This paper presents a simple analytical method for determining sugars in soybean (
Soybeans are naturally enriched with oil, protein, and carbohydrates. In food industries, soybeans are processed to several consumable soya products, such as soy milk, tofu, miso, soy sauce, natto, and soy meals. Soybeans are also important constituent of animal feeds. Flavors and nutritive values of soya foods are greatly affected by sugar contents of soybeans. Major sugar components in soybean are fructose, glucose, sucrose, raffinose, and stachyose. Fructose and glucose are monosaccharide, while sucrose is a disaccharide and these three sugars are easily digestible. Raffinose and stachyose are trisaccharide and tetrasaccharide, respectively, and belong to raffinose family oligosaccharides (RFOs). Human and other monogastric animals are not able to digest RFOs due to lack of an enzyme called alpha-galactosidase in their small intestine [
Sugar analysis has been primarily carried out by high performance liquid chromatography (HPLC) because of simplicity, accuracy, and separation ability. Chromatographic separation is achieved by appropriate combination of stationary phase and mobile phase. Stationary phases or HPLC columns for separating sugars are mainly categorized in three types with regard to packing materials: ligand exchange, normal phase, and anion-exchange. Mobile phases are prepared solutions that facilitate separation of sugars when they carry a sample through a column. Ligand-exchange columns are backboned with sulfonated polystyrene carrying a metal counter ion, such as calcium ions or sodium ions. They are environment friendly because of the use of pure water as the mobile phase [
Preferred chromatographic detection of sugars includes refractive index, UV-visible, fluoresce, pulsed amperometric detector, mass spectrometry, and evaporative light scattering. Refractive index (RI) detector has been used for analyzing oligosaccharides from soybean since the 70’s [
Sample preparation is an important step in method development for soybean and other plants. The procedure normally consists of sample extraction and sample purification. In some studies, soybean flour or powder was first defatted using hexane or other appropriate solvents. The defatted samples were then refluxed in ethanol aqueous solutions at elevated temperatures for a certain length of time [
In this paper we report a simplified method to analyze sugars from soybean. Sample preparation was successively conducted in the same sample vial. It overcame long and elaborate procedures engaged in early methods, such as to separate soybean tissues from the extract, to allocate, to dry, and to redissolve the extracted solution. The HPLC conditions of this method were optimized for high chromatographic resolution and short run time. Addition of 25% acetone to acetonitrile mobile phase was found critical to improve separation of galactose from its adjacent epimer glucose. This method is highly useful for screening large scale soybean populations in genetic and breeding programs for crop improvement with value-added traits.
Sugar standards, D-fructose, D-(+) galactose, D-(+) glucose, sucrose, D-(+) melibiose, D-(+) raffinose pentahydrate, and stachyose hydrate, were purchased from Sigma Aldrich (St Louis, MO, U.S.A). HPLC grade of acetonitrile, acetone, and water were purchased from Fisher Scientific (Hampton, NH, U.S.A). Compressed nitrogen of ultrahigh-purity (UHP) grade was purchased from Praxair (Danbury, CT, U.S.A). The HPLC-ELSD system was Agilent 1200 series (Agilent, U.S.A). The Prevail Carbohydrate ES columns, 5
Approximately one gram of soybean seeds was ground using Thomas Wiley Mini-Mill fitted with 20-mesh screen. The ground powder was lyophilized for two days in a Labconco Freeze Dry System (Labconco, U.S.A.). Dried soybean powder of 90.25 (
Two kinds of mobile phase were prepared: mobile phase A was pure water, and mobile phase B was acetonitrile: acetone mixture of 75 : 25 (v/v). Flow rate and gradient of phases A and B (Table
Gradient-elution of mobile phases.
Time (minutes) | A : B | Flow rate (mL/min) |
---|---|---|
0 | 20 : 80 | 1.2 |
5 | 20 : 80 | 1.2 |
12 | 50 : 50 | 1.2 |
14 | 50 : 50 | 1.2 |
14.1 | 20 : 80 | 0.2 |
20 | 20 : 80 | 1.2 |
Soybean is known for high protein. A substantial amount of protein concurrently dissolves in water during extraction. Purification of the extract is therefore very important to eliminate matrix interference from the dissolved proteins. Protein was commonly precipitated with 95% or pure acetonitrile. Earlier methods required four to seven steps to conduct purification, which included preparation of 95% acetonitrile, separation of suspended sugar solution from soybean tissues, transferring a given volume of supernatant to a new tube, blending in acetonitrile, removing protein, drying, and redissolving sugars. Our method requires only two steps, that is, directly adding pure acetonitrile to the same sample vial after the completion of extraction and removing soybean tissue and precipitated proteins simultaneously. This modified method effectively eliminates process variables as less preparation steps were involved.
Dilution is another key factor in this method. Low concentration of sugars is desirable not only for optimum peak shape and separation but also for an extended column life cycle. However, abundance of different sugars varies greatly in soybean; for example, Hou et al. showed that soybean is composed of 0.07–0.15% glucose, 0.08–0.19% fructose, 5.6–9.4% sucrose, 0.3–1.4% raffinose, and 0.3–6% stachyose [
The HPLC mobile phase plays a crucial role in sugar separation. Addition of acetone to acetonitrile mobile phase differentiates this method from other published methods. As shown in Figure
Chromatograms obtained from (a) acetone in mobile phase; (b) no acetone in mobile phase.
Each sugar component was recognized by its distinctive retention time. Commercial references were used for confirmation. Under the chromatographic conditions described in Section
Chromatogram of sugar standards (concentration of each sugar is 300
Figure
Distribution of sugars in different soybean germplasm lines.
