FORMULATION OF AMYLOLYTIC STARTER USING YEASTS

AND MOLDS SCREENED FROM TRADITIONAL MURCHA

by

Sangen Ruma Rai

Dharan Multiple Campus

Institute of Science and Technology

Tribhuvan University, Nepal

2016
FORMULATION OF AMYLOLYTIC STARTER USING YEASTS
AND MOLDS SCREENED FROM TRADITIONAL MURCHA

by

Sangen Ruma Rai

Dharan Multiple Campus

Institute of Science and Technology

Tribhuvan University, Nepal

2016
 Formulation of Amylolytic Starter Using Yeasts and Molds Screened
from Traditional Murcha

A dissertation submitted to Dharan Multiple Campus, Tribhuvan University, in partial

fulfillment of the requirements for the degree of B. Tech. in Food Technology

by

Sangen Ruma Rai

Dharan Multiple Campus

Institute of Science and Technology

Tribhuvan University, Nepal

April, 2016
 Acknowledgements

It is a genuine pleasure to express my deepest sense of thanks and gratitude to my mentor

and guide teacher Assoc. Prof. Basanta Kumar Rai. It is only because of his overwhelming
attitude to help his students that work of this complexity could be accomplished. His
valuable guidance, meticulous scrutiny and unwavering support have helped me to the
greatest extent in accomplishing this task.

I owe a debt of gratitude to Dr. Dil Kumar Subba (Lecturer, Central Campus of
Technology) for his keen interest in my work at every stage of my research. I acknowledge
his untiring inspiration and suggestions from the bottom of my heart. I thank the
coordinators of Dharan Multiple Campus Prof. Pashupati Mishra and Assoc. Prof. Basanta
Kumar Rai for providing the research facilities.

Thanks are due to Prince Subba (Teaching Assistant, CCT) for the wonderful
microscopy, Nar Bahadur Limbu (a key informant) for helping with survey, Assoc. Prof.
Ganesh Bahadur Thapa (CCT) for facilitating the identification of murcha weevils.

I thank profusely my friend and classmate Ms Jeny Subba for her kind help and
cooperation throughout my research period. Last but not the least, I take this opportunity
to acknowledge the entire DMC family in general and my junior brothers Mr. Manoj
Adhikari and Mr. Navin Adhikari in particular for their kind cooperation and lending their
helping hand in this work.

iv
 Abstract

The main objective of the study was to formulate amylolytic starter (murcha) in the
laboratory using different ratios of yeasts and molds screened from traditional murcha.
Murcha samples (collected from 10 sites of Eastern Nepal, viz., Saangu, Udayapur,
Kerabari, Dhankuta, Laxmimarga, Belbari, Dandaghopa, Bishnupaduka, Panmara and
Letang) were screened for amylolytic molds and fermentative yeasts, characterized and
preserved for the formulation study. Mold was used in the form of wheat bran koji and yeast
in the form of actively growing suspension. Experimental design (Face-centered, 2 Factors, 3
Levels, 13 Runs) was generated by Design Expert to formulate murcha using koji (1108,
2108 and 3108spores) and yeast (2.5105, 5105 and 7.5105 cells ) in 50 g of rice flour
(moisture ~ 30% and particle size < 280 ). Physicochemical analysis (bulk density, amylase
activity, moisture, and pH), performance test (alcohol production), sensory analysis (taste,
liquefaction and acid balance of fermented rice beer) and statistical analysis (ANOVA and
Response Surface curve) were carried out to select the best formula. The best murcha was
once again used to prepare rice and millet jand and physicochemical properties (alcohol and
acidity) were analyzed. Ester, volatile acidy and aldehyde (in the distillate) were also
determined.

Mold (identified as Rhizopus oryzae / Amylomyces rouxii) and yeast (identified as

Saccharomyces cerevisiae) from Udayapur murcha were found to be the most suitable.
The murcha formulation with 2 g koji (equivalent to 2108 spores of Amylomyces rouxii)
and 0.5 ml yeast suspension (equivalent to 5105 live Saccharomyces cerevisiae cells) per
50 g of rice flour was found to be significantly superior (p < 0.05) to other murcha (having
other mold-yeast ratios) in terms physicochemical and sensory quality. In conclusion, the
ratio of mold to yeast in murcha formulation is critical in the preparation of murcha of very
good quality. The murcha thus produced remained free from insect infestation during the
whole dissertation period (> 9 months), which is a great improvement compared to
traditional murcha making which generally become infested with insects within a month or
two of preparation. The method developed in this work is sustainable (does not depend on
harvesting of exotic plants), reliable (in terms of culture purity), consistent (in terms of
quality), and applicable even in the rural setting.

v
 Contents

Approval Letter .................................................................................................................. iii

Acknowledgements ............................................................................................................. iv
Abstract.. .......................................................................................................................... v
List of Tables .......... ..x
List of Figures.. ......................................................................................................................
1. Introduction .................................................................................................................. 1-3
1.1 General introduction .................................................................................................... 1
1.2 Statement of the problem ............................................................................................. 2
1.3 Objectives .................................................................................................................... 2
1.3.1 General objective ................................................................................................ 2
1.3.2 Specific objectives .............................................................................................. 2
1.4 Significance of the study.............................................................................................. 2
1.5 Limitations of the study ............................................................................................... 3
2. Literature review ........................................................................................................ 4-35
2.1 Food fermentation ........................................................................................................ 4
2.1.1 Fermented cereal foods and beverages ............................................................... 6
2.1.1.1 Non-alcoholic cereal gruels and beverages ............................................ 7
2.1.1.1.1 Ogi .......................................................................................... 7
2.1.1.1.2 Mahewu .................................................................................. 8
2.1.1.1.3 Atole........................................................................................ 8
2.1.1.1.4 Pozol ....................................................................................... 8
2.1.1.2 Alcoholic cereal beverages .................................................................... 8
2.1.1.2.1 Beer ........................................................................................ 9
2.1.1.2.2 Sake ........................................................................................ 9
2.1.1.2.3 Chicha .................................................................................... 9
2.1.1.2.4 Jand/Jaanr .............................................................................. 9
2.2 Starter culture technology .......................................................................................... 14
2.2.1 Amylolytic starters of the Asian countries ....................................................... 14
 2.2.1.1 Chu ....................................................................................................... 15
2.2.1.2 Nuruk ................................................................................................... 16
2.2.1.3 Ragi ...................................................................................................... 16
2.2.1.4 Koji ....................................................................................................... 16
2.2.1.5 Murcha/Marcha ................................................................................... 17
2.2.2 Microbial aspects of amylolytic starters ........................................................... 20
2.2.3 Traditional amylolytic starter technology ........................................................ 21
2.2.3.1 Rationale for use of plants, herbs and spices in starter ........................ 22
2.2.3.2 Substrate for starter .............................................................................. 23
2.2.4 Amylolysis in starter......................................................................................... 23
2.2.4.1 Assay of amylase ................................................................................. 25
2.2.4.2 Relevance of amylase analysis in amylolytic starters .......................... 26
2.2.5 Advances in amylolytic starters........................................................................ 27
2.2.6 Upgrading traditional amylolytic starters: challenges and opportunities ......... 27
2.3 Traditional methods used for the study of microbial profile of amylolytic starters .. 31
2.3.1 Mold characterization and preservation ........................................................... 31
2.3.2 Yeast characterization and preservation ........................................................... 33
2.4 Newer methods for the study of microbial profile ..................................................... 34
2.5 Assessment of the quality of murcha ......................................................................... 34
3. Materials and Methods ............................................................................................ 36-49
3.1 Materials .................................................................................................................... 36
3.1.1 Survey and sample collection ........................................................................... 36
3.1.2 Other materials ................................................................................................. 37
3.2 Methods ..................................................................................................................... 37
3.2.1 Physicochemical and microbiological analysis of murcha samples ................. 37
3.2.2 Screening of essential microorganisms (yeasts and molds) ............................. 38
3.2.2.1 Preliminary screening of murcha sample ............................................ 38
3.2.2.2 Screening of fermentative yeasts from murcha ................................... 39
3.2.2.2.1 Testing of yeast performance ............................................... 39
3.2.2.2.2 Identification and preservation of fermentative yeasts......... 40
3.2.2.3 Screening of molds from murcha ........................................................ 41

vii
 3.2.2.3.1 Identification and preservation of amylolytic molds ............ 41
3.2.3 Preparation of murcha ...................................................................................... 42
3.2.3.1 Rice flour preparation .......................................................................... 42
3.2.3.2 Preparation of wheat bran koji ............................................................. 42
3.2.3.3 Starter preparation ................................................................................ 43
3.2.4 Testing of performance of formulated starter ................................................... 45
3.2.4.1 Physicochemical analysis of prepared starter ...................................... 46
3.2.4.2 Sensory evaluation of jand from the formulated murcha .................... 47
3.2.5 Final fermentation using the selected murcha .................................................. 47
3.2.5.1 Analysis of the final jand ..................................................................... 47
3.2.5.1.1 Acidity .................................................................................. 47
3.2.5.1.2 Alcohol content .................................................................... 48
3.2.5.1.3 Volatile acidity ..................................................................... 48
3.2.5.1.4 Ester value ............................................................................ 48
3.2.5.1.5 Total aldehyde ...................................................................... 48
3.2.6 Data analysis ..................................................................................................... 49
4. Results and discussion .............................................................................................. 50-71
4.1 Survey ........................................................................................................................ 50
4.2 Physicochemical and microbiological quality of murcha .......................................... 50
4.3 Perception of murcha quality ..................................................................................... 53
4.4 Screening of murcha .................................................................................................. 53
4.4.1 Screening of molds ........................................................................................... 54
4.4.2 Identification of mold isolate............................................................................ 56
4.4.3 Preservation of mold culture ............................................................................ 56
4.4.4 Screening of yeasts ........................................................................................... 57
4.4.5 Identification of yeast isolates .......................................................................... 58
4.4.6 Preservation of yeast isolates............................................................................ 59
4.5 Preparation of fermentation starters using selected mold and yeast isolates ............. 60
4.6 Physicochemical and microbiological quality of laboratory starters ......................... 61
4.6.1 Alcohol content ................................................................................................ 61
4.6.2 Amylase activity ............................................................................................... 67

viii
 4.6.3. Sensory properties ........................................................................................... 68
4.6.4 Final fermentation using optimized laboratory murcha ................................... 69
4.6.5 Microbial properties of lab murcha (final) ....................................................... 69
4.6.6 Physicochemical properties of optimized murcha ........................................... 70
4.6.7 Comparison of performance of optimized murcha .......................................... 70
5. Conclusions and recommendations......................................................................... 72-73
6. Summary ................................................................................................................... 73-75
References .......................................................................................................................... 75
Appendices ......................................................................................................................... 84

ix
 List of Tables
Table No. Title Page No.
2.1 Factors affecting overall sensory quality of jand 13
2.2 Nutritional composition of the Himalayan alcoholic beverages 13
2.3 Fermentation starters used in different countries 15
2.4 Main enzyme activities in koji and nuruk 25
3.1 Experimental design for the formulation of amylolytic starter 44
using yeast and mold isolates
3.2 Experimental plan for response surface analysis 45
4.1 Physicochemical properties of murcha samples 51
4.2 Microbiological properties of murcha samples 52
4.3 Summary of difference between the liquefaction properties of 54
murcha samples and sensory attributes of jand prepared using
the murcha samples
4.4 Summary of difference between the liquefaction properties of 55
mold isolates and sensory attributes of saccharified rice
prepared using the isolates
4.5 Summary of difference between sensory attributes of 58
fermented molasses broth prepared using the yeast isolates
4.7 Responses of the experimental plan 62
4.8 Summary of statistical test for alcohol production by varying 62
yeast levels
4.9 Summary of statistical test for alcohol production by varying 63
koji levels
4.10 ANOVA for Response 1 (alcohol content) for surface 64
quadratic model
4.11 ANOVA table of amylase activity (Response 2) 67
4.12 Summary of ANOVA of sensory attributes of fermented rice 68
mash
4.13 Summary of statistically analyzed data on physicochemical 70
properties of millet and rice jand
D-1 ANOVA of liquefaction properties of 10 murcha samples 99
D-2 ANOVA of alcoholic properties of 8 yeast isolates 99
D-3 ANOVA of clarity properties of 8 yeast isolates 99
D-4 ANOVA of color properties of 8 yeast isolates 99
D-5 ANOVA of smell properties of 8 yeast isolates 100
D-6 ANOVA of sourness properties of 8 yeast isolates 100
D-7 Raw data for comparision of millet and rice jand 100

xi
 List of Figures
Figure No. Title Page No.

2.1 Flow diagram for the preparation of ogi 7

2.2 Outline of jand preparation from finger millet 11

2.3 Preparation of Bhatte jaanr 12

2.4 Interdependency of various factors 12

2.5 Classification of chu, a Chinese amylolytic starter 16

2.6 Flowcharts for the preparation of solid-fermented starters in 18

different countries of the Asia-Pacific Region

2.7 Sequential and concerted action of murcha flora on cereal 20

substrate

2.8 Action of - and -amylase on starch molecule 24

3.1 Survey and murcha sample collection site (Eastern Nepal) 36

3.2 A recapitulative outline of the method followed 38

3.3 Scheme for sugar assimilation test 40

3.4 A recapitulative diagram for murcha preparation 44

3.5 Schematic outline of amylase activity determination 46

4.1 Saccharification of cooked rice by Udayapur mold 55

4.2 Preservation of mold isolates in flat bottle and plate 56

4.3 Rhizopus oryzae under microscope 57

4.4 Trial fermentation in molasses broth 57

4.5 Yeast isolated from Udayapur murcha by spread-plating 59

4.6 Yeast isolates (1000) from murcha 59

4.7 Sugar assimilation test to identify yeast 59

4.8 Murcha prepared in the laboratory 61

4.9 Contour plot showing relation of amounts of mold and yeast 65

in starter for the response alcohol content (% by volume)

4.10 3D-Response surface curve of the interaction of amounts of 67

yeasts and molds vis--vis the response alcohol
 List of Plates
Plate No. Title Page No.
1 Surveying at Saangu (Taplejung) on murcha 97
2 Yeast culture preserved in flat bottles and slants 97
3 Sieve analysis of rice flour 98
4 Murcha being prepared in the laboratory 98
5 Electrical cabinet dryer used for murcha drying 98
 Part I

Introduction

1.1 General introduction

Marcha or murcha is a mixed dough inocula prepared as a dry, round to flattened, creamy
white to dusty white, solid ball which is used as an amylolytic starter to produce ethnic
alcoholic beverages (such as jand (undistilled) and raksi (distilled)) in the Himalayan
regions of India, Nepal, Bhutan, and Tibet (Tamang, 2010). The traditional method of
murcha preparation entails use of around 38 identified wild plants, hereafter called murcha
plants. The most prized murcha plant is Polygala arillata, the semi-dried root barks of
which reportedly sell ~ 4,000 (NRs)/kg. Other murcha plants of importance are Polygala
abyssynica, Vernonia cineriae, Inula spp. These plants harbor all the microorganisms
needed for the amylolytic fermentation and it is this feature of microbial association that a
murcha maker exploits (KC et al., 1999).

Amylolytic starters similar to murcha are used in many different countries of the world
for cereal-based fermentations. However, the most dominant production and use of such
starters occur in the Asian countries. As a result of serious researches, many of them (for
instance, Chinese Chu, Korean nuruk, Malaysian ragi, and Japanese koji) have now
evolved as commercial commodities. These starters contain yeasts, molds and lactic acid
bacteria and are used for the preparation of a wide variety of cereal beers (cloudy extract,
along with live yeasts) and cereal wines (clear product) (Lee, 1999).

Methods for the preparation of different types of amylolytic fermentation starters have
been described in detail by many workers (Yokotsuka, 1985; Yoon, 1993; Tamang, 2010).
Murcha preparation is unique in that inclusion of murcha plants as the source of essential
organisms is considered very crucial (Rai and Subba, 2003). However, preparation mana
(which is a variant of murcha) relies on natural flora from paddy straw (Gajurel and
Vaidya, 1979).

Several researchers, including Rai (2006) and Rai (2015) have successfully prepared
murcha in the laboratory using isolated molds and yeasts from local murcha. Their
findings indicate that it is possible to produce good-quality murcha using defined cultures.
1.2 Statement of the problem

Review of literature reveals that researches on murcha so far are limited to survey, murcha
preparation, screening, and characterization of murcha flora. Quantitative aspects of the
inocula (proportion, number of cells and spores), which is very important in the
preparation of murcha of consistent quality, appears to have been ignored. Likewise,
suitable choice of raw-materials for substrate, particle size of substrate, drying condition
(time and temperature), shelf-life of murcha, cake/ball size have not been studied yet.
Moreover, the sanitary and phytosanitary (SPS) aspect of the starter has not been studied.

1.3 Objectives

1.3.1 General objective

To formulate amylolytic starter using pure yeast and mold cultures screened from
traditional murcha.

1.3.2 Specific objectives

1. To conduct survey and collect murcha samples from selected areas of Eastern
regions of Nepal

2. To carry out screening and characterization of fermenting yeasts and saccharifying

molds from murcha samples

3. To prepare mold koji

4. To prepare murcha using different yeast-to-mold ratio

5. To carry out physicochemical and microbiological analysis of the prepared murcha

1.4 Significance of the study

This study will help find answers to a series of basic questions regarding murcha,
including the following:

(i) What are the dominant/desired microbes and what factors in the processing and
environment exert selective pressure?

(ii) What should be the optimized proportion of isolates (essential) for a good quality
murcha?

(iii) Can the shelf-life of murcha be increased?

2
Therefore, it is expected that this finding will aid researchers in identifying areas and scope
for future research. One of the aims of this study is to devise a simplified protocol for the
improvement and preparation of murcha in the laboratory and at the same time offering
practicability in the tribal setting so that the ultimate beneficiary of this work will be the
tribes who have depended on this traditional art for so long. This work is also a small
attempt to upgrade quality of murcha such that it can meet the stringent criteria of food
safety/SPS measures laid down by developed countries in terms of trading of foods. Lastly,
the developed method will have sustainability implications whereby the producers will not
have to depend on exotic (and sometimes already rare) plants/herbs.

1.5 Limitations of the study

During the dissertation work, the following limitations were taken into account:

1. Only 10 different sites of Eastern Nepal were visited for survey purpose, because of
different limiting factors such as lack of home-stays and transportation facilities.

2. Because of time constraints and scope of the present work, only rice (non-sticky,
single variety) was used as the substrate/carrier.

3. The quality of jand prepared using laboratory murcha (because pure cultures were
used), although consistent, may not approach the highly characteristic quality
(round flavor and bouquet) of jand contributed by natural succession of mixed
microflora present in traditional murcha.

3
 PART II

Literature review

2.1 Food fermentation

Fermentation has many definitions. In a biochemical sense, fermentation is an anaerobic

process in which substrate is utilized for energy production without the involvement of
molecular oxygen. In industrial microbiology, fermentation is simply a large-scale
microbial process, it can be both aerobic and anaerobic (Rai, 2012). Contextually,
fermentation is the slow decomposition process of organic substances induced by
microorganisms or enzymes, of plant or animal origin (Marshall and Meija, 2011).

Fermented foods are food substrates that are invaded by bacteria or by fungi having
pleasant flavors, attractive aromas and textures, and are non-toxic and widely accepted by
human consumers (Steinkraus, 1996). Fermented foods and beverages are one of the
indispensable components of the dietary culture of every community in the world (Tamang
and Kailasapathy, 2010).

The preparation of fermented foods involves technology from the most primitive to the
most advanced, and achieving an astounding range of sensory and textural qualities in the
final products. All such fermentations have been or remain classified as indigenous, native
to a country or culture (Smith, 1996).

Various schemes have been used for classifying fermented foods. According to recent
classification given by Steinkraus (1996), fermented foods are classified into following
categories:

1. Leavened bread such as yeast and sourdough breads

2. Flat unleavened breads

3. Alcoholic fermented foods such as wine, sake, etc.

4. Alkaline fermented foods such as kinema, natto, etc.

5. Acetic acid fermented foods such as apple cider, tea fungus, etc.
 6. Lactic acid fermented foods such as sauerkraut, yoghurt, pickles, etc.

7. Fermentation producing textured vegetable protein meat substrate in legumes/cereal

mixture such as Indonesian tempeh and ontjom, etc.

8. High salt/savory meat flavored/amino acid/peptide sauce and paste fermented foods
such as Chinese soy sauce and Japanese miso.

Fermented foods have a very good safety record even in the developing world where
foods are manufactured by people without training in microbiology or chemistry but
fermented foods by themselves cannot solve the problems of contaminated drinking water
and improper personal hygiene in food handlers (Steinkraus, 1996).

