Study on application of gamma Co - 60 radiation for production of bioactive water - soluble low molecular weight β - glucan product from spent brewer’ yeast

) and SEM image A C D B C B A 8 3.1.3. Investigation of factors affecting to β-glucan extraction yield from yeast cell walls 3.1.3.1. The effect of temperature: The results in Table 3.1 showed that the more increase of reaction temperature, the less product yield of extracted β-glucan. At a reaction temperature of 70°C, the yield of extracted β-glucan was highest with 17.11%; and at 100°C the yield of extracted β-glucan was lowest with 14.28%. However, it can be seen that the higher reaction temperature, the lower protein content in the product and the higher purity of the product. The extraction temperature of 90°C was the most appropriate. Table 3.1. Effect of reaction temperature on β-glucan yield Temperature (oC) Yield of β-glucan product (%) Purity (%) Content of protein (%) 70 17,11 ± 0,22 85,31 2,28 90 16,13 ± 0,11 90,89 1,41 100 14,28 ± 0,16 91,12 1,08 3.1.3.2. Effect of NaOH concentration: The results in Table 3.2 indicated that the yeild of β-glucan product decreased by the increase of NaOH concentration. This yield was 17.55% when NaOH concentration increased to 3% and it was the lowest (16.82%) when using NaOH 4%. Treatment of 3% NaOH decreased β-glucan extraction yield but not significantly compared to that od the treatment with 2% NaOH. In addition, in the treatment of 1 and 2% NaOH, the protein content and the purity in products were still high (over 2%) and low (85.11%), respectively. Meanwhile, in the treatment of NaOH with a concentration of 4%, protein content was low (1.73%) and purity was about 91.99% but the the product yield was strongly reduced. Therefore, to extract β- glucan with high yield, low protein content, high product purity, NaOH with a concentration of 3% was the optimal selection. Table 3.2. Effect of NaOH concentration on β-glucan yield NaOH concentration (%) Yield of β-glucan product (%) Purity (%) Content of protein (%) 1 18,68 ± 0,29 84,98 2,30 2 18,14 ± 0,14 85,11 2,00 3 17,55 ± 0,11 91,13 1,75 4 16,82 ± 0,22 91,99 1,73 3.1.3.3. Effect of extraction time: The results in Table 3.3 showed that the β-glucan extracted yield was decreased by the increase of reaction time. 9 Table 3.3. Influence of hydrolysis time on β-glucan acquisition efficiency Extraction time (hours) Rate of -glucan product (%) Purity (%) Content of protein (%) 3 17,97 ± 0,30 84,96 1,93 6 17,12 ± 0,30 86,15 1,49 9 16,13 ± 0,11 91,52 1,41 12 14,97 ± 0,10 92,08 1,34 When extracting for 3-12 hours, the extraction yield was decreased by 3%, in which the extracted yield in treatments at 9 and 12 hours significantly decreased. In addition, the protein content in β-glucans extracted extracted from 3-12 hours was less than 2% and the purity of products extracted for 9-12 hours was quite high. These results show that the 9-hour extraction time is the most effective. 