2015 Volume 21 Issue 6 Pages 765-770
Dark common beans have beneficial nutritional and functional parameters but contain flatulence-producing α-galactosides. The aim of the research was to study the effect of modified tempe-type procedure (mixed-culture fermentation of unhulled seeds) on the antioxidant parameters and α-galactosides level in the product, as compared to raw and hydrothermally processed seeds.
Soaking and cooking significantly lowered the antiradical activity and the reducing power of common beans. Tempe showed higher activity against ·OH (IC50 9.34 mg), DPPH· and ABTS·+ (on the average of 45%) and higher reducing power than the hydrothermally processed beans. Tempe contained 29% more soluble phenols (1.76 g kg−1 DM), 140% more condensed tannins (1.03 g kg−1 DM) and 30% more flavonoids (0.35 g kg−1 DM) than soaked and cooked seeds. The level of raffinose (0.031 g kg−1 DM), verbascose (0.027 g kg−1 DM) and stachyose (0.242 g kg−1 DM) in tempe was 67%, 53% and 57% lower, respectively.
Common bean (Phaseolus vulgaris) is consumed worldwide as rich and inexpensive source of protein, dietary fibers, vitamins and minerals. Dark common bean is also considered functional food (Vadivel et al., 2011) due to the high level of phenolic compounds present in the seed coat (Boateng et al., 2008).
However, processes usually applied in order to prepare dry beans for the consumption (soaking and cooking) significantly diminish the antioxidant activity of the seeds, therefore decreasing their functional properties (Xu and Chang, 2009). Tempe-type fermentation may improve the functional and nutritional parameters of plant seeds, as compared to conventional treatments. Tempe products are sliceable ‘cakes’ consisting of seeds bound together by the mycelium of Rhizopus sp., which overgrows the substrate. Originating from Indonesia, tempe is becoming better known in Western countries due to constantly growing market of vegetarian foods. This product has a wide range of culinary applications and may be served both as a meat replacement in a main dish and a salad ingredient. What is more important, tempe is characterized by the favourable nutritional composition and a low level of antinutrients (Nout and Kiers, 2005). The substrate for tempe-type processing are usually soybeans, but other plant materials may also be used (Granito et al., 2005; Reyes-Bastidas et al., 2010; Stodolak et al. 2013). When common beans are used as the fermentation substrate, the important advantage may be the decomposition of flatulence-producing α-galactosides, which limit the consumption of common beans in Western countries. However, the results are dependent on the enzymatic capabilities of a fungal strain used (Granito et al., 2005).
Our previous study showed that the modified tempe-type fermentation with mixed inoculum consisting of Rhizopus microsporus var. chinensis tempe strain and Lactobacillum plantarum DSM 20174 improved the nutritional and some bioactive parameters of tempe from unhulled common beans, as compared to fungal fermentation alone (Starzyńska-Janiszewska et al., 2014).
The aim of present research was to study the effect of mixed-culture tempe-type fermentation of unhulled dark common beans on the antioxidant capacity and selected functional compounds (phenols and α-galactosides), as compared to raw and traditionally processed seeds (soaked and cooked).
Materials The fermentation substrate was color common bean (Phaseolus vulgaris) cultivar Małopolanka obtained from Krakowska Hodowla i Nasiennictwo Ogrodnicze POLAN Sp. z o.o. (Poland).
Microbial strains Lactobacillus plantarum DSM 20174 (German Collection of Microorganisms and Cell Cultures DSMZ) was grown on de Man, Rogosa and Sharpe broth (MRS, Sigma Aldrich, St Louis, MO, USA) at 30°C for 24 h, then the cells were centrifuged, and suspended in sterile (8 g L−1) saline. The cell density in the inoculum was assayed by the turbidimetric method with McFarland's standards. Rhizopus microsporus var. chinensis tempe strain (Institute for Microbial Resources, Taichung, Taiwan) (Kuo et al., 2000) was grown on malt extract (MEA) agar at 20°C for 12 days, then the spores were harvested with sterile saline (8 g L−1) supplemented with peptone (0.01 g L−1) and Tween 80 (0.1 cm3 L−1). The spore suspension was filtered three times by nylon net filters (Milipore, ∅ 11 µm) to remove the mycelium fragments. Next, the spore density in the inoculum was measured by spore-counting in a Thoma chamber and optical microscopy.
