Crop Science 41:250-252 (2001)
© 2001 Crop Science Society of America
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A more rapid method of total sugar analysis for soybean seed
Chandler W. Geatera,
Walter R. Fehra,
Lester A. Wilsonb and
John F. Robytc
a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
b Dep. of Food Science and Human Nutrition, Iowa State Univ., Ames, IA 50011
c Dep. of Biochemistry, Biophysics, and Molecular Biology, Iowa State Univ., Ames, IA 50011
Corresponding author (wfehr{at}iastate.edu)
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ABSTRACT
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The total sugar content of soybean [Glycine max (L.) Merr.] cultivars is important to the soyfood industry. The method of total sugar analysis reported in the literature is labor intensive and time consuming. The objective of this study was to develop a more rapid method for the determination of total sugar in soybean seed. The original method was modified by reducing the amounts of reagents used, eliminating sample filtering, reducing the number of sample dilutions, and increasing the rate of acid hydrolysis. There was a significant difference between the original (187 g kg-1) and the rapid (224 g kg-1) method for the means of 10 samples with a broad range of total sugar, but no significant sample x method interaction was observed. The phenotypic correlation between the two methods for total sugar of the 10 samples was 0.91 (P < 0.05). The number of samples that could be analyzed in 1 d increased from 12 for the original method to 70 for the rapid method, and the estimated cost per sample was reduced from $5.34 to $0.92. The rapid method can be used to determine the relative differences among soybean samples for total sugar content.
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INTRODUCTION
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THE TOTAL SUGAR CONTENT of soybean cultivars is important in the production of soyfoods (Taira, 1990). A method for evaluating total sugar has been reported by the National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries of Japan (Society for the Study of Natto, 1990). The procedure is used by the soyfood industry, but is too labor intensive and time consuming for the evaluation of a large number of genotypes in a soybean cultivar development program. The objective of this study was to develop a more rapid procedure for the determination of total sugar content of soybean seed.
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Materials and Methods
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Original Method of Total Sugar Analysis
The original method of total sugar analysis described in the literature (Society for the Study of Natto, 1990) was conducted in the following manner. A 20-g sample of seed was ground with a Cyclotec 1093 Sample Mill (Tecator AB, Hoganas, Sweden). A 3-g sample of the powder was placed in a 250-mL glass media bottle, and 200 mL of distilled water and 20 mL of 25% (w/w) HCl were added. The bottles were capped with rubber-lined phenolic screw caps. The solution was mixed by shaking the bottle by hand for 5 s before the bottles were placed into a 100°C bath for 4 h. The bottles were removed from the hot water bath and cooled to room temperature in a 1°C water bath for 20 min. A 5.5-mL aliquot of 40% (w/v) NaOH was added to each bottle. The bottles were capped, and the solution was mixed by inverting each bottle five times. The solution in each bottle was filtered through Whatman no. 4 filter paper into a 500-mL glass filter flask. The solution in each filter flask was emptied into a 500-mL volumetric flask and filled to 500 mL with distilled water. Each volumetric flask was capped with a polyethylene snap cap, and the solution was mixed by inverting the flask 25 times. To dilute each sample to a measurable concentration, 3 mL of each sample solution was pipetted into a 100-mL volumetric flask and filled to 100 mL with distilled water. The flasks were capped with polyethylene snap caps, and the solution was mixed by inverting the flask 25 times. The total sugar content of the solution was determined by the modified phenol-sulfuric acid method described by Fox and Robyt (1990). A 25-µL aliquot of the test solution and 25 µL of 5% (w/v) phenol were pipetted in triplicate into a 96-well general assay plate. In addition to the test samples, standards of known glucose concentration were placed in triplicate wells of each plate. The standards were 0 µg mL-1 (distilled water blank), 10 µg mL-1, 30 µg mL-1, 50 µg mL-1, 70 µg mL-1, and 90 µg mL-1 of glucose. After all the samples were loaded, the plate was vortexed for 30 s at a speed setting of 1 on a Vortex Genie 2 Mixer fitted with a Microwell Plate Insert (Fisher Scientific, Itasca, IL). The plate was placed on crushed ice, and 125 µL of concentrated H2SO4 was added to each well. The plate was mixed for 30 s at a speed setting of 1. The plate was sealed in a plastic zipper bag and warmed in a water bath at 80°C for 30 min. Each plate was read with a EL312e Bio-Kinetics Reader (Bio-Tek, Winooski, VT) at 490 nm. The glucose concentration of each test sample was determined by comparing the absorbance of the test sample of a plot to the absorbances of the glucose standards. Total sugar content in grams per kilogram was determined by dividing the mean concentration of glucose (µg mL-1) in a test sample by 180 µg mL-1 (the concentration of soybean powder in the test sample) and multiplying by 1000 g kg-1. To adjust the total sugar content of a sample to a moisture-free basis, total sugar content was divided by [(100 – moisture percentage of the sample)/100]. The mean total sugar of the triplicates for each test sample was used for data analysis.
