2018 Volume 24 Issue 3 Pages 493-499
The regional standard for tempeh recently established by the Codex Alimentarius defines the use of Rhizopus oligosporus, Rhizopus oryzae, and/or Rhizopus stolonifer as soybean tempeh starters. However, there has been little comparative study on the tempeh prepared with these Rhizopus species. This study compared the contents and compositions of isoflavones in tempeh prepared with these Rhizopus species. The contents of total isoflavone aglycones (daidzein and genistein) and the ratio of total aglycones to total isoflavones in the tempeh fermented with R. stolonifer were higher than those with other Rhizopus species. In the isoflavone-enriched tempeh-like fermented soybeans made using hypocotyls and dehulled soybeans, the total aglycone contents and the ratio of total aglycones to total isoflavones were also higher for R. stolonifer than for the other species. These findings highlight the importance of R. stolonifer as the most appropriate Rhizopus species for the production of isoflavone aglycone-enriched tempeh.
Tempeh is a traditional fermented soybean food consumed mainly in Southeast Asia, especially in Indonesia. It is prepared by the fermentation of dehulled and cooked soybeans with Rhizopus species (Nout and Kiers, 2005). Tempeh contains greater amounts of peptides, free amino acids, and gamma-aminobutyric acid than unfermented soybeans (Nout and Kiers, 2005; Aoki et al., 2003a). Consumption of tempeh was reported to modulate fecal secondary bile acids, mucins, immunoglobulin A, harmful enzyme activities, cecal microflora, and short-chain fatty acids (Utama et al., 2013) as well as depress the elevation of systolic blood pressure in rats (Aoki et al., 2003b).
Raw soybeans and soybean foods contain a large amount of isoflavones. Isoflavones are found in multiple chemical forms including aglycones (daidzein, genistein, and glycitein) and their corresponding β-glucosides, malonylglucosides, and acetylglucosides (Kudou et al., 1991). In raw soybeans, isoflavones mainly exist in α-glucoside or malonylglucoside form (Wang and Murphy, 1994a). Isoflavone β-glucosides and malonylglucosides are degraded to their corresponding aglycones during food processing (Wang and Murphy, 1996). Analysis of the isoflavone contents in commercial soybean foods revealed that non-fermented soybean foods had greater levels of glucosides, while fermented soybean foods had greater levels of aglycones (Wang and Murphy, 1994b). In the production of tempeh, malonylglucosides decreased after soaking and cooking, and aglycone concentrations increased after fermentation, as a result of fungal enzymatic hydrolysis (Wang and Murphy, 1996). Genistein and daidzein, the main soy isoflavone aglycones, have physiological functions including anti-tumor, anti-oxidative, estrogenic, and anti-diabetic effects (Setchell and Cassidy, 1999; Lee and Lee, 2001; Tadera et al., 2006). It is commonly assumed that isoflavone aglycones are absorbed faster and in higher amounts than their glucosides (Izumi et al., 2000; Kano et al., 2006). Moreover, daidzein is converted to equol by the intestinal flora, and equol has been reported to have even stronger biological estrogenic and anti-oxidant activities (Atkinson et al., 2005; Rafii, 2015). Therefore, some attempts have been made to enhance the generation of isoflavone aglycones in soybean foods, such as natto (Wei et al., 2008) and soymilk (Bau and Ida, 2015).
Recently, regional standards for tempeh were established by the Codex Alimentarius.i) It defines the use of Rhizopus oligosporus, Rhizopus oryzae, and/or Rhizopus stolonifer as soybean tempeh starters. Taxonomic study revealed that the majority of species associated with tempeh fermentation were R. oligosporus (Hartanti et al., 2015) and R. oryzae (Ogawa et al., 2004). Since R. oligosporus and R. oryzae have been widely used as tempeh starters, a number of studies have focused on tempeh prepared with these Rhizopus species (Utama et al., 2013; Aoki et al., 2003b). Meanwhile, R. stolonifer has been less frequently used as tempeh starters (Dwidjoseputro and Wolf, 1970; Hesseltine, 1989), and has therefore not received much attention. Additionally, there has been little comparative study on the compositions of tempeh produced with the three Rhizopus species. In terms of isoflavone composition, the previous study was limited to tempeh prepared with R. oligosporus (Ikeda et al., 1995). Thus, in the present study, we determined the contents and compositions of isoflavones in tempeh prepared with three Rhizopus species. Here, we report that the fermentation with R. stolonifer produced the most isoflavone aglycone-enriched tempeh.
