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Addis Ababa University School of Graduate Studies ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES ISOLATION AND CHARACTERIZATION OF THE DOMINANT YEAST IN THE TRADITIONAL BEVERAGES OF ETHIOPIA; TELLA AND TEJ HAIMANOT ABEBE SAFAYE A Thesis Submitted to the School of Graduate Studies of the Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Applied Microbiology in Biology Department JAN, 2011 ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES ISOLATION AND CHARACTERIZATION OF THE DOMINANT YEAST IN THE TRADITIONAL BEVERAGES OF ETHIOPIA; TELLA AND TEJ HAIMANOT ABEBE SAFAYE A Thesis Submitted to the School of Graduate Studies of the Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Applied Microbiology in Biology Department Approved by Examining Board: Dr. Fassil Assefa, Examiner ______________________________________ Dr. Tesfaye Alemu, Examiner _______________________________ Dr. Dawit Abate, Advisor _____________________________________ JAN, 2011 ACKNOWLEDGEMENTS It gives me a great pleasure to acknowledge my advisor Dr. Dawit Abate for his invaluable guidance, advice, encouragement, support, and critical comments during the course of my study. Together with this my sincere appreciation goes to Miss Zenebech and Miss Nigat, for their encouragement and cooperation in laboratory works. I also enjoyed a lot to work with all my colleagues, Roman, Gidele, Abayneh, Yonas and others. I appreciate all your support. Last but not least, I am grateful to thank my family for their financial aid and encouragement through out this study. I do love you a lot my family. I TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS………………………………………………………………… I TABLE OF CONTENTS………………………………………………………………….. II LIST OF TABLES…………………………………………………………………………. IV ABSTRACT………………………………………………………………………………… V I. INTRODUCTION………………………………………………………………………… 1 II. LITERATURE REVIEW……………………………………………………………….. 4 III. OBJECTIVES…………………………………………………………………………… 19 IV. MATERIALS AND METHODS……………………………………………………….. 20 4.1. Sampling………………………………………………………………………………….. 20 4.2. Isolation and characterization of dominant yeasts from tella, tej, bikil, gesho and honey…………………………………………………….. 20 4.2.1. Isolation of the dominant yeast of tella and tej…………………………………………. 20 4.2.2. Assessment of bikil as source of the dominant yeast of tella …………………………... 21 4.2.3. Assessment of gesho as source of the dominant yeast of tell…………………………… 21 4.2.4. Assessment of honey as source of the dominant yeast of tej……………………………..22 4.3. Isolation of active dry baker’s yeast into pure culture……………………………………..22 II 4.4. Characterization of yeasts………………………………………………………………….22 4.4.1. Microscopical appearance of yeasts…………………………………………………….. 23 4.4.1.1. Microscopical examination for vegetative cells………………………………………. 23 4.4.1.2. Microscopical examination for filamentous growth………………………………….. 23 4.4.1.3. Microscopical examination for ascospores…………………………………………… 24 4.4.2. Physiological characteristics……………………………………………………………. 25 4.4.2.1. Fermentation of carbohydrates………………………………………………………... 25 4.4.2.2. Assimilation of carbohydrates………………………………………………………… 25 V. RESULTS ………………...…..…………………………………………………………... 27 5.1. Characterization and identification of the isolated yeasts…………………………………. 27 5.1.1. Microscopical observations of the isolated yeasts………………………………………. 27 5.1.2. Physiological test of yeasts………………………………………………………………. 27 5.2. Mesophilic dominant yeast count of tella and tej………………………………………….. 29 5.3. Mesophilic dominant yeast count of bikil flour, gesho leaf powder and honey…………… 30 VI. DISCUSSION …………………………………………………………………………….. 33 VI. CONCLUSION AND RECOMMENDATIONS………………………………………... 35 REFERENCES………………………………………………………………………………… 36 III LIST OF TABLES PAGE Table 1. Physiological properties of the yeast isolates………………………………………… 28 Table 2. Counts (log cfu/ml) of yeast cells from tella samples obtained from different sources………………………………………………………. 29 Table 3. Counts (log cfu/ml) of yeast cells from tej samples obtained from different sources……………………………………………………….. 30 Table 4. Counts (log cfu/ml) of yeast cells from bikil samples obtained from different sources……………………………………………………….. 31 Table 5. Counts (log cfu/ml) of yeast cells from gesho samples obtained from different sources………………………………………………………. 32 Table 6. Counts (log cfu/ml) of yeast cells from honey samples obtained from different sources………………………………………………………. 