Anti-obesity Effect and Antioxidant Activity in High-fat Diet Mice Fed Fermented Buckwheat Products (Miso)

Tables index

Veiw figure View Table

First, 1.25 kg of raw soybeans and 1.25 kg of raw buckwheat were soaked in water (beans:distilled water = 1:3, w/w). Further, the soaked soybeans and buckwheat were placed in five 1-L beakers with the soaking water and autoclaved (KT-3045, ALP Co. Ltd, Japan) at 110°C for 20 min. The paste was prepared by crushing the steamed soybeans and buckwheat, which were mixed (KN1500, Taisho Electric MFG. Co. Ltd, Japan) with rice-koji, salt, seed water, and seed miso, as shown in Table 1. BWM was packed (10 kg) in pickle barrels (Shinkigosei Co. Ltd, Japan) and fermented at 25°C –30°C for 180 days. The content of each container was thoroughly mixed once a month for 30 min using a mixer (KN1500). The products were sampled for analysis before fermentation (0 months) and after fermentation for 3 and 6 months. A portion of the sample obtained after fermentation for 6 months was freeze-dried and used as a dietary supplement in the animal experiments.

2.2. Physicochemical Properties

The samples (5 g) were extracted with 10 mL distilled water and centrifuged three times. Soluble solids were measured in water extracts using a digital pocket refractometer (PAL-Patissier; Atago Co. Ltd) and the L-glutamic acid content was determined using an L-glutamic acid assay kit II (Yamasa Corporation Co. Ltd, Japan). The surface color of the 6 month-fermented miso was measured using a Chroma Meter (MINOLTA CR-200, Japan), and the color intensity was quantiﬁed with L* a* b* scales in the CIE LAB color space ^[13]. The L*, a*, and b* values were measured. The data were expressed as the mean based on 20 measurements.

2.3. Total Phenolic Content and Antioxidant Activity

Each miso sample was extracted three times with 80% ethanol and 70% acetone, where the final volume of the extract was 60 mL. Each miso extract was used to determine the total phenolic content (TPC) and antioxidant activity.

TPC was determined using the Folin–Ciocalteau method ^{[12, 14]} with slight modifications. Miso extracts (100 μL) were mixed with 300 μL of distilled water, 400 μL of Folin–Ciocalteu reagent (previously diluted two-fold with distilled water), and 400 μL of Na₂CO₃ (100 g/L) and was placed at 30°C for 30 min. The absorbance was measured at 760 nm and expressed as mg of gallic acid (1 g/L). The linearity range of the calibration curve was 0–125 μg (r = 0.998).

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was performed according to the improved method reported by Blois ^{[12, 15]}. Miso extracts (150 μL) were incubated with the same volume of 2 mM of DPPH in ethanol in the dark for 15 min, and the decrease in absorbance at 517 nm was measured using a 96-well microplate reader (MTP300, Corona electric Co. Ltd, Japan). Standard curves were constructed using Trolox standards (0–30 nM) and the antioxidant activity of the extracts was expressed in Trolox equivalents (nM).

2.4. Animals and Diets

Male ddY mice (7 weeks old; Japan SLC, Inc., Shizuoka, Japan) were housed in plastic cages in an animal room, which was maintained with a 12:12 h light:dark cycle (light period, 8:00–20:00) at a constant temperature of 23°C ± 2°C and relative humidity of 60% ± 5%. Mice were randomly assigned to four groups (n = 5) and fed HFD for 2 weeks. The control group was fed HFD supplemented with 10% normal diet (HFD group), whereas the other groups were fed HFD supplemented with 10% BWM (BWM-HFD group).

The mice were allowed free access to food and water, and their intake rates were recorded. After 5 weeks, the final body weight and fecal weights were measured. On the final day of the experiment, mice were fasted overnight, anesthetized using nembutal injection (0.75 μL/g body weight), and laparotomy was performed. Blood was collected from the heart and rapidly mixed with EDTA-2Na as an anticoagulant, before separating by centrifugation at 1,000 ×g for 30 min. The livers and kidneys were collected, weighed, rapidly frozen in liquid nitrogen, and stored at −20°C until their analysis.

The experimental animals were handled according to the Guide for the Care and Use of Laboratory Animals ^[16] and the regulations of the National University Corporation Obihiro University of Agriculture and Veterinary Medicine for animal experiments. The experimental design was approved by the Animal Experiment Committee of Obihiro University of Agriculture and Veterinary Medicine, and it adhered to the standard principles described in the Guide for the Care and Use of Laboratory Animals ^[17].

2.5. Analysis of Sera, Organs, and Feces

Neutral lipids, free fatty acids, phospholipids, total cholesterol, LDL cholesterol, alanine aminotransaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels in serum were measured using a TDX Analyzer (Abbott Japan Co. Ltd, Tokyo, Japan).

