Faculty of Resource Science and Technology
ANALYSES OF MICROBIAL GROWTH
ON LOCALLY PRODUCED RAGI FOR TAPE
FERMENTATION
Noren anak Wilson Brandi
Bachelor of Science with Honours
(Resource Biotechnology)
2008
ANALYSES OF MICROBIAL GROWTH ON LOCALLY PRODUCED RAGI FOR TAPE FERMENTATION
NOREN ANAK WILSON BRANDI
This project is submitted
in partial fulfillment of the requirements for the Degree of
Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARAWAK
2008
ACKNOWLEDGEMENTS
I would like to thank my supervisor, Prof. Dr. Kopli Bujang for giving me the opportunity to work on this project under his careful guidance. He is responsible for helping me to complete the writing of this paper. He also gives me valuable suggestion and encouragements. My appreciation also for Dr. Sepiah Muid for her advice and opinion that she gave that help me to identify the microbes. Thanks for the help from laboratory assistants by providing guideline in the preparation of chemical. I also appreciate my course mates especially those in the same laboratory with me. Last but not least, I thank to my family for giving support and comfort.
TABLE OF CONTENT
TITLE PAGES
ACKNOWLEDGEMENTS i
TABLE OF CONTENT ii
ABSTRACT v
LIST OF ABBREVIATION vi
LIST OF FIGURES vii
LIST OF TABLES x
CHAPTER I INTRODUCTION 1
CHAPTER II LITERATURE REVIEW 5
2.1 Tapai Fermentation 5
2.2 Fermentation Starters: Ragi 8
2.3 Microbial Growth 11
2.3.1 Fungi 12
2.4 Importance of Photographic and Visual Analysis 14 CHAPTER III MATERIALS AND METHODS 15
3.1 Ragi Samples 16
3.2 Preparation of Tapai from Cooked Rice 16
3.3 Preparation of Cultures for Analysis 16
3.3.1 Microbial Growth on Ragi 16
3.3.2 Microbial Growth on Tapai 17
3.4 Identification of Microbial Isolates 17
3.5 Analysis of Ethanol and Glucose in Tapai 18
CHAPTER IV RESULTS AND DISCUSSIONS 19
4.1 Identification of Microorganism 19
4.1.1 Surface of Ragi Samples 19
4.1.2 Growth on the Petri Dish 21
4.1.2.1 Ragi 21
4.1.2.2 Tapai 21
4.1.3 Microscopic Analysis 25
4.1.3.1 Ragi 25
4.1.3.2 Tapai 28
4.2 Ethanol and Glucose Production in Tapai 32 4.2.1 Glucose Production in Tapai 32
4.2.2 Ethanol Production in Tapai 34
CHAPTER V CONCLUSION AND RECOMMENDATIONS 36
LITERATURE CITATIONS 38
PHOTOS OF INTERVIEWS 43
APPENDIX 44
A: Tabulated Data
B: Preparation of NA (DIFCO)
C: Preparation of NB (MERCK)
D: Preparation of Methylene Blue (Hamburg)
Analyses of Microbial Growth on Locally Produced Ragi for Tape Fermentation
Noren anak Wilson Brandi
Resource Biotechnology, Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
In this study, ragi samples obtained from different local suppliers were analysed for different microbial growth on the solid surface. For the preparation of tapai, cooked rice was inoculated with the powdered ragi, incubated at 27oC and then isolates for the microbes exist every 12 hours. Identification of microbes was performed using visualizing of staining specimen with methylene blue under phase contrast microscope. The results obtained showed three fungal isolates (two moulds and one yeast) and one filamentous bacterium. The mould isolates were identified as Mucor sp. and Rhizopus sp. while the yeast isolates was identified as Saccharomyces sp.. The bacterium was identified as Streptomyces sp. that may be due to the contaminants. Within 24 hours, the production of glucose was more rapid in Maranek (121.2 g/L) with 65.68% increased compared to Tambirat (25.2 g/L) tapai with 48.41% increased while the concentration of ethanol was higher almost two times in Maranek (46.8 g/L) compared to Tambirat (24.2 g/L) tapai with 92.31% and 93.39% of increased in ethanol production respectively.
