International Journal of Food Properties, 8: 113–123, 2005

Copyright & Taylor & Francis Inc.

ISSN: 1094-2912 print/1532-2386 online

DOI: 10.1081/JFP-200048055

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QUANTIFICATION OF ISOFLAVONES IN SOYMILK ANDTOFU FROM SOUTH EAST ASIA

Molamma P. Prabhakaran, Conrad O. Perera and

Suresh Valiyaveettil

Department of Chemistry, National University of Singapore, Singapore

The concentration and distribution of isoflavones in commonly used soybean foods were

evaluated by high-performance liquid chromatography. The different forms of isoflavone

glucoside esters were converted to their corresponding glucosides by a mild saponification

of the extracts. Isoflavone levels were reported as microgram aglycone equivalents per gram

of soy food. The total isoflavone concentrations in soymilk and tofu ranged from 76 to

199lg/g and 162 to 312lg/g respectively, on a wet weight basis. A comparison of

isoflavone levels in soymilk and tofu, prepared by a standard procedure with the commer-

cially available packaged soymilk and tofu samples were also carried out. Even though

different types of tofu had different isoflavones concentrations, each type had similar values

compared to those produced by the standard procedure.

Keywords: Isoflavones, Tofu, Soymilk, Protein, Aglycone.

INTRODUCTION

Phytoestrogens are naturally occurring phenolic compounds found in severalplant-foods. Though phytoestrogens possess no nutritional properties, they havebeen reported to exhibit biological activity in humans and animals, similar to thatof the natural human hormone estradiol.[1] The main known phytoestrogensare classified into four groups according to their chemical structures, namely,isoflavones, coumestans, resorcyclic acid lactones, and lignans.[1] However, themajor sources of phytoestrogens in human diets are the isoflavones.

Soybeans and soy products are particularly abundant sources of isoflavones.There are three main types of isoflavones in soybeans, and each type exists in fourdifferent chemical forms, which include the aglycons and their b-glucosideconjugates: the glucosides, the acetylglucosides and the malonyl glucosides.[2]

Figure 1 shows the structure of the three parent isoflavones and the respectiveglucosides. There is a growing literature on the health protective effects of soy foods.

Received 18 February 2004; accepted 10 April 2004.Address correspondence to Conrad O. Perera, Department of Chemistry, FST

Programme, National University of Singapore, 3 Science Drive 3, Singapore 117543;E-mail: chmpco@nus.edu.sg

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Epidemiological studies have suggested that the consumption of soybeans and soyfoods are associated with lowered risks for several types of cancers, including breast,prostate and colon,[3,4] cardiovascular diseases,[5,6] and bone health.[7,8] However,further long-term studies are required to understand their complex mechanisms ofaction. The beneficial effects of isoflavones may be due, but only in part, to the weakestrogenic activity of isoflavones.

Soybeans have long been a staple of the human diet in Asia, especially assoymilk or tofu.[9] Soymilk entered the modern era in the 1950s as it began to bemarketed in bottles, largely due to the work by K. S. Lo of Vitasoy in Hong Kongand Yeo Hiap Seng in Singapore according to the Soyfoods Centre Chronology ofsoymilk worldwide.[10] By 1967, aseptically packed Tetra Pak cartons of soymilkstarted appearing in the market. The first such product was Beanvit, made by YeoHiap Seng Ltd., in Singapore and packaged in a disposable tetrahedron-shapedcontainer. A new wave of technology development occurred since then.

Soymilk and tofu are the most popularly consumed soy food items in SouthEast Asia, China, and Japan. They are commonly served in food courts as well as insupermarkets and the consumption of these products continue to increase, sincepeople are becoming more health conscious. Recently, the FDA (1999) allowed

Figure 1 Chemical structures of parent isoflavones and respective glucosides in soybean.

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health claims to be declared on product-labels that contained a minimum of 6.25 g of

soy protein per serving.[11] It is this potential claim that further generatedconsiderable interest in soy foods. There are ongoing clinical studies to explore therelationship between soy consumption and reduction in risk of many diseases.[12–14]

It is therefore crucial to determine the qualitative and quantitative composition ofisoflavones in selected soy foods commonly consumed by the multiethnic population

in the region.

