Pilot production of products rich in beta glucan from brewers yeast using protease

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TOPIC: PILOT PRODUCTION OF PRODUCTS RICH IN ΒETA-GLUCAN FROM BREWER’S YEAST USING PROTEASE Performer : LÊ KHÁNH PHÁP Class : CNSHE Courses : 62 Faculty : Biotechnology Specialized : Molecular Biology Instructor : MSc NGUYỄN QUỐC TRUNG PhD LÊ ĐỨC THẢO Department : Molecular Biology & Applied Biotechnology Hanoi – 2022 COMMITMENT I hereby declare that this is my own research work The data and results presented in the thesis are completely honest and have never been published by anyone in any research work I take responsibility for the data in this thesis and the information cited in the thesis have been identified Hanoi, March 21, 2022 Thesis author Lê Khánh Pháp i ACKNOWLEDGEMENTS To complete this thesis, I would like to express my deep gratitude to the teachers and teachers of the Faculty of Biotechnology - Vietnam National University of Agriculture, who have supported and facilitated me throughout the process Study and research I would like to thank the Department of Refrigeration Engineering & Air Conditioning, School of TFXtile – Leather and Fashion, Hanoi University of Science and Technology for creating favorable conditions for me to successfully complete my graduation thesis I would like to express my sincere thanks to Msc Nguyen Quoc Trung and PhD Le Đuc Thao, who have enthusiastically guided and helped me in the process of researching and completing the thesis At the same time, I would also like to express my sincere gratitude to Msc Trinh Thi Thu Thuy and Ms Phung Thi Duyen, Faculty of Biotechnology - Vietnam National University of Agriculture have supported and helped me in the process of making this thesis I would like to thank my relatives, family, friends and colleagues who have encouraged and encouraged me throughout the process of studying, researching and completing the thesis Hanoi, March 21, 2022 Thesis author Lê Khánh Pháp ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS viii Chapter I INTRODUCTION 1.1 Introduction 1.2 Objective and the meaning of the topic Chapter II LITERATURE REVIEW 2.1 OVERVIEW OF Β-GLUCAN 2.1.1 Definition, chemical structure of β-glucan 2.1.2 Applications of β-glucan 2.1.3 Sources of β-glucan 2.2 OVERVIEW OF SACCHAROMYCES CEREVISIAE 10 2.2.1 Morphological properties of yeast 10 2.2.2 Structure of yeast cells 11 2.3 PROTEASE ENZYME 12 2.3.1 Definition of protease enzyme 12 2.3.3 Classification of protease 13 2.3.4 Applications of protease 13 2.4 METHODS TO DISRUPT THE YEAST CELL WALL AND OBTAIN B-GLUCAN 15 2.4.1 Mechanical method to disrupt the yeast cell wall 16 2.4.2 Sonical method to disrupt the yeast cell wall 17 2.4.3 Autolysis method to disrupt the yeast cell wall 17 iii 2.4.4 Using chemical method to obtain β-glucan from yeast cell wall 19 Chapter III MATERIALS AND METHODS 21 3.1 MATERIALS 21 3.2 METHODS 21 3.2.1 Extraction of β-glucan from yeast residues (Nguyen Quoc Trung, 2021) 21 3.2.2 Optimization of the pilot-scale cell wall digestion process (Illustration diagram) 24 3.2.3 Nutritional analysis method 28 Chapter IV RESULTS AND DISCUSSION 35 4.1 RESULTS OF OPTIMIZATION OF WASHING SAMPLE STEP 35 4.1.1 Results of washing liter of sample 35 4.1.2 Results of washing 100 liters of sample 37 4.2 RESULTS OF OPTIMIZATION OF ENZYME INCUBATION 39 4.3 RESULTS OF OPTIMIZATION OF DRYING METHODS 41 4.3.1 Drying results by method “Convection drying” 41 4.3.2 Drying results by “ Freeze drying” method 42 4.3.3 Drying results by “Heat drying” method 43 4.4 ANALYSIS OF NUTRIENT PROPERTIES OF PRODUCTS 45 4.4.1 The content of β-glucan in the product 45 4.4.2 Other nutrient properties of products 46 Chapter V CONCLUSION - RECOMMENDATION 49 5.1 Conclusion 49 5.2 Recommendation 49 REFERENCES 50 APPENDICES 55 iv LIST OF TABLES Table 2.1: A summary of representative β-glucan present in food products Table 3.1 Components of the