Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 0252-9777(Print)
ISSN : 2287-8432(Online)
The Korean Journal of Crop Science Vol.57 No.4 pp.324-330
DOI : https://doi.org/10.7740/kjcs.2012.57.4.324

Variation of β-carotene Concentration in Soybean Seed and Sprout

Joung-Kuk Ahn, Eun-Young Kang, Eun-Hye Kim, Ill-Min Chung
Department of Applied Bio Science, College of Life and Environmental Science, Konkuk University
Received 16 July 2012, Revised 18 September 2012, Accepted 25 September 2012

Abstract

In this study, β-carotene concentrations wasdetermined in soybean cultivar according to seed size, usage,seed coat color and cotyledon color as well as the processof seed germination. The total average concentration of β-carotene was 6.6 ㎍/g in soybean seed, 33.3 ㎍/g in soybeansprout. According to seed size, the total β-carotene concentrationof soybean was 6.9 ㎍/g in large soybean seed, 6.7 ㎍/g in medium soybean seed, and 6.31 ㎍/g in small soybeanseed. In soybean sprout, the total β-carotene concentrationwas 21.4 ㎍/g in large soybean sprout, 30.5 ㎍/g in mediumsoybean sprout, and 43.5 ㎍/g in small soybean sprout.According to the utilization of seed, the total β-caroteneconcentration of soybean seed was 7.2 ㎍/g in cooked withrice soybean seed, 6.1 ㎍/g in paste and curd soybean seed,and 6.3 ㎍/g in sprout soybean seed. In soybean sprout, thetotal β-carotene concentration was 25.9 ㎍/g in cooked withrice soybean sprout, 32.4 ㎍/g in paste and curd soybean sprout,and 41.9 ㎍/g in sprout soybean sprout. When comparisonwith seed coat color, the total β-carotene concentration ofsoybean with brown seed coat (8.8 ㎍/g) was slightly higherthan those of soybean with yellow (6.1 ㎍/g). In soybeansprout, the total β-carotene concentration was 21.8 ㎍/g inblack seed coat sprout, 38.7 ㎍/g in brown seed coatsprout, 34.1 ㎍/g in green seed coat sprout, 39.5 ㎍/g inyellow seed coat sprout, and 30.5 ㎍/g in mottle seed coatsprout. The results of this study suggested the functionalcharacteristics of soybean through quantitative analysis of β-carotene.

kjcs-57-2-t1.JPG74.6KB

 Approximately 100 million tons per year or more carotenoids are produced in the natural ecosystem (Kim et al., 1997). And, the carotenoids content in plant affected by genetics and their external environment (Bauernfeind, 1973). The carotenoids belong to tetra-terpenoid (C40 ) group and shows orange color, usually fat-soluble (Kim, 2001; Simpson and Chichester, 1981). In addition, that is very unstable substance, so oxidation or isomerization takes place easily (Kim, 2001).

Nutritional importance of carotenoids are the provitamin A activity, food colorants, antioxidant activity, anticarcinogenic substance, oxygen transporters, immune activation, absorbers of light energy. Of these, β-carotene has β-rings at both ends, the most powerful active oxygen remover and the highest vitamin A activity (Kim, 2001 ; Jo and Jung, 2000 ; Stahelin et al., 1991 ; Lee and Kim, 1997 ; Kim et al., 1997). Also, it reduces the risk of prostatic cancer, related to the arteriosclerosis and cardiovascular disease, and inhibition of LDL oxidation (Kirsh et al., 2006 ; Oliver et al., 1998 ; Mascio et al., 1991). And, it has been known low carotene was related to a bronchus cancer (Stahelin et al., 1991). 

Because the human body cannot synthesize vitamin A itself, human must be obtained from the diet. In other words, no side effects as β-carotene intake is recommended, this is converted into vitamin A in the body (Provitamin A). 