Raw beans Dry common bean seeds were lyophilised and stored at 4°C for further analysis.
Hydrothermally processed (soaked and cooked) beans Seeds (60 g) were thoroughly cleaned and rinsed three times with distilled water. Next, the seeds were soaked for 18 h at 24°C in sterile distilled water (1:5 v/v) and then cooked in tap water (1:3 v/v) until soft (60 min). After that, water was discarded and the seeds were lyophilised and stored at 4°C.
Mixed-culture tempe Dry common beans were thoroughly cleaned and rinsed three times with sterile distilled water. Next, they were dried for 24 h at 55°C and then parted into halves with a universal seed grinder (Porkert, Czech Republic). Seeds portions (60 g) were soaked (18 h at 24°C) in sterile distilled water (1:5 v/v) and acidified to pH 4.5 – 5.0 with lactic acid. Next, the seeds were rinsed with 0.5 L of sterile distilled water, placed in glass Petri dishes (∅ 20 cm) and autoclaved for 40 min at 121°C. Cooled (< 35°C) seeds were thoroughly mixed with the inoculum (104 spores of R. microsporus and 104 cells of L. plantarum per 1 g of seeds). Inoculated material was put into perforated plastic bags, 3 cm in height, and fermented for 24 h at 30°C. Fresh tempe products were sliced and steamed for 10 min. Tempeh samples were prepared in triplicate and mixed within the treatment. Next, they were lyophilized and stored at 4°C for further analysis.
Analytical methods Hydroxyl radical scavenging activity was measured in phosphate buffer (0.02 M, pH 7.4) extracts according to Marambe et al. (2008). The antiradical activity was expressed as IC50 which amounts a sample concentration (mg) in the extract that is required for 50% inhibition of ·OH generated in the reaction conditions. A lower value of IC50 indicates a higher antiradical activity of a sample.
ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity was obtained in 0.5% phosphate buffer (0.1 M, pH 7.4) extracts as described by Starzyńska-Janiszewska et al. (2008), and expressed as µmol Trolox g−1 DM.
DPPH· scavenging activity, reducing power, flavonoid and condensed tannin contents were measured in 5% acetone:water (80:20, v/v) extracts. The extracts were obtained by the vigorous shaking of 1 g sample with 15 mL of acetone:water mixture for 3 hours, followed by 18 hours incubation in the darkness at room temperature, centrifugation (15000 rpm, 15 min) and filtration. The solid residue was re-extracted with 5 mL of acetone:water mixture for 30 min, centrifuged, filtered and the supernatants were combined. DPPH (1,1-Diphenyl-2-picryl-hydrazyl) radical scavenging activity was obtained as described by Pekkarinen et al. (1999) with some modifications. 0.2 mL of 5% sample extract was mixed with 2.8 mL of DPPH· solution (0.1 mmol L−1 in 80% methanol) and the absorption was measured at 516 nm after 30 min against an 80% methanol blank. DPPH· scavenging activity was expressed as µmol Trolox g−1 DM. Reducing power (RP) was obtained by the method with ferricyanide according to Ardestani and Yazdanparast (2007) and expressed as RP0.5 defined as the amount of sample concentration (mg) in the extract that produces 0.5 absorbance unit at 700 nm. The lower value of RP0.5 indicates a higher reducing power of a sample. Flavonoids (g quercetin kg−1 DM) were estimated in acetone:water (80:20, v/v) extracts using protocol A2 specified in Papoti et al. (2011) paper.
Soluble phenols (g tannic acid kg−1 DM) were measured in acetone:water (80:20, v/v) extracts and in 0.5% phosphate buffer (0.1 M, pH 7.4) extracts by the method of Swain and Hillis (1959) with Folin-Ciocalteu reagent. Condensed tannins (g catechin kg−1 DM) were estimated by the vanillin assay (Price et al., 1978).
