Antioxidant Activity and Lipase Inhibitory Activity in Rice Miso Supplementary with Black Soybean , Buckwheat and Adzuki Bean

In the present study, we manufactured rice miso supplementary with black soybean, buckwheat and adzuki bean. We analyzed DPPH radical scavenging activity, melanoidin, peptide, reducing sugar content, and lipase inhibitory activity in various rice miso products at different fermentation periods (3, 6, 24, 36 months), respectively. DPPH radical scavenging activity, melanoidin content and lipase inhibitory activity in various rice miso products increased with prolonging the fermentation period. We found positive relationships between melanoidin content and DPPH radical scavenging activity, and lipase inhibitory activity. The correlation coefficients were more than 0.75, respectively. Rice miso supplementary with black soybean (RM-BS), rice miso supplementary with buckwheat (RM-BW) and rice miso supplementary with adzuki bean (RM-AB) exhibited significant higher DPPH radical scavenging activity, melanoidin and lipase inhibitory activity than RM (rice miso; control), respectively. We considered that due to prolonging the fermentation period, more and more starch and protein contained in RM-BS, RM-BW and RM-AB were decomposed into reducing sugar, peptide and amino acid, than those of RM. So thus rice miso supplementary with black soybean, buckwheat and adzuki bean could improve DPPH radical scavenging activity and lipase inhibitory activity of traditional rice miso products, which may be because of plenty of melanoidin was produced through the amino acid-glucose reactions. These results would utilize in the research on development functionality of rice miso products.


Introduction
In recent years, dietary life is characterized by a high intake of calorie and fat [1] and low intake of fruits and vegetables [2].These unhealthy dietary habits are at high risk of developing a range of physical health conditions, such as obesity, cancer, metabolic syndrome and diabetes [3,4,5,6].In Japan, the National Health and Nutrition Survey of the Ministry of Health, Labour, and Welfare indicated that there is a tendency to increase the proportion of obese patients in recent years [7].
Rice miso is a kind of Japanese traditional seasoning, which contained abundant protein, vitamins and minerals.Rice miso is usually manufactured by fermentation of boiled soybean, salt and rice-malt.It is reported that rice miso had physiological effects, including lipid peroxidation inhibition, anti-cancer and anti-diabetic [8,9,10,11].
Black soybean is noted that contained a large amount of dietary fiber and anthocyanin.Several studies have found that black soybean has various physiological effects such as strong antioxidant activity, glycolysis-inhibitory activity, anti-obesity, anti-cancer and hepato-protective effect [12,13,14,15].
Buckwheat is one of the most important functional foods throughout the world [16].It is not only an important source of basic nutrition, but also provide other positive health benefits.Buckwheat has a high level of antioxidant activity compared to other cereal crops, and this has been attributed to its high levels of flavonoid compounds [17].It has been described that the consumption of buckwheat and buckwheat-enriched products could increase LDL peroxidation inhibitory, anti-oxidant activity and immunomodulatory activity [18,19].
Adzuki bean is cultivated throughout east Asia and used in food additives, such as rice cake, bread, soup, and other snacks due to a sweet taste.It is also known American Journal of Food Science and Technology for health-promoting and nutritional properties, such as antioxidant, renal cortex protective, anti-obesity, anti-hypotensive, and hepato-protective effects [20,21,22].
Here we fermented rice miso products supplementary with black soybean (RM-BS), buckwheat (RM-BW) and adzuki bean (RM-AB) to development and utilization of Japanese traditional rice miso, and clarify the antioxidant activity and lipase inhibitory activity in rice miso supplementary with black soybean, buckwheat and adzuki bean at different fermentation periods.

Materials
For the experiment, we purchased black soybean (Glycine max), buckwheat (Fagopyrum esculentum), adzuki bean (Vigna angularis), soybean (Glycine max), rice-malt, salt, seed miso from supermarket.Rice-malt was purchased from the Salt Industry Center (Japan).The rice miso products were manufactured by the method for industrial producing rice miso [23], and sampled for analysis at 3, 6, 24, 36 Months of fermentation.

