DOI:
10.1039/C6RA00074F
(Paper)
RSC Adv., 2016,
6, 29741-29756
Enhanced total phenolic and isoflavone aglycone content, antioxidant activity and DNA damage protection of soybeans processed by solid state fermentation with Rhizopus oligosporus RT-3
Received
2nd January 2016
, Accepted 14th March 2016
First published on 16th March 2016
Abstract
In this study, Rhizopus oligosporus RT-3, which was first isolated in our group, was used for solid state fermentation of soybeans (R. oligosporus-fermented soybeans, RFS) in a short time (22 h). The effects of fermentation on total phenolic content (TPC), isoflavone composition, antioxidant activity and DNA damage protection of soybeans were investigated. Besides, the effects of different polarity solvents in extracting antioxidant compounds were also tested. The results showed that fermentation significantly enhanced TPC and isoflavone aglycone content, antioxidant activity and DNA damage protection, but decreased the content of isoflavone glucosides. The enhanced antioxidant activity of RFS could be ascribed to the markedly higher levels of TPC and isoflavone aglycones achieved during fermentation. The study also indicated that extraction solvents had significant effects on TPC and isoflavone glucoside and aglycone content, and antioxidant activity. The water extract of RFS showed the highest TPC, ABTS˙+ and hydroxyl radical scavenging activity and chelating ability, whereas the 80% ethanol extract of RFS exhibited the strongest DPPH radical scavenging activity, reducing power and antioxidant capacity determined by a silver nanoparticle-based method. TPC and isoflavone aglycone content were highly correlated with antioxidant activity. In addition, bioaccessibility studies also demonstrated that RFS exhibited higher TPC and isoflavone aglycone bioaccessibility, as well as stronger antioxidant activity compared to non-fermented soybeans. Thus, this study demonstrated that RFS with enhanced TPC and isoflavone aglycone content, antioxidant activity and DNA damage protection which could be considered a good source of natural antioxidants in the prevention of oxidative damage-induced diseases, and it can serve as a nutraceutical and functional food/ingredient in health promotion and disease risk reduction.
1. Introduction
In recent years, increasing attention has been paid to the role of diet in human health. Scientific reports have confirmed that a high intake of plant foods (e.g., legumes, fruits and vegetables) is intimately related to a reduced risk of many diseases, such as atherosclerosis, arthritis, cardiovascular disease, cancer, diabetes and cataracts.1–5 Among the plant foods consumed by humans, soybeans have been reported to receive great attention. Soybean (Glycine max L.) is the most important legume and soybean products have been widely consumed in East Asian countries for several centuries, which are notably rich in proteins, amino acids, carbohydrate fractions, lipids, and minerals.6,7 In addition to its nutritional potential, soybeans is highlighted as an important source of phenolics and isoflavones, which are receiving a growing interest for their benefits of human health, particularly in western and chronic diseases.6,8 These soybean phenolics and isoflavones (e.g., daidzein and genistein) can function as scavengers for free radicals,9 or metal ion chelators,10 as well as inhibitors to human cancer cells.11 Therefore, they are usually used for prevention and treatment of the oxidative-induced chronic diseases, such as the prevention of certain cancers,11,12 reduction of cholesterol and hypertension,13 and an improvement of bone health.14
However, many studies have demonstrated that most of phenolics and flavonoids in nature food materials existed as conjugated forms (with one or more sugar residues bound to hydroxyl groups, or groups of compounds such as organic and carboxylic acids, lipids, and amines), which have low biological activity.15–18 Shin et al.7 and da Silva et al.6 have reported that the major phenolic compounds (i.e. soybean isoflavones) in soybeans are found predominantly in the glycosides form and in low concentrations as aglycones. In addition, many studies have reported that the biological effects of isoflavones are not due to the glycosides form but instead are mainly from their aglycones, such as daidzein and genistein.16,19 It has also been demonstrated that isoflavone aglycones can be absorbed faster and in greater amount than their glycosides in human.20
A majority of the previous studies have reported that the content and composition of the phytochemical compounds (e.g. isoflavones) in soybean-based products could be greatly affected by cooking, enzymatic hydrolysis, fermentation, germination, heat treatment and other different processing techniques.6,21 In recent years, fermentation was observed to serve as an efficient approach to improve the antioxidant compounds and antioxidant activity of legume products. It has also been reported that fermented soybean products exhibit higher antioxidant, anti-cancer, and anti-metastatic activities than non-fermented soybean products.21 There are some studies reported that the isoflavone aglycones can be achieved during fermentation by lactic acid bacteria, Aspergillus oryzae, basidiomycetes, and Bacillus subtilis with their β-glucosidase enzyme activity.6,7 Some of fermented soybean products such as natto (B. subtilis fermented soybean food), doenjang (a traditional Korean fermented soybean food) obtained by fermentation with Bacillus species, or douchi (a Chinese fermented soybean product) fermented with Aspergillus egyptiacus, is largely consumed in Asian countries, and currently an increasing consumption in Western countries.16,22 This might be mainly due to the presence of high bioactive compounds (e.g., isoflavone aglycones) which have potent health-promoting effects in the fermented soybean products.7,9,10 Dietary intake of fermented soybean products is linked to greatly reduce the risk of chronic diseases such as diabetes, atherosclerosis, arthritis, cancer and cardiovascular diseases. As a result, there is much interest in fermented soybean products worldwide, as this fermentation process often results in higher amount of isoflavone aglycones and phenolic compounds, which help to protect living body systems against oxidative damage as well as the subsequent oxidative damage-induced diseases.
Rhizopus species are commonly used as starter organisms for the preparation of fermented food products and possess β-glucosidase activity to hydrolyze isoflavone glucosides during fermentation process.23 As mentioned earlier, fermentation is a useful biotechnological strategy for legumes that produces beneficial bioactive phytochemicals. The effect of fermentation on the bioactive and physicochemical properties of legume products by Rhizopus species had been investigated previously.9,24,25 However, to the best our knowledge, previous studies have reported that Rhizopus species employed in the fermentation of legumes should be about 72 h or more than that time.24,25 In the present study, in order to shorten the fermentation time as well as obtain good fermented product, we have used our laboratory firstly isolated strain (i.e., R. oligosporus RT-3) to prepare a novel starter culture for the production of fermented soybeans (the detailed method is shown in Section 2.2 and Section 2.3, Fig. 1). The required incubation time is only 22 h which is the optimum incubation time to prepare R. oligosporus fermented soybeans. In addition, to the best of our knowledge, no information is available regarding the effect of fermentation with R. oligosporus on DNA damage protective effect of soybeans. Therefore, the main objective of this study was to evaluate the influence of soybeans fermented with R. oligosporus RT-3 for 22 h on the total phenolic content, isoflavone compositions, antioxidant activity, and DNA damage protection. Furthermore, different polarities solvent systems (i.e. 80% methanol, 80% ethanol, 80% acetone, and water) were employed to extract the antioxidant compounds, and the subsequent effect on antioxidant activity and DNA damage protection was also determined. Additionally, in the present study, a novel silver nanoparticle-based method was employed for evaluation of antioxidant activity of soybean samples and also compared with those of common antioxidant assays. The work investigated would to obtain fermented soybeans with added-value and to develop it into potential nutraceuticals as well as functional foods in a short time. Meanwhile, the present study would also help to clearly understand the relationship between content of total phenolic and isoflavone, and antioxidant activity.
 |
| Fig. 1 Schematic representation of processing and solid state fermentation of soybeans. | |
2. Materials and methods
2.1 Materials and microorganism
The compounds 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), ferrozine, silver nitrate (AgNO3), ascorbic acid (vitamin C), pUC18 plasmid DNA, pepsin, pancreatin, bile, daidzin, glycitin, genistin, daidzein, glycitein and genistein were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Gallic acid and Folin-Ciocalteu's reagent were purchased from Merck (Darmstadt, Germany). All other chemicals and reagents were of analytical grade whereas the solvents used for chromatography were HPLC-grade. Soybeans (Glycine max L.), which served as the fermentation substrate, were obtained from the Anhui Yan Fang Food Co. Ltd. (Hefei, China). The cultivated soybean used in this study belongs to the family Leguminosae, subfamily Papilionoideae, genus Glycine.
The filamentous fungus Rhizopus oligosporus RT-3, which was previously isolated in our laboratory,26,27 was used as a starter for the production of fermented soybeans. R. oligosporus RT-3 was maintained on a potato dextrose agar (PDA) (Sigma, Aldrich) slant and subculture every month. Slants were incubated at 35 °C for 7 days and subsequently stored at 4 °C.