Megazyme-ireland kit 29 Table 4.1 Nutrient properties of products 45 v LIST OF FIGURES Figure 2.1 Molecular structure and the branched level of β-glucan from several sources Figure 2.2: Sources and mechanisms of β-glucans dependent on structure In the panel (a) cereal β-glucans; in the panel (b) fungal β-glucans Figure 2.3 Mushroom Saccharomyces cerevisiae 10 Figure 2.4 Structure of yeast cells 11 Figure 2.5 Classification of protease 13 Figure 2.6 Structure of yeast cell wall 15 Figure 2.7 Classification of unit operations for microbial cell disruption 16 Figure 3.1 Brewer's yeast 21 Figure 3.2 Extraction process of β-glucan from yeast residues ) 23 Figure 3.3 Optimization of the extraction process of β-glucan from yeast residues 24 Figure 3.4 Strotease SP 100 26 Figure 3.5 Freeze drying method 27 Figure 3.6 Heat drying method 27 Figure 3.7 Convection drying method in Experiment carried out in room 109, Dept of Refrigeration Engineering & Air Conditioning, School of TFXtile – Leather and Fashion, Hanoi University of Science and Technology 28 Figure 3.8 β-Glucan Assay Kit 29 Figure 4.1 Yeast juice collected from the brewery 35 Figure 4.2 Yeast fluid after washing the 1st and 2nd times 36 Figure 4.3 Yeast biomass after washing 37 Figure 4.4 100 liters of brewer's yeast 37 Figure 4.5 The process of washing 100 liters of yeast residue 38 vi Figure 4.6 Image of yeast obtained after washing 38 Figure 4.7 Enzyme Protease Solution 39 Figure 4.8 Incubate liter of yeast solution 39 Figure 4.9 Incubate 100 liters of yeast solution 40 Figure 4.10 Yeast cells autolysis in protease enzymes 40 Figure 4.11 Drying results by convection drying method 41 Figure 4.12 Drying results by Freeze drying method 42 Figure 4.13 Heat dryer and product after initial drying 43 Figure 4.14 Products after drying and grinding 44 Figure 4.15 Obtained 1.5 kg of products rich in β-glucan 44 Figure 4.16 GEBIO's Beta Glucan Product 48 vii LIST OF ABBREVIATIONS DNA : Deoxyribonucleic acid E.coli : Escherichia coli P dendritiformis : Paenibacillus dendritiformis RNA : Ribonucleic acid S Cerevisiae : Saccharomyces Cerevisiae β-glucan : beta-glucan viii Chapter I INTRODUCTION 1.1 INTRODUCTION Currently, beer production technology and beer consumption market in Vietnam as well as other countries in the world are developing, leading to a large amount of yeast waste that has been discharged into the environment On average, the amount of yeast residue discharged from beer production accounts for 1-2% of the volume of finished beer, equivalent to 40,000-80,000 tons/year This amount of yeast residue is mainly used to make animal food or is used as a cheap source of raw materials for the process of making autolytic yeast extract, the rest is discharged directly into the environment causing environmental pollution surrounding school And the main component of brewer's yeast is Saccharomyces cerevisiae - yeast In yeast, the cell wall accounts for about 20% of the dry weight, of which β-glucan accounts for about 50-60% of the dry matter The production of products with high economic value such as β-glucan can bring profits to factories while minimizing the environmental impact of these industrial plants According to study of Nguyen Quoc Trung, 2021 The method has determined that a method of extracting β-glucan from yeast residues achieved 99% efficiency, breaking the cell wall, thereby obtaining products with β-glucan content reaching 28.425% However, the method uses a small amount of raw materials, with limitations in terms of output, cost, etc., to be used in animal husbandry Therefore, I proceed with the topic Pilot Production of Products Rich In Βeta-Glucan from Yeast Residues Using Protease Enzymes In order to increase continuously) As a result, the product of convection drying method is discolored, has a bad smell, leading to a damaged final product 4.3.2 Drying results by “ Freeze drying” method Figure 4.12 Drying results by Freeze drying method