Soybean sprout is short growing time and short relatively easy to grow, low price, and also occupies a large proportion in the daily diet (Oh et al., 2007). Mainly, the soybean sprouts grown to 100-seed weight range was 7 -15 g, and smaller seed has the higher the yield of soybean sprouts, good vitality, excellent hypocotyls growth (Lee et al., 2002 ; Bates and Matthews, 1975). Seed nutrition was changed in germination process, while the fat content was reduced, fiber and crude protein were increased and especially, remarkable increase in the content of vitamins (Kim, 1981). In particular, vitamin C is not found in soybean seed, but it is synthesized in the germination process (Kim et al., 1993 ; Kim et al., 2007). Shimoyamada et al. (1991) reported that after germination, fresh weight was increased an 8-fold, and dry weight was not changed. Kim et al. (2003) reported that isoflavone content was increased about 1 times in the process of germination. Among the soybean sprouts, green sprout had a high nutritional value, specially.

In our study, β-carotene contents of soybeans examined according to the size, usage, seed coat color and cotyledon color as well as the process of seed germination. 

MATERIALS & METHODS

Preparation of samples

of Chungnam in 2006, according to standard cultivation method. Soybean seed were harvested at triplicate replications in each cultivar and stored at 4℃ for analysis of β-carotene. The varieties were classified according to the seed coat color, cotyledon color, 100-seed weight and usage. 

In this study, soybean was classified into three types, large(above 25 g, 22 varieties), medium (15-25 g, 27 varieties), and small (below 15 g, 30 varieties) by 100-seed weight, respectively. Soybean sprout was prepared with following procedure. 4 g of soybean seed was washed and submerged in tap water at 20 ± 2℃ for 12 h. Seed was spread uniformly in conical tube with holes and gave enough water more than 6 times a day, and cultivated five days in the dark chamber. After all soybean and soybean sprout was freezedried at 47℃, and then, crushed and stored in dessicator for analysis. When the soybean sprout was grown by absorbing water, its weight was increased the four times, compared to the seed fresh-weight. But, freeze-dried to completely remove the water, weight was reduced 22%, compared to the seed fresh-weight. Similarly, Shimoyamada et al. (1991) reported that during seed germination, seed fresh weight was increased about 8 times, while seed dry weight did not changed. 

Analysis of β-carotene

The concentration of β-carotene was analyzed by the modified method of Jo and Jung (2000). The 1 g of finely ground soybean seed and sprout powder were extracted with 40 mL of acetone : petroleum ether = 1 : 1 solution and stirred 30 min at room temperature. The suspension was filtered through Whatman filter paper (No. 4). This process repeated twice in each sample. The extract was transferred to a separatory-funnel and added 50 mL of saturated sodium chloride solution, then mixed. To obtain a supernatant, wait until the layer was separated, and then was added 100 mL of distilled water, then mixed. The following process was expressed as saponification. But Oliver et al. (1998) reported that the photo-diode array detection enables the spectra of each individual peak of the chromatogram, it could allow carotenoid separation without sample saponification. And Khachik et al. (1986) reported that saponification of carotenoid extracts underestimated the individual carotenoid content (Individual carotenoid contents were reduced by saponification). For this reasons, saponification was omitted in this experiment. 

After obtaining a supernatant, residual moisture was removed by sodium sulfate anhydrous. Supernatant was transferred to a 100 mL round flask and evaporated to dryness under reduced pressure using a rotary vacuum evaporator at below 35℃, and the remaining residue was reconstituted with 4mL of 0.01% BHT (Butylated Hydroxy Toluene) in chloroform. The aliquot sample was filtered through a 0.2 ㎛ nylon filter (TITAN, USA) and analyzed by HPLC. For all of the experimental process was conducted in dark condition. All of solvents were used as HPLC grade (J. T. Baker, USA) without further purification and all of reagents were used as special grade (Junsei, Japan). 

The HPLC system was consists of SPD-M10A Photo Diode Array (PDA) Detector (Shimazu, Japan), Midas auto-injector (Younglin, Korea) and a 20 μL sample loop and LC-10AD VP pump. The separation of β-carotene was performed by ODS C18  column (250 × 4.6 mm I.D.). The maximum absorption wavelength of β-carotene was measured at 452 nm. The mobile phase was followed, methanol (MeOH) : tetrahydrofuran (THF) = 8 : 2, isocratic. In selecting the appropriate solvent ratio for analysis, several solvents combinations were done. Because a β-carotene is a non-polar substance, the percentage of THF (non-polar solvents) increased in mobile phase. Retention time (RT) was detected at 13.7 min. Running time was 25 min and flow rate was 1 mL/min. The β-carotene standard, which was purchased from Sigma –Aldrich (USA), was used to make the calibration curve. The β-carotene standard was dissolved in 0.01% BHT in 4 mL chloroform, the plotting standards were made at four concentrations of 25, 50, 100 and 250 ㎍, and a high linearity (R2 =0.996) was obtained. The interpretation of the β-carotene was supported its retention time (β-carotene : 13.7 min) and adsorption spectra in HPLC.