Key words: ragi, tapai, phase contrast microscope, moulds, yeast.
ABSTRAK
Dalam kajian ini, sampel ragi yang diperoleh daripada pembekal tempatan yang berbeza dianalis kehadiran pelbagai jenis mikrob di atas permukaan pepejalnya. Untuk persediaan tapai, nasi yang telah dimasak digaul bersama serbuk ragi, dieram pada suhu 27oC dan diasingkan mikrobnya setiap 12 jam. Pengenalpastian mikrob dilakukan melalui pemerhatian spesimen yang telah diwarnakan menggunakan metilin biru di bawah mikroskop fasa kontras. Keputusan mendapati sejumlah tiga fungal dapat diasingkan (dua dari kulat dan satu dari yis) dan satu ialah bakteria berfilamen. Kulat yang diasingkan dikenalpasti sebagai Mucor sp. dan Rhizopus sp. Manakala yis yang diasingkan pula dikenalpasti sebagai Saccharomyces sp.. Bakteria yang wujud dikenalpasti sebagai Streptomyces sp. akibat daripada dicemari. Dalam 24 jam, penghasilan glukosa berlaku dengan lebih cepat dalam tapai Maranek (121.2 g/L) dengan kenaikan sebanyak 65.68% berbanding tapai Tambirat (25.2 g/L) dengan kenaikan sebanyak 48.41% manakala kepekatan etanol dalam tapai Maranek (46.8 g/L) adalah lebih tinggi hampir dua kali ganda berbanding Tambirat (24.2 g/L) dengan kenaikan penghasilan etanol sebanyak 92.31% and 93.39% masing-masing.
Kata kunci: ragi, tapai, mikroskop fasa kontras, kulat, yis. LIST OF ABBREVIATION
g gram g/L gram per litre hr hour min minute mL milliliter mm millimeter
NA nutrient agar
NB nutrient broth rpm revolution per min oC degree celcius
µm micrometer
LIST OF FIGURES
Page
Figure 1 Tapai in various wrapping. Tapai wrapped with banana leaves (left) 6
and tapai in nipah leaves (right).
Figure 2 Flow sheet: Manufacture of traditional tapai from rice. 7
Figure 3 Locally produced ragi. 8
Figure 4 Machine for mixing the mixture to form dough before flatten into 9
Figure 5 ‘Cekor’ (left) is added in preparation of ragi and moisten with boiled 10
stock of ‘kayu manis’ (right).
Figure 6 Spices (‘das manis’ and ‘das pedas’) added in the preparation of 10
ragi.
Figure 7 Flow sheet: Ragi preparation of local manufacturers (Based on the 11
interview session with ragi manufacturers from Kampung Maranek
and Kampung Tambirat, August 2007).
Figure 8 Identification of fungus in specimens. (A) Aspergillus sp., (B) 13
Penicillium sp., (C) Geotrichum sp., (D) Tricophyton sp., (E)
Microsporum sp., (F) Epidermophyton sp., and (G) Rhizophus sp.
Figure 9 Overview of materials and methods. 15
Figure 10 Photos show the top (left) and bottom (right) of the Maranek ragi at 19
0 hour.
Figure 11 Photos show the top (left) and bottom (right) of the Tambirat ragi at 20
0 hour.
Figure 12 The surface of Maranek (left) and Tambirat (right) ragi after 2 weeks 20
at room temperature (27oC).
Figure 13 The surface view and reverse side of tapai from Maranek ragi for 0, 23
1, 2 and 3 days.
Figure 14 The surface view and reverse side of tapai from Tambirat ragi for 0, 24
1, 2 and 3 days.
Figure 15 Photomicrograph of microbe on the surface of Tambirat ragi 25
identified as Saccharomyces sp. at 600x magnification.
Figure 16 Photomicrograph of microbe on the surface of Tambirat ragi 26
identified as Mucor sp. at 200x magnification.
Figure 17 Photomicrograph of microbe on the surface of Maranek ragi 26
identified as Rhizophus sp. with their rhizoids is produced at 100x
magnification.
Figure 18 Photomicrograph of Rhizophus sp. on Maranek ragi with their 27
stolons grows along the surface of the NA medium at 40x
magnification.
