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Antioxidant capacity, phenolics and isoflavones in soybean by-products

Article in Food Chemistry · December 2010

DOI: 10.1016/j.foodchem.2010.04.074

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 Food Chemistry 123 (2010) 583–589

Contents lists available at ScienceDirect

Food Chemistry

journalhomepage:www.elsevier.com/locate/foodchem

Antioxidant capacity, phenolics and isoflavones in soybean by-products

a a a,b,
Tan Seok Tyug , K. Nagendra Prasad , Amin Ismail *
b
a Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor, Malaysia
Laboratory of Analysis and Authentication, Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

article info abstract

Article history: This study aimed to determine proximate composition, antioxidant capacity, total phenolic content, iso-flavones and free
Received 14 January 2010 phenolic compounds in soy by-products. High carbohydrate and protein contents were found in grade A soymilk powder
Received in revised form 13 March 2010 (GASP) compared to grade B soymilk powder (GBSP) and soy husk pow-der (SHP). Ash, moisture and total dietary fibre
Accepted 27 April 2010 contents were reported to be the highest in soy husk, while GBSP had the highest fat content. Antioxidant capacity as assessed
using b-carotene bleaching assay was in the order of SHP GBSP > GASP, and the ranking order of the Trolox Equivalent
Antioxidant
Keywords: Capacity (TEAC) value was GASP GBSP > SHP, while the Ferric Reducing Antioxidant Power (FRAP) value was GASP >
Antioxidant capacity
GBSP > SHP. The total phenolic content was in the range of 62.44–103.86 mg GAE/100 g wet weight, and the major phenolic
Isoflavones
compounds in free form were ferulic, vanilic as well as gallic acids. Acid hydrolysis increased the amount of total extractable
Proximate composition
Soy by-products isoflavone in all soy samples.
2010 Elsevier Ltd. All rights reserved.

1. Introduction soybean processing industry, as soy husk represents about 8% of the entire
soybean (Mateos-Aparicio, Redondo Cuenca, Villanueva-Suárez, & Zapata-
Antioxidants are compounds that when added to lipids and li-pid- Revilla, 2008). After the removal of the soy husk, the beans are dried, ground,
containing foods, can increase their shelf-life by retarding the process of lipid sieved and cooked into slurry. Finally, the cooked slurry is subjected for
peroxidation. Synthetic antioxidants such as butyl-ated hydroxyanisole vacuum drying to obtain grade A soymilk powder. Under normal
(BHA) and butylated hydroxytoluene (BHT) have limited use in foods, as circumstances, wastes produced during the production of grade A soymilk
they are suspected to be carcinogenic (Namiki, 1990). As a result, the powder will be discarded or used as animal feed. However, in Malaysia, the
importance of exploiting natural antioxidants from by-products obtained wastes are processed to a lower-quality powder, called grade B soymilk
during food processing has increased tremendously in recent years. They are powder (GBSP).
considered to be attractive due to their low cost and availability in large quan-
tity as raw material (Kong, Amin, Tan, & Rajab, 2010). The benefi-cial effect Previous studies have reported that soybean is a rich source of isoflavones
of these by-products can be attributed to various phenolic compounds present and phenolic compounds. The primary isoflavones present in the whole
in them (Amin & Mukhrizah, 2006). These phenolic compounds include soybean include glycosides of genistin and daidzin, while syringic,
phenolic acids, anthocyanins, flavonoids, and hydrocinnamic acid derivatives. chlorogenic, gallic, and ferulic acids form the major phenolic compounds
(Kim et al., 2006). To date, little study has been carried out on the proximate
composition, antiox-idant capacity, isoflavones and phenolic compounds
Soybean (Glycine max L.) is one of the most commonly con-sumed profile for soy-bean by-products. This study thus aims to conduct these
legumes worldwide, with 200 million metric tons produced per year (FAO, analyses with a hope of exploring the potential use of soy by-products in food
2006). In Asian countries, soybean is processed into various products such as fortification and as nutraceuticals.
soymilk powder, soymilk, tofu, soy sauce, soy flour, soybean oil and tempeh.
In general, the processing of soybean into soymilk powder involves three
major steps. First, soy-beans are soaked in water to remove the inedible soy 2. Materials and methods
husk. How-ever, this might represent a major disposal problem for the
2.1. Chemicals

Analytical and HPLC-grade solvents were purchased from Fisher

* Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and
Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor, Malaysia. Tel.:
Scientific (Loughborough, UK). HPLC standards (daidzein, geni-stein,
+603 89462435; fax: +603 89426769. chlorogenic acid, syringic acid, ferulic acid, vanilic acid and gallic acid), total
E-mail address: amin@medic.upm.edu.my (A. Ismail). dietary fibre assay kit, b-carotene, linoleic acid,