Extraction and Fractionation
The extraction of miso products was extracted by the method of Saito [13].Each miso sample (5 g) was added with 20 mL of 80% v/v ethanol and 70% v/v acetone, vortexed, and ultrasonicated for 30 min.The suspension was then centrifuged at 1,006 ×g for 10 min.After that, according to the Ikeda's method [24].Briefly, the mixture extract was concentrated and dissolved by 20 mL distilled water.Then, added by n-hexane and ethyl acetate to delaminate the solution.Subsequently, a part of water-soluble fraction was purified by chromatography through Diaion HP-20 column.The column was washed by distilled water and then eluted by methanol, and the methanol fractions were collected for chemical analysis.

DPPH Radical Scavenging Activity Assay
The DPPH radical scavenging activity was determined by the method of Brand-Williams [22].The extract (50 μL) was added to a microplate and mixed with 100 μL of 99.5% v/v ethanol and 150 μL DPPH solution.The solution was kept in the dark for 15 min after which its absorbance was determined at 520 nm by a microplate reader.Ten-fold diluted 2 mM trolox was used as the standard and the results were expressed as μmol trolox equivalents (TE) per gram DW miso (y = -54.07x+ 116.96,R 2 = 0.9994).

Melanoidin Content Determination
The content of melanoidin was determined by the method of Martins [25].Briefly, 0.02 M of glucose and glycine were dissolved by 0.1 M phosphate buffer (pH 6.8), and heated at 120°C for 2 hours.Then, the solution was placed in a dialysis membrane (14000 MWCO, UC 36-32-100; EIDIA Corporation, Japan) and dialyzed against distilled water (7 days).The dialysate was lyophilized for 48 hours, and took as melanoidin standard.The absorbance was measured at 450 nm.The results were expressed as the mg melanoidin equivalents (ME) per gram DW miso (y = 63.855x+ 0.4273, R 2 = 0.9989).

Peptide and Reducing Sugar Content Determination
The determination of peptides was carried out by BCA method [26].NaOH buffer (0.1 M, pH 11.25) contained 1 g sodium bicinchoninate, 2 g sodium carbonate, 0.95 g sodium tartrate and 0.16 g sodium bicarbonate.The buffer was mixed with 0.4% CuSO 4 at a ratio of 50:1.The samples (100μL) were mixed with 2 mL of the mixture, and incubated at 37°C for 30 min.The absorbance was measured at 562nm.The albumin was taken as standard, and results were expressed as the mg albumin equivalents (AE) per gram DW miso (y = 59.298x -0.4885, R 2 = 0.9968).
The reducing sugar was determined by DNS method [13] with slight modifications.Specifically, the samples (50 μL) were diluted with 950 μL distilled water, and thoroughly mixed with 100 μL of 2N-NaOH, 100 μL of 1% DNS solution, and incubated in boiling water bath for 10 min.Absorbance was measured at 540nm.The results were expressed as the mg glucose equivalents (GE) per gram DW miso (y = 333.92x+ 26.832, R 2 = 0.9922).

Lipase Inhibitory Activity Assay
Lipase inhibitory activity was determined by the improved method of Han [27].The substrate solution was prepared by adding 10 mg lecithin, 80 mg triolein, and 5 mg cholic acid to 9 mL of 0.1 M TES buffer (pH 7.0) (containing 10 mM Tris, 2.5 mM EDTA•2Na and 25 mM sucrose), and the mixture was sonicated.Add 240 μL of sample extracts with different concentration (0.05, 0.1, 0.2 g), 80 μL lipase solution and 80 μL substrate solution into a glass tube, incubate at 37°C for 30 min.Then, add 2 mL copper reagent and 4 mL chloroform, stir and centrifuge at 1,006 ×g for 5 min.Transfer 2.4 mL of chloroform layer to a new glass tube and add 400 μL of 0.1% DDTC-butanol solution and measure the absorbance at 440 nm.Standard curves were using linoleic acid, and expressed as lipase inhibitory activity (%).