Statistical analysis

The analysis of statistical was undertaken using the general linear model procedure (GLM) of the statistical analysis program (SAS, 2000). All experiments were determined as duplicates. The least significant difference (LSD) test was based on the 0.05 probability level.

RESULTS AND DISCUSSION

The total average concentration of β-carotene was 6.6 ㎍/g in soybean seed, 33.3 ㎍/g in soybean sprout. In comparison with soybean seed, soybean sprout increased about 5 times for total average of β-carotene concentration. And the average concentration of β-carotene in soybean seed and sprout was significantly different (p < 0.05) (Fig. 1). That is, β-carotene content was increased significantly in the germination process. These results is similar to those of previous studies by Kim et al. (1993) and Liu (1997), who demonstrated that β-carotene content increased 17-fold during seed germination process on the dry weight. Also, β-carotene content in the soybean sprout was 6 times higher than that of soybean seed. In soybean seed, β-carotene concentration of Cheongbang-kong (12.9 ㎍/g) was the highest content, while, Sojin-kong (5.8 ㎍/g) was the lowest content. In soybean sprout, total β-carotene concentration of Buakdarikong (75.8 ㎍/g) was the highest content, while, Ogapi-kong (6.5 ㎍/g) was the lowest content.

Fig. 1. Comparison of β-carotene concentration between soybean seed and sprout. *Each vertical bar indicates the standard error values.

The result of this study may provide plant breeders with information for selecting soybean varieties that contain high concentrations of β-carote ne. Also, this results will be helpful as basic information for food industries.

Concentration of β-carotene, according to the seed size

In this study, oybean varieties were classified as large (above 25 g, 16 varieties), medium (15 ~ 25 g, 26 varieties), and small (below 15 g, 26 varieties) based on 100-seed weight. The total β-carotene concentration of soybean seed was ranged from 5.9 to 11.8 ㎍/g in large soybean seed, from 5.8 to 12.9 ㎍/g in medium soybean seed, and from 5.8 to 7.6 ㎍/g in small soybean seed. In soybean sprout, the total β-carotene concentration was ranged from 6.5 to 41.9 ㎍/g in large soybean sprout, from 7.7 to 75.8 ㎍/g in medium soybean sprout, and from 11.4 to 73.6 ㎍/g in small soybean sprout (Table 1). According to the seed size, the total β-carotene in soybean seed was not significant different (p < 0.05). Whereas, the soybean sprout concentration according to different seed size was significant difference (p < 0.05).

Table 1. Comparison of β-carotene concentration between soybean seed and sprout on seed size difference.

In the β-carotene concentration change during germination process, large soybean seed was 14.6 ㎍/g (312%), medium soybean seed was 23.8 ㎍/g (453%), and small soybean seed showed 37.2 ㎍/g (689%). The total β-carotene was significantly increased in small soybean seed. From this study, β-carotene concentrations of soybean sprouts higher than soybean seeds and showed difference by seed size.

Concentration of β-carotene, according to the seed usage

According to seed usage, soybeans divided into three types : cooked with rice (27 varieties), paste and curd (16 varieties), and sprout (25 varieties). The total β-carotene concentration of soybean seed was ranged from 5.8 to 12.9 ㎍/g in cooked with rice soybean seed, from 5.8 to 6.5 ㎍/g in paste and curd soybean seed, and from 5.8 to 7.6 ㎍/g in sprout soybean seed. In soybean sprout, the total β -carotene concentration was ranged from 6.5 to 71.1 ㎍/g in cooked with rice soybean sprout, from 7.7 to 70.4 ㎍/g in paste and curd soybean sprout, and from 11.4 to 75.8 ㎍/g in sprout soybean sprout (Table 2). The total β -carotene in soybean seed and sprout as based on their seed usage was significantly different (p < 0.05) (Fig. 3).