3.1.3.4. Effect of sample/solvent ratio: Table 3.4 showed that the β- glucan yield decreased by the increase of sample/solvent ratio. The β- glucan extracted by the rate of 1/3 is higher than those with extracted at the rates of 1/5 and 1/7. In the treatment with the rate of 1/7, the β-glucan content was equivalent to that in the treatment with the rate of 1/5 (~16%). The protein contents of all obtained β-glucan products were below 2% but the purity products extracted by the rate of 1/5 and 1/7 were higher. It can be seen that the sample/solvent ratio of 1/5 was optimal. Table 3.3. Influence of sample/solvent ratio on β-glucan acquisition efficiency Sample/solvent ratio (g/mL) Rate of β-glucan product (%) Purity (%) Content of protein (%) 1/3 17,42 ± 0,14 85,19 1,90 1/5 16,13 ± 0,11 92,01 1,41 1/7 16,15 ± 0,08 92,98 1,34 3.1.3.5. Completion of process for praparation of β-glucan from spent brewer’s yeast a. Extract β-glucan from spent brewer’s yeast with a scale of 500 liters/batch: Table 3.5. β-glucan extraction efficiency from spent brewer’s yeast with a scale of 500 liters/batch Time Volume of beer yeast waste fluid (liters) Dry weight of yeast cell (kg) Dry weight of yeast cell wall (kg) Weight of β- glucan product Efficiency (%) 1 500 15.89 4.44 0.7122 16.02 2 500 16.10 4.35 0.7285 16.76 3 500 16.51 4.77 0.7411 15.53 TB 500 16.17±0.32 4.521±0.22 0.7273±0.01 16.11±0.62 10 From the above results, the process for β-glucan extraction was completed and tested with larger spent brewer’s yeast (500 liters/batch). Results from 3 different batches presented in Table 3.5 showed that the total β-glucan product obtained was 2.18 kg. Thus, this process showed an high yield production of β-glucan from Fig. 3.3. β-glucan sample after extracted from yeast cell wall (A), after drying at 60oC (B) and its SEM image yeast cell walls with an average yield about 16.1%. The β-glucan product was in brown color as shown in Fig. 3.3. b. Determine β-glucan content: In this study, the β-glucan content in the manufactured sample in Table 3.6 shows that the purity of the β-glucan product obtained from the process is about 91.78% of β-glucan and it contains a small amount (about 1.5%) of -glucan. Table 3.6. Content of glucan types in extracted sample Total glucan (%) -glucan β-glucan 93.34 ± 0.41 1.56 ± 0.07 91.78 ± 0.34 c. Structural characterication of extracted β-glucan product The structural characteristics of extracted β-glucan product were characterized by FTIR spectrum and compared with standard sample from Sigma. Results from Fig. 3.4 and listed in Table 3.7 showed that peaks at 3333 cm -1 indicated for O-H- linkage appeared with high intensity and broad shoulders, while the peak 2896 cm -1 with medium intensity and narrow shoulder and the weak peak 2088 cm -1 attributed to C-H bond. The peaks at 1640 and 1079 cm -1 corresponded of CO bond. Meanwhile, the Fig. 3.4. FTIR spectra of β-glucan extracted from beer yeast cell walls and standard β-glucan of Sigma characteristics of CCH, C-O-C and CC bonds were recorded by the peaks at 1371, 1156 and 1040 cm -1 , respectively. It can be seen that C B A 11 structural characteristics of extracted β-glucan product were almost similar to those of the β-glucan same from Sigma. Table 3.7. Peaks of basic functional groups of β-glucan No. Peak (cm-1) Group No. Peak (cm-1) Group 1 3383 OH 5 1156 COC 2 2896 CH2 6 1079 CO 3 1640 CO 7 1040 CC 4 1371 CCH 8 890 CO of β-glucan d. Built-up the process for extraction of β-glucan with a scale of 500 liters/batch From above results, process of extracting -glucan from beer yeast waste is completed as described in Fig. 3.5. 