Figure 19 Formation of ascospores on Maranek ragi at 100x magnification. 27
This characteristic (ascospores with 1-4 per ascus) falls within of
Saccharomyces cerevisiae (Larone, 1995).
Figure 20 Photomicrograph of microbe exist on Maranek tapai identified as 28
Saccharomyces sp. at 200x magnification.
Figure 21 Hyphae of Mucor sp. growing on NA exist on Maranek tapai at 100x 29
magnification.
Figure 22 Photomicrograph of microbe exist on Tambirat tapai identified as 29
Mucor sp. at 40x magnification.
Figure 23 The sporangium of Mucor sp. on Tambirat tapai at 200x 30
magnification.
Figure 24 The sporangium of Mucor sp. on Tambirat tapai at 400x 30
magnification.
Figure 25 Photomicrograph of microbe exist on Tambirat tapai identified as 31
Streptomyces sp. at 200x magnification.
Figure 26 Production of glucose in Maranek and Tambirat tapai within 24 33
hours of fermentation.
Figure 27 Production of ethanol in Maranek and Tambirat tapai within 24 34
hours of fermentation.
LIST OF TABLES
Page
Table 1 The changes of tapai within three days of fermentation at 27oC. 21
Table 2 Summary of the characteristic of microorganism on tapai within 24 22
hours of incubation at 27oC.
Table 3 The concentration (g/L) of glucose and ethanol during tapai 44
fermentation utilizing Maranek ragi at 27oC.
Table 4 The concentration (g/L) of glucose and ethanol during tapai 44
fermentation utilizing Tambirat ragi at 27oC.
ANALYSES OF MICROBIAL GROWTH ON LOCALLY PRODUCED RAGI FOR TAPE FERMENTATION
CHAPTER I
INTRODUCTION
Fermentation is one of the oldest and most economical means of producing and preserving foods. It is believed that fermentation of foods was originated in China for preservation of cereals and legumes (Vijayalakshmi et al., 1997). The practice has been extended to many other foods including vegetables, fruits, edible parts of plants, fish, milk and meats.
According to Vijayalakshmi et al. (1997), fermented foods are prepared by microbial action on one or more components under relatively controlled conditions. It causes changes in their physical, biochemical and nutritional qualities.
Fermented food is very popular among the people in Asia-Pacific region. Since this region is characterized by its tropical and subtropical climate, it is very important to preserve the harvest crops. The inhabitants consumption varies according to geographic and climatic condition. Primarily, the inhabitants of the tropical Southeastern regions consume rice.
Those in subtropical and temperate zones of the Northeastern region including Northern
China, Korea and Japan consume wheat, buckwheat, barley, corn, millet and soybeans in addition to rice. The consumption of rice as a staple food has resulted in at typical food processing technology.
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Food technology becomes more important discipline since the rapid increase in the world population. Fermentation of foods is a way of solution to provide the necessary quantities of foodstuffs instead of preservation of existing foods and maintenance or improvement of their quality. Fermented foods have numerous advantages over the raw materials from which they are made, not only in improved flavour and texture, appearance and aroma, but also increase in storage life of foods. The cost of preservation also rather cheap compare to the preservation by canning and freezing. Their dietary fiber content is relatively high and very desirable foods for infants, expectant and nursing mothers and invalids (Vijayalakshmi et al.,
1997). The increased content of vitamins, proteins and energy in fermented foods also makes them valuable in preventing malnutrition.
The fermented food products can be divided into two categories; products that result from bacterial fermentation and products from fungal fermentation (Lee and Fujio, 1999).
Products from bacterial fermentation include Japanese natto and Philippine nata while products resulting from fungal fermentation majority are tapai or tapai-like products. The fermented foods are produced by the local people in small scale. Formerly the preparation is passed from one generation to another generation, very often with improvement and addition of ingredients. But nowadays, fermented foods also being produced with improved equipment and using bioprocessing innovations for quality control (Kuswanto, 2008).
Tapai is a type of fermented rice, has sweet and sour alcoholic taste. It is made from various substrates such as cassava tubers, cooked rice or cooked glutinous rice (Gandjar, 2003) and mix with powdered ragi. Ragi acts as the fermentation starter and used in other countries but
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under different names. In the China ragi is called chu, in Korea nuruk, in Japan koji, in
Indonesia and Malaysia ragi, in Philippine bubod, in Thailand loopang and in India marchaa.