( )
where A sample is the absorbance of the mixture of sample, substrate solution, enzyme and DDTC-butanol solvent; A control is the absorbance of the mixture of buffer (instead of sample), substrate solution, enzyme and DDTCbutanol solvent.

Statistical Analysis
The experiments were repeated at least three times.Data were expressed as means ± standard deviation.Significant differences were determined by one-way ANOVA and Fisher's test (SAS v. 7.1, SAS Institute Inc., Cary, NC, USA).Differences were considered to be significant at Р < 0.05.

DPPH Radical Scavenging Activity in Rice Miso Products
The results of DPPH radical scavenging activity of various rice miso products showed in Table 1.DPPH radical scavenging activity was increasing with prolonging the fermentation period of all rice miso products.36 Months fermentation of RM-BS exhibited the highest DPPH radical scavenging activity, and the value was 6.9 μmol/g DW miso.Comparing with 3 Months fermentation (1.0 μmol/g DW miso), DPPH radical scavenging activity at 36 Months fermentation of RM-BS was increased by 6.9 folds.36 Months fermentation of RM-BW also exhibited the highest DPPH radical scavenging activity, and the value was 6.6 μmol/g DW miso, which increased by 7.3 folds as comparing with 3 Months fermentation.The highest DPPH radical scavenging activity of RM-AB and RM (rice miso; as control) showed at 24 Months fermentation, and the values were 5.7 and 6.6 μmol/g DW miso, respectively.As compared with 3 Months fermentation, DPPH radical scavenging activity at 24 Months fermentation of RM-AB was increased by 3.2 folds, and that of RM was increased by 4.5 folds.
Moreover, RM-BS, RM-BW and RM-AB exhibited lower DPPH radical scavenging activity than RM at 24 Months fermentation, respectively.RM-BS, RM-BW and RM-AB at 3, 6 and 36 Months fermentation exhibited significant higher DPPH radical scavenging activity than those of RM, respectively.RM supplementary with black soybean, adzuki bean and buckwheat produced more antioxidants than RM.

Melanoidin, Peptide and Reducing Sugar Content in Rice Miso Products
The results of melnoidin content in various rice miso products showed in Table 2. Melanoidin content was increasing with prolonging the fermentation of rice miso products.The increase trend of melanoidin content in various rice miso products was similar as DPPH radical scavenging activity.The highest melanoidin content of RM-BS showed at 36 Months fermentation (19.9 mg/g DW miso), which was increased by 6.4 folds, as compared with 3 Months fermentation (3.1 mg/g DW miso).As compared with RM-BW of 3 Months fermentation, melanoidin content of 36 Months fermentation (12.7 mg/g DW miso) increased by 6.1 folds.The highest melanoidin content of RM-AB and RM showed at 24 Months fermentation (10.6 mg/g DW miso; 11.6 mg/g DW miso), and increased by 4.2 folds and 4.5 folds, respectively, as compare with 3 Months fermentation.Moreover, there was high positive relationship between melanoidin content and DPPH radical scavenging activity (Figure 1).The antioxidant activity of decocted Zhenjiang Aromatic Vinegar was increasing along with the increased of melanoidin content [28].Abbreviations: M, month; DW, dry weight; RM-BS, rice miso supplementary with black soybean; RM-BW, rice miso supplementary with buckwheat; RM-AB, rice miso supplementary with adzuki bean; RM, rice miso.Data represent the mean ± SD from at least three independent studies.Values within a row followed by different capital letters and values within a column followed by different small letters are significant at p < 0.05.Peptide and reducing sugar content in various rice miso products showed in Table 3 and Table 4.The fermentation process could cause starch and protein contained in rice miso to decompose into reducing sugar, peptide and amino acid by Aspergillus oryzae [24,29,30].At the early fermentation period (3 and 6 Months fermentation) of various rice miso products, the peptide and reducing sugar increased.However, the peptide and reducing sugar content declined obviously from 6 Months fermentation.We speculated that plenty of melanoidin produced through the amino acid-glucose reaction from 6 Months fermentation of various rice miso products, and the Melanoidin content (mg/g DW miso) R = 0.9220 peptide and reducing sugar content declined from fermentation after 6 months.To compare with RM, the ratios of peptide content contained in RM-BS, RM-BW and RM-AB increased from 0.6 to 1.4 folds with prolong fermentation period, and the ratios of reducing sugar increased from 0.7 to 2.5 folds.Buckwheat and black soybean are mainly rich in their protein content, and adzuki bean (Vigna angularis) contains more starch than soybeans [31,32].Therefore, we speculated that due to RM-BS, RM-BW and RM-AB have more peptide and reducing sugar than RM, more melanoidins produced, which had antioxidant activity.Moreover, the composition of melanoidin produced from an amino acid-sugar model system also effects the strength of antioxidant activity [33].
The high molecular weight reaction products resulting from the interaction of lysine with hexanal showed a higher radical scavenging activity than glycine/glucose model [34].Abbreviations: M, month; DW, dry weight; RM-BS, rice miso supplementary with black soybean; RM-BW, rice miso supplementary with buckwheat; RM-AB, rice miso supplementary with adzuki bean; RM, rice miso.Data represent the mean ± SD from at least three independent studies.Values within a row followed by different capital letters and values within a column followed by different small letters are significant at p < 0.05.Abbreviations: M, month; DW, dry weight; RM-BS, rice miso supplementary with black soybean; RM-BW, rice miso supplementary with buckwheat; RM-AB, rice miso supplementary with adzuki bean; RM, rice miso.Data represent the mean ± SD from at least three independent studies.Values within a row followed by different capital letters and values within a column followed by different small letters are significant at p < 0.05.