The sterile rice flour was prepared as follows: the rice (Anhui Yan Fang Food Co. Ltd., Hefei, China) was firstly selected, rinsed and added an appropriate amount of water (50 mL water/100 g rice) in rice cooker cooking. After that, the cooked rice was dried out at 60 °C for 7 h on the white porcelain plate, and then it was grounded using a food grinder. The flour was then passed through a 0.1 mm sieve and sterilized at 160 °C for 2 h, and then cooled down to room temperature for use.
2.2 Preparation of inoculum starter
The inoculum starter was prepared according to the following procedures. Briefly, the R. oligosporus RT-3 was prepared for two successive transfers in PDA. The microorganism was propagated in PDA medium at 35 °C for 7 days, and then the activated culture was again inoculated into PDA plates at 35 °C. After the fungus mycelia was fully grown on the PDA plates as well as large amount of black spores were generated, the mycelia and spores were then rinsed with sterile physiological saline and mixed well. The solution was then mixed with sterile rice flours in appropriate proportion. The mixture was dried at 40 °C, then stored at 4 °C and used as culture. The culture obtained was used as the inoculum starter to prepare fermented soybeans. We adopted this starter culture to ferment soybeans not just because it can shorten the fermentation time but facilitate long-term storage.
2.3 Fermentation of soybeans
Soybeans were fermented with R. oligosporus that were prepared according to Sánchez-Magaña et al.25 and Reyes-Moreno et al.28 The soybean seeds were first thoroughly washed under tap water and then with distilled water. The seeds were soaked in distilled water at 20 °C for 12 h. Seeds were drained and seed coats were removed manually. Cotyledons were cooked at 100 °C for 30 min. After cooling down to room temperature, 1% (v/w) lactic acid and 1.5% sterile rice flours were added into the cooked soybeans, and then inoculated with 1.5% inoculum starter, and packed in perforated polyethylene bags (2 × 2 cm). The solid state fermentation process was performed at 35 °C for 12, 15, 18, 20, 22, 24 and 30 h based on preliminary experiments. Sample which showed the best growth state was used for the subsequent antioxidant capacity analysis and was named fermented soybeans (R. oligosporus-fermented soybeans, RFS). The soybeans which inoculated at 0 h (non-fermented soybeans, NFS) were used as a control. The schematic representation of solid state fermentation of soybeans is presented in Fig. 1. The for further analysis, the RFS and NFS were lyophilized using a Heto Power Dry LL3000 freeze drier (Thermo Electron Co., Bath, UK) and milled using a food grinder, the flour was then passed through a 0.2 mm-sieve, packed in plastic bags and stored at 4 °C until use.
2.4 Preparation of different solvent polarities extracts
Different solvent polarities extracts of RFS and NFS was performed based on the method of Xiao et al.18 Briefly, the milled powder of the samples was extracted by shaking with 40 folds solvents of various polarities including methanol (80%), ethanol (80%), acetone (80%) and deionized water at 50 °C for 4 h, respectively. After cooling to room temperature, the extracts were centrifuged at 15
000g at 4 °C for 15 min (CT15RT versatile refrigerated centrifuge, Techcomp Ltd., Shanghai, China) before the supernatants were collected and the residues were again re-extracted twice under the same conditions. The combined extracts were evaporated using a rotary evaporator (Heidolph Instruments Co., Ltd, Schwabach, Germany) to dryness under vacuum at 50 °C and re-dissolved in a known volume (25 mL) of the respective solvent system, and then stored in the dark at 4 °C until further analysis. All the extractions were done in triplicate.
2.5 Determination of total phenolic content (TPC)
The TPC was estimated by the Folin-Ciocalteu's method detail reported by Xiao et al.29 The TPC was determined using a standard curve prepared for gallic acid and expressed as micrograms of gallic acid equivalents per gram of dry weight sample (μg GAE g−1 d.w.).
2.6 High performance liquid chromatography (HPLC) analysis of major phenolic compounds
Isoflavones are generally considered as the major phenolics in the soybean samples. The six isoflavones, namely, daidzin, glycitin, genistin, daidzein, glycitein and genistein are the main isoflavones in the soybeans, which account for above 90% of the total isoflavones.30 Therefore, the six isoflavones of the soybean extracts were analyzed by HPLC analysis. The various extracts of NFS and RFS (obtained in Section 2.4) were filtered with a syringe filter (0.45 μm PVDF membrane, Bedford, MA, USA), then subjected to HPLC system for the qualitative and quantitative analysis of specific isoflavones as previously described.24 The HPLC system (Agilent Technologies, Wilmington, DE, USA) equipped with a ZORBAX SB-C18 reverse-phase column, 4.6 × 250 mm, 5 μm particle size (Eclipse Plus, Agilent Technologies, Wilmington, DE, USA) at 25 °C. Preceding the analytical column was a ZORBAX SB-C18 guard column (4.6 × 12.5 mm, 5 μm particle size, Eclipse Plus, Agilent Technologies, Wilmington, DE, USA), used to prevent any insoluble residues of samples from contaminating the analytical column. A G1311 Quat Pump, AutoSampler, G1314A VWD detector and Star Work Station software (version Rev.A.10.02) was conducted. The mobile phase consisted of solvent A (0.1% trifluoroacetic acid) and solvent B (acetonitrile) at a flow rate of 0.8 mL min−1. Gradient elution was employed, starting with 5% B for 3 min, then 5–10% B over 5 min, 10–25% B for 12 min, 25–50% B over 12 min, and finally solvent B increased to 100% in 8 min and maintained for 1 min. The injector volume was 20 μL. Detection was performed at 254 nm. The isoflavones were identified by a comparison of their retention times and the UV spectra with those of authentic standards. The results of the analyzed isoflavones were expressed as micrograms per gram of dry weight sample (μg g−1 d.w.).
2.7 Antioxidant activities determination
2.7.1 Determination of DPPH radical scavenging activity. The DPPH radical scavenging activity of the sample was determined according to the method of Xiao et al.29 In brief, 2 mL of test sample solution was mixed with 2 mL of DPPH solution (0.2 mM), and then the reaction mixture was shaken well and incubated for 30 min at room temperature. Then the absorbance of the sample solution was read at 517 nm against a blank in a spectrophotometer (model 4001/4, GENESYS 20 Thermo-Spectronic, Thermo Electron Corp., Waltham, MA, USA). The absorbance of the DPPH solution was also read at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. A standard curve was prepared by using different concentrations of ascorbic acid (vitamin C). The final results were expressed as micrograms of vitamin C equivalents (VCE) per gram of dry weight sample (μg VCE g−1 d.w.).
2.7.2 Evaluation of ABTS radical cation scavenging activity. The ABTS radical cation (ABTS˙+) scavenging activity of the test sample was analyzed by using the method of Zhao et al.31 with minor modifications, as detail reported by Xiao et al.32 ABTS˙+ solution was generated by oxidation of ABTS with potassium persulphate. About 1 mL of different sample extracts was mixed with 4 mL of ABTS˙+ solution, and then the absorbance was read at 734 nm after a reaction of 6 min. The vitamin C calibration curve was plotted as a function of the percentage of ABTS˙+ scavenging activity similar to DPPH radical scavenging assay. The results were expressed as μg VCE g−1 d.w.
2.7.3 Estimation of ferric reducing antioxidant power (FRAP). FRAP of the test samples was carried out according to the method of Đorđević et al.33 with slight modifications. The FRAP reagent was prepared by mixing with 10 mM TPTZ (in 40 mM HCl), 0.3 M acetate buffer (pH 3.6) and 20 mM ferric chloride (10
:
1
:
1, v/v/v). One milliliter of the test sample was added to 5 mL FRAP reagent, and then the mixture was incubated at 37 °C for 20 min. The absorbance was read at 593 nm against a blank. The measurement was compared to a calibration curve of ferrous sulfate solution, and the results were expressed as micromoles of Fe(II) equivalents per gram of dry weight sample (μmol Fe(II) g−1 d.w.).
2.7.4 Assay of reducing power. The reducing power of the samples was determined based on the method of Zhao et al.31 In brief, 0.5 mL of sample solution was mixed with 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (1%, w/v). The mixture was incubated at 50 °C for 20 min. After a portion (2.5 mL) of trichloroacetic acid (10%, w/v) was added, the mixture was then centrifuged at 420g for 10 min. The upper layer of solution (2.5 mL) was mixed with 0.5 mL ferric chloride (0.1%, w/v) for 10 min at room temperature. The absorbance was then read at 700 nm against a blank in a spectrophotometer. A higher absorbance of the reaction mixture indicates a higher reducing power. The vitamin C calibration curve was plotted as a function of the reducing power. The results were expressed as μg VCE g−1 d.w.