Flavor development is the principal reason of food fermentation. Nevertheless, among

other things, fermentation improves safety aspects of traditional fermented foods by
leaching out flatulent principles like stachyose and raffinose during cooking,
decreasing/destroying anti-nutritional factors like trypsin inhibitors, lectins and phytic acid.
Moreover, food fermentations usually improve food safety due to production of ethanol,
lactic acid, acetic acid together or separately in fermented food products (Rai, 2012).

The safety of fermented foods has been recently reviewed. Cases of food-borne
infection and intoxication due to microbial metabolites such as mycotoxins,
ethylcarbamate, and biogenic amines have been reported in fermented foods. Major risk
factors include the use of contaminated raw materials, lack of pasteurization, and use of
poorly controlled fermentation conditions (Nout, 1994). Hence, these risk factors make
safety of fermented foods more controversial and questionable in developing countries.

Fermented foods play an important socio-economic role in developing countries as well

as making a major contribution to the protein requirements of natural populations (Achi,
2005). Fermented foods are produced in our homes, villages and also in small-scale food
industries. Varieties of fermented foods are produced in developing countries, using
traditional fermented technology at the household level. The preparation of many
indigenous fermented foods and beverages today remains a household-art. Traditional
fermentation processes are typically uncontrolled and dependent on microorganisms from
the environment. Traditional fermentation process is the most available and affordable

5
food preservation method, which is of great economic importance to developing countries
(Adesulu and Awojobi, 2014).

Fermentation technologies play an important role in ensuring the food security,

enhancing livelihoods and improving the nutrition and social well-being of millions of
people around the world, particularly marginalized and vulnerable groups. Fermented
foods are popular throughout the world and the production of fermented food products is
important in many countries in providing income and employment (FAO, 1998).

Various schemes have been used for classifying fermentation process. Most indigenous
fermented foods and some enzymes are produced by solid state (also termed solid-
substrate) fermentation. It is a fermentation in which microbial growth occurs on moist,
non-soluble substrate in the absence or near-absence of free-flowing water (Rai, 2012).
The author describes solid state fermentation as a highly aerobic process where
microorganisms get their nutrients by hydrolyzing the substrates.

Some of the important examples of solid state fermentation that have achieved
commercial sources are koji (an amylolytic mold culture) of Japan, tempeh (mold
fermented soybean product) of Indonesia and natto (fermented soybean product) of Japan
(Steinkraus, 1987). Although such fermentations have disadvantage in terms of control
over relative humidity, CO2 removal, aeration and temperature (Singh, 1998) and therefore
present problem in scaling up of indigenous food fermentations (Ruskin, 1992).

2.1.1 Fermented cereal foods and beverages

Fermented cereals play a significant role in human nutrition in all parts of the world where
cereals grow. Among all food fermentations (e.g. milk, meat, fish, soy or wine) cereal
fermentations reach the highest volume. Depending on the water content during processing
and on the cereal-specific enzyme activities a broad range of fermented cereal foods and
beverages were developed by mankind (Brandt, 2013).

Cereals have a variety of uses as food. Only two cereals, wheat and rye, are suited to the
preparation of leavened bread. The most general usage of cereals is in cooking, either
directly in the form of grain, flour, starch, or as semolina, etc. Another common usage of
cereals is in the preparation of alcoholic drinks such as whiskey and beer (barley;
sorghum), vodka (wheat), American bourbon (rye) and Japanese sake (rice) (Haard, 1999).

6
 Most bacterial fermentations produce lactic acids; while yeast fermentation results in
alcohol production. Many of the indigenous fermentation products of cereals are valued for
the taste and aroma active components produced and are used as seasonings and
condiments. A summary of flavor compounds formed in such products was compiled by
Chaven and Kadam in 1989 (Haard, 1999).

Excellent accounts of traditional cereal fermentations in African countries, the Asia-

Pacific Regions and the Latin American countries are given by Haard et al. (1999).

2.1.1.1 Non-alcoholic cereal gruels and beverages

Non-alcoholic cereal gruels and beverages are traditionally produced in the South African
countries (Odunfa, 1999) and Latin America (Quintero-Ramrez et al., 1999). These are
used as staple as well as weaning food. Description of some of the non-alcoholic cereal
gruels and beverages are described in the following sections.

2.1.1.1.1 Ogi

Ogi is a porridge prepared from fermented maize, sorghum or millet in West Africa. It is a
staple of that region, and serves as a weaning food for infants. Fig. 2.1 shows the
traditional preparation of ogi. Ogi is often marketed as a wet cake wrapped in leaves or
transparent polythene bags. It is diluted to a solids content of 8-10% and boiled, or cooked
and turned into a stiff gel prior to consumption (Odunfa, 1999).

Corn

Clean

Wet mill

Sieve and discard pomace

Ferment filtrate and allow to

sediment for 1-3 days

Ogi

Fig. 2.1 Flow diagram for the preparation of ogi

7
 Odunfa (1985) determined that L. plantarum was the pre-dominant organism in the
fermentation responsible for lactic acid production. Corynebacterium hydrolyzed corn
starch to organic acids while S. cerevisiae and Candida mycoderma contributed to flavor
development.

2.1.1.1.2 Mahewu

This is a fermented maize meal commonly consumed as a staple among black South
Africans. It is traditionally prepared by adding one part of maize meal to 9 parts of boiling
water. The suspension is cooked for 10 min, allowed to cool and then transferred to a
fermentation container. At this stage, wheat flour (about 5% of the maize meal used) is
added to serve as a source of inoculum. Fermentation occurs in a warm sunny place within
24 hrs. Streptococcus lactis is the main fermenting organism in traditionally prepared
mahewu (Odunfa, 1999).

2.1.1.1.3 Atole

Atole is a sour porridge-type product prepared from maize by members of the Tzotzil
ethnic group in Southern Mexico. It is produced by steeping maize grains in water for 4
days, milling and allowing it to stand for 1 day. During atole production, lactic acid
fermentation commences during steeping and continues in the milled product (Quintero-
Ramrez et al., 1999).

2.1.1.1.4 Pozol

Pozol is a fermented maize dough formed into balls of various shapes and sizes ranging
from 10 to 12 cm in length, 5 to 8 cm in width and 70 to 170 g in weight. Pozol is
consumed by Indian and mestizo populations, mainly in the Southeastern states of Mexico.
Balls of freshly prepared pozol, or pozol at various stages of the fermentation process are
diluted with water to produce a whitish porridge which is consumed in the uncooked state
as a basic food in the daily diet of large communities (Quintero-Ramrez et al., 1999).

2.1.1.2 Alcoholic cereal beverages

Traditional alcoholic cereal beverages find widespread use in the Asian countries. Modern
cereal alcoholic beverages include Japanese sake and beer.

8
2.1.1.2.1 Beer

Beer is the fermented extract of malted barley drunk throughout the world as a refreshing
mild alcoholic beverage. Beer was produced by the Sumerians before 7000 BC (Tamang
2010). The technology for production of European barley beer, the biochemical and
microbiological changes that take place during malting, fermentation and subsequent
processing and storage are well documented in the literature. In Africa, traditional beers
differ from the western-type in that they are often sour, less carbonated and has no hops.
They are consumed unrefined, including unfermented substrates and microorganisms
(Mwesigye and Okurut, 1995).

2.1.1.2.2 Sake

Sake (rice wine) is a traditional alcoholic beverage, prepared from rice, consumed
particularly in Japan and China. The rice is polished and steamed, and part of it is steamed
and used to grow Aspergillus oryzae, which produces different types of enzymes required
for sake brewing. The seed mash is traditionally obtained by natural lactic acid
fermentation involving various aerobic bacteria, wild yeasts, lactic acid bacteria, and sake
yeasts (Lotong, 1998).

2.1.1.2.3 Chicha

Chicha is a clear, yellowish, effervescent, alcoholic beverage prepared from maize. It has a
flavor similar to that of cider. Chicha has been consumed by the Andean Indians for
centuries. When prepared from pigmented maize varieties, its color varies from red to
purple. The alcoholic content of chicha varies between 2 and 12% (v/v). The traditional
production of chicha is a somewhat unique fermentation process in which saliva serves as
the source of amylase for converting starch to fermentable sugars (Steinkraus, 1996).

2.1.1.2.4 Jand/Jaanr

Jand is a traditional undistilled alcoholic beverage prepared from solid-state fermentation

of starchy cereals like corn, rice, wheat and millet, by using locally made starter culture
known as murcha. Jand contains live yeasts and suspended particles and hence, classified
by various workers as a category of cereal beer (Rai, 2012). Jand is very popular among
the rural mass of Nepal (Rai, 1991). However, with the advent of alcoholic beverages
based on new technology, this product has earned itself social stigma among the
9
urbanities: it is regarded as poor mans wine. This fact notwithstanding, the annual
production of jand is higher than that of any other indigenous fermented products and this
trade is probably the single-most important economic activity among most ethnic groups of
low income category (Subba et al., 2005). Some aspects of jand have been reviewed by
Aidoo et al. (2005). They have described the role of mucaraceous fungi in producing
amylase needed to saccharify and liquefy starch. The amylase activity has been reported to
reaches its peak on the second day of fermentation. The authors have also mentioned the
presence of mixture of yeasts (Pichia anomala, Saccharomyces cerevisiae, Candida
galbrata) and lactic acid bacteria (Pediococcus pentosaceus, Lactobacillus bifermentans)
in numbers exceeding 105 cfu/g in matured jand.

Jand is a common drink for Sherpa, Bhote, Rai, Limbu, Magar, Thakali, Newar, Jyapu,
Damai, Kami, etc. (collectively called matwali, meaning with tradition of drinking
alcoholic beverages). Now it is getting popular among Brahmin and Chhetri also (they are
actually forbidden, by religion, to drink alcoholic beverages). The natives prepare jand for
themselves. Women, especially in rural areas, prepare and sell jand in local market. It is an
important source of income for them. The fermented mash, jand, is consumed in different
ways. It is squeezed with added water, strained in a traditional bamboo-made chhapani or
aluminum strainer, and the whitish cloudy extract thus obtained is served in deep bowls,
tumblers or other containers. Alternatively, the fermented mash is put into a wooden or
aluminum cylindrical vessels, hot water added over it and the extract sucked with a help of
bamboo or aluminum tube (Kharel et al., 2009).

The cereal of choice for jand preparation is finger-millet (Eleusine coracana Gaertn L)
but other cereals like maize, wheat and rice are also used (Rai, 1991). Finger-millet is often
termed as poor mans cereal. In this light, jand preparation from this cereal can be
viewed as an important value addition activity.

Jand finds a prominent place in Limbu and Rai culture in particular and other ethnic
groups in general. The tradition of offering jand to guests is a unique way of showing
hospitality. Jand is also used in several festive occasions, ritual rites, settling disputes and
appeasing deities (Rai, 1991).

A brief outline of jand preparation from finger-millet is shown in Fig.2.2 (Rai, 1991)
and that of bhatte jaanr (from rice) is shown in Fig.2.3 (Tamang et al., 1996).

10
 Finger millet

Dehusking in local mortar

and pestle (okhli)

Winnowing in nanglo to remove

immature grains and foreign matter

Washing in water

Cooking to soft consistency

Cooling on bamboo mat

(mandro)

Sprinking of murcha
powder (~0.5%)

Transferring to bamboo
basket (dalo) previously
lined with banana leaves

Light covering with banana

leaves or cloth rags

Aerobic fermentation for a

day and a half (until mold
growth is visible)

Transfer to earthen jar

(ghyampo) and seal

Anaerobic fermentation
(one week to several
months)

Jand

Fig. 2.2 Outline of jand preparation from finger millet

11
 The sensory quality of jand is naturally dependent on its physicochemical properties,
which in turn are dependent on several other factors, including the quality of murcha. Rai,
(2006) has given simplified relation of this interdependency in Fig. 2.4. The components of
the blocks shown in Fig. 2.4 are listed in Table 2.1. The nutritional composition of
Himalayan alcoholic beverages according to Tamang et al. (2012) is shown in Table 2.2.

Glutinous rice

Wash

Cook

Cool in mandro

Inoculate with marchaa

and mix thoroughly

Place in vessel/earthen pot

and saccharify for 1-2 days

Ferment in air-tight container for

2-8 days at ambient temperature

Bhatte jaanr

Fig. 2.3 Preparation of Bhatte jaanr

Organoleptic quality of
the product

Physicochemical properties
of the product (jand)

Raw material type Murcha quality Fermentation

for fermentation conditions

Fig. 2.4 Interdependency of various factors

12
Table 2.1 Factors affecting overall sensory quality of jand

Factors Components
Raw material Cereal substrates such as finger-millet, wheat, rice, maize,
etc., used for the fermentation
Fermentation conditions Temperature, pH, aerobicity, duration of fermentation, solid-
state or submerged-state fermentation
Murcha quality Species and strains of the essential microorganism (yeasts
and molds), presence of extracellular enzymes, amylase in
particular
Physicochemical Alcohol content, acidity, pH, reducing sugars, non-reducing
properties of jand carbohydrates, total soluble solids, esters and other congeners

Organoleptic properties Taste, smell, mouth-feel and color

of jand

Table 2.2 Nutritional composition of the Himalayan alcoholic beverages

Parameter Kodo ko jaanr Bhatte jaanr

Moisture 69.68 83.4

pH 4.04 3.5
Acidity (as % lactic acid) 0.27 0.24
Alcohol 4.8 5.9
Ash (% DM) 5.1 1.7
Fat (% DM) 2.0 2.0
Protein (% DM) 9.3 9.5
Crude Fiber (% DM) 4.7 1.5
Carbohydrate (% DM) 83.7 86.9
Food value (kcal/100g DM) 389.6 404.1
Calcium (mg/100g DM) 281.0 12.8
Magnesium (mg/ 100g DM) 118.0 50.0
Iron (mg/100g DM) 24.0 7.7

% DM = g/100 g, on a dry matter basis Source: Tamang et al. (2012)

13
2.2 Starter culture technology

Unlike fruit and milk fermentations, cereal fermentation requires a saccharification

process, which is accomplished with some difficulty. Saccharification is carried out
through the malting process. Malting occurs naturally through wet damage of cereals
during storage, and is used for beer making in Europe. However, in Asia the malting
process is rarely used in traditional fermentation processes. Instead, fermentation starters
prepared from the growth of molds on raw or cooked cereals is more commonly practiced
(Lee, 1999).

The starters used for cereal fermentations are therefore amylolytic fermentation starters
(Aidoo et al., 2005). Simply defined, starter cultures consist of microorganisms that are
inoculated directly into food materials to overwhelm the existing flora and bring about
desired changes in the finished product (Hutkins, 2006). Starter cultures help initiate the
fermentation and then sustain it in the desired direction (Prescott, 2002).

Fermentation starters are given different names in different regions e.g. Murcha is well
known in Nepal as well as in India. Similarly, it is known as loogpang in Thailand, ragi in
Indonesia, koji in Japan, bubod in Philippines, nuruk in Korea and chu in China (Nout,
1992; Lee, 1999).

Mixed-culture fermentations are those in which the inoculum always consists of two or
more organisms. Mixed cultures can consist of known species to the exclusion of all
others, or they may be composed of mixtures of unknown species. The mixed cultures may
be all of one microbial group - all bacteria - or they may consist of a mixture of organisms
of fungi and bacteria or fungi and yeasts or other combinations in which the components
are quite unrelated. All of these combinations are encountered in oriental food
fermentations (Hesseltine, 1992).

2.2.1 Amylolytic starters of the Asian countries

Amylolytic starters are used in many countries of the world for cereal-based fermentations.
However, the most dominant production and use of such starters occur in the Asian
countries. A partial list of starter cultures and countries producing them (Lee, 1999; Law et
al., 2011), with some modifications, is given in Table 2.3.

14
2.2.1.1 Chu

Chu is a general term for Chinese amylolytic starter (Lee, 1999), the classification of
which is given in Fig. 2.5. Chu is commonly used in the Asia-Pacific region as an enzyme
source for the degradation of complex plant tissue to produce cereal wines, soysauce, fish
and meat sauce, sour bread, and fermented porridges and snacks. Details regarding the
ingredients and microfloras of chu are given in Table 2.3.

Cake type ping-chu is identical to nuruk in Korea, and granular type san-chu is similar to
Japanese koji. According to Yokotsuka (1985), chu may either be yellow (huang) possibly
due to Aspergillus oryzae, or white probably due to Rhizopus and Mucor. Huang-chu was
widely used for alcoholic fermentation as well as for the fermentation of soybean foods.

Table 2.3 Fermentation starters used in different countries

Country Starter Ingredients Shape Microorganism(s)

China Chu Wheat, barley, millet, Granular Rhizopus and
rice (whole grain, grits or or cake Amylomyces spp
flour)
Korea Nuruk Wheat, rice, barley Large Aspergillus and
(whole grain, grits or cake Rhizopus spp.,
flour) Yeasts

Japan Koji Wheat, rice, (whole grain Granular Aspergillus spp.

grits or flour),
Indonesia Ragi Rice (flour) Small Amylomyces spp.
cake
Philippines Bubod Rice, glutinous rice Small Mucor, Rhizopus
(flour) cake and Saccharomyces
spp.
Nepal and Marchaa Rice, glutinous rice Flat cake Hansenula anomala
India (Murcha) (flour) Mucor fragilis,
Rhizopus arrhizus

Three types of huang-chu have been described, (i) huang-yi, (ii) huang-tcheng and
(iii) nu-chu. Huang-yi is prepared from crushed wheat, huang-tcheng from wheat flour,
and nu-chu from cooked rice (Yokotsuka, 1985). Wheat chu is originated in the Northern

15
part of China and the Korean Peninsula, while rice chu originated in the South. This is
reflected by the main ingredients of the fermentation starters prepared today in the
countries of the South-Pacific region as shown in Table 2.3.

Fen-chu (without bran):

Ping-chu Shen-chu, San-hu-mai-chu
(Cake type)
Cu-chu (with bran):
CHU Bai-lao-chu, Chun-jiu-chu
San-chu Huang-yi, Huang-tcheng
(Granular type)

Fig. 2.5 Classification of chu, a Chinese amylolytic starter

2.2.1.2 Nuruk

Nuruk is the most well-known amylolytic starter of Korea. The essential organisms of nuruk
are Aspergillus spp., Rhizopus spp. and yeasts. Nuruk has the shape of a large cake (Lee,
1999). Wheat, rice and barley are used as the carrier and substrate. The starter is used to
prepare makgeolli (makkoli), a native Korean rice or wheat wine. Nuruk, Ragi (Indonesia and
Malaysia), and bubod (Philippines) are similar in that they are prepared by natural
fermentation of raw cereal powders which are molded into the shape of a cake or ball.

2.2.1.3 Ragi

Ragi is the general term for starter. Ragi tempeh means starter for tempeh (a mold-
fermented soybean product), ragi ketan means starter for ketan/ketella, an Indonesian
fermented, mildly alcoholic, partially liquefied (juicy) rice paste having a sweet-acid taste
(Merican and Yeoh, 1989), and so on. Microbiologically, ragi for cereal fermentation is a
mixed culture of mucoraceous fungi, yeasts and bacteria (Hesseltine, 1988). According to
Ko (1972), the important microorganisms of ragi are Amylomyces rouxii (mold) and
Endomycopsis burtonii (yeast).

2.2.1.4 Koji

Koji is a mold culture and is prepared from steamed-cooked cereals in Japan. The term koji
is Japanese, meaning naturally, spontaneously or artificially molded cereals and beans
(Yokotsuka, 1991). The Chinese call chu, shi or qu for koji. Rice is steamed, cooled on
bamboo-made trays, stacked with gaps of about 10 cm in between to allow air circulation,

16
inoculated with 0.1% mold spores locally called tane-koji, and incubated at 23C-25C.
The rise in temperature due to growth of mold is kept within the range of 35C-45C by
stirring and turning the koji from the top to bottom on trays at about 20-40 hrs, normally
fermented for 3 days, when mold mycelium spread throughout mass before sporulation
(Lotong, 1985).

2.2.1.5 Murcha/Marcha

Murcha (also spelt marchaa, marcha) is a traditional amylolytic starter used to produce
sweet-sour alcoholic drinks, commonly called jand in the Himalayan regions of India,
Nepal, Bhutan, and Tibet (China) (Tsuyoshi et al., 2005). Rai and Subba (2003) have
described murcha as a starter cake employed as an inoculum in the production of
traditional, cereal-based alcoholic beverages, viz., jand (undistilled) and raksi (distilled).
Murcha is a Nepali word; the Lepchas call it thamik, the Limbu call it khesung, and the
Bhutias use the word phab (Tamang and Sarkar, 1995).

