in samples were analyzed by HPLC. The data were expressed as an average of three replications.

UV-light Stability

Mayonnaises were shaken under UV-light (Intensity = 253.7 nm, Toshiba GL15, Toshiba, Electronics Co., Tokyo, Japan) at 25^oC for 3 days to determine their UV-light stability. The concentrations of ajoene in samples were analyzed by HPLC. The data were expressed as an average of three replications.

Light Stability

Mayonnaises were shaken under light (Intensity = 600 Wm^-2, Toshiba, Electronics Co., Ltd., Tokyo, Japan) at 25^oC for three days to determine their light stability. The concentrations of samples were analyzed by HPLC. The data were expressed as an average of three replications.

Sensory Evaluation

Sensory characteristics of mayonnaise: appearance, color, flavor, texture, taste, mouthfeel and overall acceptability were evaluated by 10 trained panel on 10-point hedonic scale, 1 = the least, the lowest; 1 0= the most, the highest.

Statistical Analysis

The statistical analysis of the measured parameters was made using analysis of variance (ANOVA). Significant differences among samples were evaluated by Duncan's multiple-range test (P < 0.05) using SPSS software (v. 11.0, SPSS, Chicago, IL).

RESULTS AND DISCUSSION

Amount of Ajoene in Different Mayonnaise Samples

The concentration of E- and Z-ajoene in mayonnaise significantly increased with increasing the substitution levels, as shown in Table 1 This result showed that the amounts of E- (62.4 μg/10g mayonnaise) and Z-ajoene (74.1 μg/10g mayonnaise) in 30% substitution were significantly higher than 5 percent sample [E- (47.7 μg/10g mayonnaise) and Z-ajoene (42.0 μg/10g mayonnaise)]. These results suggested that Z-ajoene is higher than E-ajoene except 5A samples. Fresh preparations of oil-macerated garlic yielded exclusively the Z-isomer, but it gradually isomerizes to give the E-isomer.[17]

Table 1 Concentration of ajoene in mayonnaise

	E-ajoene	Z-ajoene
Samples	Conc. (μg/10g mayonnaise)	Conc. (μg/10g mayonnaise)
Control	nd	nd
5A	47.71 ± 0.37^a	42.07 ± 1.18^a
10A	51.46 ± 0.93^b	53.72 ± 2.29^b
15A	54.54 ± 0.28^c	58.38 ± 1.79^c
20A	56.57 ± 2.41^c	61.42 ± 2.91^c
25A	61.57 ± 1.28^d	70.39 ± 1.10^d
30A	62.43 ± 1.03^d	74.13 ± 1.80^e
nd: not detected. Mean ± SD values followed by the same letters on the same column are not significantly different (P < 0.05) according to Duncan's multiple range test. Data were the means of three determinations.

Previous study[11] found that Z-ajoene (2, 4, 8 mg/kg food) inhibited tumor growth by 38% and 42% in mice grafted with sarcoma 180 and hepatocarcinoma 22 in vivo. In vitro, Z-ajoene clearly showed a cell growth inhibition on several human cancer cell lines at non-toxic concentrations (lower than 50 μM). According to previous study,[21] Cell counting, DNA synthesis, and cell cycle analysis showed that ajoene (1-50 μM) interfered with the progression of the G1 phase of the cell cycle. In vitro assays for protein farnesyl-transferase and protein geranyl-geranyl-transferase type I (PGFTase-I) confirmed that ajoene inhibits protein prenylation. Ajoene causes a covalent modification of the SH group of a peptide substrate for protein PGFTase-I. Z-ajoene showed slightly higher antimicrobial (gram-positive bacteria, gram-negative bacteria, and yeasts) activity than E-ajoene.[22] Perhaps, the consumption of food with ajoene will have antimicrobial and anticancer effect in human body.

pH Values of Mayonnaise

The pH values of the mayonnaise after storage for one-day and 30 days at 4^oC are shown in Table 2. There was no significant (P < 0.05) difference in pH values of samples 30A and the control in one-day storage. However, the pH value increased (3.90–3.96) with the increasing the percentage of ajoene substitution. After 30 days storage, the pH value significantly increased (3.92–4.07), whereas the pH values of substituted mayonnaise were significantly lower than the control (4.25).

Table 2 pH analysis of mayonnaise samples

	pH of mayonnaise
Samples	One-day	30 days
Control	3.96 ± 0.02^d	4.25 ± 0.01^e
5A	3.90 ± 0.01^a	3.92 ± 0.02^a
10A	3.91 ± 0.01^ab	4.01 ± 0.01^b
15A	3.92 ± 0.01^bc	4.02 ± 0.005^bc
20A	3.93 ± 0.01^c	4.03 ± 0.005^c
25A	3.94 ± 0.01^c	4.06 ± 0.01^d
30A	3.96 ± 0.01^d	4.07 ± 0.01^d
Mean ± SD values followed by the same letters on the same column are not significantly different (P < 0.05) according to Duncan's multiple range test. Data were the means of three determinations.