2.7.5 Evaluation of ferrous ion chelating activity. The chelating ability of the extracts was measured as reported by Juan and Chou34 and Wang et al.35 Briefly, 1 mL of the sample was mixed with 3.7 mL of deionized water and then the mixture was reacted with ferrous chloride (2 mM, 0.1 mL) and ferrozine (5 mM, 0.2 mL) for 20 min at room temperature. The absorbance was then read at 562 nm. Besides, one milliliter of deionized water instead of the sample was added in the control solution. The EDTA-2Na calibration curve was plotted as a function of the chelating ability. The final results were expressed as micromoles of EDTA-2Na equivalents per gram of dry weight sample (μmol EDTA-2Na g−1 d.w.).
2.7.6 Assay of hydroxyl radical scavenging activity. Hydroxyl radical scavenging activity was measured according to the method reported by Zhao et al.36 with slight modifications. Briefly, 1 mL of test sample solution was mixed with 1 mL FeSO4 (9 mM), and then 1 mL H2O2 (8.8 mM) was added into the solution and mixed well. Subsequently, 1 mL of salicylic acid–ethanol solution (9 mM) was added in the test tubes and mixed thoroughly. The mixture was incubated at 37 °C for 60 min, and the absorbance of the mixture was measured at 510 nm against a blank. The vitamin C calibration curve was plotted as a function of the percentage of hydroxyl radical scavenging activity. The results were expressed as μg VCE g−1 d.w.
2.7.7 Silver nanoparticle-based (AgNP) method. The samples were also determined by a novel antioxidant assay which based on the proposed AgNP spectrophotometric method as detail reported by Özyürek et al.37 Fresh silver nanoparticles working solution for the assay was prepared as follows: 50 mL AgNO3 (1.0 mM) was heated to boiling for 10 min, and then 5 mL trisodium citrate (1%, w/v) was added drop by drop. During the process, the solution was vigorously mixed. The solution was then heated until its colour change was evident (pale yellow) and then cooled down to room temperature. 0.2 mL of sample solution was mixed with 2 mL of initial AgNP solution and 0.6 mL H2O, and then the mixture was placed in the dark at 25 °C for 30 min. The absorbance of pale yellow solutions was measured at 423 nm against a reagent blank. The absorbance of the reagent blank increased in the presence of antioxidants and the increment of absorbance being proportional to antioxidant concentration. Calibration curve for the AgNP method was prepared by using gallic acid as a standard. The results were expressed as μg GAE g−1 d.w.
2.8 Assessment of supercoiled plasmid DNA strand breakage inhibition
To evaluate the DNA damage protective effect of the soybean samples, the original extracts (obtained in Section 2.4) of NFS and RFS were firstly diluted with fivefold, and then performed according to the method of Xiao et al.18,29 The damage solution Fenton's reagent was prepared with FeCl3 (80 mM), ascorbic acid (50 mM) and H2O2 (30 mM). 10 μL of the sample solution and 1 μL pUC18 plasmid DNA (200 ng μL−1) was mixed, and then followed by the addition of 10 μL Fenton's reagent. The mixture was then incubated for 30 min at 37 °C and the DNA was electrophoresized on 1% (w/v) agarose gel for 45 min under 100 V condition, followed by ethidium bromide staining and visualized under UV-transilluminator using Gel Doc XR system (Bio-Rad, Hercules, CA, USA). The optical density of each DNA band was obtained using Quantity One software, version 4.6.2 (Bio-Rad). The protective effect of NFS and RFS extracts was calculated based on the method of Chandrasekara and Shahidi38 with slight modifications, as detail reported by Xiao et al.18 The supercoiled DNA (%) was calculated as follows: supercoiled DNA (%) = [As/(As + Ao)] × 100. Where As is the optical density of the supercoiled DNA, and Ao is the optical density of oxidative damaged DNA (open circular form and linear form) in each gel lane. A higher supercoiled DNA (%) indicated a higher protection effects for damaged DNA of the test samples.
2.9 In vitro gastrointestinal digestion model for bioaccessibility studies
The bioaccessibility was determined by using a simulated in vitro digestion model system previously described by He et al.39 with minor modifications. Briefly, to study gastric digestion, the soybean samples (1 g) were mixed with 20 mL deionized water and then acidified to pH 2.0 with 6 M HCl. The sample was then mixed with 3 mL of a pepsin solution (40 mg mL−1, in 0.1 M HCl). The mixture was incubated for 2 h in a shaking water bath (SHZ-28A, Huamei Biochemistry Instrument Factory, Taicang, China) at 37 °C and 120 rpm. The gastric digests were maintained in ice for 15 min to stop the pepsin digestion. To study the intestinal digestion (separate sets of experiment), the same procedures were followed till the 2 h pepsin incubation, but continued by adjusting the pH to 7.0 by drop-wise addition of 0.1 M NaOH in order to simulate the small intestinal environment. After that, 4.5 mL of pancreatin–bile solution (pancreatin 4 mg mL−1, bile 25 mg mL−1 in 20 mM phosphate buffer, pH 7.0) was added. Subsequently, the digesta was incubated in a shaking water bath at 37 °C for another 2 h. A blank (without the added sample) was prepared and underwent the above-mentioned gastrointestinal digestion process to correct the interferences. After digestion, the gastric and intestinal digesta were centrifuged at 10
000g for 50 min at 4 °C, and the supernatants (bioaccessible fraction) were obtained for the assays of isoflavones, total phenolic content and antioxidant activity as described earlier. This system was used to simulate the human gastric and small intestinal digestive process. All experiments were done in triplicate.
The bioaccessibility of isoflavones and total phenolic in the soybean samples was used to measure their digestive release and stability in the stomach and gut, and their subsequent absorption. According the previous studies reported by Fonteles et al.40 and He et al.,39 the absolute bioaccessibility was defined as the amount of phenolic compounds recovered in the supernatants of the centrifuged final digesta (bioaccessible fraction), and expressed as microgram of phenolic compounds per gram dry weight sample (μg g−1 d.w.).
2.10 Statistical analysis
All values were expressed as the mean ± standard deviation (SD) of three independent experiments with samples in triplicate. One-way analysis of variance (ANOVA) and Duncan's multiple range test or independent sample T-test were carried out to determine significant differences (p < 0.05) between the means by SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) software package for Windows. Correlations among variables were examined using a two-tailed Pearson's correlation analysis. Principal component analysis was used to gain an overview of the relationships between total phenolic content, isoflavone content and antioxidant activity.
3. Results and discussion
Fig. 2 shows the growing status of R. oligosporus RT-3 in soybeans at different incubation time (0, 12, 15, 18, 20, 22, 24 and 30 h). It was illustrated in Fig. 2 that 22 h was the optimum incubation time for soybeans when they were incubated with R. oligosporus RT-3 starter. The mycelium of R. oligosporus RT-3 covered soybeans completely after 22 h cultivation continually. In addition, soybeans were tightly linked with the mycelium of R. oligosporus as well as emitting a uniquely fresh and slight yeast aroma during that period. However, an unpleasant odor was detected when the fermentation period was over that time. Besides, a weak texture and the mycelium of R. oligosporus RT-3 did not completely cover soybeans were showed when the incubation time was less than 22 h. Therefore, 22 h was the best fermentation time based on the growing status of R. oligosporus RT-3, and was used for subsequent antioxidant activity analysis. Furthermore, the relevant fermented soybeans were named as R. oligosporus RT-3 fermented soybeans (RFS).
 |
| Fig. 2 Photograph of Rhizopus oligosporus RT-3 growth in soybeans during different incubation time. | |
3.1 Total phenolic content (TPC)
Phenolic compounds have received considerable attention because of their physiological function, including antioxidant activities.1,38,41,42 Solvent extraction is a common method used to obtain phenolic compounds from plant materials. However, it has been suggested that no single solvent can work on all compounds from food matrix due to its different solubility and polarity. According to previous studies, the different polarities solvents might influence the solubility of chemical constituents in a sample and its extraction yield.43,44 Therefore, non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) were extracted using four different polarities solvent systems that are 80% acetone, 80% ethanol, 80% methanol and deionized water. The effects of these solvent systems in extracting phenolics and antioxidants from NFS and RFS were quantitatively measured and compared.
