Optimization of ethanol extraction and further purification of isoflavones from soybean sprout cotyledon

Food Chemistry 117 (2009) 312–317

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Optimization of ethanol extraction and further purification of isoflavones from soybean sprout cotyledon Seung Yong Cho a,1, Yu Nam Lee b,1, Hyun Jin Park b,* a b

Institute of Life Sciences and Biotechnology, College of Life Sciences and Biotechnology, Korea University, 1,5-Ka, Anam-Dong, Seongbuk-Gu, Seoul 136-713, Republic of Korea Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, 1,5-Ka, Anam-Dong, Seongbuk-Gu, Seoul 136-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 September 2008 Received in revised form 16 March 2009 Accepted 1 April 2009

Keywords: Isoflavone RSM Solid phase extraction (SPE) Liquid–liquid extraction (LLE)

a b s t r a c t Isoflavones from cotyledons of soybean sprouts were extracted with aqueous ethanol and further concentrated to obtain a product with a high concentration of isoflavone. The ethanol concentration, extraction time and reaction temperature were optimized by using response surface methodology (RSM). Isoflavones in aqueous ethanol were concentrated by a three-step procedure comprised of solid phase extraction (SPE) with Diaion HP-20 and Amberlite-XAD-2 adsorption columns, acid hydrolysis, and liquid– liquid extraction. The maximum amount of isoflavone in aqueous ethanol extracts (11.6 mg/g solid) was obtained when isoflavones in cotyledons (2.18 mg/ g solid) were extracted with 80–90% (v/v) aqueous ethanol above 90 °C for more than 100 min. The isoflavone extracts, obtained by SPE with a Diaion HP-20 column contained 100 mg/g solid. The liquid–liquid extraction (LLE) with ethyl ether further concentrated the extracts up to 229 mg/g solid, retaining 63% of the initial isoflavones. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Isoflavones are a class of flavonoids in the human diet that are mainly derived from soybean-based foods. The major dietary isoflavones, daidzein and genistein, have estrogen-like activities and are classified as phytoestrogens (Hodgson, Puddey, Beilin, Mori, & Croft, 1998) since they can bind to the estrogen receptor in place of estrogen due to their structural similarities to estrogen-b (Tikkanen & Adlercreutz, 2000). Thus, isoflavones in soybean-based foods are helpful in preventing certain cancers caused by hormone therapy, reducing the risk of cardiovascular disease and improving bone health (Anderson, Johnstone, & Cooknewell, 1995; Arjmandi et al., 1996). The amounts of isoflavones in beans differ with the bean cultivars and culture environments, such as the region and length of culture (Wang & Murphy, 1994a). Development of isoflavonebased functional foods and dietary supplements requires screening of raw materials with high amounts of isoflavones and concentrating of the isoflavones to the level of efficacy. However, 100 g of bean contains only 0.1–0.5 g of isoflavones and this isoflavone level is too low for efficacious health benefits. Besides, the strong beany flavour of soybean powder is a major drawback for its utilization as a source for isoflavones (Macleod & Ames, 1988). Therefore, highly concentrated isoflavone-rich materials, with acceptable organolep* Corresponding author. Tel.: +82 2 3290 3450; fax: +82 2953 5892. E-mail address: [email protected] (H.J. Park). 1 These authors contributed equally to this manuscript as co-first authors. 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.003