MATERIALS AND METHODS

Materials

Soymilk was prepared on a commercial plant scale using a standard procedure.

Commercially available and popular brands of soymilk were selected from themarket and their isoflavone levels were compared with those in soymilk obtained by

the standard procedure. Soft tofu and a firm tofu were also prepared on a plantscale. The soft tofu so prepared was ‘‘silken tofu,’’ while the firm tofu prepared was‘‘taw kwa.’’ The different types of soft tofu commercially available in the market

include ‘‘silken tofu,’’ ‘‘Chinese tofu,’’ and ‘‘organic tofu,’’ which were analyzed andtheir isoflavone levels were compared to those found in the different tofu samples

obtained by the standard procedure. Firm tofu varieties collected for similarcomparative studies were ‘‘taw kwa,’’ deep-fried tofu, and braised tofu. These

products were randomly picked from the main supermarkets in Singapore.

Chemicals

Authentic standards of daidzin, genistin, glycitin, daidzein, genistein, and

glycitein were purchased from LC Laboratories (Woburn, MA, USA). The HPLCgrade solvents were purchased from Sigma Chemical Co. (Jefferson City, MO,

USA). Linear responses were obtained for the six standards and calibration curveswere plotted by using peak areas vs. corresponding phytoestrogens concentration.All solvents used were of HPLC grade.

Plant Scale Preparation of Soymilk and Tofu

Soybeans were soaked in water for 5 h at ambient temperature. Rinsing

followed by grinding of soybean was carried out with a bean to water ratio of 1:5.Further steps involved were cooking, and extraction of soymilk by centrifugal

separation. The raw soymilk thus obtained had an approximate soluble solidscontent of 9�Brix. The product was aseptically packed hot and immediately cooled ina chilling room. This was considered as the standard soymilk prepared for the study.

For the manufacturing of tofu, soymilk with a higher Brix value was prepared.

This was achieved by adjusting the bean to water ratio accordingly. Raw soymilkhaving approximately 11�Brix was made and it was used for the preparation of soft

tofu. This soymilk was aseptically packaged with Glucono-delta-lactone (GDL) andCaSO4 and allowed to coagulate in the package itself to obtain soft tofu. This wasconsidered as the standard soft tofu preparation.

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Raw soymilk with approximately 12�Brix was prepared and used for firm tofu

making. This soymilk was coagulated with calcium chloride and excess whey was

removed by application of pressure to obtain a standard firm tofu. The

concentration of isoflavones in the prepared soymilk, soft tofu, and firm tofu were

determined immediately after their preparation.

Extraction of Isoflavones from Soy Foods

The extraction procedure followed the methodolgy of Klump et al.[15] modified

as follows. An amount of test sample that contains approximately 1 g protein was

accurately weighed into a 250mL Erlenmeyer flask with ground-glass stopper. To

this, 40mL of the extraction solvent (80% methanol in water) was added. The flask

with its contents was shaken in a 65�C water bath for 2 h, cooled to room

temperature, and 3mL of 2M NaOH were added. They were stirred well at room

temperature for 10min and 1mL of glacial acetic acid was added. The contents were

swirled and poured into a 50mL volumetric flask, diluted to mark with the

extraction solution and mixed well. The contents were centrifuged for 10min at

5000 rpm. An aliquot of the supernatant was filtered through a 0.45 mmpolytetrafluoroethylene (PTFE) filter unit and analyzed by High Performance

Liquid Chromatography (HPLC). After the extraction of isoflavones from the soy

samples, they were immediately analyzed by HPLC. Storage of the extracted

isoflavones for extended period of time before an analysis was never done. Standard

method of moisture analysis by vacuum oven method[16] was used to determine the

moisture content of all samples.