0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.04.074
584 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589

Tween 20, butylated hydroxytoluene (BHT), Trolox, potassium per-sulfate, 2.6.2. Trolox Equivalent Antioxidant Capacity (TEAC) assay
sodium carbonate (Na2CO3), sodium nitrite (NaNO2), alu-minium chloride The Trolox Equivalent Antioxidant Capacity (TEAC) assay mea-sures the
hexahydrate (AlCl3 6H2O), sodium hydroxide (NaOH) were purchased from ability of antioxidants to scavenge the ABTS radical cat-ion. This assay was
+
Sigma Chemical Co (St. Louis, MO, USA). The 2, 2 0-azino-bis (3- carried out using the improved ABTS method described by Li, Wong,
+
ethylbenzthiazoline-6-sulphonic acid) (ABTS) was purchased from Merck Cheng, and Chen (2008) with slight modifi-cations. Briefly, ABTS radical
(Darmstadt, Germany). cation was generated by a reaction between 7 mmol/l ABTS and 2.45 mmol/l
potassium persulfate. The reaction mixture was allowed to stand in the dark
2.2. Preparation of samples +
for 16 h at room temperature. The resulting ABTS solution was diluted with
70% ethanol to an absorbance of 0.700 ± 0.050 at 734 nm before use. The
Grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and samples were diluted appropriately to provide 20–80% inhibition against the
soy husk powders were provided by Brilliant Point Sdn. Bhd, Kuala Lumpur, blank absorbance (around 50-fold dilution was required). Approximately 50 ll
Malaysia. The soy husk was ground into small pieces using a wet blender +
of the diluted sample was mixed with 1.9 ml of the diluted ABTS solution.
(MX-291 N, National, Osaka, Ja-pan). The ground soy husk was then passed The absorbance value was immediately read at 734 nm after 6 min of the
through a 0.05 lm sieve, and the filtrate obtained was considered soy husk reaction. Trolox at concentrations ranging from 200–1000 mM was used as a
powder (SHP). standard, and 100 lg/ml ascorbic acid was used as a positive con-trol. The
results were expressed as lmol Trolox/100 g wet weight.

2.3. Proximate analyses

The moisture and ash contents of GASP, GBSP and SHP were determined
2.6.3. b-Carotene bleaching assay
using the standard AOAC (1990) method. Total dietary fibre was analysed
The b-carotene bleaching assay was determined according to the method
using a commercialised assay kit from Sigma Chemical Co (St. Louis, MO,
described by Amin and Tan (2002). About 1 ml of the b-carotene solution
USA). The total available carbohydrate content was determined using Clegg
with a concentration of 0.2 mg/ml dissolved in chloroform was pipetted into a
Anthrone method (Clegg, 1955). The Soxhlet and Kjeldahl systems were used
50 ml round bottom flask con-taining 0.02 ml of linoleic acid and 0.2 ml of
to determine the fat and protein contents of the samples, respectively (Tee,
100% Tween 20. The mixture was subjected to evaporation by means of
Ra-jam, Young, Khor, & Zakiyah, 1996).
removing chlo-roform at 40 LC for 10 min using a rotary evaporator.
Approxi-mately 100 ml of distilled water was added, and the mixture was
shaken vigorously to form an emulsion. From this emulsion, 5 ml was
2.4. Preparation of extracts
pipetted out and transferred into different test tubes contain-ing 0.2 ml of
samples in 70% ethanol at 1 mg/ml. All test tubes were vortexed for 1 min
The soybean powders were extracted with 70% (v/v) ethanol in a ratio of
and placed at 45 LC in a water bath for 2 h. The absorbance of the samples
1:25 (g/v) and were incubated in an orbital shaker (Uni-max 1010, Heidolph
was measured at 470 nm using a spec-trophotometer at initial time (t = 0)
Instruments GmbH & Co. KG, Germany) set at 200 rpm for 2 h at 50 LC.
against a blank consisting of an emulsion without b-carotene. A standard
After the extraction, the mixture was fil-tered through Whatman paper No.
(ascorbic acid) at a con-centration of 10 mg/ml was used and 70% ethanol as
542, and the filtrate obtained was kept at 20 LC prior to analysis.
a control. The measurement was carried out at 20 min intervals. Antioxidant
activity (AA) was measured in terms of the successful bleaching of b-carotene
according to the following equation:
2.5. Determination of total phenolic content (TPC)