tic properties, are needed for functional food materials and dietary supplements. The soybean sprout (Kongnamool) is a low cost but highly nutritive traditional Korean vegetable food that can be produced relatively easily. Soybean sprouts have higher amounts of isoflavones than have soybeans (Kim, Hwang, & Lee, 2003; Kim, Lee, & Chee, 2004), and soybean sprout cotyledon has the highest amounts of isoflavones among the parts of the soybean sprout, including cotyledon, hypocotyls and root (Lee et al., 2007). Therefore, the use of soybean sprout cotyledon as a source of isoflavones is advantageous. In addition, the beany flavour of soybean is caused by lipoxygenase, and soybean germination decreases the lipoxygenase activity. Hence soybean sprouts have a less beany flavour. Soybean sprouts also have many functional ingredients, such as vitamins B1, B2, C, and carotene (Hofsten, 1979). Extraction is a very important process for production of isoflavone concentrate from rich sources. Among the extraction methods, solid phase extraction (SPE) is one of the most widely used techniques for separating functional materials. Because of their excellent sorption characteristics, Amberlite-XAD-2 and Diaion HP-20 have been used to separate isoflavones (Choi & Kim, 2005; Fedeniuk & Shand, 1998; Hennion, 1999). Liquid–liquid extraction (LLE) also has been used to separate functional materials. In LLE, hydrophobic ingredients in the raw materials are extracted from aqueous samples with a water-immiscible organic phase. Various volatile organic solvents are used, including pentane, hexane, ethyl ether, ethyl acetate, chloroform and methylene chloride (PedersenBjergaard, Rasmussen, & Halvorsen, 2000). The extraction pro-

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cesses are affected by process variables, such as solvent, extraction temperature, amount of solvent and extraction time. Optimized process variables are required to efficiently produce highly concentrated isoflavone, and response surface methodology (RSM) is often used for optimization. RSM is a useful statistical technique that uses sequential experimental techniques to survey a domain of interest, focusing on the most important variables and their effects, to build an empirical model. Most RSM applications come from areas such as chemical or engineering processes, industrial research, and biological investigations, with emphasis on optimizing a process or system. The main advantage of RSM is the reduced number of experimental runs needed to provide sufficient information for statistically acceptable results (Hwang et al., 2002; Kim et al., 2002). The use of soybean sprouts as an isoflavone rich source requires an established process for manufacturing the isoflavone-rich products. Therefore, the objective of the present study was to propose an optimized process for extraction and concentration of isoflavones from soybean sprout cotyledon, to produce highly concentrated isoflavone-rich materials.

2. Materials and methods 2.1. Reagents and materials HPLC-grade water, ethanol and methanol were purchased from J.T. Baker Co. The standard isoflavones for quantification were daidzin (Waco Chemical Co., Japan), genistin (Fujicco Chemical Co, Japan), daidzein and genistein (Sigma Chemical Co., USA). Amberlite XAD-2 resin (surface area 300 m2/g, pore diameter 90 Å and bead size 20–60 mesh) and Diaion HP-20 (surface area 500 m2/g, pore diameter 260 Å and bead size 20–60 mesh) were obtained from Supelco Co. (USA). Soybean sprouts were purchased from Woo-Jung Food Co. (Korea). 2.2. Soybean sprout sample preparation The soybean sprouts were separated into cotyledon, hypocotyl, and root. The separated cotyledon, hypocotyl and root were frozen at 70 °C and freeze-dried in a chamber Freeze-dryer (Ilshin Co., Korea). The freeze-dried parts of soybean sprout were ground in a cutting mill and passed through a 0.149 mm screen. The resulting cotyledon, hypocotyls and root powders were used for isoflavone analysis. 2.3. Isoflavone extraction The freeze-dried cotyledons were ground in a cutting mill (Grindomix GM 200, Retsch GmbH, Germany), passed through a 0.149 mm screen, and defatted with a fivefold ratio of hexane to produce defatted soybean sprout cotyledon powder. During the defatting process, the cotyledons and hexane (1:5, w/w) were stirred with a vertical stirrer. 200 g of defatted soybean sprout cotyledon flour were suspended in 1 l of ethanol in a three neck flask reactor. Each reactor neck was attached to a thermocouple to control the temperature, a vertical stirrer for agitation and a Liebig condenser for reflux of ethanol. The concentrations of ethanol used were varied from 60% to 100%, according to the experimental design. The reactor was heated at 75–95 °C for 20–100 min in a hot water bath during the extraction process. After extraction, the suspensions were cooled to around 60 °C by immersing the reactor in a cold water bath (4 °C) immediately, and centrifuged (5000g) to obtain the supernatants. The floating materials in the supernatants were removed by filtering the supernatants through a nylon filter membrane (0.45 lm, Sigma Chemical Co., USA). The isoflavone-

