2014 Volume 20 Issue 2 Pages 499-504
Soybean broth (SB) is a waste product in the manufacturing of boiled soybean-based foods. We cultured the SB with six pure strains of microorganisms (Aspergillus oryzae, Aspergillus sojae, Rhizopus oligosporus, Zygosaccharomyces rouxii, Bacillus subtilis (natto), and Tetragenococcus halophilus) used commercially in the production of fermented soybean foods, including miso, soy sauce, natto and tempeh, and examined their cytotoxicity and apoptosis-inducing effect on U937 cells. Ethyl acetate extracts of all cultures showed cytotoxicity against U937 cells, although that of SB did not in the range of concentrations tested. Further, ethyl acetate extracts of SB after culturing with A. sojae (ESBA) and ethyl acetate extracts of SB after culturing with B. subtilis (natto) (ESBB) elicited DNA fragmentation. The DNA fragmentation elicited by ESBA was prevented by pretreatment with a general caspase inhibitor (Z-Asp-CH2-DCB), implying induction of caspase-dependent apoptosis. Assays for caspase activity showed that apoptosis induction was associated with caspase-3, -8, and 9, which are the principal caspases involved in apoptosis. Thus, SB was conferred cytotoxicity upon culturing with the microorganisms used in this study; in particular, SB cultured with A. sojae induced apoptosis in U937 cells.
Soybean broth (SB), known in Japanese as nijiru, is a waste product in the production of processed soybean foods such as miso, soy sauce, and natto. Recently, methods for reuse of SB have been developed to take advantage of its abundant nutrients. For instance, nicotianamine, an inhibitor of angiotensin I-converting enzyme, was isolated from SB, and its use as an anti-hypertensive agent was proposed (Takenaka, 2009). Utilizing SB as a culture medium for the production of γ-amino butyric acid by fermentation with lactic acid bacteria has also been devised (Furuta et al., 2008). In addition, we produced vinegar using SB, and demonstrated its growth-inhibitory and apoptosis-inducing effects on U937 cells (Inagaki et al., 2005). Subsequently, we isolated tryptophol from the vinegar as an active component and revealed that this compound was produced during ethanol fermentation by Saccharomyces cerevisiae in the production of the vinegar (Inagaki et al., 2007a, 2007b). Besides this, however, there are no reports addressing the health benefits of fermented products using soybean broth.
Apoptosis, or programmed cell death, is a highly regulated process involved in many physiological and pathological processes. It is characterized by cellular events such as formation of apoptotic bodies and fragmentation of DNA (Arends et al., 1990; Cohen, 1993; Kerr et al., 1972; Steller, 1995). Caspases, a class of cysteine aspartic acid-specific proteases, play a crucial role in apoptosis induction; the apoptosis signal is transduced by sequential activation of these enzymes (Cohen, 1997; Villa et al., 1997). Some reports have shown that natural compounds found in plants, plant products and their associated products of fermentation induce apoptosis in cancer cells and can be effective chemopreventive agents against various cancers (Bonnesen et al., 2001; Funayama et al., 1989; Graham, 1992; Moongkarndi et al., 2004; Saeki et al., 2002).
Various microorganisms, including fungi such as Aspergillus oryzae, Aspergillus sojae, and Rhizopus oligosporus, the yeast Zygosaccharomyces rouxii, and bacteria such as Bacillus subtilis (natto) and Tetragenococcus halophilus, are used in the production of fermented soybean foods: generally, A. oryzae, A. sojae, Z. rouxii, and T. halophilus are used in the production of miso and soy sauce; B. subtilis (natto) in the production of natto; and R. oligosporus in the production of tempeh, a traditional Indonesian soybean product. These soybean-based products reportedly have beneficial effects on human health, such as anti-oxidant, antitumor, and anti-hypertensive activities (Iwai et al., 2002; Matsuo et al., 1997; Watanabe, 2010); however, no reports have focused on the physiological effects of SB cultured with these microorganisms.
The purpose of this study was to expand the use of SB by investigating the health benefits of products formed by culturing with different microorganisms. In this study, we cultured SB with pure strains of the six microorganisms mentioned above, and evaluated the cytotoxicity and apoptosis-inducing effect on U937 cells.
