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Antioxidant capacity, phenolics and isoflavones in soybean by-products
Food Chemistry 123 (2010) 583–589
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Antioxidant capacity, phenolics and isoflavones in soybean by-products Tan Seok Tyug a, K. Nagendra Prasad a, Amin Ismail a,b,* a b
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
a r t i c l e
i n f o
Article history: Received 14 January 2010 Received in revised form 13 March 2010 Accepted 27 April 2010
Keywords: Antioxidant capacity Isoflavones Proximate composition Soy by-products
a b s t r a c t This study aimed to determine proximate composition, antioxidant capacity, total phenolic content, isoflavones and free phenolic compounds in soy by-products. High carbohydrate and protein contents were found in grade A soymilk powder (GASP) compared to grade B soymilk powder (GBSP) and soy husk powder (SHP). Ash, moisture and total dietary fibre 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 Capacity (TEAC) value was GASP GBSP > SHP, while the Ferric Reducing Antioxidant Power (FRAP) value was GASP > 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 compounds in free form were ferulic, vanilic as well as gallic acids. Acid hydrolysis increased the amount of total extractable isoflavone in all soy samples. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Antioxidants are compounds that when added to lipids and lipid-containing foods, can increase their shelf-life by retarding the process of lipid peroxidation. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have limited use in foods, as they are suspected to be carcinogenic (Namiki, 1990). As a result, the importance of exploiting natural antioxidants from by-products obtained during food processing has increased tremendously in recent years. They are considered to be attractive due to their low cost and availability in large quantity as raw material (Kong, Amin, Tan, & Rajab, 2010). The beneficial effect of these by-products can be attributed to various phenolic compounds present in them (Amin & Mukhrizah, 2006). These phenolic compounds include phenolic acids, anthocyanins, flavonoids, and hydrocinnamic acid derivatives. Soybean (Glycine max L.) is one of the most commonly consumed legumes worldwide, with 200 million metric tons produced per year (FAO, 2006). In Asian countries, soybean is processed into various products such as 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, soybeans are soaked in water to remove the inedible soy husk. However, this might represent a major disposal problem for the
* Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor, Malaysia. Tel.: +603 89462435; fax: +603 89426769. E-mail address: [email protected] (A. Ismail). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.074
soybean processing industry, as soy husk represents about 8% of the entire soybean (Mateos-Aparicio, Redondo Cuenca, Villanueva-Suárez, & Zapata-Revilla, 2008). After the removal of the soy husk, the beans are dried, ground, sieved and cooked into slurry. Finally, the cooked slurry is subjected for vacuum drying to obtain grade A soymilk powder. Under normal circumstances, wastes produced during the production of grade A soymilk powder will be discarded or used as animal feed. However, in Malaysia, the wastes are processed to a lower-quality powder, called grade B soymilk powder (GBSP). Previous studies have reported that soybean is a rich source of isoflavones and phenolic compounds. The primary isoflavones present in the whole soybean include glycosides of genistin and daidzin, while syringic, 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, antioxidant capacity, isoflavones and phenolic compounds profile for soybean by-products. This study thus aims to conduct these analyses with a hope of exploring the potential use of soy by-products in food fortification and as nutraceuticals. 2. Materials and methods 2.1. Chemicals Analytical and HPLC-grade solvents were purchased from Fisher Scientific (Loughborough, UK). HPLC standards (daidzein, genistein, chlorogenic acid, syringic acid, ferulic acid, vanilic acid and gallic acid), total dietary fibre assay kit, b-carotene, linoleic acid,
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Tween 20, butylated hydroxytoluene (BHT), Trolox, potassium persulfate, sodium carbonate (Na2CO3), sodium nitrite (NaNO2), aluminium chloride hexahydrate (AlCl36H2O), sodium hydroxide (NaOH) were purchased from Sigma Chemical Co (St. Louis, MO, USA). The 2, 20 -azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was purchased from Merck (Darmstadt, Germany). 2.2. Preparation of samples Grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powders were provided by Brilliant Point Sdn. Bhd, Kuala Lumpur, Malaysia. The soy husk was ground into small pieces using a wet blender (MX-291 N, National, Osaka, Japan). The ground soy husk was then passed through a 0.05 lm sieve, and the filtrate obtained was considered soy husk powder (SHP). 2.3. Proximate analyses The moisture and ash contents of GASP, GBSP and SHP were determined using the standard AOAC (1990) method. Total dietary fibre was analysed using a commercialised assay kit from Sigma Chemical Co (St. Louis, MO, USA). The total available carbohydrate content was determined using Clegg Anthrone method (Clegg, 1955). The Soxhlet and Kjeldahl systems were used to determine the fat and protein contents of the samples, respectively (Tee, Rajam, Young, Khor, & Zakiyah, 1996). 2.4. Preparation of extracts The soybean powders were extracted with 70% (v/v) ethanol in a ratio of 1:25 (g/v) and were incubated in an orbital shaker (Unimax 1010, Heidolph Instruments GmbH & Co. KG, Germany) set at 200 rpm for 2 h at 50 °C. After the extraction, the mixture was filtered through Whatman paper No. 542, and the filtrate obtained was kept at 20 °C prior to analysis. 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 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, 1.5 ml of sodium carbonate solution (0.566 M) was added into the mixture and incubated at room temperature for 90 min. The absorbance was measured at 725 nm using a spectrophotometer (Anthelie Junior Advanced 5 nm, Prim, SECOMAM, France), and the results were expressed as gallic acid equivalents (GAE) in mg/100 g wet weight. 2.6. Determination of antioxidant capacity 2.6.1. Ferric Reducing Antioxidant Power (FRAP) assay The Ferric Reducing Antioxidant Power (FRAP) assay was estimated according to the method described by Benzie and Strain (1996) with slight 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 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. Ferrous sulphate (FeSO47H2O) with a concentration from 200–1000 lM was used to plot a standard curve, and ascorbic acid at a concentration of 10 lg/ml was used as a positive control. The results were expressed as lmol Fe (II)/100 g wet weight.