Table 2. Comparison of β-carotene concentration between soybean seed and sprout on seed usage.

Fig. 2. The chromatogram of β-carotene in Akjongdari-kong soybean seed and sprout. (A) β-carotene standard; (B) Akjongdarikong soybean; (C) Akjongdari-kong soybean sprout.

Fig. 3. Comparison of β-carotene concentration in soybean seed and sprout on seed usage. *Each vertical bar indicates the standard error values. A, soybean seed; B, soybean sprout; CR, cooked with rice ; PC, paste and curd ; ST, sprout.

β -carotene concentration increased during germination process. Cooked with rice soybean seed was 18.7 ㎍/g (359%), paste and curd soybean seed was 26.4 ㎍/g (535%), sprout soybean seed was 35.6 ㎍/g (664%). The total β-carotene significantly increased in sprout soybean seed.

As a result, the concentration of total β-carotene was increased with germination process, and depends on the usage of soybean seed. The large and medium soybean has been mainly used as a cooked with rice soybean, and paste and curd soybean, small soybean seed has been used as sprout seed. In this point, it could be connected with the result of on the seed size.

Concentration of β-carotene, according to the seed coat color

Soybean seeds were divided into five types according to seed coat colors : black (17 varieties), brown (3 varieties), green (13 varieties), yellow (30 varieties), and mottle (5 varieties). The total β-carotene concentration of soybean with brown seed coat (8.8 ㎍/g) was slightly higher than those of soybean with yellow (6.1 ㎍/g) (Fig. 4). In soybean sprout, the total β-carotene concentration was 21.8 ㎍/g in black seed coat sprout, 38.7 ㎍/g in brown seed coat sprout, 34.1 ㎍/g in green seed coat sprout, 39.5 ㎍/g in yellow seed coat sprout, and 30.5 ㎍/g in mottle seed coat sprout.

Fig. 4. Comparison of β-carotene concentration in soybean seed and sprout on seed coat color. *Each vertical bar indicates the standard error values. (A) soybean seed; (B) soybean sprout; BL, black; BR, brown ; GR, green; YE, yellow; MO, mottle.

In germination process, β-carotene concentration change of yellow seed coat soybean seed was 33.5 ㎍/g (652%), brown was 29.9 ㎍/g (440%), green was 27.8 ㎍/g (539%), mottle was 23.5 ㎍/g (434%), and black was 14.5 ㎍/g (298%). The total β-carotene content significantly increased in yellow seed coat soybean.

Consequently, the total β-carotene accumulation of soybeans showed differnce according to their seed oat color. 

Concentration of β-carotene, according to the seed cotyledon color

The soybean samples divided into two types according to cotyledon color, that green (14 varieties) and yellow (54 varieties). The total β-carotene concentration of soybean with green cotyledon (7.7 ㎍/g) was higher than those of soybean with yellow (6.3 ㎍/g), and content difference was significant (p < 0.05). In soybean sprout, the total β-carotene concentration was 24.5 ㎍/g in green cotyledon sprout, and 35.6 ㎍/g in yellow cotyledon sprout, however, content difference was not significant (p < 0.05) (Table 3, Fig. 5). During germination, the content of total β-carotene increased in green (16.9 ㎍/g, 319%), yellow (29.3 ㎍/g, 562%). The total β-carotene was significantly increased in yellow seed coat soybean.

Table 3. Comparison of β-carotene concentration between soybean seed and sprout on seed cotyledon color.

Fig. 5. Comparison of β-carotene concentration in soybean seed and sprout on seed cotyledon color. *Each vertical bar indicates the standard error values. (A) soybean seed; (B) soybean sprout; GR, green ; YE, yellow.

From this study, the average total β-carotene concentrations affected by seed cotyledone color. And β-carotene concentrations increased during germination process.

ACKNOWLEDGEMENT

This paper was written as part of Konkuk University’s research support program for its faculty on sabbatical leave in 2011. 