12 Fig. 3.5. The diagram of process for extraction of β-glucan from spent brewer’s yeast at 500 L/batch 3.2. Degradation of β-glucan by gamma Co-60 irradiation method 3.2.1. Determination of the yield of water-soluble β-glucan by irradiation method: Fig. 3.6 showed that the content of water-soluble β- Spent brewer’s yeast Yeast cells Yeast cell walls Raw β-glucan Semi-pure β-glucan product Wet pure β-glucan 1 2 Final β-glucan product Removal of impurities Break down the cell 3 4 5 6 Treatmint with NaOH Treatment with HCl Lipid extraction Final process & quality control Step 1: Removal of impurities and collect yeast cells - Filter through a 0.5 mm filter to remove solid impurities; - Centrifugate at 5000 rpm and washing 3 times with distilled water. Step 2: Break down the cells and collect cell walls - Dilute of 15% yeast cell walls in distilled water and heat at 50°C under stirring conditions of 200 rpm for autolyzing in 20 hours. - Autoclave at 121°C, 15 minutes and centrifuge at 5500 rpm for removing the supernatant. The sediment is washed 3 times with distilled water to collect yeast cell walls. Step 3: Alkaline extraction - Yeast cell walls are suspended in 3% NaOH solution with ratio of 1/5 (w/v) and boiled at 90°C for 9 hours, - Centrifuge at 5500 rpm for collecting the sediment, - Wash the sediment with distilled water and centrifuge at 5500 rpm for receiving the raw β-glucan. Step 4: Acidic extraction - β-glucan with concentration of 15 (w/v) is extracted in HCl solution with concentration of 2.45, 1.75 and 0.94 M, respectively, at 75oC for 2 hours, - Centrifuge at 7000 rpm for collecting the sediment, - Wash with distilled water and centrifuge at 7000 rpm for receiving semi-pure β-glucan. Step 5: Lipid extraction - Wash the semi-pure β-glucan with absolute ethanol (with sample rate of 15%, w/v) and then centrifuge at 7000 rpm for obtaining sediment. - Wash with diethyl ether solvent (with sample rate of 15%, w/v) and centrifuging at 8000 rpm for 20 minutes to obtain pure β-glucan. Step 6: Dry, grind and check product quality - Dry pure β-glucan at 60°C for, then grind and filtered through 0.5 mm stainless steel filter to obtain ~0,7273 kg pure β-glucan product. - Determine β-glucan content in product by KIT (K- YBGL, Megaenzyme, Ireland), protein content by AOAC 987.04-1997 method, Mw by GPC, and structural 13 glucan was linear increase with the increase in radiation doses. Particlarly, when sample was irradiated at 100 kGy, the soluble β-glucan content was found ~25.8%, at 200 kGy this content was increased by ~23.2% compared to that of 100 kGy, and at 300 kGy, this content was obtained ~66,7%. 3.2.2. Decrease of molecular weight: Fig. 3.7 showed that the Mw of water-soluble β-glucan was gradually decreased and it was inversely proportional to the increase of radiation doses. At the irradiation range of 100 kGy, the Mw of water-soluble β- glucan was sharply decreased (from over 64 kDa to ~31 kDa), it was then slowly decreased and reached to about 11 kDa at 300 kGy. Fig. 3.6. The yield of water-soluble β- glucan content in 10% β-glucan mixture irradiated at various doses Fig. 3.7. The Mw reduction of soluble β-glucan by irradiation dose 3.2.3. UV spectrum analysis Figure 3.8. Water-soluble β-glucans from 10% β- glucan samples irradiated at different doses Fig. 3.9. UV-vis spectra of β-glucan prepared by irradiation method Fig. 3.8 showed that the soluble β-glucan solution after irradiation had changed its color from brown to dark brown. The results from Fig. 3.9 showed that there is no peak in the wavelength range of 200-400 mm in the spectrum of unirradiated sample, because