2.6.2. Trolox Equivalent Antioxidant Capacity (TEAC) assay The Trolox Equivalent Antioxidant Capacity (TEAC) assay measures the ability of antioxidants to scavenge the ABTS radical cation. This assay was carried out using the improved ABTS+ method described by Li, Wong, Cheng, and Chen (2008) with slight modifications. Briefly, ABTS+ radical 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 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 samples were diluted appropriately to provide 20–80% inhibition against the blank absorbance (around 50-fold dilution was required). Approximately 50 ll of the diluted sample was mixed with 1.9 ml of the diluted ABTS+ solution. The absorbance value was immediately read at 734 nm after 6 min of the reaction. Trolox at concentrations ranging from 200–1000 mM was used as a standard, and 100 lg/ml ascorbic acid was used as a positive control. The results were expressed as lmol Trolox/100 g wet weight. 2.6.3. b-Carotene bleaching assay The b-carotene bleaching assay was determined according to the method described by Amin and Tan (2002). About 1 ml of the b-carotene solution with a concentration of 0.2 mg/ml dissolved in chloroform was pipetted into a 50 ml round bottom flask containing 0.02 ml of linoleic acid and 0.2 ml of 100% Tween 20. The mixture was subjected to evaporation by means of removing chloroform at 40 °C for 10 min using a rotary evaporator. Approximately 100 ml of distilled water was added, and the mixture was shaken vigorously to form an emulsion. From this emulsion, 5 ml was pipetted out and transferred into different test tubes containing 0.2 ml of samples in 70% ethanol at 1 mg/ml. All test tubes were vortexed for 1 min and placed at 45 °C in a water bath for 2 h. The absorbance of the samples was measured at 470 nm using a spectrophotometer at initial time (t = 0) against a blank consisting of an emulsion without b-carotene. A standard (ascorbic acid) at a concentration of 10 mg/ml was used and 70% ethanol as 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:
AA ¼ ½1 ðA0 At Þ=ðA0 At Þ 100%; where A0 and A0 are the absorbance values measured at the initial incubation time for the samples and control, respectively, while At and A0 are the absorbance values measured in the samples or the standard and control at t = 120 min. 2.7. Determination of total isoflavone content In the present study, the total isoflavone content in the soy extracts was quantified based on the sum of the genistein and daidzein contents. The daidzein and genistein contents were determined according to the method of Penalvo, Nurmi, and Adlercreutz (2004). The isoflavone content in acid-hydrolysed powders was compared with that in unhydrolysed powders. 2.8. Extraction of isoflavones 2.8.1. Preparation for unhydrolysed extracts Intially, 1 g of the sample powder was added to 25 ml of 70% (v/ v) ethanol. The mixture was shaken vigorously for 2 min and followed by centrifugation at 2140g. After 10 min, the clear supernatant was passed through Whatman filter paper No. 4 and a 0.22lm polytetrafluoroethylene microfilter (Millipore, USA) before being injected into a reversed-phase HPLC system.
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2.8.2. Preparation for acid-hydrolysed extracts 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 centrifuged for 10 min at 2140g. The clear supernatant obtained was passed through a Whatman filter paper No. 4 and 0.22 lm polytetrafluoroethylene microfilter before being injected into a reversed-phase HPLC. 2.8.3. Chromatographic conditions The chromatographic condition used was as previously described by 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 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 sample extract was injected into the column set at room temperature (25 °C). The UV–Vis absorption spectra were monitored by DAD at 200–400 nm, and isoflavones were detected at a wavelength of 250 nm. 2.9. Identification of free phenolic compounds 2.9.1. Extraction of free phenolic compounds In brief, 1 g of GASP, GBSP and SHP were mixed with 25 ml of 70% ethanol. The mixtures were placed in an orbital shaker at 50 °C overnight in order to extract free phenolic compounds from the samples. The supernatants obtained were passed through a 0.22 lm polytetrafluoroethylene microfilter before being injected into a reversed-phase HPLC. 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) was utilised for the separation. A gradient elution was performed with 0.5% (v/v) acetic acid (solvent A) and 100% methanol (solvent B) at a constant flow rate of 0.6 ml/min. The linear gradient profile was set as follows: 100% A and 0% B at start, 10% A and 90% B at 20–25 min, and back to 100% A and 0% B at 30 min. Twenty microlitre of the sample extract was injected into the column set at room temperature (25 °C). The UV–Vis absorption spectra were monitored by DAD at 200– 600 nm, and phenolics were detected at the wavelength of 280 nm. 2.9.3. Standard calibration curve The standard of the respective phenolic compounds (syringic, chlorogenic, gallic, vanilic and ferulic acids) and isoflavones (genistein and daidzein) were dissolved in 70% ethanol (v/v). The calibration curves of the phenolic compounds were plotted at five different concentrations ranging from 20 to 100 lg/ml, while the calibration curves of genistein and daidzein were plotted in the range of 0–150 lm. 2.10. Statistical analysis The results were analysed using the Statistical Package for the Social Sciences (SPSS version 16). Data were expressed as means ± standard deviation (SD) of triplicate determinations. Independent t-test, ANOVA and Tukey tests were used to determine the mean differences. A Pearson correlation test was used to evaluate the correlation between the antioxidant capacity and the phenolic compounds. A p-value of less than 0.05 was considered to be statistically significant.
3. Results and discussion The recycling of by-products derived from various industries is crucial, as many studies have reported that they contain abundant health-beneficial compounds (Amin & Mukhrizah, 2006; Kong et al., 2009). For instance, soy husk derived from the soymilk industry has been proven to be an excellent source of insoluble fibre (Cole et al., 1999). To the best of our knowledge, the consumption of insoluble fibre helps to relieve constipation and prevent colon cancer. It would thus be beneficial if soy husk is reutilised after soymilk processing. 3.1. Proximate composition analyses Despite years of study on soybean, there is a lack of research on the by-products derived from the soymilk processing industry. Hence, the proximate compositions of GASP and GBSP were compared with raw soybean reported in the Malaysian Food Composition 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 content (22.39 ± 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 samples tested. The ash, moisture and total dietary fibre contents were reported to be the highest in SHP, with values of 4.21 ± 0.02%, 9.95 ± 0.04% and 74.41 ± 0.19%, respectively. A statistical analysis of One-way ANOVA revealed that there were significant differences among all tests (p < 0.05). In other words, GASP had significantly higher contents of carbohydrate and protein compared to GBSP and SHP. In comparison, SHP had significantly higher ash, moisture and total dietary fibre contents, whereas fat content in GBSP was statistically higher than in GASP and SHP. The soymilk powders in the present study were lower in crude fat and protein but had higher fibre contents compared with the reported raw soybean. Carbohydrate and ash in the studied soymilk powders were similar to raw soybean. Riaz (2006) reported that dried soy husk contained approximately 8% moisture, 86% carbohydrate, 9% protein, 4% ash, 1% lipid. Fibre content of 76% was also reported in soy husk (Anonymous, 1987), which is in good agreement with our results. The studied SHP had similar contents of ash, moisture, and fat but showed a lower carbohydrate 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).
Table 1 Proximate composition of grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powder (SHP). Analyses (%)
Ash Moisture Carbohydrate ProteinD Fat Total dietary fibre
Composition GASPA
GBSPB
SHPC
3.37 ± 0.16a 3.77 ± 0.03a 32.79 ± 0.25a 22.39 ± 0.34a 0.27 ± 0.01a 13.90 ± 0.17a
3.77 ± 0.01b 2.95 ± 0.07b 23.70 ± 1.42b 20.63 ± 0.34b 2.82 ± 0.14b 22.78 ± 0.23b
4.21 ± 0.02c 9.95 ± 0.04c 11.50 ± 1.98c 4.72 ± 0.45c 1.69 ± 0.05c 74.41 ± 0.19c
Different letters in the same row indicate significant difference at the level of p < 0.05. A Grade A soymilk powder. B Grade B soymilk powder. C Soy husk powder. D Conversion factor (5.71).