Modification of the Original Method
The most time-consuming steps in the original method were modified to develop a more rapid procedure. The amounts of soybean powder and sample reagents were reduced by a factor of 20 to decrease the amount of solid material in the final test sample, which eliminated the need for sample filtering. The procedure also was modified to dilute the sample to a measurable concentration in one dilution instead of the two dilutions used in the original method. The rate of acid hydrolysis was increased by autoclaving the sample solutions at 121°C rather than placing the samples in a 100°C water bath for 4 h. The use of autoclaving was based on the acid hydrolysis conditions for the conversion of cornstarch to D-glucose for corn syrup production (Pancoast and Junk, 1980).
To determine the autoclaving time required to hydrolize the maximum amount of glucose from soybean seed, 10 samples (replicates) of the Japanese soybean cultivar Suzumaru were autoclaved for 20, 30, and 40 min, and the total sugar content for each time was determined. Suzumaru was selected for this experiment because of its wide acceptance by the natto industry in Japan. The data were analyzed as a completely randomized design. The analysis of variance was performed by the general linear models (GLM) procedure of SAS (SAS Institute, 1992).
After the modifications of the original method had been made, the rapid method was conducted in the following manner. A 20-g sample of seed was ground with a Cyclotec 1093 Sample Mill. A 150-mg sample of the powder was placed in a 16- by 125-mm screw cap tube. A 10-mL aliquot of distilled water and 1 mL of 25% (w/w) HCl were added to each tube. The tubes were capped with rubber-lined phenolic screw caps and vortexed at a speed setting of 4 for 5 s with a Vortex Genie 2 Mixer. The tubes were autoclaved at 121°C for 20 min and cooled to room temperature in a 1°C water bath for 5 min. A 275-µL aliquot of 40% (w/v) NaOH was added to each tube. The tubes were capped, and the solution was mixed by inverting the tubes five times. The solution in each tube was emptied into a 500-mL volumetric flask. The flask was filled to 500 mL with distilled water, capped with a polyethylene snap cap, and mixed by inverting 25 times. Total sugar content was determined by the same modified phenol-sulfuric acid method of Fox and Robyt (1990) used for the original method. Total sugar was expressed in grams per kilogram on a moisture-free basis by dividing the mean concentration of glucose (µg mL-1) in a test sample by 300 µg mL-1 (the concentration of soybean powder in the test sample) and multiplying by 1000 g kg-1. Total sugar content was adjusted to a moisture-free basis by dividing the total sugar content of a sample by [(100 – moisture percentage of the sample)/100].
Method Comparisons
To compare the original and the rapid methods, a 20-g sample of Suzumaru was ground with a Tecator Cyclotec 1093 Sample Mill. The total sugar contents of 5 samples (replicates) of the powder were determined by each method. The data were analyzed as a completely randomized design with the GLM procedure of SAS (SAS Institute, 1992).
To further compare the methods, soybean genotypes were evaluated for total sugar content by the rapid method from various replicated yield tests grown by the Iowa State University soybean breeding project in 1995 and 1998. Nine samples representing one plot of each of nine genotypes were identified that had a broad range of total sugar contents. The samples of A95-686004, A95-686013, `IA2023', `IA3007', and `Kenwood 94' were from Stuart, IA in 1995, and those of `IA2017', `IA2027', `IA2028', and `IA2035' were from Griswold, IA in 1998. The seed samples had been stored at 10°C and 50% relative humidity. In addition, a sample of Suzumaru was included in the experiment. The seed of Suzumaru was obtained through H. Hasegawa of the Ibaraki Industrial Technology Center, Mito City, Japan. The producer of the seed could not be determined. A 20-g sample of each of the 10 genotypes was ground, and the powder was used for three replicates of analysis by the original and the rapid methods. The data were analyzed as a completely randomized design by the GLM procedure of SAS (SAS Institute, 1992). A phenotypic correlation analysis was made between the methods for the 10 test samples with the correlation procedure of SAS (SAS Institute, 1992).
