γ-Polyglutamic acid (γ-PGA) was purchased from Meiji Food Materia Co. Ltd. (Tokyo, Japan). The molecular weight of γ-PGA ranged from 600 kDa to 1500 kDa. The purity of γ-PGA was greater than 70%.

Male Jcl:ICR mice (6 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). All mice were specific pathogen-free (SPF) and were randomly subdivided into two groups of seven animals each. The mice were housed individually in suspended stainless-steel cages with wire mesh bottoms in a room maintained at 24˚C ± 0.5˚C under a relative humidity of 65% with 12-h periods of light and darkness. The mice were fed the AIN-93G diet for 14 days. After 14 days, the diet was replaced with a 0.1% γ-PGA—containing diet (PGA group) or a control diet (Con group) for 91 days. Table 1 presents the composition of each diet. The concentration (0.1%) of γ-PGA offered to the mice is compatible with human consumption. After 28 days from the start of the experimental diet, the maltose tolerance test was performed. After 56 days from start of feeding of the experiment diet, the starch tolerance test was performed. After the experimental diet period, the mice were euthanized by collecting blood from the heart during isoflurane anesthesia, and blood samples were taken from the heart and placed in heparinized tubes. The plasma was separated from whole blood by centrifugation and stored at −80˚C until analysis of plasma lipids. The liver, visceral fat, and caecal contents were collected. Caecal contents were stored at −80˚C until use in caecal short chain fatty acids analysis. The liver samples and visceral fat were weighed. All procedures involving mice in this study were approved by the

Animal Care Committee of Food Research Institute (Tsukuba, Japan) in accordance with the “Guidelines for Animal Care and Experimentation” of Food Research Institute, National Agriculture and Food Research Organization (Tsukuba, Japan).

After 28 days from the start of the experiment diet, the maltose tolerance test was performed. As the relatively high concentration of γ-PGA was expected to suppress the increase in blood glucose level, we used 8% γ-PGA in this experiment. All mice have fasted for 12 hrs. Immediately after measurement of initial blood glucose levels, maltose solution (1 g/kg body weight) containing 8% γ-PGA was orally administered to the PGA group using a feeding needle. Maltose solution (1 g/kg body weight) was orally administered to the Con group using a feeding needle. The blood glucose levels were measured at 15 min, 30 min, 60 min, and 120 min after maltose administration. The blood glucose levels were measured in blood collected from the tail vein using the One Touch Ultra View® blood glucose monitoring system (Johnson & Johnson Lifescan, Inc., Milpitas, CA, USA). For measurements of blood glucose, the value obtained from the maltose tolerance test was subtracted from the initial blood glucose levels. After 56 days from the start of the experiment diet, the starch tolerance test was performed. All mice have fasted for 12 hrs. Immediately after measurement of initial blood glucose levels, starch solution (1 g/kg body weight) containing 8% γ-PGA was orally administered to the PGA group using a feeding needle. Starch solution (1 g/kg body weight) was orally administered to the Con group using a feeding needle. The blood glucose levels were measured using blood collected from the tail vein, by the One Touch Ultra View® blood glucose monitoring system (Johnson & Johnson Lifescan, Inc., Milpitas, CA, USA) at 15 min, 30 min, 60 min, and 120 min after starch administration. For measurements of blood glucose, the value obtained from the starch tolerance test was subtracted from the initial blood glucose levels.

The following tests were performed using kits obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Total plasma cholesterol concentrations were measured using a cholesterol E-test kit based on cholesterol oxidase. Plasma triglyceride concentrations were determined using a triglyceride E-test kit based on the glycerol-3-phosphate oxidase method. Plasma nonesterified fatty acid (NEFA) concentrations were measured using a NEFA C-test kit utilizing acyl-coenzyme A (CoA) synthase, acyl-CoA oxidase, peroxidase. Plasma HDL-cholesterol concentrations were measured using a HDL-cholesterol E-test kit. Plasma glucose concentrations were quantified using a glucose CII-test kit involving glucose oxidase and peroxidase.

Caecal contents were diluted in distilled water, vortexed for 30 s, and then centrifuged. Supernatants were analyzed using the short chain fatty acid analysis kit (YMC Co. Ltd., Kyoto, Japan). This kit is based on the method of Miwa et al. [10] [11]. In this kit, by converting the carboxyl group of the short chain fatty acid to 2-nitrophenylhydrazine (2-NPH), high-sensitivity detection in the ultraviolet and visible regions is possible.