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from three different varieties. Compared to the previous study, the FRAP value of GASP and GBSP fall in the range reported. This assay involves a single-electron transfer by treating antioxidants as reductants in a redox-linked colorimetric reaction (Prior, Wu, & Schaich, 2005). In other words, it measures the tendency of antioxidants to give up a single electron to the FRAP reagent. The results in the present study showed that GASP had the highest tendency to transfer electrons among the extracts tested.
The ash content is an indicator of the total mineral content in food (Hasnah et al., 2009). Based on the present study, it is reflected that the removal of soy husk during the processing of soymilk powder has led to a significant loss in minerals and fibre. The differences in proximate composition could be attributed to many factors, including agronomic practices, the genotype and the growing location of the soybean. Boydak, Alpaslan, Hayta, Gercek, and Simsek (2002) found that agronomic practices, such as row spacing and irrigation are able to affect the protein content and the fatty acid composition of soybean. A recent study conducted by Bhardwaj, Hamama, Rangappa, Joshi, and Sapra (2007) also demonstrate that the genotype and growing location of soybean could have significant effects on soymilk protein and the fatty acid composition of tofu, respectively.
3.3.2. TEAC assay In the TEAC assay, the antioxidants act as hydrogen donors to terminate 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 comparable 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 among the extracts studied. The ABTS radical-scavenging activity of soymilk powder and soy husk is not well documented in previous studies. However, when compared with the TEAC value of raw soybean (6303 lmol/100 dry matter) reported by Fernandez-Orozco et al. (2007), it is found that GASP, GBSP and SHP had less ability in scavenging the ABTS cation radical. This phenomenon indicates that the processing of soybean into soymilk may bring a significant loss of antioxidant compounds.
3.2. Total phenolic content (TPC) The ability of phenolic compounds in reducing Folin–Ciocalteau (FC) reagent is quantified using a colorimetric method. The results in the present study demonstrate that GBSP had the most reducing activity toward FC reagent. However, it is important to note that the presence of reducing agents such as ascorbic acid and sugars may interfere with this assay, as these compounds are also able to reduce FC reagent. Of the extracts, GBSP had the highest TPC (103.86 ± 5.29 mg GAE/100 g wet weight), followed by GASP (96.31 ± 3.06 mg GAE/100 g wet weight) and SHP (62.44 ± 3.65 mg GAE/100 g wet weight). The TPC of SHP was statistically lower than GASP 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 free radicals. Fig. 1 indicates the degradation rate for soy powder extracts, the control and the standard. There was a decrease in the absorbance value of b-carotene in the absence of samples due to the oxidation of b-carotene and linoleic acid. As shown in the figure, all soy extracts had degradation rates higher than ascorbic acid, with SHP extract being the most degraded, followed by GBSP and GASP extracts. The above results are also supported by their respective antioxidant activity (Table 2), with the highest value found for the SHP extract (62.74 ± 2.33%) and the lowest found for the GASP extract (52.32 ± 3.76%). Based on the results obtained, it is revealed that the SHP extract had the greatest ability to neutralise free radicals. Although the SHP extract had the highest value, no significant difference (p < 0.05) was observed between the antioxidant activity of the SHP and GBSP extracts.
3.3. Antioxidant capacity Different antioxidant compounds may act through different mechanisms; consequently, one method alone cannot be utilised to fully evaluate the antioxidant capacity of foods (Pellegrini et al., 2003). For this reason, three antioxidant capacity tests with different approaches and mechanisms have been carried out. 3.3.1. FRAP assay GASP had a higher FRAP value [825.71 ± 70.18 lmol Fe (II)/ 100 g wet weight] than ascorbic acid [648.22 ± 36.99 lmol Fe (II)/ 100 g wet weight]. The FRAP value of GBSP was not significantly different from ascorbic acid, indicating that GBSP had a similar reducing power as ascorbic acid. Xu and Chang (2009) report a FRAP value range of 640–1160 lmol Fe(II)/100 g in raw soymilk 0.27
Absorbance at 470 nm
0.22
0.17
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.
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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
TPCA FRAPB TEACC BCBD
96.31 ± 3.06a 825.71 ± 70.18a 1009.50 ± 88.62a 52.32 ± 3.76c
103.86 ± 5.29a 653.10 ± 16.65b 1022.90 ± 56.38a 61.82 ± 1.72b
62.44 ± 3.65b 340.12 ± 9.43c 631.90 ± 6.24b 62.74 ± 2.33a
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 lipophilic antioxidants, as the reagent is soluble in aqueous and solvents (Apak et al., 2007). The assessment of the ranking order of FRAP, TEAC and b-carotene bleaching assays, indicates that GASP and SHP contain higher amounts of hydrophilic and lipophilic antioxidants, respectively. Further study of the correlations among antioxidant assays revealed a significant and positive correlation obtained between the FRAP and TEAC assays, while no correlation was observed between the b-carotene bleaching and TEAC assays. These two correlations indicate that hydrophilic antioxidants and not lipophilic antioxidants were the major contributors of TEAC values. 3.4. Total isoflavone Naim, Gestetner, and Zilkah (1974) report the existence of isoflavones in soybean and non fermented soybean products (such as soy protein and soymilk derivates) in the form of glycosides.
Fig. 2a. Daidzein and genistein contents without hydrolysis. Different letters indicate significant difference at the level of p < 0.05.
Fig. 2b. Daidzein and genistein contents after acid hydrolysis. Different letters indicate significant difference at the level of p < 0.05.
As only aglycones are able to be absorbed by the human digestive system, studies have been extensively carried out in an effort to transform glycosides into aglycones. Among the methods utilised, mild acid hydrolysis has been proven to be the best choice for completely hydrolysing glycosides into their 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 ± 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 daidzein content, represented by the value of 1.17 ± 0.03 mg daidzein/100 g wet weight in the respective sample, and genistein was not detected in SHP throughout the study. The addition of 1 M acid to the extraction solvent has promoted the extractability of the isoflavones daidzein and genistein from all soy extracts. As shown in Fig. 2b, the SHP extract showed a significantly higher daidzein content (19.02 ± 0.39 mg/100 g wet weight) following the acid hydrolysis. 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 significant, revealing that acid-hydrolysed GASP and GBSP contain almost identical amounts of daidzein and genistein. Both unhydrolysed and acid-hydrolysed soymilk extracts contained 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 between soymilk powders could be attributed to the variety of soybeans, 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 unhydrolysed ones. It reflects that the acidic condition favours the conversion of glycosides into aglycones, causing a significant increase of daidzein 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 suggested that a mild acid treatment may release the fibre-bound daidzein from the soy husk, yielding a significant increase in the daidzein content.