Materials and chemicals Soybeans (Glycine max (L.) Merrill.) were purchased from Kobori Industrial (Kanagawa, Japan). Unpolished rice (Oryza sativa) was from Maisen (Fukui, Japan). Microorganisms were obtained from the National Institute of Technology and Evaluation Biological Resource Center (NBRC; Chiba, Japan) and a commercial strain developer; A. oryzae NBRC 4134, A. sojae NBRC 33084, R. oligosporus NBRC 8631, Z. rouxii NBRC 0846 and T. halophilus NBRC 12172, which were derived from fermented soybean product resources, were from NBRC, while B. subtilis (natto) was from the Takahashi Yuzo Research Institute (Yamagata, Japan). Potato dextrose agar medium used for culturing fungal starters was from Kanto Kagaku (Tokyo, Japan). YM medium (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract) was prepared for culturing Z. rouxii NBRC 0846; glucose, peptone, and yeast extract were purchased from Nacalai Tesque (Kyoto, Japan) and malt extract was from Difco (Detroit, MI, USA). Luria-Bertani medium used for culturing B. subtilis (natto) was from Nacalai Tesque. de Man Rogosa Sharpe medium used for T. halophilus NBRC 12172 was from Difco. Human leukemic U937 cells were obtained from the Health Service Research Resources Bank (Osaka, Japan). RPMI 1640 medium and antibiotics (penicillin and streptomycin) were from Nacalai Tesque. Fetal bovine serum was from Veritas (Tokyo, Japan). A general caspase inhibitor (Z-Asp-CH2-DCB) was from the Peptide Institute (Osaka, Japan). All other reagents were of analytical grade and were obtained from either Wako Chemical or Nacalai Tesque.
Preparation of SB and cultivation of microorganisms The concentrations of industrial SBs from factories producing processed soybean foods are very inconsistent (e.g., the solid matter concentration of SB from a miso factory was ca. 20 mg/mL, while that from a natto factory was ca. 100 mg/mL). Therefore, SB was prepared in the laboratory to ensure a uniform concentration. First, 40 g of soybeans and 100 mL of distilled water were added to an Erlenmeyer flask, which was autoclaved at 121°C for 1 min for the soybeans to absorb the bulk of the water. After removing excess water, the soybeans were autoclaved at 121°C for 20 min to steam-cook. Then, 100 mL of distilled water was added, and the mixture was autoclaved at 121°C for 20 min. The resulting extract was removed and saved, and an additional 100 mL of distilled water was added, followed by autoclaving at 121°C for 20 min. The resulting extracts were pooled; by this method, SB was obtained with a solid matter concentration of 39 g/L. Rice broth (RB) was prepared from unpolished rice in the same manner; freeze-dried RB was dissolved in distilled water to obtain an equivalent solid matter concentration (39 g/L). After sterilizing 200-mL aliquots by autoclaving, the SB and RB were cooled, and, in separate flasks for each microorganism, evenly inoculated with a 1.0 mL spore suspension (106 spores/mL), then agitated in an incubator at the optimal temperature for each microorganism.
Preparation of ethyl acetate extracts The cultures were centrifuged at 3,000 x g for 10 min to remove mycelia. The supernatants were moved to a separation funnel and an equal volume of ethyl acetate was added. After the resulting mixture was shaken using a funnel shaker (Yamato Scientific, Tokyo, Japan) for 10 min at room temperature, the ethyl acetate layer was transferred to an Erlenmeyer flask and an equal volume of fresh ethyl acetate was added to the funnel; these procedures were repeated two more times. An ethyl acetate extract was obtained by evaporation of the combined solvent. The remaining water layer was freeze-dried to prepare a water-soluble fraction. For preparation of test samples, each ethyl acetate extract was dissolved in dimethyl sulfoxide and each water-soluble fraction was dissolved in water.
Cell culture U937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C under 5% CO2. For each test, U937 cells (1 × 105 cells/mL) were seeded and cultured in the presence or absence of test sample.