Materials All reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The isoflavone chemicals (daidzein, genistein, glycitein, daidzin, genistin, glycitin, malonyldaidzin, malonylgenistin, and malonylglycitin) were used as standards. The dehulled yellow soybeans (Glycine max) used in this experiment were of the variety Ryuuhou, which is cultivated in Akita Prefecture, Japan. Soybean hypocotyls (hypocotyl purity > 80%) were provided by K. I. Tech International Co., Ltd. (Tokyo, Japan).
Microorganisms R. microsporus var. oligosporus (hereinafter referred to as R. oligosporus) NBRC 8631, 32002, and 32003; R. oryzae NBRC 4706, 4716, and 5780; and R. stolonifer var. stolonifer (hereinafter referred to as R. stolonifer) NBRC 5411, 6188, and 30816 were used for the experiments. All these strains were transferred from the Biological Resource Center, NITE (NBRC) under an agreement. Among them, the strain R. oligosporus NBRC 8631 is used frequently as a starter in Japan. R. oligosporus NBRC 32002 and NBRC 32003 are the authorized strains as starters for tempeh in Indonesia. The other strains were selected from the NBRC culture collection as appropriate. The strains were incubated on potato dextrose agar (PDA) plates at 30°C until sporulation. Spore suspensions of each Rhizopus species were prepared by harvesting the spores on plates using sterilized water. Measurements of spore concentration were conducted using a hemocytometer.
Tempeh preparation Dehulled yellow soybeans were soaked in 0.2% acetic acid at room temperature overnight. After draining the acetic acid solution, the soaked soybeans were boiled in hot water at 100°C for 10 min. The boiled soybeans were drained and cooled to below 40°C, and subsequently inoculated with spore suspensions of each Rhizopus species (3×106 spores/100 g boiled soybeans). The inoculated soybeans were packed into polyethylene bags with small pinholes and incubated under each condition.
In the first experiment, the inoculated soybeans were incubated at 32°C for 40 h to examine the differences in isoflavone composition in fermented soybeans with each Rhizopus strain. In the second experiment, the soybeans were incubated at 20, 25, 32, and 37°C for 40 h with the representative strains of each Rhizopus species to examine the effect of fermentation temperature on the composition of isoflavones. Finally, the soybeans were incubated at each optimal temperature for 24, 40, 48, and 72 h to examine the changes in the composition of isoflavones and enzyme activities during fermentation.
The fermentation was stopped immediately by freezing. In order to evaluate the direct effects of microbial fermentation on isoflavone composition, the fermented soybeans were lyophilized without heat sterilization and used for analysis, because the composition of isoflavones changes with heat processing (Wang and Murphy, 1996).
Isoflavone-enriched fermented soybeans preparation Tempeh is produced using dehulled soybeans without the soybean germ (hypocotyl), which contains high amounts of isoflavones. Meanwhile, for the production of tempeh-like fermented soybeans containing high amounts of isoflavones, we used hypocotyls, as previously studied (Nakajima et al., 2005). Isoflavone-enriched tempeh-like fermented soybeans were prepared by using hypocotyls and dehulled soybeans at ratios of 20:80 and 100:0. Hypocotyl and dehulled soybean mixtures were soaked, boiled, and inoculated with spores of each Rhizopus species (3×106 spores/100 g boiled soybean mixture). Inoculated soybean mixtures were packed in polyethylene bags with small pinholes and incubated at 32°C for 40 h. The prepared fermented soybean mixtures were lyophilized without sterilization and used for analysis.