Vacuum freeze dryer dries the product at low temperature and drying in a lowpressure environment (usually the pressure in the drying chamber is from 0.05-0.1 bar atmospheric pressure is bar) The low-pressure drying medium is intended to lower the boiling point of the water so that the water evaporates faster when drying at low temperatures In this experiment, the product was dried at -24°C, with a pressure of 0.06 bar According to my observation, freeze drying method is very effective and saves time in drying products However, with limited equipment and a large amount of product, the dryer was damaged due to overload 42 4.3.3 Drying results by “Heat drying” method Figure 4.13 Heat dryer and product after initial drying The heat dryer blows hot air directly into the product, separating the moisture from the product The yeast residue solution after treatment is dried at 60°C and the time depends on the condition of the product PROCESS Step 1: Pour the solution into the tray (just enough solution, so that the solution is spread and thin) and dry at 60°C Step 2: After hours of drying, the yeast residue solution (liquid) has a colloidal phenomenon (brown color) then inverting the product Step 3: Continue drying and inverting the product continuously at 60°C When drying a small amount of solution and a large contact surface It will help the product dry faster and shorten the drying time 43 Figure 4.14 Products after drying and grinding After completely drying, obtained a solid product (black brown color), after grinding the product, we obtained a powdered product (brown color) Figure 4.15 Obtained 1.5 kg of products rich in β-glucan β-glucan is the main component of yeast cell wall and they have many uses in fields such as medicine, food, etc Therefore, extracting β-glucan from 44 yeast residue not only brings economic efficiency It also helps to reduce and solve environmental pollution problems After the extraction process of β-glucan from yeast residues by the method of autolysis in protease enzyme and the steps of washing, filtering and collecting residues get a product rich in β-glucan will be obtained as shown in Figure 4.15 4.4 ANALYSIS OF NUTRIENT PROPERTIES OF PRODUCTS Table 4.1 Nutrient properties of products No Parameter(s) Unit Results 46,1 Protein % Fiber % Lipid g/kg 0,68 Carbohydrate % 44,2 Vitamin B1 mg/kg 12,46 Vitamin B2 mg/kg 9,31 Vitamin B3 mg/kg Vitamin D3 mg/kg 0,746 Canxi (Ca) % < 0,03 10 Photpho (P) % 0,85 11 β-Glucan % 13,03 KPH (LOD = 0,05) KPH (LOD = 1) 4.4.1 The content of β-glucan in the product After obtaining the preparation, measure the β-glucan content according to the Megazyme-ireland kit The results showed that the preparation had a βglucan content of 13.03% 45 According to a study by Ly Thi Minh Hien et al., 2015 studying the process of breaking down yeast cell walls from Saccharomyces cerevisiae beer yeast residues of Hoang Long Beer Company (Binh Duong), the method of using enzymes derived from micro-organisms The organism (Bacillus subtilis) is a protease combined with the treatment of raw materials with NaOH and high temperature to obtain the purified β-glucan, accounting for 9.25% of the dried yeast residue It was found that the optimization process obtained a product with an increase in beta glucan content of 1.41 times compared with the study of Ly Thi Minh Hien et al., 2015 Comparison of the β-glucan content obtained from the preparation after breaking the cell wall by the according to Nguyen Quoc Trung, 2021 method and the optimization method for the pilot process and the β-glucan content from the unprocessed yeast residue: (Nguyen Quoc Trung, 2021) (28.43%); optimization (13.03%); Unprocessed (5.89%) It was found that the optimization process of obtaining the preparation gave an increase of β-glucan by 2.21 times compared with untreated yeast residue 2.18 times reduction compared to comparison of the β-glucan content obtained from the preparation after