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THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI HOA TITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN BACHELOR THESIS Study Mode : Full – time Major : Food Technology Faculty : Advance Education Program Batch : 2016 - 2020 Thai Nguyen, November 11th 2020 download by : skknchat@gmail.com ii THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI HOA TITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN BACHELOR THESIS Study Mode : Full – time Major : Food Technology Faculty : Advance Education Program Batch : 2016 - 2020 Thai Nguyen, November 11th 2020 download by : skknchat@gmail.com iii DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University Of Agriculture And Forestry Degree program Bachelor of Food Technology Student name Nguyen Thi Hoa Student ID DTN1653170035 Thesis title Isolation of some yeast strains with high proliferation ability from natural sources in Thai Nguyen Supervisor Msc Vi Dai Lam Supervisor’s signature Abstract : The reasearch purpose to isolate the yeast strains from fruit that can be used in food production A total 10 samples including locally availible source such as bananas, organes, sugarcane juice, grapes, guava are collected from other area of the Thai Nguyen province After culture proliferates, 12 yeast strains were isolated using selective medium Yeast Extract Peptone Dextrose Agar From the differential tests which included morphological and microscopic observation and physiological and biochemical characterization such as tolerant ethanol, strains were selected as possible Saccharomyces strain The result sequencing show S1( from surgarcane juice) strain is download by : skknchat@gmail.com iv Clavispora lusitaniae Keywords: Isolation, Yeast, fruits Number of page: 36 Date of submission: download by : skknchat@gmail.com v ACKNOWLEGEMENT First of all, I am grateful to the Thai Nguyen University of Agriculture and Forestry, the Advance Education Program, the Faculty of Biotechnology and Food Technology, who have taught and imparted necessary knowledge and valuable experience to me during the time I studied and researched at Thai Nguyen University of Agriculture and Forestry I am extremely thankfull to my supervisor Mr.Vi Dai Lam, who supported me for the completion of this thesis I also acknowledge with a deep sense of reverence with all members of my family and friends who are always to help and encourage me on the completion of this thesis However, due to the limited knowledge and the lack of practical experience, the content of the thesis cannot avoid short comings, I would like to receive further suggestions and advice to make my this thesis is more complete Best regards Thai Nguyen,November 11th ,2020 Nguyen Thi Hoa download by : skknchat@gmail.com TABLE OF CONTENT List of Figures………………………………………………………………….vi List of Tables ……………………………………………………………… vii List of Abbreviations…………………………………………………………viii PART I INTRODUCTION…………………………………………………….1 1.1 Research rationale…………………………………………………… 1.2 Research’s objectives………………………………………………… 1.3 Meaning of the reseach ………………………….…………………….2 1.3.1 The scientific meaning of the topic………………………………… 1.3.2 The practical meaning of the topic…………………………………….2 PART II LITERATURE REVIEW…………………………………………….4 2.1 Yeast …………………… …………………………………………….4 2.1.1 Yeast biology properties………………………………………………4 2.1.2 Metabolism in Yeast………………………………………………… 2.1.3 Yeast habitat………………………………………………………… 2.1.4 Application of yeast……………………………………………………8 2.2 Overview of research in the field of the topic………………………….14 2.2.1 In the world………………………………………………………… 14 download by : skknchat@gmail.com ii 2.2.2 In the country…………………………………………………………15 PART III METHODS ……………………………………………………… 17 3.1 Objective of study……………………………………………………17 3.1.1 Objective of study…….