Isoflavone extraction and HPLC analysis The content and composition of isoflavones in the fermented soybeans were measured by an HPLC system (Shimadzu, Ltd., Kyoto, Japan) according to the method described previously (Kudou et al., 1991), with slight modifications. The column used was an InertSustain C18 (250×4.6 mm, GL Science Inc., Tokyo, Japan). The mobile phase consisted of solvent A (acetic acid/acetonitrile/water = 0.1/15/85) and solvent B (acetic acid/acetonitrile/water = 0.1/35/65). Solvent B was increased from 0% to 100% over 50 min and subsequently held for 10 min. The solvent flow rate was 1 mL/min and the absorption was measured at 254 nm. The column temperature was held constant at 35°C.
Isoflavones were extracted from dried samples with 70% aqueous ethanol for 1 h at 25°C. The mixtures were then centrifuged at 10 000×g for 5 min, and the resulting supernatants were filtered through a 0.45–µm filter before HPLC analysis. Isoflavones were quantified based on calibration curves prepared from the HPLC peaks and peak areas of each isoflavone standard.
Assay of enzyme activity β-Glucosidase activity was assayed by hydrolysis of the synthetic substrate p-nitrophenyl-β-D-glucopyranoside (pNPG), using a slight modification of the method reported previously (Yin et al., 2005). Ten grams of each sample were added to 100 mL of 0.1 M sodium phosphate buffer (pH 6.0). The mixtures were stirred at 4°C overnight and centrifuged at 10 000×g for 10 min. The supernatants were used as the enzyme solutions. A 0.9 mL solution of 1 mM pNPG in 0.1 M sodium phosphate buffer (pH 6.0) was first incubated at 30°C for 5 min. After the addition of 0.1 mL of the enzyme solution, the reaction mixture was incubated at 30°C for 30 min. The reaction was stopped by the addition of 2 mL of 0.5 M sodium carbonate. The resulting yellow color was measured at 405 nm with a spectrophotometer (UV-1200, Shimadzu, Ltd., Kyoto, Japan). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 µmol of p-nitrophenol per min.
Statistical analysis Data were expressed as means ± standard deviations (n=3). Statistical analysis was performed by a one-way ANOVA, followed by Tukey's multiple-range test (Excel Statistics 2012 for Windows, Social Survey Research Information Co., Ltd., Tokyo, Japan). For all tests, P < 0.05 was considered to indicate a statistically significant difference.
Content and composition of isoflavones in the fermented soybeans with each Rhizopus species The content and composition of isoflavones in the fermented soybeans with each Rhizopus species are shown in Table 1. Glycitein and its derivatives were not detected in HPLC chromatograms. Similar alterations in the composition of isoflavones were found among the fermented soybeans prepared with the same species. The content of total aglycones (daidzein and genistein) in the boiled soybeans was 11 mg per 100 g dry weight. The content of total aglycones increased to 33–35 mg, 28–44 mg, and 67–83 mg per 100 g dry weight in the fermented soybeans with R. oligosporus, R. oryzae, and R. stolonifer, respectively, after 40 h of fermentation. The ratio of total aglycones to total isoflavones increased from 5% to 16%, 17–43%, and 35–51% by fermentation with R. oligosporus, R. oryzae, and R. stolonifer, respectively. In all the fermented soybeans, the content of total isoflavones was decreased by the fermentation. This may be because the molecular weights of isoflavone aglycones are lower than those of the corresponding glucosides by the amounts of sugar moieties. The contents of both β-glucosides (daidzin and genistin) and malonylglucosides (malonyldaidzin and malonylgenistin) in the soybeans fermented with R. oligosporus were significantly higher than those in the soybeans fermented with the other two Rhizopus species. In the following studies, R. oligosporus NBRC 32002, R. oryzae NBRC 4716, and R. stolonifer NBRC 30816 were used.