there is no low Mw β- glucan in the solution. Meanwhile, the spectra of irradiated β-glucan samples appeared peak at 273 mm. 3.2.4. FTIR analysis Wavenumber, nm 14 The results from Fig. 3.10 showed that irradiated β-glucan had almost no change in the number and position of the peaks compared those of in unirradiated sample. However, the peak at 1731 cm -1 corresponded to the C=O bond in the molecule after degradation was appeared with the intensity increased by the increase of radiation dose, while the intensity of 1156 cm -1 peak expressed by C-O-C linkage (glucoside bond) in the spectra of irradiated samples was decreased by the increase of radiation dose. Fig. 3.10. IR spectra of β-glucan samples irradiated in10% concentration at different doses Fig. 3.11. The change in ratio of C-O-C peak intensity (1156 cm-1)/C-C peak intensity (1040 cm-1) in the spectrum of β-glucan sample by radiation In the comparation of the intensity of this peak and the peak of 1040 cm -1 corresponded to the C-C bond, which was proposed to be stable by radiation, it can be seen that the ratio between the peak intensity of C-O- C peak (1156 cm -1 ) and of C-C (1040 cm- 1 ) decreased by the increase of irradiation dose (Fig. 3.11). This resuts suggest that the cission was mainly orcurred at glucoside bonds. 3.2.5. Analysis of NMR- 1 H and 13 C spectra NMR- 1 H and 13 C spectra of water-soluble β-glucan sample with Mw~25 kDa (Fig. 3.12a & b) showed the chemical shifts at 4.48; 3.43; 3.59; 3.45; 3.47 and 3.82 ppm were respectively representing for H-l, H-2, H-3, H-4, H-5 and H-6 bonds in β-glucan molecule after irradiation. While the chemical shifts represented for the atoms C-l, C-2, C-3, C-4, C-5 and C-6 respectively were observed at 102.55; 72.32; 84.12; 68.14; 75.56 and 60.73 ppm. These results indicated that the main component of sample is β- glucan. 400 800 1200 1600 2000 2400 2800 3200 3600 4000 A b s 1/cm 0kGy 250kGy 200kGy 150kGy 100kGy 300kGy 3 3 8 3 2 8 9 6 1 6 4 0 8 9 0 1 0 4 0 1 0 7 9 1 1 5 6 1 3 7 1 1 7 3 1 15 Fig. 3.12. NMR-1H (a) and 13C (b) spectra of water-soluble β-lucan with Mw ~ 25 kDa 3.2.6. β-glucan content analysis: The results in Table 3.8 showed that compared to unirradiated sample, the total glucan content after irradiation was slightly increased (to ~97.88%). Table 3.8. Content of glucan in water-soluble β-glucan with Mw~ 25 kDa Kinds of glucan Content in sample (%) Before irradiation After irradiation Total glucan 93.34 ± 0.41 97.88 ± 0.89 α-glucan 1.56 ± 0.07 0.91 ± 0.36 β-glucan 91.78 ± 0.34 96.97 ± 0.25 3.3. Biological activity of water-soluble β-glucan prepared by irradiation method 3.3.1. In vitro antioxidant activity of irradiated β-glucan The results in Fig. 3.13 showed that the antioxidant activity of β- glucan increased by the decrease of β-glucan Mw. At the same concentration of 100 ppm, DPPH free radical capture activity of β-glucan with Mw > 64, 48, 25 and 11 kDa was found at 5.2, 47.6, 58.2 and 60.7%, respectively. The antioxidant activity of unirradiated β- glucan (Mw > 64 kDa) was 9-12 folds lower those of irradiated samples. 