……………………………………………….17 3.1.2 Chemicals, Instruments And Machines……………………………….17 3.2 Methods … …………………………………………………………19 3.2.1 Isolation of yeast from samples……………………………………….19 3.2.2 Identification of yeast………………………………………………….24 PART IV RESULTS………………………………………………………….26 4.1 Isolation of yeast from samples…………………………………… 26 4.2 Identifiacation of yeast ………………………………………… 28 PART V CONCLUSION AND PEPTITION……………………………… 31 5.1 Conclusion……………………………………… ………………….31 5.2 Peptition …………………………………………………………… 31 REFERENCES ……………………………………………………………… 32 APENDENCES download by : skknchat@gmail.com iii LIST OF FIGURES Figure 1: Yeast Saccharomyces cerevisiae Figure 2: Ten-fold serial dilution Figure 3: Colonies of yeast isolated from fruits Figure 4: Sequencing result Figure 5: Sequencing result of S1 strain download by : skknchat@gmail.com iv LIST OF TABLES Table 1: Nutural yeast habitats Table 2: Morphological characteristic of B1, S1 and O1 strains Table 3: Growth ability of isolated strains Table 4: Ethanol tolerance test download by : skknchat@gmail.com v LIST OF ABBREVIATION Yeast Extract Peptone Agar( YEPD) Saccharomyces cerevisiae ( S cerevisiae) Carbon dioxide ( CO2) Energy( ATP) Deoxyribonucleic acid( DNA) Polypropylene( PP) Low density polyethylene( LDPE) Biaxially Oriented Polypropylene (BOPP) Gram( g) 10 Miligram(mg) 11 Mililit(ml) download by : skknchat@gmail.com 26 PART 4: RESULT AND DISCUSSION Isolation of yeasts colonies from samples In this study, 12 microbial isolates were picked up from different sources on YEPD solid media Only five ( B1 from banana, G1 and G2 from grape, O1 from orange, and S1 from sugarcane juice) were selected A B C D Figure 3: Colonies of yeasts isolated from fruits A- Spread method: Colonies of S1 strain; B- Streak method: Colonies of B1 strain; C- Streak method: Colonies of O1 strain; D- Cell morphology of yeast strain download by : skknchat@gmail.com 27 The isolate CM was obtained from commercial yeast sample from the local market to be used as a positive control These strains can grow on the selection medium YEPD with antibiotic chloramphenicol (10 mg/l) (Figure 3) The three strains B1, O1 and S1 isolated from banana, orange and sugarcane juice create visible colonies after 15 hours of incubation Five isolates were purified by streak method, then morphologically examined further on solid medium and microscope ( Figure 3) Most of the isolated colonies exhibited smooth surfaces, and color of colonies showed cream to white (Figure 3A, 3B and 3C) 3.2 Identification of yeast strains 3.2.1 Observation of cell morphology At 40X magnification, the strains, which showed morphology similar to commercial yeast CM, were oval in shape, and showed high budding rate, were selected (Figure 3D) (Table 2) These properties are suitable with the description of Tika (2017) about yeasts strains[18] Table 2:Morphological characteristic of B1, S1 and O1 strains Source Yeast Surface Margin Color Cells strains Banana B1 Smooth Circular White Ellipsoid Sugarcane S1 Smooth Circular Creamy Oval juice white download by : skknchat@gmail.com 28 Orange O1 Commercial CM Smooth Circular White Oval Smooth Circular White Ellipsoid yeast Depending on the color and the shape of colonies, the shape of the cells, the budding reproduction and the tolerance ability to antibiotic in medium, the strains B1, S1 and O1 were selected for next experiments 3.2.2 Determination of growth ability The cells of isolated strains were cultivated in 100ml liquid YEPD medium at room temperature, 180 rpm in 24 hours The density cells will be identified by hemocytometer The number of cells in 1ml is shown in table Table 3: Growth ability of isolated strains Strains 15h 24h CM 2,14×104 2,28 × 108 B1 2,1 × 104 2,52 × 108 S1 1,36×104 3,36 × 108 O1 2,1 × 104 4,68 × 108 The result indicated that isolated strains have high ability in proliferation The strains O1, S1, B1 reach more than 100.000.000 cells in ml after 24 h, faster than the commercial strain These strains may become candidates for biomass production processes in the future download by : skknchat@gmail.com 29 3.2.2 Ethanol tolerance test Many yeast cells in food production can secrete ethanol as secondary metabolites This ethanol can inhibit the development of other microorganisms When the concentration of ethanol keep increasing, yeast cells can be affected Ethanol can destroy mitochondrial DNA in yeast cells and cause inactive of