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FRAP assay tends to show a strong positive correlation with chlorogenic acid (r = 0.984, p < 0.01), daidzein (r = 0.951, p < 0.01) and genistein (r = 0.975, p < 0.01). On the other hand, chlorogenic acid (r = 0.775) and genistein (r = 0.796) appear to have a negative correlation with the b-carotene bleaching assay (p < 0.05). All of the individual free phenolic compounds and isoflavones have a strong correlation with the total phenolic content (TPC) (p < 0.01). Vanilic acid (r = 0.972), ferulic acid (r = 0.973), and daidzein (r = 0.921) were positively correlated with TPC. Additionally, the measuring of the correlation between TPC and antioxidant capacity was also carried out. A 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). The FRAP assay measures the chain-breaking potential, and TEAC measures the ability to scavenge the ABTS radical cation, while the b-carotene bleaching assay measures the ability to neutralise the free radicals. Based on the Pearson correlation tests, it is clearly demonstrated that vanilic acid, ferulic acid, chlorogenic acid, daidzein and genistein are the potential chain breakers and ABTS the radical cation scavenger. Chlorogenic acid and genistein showed a negative correlation with the b-carotene bleaching assay, which indicates that they are a weak neutralizer of free radicals. Furthermore, the present results have also shown that vanilic acid, ferulic acid, chlorogenic acid, daidzein and genistein contribute the most to the value of TPC. In other words, they have a strong reducing ability toward the FC reagent. The FRAP and TEAC assays exhibited a high positive correlation (r = 0.916, p < 0.01), suggesting that the two assays are recommendable for evaluating antioxidant capacity in soy extracts. The correlation obtained is in 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 negative correlation (r = 0.686, p < 0.05) was demonstrated between the FRAP and b-carotene bleaching assays.
Table 3 Individual free phenolic acid composition in grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powder (SHP). Free phenolic acids (mg/ 100 g wet weight)
GASP
GBSP
SHP
Chlorogenic acid Ferulic acid Gallic acid Syringic acid Vanilic acid
32.16 ± 0.05a 294.65 ± 0.01a 99.81 ± 0.03a 18.67 ± 0.01a 191.19 ± 0.13a
25.71 ± 0.03b 310.84 ± 0.01b 85.40 ± 0.06b 18.75 ± 0.01b 235.76 ± 0.02b
16.75 ± 0.03c 259.56 ± 0.01c 108.34 ± 0.01c 20.83 ± 0.01c 97.24 ± 0.10c
Different letters in the same row indicate significant difference at a level of p < 0.05.
3.5. Phenolic compounds analysis Referring to Table 3, it is clearly shown that ferulic acid is the predominant phenolic compound identified in free form in all extracts. Among the extracts, GASP had the highest amount of chlorogenic acid (32.16 ± 0.05 mg/100 g wet weight) whereas GBSP had the highest amount of vanilic (235.76 ± 0.02 mg/100 g wet weight) and ferulic acids (310.84 ± 0.01 mg/100 g wet weight). Syringic and gallic acids were found to be highest in SHP. The phenolic compounds identified in free form in all extracts were significantly different from each other at the level of p < 0.05. Kim et al. (2006) report that soybean contains chlorogenic, ferulic, syringic, gallic and vanilic acids. Soymilk powder, which originates from the soybean, might also have a significant amount of these phenolic compounds. Compared to GASP and GBSP, gallic and syringic acid are predominantly found in the soy husk; the removal of the soy husk has thus led to a loss of valuable free forms of phenolic compounds.
3.6. Correlations among individual free phenolic compounds, isoflavones, TPC and antioxidant capacity Correlations among antioxidant capacity (TEAC, FRAP and bcarotene bleaching assays), individual free phenolic compounds and isoflavones are statistically analysed and depicted in Table 4. A strong and positive 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 genistein (r = 0.849). Also, the
4. Conclusions Although GBSP and SHP are the by-products derived from the soybean processing industry, the present study has shown that
Table 4 Correlations among free phenolic acids, total phenolic content and antioxidant capacity. Correlations A
GA VAB SAC FAD CAE DaF GeG TPCH FRAPI TEACJ * ** A B C D E F G H I J K
VAB 0.939
SAC **
FAD *
0.763 0.938**
Correlation is significant at the level of p < 0.05. Correlation is significant at the level of p < 0.01. Gallic acid. Vanilic acid. Syringic acid. Ferulic acid. Chlorogenic acid. Daidzein. Genistein. Total phenolic content. Ferric Reducing Antioxidant Power. Trolox Equivalent Antioxidant Capacity. b-carotene bleaching.