3.3.2. In vitro liver protection of irradiated β-glucan Fig. 3.13. Antioxidant activity of β- glucan with diferent Mw 3.3.2.1. Effect of irradiated β-glucan on the AST index in hepatotoxic- induced mice: 16 Fig. 3.14. The AST index of treatment mice in normal (without CCl4 injection) and hepatotoxic groups supplemented with different Mw β-glucan (a) and the net change compared to that of the control mice (ĐC) (b) Fig. 3.14a showed the AST index in blood of mice in Normal group was 58.47-84.24 U/L. The AST index in blood of mice supplemented with irradiated β-glucan was significant difference compared to that in non-supplemented mice. Mice fed with Mw~11 and 25 kDa β-glucan showed the strongest decrease of AST index (Fig. 3.14b). Water-soluble β-glucan with Mw~25 had the lowest effect on the reduction of AST in blood of mice with 61.81 U/L (similar to that in blood of the Normal group). 3.3.2.2. Effect of irradiated β-glucan on the AST index in hepatotoxic- induced mice: Fig. 3.15a showed that irradiated β-glucan also reduced ALT and this index was the lowest in group of mice supplemented by β- glucan with Mw ~ 25 kDa. In the hepatotoxic group, the results from Fig. 3.15b showed that the reduction of ALT index was reciprocal to the Mw of β-glucan. The treatment supplemented with water-soluble β - glucan with Mw~11 and 25 showed a low ALT level, and almost similar to that in non-hepatotoxicity control group. Fig. 3.15. The AST index of treatment mice in normal (without CCl4 injection) and hepatotoxic groups supplemented with different Mw β-glucan (a) and the net change compared to that of the control mice (ĐC) (b) 3.3.3. Effect of irradiated β-glucan on immune index in mice 17 3.3.3.1. Blood cell count and immune cells: Table 3.9. Total red blood cells, white blood cells, lymphocytes and neutrophils in blood of mice supplemented with different Mw β-glucan Mw β-glucan (kDa) Red blood cells (106 cells/mm3) Leukocyte (103 cells/mm3) Lymphocyte (%) Neutrophils (%) Control 5.44a±0.22 4.95b±0.18 57.23b±2.18 10.08b±1.04 > 64 5.46a±0.12 5.05ab±0.13 59.92ab±1.91 14.70a±0.52 48 5.64a±0.24 5.20ab±0.12 62.55ab±0.98 15.73a±1.43 25 5.6a±0.28 5.50a±0.15 65.97a±1.84 16.37a±0.66 11 5.48a±0.28 5.40b±0.16 64.42a±2.86 15.22a±0.67 ns * * * Mean values followed by the same letter within a column are not statistically significantly different, * significant with P <0.05, ns: none significant different The results from Table 3.9 showed that the number of red blood cells was almost the same between treatments, but the number of total white blood cells (WBC) was significant different. The WBC in mice fed with irradiated β-glucan was higher and the highest value was found in mice supplemented by water-soluble β-glucan with Mw~25 kDa (5.5x103 cells/mm 3 ). 3.3.3.2. Humoral immunity factor (IgG and IgG): The results from Fig. Fig. 3.16. Content of IgG (a) and IgM (b) in blood of mice supplemented with β-glucan. ĐC: Control mice 3.16 showed that the OD values measured at 405 nm of the mice supplemented by irradiated β-glucan with Mw from 11-25 kDa were higher than that of the control one without administration and other groups as well. 3.3.4. The in vivo ability on reducing blood lipid and glucose levels of β-glucan irradiated in mice 3.3.4.1. Preparation of obese mice Table 3.10. Body weight of mice in 2 groups of after 8 weeks fed by different diets ND Group HFD Group 0 week (g/mouse) 19.45 ± 0,28 19.54 ± 0.23 After 8 weeks (g/mouse) 32.53 ± 0,95 48.87 ± 0.71 0.6 0.7 0.8 0.9 1.0 ĐC >64 48 25 11 O D 4 0 5 n m Mw, kDa (a) 0.0 0.1 0.2 0.3 0.4 ĐC >64 48 25 11 O D 4 05 n m Mw, kDa (b) 18 Weight gain (%) ↑ 67.23 ↑ 150.15 Weight increase of HFD group (%) ↑ 124.24 Results from Table 3.10 and Fig. 3.17 showed that