Murcha cakes are of two types, manapu and mana (Gajurel and Baidya, 1979; Karki,
1986; Shrestha et al., 2002). Manapu is prepared from rice flour and millet grains, whereas
mana is prepared from wheat flakes. The preparation of mana is given by Tamang (2010)
is as follows:

Wheat grains Soak in water overnight Steam for 30 min Transfer to a bamboo
basket and drain Grind into a lump Spread on a bedding of paddy straw Cover
with paddy straw or straw mat Ferment for 67 days (until green mold appears on the
wheat grains) Dry in the sun Mana Store.

Flowcharts for the preparation of solid-fermented starters in different countries of the Asia-
Pacific Region are given in Fig. 2.6.

Hesseltine et al. (1988) isolated three genera of Mucarales (Rhizopus, Mucor and
Amylomyces), yeasts and bacteria in 41 murcha and related starters of seven Asian
countries. However, Tamang and Sarkar (1995) isolated a total of 194 bacterial, 190 yeast
and 80 molds strains from 30 samples of murcha. Pediococcus pentosaceous,
Saccharomyces fibuligera, Rhizopus chinensis were the dominant microorganisms. S.
fibuligera had amylolytic activity. This shows that the microbial profile of amylolytic
starters is not very consistent.

17
 Rice flour

Moisten with water Glutinous rice

or sugarcane juice to
form a thick paste Grind while wet

Inoculate with ragi Mix with water, unwad roots

powder from a former batch and ginger

Flatten into cakes or

mold into hemispheres Mold into flattened
cakes or balls
Place on bamboo tray
and cover with muslin Sprinkle with
powdered bubod
Incubate at 25-30oC
for 2-5 days Incubate for 3 days

Dry Dry

Ragi (Indonesia) Bubod (Philippines)

Whole wheat flour or grits Polished rice

Add water to 30-40% Soak in water for

moisture content 17 hrs at 25 oC

Wrap in a cloth and press Drain excess water

in a mold to make cakes
(5 cm thick; 10-30 cm dia) Steam for 70 min and
cool to 35oC
Incubate 10 days
at 30-45 oC
Inoculate with
Aspergillus oryzae
Incubate 7 days
at 35-40 oC
Incubate at 27-28oC
for 50 hrs
Dry 2 weeks at 30oC
Dry
Age 1-2 months at room
temperature
Koji (Japan)
Nuruk (Korea)

Fig. 2.6 Flowcharts for the preparation of solid-fermented starters in different countries of
the Asia-Pacific Region

18
 Unlike other amylolytic starters, with possible exception to starter cultures of Northeast
India, viz, Assam, Manipur, Nagaland, Sikkim and Arunachal Pradesh (Tanti et al., 2010)
murcha preparation involves use of over 40 wild plants (KC et al., 2001). Rai and Subba
(2003) and Poudel (n.d.) also describe about use of murcha plants for preparing murcha.
KC et al. (2001) has recently documented 38 murcha plants from eastern region of Nepal
(Appendix A-1).

KC et al., (2001) reported that Polygala arillata is the most prized murcha plant, with its
semi-dried root-bark and flowers selling at 270 (NRs) per kg (as of 1999). The authors have
also reported that murcha makers are very particular about the choice of plants (or their
parts) for murcha making. A recent personal communication (Shrestha, 2015), however, has
revealed that the price of the same item has reached 4000 (NRs) per kg (as of 2015).

According to earlier report by Rai and Subba (2003), the top ten murcha plants (in
descending order of importance) are Polygala arillata, Vernonia cinerea, Clematis
grewiaeflora, Buddleja asiatica, Christella appendiculata, Polygala sp. and Inula sp.
Poudel (nd) has dubbed Polygala arillata as marcha [sic] plant and mentions its use as
compulsory and effective in murcha making. However, the illegal trade, high demand, an
unsustainable harvesting practice in Nepal has made it an indeterminate plant toward the
risk of extinction.

KC et al. (1999) have isolated Saccharomyces cerevisiae as the dominant yeast in these
murcha plants. Other yeast types identified include Zygosaccharomyces bailii, Hansenula
anomala and Filobasidium capsuligenum. In a latter study, the authors (2003) have
reported murcha trade as being a very lucrative business. They have shown concern on the
issues of overharvesting of murcha plants and offered some viable alternatives as control
measures. The workers, however, did not isolate molds.

It is clear that cereal fermentation resulting from use of murcha is simply the result of
concerted action of molds, yeasts and bacteria on the cooked substrate. The generalized
scheme of the actions of murcha flora on the cooked substrate (Subba et al., 2005) is given
in Fig. 2.7.

19
 MURCHA
Molds Yeast enzymes
+ Bacterial
(zymase) enzymes
Lactic acid bacteria
+
Yeasts
Mold enzyme Ethanol
(amylase) +
SUBSTRATE SIMPLE Lactic acid
SUGARS +
Saccharification Flavor compounds

Anaerobic reaction
Aerobic reaction (formation of jand)

Fig. 2.7 Sequential and concerted action of murcha flora on cereal substrate

Methods for preparing murcha vary a great deal. A general method used for murcha
preparation in Nepal according to Rai (2006) is as follows:

Steeping of cereals (maize grits, rice, or finger millet) in water Draining

Pounding the soaked cereals along with sun-dried murcha plants and a small amount
(~ 1%) of murcha seed from the previous batch Moist powder obtained Addition of
water Mixing to form a stiff dough of around 50% moisture content Dividing the
dough into small units, forming into balls (~ 1.5 cm dia) or flat cakes (0.5-1 cm thick, 3-10
cm dia) Placing the cakes (in a single layer) on a mat previously lined with fern leaves
or straw Blanketing with fern leaves, straw or cloth Incubation for 2-3 days until the
cakes swell and become distinctly moldy Sun-drying.

2.2.2 Microbial aspects of amylolytic starters

According to Nout and Aidoo (2010), studies on the microflora of ragi and Chinese yeast
cake date from work by Went and Prinsen Geerlings in 1895. Several researchers
investigated the microflora of amylolytic starters. From their combined efforts, it can be
generally concluded that there are three components of the microbiota. The first
component comprises filamentous fungi that are able to degrade the native starch in the
raw rice flour, by forming amylases especially amyloglucosidase, which degrades starch
directly into glucose (Dung et al., 2006). The second component consists of fermentative
yeasts, whereas some of these yeasts can degrade starch as well, their main function is in
the alcoholic fermentation and the production of flavor components such as esters. The
third component comprises LAB, which do not seem to have a positive contribution to the

20
quality of the fermented wine. However, they present as a natural accompaniment of yeasts
in the manufacture of amylolytic starters (Aidoo and Nout, 2010).

Hesseltine et al. (1985) stated that starters used for fermentations based on rice or
cassava, such as ragi, murcha, loog-paeng and chiu, regularly contain species of Mucor,
Rhizopus and Amylomyces with concentration from 106 to 108 cfu/g. Rhizopus oryzae
(many authors classify it as Amylomyces rouxii) is the dominant molds present in starters
based on rice and cassava. The name Amylomyces rouxii has been proposed for the variants
of Rhizopus that produce abundant chlamydospores. They hardly produce mature
sporangia or sporangiospores (Kito et al., 2009).

Microbiologically, murcha is a mixed culture containing saccharifying molds,

fermenting yeasts, and acidifying bacteria (KC et al., 2001). Some researches on murcha
and similar starters are available (Gautam, 1987; Tamang and Sarkar, 1988, Verma, 1991;
Tamang and Sarkar, 1995; Hesseltine and Ray, 1998; Shrestha et al., 2002; Shrestha and
Rati, 2003; Dung et al., 2005; Tsuyoshi et al., 2005). These workers found Saccharomyces
species as the dominant yeast. Other yeasts reported to occur were Saccharomyces
bayanus, Saccharomycopsis fibuligera, Pichia anomala, Candida glabrata, Candida
versatilis, Saccharomyces capsularis, and Pichia burtonii. The dominant bacterium was
Pediococcus pentosaceus.

Mana contains 106 cfu/g of mucorales, 107 cfu/g of Aspergilli, 103 cfu/g of yeasts, and
105 cfu of LAB (Nikkuni et al., 1996). Aspergillus oryzae and Rhizopus spp. are present in
mana (Nikkuni et al., 1996; Shrestha et al., 2002). None of the amylolytic starters of the
Himalayas have Aspergillus except mana, which is very significant (Tamang, 2010).
Aspergillus oryzae (107 cfu/g), Aspergillus niger (107 cfu/g), Rhizopus (106 cfu/g), bacteria
(107 cfu/g) and yeasts (10 5 cfu/g) were identified in nuruk. The number of molds
(103-107/g), yeasts (105-107/g) and lactic acid bacteria (105-107/g) were observed to vary
with the source and district of collection of bubod (Lee, 1999).

2.2.3 Traditional amylolytic starter technology

The use of herbs for preparing cereal-based alcoholic beverages probably dates back to
ancient times (Hill, 1937). This ancient practice has been handed down to the generation
and has become a subject of renewed interest in the scientific community. The use of
plants for the preparation of traditional amylolytic cultures in the Indian subcontinent has
21
been reported by many workers, including KC et al. (1999), Sekar and Mariapan (2007),
Das and Deka (2012), Tamang et al., (2012) and Rai et al. (2016).

Marcha-making technology reflects the native skill of ethnic people on subculturing of

desirable inocula (microorganisms consisting of filamentous molds, amylolytic and
alcohol-producing yeasts and species of LAB) from previous batch to a new culture using
rice or wheat as a starchy base or medium. This indigenous technique preserves the
functional microorganisms necessary for fermentation of starchy substrates to alcoholic
beverages in the Himalayas (Tamang, 2010).

The prepared murcha can be stored for more than a year. This art and technology is
protected as hereditary trade and passes from mother to daughters. The murcha-making
villages have linkages to nearby markets where murcha-makers sell the products once or
twice in a week in Sikkim (Tamang et al., 2012).

2.2.3.1 Rationale for use of plants, herbs and spices in starter

Various herbs and/or spices are usually included in recipes for dry starters but their role(s)
in the preparation of the starters has not been clearly established. It is generally considered
that plants used in the preparation of amylolytic starters are an inexhaustible source of
essential microorganisms (yeasts, molds and bacteria) (KC et al., 2001) but some authors
(Saikia et al., 2007) propose that leaves of few wild plants act as attracting agent of yeast
Saccharomyces cerevisiae. Murcha and ragi producers believe that addition of wild herbs
gives more sweetness to the product, and they also believe that adding chillies and ginger
get rid of devils that may spoil the product during preparation. This is actually to check the
growth of undesirable microorganisms that may inhibit the growth of native
microorganisms of ethnic starters (Soedarsono, 1972) and the addition of sweet herbs is to
supplement the carbon source for growing organisms in murcha (Tamang and Delwen,
2010). However, no comprehensive research has been conducted on either the
development of the microbial population or the effects of spices during the preparation of
starters (Nuraida and Krusong, 2015). It is also possible that the small amount of herbs
added provide some nutrients for microbial growth (Tamang and Delwen, 2010) and their
presence was observed to stimulate growth of Aspergillus rouxii and Saccharomyces
cerevisiae (Dung et al., 2005). However, there do not appear to be any reports on the
nutrients utilized in loog-paeng lao or similar types of starter cultures (Krusong, 2015).

22
 Thakur and Bhalla (2004) state that herbal mixtures in traditional alcoholic beverages
provide bioactive compounds as well as stimulatory effect. Study carried out by Panda and
Bastia (2014) in Odisha (India) revealed that the tribals had no idea about the authentic
role of plants in the fermentation. According to their (natives) knowledge, either yeast is
formed from these plants or these plants are responsible for the yeasts action in
fermentation. These authors also carried out quantitative phytochemical analysis of several
plants used for starter preparation and found presence of alkaloid, flavonoid, carbohydrate,
protein and amino acids, tannins and phenolic compounds and saponins in most plants. The
authors concluded that the plants they studied had antimicrobial properties. Rai (2006)
states in his dissertation work that inclusion of plant parts (murcha plants) not only
supplies fermentation flora but also contributes to increased surface area (by giving porous
structure) of the starter cake, thereby providing adequately aerobic environment for the
microorganisms.

Literatures abound on the different types of plants used by native peoples of different
countries of South Asia and so do on different amylolytic starters and the range of micro-
organisms they (the starters) harbor. It is very strange, except for the article by Rai and
Subba (2003), none of the work so far available report presence of yeasts and molds in the
ingredient plants.

2.2.3.2 Substrate for starter

The substrate is almost entirely raw rice starch which, in its uncooked, ungelatinized state
is relatively resistant to the activity of most amylolytic enzymes. Starch from non-sticky
rice is more popular than starch from sticky rice (Sanchez et al., 1988). In Nepal, the
substrate for murcha is usually raw rice starch (Rai and Subba, 2016). Sometimes, millet
and maize powders are also used (Limbu, 2015).

2.2.4 Amylolysis in starter

Amylolysis or amylolytic process is the hydrolysis of starch into simple sugars by the action
of acids or enzymes such as amylase (Rai and Subba, 2016). In the oriental alcoholic cereal
beverage fermentation, amylolysis occurs due to amylase enzymes elaborated by molds,
notably, species of Aspergillus, Mucor and Rhizopus (Amylomyces) (Haard et al., 1999).
These molds are known to produce -, -, and glucoamylase, -amylase being the most

23
dominant (Saranraj and Stella, 2013). Because of the important roles of fungal amylases in
cereal fermentation, a brief description of these enzymes is in order.

-amylases hydrolyze the -1,4-glycosidic linkage in starch molecule at random from the
interior part of the starch (Fig.2.8) and are therefore termed endoamylases. They rapidly
decrease the viscosity of the starch solution. -amylases sequentially hydrolyze starch
molecule from the non-reducing end to produce maltose units. The viscosity is not reduced
rapidly but the hydrolyzed product is sweet in taste. Glucoamylase (-1,6-glucosidase)
breaks down -1,6-glycosidic linkage in the branched portion of the starch molecules. Both
- and -amylases cannot attack the -1,6 linkages. They also cannot attack a few -1,4-
linkages in the vicinity of the -1,6 branch points. This inability to attack branch points of
starch by amylases results in the formation of short, highly branched fragments (residues) of
glucose polymer called limit dextrin. Before amylases can act on starch, the latter must be
converted into susceptible, gelatinized form. This is where the role of cooking cereals (in the
homes and in fermentation industries) comes in (KC and Rai, 2007).

Haard (1999) has compared the enzyme activities of koji and nuruk (Fig 2.6) from data
collected from various workers (Table 2.4). The authors conclude that enzyme activities in
koji are generally higher than those in nuruk. They opine that this may be due to the fact
that the pure culture of Aspergillus oryzae on loose cereal granules allows maximum
growth during the preparation of koji, while in nuruk manufacture, mold growth is
confined mainly to the surface of the cake or ball, thus allowing yeasts and lactic acid
bacteria to grow simultaneously, and contribute to the deeper flavor notes of Korean rice-
wine in later alcoholic fermentation stages.

Branched region
(resistant to both amylases)

Action of -amylase Action of -amylase

Fig. 2.8 Action of - and -amylase on starch molecule

24
Table 2.4 Main enzyme activities in koji and nuruk

Enzyme Koji Nuruk

-Glucosidase (Wohlgemuth value D4030/g) 1225 256

Glucoamylase (mg glucose produced/hr/g) 201 260

Acid protease (mg tyrosine produced/hr/g) 3674 181

2.2.4.1 Assay of amylase

A number of methods have been developed for the assay of amylase activity on starch.
Quantitative procedures may involve the measurement of new reducing groups, hemiacetal
or aldehyde groups that result from the hydrolysis of the glycosidic acetal linkages. The
colorimetric measurement of the formation of reducing groups has mostly been by the use
of alkaline copper, alkaline ferricyanide or alkaline 3,5-dinitrosalicylic acid. A semi-
quantitative determination of starch hydrolysis by -amylase involves measurement of the
decrease in the blue iodine color. This method is especially useful in survey of biological
samples for -amylolytic activity. The procedure reflects the endo cleavage of relatively
large starch chains and cannot be used to assay exo-acting amylase. Measurement of the
decrease in the viscosity of a starch solution has also been used to measure -amylase
activity. This method, like the iodine procedure only measures -amylase activity and the
results cannot be readily expressed in international units (Mulimani and Lalitha, 1996).

Some workers have used a modification of agar diffusion method for the assay of
amylolytic activity. Clear radial diffusion zones are measured after 4 hrs of incubation at
20C. A linear relationship between the logarithm of enzyme activities and the area of
clear zones is obtained (Mulimani and Lalitha, 1996).

Other tests include use of 3,5-dinitrosalicylic acid (DNS) reagent. Maltose (a reducing
sugar) reacts with and reduces the pale yellow colored alkaline DNS to the orange-red
colored, 3-amino, 5-nitrosalicylic acid after being heated for 5 min. Essentially, the
intensity of the color (at = 546 nm) is proportional to the concentration of maltose
present in the solution. Thus, increased amylase activity produces more maltose, which
reduces more DNS, which then turns the solution a darker orange-red (Mulimani and
Lalitha, 1996).

25
 The -amylase activity has been variously defined. According to Moulin and Galzy
(1978), one -amylase unit is defined as the amount of enzyme that hydrolyzes 10 mg
starch in 30 min under the given conditions. Mulimani and Lalitha (1996) define one unit
of enzyme activity as the amount of enzyme that liberates 1 M of maltose equivalent in 1
min, which is more generally used.

Assay of amylolytic activity is a routine test in fermentation-, pharmaceutical- and

allied industries. However, there is no standard protocol for the determination of
amylolytic activity of amylolytic starters, murcha for instance. Modifications of the
aforementioned methods have been used by some workers (Rai and Subba, 2016). A
simple method for the assay of murcha has recently been described by Rai and Subba
(2016), the principle of which is as follows:

Amylases are extracted from a known quantity of murcha sample in malate buffer and
the rate of release of reducing sugar (as maltose) by the extract from starch solution under
standard conditions of incubation (for reaction) is determined by Lane and Eynon
(Fehlings solution) method.

2.2.4.2 Relevance of amylase analysis in amylolytic starters

Amylolytic starters obviously contain small amounts of pre-formed amylases, produced

during the fermentation. Unlike malt, the primary purpose of the starter is not to supply
amylase but to include sufficient inoculum for the main fermentation (Lee, 1984; Batra and
Millner, 1974) wherein growth of microorganism, production of enzymes, and breakdown
of starch proceed in parallel (Haard et al., 1999; Rai and Subba, 2016). It is thus clear that
the pre-formed enzymes have only negligible role (unless the starter is used in very large
amounts!) in the fermentation. The assay of amylolytic activity vis--vis pre-formed
amylase, therefore, does not seem relevant for quality tests of starters. In breweries and
malting industries, however, the quality of malt is judged by analyzing amylolytic power in
terms of diastatic power or degree Lintner. For amylolytic starters, of more relevance
could be the assessment for amylolytic potential of the starter flora. However, literature on
this aspect is almost nonexistent (Rai and Subba, 2016).

26
2.2.5 Advances in amylolytic starters

Although most of the amylolytic starters are still produced under poorly controlled
conditions a few of them have reached significant success, as exemplified by Japanese koji.
Methods for preparing starters have also been standardized. For instance, Krusong (2015)
has described the preparation of Thai loog-paeng lao starter for rice wine using pure
culture, the principle steps of which is as follows:

Mix coarse and fine rice brans Moisten to 45-50% water content Autoclave the
paste at 121C for 1 h Cool Autoclave and cool the paste once more Inoculate
with Amylomyces rouxii (a mold) suspension Incubate at 35-37C for 3-5 days (until the
mycelia cover the bran) Dry at 45C Loog-paeng lao Cool and pack in bottles.
The product is stable for 4-6 months at room temperature.

Some examples of industrialized fermented foods are: Alcoholic pastes and rice wines
in Asia, such as tapai, is now produced using commercially available pure culture starters
(Merican and Yeoh, 1989); palm wine and sorghum beer in South Africa are prepared at
industrial scale using pure strains of Lactobacillus delbrueckii and Saccharomyces
cerevisiae (Haggblade and Holzapfel, 1989). Modern molecular biotechnology for starter
culture development resulted in the insertion of the -amylase gene of Saccharomycopsis
fibuligera into Saccharomyces cerevisiae with the advantage of more rapid growth and
fermentation (Yip et al., 1997). Another example is the insertion of a synthetic gene for
lysine into Saccharomyces sp. (Okafor et al., 1999). It is shown that lysine was
overproduced and excreted. When used as a fermentation starter culture, the yeast could be
used to enrich protein-poor products such as fufu. However, Nout (2003) claimed that such
expensive GMO techniques could be doubtful for practical application in view of the cost
aspects of industrial production.