One possible reason to explain our results is as follows: undissociated acetic acid is slightly soluble in oil, therefore, the pH of mayonnaise would increase as the percentage of oil increase, particularly after the oil reach 30% level. These results are agreed with the conclusions of previous study.[23] From a microbial safety point of view, it is generally recommended that mayonnaise made with un-pasteurized egg is prepared with vinegar to a pH of 4.1 or less and held at room temperature (18–22^oC) for at least 24 h to reduce the risk from microorganisms.[24]

Storage Stability of E- and Z-Ajoene in Mayonnaise

The stability of E- and Z-ajoene was dependent on the storage time. Figure 2 shows the concentration of E- and Z-ajoene in various storage times. The degradation of E- and Z-ajoene was dependent on the storage time. Generally, the amount of Z-ajoene was higher than E-ajoene in freshly prepared garlic. The amount of both E-ajoene and Z-ajoene found after 30 days of storage period were significantly different from the amount found in first day. Our previous study showed that amount of Z-ajoene was higher than E-ajoene up to three months, but the amount of Z-ajoene decreases due to isomerization after three months of storage period. E-isomer is more stable than Z-isomer.[25]

Figure 2 Stability of E- and Z-ajoene in mayonnaise toward storage period. Mayonnaise samples were stored at 4^oC temperature for 30 days. The results represent the mean values ± SD from three independent experiments. Error bars indicate standard deviations. The same letters on the same parameter are not significantly different (P < 0.05) according to Duncan's multiple range test.

Temperature Stability of E- and Z-Ajoene in Mayonnaise

The stability of E- and Z-ajoene was significantly influenced by the temperature of incubation, in which incubation at 80^oC degraded first, when compared with those at 30 and 60^oC (Table 3). After incubation at 80^oC for three days, the concentration of E-ajoene remained about 61, 62, 69, 72, 74, and 77% depending on the garlic oil substitution of 5A, 10A, 15A, 20A, 25A, and 30A samples respectively, whereas those at 30^oC were about 89, 90, 91, 92, 93, and 94%, respectively. The 30A sample remained highest percent of E- (77%) and Z-ajoene (73%) after three days incubation at 80^oC, whereas 5A remained the lowest percent of E-ajoene (61%) and Z-ajoene (52%). E-ajoene was fairly stable than Z-ajoene. Perhaps, less degradation occurred in high ajoene concentration samples. Perhaps, the decrease of E- and Z-ajoene during storage at different temperatures occurred due to thermal degradation.

Table 3 Temperature stability of ajoene in mayonnaise samples

Samples	Temperature (^oC )	Conc. (%) of ajoene
		E-ajoene	Z-ajoene
	30	89.43 ± 1.97^f	84.72 ± 3.35^g
5A	60	61.13 ± 2.44^a	56.43 ± 3.18^bc
	80	60.71 ± 1.97^a	51.79 ± 2.90^a
	30	89.96 ± 2.97^f	87.17 ± 0.92^gh
10A	60	65.34 ± 1.87^b	58.28 ± 0.99^c
	80	61.78 ± 3.17^ab	53.99 ± 2.06^ab
	30	90.84 ± 1.61f^g	88.94 ± 1.13^hi
15A	60	74.91 ± 2.94^de	65.92 ± 3.42^de
	80	69.15 ± 0.94^c	64.16 ± 1.21^d
	30	91.56 ± 2.86f^g	89.62 ± 2.84^hi
20A	60	74.21 ± 2.82^de	71.92 ± 2.68^f
	80	71.59 ± 1.70^cd	69.49 ± 1.64^ef
	30	93.04 ± 2.46f^g	91.32 ± 1.28^i
25A	60	75.06 ± 1.45^de	72.43 ± 1.33^f
	80	73.60 ± 1.77^de	71.28 ± 1.99^f
	30	93.82 ± 2.27^g	92.55 ± 2.48^i
	60	75.60 ± 1.75^de	73.55 ± 1.41^f
30A	80	77.09 ± 1.78^e	73.30 ± 1.29^f
Mean ± SD values followed by the same letters on the same column are not significantly different (P < 0.05) according to Duncan's multiple range test. Data were the means of three determinations.

UV-Light Stability of E- and Z-Ajoene in Mayonnaise

Total concentrations of all samples decreased after three days of incubation under UV-light as shown in Fig. 3. The concentration of Z-ajoene remained was 85, 87, 88, 88, 89, and 90% depending on the garlic oil substitution of 5A, 10A, 15A, 20A, 25A, and 30A samples after three days incubation. E-ajoene was fairly stable, and the remaining amount was 90, 90, 92, 92, 93, and 93% accordingly. The 30A samples remained significantly higher amount of ajoene than 5A sample. Perhaps, high degradation occurred in low ajoene concentration samples.

Figure 3 Stability of E- and Z-ajoene in mayonnaise toward UV-light. Mayonnaise samples were shaken under UV-light at room temperature for 3 days. The results represent the mean values ± SD from three independent experiments. Error bars indicate standard deviations. The same letters on the same parameter are not significantly different (P < 0.05) according to Duncan's multiple range test.