HPLC Analysis of Isoflavones

The HPLC apparatus was a Waters model 600E with inline degasser, a

Waters 2996 photodiode array detector capable of monitoring UV absorbance from

200 to 350 nm and the Waters ‘‘Empower Software System’’ (Waters Corp., Milford,

MA, USA). Reversed phase HPLC analyses of isoflavones were carried out on

YMC-pack ODS-AM 303 (5 mm, 25 cm� 4.6mm id) column (Waters Corp.,

Milford, MA, USA).A linear HPLC gradient consisting of (A) 0.1% glacial acetic acid in filtered

MilliQ water and (B) 0.1% glacial acetic acid in acetonitrile was used. The injection

volume was 20 mL, and the components were eluted using the following solvent

gradient: from 0 to 5min 10% B and 90% A; from 5 to 50min 10–35% B and

90–65% A; then held at 35% B and 65% A for another 10min and re-equilibrated

back to 10% B and 90% A. The flow rate was 0.8mL/min and the UV detector

wavelength was set at 260 nm. The total run time was 80min including the

equilibration of column. However, this long run time allowed a clean and clear

separation of the six isoflavone conjugates good enough for quantitation. The

identity and purity of isoflavones in the samples were confirmed by matching

the retention times in combination with the mass spectrum analyses (LC-MS) of

the standards.

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Protein Assay

The protein contents of soy foods were determined by the BioRad assay.[17]

This was carried out in order to establish a correlation between the isoflavone

concentration and protein content of the samples. A calibration curve was preparedby using a series of standard solution of bovine serum albumin. Absorbance was

measured at 595 nm. The protein concentrations of samples were determined bycomparison with the calibration curve.

Calculation of Isoflavone Levels in Soy Foods

The concentration of isoflavone glucosides daidzin, glycitin, and genistin wereconverted to aglycon equivalents using the following equation:

Cae ¼ ½MWa=MWg� � Cg

where Cae¼ isoflavone aglycon equivalents (mg/g); MWa¼molecular weight ofaglycon; MWg¼molecular weight of glucoside; and Cg¼ concentration of daidzin,

glycitin, and genistin (mg/g). The total isoflavones in microgram aglycon equivalents/g of sample was calculated, by summing the concentrations of daidzein, glycitein and

genistein, and adding this total to the sum of aglycon equivalent concentrations ofdaidzin, glycitin, and genistin.

T1 ¼ CaðdaidzeinÞ þ CaðglyciteinÞ þ CaðgenisteinÞ

T2 ¼ CaeðdaidzinÞ þ CaeðglycitinÞ þ CaeðgenistinÞ

where T1¼ sum of the concentrations of aglycons, and T2¼ sum of the aglycon

equivalent concentrations of glucosides. Thus total isoflavones were expressed asmicrogram aglycon equivalents per gram of sample¼T1þT2.

Statistical Analysis

Pearson’s correlation coefficient (r) between the protein content and the

isoflavone concentrations in soymilk samples are calculated using SPSS version 12.0

(SPSS Inc., Chicago, USA). The correlations output from SPPS software obtainedfor soymilk samples are shown in Table 4. The value of r usually ranges from�1.00

toþ1.00. High positive correlation is obtained if r is closer toþ1.00, which is wellobserved in this case. The Pearson’s correlation strongly supported the research

hypothesis that there is a significant positive relationship (r¼ 0.984) between theprotein content and isoflavone concentration in soymilk samples. In a similar way,

the correlation coefficient between the protein content and isoflavone concentrationsin tofu samples were calculated (r¼ 0.942). However, braised tofu and deep-fried

tofu were exempted during this calculation due to the addition of nonsoycomponents into them.