Oligosaccharides were extracted according to Muzquiz et al. (1992) with some modifications. Powdered sample (0.5 g) was sonificated with 70% methanol, then centrifuged and the supernatant was decanted. This extraction procedure was repeated twice and the combined supernatants evaporated at 35°C to dryness. The residuum was dissolved in 2.5 mL deionized water and passed through Dowex 50 W X 8 (200 – 400 mesh). Nine mL eluate was collected. Filtered samples (nylon 0.45 µm syringe filter) were analyzed using IEC-PAD system equipped with isocratic pump ISO-3100A, CarboPack PA 100 (4x250 mm) column, electrochemical detector DIONEX ED50A and Chromeleon 6.8 software. 70 mM NaOH (1 mL/min) was used as a mobile phase. Oligosaccharides contents were determined based on raffinose, stachiose and verbascose (Sigma Chemical) standards.
Dry matter (DM) was obtained with a moisture analyser (type WPS 110S, Radwag, Radom, Poland).
Statistical analysis For each determination, six replications were made, with the exception of oligosaccharides (five). The results were statistically evaluated using one-way analysis of variance. To determine statistically significant differences, the least significant difference test was used at p ≤ 0.05. Data were processed using the software STATISTICA from StatSoft, Inc. (Tulsa, OK, USA), version 9.1, 2010.
Radical scavenging activity (·OH, ABTS·+ and DPPH· scavenging activities) and reducing power In order to provide an overall assessment of the antioxidant/antiradical activity of samples, a few different tests were performed (Table 1). Hydroxyl radicals are the most reactive among free radicals generated endogenously during aerobic metabolism. They are formed in the presence of metal ions (Cu, Fe) and cause the ageing of cells and some diseases (Aruoma, 1998). ABTS·+ and DPPH· are synthetic free radicals commonly used in assays of the antiradical potential of foods (Huang et al., 2005). The reducing power of a compound may be considered as an indicator of its potential antioxidant activity, because antioxidants are often reductants (Prior and Cao, 1999). All the activities chosen for the characteristics of antioxidant potential of the samples were highly correlated with each other (Table 2).
| Raw seeds | Cooked seeds | Tempe | |
|---|---|---|---|
| Buffer extracts | |||
| ABTS·+ (µmol Trolox g−1 DM) | 39.55 c | 10.94 a | 14.36 b |
| ·OH (IC50) | 2.51 a | 16.85 c | 9.34 b |
| Acetone/water extracts | |||
| DPPH· (µmol Trolox g−1 DM) | 8.04 c | 2.11 a | 3.08 b |
| Reducing power (RP0.5) | 23.03 a | 63.51 c | 55.22 b |
IC50 – the amount of lyophilised sample (mg) used for the extraction that causes 50% inhibition of hydroxyl radical; a lower value indicates a higher antiradical activity; RP0.5 - the amount of lyophilised sample (mg) used for the extraction that produces 0.5 absorbance unit at 700 nm; a lower value indicates a higher reducing power.
Means in a raw with different letters differ significantly (P≤0.05)
| ABTS·+ | ·OH | DPPH· | RP | |
|---|---|---|---|---|
| ABTS·+ | - | −0.899 | 0.994 | −0.991 |
| ·OH | - | - | −0.920 | 0.936 |
| RP | - | - | −0.997 | - |
| Soluble phenols | 0.992 | −0.859 | 0.999 | −0.997 |
| Flavonoids | −0.166 | −0.263 | −0.117 | 0.081 |
| Condensed tannins | 0.990 | −0.944 | 0.997 | −0.998 |
RP – reducing power
Hydroxyl radical scavenging activity (IC50) of buffer extracts from the raw common beans was about 20 times lower than that of Trolox (0.127 mg, data not shown). ABTS·+ scavenging activity (37 µmol Trolox · g−1 DM) exceeded value previously reported by the authors for buffer extracts from the raw grass pea seeds (Starzyńska-Janiszewska et al., 2008). DPPH· scavenging capacity of extracts from the raw beans was lower than the one obtained for ABTS·+ assay, which confirms earlier reports (Starzyńska-Janiszewska et al., 2008; Floegel et al., 2011). Acetone/water extracts from the raw seeds of common bean showed low reducing power value (RP0.5 23 mg), when compared to that of BHT (RP0.5 1.1 mg, data not shown). The observed RP was also much lower than presented by Lee et al. (2008) for the raw seeds of soybean (Glycine max (L.) Merr.).