Light Stability of E- and Z-Ajoene in Mayonnaise

Mayonnaise samples were incubated in fluorescent light for three days and the results of ajoene concentration are shown in Fig. 4. The 5A samples remained 95% E-ajoene and 90% Z-ajoene, whereas 30A remained 98% E-ajoene and 98% Z-ajoene, respectively. These results proved that fluorescent light stability of ajoene is higher than UV-light and temperature (30^oC). The ajoene stability in mayonnaise was in the following order: fluorescent light> UV-light> temperature (30^oC). Therefore, during storage in fluorescent light, UV-light and specific temperature of E- and Z-isomer, isomerization will occur, and then they gradually degraded to other components. These results agreed with the conclusions of previous study.[26] Therefore, these phenomena should be taken into consideration when the E- and Z-ajoene were used as an additive in food.

Figure 4 Stability of E- and Z-ajoene in mayonnaise toward light. Mayonnaise samples were shaken under light at room temperature for 3 days. The results represent the mean values ± SD from three independent experiments. Error bars indicate standard deviations. The same letters on the same parameter are not significantly different (P < 0.05) according to Duncan's multiple range test.

Sensory Evaluations

Sensory evaluation scores of the control and substituted mayonnaise are shown in Table 4. The color and appearance scores significantly (P < 0.05) increased with increasing the substitution levels of ajoene. Color of substitute mayonnaise was evaluated as yellowish. 10A, 15A, and 30A samples were not significantly different from the control in appearance score. The flavor attribute was significantly influenced by the ajoene substitution. The flavor scores were significantly increased with increasing the substitution levels up to 20A, whereas those of 25A and 30A significantly decreased, because fresh, crushed heated garlic has a strong smell.[27]

Table 4 Sensory evaluation of mayonnaise samples

Samples	Color	Appearance	Flavor	Taste	Texture	Mouthfeel	Acceptability
Control	5.2 ± 0.9^a	6.0 ± 0.8^ab	5.5 ± 0.8^c	6.0 ± 0.6^b	5.7 ± 0.9^a	6.3 ± 0.8^c	6.5 ± 0.4^c
5A	6.6 ± 0.6^b	5.71 ± 0.8^a	6.2 ± 0.6^d	6.7 ± 0.8^de	6.1 ± 0.5^ab	6.3 ± 0.6^c	6.5 ± 0.5^c
10A	6.2 ± 0.6^b	6.0 ± 0.6^ab	6.3 ± 0.4^d	6.2 ± 0.6^bc	6.0 ± 0.6^ab	6.3 ± 0.6^c	6.7 ± 0.9^c
15A	6.4 ± 0.5^b	6.5 ± 0.8^bc	6.6 ± 0.5^d	6.5 ± 0.5^bcd	6.6 ± 0.5^bc	6.4 ± 0.5^c	6.8 ± 0.9^c
20A	7.9 ± 0.7^c	8.0 ± 0.8^d	7.9 ± 0.7^e	7.3 ± 0.6^e	7.1 ± 0.7^c	8.4 ± 0.6^d	8.4 ± 0.6^d
25A	7.9 ± 0.9^c	7.2 ± 0.7^c	4.7 ± 0.9^b	6.0 ± 0.6^b	5.6 ± 0.6^a	4.5 ± 0.5^b	5.8 ± 0.7^b
30A	8.0 ± 0.8^c	6.5 ± 0.8^bc	3.3 ± 0.9^a	3.4 ± 0.9^a	7.2 ± 0.9^c	3.1 ± 0.8^a	3.5 ± 0.8^a
Mean ± SD values followed by the same letters on the same column are not significantly different (P < 0.05) according to Duncan's multiple range test. Data were the means of three determinations.

The taste attribution was significantly influenced by the ajoene substitution. 10A, 15A, and 25A samples showed no statistical difference in taste score from the control. The texture scores significantly increased with increasing the substitution levels 15A, 20A, and 30A. The 25A and 30A samples showed significantly lower mouthfeel cores than the control. The overall acceptability scores were significantly lower when the substitution levels were 25A and 30A. This is mainly contributed by the performance of flavor, taste, and mouthfeel. It is reasonable that the sensory attributed with higher scores than 6 are considered to be acceptable when compared with the control. Thus, the garlic oil substituted mayonnaise with the ajoene level up to 20% were judged to be sensorial acceptable.

CONCLUSIONS

This work clarified that the nutritional food value of garlic oil substituted mayonnaise can be improved by the addition of ajoene. The ajoene stability in mayonnaise was observed in the following order: fluorescent light> UV-light> temperature (30 ^oC). However, the 20% garlic oil substituted mayonnaise significantly increased sensory quality as compared with the control sample. However, up to 20% garlic oil substitution, the mayonnaise was found sufficient amount of E- and Z-ajoene that will provide both nutritional and medicinal health benefits to consumers.

ACKNOWLEDGMENTS

The authors are greatly indebted to the Ministry of Education, Science, Sports, and Culture, Japan (Monbusho) for the award of scholarship to most Tahera Naznin. We reverentially thank Dr. Mitsugu Akagawa (Department of Biological Chemistry, Osaka Prefecture University) for his valuable suggestions. We especially thank Fine Foods Co., Ltd. (Osaka, Japan) for supplying oil.