| Isoflavones contents (mg/100 g dry weight) | Total aglycones1 (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Species | Strain | β-Glucosides | Malonylglucosides | Aglycones | Total isoflavones | Total aglycones | ||||
| Daidzin | Genistin | Malonyldaidzin | Malonylgenistin | Daidzein | Genistein | |||||
| Unfermented (Boiled soybean) | - | 21.2±0.2a | 37.7±0.7a | 51.9±0.4a | 107.6±0.6a | 4.4±0.1f | 6.9±0.3f | 230±1a | 11.2±0.4g | 4.9±0.2g |
| R. oligosporus | NBRC8631 | 6.2±0.4b | 17.5±0.8c | 48.5±1.4a | 102.5±2.9ab | 13.6±0.1de | 19.4±0.2d | 208±6b | 33.0±0.3def | 15.9±0.3f |
| NBRC 32002 | 5.1±0.4c | 18.6±1.3bc | 51.3±3.2a | 106.8±5.6a | 15.3±0.9cd | 19.1±1.0d | 216±2ab | 34.5±.9de | 15.9±0.1f | |
| NBRC 32003 | 5.5±0.2c | 19.3±0.4b | 52.2±1.0a | 107.6±2.1a | 15.6±0.3cd | 19.5±0.7d | 220±4ab | 35.2±1.0d | 16.0±0.2f | |
| R oryzae | NBRC 4706 | n.d. | 3.5±0.1fg | 14.0±0.5e | 41.1±1.6e | 17.5±0.4c | 26.2±0.8c | 102±3e | 43.7±1.3c | 42.8±0.4c |
| NBRC 4716 | n.d. | 3.8±0.1fg | 20.2±1.0d | 54.5±2.3d | 12.6±0.5e | 15.7±0.8e | 107±4e | 28.3±1.2f | 26.5±0.5e | |
| NBRC 5780 | n.d. | 3.0±0.2g | 44.2±0.8b | 97.0±1.0b | 12.9±0.3e | 16.9±0.4de | 174±2cd | 29.7±0.7ef | 17.1±0.2f | |
| R stolonifer | NBRC 5411 | n.d. | 9.4±0.6d | 21.5±0.7d | 56.4±1.3d | 29.5±1.3a | 53.1±1.1a | 170±5d | 82.6±2.3a | 48.6±0.3b |
| NBRC 6188 | n.d. | 6.6±0.0e | 35.3±1.3c | 79.9±1.6c | 23.5±0.9b | 43.0±1.7b | 188±2c | 66.5±2.6b | 35.3±1.4d | |
| NBRC 30816 | n.d. | 5.0±0.1ef | 20.9±0.8d | 53.3±1.3d | 28.3±1.5a | 52.5±1.7a | 160±5d | 80.7±3.2a | 50.5±0.6a | |
Soybeans were fermented at 32°C for 40 h. Values are means ± standard deviations (n=3).
Different lowercase letters in the same column indicate significant difference (P<0.05) according to Tukey's multiple-range test.
n.d.; not detected (<0.4 mg/100g dry weight)
Effect of fermentation temperature on isoflavone composition The composition of isoflavones in the fermented soybeans at each temperature is shown in Table 2. In the soybeans fermented with R. oligosporus, the content of β-glucosides (daidzin and genistin) significantly decreased with the elevation of fermentation temperature. However, the content of malonylglucosides (malonyldaidzin and malonylgenistin) did not significantly differ with fermentation temperature, implying that R. oligosporus has little ability to convert malonylglucosides into other forms of isoflavones. The content of total aglycones and the ratio of total aglycones to total isoflavones were the highest at 37°C (33 mg/100 g dry weight and 16%, respectively). On the other hand, in the soybeans fermented with R. oryzae, the contents of both β-glucosides and malonylglucosides decreased as the temperature increased. The content of total aglycones was the highest at 25°C (34 mg/100 g dry weight), while the ratio of total aglycones to total isoflavones was the highest at 32°C (27%). The soybeans fermented with R. stolonifer showed the highest content and ratio of total aglycones to total isoflavones at 32°C (94 mg/100 g dry weight and 60%, respectively). R. oryzae could grow at all temperatures tested; however, R. oligosporus and R. stolonifer could not grow at 20°C and 37°C, respectively. Consequently, the isoflavone compositions of the soybeans fermented by R. oligosporus at 20°C and by R. stolonifer at 37°C did not differ from those of boiled soybeans (Table 2). From these results, the changes in the composition of isoflavones and enzyme activities during fermentation were examined at 37°C for R. oligosporus, and at 32°C for R. oryzae and R. stolonifer in the next experiment.