Reference

1.Bates, R. P. and R. F. Matthews. 1975. Ascorbic acid and β -carotene in soybeans as influenced by maturity, sprouting, processing, and storage. Florida State Horticultural Society. 266-271.
2.Bauernfeind, J. C. 1973. Carotenoid vitamin A precursors and analogs in foods and feeds. J. Agric. Food Chem. 20(3) : 456-473.
3.Jo, J. O. and I. C. Jung. 2000. Changes in carotenoid contents of several green-yellow vegetables by blanching. Kor. J. Food Cookery Sci. 16(1) : 17-21.
4.Khachik, F., G. R. Beecher, and N. F. Whittaker. 1986. Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J. Agric. Food Chem. 34(4) : 603-616.
5.Kim, J. W. 2001. Methodology of Carotenoids Chemistry. Kor. J. Food Nutr. 14(4) : 360-366.
6.Kim, K. H. 1981. Studies on the growing characteristics of soybean sprout. Kor. J. Food Sci. Technol. 13(3) : 247-252.
7.Kim, M. J., J. H. Kim, H. K. Oh, M. J. Chang, and S. H. Kim. 2007. Seasonal variations of nutrients in Korean fruits and vegetables : Examining water, protein, lipid, ascorbic acid, and β-carotene contents. Kor. J. Food Cookery Sci. 23(4) : 423-432.
8.Kim, S. D., S. H. Kim, and E. H. Hong. 1993. Composition of soybean sprout and its nutritional value. Kor. Soybean Dig. 10(1) : 1-9.
9.Kim, S. J., J. W. Rhim, S. T. Jung, Y. S. Ahn, and Y. B. Oh. 1997. Carotenoid contents of yellow sweet potatoes. Kor. J. Food Sci. Technol. 29(2) : 218-222.
10.Kim, Y. H., Y. H. Hwang, and H. S. Lee. 2003. Analysis of isoflavones for 66 varieties of sprout beans and bean sprouts. Kor. J. Food Sci. Technol. 35(4) : 568-575.
11.Kirsh, V. A., R. B. Hayes, S. T. Mayne, N. Chatterjee, A. F. Subar, L. B. Dixon, D. Albanes, G. L. Andriole, D. A. Urban, and U. Peters. 2006. Supplemental and dietary vitamin e, β -carotene, and vitamin c intakes and prostate cancer risk. Journal of the National Cancer Institute 98(4) : 245-254.
12.Lee, H. S. and Y. N. Kim. 1997. Beta-carotene and lutein contents in green leafy vegetables. Journal of the East Asian of Dietary Life. 7(2) : 175-180.
13.Lee, J. D., Y. H. Hwang, H. Y. Cho, D. U. Kim, and M. G. Choung. 2002. Comparison of characteristics related with soybean sprouts between Glycine max and G. soja. Kor. J. Crop Sci. 47(3) : 189-195.
14.Liu, K. S. 1997. Soybeans: chemistry, technology, and utilization. An aspen publication. 203-205.
15.Mascio, P. D., M. E. Murphy, and H. Sies, H. 1991. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am. J. Clin. Nutr. 53 : 194S-200S.
16.Oh, B. Y., B. H. Park, and K. S. Ham. 2007. Effects of chitosan treatment on changes of soyasaponin contents in soybean sprouts. J. Kor. Soc. Food Sci. Nutr. 36(5) : 584-588.
17.Oliver, J., A. Palou, and A. Pons. 1998. Semi-quantification of carotenoids by high-performance liquid chromatography: saponification-induced losses in fatty foods. J. Chromatography A. 829(1-2) : 393-399.
18.Shimoyamada, M., K. Harada, and K. Okubo. 1991. Saponin composition in developing soybean seed (Glycine max (L.) MERRILL, cv. Mikuriyaao). Agric. Biol. Chem. 55(5) : 1403-1405.
19.Simpson, K. L. and C. O. Chichester. 1981. Annual Review of Nutrition. Metabolism and nutritional significance of carotenoids. Vol. 1 : 351-374.
20.Stahelin, H. B., K. F. Gey, M. Eichholzer, and E. Ludin. 1991. β-carotene and cancer prevention: the Basel Study. Am. J. Clin. Nutr. 53 : 265S-269S.