rich alcohol extract was obtained by removing the alcohols in the supernatants by vacuum evaporation with a rotary evaporator (Yamato Scientific Co., Japan). 2.4. Measurement of isoflavone The amount of isoflavones in the extracts was analyzed using a HPLC (Waters Alliance 2690, Waters, USA) equipped with a photodiode array detector (Waters 996, Waters, USA) and Millennium32 chromatography manager software. A 10 ll sample was loaded onto a symmetry C18 column (Hypersil ODS, 250  4.6 mm, 5 lm particle size, Waters, Ireland) through an auto-sampler. The mobile phase was composed of 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). The elution was performed with a linear gradient of A against B from 80:20 to 20:80 (A:B). The eluent flow rate was 1.0 ml/min and absorption was measured at 254 nm. 2.5. Experimental design for ethanol extraction The process conditions for aqueous ethanol extraction of isoflavones from soybean sprout cotyledons were optimized by central composite design. Response surface methodology (RSM) was used to optimize the extraction process to yield the highest amount of total isoflavones (TI) by controlling the process variables: ethanol concentration (v/v, EC), extraction time (min, ET) and reaction temperature (°C, RT). A five-level three-factor factorial design was adopted to optimize the extraction conditions, as shown in Table 1. Total isoflavones, including daidzein and genistein, were fitted to the quadratic response surface model as follows:

Y ¼ b0 þ b1 X 1 þ b2 X 2 þ b3 X 3 þ b12 X 1 X 2 þ b13 X 1 X 13 þ b23 X 2 X 3 þ b11 X 21 þ b22 X 22 þ b33 X 23

ð1Þ

where Y is the amount of total isoflavones, bi are regression coefficients for linear effects, bik are regression coefficients for effects from interaction, bii are regression coefficients for quadratic effects, and Xi are coded experimental levels of the variables. Measurements were performed in duplicate and a Statistical Analysis System was used to fit the second-order polynomial equation to the experimental data. 2.6. Concentration of isoflavones by solid phase extraction and liquid– liquid extraction The isoflavones in aqueous ethanol were concentrated by a three-step procedure comprised of solid phase extraction, acid hydrolysis, and liquid–liquid extraction. The first step was solid phase extraction, which concentrated the isoflavones by passing through Amberlite XAD-2 and Diaion HP-20 adsorbents. The adsorbents were used as adsorption resins to separate the isoflavones. Adsorbents were subsequently washed with 2 l of methanol and 2 l of distilled water and dried at 60 °C. The dried resins were swollen in methanol (1 l) overnight, and excess methanol was removed by rinsing the swollen adsorbent with distilled water. The resins were packed in a glass column (5 cm internal diameter and

Table 1 Coded and actual levels of independent variables for experimental design. Variable

Ethanol concentration (%) Extraction time (min) Reaction temperature (°C)