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RESULTS AND DISCUSSION

Soymilk is a colloidal solution that is obtained as a water extract from swelledand ground soybeans. It is popular among the Chinese people as a breakfast drink.The soymilks commercially available in Singapore were either imported fromAustralia and Malaysia or were manufactured locally. The typical HPLCchromatogram obtained for the soy samples is shown in Fig. 2. No publishedreports are available on the isoflavone content of Singapore soy foods apart from theone reported by Franke et al.[13] They did a comparative study on the isoflavonelevels in raw and cooked forms of ‘‘taw kwa,’’ raw tofu, fried ‘‘taw kwa,’’ driedsoybean curd sticks, and soybean. Cooking was carried out for 5min for all these soyfoods except for soybean (10min). Since there are many different types of tofu andtheir methods of preparation are different, the results they reported were a littleconfusing. For example, raw ‘‘taw kwa’’ and raw tofu showed nearly an equalamount of isoflavones (297 mg/g) in their study. However, it was not understood,how raw ‘‘taw kwa’’ differed from raw tofu and why both showed an equal amountof isoflavones. In general, cooked products showed lesser isoflavone levels than theraw products probably because of the leaching of isoflavones into the water. Thoughthe authors compared the isoflavone levels between the same food groups fromSingapore and Hawaii, they found no consistent results. In our study, wedifferentiated between the soft and firm tofu types available in the market andfurther determined the isoflavone concentrations in them. We prepared different soyfoods like soymilk, soft tofu, and firm tofu on a commercial plant scale by astandard procedure. Isoflavone levels in soy foods prepared by standard procedurewere compared with those in commercially available and branded soy foods of thesame group. We believe our study reduces the confusion between different types oftofu and enabled the comparison of their isoflavone levels with the same food groupsprepared by a standardized procedure.

The mean isoflavone concentration levels on a wet weight basis in soymilk, softtofu, and firm tofu prepared by the standard method, were 126.29, 167.13, and

Figure 2 Reversed-phase HPLC chromatogram of the six isoflavones in soy samples.

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241.35 mg/g respectively (Table 1). The isoflavone concentration in soybeans used for

the preparation of soy foods was 1467.23 mg/g on a dry weight basis. The isoflavone

concentration in soymilks collected from supermarkets varied between 76 and

199 mg/g, on a wet weight basis (Table 2). Among the different brands of soymilk

analyzed, Brand E contained the highest amounts of isoflavones and Brand A had

the lowest. Within the soymilk group, a higher isoflavone concentration was found

in soymilks containing higher protein levels than those with lesser protein levels

(Fig. 3). Such trends were also noted by other workers.[12,18–20] The concentration of

isoflavones in commercially available branded soymilks were comparable with those

Table 2 Total isoflavone data (on wet weight basis) reported as microgram aglycon equivalents per gram

of sample for the commercially available soymilks

Milk

type

Moisture

(%�SDb)

Proteina

(%)

Daidzein

(mg/gm)

Glycitein

(mg/gm)

Genistein

(mg/gm)

Total

aglycons� SDb

Brand A 96.0� 0.01 2.048 6.857 1.112 4.867 12.836� 0.17

Brand B 90.6� 0.01 2.858 3.635 0.117 1.717 5.469� 0.71

Brand C 85.2� 0.01 3.675 4.090 0.360 3.740 8.190� 0.04

Brand D 82.6� 0.01 3.700 1.327 0.092 0.957 2.377� 0.19

Brand E 86.2� 0.01 4.200 12.357 0.775 7.402 20.535� 0.08

Milk

type

Daidzin

(mg/gm)

Glycitin

(mg/gm)

Genistin

(mg/gm)

Glucosides in

aglycon-equivalents� SDbTotal

isoflavones� SDb

Brand A 44.23 8.41 49.92 63.58� 0.11 76.41� 0.28

Brand B 69.77 11.58 76.88 98.05� 1.54 103.52� 0.83

Brand C 102.56 11.56 143.18 159.52� 1.63 167.71� 1.60

Brand D 115.07 24.81 142.37 175.09� 1.17 177.46� 0.97

Brand E 136.72 11.95 140.12 178.72� 0.91 199.25� 0.99

aProtein calculated from the BioRad protein assay.bSD is standard deviation and all values are the mean of four replicates.

