2014 Volume 20 Issue 2 Pages 401-407
The palatability of boiled soybean vegetable with respect to maltose production was investigated using seven cultivars of soybean vegetables. Although sucrose is the most abundant free sugar (ca. 90%) in raw soybean vegetables, maltose was also shown to contribute to palatability (sweetness). Maltose production was found to be related to both β-amylase activity and starch gelatinization temperature. Of the seven cultivars studied, ShinTamba-Guro' had the lowest starch gelatinization temperature (55°C) and the highest maltose content, while ‘Fuki’ had the highest starch gelatinization temperature (> 70°C), lowest β-amylase activity and did not produce maltose, with boiling treatment. Sweetness was proposed as one of the factors related to the palatability of soybean vegetable, and was affected by the content and composition of sucrose and/or maltose.
Soybean (Glycine max (L.) Merr.) is used worldwide, not only as the mature bean but also as an ingredient in various kinds of processed foods, including tofu (soy bean curd), soymilk, natto (fermented soybean), tempeh, miso (soy bean paste) and soy sauce. Compared to mature soybean, immature soybean (soybean vegetable) is rich in vitamin C (Miyake et al., 2007) and folic acid (Masuda and Nakamura, 2010), and is recognized as a healthy Japanese food (Sasahara, 2000). For these reasons, consumption of soybean vegetable has increased both in Japan and abroad.
Unlike mature soybean, soybean vegetable is typically ingested after boiling without seasoning. The palatability of soybean vegetable depends upon the production of taste substances in the vegetable (i.e., free amino acids and sugars) and texture with boiling treatment. A positive correlation has been reported between the total free sugar content of foods and the results of sensory evaluation (Konishi et al., 1996; Sugimoto et al., 2004; Miyagi et al., 2011).
In Kyoto, there is a well-known large black soybean called Tamba-Guro (MAFF, 2010), and its soybean vegetable produces large amounts of maltose (Masuda, 2003; Furutani et al., 2012). The Kyoto Prefectural Agricultural Experiment Station conducted pure-line selection of the ‘Shin-Tamba-Guro’ cultivar from Tamba-Guro. ‘Murasaki-Zukin’ was produced by radiation-induced mutation of ‘Shin-Tamba-Guro’. Moreover, ‘Murasaki-Zukin 2go’ was produced by crossbreeding between ‘Murasaki-Zukin’ and ‘Tamadaikoku’, another black soybean cultivar, in order to extend the harvest season. Currently, these three cultivars (‘Shin-Tamba Guro’, ‘Murasaki-Zukin’ and ‘Murasaki-Zukin 2go’) form “Murasaki-Zukin®”, which is a guaranteed Kyoto brand item, a flagship item, and is produced from early September to mid-October. It has been reported that 60% of the free sugar in boiled soybean vegetable is sucrose (Abe et al., 2004) and that sucrose contributes to the palatability of soybean vegetable (Masuda, 2003 and 2004); however, sucrose levels in soybean vegetable were reported to decrease rapidly after harvesting (Hirota et al., 2003). Therefore, in order to prevent decreases in sucrose levels, modified atmosphere (MA) packaging and controlled storage temperature during shipping (Ishikawa, 2008; Sone, 2011) were introduced.
The sweetness of soybean vegetable originates not only from sucrose, but also from the maltose (Kawai, 2004; Honjo et al., 2007; Furutani et al., 2012) produced from starch by β-amylase during boiling. The palatability of soybean vegetable is thought to be related to starch gelatinization properties and the heat stability of β-amylase. However, maltose production upon boiling was reported to be lower in early cultivars than in later ones, and the amount of maltose production in the three “Murasaki-Zukin®” cultivars differed from each other (Furutani et al., 2012). In addition, maltose production of ‘Murasaki-Zukin 2go’ with boiling treatment varied widely depending on the year (Kawai, 2004).
As there are few reports on maltose production in soybean vegetable when boiled, the relationship between palatability and maltose production was investigated in various soybean vegetable cultivars, including black soybean vegetables.