CAE **
0.937 1.000** 0.941**
0.453 0.731* 0.922** 0.736*
DaF
GeG *
0.675 0.885** 0.989** 0.888** 0.959**
0.440 0.721* 0.916** 0.726* 0.999** 0.957**
TPCH
FRAPI **
0.863 0.972** 0.962** 0.973** 0.810** 0.921** 0.798**
0.508 0.767* 0.934** 0.771* 0.984** 0.951** 0.975** 0.850**
TEACJ
BCBK *
0.773 0.924** 0.962** 0.926** 0.865** 0.936** 0.849** 0.950** 0.916**
0.060 0.252 0.535 0.258 0.775* 0.640 0.796* 0.353 0.686* 0.437
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these powders have an antioxidant capacity comparable with that of GASP. This study also revealed similar isoflavone content between GASP and GBSP. Furthermore, it also proved that soy byproducts contain a significant amount of phenolics. Therefore, an in-depth study can be carried out in the future for exploring the possibility of using soy by-products to develop health-beneficial nutraceuticals. Acknowledgements We would like to acknowledge Brilliant Point Sdn. Bhd, Kuala Lumpur, Malaysia for their generosity in providing samples for this study. We also like to extend our thanks to laboratory staff from the Department of Nutrition and Dietetics at UPM for their guidance and assistance. References Amin, I., & Tan, S. H. (2002). Antioxidant activity of selected seaweeds. Malaysian Journal of Nutrition, 8, 167–177. Amin, I., & Mukhrizah, O. (2006). Antioxidant capacity of methanolic and water extracts prepared from food-processing by-products. Journal of the Science of Food and Agriculture, 86, 778–784. Anonymous (1987). Soy protein products: Characteristic, nutritional aspect and utilization. Washington, DC: Soy Protein Council. AOAC (1990). Official methods of analysis (16th ed.). Gaithersburg, MD: AOAC International. Apak, S., Güçlü, K., Demirata, B., Özyürek, M., Çelik, S. E., Bektasßog˘lu, B., et al. (2007). Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules, 12, 1496–1547. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma as a measure of antioxidant power, the FRAP assay. Analytical Biochemistry, 239, 70–76. Bhardwaj, H. L., Hamama, A. A., Rangappa, M., Joshi, J. M., & Sapra, V. T. (2007). Mineral composition of toufu made from twelve soybean genotypes grown at three locations in USA. World Journal of Agricultural Sciences, 3(2), 140–144. Boydak, E., Alpaslan, M., Hayta, M., Gercek, S., & Simsek, M. (2002). Seed composition of soybeans grown in Harran region of Turkey as affected by row spacing and irrigation. Journal of Agricultural and Food Chemistry, 50, 4718–4720. Clegg, K. M. (1955). The application of the anthrone reagent to the estimation of starch in cereals. Journal of the Science of Food and Agriculture, 7, 40–44. Cole, J. T., Fahey, G. C., Merchen, N. R., Jr., Patil, A. R., Murray, S. M., Hussein, H. S., et al. (1999). Soybean hulls as a dietary fiber source for dogs. Journal of Animal Science, 77, 917–924. Food and Agriculture Organization of the United Nations (FAO) (2006). Available at:
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Antioxidant capacity, phenolics and isoflavones in soybean by-products Tan Seok Tyug a, K. Nagendra Prasad a, Amin Ismail a,b,* a b
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
a r t i c l e
i n f o
Article history: Received 14 January 2010 Received in revised form 13 March 2010 Accepted 27 April 2010
Keywords: Antioxidant capacity Isoflavones Proximate composition Soy by-products
a b s t r a c t This study aimed to determine proximate composition, antioxidant capacity, total phenolic content, isoflavones and free phenolic compounds in soy by-products. High carbohydrate and protein contents were found in grade A soymilk powder (GASP) compared to grade B soymilk powder (GBSP) and soy husk powder (SHP). Ash, moisture and total dietary fibre 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 Capacity (TEAC) value was GASP GBSP > SHP, while the Ferric Reducing Antioxidant Power (FRAP) value was GASP > 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 compounds in free form were ferulic, vanilic as well as gallic acids. Acid hydrolysis increased the amount of total extractable isoflavone in all soy samples. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Antioxidants are compounds that when added to lipids and lipid-containing foods, can increase their shelf-life by retarding the process of lipid peroxidation. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have limited use in foods, as they are suspected to be carcinogenic (Namiki, 1990). As a result, the importance of exploiting natural antioxidants from by-products obtained during food processing has increased tremendously in recent years. They are considered to be attractive due to their low cost and availability in large quantity as raw material (Kong, Amin, Tan, & Rajab, 2010). The beneficial effect of these by-products can be attributed to various phenolic compounds present in them (Amin & Mukhrizah, 2006). These phenolic compounds include phenolic acids, anthocyanins, flavonoids, and hydrocinnamic acid derivatives. Soybean (Glycine max L.) is one of the most commonly consumed legumes worldwide, with 200 million metric tons produced per year (FAO, 2006). In Asian countries, soybean is processed into various products such as 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, soybeans are soaked in water to remove the inedible soy husk. However, this might represent a major disposal problem for the
* Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor, Malaysia. Tel.: +603 89462435; fax: +603 89426769. E-mail address: [email protected] (A. Ismail). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.074
soybean processing industry, as soy husk represents about 8% of the entire soybean (Mateos-Aparicio, Redondo Cuenca, Villanueva-Suárez, & Zapata-Revilla, 2008). After the removal of the soy husk, the beans are dried, ground, sieved and cooked into slurry. Finally, the cooked slurry is subjected for vacuum drying to obtain grade A soymilk powder. Under normal circumstances, wastes produced during the production of grade A soymilk powder will be discarded or used as animal feed. However, in Malaysia, the wastes are processed to a lower-quality powder, called grade B soymilk powder (GBSP). Previous studies have reported that soybean is a rich source of isoflavones and phenolic compounds. The primary isoflavones present in the whole soybean include glycosides of genistin and daidzin, while syringic, 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, antioxidant capacity, isoflavones and phenolic compounds profile for soybean by-products. This study thus aims to conduct these analyses with a hope of exploring the potential use of soy by-products in food fortification and as nutraceuticals. 2. Materials and methods 2.1. Chemicals Analytical and HPLC-grade solvents were purchased from Fisher Scientific (Loughborough, UK). HPLC standards (daidzein, genistein, chlorogenic acid, syringic acid, ferulic acid, vanilic acid and gallic acid), total dietary fibre assay kit, b-carotene, linoleic acid,
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Tween 20, butylated hydroxytoluene (BHT), Trolox, potassium persulfate, sodium carbonate (Na2CO3), sodium nitrite (NaNO2), aluminium chloride hexahydrate (AlCl36H2O), sodium hydroxide (NaOH) were purchased from Sigma Chemical Co (St. Louis, MO, USA). The 2, 20 -azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was purchased from Merck (Darmstadt, Germany). 2.2. Preparation of samples Grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powders were provided by Brilliant Point Sdn. Bhd, Kuala Lumpur, Malaysia. The soy husk was ground into small pieces using a wet blender (MX-291 N, National, Osaka, Japan). The ground soy husk was then passed through a 0.05 lm sieve, and the filtrate obtained was considered soy husk powder (SHP). 2.3. Proximate analyses The moisture and ash contents of GASP, GBSP and SHP were determined using the standard AOAC (1990) method. Total dietary fibre was analysed using a commercialised assay kit from Sigma Chemical Co (St. Louis, MO, USA). The total available carbohydrate content was determined using Clegg Anthrone method (Clegg, 1955). The Soxhlet and Kjeldahl systems were used to determine the fat and protein contents of the samples, respectively (Tee, Rajam, Young, Khor, & Zakiyah, 1996). 2.4. Preparation of extracts The soybean powders were extracted with 70% (v/v) ethanol in a ratio of 1:25 (g/v) and were incubated in an orbital shaker (Unimax 1010, Heidolph Instruments GmbH & Co. KG, Germany) set at 200 rpm for 2 h at 50 °C. After the extraction, the mixture was filtered through Whatman paper No. 542, and the filtrate obtained was kept at 20 °C prior to analysis. 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 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, 1.5 ml of sodium carbonate solution (0.566 M) was added into the mixture and incubated at room temperature for 90 min. The absorbance was measured at 725 nm using a spectrophotometer (Anthelie Junior Advanced 5 nm, Prim, SECOMAM, France), and the results were expressed as gallic acid equivalents (GAE) in mg/100 g wet weight. 2.6. Determination of antioxidant capacity 2.6.1. Ferric Reducing Antioxidant Power (FRAP) assay The Ferric Reducing Antioxidant Power (FRAP) assay was estimated according to the method described by Benzie and Strain (1996) with slight 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 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. Ferrous sulphate (FeSO47H2O) with a concentration from 200–1000 lM was used to plot a standard curve, and ascorbic acid at a concentration of 10 lg/ml was used as a positive control. The results were expressed as lmol Fe (II)/100 g wet weight.