As shown in Table 1, the extraction of phenolic compounds in NFS and RFS was significantly influenced by the polarity of extracting solvents. Depending on the solvent used for extraction, TPC ranged from 1355.21 ± 68.31 to 1685.07 ± 43.37 μg GAE g−1 d.w. and from 3348.26 ± 39.44 to 7768.40 ± 171.27 μg GAE g−1 d.w. with the extracts of NFS and RFS, respectively. These results demonstrated that the extraction solvents had an obvious effect on TPC. The apparent variation of TPC in four different extracts of NFS and RFS revealed that these different polarities solvent could affect the solubility of the phenolics in the samples and the extraction yields, depending on their chemical structures and polarities. Furthermore, it was found that the water extracts contained the highest TPC among the various solvent extracts of NFS and RFS, respectively. Hence, water was considered as the most efficient solvent system for extracting phenolic compounds from the soybean samples in the present study when TPC was used as evaluation index.
Table 1 Total phenolic content (TPC) of non-fermented soybeans and R. oligosporus-fermented soybeans extracted with various solventsa
Solvent |
Total phenolic content (μg GAE g−1 d.w.) |
Non-fermented soybeans |
R. oligosporus-fermented soybeans |
Each value was expressed as the mean ± standard deviation (n = 3). Means with different lower case letters (a, b) within a row indicate significant differences (p < 0.05). Means with different upper case letters (A, B, C, D) within a column demonstrate significant differences (p < 0.05). |
80% methanol |
1355.21 ± 68.31Bb |
3348.26 ± 39.44Da |
80% ethanol |
1389.93 ± 43.37Bb |
3553.13 ± 47.74Ca |
80% acetone |
1403.82 ± 12.03Bb |
3754.51 ± 51.38Ba |
Water |
1685.07 ± 43.37Ab |
7768.40 ± 171.27Aa |
Additionally, it was also noted that the TPC of the extracts of RFS were significantly higher (p < 0.05) than the respective extracts of NFS, regardless of the extraction solvents employed in the present study. The TPC of the methanol, ethanol, acetone, and water extracts of RFS were 3348.26, 3553.13, 3754.51 and 7768.40 μg GAE g−1 d.w., respectively, which were approximately 2.47-, 2.56-, 2.67-, and 4.61-fold higher, respectively, compared with the respective extracts of NFS. The results clearly demonstrated that SSF with R. oligosporus enhanced the TPC of soybeans. Previous studies also reported that TPC of the legumes, namely, black soybeans, chickpeas and common beans were apparently enhanced by SSF with R. oligosporus.24,25,45 In addition, other investigators demonstrated that cereals (e.g., wheat, brown rice, oat and maize) fermented with R. oligosporus or Aspergillus oryzae could also improve their TPC.17,46 In plants, phenolic compounds are usually presented as simple soluble-free esters and, to a greater extent, as complex insoluble bound esters with proteins, lipids, polysaccharides, or cell walls.17,47 SSF is a complex biochemical process, many enzymes (such as xylanases, amylases, and proteases) are produced and contribute to the modification of sample composition, and bound phenolics are released through the enzymatic treatment of samples and prior to extraction. Cheng et al.24 stated that the β-glucosidase produced by R. oligosporus could hydrolyze conjugated phenolics to free phenolics and enhanced the TPC of fermented black soybeans during SSF. In addition, fermentation-induced structural breakdown of plant cell walls also leads to the liberation of phenolic compounds.15,48 Therefore, the increased in TPC of RFS can be attributed to fungal hydrolytic enzymes (e.g. β-glucosidase) produced by R. oligosporus, which can lead to a soft kernel structure and liberation of phenolics during SSF.
3.2 Content of major phenolic compounds in NFS and RFS
Table 2 shows the content of major phenolic compounds in the NFS and RFS. In the present investigation, the six major isoflavones in the various extracts of NFS and RFS, namely, daidzin, glycitin, genistin, daidzein, glycitein and genistein, were identified and quantified by HPLC analysis (Table 2 and Fig. 3). Similar to TPC, Table 2 revealed that the changes in solvent polarity resulted in significantly different extraction capacities and altered the solvent's ability to dissolve isoflavones in soybean samples. In addition, it was found that the total glucoside forms were much greater than the aglycone forms in NFS. Depending on the solvent employed for extraction, the isoflavone concentrations of total glucosides and total aglycones ranged from 985.01 to 1648.92 and from 38.60 to 80.91 μg g−1 d.w., respectively, with the extracts of NFS (Table 2). However, the glucoside aglycones were significantly decreased whereas their corresponding aglycone isoflavones were remarkably increased after SSF with R. oligosporus. For example, the dadzein, glycitein and genistein contents of the methanol extract of RFS were 304.45, 61.55 and 559.85 μg g−1 d.w., respectively, which were approximately 13.14-, 5.49-, and 18.79-fold higher, respectively, compared with those of NFS. The accumulation of isoflavone aglycones via microbial fermentation has been observed in many previous studies.6,19 Cheng et al.24 reported that black soybean processed by SSF with R. oligosporus BCRC 31996 significantly (p < 0.05) enhanced dadzein and geistein. da Silva et al.6 and Marazza et al.49 attributed the increment of aglycone forms to the action of the β-glycosidase produced by the microorganisms, which could hydrolyze the glucoside forms into aglycone forms during fermentation. Previous studies also have demonstrated that R. oligosporus could produce sufficient β-glycosidase during fermentation.23,24,47 Therefore, the significantly enhanced aglycones of RFS might be attributed to the produced β-glucosidase, which can promote the transformation of isoflavone glucosides into their corresponding aglycones.
Table 2 Major phenolics composition (i.e. glucoside and aglycone isoflavones) of non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) extracts. The results were expressed as μg g−1 of dry weight samplea
Isoflavones |
80% methanol |
80% ethanol |
80% acetone |
Water |
NFS |
RFS |
NFS |
RFS |
NFS |
RFS |
NFS |
RFS |
Determinations were performed in triplicate, and data correspond to mean values. Different small letters (a, b) within a row at the same solvent extraction indicate significant differences (p < 0.05) between NFS and RFS. Means with different capital letters (A, B, C, D) in the same row indicate significant differences (p < 0.05) between 80% methanol, 80% ethanol, 80% acetone, and water extracts from NFS or RFS. |
Glucosides |
Daidzin |
684.70 ± 19.70Aa |
222.53 ± 27.10Ab |
626.80 ± 7.46Ba |
117.11 ± 12.52Bb |
672.97 ± 36.28ABa |
99.07 ± 11.93Bb |
464.20 ± 13.12Ca |
83.04 ± 7.05Bb |
Glycitin |
113.99 ± 5.77Aa |
38.40 ± 4.48Ab |
101.38 ± 3.28Aa |
19.70 ± 2.03Bb |
108.05 ± 11.68Aa |
19.08 ± 2.86Bb |
96.96 ± 0.93Aa |
41.91 ± 6.27Ab |
Genistin |
850.23 ± 23.42Aa |
314.08 ± 39.68Ab |
786.64 ± 9.91Aa |
190.55 ± 16.17Bb |
845.68 ± 41.62Aa |
164.77 ± 9.02Bb |
423.84 ± 20.04Ba |
54.70 ± 6.59Cb |
Total glucosides |
1648.92 ± 48.79Aa |
575.00 ± 69.92Ab |
1514.82 ± 20.27Aa |
327.36 ± 30.64Bb |
1626.70 ± 89.15Aa |
282.92 ± 23.74Bb |
985.01 ± 34.05Ba |
179.64 ± 7.81Cb |
![[thin space (1/6-em)]](https://www.rsc.org/images/entities/char_2009.gif) |
Aglycones |
Daidzein |
23.17 ± 1.46Bb |
304.45 ± 1.69Ca |
26.03 ± 0.74ABb |
333.42 ± 6.97Ba |
29.30 ± 1.52Ab |
355.10 ± 13.36Aa |
23.23 ± 2.31Bb |
240.35 ± 3.48 Da |
Glycitein |
11.21 ± 0.76Ab |
61.55 ± 1.66Ba |
11.44 ± 0.72Ab |
63.75 ± 1.08Ba |
13.24 ± 2.13Ab |
88.06 ± 15.26Aa |
8.22 ± 0.49Bb |
25.14 ± 0.83Ca |
Genistein |
29.79 ± 1.14Bb |
559.85 ± 16.87Ba |
34.86 ± 1.00ABb |
617.72 ± 7.88ABa |
38.38 ± 1.77Ab |
645.56 ± 11.31Aa |
7.15 ± 4.10Cb |
414.93 ± 53.99Ca |
Total aglycones |
64.17 ± 2.19Bb |
925.85 ± 15.22Ca |
72.32 ± 1.64ABb |
1014.88 ± 14.48Ba |
80.91 ± 4.81Ab |
1088.73 ± 13.24Aa |
38.60 ± 14.95Cb |
680.42 ± 58.23 Da |
 |
| Fig. 3 High performance liquid chromatographic (HPLC) chromatogram of the major six isoflavones presented in the non-fermented soybeans and R. oligosporus-fermented soybeans. Numbers show the following standard chemicals: 1, daidzin; 2, glycitin; 3, genistin; 4, daidzein; 5, glycitein; 6, genistein. | |
The bioactive activity (e.g., antioxidant activity) of isoflavone glucosides and aglycones is different. Previous studies have reported that the isoflavone aglycones showed stronger antioxidant activity than their glucosides.9,19,50 Many studies demonstrated that most isoflavones in soybean are presented in glucoside forms and are converted into aglycones during fermentation, and thereby the fermented soybeans showed stronger bioactivity activity. Cheng et al.,24 Xiao et al.32 and Landete et al.51 reported that microbial fermentation was able to efficiently bio-transform isoflavone glycosides to aglycones, and thus increased the antioxidant activity of soybean products. Hence, RFS rich in isoflavone aglycones may be more efficient for the prevention of oxidative damage-induced chronic diseases (such as coronary heart, cardiovascular and cancer) than NFS rich in isoflavone glucosides.