The term ragi will be used in this project. Ragi is made of rice flour and other ingredients
(mostly spices) with the presence of mould and yeast through natural infections from the surroundings and from the equipment used by the manufacturer (Djien, 1972). The ingredient of ragi may vary according to the countries.
During fermentation of foods, complex materials such as carbohydrates, proteins and fats are metabolized to yield others such as ethanol, organic acids and pectic hydrolysates
(Vijayalakshmi et al., 1997). The flavour of the foods may be formed through condensation of fatty acids and alcohols into esters and oxidation of fatty acids to carbonyl compounds
(Rose, 1982; Reed, 1983; Wood and Hodge, 1985, cited from Spencer and Spencer, 1997).
According to Cronk et al. (1977), Went and Geerligs (1895) were the first to isolate and identify the microorganisms that thought to be essential for the fermentation. From their discovery, at least one amylolytic filamentous fungus and one or more alcohol-producing yeast were present during the tapai fermentation. In tapai fermentation, the substrate becomes soft and some acid are formed as shown by the soft alcoholic smell produced. The fermentation requires microorganisms in order to convert the starch into glucose. Since there are spices added during the ragi preparation, it may contribute to the growth of other microorganisms or may inhibit development of undesirable microorganisms (Sie, 1962;
Soedarsono, 1972; cited from Steinkraus, 1983).
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Therefore the objective of this study is to analyse visually the different microbes exist on ragi and tapai hence giving rise to different degree of sweetness and flavours during tapai fermentation.
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CHAPTER II
LITERATURE REVIEW
2.1 Tapai Fermentation
Tapai is a traditional fermented food. It is partially liquefied (Cronk et al., 1977), with a sweet-acid taste and mild alcoholic flavour (Djien, 1972). It is a popular delicacy especially during the traditional ceremonies or just consumed as snack item without further processing after the fermentation (Cronk et al., 1977). It is prepared among the traditional manufacturers using cassava, rice or glutinous rice as the substrates. The preparations were transferred from generation to generation. According to Gandjar (2003), a carbohydrate source and an inoculum containing the microorganism is necessary to prepare tapai. The inoculum is called ragi and the success of a good tapai depends on the preparation of substrate and ragi itself.
In Sarawak, tapai from cooked rice is popular among the local. The preparation of tapai may vary from one place to another place but usually common ingredients such as powdered ragi are added to the substrate. In the traditional method, the fermentation of tapai initiated by addition of powdered ragi while in modern method, the starter used consist of a single organism or a combination of organism produced by microbiological method (Steinkraus,
1983). According to local manufacturer from Kampung Maranek and Kampung Tambirat, the amount of ragi added is about four to five pieces per one kilogram of rice. Local
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manufacturers usually used traditional method in producing tapai hence the quantity also in small scale. Powdered ragi is mixed with cool cooked rice before the mixture is wrapped and allowed to ferment at room temperature (25-30oC) for one day and after that it can be consumed. For the purpose of wrapping, various types of wrapping (Figure 1) such as banana leaves, nipah leaves, cocoa leave and ‘buan’ leaves were used (Based on interview session with the manufacturers from Kampung Maranek and Kampung Tambirat, 2007). A flow sheet of the process is given in Figure 2.
Figure 1. Tapai in various wrapping. Tapai wrapped with banana leaves (left) and tapai in nipah leaves (right).
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Rice
Wash
Cooked
Cool to room temperature
Mix with powdered ragi
Wrapped with banana or other plant leaves
Incubate for 12 hours
Fermented rice (Tapai)
Figure 2. Flow sheet: Manufacture of traditional tapai from rice.
Tapai is a very perishable product since the fermentation continues even after the optimum stage of fermentation has been reached. Therefore it must be consumed immediately, but if it is chilled, it may be kept for about two weeks (Kuswanto, 2008). If tapai is allowed to ferment longer, it will result in greater liquefaction of the rice and is consumed as an alcoholic beverage, upon filtration.
