Antioxidative Activity of Douchi (A Chinese Traditional Salt-Fermented Soybean Food) Extracts During Its Processing

Abstract

Salt-fermented soybean foods are popular in Asia as an alternative food seasoning material. The α,α-Diphenyl-β-pricrylhydrazyl (DPPH) radical-scavenging activity, 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity and reducing power of the 50% ethanol extracts from one variety called in layman term as douchi were evaluated in this study. TEAC on DPPH radical and ABTS radical of extracts and the reducing powers of the extracts was found to increase significantly during the prefermentation (p < 0.05), through to the postfermentation. We investigated the effect of adding salt to the antioxidative activity during postfermentation. It was found that high salt content was not favorable for high TEAC on DPPH radical and the reducing powers. The effect of salt content on TEAC on ABTS radical of extracts was not significant (p > 0.05). The results showed that the antioxidative compounds released during the douchi fermentation were inhibited by the high salt addition.

Keywords:

• Douchi
• Antioxidative activity
• DPPH
• ABTS
• Reducing power

INTRODUCTION

The traditional fermented soybean foods are divided in two types: salt-fermented and non-salt fermented. The non-salt fermented soybean foods include natto, tempeh and kinema, and so on; sufu, douchi (salt-fermented soybean), miso, and soy sauce are representative of the salt-fermented soybean foods. These fermented soybean foods play a very important role in people's diet in many centuries. Douchi is a traditional salt-fermented soybean product that originated in China, which has been consumed in large quantities since ancient times as the seasoning for dishes (see Fig. 1). There are three types of douchi that are fermented by Mucor, Bacteria, and Aspergillus strains, respectively. Among them, Aspergillus-type douchi is the earliest, and has been the most popular type and is produced widely in China.

Figure 1 A Aspergillus-type Douchi product made by a Douchi manufacturing company.

The traditional fermented soybean foods have been found to exhibit remarkably strong antioxidative activity, such as sufu,[1–2] miso,[3] natto,[4–6] tempeh.[7–9] Douchi is a similar fermented soybean food as natto, and tempeh, which were incubated with Baccillus natto, and Rhizopus oligosporus, respectively.[10] One would expect douchi should also have reasonable antioxidative activity. Therefore, it is intended here to evaluate the antioxidative properties during the processing of douchi. Although there have been some reports relating to elicit dose-dependent antiglycemic effects in humans of douchi by Aspergillus cultured,[11–12] it appears that no one has paid attention to the changes in the antioxidative activity of during douchi making.

Like in other salt-fermented soybean foods such as sufu,[13] miso,[14] salt has a multiple role in douchi. During the postfermentation of douchi, a salty taste to douchi by adding salt. The salt will control the microbial growth and enzyme activity in douchi as well. In this article, we analyzed the antioxiant activity of the 50% ethanol extracts from douchi, by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging assay, the ABTS (2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) radical-scavenging assay and the reducing power. We also investigated the the effect the salt supplementation during douchi postfermentation on the antioxiant activity of the extracts. By establishing the relationship between the function and process of douchi making, it would be possible to establish an improved procedure for producing highly functional douchi, and increasing its value as a healthy food product.

MATERIALS AND METHODS

Materials

Mold culture Aspergillus oryzae 3.951 was provided by the Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). Soybeans named of Kefeng No. 6 (2003) were provided by the Agricultural Academy of Jilin Province (Jilin, China). ABTS, DPPH and Trolox ((+/−)-6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid, 97%) were purchased from Sigma Chemical Co (U.S.A.). All other chemicals were of the reagent grade.

Preparation of Douchi

Douchi preparation was performed as previous description,[15] as illustrated in Fig. 2. The steps and parameters were as follows:

1. Pretreatment. Soybeans were washed, and soaked in tap water for 8 hours at room temperature (24°C ± 2°C). After draining, the soybeans were steamed for 30 minutes at 121°C in a retort (YMQ.L31.400, Beijing Jiangtai Medical Instrument CO., Ltd, Beijing, China).

2. Prefermentation. The cooked soybeans were cooled to 30°C–35°C, and then inoculated quickly with Aspergillus oryzae 3.951 incubated at 30°C for 60 hours, around 80% relative humidity in an incubator (LTI-601SD; Tokyo Rikakikai CO., Ltd, Tokyo, Japan). Semifinished products were called the douchi qu (koji).

3. Postfermentation. Aliquots of the douchi qu deposited into a series of glass jars were salted until the content of NaCl reached about 5, 7.5, 10, and 12.5% (w/w). Each sample was ripened for 1 month at 35°C in the same incubator.