32 IV ABSTRACT To date the source and type of the dominant yeast in tej are not known. In this study yeasts were isolated from gesho powder and honey. Both the ingredients contained the dominant yeast, S.cerevisiae, of tej at its consumption stage. However, gesho contained 102 to 103 cfu/g while honey contained 0 to 102 cfu/g. Thus, gesho was considered the major source of the dominant yeast in tej. The source and type of the dominant yeast in tella are not known also. Yeasts were isolated from gesho and bikil powder. S.cerevisiae, the dominant yeast at consumption stage of tella was found in both the ingredients. Bikil contained 103 to 105 cfu/g yet gesho contained 102 to 103 cfu/g. Thus, bikil was considered the major source of the dominant yeast in tella. In this study, the source and type of the dominant yeast in the beverages at consumption stage was identified. The findings of this study help to define and standardize the fermentation of the traditional beverages of Ethiopia, tella and tej. Key words: tella, tej, bikil, gesho, honey V I. INTRODUCTION In nearly all areas of the world, some type of alcoholic beverage native to its region is prepared and consumed. In Africa, fermented alcoholic beverages are consumed in different occasions such as marriage, naming and rain making ceremonies (Zvauya et al., 1997), at festivals and social gatherings, at burial ceremonies and settling disputes (Steinkraus, 1983). They are also used as medicines for fever and other ailments by adding barks or stems of certain plants (Okafor, 1972). Fermented beverages produced from cereals usually referred to as beers while those produced from fruits are classified as wines (Pederson, 1979). Indigenous fermented alcoholic beverages from different parts of the world are described (Steinkraus, 1983). Among these, information on the microbiology and biochemical properties of varieties of the indigenous African fermented alcoholic beverages is available. These include Egyptian bouza, Tanzanian wanzuki, gongo, tembo-mnazi and gara, Nigerian palm-wine, Kenyan muratna and uragela, and South African kaffir beer (Bahiru et al., 2001). Indigenous Ethiopian fermented beverages include tej (Vogel and Gobezie 1983; Fite et al., 1991; Bahiru, 2000; Bahiru et al., 2001), tella (Sahle and Gashe, 1991; Negussie, 2008), borde and shamita (Ashenafi and Mehari, 1995; Bacha et al., 1998), areki, traditional liquor (Desta, 1977; Fite et al., 1991). Alcohol in traditional beverages serves as source of calories valuable to the calorie-deficient villager. Ethanol has gross calorific values of 30 MJ/Kg (James and Lord, 1992). The primitive beverages provide not only calories but also B vitamins and proteins due to residues of the substrates, the fermenting yeasts and other microorganisms (Steinkraus, 1983; Bahiru, 2000). Riboflavin and nicotinic acid are found in kaffir beer due to synthesis of vitamins during malting and fermentation (Platt and Webb, 1964). Mexican pulque is 1 consumed as a nutritional supplement because of its vitamin content and protein. Consumption pulque three times a day by under-school-age children provides 2.2 to 12.4% of their calories (Sanchez-Marroquin et al., 1977; Herera et al., 1977). The yeasts synthesize B vitamins in Kenyan uragua and the alcohol provides calories (Harkishor, 1977). Traditional recipes are handed down through generation and are still used for food processing in many developing countries (Kebede et al., 2002). The traditionally fermented beverages are low-cost product in all aspect as they are usually manufactured using only rudimentary equipment. Because of their cheapness, low-income groups mostly consume them. Thus their handling and consumption often takes place under conditions of poor hygiene (Steinkraus, 1983). In Ethiopia villagers prepare a wide range of traditional fermented foods and beverages from different raw materials such as cereals, ensete (false banana), honey, milk, etc. Some of the known Ethiopian traditional fermented foods are injera, ambushes, kocho, bulla, ergo, and siljo. These products, if properly exploited, could be of significant economic importance for the country (Kebede et al., 2002). Most of the customs and rituals involving the Ethiopian traditional fermented foods and beverages are still prevailing today in urban areas, village communities and rural households. Fermentation of tej and tella like other traditionally fermented alcoholic beverages relies on the microorganisms present in the substrates, fermentation vats, or equipments. So, with the variable microflora of such spontaneous fermentation variability of the product is imminent. In this study, the source of dominant yeast in tej and tella at the time the beverages were ready for consumption and characterization of the dominant yeast were studied. The findings of this study may indicate the need to isolate and characterize the 2 dominant yeast for subsequent modernization or industrialization, and serve as a basis for further studies. 3 II. LITERATURE REVIEW 2.1. Ethiopian traditional alcoholic beverages Ethiopia is one of the countries where a wide variety of traditional
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