the body weight of mice in HFD group increased of 29.33 g/head (increase by 150.15%) compared to that of mice in ND group (increase by 124,24%). Results on analysis of blood lipid such as total cholesterol, triglyceride, LDL and glucose in blood of tested mice from Table 3.11 showed Figure 3.17. Mice in HFD group (A) and ND group (B) after 8 weeks that the lipid levelin blood of mice fed with high fat feed was much higher than that in mice fed by normal food. Particularly, cholesterol levels increased in 84.1%, triglyceride increased by 69.5%, LDL increased by 56.5% and blood glucose increased by 61%. Table 3.11. Biochemical indexes in blood of tested mice after 8 weeks Index ND Group HFD Group Increase of HFD Group (%) Total cholesterol (mg/dL) 78.79±4.21 145.04±6.21 ↑ 84.08 Triglyceride (mg/dL) 71.85±3.04 121.78±6.27 ↑ 69.48 LDL (mg/dL) 20.28±1.32 31.73±2.69 ↑ 56.49 Glucose (mg/dL) 102.79±8.86 165.46±7.39 ↑ 60.97 3.3.4.2. Effect of irradiated β-glucan on body weight, lipid and glucose: Table 3.12. Body weight of mice before and after 20 days fed with different Mw β-glucans Mw β-glucan (kDa) Before treatment (g/head) After 20 days (g/head) Net change rate (%) ĐC1 33.41a ± 0.71 34.84a ± 0.77 ↑ 4.35a ĐC2 45.12a ± 1.43 51.73c ± 1.03 ↑ 15.17b > 64 44.51a ± 1.01 47.51b ± 1.34 ↑ 6.72a 48 44.85a ± 1.07 47.48b ± 0.7 ↑ 6.14a 25 45.10a ± 0.78 47.16b ± 1.06 ↑ 4.53a 11 44.71a ± 1.21 46.51b ± 1.02 ↑ 4.22a ns * * ĐC1: Normal mice without feeding β-glucan; ĐC2: Obese mice without feeding β-glucan. The results from Table 3.12 showed that after 20 days administrated with irradiated β-glucan and fed by high fat feed, the body weight of mice in all treatments were increased. However, the weight gain of mice supplemented with β-glucan was much lower than that of control-2 mice (obese mice). The results of Table 3.13 showed that the blood lipid index 19 in mice in the control-2 group was significantly increased and the cholesterol index reached to 198.15 mg/dL, while this level in blood of mice supplemented with β-glucan was only 89.79-129.05 mg/dL. Table 3.13. Lipid and glucose levels in blood of mice after 20 days fed with different Mw β-glucans Mw β-glucan (kDa) After 20 days of treatment Cholesterol (mg/dL) Triglyceride (mg/dL) LDL (mg/dL) Glucose (mg/dL) Control 89.18a ± 5.44 85.27a±3.86 23.85ab±1.55 126.01b±6.92 Control-2 198.15d±5.46 164.82b±6.32 38.53c±2.80 212.31c±6.44 > 64 129.05c±2.96 90.16a±4.00 28.9b±2.82 139.74b±4.56 48 103.64b±4.01 79.62a±4.95 20.68a±2.06 124.72b±7.19 25 89.79a±3.54 83.10a±4.80 18.9a±1.69 106.8a ± 2.17 11 109.28b±4.29 88.68a ± 4.40 26.35ab±3.23 127.66b±6.63 * * * * Mice fed by water-soluble β-glucan with Mw~25 kDa had the lowest cholesterol level and almost the same as that in blood of control mice. The results of Fig. 3.18 and Table 3.14 also showed that the treatment by β-glucan with Mw~25 kDa showed the best results on cholesterol reduction after 40 days of supplementation and this level was almost similar to that of control mice. After the following 20 days of stop feeding β-glucan (only distilled water), the results from Fig. 3.18 and Table 3.15 also showed that after 60 days, the administration of β-glucan with Mw~25 kDa still showed the best effect with only 89.67 mg/dL cholesterol. Results from Table 3.13 and Fig. 3.19 indicated that, after 20 days, the triglyceride index in β-glucan administrated mice decreased from 25.27 to 33.14%, while