Figure 2 Flow diagram for processing soybeans to make Douchi.[15]

Raw soybean, soaked soybean, and cooked soybean were sampled. Samples were taken at 12-hour intervals during the prefermentation, and at one-week intervals during the postfermentation. All the samples were lyophilized and ground using a coffee mill (TSK-U928S, CANKUN Co., China) to pass a 50-mesh size screen.

Preparation of the Douchi Extracts

One gram of freeze-dried sample powder was extracted with 10 mL 50% ethanol in a shaking incubator for 2 hours at room temperature. The suspension was centrifuged at 2000 × g for 15 minutes at room temperature. The supernatant was filtered and the serial dilutions were used for assaying. The antioxidative extracts were kept in the dark for further analysis.

DPPH Free Radical Scavenging Activity

The radical-scavenging activities of the extracts of douchi were determined according to the methods described by Suda[16] with some modification. Diluted douchi extracts (100 μL) was mixed with 100 μL of a 200 μM DPPH solution in 0.1 M MES buffer (pH 6.0) containing 50% ethanol was then added. The mixture was kept at room temperature for 20 minutes, then the absorbance of the resulting solution was measured at 520 nm using a 96-well plate reader (model 550, Bio-Rad Laboratories, Japan). Trolox was used as an antioxidative standard to prepare the standard curve and calculate the trolox equivalent for each sample. The DPPH scavenging activity of the douchi extracts were expressed as Trolox Equivalent Antioxidative Capacity (TEAC).

ABTS Free Radical Scavenging Activity

ABTS free radical-scavenging assay was based on a method developed by Robert et al.[17] with some modification. ABTS· + was generated by reacting ABTS (7.4 mM) with potassium persulphate (2.6 mM). The solution was diluted to obtain an absorbance of 1.4 units at 414 nm with ethanol. The ABTS· + antioxidative reaction mixture contained 200 μL of ABTS· + solution, and 50 μL of douchi extracts or 50 μL of 50% Ethanol for the control. The absorbance at 420 nm was measured at 6 min of the reaction by a 96-well plate reader (model 550, Bio-Rad Laboratories, Japan). Trolox was used as an antioxidative standard to prepare the standard curve and calculate the trolox equivalent for each sample. The radical scavenging activity of douchi extract was expressed as Trolox Equivalent Antioxidative Capacity (TEAC).

Reducing Power

The reducing power of douchi samples extracts was determined according to the method of Oyaizu[18] with some modifications. Douchi extracts (5 mg/mL) 0.4 ml was mixed with phosphate buffer (1 mL, 0.2 mol/L, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (1 mL, 1%). The mixture was incubated at 50°C for 20 minutes. A portion (1 mL) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 10000 × g for 10 minutes. The upper layer of solution (1 mL) was mixed with distilled water (2 mL) and FeCl₃ (0.4 mL, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Statistical Analysis

All experiments were conducted in triplicates. The mean values of each experiment were calculated. The SAS system (SAS for Windows 6.12, SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. Duncan's mulitiple range test was used to estimate significant differences among the mean values at the 5% probability level.

RESULTS AND DISCUSSION

Changes in the Antioxidative Properties during Douchi Pretreatment Processing

The antioxidative properties of the extracts during douchi pre-treatment processing are shown in Table 1. TEAC on DPPH radical and ABTS radical were no significant changes (p > 0.05) after soaking and cooking comparing with raw soybean, but the reducing powers were significant increased (p < 0.05).

Table 1 Changes in the antioxidative properties for pretreatment of Douchi processing

Step	TEAC (mg trolox/g dry matter) ^a		The reducing power (absorbance at 700 nm) ^a
	DPPH radical	ABTS radical
Row soybean	1.54 ± 0.03A	13.72 ± 6.74A	0.177 ± 0.004C
Soaked soybean	1.26 ± 0.17A	16.65 ± 1.35A	0.243 ± 0.003B
Cooked soybean	1.36 ± 0.32A	16.84 ± 5.59A	0.271 ± 0.002A
^aMean of three determinations ± S. D. (standard deviation). Means with the same column with different letters were significantly (p < 0.05).

Changes in the Antioxidative Properties during Douchi Prefermentation Processing

Figure 3 shows that the scavenging activity of extracts on DPPH radical and ABTS radical during the prefermentation. TEAC on DPPH radical and ABTS radical increased significantly (p < 0.05) from 0.61 to 7.21 and from 19.60 to 45.09 during the fermentation, respectively. Arnao[19] reported the DPPH assay could significantly underestimate TEAC compared to ABTS assay. Our results were agreed with his.

Figure 3 Scavenging activity of Douchi extracts by DPPH method and ABTS method during the prefermentation. Results are the mean ± S. D. of three determinations.