3.3 Antioxidant activities
Several methods have been used to evaluate the antioxidant effect of plants and plant products. However, the specificity and sensitivity of one single method do not lead to complete examination of all antioxidant compounds in the sample.52 Therefore, it is necessary to perform more than one type of antioxidant activity measurement to take into account the variant mechanisms of antioxidant action. In this study, six complementary antioxidant activity methods (i.e. DPPH radical scavenging activity, ABTS˙+ scavenging ability, ferric reducing antioxidant power, reducing power, chelating ability, hydroxyl radical scavenging activity) and a novel silver nanoparticle-based (AgNP) method with different approaches and mechanisms were followed to evaluate the antioxidant properties of NFS and RFS. These antioxidant assays were widely used due to their simplicity, stability and accuracy.
The antioxidant activities of the different polarities solvent extracts of NFS and RFS are presented in Fig. 4A–G. As shown in Fig. 4, similar to TPC, it was noted that SSF with R. oligosporus significantly (p < 0.05) enhanced the antioxidant activities of soybeans regardless of the extraction solvents used. For example, DPPH radical scavenging activity, ABTS˙+ scavenging activity, ferric reducing antioxidant power, reducing power, chelating ability, hydroxyl radical scavenging activity and AgNP method of methanolic extract of RFS was 781.21 ± 36.74 μg VCE g−1 d.w., 5939.61 ± 140.15 μg VCE g−1 d.w., 12.08 ± 0.12 μmol Fe(II) g−1 d.w., 2016.19 ± 81.23 μg VCE g−1 d.w., 13.81 ± 0.32 μmol EDTA-2Na g−1 d.w., 7521.59 ± 480.54 μg VCE g−1 d.w. and 188.75 ± 2.71 μg GAE g−1 d.w., respectively, which was about 8.81-, 2.52-, 2.58-, 3.25-, 9.03-, 2.95- and 2.00-fold higher, respectively, compared to the respective extract of NFS (Fig. 4). SSF with fungi enhanced antioxidant activities compared with the non-fermented legume products; similar results were also found in black soybean fermented with Rhizopus spp.,24 soybean fermented with T. harzianum,10 chickpeas or oats fermented with C. militaris.18,29 It was well documented in previous studies that the enhanced antioxidant property of fermented products was mainly due to the produced phenolic compounds during fermentation process.7,10,15,53 Furthermore, several studies have demonstrated that the isoflavone aglycones (i.e. daidzein, glytitein and genistein) formed during the fermentation process exhibited stronger antioxidant activity than their β-glucoside precursors (i.e. daidzin, glycitin and genistin).9,24 Shin et al.7 also stated that the enhanced antioxidant activity of fermented soybean positively correlated to the markedly higher TPC and isoflavone aglycones content achieved during SSF. Therefore, the enhanced antioxidant activity of fermented soybeans might be due to the increased TPC and isoflavone aglycones during the fermentation process. Thus, Pearson's correlation analysis was performed to further investigate the interrelationship among TPC, isoflavone content and antioxidant activities, and the results are summarized in Table 3. Strong positive correlations (p < 0.05) were observed between TPC, isoflavone aglycone contents and antioxidant activities. For instance, the positive correlation coefficients among daidzein and different antioxidant analytical methods ranged from 0.524 to 0.998. These results were supported by previous studies that the phenolics and isoflavone aglycones might be responsible for a large proportion of the antioxidant activity in soybean samples.7,19,24,32 Therefore, the presence of higher phenolics and isoflavone aglycones in the extracts of RFS significantly contributed to their stronger antioxidant capacity than NFS.
 |
| Fig. 4 DPPH radical scavenging effects (A), ABTS radical cation scavenging ability (B), ferric reducing antioxidant power (C), reducing power (D), chelating ability (E), hydroxyl radical scavenging activity (F) and a silver nanoparticle-based method for determination of antioxidant capacity (G) of the various extracts from non-fermented soybeans and R. oligosporus-fermented soybeans. ( ) and ( ) represent non-fermented soybeans and R. oligosporus-fermented soybeans, respectively. Each value was expressed as mean ± standard deviation (n = 3). Means with different small letters (a, b, c and d) indicated significantly different (p < 0.05) among the different solvent extracts of non-fermented soybeans. Means with different capital letters (A, B, C and D) indicated significantly different (p < 0.05) among the different solvent extracts of R. oligosporus-fermented soybeans. The symbol (**) indicated a significant difference (p < 0.05) between non-fermented soybeans and R. oligosporus-fermented soybeans. | |
Table 3 Pearson's correlation coefficients among total phenolic content (TPC), contents of glucoside isoflavones and aglycone isoflavones, and antioxidant activitya
|
TPC |
Daidzin |
Glycitin |
Genistin |
Daidzein |
Glycitein |
Genistein |
TGI |
TAI |
DPPH |
ABTS |
FRAP |
RP |
CHA |
OH |
AgNP |
*Correlation was significant at the 0.05 level (two-tailed). **Correlation was significant at the 0.01 level (two-tailed). DPPH, DPPH radical scavenging activity; ABTS, ABTS radical cation scavenging activity; FRAP, ferric reducing antioxidant power; RP, reducing power; CHA, chelating ability; OH, hydroxyl radical scavenging activity; AgNP, a silver nanoparticle-based method for determination of antioxidant capacity; TGI, total glucoside isoflavones; TAI, total aglycone isoflavones. |
TPC |
1 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Daidzin |
−0.802** |
1 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Glycitin |
−0.676** |
0.964** |
1 |
|
|
|
|
|
|
|
|
|
|
|
|
|
Genistin |
−0.814** |
0.980** |
0.899** |
1 |
|
|
|
|
|
|
|
|
|
|
|
|
Daidzein |
0.636** |
−0.928** |
−0.982** |
−0.843** |
1 |
|
|
|
|
|
|
|
|
|
|
|
Glycitein |
0.343 |
−0.762** |
−0.874** |
−0.657** |
0.915** |
1 |
|
|
|
|
|
|
|
|
|
|
Genistein |
0.616** |
−0.913** |
−0.978** |
−0.822** |
0.997** |
0.925** |
1 |
|
|
|
|
|
|
|
|
|
TGI |
−0.807** |
0.996** |
0.941** |
0.994** |
−0.896** |
−0.722** |
−0.878** |
1 |
|
|
|
|
|
|
|
|
TAI |
0.608** |
−0.913** |
−0.978** |
−0.823** |
0.998** |
0.931** |
1.000** |
−0.878** |
1 |
|
|
|
|
|
|
|
DPPH |
0.461* |
−0.868** |
−0.939** |
−0.798** |
0.934** |
0.874** |
0.936** |
−0.844** |
0.936** |
1 |
|
|
|
|
|
|
ABTS |
0.987** |
−0.817** |
−0.705** |
−0.819** |
0.678** |
0.379 |
0.659** |
−0.818** |
0.650** |
0.504* |
1 |
|
|
|
|
|
FRAP |
0.492* |
−0.863** |
−0.947** |
−0.771** |
0.952** |
0.941** |
0.954** |
−0.828** |
0.957** |
0.940** |
0.504* |
1 |
|
|
|
|
RP |
0.305 |
−0.738** |
−0.878** |
−0.615** |
0.909** |
0.945** |
0.920** |
−0.690** |
0.923** |
0.928** |
0.345 |
0.958** |
1 |
|
|
|
CHA |
0.868** |
−0.754** |
−0.585** |
−0.828** |
0.524** |
0.211 |
0.498* |
−0.788* |
0.490* |
0.454* |
0.890** |
0.337 |
0.17 |
1 |
|
|
OH |
0.792** |
−0.796** |
−0.620** |
−0.894** |
0.539** |
0.294 |
0.510* |
−0.842* |
0.508* |
0.516** |
0.782** |
0.431 |
0.226 |
0.931** |
1 |
|
AgNP |
0.352 |
−0.764** |
−0.877** |
−0.657** |
0.911** |
0.887** |
0.921** |
−0.723** |
0.921** |
0.958** |
0.425* |
0.895** |
0.949** |
0.333 |
0.329 |
1 |
