Antioxidant activity of tempe fermented with three different Rhizopus species

Abstract

Tempe, a traditional Indonesian fermented soy-based food, reportedly has higher antioxidant activity than unfermented soybeans. In 2015, the Codex Alimentarius defined the starters of soybean tempe to be Rhizopus microsporus var. oligosporus, Rhizopus oryzae, and/or Rhizopus stolonifer. Most previous studies have focused only on tempe prepared with R. oligosporus. In this study, we compared the antioxidant activities of tempe fermented with three Rhizopus species and unfermented soybeans using assays for β-carotene bleaching (antioxidant activity), antioxidative potential, and oxidative stabilization. The β-carotene bleaching and antioxidative potential assays indicated that the tempe fermented with R. stolonifer had higher antioxidant activity than the tempe fermented with R. oligosporus and R. oryzae. We speculate that the high antioxidant properties of this product are attributable to the isoflavone aglycones and hydroxylated compounds produced by fermentation.

Introduction

Reactive oxygen species (ROS), superoxide anions, and hydrogen peroxide are produced by four-electron reduction during energy production by cellular respiration. In general, ROS are scavenged by enzymes, namely, superoxide dismutase and catalase, which are present in vivo (Wang et al., 2017; Milisav et al., 2018). However, activities, such as smoking (Armstrong et al., 2011) and intense exercise (Pingitore et al., 2015), have the potential to increase ROS production. Excessive ROS production has been reported to be a risk factor for the onset of many diseases, such as atherosclerosis (Kattoor et al., 2017), cancer (Prasad et al., 2017), and dementia (Kryscio et al., 2017). The intake of antioxidants through food is recommended to prevent such diseases.

Tempe is a traditional Indonesian soy-based food fermented with fungi belonging to the Rhizopus genus, and its consumption in Indonesia is similar to that of miso in Japan. Tempe has been reported to have higher antioxidant activity than unfermented soybeans due to the presence of isoflavones, peptides, amino acids, and novel polyphenol compounds produced during fermentation (Watanabe et al., 2007; Surya et al., 2020). In 2015, the Codex Alimentarius established a regional standard for tempe (Codex Alimentarius, 2015), defining the starters of tempe to be spores of Rhizopus microsporus var. oligosporus, Rhizopus oryzae, and/or Rhizopus stolonifer. Most previous studies have focused on tempe's antioxidant activity using tempe prepared with R. oligosporus, as it is a major Rhizopus species, whereas R. oryzae and R. stolonifer are considered minor species. Consequently, although R. oryzae and R. stolonifer are designated tempe starters, their antioxidant activities have yet to be conclusively established. Thus, a comparative study using tempe fermented with R. oligosporus, R. oryzae, and R. stolonifer is needed. In our previous study on the benefits of tempe for atopic dermatitis in mice, we showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (Om and Tej, 2009) of R. stolonifer is higher than that of R. oligosporus and R. oryzae (Aoki et al., 2020). To verify this conclusion, antioxidant assays of different systems are needed.

In this study, we compared the antioxidant activities of three types of tempe fermented with different Rhizopus species using assays to measure β-carotene bleaching (antioxidant activity), antioxidative potential, and oxidative stabilization.

Materials and Methods

Microorganisms  R. microsporus var. oligosporus (hereafter, R. oligosporus) NBRC 32002, R. oryzae NBRC 4716, and R. stolonifer var. stolonifer (hereafter R. stolonifer) NBRC 30816 were used in the experiments. All strains were transferred from the Biological Resource Center, NITE (NBRC) with permission.

Tempe production  Dehulled yellow soybeans were soaked in 0.2 % acetic acid at 25 °C overnight. After drying, the soaked soybeans were boiled at 100 °C for 10 min and cooled to 40 °C. Then, 100 g of the boiled soybeans were inoculated with spore suspensions (approximately 3 × 10⁶ spores) of each Rhizopus species, namely, R. microsporus var. oligosporus (Tempe RM), R. oryzae (Tempe RO), or R. stolonifer (Tempe RS). The inoculated soybeans were spread on polyethylene bags with small pinholes and incubated at 32 °C for 40 h for fermentation. The obtained tempe was sterilized by heat treatment at 90 °C for 30 min, then dried in a lyophilizer (Kameda et al., 2018).

Sample preparation  Antioxidative compounds were extracted from 3 g each of freeze-dried powdered unfermented soybeans and the three types of tempe using 30 mL of a methanol:acetone:distilled water mixture (7:7:6, v/v/v), which has been reported to be effective for extracting polyphenols and flavonoids (Teh et al., 2014).