Hydrothermal processing (soaking followed by cooking) of beans resulted in the significant decrease of activity towards ·OH (6.7-fold), ABTS·+ and DPPH· (over 3-fold), as well as RP (2.7 fold) (Table 1). The reduction of activity against ABTS·+ caused by cooking (lentil, chickpea, soybean) and soaking (lentil) was previously observed by Han and Baik (2008). Pressure steaming was shown to diminish the activity towards DPPH· of black and pinto beans (Xu and Chang, 2009). The obtained results are however inconsistent with observations of Nithiyanantham et al. (2012), who reported that soaking followed by autoclaving did not significantly change the reducing power (FRAP assay) of Cicer arietinum and Pisum sativum seeds.
As compared to hydrothermally processed seeds, mixed-culture tempe was characterized by the enhanced scavenging capacity towards free radicals. The amount of mg sample causing 50% inhibition of ·OH (IC50) was 45% lower in tempe, while the activity towards ABTS·+ and DPPH· − 31% and 46% higher, respectively. This tendency is in agreement with observations of Reyes-Bastidas et al. concerning tempe flour from common beans (2010). The reducing power of the fermented beans was also slightly improved (Table 1). Both fungal (Rhizopus) and bacterial (Bacillus) solid-state fermentation was shown to significantly increase the RP of soybean meal (Wongputtisin et al., 2007). However, as stated by Lee et al. (2008), the reducing activity of soybeans after fermentation with Rhizopus sp. may vary according to starter microorganism employed.
Changes in RSA and RP after hydrothermal and biological processing were highly dependent on both the soluble phenols and the condensed tannins content in seeds (Table 2), which is consistent with the findings of Xu and Chang (2007). Phenols are proven to show redox properties, which allow them to act as reducing agents (Rice-Evans et al., 1996). Condensed tannins are considered the important component of the overall antioxidant activity of extracts from common beans (Beninger and Hosfield, 2003). These compounds are important dietary antioxidants which may protect other molecules (proteins, carbohydrates, lipids) from oxidative damage during digestion, e.g. acting as ‘replacement targets’ for free radicals (Carbonaro et al., 1996; Hagerman et al., 1998).
Soluble phenols, flavonoids and condensed tannins The content of soluble phenols in buffer extracts from raw common beans was 70% higher than the one measured in acetone/water extracts (Table 3). However, the extraction efficiency of both solvents was similar in the case of hydrothermally processed and fermented seeds. Raw seeds contained 0.29 mg flavonoids in g DM. The analytical protocol applied in our study is specific for flavonoids as co-determination of other groups of phenols via this method is considered negligible (Papoti et al., 2011). As shown by Heimler et al. (2005) for 12 Italian landraces of common beans, the amounts of flavonoids in dry seeds may vary widely (traces — 1.27 mg/g). The level of condensed tannins in the raw seeds of ‘Małopolanka’ (Table 3) was high for the common beans (Heimler et al., 2005) and similar to that measured by Boateng et al. (2008) in pinto bean seeds.
| Compounds | Raw seeds | Cooked seeds | Tempe | |
|---|---|---|---|---|
| Soluble phenols (g kg−1 DM) | buffer extract | 5.61 b | 1.32 a | 1.39 a |
| acetone/water extract | 3.91 c | 1.36 a | 1.76 b | |
| Flavonoids (g kg−1 DM) | 0.29 b | 0.27 a | 0.35 c | |
| Condensed tannins (g kg−1 DM) | 3.05 c | 0.43 a | 1.03 b | |
| Oligosaccharides (g kg−1 DM) | verbascose | 0.261 c | 0.058 b | 0.027 a |
| stachyose | 2.098 c | 0.563 b | 0.242 a | |
| raffinose | 0.240 c | 0.095 b | 0.031 a | |
Means in a raw with different letters differ significantly (P≤0.05)
Hydrothermal treatment resulted in the significant decomposition of total phenols and condensed tannins (over 7-fold decrease) in the unhulled common beans (Table 3). The decrease of total phenols after hydrothermal processing of legumes was observed previously by other authors (Granito et al., 2008; Xu and Chang, 2009). It could be attributed to water-soluble compounds leaching into the water during soaking and cooking as well as the breakdown of phenol molecules in the latter process. However, the level of flavonoids in hydrothermally processed beans was only slightly lowered. Flavonoids decomposition after cooking and pressure steaming of legumes was previously observed (Xu and Chang, 2009; Jiratanan and Liu, 2004).