| Isoflavones contents (mg/100 g dry weight) | Total aglycones1 (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Strain | Temperature | β-Glucosides | Malonylglucosides | Aglycones | Total isoflavones | Total aglycones | ||||
| Daidzin | Genistin | Malonyldaidzin | Malonylgenistin | Daidzein | Genistein | |||||
| Unfermented (Boiled soybean) | - | 22.6±0.2b | 39.9±1.0b | 52.8±0.5a | 105.9±0.8a | 3.3±0.1h | 5.5±0.2g | 230±2a | 8.8±0.3h | 3.8±0.2i |
| R. oligosporus NBRC 32002 | 20 °C | 23.7±0.6b | 41.7±0.9b | 52.7±2.5a | 104.7±0.9ab | 3.4±0.2h | 5.5±0.3g | 232±4a | 9.0±0.6h | 3.9±0.2i |
| 25 °C | 15.5±1.0c | 34.9±1.9c | 51.3±3.7a | 103.8±5.4ab | 7.8±0.4g | 9.8±0.4f | 223±13a | 17.5±0.8g | 7.9±0.1h | |
| 32 °C | 9.0±0.4d | 26.2±0.8d | 52.1±1.1a | 103.2±0.6ab | 12.2±0.2e | 14.0±0.2e | 217±3ab | 26.1±0.4f | 12.0±0.1g | |
| 37 °C | 6.7±0.3e | 16.5±0.1e | 48.4±1.1ab | 96.7±1.2ab | 14.6±0.2d | 17.9±0.3d | 201±3b | 32.5±0.5de | 16.2±0.0f | |
| R. oryzae NBRC 4716 | 20 °C | 7.1±0.8e | 17.5±1.7e | 49.5±3.5ab | 96.0±5.0b | 12.0±1.0e | 18.1±1.2d | 200±13b | 30.1±2.2e | 15.0±0.1f |
| 25 °C | 3.4±0.2f | 10.9±0.6f | 32.6±0.5c | 74.4±1.1c | 14.8±0.1d | 19.3±0.2d | 155±3c | 34.1±0.3d | 22.0±0.3e | |
| 32 °C | n.d. | 6.3±0.2g | 23.2±0.7de | 57.6±1.4d | 13.8±1.1d | 17.9±1.3d | 119±4d | 31.6±2.4de | 26.6±1.5d | |
| 37 °C | n.d. | 5.3±0.5g | 18.7±1.5ef | 48.5±3.5e | 10.0±0.3f | 14.2±0.4e | 97±6e | 24.2±0.7f | 25.1±1.3d | |
| R. stolonifer NBRC 30816 | 20 °C | n.d. | 7.8±0.7g | 27.6±1.6cd | 65.9±2.7cd | 24.3±0.1c | 42.7±0.4c | 168±5c | 67.1±0.4c | 39.8±1.1c |
| 25 °C | n.d. | 4.3±0.1g | 16.8±0.6ef | 46.0±1.1e | 29.5±0.6b | 56.1±0.8b | 153±1c | 85.7±1.4b | 56.1±1.0b | |
| 32 °C | n.d. | n.d. | 20.6±0.6ef | 42.8±1.4e | 32.0±0.5a | 61.7±0.6a | 157±1c | 93.7±1.0a | 59.6±1.0a | |
| 37 °C | 26.6±1.0a | 52.0±3.0a | 43.4±4.1b | 96.0±6.7b | 3.7±0.2h | 6.6±1.1g | 228±6a | 10.3±1.3h | 4.5±0.7i | |
Soybeans were fermented at 20, 25, 32 or 37°C for 40 h. Values are means ± standard deviations (n=3).