breaking the cell wall by the according to Nguyen Quoc Trung, 2021 method 4.4.2 Other nutrient properties of products According the nutritional index of yeast from the source “USDA Food and nutrient database” Yeast contains 39 calories per 12 g serving This serving contains 0.9 g of fat, 4.9 g of protein and g of carbohydrate The latter is g sugar and 3.2 g of dietary fiber, the rest is complex carbohydrate Yeast contains 0.1 g of saturated fat and mg of cholesterol per serving 12 g of Yeast contains IU vitamin A, 0.0 mg of vitamin C and 0.00 mcg of vitamin D as well as 0.26 mg of iron, 3.60 mg 46 of calcium and 115 mg of potassium Yeast belongs to 'Not included in a food category' food category In addition, other nutritional ingredients make an important contribution to the introduction of β-glucan-rich preparations into animal feed: Protein (Provides building materials for organs and organ systems of the animal body); Carbohydrates (Used by the body for energy); Lipids (Provides Energy); Minerals Ca, P (Building cells, organs, organ systems); Vitamins B, D (Helps the body develop against disease-causing germs, helps digestion and balances the nervous system) From table 5.1 I see many high nutritional indicators in the preparation obtained from the optimization process, especially: Protein accounted for 46.1%, Carbohydrate accounted for 44.2% and other nutritional components: Lipid (0,68 g/kg); Vitamin B1 (12,46 mg/kg); Vitamin B2 (9,31 mg/kg); Vitamin D3 (0,746 mg/kg); Canxi (< 0,03%); Photpho (0,85%) and β-Glucan (13,03%) Some nutritional indicators are not included in the preparation such as: Fiber, vitamin B3 I found that, after optimizing the extraction process of β-glucan from yeast residues, get the product with high Protein and Carbohydrate content were obtained Specifically, Protein increased 4.61 times, Carbohydrate increased 22.1 times compared to the nutritional index of yeast from the source "USDA Food and Nutrition Database" In addition, the product also has a few other nutritional ingredients such as: Lipid, vitamin B1, B2, vitamin D, calcium, phosphorus 47 Figure 4.16 GEBIO's Beta Glucan Product Beta Glucan is known as a biological supplement because of its ability to stimulate the antibody system These compounds are present in the bran husks of cereal grains, barley, and the cell walls of yeasts, fungi, and bacteria Products rich in beta glucan are currently being applied in many animal feeds, for example: GEBIO's Beta Glucan product is extracted from the cell wall of yeast with a beta glucan content of 9.7% Uses of Beta Glucan – GEBIO: Increase immunity, increase resistance, detoxify, gain weight fast Promotes the maturation of the gut microbiota in young cattle and poultry  Compare that with my pilot optimized beta glucan product (13.03%) and GEBIO's Beta Glucan Product (9.7%) It was found that the process of pilotscale optimization increased the amount of β-glucan by 1.34 times compared to the product on the market In addition, the product also has many other nutritional ingredients such as: Protein, Calcium, Lipid, vitamin B1, B2, vitamin D, calcium, phosphorus 48 Chapter V CONCLUSION - RECOMMENDATION 5.1 CONCLUSION Through the obtained results, I had the following conclusions: Washing 100 liters sample was caried out with RO water mixing and settle and receive clean yeast biomass with milky white color Optimization of enzyme incubation using enzyme Strotease SP 100 (with annealing temperature of 55°C for hours) can break down yeast cell effectively Optimization of drying methods by heat drying method was most suitable with good quality of dried powder Nutrient properties of yeast β-glucan content in the product: Protein (46.1%); Carbohydrate (44.2%); Lipid (0,68 g/kg); Vitamin B1 (12,46 mg/kg); Vitamin B2 (9,31 mg/kg); Vitamin D3 (0,746 mg/kg); Canxi (< 0,03%); Phosphorus (0,85%) and β-Glucan (13,03%) 5.2 RECOMMENDATION Continue to standardized pilot method for large scale