Tempe contained the significantly higher levels of soluble phenols (29% in acetone/water extracts), condensed tannins (140%) and flavonoids (30%) than hydrothermally processed seeds (Table 3). Both Rhizopus sp. and L. plantarum strains are proven to produce β-glucosidase, which participates in releasing the aglycones from phenolic glycosides in plant cell walls (McCue and Shetty, 2003; Pyo et al., 2005). In the case of tannins, the increase in their percentage share in product dry matter was most probably the consequence of the utilization of other plant compounds by the microorganisms. It may also be mentioned that however tannins are known to limit the bioavailability of legume proteins (Carbonaro et al., 1996), we did not observe this effect in the case of tempe from common beans (Starzyńska-Janiszewska et al., 2014). The level of flavonoids in tempe was higher than in raw seeds, by 21%. The share of flavonoids in total soluble phenols increased from 5% in the raw beans to 25% in tempe. This is favorable in terms of possible biological effects of these compounds and/or their metabolites within the gastrointestinal tract and other body tissues (Halliwell et al., 2005). However, we did not find correlations between flavonoid contents and the changes in antioxidant potential of raw and processed common beans (Table 2). It should also be mentioned that the extracts from tempe, apart from phenolic compounds, most probably contained free amino acids and peptides released from seed proteins as well as synthesized by Rhizopus and Lactobacillus during the fermentation. It has been established that both aromatic amino-acids and low-molecular weight peptides liberated by microbial proteases from plant substrates are partially responsible for antioxidant properties of fermented plant-based foods (Hur et al. 2014). The plant seeds peptides are capable of the antiradical activity against e.g. ABTS·+ and ·OH, as shown in the case of buckwheat extracts by May et al. (2010).
α-Galactosides Hydrothermal processing resulted in the significant decrease in flatulence-inducing sugars level in beans (Table 3). These changes were most drastic for stachyose and verbascose, which were reduced on average by 75%, as compared to the raw seeds. Fermentation procedure was more effective in decreasing the oligosaccharides content, by about 53%, 57% and 67% in case of verbascose, stachyose and raffinose, respectively. Both Rhizopus microsporus var chinensis and Lactobacillus plantarum strains are capable to hydrolyze α-galactosaccharides (Schwertz et al.,1997; Silvestroni et al.,2002). Reduction in the oligosaccharides level was observed previously by Bieżanowska-Kopeć et al. (2006) during single-strain fermentation of common beans with Rhizopus microsporus.
Hydrothermal processing (soaking followed by cooking) diminished the radical scavenging activity and the reducing power of unhulled common beans, thereby decreasing their functional properties. Tempe-type fermentation procedure with mixed inoculum consisting of Rhizopus and Lactobacillus resulted in the more advantageous antioxidant potential of the product. Tempe contained more soluble phenols and condensed tannins, as compared to processed seeds. It was also enriched in flavonoids in terms both their level in product dry mass and their share in total soluble phenols. Moreover, common bean tempe had significantly less flatulence-inducing oligosaccharides of the raffinose family than hydrothermally treated seeds.
On the basis of the obtained results, modified tempe-type procedure with mixed-culture inoculum may be recommended as the method of alternative processing of unhulled dark common beans, as compared to traditionally cooked seeds. The mixed-culture fermentation of beans was also proven to be more effective than the single-culture method using R. microsporus vas. chinensis, as described in Starzyńska-Janiszewska et al. (2014).