The total phenolic content was determined using the method of Velioglu,
Mazza, Gao, and Oomah (1998) with slight modifications. Approximately 1.5 AA ¼ ½1 A0 At Þ=ðA0 At Þ 100%;
ml of Folin–Ciocalteau reagent was added into the 0.2 ml of sample extract,
and the mixture was allowed to stand at room temperature for 5 min. Later, where A0 and A0 are the absorbance values measured at the initial incubation
1.5 ml of sodium carbonate solution (0.566 M) was added into the mixture time for the samples and control, respectively, while A t and A0 are the
and incubated at room temperature for 90 min. The absorbance was measured absorbance values measured in the samples or the standard and control at t =
at 725 nm using a spectrophotometer (Anthelie Junior Advanced 5 nm, Prim, 120 min.
SECOMAM, France), and the results were expressed as gallic acid
equivalents (GAE) in mg/100 g wet weight.
2.7. Determination of total isoflavone content

In the present study, the total isoflavone content in the soy ex-tracts was
2.6. Determination of antioxidant capacity
quantified based on the sum of the genistein and daidz-ein contents. The
daidzein and genistein contents were determined according to the method of
2.6.1. Ferric Reducing Antioxidant Power (FRAP) assay
Penalvo, Nurmi, and Adlercreutz (2004). The isoflavone content in acid-
The Ferric Reducing Antioxidant Power (FRAP) assay was esti-mated
according to the method described by Benzie and Strain (1996) with slight hydrolysed powders was compared with that in unhydrolysed powders.
modifications. Firstly, fresh FRAP reagent was prepared by mixing 2.5 ml of
10 mM TPTZ solution in 40 mM hydrochloric acid with 2.5 ml of a 20 mM
FeCl3 solution and 25 ml of a 0.3 M acetate buffer (pH 3.6). Later, 50 ll of the 2.8. Extraction of isoflavones
sample solution, 150 ll of water and 1.5 ml of the FRAP reagent were mixed.
The absorbance was read after 4 min at 593 nm using a spectrophotometer. 2.8.1. Preparation for unhydrolysed extracts
Ferrous sulphate (FeSO4 7H2O) with a concen-tration from 200–1000 lM was Intially, 1 g of the sample powder was added to 25 ml of 70% (v/
used to plot a standard curve, and ascorbic acid at a concentration of 10 lg/ml v) ethanol. The mixture was shaken vigorously for 2 min and fol-lowed by
was used as a positive control. The results were expressed as lmol Fe (II)/100 centrifugation at 2140g. After 10 min, the clear superna-tant was passed
g wet weight. through Whatman filter paper No. 4 and a 0.22-lm polytetrafluoroethylene
microfilter (Millipore, USA) before being injected into a reversed-phase
HPLC system.
 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589 585