production of product rich in beta glucan 49 REFERENCES Vannucci L., Krizan J., Sima P., Stakheev D., Caja F., Rajsiglova L., Horak V & Saieh M (2013) Immunostimulatory properties and antitumor activities of glucans (Review) Int J Oncol 43(2): 357-64 Volman J J., Ramakers J D & Plat J (2008) Dietary modulation of immune function by β-glucans Physiology & Behavior 94(2): 276-284 Zimmerman J W., Lindermuth J., Fish P A., Palace G P., Stevenson T T & Demong D E (1998) A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes J Biol Chem 273(34): 22014-20 Sasaki T & Takasuka, N (1976) Further study of the structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes Carbohydr Res 47(1): 99-104 Kim Y T., Kim E H., Cheong C., Williams D L., Kim C W & Lim S T (2000) Structural characterization of beta-D-(1 > 3, > 6)-linked glucans using NMR spectroscopy, Carbohydr Res 328(3): 331-41 Roger Manson (2011) What is beta glucan? A Concise Guide to the Benefits and Uses of the Most Powerful Natural Immune Enhancer Known to Science Vesna Zechner-Krpan, Vlatka Petravić-Tominac, Ines Panjkota-Krbavčić, Slobodan Grba & Katarina Berković (2009) Potential Application of Yeast β-Glucans in Food Industry Review article Jalil, Abbe & Edwards, Christine & Combet, Emilie & Ibrahim, Muhammad & Garcia, Ada (2015) Combined effects of added beta glucan and black tea in breads on starch functionality International journal of food sciences and nutrition 66 1-7 10.3109/09637486.2014.971225 Finocchiaro, Franca & Ferrari, Barbara & Gianinetti, Alberto & Scazzina, Francesca & Pellegrini, Nicoletta & Caramanico, Rosita & Salati, Claudia & 50 Shirvanian, Vigen & Stanca, Antonio (2011) Effects of barley β-glucanenriched flour fractions on the glycaemic index of bread International journal of food sciences and nutrition 63 23-9 10.3109/09637486.2011.593504 10.Lazaridou, Athina & Biliaderis, Costas & Izydorczyk, Marta (2006) Cereal beta-Glucans: Structures, Physical Properties, and Physiological Functions Functional Food Carbohydrates 1-72 11.Rieder, Anne & Holtekjølen, Ann & Sahlstrøm, Stefan & Moldestad, Anette (2012) Effect of barley and oat flour types and sourdough on dough rheology and bread quality of composite wheat bread Journal of Cereal Science - J CEREAL SCI 55 10.1016/j.jcs.2011.10.003 12.Palić, Dušan & Herolt, Dawn & Andreasen, Claire & Menzel, Bruce & Roth, James (2006) Anesthetic efficacy of tricaine methanesulfonate, metomidate and eugenol: Effects on plasma cortisol concentration and neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820) Aquaculture 254 675-685 10.1016/j.aquaculture.2005.11.004 13.Babincová M., Bacová, Z., Machová E & Kogan G (2002) Antioxidant properties of carboxymethyl glucan: comparative analysis, J Med Food, 5(2): 79-83 14.Vetvicka, Vaclav & Vannucci, Luca & šíma, Petr (2013) The Effects of β – Glucan on Fish Immunity North American journal of medical sciences 580-588 10.4103/1947-2714.120792 15.Milewski, Stanisław & Wojcik, Roman & Zaleska, Bozena & Małaczewska, Joanna & Tanski, Zenon & Siwicki, Andrzej (2013) Effect of Saccharomyces cerevisiae dried yeast on the meat performance traits and selected indicators of humoral immunity in lambs Acta Veterinaria Brno 82 147-151 10.2754/avb201382020147 51 16.Artūras Javmen S G & Raimonda Gliebutė (2012) β-glucan extraction from Saccharomyces cerevisiae yeast using Actinomyces rutgersensis 88 yeast lyzing enzymatic complex Biologija 58(2): 51-59 17.Bacon J S., Farmer V C., Jones D & Taylor I F (1969) The glucan components of the 18.cell wall of baker's yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure Biochem J 114(3): 557-67 19.Dallies N., Franỗois J & Paquet V (1998) A new method for quantitative determination of polysaccharides in the yeast cell wall Application to the cell wall defective mutants of Saccharomyces cerevisiae Yeast 14(14): 1297-306 20.Hasan Tangüler H E (2009) The Effect of Different Temperatures on Autolysis of Baker’s Yeast