2015 Volume 21 Issue 6 Pages 787-791
We sought a method to increase γ-aminobutyric acid (GABA) levels in sesame seeds. When water was added to the seeds and they were heated at 60°C for 15 min, the GABA content increased over 8-fold compared to the starting seeds. The amount of water added to the seeds affected the production of GABA, with the addition of more water leading to production of more GABA. GABA production occurred during the first 10 min of treatment at 60°C. Moreover, the addition of l-glutamate resulted in further GABA enrichment. The results indicate that our method using a short-duration heat treatment can form GABA-rich products from sesame seeds.
γ-Aminobutyric acid (GABA) is a non-protein amino acid found in bacteria, plants and vertebrates. In mammals, GABA is present at high levels in the brain and is the predominant inhibitory neurotransmitter. It also has roles in lowering blood pressure and in mental stabilization (Hayakawa et al., 2004; Abdou et al., 2006; Guyenet, 2006). GABA is mainly metabolized via a short pathway called the GABA shunt. In this pathway, GABA is synthesized through the α-decarboxylation of l-glutamate (l-Glu) catalyzed by l-Glu decarboxylase (GAD) (Bown and Shelp, 1997; Shelp et al., 1999; Bouché and Fromm 2004; Fait et al., 2008). In plant seeds, GAD expression and activity increase during germination (Kuo et al., 2004; Xu and Hu, 2014). GABA is contained in high quantities in germinated brown rice and soybean sprouts, which are eaten on a daily basis in Japan (Martínez-Villaluenga et al., 2006; Iwaki et al., 2007).
In plants, GABA is accumulated as a metabolite in response to various stress conditions (Wallace et al., 1984; Kaplan et al., 2004; Chung et al., 2009). The GABA-accumulation phenomenon has been utilized to enrich GABA content of fruits, vegetables and other plant materials. The process includes anaerobic CO2, high-pressure and immersion treatments (Saikusa et al., 1994; Sasagawa et al., 2006; Wang et al., 2006; Komatsuzaki et al., 2007; Akihiro et al., 2008; Watanabe et al., 2013).
Sesame seeds have high nutrient content and health-promoting functions. Therefore, the seeds have been eaten for generations on a daily basis in many countries. However, the seeds contain only trace levels of GABA as a functional amino acid. Nakashima et al. (2005) reported that the GABA content in sesame seeds was increased by a 48-h germination treatment. However, GABA enrichment by germination is not of practical use because it is time-consuming and the flavor deteriorates during the process. Generally, roasted seeds are successfully processed by removing foreign substances, washing, dehydrating, roasting, cooling, again removing foreign substances, and finally, packing. On a commercial scale, GABA enrichment might be incorporated into this process. In this study, we investigated ways to enrich for GABA in sesame seeds with high efficiency before roasting.
Materials White sesame seeds (Escoba), harvested in Paraguay, were obtained from Shirosawa Co. SAIC (Limpio, Paraguay).
Measurements of GABA and l-Glu Sesame seeds were ground into a paste and the paste was defatted using n-hexane. GABA and l-Glu were extracted from the defatted powder at 4°C for 3 h after adding distilled water. The insoluble matter in the extract was removed by centrifugation at 1600 × g for 15 min. GABA and l-Glu in the extract were analyzed by high-performance liquid chromatography (HPLC) using the Waters Pico-Tag method (White et al., 1986). Namely, GABA and l-Glu were treated with phenylisothiocyanate to form phenylthiocarbamyl derivatives (PTC amino acids). The PTC amino acids were analyzed on a 3.9 × 300-mm Pico-Tag free amino acid column at 51°C using a JASCO system with a PU-2089 pump, a UV-2075 detector and a CO-2065 column heater. A linear-gradient system was used, with elution from a 70-mM sodium acetate solution containing 2.5% acetonitrile (pH 6.45) to a mixture of water/methanol/acetonitrile (40:15:45, by vol.) at 1.0 mL/min. GABA and amino acid standard solution H (Wako) were used as the standards.
Conditions for the GABA enrichment processes
(1) Heating temperature Three types of sesame seed samples were used for the heating experiments. In the first, whole sesame seeds were heated at 40 – 160°C. The optimum heating time was determined to be 15 min based on the results of a preliminary experiment. In the second, water was added to the seeds at a ratio of 100 µL/g and heated at 40 – 100°C for 15 min. The third used a paste of the seeds. The seeds were ground and added to 10 volumes of water. This paste was then heated at 40 – 100°C for 15 min. The heated seeds were immediately cooled, and the GABA content was measured. Seeds or seeds with water added were used as controls. Control seeds were maintained at room temperature for 15 min.
