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 AND TRAINING VIETNAM ACADAMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY --- Nguyen Thanh Long STUDY ON APPLICATION OF GAMMA Co-60 RADIA

MYNISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADAMY OF 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

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

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

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

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

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:

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

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

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 glucan

β-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

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 fat feeds; the stage 2: Continuing daily administration with β-glucan for

high-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)

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

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)

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

were centrifugated for collection

insoluble part consisted of

aremostly cell walls with

ivory-white color (Fig 3.2) Fig 3.2 Yeast call wall before (A) and after centrifugation (B) and SEM image

C

B

A

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 ( o C) Yield of β-glucan product (%) Purity (%) Content of protein (%)

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 (%)

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

Table 3.3 Influence of hydrolysis time on β-glucan acquisition efficiency

Extraction time (hours) Rate of -glucan product (%) Purity (%) Content of protein (%)

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

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 glucan product

β-Efficiency (%)

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 60 o C (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

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

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

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

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-

Break down the cell

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 75 o C 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 obtainpure 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

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-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 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 glucan in the solution Meanwhile, the spectra of irradiated β-glucan

β-samples appeared peak at 273 mm

3.2.4 FTIR analysis

Wavenumber, nm