The reducing powers (absorbance at 700 nm) of the extracts during the pre-fermentation are shown in Fig. 4. The reducing power increased significantly from 0.27 to 0.96. The antioxidative properties during douchi prefermentation processing were increased significantly (p < 0.05). This might have been due to the fact that antioxidative compounds were released during the fermentation. The antioxidative effects of tempeh and natto were increased during fermentation, and were found to a significant influence by fermentation time.[4, 8]

Figure 4 Reducing power of Douchi extracts during the prefermentation. Results are the mean ± S. D. of three determinations.

Changes in the Antioxidative Properties during Douchi Postfermentation Processing

The scavenging activities the extracts on DPPH radical during the post-fermentation are shown in Fig. 5. TEAC increased significantly continually with the fermentation time of douchi (p < 0.05). TEAC were influenced significantly by the salt contents (p < 0.05). TEAC were increased with the decrease of the salt contents. TEAC were increased from 7.18 (5% salt content), 7.30 (7.5% salt content), 6.66 (10% salt content), and 6.45 (12.5% salt content) at 0 week (week) to 11.54, 10.46, 8.89, 7.40 at 1 week in postfermentation, respectively. The increasing rates of TEAC were evidently different with the different salt contents during fermentation.

Figure 5 Scavenging activity of Douchi extracts by DPPH method during the postfermentation. Symbols denoted the experimental data, the mean ± S. D. of three determinations; Lines denoted the predicted data according to the Eq. (1).

The scavenging activities of the extracts on ABTS radical during the postfermentation are shown in Fig. 6. The further increase of TEAC was significant, and reached the maximum point at 2 weeks in postfermentation. The effect of salt contents on TEAC were not significant (p > 0.05).

Figure 6 Scavenging activity of Douchi extracts by ABTS method during the postfermentation. Results are the mean ± S. D. of three determinations.

Figure 7 shows the reducing powers of the extracts during the post-fermentation, suggested that the reducing powers of the extracts were increased significantly during the first 2 weeks of postfermentation (p < 0.05), and the level of salt content did affect the reducing powers of the extracts, which decreased with increases in salt content, however, the effect of salt contents on the reducing powers were not significant at 3 weeks (p > 0.05). These results suggested that the antioxidative compounds on DPPH radical, on ABTS radical and reducing powers were released further during the postfermentation. The compounds on DPPH radical and reducing power were inhibited by higher salt addition. However, the compounds on ABTS radical were not affected by salt addition.

Figure 7 Reducing power of Douchi extracts during the postfermentation. Symbols denoted the experimental data, the mean ± S. D. of three determinations; Lines denoted the predicted data according to the Eq. (1).

Fermentation Time-Dependent Kinetics for Antioxidative Activity

As shown in Figs. 5 and 7, the antioxidative activities of the extracts depended strongly on its fermentation time and the contents of salt. In general, the antioxidative activity increased with fermentation time to a certain extent, then leveled off with further increase in time. The antioxidative activities of the extracts have been found to be represented by the following equation:

(1)

where A is the antioxidative activity at any given fermentation time (weeks) in different the contents of salt; A ₀ is the initial the antioxidative activity at 0 wk of postfermentation; A∗ is the equilibrium the antioxidative activity, which is as summed to remain constant for the processes; t is the time of postfermentation (wks); and k is a rate constant (1/wk). The proportional constant (k) can be obtained from the slope of a Ln(1 − (A − A ₀) / (A − A∗)) against time (t) plot. The proportional constant (k) has therefore the unit of reciprocal time, and can be viewed as an indication for the antioxidative activity. A higher k value represents a higher rate of the growth of the antioxidative activity for the specific process.

The rate constants (k) were plotted in Fig. 8. It has been found that the rate constant (k) for the DPPH radical scavenging activity and for the reducing power all decrease with the increase of the contents of salt. This indicated that the higher content of salt could result in lower reaction rates. The rate constant (k) for the reducing powder was higher than that of for the DPPH radical scavenging activity at the same salt content. According to simple regression analysis, the correlation coefficient of the TEAC on ABTS radical correlated with TEAC on DPPH radical was 0.96 and 0.64 in the prefermentation and the postfermentation, respectively. There were some reports about the correlation of ABTS assay and DPPH assay. A good correlation of TEAC between the ABTS model and DPPH model was observed in fruits.[20] However, Significant differences by employing ABTS and DPPH scavenging assay were reported by Lissi et al.[21] Wang et al.[22] found that some compounds, which have ABTS radical scavenging activity may not show DPPH radical scavenging activity. The results obtained in this study showed a high correlation coefficient between the two assays in prefermentation, and a comparatively low correlation coefficient in postfermentation. These results indicated the compounds contributed the TEAC between the two methods were different at various stages of douchi fermentation.