this level still increased by 18.62% in the control mice and 35.84% in obese mice. After 40 days, the triglyceride index was also significantly reduced. The cholesterol index in the control mice was 3 folds higher than that in β-glucan supplemented mice. After 60 days, the triglyceride index was 70.59 - 82.79 mg/dL in β-glucan administrated mice. Thereby, despite of stopping β-glucan administration, the effect on reduction of triglyceride was still remained. During the treatment periods, the triglyceride index 20 Figure 3.18. The net change of cholesterol index in blood of tested mice compared to before supplement with different Mw β-glucan Figure 3.19. The net change of triglyceride index in blood of tested mice compared to before supplement with different Mw β-glucan Table 3.14. Lipid and glucose levels in blood of mice after 40 days fed with different Mw β-glucans Mw β-glucan (kDa) After 40 days of treatment Cholesterol (mg/dL) Triglyceride (mg/dL) LDL (mg/dL) Glucose (mg/dL) Control 88.14ab ± 5.62 86.67b ± 4.10 22.76b ± 1.62 127.28c ± 7.77 Control-2 210.74d ± 9.05 190.41c±10.79 38.48c ± 1.14 239.74d±13.02 > 64 119.54c ± 4.55 78.85ab ± 4.12 22.06b ± 1.56 134.11c ± 6.03 48 91.5ab ± 3.26 69.53a ± 1.77 19.09ab ± 1.23 97.98ab ± 2.42 25 75.97a ± 2.36 62.58a ± 3.15 17.25a ± 0.79 86.13a ± 2.94 11 96.08b ± 6.28 77.77ab ± 3.01 21.31ab ± 1.63 114.25bc ± 3.43 * * * * was highest decreased in blood of mice administrated water-soluble β- glucan with Mw~25 kDa. For LDL index, after 20 days, mice fed by β- glucan with Mw~48 and 25 kDa had the best effect on the reduction of LDL index. Particularly, the LDL indexes in mice fed with β-glucan were 18.9-28.9 mg/dL, while this level in the control-2 mice was increased into 38.53 mg/dL. After 40 days, the LDL index in mice fed with β-glucan continued to reduce almost similar to that in control mice. When β-glucan was stopped feeding in the following 20 days, the result on LDL reduction was still effective (Table 3.15). The best effect on the reduction of LDL Table 3.15. Lipid and glucose levels in blood of mice after 60 days fed with different Mw β-glucans Mw β-glucan (kDa) After 60 days of treatment Cholesterol (mg/dL) Triglyceride (mg/dL) LDL (mg/dL) Glucose (mg/dL) Control 93.83a ± 3.87 98.61b ± 2.94 24.55b ± 2.49 135.24b ± 3.04 Control-2 212.92c ± 7.71 195.43c ± 8.66 41.38c ± 1.03 245.39c ± 9.07 > 64 122.79b ± 3.80 82.79a ± 2.91 24.04ab ± 2.61 137.54b ± 3.68 48 99.78a ± 3.53 77.73a ± 3.74 19.76ab ± 1.46 110.81a ± 4.12 25 89.67a ± 3.18 70.59a ± 3.17 18.85a ± 0.92 105.90a ± 2.71 11 103.95a±5.14 79.52a ± 3.96 21.82ab ± 1.16 119.81a ± 3.01 * * * * 21 was found in 2 treatments supplemented by β-glucan with Mw~48 and 25 kDa (Fig. 3.20). The results in Table 3.13 and Fig. 3.21 also showed that mice in the group fed with β-glucan, the blood glucose levels decreased by15.62-34.12% compared to those of before testing.The treatment using water-soluble β-glucan with Mw~25 kDa showed the highest ability for reducing the blood glucose index (34.12%) but this reduction was not significant different from the treatments using β-glucan Mw~11 and 48 kDa. After 40 days of treatment, this index continued to decrease in the mice fed with β-glucan, while in the control-2 mice, this index increased into 239.74 mg/dL. After 60 days, glucose index slightly increased compared to that of 40 days and reached to 105.9-137.54 mg/dL in mice fed with diff