for the Production of Yeast Extract 21.Klis F M., Boorsma A & De Groot P W (2006) Cell wall construction in Saccharomyces cerevisiae Yeast 23(3): 185-202 22.Klis F M., Mol P., Hellingwerf K & Brul S (2002) Dynamics of cell wall structure in Saccharomyces cerevisiae FEMS Microbiol Rev 26(3): 239-56 23.Laroche C & Michaud P (2007) New developments and prospective applications for beta (1,3) glucans Recent Pat Biotechnol 1(1): 59-73 24.Lipke P N & Ovalle R (1998) Cell wall architecture in yeast: new structure and new challenges J Bacteriol 180(15): 3735-40 25.Mala B Rao, Aparna M Tanksale, Mohini S Ghatge & Vasanti V Deshpande (1998) Molecular and Biotechnological Aspects of Microbial Proteases Microbiol Mol Biol Rev 1998 Sep 62(3): 597-635 26 Monroy Salazar H A.Z.M, S Kholif A., Monroy H., Pérez L S., Zamora J L & Gutiérez A (2016) YEAST: DESCRIPTION AND STRUCTURE 4-13 27.Murli Dharmadhikari (2011) Yeast Autolysis Lowa State University Extension and Outreach 52 28.R.J Wheatcroft J K., R.W Gilbert, K.J Sime, C.G Smith & W.H Langeris (2002) Production of β-glucan-mannan preparations by autolysis of cells under certain pH, temperature and time conditions, USOO6444448B1 29.Shokri H., Asadi, F & Khosravi, A R (2008) Isolation of beta-glucan from the cell wall of Saccharomyces cerevisiae Nat Prod Res 22(5): 414-21 30.Suphantharika M., Khunrae P., Thanardkit P & Verduyn C (2003) Preparation of spent brewer's yeast beta-glucans with a potential application as an immunostimulant for black tiger shrimp, Penaeus monodon Bioresour Technol 88(1): 55-60 31.Vetvicka V & Vetvickova J (2015) Glucan supplementation enhances the immune response against an influenza challenge in mice Ann Transl Med 3(2): 22 32.Bashir K M I & Choi J S (2017) Clinical and Physiological Perspectives of β-Glucans: The Past, Present, and Future Int J Mol Sci 18(9) 33.Bhatty R S (1993) Extraction and enrichment (1 leads to 3), (1 leads to 4)beta-D-glucan from barley and oat brans Cereal chemistry (USA) 34.Bzducha-Wróbel A., Błażejak S., Kawarska A., Stasiak-RóżańskaL., Gientka I & Majewska E (2014) Evaluation of the efficiency of different disruption methods on yeast cell wall preparation for β-glucan isolation Molecules 19(12): 20941-61 35.Du B., Meenu M., Liu H & Xu B (2019) A Concise Review on the Molecular Structure and Function Relationship of beta-Glucan Int J Mol Sci 20(16) 36.Liu X.-Y., Wang, Q., Cui, S W & Liu, H.-Z (2008) A new isolation method of β-d-glucans from spent yeast Saccharomyces cerevisiae Food Hydrocolloids 22(2): 239-247 37.BORCHANI, C., FONTEYN, F., JAMIN, G., PAQUOT, M., THONART, P and BLECKER, C (2016) Physical, functional and structural characterisation 53 of the cell wall fractions from baker's yeast Saccharomycescerevisiae.Food Chemistry 194: 1149-1155 Phạm Việt Cường (2005) Nghiên cứu xây dựng công nghệ sản xuất β-glucan từ thành tế bào nấm men dùng công nghiệp thực phẩm, dược phẩm mỹ phẩm, (thuộc đề tài KC 04-28) 38.KLIS, F.M., MOL, P., HELLINGWERF, K and BRUL, S (2002) Dynamics of cell wall structure inSaccharomyces cerevisiae FEMS Microbiology Reviews 26: 239-256 39.KLIS, F.M., BOORSMA, A and DE GROOT, P.W.J (2006) Cell wall construction in Saccharomycescerevisiae.Yeast 23: 185-202 Zhu F., Du B & Xu B (2016) A critical review on production and industrial applications of beta-glucans Food Hydrocolloids 52: 275-288 40.KIM, Y.H., KANG, S.W., LEE, J.H., CHANG, H.L., YUN, C.W., PAIK, H.D., KANG, C.W and KIM, S.W (2007) High density fermentation of Saccharomyces cerevisiae jul3 in fed-batch culture for the productionof βglucan Journal of Industrial and Engineering Chemistry 13: 153-158 41.Nguyễn Quốc Trung (2021) Phương pháp phá vách tế bào nấm men tách chiết beta-glucan (β-glucan) từ bã men bia sử dụng protease bền nhiệt chịu kiềm Tạp chí Khoa Học Chăn Ni (chờ in) 54 APPENDICES Some images of the experiment Separation of enzymes by decantation Experiment with acetone and ethanol 55 Tested on a laboratory scale 56