Nghiên cứu ứng dụng bức xạ gamma co 60 để chế tạo b glucan khối lượng phân tử thấp tan trong nước có hoạt tính sinh học từ bã men bia tt tiếng anh

MYNISTRY OF EDUCATION VIETNAM ACADAMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY Nguyen Thanh Long STUDY ON APPLICATION OF GAMMA Co-60 RADIATION FOR PRODUCTION OF BIOACTIVE WATER-SOLUBLE LOW MOLECULAR WEIGHT β-GLUCAN PRODUCT FROM SPENT BREWER’ YEAST Major: BIOTECHNOLOGY Code: 42 02 01 SUMMARY OF BIOLOGY DOCTORAL THESIS Ho Chi Minh City - 2020 The thesis was completed at: Academy of Science and Technology Vietnam Academy of Science and Technology Supervisor 1: Associate Prof Le Quang Luan Supervisor 2: Associate Prof Hoang Nghia Son Opponent 1: … Opponent 2: … Opponent 3: … The thesis is submitted before the Thesis Examination Committee Meeting at Academy of Science and Technology - Vietnam Academy of Science and Technology at [time][date] [month] [year] Thesis can be found at: - Library of Academy of Science and Technology - National Library INTRODUCTION The thesis "Study on application of gamma Co-60 radiation for production of bioactive water-soluble low molecular weight β-glucan product from spent brewer’s yeast" has been carried out at Biotechnology Center of Ho Chi Minh City and Institute of Tropical Biology from November 2015 to May 2019 The urgency of the thesis β-glucan has been widely known as a strongly immune stimulant, cholesterol and triglyceride reduction, blood sugar regulator, wound healing, skin rejuvenation, etc β-glucan also has the effect on increasing the number of immune cells and inhibiting the growth of tumors in humans so it has a very strong activity for tumor prevention which help improving the effectiveness of cancer treatment, minimizing the side effects from chemical therapy, etc In animal husbandry, β-glucan strengthens the immune system and helps animals resisting to some diseases, thereby increasing product yield and quality without using antibiotics or stimulant However, β-glucan has a high molecular weight (Mw), high viscosity and low solubility leads to a poor absorption which is a barrier for application Many studies have shown that low Mw β-glucans have better biological effects those of β-glucans and water-soluble β-glucans with Mwin range about 1-30 kDa have been shown a higher immune enhancement effect than that of high Mw β-glucans Water-solube and low Mw β-glucans are short-circuiting molecules and easily disolved They can be easily absorbed and have highly biological activities, so its effectively in use are higher For preparing the water soluble and low Mw β-glucans, the degradation by irradiation method has been proven as a very effective method due to its outstanding advantages such as simple process, adjustably of Mw as expected, high purity product, without purification and environmentally friendly β-glucan is one of the main compounds of the cell wall of brewer’yeast and there are more than 300 beer factories with a capacity of 1.7 billion liters per year and the spent is about 1% Currently, this spent is only partially used and the remainder is treated and discharged into the environment Therefore, the use of the discard spent brewer’ yeast to extract and prepare β-glucan as a raw material for production of highly bioactive water-soluble low molecular weight β-glucan product from spent brewer’ yeast is very effective and practical in order to reuse the discard waste for preparing high-value products and contributing to the reduction of the waste causing environmental pollution This thesis has studied on the completion process for extraction of βglucan from cell wall of spent brewer’ yeast and the establishment process for production of water-solube and low Mw β-glucan by irradiation method In addition, it has also studied biological effects of radiation degraded β-glucan in vitro and in vivo using chickens and mice in order to prepare the β-glucan product with an appropriate