Figure 2.
Lipase inhibitory activity contained in different weights of rice miso products with different fermentation period.A, RM-BS.B, RM-BW.C, RM-AB.D, RM. for the 0.05 g DW miso; for the 0.1 g DW miso; for the 0.2 g DW miso.Values within a column followed by different capital letters and values within a row followed by different small letters are significant at p < 0.05.

Lipase Inhibitory Activity in Rice Miso Products
Lipase inhibitory activity in various rice miso products at different concentrations showed in Figure 2. Lipase inhibitory activity in various rice miso products increased significantly with prolonging fermentation period.The strongest lipase inhibitory activity in RM-BS was at 36 Months fermentation and RM-BW, RM-AB and RM was at 24 Months fermentation.Lipase inhibitory activity of RM-BS, RM-BW and RM-AB were also significant higher than RM (rice miso; as control) at different fermentation period, respectively.Moreover, we found positive relationships between melanoidin content and lipase inhibitory activity at the concentrations of 0.05, 0.1, 0.2 g rice miso products, the correlation coefficients were 0.7518, 0.7883 and 0.8016, respectively.Therefore, we concluded that supplementing with black soybean, buckwheat and adzuki bean could improve lipase inhibitory activity of traditional rice miso products.Doenjang intake resulted in significant reduction of body weight gain in high fat-fed mice [35], and HFD-fed rats drinking coffee or melanoidin had an effect of reduced fat [36].

Conclusion
In the present study, DPPH radical scavenging activity, lipase inhibitory activity and melanoidin content in various rice miso products increased with prolonging fermentation periods, and RM-BS, RM-BW and RM-AB were significant higher than those of RM, respectively.We found positive relationships between melanoidin content and DPPH radical scavenging activity, and lipase inhibitory activity, respectively.Due to prolonging the fermentation period, more and more starch and protein contained in RM-BS, RM-BW and RM-AB to decompose into reducing sugar, peptide and amino acid than those of RM.So thus we concluded that traditional rice miso supplementary with black soybean, buckwheat and adzuki bean could improve DPPH radical scavenging activity and lipase inhibitory activity of traditional rice miso products, which were because of plenty of melanoidin was produced through the amino acid-glucose reactions.
These observations also might provide important information for further research on developing health benefits of rice miso products.

Figure 1 .
Figure 1.Relationship between DPPH radical scavenging activity and melanoidin in rice miso products with different fermentation period.