some enzyme, like hexokinases and dehydrogenase [31] According to experiment of Tika in 2017, yeast strains isolated from fruits can resist ethanol at concentration 14% In this study, yeast strains were cultured at alcohol concentration 14% in liquid media The result are shown in Table At 14 % alcohol concentration, the strains B1 showed the tolerance to alcohol that was equivalent to CM while the strains S1 and O1 that was isolated from sugarcane juice and orange reach the cell concentration as 3,36 × 108 and 4,68 × 108, higher than commercial yeast with 2,28 × 108 cell in ml These indicated that these microorganisms possess the characteristics which characterize for yeasts strains which can be used for food production Strain S1 and O1 were analyzed by sequencing method for species identification Table 4: Ethanol tolerance test Ethanol (%) Strains Number of cell/ml 14 CM 1,28 × 108 14 B1 1,52 × 108 14 S1 2,36 × 108 download by : skknchat@gmail.com 30 14 O1 2,68 × 108 3.2.2 Identification by molecular method The gene sequencing of strain S1 and O1 was conducted by Phu Sa company ( Can Tho province, Viet Nam) The primers are NL1 and NL4 Figure 4: Sequencing result The result showed that both strain S1 and O1 are yeasts strains, positive with the specific primers in Polymerase Chain Reaction The S1 strain is Clavispora lusitaniae (Figure 3) This yeast strain can produce ethanol, amylase and can be use as model for antibiotic sensitive tests Figure 5: Sequencing result of S1 strain download by : skknchat@gmail.com 31 The strain O1 is positive in PCR reaction, but because of equipment error, the sequencing process need to be repeated in the future download by : skknchat@gmail.com 32 PART V: CONCLUSION AND PETITION 5.1 Conclusion The research isolated yeast strain S1, scientific name as Clavispora lusitaniae, which is resistant with ethanol 14%, grow on selective medium with antibiotic Chloramphenicol 10 mg/l, reach 2,36 × 108 cell/ml after 24h at room temperature 5.2 Petition - Continue to evaluate the physiological and biochemical characteristics of the yeast strains - Continue isolate yeast strains on different natural sources - Sequencing to identify strain O1 download by : skknchat@gmail.com 33 REFERENCES I Vietnamese references Tran Van Chi, Pham Thi Tuyet Mai, Nguyen Xuan Ninh, Dinh Thi Kim Hoa, Nguyen Thi Huong,2015, Reseach on fermentation of yeasts and lactic acid bacteria from maize stalks, rice and grass for fodder Science and Technology magazine, Thai Nguyen University, pp111-114 Luong Duc Pham,2005, Industrial yeast, Publishing scientific and technical, Hanoi Nguyen Thi Niem, Huynh Ngoc Thanh Tam, Nguyen Duc Do,2018,Isolation, selection of yeast strain (Saccharomyces sp.) for Canarium album wine fermentation, science magazine,Can Tho university II English references Akbar Ali, Aamir Shehzad, Moazzam Rafiq Khan, Muhammad Asim Shabbir, Muhammad Rizwan Amjid,2012, Yeast, its types and role in fermentation during bread making process-A Review ,p 171-179 Arias C R., Burns J K., Friedrich L M., Goodrich R M., Parish M E Yeast species associated with orange juice: evaluation of different identification methods Applied and Environmental Microbiology 2002;68(4):1955–1961 Boulton,C & Quain, 2001 Brewing yeast & fermentation Briggs,.D.E.Boulton,C.A.Brooks,P.A & Stevens,R(2004) download by : skknchat@gmail.com Brewing: 34 science and practice Ceccato-Antonini S R., Tosta C D., Da Silva A C Determination of yeast killer activity in fermenting sugarcane juice using selected ethanolmaking strains Brazilian Archives of Biology and Technology 2004;47(1):13–23 Fleury Rey Y., Bel-Rhlid R., Juillerat M.-A.(2002) Biogeneration of 2-(1hydroxyethyl)-4,5-dihydrothiazole as precursor of roasted and popcornlike aroma for bakery products ;pp19-20:pp473–477 10 Jeffery Hamelan,2013, Bread A baker’s book of techniques and recipes 2nd edition 11 Phaí H J., Miller M W., Mark E M.(1978) The Life of Yeast 2nd Industrial uses of yeast; pp 238–266 12 Psaltakis, G 2002 “Old Testament.” Volume B 'Exit-Lefkion Ed SOTIR Crop: Exit Book: 16: 12-5, ISBN 978-960-9575-42-3 13 Spencer J F., Spencer D M.