Mw for inducing highly biological effects and suitable for application as a functional food or a supplement in livestock production Objectives of the study The objective of the thesis is to successfully build up a process for preparation of bioactive water-soluble low molecular weight β-glucan product from from spent brewer’ yeast by irradiation method The main research contents of the thesis - Extraction of β-glucan from cell wall in spent brewer’s yeast - Degradation of β-glucan by the gamma Co-60 irradiation method - Investigation of biological activities of radiation-degraded β-glucan - Build-up of the process for producing water-soluble low molecular weight β-glucan by irradiation method CHAPTER LITERATURE REVIEW 1.1 Overview introduction of β-glucan This section provides an overview of the structure and sources for preparation of β-glucan 1.2 Summary of Saccharomyces cerevisiae yeast This section gives an overview of the S cerevisiae and the structure of its cell wall, in which β-glucan is emphasized 1.3 Method of obtaining cell walls from beer yeast This section presents the common methods used to break down Saccharomyces cell for obtaining cell walls 1.4 Method of extracting β-glucan from beer yeast cells This section presents common methods of cell disruption, protein extraction and purification to obtain β-glucan 1.5 Methods for degradation β-glucan This section presents the common methods used for degradation of βglucan 1.6 Biological activity of β-glucan Describsion of the biological properties and action mechanism of βglucan 1.7 Applications of β-glucan Describsion of the main applications of β-glucan in various fields 1.8 Applications of low molecular weight β-glucan This section prsents the main applications of water soluble and low molecular weight β-glucan in various fields CHAPTER MATERIALS AND METHODS 2.1 Materials - Spent brewer’ yeast (Saigon Binh Duong brewery), standard βglucan from yeast cell (Sigma, USA), and KIT for determination of content of (1-3, l-6)-glucan (Megazyme) and other pure chemicals (Meck) - Luong Phuong chicken (Gallus gallus domesticus) (Ho Chi Minh City University of Agriculture and Forestry), Swiss mice (Pasteur Institute, Ho Chi Minh City), Anti-mouse IgG primary antibody produced in goat and secondary Anti-goat IgG - Alkaline phosphatase (Sigma-Aldrich, USA) and 96-well ELISA plate (Santa Cruz Biotechnology, Canada) 2.2 Contents and methods 2.2.1 Extraction of β-glucan from spent brewer’s yeast 2.2.1.1 Collection of Saccharomyces yeast cell walls: Spent brewer’ yeast was centrifuged at 5000 rpm, washed and autolyzed for 20 hours at 50°C It was then centrifugated for receive insoluble part 2.2.1.2 Extraction of total β-glucan a Effect of temperature: Conducting at 70, 90 and 100°C 400 g of cell walls were stirred with 2000 mL of 3% NaOH solution before boiling for hours and centrifuged to collect the solid portion The solid portion was further extracted for times with 2000 mL of HCl with concentrations of 2.45; 1.75 and 0.94 M at 75°C for hrs The mixture was then centrifuged at 7000 rpm, collected the insoluble portion, washed times with alcohol 98° and triple extract with diethyl ether Dry and calculate the content of β-glucan as follows: Content of β-glucan (%) = The β-glucan sample was then analyzed the content of protein (according to AOAC 987.04-1997 method) and β-glucan (using KYBGL kit) b Effect of NaOH concentration: This expriment were conducted in the same steps in section a but the NaOH concentration changed with 1, 2, and 4% c Effect of extraction time: Steps of this expriment were similar as those in section a but using optimal NaOH concentration from section b and extraction times of 3, 4, 6, and 12 hrs d Effect of sample/solvent ratio: This experiemnt was also desinged in the the way in section a but the volume of NaOH solution with the optimal concentration (from section b) was 1200, 2000 and 2800 mL, and with