2.8.2. Preparation for acid-hydrolysed extracts 3. Results and discussion

One gram of the sample powder was added to 25 ml of 1 M acidified 70%
ethanol. The mixture was shaken vigorously for 2 min. The mixture was then The recycling of by-products derived from various industries is crucial, as
centrifuged for 10 min at 2140g. The clear supernatant obtained was passed many studies have reported that they contain abundant health-beneficial
through a Whatman fil-ter paper No. 4 and 0.22 lm polytetrafluoroethylene compounds (Amin & Mukhrizah, 2006; Kong et al., 2009). For instance, soy
microfilter before being injected into a reversed-phase HPLC. husk derived from the soymilk industry has been proven to be an excellent
source of insoluble fi-bre (Cole et al., 1999). To the best of our knowledge,
the consump-tion of insoluble fibre helps to relieve constipation and prevent
2.8.3. Chromatographic conditions colon cancer. It would thus be beneficial if soy husk is reutilised after soymilk
The chromatographic condition used was as previously de-scribed by processing.
Hasnah, Amin, Azrina, Suzana, and Loh (2009). HPLC (Agilent 1100, Palo
Alto, CA, USA) with a diode array detector (DAD) was utilised in this study.
The reversed-phase C18 column (Nova-Pak, 150 4 mm, 5 lm) was from 3.1. Proximate composition analyses
Waters (Milford, MA, USA). The isocratic mobile phase consisted of
acetonitrile–water (33:67, v/v) at a flow rate of 0.8 ml/min. About 20 ll of the Despite years of study on soybean, there is a lack of research on the by-
sample extract was injected into the column set at room temperature (25 LC). products derived from the soymilk processing industry. Hence, the proximate
The UV–Vis absorption spectra were monitored by DAD at 200–400 nm, and compositions of GASP and GBSP were com-pared with raw soybean reported
isoflavones were detected at a wavelength of 250 nm. in the Malaysian Food Composi-tion Database (Tee, Mohd Ismail, Mohd
Nasir, & Idris, 1997). The total available carbohydrate, protein, fat, moisture,
ash and total dietary fibre contents in GASP, GBSP and SHP are depicted in
Table 1. The total available carbohydrates ranged from 11.50 ± 1.98% in SHP
to 32.79 ± 0.25% in GASP. GASP had the highest protein con-tent (22.39 ±
2.9. Identification of free phenolic compounds
0.34%), followed by GBSP (20.63 ± 0.34%) and SHP (4.72 ± 0.45%). On the
other hand, GBSP had the highest value of fat (2.82 ± 0.14%) among the
2.9.1. Extraction of free phenolic compounds
samples tested. The ash, moisture and total dietary fibre contents were
In brief, 1 g of GASP, GBSP and SHP were mixed with 25 ml of 70%
reported to be the highest in SHP, with values of 4.21 ± 0.02%, 9.95 ± 0.04%
ethanol. The mixtures were placed in an orbital shaker at 50 LC overnight in
and 74.41 ± 0.19%, respectively. A statistical analysis of One-way ANO-VA
order to extract free phenolic compounds from the samples. The supernatants
revealed that there were significant differences among all tests (p < 0.05). In
obtained were passed through a 0.22 lm polytetrafluoroethylene microfilter other words, GASP had significantly higher contents of carbohydrate and
before being injected into a reversed-phase HPLC. protein compared to GBSP and SHP. In compari-son, SHP had significantly
higher ash, moisture and total dietary fi-bre contents, whereas fat content in
GBSP was statistically higher than in GASP and SHP.
2.9.2. Chromatographic conditions
The individual free phenolic compounds in soy extracts were quantified
using the HPLC method described by He and Xia (2007). A reversed-phase
Lichrospher C18 column (250 4 mm, I.D. 5 lm, Merck, Darmstadt, Germany) The soymilk powders in the present study were lower in crude fat and
was utilised for the separa-tion. A gradient elution was performed with 0.5% protein but had higher fibre contents compared with the reported raw
(v/v) acetic acid (solvent A) and 100% methanol (solvent B) at a constant soybean. Carbohydrate and ash in the studied soy-milk powders were similar
flow rate of 0.6 ml/min. The linear gradient profile was set as follows: 100% to raw soybean. Riaz (2006) reported that dried soy husk contained
A and 0% B at start, 10% A and 90% B at 20–25 min, and back to 100% A approximately 8% moisture, 86% car-bohydrate, 9% protein, 4% ash, 1%
and 0% B at 30 min. Twenty microlitre of the sample extract was injected into lipid. Fibre content of 76% was also reported in soy husk (Anonymous,
the column set at room temperature (25 LC). The UV–Vis absorption spectra 1987), which is in good agreement with our results. The studied SHP had
were monitored by DAD at 200– 600 nm, and phenolics were detected at the similar contents of ash, moisture, and fat but showed a lower carbohydrate
wavelength of 280 nm. content. This might probably be due to the fact that Riaz (2006) reported a
total carbohydrate content that included both available and non available
carbohydrates (fibre).
2.9.3. Standard calibration curve
The standard of the respective phenolic compounds (syringic, chlorogenic,
gallic, vanilic and ferulic acids) and isoflavones (geni-stein and daidzein)
were dissolved in 70% ethanol (v/v). The cali-bration curves of the phenolic Table 1
compounds were plotted at five different concentrations ranging from 20 to Proximate composition of grade A soymilk powder (GASP), grade B soymilk powder (GBSP)
and soy husk powder (SHP).
100 lg/ml, while the calibration curves of genistein and daidzein were plotted
in the range of 0–150 lm. Analyses (%) Composition
A B C
GASP GBSP SHP
Ash 3.37 ± 0.16a 3.77 ± 0.01b 4.21 ± 0.02c
2.10. Statistical analysis Moisture 3.77 ± 0.03a 2.95 ± 0.07b 9.95 ± 0.04c
Carbohydrate 32.79 ± 0.25a 23.70 ± 1.42b 11.50 ± 1.98c
D
Protein 22.39 ± 0.34a 20.63 ± 0.34b 4.72 ± 0.45c
The results were analysed using the Statistical Package for the Social Fat 0.27 ± 0.01a 2.82 ± 0.14b 1.69 ± 0.05c
Sciences (SPSS version 16). Data were expressed as means ± standard Total dietary fibre 13.90 ± 0.17a 22.78 ± 0.23b 74.41 ± 0.19c
deviation (SD) of triplicate determinations. Inde-pendent t-test, ANOVA and
Different letters in the same row indicate significant difference at the level of p < 0.05.
Tukey tests were used to determine the mean differences. A Pearson
correlation test was used to evaluate the correlation between the antioxidant A Grade A soymilk powder.
B

capacity and the phenolic compounds. A p-value of less than 0.05 was Grade B soymilk powder.
C
Soy
considered to be statis-tically significant. husk powder.
D
Conversion factor (5.71).
586 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589