(1997) Yeasts in Natural and Artificial Habitats P 381 14 Stewart& Russell,1998, An introduction to brewing science &technology series III Journal of the Institute of b rewing,1-6 15 Struyf N., Van der Maelen E., Hemdane S., Verspreet J., Verstrepen K., Courtin C.(2017) Bread Dough and Baker’s Yeast;16:850–867 16 T.Deák,2003, Encyclopedia of Food Sciences and Nutrition (Second download by : skknchat@gmail.com 35 Edition), Pages 6233-6239 17 Theophilus Abonyi Mensah1and Sheila Matilda Ayorkor Tagoe (2019) Measurement of yeast growth using spectrophotometer 18 Tika B Karki, ParashMani Timilsina, Archana Yadav, Gyanu Raj Pandey, Yogesh Joshi, Sahansila Bhujel, Rojina Adhikari, and Katyayanee Neupane1(2017) Selection and Characterization of Potential Baker’s Yeast from Indigenous Resources of Nepal, Biotechnology Research Internationa, Volume 2017, Article ID 1925820 19 W.Y.Mo,W.M.Choi ,K.Y.Man ,M.H.Wong,2019, Food waste-based pellets for feeding grass carp (Ctenopharyngodon idellus): Adding baker’s yeast and enzymes to enhance growth and immunity 20 Walker,G.M,2009 Yeast In: M Schaechter(ed_ Desk encyclopedia of microbiology.(pp.1174-1187)2nd ed 21 White,C & Zainasheff, 2010.Yeast: the partical guide to beer fermentation.Boulder CO: Brewers Publication 22 Yates, G T., & Smotzer, T (2007) On the lag phase and initial decline of microbial growth curves Journal of Theoretical Biology, 244(3), 511-517 23 Z Tsegay,2016 Isolation, Identification and Characterization of ethanol Tolerant Yeast Species From Fruits Production of Bio- ethanol 24 Chiranjeevi Tikka, Hari Prasad Osuru, Navya Atluri,Praveen Chakravarthi Veera Raghavulu, Nanda Kumar yellapu, Ismail Shaik Mannur, Uppu download by : skknchat@gmail.com 36 Venkateswara Prasad, Sudheer Aluru, Narasimha Varma K, and Matcha Bhaskar,2013, Isolation and characterization of ethanol tolerant yeast strains 25 Best Bread Production Hanbook,2018 26 J I Ibeas , J Jimenez(1997) Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts Appl Environ Microbiol 63(1):pp 7-12 27 Burrows, S 1970 Baker’s yeast In The Yeasts In Rose, edited by A.H & Harrison J.S p, 349-420 London: Academic Press, Inc 28 UC Davis - College of Biological Science - Department of Microbiology & Molecular Genetics, Cell Counting with a Hemocytometer 29 https://microbenotes.com/serial-dilution/ 30 Sadat Khattab, Ahmed Abdel-Hadi, Nageh Fathy Abo-Dahab, Omar Atta (2016) Isolation, Characterization, and Identification of Yeasts Associated with Foods, British Microbiology Research Journal, 13 31 Ewa Żymańczyk-Duda, Małgorzata Brzezińska-Rodak, Magdalena Klimek-Ochab, Maciej Duda and Agata Zerka, 2016,Yeast as a Versatile Tool in Biotechnology 32 Kwak S., Jo J.H., Yun E.J., Jin Y.S., Seo J.H.(2019) Production of biofuels and chemicals from xylose using native and engineered yeast strains pp37:271–283 download by : skknchat@gmail.com 37 APPENDICES Result of S1( Sugarcane juice) sequencing download by : skknchat@gmail.com 38 download by : skknchat@gmail.com 39 download by : skknchat@gmail.com 40 The number of cells in mililiter is calculated as the following fomula: A x 104 x F = Cell number /ml A: Cell number in mm2 square 104: The volumn of 1 mm2 square is 0.1 µl (10-4 ml) x F: dilution factor Media( YEPD) Chemical Yeast Extract Peptone Yeast Extract Peptone Dextrose Agar(YEPD Dextrose Broth( YEPD Agar) Broth) Yeast Extract 3g 3g Peptone 5g 5g Glucose 20g 20g Agar 20g _ Distilled water 1000ml 1000ml Antibiotic 10mg _ (Chloramphenicol) download by : skknchat@gmail.com ...ii THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI HOA TITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN. .. Technology Student name Nguyen Thi Hoa Student ID DTN1653170035 Thesis title Isolation of some yeast strains with high proliferation ability from natural sources in Thai Nguyen Supervisor Msc Vi... Laboratory of Faculty of Biotechnology & Food Technology, Thai Nguyen University Of Agriculture And Forestry 3.2 Reaseach content Isolation of some yeast strains with hight proliferating ability from natural
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