the optimal temperature and reaction time were determined respectively in section a and c 2.2.1.3 FTIR measurement: β-glucan samples were grinded and mixed with KBr before forming pellets The measurement was performed on a FTIR spectrophotometer model FT/IR-4700 (Jasco, Japan) 2.2.2 Degradation of β-glucan by the gamma Co-60 irradiation method 2.2.2.1 Irradiation for preparation of water-soluble low Mw β-glucans: 100 g of β-glucan were dissolved in 100 mL of distilled water to form a 10% β-glucan suspension mixture (w/v) and then irradiated in a Co-60 gamma source at various doses with a dose rate of kGy/h 2.2.2.2 Determine of the water-soluble content in irradiated β-glucan: The irradiated β-glucan sample was centrifuged at 11,000 rpm to collect supernatant The supernatant was then precipitated by ethanol with the ratio of 1/9 (v/v) and centrifuged to collect the precipitate before drying The content of water-soluble β-glucan is calculated by the formula: Water-soluble β-glucan content = 2.2.2.3 UV-vis measurement: The UV-vis spectra of β-glucan samples were measured on a GENESY 10S UV-Vis spectrophotometer (Thermo, USA) at a concentration of 0.025% and wavelengths of 200-600 nm 2.2.2.4 Mw determination: Mw of the sample β-glucan was measured on the GPC e2695 system using the Ultrahydrogel column (Water, USA) and the β-glucan solution of 0.1% (20 µL) was injected at mL/minute at 40°C 2.2.2.4 FTIR measurement: Proceed as described in section 2.1.2.3 2.2.2.5 NMR spectrum measurement: 1H- and 13C-NMR spectra of βglucan are measured on a Utrashield 500 plus (Brucker, USA) at frequencies of 500 MHz and 125 MHz using D2O (Cambridge, USA) with a sample concentration of mg/L 2.2.3 Investigation of biological activities of radiation-degraded βglucan 2.2.3.1 In vitro antioxidant activity: 1.5 mL of 100 ppm β-glucan solution was added into 1.5 mL of 0.1 mM DPPH solutio The mixture was shaken and kept in dark condition for 30 minutes before measuring the OD at 517 nm (distilled water was used as the control sample) The free radical scavenging activity was calculated by the formula: H (%) = (1 - A/Ao) x 100 Where as: A is the OD value at 517 nm of sample and Ao is the OD value at 517 nm the control sample 2.2.3.2 In vivo antioxidant activity in mice: Mice were divided into two groups (each group consisted of 45 mice with treatments, each with mice and repeated times): The mice in Normal group were not injected with CCl4, while the mice in hepatotoxic group were intraperitoneal injection for times by CCl4 at a dose of 10 mL/kg of body weight (the mice were fasted for 15 hours before injecting every days) After injection for 60 minutes, β-glucan samples (2 mg/mouse) were daily oral administrated for week The control mice only supplied with distilled water After days, the AST and ALT indexes in blood of tested mice were analyzed by a BioSystem AI (Belgium) 2.2.3.3 Investgation of blood formula and immunity indexes in mice: The experiment consisted of treatments and triplicated Every day, mice oral administrated with 100 µL of 2% β-glucan solution (2 mg/mouse) After 28 days, blood was collected for analyzing the blood formula (erythrocytes, total white blood cells, neutrophils and lymphocytes) and immune factors (IgG and IgM) 2.2.3.4 The activity on reduction of lipid and glucose in blood of mice a Preparation of obese mice: Mice in Fat-fed groups were fed with high-fat feed (HFD-high fat diet) and normal-diet groups were fed with standard fees (ND-normal diet) for weeks The blood was then collected for analyzing the glucose, cholesterol, triglyceride and LDL indexes b Investigation of the effect on clinical chemistry indexes in blood of obese mice: The obese mice were fed daily with 100 µL of 2% β-glucan solusion The control ones were supplemented with only DW The clinical chemistry indexes in blood were analyzed at stages (the stage 1: After daily administrating β-glucan for 20 days and