2.6.2. Trolox Equivalent Antioxidant Capacity (TEAC) assay The Trolox Equivalent Antioxidant Capacity (TEAC) assay measures the ability of antioxidants to scavenge the ABTS radical cation. This assay was carried out using the improved ABTS+ method described by Li, Wong, Cheng, and Chen (2008) with slight modifications. Briefly, ABTS+ radical 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 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 samples were diluted appropriately to provide 20–80% inhibition against the blank absorbance (around 50-fold dilution was required). Approximately 50 ll of the diluted sample was mixed with 1.9 ml of the diluted ABTS+ solution. The absorbance value was immediately read at 734 nm after 6 min of the reaction. Trolox at concentrations ranging from 200–1000 mM was used as a standard, and 100 lg/ml ascorbic acid was used as a positive control. The results were expressed as lmol Trolox/100 g wet weight. 2.6.3. b-Carotene bleaching assay The b-carotene bleaching assay was determined according to the method described by Amin and Tan (2002). About 1 ml of the b-carotene solution with a concentration of 0.2 mg/ml dissolved in chloroform was pipetted into a 50 ml round bottom flask containing 0.02 ml of linoleic acid and 0.2 ml of 100% Tween 20. The mixture was subjected to evaporation by means of removing chloroform at 40 °C for 10 min using a rotary evaporator. Approximately 100 ml of distilled water was added, and the mixture was shaken vigorously to form an emulsion. From this emulsion, 5 ml was pipetted out and transferred into different test tubes containing 0.2 ml of samples in 70% ethanol at 1 mg/ml. All test tubes were vortexed for 1 min and placed at 45 °C in a water bath for 2 h. The absorbance of the samples was measured at 470 nm using a spectrophotometer at initial time (t = 0) against a blank consisting of an emulsion without b-carotene. A standard (ascorbic acid) at a concentration of 10 mg/ml was used and 70% ethanol as 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:
AA ¼ ½1 ðA0 At Þ=ðA0 At Þ 100%; where A0 and A0 are the absorbance values measured at the initial incubation time for the samples and control, respectively, while At and A0 are the absorbance values measured in the samples or the standard and control at t = 120 min. 2.7. Determination of total isoflavone content In the present study, the total isoflavone content in the soy extracts was quantified based on the sum of the genistein and daidzein contents. The daidzein and genistein contents were determined according to the method of Penalvo, Nurmi, and Adlercreutz (2004). The isoflavone content in acid-hydrolysed powders was compared with that in unhydrolysed powders. 2.8. Extraction of isoflavones 2.8.1. Preparation for unhydrolysed extracts Intially, 1 g of the sample powder was added to 25 ml of 70% (v/ v) ethanol. The mixture was shaken vigorously for 2 min and followed by centrifugation at 2140g. After 10 min, the clear supernatant was passed through Whatman filter paper No. 4 and a 0.22lm polytetrafluoroethylene microfilter (Millipore, USA) before being injected into a reversed-phase HPLC system.
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2.8.2. Preparation for acid-hydrolysed extracts 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 centrifuged for 10 min at 2140g. The clear supernatant obtained was passed through a Whatman filter paper No. 4 and 0.22 lm polytetrafluoroethylene microfilter before being injected into a reversed-phase HPLC. 2.8.3. Chromatographic conditions The chromatographic condition used was as previously described by 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 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 sample extract was injected into the column set at room temperature (25 °C). The UV–Vis absorption spectra were monitored by DAD at 200–400 nm, and isoflavones were detected at a wavelength of 250 nm. 2.9. Identification of free phenolic compounds 2.9.1. Extraction of free phenolic compounds In brief, 1 g of GASP, GBSP and SHP were mixed with 25 ml of 70% ethanol. The mixtures were placed in an orbital shaker at 50 °C overnight in order to extract free phenolic compounds from the samples. The supernatants obtained were passed through a 0.22 lm polytetrafluoroethylene microfilter before being injected into a reversed-phase HPLC. 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) was utilised for the separation. A gradient elution was performed with 0.5% (v/v) acetic acid (solvent A) and 100% methanol (solvent B) at a constant flow rate of 0.6 ml/min. The linear gradient profile was set as follows: 100% A and 0% B at start, 10% A and 90% B at 20–25 min, and back to 100% A and 0% B at 30 min. Twenty microlitre of the sample extract was injected into the column set at room temperature (25 °C). The UV–Vis absorption spectra were monitored by DAD at 200– 600 nm, and phenolics were detected at the wavelength of 280 nm. 2.9.3. Standard calibration curve The standard of the respective phenolic compounds (syringic, chlorogenic, gallic, vanilic and ferulic acids) and isoflavones (genistein and daidzein) were dissolved in 70% ethanol (v/v). The calibration curves of the phenolic compounds were plotted at five different concentrations ranging from 20 to 100 lg/ml, while the calibration curves of genistein and daidzein were plotted in the range of 0–150 lm. 2.10. Statistical analysis The results were analysed using the Statistical Package for the Social Sciences (SPSS version 16). Data were expressed as means ± standard deviation (SD) of triplicate determinations. Independent t-test, ANOVA and Tukey tests were used to determine the mean differences. A Pearson correlation test was used to evaluate the correlation between the antioxidant capacity and the phenolic compounds. A p-value of less than 0.05 was considered to be statistically significant.
3. Results and discussion The recycling of by-products derived from various industries is crucial, as many studies have reported that they contain abundant health-beneficial compounds (Amin & Mukhrizah, 2006; Kong et al., 2009). For instance, soy husk derived from the soymilk industry has been proven to be an excellent source of insoluble fibre (Cole et al., 1999). To the best of our knowledge, the consumption of insoluble fibre helps to relieve constipation and prevent colon cancer. It would thus be beneficial if soy husk is reutilised after soymilk processing. 3.1. Proximate composition analyses Despite years of study on soybean, there is a lack of research on the by-products derived from the soymilk processing industry. Hence, the proximate compositions of GASP and GBSP were compared with raw soybean reported in the Malaysian Food Composition 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 content (22.39 ± 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 samples tested. The ash, moisture and total dietary fibre contents were reported to be the highest in SHP, with values of 4.21 ± 0.02%, 9.95 ± 0.04% and 74.41 ± 0.19%, respectively. A statistical analysis of One-way ANOVA revealed that there were significant differences among all tests (p < 0.05). In other words, GASP had significantly higher contents of carbohydrate and protein compared to GBSP and SHP. In comparison, SHP had significantly higher ash, moisture and total dietary fibre contents, whereas fat content in GBSP was statistically higher than in GASP and SHP. The soymilk powders in the present study were lower in crude fat and protein but had higher fibre contents compared with the reported raw soybean. Carbohydrate and ash in the studied soymilk powders were similar to raw soybean. Riaz (2006) reported that dried soy husk contained approximately 8% moisture, 86% carbohydrate, 9% protein, 4% ash, 1% lipid. Fibre content of 76% was also reported in soy husk (Anonymous, 1987), which is in good agreement with our results. The studied SHP had similar contents of ash, moisture, and fat but showed a lower carbohydrate 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).