The ash content is an indicator of the total mineral content in food from three different varieties. Compared to the previous study, the FRAP
(Hasnah et al., 2009). Based on the present study, it is re-flected that the value of GASP and GBSP fall in the range reported. This assay involves a
removal of soy husk during the processing of soy-milk powder has led to a single-electron transfer by treating antioxidants as reductants in a redox-
significant loss in minerals and fibre. The differences in proximate linked colorimetric reaction (Prior, Wu,
composition could be attributed to many factors, including agronomic & Schaich, 2005). In other words, it measures the tendency of anti-oxidants to
practices, the genotype and the grow-ing location of the soybean. Boydak, give up a single electron to the FRAP reagent. The re-sults in the present
Alpaslan, Hayta, Gercek, and Simsek (2002) found that agronomic practices, study showed that GASP had the highest tendency to transfer electrons among
such as row spacing and irrigation are able to affect the protein content and the extracts tested.
the fatty acid composition of soybean. A recent study conducted by Bhard-
waj, Hamama, Rangappa, Joshi, and Sapra (2007) also demonstrate that the
3.3.2. TEAC assay
genotype and growing location of soybean could have sig-nificant effects on
In the TEAC assay, the antioxidants act as hydrogen donors to terminate
soymilk protein and the fatty acid composition of tofu, respectively.
the oxidation process (Tachakittirungrod & Okonogi, 2006). It is clearly
demonstrated that ascorbic acid is more prone to donate a hydrogen atom to
the ABTS cation radical, giving rise to the highest scavenging activity among
extracts (1866.9 ± 17.1 lmol Trolox/100 g wet weight). GASP and GBSP had
3.2. Total phenolic content (TPC)
compara-ble scavenging abilities. On the other hand, SHP (631.90 ± 6.24 lmol
Trolox/100 g wet weight) had the lowest tendency to act as a hydrogen donor
The ability of phenolic compounds in reducing Folin–Ciocalteau (FC)
among the extracts studied.
reagent is quantified using a colorimetric method. The results in the present
study demonstrate that GBSP had the most reducing activity toward FC
The ABTS radical-scavenging activity of soymilk powder and soy husk is
reagent. However, it is important to note that the presence of reducing agents
not well documented in previous studies. However, when compared with the
such as ascorbic acid and sugars may interfere with this assay, as these
TEAC value of raw soybean (6303 lmol/100 dry matter) reported by
compounds are also able to reduce FC reagent. Of the extracts, GBSP had the
Fernandez-Orozco et al. (2007), it is found that GASP, GBSP and SHP had
highest TPC (103.86 ± 5.29 mg GAE/100 g wet weight), followed by GASP
less ability in scavenging the ABTS cation radical. This phenomenon
(96.31 ± 3.06 mg GAE/100 g wet weight) and SHP (62.44 ± 3.65 mg
indicates that the processing of soybean into soymilk may bring a significant
GAE/100 g wet weight). The TPC of SHP was statistically lower than GASP
loss of antioxidant compounds.
and GBSP. On the other hand, differences were not observed between the
TPC of GASP and GBSP. In other words, GASP and GBSP had comparable
total phenolic content.
3.3.3. b-Carotene bleaching assay
The b-carotene bleaching assay tests the ability of extracts to neutralise
3.3. Antioxidant capacity free radicals. Fig. 1 indicates the degradation rate for soy powder extracts, the
control and the standard. There was a de-crease in the absorbance value of b-
Different antioxidant compounds may act through different mechanisms; carotene in the absence of sam-ples due to the oxidation of b-carotene and
consequently, one method alone cannot be utilised to fully evaluate the linoleic acid. As shown in the figure, all soy extracts had degradation rates
antioxidant capacity of foods (Pellegrini et al., 2003). For this reason, three higher than ascorbic acid, with SHP extract being the most degraded,
antioxidant capacity tests with different approaches and mechanisms have followed by GBSP and GASP extracts.
been carried out.
The above results are also supported by their respective antiox-idant
3.3.1. FRAP assay activity (Table 2), with the highest value found for the SHP extract (62.74 ±
GASP had a higher FRAP value [825.71 ± 70.18 lmol Fe (II)/ 100 g wet 2.33%) and the lowest found for the GASP extract (52.32 ± 3.76%). Based on
weight] than ascorbic acid [648.22 ± 36.99 lmol Fe (II)/ 100 g wet weight]. the results obtained, it is revealed that the SHP extract had the greatest ability
The FRAP value of GBSP was not signifi-cantly different from ascorbic acid, to neutralise free radicals. Although the SHP extract had the highest value, no
indicating that GBSP had a sim-ilar reducing power as ascorbic acid. Xu and significant dif-ference (p < 0.05) was observed between the antioxidant
Chang (2009) report a FRAP value range of 640–1160 lmol Fe(II)/100 g in activity of the SHP and GBSP extracts.
raw soymilk