Figure 8 The rate constants (k) for the DPPH radical scavenging activity and for the reducing power in different salt contents. Results are the mean + S. D. of at 1 week and 2 weeks in postfermentation.

The effect of antioxidative activity on DPPH radical scavenging and the reducing power of the douchi extracts were all thought to be due to its hydrogen donating ability.[23] From our result, a good relationship (r = 0.99 in prefermentation, and r = 0.94 in postfermentation) was found between this two methods during fermentation. Hence, we suggest that the compounds contributed to the antioxidative properties and might be the same between the two methods.

Antioxidative peptides were found in the hydrolysates of soy proteins.[24–25] Fan et al.[26] reported the peptides by hydrolysate from soybean protein isolate for 4 hours showed the highest DPPH radical-scavenging activity, which the molecular weights were less than 33 kDa. Esaki et al.[4] reported that the antioxidative activity of natto was dependent on the increase of water-soluble fractions of natto with fermentation time, rather than on the increase of isoflavone agalycons, such as daizen and genistein. The protein (albumin, globulin, and alkaline soluble) were rapidly degraded to amino acids and low-molecular-weight peptides during the tempeh fermentation.[27] Markus et al.[28] suggested the antioxidative activity of tempeh was a synergistic effect of tocopherols (present in the soybeans) and amino acids (liberated during the fermentation). Therefore, it was expected that peptides and amino acids were liberated during douchi fermentation process,[29] contributing to the antioxidative activity of douchi extracts.

It was known that isoflavones participate in the antioxidative activity of soybean. Isoflavone aglycones showed higher physiological properties than their glucoside form. Daidzein and genistein were known for their antioxidative activity in biological systems with an emphasis on the scavenging activity on ABTS radical.[30] These polyphenols can scavenge a peroxyl radical and protect against iron-induced free radical reaction.[31] Daidzein and genistein were confirmed the main components responsible for the antioxidative activity of tempeh.[7, 32] In our previous study,[15] isoflavone glucosides were hydrolysed into their corresponding aglycones during Douchi fermentation. Hence, we presumed isoflavone transformation during douchi fermentation was one of the major reasons for the increase of antioxidative activity of douchi extracts.

Melanoidin, the end products of Maillard reaction process, was emphasized as one of the important factors on antioxidative activity of soy sauce.[33] Maillard reaction was found during the douchi processing.[34] This means that the melanoidin in douchi may play a more important role on its antioxidative activity effect. Although, peptide, amino acid, isoflavones, melanoidin, and tocopherols were all expected to be active ingredients in the Douchi extracts, isolation and identification of the antioxidative substances would have to be conducted to explore their contributions .This work is in progress.

A high NaCl addition may result in inhibiting the enzyme activity and preventing some compound contributing antioxidative active release during douchi fermentation. Some research about this phenomenon have been reported, such as that isoflavone transformation was inhibited during the fermentation of miso, sufu, and douchi;[15, 35–36] protein degradation was reduced during the fermentation of Sufu.[13] Furthermore, high NaCl content of food could increase the dietary sodium intake.[37] Therefore, douchi with a low NaCl content may be preferable from the viewpoint of public nutritional health. Further investigation is needed to clarify the flavour, taste, texture, consumer acceptability, and shelf life of douchi with a lower NaCl content.

CONCLUSION

The antioxidative properties of the 50% ethanol extracts from douchi were effected during douchi processing. The antioxidative properties (TEAC on DPPH radical and ABTS radical) of the extracts during douchi pretreatment processing showed no significant changes (p > 0.05), but the reducing powers significantly increased (p < 0.05). The antioxidative properties of the extracts during douchi prefermentation processing were increased significantly (p < 0.05), through to the postfermentation. The content of NaCl did affect the antioxidative properties of the extracts. The higher the salt content, the lower the DPPH radical scavenging activity and the reducing power. These results indicate that the antioxidative compounds were released during the douchi fermentation and that they are inhibited by high salt addition. Furthermore, a simple model has been found to be useful for correlating the data of the effects of salt content on the DPPH radical scavenging activity and the reducing power.

ACKNOWLEDGMENTS

This study was conducted within the framework of the collaborative research project between Japan and China titled, “Development of sustainable production and utilization of major food resources in China,” supported by Japan International Research Center for Agricultural Sciences (JIRCAS). This work was supported using the fund (DIC2003-04) called, “Study on the antioxidative activities of the Chinese traditional fermented soybean foods,” from the Research/Communication Proposal of Danone Institute China.