feeding with highfat feeds; the stage 2: Continuing daily administration with β-glucan for 20 days (after 40 days); and the stage 3: Stopping administration of βglucan for 20 days (after 60 days) The analyzed indexes including Cholesterol, triglycerides, LDL and blood glucose 2.2.3.5 Test of growth promotion and immune stimulation effects in chickens: The experiments were designed with treatments, each treatement containing 18 chickens with tripplicated Chickens were supplemented with 500 ppm of different Mw β-glucan The monitoring indexes include: Average weight, the average weight gain, feed conversion rate (FCR), cumulative survival rate, and cellar immunity indexes (total white blood cells/1 mm3, lymphocyte and neutrophil ratios), antibody titer related to anti-Newcastle disease virus (NDV), anti-infectious bursal disease virus (IBDV), meat quality (eviscerated rate, carcass yield, chesk yield and thigh yield) 2.2.4 Built-up of the process for producing water-soluble and low Mw β-glucan at a dose range below 100 kGy 2.2.4.1 Degradation of β-glucan by irradiation method at various pH: The pH of 10% glucan mixture were adjusted to 3, 5, and before irradiation as the same conditions in section 2.2.2.1 2.2.4.2 Degradation of β-glucan by irradiation method in condition with H2O2: The experiment was conducted as descibtion in section 2.2.4.1 but the concentrations of β-glucan were 5, 10 and 15% (w/v) in 1% H2O2 solution 2.2.4.3 X-ray diffraction: X-ray diffraction (XRD) diagrams of β-glucan samples were measured by an D8 Advance ECO (Bruker, Germany) using CuKα radiation (lq = 1,5406 A, u = 40 kV, I = 25 mA) over the angular range of 30-100° (2θ), with a step size of 0.05° (2θ) and a counting time of 0.5/s 2.2.5 Data analysis Data were statistically analyzed by Excel software and one-way variance analysis (ANOVA) using SPSS 16.0 software CHAPTER RESULTS AND DISCUSSION 3.1 Extraction of β-glucan from spent brewer’s yeast cell 3.1.1 Collection of yeast from brewer’s yeast slurry Brewer’s yeast slurry after collection were centrifuged at 5000 rpm to receive precipitate, washings and centrifuging to collect yeast cells (Fig 3.1) A B C D Figure 3.1 Brewer’s yeast slurry (A) and its SEM image (B), yeast cell (C) and its SEM image (D) 3.1.2 Separation and collection of yeast cell walls A B C After autolysating, yeast cells were centrifugated for collection insoluble part consisted of aremostly cell walls with ivoryFig 3.2 Yeast call wall before (A) and after white color (Fig 3.2) centrifugation (B) and SEM image 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 Temperature (oC) 70 90 100 Table 3.1 Effect of reaction temperature on β-glucan yield Yield of β-glucan product (%) Purity (%) Content of protein (%) 17,11 ± 0,22 85,31 2,28 16,13 ± 0,11 90,89 1,41 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 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 βglucan 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 (%) 18,68 ± 0,29 84,98 2,30 18,14 ± 0,14 85,11 2,00 17,55 ± 0,11 91,13 1,75 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 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 Content in sample (%) Kinds of glucan 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 βglucan 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 βglucan (Mw > 64 kDa) was 9-12 folds lower those of irradiated samples 3.3.2 In vitro liver protection of Fig 3.13 Antioxidant activity of βglucan with diferent Mw irradiated β-glucan 3.3.2.1 Effect of irradiated β-glucan on the AST index in hepatotoxicinduced 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 hepatotoxicinduced mice: Fig 3.15a showed that irradiated β-glucan also reduced ALT and this index was the lowest in group of mice supplemented by βglucan 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 Red blood cells (106 Leukocyte (103 Lymphocyte Neutrophils (kDa) cells/mm3) cells/mm3) (%) (%) 