2.2.6 Upgrading traditional amylolytic starters: challenges and opportunities

Going by the review, it appears that the traditional fermented cereal products are well
accepted, affordable and use local resources. It is important to ensure that their quality and
safety meet the requirements of present-day and future consumers. Upgrading traditional
home-scale processes is needed so that they can compete successfully with imported
products. Whereas small-scale manufacture has advantages of short distribution lines,

27
income generation for families etc., urbanization and the resulting growing demand for
ready-to-consume foods requires larger-scale industrial production. Ogi, a sour fermented
starch cake processed from maize, sorghum or millet grains has been industrialized in
Nigeria (Onyekwere, et al., 1989) where the fermentation is not inoculated and depends on
the natural fermenting flora. A cost analysis showed that inoculation with pure culture
starters would be unacceptably expensive, considering the infrastructure needed to
propagate and maintain appropriate quality and safety of such cultures (Nout, 2003).

Specific microflora involved with indigenous fermentations is, in many cases, not
known at this time. Specific information on microflora appears to be lacking for several
indigenous fermented cereal products. The microbiology of many of these fermentations is
undoubtedly quite complex. In this light, identifying and providing a practical means of
using appropriate starter cultures is advantageous due to the competitive role of
microorganisms and their metabolites in preventing growth and metabolism of unwanted
microorganisms. A strong starter may reduce fermentation times, minimize dry matter
losses, avoid contamination with pathogenic and toxigenic bacteria and molds, and
minimize the risk of incidental microflora causing off-flavor, etc. According to Nout
(1994) optimization of starter cultures may be achieved by either conventional selection
and mutation, or by recombinant-DNA techniques to result in increased levels of safety.
Relatively little is known of the contribution of microflora to the formation of desired
flavor notes during such fermentations. Genes for flavor and other beneficial enzymes that
come from incidental microflora may be incorporated into starter bacteria to facilitate more
subtle and ancillary aspects of the fermentation along with primary events such as lactic
acid production, thus preserving the distinctive nature of products made in different
regions (Haard, 1999).

Knowledge of domestication of wild strains of microbes has become a priority area in

food science research because of their importance in food quality, safety and shelf-life. In
an era of industrial food production, it has become necessary to identify and optimize these
naturally occurring microorganisms to produce starter cultures that contain defined types
and amounts of particular organisms to ensure the consistency, safety and quality of the
final product. Analyzing microorganisms in different environments allows quantification
of metabolic fluxes that can be used to predict and optimize starter culture performance.
Evaluating the diversity of strains within a species with similar metabolic functions is also

28
used to speed up the process of target strain improvement or produce foods with unique
sensory qualities (Borresen et al., 2012).

Techniques to stabilize fermentations operating under non-sterile conditions would

therefore be appropriate in the control of natural fermentations. For this purpose the use of
pure cultures, obtained either by laboratory selection procedures or genetic engineering,
offers no realistic solutions because they are expensive and require sterile processing
conditions. A more feasible approach is to exploit the ecological principle of inoculum
enrichment by natural selection. In the exercise of upgrading traditional food fermentation
techniques, it would therefore be worthwhile to investigate the effect of inoculum
enrichment on product characteristics and consumer acceptance (Nout, 1992).

A different tool to stabilize fermentations under non-sterile conditions is the use of

multistrain dehydrated starters, which can be stored at ambient temperatures, enabling
more flexibility. Such homemade starters are widely used in several Asian food
fermentations. Examples are the manufacture of tempeh (mainly from soybeans) and tap
(from glutinous rice or cassava). These starters are more homogenous and their dosage is
convenient, but because they are manufactured under non-sterile conditions, some are
heavily contaminated with spoilage microorganisms. This requires quality monitoring of
the inoculum and of the fermentation process in which it is used (Nout, 1992).

Food safety is an emerging frontline yardstick in the realm of food trade. The
globalization and economic liberalization have placed certain obligations to enhance
capacity building in the area of quality assurance. The food chain is considerably longer in
developing world where infrastructures are poorly developed. Hence, the food product
should be safely handled, transported and stored not to cause any deleterious effect to the
consumers (Karki, 2002). Against this backdrop, mention of the final Sanitary and
Phytosanitary (SPS) agreement developed in the Uruguay Round of GATT (General
Agreement on Tariffs and Trade) 1994 to elaborate rules for the applications which relate
to the use of sanitary or phytosanitary measures becomes pertinent (Karki et al., 2004).

The SPS agreement reinforces the right of World Trade Organization (WTO) member
countries to apply measures necessary to protect human, animal and plant life and health.
Generally the developing countries apply lower SPS standards, qualitatively or
quantitatively, than developed countries (Karki et al., 2004).

29
 To this end, in exercise of the powers conferred by Section 35 of the Plant Protection
Act, 2007, the Government of Nepal has framed Plant Protection Rules, 2010. The rule
and regulations deal, among others, with genetically modified organisms, transgenic
substances, insect pests and their quarantine importance (Nepal Gazette, 2010).

Virtually all agricultural and many non-agricultural commodities can be the subject of
phytosanitary measures at some time or another because they are either hosts or incidental
carriers of unwanted pest organisms. For instance despite the cosmopolitan nature of most
pests of durable commodities a few remain that are subject to wide-ranging, quarantine-
based prohibitions. Of these, the khapra beetle and the larger grain borer are possibly of
greatest importance. Other quarantine pests include warehouse beetle (Trogoderma
variabile) and rice weevil (Sitophilus oryzae) (Heather and Hallman, 2008).

Amylolytic starters of Nepal have a shelf-life of about 1 year (Tamang et al., 2012). It is
well known that these starters kept in the homes become unsightly within a few months
because of the infestation by weevils (Fig. 2.9). An earlier survey by the present worker
has revealed development of large numbers of weevils in murcha samples stored for
duration of 3 months (Fig. 2.9). The weevils were identified to be Sitophilus oryzae species
of Callosobruchus. This indicates that there is ample scope for study on industrialization
and cross-border trade vis--vis Sanitary and Phytosanitary aspects of murcha.

The traditional method of murcha preparation has many shortcomings. The quality of
murcha is never consistent and thus the quality of jand. Maintenance and use of pure
cultures in jand preparation is an attractive option but at the same time it offers less
practicability in the tribal setting where jand is produced with very little technical
proficiency. A better option would be to develop a mixed culture that is relatively easy to
maintain and propagate (Hesseltine, 1992).

Available literatures on amylolytic fermentation starters to date indicate that starter

preparation can entail several levels of complexity. At the simplest, a good quality starter
can be screened and propagated by traditional method (KC et al., 2004). At the most
complex, individual organisms can be isolated, characterized, improved (by mutation,
genetic engineering, etc.), and developed into multistrain starter. The latter option is of
significance for large-scale production of good quality starter. However, this is of remote
possibility for underdeveloped countries because of the high level of technical know-how

30
and capital outlay required. Underdeveloped countries require options of intermediate
complexity, which can be addressed with appropriate technology. Since the basic
principles underlying all amylolytic fermentations are the same, this understanding can be
gainfully employed for developing starters that are safe and of consistent and reliable
quality. Basic concepts of microbiology, biochemistry, food processing, asepsis, etc., are of
course very essential.

Sitophilus oryzae in Callosobruchus sp. in Packaging materials destroyed by weevils

murcha murcha

Fig. 2.9 Infestation of murcha with insects

2.3 Traditional methods used for the study of microbial profile of amylolytic starters

Various methods have been described by different authors, including Hesseltine et al.
(1988), KC et al. (1999), Tamang and Sarkar (1995), Rai (2006), Rai and Subba (2016) for
the study of microbial profile of amylolytic starters. A brief description for the same is
given in following paragraphs:

2.3.1 Mold characterization and preservation

An excellent account of mold isolation techniques have been given by Malloch (1997).
These techniques can be divided into two broad categories: (1) Direct methods, and
(2) Selective methods. Both are routinely used in mycology laboratories and can be further
divided into number of subtypes.

31
 It is often most convenient to use the direct plating method for the mold isolation from
food samples, which entails direct inoculation of the material on nutrient agar amended
with Martins Rose Bengal Medium and then incubation for a few days (Malloch, 1997).

KC et al. (2004) has described a very simple method for the isolation of saccharifying
molds from murcha. The method involves spread-plating of broth sample on MYGP, and
incubation at 30C for 2-3 days to get mold colonies. The saccharification ability is tested
by inoculating the isolate on to cooked rice substrate and incubation for some days for the
evidence of liquefaction.

Identification of molds is based almost entirely on the morphological characteristics

including spore-bearing structure and on the spores themselves. The most common means
of identifying molds at the level of genera is by the use of dichotomous keys, a system
presenting a series of alternatives for consideration. The text keys are often accompanied
by picture keys. Keys to sixty common genera of molds prepared by Malloch (1997)
appear in Appendix B-1. The original version published in the internet is fully interactive,
and provides faster identification. Harrigan and McCance (1976) have also given an
excellent account of mold identification procedures.

Mold identification requires preparation of slides for microscopic examination. Some

basic techniques have been described by several authors, including Aneja (1996), Malloch
(1997), and KC et al. (2004), the last one being probably the simplest method. This
method, termed tape culture, basically involves inverting a cello-tape over the colony to
stick the spores and mycelia followed by adhesion on a clean slide for microscopic
examination under objectives of different magnifications.

The mold cultures can be managed by many methods, such as in soil, in slants, in flat
bottles (FAO, 2016) or in agar plates. The stock plates are packed in air-tight polyethylene
bag and stored in refrigerator. Alternatively, masking tape is used around the edge of the
plate to seal it and then stored in refrigerator (KC et al., 2004). In most traditional
fermentations, the cultures are maintained as starter cultures such as ragi, koji, and murcha
(Lee, 1999).

32
2.3.2 Yeast characterization and preservation

Various techniques for yeasts isolation from natural sources have been described by
different authors (Kirsop, 1987; Barnett et al., 1990; Hayford and Jespersen, 1998;
Kaufmann, 1998), the protocols largely depending on the purpose and objective. For the
most part, fermentative yeasts are cultured in Wickerham medium (Prescott & Dunn,
1959), commonly called Malt Yeast extract Glucose Peptone (MYGP) medium. Inclusion
of malt promotes faster yeast growth and hence better isolation. The pH of the medium is
kept at around 4.0 so that the condition becomes selective for yeast growth. Rai and Subba
(2016) have used molasses broth and agar for same.

Fermentative yeasts from murcha samples can be easily isolated by the method
described by KC et al. (1999) and Rai and Subba (2016). The isolation process consists of
pre-fermentation, separation of molds, microscopic examination of broth for the presence
of yeasts, spread-culturing on MYGP agar, further microscopic examination of isolated
colonies, and subculturing. Pre-fermentation exerts a selective pressure against the non-
fermentative organisms. Molds can be easily separated from the yeast suspension. Finally,
spread-plating provides equal chance for all fermentative yeast-types to be isolated.

Selective isolation of wild yeast types (for differentiation from culture yeasts) have also
been described by different investigators (Van der Aa Kuhle and Jespersen, 1998; Helbert,
1987).

Traditionally, yeasts are identified by morphological and physiological criteria (Kreger

van Rij, 1984; Lachance, 1987; Barnett et al., 1990; Suh et al., 2008; Yarrow, 1998).
Auxanography and additional tests such as sugar fermentation test, urea hydrolysis and
nitrogen assimilation test are used for identification of species.

Auxanography (sugar-assimilation test) is routinely used for characterizing yeasts at the

level of species (Lachance, 1987). The most frequently used test sugars for the assimilation
test are glucose, galactose, sucrose, maltose, lactose and raffinose. However, in some cases
other sugars are also tested. Rai and Subba (2004) have described modified auxanogram for
sugar-substrates. The authors claim that the method is much faster and more economic than
conventional method and is particularly suitable when dealing with a small number of yeast
types (less than 8). The modification of the routine method described here entails preparation

33
of sugar plates on which yeast isolates are inoculated. The response of yeast cells to different
sugars are consistently and quickly established by this method.

Yeasts are identified at the level of genera on the basis of vegetative features and cell
morphology including cell shape and size, the mode of budding and the presence of
unusual organelles (Lachance, 1987). Most methods use dichotomous keys for the
differentiation of yeast genera. An example of key described by Adams and Moss (1996) is
given in Appendix B-2. The keys indicate how some of the important genera of yeasts
differ from each other. The key to differentiation of Saccharomyces cerevisiae is given in
the Appendix B-3 (Harrigan and McCance, 1976).

A good review on yeast preservation and maintenance is given by Kirsop (1987) which
includes subculturing/active transfer, drying/desiccation, freeze-drying and freezing/
cryopreservation. Although subculturing is not very reliable for long-term purpose, it is the
simplest and easiest method. Yeast isolates from plant sources can be maintained by active
transfer (subculturing every 3-6 months) and storage at refrigeration temperature without
any significant change in functional properties (Rai and Subba, 2003).

2.4 Newer methods for the study of microbial profile

Modern methods of yeast identification (at the level of species) are more sophisticated in
that they utilize molecular techniques and have been described by several investigators
(Granchi et al., 1999; Hayford and Jespersen; 1998; Vasdinyei and Deak, 2003).

2.5 Assessment of the quality of murcha

Several authors have assessed the quality attributes of murcha in terms of

physicochemical and microbiological properties of murcha cake. According to Tamang and
Sarkar, (1995), murcha cakes are mildly acidic (pH 5.2), contain 13% (w/w) moisture, and
0.7% ash (w/w, dry basis). They have isolated a total of 194 bacterial, 190 yeast, and 80
mold strains from 30 samples of murcha. The counts (cfu/g fresh weight) of microorganisms
in the samples were 2.0107-4.2108 for Pediococcus pentosaceus, 4.0107-6.8108 for
Saccharomyces fibuligera, 2.0106-4.2107 for Pichia anomala, 1.0106-4.1107 for Mucor
circinelloides and less than 106 for Rhizopus chinensis. However, standard value for these
parameters for good quality murcha has not been established as yet. Moreover, there is no
any standard protocol available for the quality assessment of murcha till date. Therefore,

34
quality assessment of murcha almost totally depends on perception of people, experience
shared by murcha makers and final quality of jand.

Physical appearance of murcha such as shape, size and color cannot indicate the quality
of murcha, as standard shape and size for murcha cakes are not established while color may
vary according to raw materials used. The microbiological analysis (for mold and yeast
quality) and test fermentation are of course important but are not feasible in the rural setting.

35
 PART III

Materials and Methods

3.1 Materials

3.1.1 Survey and sample collection

Ten sites (Saangu, Udayapur, Kerabari, Dhankuta, Belbari, Laxmimarga, Dandaghopa,

Bishnupaduka, Panmara and Letang) representing 5 districts (Morang, Sunsari, Dhankuta,
Udayapur and Taplejung) (Fig. 3.1) were chosen for survey and murcha samples prepared by
the natives were purchased. Since the shelf life of murcha has been reported to be 1 year
(Tamang et al., 2012) tentative date of murcha production was confirmed from the murcha
seller to ensure that the sample was not too old (less than 3 weeks of production).

Solukhumbu
Sankhuwasabha Taplejung

1
Okhaldhunga
Khotang Bhojpur
N
3
Udayapur Dhankuta
W E Ilam
2

Siraha 4
S Sunsari Morang
Saptari 6 7
10 Jhapa
5
8 9

0 25 km
1 Saangu 2 Udayapur 3 Dhankuta 4 Bishnupaduka 5 Dandaghopa
6 Panmara 7 Kerabari 8 Belbari 9 Laxmimarga 10 Letang

Fig. 3.1 Survey and murcha sample collection site (Eastern Nepal)

General survey was carried out using semi-structured questionnaire (Appendix A-2).
From each site 3 respondents (who were either murcha producers/murcha sellers) were
picked upon consultation with the key informants (consisting of village heads, school
teachers, senior citizens belonging to ethnic group). To wrest honest response, help was
taken from local people as mediators.

3.1.2 Other materials

Cane molasses (high-test) needed for the preliminary screening of the yeast culture (Rai and
Subba, 2003) was bought from local market of Dharan. Media needed for isolation and
identification were obtained from the campus and confirmed to be of Hi-Media, India.
Photomicrographs of molds and yeasts were taken by high resolution digital camera. Utensils
(culture vessels), equipment (blender, mortar-pestle, cabinet dryer, oil-immersion microscope,
autoclave, oven, incubator, sieve, humidity chamber, IR moisture meter, etc.) and glassware
needed for the work was obtained from the campus. Chemicals intended for use as extractant,
solvent, and standard were obtained the campus and confirmed to be of Merck, India.

3.2 Methods

3.2.1 Physicochemical and microbiological analysis of murcha samples

After collection, the samples were packed in reclosable plastic pouches in order to avoid
cross-contamination. Murcha samples were tested for three important quality attributes,
viz., (i) physicochemical properties, which relate to shelf -life and standardization,
(ii) microbial profile, which relates to safety, contamination, and the profile of essential
microorganisms, (iii) performance which relates to quality of jand.

Moisture content of murcha samples were determined by routine IR method (KC and
Rai, 2007). Bulk density was determined indirectly by immersing the sample cakes (with
known weight) in a measuring cylinder filled with rapeseed (Rai and Subba, 2016). pH was
measured on 10% aqueous suspension of the cakes with a portable digital pH meter (Hanna
make, sensitivity 0.01 unit). Dimensions (length, breadth, height) of murcha cakes and
balls were determined directly with the help of measuring scale.

Microbial profile was determined giving emphasis to the enumeration of essential

molds and yeasts, aerobic mesophiles, and coliforms. Yeast count was done in 4%
molasses agar by serial dilution of murcha cake (Rai, 2013) whereas mold count was done
in Potato Dextrose Agar (PDA) amended with Rose Bengal (35 ppm). Coliform and total
plate counts were carried out on (Violet Red Bile Agar) VRBA and Plate Count Agar
(PCA), respectively, following the methods described by Harrigan and McCance (1976).
37
 Performance of murcha samples were determined by trial fermentation test, followed by
sensory analysis of the product. Trial fermentation was done on cooked rice in small batches
(~ 100 g cooked rice) using 1 g of murcha. Fermentation was carried out in transparent PET
jar securing lid for 7 days and assessed for sensory quality in order to test the workability of
the murcha or starter. An overview of the method used is given in Fig. 3.2.

Survey

Collection of murcha samples

General analysis of murcha

(Physicochemical and microbiological)

Screening of amylolytic
molds and yeasts

Characterization of isolates

Culture propagation
(Mold koji and yeast suspension)

Formulation of murcha

Testing of murcha quality

(Physicochemical, performance, sensory)

Data analysis

Fig. 3.2 A recapitulative outline of the method followed

3.2.2 Screening of essential microorganisms (yeasts and molds)

Traditional murcha contains a plethora of microorganisms, both desirable and undesirable.

Although the fermentation process tends to select the hardiest ones naturally, the process is
not always reliable. It is therefore important to screen for only the useful microorganisms
for producing murcha of consistent quality.

3.2.2.1 Preliminary screening of murcha sample

The collected murcha samples were first tested for suitability by inoculating cooked rice to
produce jand. This step was also thought necessary to avoid the screening load.
Accordingly, ~ 100 g of cooked rice was inoculated with 1 g of murcha sample left for
fermentation left for fermentation (in small plastic jars) of 15 days. Jand produced in this

38
preliminary step were subjected to general analysis (liquefaction, taste, smell, sourness and
overall) as the important criteria for selection. The number of murcha samples was reduced
after ANOVA of the test data.

3.2.2.2 Screening of fermentative yeasts from murcha

The murcha samples selected in Section 3.2.2.1 were used for screening of fermentative
yeasts by the technique described by KC et al. (1999). Accordingly, 10 g of murcha
powder was suspended in 100 ml of sterile enrichment broth (15% molasses broth, pH 4.5
with H2SO4, boiled and cooled) and incubated at 30C for a week (with occasional
agitation for aeration). Alcoholic smell and gas formation were observed for evidence of
fermentation. Only those broths that showed evidence of alcoholic fermentation and
exhibited rapid drop in TSS were selected for microscopic examination. Microscopic
examination of the fermenting broth was carried out by negative staining (for ascertaining
the presence of yeast cells), the procedure being described in Section 3.2.2.2.2. The yeast-
positive broths were spread-plated on a series of 5-6 molasses agar plates and incubated
again at 30C for 2-3 days. The plates that bore the well-isolated colonies were selected
and subcultured in molasses agar (flat bottles and slants, Appendix C) for stock culture.