Assay for cell viability Cells were subcultured for 4 h in 96-well plates and then treated with different doses of test sample. After incubation for 24 h, cell viability was determined based on a WST-8 assay, using a commercial cell counting kit (Kishida Chemical, Tokyo, Japan) according to the manufacturer's instructions. The cytotoxicity of test samples on U937 cells was evaluated by measuring absorbance at 490 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Determination of DNA fragmentation DNA in U937 cells was extracted according to the method described by Ishizawa et al. (1991). Cells were suspended in 200 µL lysis buffer (0.05% SDS, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA) and treated with 1 mg/mL proteinase K, followed by incubation for 60 min at 37°C. DNA was then precipitated for 2 h at −20°C in 50% isopropanol. The precipitate was pelleted by centrifugation for 10 min at 15,000 x g, dried, and resuspended in TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) buffer. The DNA solutions were treated with 10 µg/mL RNase A for 30 min at 37°C. Equivalent amounts of DNA (1 µg) were loaded into the wells of 1.0% agarose gels, electrophoresed in TAE buffer (Tris-acetic acid (pH 8.0) and 2 mM EDTA), stained with ethidium bromide, and imaged using an AE-9000 E-Graph system (Atto, Tokyo, Japan) and ImageSaver5 software (Atto).
Determination of caspase activity The time course of caspase-3, -8, and -9 activities was determined using a Caspase Colorimetric Protease Assay Kit (MBL, Aichi, Japan), according to the manufacturer's instructions. After treatment with sample for 2, 4, 6, 8, 12, 16, or 24 h, cells were collected, washed with phosphate-buffered saline, and lysed in lysis buffer (provided in the kit) for 15 min at 4°C, followed by centrifugation at 15,000 × g for 10 min to remove cell debris. Reaction mixtures containing cytosolic extract (supernatant after the centrifugation), substrate peptides, including IETD-, LEHD-, or DEVE-p-nitroanilide (pNA), specific for caspase-8, -9, and -3, respectively, and reaction buffer were incubated in 96-well plates at 37°C for 2 h. Absorbance at 405 nm was measured using an iMark plate reader, and the measurements were compared to a standard curve generated using pNA solution of known concentration. The caspase activities were expressed as units per protein (µg). Units were defined as the following equation:
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Statistical analysis Values are shown as means ± standard error (SEM). The difference between groups was evaluated by a two-sided Student's t-test. Differences were considered significant at p < 0.05.
Cytotoxicity against U937 cells In an initial experiment, cell viability of U937 cells treated with each sample was examined. As shown in Fig. 1, ethyl acetate fractions of all SB cultures exhibited a significant cytotoxic effect, while that uncultured had a negligible effect in the concentrations tested. Water-soluble fractions of the cultures did not have any effect (data not shown). These results suggested that the cytotoxic compounds were produced during culturing of the microorganisms in SB. Of future interest is whether the same or distinct growth-inhibitory compounds were produced in cultures of different microorganisms. In contrast, culturing in RB showed the reverse effect; that is, the ethyl acetate extracts of RB cultured with the microorganisms showed less cytotoxicity than RB itself (the results of RB and RB cultured with A. sojae are shown in Fig. 1H and 1I). The cytotoxic effect of extract from rice bran on various cancer cells has been reported (Forster et al., 2013). The cytotoxicity of RS may be attributable to the functional ingredients in rice bran, such as caffeic acid, ferulic acid, and vitamin E, and these active compounds may have been lost during culturing. These results suggest that SB contains raw materials that can be converted to compounds with cytotoxic effects on U937 cells after cultivation, but RB does not. Because soybean has a high protein content, fermented soybean products contain various amine compounds produced by decarboxylation of amino acids (Shukla et al., 2011). In the present research, cytotoxicity against U937 cells was found in ethyl acetate fractions of SB cultures. Therefore, we speculate that the low polarity of aromatic amine compounds such as tryptamine and tyramine in the cultures may contribute to the effect.
Cell viability in U937 cells treated with each sample. Cells were treated with different doses of ethyl acetate extracts for 24 h. Cell viability (%) was measured using a WST-8 assay. A to G indicate samples related to soybean broth: A, uncultured; B, cultured with A. oryzae; C, A. sojae; D, R. oligosporus; E, Z. rouxii; F, B. subtilis; and G, T. halophilus. H and I indicate samples related to rice broth: H, uncultured; I, cultured with A. sojae. Values are means (± SEM) of triplicate experiments. *p < 0.05, **p < 0.01 (untreated versus sample-treated cells).