β-carotene bleaching antioxidant assay  The β-carotene bleaching antioxidant assay (Miller, 1971; Tsushida, 1994) was performed as follows: initially, 0.1 mL of linoleic acid solution (1 g of linoleic acid in 10 mL of chloroform), 0.25 mL of β-carotene solution (10 mg of β-carotene in 10 mL of chloroform), and 0.5 mL of Tween 40 solution (2 g of Tween 40 in 10 mL of chloroform) were mixed; chloroform was then completely removed by nitrogen spraying. Finally, 45 mL of distilled water and 5 mL of 0.2 M phosphate buffer (pH 6.8) were added to the solution, and the mixture was used as the linoleic acid and β-carotene emulsion. For each 100-µL sample, 4900 µL of this emulsion was added. The mixture was then incubated at 50 °C and absorbance was measured at 490 nm at 0, 30, 60, 90, and 120 min. Butylated hydroxyanisole was used as a positive control.

Antioxidative potential assay  The antioxidative potential was measured using a free radical elective evaluator (FREE; Diacron International srl, Italy) according to the manufacturer's instructions. The antioxidative potential was evaluated using the ferric reducing ability (Benzie and Strain, 1996).

Oxidative stabilization assay  Freeze-dried, powdered, unfermented soybeans and the three types of tempe were incubated at 50 °C for 21 days. Total lipids were extracted using diethyl ether, after which peroxide value (POV) and conjugated diene were measured as follows. POV measurements were performed on days 0, 7, 14, and 21. Total lipids from the samples were dissolved in 8 mL of chloroform, after which 12 mL of acetic acid and 0.8 mL of saturated potassium iodide solution were added and mixed. After the mixture was maintained at room temperature (25 °C) in the dark for 5 min, 5 % starch was added as an indicator. The mixture was titrated with a 0.01 N sodium thiosulfate solution. The conjugated diene measurements were carried out on day 21; 0.01 g of total lipid was dissolved in 5 mL of petroleum ether, then the absorbance was measured at 234 nm.

Statistical analysis  All data are expressed as the mean ± standard error (SE). Statistically significant differences (p < 0.05) between groups were evaluated using Tukey's test. Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA).

Results and Discussion

The results of the β-carotene bleaching antioxidant assay are shown in Fig. 1. The order of the antioxidant activity was tempe RS > tempe RM > tempe RO > unfermented soybeans. Significant differences between these experimental samples were found at the 30, 60, 90, and 120-min time points. The results of the antioxidant potential assay are shown in Fig. 2. The order of the antioxidative potential was tempe RS > tempe RM > tempe RO > unfermented soybeans. Significant differences were found between all experimental samples. Furthermore, a previous study has reported that the order of the Trolox equivalents (measured by the DPPH radical scavenging activity assay) was tempe RS = tempe RM > tempe RO > unfermented soybeans (Aoki et al., 2020).

Fig. 1.

Antioxidant activities evaluated using β-carotene breaching antioxidant assay. Data are expressed as the mean ± SE (n = 3). Error bars (SE) are too small to be shown.

Fig. 2.

Antioxidant activities evaluated using antioxidative potential assay. Data are expressed as the mean ± SE (n = 3). a, b, c, d: Different letters express significant differences (p < 0.05).

The results of the oxidative stabilization assay, that is, the changes in the POV of unfermented soybean, tempe RM, tempe RO, and tempe RS, are shown in Fig. 3. The POV of all three types of tempe did not increase over time, whereas that of the unfermented soybeans increased. The relative conjugated diene contents of the unfermented soybeans and the three types of tempe are shown in Fig. 4. The conjugated diene contents of all tempe types were significantly lower than that of the unfermented soybeans. No significant differences were found between the different tempe types.

Fig. 3.

The results of oxidative stabilization assay. Data are expressed as the mean (n = 3).

Fig. 4.

Relative conjugated diene contents. Data are expressed as the mean ± SE (n = 3). a, b: Different letters express significant differences (p < 0.05).

The total polyphenols, isoflavones, isoflavone glucosides, isoflavone malonylglucosides, and isoflavone aglycones in unfermented soybeans and the three tempe types are shown in Fig. 5, which was reproduced from Aoki et al. (2020). The total polyphenol content was measured by the Folin-Ciocalteu method with gallic acid as the reference standard, and the isoflavone content was measured using high-performance liquid chromatography with ultraviolet detection. The order of the total polyphenol content was tempe RS = tempe RM > tempe RO > unfermented soybeans. The order of the total isoflavone aglycone content was tempe RS > tempe RM = tempe RO > unfermented soybeans. The order of the total isoflavone content was unfermented soybeans = tempe RM > tempe RS > tempe RO. The order of the total isoflavone glycoside content was tempe RM = unfermented soybeans > tempe RS = tempe RO. The order of total isoflavone malonylglucosides was unfermented soybeans > tempe RM > tempe RO = tempe RS.

Fig. 5.