Crotonic acid was used as the internal standard. Crotonic acid was added diluted caecal contents. A total of 150 μL of diluted caecal contents was added to 100 μL of 2-nitrophenylhydrazine hydrochloride (2-NPH∙HCl) solution and 100 μL of working 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1-EDC∙HCl) solution. The mixture was incubated at 60˚C for 20 min. After addition of 100 μL of potassium hydroxide solution, the mixture further incubated for 15 min. After cooling in room temperature, 2 mL of phosphate buffer-0.5M hydrochloric acid was added to neutralize the solution. The mixture was washed with 2.5 mL of n-hexane to remove the long-chain fatty acid hydrazides and interfering substances. About 1.5 mL of aliquot of the aqueous layer was taken. The short chain fatty acid hydrazides in aliquot of the aqueous layer were extracted with 2.5 mL of diethyl ether. The ether layer was taken. The ether layer was evaporated with a stream of nitrogen gas. The residue was dissolved in 200 μL of methanol. For HPLC analysis, 20 μL of each preparation was injected into a 250 × 6.0 mm YMC-Pack FA (YMC, Kyoto, Japan). To detect the hydrazides of short chain fatty acids, a photodiode array detector (MD-1515; JASCO, Co., Ltd., Tokyo, Japan) was used to monitor the spectral data from 200 to 448 nm for each peak. Acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid were used as standard samples. Crotonic acid was used as the internal standard. The spectral data at 400 nm was used to quantify the hydrazides of short chain fatty acid. The mobile phase consisted of acetonitrile/methanol/water (30:16:54, v/v/v, pH 4.5). The running conditions of HPLC were a column temperature of 50˚C and a flow rate of 0.9 mL/min. HPLC chromatogram of acetic acid hydrazide, propionic acid hydrazide, butyric acid hydrazide, crotonic acid hydrazide, isovaleric acid hydrazide, and valeric acid hydrazide in standard sample obtained from at 400 nm is shown in Figure 2.

Data are expressed as the mean ± standard error (SE). All data were analyzed using Sigma Plot 11 (Systat Software, Inc., San Jose, CA, USA). The data were analyzed by the Student’s t-test, Mann-Whitney rank sum test, and/or Pearson Product-Moment Correlation. Statistical significance was confirmed at p-value < 0.05.

No significant differences were observed between the PGA and Con groups in

regard to final body weight (PGA [48.7 g ± 1.4], Con [50.3 g ± 2.0], liver weight (PGA [1.99 g ± 0.11], Con [2.25 g ± 0.13], and caecal contents (PGA [0.13 g ± 0.02], Con [0.15 g ± 0.02]. The average visceral fat per final body weight in the PGA group (0.066 ± 0.004) was lesser than that in the Con group (0.081 ± 0.010). The perigenital visceral fat per final body weight in the PGA group (0.034 ± 0.002) also tended to be lesser than that in the Con group (0.046 ± 0.006) (P = 0.097). There were no significant differences in food consumption (g/day) between the PGA group (4.66 ± 0.06) and the Con group (4.67 ± 0.07).

The plasma concentrations of triglyceride, total cholesterol, and HDL-cholesterol were investigated, and are presented in Table 2. No significant differences were observed in the plasma concentrations of triglyceride, total cholesterol, and HDL-cholesterol between two groups. The plasma glucose levels (Figure 3) and the plasma nonesterified fatty acids (NEFA) levels (Figure 4), however, were significantly lower in the PGA group compared to those in the Con group (P < 0.05).

The results of the maltose tolerance test are presented in Figure 5. All data are representative of values deducted from the blood glucose levels immediately prior to administration of maltose. In the PGA group, the average blood glucose levels measured at 15 min, 30 min, 60 min, and 120 min after maltose loading

were lower than those in the Con group. Incremental AUC determined from 0 min to 120 min was lower in the PGA group than in the Con group (P = 0.086). The results of starch tolerance test are provided in Figure 6. All data are representative of values deducted from the blood glucose level immediately prior to administration of starch. Blood glucose levels at 15 min were significantly lower in the PGA group than in the Con group (P < 0.05). Co-administration of

γ-PGA and starch to mice is believed to suppress the initial rise in blood glucose levels. In the PGA group, the average blood glucose levels measured at 30 min, 60 min, and 120 min after starch loading were lower than those observed in the Con group. Incremental AUC determined from 0 min to 15 min, 0 min to 30 min, and 0 min to 60 min was significantly lower in the PGA group than in the Con group (P < 0.05). These results suggest that PGA decreases postprandial glucose rise in adult male mice.

The caecal short chain fatty acid concentrations observed in the two dietary groups are shown in Figure 7. The average caecal acetic acid concentration was higher in the PGA group than in the Con group. The average total caecal short chain fatty acid concentration (acetic acid+ butyric acid + propionic acid + valeric acid + isovaleric acid) was higher in the PGA group than in the Con group. The ratio of caecal acetic acid/total caecal short chain fatty acids was significantly greater in the PGA group (P < 0.05) than in the Con group (Figure 8). In contrast, the ratio of caecal butyric acid/total caecal short chain fatty acids was significantly lower in the PGA group (P < 0.05) (Figure 8). These results suggest that the production rates of each of the short chain fatty acids differ between the two groups. The ratio of caecal acetic acid/propionic acid was related to the weight of visceral fat/BW. From the Pearson Product-Moment Correlation analysis, significant negative correlations were observed between the ratio of caecal acetic acid/propionic acid and the weight of visceral fat/BW (r = −0.57) (Figure 9).