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2.2 Fermentation Starters: Ragi
Ragi is a dry circular cakes prepared locally from rice flour with distinctive spices (Cronk et al., 1977). It has many varieties of shapes and sizes; some spherical balls or round flattened cakes (Figure 3). Ragi is used as one of the materials to make tapai. Ragi is produced using traditional method by household or village manufacturer using closely recipes and available in local market.
Figure 3. Locally produced ragi.
The production of ragi can be seen in the area of Asajaya and Kota Samarahan, Sarawak.
The manufacturers still using traditional ingredients with a little adjustment in mixing the ingredients. Equipment such as a machine (Figure 4) was used to mix the ingredients and formed the dough.
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Figure 4. Machine for mixing the mixture to form dough before flatten into cakes.
The production of ragi may vary from one place based on the ingredients that they used.
Usually basic ingredient such as ginger, onion, garlic, white pepper, ‘lengkuas’, ‘cekor’
(Figure 5), ‘akar kayu manis’ (Figure 5) and ‘kulit kayu manis’ were used. Some addition spices such as ‘das manis’ and ‘das pedas’ (Figure 6) were used by the manufacturer from
Kampung Tambirat. The starters are prepared under non-sterile conditions by the manufacturers (Based on the interview session with the manufacturers from Kampung
Maranek and Kampung Tambirat).
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Figure 5. ‘Cekor’ (left) is added in Figure 6. Spices (‘das manis’ and ‘das preparation of ragi and moisten with boiled pedas’) added in the preparation of ragi. stock of ‘kayu manis’ (right).
Ragi is prepared from rice flour that is mix with grinded spices as above. The mixture is moisten with the boil water that already been cold taken from ‘akar kayu manis’ and ‘kulit kayu manis’. It is then mix to form a dough. The flattened cakes are incubated for three to four days at room temperature followed by drying under the sun to preserve the cakes. A flow sheet of the process is given in Figure 7.
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Rice flour
Add grinded spices
Moisten with boil stock of ‘akar kayu manis’ and ‘kulit kayu manis’
Flatten into cakes
Incubated for 3-4 days at room temperature
Dried ragi
Figure 7. Flow sheet: Ragi preparation of local manufacturers (Based on the interview session with ragi manufacturers from Kampung Maranek and Kampung Tambirat, August 2007).
2.3 Microbial Growth
Essential microorganisms are preserved during the air- or sun-dried ragi cakes for several months at room temperature in the tropics. Since there are spices added during the ragi preparation, it may contribute to the growth of other microorganisms or may inhibit development of undesirable microorganisms (Sie, 1962; Soedarsono, 1972; cited from
Steinkraus, 1983). Raw dry spices contain a wide variety of microorganisms and some inhibitory properties also exist due to the essential oils that can be found in spices. There were studies about the role of oriental herbs in the traditional starter preparation to their effect on the microflora of starter during the manufacture. It was observed that oriental herbs stimulate the growth of yeasts and moulds (Dung, 2004). Under certain condition for the
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production of ragi, the growth of amylolytic yeasts and moulds may be favored while growth of undesirable bacteria is inhibited.
There are various studies done by the researchers about the fermentation starter in different countries including Thailand, Vietnam, Indonesia and Malaysia. Savitree et al. (2008) reported that in Thai traditional fermentation starter (Loog-pang), it comprised of
Saccharomycopsis fibuligera which showed strong amylolytic activity. In addition, according to Lee and Fujio (1999), among the yeast species isolated from banh men, a fermentation starter from Vietnam is S. fibuligera that has the ability to hydrolyse starch.
Gandjar (2003) stated that the moulds in the ragi are strong amylolytic and degrade mainly the carbohydrate of the rice or glutinous rice into simple sugars which are then further decomposed by the yeasts into alcoholic compounds.
2.3.1 Fungi
Fungi regarded as filamentous organisms, form dry colonies when growing on agar or natural substrates and their colour is generally depend on the pigmentation of their spores
(Wainwright, 1992). Most industrially important fungi are either single-celled yeasts or grow as filaments called hyphae. These usually measure from 0.5 µm to 1.0 mm in diameter and can aggregate to form a mycelium (Deacon, 1984; cited in Wainwright, 1992).
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