Table 1 Total isoflavone data (on wet weight basis) reported as microgram aglycon equivalents per gram

of sample for soymilk, soft tofu, and firm tofu prepared by standard procedure

Soy

product

Moisture

(%�SDb)

Proteina

(%)

Daidzein

(mg/gm)

Glycitein

(mg/gm)

Genistein

(mg/gm)

Total

aglycons� SDb

Soymilk 86.4� 0.01 3.30 2.746 2.170 1.771 6.687� 0.118

Soft tofu 89.8� 0.01 6.10 3.443 4.133 2.070 9.646� 0.183

Firm tofu 79.2� 0.01 10.05 7.306 4.153 4.916 16.376� 0.045

Milk

type

Daidzin

(mg/gm)

Glycitin

(mg/gm)

Genistin

(mg/gm)

Glucosides in

aglycon-equivalents�SDbTotal

isoflavones� SDb

Soymilk 84.14 9.84 99.08 119.60� 1.73 126.287� 1.70

Soft tofu 111.71 12.84 129.67 157.48� 0.340 167.13� 0.456

Firm tofu 147.80 17.34 197.78 224.97� 1.09 241.34� 1.05

aProtein calculated from the BioRad protein assay.bSD is standard deviation and all values are the mean of 6 replicates from two plant trials of the

respective soy food making.

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of the concentration of isoflavones in soymilk prepared by the standard procedure.Soymilks possessed an appreciable amount of isoflavones with regard to theirprotein contents. Though different manufacturers use different variety of soybeanand different processing methods for soymilk manufacture, the concentration ofisoflavones was high in soymilk with high protein content.

For the basic tofu production, soymilk is coagulated with salts or acids(coagulants) either alone or in combination to produce a protein gel which trapswater, soy lipids, and other constituents in the matrix. After addition of thecoagulant to the soymilk, a curd was formed and excess whey (supernatant) may beremoved depending on the type of tofu prepared. Texture (soft and firm) of tofudepends on the type of coagulant used, amount of pressure employed on the curd toexpel excess whey, pressing time, and the amount of curd obtained. For example,soft or silken tofu is an aseptic tofu, where processed soymilk is aseptically packagedwith GDL and allowed to coagulate in the package, while a firm tofu is obtainedafter an application of pressure on the coagulated soymilk to remove some of thewhey from the coagulum. The higher the pressure applied, the firmer the tofu andlesser the water content. ‘‘Taw kwa’’ is an example of firm tofu.

Based on the methods of subsequent processing or preparation, tofu is alsoclassified into frozen tofu, deep-fried tofu, grilled tofu, braised tofu etc. Theirpreparations involve either exposure to severe cold temperatures, further expulsionof water or even addition of seasoning ingredients and nonsoy components like spicepowder or sweet and sour sauce. These are the many different types of tofu that haveappeared in the market.

In this study, a soft variety of tofu and a firm variety of tofu were prepared ona commercial plant scale and were considered as bench-marks to compare theisoflavones of samples obtained from supermarkets in Singapore. The isoflavoneconcentrations in the prepared soft tofu were compared with the isoflavoneconcentrations in commercially available ‘‘silken,’’ ‘‘Chinese,’’ and ‘‘organic’’ tofu.Analysis showed that ‘‘silken,’’ ‘‘Chinese,’’ and ‘‘organic’’ tofu differed in their waterand protein contents. Also the types of coagulants used for their preparation weredifferent. However, the concentrations of isoflavones in these commercially available

Figure 3 Correlation of protein and isoflavone levels for soymilk samples.

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soft tofu varieties were comparable with the isoflavone levels in the soft tofu preparedby the standard procedure. The minor differences in the isoflavone concentrationscould be partly due to the variety of beans used or the manner in which the tofu wasprepared using different coagulants. Some coagulants might be better and moreefficient than the other in terms of retaining isoflavones in tofu from soymilk.[21]

In a similar fashion, the isoflavone levels in the prepared firm tofu werecompared with the isoflavone levels in commercially available ‘‘taw kwa,’’ deep-fried, and braised tofu. Since the raw material for deep-fried and braised tofu is ‘‘tawkwa,’’ it was ideal to compare their isoflavone levels with the firm tofu prepared bythe standard procedure. Deep-fried tofu is usually made by frying the firm tofu in oilat high temperature of 180�C. For braised tofu making, the firm tofu is marinatedwith a sauce (sweet or spicy), whereby the pH will be reduced and it furtherundergoes a pasteurization procedure. These procedures cause the isoflavone levelsin braised tofu to be much lower than that of ‘‘taw kwa’’ and deep-fried tofu.The presence of other nonsoy components in braised tofu influence the isoflavoneconcentration in the total food consumed.