3.2.2.2.1 Testing of yeast performance

The performance of each yeast isolate was tested by pitching 4 loopful of yeast culture
from the preserved slant (Section 3.2.2.2) in 750 ml of sterile high-test cane molasses
adjusted to 15 brix and pH 4.5 (with citric acid). Fermentation was carried out in cotton
plugged 1-lit PET bottle at 30C until the TSS ceased to decrease further. The
characteristics of the isolates (e.g., flocculation, foaming) and the beer itself (taste, smell,
etc.) were noted to get an idea regarding their suitability in starter preparation. The beer
was analyzed for sensory attributes (perceived alcoholic property, clarity, smell, color and
sourness) using 9-point hedonic rating (Rai and Subba 2016) and data analyzed for
ANOVA using Genstat v12 (Payne et al, 2009) to select only two isolates (the best and the
second best) for further study.

Many authors have reported that microorganisms growing in natural substrates exhibit
natural selection. A particular microorganism seems to be predominant at the beginning of
growth may be suppressed over time by the other microorganism which ultimately

39
becomes the dominant one (Nout, 1992). This ultimate dominant microorganism was
assumed to produce better quality end product. In this light, new approach for yeast
screening can also be carried out (spread plating) which entails the use of inoculum from
fermented mash of matured jand that selects for dominant yeasts.

3.2.2.2.2 Identification and preservation of fermentative yeasts

The yeasts (the two most potential isolates) selected in Section 3.2.2.2.1 were subjected to
microscopic examination (oil immersion objective) and pictures were taken in high-
resolution trinocular microscope. For this purpose, negative staining of yeast with
nigrosine was carried out as described by Aneja (2003) and Rai and Subba (2016). The
colony characteristics of isolated yeasts were studied and noted following method in
Barnett et al. (1990). Also the yeast isolates were further characterized by modified
auxanography (sugar assimilation test) given by Rai and Subba (2004) and sugar
fermentation test described by Suh et al. (2008) with some modification.

The modified auxanography method mentioned earlier entailed preparation of seeded

Yeast Nitrogen Base (YNB) media plates (0.1% KH2PO4, 0.05% MgSO4, 0.5% (NH4)2SO4,
2% agar) on to which test-sugars (~ 100 mg) were added. The sugars used were dextrose,
galactose, sucrose, xylose, maltose and lactose. Care was taken not to place the sugars too
near as this causes cross-feeding problem. Then, the plates were incubated for 2-3 days at
30oC and the growth responses of yeast cells to different sugars were observed (Rai and
Subba, 2004).The yeast was tentatively characterized using key due to Kurtzman et al.
(1998), a truncated form of which is given in Appendix B-3. Fig. 3.3 shows an
arrangement for sugar assimilation test used in this study.

Fig. 3.3 Scheme for sugar assimilation test

(1, 2, 3, 4, 5, and 6 imply sugars (glucose, galactose, sucrose, xylose, maltose and lactose)

Sugar fermentation test was conducted in order to determine the type of yeast that was
isolated in lab. This method entailed preparation of basal medium (0.1% KH2PO4, 0.05%

40
MgSO4, 0.5% (NH4)2SO4, 0.45% peptone and 2% sugar) with phenol red (1 mg/ml) as
indicator. The sugars used were dextrose, galactose, sucrose, xylose, maltose and lactose.
The medium was dispensed into test tubes containing Durham tube (inverted) and
sterilized by autoclaving at 121C for 15 min. After cooling the media to room
temperature, 0.1 ml suspension of pure yeast isolate was inoculated to each test tube and
incubated at 25C up to 72 hr. Yeast activity and fermentation was indicated by gas
formation and change in color from red to yellow. Control tubes were used in each set to
monitor contamination and also compare change in color (Tiwari et al., 2007). An abridged
version of the key for sugar assimilation- and fermentation tests is given in Appendix B-3.
The isolated yeasts were preserved following method given by Kirsop (1987) but using
molasses agar in normal slants. The slants were streaked with yeast culture and incubated
at 273C for 2-5 days till luxurious growth was observed. Finally they were stored under
refrigeration at 4C.

3.2.2.3 Screening of molds from murcha

Murcha sample screened in 3.2.2.1 was used for the screening of molds. Screening was
carried out on molasses agar using a method described by Rai (2016). Small pieces of
murcha (~ 3 mm 3 mm) were embedded at 2-3 places with the help of tweezers in
molasses agar, keeping at least 1.5 cm space between the pieces.

The plates were incubated at 27-28C for 2-3 days and observed daily for cottony
growth. The distinctly different colonies were subcultured on a fresh molasses agar plate
by spot-culturing with the help of inoculating needle.

3.2.2.3.1 Identification and preservation of amylolytic molds

The most desirable type of mold (only one type) was selected by first testing the
performance of the mold following rapid method described in (Rai and Subba, 2016). The
test was done by inoculating small lots of cooked rice (~ 100 g) in transparent PET jars
with mold isolates from the plate (Section 3.2.2.3) and incubating for 10 days at 28-30C.
The amylolytic molds were selected based on sensory (sweetness, sourness, smell, overall)
and liquefaction (visual observation of limpid liquid in the mash).

The selected mold was preserved in molasses agar plates and flat bottles for future use
(characterization and koji preparation). The stock plates were sealed with a masking tape

41
by running the tape around the plate perimetrically and stored in refrigerator at 4C. The
selected mold was examined under microscope (at 10 objective) by preparing tape culture
(Rai and Subba, 2016). Dichotomous keys (Malloch, 1997; Harrigan and McCance, 1976)
were used for the identification (Appendix B-1).

3.2.3 Preparation of murcha

Murcha was prepared aseptically following a slight modification of the method described
for loog- paeng lao by Krusong (2015) and Rai (2016), using wheat bran instead of rice
bran. The details of the process are given in the sections to follow.

3.2.3.1 Rice flour preparation

Non-sticky variety of rice (2 kg) was washed and steeped for 3 hrs in warm water (40C)
that was acidified to pH 2.5 with 2% aqueous citric acid solution. The low pH was used to
discourage bacterial growth during steeping (Rai and Subba, 2016). The steeped rice was
drained in a clean flour sieve and ground in an iron mortar pestle followed by an electric
grinder to give a moist rice flour containing ~ 30% moisture. The flour used for murcha
preparation was subjected to sieve analysis to get an idea about the distribution of particle
size. The standard test sieve manufactured by Associated Scientific and Engg. Works
(ASEW), India was used for sieve analysis. Only the flour fraction that was < 280 was
used for murcha making. Course particles do not stick well and murcha easily crumbles
during handling.

3.2.3.2 Preparation of wheat bran koji

Molds selected in 3.2.2.4 were propagated aseptically in sterile (autoclaved) wheat bran to
produce koji. The process involved profuse sporulation of mold on molasses agar (at 28-
30C, for 4-5 days) before propagation. The details of the method have been provided by
Rai and Subba (2016). Propagation was done in sterilized, moist wheat bran in a 1-lit
conical flask for 5-6 days at 28-30C and ~ 95% RH (Fig. 3.4). The koji was taken out,
mycelial network broken, dried in an electronic dryer at 501C until around 7% moisture.
The koji was then packed in a reclosable polyethylene bag and stored at 5C until needed.
Asepsis was maintained throughout the operation. In the meantime, mold count was
performed in the koji by plating on PDA amended with 35 ppm of Rose Bengal. A
recapitulative diagram for murcha preparation is given in Fig.3.4.
42
3.2.3.3 Starter preparation

Wheat bran koji (Section 3.2.3.2) was mixed with yeast isolates (Section 3.2.2.2.2) and
propagated in moist rice flour (Section 3.2.3.1) as a carrier-cum-medium. For every 50 g of
moist rice flour, calculated amount of dry koji (~ 7% moisture content) and thick yeast
suspension (obtained by propagating in the laboratory in 10% molasses broth in conical
flask) were added.

The experimental design (Central Composite, Face-centered, 2 Factors, 3 Levels and 13

Runs, Table 3.1) was done with Design Expert V7.25. The experimental plan for Response
Surface analysis is given in Table 3.2. For every 50 g of rice flour three levels of koji (1, 2
and 3 g) and 3 levels of yeast suspension (0.25, 0.5 and 0.75 ml) were used. The details of
the runs are given in the experimental plan (Table 3.2).

Preparation of the cakes was done according to Rai and Subba (2016). An exact outline
of the preparation is given in Fig. 3.4. Briefly, the admixture was formed into a stiff dough
(~ 43% moisture, adjusted by adding 10 ml distilled water). The dough was then molded
into circular cakes by placing over muslin-lined regular Petri plate. The plate was inverted
on a muslin-lined wire-mesh to get circular cakes (dia, thickness). The molded cakes were
finally covered with sterile wet muslin cloth and allowed to ferment for 2 days at 28-30C.

The swelling of cake, appearance of profuse mycelia, and prevalence of sweet alcoholic
smell were taken as indicator for the completion of fermentation. The cakes were dried at
501C in a cabinet drier (Appendix C) having provision for hot air circulation for 6 hrs to
reduce moisture content to ~ 9%. The cakes (Fig. 3.4) were finally packed in polyethylene
bag, sealed and stored in refrigerator.

43
 Soaking of rice in
acidulated water
(45oC/~3 hrs) Slant culture

Grinding Broth culture

Water

Kneading Koji
(43% mc)
Dough
Muslin lining
Petri plate

Meshed rack

Cake
Incubation

Drying

Murcha

Fig. 3.4 A recapitulative diagram for murcha preparation

Table 3.1 Experimental design for the formulation of amylolytic starter using yeast and
mold isolates

Mold (g) 2 2 1 2 3 1 3 1 2 3 2 2 2

Yeast (ml) 0.5 0.5 0.5 0.5 0.75 0.75 0.50 0.25 0.50 0.25 0.50 0.25 0.75

44
Table 3.2 Experimental plan for response surface analysis (Format generated by Design
expert 7.25)

Factor 1 Factor 2 Response 1 Response 2

Standard Run A: Mold B: Yeast Alcohol Amylase
(g)a (ml)b (% abv) (Unit)c
10 1 2 0.5
11 2 2 0.5
8 3 2 0.75
3 4 1 0.75
1 5 1 0.25
5 6 1 0.5
7 7 2 0.25
2 8 3 0.25
12 9 2 0.5
13 10 2 0.5
9 11 2 0.5
6 12 3 0.5
4 13 3 0.75

(g)a = g of koji (1 g koji ~ 108 Rhizopus oryzae spores): (ml)b = ml of suspension of yeast
isolate (Saccharomyces cerevisiae), where 1 ml ~ 106 cells; (Unit)c = amount of enzyme
(murcha) needed to liberate 1 M of maltose equivalent in 1 min at 40C.

The spore count and cell counts were carried out as described in Section 3.2.3.2.

3.2.4 Testing of performance of formulated starter

Performance of the formulated starter was tested by trial fermentation (15 days at 28-30C)
on cooked rice (100 g cooked rice inoculated with 1% prepared murcha), followed by
sensory and chemical analysis of the product. The best formulation was selected on the
basis of sensory (taste, acidity balance, liquefaction of fermented mash), diastatic activity,
and alcohol content (Section 3.2.4.1 3.2.4.3).

45
3.2.4.1 Physicochemical analysis of prepared starter

The relevant physicochemical analysis of formulated starters included amylase activity and
alcohol content. Amylase activity was carried out using modified method given by Rai and
Subba (2016), a working outline of which is given in Fig. 3.5. The amylase activity was
calculated as amount of enzyme that liberates 1 M of maltose equivalent in 1 min at 40C
(Mulimani and Lalitha, 1996). Alcohol content was determined by pycnometric method,
also described by Rai and Subba (2016).

Murcha
(5 g)

Suspension in
50 ml maleate
buffer (pH 5.4)

Centrifuge for 10 min

(1000 rpm)

Distilled water Supernatant

(5 ml) 5 ml 5 ml

Conical flask Conical flask

(Control 1) Conical flask (Sample)
(Control 2)
Add starch solution
Add starch solution (20 ml, 1% starch solution)
(20 ml, 1% starch solution) Add warm water
(45 ml) Add warm water
Add warm water (25 ml)
(25 ml)
Add NaOH Incubate
(5 ml, 1N) (15 min, 40oC)
Add NaOH
(5 ml, 1N)
Add NaOH
Bring to boil (5 ml, 1N)
Bring to boil
Bring to boil
Incubate
Incubate (15 min, 40oC)
(15 min, 40oC) Cool to ~ 40oC

Titration with Titration with Titration with

Fehling solution Fehling solution Fehling solution

Fig. 3.5 Schematic outline of amylase activity determination

46
3.2.4.2 Sensory evaluation of jand from the formulated murcha

Sensory evaluation of jand (whole mash) was done using selected panelists (familiar to the
taste of jand) on a 5-point hedonic rating (5 = like extremely, 1 = dislike extremely) for
liquefaction, acid-balance, and taste. The evaluation method described by Ranganna (1986)
and Mabesa (1986) was adopted for the purpose. The specimen evaluation card is given in
Appendix A-3.

3.2.5 Final fermentation using the selected murcha

The selected starter was finally tested on both cooked rice and cooked finger millet (~ 2 kg
each) for the comparative study. Finger millet was chosen for the final test because of two
reasons, viz., (i) finger millet is supposed to give jand of unmatchable quality, and (ii) it is
tougher for murcha organisms to ferment finger millet. The latter reason relates to
hardiness of the murcha flora. It was assumed (in the study) that a murcha that is capable
of quickly fermenting millet will almost easily ferment the common cereals like rice, wheat
and maize (Rai, 2006).

Because of the increase in size of the mash, a biomass build-up period (aerobic
fermentation) of 1 day was used (Rai and Subba, 2016). The preparation of millet for jand
fermentation was done according to the method described by Rai and Subba (2016). The
appearance of fuzzy mycelial growth and prevalence of sweet smell was taken as an
indicator for completion of aerobic growth of essential organisms. The mash was then
transferred to a clean, transparent glass jar, bringing it almost to full, and the lid secured.
The mash was observed at weekly interval for the evidence of saccharification and
formation of alcohol.

3.2.5.1 Analysis of the final jand

Both millet and rice jand were subjected to physicochemical analysis for comparison.
Acidity was performed on the jand mash whereas other properties (volatile acidity, ester,
aldehydes) were performed on the distillate.

3.2.5.1.1 Acidity

Acidity was determined on 10 g mash by titrimetric method (Ranganna, 1986) using 0.1N
NaOH as the base.

47
3.2.5.1.2 Alcohol content

Alcohol content was determined by pycnometric method (KC and Rai, 2016; AOAC,
2005) taking 200 g of jand and 200 ml of water in a 1-liter distillation flask where 90 ml
distillate (raksi) was collected and volume was made up to 100 ml. The apparent density of
the distillate was compared with the standard chart to % obtain alcohol by volume.

3.2.5.1.3 Volatile acidity

Volatile acidity was determined on 50 ml alcohol distillate using 0.05 N NaOH as the base
(FSSAI, 2012).

3.2.5.1.4 Ester value

Ester value of distillate was determined as per FSSAI method (FSSAI, 2012). Briefly, 50
ml of neutralized distillate was taken in a reflux flask to which 10 ml 0.1 N NaOH was
added and refluxed for 1 hr. Then, it was cooled and titrated with 0.1 N H2SO4. Blank was
carried out simultaneously taking 50 ml of water instead of distillate in the same way.
Finally, ester content was calculated using following expression:

Ester expressed as ethyl acetate, V 0.0088 100 1000 2

g/100 L absolute alcohol V1

Where, V= difference of titer value of std. H2SO4 used for blank and sample, in ml.
V1= alcohol % by volume

3.2.5.1.5 Total aldehyde

Total aldehyde as g acetaldehyde / 100 lit alcohol was determined by the method given by
FSSAI, (2012). 50 ml of distillate was taken in 250 ml iodine flask and 10 ml of bisulphite
solution was added. The flask was kept in dark place for 30 min with occasional shaking.
Then, 25 ml of standard iodine solution was added and then it was back titrated with
excess iodine against standard thiosulfate solution using starch indicator to light green end
point. A blank was similarly carried out using 50 ml of distilled water in place of the
distillate. The difference in titer value (in ml), of sodium thiosulfate solution gives the
equivalent aldehyde content, which is calculated using the following expression:

48
 Aldehydes expressed acetaldehyde V 0.0011 100 1000 2
=
(g per 100 lit of absolute alcohol) V1

Where, V = difference in titer of blank and sample in ml of sodium thiosulfate solution

V1 = alcohol % by volume

3.2.6 Data analysis

The work was carried out in duplicate (starting from the research culture) while the
analyses were carried out in triplicate. Data on microbial and physicochemical parameters
of starter and murcha were tabulated for comparison and a descriptive treatment given.
Data on sensory quality of jand from different starters (laboratory) and murcha were
statistically processed by Genstat Release v12 (Payne et al., 2009) for Two Way Analysis
of Variance (ANOVA). Means of the data were compared using LSD (least significance
difference) method at 5% level of significance. Data on alcohol content was processed
using Design expert for response surface curves, contour plots, and model equations.

49
 PART IV

Results and discussion

4.1 Survey

Murcha samples were collected from 10 different selected sites (Saangu, Udayapur,
Kerabari, Dhankuta, Belbari, Laxmimarga, Dandaghopa, Bishnupaduka, Panmara and
Letang) representing 5 districts (Morang, Dhankuta, Sunsari, Udayapur and Taplejung) of
Eastern Nepal. The results showed that 90% of the sellers were ethnic females. The
production of murcha ranged from 100-200 cakes per week per seller (earning NRs 1000-
2000) which translates into NRs 48,000-96,000 annually. This sum of money is important
for the livelihood of murcha traders, as this is the sole source of income for many of them.
A huge part of their income was found to be invested for schooling of their children.
Murcha trade reflects important socio-economic activity in enhancing livelihood and
improving social well-being of many ethnic groups of Nepal. The survey results showed
that majority of ethnic people engaged in murcha trade were farmers and among them most
were illiterate. Some of them were found to be literate having academic qualification
ranging from school level to diploma level.

4.2 Physicochemical and microbiological quality of murcha

Murcha samples were tested for three important quality attributes (Rai, 2006), viz.,
(i) physicochemical properties, which relate to shelf-life and standardization, (ii) microbial
profile, which relates to safety, contamination, and the profile of essential microorganisms,
and (iii) performance which relates to quality of jand (already covered in Section 3.2.1).
The physicochemical properties of murcha are given in Table 4.1.

From Table 4.1, it is evident that most murcha prepared in the Eastern Nepal are in the
form of flat cakes with highly variable sizes (4-20 cm dia). The thickness is less variable
(1 cm on average). The moisture content was also found to vary a lot, ranging from 7.7-
15.4%. The bulk density was always less than 0.75 kg/lit. The pH ranged from 4.6-5.5. The
value of pH and moisture was found to be in the range given by Tamang and Sarkar (1995)
(pH 5.2 and moisture 13%). The implications of these physicochemical properties are
largely a matter of conjecture because literatures dealing with these aspects are not
available. Low moisture is important in the preservation of foods but may be detrimental to
the survival of yeasts and molds that have certain requirements for water activity. The low
pH is presumably due to accumulation of metabolic organic acids. As such, low pH is
favorable for the growth of yeast and inhibitory to most bacterial contaminants. Bulk
density is a measure of intragranular porosity. Higher bulk density leads to better growth of
molds but the starter may require more space for storage when produced in mass scale.

Table 4.1 Physicochemical properties of murcha samples

Murcha Moisture Bulk density pH Dimension Remarks

source (%) (kg/lit) (10% aq) (l, b, h) cm
Letang 13.1 0.65 5 13, 11.6, 1 Irregular cake

Bishnupaduka 12.8 0.5 5.3 7, 7, 1.5 Thick, circular cake

Laxmimarga 7.7 0.6 5.5 11, 12, 0.5 Thin cake

Udayapur 14.5 0.6 5.5 4, 6.6, 2 Cylindrical

Saangu 12.5 0.6 4.9 17, 20, 1 Irregular large cake

Dandaghopa 12 0.4 5.5 8.5, 8.5, 1.7 Thick cake

Panmara 11.5 0.8 4.9 10.2, 10.5, 1 Circular cake

Belbari 13.3 0.74 5.6 7.5, 7.5, 0.8 Circular cake

Kerabari 15.4 0.54 5.5 13, 12.5, 1 Circular cake

Dhankuta 13.55 0.54 4.6 9, 9, 1 Circular cake

The very often studied microbiological properties of starters are total plate count, yeast
and mold count, and coliform count. Coliform count indicates the sanitary history of
murcha, total plate count indicates (generally) the bacterial load, and yeast and mold
counts give an idea about the abundance of essential yeasts and molds present. The
microbiological analysis of 8 murcha samples, 2 being rejected during the preliminary
screening, is given in Table 4.2 (Section 4.4).