Determination of apoptosis induction To examine the trigger for cytotoxicity against U937 cells, we assessed DNA fragmentation and observed morphological changes in U937 cells. ESBA and ESBB elicited DNA fragmentation (Fig. 2A), while none of the other samples did so in the range of concentrations tested (data not shown). These DNA fragmentations occur at the sample concentrations cytotoxic against U937 cells; that is, at the concentrations of 0.4 to 1.0 mg/mL. DNA fragmentation elicited by ESBA was prevented by a general caspase inhibitor (Z-Asp-CH2-DCB), while fragmentation elicited by ESBB was not (Fig. 2A, lanes 7 and 8). Formation of apoptotic bodies in U937 cells treated with ESBA was observed by phase contrast microscopy (Fig. 2B-b). On the other hand, scattered U937 cells treated with ESBB appeared (Fig. 2B-c). These results were reproduced in tests repeated three times. Based on these results, ESBA induces apoptosis involving caspases in U937 cells. ESBB could give some other chemical stimulus that leads to caspase-independent cell death. We noted that the morphology of dying cells differed depending on the species of microorganism used in the cultures, suggesting that constituents with different mechanisms of cytotoxicity against U937 cells were produced during culturing in SB.
DNA fragmentation analysis and microscopic observation. U937 cells were treated in the absence (lane 1) or presence of 0.2 (lane 2), 0.4 (lane 3), 0.6 (lane 4), 0.8 (lane 5), and 1.0 mg/mL (lane 6) ethyl acetate extracts of SB after culturing with A. sojae (ESBA) (a) and ethyl acetate extracts of SB after culturing with B. subtilis (natto) (ESBB) (b) for 16 h. Cells were pretreated with general caspase inhibitor Z-Asp-CH2-DBC (100 µM) for 30 min prior to the treatment with each sample (lane 8); lane 7 (a) and (b) are the results with 0.4 mg/mL ESBA and 0.6 mg/mL ESBB, respectively. DNA was extracted and separated. M, λDNA digested with Hind III, used as size marker. B, U937 cells were treated in the absence (a) or presence of 0.4 mg/mL ESBA (b) or 0.6 mg/mL ESBB (c) for 16 h, and morphological changes were observed. Arrows indicate apoptotic bodies. All magnifications are × 100.
Caspase activities In order to determine the activation pathway for apoptosis induced by ESBA, we investigated the activation of caspase-8, -9, and -3, the principal caspases controlling apoptosis, in U937. The activities of all three caspases increased after adding 0.4 mg/mL ESBA, the concentration at which DNA fragmentation was observed most clearly, and peaked at 12 h (for caspase-3 and -9) or 16 h (for caspase-8); thereafter, the activities decreased (Fig. 3). ESBB did not activate these caspases in U937 cells (data not shown). Caspase-8-dependent apoptosis is initiated by cell-surface death receptors such as FS7-associated cell surface antigen and death receptors 4 and 5 (Mandal et al., 2005). On the other hand, the involvement of mitochondria has been implicated in caspase-9-dependent apoptosis (Nakamura et al., 2002). Since ESBA-induced apoptosis was accompanied by activation of both caspase-8 and -9, multiple active constituents for the induction of apoptosis may be present in ESBA.
Caspase activities induced by ethyl acetate extracts of SB after culturing with A. sojae (ESBA). U937 cells were treated with 0.4 mg/mL ESBA for the indicated times. The time course of caspase activity was determined: ●, caspase-3; ■, caspase-8; ▲, caspase-9. Values are means (± SEM) of triplicate experiments. **p < 0.01 (not time-progressed versus progressed cells after sample treatment).
We cultured SB with various microorganisms used in the production of fermented soybean foods, and revealed that ethyl acetate extracts of all the cultures exhibited cytotoxicity against U937 cells, while uncultured SB did not have such an effect in the range of concentrations tested. ESBA induced apoptosis by activating caspase-8, -9, and -3 in U937 cells. To our knowledge, this is the first report showing that after culturing with microorganisms used in the production of fermented soybean foods, SB has cytotoxicity against cancer cells, and that one or more apoptosis-inducing ingredients are formed in SB cultured with A. sojae. We are currently attempting to identify these apoptosis-inducing compounds. Even though further research is needed, such as identifying active compounds involved in cytotoxicity against U937 cells and safety assessments including the effect against normal lymphocytes, our findings should lead to the development of diverse anti-cancer agents from SB.