Samples Soybean vegetables were harvested in 2011 at the Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Center. Samples harvested included four varieties of black soybean vegetable, ‘Murasaki-Zukin 2go’ (M2), ‘Murasaki-Zukin’ (MU), ‘Shin-Tamba-Guro’ (ST), and ‘Tamadaikoku’ (TD), and three varieties of yellow soybean vegetable, ‘Kyo-Shiro-Tamba’ (KT), ‘Otsuru’ (OT), and ‘Fuki’ (FK) (Table 1). ‘Kyo-Shiro-Tamba’ (Minamiyama et al., 2012) was obtained by breeding ‘Murasaki-Zukin’ and ‘Tamadaikoku’.
| Seed color | Brand name | Cultivar1) | Flowering date | Harvesting date | g f.w./ seed pod2) | g f.w./ grain |
|---|---|---|---|---|---|---|
| Black soybean | Murasaki-Zukin® | M2 | 21 July | 10 September | 1.79 ± 0.13 | 1.14 ± 0.12 |
| MU | 27 July | 26 September | 2.39 ± 0.06 | 1.39 ± 0.13 | ||
| ST | 6 August | 11 October | 2.25 ± 0.03 | 1.49 ± 0.09 | ||
| TD | 16 July | 9 September | 1.38 ± 0.12 | 0.79 ± 0.06 | ||
| Yellow soybean (grain-type) | KT | 19 July | 26 September | 1.61 ± 0.15 | 0.91 ± 0.05 | |
| OT | 24 July | 22 September | 0.80 ± 0.02 | 0.49 ± 0.05 | ||
| 〃 (vegetable-type) | FK | 13 July | 30 August | 1.16 ± 0.06 | 0.59 ± 0.07 |
1)Abbreviations are shown following the name of each cultivar.
M2: Murasaki-Zukin 2go; MU: Murasaki-Zukin; ST: Shin-Tamba-Guro;
TD: Tamadaikoku; KT: Kyo-Shiro-Tamba; OT: Otsuru; FK: Fuki
2)Each value represents mean ± S.D. Seed pod indicates the weight of pods containing two seeds/pod.
Sixty soybean vegetable pods of each variety were harvested randomly from 16 plants and were either directly stored at −30°C or boiled for 10 min (6 min in the case of ‘Otsuru’ and ‘Fuki’ because of their small size) and then stored at −30°C until analysis. The flowering and harvesting dates are shown in Table 1.
Analysis of free sugar composition The water-soluble fraction of raw or boiled seeds of soybean vegetables was obtained by homogenizing in eight volumes of 80% ethanol for 1 min at room temperature, and then the extraction was repeated in another eight volumes of 80% ethanol. The extract obtained after centrifugation was then filtered through a 0.45 µm membrane filter. The sugar composition of the filtrate was analyzed using an HPLC system (1260 Infinity, Agilent Technologies, Palo Alto, CA, USA) equipped with a RID detector (1260 RID, Agilent Technologies, Palo Alto CA, USA). The analytical conditions were as follows: column, Asahipak NH2-50 4E (SHOWA DENKO, Tokyo, Japan); solvent, 70% acetonitrile; flow rate, 1 mL/min; column temperature, 40°C.
β-Amylase activity of soybean vegetables The β-amylase-containing fraction was obtained by homogenizing soybean vegetable (0.5 g fresh weight) in 5 mL of 1 M Tris/HCl buffer, pH 8.0. β-Amylase activity in the crude extract was determined by using a BETAMYL-3® kit according to the manufacturer's instructions (Megazyme, Bray, Ireland). Briefly, crude extract (100 µL) was incubated with 100 µL of 1 mM PNP β-G3 for 10 min at 40, 60, 65, 70 or 75°C in 0.1 M MES buffer, pH 6.2, containing 1 mM EDTA, 1.0 mg/mL of BSA and 0.02% sodium azide. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µM of p-nitrophenol from 1 mM of PNP β-G3 in 1 min.
Starch content The starch content was determined using a modified method of McCready et al. (1950). The lyophilized raw sample was washed four times with 80% ethanol to remove water-soluble free sugars, and then hydrolyzed with 52% perchloric acid at room temperature for 10 min. After hydrolysis, the sugar content was determined using the phenol-sulfuric acid method (Dubois et al., 1956). The starch content was obtained by multiplying the sugar content by a factor of 0.9.
Fractionation of starch Fractionation of starch was performed according to the method of Matsunaga et al. (2003). Each soybean vegetable sample (30 g f.w.) was first deproteinized by homogenizing in 0.05 M NaOH. After centrifugation (5500 × g, 10 min), the precipitates were neutralized with 1M HCl, and washed several times with water. The precipitates were filtered through a 325-mesh particle size screen and the starch fraction was obtained after drying at 30°C for 24 hr.
Thermal transition of starch In order to investigate the starch gelatinization properties, the heat of onset temperature (To), peak temperature (Tp), and end set temperature (Te) were analyzed by DSC (DSC-60, SHIMADZU, kyoto, Japan). The temperature was increased at a rate of 2°C/min. Each sample (4 mg) was suspended in 20 µL of water for analysis.
Statistical analysis To analyze the significance of differences in results of free sugar content, β-amylase activity, starch content, and starch gelatinization temperature, Tukey-Kramer's test was performed using Excel Statistics-third edition (OMS, Saitama, Japan).
Characterization of soybean vegetables Characteristics of the soybean vegetables are shown in Table 1. Although the seven cultivars were sown on the same date (23 June), the flowering and harvesting dates differed among cultivars. The flowering and harvesting dates between the early cultivar, ‘Fuki’, and the late cultivar, ‘Shin-Tamba-Guro’, were 24 and 42 days apart, respectively.
The seed weights ranged from 0.49 g–1.49 g; the beans of ‘Otsuru’ were the lightest, and those of ‘Shin-Tamba-Guro’ were the heaviest. The weight of ‘Murasaki-Zukin’ seed pods were the heaviest.
Free sugar composition The sugar composition of the water-soluble fractions of raw and boiled soybean vegetables is shown Fig. 1. The total sugar content was highest in ‘Fuki’ (3.87 g/100 g f.w.), followed by ‘Shin-Tamba-Guro’ (3.02 g), ‘Murasaki-Zukin 2go’ (2.89 g), ‘Otsuru’ (2.78 g), ‘Murasaki-Zukin’ (2.46 g), ‘Tamadaikoku’ (2.20 g) and ‘Kyo-Shiro-Tamba’ (2.05 g). The main sugar component in the raw soybean vegetables was sucrose, which accounted for about 90% of the total free sugar, along with small amounts of glucose and fructose. After boiling, a 10–20% reduction in sucrose content was observed, and a considerable amount of maltose was produced except for in the case of ‘Fuki’. The maltose content per 100 g f.w. was 1.52 g in ‘Shin-Tamba-Guro’, 1.33 g in ‘Kyo-Shiro-Tamba’, 1.11 g in ‘Otsuru’, 1.08 g in ‘Murasaki-Zukin’, 0.95 g in ‘Murasaki-Zukin 2go’ and 0.91 g in ‘Tamadaikoku’. In particular, the maltose content in ‘Shin-Tamba-Guro’ and ‘Kyo-Shiro-Tamba’ was significantly higher (p < 0.05) than in other cultivars. The total free sugar content in boiled soybean vegetable was highest in ‘Shin-Tamba-Guro’ (4.26 g/100 g f.w.), followed by ‘Otsuru’ (3.74 g), ‘Murasaki-Zukin 2go’ (3.59 g), ‘Murasaki-Zukin’ (3.53 g), ‘Kyo-Shiro-Tamba’ (3.26 g), ‘Tamadaikoku’ (2.84 g) and ‘Fuki’ (2.83 g).
Free sugar contents of soybean vegetables in various cultivars.
a) Raw seeds and b) Boiled seeds
: M2 (Murasaki-Zukin 2go); 
: MU (Murasaki-Zukin); 
: ST (Shin-Tamba-Guro); 
: TD (Tamadaikoku); 
: KT (Kyo-Shiro-Tamba); 
: OT (Otsuru); 
: FK (Fuki)
The different letters within raw and boiled soybean vegetables indicate statistical significance in each sugar ( p < 0.05). Error bars indicate the standard deviation of three independent measurements.
β-Amylase activity To examine if the production of maltose after boiling might be related to β-amylase activity, the β-amylase activity in crude extracts at various temperatures was determined. The β-amylase activities at each temperature are shown in Fig. 2. In all cultivars, β-amylase activity was highest at 65°C and decreased at 70°C. When comparing β-amylase activities at 65°C, that of ‘Otsuru’ (470 U/g f.w.), a yellow soybean grain-type cultivar, was significantly higher than other cultivars, indicating that this cultivar had a high β-amylase content. There was no significant difference in total β-amylase activity at 65°C among the other cultivars, except for ‘Fuki’, in which no maltose production was observed after boiling, i.e., ‘Tamadaikoku’ (295U), ‘Kyo-Shiro-Tamba’ (293U), ‘Murasaki-Zukin’ (240U), ‘Murasaki-Zukin 2go’ (241U), ‘Shin-Tamba-Guro’ (250U) and ‘Fuki’ (201U). In addition, there was no correlation between the maltose content after boiling and total β-amylase activity at 65°C.
Total activities of β-amylase per 1g fresh weight (f.w.) of soybean vegetable at various temperatures.
M2: Murasaki-Zukin 2go; MU: Murasaki-Zukin; ST: Shin-Tamba-Guro;
TD: Tamadaikoku; KT: Kyo-Shiro-Tamba; OT: Otsuru; FK: Fuki
Error bars indicate the standard deviation of three independent measurements.
In order to investigate the absence of maltose production in ‘Fuki’, starch characterization was performed.
Starch content and gelatinization temperature The starch content of each cultivar is shown in Fig. 3. Tamba-Guro was previously reported to contain high sucrose and starch contents (Masuda, 2004). In the present study, the starch content was highest in ‘Shin-Tamba-Guro’ (5.3 g/100 g f.w.) when compared to other cultivars: ‘Murasaki-Zukin 2go’ (4.74 g), ‘Otsuru’ (4.65 g), ‘Tamadaikoku’ (4.03 g), ‘Murasaki-Zukin’ (3.99 g) and ‘Fuki’ (3.93 g). The starch content of ‘Fuki’ was similar to that of ‘Tamadaikoku’ and ‘Murasaki-Zukin’. In addition, there was no correlation between starch content and maltose production after boiling. Thus, the starch gelatinization properties of each cultivar were investigated using DSC, and the results are shown in Table 2.
Starch content in soybean vegetable cultivars.
M2: Murasaki-Zukin 2go; MU: Murasaki-Zukin; ST: Shin-Tamba-Guro;
TD: Tamadaikoku; KT: Kyo-Shiro-Tamba; OT: Otsuru; FK: Fuki
Error bars indicate the standard deviation of three measurements.
Superscript letter indicate the level significance (*p < 0.05)
| Cultivar1) | Endothermic transition (°C) | ||
|---|---|---|---|
| To2) | Tp | Te | |
| M2 | 62.7 ± 0.4abc | 69.4 ± 0.7a | 75.1 ± 0.3a |
| MU | 61.2 ± 0.3ade | 65.8 ± 0.5bc | 71.6 ± 0.6bc |
| ST | 55.2 ± 0.2g | 57.5 ± 0.2e | 61.9 ± 0.5g |
| TD | 64.9 ± 1.0h | 68.3 ± 1.1a | 73.4 ± 0.9ade |
| KT | 61.2 ± 0.5bdf | 65.8 ± 0.1bd | 71.8 ± 0.5bdf |
| OT | 61.7 ± 0.6cef | 66.7 ± 0.3cd | 72.0 ± 0.6cef |
| FK | 69.5 ± 0.2i | 73.4 ± 0.1f | 77.2 ± 0.3h |
1)M2: Murasaki-Zukin 2go; MU: Murasaki-Zukin; ST: Shin-Tamba-Guro;
TD: Tamadaikoku; KT: Kyo-Shiro-Tamba; OT: Otsuru; FK: Fuki
2)Each value represents mean ± S.D.
The different letters within a column indicate statistical significance (p < 0.05).
There were significant differences in To, Tp and Te values between ‘Shin-Tamba-Guro’, which had a high maltose content, and ‘Fuki’, which did not produce maltose when boiled. The To, Tp and Te values of ‘Fuki’, which were the highest among the seven cultivars, were 14.3, 15.9, and 15.3°C higher, respectively, than those of ‘Shin-Tamba-Guro’, which had the lowest values among the seven cultivars.
Soybean vegetable is generally ingested after boiling and without seasoning. The palatability of soybean vegetable depends on the production of taste substances (i.e., free amino acids and sugars) and texture after boiling. As the palatability of rice, chestnut and Japanese bunching onion was reported to be positively correlated with free sugar content (Konishi et al., 1996; Sugimoto et al., 2004; Miyagi et al., 2011), the palatability of soybean vegetable with respect to sugar composition after boiling was investigated in this study.
In the case of fresh soybean vegetable, sucrose was the main free sugar in all cultivars investigated, as previously reported by Masuda (2004). Among the samples, the ‘Fuki’ cultivar had the highest sucrose content, 3.8 g/100 g f.w. In the case of boiled soybean vegetable, both sucrose and maltose were shown to contribute to sweetness, although sucrose contents were decreased to about 80% of that observed in the fresh soybean vegetable. On the other hand, while no maltose was detected in ‘Fuki’, it contained the highest sucrose content when compared to the other varieties in this study. Therefore, ‘Fuki’ is a high-sucrose type of vegetable soybean (Masuda, 2004). The sweetness of maltose is known to be 30% that of sucrose, and is mellower than sucrose (Takasaki, 1980; Sakai, 1981). Thus, even if the free sugar contents are equal, the sweetness and its quality might vary depending on the ratio of sucrose to maltose. In the case where the sugar contents of samples are similar, samples with a high sucrose content would score high in sweetness (Inukai et al., 2007; Masuda et al., 2007). The sucrose content was found to decrease soon after harvesting, but the decrease in maltose was small compared to that of sucrose (Sugimoto et al., 2010). Therefore, maltose production when boiling might play an important role in the palatability of soybean vegetable.
Soybean contains large amounts of β-amylase and negligible amounts of α-amylase (Kono et al., 2011). Therefore, in the present study the production of maltose after boiling was investigated with respect to β-amylase and starch contents, as well as starch gelatinization properties, using seven cultivars of soybean vegetables. Maltose production from starch during boiling is thought to depend upon the content of β-amylase and its thermal stability, as well as the content and gelatinization properties of starch. Although the optimum temperature for β-amylase activity in sweet potato is 40°C (Ogura et al., 2001), that of soybean vegetable was shown to be 65°C. When considering the relation between starch gelatinization temperature (around 60 ∼ 70°C) and the temperature at which β-amylase activity declined (over 70°C), maltose might be produced by the degradation of gelatinized starch by β-amylase, with the exception of ‘Fuki’. In the case of ‘Fuki’, the reason why maltose was not produced upon boiling may be that the gelatinization temperature was higher than the temperature at which enzyme activity declined. Masuda (2003 and 2004) suggested that the differences in maltose content after boiling of soybean vegetable cultivars were determined by the differences in gelatinization temperature rather than β-amylase heat stability. The large amount of maltose produced by ‘Shin-Tamba-Guro’ may be due to it having the lowest starch gelatinization temperature. ‘Kyo-Shiro-Tamba’, which showed very high β-amylase activity, produced large amounts of maltose. Overall, the amount of maltose produced with boiling was affected by the relation between gelatinization temperature and β-amylase activity.
In addition, pod size might be associated with maltose production; the size of ‘Shin-Tamba-Guro’ was twice as large as that of ‘Fuki’ (Table 1). Therefore, the temperature of the seeds in the pod might increase more slowly in the case of ‘Shin-Tamba-Guro’, which was thought to contribute to starch degradation upon boiling. In contrast, in the case of ‘Fuki’, the temperature of the seeds in the pod may have increased rapidly, possibly preventing β-amylase from degrading the starch.
The new cultivar “Murasaki-Zukin®” is currently under investigation in an effort to extend the harvest period. From the viewpoint of palatability, the new cultivars are expected to contain high sucrose as well as maltose after boiling. ‘Shin-Tamba-Guro’ was shown to be a very promising cultivar in terms of large size of seed and pod, good taste and characteristic texture (Furutani et al., 2012), in addition to producing high levels of maltose. It has been suggested that the starch gelatinization temperature of soybean vegetable might be affected by growth temperature (Masuda, 2004; Honjo et al., 2007).
The results of this study suggest a relationship between soybean vegetable palatability and maltose production upon boiling, which is useful information for breeding programs.