Coded level of variables 1.68

1

0

1

1.68

60.0 20.0 75.0

68.1 36.2 79.1

80.0 60.0 85.0

91.9 83.8 90.9

100. 100 95.0

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50 cm length). 500 ml of isoflavone-rich aqueous ethanol extracts were passed through the column at a flow rate of 1.2 ml/min and the column was rinsed with 2 l of distilled water to remove impurities. Isoflavones adsorbed by the column were eluted with 1.5 l of 80% aqueous ethanol at a flow rate of 1 ml/min. The glycosidic linkages were acid-hydrolyzed with 2.5 mol/l of hydrochloric acid at 85 °C for 1 h to obtain aglycones for further concentration of isoflavone. The acid-hydrolyzed isoflavones were mixed with diethyl ether in a separate funnel at a ratio of 8:2 for liquid–liquid extraction. The ether-extracted fractions were separated and dried with nitrogen gas to produce concentrated isoflavone. 3. Results and discussion 3.1. Amount of isoflavone in soybean sprout The amounts of isoflavones in soybean sprout cotyledons, hypocotyls and roots are presented in Table 2 and are also compared with those from soybean. The isoflavone contents in soybean sprout ranged from 1.14–2.18 mg/g of dry matter and differed according to the part from which they were obtained. For example, soybean sprout cotyledon had 2.18 mg/g of dry matter, which was four times higher than the amount of isoflavones in hypocotyls. The amount of isoflavones has been reported to vary according to the type of product, as well as their cultivar, place of growth, cultivation conditions and harvest time (Kim et al., 2003; Kim et al., 2004; Wang & Murphy, 1994a; Wang & Murphy, 1994b). Likewise, the isoflavone content was higher in soybean sprouts than in soybean in the present study. This result is consistent with the report issued by Choi, Kwon, and Kim (1996), who reported an increase in total isoflavones during sprouting. 3.2. Optimization of extraction procedure The concentration (%) of aqueous ethanol solution, the extraction time, and the reaction temperature were chosen as independent variables for optimization of the isoflavone extraction procedure. The total isoflavones (TI) in the aqueous ethanol extracts of cotyledon, prepared by controlling the independent variables at the predetermined combinations, are shown in Table 3. The combinations of coded variables were predetermined according to the central composite experimental design, and the specific coded values are shown in Table 1. The total amounts of isoflavones in ethanol extracts ranged from 9.14 to 11.6 mg/g of extracts with respect to variations in extraction conditions. Table 4 summarizes the results of multiple regression analysis of three independent variables (X1 = EC, X2 = ET, and X3 = RT) on TI in aqueous alcohol extracts, along with the results of analysis of variance (ANOVA). The regression analysis showed a significant probability of F-value (p < 0.05) in estimating TI values, which means that the three independent variables had significant effects on TI in aqueous ethanol extracts. At the 5% significance level, the significant independent variables in the TI model were the inter-

Table 2 Amounts of isoflavones in soybean and soybean sprouts. Varieties

Coded level of variableb EC (%)

ET (min)

RT (°C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0 0 0 0

1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0 0 0 0

a b c

TIc (mg/g)

9.14 10.0 10.5 10.4 11.3 11.2 11.5 11.6 10.8 11.1 10.7 11.6 9.46 10.7 11.5 11.4 11.3 11.5 11.2 11.2

Test runs were performed in random order. EC: Ethanol concentration, ET: extraction time, RT: reaction temperature. TI: Total isoflavone.

Table 4 Regression coefficients of the quadratic regression modela for the determination of total isoflavones in the cotyledon of bean sprouts. Coefficient

Regression coefficient

T value

b0 Linear b1 b2 b3

59.76

3.88***b

0.6880 0.1298 0.8648

5.73*** 2.35** 2.82**

Quadratic b11 b22 b33

0.003095 0.000139 0.004081

7.26*** 1.31 2.39**

Two-factor cross b12 b13 b23

0.000490 0.001374 0.000733

1.71 1.20 1.28

Regression R square F value

0.9426 18.24

P < 0.0001

a

Model for regression analysis of total isoflavones (Y) used X1 = Ethanol concentration of aqueous ethanol solution (EC,%, v/v), X2 = Extraction time (ET, min), and X3 = Reaction temperature (RT, °C), and Y = b0 + b1X1 + b2X2 + b3X3 + b12X1 X2 + b13X1X13 + b23X2X3 + b11X 21 + b22X 22 + b33X 23 . b ** Significant at 5% level (p < 0.05), and ***Significant at 1% level (p < 0.01).

cept (p = 0.003), EC (p < 0.001), ET (p = 0.0408) and RT (p = 0.018) in the linear term, EC2 (p < 0.001) and RT2 (p = 0.0378) in the quadratic term. The model could be expressed as:

 0:003095X 21  0:004081X 23

Isoflavone (mg/g) Genistein

Total

Soybean

0.49 ± 0.02a

0.65 ± 0.16

1.14 ± 0.08

Soybean sprout – whole Cotyledon Hypocotyl Root

0.68 ± 0.03 0.80 ± 0.08 0.43 ± 0.13 1.81 ± 0.15

0.76 ± 0.02 1.39 ± 0.17 0.10 ± 0.08 0.20 ± 0.02

1.44 ± 0.03 2.18 ± 0.18 0.53 ± 0.16 2.02 ± 0.13

Experiments were performed in triplicate.

Test runa no.

TI ¼ 59:76 þ 0:6880X 1 þ 0:1298X 2 þ 0:8648X 3

Daidzein

a

Table 3 Coded level combinations of three variables for central composite orthogonal and rotatable design.

ð2Þ

The proposed model had a sufficiently high R-square value (R2 = 0.9426) to indicate that the TI data were adequately explained. Therefore, the model can be used as an estimate of tendency. The contour and three-dimensional plots that account for the effects of EC, RT and ET on TI were produced by the above regression equation and are presented in Fig. 1. The plots in Fig. 1 were produced for each pair of factors, whereas the third factor was taken as a constant

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100

a

Total isoflavone

11 10

(% n

ra

nt

ce

on

n)

11.0

60

11.0

50 40 30 20

lc

me (m i

20

b

10.0 10.5

11.0

10.0 10.5

9.0

9.5 8.5

11.0

20

30

ha

ion ti

11.5

70 9.5

no

90 80 70 60 50 40 Extra 30 ct

80

40

50

60

70

90

80

100

Ethanol concentration (%)

Et

7

t io

8

95 90 85 80 75 70 65 60

)

9

11.5

11.0

90

Extraction time (min)

(mg/g)

12

95

c

11 10

(%

95 90 85 80 75 70 65 60

90 9.5 10.0

11.0

9.5 10.0

80

9.0

11.0

ce

on

80

empe

rature

75

(ºC)

11.0

75 20

lc

tion t

30

40

50

11 10 9

tim e(

mi n)

100 90 80 70 60 50 40 30 20

rature

on

80

empe

75

(ºC)

cti

tion t

tra

Reac

85

Ex

90

90

80

11.0 11.0

Reaction temperature (ºC)

12

7

70

10.8

e

8

60

100

Ethanol concentration (%) 95

(mg/g) Total isoflavone

10.5

8.5

ha

85

Et

90

Reac

11.0

nt

7

d

10.5

85

ra

tio

n

8

)

9

no

(mg/g) Total isoflavone

12

Reaction temperature (ºC)

11.0 11.0

90

f

11.2 10.8 10.6

11.0

11.2

85 10.4

80 10.6

10.2 10.0 9.8

75 20

30

10.8 11.0

11.2

10.4

40

50

60

70

80

90

100

Extraction time (min)

Fig. 1. Response surface and contour plots for the effects of variables on the total isoflavones (TI) in the ethanol extracts of soybean sprout cotyledon: (A and B) ethanol concentration (EC) and extraction time (ET); (C and D) ethanol concentration (EC) and reaction temperature (RT); (E and F) extraction time (ET) and reaction temperature (RT).

at its middle level. The plot a and b in Fig. 1 represent the effects of EC and ET on TI in the ethanol extracts of soybean sprout cotyledon. The maximum TI could be observed at optimized ethanol concentrations, and an increase of TI was observed with increased extraction time. The maximum TI was obtained with 80–90% aqueous ethanol solution and more than 90 min of extraction time. EC higher than 90% decreased TI due to the poor solubility of isoflavone in pure ethanol. Plots c and d in Fig. 1 illustrate the effects of EC and RT on the TI of aqueous ethanol extracts. The maximum TI was obtained with EC between 85% and 90% and RT around 85 °C. The de-