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Results and Discussion
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Significant differences in total sugar content were not observed among the three lengths of autoclaving. The mean total sugar content and standard deviation was 292 ± 31 g kg-1 for the 10 samples autoclaved for 20 min, 314 ± 45 g kg-1 for 30 min, and 307 ± 69 g kg-1 for 40 min. An autoclaving time of 20 min was considered suitable for the new procedure. Approximately 3.5 h were saved by substituting autoclaving for the hot water bath in the original procedure.
The total sugar contents obtained for Suzumaru by the original and the rapid extraction methods were not significantly different. The mean total sugar content by the original method was 230 ± 17 g kg-1 and by the rapid method was 235 ± 15 g kg-1. The coefficient of variability (CV) was 7% for the original method and 6% for the rapid method, which indicated that the error associated with the two methods was comparable.
Significant differences between the two methods were observed when seed from one plot of 10 genotypes was evaluated (Table 1). The mean total sugar determined by the rapid method was significantly greater than for the original method. The difference was attributed to greater hydrolization of the sugars in the seed by the rapid than by the original method. Whistler and Daniel (1985) reported that the rate of hydrolysis can be affected by several factors, including pH, temperature, and the length of acid hydrolysis. All of the samples had greater total sugar when analyzed by the rapid method, except for Suzumaru. It is not clear why the total sugar content of Suzumaru was not affected by the method of analysis. The sample x method interaction was not significant, indicating that differences among samples were relatively consistent regardless of the method of analysis used to determine total sugar content (Table 1). The means and ranks of the 10 samples for the two methods were significantly correlated (P < 0.01). The phenotypic correlation coefficient for sample means was 0.91, and the rank correlation coefficient was 0.82. The CV was 8% for the original method and 11% for the rapid method.
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Table 1. Mean and rank of the total sugar contents of one sample for each of 10 soybean genotypes analyzed by two methods
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There were important differences in the analysis time and cost per sample between the two methods. The maximum number of samples that could be extracted and analyzed in a day by one person was 12 for the original method and 70 for the rapid method. The reasons that more samples could be analyzed by the rapid method were the reduction of the extraction time from 4 h to 20 min and the elimination of sample filtering. The cost of equipment for the two methods was similar because the same analytical instrumentation was used by both methods. With a labor cost of $8.00 an hour, the estimated cost per sample was $5.34 for the original method and $0.92 for the rapid method. The reduced cost of analysis will make it more feasible to analyze a large number of genotypes in a soybean breeding program.
The results of the study indicated that the amount of total sugar obtained from a soybean sample depends on the method of analysis. When soybean genotypes are compared, the seed sample of each should be produced in the same environment to avoid environmental effects and the same method of total sugar analysis should be used (Geater and Fehr, 2000; Geater et al., 2000). When total sugar of commercial soybean grain is determined, the analysis procedure should be defined.
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NOTES
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Journal Paper no. J-18744 of the Iowa Agric. and Home Econ. Exp. Stn. Project no. 3107, and supported by the Hatch Act, State of Iowa, and Iowa Soybean Promotion Board.
Received for publication February 7, 2000.
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REFERENCES
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- Geater, C.W., and W.R. Fehr. 2000. Association of total sugar content with other seed traits of diverse soybean cultivars. Crop Sci. 40(6):1552–1555.[Abstract/Free Full Text]
- Geater, C.W., W.R. Fehr, and L.A. Wilson. 2000. Association of soybean seed traits with physical properties of natto. Crop Sci. 40(6):1529–1534.[Abstract/Free Full Text]
- Fox, J.D., and J.F. Robyt. 1990. Miniaturization of three carbohydrate analyses using a microsample plate reader. Anal. Biochem. 195:93–96.
- Pancoast, H.M., and W.R. Junk. 1980. Manufacture of corn syrups and sugars. p. 157–170. In H.M. Pancoast and W.R. Junk (ed.) Handbook of sugars. AVI Publishing Co., Inc., Westport, CT.
- SAS Institute. 1992. The SAS system for Windows. Release 6.12. SAS Inst., Cary, NC.
- Society for the Study of Natto. 1990. Methods of natto research. 1st ed. (In Japanese). National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Japan.
- Taira, H. 1990. Quality of soybeans for processed foods in Japan. Japan Agric. Res. Quarterly 24:224–230.
- Whistler, R.L., and J.R. Daniel. 1985. Carbohydrates. p. 69–137. In O.R. Fennema (ed.) Food Chemistry. 2nd ed. Marcel Dekker, Inc., New York.
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ABSTRACT
INTRODUCTION