Table 1 Proximate composition of grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powder (SHP). Analyses (%)
Ash Moisture Carbohydrate ProteinD Fat Total dietary fibre
Composition GASPA
GBSPB
SHPC
3.37 ± 0.16a 3.77 ± 0.03a 32.79 ± 0.25a 22.39 ± 0.34a 0.27 ± 0.01a 13.90 ± 0.17a
3.77 ± 0.01b 2.95 ± 0.07b 23.70 ± 1.42b 20.63 ± 0.34b 2.82 ± 0.14b 22.78 ± 0.23b
4.21 ± 0.02c 9.95 ± 0.04c 11.50 ± 1.98c 4.72 ± 0.45c 1.69 ± 0.05c 74.41 ± 0.19c
Different letters in the same row indicate significant difference at the level of p < 0.05. A Grade A soymilk powder. B Grade B soymilk powder. C Soy husk powder. D Conversion factor (5.71).
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from three different varieties. Compared to the previous study, the FRAP value of GASP and GBSP fall in the range reported. This assay involves a single-electron transfer by treating antioxidants as reductants in a redox-linked colorimetric reaction (Prior, Wu, & Schaich, 2005). In other words, it measures the tendency of antioxidants to give up a single electron to the FRAP reagent. The results in the present study showed that GASP had the highest tendency to transfer electrons among the extracts tested.
The ash content is an indicator of the total mineral content in food (Hasnah et al., 2009). Based on the present study, it is reflected that the removal of soy husk during the processing of soymilk powder has led to a significant loss in minerals and fibre. The differences in proximate composition could be attributed to many factors, including agronomic practices, the genotype and the growing location of the soybean. Boydak, Alpaslan, Hayta, Gercek, and Simsek (2002) found that agronomic practices, such as row spacing and irrigation are able to affect the protein content and the fatty acid composition of soybean. A recent study conducted by Bhardwaj, Hamama, Rangappa, Joshi, and Sapra (2007) also demonstrate that the genotype and growing location of soybean could have significant effects on soymilk protein and the fatty acid composition of tofu, respectively.
3.3.2. TEAC assay In the TEAC assay, the antioxidants act as hydrogen donors to terminate 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 comparable 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 among the extracts studied. The ABTS radical-scavenging activity of soymilk powder and soy husk is not well documented in previous studies. However, when compared with the TEAC value of raw soybean (6303 lmol/100 dry matter) reported by Fernandez-Orozco et al. (2007), it is found that GASP, GBSP and SHP had less ability in scavenging the ABTS cation radical. This phenomenon indicates that the processing of soybean into soymilk may bring a significant loss of antioxidant compounds.
3.2. Total phenolic content (TPC) The ability of phenolic compounds in reducing Folin–Ciocalteau (FC) reagent is quantified using a colorimetric method. The results in the present study demonstrate that GBSP had the most reducing activity toward FC reagent. However, it is important to note that the presence of reducing agents such as ascorbic acid and sugars may interfere with this assay, as these compounds are also able to reduce FC reagent. Of the extracts, GBSP had the highest TPC (103.86 ± 5.29 mg GAE/100 g wet weight), followed by GASP (96.31 ± 3.06 mg GAE/100 g wet weight) and SHP (62.44 ± 3.65 mg GAE/100 g wet weight). The TPC of SHP was statistically lower than GASP 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 free radicals. Fig. 1 indicates the degradation rate for soy powder extracts, the control and the standard. There was a decrease in the absorbance value of b-carotene in the absence of samples due to the oxidation of b-carotene and linoleic acid. As shown in the figure, all soy extracts had degradation rates higher than ascorbic acid, with SHP extract being the most degraded, followed by GBSP and GASP extracts. The above results are also supported by their respective antioxidant activity (Table 2), with the highest value found for the SHP extract (62.74 ± 2.33%) and the lowest found for the GASP extract (52.32 ± 3.76%). Based on the results obtained, it is revealed that the SHP extract had the greatest ability to neutralise free radicals. Although the SHP extract had the highest value, no significant difference (p < 0.05) was observed between the antioxidant activity of the SHP and GBSP extracts.
3.3. Antioxidant capacity Different antioxidant compounds may act through different mechanisms; consequently, one method alone cannot be utilised to fully evaluate the antioxidant capacity of foods (Pellegrini et al., 2003). For this reason, three antioxidant capacity tests with different approaches and mechanisms have been carried out. 3.3.1. FRAP assay GASP had a higher FRAP value [825.71 ± 70.18 lmol Fe (II)/ 100 g wet weight] than ascorbic acid [648.22 ± 36.99 lmol Fe (II)/ 100 g wet weight]. The FRAP value of GBSP was not significantly different from ascorbic acid, indicating that GBSP had a similar reducing power as ascorbic acid. Xu and Chang (2009) report a FRAP value range of 640–1160 lmol Fe(II)/100 g in raw soymilk 0.27
Absorbance at 470 nm
0.22
0.17
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.
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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
TPCA FRAPB TEACC BCBD
96.31 ± 3.06a 825.71 ± 70.18a 1009.50 ± 88.62a 52.32 ± 3.76c
103.86 ± 5.29a 653.10 ± 16.65b 1022.90 ± 56.38a 61.82 ± 1.72b
62.44 ± 3.65b 340.12 ± 9.43c 631.90 ± 6.24b 62.74 ± 2.33a
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 lipophilic antioxidants, as the reagent is soluble in aqueous and solvents (Apak et al., 2007). The assessment of the ranking order of FRAP, TEAC and b-carotene bleaching assays, indicates that GASP and SHP contain higher amounts of hydrophilic and lipophilic antioxidants, respectively. Further study of the correlations among antioxidant assays revealed a significant and positive correlation obtained between the FRAP and TEAC assays, while no correlation was observed between the b-carotene bleaching and TEAC assays. These two correlations indicate that hydrophilic antioxidants and not lipophilic antioxidants were the major contributors of TEAC values. 3.4. Total isoflavone Naim, Gestetner, and Zilkah (1974) report the existence of isoflavones in soybean and non fermented soybean products (such as soy protein and soymilk derivates) in the form of glycosides.