Lipid peroxidation in serum was assessed by determining the thiobarbituric acid reactive substances (TBARS), where 400 μL of 8.1% sodium dodecyl solution was added to 100 mg of liver, before adding 300 μL of 20% acetic acid buffer (pH 3.5) and 500 μL of distilled water. The mixture was homogenized using a Teflon homogenizer and 600 μL was transferred to a clean dry glass tube containing an equal volume of 25 ppm aqueous solutions of copper (II) sulfate, and the reaction mixture was incubated for 1 h at 37°C. After the reaction, 950 μL was placed in a capped test tube, before adding 25 μL of 0.8% butylated hydroxytoluene acetic acid and 750 μL 0.8% tertiary butyl alcohol. After placing the reaction mixture for 1 h on ice, it was incubated at 100°C for 1 h in a block heater and cooled in running water, following which 500 μL of distilled water and 2.5 mL of n-butanol:pyridine (15:1) were added with vigorous stirring. The mixture was subjected to centrifugation at 1,500 ×g for 10 min, and the absorbance of the supernatant was measured at 532 nm. Lipid peroxidation was calculated as the amount of 1,1,3,3- tetraethoxypropane ^{[18, 19]}.

The freeze-dried fecal masses were degreased, redissolved in isopropanol, and analyzed to determine their cholesterol contents using a Wako cholesterol-E Test kit (Wako Pure Chemical Industries Ltd, Osaka, Japan).

The bile acid content was measured by extracting 100 mg of feces with three volumes of distilled water for 20 min by sonication. The extract was centrifuged at 1,000 ×g, and the supernatant was collected. The procedure was performed three times and the supernatants were combined. The bile acid content was measured using a Total Bile Acid Test Wako kit (Wako) and expressed as the amount of cholic acid ^[19].

2.6. Statistical Analyses

The data were expressed as the mean ± standard error. Significant differences were determined by ANOVA and Tukey’s studentized range (Honestly Significant Difference) test (SAS 9.3, SAS Institute Inc.). Differences were considered to be significant at p < 0.05.

3. Results and Discussion

3.1. Physicochemical Properties, TPC, and Antioxidant Activity of Miso

Buckwheat fermentation (miso) includes the hydrolysis of proteins to amino acids and starch to glucose ^[20]. We determined the L-glutamic acid and soluble solids content of miso, which increased with the time of fermentation (Table 2).

Table 2. Changes in the L-glutamic acid and soluble solids contents in the miso produced using buckwheat and soybeans

Download as

Tables index

Veiw figure View Table

L* and b* values obtained for the samples decreased with the time of fermentation (Figure 1), whereas a* values decreases with the time of fermentation (Figure 1).

The fermentation process used to obtain miso caused proteins to decompose into peptides and amino acids ^{[21, 22]}. Thus, we suggest that the protein and starch contents of buckwheat miso were broken down into amino acids and glucose on fermentation.

Download as

Veiw figureFigures index

NEW
View larger figure in new window

View next figure

Figure 1. Changes of the L*(

), a* (

) and b *(

) in the miso mixed with buckwheat and soybeans. The superscript * indicates significant difference in the data presented in the seam internal compared with the values for 0 month (p<0.05)

In the fermented buckwheat product, TPC and DPPH radical scavenging activity increased with the time of fermentation (p < 0.05, Table 3).

Table 3. Changes of the total phenolic content (TPC) and diphenyl - 2 - picryl-hydrazyl (DPPH) content in the miso produced using buckwheat and soybeans

Download as

Tables index

Veiw figure View Table

The correlation between DPPH radical - scavenging activity and TPC in the fermented buckwheat product was calculated using Pearson’s correlation coefficient (r = 0.961; p < 0.01). Takasaki et al. reported a high positive correlation between the total polyphenols content and DPPH radical-scavenging activity [r = 0.990 (p < 0.01)] ^[23], which is consistent with the result of the present study. Therefore, we hypothesize that the antioxidant activity of buckwheat miso is closely associated with polyphenols.

3.2. Effects of Buckwheat Miso on Food Intake and the Body, Organ, and Fecal Weights in HFD Mice

There were no significant differences in food intake, kidney weight, and fecal weight between the groups (Table 4).

Table 4. Food intake, body weight, fecal weight, and organ weight in HFD mice

Download as

Tables index

Veiw figure View Table

However, HFD mice fed a diet supplemented with buckwheat miso had lower final body and liver weights (13.6% and 17.5%, p < 0.05), and increased fecal lipid contents (1.34-fold, p < 0.05) compared with the control group (HFD). Shen et al. ^[24] showed that dietary supplementation with green tea polyphenols inhibited weight gain and decreased the fat mass. Gu et al. ^[25] reported that dietary cocoa supplementation of HFD for 10 weeks suppresses increased liver weight and ameliorates fatty liver disease symptoms in mice. Orihashi ^[26] showed that dietary supplementation with 1-year fermented miso for 6 weeks prevented fatty liver disease in cows. Thus, the hepatoprotective effect of miso may depend on the time of fermentation and vary according to the experimental animal species and feeding period tested. Hsu et al. ^[27] reported that supplementation with rutin suppressed increases in body weight, liver weight, and adipose tissue weight in HFD rats. Here we found that dietary supplementation with buckwheat miso for 5 weeks increased the excretion of lipids in feces, suppressed body weight increases, and reduced liver damage in HFD mice.

3.3. Effects of Buckwheat on Serum LDH, AST, and ALT, and Lipid Peroxidation in HFD Mice

Dietary supplementation with buckwheat miso reduced serum Neutral lipids, AST and ALT levels by 40.9%, 54.2% and 40.7%, respectively, compared with HFD (p < 0.05, Table 5).

Table 5. Serum lipid contents after 5 weeks of the dietary treatment (high fat diet : miso = 9:1) in HFD mice

Download as

Tables index

Veiw figure View Table