Different lowercase letters in the same column indicate significant difference (P<0.05) according to Tukey's multiple-range test.
n.d.; not detected (< 0.4 mg/100g dry weight)
Changes in isoflavone composition during fermentation Changes in isoflavone composition in the fermented soybeans with each Rhizopus species during fermentation are shown in Table 3. As the fermentation time was prolonged, the content of total aglycones was increased in the fermented soybeans with R. oligosporus and R. stolonifer. However, the prolongation of fermentation time did not affect the content of total aglycones in the fermented soybeans with R. oryzae. After 72 h of fermentation, the contents of total aglycones were 40 mg, 25 mg, and 103 mg per 100 g dried fermented soybeans with R. oligosporus, R. oryzae, and R. stolonifer, respectively. The ratios of total aglycones to total isoflavones increased to 20%, 33%, and 65% by the fermentation for 72 h with R. oligosporus, R. oryzae, and R. stolonifer, respectively.
| Isoflavones contents (mg/100 g dry weight) | Total aglycones1 (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Strain | Temperature | β-Glucosides | Malonylglucosides | Aglycones | Total isoflavones | Total aglycones | ||||
| Daidzin | Genistin | Malonyldaidzin | Malonylgenistin | Daidzein | Genistein | |||||
| Unfermented (Boiled soybean) | - | 22.5±0.2a | 40.3±0.5a | 56.4±0.5a | 115.1±0.7a | 3.3±0.0g | 4.9±0.1h | 243±1a | 8.2±0.1h | 3.4±0.0j |
| R. oligosporus NBRC 32002 | 24h | 11.1±0.0b | 28.0±0.2b | 51.3±1.3b | 109.6±3.4ab | 11.3±0.4f | 14.2±0.7fg | 226±6ab | 25.5±1.1g | 11.3±0.2i |
| 40h | 7.2±0.6cd | 21.3±0.9c | 47.2±0.5c | 103.0±0.8bc | 13.9±0.4de | 16.7±0.6fg | 209±2bc | 30.7±1.0fg | 14.7±0.5h | |
| 48h | 7.7±0.2cd | 22.1±0.3c | 47.2±2.8c | 102.5±5.4bc | 15.1±1.4d | 17.7±1.3f | 212±11bc | 32.8±2.7f | 15.5±0.5h | |
| 72h | 6.8±0.3d | 19.0±0.3d | 42.4±2.6d | 92.4±4.6d | 18.1±1.4c | 21.9±1.5e | 201 ±9cd | 39.9±2.9e | 19.9±0.5g | |
| R. oryzae NBRC 4716 | 24h | 6.2±0.1e | 18.9±0.4d | 41.0±1.1d | 92.8±3.0d | 12.7±0.6def | 14.7±0.8fg | 186±6de | 27.4±1.3fg | 14.7±0.3h |
| 40h | n.d. | 7.0±0.2f | 20.5±0.7ef | 57.8±1.6ef | 12.3±0.5ef | 15.1±0.5fg | 113±3g | 27.4±1.1fg | 24.3±0.8f | |
| 48h | n.d. | 4.1±0.0g | 16.4±0.1gh | 51.9±0.8f | 12.1±0.6ef | 15.6±0.6fg | 100±2g | 27.8±1.1fg | 27.7±0.8e | |
| 72h | n.d. | n.d. | 11.3±0.8i | 39.7±2.0g | 11.4±0.8ef | 13.4±0.9g | 76±4h | 24.8±1.7g | 32.8±1.5d | |
| R. stolonifer NBRC 30816 | 24h | n.d. | 10.1±0.6e | 42.1±0.9d | 95.7±1.6cd | 19.5±0.1c | 33.3±0.6d | 201 ±2cd | 52.8±0.6d | 26.3±0.5ef |
| 40h | n.d. | 4.6±0.4g | 23.3±1.7ef | 61.4±3.4ef | 27.7±1.4b | 51.4±2.5c | 168±9f | 79.1±3.9c | 47.0±1.1c | |
| 48h | n.d. | 3.9±0.1g | 18.8±0.6fg | 52.8±1.1f | 33.1±1.0a | 62.6±2.2b | 171±4ef | 95.7±3.1b | 55.9±1.0b | |
| 72h | n.d. | 3.5±0.5g | 12.8±1.2hi | 39.9±3.0g | 35.4±1.2a | 67.5±1.6a | 159±6f | 102.9±2.8a | 64.7±1.8a | |
Soybeans were fermented at 37°C for R. oligosporus, and at 32°C for R. oryzae and R. stolonifer. Values are means ± standard deviations (n=3).