Table 4.2 shows that the bacterial load ranging from 4.76106 1.25108 cfu/ml. These
values are far greater than those reported by Rai (2006) (a maximum of 2103 cfu/ml) but
similar to the findings of Tamang and Sarkar (1995) (a maximum of 6.8108 cfu/ml). The

51
yeast and mold counts were also similar to those reported by Tamang and Sarkar (2010).
Every murcha samples were found to harbor greater number of yeast population than mold.
Molds, being strict aerobes are only capable of growing on the surface of starter cakes
whereas yeasts (needs minimum oxygen content for growth) can grow both on the surface
and inside the compact starter cakes. This has most probably resulted in greater yeast count
than that of mold count. Out of the 8 murcha samples analyzed, coliforms were detected in
2 samples (Table 4.2, Bishnupaduka and Panmara) in 10-fold dilute samples. It is not
known whether or not coliforms can survive the murcha fermentation (a temperature of as
high as 40C). The coliforms detected in the samples could also be due to post-handling
contamination.

Table 4.2 Microbiological properties of murcha samples

Murcha source TPC Yeast count Mold count Coliform count

(cfu/g) (cfu/g) (cfu/g)
Bishnupaduka 1.35107 4.14107 1.54106 +

Laxmimarga 3.9107 4.58107 3.08105 ND

Udayapur 1.25108 2.12108 8104 ND

Saangu 6.05107 2.39107 6104 ND

Dandaghopa 4.48107 2.28107 1.25106 ND

Panmara 4.76106 8.97106 1.64105 +

Belbari 1.41107 5.02107 3.8105 ND

Kerabari 1.21107 5.6107 4.8105 ND

ND = not detected, cfu = colony forming units, + = detected.

The yeast count of Udayapur murcha had the highest yeast and bacteria count but
comparatively low mold count. Udayapur murcha was also found to be significantly
superior to the rest (Table 4.3). This finding is probably a coincidence because murcha
quality depends more on yeast quality than the quantity.

52
4.3 Perception of murcha quality

Physical appearance of murcha such as shape, size and color cannot indicate the quality
of murcha, as standard shape and size for murcha cakes are not established while color
may vary according to raw materials used. The microbiological analysis (for mold and
yeast quality) and test fermentation are of course important in confirming murcha quality
but the former one is not feasible in the rural setting.

In the survey, murcha quality was found to be assessed at two levels, viz., (i) by the
producer, and (ii) by the customer. Based on information collected from murcha-makers
and buyers, a good (presumptive) quality murcha has puffed appearance, sweet alcoholic
smell, and shows effervescence (air bubbles) when murcha powder is added to saliva. The
latter property relates to a scientific reason: saliva contains amylase and some traces of
sugars which readily activates yeasts when murcha powder is added to the saliva.

4.4 Screening of murcha

Jand produced in this preliminary step were subjected to liquefaction and sensory tests
(taste, smell, sourness and overall) as the criteria for selection. ANOVA of samples for
liquefaction, taste, smell, sourness and overall showed significant difference (p < 0.5)
between murcha samples whereas there was no difference among the panelists. Murcha
samples from Kerabari and Dandaghopa were superior to other samples in terms of
liquefaction property (Table A-1 of the Appendix D). Since liquefaction property comes
from the amylase elaborated by molds (Rai, 2016), it is apparent that the molds present in
the murcha have different amylolytic ability. Similarly, in case of taste attributes murcha
from Saangu and Udayapur were found to be superior to other samples. Likewise, in case
of sensory attributes like smell and sourness, murcha samples from Udayapur and
Laxmimarga were significantly superior (p < 0.05) to other samples. A summary of
difference in liquefaction properties and sensory attributes between the murcha samples is
given in Table 4.3.

Based on the statistical evidence, two murcha samples, viz., murcha from Dhankuta and
Letang were rejected while the remaining 8 were retained for subsequent tests. Murcha
from Dhankuta was rejected because of strange flavor and strong acetone taste despite
good liquefaction test. Murcha from Letang was rejected because of very sour taste.
Both acetone flavor and excessive sourness are not desirable. The reason behind this
53
property, however, is still not clear.

The acidity and strange flavors may well have been due to dominance of undesirable
bacteria in murcha, (in spite of presence probably good yeasts and molds) but because of
the scope of the present study, these aspects were not attempted.

Table 4.3 Summary of difference between the liquefaction properties of murcha samples
and sensory attributes of jand prepared using the murcha samples

Murcha sample Liquefaction# Taste Smell Sourness Overall

Belbari 3.8a (0.45) 3.8c (0.45) 3.8cd (0.45) 4.2b (0.45) 3.8b (0.45)

Dhankuta 3.8a (0) 2.2b (0.45) 1.2a (0.45) 1.2a (0.45) 1.2a (0.45)

Panmara 3.8a (0.45) 3.8c (0.45) 3.8cd (0.45) 3.8b (0.45) 3.8b (0.45)

Saangu 3.8a (0.45) 4.8d (0.45) 3.6c (0.55) 3.8b (0.45) 4.2bc (0.45)

Udayapur 3.8a (0.45) 5.0d (0) 4.8e (0.45) 4.8c (0.45) 4.4cd (0.55)

Laxmimarga 4.0a (0.45) 4.2c (0.45 4.2d (0.45) 4.8c (0.45) 4.2bc (0.45)

Letang 4.0a (0) 1.2a (0.45) 1.2a (0.45) 1.4a (0.55) 1.2a (0.45)

Bishnupaduka 4.2a (0.45) 4.2c (0.45) 3.0b (0) 4.8c (0.45) 4.0bc (0)

Dandaghopa 4.8b (0.45) 4.8d (0.45) 4.0cd (0) 4.8c (0.45) 4.8d (0.45)

Kerabari 4.8b (0.45) 3.8c (0.45) 3.8cd (0.45) 3.8b (0.45) 3.8b (0.45)

#
Visually assessed for limpid liquid in the mash, no physicochemical tests done.

Figures are arithmetic means of 5 responses (by 5 panelists). Values in the parenthesis
indicate sample standard deviation (n = 5). Means with similar superscripts in the column
are not significantly different (p < 0.05).

4.4.1 Screening of molds

Molds were successfully screened from the screened murcha samples by using a
modification of the screening techniques described by Malloch (1997) and KC et al.
(2004). In each of the samples, screening showed the dominance of a single-type,
morphologically distinct mold colony. The screened molds differed slightly with respect to
abundance and color of spores. Under conditions described in the methodology, all of

54
those mold isolates were found to saccharify cooked rice (Fig. 4.1) and sensory analysis of
the saccharified rice was carried out. ANOVA of samples for liquefaction, sweetness,
smell, sourness and overall shows significant difference (p < 0.5) between mold isolates
whereas there is no difference among the panelists. The statistical part of the test data is
given in Table 4.4.

Fig. 4.1 Saccharification of cooked rice by Udayapur mold

Table 4.4 Summary of difference between the liquefaction properties of mold isolates and
sensory attributes of saccharified rice prepared using the isolates

Mold isolates Liquefaction Sweetness Smell Sourness Overall

Belbari 4.2b (0.45) 3.2a (0.45) 4.2c (0.45) 3.2cd (0.45) 3.8c (0.45)
Laxmimarga 2.8a (0.45) 4.0c (0) 3.0b (0) 2.0b (0) 3.0b (0)
Panmara 3.8b (0.45) 4.0c (0) 2.8b (0.45) 2.8c (0.45) 2.8b (0.45)
Udayapur 5.0c (0) 4.0c (0) 4.0c (0) 3.8e (0.45) 5.0d (0)
Kerabari 4.8c (0.45) 4.0c (0) 1.2a (0.45) 1.0a (0) 1.2a (0.45)
Dandaghopa 5.0c (0) 3.6b (0.55) 3.8c (0.45) 3.4de (0.55) 4.2c (0.45)
Bishnupaduka 3.8b (0.45) 4.0c (0) 3.2b (0.45) 1.0a (0) 2.8b (0.45)
Saangu 5.0c (0) 4.0c (0) 4.0c (0) 3.2cd (0.45) 4.0c (0)

Values are arithmetic means of scores given by 5 panelists. Figures in parenthesis denote
sample standard deviations (n = 5). Means in the column bearing the same superscript(s)
are not significantly different at 5% level of significance.

Table 4.4 indicates that the overall score heavily depends on smell and sourness of the

55
product. Liquefaction and sweetness are both related to amylolytic property of the mold.
Although, this has an important bearing on the alcohol content of the product by
subsequent yeast fermentation, mold isolates from Panmara, Bishnupaduka, Kerabari and
Laxmimarga murcha samples were rejected by the panelists as these mold isolates failed to
meet the criteria set up for smell and sourness attributes of jand. Hence the best (Udaypur)
was taken for koji making and murcha formulation. Koji and murcha were prepared as
given in Section 3.2.3.2 3.2.3.3.

4.4.2 Identification of mold isolate

The mold isolate rom Udayapur murcha was characterized by microscopic examination
and colony characteristics. The isolated mold was identified to be Rhizopus oryzae
(Amylomyces rouxii). The appearances of preserved culture in flat bottles are given in Fig.
4.2. The KEYS to the mold genera (Malloch, 1997) referred for the identification are given
in Appendix B-1. The microscopic photographs of the isolated molds are shown in Fig.
4.3.

4.4.3 Preservation of mold culture

The mold isolate was successfully preserved by sporulation in molasses agar plates and
storage at both refrigeration and room temperature. The cultures remained stable even after
9 months of storage. Two-month old plate and bottle cultures are shown in Fig. 4.2. The
mold culture could also be maintained as dried koji in sterile wheat bran. This later served
as a stock culture.

Udayapur mold

Fig. 4.2 Preservation of mold isolates in flat bottle and plate

56
 At 45 objective At 20 objective

Fig. 4.3 Rhizopus oryzae under microscope

4.4.4 Screening of yeasts

Yeasts were successfully screened from the murcha samples by using a technique given by
Rai (2016). Fig. 4.5 shows spread-plating of molasses agar medium for the isolation. All of
the murcha samples were found to harbor different type of fermentative yeasts with
different morphological characteristics and fermentation vigor. Fermentation vigor was
determined by trial fermentation test. Thus, the fermented molasses broth was subjected to
sensory analysis (9-point hedonic rating: 9 = like extremely, 1 = dislike extremely) and the
statistical part for which is given in Table 4.5. The specimen card for the score appears in
the Appendix A-3. ANOVA of samples for alcoholic property, clarity, smell, color and
sourness attributes of fermented broth shows significant difference (p < 0.5) between yeast
isolates whereas there is no difference among the panelists (Appendix D). Photograph of
some samples kept for fermentation during the study is shown in Fig. 4.4 (black color and
flocculation at the bottom indicates efficient fermentation).

A B C D E F G H

Fig. 4.4 Trial fermentation in molasses broth

A, B, C, D, E, F, G and H refer to yeast isolates from Bishnupaduka, Udayapur,
Laxmimarga, Kerabari, Belbari, Saangu, Dandaghopa and Panmara

57
 Based on the test data, yeast isolates from Laxmimarga and Udayapur murcha were
found to be reliable and efficient to carry out the work further. To correlate these results,
new approach for yeast screening was carried out which entails the use of inoculum from
fermented mash of matured jand that selects for dominant yeasts. These screened yeasts
were used for trial fermentation test and the results thus obtained from this test were tallied
with the previous results. The results showed no variation between them and hence, it was
confirmed that the yeast growing at the early stage of screening remained dominant
throughout the entire growth period.

Table 4.5 Summary of difference between sensory attributes of fermented molasses broth
prepared using the yeast isolates

Yeast isolates Alcohol Clarity Color Smell Sourness

Bishnupaduka 1.20a (0.45) 5.20a (0.45) 6.20a (0.45) 4.80b (0.45) 5.60c (0.55)

Kerabari 1.20a (0.45) 5.00a (0.71) 6.00a (0.71) 3.60a (0.55) 5.60c (0.55)

Panmara 1.20a (0.45) 4.80a (0.45) 5.60a (0.55) 5.60c (0.55) 2.40a (0.55)

Belbari 1.40a (0.54) 5.40a (0.55) 7.00b (0.71) 6.40d (0.55) 4.80b (0.45)

Dandaghopa 2.80b (0.45) 5.40a (0.55) 6.00a (0.71) 3.80a (0.45) 5.80c (0.45)

Saangu 2.80b (0.45) 5.40a (0.55) 5.60a (0.55) 5.80cd (0.45) 5.80c(0.45)

Laxmimarga 8.20c (0.45) 7.40b (0.55) 8.40c (0.55) 8.40e (0.55) 7.40d (0.55)

Udayapur 8.20c (0.45) 8.40c (0.55) 8.20c (0.45) 8.60e (0.55) 7.40d (0.55)

4.4.5 Identification of yeast isolates

Yeasts were isolated by spread plating on molasses agar as described by Rai and Subba
(2016) (Fig. 4.5). The yeasts isolated from murcha samples of Laxmimarga and Udaypur
were identified to be strains of Saccharomyces cerevisiae. The KEYS used for the
identification (Adam and Moss, 1996) are given in Appendix B-2. The photomicrographs
(1000) of the yeast cells are shown in Fig. 4.6 The auxanographic (modified) and sugar
fermentation testing is shown in Fig. 4.7.

58
 Fig. 4.5 Yeast isolated from Udayapur murcha by spread-plating

10 m

dayapur yeast La mimarga yeast

Fig. 4.6 Yeast isolates (1000) from murcha

Mal Xyl
Gal

Xyl
Lac
Suc Mal

Glu Gal
Lac
Glu
Suc

Udayapur yeast Laxmimarga yeast

Fig. 4.7 Sugar assimilation test to identify yeast

Notations: Suc = sucrose, Glu = glucose, Xyl = xylose;
Lac = lactose, Mal = maltose, Gal = gal.

4.4.6 Preservation of yeast isolates

The yeast isolates were successfully preserved by subculturing in molasses agar plates and
slants and storage at 4C (under refrigeration). They could also be maintained at room

59
temperature (~ 30C) for 15 days in sterile (autoclaved) molasses broth (5 brix) without
any decrease in functional properties.

4.5 Preparation of fermentation starters using selected mold and yeast isolates

The starter types prepared in the laboratory (using the techniques described in PART III,
Section 3.2.3.3) are shown in Fig. 3.4. Using the selected mold (Amylomycess rouxii) and
yeast (Saccharomyces cerevisiae), both screened from Udayapur murcha, starters were
formulated in terms of their proportion. The formulated starters were prepared using
calculated amounts of yeast and mold isolates derived from experimental design (Table
4.6). Among those prepared starters, the best formulation was selected on the basis of
fermentation performance (alcohol), sensory (taste, acidity balance, liquefaction) and
diastatic (amylase) activity. The best formulation was found to be 2.00 g yeast and 0.50 ml
mold isolates. The analyzed statistical data for the same is shown in Table 4.7- 4.10. The
contour plot and Response Surface are given in Fig. 4.8 and 4.9, respectively.

Incubation at 28-30C in a moist chamber (RH ~ 95%) for 48 hrs produced highly
characteristic puffy cakes overgrown by white mycelia. Incubation for more than 3 days
was found to be unsuitable as far as the appearance and handling characteristics of starters
were concerned. Longer incubation periods led to profuse spore formation, thereby making
the cakes black and also rate of respiration increases over time therefore, the temperature
of cake thoroughly rises making them highly humid in nature which may spoil the starter
cakes. The cakes were also difficult to handle because of the tendency of spores to spread
in air readily. Naturally, such propagules not only contaminate the environment (Malloch,
1997) but may also be a cause of allergy upon inhalation.

The green murcha was therefore subjected to drying in a cabinet dryer at 501C to
around 9% moisture content and finally packed in plastic bags. This drying temperature
was found to be non-detrimental to murcha flora. The shelf life of murcha has been
reported to be ~1 year (Tamang et al., 2012) but this could not be ascertained in the present
study because of time constraint. However, the murcha cake developed in the present work
remained infestation-free during the whole dissertation period (~ 9 months), which can be
considered a significant achievement. The circular murcha cakes formulated in the study
are shown in Fig. 4.8. Appendix C shows murcha cakes being made ready for incubation.

60
 Fig. 4.8 Murcha prepared in the laboratory

4.6 Physicochemical and microbiological quality of laboratory starters

The laboratory murcha formulated according to the experimental plan (Table 4.7) using the
chosen yeast (from Udayapur murcha) and mold (also from Udayapur murcha, Section
4.4.1) was analyzed for amylase activity, alcohol content and sensory properties (taste, acid
balance, and liquefaction).

4.6.1 Alcohol content

The alcohol content in in cooked rice after fermenting for 15 days at 30C using 1% starter
in 100 g of cooked rice ranged from 4.3 12.35% abv (Table 4.8). The ANOVA table of
the variate (Response 1: % abv) is given in the Appendix D. A summary of statistical test
for alcohol production by varying yeast levels is given in Table 4.8. The same by varying
koji levels in given in Table 4.9.

61
Table 4.7 Responses of the experimental plan (Table 3.2, Section 3.2.3.3)

Factor 1 Factor 2 Response 1 Response 2

Std Run A: Mold B: Yeast Alcohol Amylase
(g)a (ml)b (% abv) (Unit)c
10 1 2 0.5 12.35 12.46

11 2 2 0.5 12.28 12.46

8 3 2 0.75 8.12 6.23

3 4 1 0.75 5.31 15.58

1 5 1 0.25 4.3 12.46

5 6 1 0.5 7.1 18.69

7 7 2 0.25 7.5 12.46

2 8 3 0.25 8.65 17.14

12 9 2 0.5 12.34 12.46

13 10 2 0.5 12.4 12.46

9 11 2 0.5 12.3 12.46

6 12 3 0.5 11.45 14.02

4 13 3 0.75 11.6 14.02

(g)a = g of koji (1 g koji ~ 108 Rhizopus oryzae spores): (ml)b = ml of suspension of yeast
isolate (Saccharomyces cerevisiae), where 1 ml ~ 106 cells; (Unit)c = amount of murcha
required to liberate 1 M equivalent of maltose in 1 min at 40C.

Table 4.8 Summary of statistical test for alcohol production by varying yeast levels

Source of variation Amount of yeast (ml) Alcohol (% abv) LSD (5%)

0.25 6.82 (2.25)a

Yeast 0.5 6.34 (2.81)ab 2.58

0.75 10.3 (3.15)b

Values of alcohol in the column are arithmetic means of three data at corresponding yeast
levels (but varying mold levels). Figures in the parenthesis are sample standard deviation

62
of three data. Values in the alcohol column having the same superscript are not
significantly different at 5% level of significance.

Table 4.9 Summary of statistical test for alcohol production by varying koji levels

Source of variation Amount of koji (g) Alcohol (% abv) LSD (5%)

1 5.57 (1.42)a

Mold 2 9.32 (2.64)b 2.58

3 10.57 (1.66)b

Values of alcohol are arithmetic means of three data at corresponding mold levels (but
varying yeast levels). Figures in the parenthesis are sample standard deviation of three
data. Values in the alcohol column having the same superscript are not significantly
different at 5% level of significance.

Table 4.8 and 4.9 show that the alcohol content is influenced by both mold and yeast
concentration in murcha. The analyzed data show that koji content at concentrations of 2-3
g and yeast concentrations at 0.5-0.75 ml give significantly higher amounts of alcohol.
Both lower levels of koji and yeast gave significantly poorer results. Thus there exists a
range of mold-yeast ratios where alcoholic fermentation is most efficient.

The results can be partly explained on the basis of the roles of amylolytic molds and
fermentative yeasts described by several authors including Haard (1999), Lee (1999),
Tamang (2010), and Rai (2012). In traditional cereal beverages, both alcohol content and
other congeners are important, which again depend on the quality and composition of the
essential microorganisms (their ratio in particular), the type of raw material, and the
process (asepsis in particular) used. This part has been reviewed by Lee (1984), Baltra and
Millner (1974), Haard (1999) and Rai (2006).

ANOVA for response 1 (alcohol content) for surface quadratic model show that model
is significant (Table 4.10). In this case, A, A2, and B2 are significant model terms (Fpr
values bearing the asterisk *).