0.27
0.22

0.17
Absorbance at 470 nm

0.12

0.07

0.02

0 20 40 60 80 100 120 140

Time (min)

Control Ascorbic acid Grade A soymilk powder Grade B soymilk powder Soy husk

Fig. 1. Degradation rate in absorbance of soy powders and ascorbic acid at a concentration of 1 mg/ml using b -carotene bleaching assay.
 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589 587

Table 2
Total phenolic content and antioxidant capacity of grade A soymilk powder (GASP), grade B
soymilk powder (GBSP) and soy husk powder (SHP).

Analyses GASP GBSP SHP

A 96.31± 3.06a 103.86 ± 5.29a 62.44 ± 3.65b
TPC
B
FRAP 825.71± 70.18a 653.10 ± 16.65b 340.12 ± 9.43c
C
TEAC 1009.50± 88.62a 1022.90 ± 56.38a 631.90 ± 6.24b
D 52.32± 3.76c 61.82 ± 1.72b 62.74 ± 2.33a
BCB

Different letters in the same row indicate significant difference at a level of p < 0.05.
A Total phenolic content in mg GAE/100 g wet weight.
B Ferric Reducing Antioxidant Power in lmol Fe(II)/100 g wet weight.
C Trolox Equivalent Antioxidant Capacity in lmol Trolox/100 g wet weight. D b-
carotene bleaching assay in %.

3.3.4. Comparison of FRAP, TEAC and b-carotene bleaching assays The

three antioxidant assays used in the present study can be
differentiated by their hydrophilic and lipophilic properties. The FRAP assay
is limited to water-soluble antioxidants (hydrophilic antioxidants), while the
b-carotene bleaching assay is suitable for determining fat-soluble antioxidants
(lipophilic antioxidants). The TEAC assay has an advantage over both
hydrophilic and lipo-philic antioxidants, as the reagent is soluble in aqueous
and sol-vents (Apak et al., 2007). The assessment of the ranking order of
Fig. 2b. Daidzein and genistein contents after acid hydrolysis. Different letters indicate
FRAP, TEAC and b-carotene bleaching assays, indicates that GASP and SHP
significant difference at the level of p < 0.05.
contain higher amounts of hydrophilic and lipophilic anti-oxidants,
respectively. Further study of the correlations among antioxidant assays
revealed a significant and positive correlation obtained between the FRAP As only aglycones are able to be absorbed by the human digestive system,
and TEAC assays, while no correlation was observed between the b-carotene studies have been extensively carried out in an effort to transform glycosides
bleaching and TEAC assays. These two correlations indicate that hydrophilic into aglycones. Among the methods utilised, mild acid hydrolysis has been
antioxidants and not lipophilic antioxidants were the major contributors of proven to be the best choice for com-pletely hydrolysing glycosides into their
TEAC values. corresponding aglycones (Penalvo et al., 2004). Therefore, a pre-treatment of
the studied samples with mild acid was carried out in the present study.

The daidzein and genistein contents of soy extracts without hydrolysis are
shown in Fig. 2a. The GASP extract had significant higher daidzein (3.13 ±
3.4. Total isoflavone
0.15 mg/100 g wet weight) and genistein (0.84 ± 0.31 mg/100 g wet weight)
contents compared to GBSP and SHP extracts, SHP extract exhibited the least
Naim, Gestetner, and Zilkah (1974) report the existence of iso-flavones in
daidzein content, represented by the value of 1.17 ± 0.03 mg daidzein/100 g
soybean and non fermented soybean products (such as soy protein and wet weight in the respective sample, and genistein was not detected in SHP
soymilk derivates) in the form of glycosides. throughout the study. The addition of 1 M acid to the extraction solvent has
promoted the extractability of the isoflav-ones daidzein and genistein from all
soy extracts. As shown in Fig. 2b, the SHP extract showed a significantly
higher daidzein con-tent (19.02 ± 0.39 mg/100 g wet weight) following the
acid hydro-lysis. The GASP extract had higher daidzein and genistein
contents compared to the GBSP extract. However, the statistical result of
One-way ANOVA analysis revealed that the difference was not sig-nificant,
revealing that acid-hydrolysed GASP and GBSP contain al-most identical
amounts of daidzein and genistein.