(2) Water addition Water in the range of 10 – 1000 µL/g was added to the seeds, which were heated at 60°C for 15 min. After immediate cooling, the GABA content was measured.
(3) Time-course experiment Whole sesame seeds were heated at 60, 100 and 160°C. Periodically, aliquots of the sample were withdrawn, cooled immediately, and the GABA content measured. In another experiment, water was added to the seeds at 100 µL/g, and the seeds were heated at 60°C. Periodically, the GABA and l-Glu contents were measured.
(4) Addition of l-Glu The effect of l-Glu was investigated by running three experiments. In experiment I, a 17 mM l-Glu solution was added to the seeds at 200 µL/g, and the seeds were heated at 60°C for 15 min. Experiment II consisted of two steps. Initially, water was added to the seeds at 100 µL/g, and the seeds were heated at 60°C for 15 min. Subsequently, a 34 mM l-Glu solution was added to the seeds at 100 µL/g, and the seeds were heated again at 60°C for 15 min. In experiment III (the control for experiment I), water was added to the seeds at 200 µL/g, and the seeds were heated at 60°C for 15 min. After heating, the GABA content of each sample was measured.
Optimum temperature for GABA production Sesame seeds should be treated before roasting to enrich the GABA content because the endogenous GABA-producing enzyme, GAD, is deactivated by roasting. Figure 1 shows the relationship between the heating temperature and the GABA content of sesame seeds after heating for 15 min. An increase in the GABA content was observed for all of the tested temperatures. In particular, GABA enrichment was remarkable at 80°C or above. The maximum GABA content was 0.84 µmol/g when the seeds were heated at 100°C.
Effect of heating temperature on GABA production in sesame seeds without added water. Sesame seeds were heated at the indicated temperature for 15 min. C, control, was maintained at room temperature. Error bars represent standard deviations of the means (n = 3).
A time-course experiment was performed at 60, 100, and 160°C (Fig. 2). Rapid production of GABA was observed in treatment at 100 and 160°C. The maximum GABA production depended on the heating temperature: at higher temperatures, production of GABA was faster. This indicates that GABA is produced until the inside temperature of the seeds reaches the temperature of GAD deactivation. After the maximum production of GABA, the decrease was gradual, probably by an aminocarbonyl reaction.
Changes in GABA in sesame seeds during heating. Seeds without added water were heated at 60, 100 and 160°C. Error bars represent standard deviations of the means (n = 3).
Non-enzymatic production of GABA was confirmed by heating of l-Glu alone, which did not generate GABA (data not shown). Nagaoka (2005) and Matsuyama et al. (2009) reported that GABA is generated by GAD during germination. Therefore, GABA would be generated by an enzymatic reaction during heating of the seeds.
When water was added to sesame seeds, heating treatment caused more GABA production (Fig. 3). The maximum GABA content was 4.2 µmol/g when the seeds were heated at 60°C. This means that the GABA content is 70 times greater than that of untreated raw seeds. The inside temperature of the seeds would be important for the GABA enrichment process. The rate of increase of the inside temperature varies depending on whether water is added. The inside temperature would become suitable for enriching the GABA content when seeds are heated with water at 60°C. Without heating (at room temperature), the seeds with water added contained a significantly greater amount of GABA compared to the control of Fig. 1 (p < 0.01). Bewley (1997) reported that the first change upon imbibition of seeds is the resumption of respiratory activity, which can be detected within minutes. Upon imbibition, sesame seeds should resume metabolic activity, including the GABA shunt.
Effect of heating temperature on the production of GABA in sesame seeds with water added. Sesame seeds with added water (100 µL/g of seeds) were heated at the indicated temperature for 15 min, or for Cwater, the control, was maintained at room temperature. Error bars represent standard deviations of the means (n = 3).
The paste state of seeds with 10 times the volume of water (w/v) was incubated at different temperatures (Fig. 4). The maximum GABA production of 0.74 µmol/g was obtained when the paste was incubated at 40°C. This temperature was near the optimum temperature of GAD of rice germ and potatoes, for example (Zhang et al., 2007; Satyanarayan and Nair, 1985).