Different lowercase letters in the same column indicate significant difference (P< 0.05) according to Tukey's multiple-range test.
n.d.; not detected (< 0.4 mg/100g dry weight)
Changes in β-glucosidase activity during fermentation To examine the underlying cause of differences in the isoflavone composition, β-glucosidase activity was determined. β-Glucosidase is considered to be the key enzyme to release isoflavone aglycones via hydrolysis of the isoflavone glucosides. The β-glucosidase activity in fermented soybeans with each Rhizopus species is shown in Table 4. In all Rhizopus species, the β-glucosidase activity increased with increasing fermentation time. The β-glucosidase activity of the fermented soybeans with R. oligosporus was the lowest among all the fermented soybeans (45 mU/g dry weight for 72 h). Meanwhile, the β-glucosidase activity of soybeans fermented with R. oryzae (373 mU/g dry weight for 72 h) was significantly higher than that with R. stolonifer (337 mU/g dry weight for 72 h) during fermentation.
| Strain | β-glucosidase activity (mU/g dry weight) | |||
|---|---|---|---|---|
| 24h | 40h | 48h | 72h | |
| R. oligosporus NBRC 32002 | 16±4h | 31±4gh | 38±1g | 45±3g |
| R. oryzae NBRC 4716 | 136±6e | 298±6c | 307±4c | 373±7a |
| R. stolonifer NBRC 30816 | 98±3f | 151±3e | 198±4d | 337±13b |
Soybeans were fermented at 37°C for R. oligosporus, and at 32°C for R. oryzae and R. stolonifer. Values are means ± standard deviations (n=3).
Different lowercase letters indicate significant difference (P<0.05) according to Tukey's multiple-range test.
Content and composition of isoflavones in isoflavone-enriched fermented soybeans The content and composition of isoflavones in the isoflavone-enriched tempeh-like fermented soybeans are shown in Fig. 1. The boiled hypocotyls used in this study had a higher ratio of glycitein analogs and the β-glucoside form of isoflavones compared to the boiled soybeans. The contents of total aglycones (daidzein, genistein, and glycitein) in both fermented soybeans (using dehulled soybeans and hypocotyls at ratios of 20:80 and 0:100) were in the rank order of R. stolonifer > R. oryzae > R. oligosporus. The ratios of total aglycones to total isoflavones in the fermented soybeans using 20% and 100% hypocotyls with R. stolonifer reached 83% (Fig. 1(a): total aglycones, 241 mg/100 g dry weight; total isoflavones, 292 mg/100 g dry weight) and 95% (Fig. 1(b): total aglycones, 964 mg/100 g dry weight; total isoflavones, 1015 mg/100 g dry weight), respectively. Conversely, in the fermented soybeans using 100% hypocotyls with R. oligosporus, the content of total aglycones decreased from 363 mg to 265 mg per 100 g dry weight, while that of β-glucosides increased from 763 mg to 968 mg per 100 g dry weight with fermentation.
Isoflavones contents and compositions in the isoflavone-enriched tempeh-like fermented soybeans. The fermented soybeans were prepared by using hypocotyls and dehulled soybeans in the ratio of 20:80 (%) (a), and 100:0 (%) (b). Each bar shows the total contents of β-glucosides, malonylglucosides, and aglycones, in order from the left. Values are means ± standard deviations (n=3). Different lowercase letters on the top of bar of the same pattern indicate significant difference (P < 0.05) according to Tukey's multiple-range test.