Comparison of different polyphenol contents. Data are expressed as the means (n = 3). a, b, c: different letters express significant differences between the samples (p < 0.05). The data for total polyphenols and total isoflavones are reproduced from Aoki et al. (2020). Total polyphenol content was measured by the Folin-Ciocalteu method; meanwhile, isoflavone content was measured using high-performance liquid chromatography with ultraviolet detection. Because quantification mechanism is different for total polyphenol and isoflavone content, careful interpretation is required to compare them with each other.

In the present study using β-carotene bleaching antioxidant activity, antioxidative potential and oxidative stabilization assays, we revealed that tempe fermented with all three Rhizopus species showed higher antioxidant activity than the unfermented soybeans. A comparison of the three types of tempe showed that the order of the antioxidant activity was tempe RS > tempe RM > tempe RO. In regard to the relationship between the antioxidant activity and the total polyphenol or partial isoflavone content, we speculate that the pattern of the antioxidant activities may be associated with the amount of total isoflavone aglycones and total polyphenols.

Isoflavone is considered the main antioxidant found in soy products (Kim et al., 2020). The antioxidant activity of isoflavone aglycones, such as genistein and daidzein, has been reported to be stronger than that of glycosides, such as genistin and daidzin (Pratt et al., 1979; Benediktus at al., 2022). The strong antioxidant activity of isoflavone aglycones is reportedly attributed to their high reactivity with free radicals, their action as an electron donor, or their ability to terminate free radical chain reactions (Zuo et al., 2011). A previous study on the changes in antioxidant activity during fermentation of miso and tempe suggested that the highest isoflavone aglycone levels (produced from glycosides by β-glucosidase) coincide with the highest antioxidant activity (Ikeda et al., 1995). Our findings in the present study also indicate that the total isoflavone aglycone content is closely associated with the strength of the antioxidant activity of tempe. However, the amount of isoflavone aglycones alone cannot account for the strength of the antioxidant activity of tempe.

Although the isoflavone aglycone content in tempe RM and tempe RO (both 25 mg/100 g dry weight) was approximately half of that in tempe RS (65 mg/100 g dry weight), the antioxidant activity of tempe RM and tempe RO was not half of that of tempe RS. Furthermore, although the contents of isoflavone aglycones in tempe RM and tempe RO (both 25 mg/100 g dry weight) were similar, we found the antioxidant activity of tempe RM to be higher than that of tempe RO.

It has been reported that tempe prepared using R. oligosporus, i.e., tempe RM in this study, produces hydroxylated compounds, such as 6,7,4′-trihydroxyisoflavone and 3-hydroxyanthranilic acid (Esaki et al., 1996; Ikehara et al., 1968). The reduction in the total isoflavone content of tempe when compared to unfermented soybean may be attributed to the conversion of isoflavones to hydroxylated compounds during fermentation. In addition, soyasaponins in soybeans are also known to have strong antioxidant activities, and have been reported to be degraded to hydroxy compounds, such as soyasapogenol B, during fermentation (Kamo et al., 2014). The Folin–Ciocalteu method used in this study for the measurement of total polyphenols can detect hydroxylated compounds. Thus, a reduction in total isoflavones and an increase in total polyphenols in tempe may be associated with an increase in hydroxylated compounds derived from isoflavones and soyasaponins. Consequently, it is conceivable that an increase in these hydroxylated compounds could also be associated with the strength of the antioxidant activity of tempe.

In addition, peptides and free amino acids produced during fermentation may be responsible for the antioxidant activity of tempe. Peptides isolated from digests of soybean protein (Chen et al., 1996), peptides in okara, residues of soymilk production, proteins (Yokomizo et al., 2002), digested products from tempe protein (Tamam et al., 2021), and free amino acids, such as ornithine, lysine, arginine, and histidine (Tamura et al., 1998), possess high antioxidant activity. The antioxidant activity of peptides and free amino acids is attributed to their loose binding to lipid peroxides (Murase et al., 1993), radical scavenging (Saito et al., 2003), and metal chelate formation (Uchida and Kawashima, 1992). The order of the free amino acid production capacity has been reported to be tempe RM > tempe RO > tempe RS (Baumann and Bisping, 1995), whereas the order of the antioxidant activity has been reported to be tempe RS > tempe RM > tempe RO. Therefore, the free amino acids and peptides present in tempe likely exerted little influence on the strong antioxidant activity of tempe RS in this study.

These results suggest that the antioxidant activity of tempe fermented with R. stolonifer is higher than that of tempe fermented with R. oligosporus and R. oryzae. Therefore, the use of R. stolonifer as a starter is preferable for tempe production to obtain the benefit of stronger antioxidant activity. Isoflavone aglycones and hydroxylated compounds produced during fermentation may contribute to this high antioxidant potential of tempe fermented with R. stolonifer, but more detailed structural analyses of the hydroxylated compounds are required.

Conflict of interest  The authors declare no conflicts of interest.

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