63
Table 4.10 ANOVA for Response 1 (alcohol content) for surface quadratic model

Source of variation SS df MS F Fpr

Model 98.97 5 19.79 23.02 0.0003*

A-Mold 37.45 1 37.45 43.56 0.0003*

B-Yeast 3.5 1 3.5 4.07 0.0836

AB 0.94 1 0.94 1.09 0.3303

A2 8.45 1 8.45 9.82 0.0165*

B2 28.53 1 28.53 33.18 0.0007*

Residual 6.02 7 0.86

Lack of fit 6.01 3 2.00 918.96 < 0.0001*

Pure error 0.00872 4 0.00218

Cor Total 104.99 12

The Lack of Fit Fpr of 918.96 implies the Lack of Fit is significant. There is 0.01% chance
that a Lack of Fit Fpr this large could occur due to noise. Significant lack of fit is bad, we
want the model to fit. Hence there is room for improvement of experiment and the data.

The general inference from the ANOVA table is that the amounts of mold and yeast in
the starter can significantly alter the amount of alcohol in the cereal mash (hereafter
referred to as beer). This is as e pected because amylolytic molds are responsible for the
saccharification of starch into simple sugars which are simultaneously utilized by the yeast
to produce ethanol as the main product, a combined process also described as parallel
fermentation (Rai and Subba, 2016).

In the present study, the interaction between the amounts of molds and yeasts in the
starter, as far as alcohol yield is concerned, was not found to be significant. Again, this
result can be partly attributed to the comple parallel fermentation pattern that occurs in
cereal beer fermentation. Simple sugars are utilized for growth and deriving energy by
both yeasts and molds. This apart, yeasts also utilize the sugars to produce ethanol, the
primary metabolite. The synthesis of alcohol occurs only under Crabtree effect", which
implies that the reducing sugar level must always be more than 5% in the fermenting mash
(Rai, 2012). Considering the varying fates and rates of utilization of simple sugars, it is

64
presumably difficult to predict the nature of interaction(s) between the quantities of
amylolytic molds and fermentative yeasts in cereal fermentation that is predominantly of
solid-substrate nature. Thus, although the interaction between molds and yeasts in cereal
fermentation is clear, the effect(s) of their quantities in the starter is still elusive.

Nevertheless, equations of the quadratic model obtained from response surface can still
be useful. The contour plot (iso-response curve) and 3D response curve of the interaction
of amounts of yeasts and molds vis--vis the response alcohol are given in Fig. 4.9 and
4.10. The final equations of the quadratic model are:

(i) In terms of coded factors (where A = mold, B = yeast, Table 4.8)

Alcohol (% abv) 11.96 2.50 A 0.76 B 0.49 A B 1.75 A2 3.21 B2

(ii) In terms of actual factors

Alcohol (% abv) 12.47 8.52 Mold 50.59 Yeast 1.94 Mold Yeast
1.75 Mold 2 51.42 Yeast 2

Design-Expert Software Alcohol (% abv)

0.75
Alcohol 6.40
Design Points 7.49

0.63
X1 = A: Mold
X2 = B: Yeast 11.86 12.40 12.77 12.95
10.77
0.50 5

9.68
8.58
0.38
7.49
6.40
5.31
0.25
1.00 1.50 2.00 2.50 3.00
A: Mold concentration (g)
Fig. 4.9 Contour plot showing relation of amounts of mold and yeast in starter for the
response alcohol content (% by volume)

Fig. 4.9 shows that some relations exist (although not statistically significant) between
the amounts of mold and yeast in the starter for alcohol production. Under the short
duration of fermentation carried out in the experiment, the plot shows that 12.95% abv is
the maximum that can be achieved. The corresponding mold and yeast contents are 3 g and

65
0.55 ml. In the trade (semi-commercial) fermentation, however fermentation time is
usually more than 15 days, and often several months (Rai, 1991). The alcohol content may
then slowly reach up to 18% (because of parallel fermentation, with no role of mold in the
later phases).

During preparation of murcha, in commercial scale in particular, the amounts of koji

and yeast to be used become critical from cost perspective. Setting up the goal (in Design
expert) to minimize amounts of mold and yeast in murcha while at the same time
ma imize alcohol content in the beer to around 12.5% (which is the normal
concentration in jand), the predicted data for maximum desirability, as generated by
Design expert are:

Constraints
Name Goal Lower Limit Upper Lower Upper Importance
limit Weight Weight

Mold minimize 1 3 1 1 5

Yeast minimize 0.25 0.75 1 1 5

Alcohol maximize 10 12.4 1 1 5

Solutions

Number Mold Yeast Alcohol Desirability

1 2.08 0.46 11.97 0.60 Selected

1 Solution found

Fig, 4.10 is a 3D curve of the contour plot given in Fig, 4.9. The design points X1=2.00
and X2=0.5 gives 12.3% abv while the predicted points for desirable solution (for
maximizing alcohol) are X1 = 2.08 and X2 = 0.46.

The result indicates that there is ample scope for manipulating the amounts of mold and
yeast for preparing murcha. However, since the Lack of fit in the model is significant, it is
evident that the final model can be reached only after several replicated studies.

66
 Alcohol = 12.3%
Std#9, Run#1
X1= 2.00
X2 = 0.5
Design-Expert Software
Alcohol
Design points above 13
predicted value
10.8
Design points below
predicted value 8.6

X1 = A: Mold 6.4
X2 = B: Yeast
4.2
3.00
0.75 2.50
0.63
0.50 2.00
0.38 1.50
0.25 1.00

Fig. 4.10 3D-Response surface curve of the interaction of amounts of yeasts and molds vis-
-vis the response alcohol

4.6.2 Amylase activity

The highest and the lowest amylase activity (Response 2, Table 4.7) in the laboratory
murcha were 18.69 (at X1 = 1, X2 = 0.5) and 6.23 (at X1 = 2, X2 = 0.75). However, the
amylase content of murcha was not found to significantly change (at 5% level of
significance) with the change in mold-to-yeast ratio in the formulation (Table 4.11).

Table 4.11 ANOVA table of amylase activity (Response 2)

Source of variation df SS MS VR F pr.

Mold 2 49.109 24.554 2.68 0.183

Yeast 2 15.08 7.54 0.82 0.502

Residual 4 36.691 9.173

Total 8 100.88

Explanation similar to that given for alcohol concentration can be offered here also. It is
obvious that the parallel and solid-state natures of fermentation are the main sources of
complication (unpredictability). The highly variable micro-environment existing in the
67
murcha cake during incubation may be another reason for variations in amylase activity.
As such, the amylase level in murcha is of minor significance (Rai and Subba, 2016).
Unlike koji used in sake (Japanese wine) fermentation, the purpose of murcha is to serve as
an inoculum rather than as an enzyme source. In jand production, both the growth of mold
and the concomitant production of amylase are thus obligatory (Rai and Subba, 2016). On
the contrary, sake production uses koji that already contains preformed amylase
(Steinkraus, 1995), which means that the growth of molds has no role in the actual sake
fermentation: koji is merely used as a source of preformed amylase.

4.6.3. Sensory properties

The sensory property of the formulated starters (murcha) were evaluated by sensory
analysis of the fermented rice mash prepared in (Section 4.3.1) by 5 panelists using 5-point
rating. The results consistently show that formula A (containing 2 g mold and 0.5 ml yeast
suspension in 50 g rice flour) that has the best property. Summary of ANOVA test appears
in Table 4.12. This is an ample evidence that mold-yeast ratio in starter formulation does
indeed matter.

Table 4.12 Summary of ANOVA of sensory attributes of fermented rice mash

Sample Liquefaction# Acid balance* Taste Overall

A 5.0c (0) 5.0f (0) 4.8e (0.45) 4.8d (0.45)

B 4.2b (0.45) 4.8ef (0.45) 4.0cd (0) 4.0c (0)

C 3.8a (0.45) 3.8bc (0.45) 3.6bc (0.55) 3.8bc (0.45)

D 4.0ab (0) 3.4ab (0.55) 3.4ab (0.55) 3.4ab (0.55)

E 4.2b (0.45) 4.4de (0.55) 4.0cd (0) 4.0c (0)

F 4.0ab (0) 4.2cd (0.45) 4.4de (0.55) 4.2c (0.45)

G 4.0ab (0) 3.2a (0.45) 3.2ab (0.45) 3.2a (0.45)

H 4.0ab (0) 3.2a (0.45) 3.0a (0) 3.2a (0.45)

I 4.0ab (0) 5.0f (0) 4.0cd (0) 4.0c (0)

LSD (5%) 0.35 0.57 0.50 0.50

Liquefaction# and Acid balance* was a subjective, sensory evaluation

68
 A = Starter prepared using 2 g mold and 0.50 ml yeast isolates
B = Starter prepared using 1 g mold and 0.50 ml yeast isolates
C = Starter prepared using 3 g mold and 0.75 ml yeast isolates
D = Starter prepared using 1 g mold and 0.75 ml yeast isolates
E = Starter prepared using 3 g mold and 0.50 ml yeast isolates
F = Starter prepared using 1 g mold and 0.25 ml yeast isolates
G = Starter prepared using 3 g mold and 0.25 ml yeast isolates
H = Starter prepared using 2 g mold and 0.25 ml yeast isolates
I = Starter prepared using 2 g mold and 0.75 ml yeast isolates

Figures are arithmetic means of 5 responses (by 5 panelists). Values in the parenthesis
indicate sample standard deviation (n = 5). Means in the column bearing the same
superscript(s) in the column are not significantly different (p < 0.05).

4.6.4 Final fermentation using optimized laboratory murcha

The optimized murcha (prepared in the laboratory) that was once again subjected to
analysis for physicochemical (moisture, pH, dimension), microbiological (yeast, coliform,
mold and aerobic count) and fermentation (alcohol content, esters, aldehydes) properties.
The method is described in detail in Section 3.2.5.

4.6.5 Microbial properties of lab murcha (final)

The coliforms were found to be absent in all the laboratory murcha. The bacterial count of
the prepared murcha ranged from 100-153 cfu/g, probably due to post-handling
contamination (during drying, packaging, etc.). The study shows how the practice of simple
hygiene and sanitary principles can lead to significant improvement in the quality of murcha.

Several authors, including Nout (1992), Tamang and Sarkar (1995), Rai (2006) have
assessed the quality attributes of murcha in terms of physicochemical and microbiological
properties of murcha cake. However, standard value for these parameters for good quality
murcha has not been established as yet. Moreover, there is no any standard protocol available
for the quality assessment of murcha till date. Therefore, quality assessment of murcha almost
totally based on perception of people and experience shared by the murcha makers.

69
4.6.6 Physicochemical properties of optimized murcha

The optimized murcha had following physicochemical properties:

pH : 5.4
Moisture : 9%
Dimension (l, b, h) cm : 8.5, 8.5, 1.0
Yeast count : 8.88107 cfu/g
Mold count : 1.3104 spores/g

The above values, pH and microbial counts are within the values reported by Tamang and
Sarkar (1995).

4.6.7 Comparison of performance of optimized murcha

The optimized starter was finally tested on both cooked rice and cooked finger millet (~ 2 kg
each) for the comparative study. The methodology is described in Section 3.2.5. The raw
data and ANOVA for the variables acidity, alcohol content, ester, volatile acidity and
aldehyde are presented in the Appendix D. A summary of the same is given in Table 4.13.

Table 4.13 Summary of statistically analyzed data on physicochemical properties of millet

and rice jand.

Sample Acidity(a) Alcohol Volatile Ester(d) Total

content(b) Acidity(c) aldehydes(e)

Millet jand 1.07 (0.11) 10.09 (0.52) 0.57 (0.04) 152.25 (2.9) 24.15 (0.59)

Rice jand 0.7 (0.02) 14.18 (0.33) 0.35 (0.02) 181 (2.7) 18.28 (0.31)

LSD (5%) 0.133 0.753 0.054 4.83 0.824

Difference 0.367 4.09 0.233 28.75 5.87

Acidity(a): % lactic acid; Alcohol content(b): % abv; Volatile acidity(c): g of acetic acid/100
lit absolute alcohol; Ester(d): g of ethyl acetate/100 lit absolute alcohol; Total aldehydes(e):
g of acetaldehyde/100 lit absolute alcohol.

Figures are arithmetic means of 4 replicates. Values in the parenthesis indicate sample
standard deviation (n = 4).

70
 As is evident from Table 4.13, all the chemical properties of millet and rice jand differ
significantly (p < 0.05). Although literatures explaining these differences are not available,
it is easy to speculate that raw material type is one of the chief reasons for the difference.
Acidity in millet jand is almost always greater than that in rice jand, which has been
observed by other authors also (Rai, 1991; Rai, 2015), although the reason for this has not
been given.

71
 PART V

Conclusions and recommendations

The present study was aimed at formulating starter (murcha) in the laboratory by
incorporating different ratios of pure mold and yeast cultures from traditional murcha.
Based on the study, following conclusions have been drawn:

1. Traditional murcha is very prone to insect infestation, even when carefully packed
and stored.

2. Amylomyces rouxii (Rhizopus oryzae) and Saccharomyces cerevisiae are the

dominant microfloras present in murcha from Eastern region of Nepal.

3. Murcha of very consistent and good quality can be easily prepared using the
method developed in this work. This entails use of pure cultures of mold (in the
form of koji) and yeast suspension in a carefully worked out proportion.

4. The new method developed in the present work can have following far-reaching
implications:

a. Murcha makers do not have to depend on herbs/plants (which are

sometimes rare and thus pose threat to floral diversity)

b. Murcha of consistent quality can be prepared (no failure)

c. Very easy to prepare

d. Prolonged shelf-life of murcha (reduced or no infestation)

e. Possibility for cross-border trade

Based on the present work, following recommendation can be made:

1. Emphasis must be placed on sanitary and phytosanitary aspects of murcha (from

quality and commercialization perspectives).

2. Attempts can be made to develop multistrain starters for increased stability.

3. Further study on genetic stability of the pure wild culture is needed (as this may
change overtime because of a host of reasons).
 PART VI

Summary

The main objective of the study was to formulate amylolytic starter (murcha) in the
laboratory using different ratios of yeasts and molds screened from traditional murcha.
Murcha samples for the work were collected from 10 sites (districts in parenthesis) of
Eastern Nepal, viz., Saangu (Dhankuta), Udayapur (Udayapur), Kerabari (Susari),
Dhankuta (Dhankuta), Laxmimarga (Morang), Belbari (Morang), Dandaghopa (Sunsari),
Bishnupaduka (Susanri), Panmara (Sunsari) and Letang (Morang). Specific objectives
included characterization and preservation of the isolated cultures, koji development, and
performance tests of the prepared murcha. Amylolytic molds and fermentative yeasts were
screened (in a series of trial fermentations in molasses broth as well as cooked rice),
characterized (microscopic examination, sugar assimilation/ fermentation tests) and
preserved (subculturing in molasses agar) for the formulation study. Koji (containing 108
spores of Amylomyces rouxii/g koji) and yeast suspension (containing 106 actively growing
cells of Saccharomyces cerevisiae/ml) were used for the preparation of murcha in the
laboratory.

Experimental design (Face-centered, 2 Factors, 3 Levels, 13 Runs) was set up using

Design Expert to formulate murcha using koji (1, 2, and 3 g) and yeast (0.25, 0.50, and
0.75 ml) in 50 g of rice flour (moisture ~ 30% and particle size < 280 ). The murcha
cakes were incubated at ~ 30C for 2 days and then dried in an electrical cabinet dryer at
501C until ~ 9% moisture content. The best formulation was selected on the basis
physicochemical analysis (bulk density, amylase activity, moisture, and pH), performance
test (alcohol production), sensory analysis (taste, liquefaction and acid balance of
fermented rice beer) and statistical analysis (ANOVA and Response Surface curve). The
best murcha was once again used to prepare rice and millet jand and physicochemical
(alcohol and acidity) analysis carried out. Ester, volatile acidy and aldehyde were also
determined.

Mold (identified as Rhizopus oryzae / Amylomyces rouxii) and yeast (identified as

Saccharomyces cerevisiae) from Udayapur murcha were found to be the most suitable.
The murcha formulation with 2 g koji (equivalent to 2108 spores of Amylomyces rouxii)
and 0.5 ml yeast suspension (equivalent to 5105 live Saccharomyces cerevisiae cells) per
50 g of rice flour was found to be significantly superior (p < 0.05) to other murcha (having
other mold-yeast ratios) in terms physicochemical and sensory quality. In conclusion, the
ratio of mold to yeast in murcha formulation is critical in the preparation of murcha of very
good quality. The murcha thus produced remained free from insect infestation during the
whole dissertation period (> 9 months), which is a great improvement compared to
traditional murcha making which generally become infested with insects within a month or
two of preparation. The method developed in this work is sustainable (does not depend on
harvesting of exotic plants), reliable (in terms of culture purity), consistent (in terms of
quality), and applicable even in the rural setting.

74
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 Appendices

Appendix A-1
Database of murcha plants from Eastern Nepal
Plant Vernacular name Plant part(s) used
Ananas comosus (L) Mer. Anaras Leaf
Anaphalis triplinevis Buki phool Whole
Artocarpus heterolphyllus Lamk Rukh katahar Fruit-stalk, leaf and bark
Asparagus racemosus Willd Kurilo Root
Buddleja asiatica Lour Bhimsenpati Leaf and tender shoot
Capsicum annum L. Khursani Fruit
Carica papaya L. Mewa Root
Centella asiatica L. Ghodtapre Whole
Clematis grewiaeflora Mahagagro Whole
Clerodendrum indicum (L.) Kize Bhatu Tender shoot
Christella appendiculata (Bl.) Holtt Pire uneu Tender leaf
Dolichos lab-lab L. Hiude simi Root
Drymaria cordata Willd. Abhijalo Whole
Elephantopus scaber L. Mulapate Root
Elephantopus sp. Anglah lena Whole
Geniosporum coloratum D. Don Sengreng Tender shoot and flower
Ichnocarpus fructescens R. Br Dudhe Whole
Ichnocarpus sp. Tite Root bark
Inula sp. Chhatre Whole
Juglan regia L. Okhar Bark
Ophiopogon parviflorus (Hook.f) Hara Lasunpate Root-tuber
Piper chaba Hunder Chabo Whole
Piper longum L. Pipla Whole
Piper nigrum L. Marich Whole
Plumbago zeylanica L. Chitu Whole
Polygala abyssinica Buch. Hum. Gahate jhar Whole
Polygala arillata Buch. Hum Khedeii Root-bark and flower
Polygala triphyla Buch. Hum. Pulukna Whole
Polygala sp. Angtellek Whole
Pteridium revolutum (Bl.) Nakai Uneu Tender leaf
Scoparia dulcis L. Chini jhar Whole
Sida acuta Burm F Khareto Leaf
Spergula arvensis L. Lwangta Whole
Spilanthus acmella (L.) Less Pire jhar Whole
Vernonia cinerea (L.) Less Phulange Whole
Vernonia sp. Nighare Whole
Woodfordia fructicosa Kurtz Dhaenro Flower
Zingiber officinale Roxb. Aduwa Rhizome

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Appendix A-2
Semi-structured questionnaire

Survey of murcha-making site

Objectives
1. Collect information on murcha plants Survey site: ..................................
2. Indigenous murcha making protocols Geographical coordinates: ............
3. Murcha trade and socio-economic contribution Population: ...................................
Main occupation: ..........................
Ethnic distribution: .......................

Information on key informants Information on respondent

Name:............................... Name:...........................................
Age and sex: .................... Age and sex: ................................
Education: ........................ Education: ....................................
Address: ........................... Address: .......................................
Profession:........................ Profession:....................................

Questionnare for respondents

1. How long have you been in this business?
.......................................................................................................................................
2. Who taught you this art?
......................................................................................................................................
3. How many other murcha makers do you know?
.....................................................................................................................................
4. What is your annual income from murcha?
.....................................................................................................................................
5. How much do you sell murcha weedly
.....................................................................................................................................
6. How do you use the earning from murcha?
.....................................................................................................................................
7. What plants do you use for murcha making?
.....................................................................................................................................

85
Appendix A-3.1
Sensory evaluation card for 5-point hedonic rating

Specimen Evaluation Card

HEDONIC RATING TEST

Name: .. Product: ..
Date:

Taste these samples and check how much you like or dislike each one. Use the appropriate
scale to show your attitude by checking at the point that best describes your feeling about
the sample. Please give a reason for this attitude. Remember you are the only one who can
tell what you like. An honest expression of your personal feeling will help us.

Like extremely = 5; Like moderately = 4; Neither like nor dislike = 3; Dislike moderately
= 2; Dislike extremely = 1.

Sample Liquefaction Acid balance Taste Overall

A
B
C
D
E
F
G
H
I

Reason: Signature: .