crease of TI with RT above 90 °C seemed be due to the unstable concentration of solvent mixture above the boiling temperature of ethanol. The effects of ET and RT on the TI of aqueous ethanol extracts are shown in plot e and f. The maximum TI could be obtained with RT around 85 °C, and an increase of TI was observed with increased extraction time. The maximum TI was obtained when the extraction procedure was operated at 85 °C for 95 min or more. The amount of total isoflavone in extracts prepared with the above extraction conditions was 11.6 mg of isoflavone/g of extract (Table 5). The TI in aqueous ethanol extracts was 5.3 times higher

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Table 5 The yield, retention and content of isoflavone in cotyledon, ethanol extracts and further concentrated isoflavone extracts. Isoflavone (mg/g)a

Yieldb (%)

Retentionc (%)

Content (g/100 g)

Daidzein

Genistein

Total

Extraction Cotyledon Ethanol extracts

0.80 ± 0.08 3.93 ± 0.39

1.39 ± 0.17 7.25 ± 0.95

2.18 ± 0.18 11.6 ± 0.77

100 17.7

100 90.3

0.22 1.16

Purification Amberlite XAD-2 Diaion HP-20 Liquid–liquid xtraction

22.1 ± 3.22 29.7 ± 1.47 60.7 ± 4.28

59.6 ± 5.61 70.1 ± 2.94 170 ± 3.96

81.7 ± 4.18 100 ± 3.91 229 ± 4.86

2.42 1.95 0.60

86.7 88.5 63.0

8.17 10.0 22.9

a

Experiments were performed in triplicate. Yield is the weight of dried extracts from 100 g of soybean sprout cotyledon feed (=Wextract  100/Wsoybean sprout cotyledon feed). c Retention is the weight of total isoflavone in dried extracts from 100 g of total isoflavones in soybean sprout cotyledon feed (=(WTI)extract  100/(WTI) soybean sprout cotyledon feed). b

than the amount of isoflavones in cotyledon (2.18 mg/g of dry weight). Using this optimized extraction process, 90.3% of isoflavones in the soybean sprout cotyledon were retained in the aqueous ethanol extracts. Several researchers have explored the optimal conditions for extraction of isoflavones from beans. Choi et al. (1996) compared the amounts of isoflavones in extracts according to extraction temperature, solvents and time. They reported that the optimal conditions for isoflavone extraction were 60% and 80% ethanol, 90 °C extraction temperature and 1 h extraction time. Rostagno, Palma, and Barroso (2003) optimized the solvent condition for extraction of soy isoflavones and developed an ultrasound-assisted extraction method for isoflavone determination. Their optimized condition for ultrasound-assisted isoflavone extraction was 50% ethanol concentration, 60 °C reaction temperature and 20 min of ultrasoundassisted extraction. The ultrasound-assisted extraction might be responsible for the alteration of optimal extraction conditions between their work and the present study. Zhang, Ng, and Luo (2007) optimized extraction and purification parameters for manufacturing soy isoflavone products on the basis of the yields of isoflavones. The optimum extraction conditions of their study were determined to be 80 °C for 8 h with a 96% ethanol over soybean flour ratio of 3:1 to achieve the best extraction of isoflavones, and the yield was about 0.87 mg of total isoflavones from 1 g of soybean flour. Their optimal conditions for soy isoflavone extraction are similar to those of the present study, except for extraction time. In this study, the extraction time over 90 min had only a small effect on extraction of isoflavone when isoflavone was extracted with 85% ethanol at 85 °C. Based on these studies, ethanol concentration and extraction temperature had the most profound effects on the amounts of isoflavones in aqueous ethanol extracts of soybean products. 3.3. Purification of isoflavones The aqueous ethanol-extracted isoflavone solution was further purified by solid phase extraction (SPE) with adsorption columns packed with Diaion HP-20 and Amberlite XAD-2. The results are presented in Table 5. Solid phase adsorption with a Diaion HP-20 column performed better in separating isoflavones than did the Amberlite-XAD-2 resin column. The amount of isoflavone after SPE with Diaion HP-20 increased to 100 mg/g of sample. The isoflavone-rich fraction retained 88.5% of the soybean sprout cotyledon isoflavone with 1.95% yield. This result is in accordance with the previous work of Choi and Kim (2005) who reported the effectiveness of Diaion HP-20 in extracting phytochemicals. After SPE, the isoflavone-rich sample was acid-hydrolyzed for 1 h at 95 °C to yield the aglycone forms of isoflavones, which had increased hydrophobicity. The presence of aglycone isoflavones is preferable, due to the ease of separation and their biological ef-