Both unhydrolysed and acid-hydrolysed soymilk extracts con-tained lower

unbound daidzein and genistein contents compared to a study conducted by
Hutabarat, Greenfield, and Mulholland (2001). The variation in the daidzein
and genistein contents be-tween soymilk powders could be attributed to the
variety of soy-beans, techniques to produce soymilk powder and differences
in the analytical procedure used to analyse isoflavones (Hutabarat et al., 2001;
Hasnah et al., 2009). The total isoflavone content in acid-hydrolysed soy
powders were much higher than unhydroly-sed ones. It reflects that the acidic
condition favours the conversion of glycosides into aglycones, causing a
significant increase of daidz-ein and genistein levels. For soy husk, genistein
was not detected in either condition; however, there was a significant increase
in the daidzein content following the hydrolysis process. It is thus sug-gested
that a mild acid treatment may release the fibre-bound daidzein from the soy
husk, yielding a significant increase in the daidzein content.

Fig. 2a. Daidzein and genistein contents without hydrolysis. Different letters indicate significant
difference at the level of p < 0.05.
588 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589

Table 3 FRAP assay tends to show a strong positive correlation with chlor-ogenic acid
Individual free phenolic acid composition in grade A soymilk powder (GASP), grade B soymilk (r = 0.984, p < 0.01), daidzein (r = 0.951, p < 0.01) and genistein (r = 0.975, p
powder (GBSP) and soy husk powder (SHP).
< 0.01). On the other hand, chlorogenic acid (r = 0.775) and genistein (r =
Free phenolic acids (mg/ GASP GBSP SHP 0.796) appear to have a negative correlation with the b-carotene bleaching
100 g wet weight) assay (p < 0.05). All of the individual free phenolic compounds and
Chlorogenic acid 32.16 ± 0.05a 25.71 ± 0.03b 16.75 ± 0.03c isoflavones have a strong correlation with the total phenolic content (TPC)
Ferulic acid 294.65 ± 0.01a 310.84 ± 0.01b 259.56 ± 0.01c
Gallic acid 99.81 ± 0.03a 85.40 ± 0.06b 108.34 ± 0.01c
(p < 0.01). Vanilic acid (r = 0.972), ferulic acid (r = 0.973), and daidzein (r =
Syringic acid 18.67 ± 0.01a 18.75 ± 0.01b 20.83 ± 0.01c
Vanilic acid 191.19 ± 0.13a 235.76 ± 0.02b 97.24 ± 0.10c 0.921) were positively correlated with TPC. Addition-ally, the measuring of
the correlation between TPC and antioxidant capacity was also carried out. A
Different letters in the same row indicate significant difference at a level of p < 0.05. strong and positive correlation (p < 0.01) was observed between TPC and
FRAP (r = 0.850) as well as between TPC and TEAC (r = 0950).

3.5. Phenolic compounds analysis The FRAP assay measures the chain-breaking potential, and TEAC
measures the ability to scavenge the ABTS radical cation, while the b-
Referring to Table 3, it is clearly shown that ferulic acid is the
carotene bleaching assay measures the ability to neu-tralise the free radicals.
predominant phenolic compound identified in free form in all ex-tracts.
Based on the Pearson correlation tests, it is clearly demonstrated that vanilic
Among the extracts, GASP had the highest amount of chlor-ogenic acid
acid, ferulic acid, chlorogenic acid, daidzein and genistein are the potential
(32.16 ± 0.05 mg/100 g wet weight) whereas GBSP had the highest amount of
chain breakers and ABTS the radical cation scavenger. Chlorogenic acid and
vanilic (235.76 ± 0.02 mg/100 g wet weight) and ferulic acids (310.84 ± 0.01
genistein showed a negative correlation with the b-carotene bleaching assay,
mg/100 g wet weight). Syringic and gallic acids were found to be highest in
which indicates that they are a weak neutralizer of free radicals. Furthermore,
SHP. The phenolic com-pounds identified in free form in all extracts were
the present results have also shown that vanilic acid, ferulic acid, chlorogenic
significantly dif-ferent from each other at the level of p < 0.05.
acid, daidzein and genistein contribute the most to the value of TPC. In other
words, they have a strong reduc-ing ability toward the FC reagent.
Kim et al. (2006) report that soybean contains chlorogenic, feru-lic,
syringic, gallic and vanilic acids. Soymilk powder, which origi-nates from the
soybean, might also have a significant amount of these phenolic compounds.
The FRAP and TEAC assays exhibited a high positive correlation (r =
Compared to GASP and GBSP, gallic and syringic acid are predominantly
0.916, p < 0.01), suggesting that the two assays are recom-mendable for
found in the soy husk; the re-moval of the soy husk has thus led to a loss of
evaluating antioxidant capacity in soy extracts. The correlation obtained is in
valuable free forms of phenolic compounds.
agreement with those reported by Moon, Lee, Lee, and Trakoontivakorn
(2002) in soy sauce. No significant correlation has been observed between
TEAC and b-carotene bleaching assays, whereas a significant and moderate
3.6. Correlations among individual free phenolic compounds, negative cor-relation (r = 0.686, p < 0.05) was demonstrated between the
isoflavones, TPC and antioxidant capacity FRAP and b-carotene bleaching assays.