Effect of heating temperature on GABA production in sesame seed paste. Water was added to ground sesame seeds (10 mL/g of seeds) and heated at the indicated temperature for 15 min. Cwater, samples maintained at room temperature. Error bars represent standard deviations of the means (n = 3).
Effect of added water Figure 5 shows the relationship between the amount of added water and the GABA content of the seeds after heating. The GABA content increased in proportion to the added water up to 100 µL/g. When water was added to seeds at a range of 100 to 1000 µL/g, no significant difference in the GABA content was observed. These results indicate that addition of water is necessary to increase the GABA content in sesame seeds.
Effect of water addition on the production of GABA. The indicated amount of water was added to sesame seeds and heated at 60°C for 15 min. C, control. Error bars represent standard deviations of the means (n = 3).
Changes in GABA and l-Glu contents during heating The changes in the amounts of l-Glu, a precursor of GABA, and of GABA in seeds during heating are shown in Fig. 6. The GABA content was increased after the first 10 min of heating, and then maintained a constant value.
Changes in GABA and l-Glu in sesame seeds during heating. Seeds with water added (100 µL/g seeds) were heated at 60°C. Error bars represent standard deviations of the means (n = 3).
The l-Glu content also decreased by the first 15 min of heating, and then became constant. The amount of increased GABA was almost the same as that of the decreased l-Glu. This observation suggests that GABA was synthesized enzymatically from l-Glu during heating.
Germination of sesame seeds also results in the production of GABA, and the optimum germination temperature has been reported to be 20 – 30°C (Kyauk, et al., 1995). This is different from the temperature used for GABA enrichment in this study. We assumed that the GABA enrichment in the 60°C treatment might be controlled by a different mechanism from that in seed germination.
The accumulation of GABA in plant cells has roles in pH regulation, protection against oxidative stress and in osmoregulation (Chung et al., 2009; Wallace et al., 1984; Kaplan et al., 2004). A high accumulation of GABA in sesame plants under heat stress has been reported (Bor et al., 2009). In this study, a high accumulation of GABA was recognized in sesame seeds heated at 60°C. Therefore, it is presumed that heat stress induced the GABA accumulation.
GABA enrichment of sesame seeds by conventional germination requires 2 days of processing time. On the other hand, our new GABA-enrichment method of heating at 60°C for 10 min brings about sufficient efficiency for application in industry.
There are many reports concerning the enrichment of GABA in fruits, vegetables and other plant materials. For example, the GABA levels of GABA-rich tomatoes and pumpkins have been reported to be approximately 1000 mg/100 g and 500 mg/100 g, respectively (Watanabe et al., 2013; Akihiro et al., 2008). On the other hand, the levels in GABA-rich rice have been reported to be 20 – 40 mg/100 g (Sasagawa et al., 2006; Lu et al., 2010). The GABA content in our prepared sesame seeds was calculated to be 43.3 mg/100 g. This content is almost the same as that of GABA-rich rice. Typical consumption is about 10 g of sesame seeds per meal, and therefore GABA-rich sesame seeds might be one source of GABA.
Effect of l-Glu on GABA enrichment The possibility of further GABA enrichment was examined by adding l-Glu to sesame seeds (Table 1). We compared the GABA content of experiments I, II and III. Experiment I showed that the GABA content in seeds increased after equimolar addition of l-Glu (3.4 µmol/g) compared with that of the control experiment III. Experiment II consisted of two steps. The first step was enriching for GABA without l-Glu by heating to 60°C. The second step was the addition of l-Glu to the seeds, followed by heating again. In the second step, the GABA content increased again. There were no significant differences between the GABA contents in experiments I and II (p < 0.05). This result suggests that heating at 60°C does not affect the deactivation of enzymes related to GABA enrichment. The reason why the GABA content became constant after 10 min of heating (Fig. 6) is likely because the concentration of l-Glu, a precursor of GABA, was lowered by the enzymatic reaction.
| Experiment | GABA(µmol/g)a) |
|---|---|
| I | 8.38 ± 1.13 |
| II | 8.53 ±1.25 |
| III | 4.55 ±0.39 |
The GABA content could be further increased by selecting raw sesame seeds containing a high percentage of l-Glu. However, there is a limitation to GABA enrichment using endogenous l-Glu in the seeds. According to this study, enriching GABA by adding l-Glu is possible, resulting in a more efficient method for enriching the GABA content.
We investigated a quick, high-efficiency method for GABA enrichment in sesame seeds without germination. The results were as follows:
