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Development of a Simple Enzymatic Method for Screening Sucrose Content in Legume Seeds
Plant Breed. Biotech. 2021;9:250-258
Published online September 1, 2021
© 2021 Korean Society of Breeding Science.

Gyutae Kim1, Aron Park1, Woon Ji Kim1, Chang Yeok Moon1,2, Byeong Hee Kang1,2, Seong-Hoon Kim3, Yu-Mi Choi3*, Bo-Keun Ha1,2*

1Department of Applied Plant Science, Chonnam National University, Gwangju 61186, Korea
2BK21 Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju 61186, Korea
3National Agrobiodiversity Center, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea
Corresponding author: Bo-Keun Ha, bkha@jnu.ac.kr, Tel:+82-62-530-2055, Fax:+82-62-530-2059
Yu-Mi Choi, cym0421@korea.kr, Tel: +82-63-238-4911, Fax:+82-63-238-4909
Received July 29, 2021; Revised August 10, 2021; Accepted August 17, 2021.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The soluble sugar content of legume seeds affects the final flavor of the legume and legume products. The purpose of this study was to develop a rapid, simple, and low-cost colorimetric method for high-throughput screening of sucrose content in legume seeds. This colorimetric method was based on the enzymatic reactions of invertase (INV) and glucose oxidase (GOD). Briefly, 20 different soybean and cowpea varieties were used in this study. For sucrose extraction, solvent-to-sample ratios of 10:1 and 5:1 were found to result in optimal absorbance values for determining sucrose content in soybean and cowpea, respectively. The extraction efficiency was also evaluated under various extraction temperatures (25℃ and 50℃) and incubation times (15 minutes, 2 hours, 8 hours, and 24 hours) and the sucrose content was found to increase with increasing temperature and time. Accordingly, the optimal extraction conditions were 24 hours of incubation at 50℃. Under this condition, the GOD/INV method had significant positive correlations (r = 0.91** for soybean and r = 0.87** for cowpea) with the high-performance liquid chromatography method. Overall, this colorimetric method is a fast, simple, and inexpensive tool for quantitative determination of sucrose content in legumes.
Keywords : Sucrose quantification, Soybean, Cowpea, Enzymatic Method, Invertase, Glucose oxidase
INTRODUCTION

Legume crops have been cultivated for thousands of years for human nutrition and animal feed (Bennetau-Pelissero 2018). Legume is nutritionally valuable and provides protein (20%-45%) with essential amino acids, complex carbohydrates (± 60%), and dietary fiber (5%-37%) (Duranti and Gius 1997; Messina 1999; Maphosa and Jideani 2017). Soybean (Glycine max (L.) Merr.), which is the most important legume crop, contains approximately 40% protein, 20% oil, and 33% carbohydrates (Hymowitz et al. 1972; Bellaloui et al. 2015; Akond et al. 2018). The world’s soybean production area is approximately 120 million hectares and the annual production is 333 million metric tons. In 2019, the U.S. was the top producer of soybean (34%), followed by Brazil (33%), Argentina (16%), China (4%), and Paraguay (3%) (Faostat 2019). Cowpea (Vigna unguiculata (L.) Walp) is also an important legume crop that contains high levels of carbohydrates (~64%), proteins (~25%), vitamins, and minerals such as iron, calcium, thiamine, and folic acid (Boukar et al. 2011). In 2019, the total production area for cowpea was approximately 14.4 million hectares and the annual output was more than 8.9 million tons worldwide (Faostat 2019). Cowpea is widely distributed in the tropics. In Central and West Africa, its cultivation is carried out on approximately 8 million hectares of land, covering 55.5% of the area, followed by Latin America (16.7%), Asia (9%), and East Asia (5.6%) in cowpea coverage (Faostat 2019).

Mature legume seeds contain high levels of soluble carbohydrates, which are important physiological traits that influence food quality in seeds and seed production (Hou et al. 2009b; Obendorf and Górecki 2012; Nassourou et al. 2017). Sucrose is the most abundant soluble carbohydrates in grain legumes. In fact, the soybean sucrose content ranges from 1.5% to 10.2% (Hou et al. 2009a; Taira 1990) and the soluble sugar content of cowpea ranges from 0.5% to 1.3% (Nassourou et al. 2017). Other carbohydrates include raffinose and stachyose, which belong to the raffinose family of oligosaccharides (RFOs), are not readily digested by humans, and cause flatulence or diarrhea (Rackis 1975). Therefore, several breeding efforts have been made to increase the sucrose content and decrease the RFO content.

Rapid and reliable methods to quantify sucrose content are essential for large breeding programs that aim to develop new legume cultivars with high sucrose content. Generally, high-performance liquid chromatography (HPLC) has been commonly used for qualitative and quantitative analysis of sugars (Kuo et al. 1988; Lowell and Kuo 1989). However, HPLC requires expensive equip-ment and the high costs per sample hinders its use in screening large numbers of plants in breeding populations and genetic resources. Therefore, simple and non-expensive enzyme methods that use invertase (INV) and glucose oxidase (GOD) were developed for sucrose quantification (Kumar et al. 2010; Teixeira et al. 2012). This enzyme method involves the conversion of sucrose to glucose and fructose using INV. Thereafter, GOD is sequentially used to convert glucose to gluconic acid and hydrogen peroxide. The sucrose content in samples is directly related to hydrogen peroxide production, which causes a change in the reaction color that is measured by a spectrophotometer (Teixeira et al. 2012). The enzyme method exhibits a high correlation coefficient (r = 0.9854) with the HPLC method for determining sucrose content in soybean seeds extracted with 80% ethanol (Teixeira et al. 2012).

Several extraction conditions have been reported for the analysis of sugar from dried seeds. Generally, in sugar extraction, 50%-80% ethanol is used as a solvent with heating or repeated extraction steps (Hymowitz et al. 1972; Kuo et al. 1988; Kim et al. 2005; Giannoccaro et al. 2006). Giannoccaro et al. (2006) reported that, for soybean, a sample size of 0.1 g, a water solvent-to-sample ratio of 10:1, and an extraction time of 15 minutes at 25℃ or 50℃ were optimal extraction conditions for the HPLC method. However, a specific extraction condition for the enzyme method has not been reported in legume seeds.

Thus, the purpose of this study was to optimize the GOD/INV enzyme method for rapid and simple quantification of sucrose in soybean and cowpea seeds.

MATERIALS AND METHODS

Plant materials

Twenty genotypes of soybean and cowpea were used in the study. Seeds were harvested from the field in 2018 at the Chonnam National University in Gwangju, Korea. Five grams of each sample was ground using a mill (HANIL ELECTRIC CO., LTD., Seoul, Korea) for 30 seconds and filtered through a 500 µm sieve (DAIHAN Scientific Co., Ltd., Seoul, Korea). Thereafter, samples were dried in a chamber at 105℃ for 1 hour and stored in a desiccator.

Sucrose extraction

Soluble sugar was extracted by modifying the methods reported by Giannocaro et al. (2006) and Hou et al. (2009b). For soybean, 150 mg of sample was mixed with deionized (DI) water at two different solvent-to-sample ratios (5:1 and 10:1 (v/w)). The mixtures were then incubated at two different extraction temperatures (25℃ and 50℃) and four different extraction times (15 minutes, 2 hours, 8 hours, and 24 hours). For cowpea, 100 mg of sample was mixed with 80% methanol at a solvent-to-sample ratio of 5:1 (v/w). Extraction was conducted at 50℃ with two different extraction times (30 minutes and 24 hours) (Sosulski et al. 1982).

Samples were placed in 2 mL centrifuge tubes with the selected solvent. The mixture was vortexed and incubated at different temperatures in a shaker at 200 rpm for 15 minutes to 24 hours. After this period, the tubes were centrifuged for 10 minutes at 13,000 × g. The supernatant was transferred to a fresh tube and centrifugation was repeated with the above conditions. The extracts were tested using the GOD/INV method in this study (Fig. 1).

Figure 1. Flow chart of the glucose oxidase (GOD)/invertase (INV) method.

GOD/INV method

Sucrose content was determined by the GOD/INV method reported by Teixeira et al. (2012). GOD and INV were purchased as part of the Sucrose/D-Glucose Assay Kit (K‐SUCGL, Megazyme International Ireland Ltd., Wicklow, Ireland). 5 mL of sample extract from each genotype was placed in a 96-well cell culture plate and mixed with 85 mL DI water and 10 mL INV. The plate was then covered with a lid and placed in an incubator at 55℃ for 10 minutes. After the plate was removed from the incubator, 200 mL of GOD reagent was added. The plate was then covered and placed in an incubator at 50℃ for 20 minutes. After the plate was taken out of the incubator, it was placed at room temperature for 5 minutes. The absorbance at 490 nm was read using a microplate reader (EpochTM; BioTek Instruments, Inc., Winooski, VT, USA). Standard sucrose solutions were also added to each plate in separate wells. Six concentrations of sucrose were prepared for soybean (0%, 0.15%, 0.25%, 0.5%, 0.75%, and 1%) and cowpea (0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%), enabling a standard curve to be constructed to determine the sucrose concentration in each sample. Thereafter, the dilution factor was calculated. Each analysis was performed in duplicate.

HPLC method

In the HPLC analysis, 1 g of the powdered sample was mixed with 30 mL of 75% ethanol in a 50 mL tube. The mixture was sonicated for 1 hours and incubated for 15 minutes and 24 hours in the refrigerator. The extract was filtered using a 0.22 mm PVDF syringe filter (Futecs Co., Ltd., Daejeon, Korea). 20 mL of the filtered solution was injected into the HPLC system, Dionex ultimate 3000 (Dionex CO., Sunnyvale, CA, USA), equipped with the refractive index detector, Shodex RI 101 (Showa Denko KK, Kawasaki, Japan). The Sugar-Pak (6.5 × 300 mm; Waters, Milford, MA, USA) column was used and maintained at 70℃. Sugars were eluted with degassed distilled water at a flow rate of 0.5 mL/minutes. Peaks detected by the refractive index detector were identified and quantified by comparing the retention times (RT) with those of analytical standards: sucrose, raffinose, and stachyose (St. Louis, MO, USA).

Statistical analysis

Sample extraction was carried out in duplicate. All measurements for GOD/INV and HPLC analysis were also performed in duplicate. The data are presented as mean ± standard deviation (SD). Pearson correlation analysis and linear regression analysis were used to determine the correlation coefficient and regression equation between GOD/INV and HPLC. A P-value < 0.01 was considered to indicate statistical significance. All statistical calculations were conducted using the standard R package (Team 2013).

RESULTS

Optimal sucrose extraction conditions: Solvent-to-sample ratio

For the GOD/INV method, the sucrose content is based on the color change measured by spectrophotometer at 490 nm. Thus, the absorbance value should be directly proportional to the sucrose content. In soybean seeds, extractions with two different solvent-to-sample ratios, including 5:1 and 10:1, were tested via the GOD/INV method. At a ratio of 5:1, all soybean genotypes had similar absorbance values ranging from 1.74 to 1.90, which corresponded with 5.24%-5.75% of sucrose (Table 1). There was no discrimination among soybean genotypes. In contrast, the ratio of 10:1 reduced the absorbance values that increased the variations among soybean genotypes. Absorbance values ranged from 1.05 to 1.44, which corresponded with 5.71%-7.96% of sucrose. As expected, Ilmikong had higher sucrose content than other cultivars. Therefore, the 10:1 ratio enabled better discrimination of soybean genotypes than the 5:1 ratio. Cowpea has a lower total sucrose than soybeans. As a result, it did not exceed the maximum absorbance. Therefore, cowpea showed a distinct difference at a ratio of 5:1 (data not shown). Therefore, the 10:1 and 5:1 solvent-to-sample ratios were selected for further studies with soybean and cowpea, respectively.

Table 1 . Sucrose content andmaximum absorbance of soybean genotypes according to the ratio of sample and solvent.

Sample5:110:1
AbsorbanceSucrose (%)AbsorbanceSucrose (%)
IT2241831.745.241.136.14
IT1951551.755.261.146.19
Shingi1.815.461.065.74
Josaengseori1.905.751.055.71
Sinhwa1.815.481.116.07
Cheongja31.795.411.337.31
Ilmikong1.765.301.447.96


Optimal sucrose extraction conditions: extraction time and temperature

10 soybean genotypes were used to determine the effect of various extraction times (15 minutes, 2 hours, 8 hours, and 24 hours) and temperatures (25℃ and 50℃) on sucrose content at a solvent-to-sample ratio of 10:1 (Fig. 2, Supplementary Table S1). The sucrose content was found to be affected by the extraction time and temperature. As extraction time increased, the sucrose content in all soybean genotypes increased. At 25℃, the average sucrose content was 4.7% for 15 minutes, 5.2% for 2 hours, 5.8% for 8 hours, and 7.3% for 24 hours (Supplementary Table S1). At 50℃, the average sucrose content was 5.7% for 15 minutes, 6.3% for 2 hours, 6.7% for 8 hours, and 7.2% for 24 hours (Supplementary Table S1). At each extraction time, the sucrose content was high at 50℃. Further, a large difference was found between genotypes. High (Ilmikong and Taekwangkong) and low sucrose genotypes (Kwangankong, Sinapldalkong2, and Danbaekkong) had the same rank orders across extraction times at 50℃ (Fig. 2, Supplementary Table S1). However, most genotypes showed a change in their rankings between 15 minutes and 2 hours at 25℃ (Fig. 2, Supplementary Table S1). Therefore, 50℃ was found to produce more stable and higher sucrose yield in the GOD/INV method.

Figure 2. Sucrose content in soybean seeds based on different extraction times at 25℃ (A) and 50℃ (B).

Correlation between the GOD/INV method and HPLC method

The sucrose content measured by the GOD/INV method was validated through HPLC analysis using 20 soybean samples. The same extraction condition, such as incubation at 50℃ for 15 minutes and 24 hours, was used for the two methods. At 50℃ for 15 minutes, the GOD/INV method showed a highly significant correlation (r = 0.77**) with the HPLC method (Fig. 3). The Sorog soybean cultivar, which had the lowest sucrose content (3.29%) in the GOD/INV method, showed a similar sucrose content (4.45%) in the HPLC method (Supplementary Table S2). In addition, the other low sucrose genotypes (Galchae, Hoseo, and Kwangankong) with 4.5% or less sucrose by the HPLC method also showed a 4.1% or less sucrose content in the GOD/INV method. The high sucrose genotype, Taekwangkong, had 7.28% sucrose with GOD/INV and 7.08% with HPLC (Supplementary Table S2). A highly significant correlation (r = 0.91**) was found between the GOD/INV method and HPLC method at 50℃ for 24 hours (Fig. 3). In particular, most soybean genotypes, except Daewonkong and Bosug, had less than a 1% difference in sucrose content when measured by the two methods (Supplementary Table S2).

Figure 3. Scatter plot of sucrose content in soybean seeds measured by HPLC vs glucose oxidase (GOD)/invertase (INV) methods using extraction times of 15 minutes (A) and 24 hours (B) at 50℃.

In cowpea, two different extraction times (30 minutes and 24 hours) for the GOD/INV methods were compared with 24 hours for the HPLC method. The 30 minutes GOD/INV method and the 24 hours HPLC method showed a highly significant correlation (r = 0.934 **, Fig. 4). However, the regression coefficient was 0.5367, indicating that the sucrose content of the GOD/INV method was approximately half the percentage of the HPLC method (Supplementary Table S3). The increased extraction time (24 hours) of the GOD/INV method led to similar sucrose content to that of the HPLC method. Further, most cowpea genotypes, except IT 210005 and IT 261863, had less than 0.3% difference in sucrose content measured by the two methods (Fig. 4, Supplementary Table S3).

Figure 4. Scatter plot of sucrose content in cowpea seeds measured via 24 hours HPLC vs 30 minutes (A) and 24 hours (B) glucose oxidase (GOD)/invertase (INV) methods at 50℃.
DISCUSSION

Sucrose can provide several desirable properties for the taste and flavor of soybean-derived foods, such as tofu, soymilk, and natto (Taira 1990). Previously, some trials sought to select high sucrose plants from 20 diverse soybean germplasm (Hou et al. 2009a) and identify the quantitative trait loci responsible for the high sucrose content in soybeans (Zen et al. 2014). However, there have been no further studies on the screening of high sucrose genotypes from massive genetic resources and the success of any breeding program for the development of high sucrose cultivars. Quantification of sucrose content requires expensive equipment and is time-consuming, which are major problems in the breeding procedure (Tchiagam et al. 2011; Wang et al. 2014; Zeng et al. 2014; Patil et al. 2018). In the present study, we optimized the sucrose extraction condition and GOD/INV method to enable rapid estimation of sucrose content using basic laboratory tools and reagents (Fig. 1).

First, we optimized the solvent-to-sample ratio for the GOD/INV method. Based on the study of Giannocaro et al. (2006), water was used as an extraction solvent with two different solvent-to-sample ratios (5:1 and 10:1). For soybean seeds, the 10:1 ratio had broader absorbance values and higher sensitivity to sucrose content than the 5:1 ratio (Table 1). In the GOD/INV method, the sucrose content is determined using absorbance values for a pink-red quinoneimine dye based on the amount of hydrogen peroxide (Outlaw 1984). However, the detection sensitivity was low when the absorbance value was almost saturated with excess hydrogen peroxide (Zhang et al. 2018). In the present study, the 5:1 solvent-to-sample ratio for soybean seeds containing 1.5%-10.24% sucrose content produced sufficient hydrogen peroxide content that led to saturated absorbance value. However, cowpea seeds containing low sucrose content (0.5%-1.3%) showed high detection sensitivity under the 5:1 solvent-to-sample ratio. Depending on the sucrose content in the seed, a suitable extraction volume must be selected for the GOD/INV method to prevent saturation of the absorbance value.

The extraction efficiency was found to differ according to various extraction temperatures (25℃, 50℃) and time (15 minutes, 2 hours, 8 hours, 24 hours). In this study, sucrose content increased with increasing extraction time and temperature (Fig. 2). On average, an extraction time of 24 hours yielded approximately 40% higher sucrose content than 15 minutes, and a temperature of 50℃ also yielded approximately 14% higher sucrose content than 25℃ in the GOD/INV method. The results of this study were similar to those of a previous report, where higher temperature up to 50℃ and longer extraction time increased the extracted amounts of fructose, sucrose, and oligosaccharides from defatted soybean meal (Kim et al. 2003). However, sucrose content was reduced by 30%-43% at 65℃ and 80℃ (Kim et al. 2003). Comparison of the effect of longer extraction times (from 15 minutes to 24 hours) between the GOD/INV and HPLC methods showed that the GOD/INV method resulted in a 31% increase in sucrose content. However, the HPLC method only resulted in a 13% increase in sucrose content (Supplementary Table S2). These results indicate that the extraction time had a more significant impact on the GOD/INV method than the HPLC method.

When the same extraction condition was analyzed, the GOD/INV method showed a good agreement with the HPLC method, with a correlation coefficient of 0.77 at 15 minutes and 0.91 at 24 hours in soybean seeds (Fig. 2). In addition, the detected range (4.51%-9.89%) of sucrose content using the GOD/INV method was almost the same as that with the HPLC method (4.7%-9.3%) at 24 hours (Supplementary Table S2). The high and significant correlation (r = 0.97) between GOD/INV and HPLC was also reported by Teixeira et al. (2012). In this study, the GOD/INV method also showed high sensitivity to low levels of sucrose content in cowpea seeds. With a highly significant correlation (r = 0.873), the two methods had similar detected ranges of 0.64%-1.79% for GOD/INV and 0.65%-1.75% for HPLC at 24 hours (Supplementary Table S3). This finding suggested that the GOD/INV method could be employed for various legume crops with a wide range of sucrose content.

In conclusion, we optimized methods for determining sucrose content in soybean and cowpea using an enzyme colorimetric reaction. The optimal extraction condition for the GOD/INV method was as follows: 150 mg of soybean seeds was extracted with 1.5 mL of water in a 10:1 solvent-to-sample ratio over 24 hours at 50℃. For cowpea, 1 g of crushed seeds was extracted with 5.0 mL of 80% methanol at a 5:1 solvent-to-sample ratio over 24 hours at 50℃. After sucrose extraction, GOD/INV reactions were conducted in a 96-well cell culture plate and sucrose content was determined within 1 hours. The GOD/INV method can quantify sucrose content using basic laboratory tools without equipment, such as HPLC or near-infrared (NIR). Thus, this method is a low-cost alternative for sucrose quantification in legume breeding programs and is readily adaptable to other species.

SUPPLEMENTARY MATERIALS
pbb-9-3-250-supple-tables.docx
ACKNOWLEDGEMENTS

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01416805 2021)” Rural Development Administration, Republic of Korea.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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