fects. The aglycones of isoflavones were further purified by ethyl ether extraction, and the results are presented in Table 5. The liquid–liquid extraction procedure increased the amount of isoflavone in extracts up to 229 mg/g of sample, and the extract retained 63% of the initial isoflavones. The performance of liquid–liquid extraction in the present study was comparable to that in previous works on the concentration and purification of isoflavones (Chang, Cheng, & Chang, 2004; Choi et al., 2003). Chang et al. (2004) used Amberlite 16-HP resin for purification and obtained 24.7% of isoflavones when they scaled up the liquid–liquid extraction. They suggested that production of high purity total isoflavone might be significantly influenced by the feed concentration of total isoflavones. Thus, using the feed extracts obtained under optimum conditions is beneficial for purifying isoflavones (Chang et al., 2004). 4. Conclusion The soy isoflavone concentrate was prepared by a series of procedures, comprised of ethanol extraction at optimized condition and followed by a three step concentration process, including solid phase extraction, acid hydrolysis and liquid–liquid extraction. 0.22 mg of isoflavone/g of soybean sprout cotyledon was concentrated to 229 mg/g of concentrate through a series of concentration procedures with 63% recovery of the total isoflavones. The production of highly concentrated isoflavone might allow its application as a biologically active food material since the trace amount of isoflavone in soybean sprout has limited its use in health food. References Anderson, J. W., Johnstone, B. M., & Cooknewell, M. E. (1995). Metaanalysis of the effects of soy protein-intake on serum-lipids. New England Journal of Medicine, 333(5), 276–282. Arjmandi, B. H., Alekel, L., Hollis, B. W., Amin, D., StacewiczSapuntzakis, M., Guo, P., et al. (1996). Dietary soybean protein prevents bone loss in an ovariectomized rat model of osteoporosis. Journal of Nutrition, 126(1), 161–167. Chang, L.-H., Cheng, Y.-C., & Chang, C.-M. (2004). Extracting and purifying isoflavones from defatted soybean flakes using superheated water at elevated pressures. Food Chemistry, 84(2), 279–285. Choi, Y.-B., & Kim, K.-S. (2005). Purification of isoflavone from soybean hypocotyls using various resins. Korean Journal of Environmental Health, 31(3), 221–226. Choi, Y.-B., Kim, M. J., Lee, Y. B., Sohn, H. S., Lee, O. H., & Kim, K. S. (2003). Purification of isoflavone from soybean hypocotyl using different solvents. Korean Journal of Environmental Biology, 21(3), 245–250. Choi, J.-S., Kwon, T.-W., & Kim, J.-S. (1996). Isoflavone contents in some varieties of soybean. Food Science and Biotechnology, 5(2), 167–169. Fedeniuk, R. W., & Shand, P. J. (1998). Theory and methodology of antibiotic extraction from biomatrices. Journal of Chromatography A, 812(1–2), 3–15. Hennion, M. C. (1999). Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography. Journal of Chromatography A, 856(1–2), 3–54. Hodgson, J. M., Puddey, I. B., Beilin, L. J., Mori, T. A., & Croft, K. D. (1998). Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: A randomized controlled trial in humans. Journal of Nutrition, 128(4), 728–732.

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