Fig. 2a. Daidzein and genistein contents without hydrolysis. Different letters indicate significant difference at the level of p < 0.05.
Fig. 2b. Daidzein and genistein contents after acid hydrolysis. Different letters indicate significant difference at the level of p < 0.05.
As only aglycones are able to be absorbed by the human digestive system, studies have been extensively carried out in an effort to transform glycosides into aglycones. Among the methods utilised, mild acid hydrolysis has been proven to be the best choice for completely hydrolysing glycosides into their 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 ± 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 daidzein content, represented by the value of 1.17 ± 0.03 mg daidzein/100 g wet weight in the respective sample, and genistein was not detected in SHP throughout the study. The addition of 1 M acid to the extraction solvent has promoted the extractability of the isoflavones daidzein and genistein from all soy extracts. As shown in Fig. 2b, the SHP extract showed a significantly higher daidzein content (19.02 ± 0.39 mg/100 g wet weight) following the acid hydrolysis. 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 significant, revealing that acid-hydrolysed GASP and GBSP contain almost identical amounts of daidzein and genistein. Both unhydrolysed and acid-hydrolysed soymilk extracts contained 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 between soymilk powders could be attributed to the variety of soybeans, 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 unhydrolysed ones. It reflects that the acidic condition favours the conversion of glycosides into aglycones, causing a significant increase of daidzein 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 suggested that a mild acid treatment may release the fibre-bound daidzein from the soy husk, yielding a significant increase in the daidzein content.
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FRAP assay tends to show a strong positive correlation with chlorogenic acid (r = 0.984, p < 0.01), daidzein (r = 0.951, p < 0.01) and genistein (r = 0.975, p < 0.01). On the other hand, chlorogenic acid (r = 0.775) and genistein (r = 0.796) appear to have a negative correlation with the b-carotene bleaching assay (p < 0.05). All of the individual free phenolic compounds and isoflavones have a strong correlation with the total phenolic content (TPC) (p < 0.01). Vanilic acid (r = 0.972), ferulic acid (r = 0.973), and daidzein (r = 0.921) were positively correlated with TPC. Additionally, the measuring of the correlation between TPC and antioxidant capacity was also carried out. A 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). The FRAP assay measures the chain-breaking potential, and TEAC measures the ability to scavenge the ABTS radical cation, while the b-carotene bleaching assay measures the ability to neutralise the free radicals. Based on the Pearson correlation tests, it is clearly demonstrated that vanilic acid, ferulic acid, chlorogenic acid, daidzein and genistein are the potential chain breakers and ABTS the radical cation scavenger. Chlorogenic acid and genistein showed a negative correlation with the b-carotene bleaching assay, which indicates that they are a weak neutralizer of free radicals. Furthermore, the present results have also shown that vanilic acid, ferulic acid, chlorogenic acid, daidzein and genistein contribute the most to the value of TPC. In other words, they have a strong reducing ability toward the FC reagent. The FRAP and TEAC assays exhibited a high positive correlation (r = 0.916, p < 0.01), suggesting that the two assays are recommendable for evaluating antioxidant capacity in soy extracts. The correlation obtained is in 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 negative correlation (r = 0.686, p < 0.05) was demonstrated between the FRAP and b-carotene bleaching assays.
Table 3 Individual free phenolic acid composition in grade A soymilk powder (GASP), grade B soymilk powder (GBSP) and soy husk powder (SHP). Free phenolic acids (mg/ 100 g wet weight)
GASP
GBSP
SHP
Chlorogenic acid Ferulic acid Gallic acid Syringic acid Vanilic acid
32.16 ± 0.05a 294.65 ± 0.01a 99.81 ± 0.03a 18.67 ± 0.01a 191.19 ± 0.13a
25.71 ± 0.03b 310.84 ± 0.01b 85.40 ± 0.06b 18.75 ± 0.01b 235.76 ± 0.02b
16.75 ± 0.03c 259.56 ± 0.01c 108.34 ± 0.01c 20.83 ± 0.01c 97.24 ± 0.10c
Different letters in the same row indicate significant difference at a level of p < 0.05.
3.5. Phenolic compounds analysis Referring to Table 3, it is clearly shown that ferulic acid is the predominant phenolic compound identified in free form in all extracts. Among the extracts, GASP had the highest amount of chlorogenic acid (32.16 ± 0.05 mg/100 g wet weight) whereas GBSP had the highest amount of vanilic (235.76 ± 0.02 mg/100 g wet weight) and ferulic acids (310.84 ± 0.01 mg/100 g wet weight). Syringic and gallic acids were found to be highest in SHP. The phenolic compounds identified in free form in all extracts were significantly different from each other at the level of p < 0.05. Kim et al. (2006) report that soybean contains chlorogenic, ferulic, syringic, gallic and vanilic acids. Soymilk powder, which originates from the soybean, might also have a significant amount of these phenolic compounds. Compared to GASP and GBSP, gallic and syringic acid are predominantly found in the soy husk; the removal of the soy husk has thus led to a loss of valuable free forms of phenolic compounds.
3.6. Correlations among individual free phenolic compounds, isoflavones, TPC and antioxidant capacity Correlations among antioxidant capacity (TEAC, FRAP and bcarotene bleaching assays), individual free phenolic compounds and isoflavones are statistically analysed and depicted in Table 4. A strong and positive 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 genistein (r = 0.849). Also, the
4. Conclusions Although GBSP and SHP are the by-products derived from the soybean processing industry, the present study has shown that
Table 4 Correlations among free phenolic acids, total phenolic content and antioxidant capacity. Correlations A
GA VAB SAC FAD CAE DaF GeG TPCH FRAPI TEACJ * ** A B C D E F G H I J K
VAB 0.939
SAC **
FAD *
0.763 0.938**
Correlation is significant at the level of p < 0.05. Correlation is significant at the level of p < 0.01. Gallic acid. Vanilic acid. Syringic acid. Ferulic acid. Chlorogenic acid. Daidzein. Genistein. Total phenolic content. Ferric Reducing Antioxidant Power. Trolox Equivalent Antioxidant Capacity. b-carotene bleaching.
CAE **
0.937 1.000** 0.941**
0.453 0.731* 0.922** 0.736*
DaF
GeG *
0.675 0.885** 0.989** 0.888** 0.959**
0.440 0.721* 0.916** 0.726* 0.999** 0.957**
TPCH
FRAPI **
0.863 0.972** 0.962** 0.973** 0.810** 0.921** 0.798**
0.508 0.767* 0.934** 0.771* 0.984** 0.951** 0.975** 0.850**
TEACJ
BCBK *
0.773 0.924** 0.962** 0.926** 0.865** 0.936** 0.849** 0.950** 0.916**
0.060 0.252 0.535 0.258 0.775* 0.640 0.796* 0.353 0.686* 0.437
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