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 64 48 Mw, kDa 25 11 ĐC >64 48 Mw, kDa 25 11 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 groups of after weeks fed by different diets week (g/mouse) After weeks (g/mouse) ND Group 19.45 ± 0,28 32.53 ± 0,95 HFD Group 19.54 ± 0.23 48.87 ± 0.71 18 Weight gain (%) Weight increase of HFD group (%) ↑ 67.23 ↑ 150.15 ↑ 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 Figure 3.17 Mice in HFD group (A) glucose in blood of tested mice from Table 3.11 and ND group (B) after weeks showed 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 weeks Index Total cholesterol (mg/dL) Triglyceride (mg/dL) LDL (mg/dL) Glucose (mg/dL) ND Group 78.79±4.21 71.85±3.04 20.28±1.32 102.79±8.86 HFD Group 145.04±6.21 121.78±6.27 31.73±2.69 165.46±7.39 Increase of HFD Group (%) ↑ 84.08 ↑ 69.48 ↑ 56.49 ↑ 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 a c ĐC2 45.12 ± 1.43 51.73 ± 1.03 ↑ 15.17b > 64 44.51a ± 1.01 47.51b ± 1.34 ↑ 6.72a a b 48 44.85 ± 1.07 47.48 ± 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 After 20 days of treatment Mw β-glucan (kDa) 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 d b c Control-2 198.15 ±5.46 164.82 ±6.32 38.53 ±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 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 After 40 days of treatment Mw β-glucan (kDa) 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 d c c Control-2 210.74 ± 9.05 190.41 ±10.79 38.48 ± 1.14 239.74d±13.02 > 64 119.54c ± 4.55 78.85ab ± 4.12 22.06b ± 1.56 134.11c ± 6.03 ab a ab 48 91.5 ± 3.26 69.53 ± 1.77 19.09 ± 1.23 97.98ab ± 2.42 25 75.97a ± 2.36 62.58a ± 3.15 17.25a ± 0.79 86.13a ± 2.94 b b ab 11 96.08 ± 6.28 77.77a ± 3.01 21.31 ± 1.63 114.25bc ± 3.43 * * * * was highest decreased in blood of mice administrated water-soluble βglucan with Mw~25 kDa For LDL index, after 20 days, mice fed by βglucan 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 After 60 days of treatment Mw β-glucan Triglyceride (kDa) Cholesterol (mg/dL) LDL (mg/dL) Glucose (mg/dL) (mg/dL) a b Control 93.83 ± 3.87 98.61 ± 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 b a ab > 64 122.79 ± 3.80 82.79 ± 2.91 24.04 ± 2.61 137.54b ± 3.68 48 99.78a ± 3.53 77.73a ± 3.74 19.76ab ± 1.46 110.81a ± 4.12 a a a 25 89.67 ± 3.18 70.59 ± 3.17 18.85 ± 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 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 Figure 3.20 The net change of LDL index in blood of tested mice compared with before significant different from the treatments taking supplement of different Mw β-glucan 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 Figure 3.21 The net change of glucose index in mg/dL in mice fed with different Mw blood of tested mice compared with before taking supplement of different Mw β-glucan β-glucan 3.3.5 Effect of irradiated β-glucan on growth and immunity of Luong Phuong chicken Table 3.16 showed that the total body weight of chicken supplemented water-soluble β-glucan with Mw~11-25 kDa was the highest (1260 & 1256 g/chicken) and it was the lowest for the control one with only 1013 g/chicken The weekly weight gain in chicken supplemented with water-soluble β-glucan with Mw~11-25 kDa was still highest The consumed Table 3.16 Effect of β-glucan with different Mw on weight gain and meat quality in chickens after weeks of feeding Index Survival ratio (%) The weekly weight gain (g/bird/week) Control 61.1b±3.2 d 16.3 ±0.5 > 64 kDa 49 kDa 66.7b±6.4 72.2b ± 5.5 cd 17.8 ±0.3 bc 18.8 ±0.2 25 kDa 94.4a ± 5.6 ab 20.6 ±0.7 11 kDa 94.4a ± 5.6 20.7± 0.9 * * 22 The used feed (g/bird/week) 491.1a±5.4 508.3a±1.7 480.4ab±9.2 453.3b±4.4 479ab±15.7 * FCR (kg feed/kg body weight) 4.8b±0.7 3.8ab±0.4 3.7ab±0.2 3.1a±0.2 3.4a±0.2 * Total body weight (g/bird) 1012.7d±10.3 1096c±6.7 1152.3b±17.1 1255.7a±5.7 1259.7a±21.1 * Mean values followed by the same letter within a row are not statistically significantly different, * significant with P