The isoflavone content and composition in fermented soybean foods vary depending on the thermal processes or fermentation conditions (Wang and Murphy, 1996). In our study, the ratios of total aglycones to total isoflavones in fermented soybeans with R. oligosporus, R. oryzae, and R. stolonifer were 16%, 29%, and 45% on average with fermentation at 32°C for 40 h, respectively. As non-salt fermented soybean foods, natto and cheonggukjang are widely consumed in Japan and Korea, respectively. Both soybean foods are made by fermentation with Bacillus species. In commercial natto products, the ratio of total aglycones to total isoflavones was 24% at most (Wei et al., 2008). In cheonggukjang, the same ratio increased from 3% to 25% during 45 h of fermentation (Jang et al., 2006). Thus, the soybeans fermented with R. stolonifer appear to have the highest content of isoflavone aglycones as compared to those with other Rhizopus species and other non-salt fermented soybean foods with Bacillus species. Nakajima et al. (2005) developed a procedure for the preparation of isoflavone-enriched fermented soybeans with R. oligosporus, in which the ratio of total aglycones to total isoflavones was 44%, by adding soybean hypocotyls. In the present study, we found that the ratio of total aglycones to total isoflavones in the fermented hypocotyls prepared with R. stolonifer reached 95%, which was much higher than that reported by Nakajima et al. (2005).
We examined the β-glucosidase activity to clarify the reason for differences in the isoflavones composition in the soybeans fermented with the three Rhizopus species. Ismail and Hayes (2005) showed that β-glucosidase was able to hydrolyze malonylglucosides as well as β-glucosides, but at a much lower rate compared to their β-glucosides in vitro. In the present study, β-glucosidase activity and the ratio of total aglycones to total isoflavones in the fermented soybeans with R. oligosporus were the lowest among all the fermented soybeans (Tables 4 and 3). Thus, R. oligosporus may have a lower ability to generate aglycones from isoflavone glucosides. On the other hand, β-glucosidase activity in the fermented soybeans with R. oryzae was the highest among all the fermented soybeans (Table 4). Nevertheless, the content of total isoflavone aglycones in the fermented soybeans with R. oryzae was lower than that with R. stolonifer (Table 3). This suggests that high β-glucosidase activity does not always result in a higher level of isoflavone aglycones. Further study is necessary to investigate this issue using isoflavone glucosides instead of pNPG as the substrate for the β-glucosidase assay.
One possible reason for this discrepancy is that isoflavones were converted to other analogs in the soybeans fermented with R. oryzae by the action of enzymes other than β-glucosidase, since there were unidentified peaks in the chromatograph obtained from HPLC of the fermented soybeans with R. oryzae (data not shown). The unidentified peaks did not appear in the chromatograph obtained from HPLC of the fermented soybeans with the other Rhizopus species. In addition, esterase has also been suggested to be involved in the generation of isoflavone aglycones, which cleaves off the malonyl moieties of malonylglucosides and facilitates the formation of aglycones (Hiderer et al., 1986). Thus, the possibility remains that higher production of isoflavone aglycones by fermentation with R. stolonifer is due to higher esterase activity. Further investigation is required to test this possibility.
Meanwhile, in the fermented soybeans using 100% hypocotyls with R. oligosporus, the content of aglycones decreased, while that of β-glucosides increased by fermentation (Fig. 1(b)). However, this was not the case with the other Rhizopus species (Fig. 1(b)). β-Glucosidase is also known to synthesize oligosaccharides by the transglycosylation reaction (Park et al., 2010). Thus, the possibility cannot be ruled out that β-glucosidase produced by R. oligosporus causes the reverse conversion of isoflavone aglycones to β-glucosides under certain conditions.
To date, there has been little comparative study on tempeh produced using Rhizopus species. It is known that R. stolonifer has the lowest amylase and protease activities among Rhizopus species (Shurtleff and Aoyagi, 1979; Baumann and Bisping, 1995). However, the advantage of preparing tempeh with R. stolonifer has not been clarified yet. To the best of our knowledge, this study provides the first evidence suggesting that among the three Rhizopus species, R. stolonifer might be the most suitable for the production of tempeh with a higher content of isoflavone aglycones. Further study in our group is in progress to elucidate the health benefits of the consumption of tempeh fermented with R. stolonifer.
Acknowledgment We are grateful to Ms. Fumi Sato and Ms. Aya Miyajima for their outstanding technical assistance.
