Summary of Biology doctoral 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

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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.

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Nội dung Text: Summary of Biology doctoral 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

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: 9 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
1 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. 1. 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
2 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 β- glucan 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. 2. 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. 3. 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 1. 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.
3 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 β- glucan. 1.6. Biological activity of β-glucan Describsion of the biological properties and action mechanism of β- glucan. 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 2. MATERIALS AND METHODS 2.1. Materials - Spent brewer’ yeast (Saigon Binh Duong brewery), standard β- glucan 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
4 boiling for 9 hours and centrifuged to collect the solid portion. The solid portion was further extracted for 3 times with 2000 mL of HCl with concentrations of 2.45; 1.75 and 0.94 M at 75°C for 2 hrs. The mixture was then centrifuged at 7000 rpm, collected the insoluble portion, washed 3 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 K- YBGL 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, 3 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, 9 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 3 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
5 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 1 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 β- glucan 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 5 mg/L. 2.2.3. Investigation of biological activities of radiation-degraded β- glucan 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 5 treatments, each with 3 mice and repeated 3 times): The mice in Normal group were not injected with CCl4, while the mice in hepatotoxic group were intraperitoneal injection for 3 times by CCl4 at a dose of 10 mL/kg of body weight (the mice were fasted for 15 hours before injecting every 2 days). After injection for 60 minutes, β-glucan samples (2 mg/mouse) were daily oral administrated for 1 week. The control mice only supplied with distilled
6 water. After 8 days, the AST and ALT indexes in blood of tested mice were analyzed by a BioSystem AI 5 (Belgium). 2.2.3.3. Investgation of blood formula and immunity indexes in mice: The experiment consisted of 5 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 8 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 3 stages (the stage 1: After daily administrating β-glucan for 20 days and feeding with high- fat feeds; the stage 2: Continuing daily administration with β-glucan for 20 days (after 40 days); and the stage 3: Stopping administration of β- glucan 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 5 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).
7 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, 7 and 9 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 3. 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 After autolysating, yeast cells A B C were centrifugated for collection insoluble part consisted of aremostly cell walls with ivory- Fig. 3.2. Yeast call wall before (A) and after white color (Fig. 3.2). centrifugation (B) and SEM image
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 Dry weight of Dry weight of Weight of β- Efficiency yeast waste fluid yeast cell (kg) yeast cell wall glucan product (%) (liters) (kg) 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 A B C presented in Table 3.5 showed that the total β-glucan product obtained was 2.18 kg. Thus, this Fig. 3.3. β-glucan sample after extracted from yeast cell process showed an high yield wall (A), after drying at 60oC (B) and its SEM image production of β-glucan from 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 Fig. 3.4. FTIR spectra of β-glucan extracted from beer 1079 cm-1 corresponded of yeast cell walls and standard β-glucan of Sigma CO bond. Meanwhile, the 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
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 Removal of Break impurities down the cell Spent brewer’s yeast Yeast cells Yeast cell walls 1 2 Treatmint 3 Step 1: Removal of impurities and collect yeast cells with NaOH - Filter through a 0.5 mm filter to remove solid impurities; - Centrifugate at 5000 rpm and washing 3 times with distilled water. Raw β-glucan 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 Treatment autolyzing in 20 hours. 4 with HCl - 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 Semi-pure with ratio of 1/5 (w/v) and boiled at 90°C for 9 hours, β-glucan product - 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. Lipid Step 4: Acidic extraction 5 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 Wet pure for receiving semi-pure β-glucan. 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 Final process 7000 rpm for obtaining sediment. 6 & quality - Wash with diethyl ether solvent (with sample rate of 15%, w/v) and centrifuging at 8000 rpm for 20 minutes control 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 Final β-glucan pure β-glucan product. 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 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-
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%. Fig. 3.6. The yield of water-soluble β- 3.2.2. Decrease of molecular weight: Fig. glucan content in 10% β-glucan mixture irradiated at various doses 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 Fig. 3.7. The Mw reduction of soluble decreased and reached to about 11 kDa at β-glucan by irradiation dose 300 kGy. 3.2.3. UV spectrum analysis Figure 3.8. Water-soluble β-glucans from 10% β- Fig. 3.9. UV-vis spectra of β-glucan glucan samples irradiated at different doses Wavenumber, nm 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
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. 1079 1040 3383 1156 2896 1371 1640 1731 890 300kGy Abs 250kGy 200kGy 150kGy 100kGy 0kGy 4000 3600 3200 2800 2400 2000 1600 1200 800 400 1/cm Fig. 3.11. The change in ratio of C-O-C peak Fig. 3.10. IR spectra of β-glucan samples irradiated intensity (1156 cm-1)/C-C peak intensity (1040 cm-1) in the spectrum of β-glucan sample by radiation in10% concentration at different doses In the comparation of the intensity of this peak and the peak of 1040 cm-1corresponded 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- 1H and 13C spectra NMR-1H and 13C 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.
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 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 Fig. 3.13. Antioxidant activity of β- irradiated β-glucan 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 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 25 11 ĐC >64 48 25 11 Mw, kDa Mw, kDa 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
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 Figure 3.17. Mice in HFD group (A) glucose in blood of tested mice from Table 3.11 and ND group (B) after 8 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 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 a 45.12 ± 1.43 c 51.73 ± 1.03 ↑ 15.17b > 64 44.51a ± 1.01 47.51b ± 1.34 ↑ 6.72a 48 a 44.85 ± 1.07 b 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