Our analyses showed that the protein content in firm variety of tofu washigher than that of soft variety of tofu. High concentrations of isoflavones were alsopresent in tofu with high protein content. Thus ‘‘taw kwa’’ and deep-fried tofu hadhigher protein content and also had higher isoflavone concentrations whencompared to ‘‘silken,’’ ‘‘Chinese,’’ and ‘‘organic’’ tofu, which had lower proteincontent (Table 3).

Table 3 Total isoflavone data (on wet weight basis) reported as microgram aglycon equivalents per gram

of sample for the commercially available tofu varieties

Tofu

type

Moisture

(%�SDb)

Proteina

(%)

Daidzein

(mg/gm)

Glycitein

(mg/gm)

Genistein

(mg/gm)

Total

aglycons� SDb

Silken tofu 85.90� 0.01 5.280 2.440 ND 2.820 5.260� 0.24

Chinese tofu 89.60� 0.01 6.769 1.943 0.050 2.280 4.273� 0.02

Organic tofu 88.60� 0.01 6.536 4.260 0.040 2.500 6.800� 1.49

Braised tofu 67.40� 0.01 9.690 6.045 0.250 4.825 11.120� 0.07

Deep fried 74.40� 0.01 11.70 10.885 0.490 7.840 19.215� 0.72

Taw Kwa 74.40� 0.01 9.674 5.500 0.210 3.680 9.390� 0.12

Tofu

Daidzin

(mg/gm)

Glycitin

(mg/gm)

Genistin

(mg/gm)

Glucosides in

aglucon equivalents�SDbTotal

isoflavones� SDb

Silken tofu 131.73 15.40 109.28 158.59� 2.77 163.85� 2.45

Chinese tofu 129.92 16.95 109.53 158.63� 3.26 162.91� 3.24

Organic tofu 139.77 16.14 120.67 171.10� 0.72 177.90� 2.22

Braised tofu 109.36 19.75 123.82 156.79� 9.23 167.91� 9.31

Deep fried 217.32 26.42 205.96 278.34� 4.84 297.55� 4.12

Taw Kwa 236.66 23.98 229.06 303.03� 2.74 312.42� 2.33

aProtein calculated from the BioRad protein assay.bSD is standard deviation and all values are the mean of four replicates. ND¼not detectable. All tofu

samples analyzed were from the same manufacturer. ‘‘Silken’’ tofu, ‘‘chinese’’ tofu, and ‘‘organic’’ tofu

are soft tofu varieties which is incorporated with whey and the difference between them being the type

of coagulants used for its coagulation. ‘‘Organic’’ tofu uses organic soybean seeds. ‘‘Braised’’ tofu,

‘‘deep-fried’’ tofu, and ‘‘taw kwa’’ are firm tofu varieties.

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Since isoflavones are absorbed in the body as the aglycone, the totalconcentration of isoflavones in food products should not be expressed as thearithmetic sum of the aglycons and glucosides.[22] Therefore, the total isoflavonecontents were adjusted for their molecular weight differences and expressed inaglycone equivalents to avoid erroneous conclusions. Such information will also beuseful for food processors for the correct labeling of isoflavones on soy foodproducts in the future. Display of isoflavone concentrations on the labels of soyfoods would also be advantageous since it could provide additional nutritionalinformation to the consumers. From these results, it was apparent that isoflavoneswere found in higher concentration in soy foods containing higher levels of proteincontent than those with lesser protein content.

ACKNOWLEDGMENT

We would like to thank Ms. Ravinder Kaur from Unicurd Food Company,Singapore for helping to carrying out the plant scale preparations of soymilk andtofu. This research was supported by a grant from The National University ofSingapore R-143-000-179-112.

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Table 4 Correlations output from SPSS software obtained for soymilk samples

Protein Isoflavone

Protein Pearson Correlation 1 0.984a

Sig. (2-tailed) — 0.002

N 5 5

Isoflavone Pearson Correlation 0.984a 1

Sig. (2-tailed) 0.002 —

N 5 5

aCorrelation is significant at the 0.01 level (2-tailed).

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