Quantification of the sugars was accomplished by calibration curves that ranged from 50 to 1000
Calibration curves of seven sugars. (a) Quadratic regression; (b)
Recovery rate of each sugar was obtained by spiking known amount of standards at different steps of sample preparation. In one of the spiking experiments, standards were added to the dried soybean samples. In another experiment spike standards were added to the sugar extracts. In both cases, 500
Method validation including detection limit, repeatability and spike recovery.
Detection limit |
Repeatability | Recovery (%) ± RSD | |||
---|---|---|---|---|---|
Intraday (RSD%) | Interday (RSD%) | Spike to dried sample | Spike to sugar extract | ||
|
|
|
|
||
Fructose | 60.0 | 2.2 | 6.0 | 103 ± 1.5 | 101 ± 2.0 |
Galactose | 135.0 | — | — | 105 ± 3.2 | 111 ± 1.2 |
Glucose | 30.0 | 2.0 | 9.0 | 101 ± 1.1 | 99 ± 1.0 |
Sucrose | 9.3 | 1.2 | 2.9 | 99 ± 3.0 | 98 ± 4.4 |
Melibiose | 19.4 | 1.7 | 12.0 | 73 ± 1.4 | 92 ± 0.5 |
Raffinose | 12.1 | 1.7 | 2.9 | 102 ± 2.8 | 101 ± 1.6 |
Stachyose | 10.0 | 1.2 | 4.7 | 102 ± 0.9 | 102 ± 3.9 |
Relative standard deviation (RSD%) of both interday and intraday was determined and reported in Table
Accuracy of the method was further evaluated on an HPAEC-PAD system from an external lab. Six finely ground and dried soybean seed samples were verified by the HPAEC-PAD for sucrose, raffinose, and stachyose. Table
Comparison of sugar content generated from the present method with the HPAEC-PAD method.
Present |
HPAEC- |
Present |
HPAEC- |
Present |
HPAEC- | |
---|---|---|---|---|---|---|
Sucrose% (dry matter) | Stachyose% (dry matter) | Raffinose% (dry matter) | ||||
|
|
|
||||
Sample-1 | 7.1 | 6.8 | 0.9 | 0.5 | 0.8 | — |
Sample-2 | 5.8 | 4.8 | 3.5 | 3.1 | 0.6 | 0.3 |
Sample-3 | 4.7 | 3.4 | 4.3 | 3.2 | 0.6 | 0.3 |
Sample-4 | 2.8 | 2.1 | 3.7 | 2.6 | 1.1 | 0.9 |
Sample-5 | 5.7 | 4.2 | 1.4 | 1.0 | 0.6 | — |
Sample-6 | 3.1 | 2.1 | 4.4 | 3.2 | 0.9 | 0.5 |
This analytical method has played a significant role in the research projects on the natural genetic variation of sugar contents and components in soybean seeds and other tissues conducted at the University of Missouri. The soy industry and soy food markets need soybeans with high-sucrose, low-raffinose, and stachyose content and with low trypsin inhibitor activity as one package. This will provide high levels of energy and yield better tasting food and feed without inducing indigestion problems. The extensive screening of soybean plant introductions (PIs) and current soybean varieties for natural genetic variation will help identifying new sources for gene discovery and further application in molecular breeding and crop improvement programs. More than 5000 lines have been screened. Several soybean PIs with low raffinose and stachyose and high sucrose in seed tissues have been identified. Table
Maximum and minimum sugar component concentration in the 540 soybean germplasm lines.
Fructose | Glucose | Sucrose | Melibiose | Raffinose | Stachyose | Total sugar | |
---|---|---|---|---|---|---|---|
(sugar content in soybean mg/g (dry matter)) | |||||||
Maximum | 16.6 | 13.7 | 73.4 | 7.7 | 33.4 | 70.9 | 160.3 |
Minimum | 2.1 | — | 15.0 | — | 3.4 | 3.2 | 56.7 |
Average | 8.1 | 5.7 | 42.2 | 3.8 | 8.0 | 41.1 | 108.8 |
Distribution of sucrose contents in the seeds of 1500 soybean germplasm.
Developing an HPLC method that can resolve a mixture of mono-, di-, tri-, and oligosaccharides in a short elution time is a challenge. Earlier described methods had several shortcomings and some of the methods have shown an unacceptable long retention time for raffinose and stachyose while some others had poor resolution or broad peaks. The method presented in this paper is simple, fast, reliable, and successful in the case of high throughput screening. The HPLC of this method is comprised of a column, specially packed for carbohydrate assay and equipped with an ELSD detector. The mobile phase is 25% acetone in acetonitrile and water. All major sugars in soybean, that is, fructose, glucose, sucrose, raffinose, and stachyose, are completely resolved in less than 14 minutes. Sample preparation procedure is much simplified and improved from known methods. The optimal low sample-to-solvent ratio (10 mg : 1 mL) lessened sample load and extended HPLC column life. More than 1500 samples can be analyzed on a single HPLC column. The detection limit in this method is low; for example, sucrose is 9.3 ppm and others are lower than 60 ppm. The selection of calibration range allows direct quantification from the nonlinear detector output. Robustness and high throughput application of this method help soybeans breeding programs including the one for developing soybean varieties with reduced RFOs and higher metabolizable energy.
Raffinose family oligosaccharides
High performance liquid chromatography
Ultraviolet
Refractive index
High-performance anion-exchange chromatography with pulsed amperometric detection
Evaporative light scattering detector
Plant introduction.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the United Soybean Board, Grant no. 1236. The authors acknowledge Dr. Grover J. Shannon for soybean germplasm seed increase at the Delta Research Center, University of Missouri, Portageville, Missouri, and Dr. Tri D. Vuong, Department of Plant Science, University of Missouri, for helping collection of more than 5000 data using this method.