Correlations among antioxidant capacity (TEAC, FRAP and b-carotene

bleaching assays), individual free phenolic compounds and isoflavones are
statistically analysed and depicted in Table 4. A strong and positive 4. Conclusions
correlation (p < 0.01) existed between TEAC and vanilic acid (r = 0.924),
ferulic acid (r = 0.926), chlorogenic acid (r = 0.865), daidzein (r = 0.936) and Although GBSP and SHP are the by-products derived from the soybean
genistein (r = 0.849). Also, the processing industry, the present study has shown that

Table 4
Correlations among free phenolic acids, total phenolic content and antioxidant capacity.

Correlations B C D E F G H I J K
VA SA FA CA Da Ge TPC FRAP TEAC BCB
A ** * ** 0.453 * 0.440 ** 0.508 * 0.060
GA 0.939 0.763 0.937 0.675 0.863 0.773
B ** ** * ** * ** * **
VA 0.938 1.000 0.731 0.885 0.721 0.972 0.767 0.924 0.252
C ** ** ** ** ** ** **
SA 0.941 0.922 0.989 0.916 0.962 0.934 0.962 0.535
D * ** * ** * **
FA 0.736 0.888 0.726 0.973 0.771 0.926 0.258
E ** ** ** ** ** *
CA 0.959 0.999 0.810 0.984 0.865 0.775
F ** ** ** **
Da 0.957 0.921 0.951 0.936 0.640
G ** ** ** *
Ge 0.798 0.975 0.849 0.796
H ** **
TPC 0.850 0.950 0.353
I ** *
FRAP 0.916 0.686
J 0.437
TEAC
* Correlation is significant at the level of p < 0.05.
* Correlation is significant at the level of p < 0.01.
A

Gallic acid.
B C
Vanilic acid.
D
Syringic acid.
Ferulic acid.
E F
Chlorogenic acid.
Daidzein.
G
Genistein.
H
Total phenolic content.
I
Ferric Reducing Antioxidant Power.
J K
Trolox Equivalent Antioxidant Capacity. b-
carotene bleaching.
 T.S. Tyug et al. / Food Chemistry 123 (2010) 583–589 589

these powders have an antioxidant capacity comparable with that of GASP. He, Z., & Xia, W. (2007). Analysis of phenolic compounds in Chinese olive (Canarium album
L.) fruit by RPHPLC-DAD-ESI-MS. Food Chemistry, 105, 1307–1311.
This study also revealed similar isoflavone content be-tween GASP and
Hutabarat, L. S., Greenfield, H., & Mulholland, H. (2001). Isoflavones and coumestrol in
GBSP. Furthermore, it also proved that soy by-products contain a significant soybeans and soybean products from Australia and Indonesia. Journal of Food Composition
amount of phenolics. Therefore, an in-depth study can be carried out in the and Analysis, 13, 43–58.
Kim, J. A., Jung, W. S., Chun, S. C., Yu, C. Y., Ma, K. H., Gwag, J. G., et al. (2006). A
future for exploring the possibility of using soy by-products to develop
correlation between the level of phenolic compounds and the antioxidant capacity in
health-beneficial nutraceuticals. cooked-with-rice and vegetable soybean (Glycine max L.) varieties. European Food
Research and Technology, 224, 259–270.
Kong, K. W., Amin, I., Tan, C. P., & Rajab, N. F. (2010). Optimization of oven drying
conditions for lycopene content and lipophilic antioxidant capacity in a by-product of the
Acknowledgements pink guava puree industry using response surface methodology. LWT – Food Science and
Technology, 43(5), 729–735.
We would like to acknowledge Brilliant Point Sdn. Bhd, Kuala Lumpur, Li, H. B., Wong, C. C., Cheng, K. W., & Chen, F. (2008). Antioxidant properties in vitro and
total phenolic contents in methanol extracts from medicinal plants. LWT – Food Science
Malaysia for their generosity in providing samples for this study. We also like and Technology, 41, 385–390.
to extend our thanks to laboratory staff from the Department of Nutrition and Mateos-Aparicio, I., Redondo Cuenca, A., Villanueva-Suárez, M. J., & Zapata-Revilla, M. A.
Dietetics at UPM for their guid-ance and assistance. (2008). Soybean, a promising health source. Nutricion Hospitalaria, 23, 305–312.

Moon, G., Lee, M., Lee, Y., & Trakoontivakorn, G. (2002). Main component of soy sauce
representing antioxidative activity. International Congress Series, 1245, 509–510.
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