Fig. 4 also illustrates that various polarities solvent extracts from NFS and RFS showed significant (p < 0.05) differences in their antioxidant activities. Despite the variant antioxidant activity exerted by the various solvent extracts examined, this antioxidant activity was found to be related to the content of phenolics and isoflavone aglycones in the extracts (Tables 1 and 2). A selected solvent system might have different efficacy for extracting individual antioxidant compounds. Changes in solvent polarity alter the ability to dissolve a selected group of antioxidant compounds and influence the antioxidant activity estimation.34,54 As TPC, isoflavone aglycones contents and antioxidant activities were markedly correlated (Table 3),9,24 the differences in antioxidant activities of the four kinds of solvent extracts from NFS and RFS might be probably due to the difference in solvent selectivity for extracting phenolics and isoflavone aglycones (Tables 1 and 2). Therefore, the discrepant levels of TPC and isoflavone aglycones in the studied extracts contributed to their variant antioxidant capacity of the various extracts of NFS and RFS. In addition, Table 3 also showed that there is significantly positive correlations between the novel AgNP assay and DPPH, ABTS, FRAP, RP, daidzein, glycitein, genistein, TAI (the correlation coefficients ranging from 0.425 to 0.949, p < 0.05). To the best of our knowledge, this is the first study to report on the antioxidant activity of soybean samples determined by AgNP assay, and also demonstrated the positive correlations between AgNP assay and DPPH, ABTS, FRAP, RP, daidzein, glycitein, genistein, TAI.
3.4 Inhibition of hydroxyl radical induced supercoiled plasmid DNA strand breakage
Free radicals are known for breaking and damaging DNA strand which might eventually contribute to many diseases, including mutagenesis, carcinogenesis and cytotoxicity.38 It is well documented that frequently associated oxidative stress occurring in biological systems is attributed to hydroxyl radical. Hydroxyl radical is reactive oxygen species that possess deleterious effects on biological systems. The negative effects may take place in both extra- and intra-cellular media. The inhibition of supercoiled plasmid DNA strand breakage is a useful biological marker to evaluate the potential bioactivity of the sample. Hydroxyl radical generated by the Fenton reaction are known to cause DNA damage and yield fragmented forms.18,29,55 The treatment of pUC18 plasmid DNA with Fenton's reagent directed the alteration of supercoiled DNA (native form) to open circular form and linear form (oxidative damaged DNA). In this study, the change of DNA conformation was evaluated by electrophoresis (Fig. 5).
 |
| Fig. 5 DNA damage protecting effect of 80% methanolic, 80% ethanolic, 80% acetone and water extracts of non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) against hydroxyl radical induced DNA damage of pUC18. M-, E-, A- and W-represents the samples were extracted with 80% methanol, 80% ethanol, 80% acetone and water, respectively. Lane 1: native pUC18 plasmid DNA (control); lane 2: DNA + Fenton's reagent (DNA damage control, blank); lane 3: DNA + Fenton's reagent + M-RFS; lane 4: DNA + Fenton's reagent + E-RFS; lane 5: DNA + Fenton's reagent + A-RFS; lane 6: DNA + Fenton's reagent + W-RFS; lane 7: DNA + Fenton's reagent + M-NFS; lane 8: pUC18 DNA + Fenton's reagent + E-NFS; lane 9: pUC18 DNA + Fenton's reagent + A-NFS; lane 10: pUC18 DNA + Fenton's reagent + W-NFS. The gels were visualized under UV-transilluminator using Gel Doc XR system (Bio-Rad, Hercules, CA, USA). Assessment of the protective of DNA damage gels were analyzed as three separate gels and all samples were analyzed in triplicate. | |
In the hydroxyl radical-induced DNA damage assay, the control plasmid pUC18 DNA was mainly composed of the supercoiled form (approximately 78.26%) in the absence of Fenton's reagent (Fig. 5, lane 1, control). During the addition of Fenton's reagent, the supercoiled form of DNA was shown to totally breakdown and converted into the relaxed circular and linear forms (Fig. 5, lane 2, DNA damage control). However, the addition of various extracts of NFS and RFS provided protection to plasmid DNA, resulting in the retention of the native form (supercoiled form, lanes 3–10 of Fig. 5, and 6). The NFS and RFS samples significantly inhibited the oxidation of DNA induced by Fenton's reagent, possibly due to the high phenolic and flavonoid content in the extracts. Liyana-Pathirana et al.56 stated that phenolics effectively inhibited hydroxyl radical-mediated DNA scission. The inhibition of supercoiled DNA scission induced by hydroxyl radical may be explained by two mechanisms as previously demonstrated. Previous studies have reported that redox-active metals in solution might bind to phenolic and flavonoid compounds and form the complexes, consequently preventing the reduction of redox-active metal ions with H2O2, and then preventing the generation of hydroxyl radical.18,38 In addition, phenolic extracts might directly quench of hydroxyl radical by donating hydrogen-atom or electron, and therefore protecting the supercoiled plasmid DNA from hydroxyl radical dependent strand breaks.18,38 Thus, NFS and RFS extracts are rich in phenolic compounds which scavenge free radicals and provide protection against DNA damage caused by Fenton's reagent. Besides, the results observed in Fig. 5 and 6 revealed that various extracts of NFS and RFS showed significant (p < 0.05) differences in their DNA damage protection effect. Different phenolics content and flavonoids in the extracts might result in their discrepancies in DNA damage protection. In addition, for NFS, the acetone extract showed the weakest DNA damage protection (Fig. 5 and 6) whereas the phenolic content in the extract was not the lowest (Tables 1 and 2). These findings indicated that not only the phenolics content, but also the type of phenolic constituent, and the chemical structures present in the phenolic extract might be responsible for DNA damage protective activity.57 Furthermore, Apostolou et al.58 demonstrated that there was a synergism between some phenolics or between phenolics and other phytochemical compounds, which should also be considered for DNA damage protection.
 |
| Fig. 6 Protective effect of the various extracts from non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) upon DNA oxidation induced by Fenton's reagent. M-, E-, A- and W-represents the samples were extracted with 80% methanol, 80% ethanol, 80% acetone and water, respectively. The optical density of the band was quantified using Quantity One software, version 4.6.2 (Bio-Rad, Hercules, CA, USA). Data were presented as the mean ± standard deviation of three experiments. Bars with different letters indicated significantly (p < 0.05) different among the samples. The letter ‘a’ represents the highest value. ND, no detected. A higher supercoiled DNA (%) indicated a higher protection effects for damaged DNA of the test samples. | |
Furthermore, the extracts of RFS exhibited significantly (p < 0.05) greater protection of supercoiled DNA than the respective extracts of NFS, as shown in Fig. 5 and 6. For instance, the water extract of RFS showed supercoiled DNA protection of 66.50 ± 1.90%, which was about 2.84-fold higher compared to the respective extract of NFS (Fig. 6). In addition, the methanolic extract of NFS and RFS both exhibited strong protection of supercoiled DNA for the higher supercoiled DNA (>60%). It was also reported by Singh et al.10 and Xiao et al.29,32 that fermented legume products showed higher DNA damage protection than unfermented legume products due to higher phenolics and isoflavone aglycone contents in the fermented samples. Marazza et al.19 also reported that the enhanced inhibition of oxidative-damage DNA of fermented products mainly due to a higher concentration of isoflavone aglycones. The isoflavone aglycones contained free phenolic groups in their structures cable of scavenging hydroxyl radical generated by Fenton's reaction. Therefore, higher accumulated phenolics and isoflavone aglycones of RFS is thus an indicative of enhanced protection of supercoiled DNA. These results are greatly interest because this is the first time that the ability of fermented soybeans using R. oligosporus to inhibit the plasmid pUC18 DNA oxidation has been reported, and RFS exhibited stronger DNA damage protection than NFS.
