ANALYSES OF MICROBIAL GROWTH

ON LOCALLY PRODUCED RAGI FOR TAPE

FERMENTATION

Noren anak Wilson Brandi

Bachelor of Science with Honours

(Resource Biotechnology)

2008

Faculty of Resource Science and Technology

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)

and tapai in nipah leaves (right).

6

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

cakes.

9

Figure 5 ‘Cekor’ (left) is added in preparation of ragi and moisten with boiled

stock of ‘kayu manis’ (right).

10

Figure 6 Spices (‘das manis’ and ‘das pedas’) added in the preparation of

ragi.

10

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).

11

Figure 8 Identification of fungus in specimens. (A) Aspergillus sp., (B)

Penicillium sp., (C) Geotrichum sp., (D) Tricophyton sp., (E)

Microsporum sp., (F) Epidermophyton sp., and (G) Rhizophus sp.

13

Figure 9 Overview of materials and methods. 15

Figure 10 Photos show the top (left) and bottom (right) of the Maranek ragi at

0 hour.

19

Figure 11 Photos show the top (left) and bottom (right) of the Tambirat ragi at

0 hour.

20

Figure 12 The surface of Maranek (left) and Tambirat (right) ragi after 2 weeks

at room temperature (27oC).

20

Figure 13 The surface view and reverse side of tapai from Maranek ragi for 0,

1, 2 and 3 days.

23

Figure 14 The surface view and reverse side of tapai from Tambirat ragi for 0,

1, 2 and 3 days.

24

Figure 15 Photomicrograph of microbe on the surface of Tambirat ragi

identified as Saccharomyces sp. at 600x magnification.

25

Figure 16 Photomicrograph of microbe on the surface of Tambirat ragi

identified as Mucor sp. at 200x magnification.

26

Figure 17 Photomicrograph of microbe on the surface of Maranek ragi

identified as Rhizophus sp. with their rhizoids is produced at 100x

magnification.

26

Figure 18 Photomicrograph of Rhizophus sp. on Maranek ragi with their

stolons grows along the surface of the NA medium at 40x

magnification.

27

Figure 19 Formation of ascospores on Maranek ragi at 100x magnification.

This characteristic (ascospores with 1-4 per ascus) falls within of

Saccharomyces cerevisiae (Larone, 1995).

27

Figure 20 Photomicrograph of microbe exist on Maranek tapai identified as

Saccharomyces sp. at 200x magnification.

28

Figure 21 Hyphae of Mucor sp. growing on NA exist on Maranek tapai at 100x

magnification.

29

Figure 22 Photomicrograph of microbe exist on Tambirat tapai identified as

Mucor sp. at 40x magnification.

29

Figure 23 The sporangium of Mucor sp. on Tambirat tapai at 200x

magnification.

30

Figure 24 The sporangium of Mucor sp. on Tambirat tapai at 400x

magnification.

30

Figure 25 Photomicrograph of microbe exist on Tambirat tapai identified as

Streptomyces sp. at 200x magnification.

31

Figure 26 Production of glucose in Maranek and Tambirat tapai within 24

hours of fermentation.

33

Figure 27 Production of ethanol in Maranek and Tambirat tapai within 24

hours of fermentation.

34

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

hours of incubation at 27oC.

22

Table 3 The concentration (g/L) of glucose and ethanol during tapai

fermentation utilizing Maranek ragi at 27oC.

44

Table 4 The concentration (g/L) of glucose and ethanol during tapai

fermentation utilizing Tambirat ragi at 27oC.

44

1

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.

2

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

3

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).

4

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.

5

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

6

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).

7

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.

8

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.

9

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).

10

Figure 5. ‘Cekor’ (left) is added in

preparation of ragi and moisten with boiled

stock of ‘kayu manis’ (right).

Figure 6. Spices (‘das manis’ and ‘das

pedas’) added in the preparation of ragi.

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.

11

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

12

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).