Source: Ranganna (1986)

86
Appendix A-3.2
Sensory evaluation card for 9-point hedonic rating

Specimen Evaluation Card

HEDONIC RATING TEST

Name:.. Product:.
Date:

Taste these samples and check how much you like or dislike each one. Use the appropriate
scale to show your attitude by checking at the point that best describes your feeling about
the sample. Please give a reason for this attitude. Remember you are the only one who can
tell what you like. An honest expression of your personal feeling will help us.

Like extremely = 9; Like very much = 8; Like moderately = 7; Like slightly = 6; Neither
like nor dislike = 5; Dislike slightly = 4; Dislike moderately = 3; Dislike very much = 2;
Dislike extremely = 1.

Sample Alcohol Clarity Color Smell Sourness

1
2
3
4
5
6
7
8

Reason: Signature: .

Source: Ranganna (1986)

87
Appendix B-1
Keys to some common genera of molds

GROUP I
1. Spores single-celled 2
1. Spores with more than one cell 12
Usually this is obvious. Occasionally spores may have darkened areas resembling
septa, but these will not be visible in optical section when you focus on the centre of
the spore
2. Colonies, spores, and other tissues colorless or brightly colored 3
2. Colonies, spores, and/or other tissues dark colored 8
The dark color is either brown or black. The best indication of dark colors comes
from looking at the culture itself, either directly or with a dissecting microscope.
Under the compound microscope many brown structures will appear nearly colorless.
3. Spores produced in chains 4
Often chains of spores break apart thoroughly when placed in a water mount. In
many species of Aspergillus and Penicillium a few spores will remain together in
a group, so that you can assume that they were originally in chains. On the other
hand, species of Cladosporium produce chains of spores that completely disassociate
on contact and leave no clue about their original orientation. The easiest way to
check this is to examine the colony under the 10 objective of a compound
microscope, being careful not to get spores on the lens.
3. Spores not produced in chains 6
4. Conidiophores with a swollen head or vesicle bearing bottle-shaped phialides
Aspergillus
4. Conidiophores not swollen at apex 5
5. Spores in unbranched chains, borne from clusters of cylindrical to bottle-
shaped phialides; colonies usually green Penicillium
Compare with Paecilomyces (GROUP II), Gliocladium (GROUP III), and
Scopulariopsis (GROUP III).
5. Spores borne in branching chains from undifferentiated conidiophores; colonies
often very fast growing and pink Chrysonilia
6. Spores borne in a sporangium with a columella; often with only the columella
evident as a swollen hyphal tip; hyphae not septate Mucor
Compare Rhizopus (GROUP I), Mortierella (GROUP II), Absidia, Circinella
(GROUP V), and Zygorhynchus
6. Spores produced externally; hyphae septate 7
7. Conidiophores well-developed and usually with a central axis; very fast growing and
with conidiophores usually produced in small cushions of hyphae; often green
Trichoderma
Compare with Verticillium (GROUP II) and Gliocladium (GROUP III)
7. Conidiophores poorly developed or lacking; phialides produced singly along the
88
 vegetative hyphae; hyphae often aggregated into ropes; seldom or never green
Acremonium
Compare with Verticillium (GROUP II), Sporothrix (GROUP IV), and Phialophora
(GROUP IV)
8. Spores in chains, produced externally 9
8. Spores not in chains, produced inside sporangia or fruiting bodies (pycnidia) 10
9. Conidiophores with a swollen head or vesicle bearing bottle-shaped
phialides; conidial chains unbranched Aspergillus
9. Conidiophores lacking a swollen apex; spore chains often branched; spores often
both 1- and 2-celled Cladosporium
10. Spores produced inside a fruiting body (pycnidium) with a cellular wall;
hyphae septate Phoma
Compare Pyrenochaeta (GROUP IV) and Microsphaeropsis (GROUP IV) and
also be sure that asci are not present at a very early stage
10. Spores produced within a sporangium with a columella, often with only the
columella evident as a swollen hyphal tip; hyphae not septate 11
11. Sporangiophores with rhizoids (branched roots) at base Rhizopus
Compare with Absidia
11. Sporangiophores lacking rhizoids Mucor
Compare with Mortierella (GROUP II), Absidia, Circinella (GROUP V), and
Zygorhynchus
12. Spores with transverse septa only 13
12. Spores with both transverse and vertical septa 14
13. Spores dark, produced in branched chains Cladosporium
13. Spores colorless or brightly colored, mostly with more than two cells, often canoe-
shaped, usually produced in slimy masses; colonies often pink Fusarium
Compare Cylindrocarpon (not treated here), Candelabrella, Monacrosporium (all
GROUP III), and Trichophyton (GROUP V)
14. Spores usually in chains, usually club-shaped; colonies grey to brown Alternaria
Compare Ulocladium and Stemphylium (GROUP II)
14. Spores in clusters but not in chains, usually spherical; colonies often (but not
always) bright orange or yellow and purplish in reverse Epicoccum
Compare with Stemphylium (GROUP II)
GROUP II
1. Colonies composed of hyphae, or at least with some hyphae present 2
1. Colonies lacking hyphae; short chains of "budding" cells may be produced 16
2. Spores 1-celled 3
2. Spores with more than one cell 14
3. Spores and hyphae colorless or brightly colored 4
3. Spores and/or hyphae dark colored 10

89
4. Spores produced in chains 5
4. Spores not produced in chains 7
5. Spores produced from small clusters of tapering phialides, often rather pointed at
the ends Paecilomyces
Compare with Penicillium (GROUP I) and Verticillium (GROUP II)
5. Spores produced by the simple fragmentation of hyphal segments into individual
cells 6
6. Colonies very slow growing (slower than 5 mm/week), often grey, often with a
strong earthy odour; hyphae usually less than 1 in diameter Streptomyces
6. Colonies growing faster, with a fruit-like odor or odorless, hyphae larger
Geotrichum
Compare with Geomyces (GROUP IV)
7. Spores produced in sporangia, with sporangia often broken and represented only
by simple blunt sporangiophores (no swollen columella); colonies often velvety in
texture and pink to brown Mortierella
Compare with Mucor (GROUP I) and Absidia
7. Spores produced externally 8
8. Spores produced in large numbers and completely covering the surface of large
terminal cells; cells of conidiophores often flattening in alternating planes as they dry;
colonies often producing black stony sclerotia Botrytis
Compare with Chromelosporium (not treated here)
8. Spores produced at the tips of terminal cells and never covering them; cells of the
conidiophore not flattening characteristically upon drying 9
9. Conidia produced in small round masses at the tips of phialides; phialides in
whorls, tapering gradually to a very narrow tip Verticillium
Compare with Acremonium (GROUP I)
9. Conidia produced singly at the ends of short branches; or in short chains, not on
phialides; spore-producing cells not in whorls Chrysosporium, Sepedonium and
Trichophyton (both GROUP V) and Geomyces (GROUP IV) are similar
10. Spores produced in sporangia or in fruiting bodies 11
10. Spores produced externally 12
11. Spores produced within densely hairy fruiting bodies (perithecia), very dark;
asci present when young Chaetomium
11. Spores produced in sporangia (Go back to 7)
12. Conidiophores united to form large synnemata that have a sterile base and a spore-
bearing upper part, often accompanied by spores of Echinobotryum (GROUP V)
Doratomyces
Compare with Trichurus and Graphium (both GROUP III)
12. Conidiophores never united to form such structures 13
13. Spores arising in dense masses directly from swellings on the vegetative mycelium;
colonies usually rather flat and moist Aureobasidium
Compare with Exophiala (not treated here)
90
13. Spores completely covering the terminal cells of erect conidiophores; colonies
cottony and rather dry; black sclerotia often present Botrytis
14. Spores with transverse walls only, colorless; colonies often pink; often associated
with eelworms Arthrobotrys
Compare with Trichothecium (GROUP V), Candelabrella and Geniculifera
(both GROUP III)
14. Spores with transverse and vertical walls, dark brown 15
15. Conidiophores more or less straight because of their elongation directly through the
scar of the previous spore, bearing only one spore at a time Stemphylium
Compare with Pithomyces (GROUP IV)
15. Conidiophores often with a slight zigzag appearance due to new growth from just
below the tip, often bearing a spore at each bend Ulocladium
Compare with Pithomyces (GROUP IV) and Curvularia (not treated here)
16. Cells very small, seldom more than 1-2 in diameter, dividing by simple fission
into two equal-sized daughter cells, sometimes containing a single internal spore
Bacteria
16. Cells usually larger than 1-2 in diameter, dividing by budding, with the
daughter cell seen as a small "bubble" arising from the wall of the parent cell,
sometimes containing one or more internal spores (ascospores) Yeasts
Compare Aureobasidium (GROUP II), Candida (GROUP III), and Exophiala
GROUP III
1. Spores 1-celled 2
1. Spores with more than one cell 10
2. Conidiophores united into complex synnemata with a sterile base and fertile
upper part 3
2. Conidiophores solitary, never forming complex structures, sometimes not present 4
3. Spores produced in a large colorless drop of fluid at the tip of the synnema
Graphium
Compare with Pesotum (not treated here)
3. Spores produced along the sides of the upper part of the synnema, dry, interspersed
with loosely coiled hairs Trichurus
4. Spores produced in chains 5
4. Spores not produced in chains 6
5. Spores brown, produced from a cluster of strongly swollen cells (phialides)
Memnoniella (See Stachybotrys)
5. Spores usually grey, tan, or colorless, produced from clusters of bottle-shaped cells
(annellides) Scopulariopsis
Compare with Penicillium (GROUP I) and Scedosporium
6. Spores produced in small clusters at several "nodes" along the length of erect
conidiophores Gonatobotrys
6. Spores apical or conidiophores not obviously well-developed 7

91
7. Spores borne along the length of the hyphae from apparently undifferentiated cells;
colonies white, moist, and flat Candida
7. Spores produced at the apex of distinct conidiophores or phialides; colony appearance
various - 8
8. Conidiophores small and inconspicuous, usually consisting of short cells or branches
functioning as annellides; colonies often black and yeast-like; spores collecting in wet
masses at the apex of the conidiophores, sometimes budding Exophiala
8. Conidiophores large and conspicuous; colonies never yeast-like 9
9. Conidiophores unbranched or rarely very simply so; spores arising from an apical
cluster of swollen cells (phialides) Stachybotrys
9. Conidiophores highly branched; spores borne from clusters of narrow cells
(phialides), produced in a slimy mass Gliocladium
Compare with Penicillium (GROUP I), Scedosporium and Leptographium
(GROUP V)
10. Spores dark brown, rather large, several-celled Bipolaris
Compare with Pithomyces (GROUP IV) and Trichocladium (GROUP V)
10. Spores colorless; usually associated with eelworms 11
11. Spores solitary at the tip of a long unbranched to weakly branched conidiophore
Monacrosporium
11. Several spores on each conidiophore 12
12. Spores produced along the length of an elongating and more or less zigzag
conidiophore Geniculifera
12. Conidiophores producing a series of short branches from a single locus (candelabrum-
like), with each branch bearing a spore Candelabrella
GROUP IV
1. Spores 1-celled 2
1. Spores with more than one cell 11
2. Spores produced within a distinct fruiting body having a hyphal or cellular wall 3
2. Spores borne externally 6
3. Fruiting bodies or spore mass brown or black 4
3. Fruiting bodies and spore mass colorless or brightly colored 5
4. Spores brown; fruiting bodies (pycnidia) lacking spines Microsphaeropsis
Compare with Myrothecium (GROUP V)
4. Spores colorless or brightly colored; fruiting bodies (pycnidia) with spines around the
apical opening Pyrenochaeta
5. Fruiting bodies (cleistothecia) composed of hyphae; usually with an associated
Penicillium (GROUP I) anamorph Talaromyces Compare with Gymnoascus
(GROUP V) and Arachniotus (not treated here)
5. Fruiting bodies (cleistothecia) with a distinctly cellular wall; usually with an
associated Aspergillus (GROUP I) anamorph Eurotium
Compare with Neosartorya and Emericella (neither treated here). Eupenicillium
92
 species (also not treated here) are similar molds with Penicillium anamorphs.
6. Spores distinctly dark brown or black 7
6. Spores colorless or quite pale 8
7. Spores usually spherical and roughened, with two hyphal connections; hyphae not
septate Zygospores of Mucorales
Usually associated with Mucor, Zygorhynchus (see discussion under Mucor,
Rhizopus, etc.).
7. Spores discoid or egg-shaped, often with a colorless band, usually smooth, with only
one connection to the conidiophore; hyphae septate Arthrinium
Compare with Wardomyces and Nigrospora (both GROUP V)
8. Spores in chains (sometimes interrupted by sterile cells) 9
8. Spores not in chains 10
9. Spore chains often characterized by an alternating series of spores and narrow sterile
cells (bead-like in appearance); filaments never dark Geomyces
Compare with Chrysosporium (GROUP II)
9. Spore chains composed of uniformly cylindrical spores, never with alternating sterile
cells; conidiophores often dark Oidiodendron
10. Spores borne from the apex of flask-shaped phialides with a flaring collar
Phialophora
10. Spores borne at the tips of somewhat jagged conidiophores Sporothrix
11. Spores borne in fruiting bodies (pycnidia), 2-celled Diplodia
11. Spores borne externally, with more than two cells Pithomyces
Compare with Trichocladium (GROUP V)
GROUP V
1. Spores 1-celled 2
1. Spores with more than one cell 11
2. Spores borne in dense masses within some kind of structure 3
2. Spores produced externally, never from any kind of compound structure 5
3. Spores produced inside thin-walled sporangia that are recurved on short hooks
Circinella
Compare with Mucor (GROUP I)
3. Spores never produced in recurved sporangia 4
4. Fruiting structure a sporodochium containing a layer of conidiophores; spores in
slimy masses, very dark green Myrothecium
4. Fruiting structure a cleistothecium containing asci (when young) and ascospores;
spores dry at maturity; never associated with phialides Gymnoascus
5. Spores brown to black 6
5. Spores brightly colored or colorless 8
6. Spores roughened, with a prolonged apical snout; often associated with Doratomyces
(GROUP II) Echinobotryum
93
6. Spores smooth 7
7. Spores usually borne in small clusters, usually egg- or bullet-shaped, with a narrow
colorless band (germ slit); may be associated with Scopulariopsis (GROUP III)
Wardomyces
7. Spores solitary, usually spherical to somewhat flattened spherical, often with a germ
slit Nigrospora
8. Conidiophores dark brown, densely branched at the apex and bearing the colorless
spores in a drop of fluid Leptographium
Compare with Verticicladiella and Phialocephala (not treated here)
8. Conidiophores colorless 9
9. Spores completely covering a large swelling at the apex of an erect conidiophore
Oedocephalum
Compare with Cunninghamella
9. Conidiophores not well-developed and lacking a terminal swelling 10
10. Spores relatively large, usually spherical, roughened Sepedonium
Several fungi produce large spherical scultured spores. Important among these are
Histoplasma capsulatum and some Mortierella species. Histoplasma capsulatum is a
serious human pathogen; if you believe that it is present in your culture, do not open
the lid.
10. Spores quite small, usually egg-shaped, smooth; often causing skin infections in
animals, including humans Trichophyton
11. Spores dark (at least some of the cells) 12
11. Spores colorless 13
12. Spores with long appendages at the apex, borne in sporodochia Pestalotiopsis
12. Spores lacking appendages, not in sporodochia Trichocladium
13. Spores 2-celled, produced in chains from the apex of erect conidiophores
Trichothecium
13. Spores usually with more than 2-cells or irregularly 1- to several-celled, not arising
from distinct conidiophores; often causing skin infections in animals, including
humans Trichophyton Compare with Microsporum
Source: Malloch (1997)

94
Appendix B-2
Dichotomous key indicates for common yeast genera

1. Vegetative reproduction by cross-wall formation followed by fission

Schizosaccharomyces*

1. Vegetative reproduction by budding 2

2. Ascospores not formed Candida

2. Ascospores formed 3

3. Nitrate assimilated Hansenula

3. Nitrate not assimilated 4

4. Abundant true mycelium as well as budding 5

4. True mycelium scarce or absent 6

5. Asci formed exclusively on the true hyphae Saccharomycopsis

5. Asci not formed exclusively on the true hyphae Pichia

6. Asci dehiscent Kluyveromyces

6. Asci persistent 7

7. No conjugation preceding ascus formation Saccharomyces

7. Conjugation preceding ascus formation 8

8. Ascospores warty or with ridges Debaryomyces

8. Ascospores spherical and smooth Zygosaccharomyces

*Schizosaccharomyces belongs to the Archiascomycetes

Source: Moss and Adams (1996)

95
Appendix B-3
Key to the identification yeast species

Fermentation of

Saccharomyces cerevisiae + + V + + + +
S. cerevisiae var. ellispsiodeus + + V + + + +
S. fragilis + - + + + + +
S. lactis + - + + + + +
S. rouxii + + - - - + +
S. mellis + - - - - - -
S. carlsbergensis + + + + + + +

Source: Harrigan and McCance (1997)

Summary of species characteristics

Growth and other
Fermentation characteristics

Ln.
no. Species
1068 Saccharomyces
1068 S. arboricolus + + + - - + n + - + + + + - -
1070 S. bayanus var. bayanus + V + + - + - + - + + V V - -
1071 S. bayanus var. uvarum + V + + - + - + - + + V V - -
1072 S. cariocanus + + + - - + - + - + + - + - -
1073 S. cerevisiae + V + + - + - + - + + - V - -
1074 S. kudriavzevii + - + + - + - + + V + - + - -
1075 S. mikatae + + + + - + - + - + + + + - -
1076 S. paradoxus + + + V - V - + - + + - + - -
1077 S. pastorianus + V V + - + - + - V + - + - -

Source: Kurtzman et al., (1998)

96
Appendix C

Plates

Plate-1 Surveying at Saangu (Taplejung) on murcha

Plate-2 Yeast culture preserved in flat bottles and slants

97
 Plate-3 Sieve analysis of rice flour

Plate-4 Murcha being prepared in the laboratory

Plate-5 Electrical cabinet dryer used for murcha drying

98
Appendix D

Tables
Table D-1 ANOVA of liquefaction properties of 10 murcha samples

Source of d.f. s.s. m.s. v.r. F pr.

variation
Sample 9 7.28 0.81 4.76 <0.001
Panelist 4 0.28 0.07 0.41 0.80
Residual 36 6.12 0.17
Total 49 13.68

Table D-2 ANOVA of alcoholic properties of 8 yeast isolates

Source of variation d.f. s.s. m.s. v.r. F pr.

Sample 7 326.58 46.65 207.36 <0.001
Panelist 4 0.5 0.13 0.56 0.697
Residual 28 6.3 0.26
Total 39 333.38

Table D-3 ANOVA of clarity properties of 8 yeast isolates

Source of variation d.f. s.s. m.s. v.r. F pr.

Sample 7 58.78 8.4 28.16 <0.001
Panelist 4 1.25 0.31 1.05 0.4
Residual 28 8.35 0.3
Total 39 68.38

Table D-4 ANOVA of color properties of 8 yeast isolates

Source of variation d.f. s.s. m.s. v.r. F pr.

Sample 7 44.18 6.31 17.77 <0.001
Panelist 4 1.25 0.31 0.9 0.49
Residual 28 9.95 0.36
Total 39 55.38

99
Table D-5 ANOVA of smell properties of 8 yeast isolates

Source of variation d.f. s.s. m.s. v.r. F pr.

Sample 7 123 17.71 60.85 <0.001
Panelist 4 0.25 0.06 0.21 0.93
Residual 28 8.15 0.3
Total 39 132.38

Table D-6 ANOVA of sourness properties of 8 yeast isolates

Source of variation d.f. s.s. m.s. v.r. F pr.

Sample 7 87.2 12.46 43.33 <0.001
Panelist 4 0.35 0.09 0.3 0.88
Residual 28 8.05 0.29
Total 39 95.6

Table D-7 Raw data for comparision of millet and rice jand

Sample Acidity Alcohol content Volatile acidity Ester Aldehyde

Millet 1.16 9.77 0.55 150 24.77
Millet 1.13 10.5 0.63 156 24.53
Millet 0.92 9.89 0.58 150 23.5
Millet 1.05 10.2 0.55 153 23.8
Rice 0.69 14.61 0.33 180 18.03
Rice 0.68 14.54 0.34 185 18.57
Rice 0.70 13.98 0.38 180 18
Rice 0.72 13.5 0.33 179 18.53

100
About the researcher

Name: Sangen Ruma Rai

Mail address: senmingrai@gmail.com
Dharan

Guide: Basanta Kumar Rai, Assoc. Prof.

Co-guide: Dr. Dil Kumar Subba