3.5 Principal component analysis (PCA) of different antioxidant analytical methods, TPC, and isoflavones in soybean samples
Phenolics and isoflavones have been reported to be mainly responsible for the antioxidant activities of soybean materials. DPPH radical scavenging activity, ABTS˙+ scavenging ability, ferric reducing antioxidant power, reducing power, chelating ability, hydroxyl radical scavenging activity and a silver nanoparticle-based method all have been used to evaluate antioxidant activity of NFS and RFS. Therefore, PCA was performed to gain an overview of the relationships among TPC, isoflavones content and antioxidant activities, and the results are shown in Fig. 7A. The first two principal components accounted for 95.22% of the total variance in the data set. The remaining principal components (PC) were probably ineffective for the very small proportion (4.78%) of total variability. It was well documented in previous studies that the loadings in the PCA loading plot described not only how well the principal components were correlated with the original variables, but also how correlated variables were explained by the same principal component and less correlated variables were explained by different principal component.18,31 As shown in Fig. 7A, the first principal component (PC1) positively correlated well with DPPH, FRAP, RP, AgNP and all aglycone isoflavones (daidzein, glycitein and genistein) due to their high loading on PC1, which indicated significant correlations between the aglycone isoflavones and DPPH, FRAP, RP and AgNP. In addition, it can be noted in Fig. 7A that ABTS, CHA, OH and TPC were found to be similarly loaded on PC1 suggested that there was close correlation among ABTS, CHA, OH and TPC. Besides, TPC, aglycone isoflavones and all different antioxidant evaluated methods loaded on the right side of PC1, which suggested that the antioxidant activities of the assayed samples was mainly ascribed to the TPC and aglycone isoflavones. Furthermore, the correlation among the antioxidant activity evaluation indices, TPC and isoflavones observed by PCA were similar to the conclusions drawn from Pearson's correlation analysis (Table 3), which indicated that TPC and aglycone isoflavones are potent antioxidants. On the other hand, aglycone isoflavones also has strong antioxidant activity from some other reports,9,19,24 and this is also similar to our correlation analysis results. In addition, according to the direction of the variables, aglycone isoflavones (daidzein, glycitein, genistein and TAI) was found to be highly inversely correlated with glucoside isoflavones (daidzin, glycitin, genistin and TGI), suggesting a certain contribution of this conjugated glucoside isoflavones to the release of free aglycone isoflavones during the fermentation process.
 |
| Fig. 7 (A) Principal component analysis (PCA) loading plot of total phenolic content (TPC), contents of glucoside isoflavones and aglycone isoflavones, and antioxidant activity of non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS). All of abbreviations are shown in Table 3. (B) Principal component analysis (PCA) score plot of different solvent extracts of NFS and RFS. M-, E-, A- and W-represents the samples were extracted with 80% methanol, 80% ethanol, 80% acetone and water, respectively. | |
PCA was also performed to gain an overview of the similarities and differences among the various extracts of NFS and RFS. The scores of the first two principal components for the studied extracts of NFS and RFS are presented in Fig. 7B. The distance between the locations of the samples on the score plot directly reflected the degree of differences or similarity of the antioxidant compounds content and antioxidant activities for the studied samples. It is noteworthy that M-RFS, E-RFS, A-RFS and W-RFS were located to the right part in the score plot of the PCA, these samples can be best described as having high antioxidant activity, TPC and aglycone isoflavones content. Furthermore, close relationships among M-RFS, E-RFS and A-RFS were observed in Fig. 7B, which indicated that they have similar antioxidant activity, TPC and aglycone isoflavones content, these explanations are also further supported by the results revealed in Tables 1, 2 and Fig. 4. However, M-NFS, E-NFS, A-NFS and W-NFS had negative score of PC1, which indicated that these samples exhibited weak antioxidant activity and low TPC and aglycone isoflavones content. Thus, there are differences in the total amount of antioxidant compounds extracted by different solvents from NFS and RFS. Therefore, this finding suggested the PCA could be helpful to provide valuable information on classification and discrimination of the diverse soybean samples, as well as the relationships among different antioxidant activity evaluation indices, TPC and isoflavone of soybean samples.
3.6 Bioaccessibility studies of NFS and RFS
The biological activity of phytochemicals found in the samples is mostly affected by the metabolism and bioavailability of these molecules in the body. In other words, molecules are required to be bioaccessible in order to exert their bioactivity after biotransformation. Therefore, the bioaccessibility studies were carried out in this paper by using a simulated in vitro gastrointestinal digestion method. The in vitro digestion model simulates the physiological processes (transit time, pH and enzymatic conditions) occurring in the gastrointestinal tract of the human digestive system and has been widely used to study the complex multistage process of human digestion.39,59 These models deliver a beneficial choice to animal and human models by quickly evaluating the bioaccessibility of phenolic compounds.39,40,60 The bioaccessibility of soybean isoflavones in NFS and RFS are displayed in Table 4. It was found that the bioaccessible isoflavones (daidzin, glycitin, genistin, daidzein, glycitein and genistein) of NFS and RFS all decreased after gastrointestinal digestion when compared to their initial contents (Table 2). Nevertheless, the isoflavones were still bioavailable and also showed high amounts. In addition, the bioaccessible fraction of NFS and RFS was mainly composed of glucosides and aglycones, respectively, which was in agreement with their initial compositions (Table 2). After intestinal digestion, the total aglycone isoflavones bioaccessibility of NFS and RFS was 7.46 and 148.13 μg g−1 d.w., respectively, whereas the values for the total glucoside isoflavones bioaccessibility of NFS and RFS was 284.30 and 66.23 μg g−1 d.w., respectively. The observation suggested that both isoflavones and glucosides isoflavones could be resistant to gastrointestinal digestion, and then to reach the target tissue in an active form. Previous researches also reported the similar results that soybean isoflavones were quite stable during digestion.61–64
Table 4 Absolute bioaccessibility (μg g−1 d.w.) of isoflavones from non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) subjected to simulated in vitro gastrointestinal digestiona
Isoflavones |
Gastric digestion |
Intestinal digestion |
NFS |
RFS |
NFS |
RFS |
Each value was expressed as mean ± standard deviation (n = 3). Means with different lower case letters (a, b) within a row indicate significant differences (p < 0.05) between NFS and RFS. Means with different upper case letters (A, B) within a row indicate significant differences (p < 0.05) between gastric digestion and intestinal digestion from NFS or RFS. |
Glucosides |
Daidzin |
165.91 ± 0.91Aa |
28.52 ± 4.83Ab |
152.49 ± 3.70Ba |
27.74 ± 3.58Ab |
Glycitin |
30.81 ± 3.99Aa |
14.16 ± 1.50Ab |
22.41 ± 1.42Ba |
7.96 ± 1.39Bb |
Genistin |
141.81 ± 2.76Aa |
52.67 ± 1.58Ab |
109.41 ± 4.28Ba |
30.53 ± 1.97Bb |
Total glucosides |
338.52 ± 4.52Aa |
95.35 ± 4.59Ab |
284.30 ± 2.09Ba |
66.23 ± 4.24Bb |
![[thin space (1/6-em)]](https://www.rsc.org/images/entities/char_2009.gif) |
Aglycones |
Daidzein |
4.89 ± 0.09Ab |
94.42 ± 3.65Aa |
2.54 ± 0.0.41Ba |
88.37 ± 3.65Aa |
Glycitein |
4.45 ± 0.19Ab |
12.3 ± 0.17Aa |
4.01 ± 0.57Ab |
10.07 ± 1.18Ba |
Genistein |
1.66 ± 0.34Ab |
53.02 ± 0.63Aa |
0.91 ± 0.64Ab |
49.69 ± 0.26Ba |
Total aglycones |
11.00 ± 0.12Ab |
159.74 ± 4.12Aa |
7.46 ± 1.37Bb |
148.13 ± 4.87Ba |
The total phenolic content (TPC) and antioxidant activity of bioaccessible fraction of NFS and RFS were also investigated after gastrointestinal digestion, and the results are summarized in Table 5. As shown in Table 5, the total phenolic bioaccessibility of NFS and RFS showed high after gastrointestinal digestion. Furthermore, the fermentation processed soybeans showed better total phenolic bioaccessibility compared to the non-fermented soybeans either in the gastric or intestinal phase. The results demonstrated that fermentation improved the total phenolic bioaccessibility of soybeans. In addition, the total phenolic bioaccessibility of NFS and RFS was 6619.43 and 9017.14 μg GAE g−1 d.w., respectively, after intestinal digestion, which was higher than their initial values (Table 1). Our findings are in accordance with the results reported by Szawara-Nowak et al.60 who also found that an increase in total phenolic of wheat bread after gastrointestinal digestion. This could be explained by the effect of gastric and intestinal digestive enzyme on the complex food matrix, facilitating the release of phenolics bound to the matrix.65 It is known that both pH and the digestion process result in starch hydrolysis, proteolysis and releasing phenolics from their conjugation forms as well as cell wall matrix. Chandrasekara and Shahidi66 also demonstrated that digestion process released the phenolic compounds bound to the insoluble fibre in the grain.
Table 5 Total phenolic content and antioxidant activity of bioaccessible fraction of non-fermented soybeans (NFS) and R. oligosporus-fermented soybeans (RFS) subjected to simulated in vitro gastrointestinal digestiona
Antioxidant capacity |
Gastric digestion |
Intestinal digestion |
NFS |
RFS |
NFS |
RFS |
Each value was expressed as mean ± standard deviation (n = 3). DPPH, DPPH radical scavenging activity; ABTS, ABTS radical cation scavenging activity; FRAP, ferric reducing antioxidant power; RP, reducing power; CHA, chelating ability; OH, hydroxyl radical scavenging activity; AgNP, a silver nanoparticle-based method for determination of antioxidant capacity; TPC, total phenolic content. Means with different lower case letters (a, b) within a row indicate significant differences (p < 0.05) between NFS and RFS. Means with different upper case letters (A, B) within a row indicate significant differences (p < 0.05) between gastric digestion and intestinal digestion from NFS or RFS. |
DPPH (μg VCE g−1 d.w.) |
262.12 ± 9.61Ab |
547.77 ± 5.07Aa |
188.05 ± 11.76Bb |
266.33 ± 20.21Ba |
ABTS (μg VCE g−1 d.w.) |
1247.29 ± 30.10Bb |
1937.16 ± 36.67Ba |
5360.99 ± 60.84Ab |
5806.53 ± 174.78Aa |
FRAP (μmol Fe(II) g−1 d.w.) |
13.72 ± 0.20Ab |
22.22 ± 0.33Aa |
11.11 ± 0.23Bb |
19.06 ± 0.37Ba |
RP (μg VCE g−1 d.w.) |
869.40 ± 36.51Ab |
1732.67 ± 18.59Aa |
876.80 ± 23.08Ab |
1529.60 ± 11.09Ba |
CHA (μmol EDTA-2Na g−1 d.w.) |
0.93 ± 0.07Bb |
1.14 ± 0.06Ba |
11.27 ± 0.14Ab |
14.89 ± 0.11Aa |
OH (μg VCE g−1 d.w.) |
9755.38 ± 160.46Ab |
12 546.60 ± 890.05Aa |
9289.55 ± 84.38Bb |
13 059.26 ± 234.03Aa |
AgNP (μg GAE g−1 d.w.) |
403.52 ± 30.98Ab |
590.08 ± 4.43Aa |
45.87 ± 10.67Bb |
173.87 ± 18.48Ba |
TPC (μg GAE g−1 d.w.) |
4429.14 ± 158.43Bb |
7982.64 ± 174.83Ba |
6619.43 ± 82.41Ab |
9017.14 ± 75.59Aa |
By measuring the antioxidant activity of bioaccessible fraction of NFS and RFS, it is possible to quickly and cost-effectively provide more biologically relevant data detailing the antioxidant properties of these soybean samples. Crucially the results of this study showed that antioxidant capacity of NFS and RFS is relatively stable and exhibited high value throughout the digestive process (Table 5). This is in consistence with the previous research examining soybean products which have consistently shown stable antioxidant activity during gastrointestinal digestion.61 Interestingly, in the present study, it was found that the bioaccessible fraction of RFS exhibited stronger antioxidant activity than NFS, regardless of the gastric or intestinal phase's digestion, suggesting that RFS may be a greater source of bioaccessible antioxidants than NFS. For example, the DPPH radical scavenging activity of bioaccessible fraction of RFS was 547.77 and 266.33 μg VCE g−1 d.w. after gastric and intestinal digestion, respectively, which was about 2.09- and 1.42-folds higher compared to the respective bioaccessible fraction of NFS. These results clearly demonstrated that the RFS would show greater antioxidant activities than NFS after gastrointestinal digestion. In addition, the results also showed that the ABTS, OH and AgNP value of the bioaccessible fraction of NFS and RFS (Table 5) were increased after gastrointestinal digestion when in comparison with their initial values regardless of the different solvent extraction employed (Fig. 4C, F and G). Some studies demonstrated that structural transformations may occur in the antioxidants following the gastrointestinal digestion, and hence enhancing the antioxidant activity with respect to the ABTS, OH and AgNP assays.65
4. Conclusions
Consumption of natural and healthy foods is nowadays the major interest of consumers. Soybean products are very important foods due to their numerous nutritional benefits and their global availabilities. Results obtained from the present study demonstrated that the isolated strain of Rhizopus oligosporus RT-3 can well ferment soybeans in a short time (22 h). Furthermore, SSF with R. oligosporus RT-3 significantly enhanced the TPC and isoflavone aglycones content of soybean samples. Fermentation by R. oligosporus RT-3 promoted a significant transformation of isoflavone glucosides into aglycones. Besides, the enhanced antioxidant activity and DNA damage protection of soybeans processed by SSF with R. oligosporus RT-3 was also demonstrated in this work. This is the first study which reported the enhanced DNA damage protection of soybeans processed by SSF with R. oligosporus RT-3. Moreover, the present study also showed that extraction solvent systems of varying polarities differed significantly in their extraction capacity and selectivity for phenolic and flavonoids contents, and antioxidant activities evaluation of soybean samples. The antioxidant activities of the samples were closely correlated with TPC, isoflavone aglycones by both Pearson's correlation analysis and PCA. In addition, bioaccessibility studies carried out by simulated in vitro gastrointestinal digestion model further demonstrated that RFS contained higher total phenolic and isoflavone aglycones bioaccessibility, as well as showed greater source of bioaccessible antioxidants than NFS. Therefore, SSF with R. oligosporus RT-3 is effective for the enhancement of phenolics, isoflavone aglycones, and antioxidant activities of soybeans in a short time. Thus, use of fermented soybeans as nutraceutical and functional food ingredients or inclusion in therapeutic diets for patients for the treatment of oxidative-related diseases (e.g., inflammation, arthritis, cardiovascular diseases, and cancer) may also be recommended, and it may have great potential for the nutraceutical and pharmaceutical industries.
Abbreviation
NFS | Non-fermented soybeans |
RFS | Rhizopus oligosporus-fermented soybeans |
DPPH | 2,2-Diphenyl-1-picrylhydrazyl |
ABTS | 2,2-Azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt |
TPTZ | 2,4,6-Tris(2-pyridyl)-S-triazine |
EDTA-2Na | Ethylenediaminetetraacetic acid disodium salt |
PDA | Potato dextrose agar |
Vc | Ascorbic acid |
FRAP | Ferric reducing antioxidant power |
RP | Reducing power |
CHA | Chelating ability |
TPC | Total phenolic content |
SSF | Solid state fermentation |
PBS | Phosphate buffer saline |
HPLC | High performance liquid chromatography |
AgNP | Silver nanoparticle-based method |
TGI | Total glucoside isoflavones |
TAI | Total aglycone isoflavones |
PCA | Principal component analysis |
Acknowledgements
This work was co-financed by the National Natural Science Foundation of China (no. 31371807; 31201422), High-Tech Research and Development Program of China (no. 2011AA100903; 2013BAD18B01-4) and Jiangsu Provincial Postgraduate Innovation Project of China (no. KYLX15_0594).
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